Untitled - Latin American Journal of Aquatic Research

Transcripción

www.lajar.cl
Latin American Journal of Aquatic Research
ISSN 0718 -560X
www.scielo.cl
CHIEF EDITOR
Sergio Palma
Pontificia Universidad Católica de Valparaíso, Chile
[email protected]
ASSOCIATE EDITORS
Cristian Aldea
Universidad de Magallanes
Chile
Dr. Álvaro J. Almeida Bicudo
Universidad Federal Rural de Permambuco
Brasil
José Angel Alvarez Perez
Universidade do Vale do Itajaí
Brasil
Patricio Arana
Pontifícia Universidad Católica de Valparaíso
Chile
Eduardo Ballester
Universidade Federal do Paraná
Brasil
Claudia S. Bremec
Instituto de Investigación y Desarrollo Pesquero
Argentina
Enrique A. Crespo
Centro Nacional Patagónico, Argentina
Patricio Dantagnan
Universidad Católica de Temuco, Chile
Enrique Dupré
Universidad Católica del Norte
Chile
Diego Giberto
Instituto de Investigación y Desarrollo Pesquero
Argentina
Maurício Laterça-Martins
Universidade Federal de Santa Catarina
Brasil
César Lodeiros-Seijo
Instituto Oceanográfico de Venezuela
Universidad de Oriente, Venezuela
Beatriz E. Modenutti
Universidad Nacional del Comahue
Argentina
Guido Plaza
Pontificia Universidad Católica de Valparaíso
Chile
Luis M. Pardo
Universidad Austral de Chile, Chile
Jesús T. Ponce
Universidad Autónoma de Nayarit, México
Ricardo Prego
Instituto de Investigaciones Marinas, España
Erich Rudolph
Universidad de Los Lagos, Chile
Nelson Silva
Pontificia Universidad Católica de Valparaíso
Chile
Oscar Sosa-Nishizaki
Centro de Investigación Científica y Educación Superior
de Ensenada, México
Ingo Wehrtmann
Universidad de Costa Rica
Costa Rica
Financiamiento parcial de CONICYT obtenido en el Concurso
“Fondo de Publicación de Revistas Científicas año 2015”
Escuela de Ciencias del Mar, Pontificia Universidad Católica de Valparaíso
Casilla 1020, Valparaíso, Chile - Fax: 56-32-2274206, E-mail: [email protected]
LATIN AMERICAN JOURNAL OF AQUATIC RESEARCH
Lat. Am. J. Aquat. Res., 43(4) 2015
CONTENTS
Research Articles
Daniel Arceo-Carranza & Xavier Chiappa-Carrara
Feeding ecology of juvenile marine fish in a shallow coastal lagoon of southeastern Mexico. Ecología alimentaria de peces marinos juveniles en un sistema lagunar somero del sureste de México……………………………………….…………621-631
Manuel Alvarado, Edison Serrano, Juan Carlos Sánchez & Luis Valladares
Changes in plasma steroid hormones and gonadal histology associated with sexual maturation in wild southern hake
(Merluccius australis). Cambios en las hormonas esteroidales plasmáticas y en la histología gonadal asociados a la maduración sexual de la merluza austral (Merluccius australis)………………………………………………………………………..632-640
Emyr Peña, Crisantema Hernández, Carlos Alfonso Álvarez-González, Leonardo Ibarra-Castro, Ana Puello-Cruz & Ronald W.
Hardy
Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus).
Caracterización comparativa de la actividad de la proteasa en juveniles cultivados de pargo flamenco (Lutjanus
guttatus)………………………………………………………………………………………………………………………………..641-650
Luis César Almendarez-Hernández, Germán Ponce-Díaz, Daniel Lluch-Belda, Pablo del Monte-Luna & Romeo Saldívar-Lucio
Risk assessment and uncertainty of the shrimp trawl fishery in the Gulf of California considering environmental variability. Evaluación de riesgo e incertidumbre de la pesquería de camarón de alta mar del golfo de California considerando la variabilidad ambiental………………………………………………………..……………………………………………...…………..651-661
Laura María Sánchez, María Romina Schiaffino, Haydée Pizarro & Irina Izaguirre
Periphytic and planktonic bacterial community structure in turbid and clear shallow lakes of the Pampean Plain
(Argentina): a CARD-FISH approach. Estructura de las comunidades bacterianas perifíticas y planctónicas en lagunas turbias y claras de la llanura pampeana (Argentina): un enfoque aplicando CARD-FISH…………….…………………….…..662-674
Aline Lopes-Souza, Alexandre Schiavetti & Martín Roberto Álvarez
Analysis of marine turtle strandings (Reptilia: Testudine) occurring on coast of Bahia State, Brazil. Análisis de varamientos de tortugas marinas (Reptilia: Testudine) ocurridas en la costa del Estado de Bahía, Brasil……….……………..675-683
Olimpia Chong-Carrillo, Fernando Vega-Villasante, Ricardo Arencibia-Jorge, Shehu L. Akintola, Layla Michán-Aguirre & Fabio
G. Cupul-Magaña
Research on the river shrimps of the genus Macrobrachium (Bate, 1868) (Decapoda: Caridea: Palaemonidae) with
known or potential economic importance: strengths and weaknesses shown through scientometrics. Investigaciones
sobre camarones de río del género Macrobrachium (Bate, 1868) (Decapoda: Caridea: Palaemonidae) con importancia económica conocida o potencial: fortalezas y debilidades mostradas a través de la cienciometría………….…………..………..684-690
Jorge Paramo, Daniel Pérez & Arturo Acero
Estructura y distribución de los condrictios de aguas profundas en el Caribe colombiano. Structure and distribution of
deep-water chondrichthyans in the Colombian Caribbean……..…………………..…………………………………………….691-699
Manuel Estay & Carlos Chávez
Decisiones de localización y cambios regulatorios: el caso de la acuicultura en Chile. Location decisions and regulatory
changes: the case of the Chilean aquaculture……..…………….………………….………………………………...…………..700-717
Esteban Avigliano, Guy Comte, Juan José Rosso, Ezequiel Mabragaña, Paola Della Rosa, Sebastian Sanchez, Alejandra
Volpedo, Franco del Rosso & Nahuel Federico Schenone
Identificación de stocks pesqueros de la corvina de río (Plagioscion ternetzi) de los ríos Paraguay y Paraná, mediante
el análisis morfométrico de sus otolitos. Identification of fish stocks of river crocker (Plagioscion ternetzi) in Paraná andParaguay rivers by using otolith morphometric analysis………….….…………….……………………………………………..718-725
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Natalia Leiva, Mario George-Nascimento & Gabriela Muñoz
Carga parasitaria en crustáceos decápodos de la costa central de Chile: ¿existe alguna asociación con la abundancia
de los hospedadores definitivos?. Parasite burden in decapod crustaceans from the central coast of Chile: is there any
association with the relationship with definitive host abundances?………….….………………….…………..……….…..…..726-738
Wanessa de Melo-Costa, Cristina Vaz Avelar de Carvalho, Gabriel Passini, Andressa Teles, Manuela Sozo-Cecchini & Vinicius
Ronzani-Cerqueira
Inclusion of copepod Acartia tonsa nauplii in the feeding of Centropomus undecimalis larvae increases stress
resistance. La inclusión de nauplios del copépodo Acartia tonsa en la alimentación de larvas de Centropomus undecimalis
aumenta su resistencia al estrés……….…………………………………………….……………….…………….……….….…..739-744
Jorge E. Moreno-Reyes, Carlos A. Méndez-Ruiz, Gina X. Díaz, Jaime A. Meruane & Pedro H. Toledo
Chemical composition of the freshwater prawn Cryphiops caementarius (Molina, 1782) (Decapoda: Palaemonidae) in
two populations in northern Chile: reproductive and environmental considerations. Composición química del camarón
de río Cryphiops caementarius (Molina, 1782) (Decapoda: Palaemonidae) en dos poblaciones del norte de Chile: consideraciones reproductivas y ambientales………….….…………………………………………………..….……………….……..…..745-754
José Luis Ochoa, Norma Ochoa-Alvarez, María Antonia Guzmán-Murillo, Sergio Hernández & Felipe Ascencio
Isolation and risk assessment of Geotrichum spp. in the white shrimp (Litopenaeus vannamei Boone, 1931) from
culture ponds. Aislamiento y evaluación de riesgos de Geotrichum spp. en el camarón blanco (Litopenaeus vannamei
Boone, 1931) en estanques de cultivo………….………………………………………………..….…………………………..755-765
Irasema E. Luis-Villaseñor, Domenico Voltolina, Bruno Gómez-Gil, Felipe Ascencio, Ángel I. Campa-Córdova, Juan M. AudeloNaranjo & Olga O. Zamudio-Armenta
Probiotic modulation of the gut bacterial community of juvenile Litopenaeus vannamei challenged with Vibrio
parahaemolyticus CAIM 170. Modulación por probióticos de la comunidad bacteriana intestinal de juveniles de Litopenaeus
vannamei infectados con Vibrio parahaemolyticus CAIM 170…………………………..…..….…………….……..………..766-775
Short Communications
José Manuel Grijalva-Chon, Reina Castro-Longoria, Tania Lizbeth Enríquez-Espinoza, Alfonso Nivardo Maeda-Martínez &
Fernando Mendoza-Cano
Molecular evidence of the protozoan parasite Marteilia refringens in Crassostrea gigas and Crassostrea corteziensis
from the Gulf of California. Evidencia molecular del parásito protozoario Marteilia refringens en Crassostrea gigas y Crassostrea corteziensis del Golfo de California……………………………..…………………..…..….…………….……………..776-780
Fernando Vega-Villasante, José J. Ávalos-Aguilar, Héctor Nolasco-Soria, Manuel A. Vargas-Ceballos, José L. BortoliniRosales, Olimpia Chong-Carrillo, Martín F. Ruiz-Núñez & Julio C. Morales-Hernández
Wild populations of the invasive Australian red claw crayfish Cherax quadricarinatus (Crustacea, Decapoda) near
the northern coast of Jalisco, Mexico: a new fishing and profitable resource. Poblaciones silvestres invasoras de langosta australiana de pinzas roja Cherax quadricarinatus (Crustacea, Decapoda) cerca de la costa norte de Jalisco, México:
un nuevo y rentable recurso pesquero……………………………..………………….…..…..….…………….……..………..781-785
Filipe dos Santos-Cipriano, Kauana Santos de Lima, Érica Bevitório-Passinato, Raildo Mota de Jesus, Francisco Oliveira de
Magalhães Júnior, William Cristiane Teles-Tonini & Luis Gustavo Tavares-Braga
Apparent digestibility of energetic ingredients by pirarucu juveniles, Arapaima gigas (Schinz, 1822). Digestibilidad
aparente de ingredientes energéticos por juveniles de pirarucu, Arapaima gigas (Schinz, 1822)……….………………..786-791
María Fernanda da Silva-Souza, Juliet Kiyoko-Sugai & Mônica Yumi-Tsuzuki
Anticipation of Artemia sp. supply in the larviculture of the barber goby Elacatinus figaro (Gobiidae: Teleostei)
influenced growth, metamorphosis and alkaline protease activity. La anticipación del suministro de Artemia sp. en la
larvicultura del neón gobi Elacatinus figaro (Gobiidae: Teleostei) influenció el crecimiento, metamorfosis y actividad de
proteasas alcalinas………………………………………….………………………………………………..……….……..……..792-797
Víctor M. Aguilera, Rubén Escribano & José Martínez-Oyanedel
Electrophoretic protein profiles of mid-sized copepod Calanoides patagoniensis steadily fed bloom-forming
diatoms. Perfiles electroforéticos de proteínas del copépodo de talla media Calanoides patagoniensis alimentado
sostenidamente con diatomeas formadoras de florecimientos…………………..………….……..…..…….……………..798-806
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Lat. Am. J. Aquat. Res., 43(4): 621-631, 2015
Feeding ecology of marine fishes in a shallow coastal lagoon
DOI: 10.3856/vol43-issue4-fulltext-1
Research Article
Feeding ecology of juvenile marine fish in a shallow coastal lagoon
of southeastern Mexico
1
Daniel Arceo-Carranza1 & Xavier Chiappa-Carrara1
Unidad Multidisciplinaria de Docencia e Investigación, Universidad Nacional Autónoma de México
Laboratorio de Ecología, Puerto de Abrigo s/n, CP 97356, Sisal, Hunucmá, Yucatán, México
Corresponding author: Daniel Arceo-Carranza ([email protected])
ABSTRACT. Many species of marine fish use coastal lagoons during early stages of their life cycles due to the
protection provided by their turbid waters and complex structure of the environment, such as mangroves and
mudflats, and the availability of food derived from the high productivity of these sites. In this study, we analyzed
the diet of six species of juvenile marine fishes that use a karstic lagoon system in the northwest portion of the
Yucatan Peninsula, Mexico. Through stomach contents analysis we determined the trophic differences among
Caranx latus, Oligoplites saurus, Trachinotus falcatus, Synodus foetens, Lutjanus griseus, and Strongylura
notata. C. latus, O. saurus, S. foetens, and S. notate, which are ichthyophagous species (>80% by number). L.
griseus feeds mainly on crustaceans (>55%) and fish (35%), while T. falcatus feeds on mollusks (>50% bivalves,
>35% gastropods). The analysis of similarities (ANOSIM) showed differences in the diet of all species. Cluster
analysis, based on the Bray-Curtis similarity matrix revealed three groups; one characterized by the
ichthyophagous guild (S. notata, S. foetens, C. latus, and O. saurus), other group formed by the crustacean
consumers (L. griseus), and the third, composed by the mollusk feeder (T. falcatus). Species of the
ichthyophagous guild showed overlap in their diets, which under conditions of low prey abundance may trigger
competition, hence affecting juvenile stages of these marine species that use coastal lagoons to feed and grow.
Keywords: juvenile fish, stomach contents, diet breadth, piscivory, trophic overlap.
Ecología alimentaria de peces marinos juveniles en un sistema
lagunar somero del sureste de México
RESUMEN. Muchas especies de peces marinos utilizan las lagunas costeras durante los estadios juveniles de
su ciclo de vida, por la protección que les provee las aguas turbias y la complejidad estructural de ambientes
como los manglares y planicies lodosas, además de la disponibilidad de alimento originada por la alta
productividad de estos sitios. En el presente estudio se analizó la dieta de juveniles de seis especies de peces
marinos que utilizan una laguna cárstica en el noroeste de la Península de Yucatán, México, para determinar las
diferencias alimentarias en los contenidos estomacales de Caranx latus, Oligoplites saurus, Trachinotus
falcatus, Synodus foetens, Lutjanus griseus y Strongylura notata. C. latus, O. saurus, S. foetens y S. notata, que
son especies ictiófagas (>80% en número). L. griseus se alimenta principalmente de crustáceos (>55%) y peces
(35%), mientras que T. falcatus se alimenta de moluscos (>50% bivalvos y >35% gasterópodos). El análisis de
similitud (ANOSIM) mostró diferencias en la dieta de todas las especies. El análisis de agrupación, basado en
la matriz de similitud de Bray-Curtis mostró tres grupos; uno caracterizado por peces ictiófagos (S. notata, S.
foetens, C. latus y O. saurus), otro grupo formado por consumidores de crustáceos (L. griseus) y el tercer grupo,
conformado por consumidores de moluscos (T. falcatus). Los resultados indican que existe traslape en las dietas
de las especies que forman el grupo de peces piscívoros. Por lo tanto, en condiciones de baja abundancia de
presas puede desencadenar la competencia por el alimento afectando las etapas juveniles de estas especies
marinas que utilizan las lagunas costeras para alimentarse y crecer.
Palabras clave: peces juveniles, contenidos estomacales, amplitud de dieta, piscivoría, traslape trófico.
__________________
Corresponding editor: Claudia Bremec
621
622
INTRODUCTION
Coastal lagoons are recognized as high productivity
systems that provide shelter for early-life stages of
many marine fish species. Juvenile fish spend time in
shallow coastal waters where they feed and grow to
sub-adults before migrating into deeper waters (Blay et
al., 2006). Shallow soft-bottom habitats, including
mangroves and mudflats, are important nurseries for
juvenile fish (Laegdsgaard & Johnson, 1995;
Nagelkerken & Van der Velde, 2002; Verweij et al.,
2006; Tse et al., 2008; Arceo-Carranza et al., 2009).
The productive and structurally complex environment
provided by mangrove stands is used as feeding
grounds and refuge by juvenile fishes (Ruiz et al., 1993;
Laegdsgaard & Johnson, 2001) and many juveniles and
sub-adults of nocturnal species use these areas as refuge
habitats during the day (Cocheret de la Morinière et al.,
2004; Verweij et al., 2006). Even if mudflats are
structurally less complex, they hold a great abundance
and diversity of invertebrates and are used as feeding
grounds for juvenile fishes (Laegdsgaard & Johnson,
2001; Tse et al., 2008).
To assess the feeding habits is fundamental for
understanding the role of fish within their ecosystems,
since they indicate relationships among species based
on feeding resources and they indirectly indicate
community energy flux (Yáñez-Arancibia & Nugent,
1977; Hajisamaea et al., 2003). This allows inferences
to be drawn regarding the effects of competition and
predation on the community structure (Krebs, 1999).
Other resources, such as space, are also important for
community ecology. Ecological theory predicts that
resource partitioning at the spatial, temporal, and
trophic level may reduce niche overlap and thereby
reduce competition pressure between co-occurring
species. Ross (1986) identified that food is the main
limiting factor in aquatic environments, and suggested
that the use of similar prey types defines functional
groups within the community, so species can be
grouped in guilds according to their trophic similarity.
Trophic guilds (Root, 1967) seem to be a consequence
of resource partitioning and could explain how several
species can co-exist in the same space by the
differential use of resources in several dimensions,
including time. Studies of competitive exclusion and
resource partitioning in teleost fishes (Hixon, 1980;
Ross, 1986) have found that habitat partitioning could
be related to high dietary overlap among competing
species or to interactive competition, when competing
species have the same preference for prey species
(Jansen et al., 2002; Ramírez-Luna et al., 2008). Under
the assumption that juvenile marine fish species use
shallow coastal waters as feeding and refuge grounds,
the goal of this study was to analyze and compare the
diet of six marine fish species that use a shallow karstic
lagoon and to assess the trophic overlap among them.
MATERIALS AND METHODS
La Carbonera Lagoon is located in the northeastern part
of the Yucatán Peninsula. It is a semi-enclosed water
body with an average depth of 30 cm and is surrounded
by mangroves, mainly Rhizophora mangle and
Avicennia germinans. The lagoon bottom is dominated
by mud flats. It also contains freshwater seeps, although
the average salinity is 35. Samples were obtained
bimonthly from beach seine landings (40 m in length
and mesh size of 2.5 cm) in nine soft-bottom sites (Fig.
1), from April 2008 to December 2010. Collected
specimens were euthanized in ice slurry and preserved
in formaldehyde (10%). In the laboratory, standard
length (SL ± 0.1 cm) of each individual was measured,
and the body weight (g ± 0.1) was obtained. Among all
landings, juvenile marine species that use the lagoon to
feed (Gallardo et al., 2012) were selected, since they
are important in the transport of matter and energy
between coastal environments. Maturity stages were
assessed from the available data on length at first sexual
maturity (Tzeek-Tuz, 2013; Froese & Pauly, 2014).
Furthermore, we choose those species whose abundance was consistently greater than 30 individuals to
analyze their stomach contents. According to these
criteria, the species analyzed in this study were: Caranx
latus, Oligoplites saurus, Trachinotus falcatus,
Synodus foetens, Lutjanus griseus and Strongylura
notata. A species accumulation curve was obtained to
assess the sampling effort representativeness, measured
as the number of stomachs analyzed. Parameters of the
Clench model (1979) were obtained using Statistica 7.0
(Jiménez-Valverde & Hortal, 2003) considering 500
permutations of data obtained with EstimateS 8.0
(Colwell, 2006). The coefficient of determination was
used as an indicator of the goodness of fit, and slope
values below 0.1 were considered asymptotic.
Stomach contents
The number (N), weight (W), and frequency of
occurrence (FO) of each dietary component were
quantified and expressed as percentages (Hyslop,
1980). The index of relative importance (IRI) was
calculated for each dietary component as (Pinkas et al.,
1971).
IRI = (%N + %W)% FO
Cortés (1997) suggested that IRI values should be
expressed as percentages (% IRI).
623
and a percent measure of abundance (%N) to provide a
description of prey importance (dominant or rare),
predator feeding strategy (specialized or generalized),
and the degree of homogeneity of feeding in the
predator population (Bacha & Amara, 2009). To
determine the feeding strategy, prey species were
grouped as fish, crustaceans, mollusks, insects, and
“others”.
Diet overlap index
Shoener's index (Shoener, 1971) of niche overlap (α)
was used to assess dietary overlap considering that α =
0 indicates that diets have no common items and α = 1
indicates identical diets (Wallace, 1981).
RESULTS
Figure 1. La Carbonera Lagoon (Yucatán, Mexico) and
the sampling sites.
Data analysis
Trophic guilds
One-way analysis of similarity (ANOSIM) was used to
test the null hypothesis of no differences in the diet
composition between the studied species over a BrayCurtis rank similarity matrix constructed with the
fourth-root transformed data. A cluster (using groupaverage linkage) was generated to determine the trophic
guilds based on the diet similarities among species.
Analyses were performed using the statistical package
PRIMER 6 (Clarke & Warwick, 2001).
Diet breadth
The diet breadth was calculated using the Levin´s
standardized index (Krebs, 1999) as:
Bi = 1/n – 1 {(1/Σ pij2) – 1)}
where Bi is the Levin index for species i; pij is the
proportion of the diet of predator i that is made up of
prey j, and n is number of prey species. Bi values range
between 0 and 1. Zero indicates that fish feed on only
one prey type, representing the minimum diet breadth
and high feeding specialization. As the index
approaches 1, the species consumes all food resources
in the same proportion (pj = 1/n), representing no
selection among prey types and the widest possible
trophic niche (Krebs, 1999).
Feeding strategy
The Amundsen et al. (1996) method, which is a
modification of Costello´s (1990) graphical method,
was used to obtain the feeding strategy of each species.
This method uses the frequency of occurrence (%FO)
We analyzed 223 individuals belonging to six species
(C. latus, O. saurus, T. falcatus, S. foetens, L. griseus
and S. notata). Of them, 185 (83%) presented some
type of food in their stomachs while 38 (17%) were
empty (Table 1). C. latus, O. saurus, T. falcatus, and S.
foetens were caught only as juvenile fish, according to
data on length at first sexual maturity (Froese & Pauly,
2014); S. notata was captured in juvenile and adult
stage (Tzeek-Tuz, 2013), and data of length at first
maturity of the lizardfish S. foetens, are not available
but, according to their sizes (4.0-15 cm), they were
considered as juvenile fish (Fig. 2).
Diet composition
Species accumulation curves for each species are
shown in Figure 3; slope values of the Clench (1979)
model are greater than 0.1, with R2 values >0.9. C. latus
fed mainly on fish (78% IRI), but crustaceans were also
consumed (20% IRI). O. saurus fed on small fish (97%
IRI), principally Engraulidae. T. falcatus fed on
mollusks, mainly bivalves (57% IRI) and gastropods
(34% IRI). Crustaceans (7% IRI) such as amphipods,
tanaids, and ostracods, were also present. S. foetens fed
primarily on small fish (86% IRI) such as Gerreidae
(Eucinostomus spp.), Cyprinodontidae (Floridich
polyommus), and Clupeidae (Opisthonema oglinum);
penaeid crustaceans were also present (8% IRI). Small
Synodontidae were also found in their stomachs (10%
IRI), which may constitute an evidence of cannibalism.
The grey snapper (Lutjanus griseus), which is a
commercially important species, fed on a wide variety
of crustaceans (59% IRI), such as penaeid shrimps,
amphipods, mysids, and isopods but also on fishes
(35% IRI) such as Ariidae, Clupeidae and Cyprinodontidae. The Needlefish S. notata also consumed
fishes (91% IRI) (Cyprinodontidae, Clupeidae,
Gerreidae, and Atherinopsidae, mainly Menidia sp.), as
well as decapod crustaceans (8% IRI) (Table 2).
624
Table 1. Size interval of captured specimens (standard length) and sample size (number of stomachs) of six marine fish
species in La Carbonera Lagoon.
Species
C. latus
O. saurus
T. falcatus
S. foetens
L. griseus
S. notata
Total number of
stomachs
42
36
35
34
39
37
Number of empty
stomachs
12
0
10
1
5
10
Percentage of empty
stomachs (% )
28.57
0
28.57
2.94
12.82
27.02
Standard
length (cm)
4.70-23.50
5.09- 8.82
4.70-18.00
4.02-15.10
3.15-22.50
3.27-41.50
Figure 2. Length-frequency distributions of the fish species analyzed. First maturity and maximum size are shown (TzeekTuz, 2013; Froese & Pauly, 2014).
Trophic guilds
The Bray-Curtis similarity index formed three groups
(Fig. 4). The first includes C. latus, O. saurus, S.
foetens, and S. notata which form the piscivorous
trophic guild; the second has Lutjanus griseus that
displayed a mixed diet composed of fish and
crustaceans; while in the third group appears T.
falcatus, a specialized carnivore whose mouth and
dentine adaptations allow it to crush the calcareous
shells of mollusks, mainly bivalves.
In the ichthyophagous guild, the analysis of
similarities (ANOSIM) showed significant differences
between each species (R Global = 0.109; P = 0.001)
indicating that the most important prey types in the diet
of each species differ (Table 3) even if fish are their
main food.
Diet breadth
The diet breadth values indicate that all species display
a specialized type of feeding behavior. Values of the
Levin’s index fall below 0.6 (O. saurus, Bi = 0.026; T.
falcatus, Bi = 0.123; L. griseus, Bi = 0.124; S. notata,
Bi = 0.036; S. foetens, Bi = 0.163; C. latus, Bi = 0.042),
indicating that only a few prey types dominate the diet
and predators can be classified as stenophagous
species.
625
Figure 3. Prey type accumulation curve (scaled by the number of stomachs) of each of the six marine fish species in the La
Carbonera Lagoon (Os: Oligoplites saurus, Tf: Trachinotus falcatus, Lg: Lutjanus griseus, Sn: Strongylura notate, Sf:
Synodus foetens, Cl: Caranx latus).
Feeding strategy and trophic overlap
Values of the Shoener’s index of trophic overlap were
greater than 0.6, indicating that the piscivorous species
display a significant diet overlap (Table 4).
The feeding strategy displayed by the specialist fish
analyzed was confirmed by the Costello method (Fig.
5). O. saurus, S. notate, S. foetens, and C. latus were
classed as piscivorous. L. griseus specializes in the
consumption of penaeid crustaceans, and T. falcatus
feeds mainly on mollusks.
DISCUSSION
Shallow soft-bottom habitats are recognized worldwide
as important nurseries for many marine fish species.
Several factors, including high structural complexity,
low predation risk, and high foraging efficiency, may
explain why juvenile of many marine species use these
shallow habitats (Tse et al., 2008).
The six species of fish examined are considered to
be marine migrants (Castro-Aguirre et al., 1999; Elliott
et al., 2007) that use shallow waters as nurseries,
refuge, and feeding grounds. They are carnivorous
species, mainly piscivores with stenophagy and trophic
specialization (Carr & Adams, 1973; Arceo-Carranza
et al., 2004; Cocheret de la Morinière et al., 2004; CruzEscalona et al., 2005; Guevara et al., 2007). Even if
many marine fish species exhibit marked changes in
their diet according to the temporal availability of prey
and life stage of individuals (Blaber, 1997; Platell et al.,
1997; Hajisamae, 2009), our observations allow to say
that five of them show a relatively homogeneous diet
over time since the overall occurrence of these species
within the study area is low (Gallardo-Torres et al.,
2012). The species accumulation curves did not reach
the asymptote, but values of r2 > 0.9 indicate a good fit
of the Clench model. Although one or two prey types
dominate the diets of the majority of the analyzed
species, representativeness of prey types was not
achieved indicating that a greater number of stomachs
ought to be analyzed. Anyhow, this is the first report on
the diet of these migratory fish species in the study area.
In general, carangid are pelagic species considered
to be piscivorous. Silvano (2001) reported that C. latus
feeds on crustaceans and fish, and Carr & Adams
(1973) place O. saurus as a piscivorous species; indeed,
juvenile O. saurus analyzed in the present study fed
almost exclusively on small fishes, principally
Engraulidae.
Qualitative studies (Carr & Adams, 1973) on the
feeding habits of T. falcatus, in the Cristal River,
Florida, indicate that juvenile fish feed primarily on fish
and benthic invertebrates, including worms, mollusks,
and crustaceans, mainly shrimps. In La Carbonera
Lagoon, juvenile fish consumed mainly mollusks,
gastropods, and bivalves. Differences in the diet
composition among geographical zones demonstrate
the flexibility of this tropical marine fish, whose
juveniles take advantage of the available resources in
626
Table 2. IRI values for each prey type of six carnivorous fish species in La Carbonera Lagoon, Yucatán.
Size class
N
Fish
Unidentified fishes
Clupeidae
Opisthonema oglinum
Belonidae
Strongylura notata
Poecilidae
Cyprinodontidae
Floridichthys polyommus
Fundulus spp.
Gerreidae
Eucinostomus spp.
Ariopsis spp.
Engraulidae
Menidia spp.
Mugilidae
Sciaenidae
Synodontidae
Crustacea
Unidentified crustaceans
Decapoda
Brachyura
Penaeidae
Farfantepenaeus
Caridea
Portunidae
Amphipoda
Gammaridae
Corophiidae
Isopoda
Cassidinidae
Tanaidacea
Mysidacea
Ostracoda
Copepoda
Mollusca
Unidentified gastropods
Odostomia
Truncatella
Caecum
Bittium
Cerithium
Bulla
Neritina
Olivella
Unidentified bivalves
Musculus
Anomalocardia
Tellina
Veneridae
Brachidontes
C. latus
O. saurus
T. falcatus
L. griseus
S. foetens
S. notata
4.7-23.5
42
5.09-8.82
36
4.7-14.2
35
3.13-22.5
39
4.02-15.1
34
9.24-41.5
37
77.6111
86.9352
0.8013
32.7878
0.2086
68.1451
1.8587
1.3828
78.5323
1.9298
0.7997
0.5665
0.0221
0.0055
0.2772
0.0118
2.0174
0.2050
0.0277
0.2050
15.5034
2.5369
0.1604
0.4246
9.4364
0.2349
0.0094
0.7997
1.5586
2.1676
0.0116
0.8203
0.0811
0.0008
0.0328
0.4874
2.3234
2.6852
9.9735
1.6876
1.2297
1.9498
0.4874
1.9498
0.4874
0.6713
0.1930
0.6713
4.1490
0.0399
0.0075
2.5901
3.3713
0.4139
0.7634
6.4368
0.0799
22.8660
0.0399
0.0316
0.3743
16.3846
0.2079
3.0710
34.6154
1.2497
1.9249
41.0167
3.4835
0.2212
4.3734
1.3034
0.7781
0.6336
4.2152
0.5106
0.4584
0.2893
2.0424
0.0556
0.5106
0.0409
0.1707
8.7165
1.9139
0.6713
2.6852
1.3182
0.6453
0.9457
0.1483
0.0510
0.0032
627
Continuation
C. latus
Annelida
Polychaeta
Insecta
Diptera
Hymenoptera
Foraminiferida
Quinqueloculina
Plant remains
Seagrass
Organic matter no identified
O. saurus
T. falcatus
L. griseus
S. foetens
S. notata
0.0777
0.4389
0.1989
0.1758
0.3481
0.7764
0.0166
0.0062
0.1901
4.6276
4.3871
0.0259
Figure 4. Diet composition and trophic guilds of six marine fish species, based on the Bray-Curtis similarity index.
the environment. It has been reported that adults feed
almost exclusively on mollusks and crabs (Carr &
Adams, 1973).
On the other hand, the snapper L. griseus, is an
euryhaline marine species that uses coastal lagoons
bordered by mangroves for feeding. In the present study
the trophic groups found in the stomachs of young
individuals were benthic organisms (caridean shrimp
and other crustaceans), which are usually considered as
evasive species (Nagelkerken et al., 2000; Cocheret de
la Morinière et al., 2004; Guevara et al., 2007). In
coastal systems of the southern Gulf of Mexico,
Guevara et al. (2007) found that juvenile L. griseus feed
on penaeid and caridean shrimp species during the
night. This probably explains the fact that few
individuals were collected since samplings for the
present study were during the day.
The inshore lizardfish, Synodus foetens, is one of the
most common coastal demersal predators on the Gulf
of Mexico´s continental shelf (Cruz-Escalona et al.,
2005). This is a piscivorous species that feeds mainly
on juvenile of other marine fishes known to be
carnivorous, so it can be considered an apex predator
on sandy bottoms of the continental shelf, which uses
different habitats during its feeding activity and hunts
various prey types, depending on resource availability
and the size of prey (Esposito et al., 2009). There are
no reports on the feeding habits of this species in the
Gulf of Mexico but, off the coast of Italy, Synodus
saurus is also a piscivorous species that feeds on small
fish like sardines and anchovies (Esposito et al., 2009).
The needlefish, S. notata, is considered a piscivore
(Carr & Adams, 1973; Arceo-Carranza et al., 2004) that
obtains prey throughout the water column, as
demonstrated by the presence of both demersal and
pelagic components in the diet (Arceo-Carranza et al.,
2004). Even if in this study juvenile and adult fish were
analyzed, the low number of fish collected prevented to
formally compare between size classes but it has been
shown that this species displays ontogenetic changes in
628
Table 3. Similarity, R statistic and P-values of ANOSIM test of prey types that contribute to differences of diets among
fish species. Only significant combinations are show.
Predators
O. saurus & C. latus
O. saurus & S. notata
O. saurus & S. foetens
O. saurus & L. griseus
O. saurus & T. falcatus
C. latus & S. notata
C. latus & S. foetens
C. latus & L. griseus
C. latus & T. falcatus
S. notata & L. griseus
S. notata & T. falcatus
S. foetens & L. griseus
S. foetens & T. falcatus
L. griseus & T. falcatus
% similarity
71.33
78.23
82.26
84.83
95.95
81.09
85.39
80.40
96.76
88.66
97.68
92.04
97.83
97.10
R value
0.051
0.081
0.088
0.171
0.344
0.035
0.056
0.062
0.359
0.046
0.199
0.060
0.149
0.254
P
0.019
0.014
0.005
0.001
0.001
0.05
0.01
0.031
0.001
0.041
0.001
0.016
0.001
0.001
Table 4. Trophic overlap (Schoener index) values for the six species of juvenile marine fish (significant values in bold).
C. latus
O. saurus
T. falcatus
S. foetens
L. griseus
S. notata
C. latus
0.8078
0.0240
0.5050
0.6413
0.8733
O. saurus
0.0212
0.8325
0.4587
0.9343
T. falcatus
0.0239
0.0192
0.0225
S. foetens
0.5648
0.8982
L. griseus
0.5212
S. notata
-
Figure 5. Graphic representation of the dominant prey types (frequency of occurrence % and weight %) of six marine fish
species.
its diet (Arceo-Carranza, 2002; Hajisamae, 2009).
Arceo-Carranza et al. (2004) found that S. notata from
the Alvarado Lagoon, Mexico, is an active predator
with great trophic plasticity to exploit the available
resources in the environment (fish, shrimp, and insects
among other prey types) while in the clearer waters of
the northwestern coast of the Yucatán Peninsula it
behaves as a piscivorous species.
Diet breadth of predators tends to increase when
food availability is low, and decreases when food
availability is high (Tse et al., 2008). The behavior of
large predators feeding on larger prey usually follows
the traditional optimal foraging theory which states that
animals should maximize their net rate of energy return
when selecting prey (Shoener, 1971; Bacha & Amara,
2009). The high diversity of prey found in the stomach
contents of the six species analyzed in this work
indicates that numerous food resources are exploited.
Piscivory is a common phenomenon in aquatic and
marine ecosystems, and is the largest cause of fish
removal in most marine ecosystems, usually larger than
fishery catches (Link & Garrison, 2002) but, within this
fish assemblage, it is difficult to determine the relative
impacts of the different piscivores on other fish
populations when considered as prey. For instance,
some carangids can consume large numbers of
demersal juveniles that use shallow nurseries, but these
predators may feed only sporadically in shallow waters,
in a manner similar to their transient feeding on coral
reefs (Hixon & Carr, 1997). The coexistence of pelagic
and demersal prey adds further complexity to the
structuring of predation pressure by carangids on
individual cohorts of recruits in shallow systems (Baker
& Sheaves, 2005).
This study provides important information about
shallow soft bottoms, including mudflats as important
feeding grounds for fish. The species studied in La
Carbonera Lagoon are marine fish that use turbid and
shallow waters that provide shelter and food for
juvenile stages. Furthermore, these results on the diet
composition of juvenile fish provide evidence on the
protection value of the mudflats adjacent to mangroves.
ACKNOWLEDGEMENTS
The authors wish to thank Maribel Badillo Alemán for
the administrative and operational support during
sampling; Juani Tzeek and Alfredo Gallardo for
technical assistance during sampling, and help in fish
identification; the POSDOC program from DGAPAUNAM for a postdoctoral scholarship to D.A.C. This
project was funded by PAPIIT (IN213012, IN219515)
and FOMIX-Yucatán (103229) research grants. Fish
629
were captured under the permit for collection awarded
by the National Commission of Aquaculture and
Fisheries number DGOPA/04031/310510.1940
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Lat. Am. J. Aquat. Res., 43(4): 632-640, 2015
Changes in sexual maturation of M. australis
Research Article
Changes in plasma steroid hormones and gonadal histology associated with
sexual maturation in wild southern hake (Merluccius australis)
Manuel Alvarado1, Edison Serrano1, Juan Carlos Sánchez1 & Luis Valladares2
Estación Experimental Quillaipe, Unidad de Gestión Tecnológica, Área de Alimentos y Biotecnología
Fundación Chile, P.O. Box 27-D, Puerto Montt, Chile
2
Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile
P.O. Box 138-11, Santiago, Chile
1
Corresponding author: Edison Serrano ([email protected])
ABSTRACT. A detailed study of gametes development and characterization of plasma sex steroid hormones
during the maturation cycle was performed for the first time in the southern hake (Merluccius australis). Fish
were caught in the inland waters of the Reloncaví Sound, Interior Sea of Chiloé, Chile. Samples of gonads and
blood were collected for histology and sex steroid hormone (17 β-estradiol, 11-ketotestosterone and 17,20 βdihydroxy-4-pregnen-3-one) analysis, respectively. Sex steroid hormone quantification was performed using
enzyme-immunoassay (ELISA). Results showed that M. australis males and females have asynchronous
development of testicles and ovaries, in all stages of maturation. Most spawning fish were found during the
spring months. Regarding the sex steroid hormones, serological fluctuations of 17 β-estradiol and 11ketotestosterone were found during gonadal maturation of M. australis. These hormones are the main hormones
responsible for vitelogenesis and spermatogenesis processes, respectively. Conversely, 17,20 β-dihydroxy-4pregnen-3-one did not show any serological fluctuation in females and males. Further studies involving
gonadotropins, 17,20 β,21-trihydroxy-4-pregnen-3-one and vitellogenin quantification are required in order to
obtain a more complete description of the reproductive physiology of wild and farmed M. australis.
Keywords: Merluccius australis, southern hake, gonad maturation, sex steroids, southern Chile.
Cambios en las hormonas esteroidales plasmáticas y en la histología gonadal
asociados a la maduración sexual a la merluza austral (Merluccius australis)
RESUMEN. El presente estudio se realizó para caracterizar el desarrollo de los gametos y el comportamiento
de las hormonas esteroidales sexuales en plasma durante el ciclo de maduración de la merluza austral
(Merluccius australis). Los peces estudiados fueron capturados en las aguas interiores del Seno de Reloncaví,
Mar Interior de Chiloé, Chile. Muestras de gónadas y sangre fueron recolectadas para histología y análisis de
hormonas esteroides sexuales (17 β-estradiol, 11-cetotestosterona y 17,20 β-dihidroxi-4-pregnen-3-ona),
respectivamente. La cuantificación de las hormonas esteroidales sexuales se realizó utilizando la enzimainmunoensayo (ELISA). Los resultados mostraron que machos y hembras de M. australis poseen un desarrollo
asincrónico de los testículos y ovarios, en todas las etapas de maduración. La mayoría de los ejemplares en etapa
de desove se encontraron durante la primavera. En cuanto a las hormonas esteroides sexuales, fluctuaciones
serológicos de 17 β-estradiol y 11-cetotestosterona se encontraron durante la maduración gonadal de M.
australis. Estas hormonas son las principales responsables de los procesos de vitelogénesis y espermatogénesis,
respectivamente. Por el contrario, 17,20 β-dihidroxi-4-pregnen-3-ona no mostró ninguna fluctuación serológica
en hembras y machos. Nuevos estudios que incluyan la cuantificación de las hormonas gonadotropinas, 17,20
β,21-trihydroxy-4-pregnen-3-one y vitelogenina son requeridos para obtener una descripción más completa de
la fisiología reproductiva de M. australis en estado silvestre y cautiverio.
Palabras clave: Merluccius australis, merluza austral, maduración gonadal, esteroides sexuales, sur de Chile.
__________________
Corresponding editor: Guido Plaza
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INTRODUCTION
Southern hake (Merluccius australis) is a demersal
gadiform fish species found in the southern hemisphere
between Argentina in the Atlantic Ocean (Tingley et
al., 1995) and New Zealand in the Pacific Ocean
(Aguayo-Hernandez, 1995; Colman, 1995). This
species supports important industrial and artisanal
fisheries in Chile, Argentina, and New Zealand, which
supply local and mainly overseas market of Japan,
USA, Spain and Portugal (Sylvia, 1995).
Global southern hake landings had historical peaks
of about 65,000 ton between 1987 and 1989 (Sylvia,
1995), but there were dramatically declining catches in
later years due to smaller fishing quotas to protect this
resource from overexploitation. Nowadays, global
southern hake landings are steady at just over 30,000
ton per year with prices around US$10 per kilo.
Nevertheless, the global demand for southern hake is
growing and the wild capture is declining, creating an
undersupplied market for this fish. Indeed, the extent of
this dependence has prompted the development of
southern hake farming in Chile.
Even though there is an advanced understanding of
the biology of the southern hake (Aguayo-Hernández,
1995; Colman, 1995; Tingley et al., 1995; Bustos et al.,
2007; Effer et al., 2013), there are some relevant
questions concerning their reproductive biology,
particularly the sexual maturation cycle and reproductive endocrinology that still remains unrevealed. To
date, it is known that fish reproduction is regulated by
a wide variety of abiotic and biotic environmental
factors that trigger internal physiological mechanisms
responsible for causing sexual maturation of fish
(Arcand-Hoy & Benson, 1998).
Wild broodstock fish can spawn naturally in the
tank when the environmental conditions are favorable,
nevertheless, several fish species exhibit reproductive
dysfunctions when they are raised in captivity
(Mylonas et al., 2010). Reproductive dysfunctions are
usually more seriously in female broodstock and can be
associated with final oocyte maturation, ovulation and
spawning (Zohar et al., 1988; Peter et al., 1993). These
dysfunctions most likely result from the combination of
the stress induced by captivity, and the lack of a suitable
environment for natural spawning (Schreck et al., 2001;
Pankhurst, 2011). Therefore, in the case of the absence
or scarcity of natural spawning, several studies have
shown that hormone induced spawning is a reliable
method of inducing reproduction in these fishes (Zohar
& Mylonas, 2001). However, this method has been
reported to exhibit negative effects on the quality of
gametes and survival rate at later stages of Salmo salar
(Crim & Glebe, 1984; Crim et al., 1986), S. trutta
(Mylonas et al., 1992), Oncorhynchus nerka (Slater et
al., 1995), and O. mykiss (Arabaci et al., 2004). In
southern hake farming, one of the most critical aspects
is to achieve the spawning of wild broodstock under
captive conditions. Wild southern hake broodstock are
spawned mainly by the use of hormones, causing a low
survival rate of their larvae during weaning from
Artemia to dry feed in culture conditions.
Considering the above-mentioned problem, the
analysis of blood steroid levels has been used to clarify
the optimum time to hormone induce spawning in fish,
which can help to obtain higher quality gametes and
therefore more suitable larvae, and also prevents the
occurrence of over-maturation and follicular atresia of
the gametes (Donaldson, 1996). Strictly, the use of
quantitative analysis of blood steroid hormones, as a
method for predicting the maturation stage of southern
hake, involves less handling of broodstock compared to
current method of gonadal biopsies, which is an
invasive method and requires large samples. Moreover,
the knowledge of the reproductive management
concepts such as maturation cycle, reproductive
endocrinology and gonadal development are scarce in
southern hake (Bustos et al., 2007; Effer et al., 2013)
and therefore, in order to scale up the commercial
farming of southern hake is relevant to research in this
important area. Hence, the aim of this study was to
identify the gamete developmental stages and
characterise plasma sex steroid hormones during the
maturation cycle of southern hake.
Sample collection
The specimens were captured by longline gear at 250300 m depth in the inland waters of the Reloncaví
Sound, Interior Sea of Chiloé in the Lagos Region,
Chile (41°31'S, 72°44'W). Fishing activities were
carried out from September 2011 to January 2012.
During this period, fishes were collected every two or
three weeks depending on weather conditions. Immediately upon reaching the surface, fishes were
sacrificed and the samples of blood and gonads were
collected and stored for later analysis.
Gonadal histology
Seventy six samples of gonads in different maturation
stages from 40 females and 36 males of M. australis
were collected for histological analysis. The dissected
tissue was fixed in 5% formalin for 24 h and stored in
70% ethanol. The fixed tissue was subsequently
dehydrated and embedded in the paraffin wax. The
waxed tissue were cut in transverse sections of 6-7 µm
thickness (Microtome, Leica Microsystems, model
6343
RM2125, Bannockburn, IL, USA) and then stained
with hematoxylineosin. Sample sections were
examined under a light microscope (Leica Microsystems model DM750, Leica, Bannockburn, IL, USA)
and classified according to their maturation status as
immature, proliferation, growth, maturation and
spawning. After the maturation stage was determined,
the samples were correlated with the levels of
serological steroid.
Analysis of hormonal steroids
Blood samples were extracted from each fish by caudal
venipuncture and immediately placed on ice, where
they were allowed to clot for 3-6 h. Blood samples were
later centrifuged for 15 min at 1500 g (MiniSpin
Centrifuge, AG 22331, Merck, Hamburg) and serum
stored at -80°C for later sex steroid hormones analysis.
Quantification of sex steroid hormones was
performed by enzyme-immunoassay technique (ELISA)
using commercial kits protocols; 17,20β-dihydroxy-4pregnen-3-one (17,20βP) (Cayman Chemicals Company, MI, USA) and 11-ketotestosterone (11-TK) and 17
β-estradiol (E2) (Mybiosource, Beijing, China).
Statistical analysis
The results were analyzed using the programme SPSS
Statistics 8.0 for Windows (SPSS Inc. Chicago, IL,
USA). Normality and homoscedasticity were assessed
using the Kolmogorov-Smirnov and Bartlett’s test
respectively. An analysis of variance (ANOVA) was
performed to determine the existence of significant
differences among sex hormones levels of each stage of
gonadal maturation. Differences in mean values were
determined by Tukey's test. The probability level for all
statistical tests was set at 0.05.
RESULTS
Gonad morphology and histology
Male
The testicles of M. australis are paired organs of similar
size and are white. They are composed of several lobes
with similar morphology, which are joined to form a Ushaped structure. These organs are located ventral to the
swim bladder of the fish.
Histological analysis of the testicles reported the
presence of individuals in all maturation states (Table
1). Five individuals were found in an immature stage,
which showed absolute dominance of germ cells (Fig.
1a). Eight individuals were found in the stage of
proliferation (spermatogenesis), which had a high
presence of spermatogonia and spermatocytes and
fewer spermatids (Fig. 1b). Ten individuals were found
Table 1. Histological classification of gonadal maturity
stage of wild southern hake (M. australis).
Gonadal maturity
stage
Immature
Proliferation
Growth
Maturation
Spawning
Males
(n = 36)
Females
(n = 40)
5
8
10
9
4
0
15
9
11
5
in the growth stage (spermiogenesis), which showed a
dominance of spermatids and a considerable amount of
sperm, spermatocytes and spermatogonia (Fig. 1c).
Nine individuals were found in the stage of maturation
(spermiation), which had the exclusive presence of free
sperm in the testis lobular lumen (Fig. 1d). Finally, ten
individuals were found in the stage of spawning, which
showed the presence of a few free spermatozoa and
occasionally spermatogonia in the testicles lumen.
Females
The ovaries of M. australis are paired organs in the
shape of elongated and bilobed sacs, which are located
ventral to the swim bladder. The ovarian wall is
transparent and thin, allowing oocytes in advanced
stages of maturity to be visible to the naked eye. In the
posterior region (caudal) is observed the fusion of the
ovaries that extend into a short oviduct, which opens in
the urogenital pore.
In the early developing stages, the ovaries showed a
light orange colour which becomes more intense with
advancing sexual maturity. Histological analysis of the
ovaries reported the presence of individuals in all
maturation stages except the immature stage (Table 1).
Fifteen individuals were found in the proliferation
stage (primary growth), showing the presence of
chromatin-nucleolar and perinuclear oocytes (Fig. 2a).
Nine individuals were found in the growth stage
(vitellogenesis), which had got oocytes in cortical
alveoli and early vitellogenic stage. Similarly, the
presence of oocytes in earlier stages of oogenesis (Fig.
2b) was also observed.
Eleven individuals were found in the maturation
stage, which showed the presence of oocytes with
nucleus migration and a noticeable size increase due to
hydration (Fig. 2c).
Finally, five individuals were found in the spawning
stage, which showed the presence of large amount of
post-ovulatory follicles, atretic oocytes and also
oocytes in earlier stages of oogenesis (Fig. 2d).
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Figure 1. Cross sections of M. australis testes showing different maturity stages. a) Testis in immature stage (10x), b) testis
in proliferation stage (40x), b) testis in the growth stage (40x), d) testis in the maturation stage (40x). The blue arrows
indicate cells in spermatogenesis.
Plasma sex steroid hormonal profile 11-ketotestosterone
Plasma concentrations of 11-KT showed significant
differences (P < 0.05) between the males found in the
stage of proliferation and those found in the others
maturity stages.
In the immature stage, the individuals reached an
average plasma 11-KT concentration of 0.23 ± 0.03 ng
mL-1. However, during the proliferation stage, the
levels of this hormones in the individuals increased
significantly (P < 0.05), reaching the highest levels at
maturational with an average of 1.04 ± 0.45 ng mL -1.
As maturation progressed, the concentrations of 11-KT
present in individuals began to decrease, progressively
reaching averages of 0.32 ± 0.24, 0.18 ± 0.07 and 0.15
± 0.04 ng mL-1 in the stage of growth, maturation and
spawning respectively.
Plasma levels of 11-KT in each of stages of
testicular maturity of male southern hake described by
the histological analysis are shown in (Fig. 3).
17β-estradiol
Plasma concentrations of E2 in M. australis females
exhibited significant differences among the different
stages of gonadal development (P < 0.05). These
differences were showed among individuals in the
growth phase (vitellogenesis) and those in the late
stages of development.
In the proliferation stage (primary growth), the
females reached an average plasma E2 concentration of
0.29 ± 0.06 ng mL-1. Afterwards, the concentration of
E2 increased significantly (P < 0.05) during the growth
stage (vitellogenesis), achieving a mean maximum
concentration of 0.62 ± 0.14 ng mL-1.
Subsequent to vitellogenesis, the E2 levels
decreased significantly (P < 0.05) as the gonadal
development progresses. Females in maturation and
spawning stages showed average concentrations of 0.32
± 0.24 and 0.13 ± 0.03 ng mL-1 respectively. Plasma
levels of E2 related to each of stage of ovarian maturity,
described by the histological analysis, are shown in
(Fig. 4).
6365
Figure 2. Cross sections of M. australis ovaries showing different maturity stages. a) Ovary in proliferation stage (4x), b)
ovary in growth stage (4x), b) ovary in the maturation stage (4x), d) ovary in the spawning stage (4x). The blue arrows
indicate oocyte cells in oogenesis.
17α,20β-dihydroxy-4-pregnen-3-one
The levels of 17α,20β-DP in the plasma of M. australis
males showed no significant differences (P > 0.05)
among different stages of gonadal development.
However, the concentration of this hormone reached
the maximum average of 0.3 ± 0.1 ng mL-1 during the
maturation stage (espermiation).
The remaining gonadal development stages had
lower levels of 17α,20β-DP, with average values of
0.07 ± 0.03, 0.11 ± 0.03, 0.09 ± 0.03 and 0.04 ± 0.01 ng
mL-1 in the immature, proliferation, growth and
spawning stages respectively.
Similar to the results reported in males, levels of
17α,20β-DP in females plasma showed no significant
differences (P > 0.05) among different stages of
gonadal development averaging 17α,20β-DP plasma
values of 0.07 ± 0.02, 0.05 ± 0.01, 0.05 ± 0.01, 0.05 ±
0.01 and 0.06 ± 0.02 ng mL-1 in immature, proliferation,
growth, maturation and spawning stages respectively.
Plasma levels of 17α, 20β-DP in each of stages of
ovarian and testicular maturity of southern hake
described by the histological analysis are shown in
Figures 3 and 4.
DISCUSSION
Studies concerning the anatomy and physiology of the
reproductive system are important in order to
understand the biology of fish reproduction. This study
represents the first attempt at a detailed histological
identification of gamete developmental stages and
characterization of plasma sex steroid hormones during
the maturation cycle of southern hake (Merluccius
australis).
Histological evaluation of the gonad development
stages in male and female specimens of M. australis
found cells that exhibit all stages of maturation,
showing clearly the type of asynchronous ovarian and
testicular development of this species.
Testicular histology showed spermatogonia, spermatocytes and spermatids in the lobular wall, whereas
spermatozoa were observed free in the lumen. At the
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6
Figure 3. Plasma steroidal profiles of M. australis males
during maturation cycle (Mean ± Standard Error) (P <
0.05).
Figure 4. Plasma steroidal profiles of M. australis females
during maturation cycle (Mean ± Standard Error) (P <
0.05).
stage of spermiation, however, the individuals showed
almost exclusively spermatozoa, probably due to the
extensive spawning period.
According to the observations in this study, ovarian
histology is very similar to that reported in studies with
Merluccius hubbsi (Cornejo, 1998; Honji et al., 2006),
and M. merluccius (Recasens et al., 2008), exhibiting
the same characteristics in each oocyte stage.
Sex steroids concentrations in M. australis were at
very low levels compared to studies on Chalcalburnus
tarichi (Ünal et al., 2005), Perca fluviatilis (Migaud et
al., 2003), Coregonus clupeamorfis (Rinchard et al.,
2001) and Pleuronectes americanus (Harmin et al.,
1995). However, studies on fish with asynchronous
gonadal development such as Gobio gudgeon and
Verasper variegatus have also reported low steroid
concentrations (Rinchard et al., 1993; Koya et al.,
2003).
The low concentration of sex steroids present in M.
australis could be attributed to the fact that this marine
fish is a partial spawner and therefore the levels of
circulating sex steroids are diluted as consequence of
extensive spawning periods.
The androgen 11-ketostestosterone has been
identified as the most important steroid hormone in
teleost testes (Borg, 1994). In the present study,
fluctuations of serological 11-KT levels were found
during testicular maturation. The levels of 11-KT were
higher in the stage of spermatogenesis compared to
other maturational stages, demonstrating the importance
of this hormone in the process of spermatogenesis. The
observed levels of 11-KT in M. australis, are consistent
with findings reported by studies with Hucho perryi
(Amer et al., 2001); Clupea pallasii (Koya et al., 2002);
Verasper variegatus (Koya et al., 2003) and Solea
senegalensis (García-López et al., 2006), where 11-KT
was the most influential androgen for the spermatogenesis stage.
In female teleosts, the level of the E2 has been
reported to increase gradually during cortical alveoli
phase, peaking in the vitellogenesis phase and then
declining prior to the ovulation phase (Mayer et al.,
1990; Schulz et al., 2010). Overall, these previous
findings are consistent with the present results, where
the levels of E2 were higher in the stage of
vitellogenesis compared to other maturational stages,
indicating that this sex hormone is essential to induce
the process of vitellogenesis in female M. australis.
Similar results were reported in Engraulis ringens
(Cisneros, 2007), Sardinops melanostictus (Murayama
et al., 1994), Salvelinus leucomaenis (Kagawa et al.,
1981), Mugil cephalus (Tamaru et al., 1991),
Acheilognathus rhombea (Shimizu et al., 1985), and
Oreochromis mossambicus (Cornish, 1998), where
oocytes development was mediated by increasing 17βestradiol during vitellogenesis stage.
The 17α,20β-DP has been identified as a
maturation-inducing steroid (MIS) in several fish
species during final oocyte maturation (Yamauchi et
al., 1984; Tamaru et al., 1991; Petrino et al., 1993;
Murayama et al., 1994), however, this hormone did not
show noticeable fluctuation during any maturation
stages in M. australis females. Similar findings
regarding the consistency in the levels of 17α,20β-DP
were reported in Engraulis ringens (Cisneros, 2007)
and Dicentrarchus labrax (Prat et al., 1990). Shortrange variations of 17α,20β-DP levels during the
process of oocyte final maturation and ovulation in M.
australis females could be explained by a lack of blood
samples at the precise moment of increase in this
hormone. A variety of experiments have shown that the
6387
increase in this steroid occurs for a short period of time,
just prior to ovulation when the germinal vesicle
membrane breaks down (Tamaru et al., 1991;
Murayama et al., 1994; Mylonas et al., 1997).
Furthermore, in vitro studies carried out by Migaud et
al. (2003) showed that 17α,20β-DP is detectable up to
two hours after being synthesized, which could also
explain the low concentration of this hormone in the
analyzed samples. Another explanation for this
phenomenon could be that 17α,20β-DP did not act as
MIS in M. australis females. Studies carried out with
Micropogonias undulatus (Trant & Thomas, 1989),
Cynoscion nebulosus (Thomas & Trant, 1989) and
Halobatrachus didactylus (Modesto & Canário, 2002)
have shown that 17,20β,21-trihydroxy-4-pregnen-3one (17,20β,21P) act as MIS instead of 17α,20β-DP.
However, the role of 17,20β,21P as MIS in M. australis
females is still unclear and further investigations are
needed.
In southern hake males, levels of 17α,20β-DP
showed a slight fluctuation during gonadal development. Conversely, studies with other fish species have
shown an increase in the levels of this steroid during the
espermiation stage (Vermeirssen et al., 1998, 2000;
Koya et al., 2002). The difference between our findings
and those reported in the literature could be due to the
low number of individuals sampled, the continuous
process of spermatogenesis, or the short duration of
17α,20β-DP in the bloodstream.
In conclusion, this study reported that there were
serological fluctuations of E2 and 11-KT during
gonadal maturation of M. australis, identifying these
hormones as the main hormones responsible for
vitelogenesis and spermatogenesis respectively. On the
other hand, the levels of 17α,20β-DP did not show
fluctuations so, apparently, this hormone is no involved
in gonadal maturation of this species. Future research
should include the entire maturation cycle of wild and
captive M. australis, in order to evaluate the
physiological effect of captivity conditions on
broodstock of this species. Similarly, additional studies
regarding the quantification of gonadotropins (FSH and
LH), 17,20β,21P and vitellogenin are required for a
complete understanding of the reproductive physiology
of M. australis.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Karl D. Shearer and
Dr. Ivan Valdebenito for their critical review of this
manuscript. This research was supported by funding
from Chilean National Commission for Scientific and
Technological Research (CONICYT) in the frame of
the project FONDEF DA09I 1001.
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Lat. Am. J. Aquat. Res., 43(4): 641-650, 2015
Proteases in spotted rose snapper juveniles
Research Article
Comparative characterization of protease activity in cultured spotted
rose snapper juveniles (Lutjanus guttatus)
Emyr Peña1, Crisantema Hernández1, Carlos Alfonso Álvarez-González3
Leonardo Ibarra-Castro1, Ana Puello-Cruz1 & Ronald W. Hardy2
1
Food Research and Development Center A.C., Mazatlán Unit
Av. Sábalo Cerritos s/n, Mazatlán, Sinaloa 89010, México
2
Laboratory of Tropical Aquaculture DACBIOL-UJAT, Carr Vhsa-Cárdenas km 0.5
Bosques de Saloya, Villahermosa, Tabasco, México
3
Hagerman Fish Culture Experiment Station, University of Idaho, Hagerman, ID 83332, USA
Corresponding author: Crisantema Hernández ([email protected])
ABSTRACT. Partial characterizations of digestive proteases were studied in three life stages of spotted rose
snapper: early (EJ), middle (MJ) and late juvenile (LJ) with corresponding average weights of 21.3 ± 2.6 g (3
months after hatching, MAH), 190 ± 4.4 g (7 MAH), and 400 ± 11.5 g (12 MAH). At sampling points, the
digestive tract was dissected into the stomach (St), pyloric caeca (PC), and the intestine in three sections
(proximal (PI), middle (MI) and distal intestine (DI)). The effect of pH and temperature and specific inhibitors
were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to
increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and
alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total
activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was
significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant
differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through
juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead
a better comprehension of changes in digestive potential of Lutjanidae fish.
Keywords: Lutjanus guttatus, spotted rose snapper, digestive enzymes, pepsin, trypsin, protease inhibitors.
Caracterización comparativa de la actividad de la proteasa en juveniles
cultivados de pargo flamenco (Lutjanus guttatus)
RESUMEN. Se caracterizaron parcialmente las proteasas ácidas y alcalinas en tres estadios juveniles del pargo
flamenco: temprano (EJ), medio (MJ) y juvenil tardío (LJ) con pesos promedios correspondientes a 21,3 ± 2,6
g (3 meses post-cultivo larvario, MAH), 190 ± 4,4 g (7 MAH) y 400 ± 11,5 g (12 MAH). El tracto digestivo fue
seccionado en estómago (St), ciegos pilóricos (PC) e intestino en tres secciones (proximal (PI), medio (MI) e
intestino distal (DI)). El efecto de la temperatura, pH e inhibidores específicos sobre proteasas ácidas y alcalinas
fue evaluado en los tres estadios juveniles. Los resultados indican una tendencia de aumento en la actividad de
proteasas ácidas y alcalinas totales con el aumento de edad, mientras que la actividad de tripsina disminuye con
la edad. Se encontraron diferencias en actividad de proteasas ácidas y alcalinas a diferentes temperaturas y pH
entre los tres estadios juveniles. Pepstatin A inhibió la actividad total de proteasas ácidas en los tres estadíos
juveniles. La inhibición de la actividad de proteasas alcalinas con los inhibidores TLCK, PMSF, SBTI, Phen y
Ovo fue significativamente mayor en el estadio EJ en comparación a MJ y LJ, mientras que no se encontraron
diferencias en inhibición con TPCK. El pargo flamenco presenta un incremento en actividad total de proteasas
ácidas y alcalinas en conjunto con su desarrollo juvenil, aunado a una sustitución o diversificación en el tipo de
proteasas alcalinas. Estos resultados permiten una mejor comprensión de los cambios en el potencial digestivo
de lutjánidos.
Palabras clave: Lutjanus guttatus, pargo flamenco, enzimas digestivas, pepsina, tripsina, inhibidores de
proteasas.
_____________________
Corresponding editor: Erich Rudolph
641
642
INTRODUCTION
The spotted rose snapper (Lutjanus guttatus) has a high
potential for intensive culture in Latin American
countries (Davis et al., 2000). In Mexico and Costa
Rica, fish farmers capture wild juveniles and stock
them in floating sea cages where they are fed until they
reach the appropriate market size (450 g) (HerreraUlloa et al., 2010). Reproduction techniques for
juvenile mass production in hatcheries have been
developed on a pilot scale for this species in Mexico
(Ibarra-Castro & Alvarez-Lajonchère, 2011). The
spotted rose snapper, similar to other members of the
Lutjanidae family, are carnivorous marine fish
distributed in tropical zones. They primarily feed on
demersal organisms, such as crustaceans and fish
(Allen, 1995). Under culture conditions, they require a
high protein diet containing between 45 and 50%
(Silva-Carrillo et al., 2012). This species has a welldefined stomach, with five to six blind sacs in a pyloric
caeca, and a very short intestine. Little information is
available regarding the digestive physiology Lutjanids
and more knowledge in this area is required to develop
appropriate feeds for rearing to market size.
Some studies describe the early ontogeny development of the digestive system in spotted rose snapper,
presenting same pattern of digestive enzyme activity as
previously reported for other species, in which
pancreatic and intestinal enzymatic activities are
present at hatching (Moguel-Hernández et al., 2013),
and maturation of digestive function occurs around 2025 days after hatching with pepsin secreted by
functional stomach, described by Galaviz et al. (2012).
Studies in others Lutjanidae species (Alarcón et al.,
2001) described the effect of plant regional protease
inhibitors on digestive proteases of yellow snapper (L.
argentiventris) and Pacific dog snapper (L.
novemfasciatus). Additionally, Khantaphant &
Benjakul (2008, 2010) reported the skin gelatin
hydrolyzation capability in brown stripe red snapper (L.
vitta) with proteases from pyloric caeca and performed
a trypsin characterization for this species. Therefore,
early development of digestive enzymes in L. guttatus
has been described, but similar research on the juvenile
or adult stage has not been performed.
Some authors have indicated that independent of
feeding habits, fish digestive system responses closely
correlate with diet and age (Pérez-Jiménez et al., 2009;
Falcon-Hidalgo et al., 2011). Differences in proteolytic
enzyme activities and zymogens in fish at different ages
have been reported, but the changes have been
attributed to feeding habitats or diet changes and not
solely influenced by age (Falcon-Hidalgo et al., 2011;
Unajak et al., 2012). Other report presents the existence
of variations of genetically trypsin-like isozymes
correlated with fish size in Salmo salar fry (Torrissen,
1987), and these variations are related and could affect
growth rate and/or feed conversion efficiency
(Torrissen & Sharer, 1992). Hence, determine possible
changes in proteases potential over juvenile ontogeny
that represents culture time period is important, which
could be useful to develop efficient specific size diets
to optimize growth of L. guttatus.
Therefore, protease activity could change during
juvenile stages of L. gutattus with different digestive
potential and possible variations in protease enzymes or
isozymes. Therefore, the objective of this study was to
compare the partial characterization of acid and
alkaline digestive proteases in the digestive tract of
three spotted rose snapper juvenile stages using
biochemical techniques to understand the protein
digestive potential variations during the culture period
of spotted rose snapper.
Experimental animals
Fish for this study were obtained from the Laboratory
of Reproduction and Marine Finfish Hatchery (CIAD),
Sinaloa, México, where all juvenile stages were
obtained from single spawning batch, conducted as
described by Alvarez-Lajonchère et al. (2012). After
one batch larval culture, all juvenile fish continued
under normal culture (nursery step) and fattening
process. Fish were collected in different times from one
cycle. When given fish stage was required, fish were
place in tanks and fed the same feed for 20 days.
According to their wet weight, fish where classified in
three groups (all considered in the juvenile stage): early
juvenile (EJ; 21.3 ± 2.6 g; 3 month after hatchery,
MAH), middle juvenile (MJ; 190 ± 4.4 g; 7 MAH) and
late juvenile (LJ; 400 ± 11.5 g; 12 MAH). Diet
adaptation was performed in fiberglass tanks (4000 L)
with a constant water flow and the fish were fed twice
at day (9:00 and 16:00 h) with a diet containing
fishmeal as a main protein source (Table 1). Fish were
starved for 24 h to ensure the emptiness of the gut,
euthanized ethically by a single puncture in the head
with scalpel and immediately dissected to extract the
digestive tract. The parameters and biometric indices of
fish used in the assays are summarized in Table 2.
Dissection and extract preparation
The digestive tract of each fish was individually
divided into five segments: stomach (ST), pyloric caeca
(PC), and intestine in three sections (proximal (PI),
middle (MI) and distal intestine (DI). All of the proce-
Table 1. Composition and proximate analyses of diet for
spotted rose snapper L. guttatus. 1Premium grade fish
meal was obtained from Selecta de Guaymas, S.A. de C.V.
Guaymas, Sonora, México. 2Marine Protein and
Agricultural, S.A. of C.V., Guadalajara, Jalisco, México.
3
PROAQUA, S.A. de C.V. Mazatlán, Sinaloa, México.
4
Droguería Cosmopolita, S.A. de C.V. México, D.F.,
México. 5Trouw Nutrition México S.A. de C.V. (by
courtesy). 6DSM Nutritional Products México S.A. de
C.V., El Salto, Jalisco, México. *Vitamin premix
composition: Vitamin A, 10 000 000 IU o mg g-1; Vitamin
D3, 2 000 000 IU; Vitamin E, 100 000 g; Vitamin K3, 4.00
g; Thiamine B1, 8.00 g; Riboflavin B2, 8.70 g; Pyridoxine
B6, 7.30 g; Vitamin B12, 20.00 mg; Niacin, 50.00 g;
Pantothenic acid, 22.20 g; Inositol, 153.80 g; Nicotinic
Acid, 160.00 g; Folic acid, 4.00 g; 80 mg; Biotin, 500 mg;
Vitamin C, 100.00 g; Choline 300.00 g, Excipient c.b.p.
2000.00 g. **Mineral premix composition: Manganese,
100 g; Magnesium, 45.00 g; Zinc, 160 g; Iron, 200 g;
Copper, 20 g; Iodine, 5 g; Selenium, 400.00 mg; Cobalt
600.00 mg. Excipient c.b.p. 1500.00 g.
Ingredient
Fishmeal
Squid meal2
krill meal3
fish oil4
Dextrine4
Wheat gluten4
Vitamin premix5*
Minerals premix5**
Carotenoids6
Antioxidant6
Soybean lecithin (70%)6
Vitamin C6
Alginate4
Proximate analyses
Dry matter
Crude protein
Crude fat
Ash
Nitrogen free extract
1
(% dry weight)
52.60
6.00
7.59
8.78
17.47
2.00
0.60
0.23
0.08
0.05
1.50
0.10
3.00
92.91
43.06
13.86
14.01
15.27
dures were conducted at temperatures of 0-4°C. All
segments were frozen individually at -64°C for 24 h and
then lyophilized for four days and stored under dry
conditions at 4°C until the assay was conducted. Prior
to analysis, each lyophilized segment, diluted at a ratio
of 1:10 (wet weight: volume) in a physiological saline
solution (NaCl 9 g L-1), was ice-cold-homogenized with
an Ultra-Turrax homogenizer. All homogenates were
centrifuged (8500 g) at 4°C for 15 min, and the
supernatant was used to perform enzyme activity assays
(Matus de la Parra et al., 2007).
643
Enzyme activity assay
The pepsin-like or total acid protease activity was
measured by a modified method of Sarath et al. (1989),
with denatured hemoglobin (2% pH 2) as substrate. The
enzymatic reaction mixture consisted of 300 µL of
substrate with 0.2 mol L-1 glycine-HCl buffer (pH 2)
and 100 µL of enzymatic extract, incubated at 37°C and
stopped by the addition of 600 µL of 5% (w/v)
trichloroacetic acid (TCA). Alkaline protease activity
was estimated by method of Walter (1984) using casein
as substrate. The enzymatic reaction mixtures consisted
of 250 µL of 0.1 mol L-1 Tris-HCl buffer, 0.01 M (pH
9) CaCl2, 100 µL of enzymatic extract and 250 µL of
1% casein in Tris-HCl buffer, incubated at 37°C and
stopped by adding 600 µL of 8% (w/v) TCA. The
trypsin activity was determined by modified method of
Erlanger et al. (1961). A Nα-benzoyl-L-arginine-4-pnitroanilide hydrochloride (BAPNA 1 mmol L-1)
substrate was used. The enzymatic reaction mixtures
consisted of 560 µL of substrate in 0.05 mol L-1 TrisHCl, 0.01 mol L-1 (pH 8.2) CaCl2 and 80 µL of
enzymatic extract, incubated at 37°C and stopped by
adding 160 µL of acetic acid at 30%. The protein
content of the supernatant solution was determined by
Bradford assay (1976) using bovine serum albumin as
the standard.
One unit (U) of enzymatic activity was defined as
the amount of enzyme that produced 1 µg of product
released per minute. Tyrosine amount liberated from
haemoglobin and casein hydrolysis was determined at
280 nm, while amount of p-nitroaniline liberated from
BAPNA was determined at 410 nm.
Total activity (Units mL-1) = [Δabs*reaction final volume
(mL)]/[MEC*time (min)*extract volume (mL)]
Specific activity (Units mg prot-1) = Total activity/
soluble protein (mg),
Tissue activity (Units wet tissue-1) = Total activity
*total tissue (g)
where Δabs represent the increase in absorbance, and
MEC represents the molar extinction coefficient of
tyrosine or p-nitroaniline (0.005 and 0.008 mL/µg/cm,
respectively).
Characterization of digestive enzymes
Pepsin-like, total alkaline protease and trypsin were
characterized by determining the relative activity (%)
as a function of pH and temperature. The temperature
effect for pepsin-like was measured from 10 to 50°C;
alkaline protease and trypsin were measured from 10 to
60°C, with similar assay conditions as previously
described. The pH effect on digestive activity was
measured at 37°C, and the following buffers were used:
644
Table 2. Biometric parameters for three juvenile stages of spotted rose snapper Lutjanus guttatus. DSI: (Digestive tract
weight (g)/fish weight (g))*100. Digestive tract represents the sum of stomach, pyloric caeca and intestine weight. EJ, MJ
and LJ represent early, middle and late stage juveniles, respectively. Different superscript within columns indicate
significant differences (P < 0.05).
Stage
Fish weight (g)
EJ
MJ
LJ
21.3 ± 2.6
190.0 ± 4.4
400.0 ± 11.5
glycine-HCl at a pH of 1 to 3; acetate buffer at a pH of
4 and 5; Tris-HCl at a pH of 7 to 9; and glycine-NaOH
at a pH of 10. The buffers molarities were 0.2 mol L-1
for acid proteases, 0.1 mol L-1 for alkaline proteases and
0.05 mol L-1 for trypsin activity, with CaCl2 (0.01 mol
L-1) for alkaline protease and trypsin activities (Matus
de la Parra et al., 2007).
In addition, characterizations of acid and alkaline
proteases were performed according to Guerrero-Zárate
et al. (2014) using specific inhibitors. Pepstatin A (1
mmol L-1) was used as an inhibitor of acid proteases
from stomach and alkaline protease activity inhibition
in pyloric caeca sections were performed using the
following inhibitors: 250 mmol L-1 soybean trypsin
inhibitor (SBT1), 10 mol L-1 N-tosyl-L-phenylchloromethyl ketone (TPCK), 100 mmol L-1
phenylmethylsulfonyl fluoride (PMSF), 10 mmol L-1
Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride
(TLCK), 10 mmol L-1 1,10-Phenanthroline (Phen) and
250 mmol L-1 Type II-Turkey egg Ovomucoid (Ovo).
Eight juveniles of each stage of spotted rose snapper
were handled individually to maintain eight replicates
per analysis. For comparison, the percent inhibition and
percent relative activity in enzyme characterization was
arcsin (x1/2) transformed. The data for each parameter
were tested for normality and homoscedasticity. Oneor two-way ANOVA analyses were run when required.
When differences were found, Tukey’s HSD test was
used (P ≤ 0.05). All of the statistical analyses were
performed using Statistica 7.0 Software for Windows
(StatSoft, USA).
RESULTS
Enzyme activity assays
The acid and alkaline proteases activities of different
digestive tract sections in the three juvenile stages are
presented in Table 3. The stomach acid proteolytic
Digestive tract
weight (g)
0.25 ± 0.03
2.5 ± 0.21
7.6 ± 0.42
DSI
1.17 ± 0.12b
1.32 ± 0.11b
1.91 ± 0.09a
activity showed significantly higher specific and tissue
activities (P ≤ 0.001) value with increasing life stage.
No significant differences in specific activity of
alkaline proteases were observed between pyloric caeca
and intestine sections for all juvenile stages (P ≤ 0.001),
however, tissue activity showed higher values in PC
than other intestine sections for all juvenile stages.
Meanwhile, significantly higher specific and tissue
activities in the LJ stage (P ≤ 0.001) were found
between stages when individual sections were
compared. The trypsin-like specific activity showed a
significantly higher (P ≤ 0.001) value in the EJ stage
than MJ and LJ stages (Table 4), nevertheless, tissue
activity values increase with increasing life stage (P ≤
0.001).
Temperature effect on acid and alkaline protease
activity
The three juvenile stages presented optimum
temperature of acid proteases at 45°C (Fig. 1a) (P ≤
0.001). Acid proteases relative activity at 30°C showed
differences between EJ, MJ (70%) and LJ (40%) (P ≤
0.001), while relative activity at 50°C showed
differences between EJ (80%) and MJ and LJ (60%) (P
≤ 0.001). The optimum temperature of total alkaline
proteases was 55°C for EJ, 50°C for MJ and LJ (Fig.
1b) (P ≤ 0.001). Differences were found in the relative
activity percent at 20, 30, 40 and 60°C between EJ and
the other stages (P ≤ 0.001). In general terms, LJ
showed higher relative activities (%) than EJ and MJ in
total alkaline protease activity, when individual
sections were compared.
Effect of pH on acid and alkaline protease activity
The optimum activity of acid proteases was measured
at pH 3 for EJ and LJ and at pH 2 for MJ, with 80 to
90% of remnant activity at pH 2 and 3, respectively
(Fig. 2a) (P ≤ 0.001). Significant differences in relative
activity at pH 4 were found between EJ, LJ (30%) and
LJ (50%) (P ≤ 0.001). Alkaline protease activity
showed high relative activity (%) over a wide pH range
645
Table 3. Protease activity in the stomach (ST), pyloric caeca (PC), proximal (PI), middle (MI) and distal intestine (DI) in
three juvenile stages of spotted rose snapper Lutjanus guttatus. EJ, MJ and LJ represent early, middle and late stage
juveniles, respectively. Lower-case show differences in columns, upper-case show differences in rows.
Stage
EJ*
**
MJ*
**
LJ*
**
*Specific activity (U mg protein-1) of crude extract
**Tissue activity (U wet tissue)
ST
PC
PI
1754.4 ± 307.8c
17.4 ± 5.9b
15.0 ± 1.1c
269.2 ± 28.7 c
2.15 ± 0.6 A,c 0.46 ± 0.1 B
b
3864.2 ± 796.0
22.2 ± 3.8b
20.0 ± 2.4b
b
A,b
1002.3 ± 161.4
5.24 ± 0.6
2.61 ± 0.7 B
6210.1 ± 657.6a
32.3 ± 4.2a
28.2 ± 3.0a
a
A,a
1746.6 ± 203.6
13.95 ± 1.6
4.33 ± 0.5 BC
MI
15.6 ± 2.9b
0.46 ± 0.9 B
27.5 ± 5.0a
2.05 ± 0.5 B
29.1 ± 6.4a
3.12 ± 0.5 C
DI
15.8 ± 3.2c
0.67 ±0.2 B
23.0 ± 3.8b
2.52 ± 0.6 B
34.0 ± 6.2a
4.59 ± 0.8 B
Table 4. Trypsin-like activity in the pyloric caeca in three
juvenile stages of spotted rose snapper Lutjanus guttatus.
EJ, MJ and LJ represent early, middle and late stage
juveniles, respectively. Different superscript within rows
indicate significant differences (P < 0.05).
*Specific activity (U mg protein-1)
**Tissue activity (U wet tissue)
EJ
MJ
LJ
a
b
*82.50 ± 2.24
*23.18 ± 2.47
*22.77 ± 9.66b
b
b
**0.35 ± 0.01
**3.68 ± 0.47
**13.11 ± 5.63a
(5-10) and an optimum at pH 9 in the three juvenile
stages (Fig. 2b) (P ≤ 0.001) Differences were found in
relative activity percent at pH 5 between LJ (80%) and
EJ, MJ (50%) (P ≤ 0.001).
Temperature and pH effect on trypsin activity
The optimum temperature of trypsin was 50°C for MJ
and LJ, while EJ presented an optimum at 60°C.
Differences were found in relative activity (%) between
almost all temperatures tested (P ≤ 0.001). In general,
EJ presented higher relative activities (%) than MJ and
LJ (Fig. 3a). Trypsin activity showed optimum activity
at pH 9 for all juvenile stages. Remnant activity showed
significant differences (P ˂ 0.001) at pH 10 (between
80 and 90%) versus pH 8 (between 40 and 60%) (Fig.
3b).
Specific inhibitors effects
Pepstatin A inhibited the total activities in stomach
extracts in all juvenile stages (Fig. 4). The percent of
alkaline protease inhibition are summarized in Table 5.
In general, the inhibited percent of activity in total
alkaline proteases was significantly higher (P ≤ 0.001)
Figure 1. Temperature effects (°C) on the relative activity
of a) acid, and b) alkaline proteases in three juvenile stages
of Lutjanus guttatus.
in EJ using TLCK, PMSF, SBTI, Phen and Ovo
compared to MJ and LJ, while no significant differences were found between inhibition percent with TPCK
(P = 0.240).
646
Figure 2. pH effects on the relative activity of a) acid and
b) alkaline proteases in three juvenile stages of Lutjanus
guttatus.
Figure 3. a) Temperature and b) pH effects on the relative
trypsin-like activities in three juvenile stages of Lutjanus
guttatus.
DISCUSSION
Previous studies in early ontogeny of the present
species report the presence of wide battery of digestive
enzymes, such as pancreatic (i.e., trypsin, chymotrypsin, amylase, and lipase) and intestinal (i.e., acid
and alkaline phosphatases and leucine aminopeptidase)
present from hatching, joined to appearance of pepsin
activity between 20-25 days after hatching, considered
as onset of juvenile period (Galaviz et al., 2012;
Moguel-Hernández et al., 2013). However, this is the
first work focused in changes over digestive proteases
during ontogeny of juvenile stages, where differences
in some parameters suggest the presence of other
proteases type in larger juvenile stages and at same
time, the use of a variety of specific inhibitors confirm
the presence of wide range of proteases in the species.
As a rule, total digestive activity increases with fish
age due to the increase of digestive tract size and
mucosa weight (activity * total intestinal mucosa weight)
Figure 4. Percent of residual activity in stomach extract
after incubation with pepstatin A in three juvenile stages
of Lutjanus guttatus. EJ, MJ and LJ represent early,
middle and late stage juveniles, respectively.
647
Table 5. The percent of activity inhibition in pyloric caeca after incubation with enzyme specific inhibitors in three juvenile
stages of spotted rose snapper Lutjanus guttatus. EJ, MJ and LJ represent early, middle and late stage juveniles, respectively.
*SBTI (soybean trypsin inhibitor), TPCK (N-tosyl-L-phenyl-chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride),
TLCK (Nα-tosyl-L-lysine chloromethyl ketone hydrochloride), Phen (1.10-Phenanthroline), Ovo (Type II-T: Turkey egg
ovomucoid). Different superscript within columns indicate significant differences (P < 0.05).
Inhibitor concentration
(mmol-1)
Inhibitor type*
EJ
MJ
LJ
Percentage of activity inhibition
10
TPCK
11.7 ± 4.8 a
9.9 ± 2.6 a
6.6 ± 2.1 a
10
TLCK
14.2 ± 1.3a
6.1 ± 0.6b
7.9 ± 1.3b
(Kuz᾽mina, 1996). In the present work a higher relation
between digestive tract weight and total fish weight was
found when increasing fish age as we detected in (Table
2), represented by higher DSI values. Therefore,
increasing activities in all digestive sections related
with increasing juvenile stage is in accordance with
above mentioned. Some reports in certain fish species
indicate that changes in specific enzyme activity (U mg
prot-1) vary at different ages (Chiu & Pan, 2002;
Falcon-Hidalgo et al., 2011), which was found in this
study and represents a higher capacity for protein
breakdown. Some authors have reported comparative
activities between fish stages, but the results are
attributed to adaptations in feeding habitats (Kuz᾽mina,
1996; Falcon-Hidalgo et al., 2011). In this work,
changes in specific activity (U mg prot-1) and tissue
activity (U wet tissue) for proteases at different juvenile
stages were present, even though the three juvenile
stages are from same batch culture and were
conditioned for 20 days with the same diet and feeding
frequency. Furthermore, differences at varying
temperatures and pH were attributed to ontogenetic
digestive changes and adaptations. L. guttatus shows
adequate adaptive changes in enzyme activities that
correspond to other carnivorous species, with
proteolytic activity increasing with growth (Kuz᾽mina,
1996; Falcon-Hidalgo et al., 2011).
The high activities found in acid proteases of all
juvenile stages of L. guttatus is an important
characteristic leading to a more efficient breakdown
and utilization of feed protein. The acid protease
activities reported for EJ are comparable with those
reported in gilthead seabream (Sparus aurata),
common dentex (Dentex dentex) (Alarcón et al., 1998)
and L. novemfasciatus and L. argentiventris (Alarcón et
al., 2001) (sampled fish weighed between 25 and 50 g).
Gastric digestion increase intestinal hydrolysis, leading
to a significant shift in soluble polypeptides to oligoand dipeptides (Yasumaru & Lemos, 2014). Therefore,
100
PMFS
15.7 ± 2.5a
13.6 ± 0.6a
5.4 ± 1.9b
250
SBTI
54.9 ± 6.6a
25.8 ± 5.4b
16.1 ± 3.9c
10
Phen
32.7 ± 2.0a
28.8 ± 1.3b
23.3 ± 1.1c
250
Ovo
18.5 ± 1.2a
7.3 ± 0.5b
6.3 ± 1.0b
because acid protease activities are higher with growth
in L. guttatus, fishmeal could be reduced in the
balanced diets of larger fish, and a higher amount of
plant or animal by-products as protein sources in feeds
could be used.
Specific activity between PC and the three intestine
sections did not show variation, however, tissue activity
showed higher activity in PC that other intestine
section, related to tissue size. Pyloric caeca in fish is an
organ with principal function of increase surface area
and hence the nutrient uptake (digestion and
absorption) capacity of fish, where PC is reported as the
major site of uptake, even than the entire remaining
alimentary tract (Buddington & Diamond, 1986), as
reported in the present study.
The total alkaline protease activity at 37°C for L.
guttatus in the three juvenile stages are comparable to
those reported for L. argentiventris (52.3 ± 3.9 U mg
prot-1) and L. novemfasciatus (17.2 ± 1.1 U mg prot-1)
(Alarcón et al., 2001). The use of a non-specific
technique (Walter, 1984) at a neutral and basic pH
enables the quantification of activities of different
proteases, such as trypsin, chymotrypsin, carboxypeptidases, aminopeptidases, elastases and collagenases as
the main proteases that acts together as reported in
several fish species (Torrissen,1987; Klomklao, 2008;
Unajak et al., 2012). This demonstrates the real
digestion capacity of the species over a wide range of
parameters. In this sense, the extracts use from the
digestive system of the species of interest is more
suitable, because a complex battery of digestive
enzymes catalyses digestion (Alarcón et al., 2002). On
the other hand, temperatures and pH used in the assays
are only operational parameters used to understand
changes in enzymatic activities among juvenile L.
guttatus stages and are not exactly the same as natural
conditions. Moreover, similar to other poikilothermic
fish species, L. guttatus possess a maximum and
minimum tolerance for some parameters.
648
Most fish species have two or three major pepsins
with an optimum haemoglobin digestion at a pH
between 2 and 4 (Gildberg & Raa, 1983; Klomklao,
2008). In this study, the optimum pepsin-like enzyme
activity occurred at pH 2 for MJ and at pH 3 for EJ and
LJ, coupled to total inhibition of pepsin with pepstatin
A in the three juvenile stages and changes in relative
activity (%) at different temperatures and pH indicates
the existence of at least two pepsin isoforms. Klomklao
et al. (2007) reported pepsin A and pepsin B
characterization from giant grenadier (Coryphaenoides
pectoralis) with different optimum pH (3.0 and 3.5,
respectively) and an optimum temperature of 45°C.
Chiu & Pan (2002) report that two pepsins, designated
PI and PII, isolated from stomach of juvenile and adult
of Japanese eel (Anguilla japonica) and differences in
optimum pH and total activity between isoforms were
found.
Alkaline proteases present a wide range of activity;
over 80% of the relative activity occurred in the pH
range of 7 to 10 for the three juvenile stages. EJ and MJ
present a relative activity that fell to 50% at a pH of 5,
while LJ conserve relative activity (80%). The presence
of other alkaline protease type such as thiol proteasetype called cathepsin, which appears to be pancreatic or
intestinal in origin (Kirschke & Barret, 1987) could
explain these results. Cathepsins from different species
display maximum activity over a broad pH range from
3.5 to 8.0 (Zeef & Dennison, 1988).
Four serine-protease inhibitor types were used
(TLCK, PMFS, SBTI and Ovo), where TLCK and
SBTI showed a more trypsin-like affinity for enzyme
inhibitors. Strong relative inhibition of SBT1 was
found in EJ (54.9 ± 6.6%), while other inhibitors
showed a lower relative contribution (14.2 ± 1.3%, 15.7
± 2.5% and 18.5 ± 1.2% for TLCK, PMFS and Ovo,
respectively). For all of the serine protease inhibitors, a
decreased tendency was found with growth. Serineproteases are found in different isoforms in the pyloric
caeca and intestine in some fishes (Falcon-Hidalgo et
al., 2011; Unajak et al., 2012); therefore, differences in
affinity with inhibitor type could exist, which could
explain variations in the relative contributions of
enzyme type over different juvenile stages in L.
guttatus.
A metalloproteinase type inhibitor (Phen) showed a
tendency to decrease with age, and fluctuated between
32.7 ± 2.0 and 23.3 ± 1.1 for EJ and LJ, respectively.
Collagenolytic serine proteases differ from muscle
collagenases, which belong to zinc metalloproteinase,
and physiological function in several organisms is
attributed to their digestive power (Kristjansson et al.,
1995), and they display both trypsin-like and
chymotrypsin-like activities (Haard, 1994) and have
been previously characterized in Atlantic cod (Gadus
morhua) (Kristjansson et al., 1995). Other authors
report in mammalian and fish pancreases existence of
two zinc carboxypeptidases, previous reported in
marine organisms (Hajjou et al., 1995; Kishimura et al.,
2006).
Total alkaline and trypsin-like optimum activities at
different temperatures show differences between the EJ
stage and other juvenile life stages. A different type of
enzyme or isozyme can be expressed in the EJ stage and
not in other juvenile stages, results that are in
accordance with other reports (Torrerissen, 1987;
Unajak et al., 2012). In conjunction with the abovementioned, total specific trypsin-like activity was four
times higher in EJ than other juvenile stages (Table 4),
where the specific activity reported for MJ and LJ are
in accordance with the trypsin-like activity reported for
L. vitta (Khantaphant & Benjakul, 2010), with 21.9 U
mg protein-1 from pyloric caeca extract. Nevertheless,
the optimum trypsin activity differs from the total
alkaline protease optimum in the EJ stage, and
combined with the decrease of inhibition average by
serine-inhibitors with fish growth, indicate that trypsinlike enzymes are not the main digestive alkaline
enzymes and other types of enzymes are present in this
species.
In this study, no gonad was found in 400 g fish
represented by the LJ stage, therefore, changes found in
protease activity under different conditions could not be
attributed to the onset of sexual maturity. In addition,
some authors report enzyme changes during the
ontogenesis of fish, suggesting that specific types of
protease could be produced at a specific fish age by
means of fish ontogenesis (Torrissen, 1987; Kuz᾽mina,
1996; Bassompierre et al., 1998; Chiu & Pan, 2002;
Rathore et al., 2005; Chakrabarti et al., 2006; Unajak et
al., 2012).
In conclusion, the digestive system of spotted rose
snapper is highly efficient in the breakdown of protein.
The high pepsin activities suggest the potential for
hydrolysis of a wide range of protein sources joined to
final alkaline digestion. This potential increases with
fish growth through juvenile stages in which a
substitution or diversification in the type of alkaline
enzymes exists. The present study represents the first
research conducted on digestive proteases activities
with comparative objective in snapper juveniles and
that will serve as a basis for future studies in SDS-Page
electrophoresis and in vitro digestibility assays with
different protein sources, that will provide more
information about the digestive physiology of L.
guttatus at different juvenile stages, which will be
useful to develop efficient diets to optimize growth
under cultural conditions.
ACKNOWLEDGMENTS
This research was co-funded by a research grant from
the National Council for Science and Technology
(CONACyT) of Mexico SAGARPA (Project 164673).
The authors are grateful to Margarita HernandezMaldonado for her technical assistance. Emyr Peña
would like to thank CONACyT for his graduate study
fellowship.
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Lat. Am. J. Aquat. Res., 43(4): 651-661,Risk
2015 assessment of shrimp trawl fishery in the Gulf of California
651
Research Article
Risk assessment and uncertainty of the shrimp trawl fishery in the Gulf of
California considering environmental variability
Luis César Almendarez-Hernández1, Germán Ponce-Díaz1, Daniel Lluch-Belda1†
Pablo del Monte-Luna1 & Romeo Saldívar-Lucio1
1
Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas
Av. IPN s/n, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, México
Corresponding autor: Luis Almendarez ([email protected])
ABSTRACT. The shrimp fishery off the Mexican Pacific coast is the country's most important fishery from the
economic standpoint. However, it faces serious problems, including the fleet’s overcapitalization and age, in
addition to the environmental variability that affects the size of catches. Thus, this activity depends on a variety
of factors that add uncertainty to the profitability of fishing vessels. This study aims to estimate the probability
of success and economic risk of "type vessels" under two different environmental variability scenarios in the
Gulf of California. The results from the economic simulation pointed to the vessel type used in Guaymas
(Sonora) as the most efficient one under a neutral climate change scenario, showing a homogeneous behaviour
in physical characteristics and mode of operation. By contrast, under a scenario of a monotonic rise in sea surface
temperature, the shrimp fishery faces a greater risk of incurring economic losses. The simulated climate
behaviour scenarios revealed that the activity involves a moderate economic profitability under the neutral
scenario; however, under the warming scenario, profitability may be low or even nil due to the risks and
uncertainty resulting from the influence of environmental phenomena.
Keywords: shrimp, sensitivity analysis, risk, uncertainty, Gulf of California, México.
Evaluación de riesgo e incertidumbre de la pesquería de camarón de alta mar
del golfo de California considerando la variabilidad ambiental
RESUMEN. La pesquería de camarón del litoral del Pacífico es la más importante del país desde el punto de
vista económico. Sin embargo, afronta serios problemas como la sobre-capitalización y antigüedad de la flota,
además está presente la variabilidad ambiental que influye en la abundancia de las capturas. Así esta actividad
depende de varios factores que generan incertidumbre en la rentabilidad económica de las embarcaciones. El
objetivo del trabajo fue estimar la probabilidad del éxito y riesgo económico de embarcaciones "tipo"
considerando dos escenarios de variabilidad ambiental en el golfo de California. Los resultados de la simulación
económica indicaron que el barco más eficiente es el de Guaymas (Sonora) bajo el escenario de cambio climático
neutral, mostrando un comportamiento homogéneo en sus características físicas y forma de operar. Por el
contrario, bajo condiciones de calentamiento monótono de la temperatura superficial del mar, la actividad
presenta mayor riesgo de incurrir en pérdidas económicas. Los escenarios simulados de comportamiento
climático mostraron que la actividad tiene una rentabilidad económica moderada para el escenario neutral. En
condiciones de calentamiento la rentabilidad llega a ser baja o inclusive nula, debido al factor de riesgo e
incertidumbre provocado por la influencia de los fenómenos ambientales.
Palabras clave: camarón, análisis de sensibilidad, riesgo, incertidumbre, golfo de California, México.
INTRODUCTION
Overview of the Mexican shrimp fishery
The shrimp fishery is not only the most complex
activity of this kind in Mexico for its broad geographic
___________________
Corresponding editor: José Ángel Álvarez P.
distribution, multi-species composition and sequentiality of captures, which altogether involve a number
of fisheries, fishing gear, social sectors and fishing
strategies; it is also the most important fishery in
Mexico as a source of foreign income. The price of shrimp
652
shrimp in the international market is high, ranging from
5 to US$9 per pound, with the United States as the
largest buyer.
The volume of wild shrimp catches reported for the
Mexican Pacific coast ranks third countrywide; with
just over 42.000 ton live weight, 92% of which come
from the Gulf of California (CONAPESCA, 2012). The
importance of this fishery is also evident from the social
standpoint, as it generates over 37,000 jobs in the
Pacific coast alone (INP, 2006).
Shrimp are exploited by both offshore and artisanal
fisheries; the latter is conducted in protected waters
(bays and lagoons). In 2012, the industrial shrimp fleet
was comprised by 906 shrimp vessels that share
common features as defined in the National Fishing
Chart for large fishing units or vessels (DOF, 2012),
and represents 70% of the total large fleet in Mexico
(CONAPESCA, 2011). These vessels have been
continuously improved both to increase their autonomy
and to enable them to operate in depths ranging from 9
to 90 m (INP, 2006). This study focused only on the
industrial fleet, which concentrates the greatest
investment and has been operating the shrimp fishery
for a longer time.
Artisanal fishing is conducted in small boats
(locally named “pangas”), with approximately 56,412
units recorded, 85% of which are estimated to be
devoted to shrimp fishing (INP, 2006). Artisanal
fishing provides most jobs, whereas the shrimp trawl
fishery generates more economic value by catching
larger specimens, which amount to 53% of the sea catch
in the Pacific coast, while the rest comes from bays and
lagoons (CONAPESCA, 2012).
The shrimp fishery relies on four different penaeid
species, known locally as white (Litopenaeus vannamei),
blue (L. stylirostris), brown (Farfantepenaeus californiensis) and red or crystal (Farfantepenaeus brevirostris) shrimp. The brown shrimp is the most abundant
species in the Pacific Ocean fishing grounds, followed
by the blue and white shrimps and, with the lowest
abundance, by the red or crystal shrimp (Lluch-Cota et
al., 2006).
The states of Sinaloa and Sonora are the top
producers along the Mexican Pacific coast. Their high
production is determined by factors such as vessels
concentration, port infrastructure and the presence of
processing plants (CONAPESCA, 2011).
The issues
The Mexican shrimp trawl fishery, particularly the one
that operates inside the Gulf of California, faces
significant problems, including over-capitalization (a
surplus of vessels in relation to those required to
optimize yield per vessel) and fleet senescence (LluchBelda, 1974; Quimbar, 2004; García-Caudillo &
Gómez-Palafox, 2005). These problems are the result
of poor management practices as well as organizational
and structural limitations arising from failed or absent
public policies to promote a proper performance of the
activity (Medina-Neri, 1982; Quimbar, 2004; INP,
2006; Almendarez-Hernández, 2008).
In addition, the fishery’s historical statistical
records show variations not entirely accounted for by
the fishing effort alone, likely because shrimp catch is
a multifactor phenomenon in which interrelationships
between biological, environmental, economic and
social factors can be expected.
This multifactor phenomenon leads to uncertainty in
terms of the fleet’s economic profitability, as the
shrimp fishery is a high-risk activity due to catch
variability. The aim of this study is to conduct a
probabilistic estimation of the economic success and
risk of shrimp trawler vessels under different scenarios
of environmental variability in the Gulf of California.
Stochastic simulation models are commonly used for
analyzing capital expenditure and generating management scenarios under uncertainty conditions (Richardson
& Mapp, 1976; Richardson et al., 2000). The model used
in this study works on a number of Microsoft® Excel
spreadsheets and utilizes a companion program named
Simulation and Econometrics of Risk Analysis,
SIMETAR©, developed at Texas A&M University
(Richardson et al., 2004, 2008). For this study we used
data from the fishing fleet operating along the Gulf of
California, particularly in the states of Sinaloa and
Sonora (Fig. 1).
Representative shrimp trawler vessels
The Representative Production Units (RPU) method is
based on a panel technique. A panel includes groups of
producers who characterize a production system; all
production units within a given production system are
similar to each other. Producers are grouped through a
consensus-building process, by identifying the main
characteristics (scale, production and marketing) that
define the region’s most representative production unit
(SAGARPA, 2010).
To define a RPU, groups of shrimp trawler vessels
sharing common characteristics were first identified
based on official information issued by the fisheries
authorities. These groups were called Representative
Shrimp Trawler Vessels (RSV) in this study. This
information was supplemented and corroborated through
Risk assessment of shrimp trawl fishery in the Gulf of California
653
Figure 1. Map of the Gulf of California, bordered by the states of Baja California Sur (BCS), Baja California (BC), Sonora
and Sinaloa. Guaymas (Sonora) and Mazatlán (Sinaloa) ports are the main shrimp producers and concentrate most of the
industrial fleet.
meetings and interviews with shrimp trawl fishermen
(owners) held in Mazatlán and Guaymas.
In order to gather representative information on the
fishery’s activity, data on the major costs incurred by
producers during the shrimp trawler vessels operation,
shrimp sale (export and domestic) prices, taxes,
subsidies, etc. were collected directly from producers
during the meetings (SAGARPA, 2010).
Two meetings with producers of offshore shrimp
fishery from the Gulf of California (AlmendarezHernández, 2013) were also held. The first meeting
aimed to gather information on the activity’s economic
aspects. These meetings were held in January 2010 in
Guaymas and Mazatlán. The second meeting was
meant to validate the data that would afterwards feed
the economic simulation model, as well as to make
adjustments according to the producers’ perspective.
These meetings were held in August 2010 in Mazatlán
and in December 2010 in Guaymas.
Economic analysis and environmental variability
Data supplied by producers were used for analyzing
and simulating the economic and financial performance
of previously characterized RSVs from analysing
Guaymas and Mazatlán. The simulation model used is
based on the analysis of Net Cash Income (NCI), as
defined by the following equation:
YN  YT  CT
where: YN = net cash income, YT = total income, and
CT = total costs.
NCI represents the average figure obtained from
subtracting total cash outflows from total income over
the 2010-2019 period. Total Income (TI) is the average
cash income from all possible sources, including sales,
subsidies and other income related to the activity.
Finally, total costs (TC) correspond to the total cash
outflow resulting from each vessel operation; i.e., the
sum of variable plus fixed costs.
The model used is based on an iterative, stochastic,
Monte Carlo simulation process that relies on empirical
probability distributions to generate random outputs.
The larger the number of iterations (i.e., the more
simulations are run), the more statistically reliable the
result (Richardson & Outlaw, 2008; Baca-Urbina,
2010). For this study, the model was set to run 500
iterations, each producing outputs for a 10-year
planning horizon.
The model used empirical probability distributions
of projected price and income for the analysis, under
different assumptions:
654
Base year information:
Total costs
Income generated by the activity
30-year historical data series of production volumes
30-year historical data series of product prices
Climate greatly influences shrimp populations, as
these species are favoured by mild El Niño events
(Lluch-Cota et al., 1995), probably due to the increase
in temperature and rainfall, rise in mean sea level, and
decreasing salinity. The input of continental fresh water
increases productivity and promotes shrimp growth
(Soto, 1969; INP, 2000; Rodríguez de la Cruz, 2000).
For the above, warm periods foster higher shrimp
productivity and increased fishery yield, thus making
the shrimp fleet more likely to obtain economic
benefits. However, severe environmental conditions
affect biological shrimp productivity (Castro-Ortiz &
Lluch-Belda, 2008), and extreme warming conditions
were considered in this investigation in order to
simulate a potential negative effect on the fishery. In
order to include environmental factors into the
simulation model and examine two output scenarios,
the projected future behaviour of two different
estimates of future shrimp catch functions-named
neutral climate change and monotonic increase of sea
surface temperature (SST) scenarios-was included.
The neutral climate change scenario portrays the
historical fishery behaviour. The monotonic warming
scenario includes potential future SST conditions as
predicted by the Japanese Atmospheric General
Circulation Model (AGCM/MRI), which is used by the
Intergovernmental Panel on Climate Change (IPCC) to
build the A1B or intermediate scenario (moderate CO2
emissions).
Catch forecasts were obtained by means of a
Generalized Additive Model (GAM). GAMs are semiparametric versions of generalized linear models that
have proved useful in identifying numerical relationships between variations in the abundance of marine
organisms and fluctuations in their environment
(Murase et al., 2009). GAMs are characterized by their
flexibility for describing complex relationships (Hastie
& Tibshirani, 1990).
The determination coefficient and the percentage of
deviance explained (DE) for by the models were used
to assess, firstly, the explanatory power of climate
variables on shrimp abundance. Subsequently, the bestfit models were chosen to exchange atmospheric for
oceanic variables in an attempt to further improve the
models’ goodness of fit. The lowest Generalized CrossValidation (GCV) figures were used as the criterion to
identify the model with the best balance between
goodness of fit and complexity (Wood, 2006). Given
the numerical discrete nature of the response variable,
a quasipoisson error distribution with a log link
function was chosen to build the model, while the scale
parameter was set to 0, denoting that the parameter was
known. GAMs were fitted using the mgcv library
(Wood, 2006) in R (R Core Team, 2013).
The GAMs chosen were built including the
following input variables: 1) the first component (PC)
of rainfall in Sonora, Sinaloa, Baja California and Baja
California Sur; 2) the cumulative sum of SST
anomalies as represented by the Pacific Decadal
Oscillation (PDO) index; and 3) the common pattern of
change in upwelling indices (NOAA, 2013), as
obtained from a minimum/maximum autocorrelation
factor (MAF) analysis that sought to isolate a common
variation pattern among time series from different
localities (Shapiro & Switzer, 1989; Solow, 1994; Zuur
et al., 2007). The output variable was the size of the
shrimp catch.
The two climate change scenarios (neutral and
monotonic warming) were incorporated into the
simulation model in terms of Catch per Unit Effort
(CPUE) figures predicted by the GAM as an indicator
of production per vessel (Csirke, 1989), assuming a
normal probability distribution for each year’s catch.
Resource variability and future availability were
analyzed by building scenarios to explore situations
that might pose a risk to the activity. To this end, a
random component was added to the outputs from the
Monte Carlo simulation model analyzed.
RESULTS
Vessels characterization and information provided
by producers
First, relevant information on the main features of the
shrimp trawler fleet in Sinaloa and Sonora was obtained
from official sources. This information allowed the
discussion of these aspects in meetings held with
representatives from the production sector, to reach an
agreement on the criteria to use for defining each RSV.
The main physical features of each RSV are shown
in Table 1, where differences such as vessel length,
width, engine power, number of trips, crew size and the
composition of species caught are evident. The
characteristics are homogeneous in both cases. Largesized blue and brown shrimp are meant for the export
market, whereas medium and small shrimp are
allocated to the domestic market.
The operating range of the two RSVs stretches
across the coasts of Sinaloa, Sonora and Baja
California. The Sinaloa RSV conducts four fishing trips
during the catch season (September to February), where
655
Table 1. Comparison of physical, operating, and catch features of the two RSV.
Feature/Port
Physical characteristics
Length (m)
Width (m)
Gross weight (m3)
Net weight (m3)
Storage capacity (m3)
Engine power (HP)
Operating characteristics
Base port
Daily diesel consumption (L)
Number of trips
Crew size
Catch
Blue shrimp (%)
Brown shrimp (%)
White shrimp (%)
Medium and small-sized shrimp (%)
as the Sonora RSV conducts five fishing trips
(September to March). Each trip lasts about 30 days at
sea plus a five-day in-port stay to download the
product. Each RSV is older than 30 years.
The base year data used as input for the simulation
model are summarized in Table 2. This table shows the
income obtained and the expenses incurred (both in
2009 US$) by each RSV in 2009. Revenues include
shrimp sales as the main income, as well as the marine
diesel subsidy and the Value Added Tax (VAT) refund,
as these two items derive from government policies set
out in the Mexican law for supporting the primary
sector. In both cases, a favourable income was earned.
Costs incurred include, firstly, the expenditure on
fuel and lubricants; second, crew wages; then, expenses
related to catch processing, packaging and marketing.
Maintenance costs include those related to the vessel
itself, plus those of the vehicles used to haul the product
at landing, using only one truck for each RSV. Finally,
there are other expenses such as payment for accounting services, telephone and electricity bills, ground
staff (secretary, fleet manager, etc.), taxes, vessel
insurance, etc.
Catch figures were expressed as CPUE for each
RSV, and were validated in meetings with producers.
The Sinaloa RSV had a historical maximum CPUE of
35 ton in 1987, and a minimum of 10 ton in 2004 (Fig.
2). The highest CPUE for the Sonora RSV was
achieved in 1986 with 25 ton, and the minimum was
only 8 ton in 1992 (Fig. 2). Figures shown in Fig. 2 for
RSV Sinaloa
RSV Sonora
22
6
100
56
50
450
24
6.3
100
60
50
425
Mazatlán
1,300
4
7
Guaymas
1,280
5
6
17
40
43
59
26
15
2010 onwards are the outputs projected by GAMs
under the two environmental variability scenarios.
Table 3 summarizes the results of fitting shrimp
catch data in Sonora and Sinaloa to GAM, using the
long-term pattern in the PDO, upwelling events and the
first component of rainfall records in the region as
predicting variables. For both regions, the fitness
parameters were similar, with R2 > 0.7 and deviance
explained >80%; both models were selected based on
the value of the GCV.
As for price, the historical trend (1980-2009) of
nominal price was obtained from fisheries and
aquaculture statistical yearbooks. Projected shrimp
prices for 2010-2019 were estimated using official
inflation rate projections (BANXICO, 2013).
Simulation for 2010-2019
A Minimum Acceptable Rate of Return (MARR) of
10.67% was used for calculating the Net Present Value
(NPV). MARR was calculated as the sum of the
inflation rate (3.57%; BANXICO, 2013) and the asset
interest rate (7.1%; World Bank, 2013) in Mexico in
2009.
The mean NCI reveals a positive balance under the
neutral climate change scenario, and a negative one
under warming conditions. The other key financial
indicators used for assessing economic risk are also
shown in Table 4. Over the study period, the mean
Cost/Benefit (C/B) ratio was greater than the decision
criterion for this indicator only under the neutral
scenario in both cases.
656
Table 2. Operating costs and revenue generated in 2009 (thousand US$) by producers from Sinaloa and Sonora. These data
were used as the base year information to feed the simulation model and as baseline for the simulation runs.
Item
Shrimp
Refund of Value Added Tax (VAT)
Subsidy to marine diesel
Fuel and lubricants
Workmanship
Processing, packaging and marketing
Maintenance
Other
Total
Net Cash Income (NCI)
Sinaloa
Income Expenditures
164.61
22.60
41.18
72.63
44.42
30.92
26.16
26.56
228.39
200.70
27.69
Sonora
Income Expenditures
239.65
23.94
38.92
98.79
62.57
30.60
21.89
24.31
302.51
238.17
64.33
Figure 2. Catch per unit effort (CPUE) in Sinaloa (solid black line) and Sonora (solid gray line). Dashed lines are CPUE
figures projected by GAMs under two scenarios: neutral climate change and monotonic warming. The gray dotted line
depicts the historical trend of shrimp price up to 2009 and, from this year onward, the projected price.
The rate of return on assets was positive for both
RSVs over the ten-year period analyzed, with lower
figures under the monotonic warming scenario, noting
that the only asset considered is the value of each
vessel. NPV was positive under the neutral scenario,
with a lower percentage of economic success for the
Sinaloa RSV, and negative for both warming scenarios.
This indicates that the established rate of return turned
out to be lower than the required one, and generates no
value over time. The Internal Rate of Return (IRR) was
higher than MARR only under the neutral scenario for
both RSVs.
The Sinaloa RSV displays a favourable behaviour
under the neutral scenario, since the probability of NCI
being lower than zero ranges between 13 and 18%.
Meanwhile, under the warming scenario, NCI has a
higher probability (between 46 and 95%) of falling
below zero (Fig. 3). Mean NCI over the simulation
period remained positive under the neutral scenario;
under the warming scenario, however, it was negative
from 2010 to 2017, and then it became positive again,
thus generating losses in seven out of the ten years of
simulation.
Figure 3 shows NCI values for each simulated year
under both scenarios, displaying the mean and 25th and
75th percentiles, with a 90% confidence interval. Under
the neutral climate change scenario, and assuming that
fishing is only conducted by the Sinaloa RSV, the
cumulative probability distribution of NCI shows a
probability lower than 8% of incurring losses; under the
monotonic warming scenario, the probability of
yielding a positive NCI is 23%.
657
Table 3. Fitness of shrimp catches data in Sonora and Sinaloa to GAM. Environmental variables where used to approximate
the curve of the response variable. Below each predictor are shown the effective degrees of freedom (edf), F-statistic and
significance P-values.
Site
Sonora
Sinaloa
Environmental variables
te(Upwelling + PDOCumSum ) + Rain(PC1)
edf = 13.2; F = 4.9; P = 1.45-5; edf = 1.3; F = 3.6; P = 0.05
te(Upwelling + PDOCumSum) + Rain(PC1)
edf = 17.9; F = 4.1; P = 1.784; edf = 1.5; F = 0.73; P = 0.04
R2
DE (%)
n
GCV
0.73
84
44
502
0.72
83
44
960
Table 4. Projected values for the key financial indicators of shrimp trawler vessels (average 2010-2019).
Indicator
Total Revenue (thousand US$)
Cash Expenses (thousand US$)
Net Cash Income (thousand US$)
C/B ratio
Return on Assets (%)
Net Present Value (thousand US$)
P (positive NPV or economic success) (%)
Internal Rate of Return (%)
Final Cash Reserves (thousand US$)
Sinaloa RSV
Neutral
Warming
286.48
235.81
240.03
258.41
46.45
-22.60
1.20
0.90
11.83
4.90
56.71
-174.04
74.73
3.20
16.74
38.30
-205.58
Sonora RSV
Neutral Warming
354.08
286.91
284.82
309.86
69.26
-22.95
1.25
0.91
12.46
6.15
142.10
-167.40
86.99
10.61
22.46
330.39
-259.25
Figure 3. Simulated Average Net Cash Income for the Sinaloa RSV, considering the 25th and 75th percentiles of the NCI,
under two different climate change scenarios, neutral (N) solid black line and monotonic warming (W) discontinued gray
line, and using the cumulative probability distributions of the net present value of the average NCI for each scenario.
The Sonora RSV also displayed favourable
conditions under the neutral scenario, with a 12-19%
probability of the NCI falling below zero; under the
warming scenario, this probability increased to 30-95%
(Fig. 4). Mean NCI was positive under the neutral
scenario; by contrast, under the warming scenario it
became negative from 2010 to 2016, then shifting to
positive figures until the end of the simulated period.
Under the neutral environmental variability
scenario, the cumulative probability distribution of NCI
showed a probability lower than 5% of incurring losses;
under the monotonic warming scenario, the probability
of achieving a positive NCI was 31%.
DISCUSSION
Slight physical differences between RSVs were
observed, including length, width and engine power.
The latter is the most important feature, as it defines the
main operating cost and the consumption of marine
658
Figure 4. Simulated Average Net Cash Income for the Sonora RSV, considering the 25th and 75th percentiles of the NCI,
under two different climate change scenarios, neutral (N) solid black line and monotonic warming (W) discontinued gray
line, and using the cumulative probability distributions of the net present value of the average NCI for each scenario.
diesel, which, in turn, determine the main government
subsidy, i.e., the volume of marine diesel to subsidize.
The Sinaloa RSV displays higher diesel consumption
per day, just above the figure for the Sonora RSV.
Differences in crew size and number of trips, which
also affect each vessel’s operating costs, were also
noted. However, although the Sinaloa RSV has an
additional crew member, the Sonora RSV devotes a
larger proportion to pay crew salaries, which are
proportionally related to catch volume. The Sonora
RSV also makes an additional trip, hence determining
the higher profitability of this RSV in the present
simulation.
In general, any extractive activity poses a risk to
investment, and the analysis of the fishing activity is
particularly challenging. Under the neutral scenario,
both RSVs showed a high probability of making profit.
By contrast, under warming conditions, such as a strong
El Niño event that negatively affects production
(López-Martínez, 2000), there is a higher probability of
incurring losses in the planning horizon for both RSVs.
Moreover, these penaeid shrimp are considered
stenothermic, with an optimum growth temperature
between 24 and 28°C (Rodríguez de la Cruz, 1981);
temperatures outside this range negatively affect their
growth and spawning (Rodríguez de la Cruz & JuárezRosales, 1976). The low tolerance of these shrimp
species to variations in temperature was confirmed in
the warming scenario, which was built based on the
GAM model by including a temperature increase from
28 to 32.6ºC, resulting in a negative effect on
production and, therefore, on profitability.
In this sense, under the neutral scenario IRR figures
for both vessels suggest that the shrimp fishery
generates attractive returns, particularly considering
that this is a high-risk extractive activity due to catch
variability and the range of factors affecting it. IRR
exceeded MARR for both vessels; however, the Sonora
vessels showed a higher profitability and also a higher
NPV, with a greater capital growth than the Sinaloa
vessels.
Under the warming scenario, both RSVs showed
losses due to a decline in production, thus increasing
the risk and uncertainty related to obtaining no profits.
However, the simulation in this analysis did not include
factors such as changes in public policy, variations in
the number of trips, etc., since the simulation was based
on the base year conditions projected over a planning
horizon.
Another indicator that does not take into account the
value of money over time is the C/B ratio. Under the
neutral scenario, C/B figures were higher than the
decision criterion for both vessel types. This means that
operating costs are covered and positive economic
returns are generated: for every dollar spent or invested,
0.20 ¢ are recovered by the Sinaloa RSV and 0.25 ¢ by
the Sonora RSV. Opposite results were obtained under
the warming scenario for both RSVs.
The analysis reported here, which considers
scenarios of environmental variability as alternatives,
provides a tool to support decision makers involved in
the shrimp fishery management, but does not imply that
the fishery will necessarily behave as described herein.
In this regard, Ramírez-Rodríguez & AlmendarezHernández (2013) suggested reducing the number of
fishing trips of a unit composed of a vessel that fishes
shrimp and squid, for NCI not to become negative. The
idea was to represent the producer behaviour in an
attempt to preserve his income or reduce his losses.
This agrees with the producers’ point of view, given
that the first fishing trip is essential to determine the
course of the shrimp fishing season (Quimbar, 2004;
FIRA, 2009).
Under environmental conditions unfavourable for
the resource, such as an anomalous and sustained rise
in temperature, the offshore shrimp fishery becomes an
unprofitable and unattractive activity. However, as in
any economic activity, the operation of each RSV will
be driven by the availability and size of catches, market
conditions, policies for vessels withdrawal, etc.
CONCLUSIONS
Type vessels of Guaymas and Mazatlán are similar in
terms of physical features; however, they differ in the
way they operate and their economic performance, and
are largely representative of the industrial shrimp fleet
operating in these two ports.
The shrimp fishery, as any marine resource, is
affected by fluctuating environmental processes which,
in turn, impose variations in catch volume and, thus, in
the expected economic return. Therefore, the climatic
behaviour scenarios simulated here provided a
sensitivity analysis of the activity for the two vessel
types, showing an activity with moderate profitability
under the neutral scenario, but with a low or nil
profitability under the warming scenario. Which of
these scenarios could be used for management and
planning purposes will depend on a more profound
understanding of past and present climatic conditions in
the Gulf of California.
While the shrimp fishery always involves certain
risk and uncertainty arising from climate fluctuations
and associated biological processes, this study
contributes to the identification of relevant factors that
should be considered in planning and management to
achieve a better performance of this activity.
ACKNOWLEDGEMENTS
LCAH thanks CICIMAR-IPN, the PIFI program and
CONACyT for scholarships awarded. GPD thanks
COFAA and EDI. The authors thank to María Elena
Sanchez Salazar for the translation of this manuscript.
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Received: 15 August 2014; Accepted: 6 May 2015
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Lat. Am. J. Aquat. Res., 43(4): 662-674, 2015
Periphyton and planktonic bacteria in shallow lakes
662
1
Research Article
Periphytic and planktonic bacterial community structure in turbid and clear
shallow lakes of the Pampean Plain (Argentina): a CARD-FISH approach
Laura María Sánchez1, María Romina Schiaffino1, Haydée Pizarro1 & Irina Izaguirre1
1
Departamento de Ecología, Genética y Evolución, IEGEBA (UBA-CONICET)
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires
Intendente Güiraldes 2160, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina
Corresponding author: María Laura Sánchez ([email protected])
ABSTRACT. Bacterioplankton and bacterioperiphyton composition was analyzed using the CARD-FISH
technique in three shallow lakes of the Pampean Plain (Argentina) with contrasting regimes: clear vegetated,
turbid due to phytoplankton and turbid inorganic, due to inorganic particles. We postulated that these differences
would influence the proportion of the main bacterial groups both in periphyton and in plankton. The turbid lake
due to phytoplankton presented the highest total abundances in both communities. Alphaproteobacteria was the
dominant group in the three lakes in both communities. Redundancy analysis (RDA) evidenced that the
bacterioplankton structure was different among lakes and mainly influenced by dissolved inorganic nitrogen and
conductivity. On the other hand, for the bacterioperiphyton, RDA showed that bacterial group abundances
increased with higher periphytic chlorophyll-a values. In the clear lake the relative abundance of
Betaproteobacteria and Cytophaga increased in the bacterioperiphyton towards the end of the colonization. Our
study suggests that the lake regime (clear or turbid) influence the structure of bacterioplankton and
bacterioperiphyton.
Keywords: bacterioperiphyton colonization, bacterioplankton, shallow lakes, CARD-FISH, Pampean Plain,
Argentine.
Estructura de las comunidades bacterianas perifíticas y planctónicas en lagunas
turbias y claras de la llanura pampeana (Argentina): un enfoque aplicando
CARD-FISH
RESUMEN. Se analizó la composición de bacterioplancton y bacterioperifiton utilizando la técnica de CARDFISH en tres lagunas de la llanura pampeana (Argentina) con regímenes contrastantes: laguna clara vegetada,
laguna turbia por fitoplancton y laguna turbia inorgánica. Se postula que estas diferencias podrían influir sobre
la proporción de los principales grupos bacterianos, tanto en el bacterioperifiton como en el bacterioplancton.
La laguna turbia por fitoplancton presentó la mayor abundancia total de bacterias en ambas comunidades. El
grupo de Alfaproteobacteria fue dominante en ambas comunidades y en las tres lagunas. El análisis de
redundancia (RDA) evidenció que la estructura del bacterioplancton fue diferente entre las lagunas y estuvo
principalmente influenciado por el nitrógeno inorgánico disuelto y la conductividad. Por otra parte, para el
bacterioperifiton el RDA mostró que la abundancia de los grupos bacterianos se incrementó a mayores valores
de clorofila-a perifítica. En la laguna clara las abundancias relativas de los grupos Betaproteobacteria y Citofaga
aumentaron hacia el final del período de estudio. Este estudio sugiere que el régimen (claro o turbio) de cada
cuerpo de agua influye sobre la estructura del bacterioplancton y bacterioperifiton.
Palabras clave: colonización del bacterioperifiton, bacterioplancton, lagunas, CARD-FISH, llanura pampeana
Argentina.
INTRODUCTION
Different bacterial taxonomic groups, which differ in
their trophic strategies and physiological capabilities,
__________________
Corresponding editor: Beatriz Modenutti
have been reported for both freshwater and marine
environments (e.g., Alonso Sáez & Gasol 2007;
Newton et al., 2011). Betaproteobacteria, well
represented in freshwaters are characterized as opportu-
2663
nistic in nutrient-enriched conditions (Glöckner et al.,
1999; Newton et al., 2011; Salcher, 2014).
Actinobacteria, whose small cell sizes confer them an
advantage under high predation pressure and UV
radiation (Pernthaler et al., 2001; Warnecke et al.,
2005) and Gammaproteobacteria, adapted to grow
under high-nutrient concentrations (Newton et al.,
2011), are also successful in freshwater systems. In
contrast, Alphaproteobacteria are more abundant in
marine than in freshwater ecosystems (Methé et al.,
1998; Glöckner et al., 1999). Finally, a large group of
bacteria constituted by Cytophaga-FlavobacteriumBacteroidetes (hereinafter Cytophaga), which are
important in biopolymer degradation, has also been
reported in freshwater systems (Lemarchand et al.,
2006).
These bacterial groups can be represented both in
bacterioplankton and bacterioperiphyton (bacterial
component of the biofilms), and the proportion of the
different groups may exhibit temporal changes. In the
bacterioplankton these changes have been mainly
studied in relation to fluctuations in algal biomass,
nutrient and grazers (e.g., Posch et al., 2007; Salcher,
2014), whereas the succession in bacterioperiphyton
community has been poorly analyzed. In freshwater
biofilms, the relative proportion of the main bacterial
groups changes according to environmental variables
(Glöckner et al., 2000), being identified Cytophaga as
pioneers and Gammaproteobacteria and Betaproteobacteria dominant in later stages of the succession
(Pohlon et al., 2010). On the other hand, the nutritional
status of the lake has influence on the bacterial
abundance in the biofilms. It has been reported that in
nutrient-enriched lakes, bacteria of the biofilms tend to
be more abundant and form a thicker matrix than under
poor nutrient conditions; however, the influence of the
different nutrients on the characteristics of bacterioperiphyton is still unclear (Stoodley et al., 2000).
The Pampa Plain (Argentina) contains thousands of
shallow lakes, thus constituting a typical lacustrine
wetland (Brinson, 2004). Due to the intensive
agriculture that takes place in this region, nowadays
most of these shallow lakes are in a turbid state,
showing high phytoplankton biomass (hereinafter
phytoplankton turbid shallow lakes) (Quirós et al.,
2002, 2006). Nevertheless, some of them still present a
clear vegetated state with high transparency and
submerged vegetation (clear shallow lakes). These two
types of shallow lakes fit well with the two alternative
equilibria stable states described by Scheffer et al.
(1993). Recently, Scheffer (2009) postulated the
concept of regime instead of alternative stable states,
since shallow lakes are in permanent slow change, and
called ´shift regime´ to the transition from one regime
to another. In addition, a third type of water body is also
present in this region -inorganic turbid shallow lakes-,
characterised by high turbidity associated with high
concentrations of inorganic suspended particles from
allochthonous sources resulting from the direct human
impact on their drainage basin (Quirós et al., 2002;
Allende et al., 2009). The variety of shallow lakes in
this region constitutes an interesting scenario to analyze
the structure of the microbial communities. In
particular, the contrasting optical properties of these
water systems (Pérez et al., 2010) have been found to
influence the phytoplankton structure (Allende et al.,
2009; Izaguirre et al., 2012) and the relative importance
of phytoplankton vs periphyton communities (Sánchez
et al., 2010, 2013). Silvoso et al. (2010) analyzed the
relative abundances of the picoplankton components,
including bacterioplankton, in shallow lakes of this
region and found that clear vegetated lakes exhibit
similar picophytoplankton/bacterioplankton ratios but
no clear trend in turbid lakes. In the same region,
Llames et al. (2013) studied the influence of
environmental factors on bacterioplankton community
composition by using molecular techniques, and found
that the regime of the systems plays a major role in
structuring the bacterial community. These authors
suggested that the patterns observed in each type of
shallow lake are probably driven by differences in the
nature of the predominant organic matter sources and
pools (macrophytes, phytoplankton and terrestrial
organic carbon). Nevertheless, none of these studies
were focused on both bacterioplankton and bacterioperiphyton. The incorporation of the bacterioperiphyton
community is essential to a better understanding of the
aquatic bacteria ecology since shallow lakes have a
well-developed littoral zone with an important growth
of the attached communities (Sánchez et al., 2013).
In this study we analyzed, applying Catalyzed
Reported Deposition Fluorescent in situ Hybridization
technique (CARD-FISH), the structure of bacterioplankton and bacterioperiphyton in shallow lakes of the
Pampa Plain (Argentina) with different regimes (clear
vegetated, phytoplankton turbid and inorganic turbid).
Moreover, we identified, by means of multivariate
analyses, which environmental variables influenced the
bacterial composition in each community. We also
experimentally analyzed the changes in bacterioperiphyton colonization in each shallow lake.
Study area
We studied three shallow lakes, which differ in their
regime: el Triunfo, a clear vegetated shallow lake with
abundant submerged vegetation (mainly Ceratophyllum
demersum) and low phytoplankton abundances
(35º51´S, 57º52´W); El Burro, a turbid shallow lake
with high abundances of phytoplankton (35º42´S,
57º55´W) and Yalca, an inorganic turbid shallow lake
with high amounts of suspended solids (35º35´S,
57º55´W). The studies were carried out from October
22 to November 1st 2010.
Experimental design
Bacterioperiphyton was studied with artificial substrata
placed near the surface in the littoral zone of each
shallow lake, using an acrylic device with 40 artificial
substrata. Artificial substrata consisted in polycarbonate pieces 1 mm thick and 2 cm wide x 7.5 cm long.
At least two devices with artificial substrata were
placed in each lake. The colonization experiments were
run along ~3 weeks in all lakes. In El Burro and Yalca,
bacterioperiphyton samples were taken at 3 (t1), 10 (t2)
and 20 days (t3), whereas in El Triunfo, samples were
taken at 3 (t1), 7 (t2), 10 (t3) and 17 days (t4).
Bacterioplankton samples were collected in a plastic
bottle simultaneously in all lakes throughout the
experiment. All samples were obtained by duplicate.
Bacterioplankton was pre-filtered through a 55-µm
pore net to exclude zooplankton. All samples were
fixed with formaldehyde 10% final concentration. The
artificial substrata were preserved in dark and cold
conditions inside hermetic bags during their transport
to the laboratory where the attached material was
scraped off with a sharp piece of polycarbonate,
suspended in milliQ water and fixed in the same way as
bacterioplankton samples.
Physical and chemical variables
To characterize each shallow lake, pH, conductivity
and temperature were measured in situ on each
sampling date with a portable sensor HORIBA D-54E
(Japan), whereas dissolved oxygen (DO) was measured
with a portable sensor HANNA HI 9146 (Hanna
Instruments, USA).
Downward irradiance profiles were obtained around
noon using a USB2000 (Ocean Optics, Florida, USA)
spectroradiometer attached to an optical fiber, and a
teflon diffuser, following the same methodology
described in Sánchez et al. (2013). Vertical diffuse
attenuation coefficients for PAR (KdPAR) were
determined from the slope of the linear regression of
the natural logarithm of downward irradiance profiles
vs. depth. Additionally, we measured the Secchi depth
of each shallow lake.
Main dissolved and total nutrients were determined
in duplicate on each sampling date. Sub-superficial
samples were taken in each shallow lake. Nitrate +
nitrite (cadmium reduction method), soluble reactive
664
3
phosphorous (SRP) (ascorbic acid method) and
ammonium (salicylate method) were analyzed with a
HACH DR/2010 spectrophotometer (HACH Company,
USA) using the corresponding kits of HACH reagents.
Dissolved inorganic nitrogen (DIN) was calculated as
nitrate + nitrite + ammonium. Total nitrogen (TN) and
total phosphorus (TP) concentrations were determined
subsequently to the digestion with boric acid and
potassium persulphate, following the methodology
described in APHA (2005) using the same HACH kits
as those used for dissolved nutrients.
Chlorophyll-a concentration (Chl-a) of phytoplankton and periphyton communities was determined
in duplicate on each sampling date. The samples were
filtered through Whatman GF/F filters. Chl-a was
estimated spectrophotometrically using hot ethanol
(60-70°C) (Marker et al., 1980), following the formulae
described in Lorenzen (1967).
Catalyzed reported deposition fluorescent in situ
hybridization (CARD-FISH)
Bacterioplankton
The CARD-FISH technique was performed following
the methodology described in Pernthaler et al. (2004).
Each sample was homogenized using a vortex Velp
Scientifica (Usmate, Italy) and then a known volume
was filtered through a 0.2-µm pore-size polycarbonate
white filter and preserved at -20ºC.
Whole-cell in situ hybridizations of polycarbonate
filter sections were performed as described by
Pernthaler et al. (2002) and Sekar et al. (2003), using
the following oligonucleotide probes: EUB338-II-III (a
mix of EUB338, EUB338(II) and EUB338(III)), to
target most Eubacteria, including Verrucomicrobia and
Planctomycetes (Amann et al., 1990; Daims et al.,
1999) -being this probe useful as a control to analyze
the hybridization percentage in relation to the total 4´,6diamidino-2-phenylindole (DAPI)-stained bacterial
density-; ALF968, specific for Alphaproteobacteria
(Amann & Fuchs, 2008); BET42a, to target Betaproteobacteria (Manz et al., 1992); GAM42a, for
Gammaproteobacteria (Manz et al., 1992); CF319a, to
target Cytophaga (Manz et al., 1996), and HGC69a for
Actinobacteria (Amann et al., 1995). Probes were
supplied by Thermo Electron Corporation (Waltham,
MA, USA) with an aminolink (C6) at the 5´ end, ligated
with a horseradish peroxidase enzyme (Urdea et al.,
1988). Each probe was incubated in the corresponding
hybridization buffer. Competitor probes, which are
specific probes that avoid miss-match in the
hybridization process between both bacterial groups,
were added to BET42a and GAM42a. Hybridizations
were run overnight at 35ºC. After hybridization, the
signal was amplified with Alexa 488-labeled tyramide
4665
and counter-stained with immersion oil containing
DAPI (Vecta Shield, USA). Filter pieces were mounted
on a slide and observed by epifluorescence microscopy
(Olympus BX40F4, Japan) under blue light and UV
excitation. Because the probe EUB338-II-III does not
give total hybridisable bacterial abundances in all
environments, the contribution of each bacterial group
to the prokaryotic community was calculated as a
percentage of DAPI counts (relative abundance or
hybridization percentage).
Bacterioperiphyton
The attached material was scraped off carefully,
suspended in milliQ water and fixed. The
bacterioperiphyton was then mechanically separated
with a vortex and subsequently subjected to the action
of a sonicator Sonics (Newtown, USA) to obtain a
complete homogenization of the samples (Velji &
Albright, 1993). Then, the samples were filtered
through a 0.2-µm pore-size polycarbonate white
membrane. The hybridization steps were identical to
those previously described for bacterioplankton and
were performed simultaneously.
Statistical analyses
To analyze variations over time, we performed repeated
measures ANOVA (RM ANOVA), using bacterial
groups (Alphaproteobacteria, Betaproteobacteria,
Gammaproteobacteria, Actinobacteria, Cytophaga)
and time as factors. Two-way ANOVA were performed
at final time to compare abundances of the different
bacterial groups among lakes. Before each analysis,
Shapiro-Wilk and Levene tests were run to check data
for normality and homocedasticity. Whenever the data
did not confirm the assumptions, the values were
transformed as necessary. RM ANOVA was run with
SPSS 17.0 (USA). To compare relative abundances
among different groups at the end of the study, we
performed chi-squared contingency tables with the
following factors: lake (El Triunfo, Yalca and El Burro)
and bacterial groups (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria,
Cytophaga). We also analyzed differences in the
hybridization percentage of bacterial groups among the
three shallow lakes between bacterioperiphyton and
bacterioplankton at the end of the study with chisquared contingency tables using Infostat (Argentina).
In addition, to analyze relations among environmental variables and bacterial composition, we
performed multivariate analyzes with planktonic and
periphytic bacteria using the software CANOCO (Ter
Braak, 1986). All data of absolute abundances were
included in the analysis. To identify the environmental
variables controlling the abundances of the bacterial
groups, we carried out a redundancy analysis (RDA),
after the application of a detrended correspondence
analysis (DCA) with the matrix of bacterioplankton and
bacterioperiphyton abundances, which indicated a
linear response of the data (Ter Braak & Smilauer,
2002).
RESULTS
Physical and chemical variables
Table 1 indicates the results of the environmental
variables measured along the study period. The clear
vegetated shallow lake showed higher pH and
conductivity values than the turbid ones. The turbid
lakes did not differ in their pH values. Yalca presented
the lowest values of conductivity. The lowest DIN
concentrations were found in the clear vegetated (El
Triunfo) and in the inorganic turbid (Yalca) shallow
lakes. Although SRP was relatively high in the three
shallow lakes, their values were highest in El Triunfo.
TN was highest in El Triunfo and lowest in Yalca and
TP showed no variations among the lakes. According
to their optical variables (Secchi depth and KdPAR), El
Burro and Yalca showed higher light limitation than El
Triunfo (Table 1).
Bacterioplankton
The hybridization percentage of EUB338-II-III
(Eubacteria) on total DAPI-stained bacteria varied
among the lakes. El Triunfo exhibited the highest
hybridization percentages (average 60.6%), intermediate values were observed in Yalca (average 47.6%),
whereas El Burro showed the lowest values (average
29.5%).
Alphaproteobacteria was the most abundant group
in the three shallow lakes and presented the highest
hybridization percentage (average values: 46.5% in El
Triunfo, 31% in El Burro and 54.2% in Yalca). The
second most important group in El Triunfo was
Betaproteobacteria (34.6%), whereas Betaproteobacteria and Actinobacteria were the second most
important groups in the other two lakes (13.7% and
13.9% in Yalca; 8.8% and 7.4% in El Burro,
respectively) (Figs. 1a-1c). Actinobacteria and
Cytophaga showed intermediate abundances in El
Triunfo (Fig. 1a) and Gammaproteobacteria was the
least represented group in the three lakes.
Total bacteria attained the highest abundances in El
Burro (two-way ANOVA factors: lake and bacterial
groups, simple effect P < 0.0001) and the lowest in El
Triunfo (Fig 1a).
At the end of the sampling period we found
significant differences in the abundance of bacterial
666
5
Table 1. Environmental variables measured in the three shallow lakes studied. Maximum and minimum values recorded
throughout the study period are shown. DO: dissolved oxygen, DIN: dissolved inorganic nitrogen, SRP: soluble reactive
phosphorus, TN: total nitrogen, TP: total phosphorus, KdPAR: vertical diffuse attenuation coefficients for PAR.
Variable
pH
Conductivity (µS cm-1)
DO (mg L-1)
Temperature (°C)
DIN (µg L-1)
SRP (µg L-1)
TN (µg L-1)
TP (µg L-1)
Secchi (cm)
KdPAR (m-1)
El Triunfo
(clear vegetated)
El Burro
(phytoplankton turbid)
Yalca
(inorganic turbid)
9.4-9.8
1108-1480
7.3-10.2
15.9-25.0
15-75
110-355
8950-10150
400-530
70-88
3.7-5.4
8.4-8.8
961-1300
8.6-12.3
17.2-23.0
375-440
90-245
1390-7490
420-550
16-16.5
8.6-16.7
8.4-8.8
428-588
8.4-10.4
17.4-27.4
25-65
105-255
3680-7060
420-490
12-16.5
11.4-26.1
groups among the three lakes (contingency table P <
0.0001). However, in each lake we did not detect
differences in the abundance of each bacterial group
throughout the period studied.
Results of RDA based on the absolute abundances
of each planktonic bacterial group and environmental
variables are shown in Fig. 2. The first two axes
accounted for 92.5% of the variance (axis 1: 69.5%,
axis 2: 23.0%). The Monte Carlo’s test indicated that
the abiotic factors were significantly correlated with the
first canonical axis (P < 0.01), and was also significant
with all canonical axes (P < 0.05). The following
variables were statistically significant: DIN (P <
0.005), conductivity (P < 0.005) and DO (P < 0.05)
(solid arrows in Fig. 2). The first axis was mainly
defined by DIN and phytoplanktonic Chl-a (correlation
coefficients: 0.89 and 0.85 respectively) and the second
axis was mainly correlated with conductivity and
KdPAR (correlation coefficients: 0.87 and -0.79
respectively). This analysis showed an almost
invariable composition of the planktonic bacterial
community throughout time. Remarkably, RDA
evidenced three well-separated groups of sites
corresponding to each shallow lake (dotted lines in Fig.
2) based on their bacterioplankton composition and the
main environmental variables. This analysis also
showed that the samples corresponding to the
phytoplankton turbid lake were associated with higher
phytoplankton Chl-a, DIN and DO values. On the other
hand, samples corresponding to El Triunfo were related
with higher SRP values, whereas those of Yalca were
ordered to lower conductivity and higher KdPAR values.
Regarding to bacterial groups, Alphaproteobacteria
and Actinobacteria were ordered to higher values of
KdPAR and DO and were more related with samples
corresponding to the inorganic turbid lake. Cytophaga
and Betaproteobacteria were more associated with
conductivity and Gammaproteobacteria with higher
values of DIN and Chl-a. These three bacterial groups
were more related with the phytoplankton turbid lake.
Bacterioperiphyton
The Eubacteria hybridization percentages were similar
in El Triunfo and El Burro (average values 77%, and
79.6% respectively), whereas Yalca showed the lowest
values (65.4% on average).
Bacterioperiphyton was mainly represented by
Alphaproteobacteria in the three shallow lakes all over
the studied period (Fig. 3). On average, the second
largest group was Betaproteobacteria in El Triunfo and
Yalca (hybridization percentage 22.6% and 14.11%
respectively), and Gammaproteobacteria in El Burro
(hybridization percentage 24.6%). Actinobacteria,
whose hybridization percentages varied between 1.3%
and 2.2%, was the least represented group in the three
lakes studied (Fig. 3).
Analyzing the variations through the time, we
detected an increase in the hybridization percentage of
Alphaproteobacteria from 55.8% to 60.2% (RM
ANOVA factor: time P < 0.01) for El Triunfo. In this
lake, Gammaproteobacteria was the second group in
importance at the beginning of the colonization period,
decreasing towards the end (from 46.2 to 9.2%). This
group was replaced by Cytophaga (which increased
from 6.6 to 19.7%) and Betaproteobacteria (which
oscillated between 22.1 and 31.8%) (Fig. 3a).
In El Burro we did not observe differences among
the abundances of the different bacterial groups at the
beginning of colonization. However, after that
Alphaproteobacteria showed a significant increase
(RM ANOVA factor: time P < 0.0001) and then
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667
Figure 1. Bacterioplankton variation throughout time in the three shallow lakes studied. Left panel: total bacteria
abundances, bars represent ± standard deviation (n = 2). Right panel: hybridization percentage of each probe regarding total
DAPI-stained bacteria.
remained constant until the end of the experiment (Fig.
3b).
In Yalca, Alphaproteobacteria showed an increase
in their abundances with time (RM ANOVA factor:
time P < 0.01; factor: bacterial group P < 0.02). The
hybridization percentage oscillated between 53.1% at
the beginning (t1) and 56.2% at the end (t3).
Furthermore, in this inorganic-turbid lake the
abundances of Betaproteobacteria and Cytophaga
increased towards the end of the study period (Fig. 3c).
At the end of the study El Burro showed
significantly higher abundances of Alphaproteobacteria and Gammaproteobacteria than the other two
lakes (two-way ANOVA factors: lake and bacterial
group, simple effects P < 0.0001; P < 0.03
respectively). Contrarily, no significant differences
Figure 2. Triplot corresponding to the redundancy
analysis (first and second axis) based on the abundance of
the different bacterioplankton groups and environmental
variables. Samples 1-4: El Triunfo (samples taken at t0,
t1, t2 and t3 respectively); samples 5-8: El Burro (samples
taken at t0, t1, t2 and t3 respectively); samples 9-12: Yalca
(samples taken at t0, t1, t2 and t3 respectively). Solid and
dotted arrows indicate significant and non-significant
environmental variables (P < 0.05), respectively. Cond:
conductivity, DO: dissolved oxygen, DIN: dissolved
inorganic nitrogen, SRP: soluble reactive phosphorus, Kd
PAR: vertical diffuse attenuation coefficients for PAR,
phyto Chl a: phytoplanktonic chlorophyll-a .The names of
the bacterial groups are abbreviated.
were recorded among the other bacterial groups. We
also found differences among the lakes in the
hybridization percentage of the bacterial groups at the
end of the study (Contingency table, P < 0.0001). These
differences were due to changes in the contribution of
the subdominant groups in each lake.
The results of the RDA based on the abundance of
bacterioperiphyton groups and environmental variables
are shown in Fig. 4. The first two axes accounted for
99.6% of the variance (first axis: 96.1%, second axis:
3.5%). Monte Carlo’s test indicated that abiotic factors
were significantly correlated with the first canonical
axis (P = 0.03) and with all canonical axes (P = 0.03).
Environmental variables that resulted significant were
periphytic Chl-a (P = 0.008) and DO (P = 0.04). The
most important variables in the first axis were
periphytic Chl-a and DIN (correlation coefficients:
0.97 and 0.74 respectively), whereas the most
important variables in the second axis were SRP and
6687
conductivity (correlation coefficients: -0.61 and 0.48
respectively). The ordination of the samples corresponding to El Burro evidenced a temporal trend, which was
related with an increase in the relative abundance of
Actinobacteria, Betaproteobacteria and Gammaproteobacteria during the colonization process. Concomitantly, these samples were ordered along an increasing
gradient of periphyton Chl-a. On the other hand,
samples of Yalca were placed following a decrease in
the relative abundance of Alphaproteobacteria during
colonization. Contrarily, for El Triunfo, no temporal
effect was detected, since almost all samples were
ordered together, with the exception of samples of t4
that was plotted separately in coincidence with an
increase in the relative abundance of Betaproteobacteria.
Finally, we compared the hybridization percentage
of the different groups between bacterioperiphyton and
bacterioplankton among the three shallow lakes at the
end of the study, and we observed that the bacterial
composition varied between both communities
(contingency table, P < 0.0001). Notwithstanding,
Alphaproteobacteria was dominant in both communities and in the three studied lakes; Actinobacteria was
more important in bacterioplankton and Gammaproteobacteria in bacterioperiphyton (Fig. 5).
DISCUSSION
Our results showed that each shallow lake (clear,
phytoplankton turbid and inorganic turbid) has
noticeable differences in the structure of their
bacterioperiphyton and, not so markedly, in the
bacterioplankton. Relative abundances of each
bacterial group changed in the periphyton during the
succession, reaching a different mature final stage in
each lake. These differences could be attributed to the
different regime but also to the inner conditions of the
matrix and to the biological interactions that occur
within it; as the dissolved organic matter production by
epiphytic algae that could be degraded by bacteria
generating mutual benefit (Hempel et al., 2008). On the
other hand, differences in bacterioplankton were
associated with variations in the relative abundances of
the subdominant groups in each lake, as was observed
in the higher contribution of Actinobacteria in the
turbid lakes, and particularly in the higher proportion of
Gammaproteobacteria, Betaproteobacteria and Cytophaga in the phytoplankton turbid one. These
differences are likely due to the contrasting optical and
nutrient conditions of these lakes. Our results are in line
with those of Van der Gucht et al. (2005), who analyzed
the bacterioplankton in eutrophic and hypertrophic
clear and turbid shallow lakes, finding a distinctive
669
8
Figure 3. Bacterioperiphyton variation throughout time in the three shallow lakes studied. Left panel: total bacterial
abundances, bars represent ± standard deviation (n = 2). Right panel: hybridization percentage of each probe regarding total
DAPI-stained bacteria.
bacterial community in each one of the lakes, and partly
attributed these differences to the lake states (clear or
turbid). Furthermore, within each shallow lake, the
comparison between bacterioplankton and bacterioperiphyton showed that both communities differed in their
bacterial composition.
Some of the most important physical and chemical
factors that regulate bacterial assemblages are tempe-
rature, UV radiation, organic matter, and nutrient
concentrations (Logue et al., 2008). In our study, the
bacterioplankton was more influenced by DIN and
conductivity; coincidently, these variables showed the
highest range of variation among the studied shallow
lakes. In particular, we recorded low levels of DIN in
the clear vegetated lake and in the inorganic turbid one.
The presence of submerged macrophytes in the clear
Figure 4. Redundancy analysis (first and second axis)
triplot Analysis based on the abundance of the different
bacterioperiphytic groups and environmental variables.
Samples1-4: El Triunfo (samples taken at t1, t2, t3 and t4
respectively); samples 5-7: El Burro (samples taken at t1,
t2 and t3 respectively); samples 8-10: Yalca (samples
taken at t1, t2 and t3 respectively). Solid and dotted arrows
indicate significant and non-significant environmental
variables (P < 0.05), respectively. Cond: conductivity,
DO: dissolved oxygen, DIN: dissolved inorganic nitrogen,
SRP: soluble reactive phosphorus, Kd PAR: vertical
diffuse attenuation coefficients for PAR, peri Chl-a:
periphytic chlorophyll-a. The names of the bacterial
groups are abbreviated.
shallow lake could explain the low availability of DIN;
low nutrient levels in vegetation stands may be due to
uptake by plants but also to uptake by periphyton and
denitrification (Villar et al., 1998; Scheffer, 1998). In
Yalca, the lower values of DIN, conductivity and
turbidity in comparison with those reported in previous
studies (Allende et al., 2009) could be tied to a dilution
effect due to an increase in the hydrometric level of the
shallow lake (personal observation). Several physical
and chemical factors (such as light, temperature and
nutrients) could regulate bacterial growth; in particular,
temporal nitrogen depletion might favour oligotrophic
ultramicrobacteria such as LD12 Alphaproteobacteria,
the most abundant and ubiquitous freshwater bacterial
lineages (Salcher, 2014).
Interestingly, we observed the dominance of
Alphaproteobacteria in the three shallow lakes and in
the two communities analyzed. Recently, Newton et al.
(2011) have pointed out that the genomic and lifestyle
plasticity of Alphaproteobacteria allows them to live in
a great variety of habitats. Moreover, De Figueiredo et
al. (2010) have described Alphaproteobacteria as a
group related with high values of temperature,
conductivity, pH and SRP in a eutrophic lake. In
670
9
another study that includes several lakes with different
limnological characteristics, Salcher et al. (2011) found
a sub-clade of Alphaproteobacteria (LD12) that was
highly abundant in all the studied water bodies.
Furthermore, and in agreement with our results, in a
study about successional changes of bacterial
community on biofilms in rivers, Alphaproteobacteria
was the most abundant group towards the last
succession stages (Lupini et al., 2011).
Regarding bacterioplankton, Betaproteobacteria
was the second most abundant group in importance in
the three shallow lakes, together with Actinobacteria in
the turbid ones. Betaproteobacteria have the ability to
take advantage under eutrophic conditions, as was
demonstrated by Bertoni et al. (2008), in an experiment
with nutrient addition in oligotrophic lakes. These
authors proposed that the successful of this group was
associated with its opportunistic ecological strategy.
Moreover, although Gammaproteobacteria seem to
prefer lakes with high nutrient concentrations, when
nutrients were added, Betaproteobacteria outcompete
the other groups. In agreement with these results
Betaproteobacteria were more abundant than
Gammaproteobacteria in the eutrophic shallow lakes
here studied.
In the bacterioperiphyton, even though Alphaproteobacteria were also dominant, an increase in the
relative abundances of Betaproteobacteria and
Cytophaga was observed towards the end of the
colonization period in the clear lake and, less evidently,
in the inorganic turbid one. In an experimental study,
Šimek et al. (2006) reported planktonic Betaproteobacteria as an opportunistic and fast-growing group.
This group may show a similar growth strategy in the
bacterioperiphyton, which could explain the increase in
its relative abundance. On the other hand, Cytophaga
have been described as principal component of biofilms
in several studies. These bacteria degrade organic
molecules of high molecular weight (Hempel et al.,
2008), thus being best represented in mature steps of
the colonization. In another study on biofilm
colonization, Araya et al. (2003) found that Betaproteobacteria and Cytophaga dominated during the whole
analyzed period. The results of the multivariate analysis
for bacterioperiphyton suggest a positive relationship
between the abundance of most of the analyzed
bacterial groups (Actinobacteria, Gammaproteobacteria, Betaproteobacteria) and the autotrophic
fraction of the biofilm (evaluated as Chl-a). This
tendency was particularly evident in the shallow lake El
Burro. However, specifically for Cytophaga we
observed an opposite trend, since higher values of this
group were associated with lower periphytic Chl-a.
671
10
Figure 5. Hybridization percentage on the final sampling occasion, in each shallow lake and in both communities.
Comparing the three types of shallow lakes here
studied, the phytoplankton turbid one (El Burro)
presented the highest bacterial abundances, dominated
by Alphaproteobacteria, in both bacterioplankton and
bacterioperiphyton. A previous study on the picoplankton of shallow lakes of the same region (Silvoso
et al., 2010) also showed that bacterioplankton
abundance was higher in phytoplankton turbid lakes
than in clear or inorganic turbid ones. In the same study,
a positive correlation was found between bacterioplankton abundance and phytoplankton Chl-a,
suggesting commensalistic interactions between phytoplankton and bacteria, since the labile organic matter
produced by phytoplankton would be the main C source
for bacterial growth in shallow lakes with high
phytoplankton biomass. Recently, Llames et al. (2013)
proposed that the differences in optical characteristics
in the shallow lakes of the same region are related to
differences in the main organic matter pools and thus,
in the substrata for the different planktonic bacterial
groups in each type of shallow lake. According to these
authors, in clear vegetated lakes, the abundant
macrophytes provide mainly organic carbon for
bacteria, while in phytoplankton turbid lakes algae
would be the principal source of organic matter. In this
sense, the highest bacterial abundances both in
plankton and periphyton observed in the phytoplankton
turbid lake in our study are probably related to a higher
amount of labile DOC in this lake. On the other hand,
in inorganic turbid lakes, the organic carbon derived
from terrestrial sources would dominate the organic
matter, which is considered a poor substrate to bacteria
because of its chemical recalcitrance (Salcher, 2014).
Probably, these different sources of organic matter
would influence the differences in bacteria abundances
in the three shallow lakes here studied.
When comparing bacterioperiphyton and bacterioplankton composition in each shallow lake, we found
differences between both communities, i.e., in El Burro
a more important presence of Gammaproteobacteria in
the bacterioperiphyton than in the bacterioplankton.
Pelagic and benthic habitats could differ considerably
regarding nutrient and light availability. Unlike
plankton, biofilms form a great diversity of complex
structures that allow them to grow under a diverse range
of conditions. One of these structures are water
channels which are present in the periphytic matrix and
may increase the supply of nutrients to the cells
(Stoodley et al., 2000), thus favouring their growth
under critical conditions.
The hybridization percentages of EUB II-III
obtained in our study varied depending on the lake and
the bacterial community. The clear vegetated lake
exhibited higher values of hybridization than the turbid
ones. It should be noted that the hybridization
efficiency could vary according to the quantity of
ribosomes present in the target cell (Pernthaler et al.,
2002). In the present study, the hybridization
percentage of Eubacteria was always higher in
bacterioperiphyton than in bacterioplankton. A similar
difference was observed by Araya et al. (2003), who
argued that this may be related to the higher activity of
the cells belonging to biofilm in comparison with the
planktonic bacteria.
It should be noted that the probes selected cover
large groups as well as several lineages within them
(Newton et al., 2011). Since this general probes could
be masking a great variability of lineages, similar
studies should be conducted using more specific probes
to detect possible differences that may have been
overlooked. Another factor that should not be discarded
is the effect of the use of artificial substrata on
bacterioperiphyton analyzes. In the first states of the
conformation of the adhering community, the presence
of organic molecules over the colonized surface is of
great importance since bacteria do not adhere to a clean
substrate (Busscher & Van der Mei, 2000). This may
affect the bacterioperiphyton community developed
over artificial or natural substrata (e.g., macrophytes,
rocks, sediments). However, artificial substrata allow
quantifying and manipulating the attached community,
which would be almost impossible by means of natural
substrata.
Our study suggest that the lake regime (clear and
turbid) influence the bacterial structure of planktonic
and periphytic communities. This influence was mainly
reflected in the subdominant bacterial groups in each
lake type. Moreover, we found that the bacterial
composition differed between the pelagic and the
attached communities.
ACKNOWLEDGMENTS
We thank to the owners of the farms for allowing us to
have access to the lakes studied and to the members of
Laboratorio de Ecología y Fotobiología Acuática from
IIB-INTECH (Chascomús, Buenos Aires, Argentina)
where part of the CARD-FISH technique was
performed. We also thank to two anonymous reviewers
and to the editor for their valuable suggestions on the
manuscript. This research was supported by a grant of
the University of Buenos Aires (UBACyT X838) and
PAMPA2 Project (CONICET).
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Lat. Am. J. Aquat. Res., 43(4): 675-683, 2015
Sea turtle stranding on Bahia, Brazil
Research Article
Analysis of marine turtle strandings (Reptilia: Testudine)
occurring on coast of Bahia State, Brazil
Aline Lopes-Souza1, Alexandre Schiavetti2 & Martín Roberto Álvarez3
1
Programa de Pós-Graduação em Zoologia, Departamento de Ciências Biológicas
Universidade Estadual de Santa Cruz, Rod. Jorge Amado, km 16
Salobrinho, 45660-900, Ilheus-Bahia, Brasil
2
Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz
Rod. Jorge Amado, km 16, Salobrinho, 45660-900, Ilheus-Bahia, Brasil
3
Departamento de Ciências Biológicas, Universidade Estadual de Santa Cruz
Rod. Jorge Amado, km 16, Salobrinho, 45660-900, Ilheus-Bahia, Brasil
Corresponding author: Alexandre Schiavetti ([email protected])
ABSTRACT. This study provides an analysis of the occurrence and the spatial and temporal distribution of
marine turtle strandings found in the south of the State of Bahia. Data was collected between January 2006 and
June 2008. This study covers an area of 220 km of the southern coast of Bahia State (northeastern Brazil), and
spatial analyses were made considering data collected in three bases suported by Petrobras-Petróleo Brasileiro
S/A distributed in the area. The records were sorted according to month and year, species, age group and sex. A
total of 260 stranding were reported: 183 of Chelonia mydas (74.1%), the most frequent species. The highest
number of strandings was recorded in Gamboa do Morro Base. Juveniles presented the highest densities, but no
differences between adults and small juveniles were detected. Males were more frequently stranded in Gamboa
do Morro Base, while females were more frequent in Ilhéus Base. An increase in the number of stranding
between 2006 and 2008 was noted; moreover, the months with more records were January, February, March,
October and December. The number of stranding events was discontinuously distributed in the study area. This
study also demonstrated the usefulness of implement different strategies of recording marine turtle strandings:
direct monitoring efforts (patrol) in remote beaches and educational campaigns applied on beaches frequented
by tourists. This study demonstrated that, despite spatial nearby, the three bases attend independent biological
systems and show different stranding dynamics, thus different conservancy actions should be implemented in
order to improve the knowledge on natural history of sea-turtles in the southern coast of Bahia State.
Keywords: turtle stranding, monitoring beaches, educational campaigns, conservation strategies, northeastern
Brazil.
Análisis de varamientos de tortugas marinas (Reptilia: Testudine)
ocurridas en la costa del Estado de Bahía, Brasil
RESUMEN. Se analiza de la incidencia y distribución espacio-temporal de los varamientos de tortugas marinas en el
sur del Estado de Bahía, nordeste de Brasil. Los datos fueron obtenidos entre enero de 2006 y junio de 2008. Este
estudio cubrió un área de 220 km de la costa sur del Estado de Bahía (noreste de Brasil) y el análisis espacial se realizó
considerando las tres bases financiadas por la empresa Petrobras-Petróleo Brasileiro S/A. Los registros fueron
ordenados según mes y año, especie, grupo etario y sexo. Se registró un total de 260 varamientos: 183 de Chelonia
mydas (74,1%), la especie más frecuente. La mayor frecuencia de varamientos se observó en la base Gamboa do
Morro. Los juveniles presentaron las mayores densidades, pero no se detectaron diferencias entre adultos y crías. Los
machos vararon con mayor frecuencia en la base Gamboa do Morro, mientras que las hembras fueron más frecuentes
en la base Ilhéus. Se observó un aumento en el número de varamientos entre 2006 y 2008 y además, los meses con
más registros fueron enero, febrero, marzo, octubre y diciembre. El número de eventos de varamiento fue distribuido
de forma discontinua en el área de estudio. Este estudio también demostró la utilidad de aplicar diferentes estrategias
de registro de varamientos de tortugas marinas: esfuerzos de monitoreo directo (patrulla) en playas remotas y campañas
educativas aplicadas en playas frecuentadas por turistas. Este estudio demostró que, a pesar de encontrarse próximas
espacialmente, las tres bases atendieron sistemas biológicos que funcionan en forma independiente y muestran diferen__________________
Corresponding editor: Sergio Palma
675
676
tes dinámicas de varamiento, por lo tanto se deben implementar diferentes acciones de conservación para mejorar el
conocimiento de la historia natural de las tortugas marinas en la costa sur del Estado de Bahía.
Palabras clave: varamiento de tortugas, monitoreo de playas, campañas educativas, estrategias de conservación,
noreste de Brasil.
INTRODUCTION
Many species of large marine vertebrates, such as
marine mammals, marine turtles and seabirds, are
subject to strandings for different reasons (Peckham et
al., 2008; Velozo et al., 2009; García-Borboroglu et al.,
2010; Williams et al., 2011). Animals can be found
dead or alive, ashore or floating in coastal waters, and
such data should be treated differently (Casale et al.,
2010). While inferences from strandings should be
carefully employed (Hart et al., 2006), when studied
across large spatio-temporal extents, the findings can
provide information about geographic and seasonal
distribution, natural and anthropogenic impacts, and
life history and natural history of marine vertebrates,
including marine turtles (Epperly et al., 1996; Tomás et
al., 2008).
Five species of marine turtles are found in Brazil:
Caretta caretta, Eretmochelys imbricata, Lepidochelys
olivacea, Chelonia mydas and Dermochelys coriacea
(Marcovaldi & Laurent, 1996). Four species of marine
turtles have been identified reproducing on the beaches
of the southern Bahia coast, which comprises one of the
three main nesting areas in Brazil (Marcovaldi &
Marcovaldi, 1999): C. caretta, E. imbricata, L. olivacea
and C. mydas (Camilo et al., 2009).
According to Wallace et al. (2010), the regional
management units for the marine turtles are areas
occupied by populations that function independently
and have different demographic processes. The
southern Bahia coast is included in the management
unit of the West Atlantic (for L. olivacea) and southwestern Atlantic (for C. caretta, E. imbricata and C.
mydas). All these management units are small in area
and almost unique to the Brazilian coast. Therefore,
there is a need to maintain the life cycle of these species
to the full extent of the Brazilian coast to prevent loss
of genetic diversity.
Each of these marine turtles species is threatened in
Brazil (MMA, 2014) as a result of environmental
degradation and pollution in their habitat, hunting, egg
collecting, fisheries bycatch and as a consequence of
fishing with trawl and drag nets (Bugoni et al., 2001;
Almeida et al., 2011; Castilhos et al., 2011; Marcovaldi
et al., 2011; Santos et al., 2011; Braga & Schiavetti,
2013).
In 1980, the National Program for Protection of
Marine Turtles, the TAMAR Project (IBAMA), was
founded in Brazil (Marcovaldi & Marcovaldi, 1999).
Currently, the project has 22 bases discontinuously
distributed along the Brazilian seacoast; however, other
partnering institutions also contribute to the conservation of the chelonian species (Camilo et al., 2009).
The Praia do Forte (northern Bahia State) is
considered by TAMAR Project the core nesting area for
marine turtle reproduction in the northeastern Brazil
(Marcovaldi & Marcovaldi, 1999). Nevertheless,
marine turtles were reported nesting in marginal
reproductive areas south (Camilo et al., 2009) and north
(Parente et al., 2006) of this core area. According to
Lesica & Allendorf (1995), the study of peripheral
populations, which is the subject of this paper, may help
in the conservation of the species as a whole. These
marginal or peripheral populations allow for the
expression and preservation of a larger set of genes,
which decreases the chance of inbreeding and thus
strengthens population viability, as well as provides a
demographic reserve for the species as a whole
(Allendorf et al., 2012). Thus, studies of marginal
reproductive areas are necessary to aid in the
development of management strategies for the
conservation of these chelonian species on the Brazilian
coast.
The aim of this study was to analyse the temporal
and spatial distribution of marine turtle strandings on
the southern coast of Bahia State, a marginal reproductive
area without any institutional conservation action
nearby.
Study area
The study area is located within the South Atlantic trade
winds belt (NE-ESE), which is related to the highpressure cell existing in this region (Dominguez et al.,
1992; Bittencourt et al., 2000, 2005, 2007) (Fig. 1).
This study covers an area of 220 km of the southern
coast of Bahia State (northeastern Brazil), between the
municipalities of Valença (13º22´26”S, 38º96´58”W)
and Una (15º19´23”S, 38º99´76”W). The coastline was
divided into three bases supported by PetrobrasPetróleo Brasileiro S/A for different periods (Fig. 1,
Table 1). The bases in Gamboa do Morro (GBM) and
Baia de Camamu (BCM) are along remote beaches
without tourism activities, and Ilhéus (IOS) base falls
677
Figure 1. Map of study area in Bahia State southern coast (northeastern Brazil), considering the three bases Gamboa do
Morro (GBM), Baia de Camamu (BCM) and Ilhéus (IOS) and the municipalities covered by each base. The numbers show
the absolute frequency of strandings in each base during the period from January 2006 to June 2008.
Table 1. Sampling effort at each base as a function of the operating time and tracking area. It is also presented the base
name, the sampling period (month), the extension of beach comprehended by the base (km), and the sampling effort
performed.
Base
Gamboa do Morro (GBM)
Baia de Camamu (BCM)
Ilhéus (IOS)
Sampled period
(month)
30
13
13
in a region of the coast mainly supported by tourism (on
a tourist and residential/urban beach).
Data collection
Marine turtles stranded were found on the beach, dead
or alive, with the help of tourist and locals’ calls/
complaints or during beach monitoring efforts between
January 2006 and June 2008. The study areas were
monitored by foot; everyday, a different stretch of 15
km of beach was monitored until all research area was
travelled. Surveyors then returned to the starting point
of monitoring.
Sampled area extension
(km)
31
72
117
Sampling effort
(month*km)
930
936
1521
Species information, the location (GPS) of each
stranding, the condition of the animal (alive or dead)
and the curved carapace length (CCL) (Bolten, 1999)
were collected. Dead animals and carcasses were
necropsied. When possible, sex was determined by
reproductive tract visual observation in adult individuals (Wyneken, 2001), for which the visual analysis is
reliable.
When GPS location was not able to be determined,
the location of the stranding was classified by taking
into account characteristics of the stranding location
(name of beach or river, county, etc.).
678
Curved carapace length (CCL) was used to classify
specimens in age classes (small juveniles, juveniles and
adults) according to Almeida et al. (2011), Castilhos et
al. (2011), Marcovaldi et al. (2011), Limpus &
Chaloupka (1997), and Santos et al. (2011) (Table 2).
The values presented in Table 2 for adults are all below
the mean value for monitored nesting females on the
shores of Brazil.
Data analysis
Marine turtle stranding records came from tourist or
locals’ calls/complaints and during beach patrols.
Moreover, both sources depend on monitoring areas
and periods of activity experienced by each base. Thus,
comparisons between bases and seasons should
consider the sampling efforts (month*km), calculated
as the total operating time (month) multiplied by the
beach extension (km) monitored by each base (Table
1). As not all bases were monitored with the same
effort, these results should be regarded as a rough
estimation for comparing these areas (Casale et al.,
2010).
Spatial analysis was conducted using ArcGIS
software. The relative frequency regarding species, age
group, sex and climatic season (rainy or dry),
standardising the records with the sampling effort, was
analysed using a Chi-square test (Siegel & Castellan Jr.,
2006).
RESULTS
Between January 2006 and June 2008 (30 months), a
total of 260 marine turtle were found stranded. Only
5.0% (13) were of unidentified species. The most
frequent species was C. mydas (74.1%), followed by L.
olivacea and C. caretta (both with 10.9%), and finally,
E. imbricata (4.1%) (Fig. 2).
Table 2. Age classes (small juveniles, juveniles and
adults) for the fourth marine turtles species found, defined
as function of curved carapace length (CCL), according to
Almeida et al. (2011), Castilhos et al. (2011), Marcovaldi
et al. (2011) and Santos et al. (2011).
Species
Chelonia mydas and
Caretta caretta
Eretmochelys
imbricata ♂
Eretmochelys
imbricata ♀
Lepidochelys
olivacea
Small
juveniles
<20 cm
Juveniles
Adults
21-80 cm
>80 cm
<20 cm
21-70 cm
>70 cm
21-75 cm
>75 cm
11-46 cm
>46 cm
<10 cm
Figure 2. Frequency of marine turtle strandings
(according to species and age group) occurred in the beach
extension between the municipalities of Valença and Una
(Brazil, BA) from January 2006 to June 2008 (n = 247).
From the 260 strandings, 226 marine turtles were
found dead (86.9%) and only 34 alive (13.1%), of
which 22 (64.7%) were reintroduced to seawaters. Most
of the marine turtles found alive were C. caretta (n =
17 strandings, 50%), followed by C. mydas (n = 15
individuals, 44%); mostly small juveniles 92.6% and
80%, respectively. The lowest number of strandings
alive were found for E. imbricata (n = 1) and L.
olivacea (n = 1), with 3% each both small juveniles.
Regarding age classes, juveniles were more
common (n = 172; 75.8%) than adults (n = 30; 13.2%)
and small juveniles (n = 25; 11%). Considering species
and age classes, we noted that the small juveniles
belonged to C. caretta (n = 19; 76%), whereas most
juveniles belonged to C. mydas (n = 157; 91.3%) and
most adults pertained to L. olivacea (n = 19; 63.3%)
(Fig. 2).
The CCLs showed intraspecific and interspecies
variations. The largest CCL mean was found in E.
imbricata (65.0 ± 15.9 cm) and the lowest in C. caretta
(32.9 ± 32.4 cm). The CCL means were 48.8 ± 15.6 cm
and 46.4 ± 31.5 cm) for C. mydas and L. olivacea,
respectively (Fig. 3).
Sex was identifiable in only 25 of the necropsied
individuals, being 17 females (12 C. mydas, 3 L.
olivacea, 2 E. imbricate) and 8 males (4 L. olivacea, 2
C. mydas and 2 E. imbricate). In any stranded C. caretta
was able to identify the sex. This sex bias (2 females: 1
male) did not necessarily represent the true proportion
of strandings among the sexes and may be due to the
difficulty in determining the sexes.
The origin of the 260 stranding records was
classified into two groups: those reported by tourist and
679
quent at the GBM and IOS bases, while juveniles and
small juveniles were concentrated at the GBM base
(Fig. 5b).
DISCUSSION
Figure 3. Mean, 25% to 75% quartiles, maximum and
minimum values of curved carapace length (CCL) for the
fourth sea turtles species stranded on coast of Bahia State,
Brazil, in the period from January 2006 to June 2008.
locals’ calls/complaints (35.4%) and those found
during beach monitoring efforts (64.6%). When
analysing the origin of the records from each base, it
was noted that the records for the IOS base mainly
originated from calls (66.3%), whereas the reports for
GBM and BCM bases mostly resulted from beach
monitoring (80.8% and 80.4%, respectively).
The stranding relative frequency varied between
years, from 0.16 strands/sampling efforts in 2006 to
0.06 in 2008 (yet the latter had only six months
evaluated). The lowest rate was 0.01 strands/sampling
effort in 2007. When analysing monthly strandings of
all species, it was noted that the months with the largest
number of events were January, February, March,
October and December, which corresponded to the
rainy season (summer) in the region (Fig. 4).
Comparing the relative frequencies of summer (N =
194) and winter (N = 66), we observed significant
differences (2 = 63.01, df = 1, P < 0.001), even
considering both record sources (calls/complaints and
beach monitoring efforts) (Fig. 3).
When analysing the spatial distribution of
strandings, we verified that they were discontinuously
distributed along the study area and that there is a
higher (2 = 47.37, df = 2, P < 0.001) absolute
frequency of stranding events at the GBM base (Fig. 1).
Despite the sampling effort in each base, the most
frequently stranded marine turtle was C. mydas at all
bases, and E. imbricata had the lowest stranding
frequency (Fig. 5a). With regard to the stranding rate
classified according to age group, adults were more fre-
Chelonia mydas accounted for the highest number of
strandings, especially juvenile-stage individuals. This
may be due to the distribution of feeding areas of this
species (Almeida et al., 2011) and the coastal geomorphology of the region (Dominguez et al., 1992). The
diet of a green marine turtle depends on its age. When
juvenile (pelagic phase), they are omnivores with a bias
toward carnivory (Guebert-Bartholo et al., 2011). After
leaving the pelagic phase, they become herbivores and
primarily feed on aquatic plants and algae, and
eventually they feed on jellyfish and sponges (Nagaoka
et al., 2012; Awabdi et al., 2013; Reisser et al.,
2013).The study area contains rock formations and
reefs parallel to the coast (Caló et al., 2009), making it
suitable for the establishment of benches of algae and
aquatic plants and thus attractive to green marine
turtles.
It is well documented that the loggerhead marine
turtle is the species that reproduces in most of the
Brazilian coast, and the coast of Bahia State represents
their main breeding site (Marcovaldi & Marcovaldi,
1985). A study reported the occurrence of C. caretta
(Camilo et al., 2009) nesting and breeding on a beach
located in southern Bahia. As we found that the
smallest juveniles stranded were C. caretta in all three
regions of the study area, our results affirm the value of
the entire southern Bahia coast as a breeding and
development area and nesting site for this species.
The strandings recorded for Lepidochelys olivacea
were almost all adults, both male and female. This is
the smallest species of marine turtle present in Brazil.
The distance from the study area to the closest
registered breeding sites (Sergipe State) (Marcovaldi &
Marcovaldi, 1985) reaches 500 km, which agrees with
the high dispersal ability of this species (Palovina et al.,
2004). Also suggested by Palovina et al. (2004), olive
ridley marine turtles forage while diving over 100 m
deep. In the study area, the continental shelf-break
occurs between 15 and 50 km away from the coastline
(Knoppers et al., 1999), which could mean that these
species co-inhabit during their non-reproductive phase,
as well as are impacted by human foraging activities.
The presence of adults in this area means that during
their non-reproductive phase, this species is distributed
along areas of the coast of Brazil where the official
program of marine turtle protection (TAMAR Project)
does not act.
680
70
Direct monitoring effort
Monthly relative frequency
60
Strands registereds by calls
50
40
30
20
10
0
JAN FEV MAR
apr
may
jun
jul
aug
sep
OCT NOV DEC
Figure 4. Monthly relative frequency of marine turtles strandings, according to sampling efforts performed in each month,
occurred in beach extensions ranging from the municipalities of Valença to Una (Brazil, BA), in the period from January
2006 to June 2008. Months on cap letter correspond to marine turtles reproductive season and rainy season in the study
area.
Figure 5. Frequency of marine turtles strands according
to species a) and age group b) in the bases Gamboa do
Morro (GBM), Baia de Camamu (BCM) and Ilhéus (IOS)
during the period January 2006 to June 2008.
Most stranded animals were found during beach
monitoring, which shows the effectiveness of this type
of activity in studies involving stranding of marine
animals (Batista et al., 2005; Meirelles et al., 2009;
Velozo et al., 2009). However, most records (66.3%) at
the IOS base came from calls by tourist or locals during
the rainy season, which corresponds to summer
vacations in Brazil. During this period, there is an
increased frequency of tourists on the beaches, which,
together with educational campaigns, may have
contributed to the increase in stranding reports. An
increase in reports after educational campaigns was
also observed in the study area for cetacean strandings
reports (Batista et al., 2012).
Of all strandings, 75% occurred in the summer (Fig.
4). This can be explained by a confluence of factors, but
primarily, this is the breeding season of these species
(Camilo et al., 2009). A higher number of adults (71%)
and small juveniles (93%) were found during this
period. Moreover, these months correspond to summer
vacation season, which should increase the probability
of finding stranded animals as described above. Finally,
the change in wind direction during this season, which
starts to blow from the ocean to the beach
predominantly from the northeast (Dominguez et al.,
1992; Bittencourt et al., 2007), can direct marine turtles
to the beach and increase the risk of stranding as well
as drift carcasses coming from the sea.
The frequency of marine turtle strandings has
increased in the study period (2006-2008) because of
the expansion of the sampling area. This expansion
enabled the identification of different population
dynamics among species. Bases covering distances of
approximately 100 km each showed strandings of
species and different life stages (Figs. 2, 5), which
illustrates the dynamics of marine coastal systems of
the State of Bahia and the need to implement different
conservation strategies for each.
CONCLUSIONS
This study showed that despite being spatially
proximate, the three bases show a variety of stranding
dynamics affecting species and age classes differentially.
Thus, different conservancy actions should be implemented to improve the understanding of the natural
history of seaturtles along the southern coast of Bahia
State.
If there are three different “systems” that operate on
one third of the coast of Bahia, as identified by the
observed variations between the bases, it is necessary
to expand the sampling area to the north and south. This
could help determine whether there are other “systems”
related to different populations of marine turtles, their
composition, temporal structure and phases of life. This
is considering the fact that all the species are recorded
in the Bahia State.
This study also showed the need to implement
different strategies of recording marine turtle
strandings. We therefore recommend that studies of
strandings use direct beach monitoring (patrols) as the
main strategy in isolated areas, complemented by
efforts with educational campaigns in areas with a
higher presence of people on the beaches.
Finally, more studies are needed to assess other
factors associated with strandings of marine turtles in
the studied region; these factors include marine
topography, the action of winds, ocean circulation and
interaction with fishing and other human activities. The
implementation of these study factors is aimed at
mitigating the impacts on the populations of these
endangered marine turtle species in Brazil.
ACKNOWLEDGMENT
The authors are thankful to Petrobras-Petróleo
Brasileiro S/A and the Instituto Mamíferos Aquáticos IMA (Aquatic Mammals Institute) for the provision of
data to staff and trainees from all the bases who have
helped in monitoring and data collection; the authors
are also thankful to the Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior-CAPES and Conselho
Nacional de Pesquisa Cientifica e Tecnologica-CNPq
for scholarships (first and second authors respectively).
Anonymous reviewers improved the manuscript.
681
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Received: 7 March 2014; Accepted: 13 May 2015
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Lat. Am. J. Aquat. Res., 43(4): 684-690, 2015
Scientometric study of Macrobrachium genus
6841
Research Article
Research on the river shrimps of the genus Macrobrachium (Bate, 1868)
(Decapoda: Caridea: Palaemonidae) with known or potential economic importance:
strengths and weaknesses shown through scientometrics
Olimpia Chong-Carrillo1, Fernando Vega-Villasante1, Ricardo Arencibia-Jorge2
Shehu L. Akintola3, Layla Michán-Aguirre4 & Fabio G. Cupul-Magaña5
1
Laboratorio de Acuicultura Experimental, Centro de Investigaciones Costeras
Universidad de Guadalajara, Puerto Vallarta, CP 48280, Jalisco, México
Centro Nacional de Investigaciones Científicas, Playa La Habana, CP 10600, Cuba
3
Fisheries Department, Lagos State University, PMB 0001, LASU, Ojo, Lagos, Nigeria
4
Laboratorio de Cienciometría, Información e Informática en Ciencias Biológicas
Departamento de Biología Comparada, Facultad de Ciencias, UNAM
Coyoacán, Distrito Federal, CP 04510, México
5
Laboratorio de Artrópodos, Centro Universitario de la Costa, Universidad de Guadalajara
Puerto Vallarta, CP 48280, Jalisco, México
2
Corresponding author: Fernando Vega-Villasante ([email protected])
ABSTRACT. This study revealed that the scientific interest in the genus Macrobrachium was not restricted to
a biological point of view, but included also social and economic aspects. Many species of the genus are subject
of traditional fisheries and culture worldwide. Several research groups across the globe have developed projects
in various subject areas on commercial or non-commercial native species of this genus. This investigation aimed
to contribute to the development of the genus Macrobrachium research through a scientometric study. The study
was based on publications (1980 to 2013) registered in the following databases: Biological Abstracts, ISI Web
of Science, SciELO Citation Index, BioOne, Science Direct, Scopus, and Redalyc. A total of 2165 publications
on Macrobrachium in the last 33 years were included in this analysis. The themes that yielded most posts were
related to culture, nutrition/feeding, and genetics with almost 60% of the total. Publications concerning M.
rosenbergii represented more than 60% of the total with the remaining 40% encompassing 22 other species.
Analysis performed by geographical regions evidenced that Latin America produced 23% of the publications,
South Asia 22%, and East Asia 16%. Brazil generated 65% of the percentage mentioned for the Latin American
region. It is necessary to strengthen research on topics of basic biology, especially those of native species. This
will allow rapid progress in the generation of production technologies sustained by a solid biological knowledge
base.
Keywords: native species, river shrimps, Macrobrachium, research, scientometrics.
Investigaciones sobre camarones de río del género Macrobrachium (Bate, 1868)
(Decapoda: Caridea: Palaemonidae) con importancia económica conocida o potencial:
fortalezas y debilidades mostradas a través de la cienciometría
RESUMEN. El interés científico por las especies del género Macrobrachium no ha sido sólo desde el punto de
vista biológico, sino también económico y social. Muchas de sus especies son objeto de pesquerías tradicionales
y cultivo. Diversos grupos de investigación del mundo han realizado trabajos sobre temáticas del conocimiento
de especies nativas comerciales o no-comerciales. El presente trabajo pretende contribuir al desarrollo de la
investigación del género Macrobrachium mediante análisis cienciométrico. Este estudio se basó en las
publicaciones registradas en las siguientes bases de datos desde 1980 a 2013: Biological Abstracts, ISI Web of
Science, SciELO Citation Index, BioOne, Science Direct, Scopus y Redalyc.
__________________
Corresponding editor: Ingo Wehrtmann
685
2
El número total de publicaciones sobre Macrobrachium en los 33 años analizados fue de 2165. Las temáticas
que más publicaciones mostraron (60% del total registrado) fueron las relacionadas con el cultivo,
nutrición/alimentación y genética. Solo de M. rosenbergii se ha publicado más del 60% del total, el restante
39% de las publicaciones corresponde a 22 especies. El análisis por regiones geográficas puso en evidencia que
Latinoamérica genera el 23%, Asia del Sur 22% y Asia Oriental 16% de los trabajos publicados. Sólo Brasil
genera el 65% del total de Latinoamérica. Es necesario reforzar la investigación dirigida a temáticas de biología
básica sobre todo en las especies nativas. Esto permitirá avanzar más rápidamente en la generación de
tecnologías de producción sustentadas en una base sólida de conocimiento biológico.
Palabras clave: especies nativas, camarones de río, Macrobrachium, investigación, cienciometría.
INTRODUCTION
Among the suborder Caridea, the family Palaemonidae
includes 36 genera (Holthuis, 1952). Macrobrachium
(Bate, 1868) (De Grave et al., 2008) constitutes the
most diverse genus among the palaemonids, with at
least 238 species distributed in tropical and subtropical
streams and rivers around the world (Bauer, 2013).
These shrimps are colloquially called prawns, acamayas,
cauque, langostino or shrimp, depending on the region
in which they are found (García-Guerrero et al., 2013).
The scientific interest in the genus Macrobrachium
is not limited to a biological point of view, but also
includes social and economic aspects. Many of these
species are subject of traditional fisheries and culture
(García-Guerrero et al., 2013). According to New
(2009), production of shrimp reaches hundreds of
thousands of tons per year, most of which are M.
rosenbergii (De Man, 1878), which is originally from
Asia. This species known as "Malaysian shrimp" or
“giant river prawn” has been the most studied one, and
its farming production technology has been exported to
many countries outside their original distribution area.
Since 1980, when the first meeting in Thailand on
the culture of M. rosenbergii was held, there has been
increased scientific research directed towards
establishing optimal conditions for controlled production of this species as well as native Macrobrachium
species of economic importance (New & Nair, 2012).
Although other native species have been studied,
knowledge about them has not matched that on M.
rosenbergii. Several research groups have developed
works in various areas on commercial or noncommercial native species. Currently, there are no
studies showing how research has evolved concerning
this important genus of decapods, the interests of
research groups and countries, and the knowledge
areas, which remain poorly understood for most of the
species.
According to Hess (1997) scientometrics can be
defined as the “quantitative study of science, communication in science, and science policy”. Therefore, this
study aimed to contribute to the development of
research on the genus Macrobrachium, through a
scientiometric analysis of scientific papers published
and archived in various databases in the past three
decades. In particular, this investigation has led to
highlighting species with clear or potential economic
value.
The study, carried out according to the methodology of
Michán & Llorente-Bousquets (2010), included publications from 1980 to 2013 and registered in the
following databases: Biological Abstracts, ISI Web of
Science, SciELO Citation Index, BioOne, Science
Direct, Scopus and Redalyc. In particular the Latin
American databases (SciELO and Redalyc) were
considered since they included some journals not
compiled on commercial international databases. These
omissions could cause a significant bias in the final
results. In each of the databases all records containing
the word Macrobrachium in the title, abstract, and
keywords fields were searched. The records were
collected from databases and systematized in a
particular database organized with the aid of the
EndNote X7 ® (Thomson Reuters ®) software. The
data were subsequently validated and standardized,
selecting the items that addressed species with known
or potential economic importance. Analysis was then
carried out by the following categories:
i) Theme: words in this selection were adjusted to
include: farming, nutrition/feeding, genetics, reproduction, ecology, physiology, pathology, taxonomy,
and behavior;
ii) Species: all species of the genus Macrobrachium
that appear in any scientific document;
iii) Countries: the entire world is covered both by
region and by individual countries;
iv) Authors: the most prolific authors were recorded;
v) Institutions: universities, research centers, and
others that have made major contribution to the
study of this genus.
The data sets obtained by this process were
transferred to Excel® spreadsheets (Microsoft®) for
further analysis.
RESULTS
The total number of publications concerning Macrobrachium in the 33 years under review and within the
above-mentioned terms was 2165. The lowest number
of publications was in the 80’ and showed a stable
trend. A significant increase was observed in the early
90’, particularly during the second half of this decade;
and the trend of increasing number of publications
continued up to 2013 (Fig. 1).
The number of publications per subject per decade
is shown in Fig. 2. The themes that yielded most posts
were related to the culture (947), nutrition/feeding
(422), and genetics (262). Considerably fewer publications referred to the other research areas. The three
above-mentioned most popular topics showed an
upward trend with steep slopes, where publications
about culture stood out. However, subjects with the
lowest number of publications also showed an increase
but less pronounced.
When the total number of publications by subject
was analyzed, it was noted that those related to culture
and nutrition/feeding accounted for more than 60% of
the total. The remaining subjects shared the other 40%
out of which genetics was about 12%. Macrobrachium
686
3
rosenbergii comprised more than 60% of the total
publications (Fig. 3). The other 22 species made up
approximately 40% of the published studies in the
databases. M. amazonicum (Heller, 1862) and M.
nipponense (De Haan, 1849) represented 6.2% and
6.5% respectively, of the published literature. M.
amazonicum was the most studied and analyzed species
in the American region followed by M. carcinus
(Linnaeus, 1758) and M. acanthurus (Wiegman, 1836)
with 14% and 13% respectively. These three species
accounted for 56% of the published literature. The
remaining nine Latin American species accounted for
44%: M. olfersii 10%, M. borelli 9%, M. tenellum and
M. americanum 5% each, M. jelskii and M. ohione 4%
each, M. heterochirus and M. crenulatum 3% each, and
M. birai with 1%.
The country analysis (Fig. 4) revealed that only four
of the 21 contributing countries provided more than
50% of published papers. India led the countries with
almost 20%, followed by Brazil with 14.4%, United
States of America with 10.4%, and China with 9.0%.
The analysis by region demonstrated that Latin
America produced 23% of the papers published, South
Asia 22%, and East Asia 16%. Therefore, these three
regions accounted for more than 50% of publications
concerning Macrobrachium. In Latin America, Brazil
produced the majority of publications (65%), followed
by Mexico (19%). The ten authors with most scientific
publications regarding the genus are indicated in Fig. 5.
Universities and research centers that are mentioned in
Figure 1. Number of manuscripts published worldwide concerning the genus Macrobrachium in 33 years. The dashed
circle indicates the unusual decline in scientific publications regarding Macrobrachium, probably caused by the global
economic crisis.
687
4
Figure 2. Number of manuscripts published per decade regarding the genus Macrobrachium by subject.
Figure 3. Worldwide publications concerning the genus
Macrobrachium by species. White bars and circle represent
the American species.
the literature are shown in Fig. 6. The University of São
Paulo is the institution that produced most of the
publications concerning economically important
prawns of the genus Macrobrachium.
DISCUSSION
As far as we know, the only previous evaluation of the
scientific literature concerning the genus Macrobrachium was published by García-Guerrero et al.
(2013); these authors discussed 195 scientific papers
Figure 4. Publications about the genus Macrobrachium
by geographical area and country.
regarding Latin American species of the genus. Their
results agreed with those presented in this study and
revealed that scientific articles published on grow-out
techniques comprised the highest percentage (21%).
However, their findings about the number of
publications focused on nutrition and genetics differed
from the trends detected in the present study. In the above-
Figure 5. Top ten authors of Macrobrachium publications
worldwide.
mentioned study (García-Guerrero et al., 2013), the
authors found that subjects regarding physiology
(15%), reproduction (9%), and phylogeny, taxonomy
and systematics (9%) showed the highest percentage of
published results, while nutrition and genetics were
represented by 5% and 2% of the total, respectively.
These findings differ from those of the present study
where nutrition and genetics are two of the three main
themes covered by published manuscripts. Nevertheless, the study of García-Guerrero et al. (2013) included
only Latin American species of Macrobrachium
whereas our study comprised species on a global level
including M. rosenbergii with a high number of
publications. Despite these differences between both
studies, culture was by far the topic that generated the
highest number of scientific publications.
After the conference on "Giant Prawn", held in
Thailand in 1980, a slight increase in the number of
publication concerning the genus was observed, but
scientific production remained stable during that
decade. The largest increase was observed in the early
‘90s. This phenomenon is probably due to the
development of new technologies, most of them related
to culture innovations, feed formulations, diagnosis and
treatments of diseases, and reproduction techniques
(Chong-Carrillo et al., unpublished data). We assume
that the 80’s had a scientific production stimulated by
the above-mentioned meeting, and investigations
regarding developing production technologies were
intense but had not yet resulted in publications. By the
90’s, new research groups began to consolidate their
studies towards production of culture techniques.
However, scientists also published their findings about
other scientific aspects working with native species as
well as non-native species (for example Brazilian
groups led by John C. McNamara and Wagner C.
Valenti, as well as many Indian groups working on M.
rosenbergii).
688
5
The explosive growth of scientific production
noticed from the first part of the 90’s has not slowed
until recently. Although there have been declines in the
published scientific production as manifested in 2010
and 2011, only two years after the onset of the global
economic crisis, there was a remarkable rebound in
2009, probably due to the inertia of scientific
production that had already been carried out in previous
years. The decrease that occurred in 2010 and 2011
might be associated with the effects of the global
economic crash, which started in 2008 and lasted until
2011, with deleterious effects in all areas of the world
development, including science and technology
country budgets (Chinn, 2010). Only three years later
the trend of scientific production got back to previous
levels.
Revising the topics addressed in the scientific
literature, it is obvious that the highest number of these
publications referred to culture aspects. Studies of
culture techniques, nutrition, and genetics were
certainly those topics related to developing a more
efficient production. Noticeable was the fact that
genetics was also an emerging topic in the field of
scientific publications on Macrobrachium. The growth
of genetic themes started at the beginning of the 90’s,
ten years later than the other two most popular themes.
If production trends remain as they are today, we are
likely to see a gradual overlap in the number of
publications of the three main topics. However, the
remaining topics (ecology, physiology, reproduction,
pathology, taxonomy and behavior), which might
provide the biological basis for the other three most
popular themes, showed a poor increase in publications
especially with regard to native species.
FAO (2012) has repeatedly mentioned the need for
increased research on native species before introducing
alien species. This suggestion is particularly
emphasized in its report: State of Fisheries and
Aquaculture 2012 (Sofia 2012 Report; FAO, 2012).
Although various research groups worldwide have
addressed this advice, it has not influenced the ongoing
increase of scientific publications based on studies
regarding M. rosenbergii. Research groups from Asia,
Oceania, USA, and Israel supported the scientific
production about this species. While this trend has not
been reversed, many countries have developed studies
on native species in actual or potential farming.
Noteworthy examples are the groups in Brazil, with the
largest number of publications on native species,
particularly concerning M. amazonicum. Another
already cultivated species that has produced a lot of
publications is M. nipponense in China and M.
malcomsonii (H. Milne-Edwards, 1844) in India. In
Latin America, there are also efforts on research and
689
6
Figure 6. Top global institutions generating scientific publications concerning the genus Macrobrachium.
publications about native species: Brazil has provided
a significant number of publications regarding M.
amazonicum, as well as about species such as M.
acanthurus, M. olfersii (Wiegmann, 1836), and M.
carcinus, among others. Argentine groups have made
significant progress in scientific knowledge about M.
borellii (Nobili, 1896), especially in the areas of basic
biology, placing this species as one of the most studied
ones in the Americas. Great research efforts, though
scattered, are being made in Mexico with M. tenellum
(Smith, 1871), M. americanum (Bate, 1868), and M.
carcinus.
Brazil has devoted considerable more human and
funding resources compared to other countries in the
region: in 2011 Brazil spent 1.6% of its gross domestic
product (GDP) in science and technology, a considerably higher percentage when compared to 0.62% of
Argentina, 0.48% of Costa Rica, 0.46% of México,
0.42% of Chile, and 0.18% of Colombia (World Bank,
2014). It is thus clear that the higher the support for
science and technology, the higher is the capacity for
knowledge generation; a premise valid in the case of
Macrobrachium research.
Worldwide, the ten most productive scientists
regarding Macrobrachium research include two
Brazilians (John C. McNamara and Wagner C.
Valenti), Amir Sagi from Israel (4.4% of GDP in
science and technology), and Jian Chu Chen from
China (1.8% of GDP in science and technology),
among others. Again, Brazil shows a considerable ad-
vantage compared to other countries with regard to
institutions and centers studying Macrobrachium. The
University of São Paulo (USP) stood out as the
foremost institution worldwide concerning scientific
publications about Macrobrachium. According to
Scimago Institutions Rank (SIR) 2013, the USP ranked
12 in the overall list, and first in the 2013 Iberoamerican
SIR.
The results shown in the Figures 5 and 6 were
obtained solely from the ISI database because it
remained the most important global reference in the
analysis and evaluation of published science. We are
aware that the inclusion of other databases may modify
results obtained in the present investigation.
The scenario described in this study allowed us to
conclude that there are clear conclusions regarding
research about Macrobrachium in the world: i)
scientific publications on Macrobrachium is on the rise
worldwide; ii) there is a strong scientific community
that focuses its research efforts on this genus; iii) main
topics address and are intended to resolve issues of
growing and breeding; iv) there is a trend of increasing
research on native species; v) compared to developed
countries,, emerging countries are devoting more efforts
to conduct research concerning Macrobrachium; vi)
renowned universities and research centers globally
support scientific productivity regarding Macrobrachium.
There are, however, also weaknesses: i) research is
mainly directed to M. rosenbergii, while the number of
studies on native species remains low; ii) the basic
themes that can sustain a solid understanding of the
biology of native species are not addressed with the
same intensity as those directed towards growth; iii)
only a few of the emerging countries maintain scientific
production; iv) although Latin America is the most
productive region, few universities and research centers
support the scientific productivity regarding Macrobrachium; v) with the exception of Brazil, apparently
there is no concerted effort in the remaining Latin
American countries to increase their knowledge on
native Macrobrachium species; and v) only two authors
and two Latin American universities appear as main
publication generators of Macrobrachium literature. It
is necessary to strengthen research on topics of basic
biology especially of native species. This will allow a
rapid progress in the generation of production
technologies sustained by a solid biological knowledge
base. Also, it will help setting priorities for the
advancement of aquaculture at local and regional levels
as well as the protection of indigenous natural
resources. There is also a need to increase research
efforts on culture of local species with commercial
potential to replace or compete with the production of
M. rosenbergii. Governments and universities in
emerging countries should devote more resources to
study the ecology and other basic subjects of native
Macrobrachium species.
ACKNOWLEDEGMENTS
This work was carried out thanks to a doctoral
fellowship from the Consejo Nacional de Ciencia y
Tecnología (CONACYT) from México, awarded to the
first author of this manuscript. The authors wish to
thank the anonymous reviewers of this manuscript,
since their suggestions significantly improved its
quality. Special thanks to Dr. Ingo Wehrtmann for his
contribution and patience as Associate Editor.
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Lat. Am. J. Aquat. Res., 43(4): 691-699, 2015Condrictios de profundidad en el Caribe colombiano
691
Research Article
Estructura y distribución de los condrictios de aguas profundas
en el Caribe colombiano
Jorge Paramo1, Daniel Pérez1 & Arturo Acero2
Grupo de Investigación Ciencia y Tecnología Tropical (CITEPT), Universidad del Magdalena
Cra. 32 Nº22-08 Avenida del Ferrocarril, Santa Marta, Colombia
2
Universidad Nacional de Colombia sede Caribe, CECIMAR/INVEMAR
Santa Marta, Colombia
1
Corresponding author: Jorge Paramo ([email protected])
RESUMEN. Si bien, aunque actualmente no existe una pesquería de aguas profundas en el Caribe colombiano,
es importante conocer la biología y ecología de la ictiofauna de aguas profundas para identificar el impacto de
la pesca sobre estas comunidades. Con fines de aportar conocimiento que sirva como línea base para su
conservación, el objetivo del presente estudio fue determinar la composición específica, y aspectos de su
estructura poblacional y ecológica tales como abundancia y distribución (espacial y batimétrica) de los
condrictios de aguas profundas en el mar Caribe colombiano. Se realizaron cuatro muestreos a bordo de un barco
de arrastre camaronero entre 200 y 550 m de profundidad, durante agosto y diciembre de 2009 y, marzo y mayo
de 2010. Se encontró un total de 331 especímenes de 13 especies correspondientes a nueve familias. Las especies
que se capturaron con más de 15% de frecuencia de ocurrencia fueron Etmopterus perryi, Galeus cadenati,
Anacanthobatis americanus y Gurgesiella atlantica. La zona donde se encontró la mayor abundancia relativa
de especies e individuos fue el norte del Caribe colombiano, denominada Ecoregión La Guajira.
Palabras clave: condrictios, aguas profundas, distribución, manejo, Mar Caribe, Colombia.
Structure and distribution of deep-water chondrichthyans in the
Colombian Caribbean
ABSTRACT. Although currently there is no deep-sea fishery in the Colombian Caribbean Sea, however it is
important to know the biology and ecology of the deep-sea ichthyofauna in order to identify the impact of the
fishing on these communities. Therefore, to produce the baseline biological knowledge for their conservation,
the objective of the present study was to determine the specific composition and describe some aspects of their
population and ecology, as their abundance and distribution (spatial and bathymetric) of the deep-sea
chondrichthyes at the Colombian Caribbean Sea. We carried out four samplings on board of a shrimp fishing
vessel, trawling between 200 and 550 m of depth, during the months of August and December 2009 and March
and May 2010. We found a total 331 specimens of thirteen species corresponding to nine families. The species
that were captured with more than 15% of appearance frequency were Etmopterus perryi, Galeus cadenati,
Anacanthobatis americanus and Gurgesiella atlantica. The higher relative abundances of species and
individuals were found in the northern area of the Colombian Caribbean Sea (La Guajira Ecoregion).
Keywords: chondrichthyans, deep waters, distribution, management, Caribbean Sea, Colombia.
INTRODUCCIÓN
El mar profundo se extiende desde los límites de la
plataforma continental, desde 200 m de profundidad, el
cual es un ambiente oscuro y frío, que depende de los
aportes de materia orgánica proveniente de los
ecosistemas superficiales, debido a su nula productivi_____________________
Corresponding editor: Oscar Sosa
dad primaria (García et al., 2008). No obstante, los
recursos pesqueros de profundidad son especialmente
vulnerables a la sobre-explotación debido a las
características de la historia de vida de las especies que
incluyen longevidad alta, tasa de crecimiento lenta,
madurez tardía y fecundidad baja (Koslow et al., 2000;
Stevens et al. 2000; Morato et al., 2006; García et al.,
692
2008; Follesa et al., 2011) y pocos años reproductivos
(Ebert, 2005). Por lo tanto, la recuperación poblacional
es mucho más lenta que en las especies de aguas
someras (Roberts, 2002). Debido a la gran vulnerabilidad de las especies y habitats de aguas profundas, se
requiere mayores medidas de protección que limiten la
pesca, y que se basen en un enfoque altamente
precautorio (Stevens et al., 2000; Roberts, 2002;
Devine et al., 2006; Hart & Pearson, 2011). Estas
medidas incluyen la posible creación de un Área
Marina Protegida (AMP) que es una herramienta de
conservación y manejo pesquero que sigue un enfoque
ecosistémico (Worm et al., 2006; Fraser et al., 2009;
Paramo et al., 2009; Jackson & Jacquet, 2011).
Existe escasa información sobre la biología de
condrictios de aguas profundas debido a la dificultad de
realizar estudios a grandes profundidades, por esto se
tiene mayor conocimiento de las especies de aguas
someras (<100 m de profundidad). La clase
Chondrichthyes se encuentra dividida en dos subclases, Holocephali que incluyen a las quimeras y
Elasmobranchii que incluye a tiburones y rayas (Kyne
& Simpfendorfer, 2010). Según Kyne & Simpfendorfer
(2010) existen 1144 especies de condrictios, de los
cuales 530 (46%) habitan en aguas profundas, siendo
254 tiburones, 236 rayas y 40 quimeras. En Colombia
se han reportado 88 especies de condrictios, de las
cuales 32 (36%) son de aguas profundas (Mejía-Falla et
al., 2007).
Los peces cartilaginosos de aguas profundas tienen
distribución restringida o pasan el mayor tiempo de su
vida a profundidades >200 m (Kyne & Simpfendorfer,
2010). Aunque se han registrado especies hasta 4500 m
de profundidad, los estudios realizados por Priede et al.
(2006) han demostrado que su desarrollo en ambientes
abisales (>3000) es menos probable debido a su gran
requerimiento energético. Sin embargo, estos peces son
poco resistentes a la presión pesquera debido a su
limitada capacidad reproductiva, combinada con una
baja biomasa poblacional (Cavanagh & Kyne, 2006).
Debido a que la mayoría de los condrictios son
depredadores tope de la trama trófica, su captura puede
causar cambios: en su abundancia, estructura de tallas,
parámetros de historia de vida y/o conllevar a la
extinción de especies (Stevens et al., 2000). La
información sobre condrictios de aguas profundas en el
Caribe colombiano es escasa, donde no se ha
desarrollado una pesquería comercial y el ecosistema se
puede considerar prístino (Paramo et al., 2012). No
obstante, se han realizado estudios preliminares donde
se describe la presencia de peces cartilaginosos que
habitan entre 200 y 800 m de profundidad (Polanco et
al., 2010).
Estudios anteriores han identificado el potencial de
una nueva pesquería de crustáceos de profundidad en el
Caribe colombiano (Paramo et al., 2011a; Paramo &
Saint-Paul, 2012a, 2012b, 2012c). Si bien, actualmente
no existe una pesquería de aguas profundas en el Caribe
colombiano, antes de su desarrollo, es importante
conocer la biología y ecología de la ictiofauna de aguas
profundas para identificar el impacto de la pesca sobre
éstas comunidades. De esta manera, el objetivo del
presente estudio es determinar la composición
específica de los condrictios, así como aspectos de su
estructura poblacional y ecológica, como abundancia y
distribución (espacial y batimétrica) en aguas profundas
en el mar Caribe colombiano, para aportar conocimientos
que sirvan como línea base para su conservación.
MATERIALES Y MÉTODOS
El área de estudio comprende el mar Caribe colombiano, el cual está clasificado en nueve Eco-regiones
naturales: La Guajira (GUA), Palomino (PAL), Tayrona
(TAY), Magdalena (MAG), Morrosquillo (MOR),
Archipiélagos coralinos (ARCO), Darién (DAR), San
Andrés y Providencia (SAN) y Caribe Oceánico (CAO)
(Díaz et al., 2005) (Fig. 1). Los peces fueron capturados
en el mar Caribe colombiano (12°40’N, 71°40’W;
8°40’N, 77°10’W) mediante pesca de arrastre en
profundidades entre 200 y 550 m (estratos de profundidad de 100 m), en agosto y diciembre 2009; marzo y
mayo 2010 (Fig. 1).
Se utilizó el barco camaronero comercial “Tee
Claude” con una red de arrastre camaronera con tamaño
de malla al final del copo de 44,5 mm entre nudos,
abertura de la red de 11,58 m, a velocidad de 2,5 nudos,
con un total de 87 estaciones y una duración promedio
del arrastre de 30 min. No fue posible colectar muestras
entre el talud frente a Cartagena y la desembocadura del
río Magdalena debido a la irregularidad del fondo. La
ubicación de fondos apropiados para los arrastres se
determinó usando un ecosonda comercial Furuno FCV
1150 con un transductor de frecuencia de 28 kHz y la
posición de inicio y final del arrastre se estimó con un
GPS Garmin MAP 76CSx.
Se utilizaron fichas de identificación para los
condrictios de profundidad (Compagno, 1999, 2002;
Didier, 2002; McEachran & de Carvalho, 2002;
Douady et al., 2003; McEachran & Aschliman, 2004;
Mejía-Falla et al., 2007). Se registró el número y peso
de cada especie de condrictio en cada estación. En cada
arrastre se calculó la densidad (ind km-2) y biomasa
íctica (kg km-2). Se determinó la abundancia relativa
(AR%), biomasa relativa (BR%), frecuencia de ocurren-
Condrictios de profundidad en el Caribe colombiano
693
Figura 1. Área de estudio en el mar Caribe colombiano. Los círculos indican las estaciones de muestreo.
cia (FO%), índice de importancia relativa (IIR%) e
importancia relativa de las especies, mediante un índice
de valoración de importancia (IVI%). Se realizó el
análisis de clasificación de la abundancia con la matriz
de similitud de Bray-Curtis y un análisis de ordenación
de escalamiento multidimensional no métrico (nMDS),
con previa transformación de los valores de la matriz en
log(x+1) (Clarke & Warwick, 1994; Clarke & Gorley,
2001), para determinar los posibles agrupamientos
entre las estaciones de muestreo similares y las áreas
donde se presentaron mayores abundancias relativas.
RESULTADOS
Se capturaron 331 especímenes de 13 especies de
condrictios correspondientes a nueve familias, pertenecientes a cuatro órdenes y dos subclases. Las especies
que se capturaron con una FO >15% de FA fueron
Etmopterus perryi, Galeus cadenati, Anacanthobatis
americanus y Gurgesiella atlantica (Tabla 1). Las
especies que mostraron mayor BR (%) fueron E. perryi,
Cruriraja rugosa, Squatina dumeril y G. atlantica,
mientras que las especies con mayor porcentaje de IIR
e IVI fueron en el mismo orden E. perryi, G. cadenati,
A. americanus y G. atlantica (Tabla 1).
En cuanto a su distribución batimétrica, las especies
con mayor intervalo batimétrico fueron E. perryi
(profundidad media (PM) = 382,1 m), Scyliorhinus boa
(PM = de 383,0 m) y A. americanus (PM = 409,4 m),
con ocupación de todos los estratos de profundidad
estudiados y en profundidades intermedias (Tabla 1,
Fig. 2). Las especies que ocuparon profundidades
medias >400 m fueron Dactylobatus clakii (PM = 421,0
m), G. atlantica (PM = 423,1 m), C. rugosa (PM =
432,8 m), Hydrolagus alberti (PM = 460,9 m) y
Anacanthobatis longirostris (PM = 501,0 m) (Tabla 1,
Fig. 2).
La distribución espacial de todos los peces
cartilaginosos mostró valores altos de abundancia
relativa (AR) (400-1195 ind km-2) en la zona norte del
Caribe colombiano hacia el norte de Santa Marta (Ecoregión Tayrona). Sin embargo, también se encontraron
valores medios de AR (100-400 ind km-2) al frente de
Cartagena y del Golfo de Morrosquillo (Eco-región
Morrosquillo) (Fig. 3).
La distribución espacial de las especies de peces
cartilaginosos de profundidad en frecuencia de
ocurrencia FO >15%, mostró que el tiburón linterna
enano E. perryi, se encuentra localizado en la zona
norte del Caribe colombiano, desde la desembocadura
del Río Magdalena (Eco-región Magdalena) hasta
Punta Gallinas (Eco-región La Guajira), pero con
valores altos de abundancia relativa (AR) (400-910 ind
km-2) hacia el norte de Santa Marta (Eco-región
Tayrona) (Fig. 4a). La especie de tiburón G. cadenati
se distribuyó espacialmente al frente de Cartagena y del
Golfo de Morrosquillo (Eco-región Morrosquillo) y
hacia el norte entre el río Magdalena y Riohacha, Ecoregiones Magdalena y La Guajira, respectivamente, con
valores medios de AR (100-200 ind km-2) (Fig. 4b). La
Tabla 1. Frecuencia de ocurrencia (FO%), abundancia (A%), biomasa (B%), índice de importancia relativa (IIR%), índice de valoración de importancia (IVI) e
intervalo de profundidad (m) de las especies de condrictios en el Caribe colombiano.
694
raya A. americanus presentó valores bajos de AR (11100 ind km-2) al frente del Golfo de Morrosquillo (Ecoregión Morrosquillo) y el río Magdalena (Eco-región
Magdalena), pero con valores medios de AR (100-200
ind km-2) hacia el norte de Santa Marta (Eco-región
Tayrona) y Punta Gallinas (Eco-región La Guajira)
(Fig. 4c). De la misma manera, la raya G. atlantica se
distribuyó al frente del Golfo de Morrosquillo (Ecoregión Morrosquillo) con valores medios de AR (200400 ind km-2) y hacia el norte de Santa Marta (Ecoregión Tayrona) con valores bajos de AR (100-200 ind
km-2) (Fig. 4d).
El análisis de similitud de Bray-Curtis mostró que
hay seis agrupaciones. Sin embargo, el grupo cuatro (4)
es el que presentó una mayor cantidad de estaciones,
todas perteneciente al nororiente del Caribe colombiano (Fig. 5), donde se encontró una mayor cantidad
de especies y mayores abundancias relativas. Mientras
que en las otras agrupaciones se presentaron pocas
estaciones, en su mayoría pertenecientes al Caribe sur.
La distribución de las agrupaciones se observa
claramente con el análisis nMDS (Fig. 6).
DISCUSIÓN
Las mayores agregaciones de condrictios de profundidad se localizaron en el Caribe nororiental colombiano. Si bien en aguas profundas hay una mayor
estabilidad ambiental (DÓnghia et al., 2004), la
biomasa de la ictiofauna de aguas profundas depende
del régimen de productividad en superficie (DÓnghia
et al., 2004; Company et al., 2008). La zona altamente
productiva de La Guajira (Paramo et al., 2011b, 2012)
presenta las mejores condiciones para la aparición de
condrictios de profundidad. Además, Polanco et al.
(2010) encontraron una mayor abundancia relativa de
peces de profundidad en esta área y Paramo et al.
(2012) detectaron los valores mayores de diversidad de
peces de profundidad, lo cual puede estar influenciado
por el proceso de surgencia tipo Ekman (Paramo et al.,
2011b). Trabajos anteriores encontraron 16 especies de
peces cartilaginosos (Roa, 2000), pero en el presente
estudio se encontraron 13 especies. Estas diferencias se
pueden atribuir al tipo de arte de captura utilizado, pues
en el trabajo de Roa (2000) se utilizó una red de arrastre
demersal tipo semibalón y en este estudio se utilizó una
red de arrastre camaronero de mayor dimensión pero
con tamaño de malla menor. Los patrones de mayor
intervalo de distribución batimétrica que presentaron E.
perryi, S. boa y A. americanus, puede estar relacionado
a la capacidad natatoria debido a la morfometría de la
aleta caudal, que les permitiría ocupar diferentes profun-
695
Figura 2. Distribución batimétrica de los condrictios de profundidad en el Caribe colombiano.
Figura 3. Distribución espacial de la abundancia (ind km-2) de todos los condrictios de profundidad en el Caribe
colombiano.
didades, hábitat, y el alto nivel trófico con
comportamiento de alimentación oportunista (Scacco et
al., 2010). No obstante, es de gran relevancia hacer
estudios más detallados, con una escala temporal
mayor, y contrastarlos con variables ambientales que
complementen y aporten mayor información sobre la
distribución de los condrictios de profundidad en el
Caribe colombiano.
En términos de abundancia, los condrictios de aguas
profundas en el Caribe colombiano ocuparon solo un
1,47% y en biomasa un 4,47% de la captura total, lo
cual los haría muy vulnerables en pesquerías de
crustáceos de profundidad. Si bien se conoce poco
sobre la biología de condrictios de profundidad, no
existen acciones de conservación en la lista roja de
especies amenazadas (The IUCN Red List of Threatened
696
Figura 4. Distribución espacial de la abundancia (ind km-2) de los condrictios de profundidad en el Caribe colombiano con
una frecuencia de ocurrencia >15%. a) Etmopterus perryi, b) Galeus cadenati, c) Anacanthobatis americanus, d) Gurgesiella atlantica.
Figura 5. Dendograma de similitud de Bray-Curtis de las estaciones de muestreo, para los condrictios de profundidad en
términos de abundancia (ind km-2).
697
recursos marinos en el Caribe colombiano, teniendo en
cuenta el enfoque ecosistémico para el manejo
pesquero (Paramo et al., 2012).
AGRADECIMIENTOS
Figura 6. Análisis de ordenación NMDS de las estaciones
de muestreo para los condrictios de profundidad en
términos de abundancia (ind km-2) en el Caribe
colombiano. N: Norte; S: Sur.
species; www.iucnredlist.org), debido a que no existe
actualmente una pesquería de aguas profundas en el
Caribe colombiano. No obstante, la mayoría de los
condrictios de aguas profundas son capturados en
pesquerías multiespecíficas o como pesca acompañante
(bycatch) en pesquerías de teleósteos y crustáceos más
abundantes y valiosos (Cavanagh & Kyne, 2006; Norse
et al., 2012). Además, los peces cartilaginosos por ser
menos abundantes y más vulnerables a la extinción son
fácilmente sobre-explotados (García et al., 2008). Por
ejemplo, las especies del género Centrophorus están
entre las menos abundantes de los condrictios. Su baja
fecundidad, con 1-2 crías, un periodo de gestación muy
largo (2 años), edad tardía de madurez sexual de las
hembras (16,5 años) y una longevidad de 39 años, se
traduce en solo 12 crías en su vida reproductiva (Kyne
& Simpfendorder, 2010). Especies amenazadas de
extinción, tales como Centrophorus harrissoni y C.
uyato han sufrido una dramática disminución
poblacional como resultado de la pesca comercial
(Cavanagh & Kyne, 2006). Por lo tanto, es de vital
importancia el monitoreo e investigación de la pesca
acompañante de condrictios (Cavanagh & Kyne, 2006),
para entender los parámetros biológicos de estas
especies, evaluar su abundancia y vulnerabilidad a las
pesquerías (Kyne & Simpfendorfer, 2010). En este
sentido, es esencial el manejo altamente precautorio si
se quiere desarrollar nuevas pesquerías de profundidad,
para mantener los stocks de peces y la biodiversidad
(Simpfendorfer & Kyne, 2009). Una alternativa es
identificar sitios donde los condrictios de profundidad
tengan una alta vulnerabilidad y valorar la implementación de un área marina protegida (AMP). La AMP o
las AMPs potenciales, darían una alta prioridad a la
conservación (García et al., 2008) y permitirían
desarrollar un manejo racional y sostenible de los
Este trabajo es una contribución del grupo de
investigación Ciencia y Tecnología Pesquera Tropical
(CITEPT) de la Universidad del Magdalena (Colombia).
Agradecemos a la tripulación del barco “Tee Claude” y
al Capitán José Guillem. Agradecemos a Fabián
Moreno por la identificación de las especies de condrictios de profundidad. El trabajo fue patrocinado por
COLCIENCIAS (código 117-452-21288), la Universidad del Magdalena, el Instituto Colombiano de
Desarrollo Rural (INCODER) a través de la Subgerencia de Pesca y Acuicultura, el Leibniz-Zentrum für
Marine Tropenökologie (ZMT), Alemania y la
Autoridad Nacional de Acuicultura y Pesca (AUNAP)
convenio Nº 790. Contribución Nº419 del CECIMAR
de la Universidad Nacional de Colombia sede Caribe.
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Lat. Am. J. Aquat. Res., 43(4): 700-717, 2015
Localización y regulación de la acuicultura en Chile
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Research Article
Decisiones de localización y cambios regulatorios: el caso
de la acuicultura en Chile
Manuel Estay1 & Carlos Chávez1,2
1
Departamento de Economía, Núcleo Milenio de Investigación en Economía Ambiental
y de Recursos Naturales, Universidad de Concepción
Victoria 471, Barrio Universitario, Concepción, Chile
2
Centro Interdisciplinario para la Investigación en Acuicultura (INCAR)
O’Higgins 1695, Concepción, Chile
Corresponding author: Manuel Estay ([email protected])
RESUMEN. Se estudia la evolución de la actividad acuícola en Chile y el impacto de los cambios regulatorios
sobre las decisiones de localización de los centros de cultivo. Este estudio considera un análisis descriptivo del
desarrollo espacio-temporal de los centros de cultivo. Luego, utilizando un panel de datos, se estimó un modelo
de elección de sitios con el objetivo de explorar los factores determinantes de la elección de ubicación de los
centros acuícolas. Los resultados del análisis sugieren la existencia de un claro patrón de desarrollo espaciotemporal de la acuicultura en Chile. Este patrón está caracterizado por el desplazamiento de la actividad
productiva hacia el sur de la Patagonia chilena cambiando su concentración desde los alrededores de Puerto
Montt hacia el sur de la isla de Chiloé. La estimación del modelo de elección de sitios sugiere que la distancia
entre los centros y la existencia de centros de la misma especie cultivada son relevantes para explicar el
fenómeno de expansión de la acuicultura hacia el sur. Los cambios regulatorios parecen ser un factor que ha
influido en el desarrollo del patrón espacio-temporal que caracteriza el uso del territorio por parte de la industria
acuícola nacional.
Palabras clave: acuicultura, localización, cambios regulatorios, sur de Chile.
Location decisions and regulatory changes: the case of the Chilean aquaculture
ABSTRACT. We study the development of aquaculture activities in Chile and the impacts of regulatory
changes on location decision for aquaculture production centers. Our study considers a descriptive analysis on
the spatial and temporal development of aquaculture production centers. Next, using a panel data we estimate a
site selection model to explore determinant factors of site choices for aquaculture production. Our results suggest
a clear pattern for the spatial-temporal development of Chilean aquaculture. The pattern is characterized by a
movement of the production centers towards the south of Chilean Patagonia, changing the concentration of the
production activities from Puerto Montt to the southern region of Chiloé Island. The estimation of a model of
site selection suggests that the distance between production centers and the presence of centers devoted to the
production of the same species are relevant in explaining the movement of the production activities towards the
southern region. The regulatory changes seem to be important determinant factors for the observed spatial and
temporal pattern of development of the aquaculture industry in the country.
Keywords: aquaculture, location, regulatory changes, southern Chile.
INTRODUCCIÓN
La acuicultura constituye una importante fuente de
producción de alimentos que contribuye a reducir la
presión sobre otros recursos. En el caso de Chile, la
__________________
Corresponding editor: Erich Rudolph
acuicultura se ha transformado en uno de los sectores
económicos de mayor dinamismo (Buschmann et al.,
2009; O’Ryan & Pereira, 2015). Esta industria puede
contribuir al desarrollo económico a través del
incremento sostenido de su producción, el desarrollo de
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2
mercados a nivel nacional e internacional, la generación
de nuevas oportunidades de empleos directos e
indirectos, así como también mediante otros efectos
positivos que se transmiten a las industrias relacionadas. Existe, no obstante, efectos no deseados como
subproductos de esta actividad económica, entre otros,
impactos negativos sobre el sistema ambiental y
natural, competencia por el uso del espacio geográfico,
y cambios sociales relacionados con fenómenos
migratorios (Engle, 2010; Phillips, 2010).
La actividad acuícola está basada en el uso intensivo
de recursos naturales: demanda de agua y sus
nutrientes, hace uso de espacio físico, que puede
competir con otras actividades productivas, utiliza
productos químicos y antibióticos para prevenir y
controlar enfermedades generando como subproducto
de la actividad, residuos, algunos de los cuales se
depositan en cuerpos receptores, principalmente en el
agua y el fondo del mar o lagos, alterando la calidad
ambiental de los sitios, pudiendo ser una fuente
potencial de impactos negativos sobre los seres
humanos y el medio natural.
La acuicultura en Chile incluye dos áreas principales
de producción: salmonicultura (otros peces), y mitilicultura. La salmonicultura es la actividad, organizada por
el hombre, destinada a la producción de salmones. La
producción se realiza a través de centros de cultivo que
intentan reproducir, al menos parcialmente, las
condiciones y desarrollo de salmón silvestre,
maximizando de paso los niveles de sobrevivencia,
reduciendo los periodos de crecimiento, realizando
manejo genético, etc. En los centros de cultivo se
intenta controlar el ciclo de vida de los peces y se
maneja su alimentación y condiciones de salud. La
mitilicultura se refiere a la actividad de cultivos de
moluscos bivalvos, principalmente choritos. Esta
actividad consiste en capturar y luego mantener grandes
cantidades de semillas de mitílidos hasta su crecimiento
apropiado para comercialización.
Es precisamente debido al uso intensivo de los
recursos naturales, y sus impactos asociados, que el
desarrollo de la actividad acuícola no ha estado exento
de controversias. Los impactos ambientales de la
acuicultura son diversos. La literatura existente sugiere
las siguientes áreas principales de impacto: 1) escape
de peces (caso salmonicultura), 2) deterioro de la
calidad del agua (uso de alimentos, productos químicos
y antibióticos, y descarga de nutrientes), y 3)
transmisión de enfermedades e infecciones. (e.g.,
Buschmann, 2001; Nordvarg & Johansson, 2002;
Gyllenhammar & Håkanson, 2005; Buschmann et al.,
2006; Mente et al., 2006; Pitta et al., 2006; Arismendi
et al., 2011; Sepúlveda et al., 2013). Los efectos de la
acuicultura en la calidad del agua, sus nutrientes y otros
ecosistemas, dependen tanto del nivel de la actividad
productiva, lo cual a su vez determina la cantidad de
descarga de nutrientes inorgánicos (nitrógeno y
fósforo), la tecnología de producción (densidad del
stock de peces), y características ambientales y físicas
de los sitios donde se localiza la producción en zonas
costeras (Buschmann et al., 2007). El uso de productos
químicos y antibióticos tiene efectos negativos sobre el
medio en que se desarrolla la actividad y podría
finalmente tener consecuencias sobre la salud humana
(Cabello, 2004). Entre los productos químicos y
antibióticos utilizados se incluyen: fungicidas,
colorantes, tetraciclina, ácido oxilínico, flumequina y
penicilina.
El presente trabajo tiene dos objetivos. En primer
lugar, realizar un análisis exploratorio de los patrones
de desarrollo espacio-temporal de la acuicultura en
Chile y los impactos de los cambios regulatorios sobre
las decisiones de localización de los centros de cultivo
acuícolas. El análisis se inicia con una descripción
general de las principales especies cultivadas y una
exploración respecto a la existencia de patrones de
ubicación entre especies y a través del tiempo. Luego,
se estudian los distintos indicadores de concentración
espacial y se analiza si los cambios en la regulación
pudieron haber influido en las decisiones de
localización de los centros en el transcurso del tiempo.
En segundo lugar, se estudian los determinantes de las
decisiones de localización de los centros de acuicultura
en Chile, utilizando información de centros acuícolas
concesionados a nivel de distritos comunales de la
región de Los Lagos, región de Aysén del General
Carlos Ibáñez del Campo (en adelante Aysén) y región
de Magallanes y de La Antártica Chilena (en adelante
Magallanes), se estima un modelo de elección de
localización para determinar cuáles son los factores
relevantes en la elección de la ubicación de un centro
de acuicultura. Las regiones de Los Lagos, Aysén y
Magallanes son tres de las 15 regiones en las cuales está
dividido el país política y administrativamente. Estas
regiones están ubicadas en el sur del país, zona también
conocida como Patagonia chilena.
Varios autores han estudiado previamente el
proceso de desarrollo de esta industria en Chile, aunque
considerando perspectivas diferentes. Por ejemplo,
Barton & Fløysand (2010) exploran, mediante un
análisis de economía política y ecología política, los
riesgos asociados al desarrollo de un sector exportador
no tradicional como la salmonicultura, enfatizando en
su análisis los conflictos entre stakeholders en el
contexto de procesos de globalización. Un análisis más
general respecto a los cambios en factores asociados a
la gobernabilidad y administración de recursos marinos
es provisto por Gelcich et al. (2010).
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En un trabajo reciente, Niklitschek et al. (2013)
revisan los posibles impactos asociados a la expansión
de la acuicultura hacia zonas de fiordos en la región de
Aysén considerando diferentes escenarios productivos.
El trabajo sugiere la importancia de generar conocimiento científico respecto de los potenciales impactos
sobre el medio ambiente de la Patagonia y propone
detener la entrega de concesiones para la actividad
acuícola mientras no se disponga de mayor conocimiento respecto a los impactos y riesgos asociados a la
actividad.
Ninguno de los esfuerzos de investigación
realizados hasta ahora ha examinado el proceso de
desarrollo espacio-temporal ni los factores determinantes de las decisiones de localización, incluidos el
marco regulatorio, en el caso de la industria acuícola
chilena. Cambios regulatorios orientados a reducir la
concentración geográfica de los centros acuícolas
fueron introducidos en Chile durante la segunda mitad
de la década pasada. Estos cambios habrían sido
motivados por la aparición del virus ISA a mediados
del año 2007. Como se ha sugerido en la literatura
existente, una empresa tendería a localizarse en lugares
donde pueda incrementar su beneficio económico,
desarrollar actividades productivas en cumplimiento de
las regulaciones, y/o donde el marco regulatorio sea
menos restrictivo (e.g., Abdalla et al., 1995; Rauscher,
1995; Levinson, 1996; Becker & Henderson, 2000; List
et al., 2003; Petrakis & Xepapadeas, 2003;
Brunnermeier & Levinson, 2004; Cabello, 2004;
Duvivier & Xiong, 2013). En el caso chileno, los
referidos cambios regulatorios podrían haber desencadenado un aumento en la dispersión de la localización
de las actividades de producción y un desplazamiento
no deseado de los centros de cultivo de peces.
cumplimiento involucra costos más bajos, y/o donde el
control regulatorio es menos exigente.
La decisión de localización de empresas y su
relación con el diseño de la regulación ambiental ha
recibido creciente atención en la literatura. Rauscher
(1995), y más recientemente Petrakis & Xepapadeas
(2003) muestran, en distintos contextos, que las
empresas podrían preferir ubicarse en zonas con
esquemas regulatorios más permisivos; incluso cuando
ya están instaladas, existiría la posibilidad que
posteriormente se muevan hacia zonas con regulaciones ambientales más débiles. Desde una perspectiva
empírica, Levinson (1996) utilizando datos de corte
transversal de un censo de manufactura en Estados
Unidos, encontró que diferencias en los niveles de
exigencia regulatorios no siempre afectan las
decisiones de localización de las empresas. No
obstante, Brunnermeier & Levinson (2004) realizaron
un análisis de las diferencias en las exigencias
regulatorias y discuten una serie de trabajos basados en
paneles de datos y otras técnicas econométricas que
controlan por problemas de endogeneidad. Tomado en
conjunto, estos estudios sugieren que el efecto de las
diferencias regulatorias entre zonas geográficas podría
ser importante para la decisión de localización de la
planta o empresas. Más recientemente en un contexto
de regulaciones ambientales descentralizadas, Duvivier
& Xiong (2013) presentan evidencias para China
respecto a que, debido a que la contaminación tiende a
cruzar las fronteras entre provincias, las empresas
tienden a localizarse preferentemente en condados
localizados en los límites provinciales. Esto sustenta la
idea de respuesta de las empresas en relación a su
localización dependiendo del contexto regulatorio que
enfrentan.
ANTECEDENTES
La regulación de la acuicultura en Chile
La autoridad reguladora de la actividad acuícola es la
Subsecretaría de Pesca y Acuicultura, dependiente del
Ministerio de Economía, Fomento y Turismo. Esta
Subsecretaría es responsable de la administración de
actividades pesqueras y acuícolas del país, así como
también de proponer normas y formular la política
pesquera nacional. El control y cumplimiento de las
regulaciones y normas que rigen la actividad pesquera
y acuícola es realizado por el Servicio Nacional de
Pesca (SERNAPESCA), organismo también dependiente del Ministerio de Economía. En el caso
específico de la acuicultura, existen también otros
organismos con atribuciones reguladoras y/o fiscalizadoras, con competencias sobre asuntos específicos
como aspectos laborales, localización y operación de
las concesiones en el territorio marítimo, etc.
Decisiones de localización y regulación ambiental
La hipótesis central para el análisis sobre la decisión de
localización de una empresa es que tal elección esté
guiada por el criterio de maximización de beneficios.
La decisión de localización puede afectar los beneficios
a través de su impacto en costos, por ejemplo, debido a
mayores y mejores oportunidades de acceso a insumos
necesarios para el proceso productivo, y a servicios
relacionados con características apropiadas, entre otros.
De igual modo, si la actividad productiva genera
impactos ambientales, los niveles de exigencia de las
regulaciones, así como el costo de cumplir con las
mismas podrían afectar también la decisión de
localización. Las empresas tenderían a localizarse en
regiones o zonas con regulaciones más laxas, y/o cuyo
4703
El marco legal general para el desarrollo de la
actividad pesquera y acuícola es provisto por la Ley
General de Pesca y Acuicultura de 1991 (LGPA) y sus
modificaciones. El texto refundido de la LGPA se
presenta en el Decreto Nº430 de 1992. Naturalmente,
existen otros textos legales y reglamentarios que son
también relevantes. Estos incluyen la Ley sobre Bases
Generales del Medio Ambiente (Ley Nº19.300 de
1994) y el cuerpo de reglamentos y resoluciones
particulares asociados a la LGPA.
Es interesante notar que aunque la LGPA contempló
regulaciones sobre la actividad acuícola, ésta era poco
significativa a nivel nacional durante la primera mitad
de la década de los 90’, periodo en que se aprobó este
cuerpo legal. No es sino hasta principios de la década
pasada que se aprueban los reglamentos básicos para el
funcionamiento de la acuicultura. Este marco regulatorio incluye el Reglamento Ambiental para la
Acuicultura (RAMA), Decreto Supremo (DS) Nº320 de
2001, y el Reglamento Sanitario (RESA) contenido en
el Decreto Supremo (DS) Nº319 del año 2002.
La acuicultura se desarrolló entonces, primeramente, bajo el marco legal provisto por la Ley y
posteriormente por reglamentos relacionados. En
contraste con la experiencia de otros países, la
regulación de la acuicultura en Chile surgió como
respuesta al desarrollo exhibido por la industria. Una
descripción breve de la experiencia regulatoria de la
industria salmonícola en Noruega, Escocia y Chile se
encuentra en Asche & Bjørndal (2011).
Una explicación para esta evolución más bien tardía
del marco regulatorio específico de la acuicultura
nacional es que las prioridades consideraron primeramente identificar las áreas susceptibles de ser asignadas
para concesión (áreas habilitadas). En este sentido, la
industria se expandió, hasta que se enfrentó a la
necesidad de generar nuevas reglamentaciones, como
las identificadas previamente.
El análisis de los distintos escenarios regulatorios
que han afectado el desarrollo de la actividad acuícola
en Chile, y en particular las decisiones de localización,
sugiere que son tres los hitos regulatorios importantes
observados durante el periodo de estudio. El primer hito
es la entrada en vigencia de la LGPA el año 1991. En
esos años se genera un marco regulatorio básico bajo el
cual se desarrollará la actividad en el futuro. A partir de
esta Ley, surgen dos reglamentos que tienden a generar
directrices conducentes a ordenar el sector; estos son:
Reglamento sobre Limitación de áreas de las
Concesiones y Autorizaciones de Acuicultura (D.S.
Nº550 de 1992) y Reglamento de Concesiones de
Acuicultura (D.S. N°290 de 1993).
El segundo hito ocurre el 2001, periodo en que se
modifica la Ley y se incluye un conjunto de normas que
tienden a ordenar al sector, dando origen a la denomi-
nada Nueva Ley de Pesca. Este nuevo marco legal
contempló varias modificaciones relevantes para la
acuicultura. Entre otras, es pertinente mencionar aquí la
modificación de los requisitos de sometimiento al
Sistema de Evaluación de Impacto Ambiental (SEIA).
Durante el 2001 se establece también el Reglamento
Ambiental de la Acuicultura (RAMA), que entre otras
cosas, reglamenta la distancia mínima por tipos de
centros para aquellos que se concesionen a partir de su
entrada en vigencia. Previo a la reglamentación
establecida en el RAMA, la Ley señalaba una distancia
mínima entre centros pero dejaba al reglamento el
establecimiento de las condiciones técnicas para dar
cumplimiento a esta limitación. El RAMA estableció
como se haría efectiva esta limitación y pone en
práctica esta norma. Un análisis de la regulación
ambiental que afectaba a la acuicultura chilena hasta el
2006 se puede revisar en Bermúdez (2007).
El tercer hito identificado como relevante para este
análisis ocurre el 2005. En este año se promulga la Ley
20.091 que introduce modificaciones a la LGPA en
materia de acuicultura. Se introduce la posibilidad de
que la concesión caduque, es decir, las concesiones
pierden la posibilidad de ser derechos perpetuos a todo
evento al incorporarse un mínimo de operación para
que tales derechos continúen vigentes. También se
incorporan otras medidas tendientes a evitar que
agentes que no desarrollen la actividad soliciten
concesiones con un fin especulativo; incluyendo, por
ejemplo, modificaciones al régimen de concesiones y
autorizaciones de acuicultura, además de la simplificación de trámites, y requerimiento de patente única de
acuicultura, entre otros. Posterior al 2005, la legislación
ha incorporado medidas principalmente sanitarias
gatilladas fundamentalmente por la irrupción del virus
ISA. Entre las medidas adoptadas se puede destacar la
zonificación del área donde se desarrolla mayormente
la actividad productora de salmónidos. Los barrios,
como se denominó a esta división, fueron establecidos
en la Resolución Nº450 del 2009 de SERNAPESCA.
Los barrios o agrupación de concesiones son zonas
donde existen concesiones de salmónidos, creadas con
un fin sanitario, permitiendo a la autoridad imponer
medidas que permitan contener la propagación del virus
ISA.
En el mismo año 2009 la Resolución Exenta Nº1449
estableció un número de peces máximo en etapa de
engorda dependiendo del tamaño en metros cúbicos de
la balsa jaula y de la especie cultivada. Además, dicho
decreto establece periodos de descanso anuales por
barrio, período en el cual los centros localizados en un
determinado barrio en periodo de descanso no podrán
ingresar peces para su cultivo.
Datos
En este estudio se utiliza una base de datos proporcionada por Subsecretaría de Pesca y Acuicultura de
Chile para los centros concesionados hasta agosto de
2012. Esta base de datos contiene información de la
especie para la cual la concesión fue autorizada, tamaño
y coordenadas geográficas del centro y año de
concesión según decreto del Servicio Nacional de Pesca
y según la Subsecretaria para las Fuerzas Armadas (Ex
Subsecretaria de Marina) del Ministerio de Defensa
Nacional del Gobierno de Chile. La Tabla 1 muestra un
resumen con el número de concesiones contempladas
en este estudio por región y tipo de organismos
autorizados para cultivar.
Análisis
El análisis inicial considera todo el territorio nacional;
sin embargo, se desarrolla con especial atención sobre
los centros localizados desde la región de Los Lagos
hacia el sur, dada la fuerte concentración de la actividad
en esa zona geográfica. Este análisis incluye un análisis
estadístico descriptivo de la evolución de las concesiones acuícolas a través del tiempo y en el espacio
geográfico.
El análisis formal de la evolución de la relación
espacial a través del tiempo se realizó utilizando el
índice I de Moran y el índice LISA. Se consideró la
zona geográfica comprendida entre la región de Los
Lagos y la región de Magallanes, porque en esa zona se
concentra la mayor cantidad de centros. Para realizar el
análisis se dividió el territorio utilizando como unidad
mínima territorial los distritos censales, que corresponden a una división geográfica de las comunas con
fines censales. El tamaño del distrito censal es asignado
en las zonas rurales de acuerdo a la población y número
de viviendas, y en el caso de las zonas rurales,
generalmente de acuerdo a la superficie. El tamaño de
los distritos es mayor para zonas no pobladas o
escasamente pobladas. De igual forma, estos distritos
muchas veces son divididos en zonas ubicadas en el
mar y tierra, existiendo la posibilidad que los distritos
ubicados en tierra contengan lagos y ríos donde se
pueda practicar la acuicultura.
Ello implica contar con un total de 335 distritos de
distinto tamaño para las regiones de Los Lagos, Aysén
y Magallanes. Los índices I de Moran y LISA se
calcularon para el número de centros acuícolas por
distrito. El índice I de Moran mide el grado de
asociación espacial en todo el territorio, valores
positivos indican que zonas con mayor número de
centros tienden a agruparse (acercarse) en el espacio,
valores negativos indican que zonas con alta cantidad
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5
de centros se alternan con zonas con pocos centros. El
índice LISA (I local de Moran) permite analizar en el
espacio, los clúster o distintos grupos de centros concesionados identificados. Luego de calcular el índice de
LISA se utiliza un mapa que permite representar los
distintos grupos de acuerdo a los valores del índice.
El análisis respecto a la evolución del uso del
espacio a través del tiempo incluye también el estudio
de la relación entre la distancia entre centros concesionados y tamaño de los centros. El análisis de estas
variables se realizó utilizando estadística descriptiva.
De manera análoga al análisis de concentración
espacial, el análisis econométrico respecto a los
factores determinantes de las decisiones de localización
utiliza información de los centros ubicados desde la
región de Los Lagos hacia el sur. Los enfoques tomados
en la literatura aplicada para estudiar la elección de un
sitio con fines productivos y, en especial, el efecto de
regulaciones sobre la elección de localización de una
empresa son variados (e.g., Guimarães et al., 2003).
Para explicar la relación entre el número de centros
observados y la zona donde se ubican, se consideró una
metodología similar a la presentada en Duvivier &
Xiong (2013), con la variante que en este caso se
consideró un modelo Poisson para panel, a diferencia
de los autores que utilizaron un modelo ZIP (Zero
Inflate Poisson). El modelo Poisson es un modelo
estadístico utilizado cuando la variable dependiente
representa el número de veces que ocurre un evento. El
modelo ZIP es un modelo Poisson con un ajuste para
corregir el sesgo generado cuando la variable
dependiente contiene muchos valores cero (ver detalles
en Green, 2003).
El número de centros existentes en un distrito censal
estaría determinado tanto por variables asociadas a
características de los distritos, como también por
características de los centros. En este caso el modelo
Poisson panel realiza un seguimiento del número de
centros por localidad a través del tiempo, permitiendo
evaluar cómo el número de centros por distrito
reacciona a las características de los centros existentes
y de los distritos.
Para el análisis descrito se cuenta el número total de
centros existentes en cada distrito separado por tipo de
organismos cultivados. Además, se rescatan características de los centros que existían en el año anterior
para evaluar cómo influyen estas características en la
elección del distrito para la localización de centros. De
igual modo, la especificación del modelo considera
características geográficas de los distritos. También se
incluyen otras variables de control, tales como zona
geográfica donde está el centro, variables que intentan
capturar las condiciones económicas imperantes en el
momento que se concesionó el centro, las variables tem-
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6
Tabla 1. Número de centros concesionados por región y tipo de organismos cultivados entre 1979 y 2012. S: salmónidos,
RB: recursos bentónicos, OP: otros peces, RB-S: recursos bentónicos y salmónidos, RB.OP: recursos bentónicos y otros
peces, O: otros cultivos.
Región
Arica y Parinacota
Tarapacá
Antofagasta
Atacama
Coquimbo
Valparaíso
Biobío
La Araucanía
Los Ríos
Los Lagos
Aysén
Magallanes
Nº total de centros por
organismos cultivados
Tipo de organismos cultivados
S
2
10
515
685
64
1276
RB
10
17
10
82
67
1
11
13
14
1554
4
OP
1783
2
porales que identifican los hitos regulatorios y variables
cruzadas para evaluar las posibles interacciones entre
los determinantes más relevantes. La Tabla 2 resume
las variables utilizadas en el modelo estimado.
Se estimaron dos regresiones para el número de
centros existentes en un distrito en un año determinado.
La primera estimación corresponde al número de
centros de salmónidos en el distrito y la segunda
considera el número de centros de moluscos en el
distrito.
El análisis está centrado en tres aspectos claves a
evaluar: a) efecto que ha tenido la regulación respecto
a la distancia entre centros sobre la localización de los
mismos. Con tal propósito, se usó en la estimación la
distancia media entre centros de aquellos centros
ubicados en un distrito determinado (variable LDmcc);
b) efecto del tamaño promedio de los centros en el
periodo anterior sobre el número de centros por distrito
en un periodo determinado (Lareacentk2). En este
sentido, el incluir el tamaño medio de los centros
permite separar el efecto del tamaño del centro del
efecto de la distancia a la que se ubican los centros. El
tamaño del centro tiene relación con la distancia a la
que se pueden ubicar los centros puesto que influye en
la disponibilidad de espacio para albergar más centros
en el distrito; c) efecto del alejamiento de centros
urbanos sobre el número de centros por distrito. El
alejamiento de centros urbanos y el movimiento hacia
el sur impone costos que las empresas deben asumir.
Para medir este efecto se usaron cuatro variables:
distancia media de los centros del distrito al puerto más
cercano (distkpuert), distancia media de los centros a
RB-S
RB-OP
O
1
1
1
1
1
34
1
1
1
37
1
2
Nº total de
centros por región
10
18
10
83
70
1
11
14
25
2104
690
65
3101
las capitales provinciales (diskcaprov), distancia del
distrito a las capitales provinciales (ddistcappr) y
distancia del distrito a Puerto Montt (ddistptomo). Las
dos primeras variables capturan el efecto de la
ubicación de los centros y la tercera variable
(ddistcappr) separa el efecto de la ubicación de los
centros del efecto de la ubicación del distrito. La cuarta
variable captura el efecto de la lejanía del distrito
respecto a Puerto Montt, por mucho tiempo el principal
centro urbano, de procesamiento de los productos
acuícolas.
RESULTADOS
Patrones de localización de la actividad acuícola en
Chile y efectos regulatorios
La evolución temporal de los centros concesionados
sugiere la presencia de periodos de fuerte expansión de
las concesiones acuícolas. Por ejemplo, se observó una
fuerte expansión en centros concesionados durante la
segunda mitad de los 90’, y luego entre 2002 y 2005.
La expansión de concesiones de centros acuícolas se
mantiene incluso durante el periodo de la crisis del
2007-2008, aunque a un ritmo significativamente
inferior al observado en periodos previos.
La Figura 1 indica la evolución de las concesiones
otorgadas para los dos principales cultivos. Se aprecia
un crecimiento sostenido en la autorización de los
centros con periodos de fuerte dinamismo e
incrementos de concesiones y otros con bajas, donde
el número de concesiones se redujo sustancialmente
respecto al año anterior. Dos hitos a notar son el incre-
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7
Tabla 2. Variables utilizadas en la estimación del modelo econométrico.
Tipo
Características de los
centros
Características geográficas
de los distritos
Hitos regulatorios
Otras variables de control
Variable
LDmcc
Lareacenk2
distkpuert
diskcaprov
ddistptomo
ddistcappr
areak2
y_92to01
y_02to05
y_06to12
psalmon
trend
Lsalmonido
region11
region12
Variables de interacción
X#Y
Descripción
Media de distancia al centro más cercano que tenían el año anterior los
centros ubicados al interior del distrito.
El tamaño medio de los centros en kilómetros cuadrados en el año
anterior.
La distancia media de los centros al puerto o muelle más cercano.
La distancia media de los centros a la capital provincial.
Distancia del centro representativo del distrito (punto central
equidistante de los límites del distrito) a Puerto Montt.
Distancia del centro representativo del distrito a la capital provincial.
km cuadrados del distrito.
Variable que toma valor 1 entre los años 1992 a 2001, cero en otro caso.
Promedio anual del precio del salmón en dólares por kg.
Variable de tendencia. 0 el primer año y crece 1 unidad cada año.
El porcentaje sobre el total de centros que eran centros de salmónidos el
año anterior.
Variable que toma valor 1 para los distritos ubicados en la región de
Aysén, cero en otro caso.
Variable que toma valor 1 para los distritos ubicados en la región de
Magallanes, cero en otro caso.
Es una variable que corresponde a la multiplicación entre la variable X
y la variable Y. X e Y corresponden a las variables descritas previamente.
Figura 1. Relación entre el número de centros con autorización de concesión y el año en que se concesionaron por grupo
de especies. Las líneas verticales muestran los hitos regulatorios identificados previamente en el texto central.
mento de las concesiones previo a la entrada en
vigencia del RAMA, años 2000 al 2003, y la posterior
caída de las concesiones entre 2006 y 2008, periodo
donde se gestó la crisis del salmón producto del virus
ISA.
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8
La mayor parte de los centros corresponden a
recursos bentónicos y salmónidos localizados en el
mar. Se observa una cantidad importante de centros
concesionados en ríos o esteros en el caso de recursos
bentónicos. Este ha sido un aspecto controversial y que
ha motivado modificaciones para evitar que lagos y ríos
de poco caudal sean utilizados para la acuicultura. La
primera modificación de la LGPA que tuvo este fin fue
introducida en las modificaciones a la Ley introducidas
el 2005. Sin embargo, sigue siendo un tema vigente
puesto que estos cuerpos de agua, ríos y lagos, son
lugares con alto potencial turístico, actividad que
compite con la acuicultura por el uso del espacio
geográfico (Tabla 3).
La acuicultura se desarrolla principalmente en las
regiones de Los Lagos, Aysén y Magallanes, sin
embargo desde su inicio, se concentra fuertemente en
la región de Los Lagos. Durante parte del periodo
considerado en el estudio, la región de Los Lagos
incluía cinco provincias. A partir del año 2007 una de
las provincias de esta región (Valdivia) se convirtió en
la región de Los Ríos. Para efectos del estudio, la región
de Los Lagos incluye las provincias de Osorno,
Llanquihue, Chiloé y Palena. De los 3.101 centros con
concesión en el periodo estudiado, 2.104 se localizan
en Los Lagos, mientras que 690 están localizados en
Aysén. Sin embargo, mientras las concesiones en la
región de Los Lagos corresponden tanto a recursos
bentónicos como a salmónidos, aquellas ubicadas en la
región de Aysén corresponden casi exclusivamente a
salmónidos. En este sentido la industria acuícolabentónica está concentrada fuertemente en Los Lagos.
Es importante notar también que mientras las
concesiones para salmónidos se han autorizado casi
exclusivamente entre Los Ríos y Magallanes, las
concesiones para producción de recursos bentónicos,
aunque concentradas en Los Lagos, se encuentran
también en el resto del territorio, incluyendo la zona
norte del país.
La alta concentración de la actividad acuícola se
reproduce también al interior de estas divisiones
político-administrativas a nivel de territorio comunal
(Fig. 2). El patrón de desarrollo territorial se caracteriza
por el surgimiento de enclaves geográficos, con alta
concentración de centros en zonas geográficas
específicas. Los Lagos presenta centros dedicados al
cultivo de distintos organismos interactuando en el
mismo espacio geográfico, ubicándose en la costa
interior desde Puerto Montt hasta más allá de Quellón,
frente al Parque Nacional Corcovado. Un análisis más
detallado de los centros sugiere que, si bien los centros
se ubican a más de 1 km de distancia entre ellos, las
concesiones cubren gran parte de las zonas frente a la
costa, (Fig. 2). Respecto a la región de Aysén, por
características propias de la zona, los centros muchas
veces rodean completamente las islas frente al territorio
continental.
Esta exploración respecto del desarrollo espaciotemporal de la acuicultura, incluye también un análisis
descriptivo del desarrollo de concesiones por regiones
a través del tiempo. El cambio en la localización de las
concesiones para producción de salmónidos es
diferente entre las décadas de los 90’ y 2000’. Mientras
que durante las dos primeras décadas del desarrollo
acuícola, la actividad acuícola de salmónidos se
concentraba en Los Lagos, durante la última década la
actividad inicia una decidida expansión hacia el sur de
la Patagonia. Es importante notar que hasta ahora, las
concesiones acuícolas en la zona patagónica tienden a
concentrarse en Aysén, pero continúan expandiéndose
hacia Magallanes (Tabla 4).
El cambio en la localización de los centros de
acuicultura es más claro observando la ubicación de los
centros en Los Lagos y Aysén (Fig. 3).
Tabla 3. Número de centros concesionados por grupo de organismos cultivados y tipo de cuerpos de agua donde se
encuentran ubicados (1979-2012).
Tipo de organismos
Tipo de cuerpos de agua
Salmónidos
Recursos bentónicos
Otros peces
Salmónidos y R. bentónicos
R. bentónicos y otros peces
Otros cultivos
Mar Lago o laguna
1182
43
1374
2
2
32
1
1
2
Total por cuerpo de agua
2593
46
Río o estero
51
407
4
462
Total
1276
1783
2
37
1
2
3101
7089
Figura 2. Mapa de centros concesionados en las regiones de a) Los Lagos y b) Aysén. Los puntos simbolizan la ubicación
del centro y el color corresponde al tipo de organismos cultivados.
En la Figura 3 se observa que los centros autorizados antes de 1990 (puntos amarillos), estaban
localizados principalmente en la región de Los Lagos y
paulatinamente, los centros comenzaron a poblar zonas
ubicadas al sur de Puerto Montt, para finalmente
comenzar a poblar las zonas ubicadas en la región de
Aysén. En Aysén también se puede distinguir que este
movimiento ha sido de este a oeste, partiendo mayoritariamente en zonas cercanas a los asentamientos urbanos
del este hacia las islas ubicadas frente a Puerto Aysén.
La evidencia sugiere que en ambas regiones, al
principio se fueron ubicando cerca de asentamientos
urbanos y a medida que la industria creció, se fueron
considerando zonas más alejadas en busca de
condiciones apropiadas para el cultivo.
Los resultados de este análisis para el tamaño
promedio de centros con autorización de concesión se
indican en la Tabla 5, donde las concesiones muestran
variaciones en su tamaño a través del tiempo. En
principio, durante los 80’, la concesión promedio tenía
un tamaño de 4 a 140 ha. Después de los 90’ el tamaño
medio pasó de 4 y 16 ha. De igual modo, existen
diferencias en el tamaño de las concesiones entre los
distintos tipos de organismos. Generalmente, las
concesiones de salmónidos tienden a ser más grandes
que las de recursos bentónicos, no obstante, cuando
dichas diferencias existen, no son sustancialmente
grandes.
El cálculo del tamaño promedio de los centros
autorizados para cada región muestra que, en general,
los centros son más pequeños a medida que se ubican
en zonas localizadas más al sur. La Tabla 6 indica el
promedio de centros con autorización de concesión por
tipo de organismos, según región. En ésta se nota la
existencia de un patrón espacial en el tamaño de los
centros que es similar entre tipo de organismos.
Mientras más al sur se encuentre el centro, menor es el
tamaño promedio del mismo. Específicamente, se
detecta que en el caso de los centros de salmónidos, los
centros pasan de un tamaño medio de 14 ha en las
regiones de Los Lagos y Los Ríos, a solo 7 ha en la
región de Magallanes. Esto indica una disminución en
el tamaño medio de los centros ubicados hacia el sur de
la Patagonia. Es importante notar que no necesariamente a menor tamaño del centro es menor la
producción que genera, esto porque la jaula puede tener
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Tabla 4. Número de centros concesionados por región para el periodo 1979-2012. AP: Arica y Parinacota, TAR: Tarapacá,
ANT: Antofagasta, ATA: Atacama, CO: Coquimbo, VAL: Valparaíso, BIO: Biobío, AR: La Araucanía, RIO: Los Ríos,
LA: Los Lagos, AYS: Aysén, MA: Magallanes.
Año de la
concesión
<1990
1991-1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Total de centros
por región
LA
112
142
56
94
69
95
277
125
79
129
136
103
236
146
83
56
28
77
51
10
AYS
5
11
29
12
8
9
50
56
4
51
73
107
66
24
24
22
18
67
33
21
MA
1
7
3
2
4
Total de
centros del
período
156
197
108
121
83
116
341
198
92
191
229
228
316
175
114
87
63
155
96
35
2104
690
65
3101
Región de concesión
AP
TAR
3
ANT
4
1
3
2
1
1
1
2
1
2
3
2
2
1
1
3
10
18
3
2
10
ATA
10
11
6
3
1
7
1
6
2
3
5
7
5
2
4
5
2
3
83
CO
14
11
3
8
3
3
8
4
6
VAL BIO
1
1
2
1
1
2
2
2
mayor profundidad. No se cuenta con información
adicional para identificar cual es la causa de la
disminución del tamaño del centro, pero es razonable
suponer que debiera existir alguna relación entre el
tamaño del centro y la producción, más aún cuando la
tecnología de producción (jaulas) es similar entre
centros (Tabla 7).
Como se mencionó previamente, la distancia entre
centros está explícitamente regulada en el RAMA. Para
analizar la evolución de la distancia entre centros, se
calculó la distancia mínima de un centro al centro más
cercano. Luego se obtuvo el promedio de dicha
distancia para los centros ubicados en una zona
geográfica específica.
La Tabla 7 indica que, en términos generales y
también según región, la distancia promedio al vecino
más cercano entre centros autorizados ha disminuido a
través del tiempo, estabilizándose a partir de 2005. Es
notable la diferencia existente entre regiones donde
claramente se destaca la distancia media a la cual se
ubican los centros en la región de Magallanes, que
alcanza a un poco más de 5 km.
En general, se observa que los centros ubicados en
ríos y en el mar están en promedio más cerca que los
RIO
5
10
4
2
1
1
1
2
1
1
1
2
1
70
3
AR
3
3
4
1
1
1
1
1
11
14
25
5
6
1
2
3
1
6
14
6
4
ubicados en lagos. Al analizar la evolución de la
distancia por región, se observa también que después de
2001 casi no ha variado la distancia media a la que se
ubicaron los centros.
Al diferenciar por tipo de organismos cultivados y
el cuerpo de agua, los resultados no cambian (Anexo 1).
En general, los centros se han tendido a concentrar; sin
embargo, existen diferencias por organismos cultivados
y según región donde se localizan los centros. Este
análisis se basa en la distancia entre todos los centros
en un año específico. No obstante, es relevante
preguntarse qué sucede cuando se considera la decisión
de las empresas al analizar la distancia a la que se
ubican sólo los nuevos centros, dejando de lado los
centros existentes. De los resultados se obtiene que los
centros concesionados después de 2005 se han ubicado
a mayor distancia que sus predecesores.
En relación con el análisis de correlación espacial a
través del tiempo, la Tabla 8 muestra el índice I de
Moran y los estadígrafos asociados al índice. Estos resultados sugieren que para los cuatro años analizados
ha existido correlación espacial leve positiva y
significativa. Los centros que existían en 1991 estaban
más concentrados que los de años posteriores, pero
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11
Figura 3. Mapa de centros concesionados en la región de a) Los Lagos y b) Aysén. Los puntos simbolizan la localización
del centro donde el color representa el año en que fue concesionado.
también eran mucho menos, y estaban agrupados y
distribuidos en la región de Los Lagos.
En 2001 el índice cae a 0,029. En este periodo ya se
habían concesionado muchos más centros y se ubicaron
más al sur. En 2005 y 2012 el índice aumentó,
duplicándose el 2005 y subió levemente el 2012. El
índice LISA y el gráfico de sus valores permiten revisar
cómo se han desplazado los centros en el tiempo. Los
resultados del índice LISA se muestran en la Figura 4
para los cuatro años de interés.
Para cada año se calculó el índice I de Moran con
los centros existentes y se graficaron sus resultados
(Tabla 8 y Fig. 4). Como se observa, en 1991 los centros
se concentraban alrededor de Puerto Montt y en Chiloé.
Sin embargo, también se presentan agrupamientos de
centros más al sur. En 2001 las zonas donde se agrupan
los centros se desplazan hacia la región de Aysén. En
ese año ya se aprecia una fuerte concentración de
centros al sur de la isla de Chiloé. En 2005 el
agrupamiento de los centros se había intensificado
expandiéndose a los distritos aledaños al sur de la isla
de Chiloé y a zonas ubicadas hacia el este. La Figura 4
para el periodo más reciente (año 2012) muestra la
intensificación de la concentración de centros en la
zona antes señalada.
Resultados del análisis econométrico de factores
determinantes de la decisión de localización
A continuación se presentan los resultados de las
estimaciones econométricas respecto de los factores
determinantes de la decisión de localización (Tabla 9).
Una lectura correcta de los resultados requiere notar
que las variables de interacción muestran el cambio en
el efecto de una variable relacionada a la variable que
la multiplica con relación también a la situación base.
Por ejemplo, el coeficiente asociado a la variable
distancia media entre los centros del periodo anterior
(LDmcc) corresponde al efecto de LDmcc, después,
LDmcc#y_92to01 muestran el efecto adicional por
sobre el de LDmcc para el periodo 1992-2001 con respecto a la situación base, correspondiente al periodo
1980-1991. De esta forma se puede revisar si el efecto
de LDmcc es distinto en el periodo 1992-2001. El
mismo cambio es obtenido por la multiplicación de las
variables Region11 y Region12 que miden la diferencia
en el parámetro que multiplica a esta variable para los
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12
Tabla 5. Tamaño promedio anual (ha) de centros por año de concesión y tipo de organismos cultivados. El valor
corresponde al promedio de todos los centros concesionados en un año para los organismos indicados. S: salmónidos, RB:
recursos bentónicos, OP: otros peces, RB-S: recursos bentónicos y salmónidos, RB-OP: recursos bentónicos y otros peces,
O: otros cultivos.
Año de concesión
1979
1980
1981
1982
1983
1984
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Tamaño medio
del período
S (ha)
RB (ha)
4,0
136,8
OP (ha)
RB-S (ha)
RB-OP (ha)
O (ha)
35,6
3,9
4,2
9,1
4,7
11,7
42,6
22,5
18,3
25,9
6,2
8,3
12,6
13,5
7,9
12,4
13,1
16,9
11,0
8,5
7,4
7,2
9,7
8,1
8,7
7,0
9,0
9,0
9,0
11,0
10,7
6,8
6,6
13,1
13,4
10,5
55,2
9,0
20,7
24,6
19,2
3,0
10,6
8,5
14,9
7,2
3,7
5,2
8,1
5,5
8,4
8,6
12,5
13,4
10,9
19,0
12,0
16,1
15,5
8,0
10,4
10,2
6,8
13,8
9,7
2,8
8,8
11,1
41,6
3,3
10,8
49,6
0,8
6,0
24,5
3,5
5,0
1,6
3,2
5,1
1,0
15,3
18,0
10,0
17,2
centros ubicados en la región de Aysén y/o región de
Magallanes con respecto a la situación base que, en ese
caso, es la región de Los Lagos.
De los resultados de los modelos presentados para
los centros de salmónidos y de moluscos, se observa
que el número de centros existentes en un distrito se
14,4
6,8
0,9
Tamaño medio anual de
las concesiones (ha)
4,0
136,8
35,6
3,9
7,3
4,7
7,8
23,0
19,0
14,9
34,6
7,7
13,5
15,4
17,8
5,8
11,7
10,4
16,0
8,4
4,6
6,0
7,9
7,0
8,2
8,6
10,8
12,1
10,5
16,0
11,4
12,9
11,8
6,9
10,3
relaciona negativamente con la distancia media que
existía el año anterior entre los centros del distrito
(LDmcc). Este efecto tiene una mayor magnitud desde
1992 en adelante, donde el mayor efecto estimado se
determinó durante el periodo 1992-2001. Este resultado
sugiere que la legislación influyó sobre la distancia que
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Tabla 6. Tamaño promedio anual de centros concesionados por región y tipo de organismos cultivados. El valor
corresponde al tamaño promedio de todos los centros de la región donde fue concesionado para el tipo de organismos
indicados. S: salmónidos, RB: recursos bentónicos, OP: otros peces, RB-S: recursos bentónicos y salmónidos, RB-OP:
recursos bentónicos y otros peces, O: otros cultivos.
Región
Arica y Parinacota
Tarapacá
Antofagasta
Atacama
Coquimbo
Valparaíso
Biobío
La Araucanía
Los Ríos
Los Lagos
Aysén
Magallanes
Tamaño promedio
por tipo de organismos
S (ha)
50,5
14,1
13,6
8,2
6,6
10,4
RB (ha)
14,7
37,6
29,3
20,9
40,3
38,9
5,4
1,6
2,2
8,1
2,4
OP (ha)
10,2
17,2
RB-S (ha)
RB-OP (ha)
O (ha)
Tamaño promedio
de la región (ha)
24,5
10,0
6,8
1,6
49,6
13,6
1,0
0,8
20,1
14,4
6,8
0,9
14,7
36,9
29,3
20,7
40,1
38,9
5,4
1,6
8,9
9,5
8,1
6,8
10,3
Tabla 7. Distancia promedio en km entre centros autorizados por cuerpo de agua y región, en 1991, 2001, 2005 y 2012. La
distancia está definida como la distancia mínima desde el punto ubicado justo en el centro de la concesión (centroide) hasta
el homónimo de la concesión más cercana.
Año
1991
2001
2005
2012
Región
Mar
Lago o laguna
Los Ríos
Los Lagos
2,9
Aysén
10,7
Magallanes 696,0
Promedio año
8,4
Los Ríos
Los Lagos
1,2
Aysén
3,3
Magallanes
18,2
Promedio año
1,9
Los Ríos
Los Lagos
0,9
Aysén
3,0
Magallanes
5,8
Promedio año
1,6
Los Ríos
Los Lagos
0,9
Aysén
2,8
Magallanes
5,0
Promedio año
1,5
separaba los centros propiciando que el número de
centros por distrito sea menor mientras mayor sea la
distancia media entre los centros. Este efecto difiere en
términos de magnitud para cada hito analizado
Río o estero
Promedio región
4,8
5,1
6,0
3,5
5,1
4,8
4,0
4,0
1,7
0,3
3,3
4,1
4,8
3,9
0,4
1,5
0,3
3,3
3,9
4,8
3,8
0,4
1,5
0,3
3,3
3,9
0,3
5,3
3,3
10,7
696,0
7,5
2,2
1,0
3,3
18,2
1,6
2,0
0,8
3,0
5,8
1,4
2,0
0,8
2,8
5,0
1,4
(Parámetros LDmcc#y_92to01, LDmcc#y_02to05 y
LDmcc#y_06to12). Sin embargo, un test estadístico
sobre la igualdad de los parámetros para los distintos
periodos regulatorios, muestra que no hay diferencias
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Tabla 8. I de Moran para los centros ubicados de la región
de Los Lagos al sur, en 1991, 2001, 2005 y 2012. E[I]:
valor esperado de I bajo la hipótesis nula de ausencia de
autocorrelación espacial, V: varianza estimada de I, z:
estadígrafo de prueba bajo la hipótesis nula de no
autocorrelación, P: valor P asociado a la hipótesis nula).
Indicador
I de Moran
E[I]
V
z
P
1991
2001
2005
2012
0,155
-0,003
0,000
13,990
0,000
0,029
-0,003
0,000
4,182
0,000
0,044
-0,003
0,000
4,711
0,000
0,049
-0,003
0,000
5,028
0,000
entre el parámetro de LDmcc#y_92to01 y el de
LDmcc#y_02to05. Por ejemplo, a la misma distancia
media entre los centros, un distrito tendrá menos
centros antes de 1991 que después de 1991, controlado
por las características del distrito y las otras variables
incluidas en las estimaciones. En términos espaciales,
el resultado indica que el efecto de la distancia entre los
centros es menor para los centros ubicados en la región
de Magallanes.
El tamaño medio de los centros no influye en el
número de centros de salmónidos del distrito hasta
1991 (Lareacenk2). Luego de este periodo, el signo
negativo de la variables Lareacenk2#y_92to01,
Lareacenk2#y_02to05 y Lareacenk2#y_06to12, sugiere
que el tamaño medio de los centros del distrito influye
negativamente en los centros existentes en la zona. Para
el caso de los centros de moluscos el efecto del tamaño
del centro tiene una dirección contraria al caso de los
salmónidos. El tamaño de los centros que prevalecía el
año anterior (Lareacenk2) se relaciona positivamente
con el número de centros del año en curso, siendo
mayor el efecto durante el periodo 2006-2012
(Lareacenk2#y_06to12).
La distancia de los centros a puertos o a la capital
provincial del distrito (distkpuert o diskcaprov,
respectivamente) indica dos efectos que no pueden ser
separados en el modelo. Si se piensa en término de
costos, menor distancia a estos lugares implica
reducción en costos de trasporte para abastecimiento,
mejor posibilidad de reclutar mano de obra y otras
economías producto de la aglomeración. Sin embargo,
otros factores podrían ser importantes para alejarse de
centros urbanos como por ejemplo condiciones
ambientales. Generalmente, zonas con mejores cualidades ambientales no se encuentran cerca de los centros
urbanos. Por lo tanto, el signo negativo de estas
variables implica que es más importante ubicarse cerca
de centros urbanos y el signo positivo implica que otros
factores son más importantes. La Tabla 9 muestra que
el signo de ambas variables es positivo tanto para
salmónidos como moluscos.
Los modelos también permiten revisar que ocurre
en términos espaciales y temporales con la evolución
de los centros. Como regla general se observa que
distritos más distanciados de Puerto Montt o de las
capitales provinciales tienen menos centros (notar el
signo negativo de las variables ddistptomo, ddistcappr);
en el espacio la distancia respecto a Puerto Montt
significa estar ubicado más al sur y la distancia respecto
a las capitales provinciales cercanas implica estar
ubicado más al este. También al comparar la magnitud
de los parámetros asociados a region11 (Región de
Aysén) y region12 (Región de Magallanes) se nota que,
en el caso de los centros de moluscos, los distritos
ubicados más al sur debieran presentar menor número
de centros dado que los parámetros de región12 son
mayores en valor absoluto.
Las variables y_92to01, y_02to05, y_06to12 y tred
muestran lo que es natural en la evolución de los
centros, a medida que ha ido pasando el tiempo los
distritos tiene mayor número de centros. Finalmente,
Lsolmonido, variable que mide el porcentaje de centros
de salmónidos del periodo anterior, puede ser
considerada una media del atractivo a formar
conglomerados entre productores del mismo tipo de
organismos, dado que su signo positivo para el número
de centros de salmónidos (y el signo negativo en
Moluscos), implica que los centros de este tipo tienden
a concentrarse en distritos donde ya existe este tipo de
centros.
DISCUSIÓN
En este estudio se ha realizado un análisis descriptivo
del desarrollo espacio-temporal de la actividad acuícola
en Chile, con particular atención a los centros
concesionados, considerado también un análisis de
correlación sobre la base de un panel de datos, a través
del cual se estimó un modelo de elección de sitios para
explorar los factores determinantes de la decisión de
localización de los centros acuícolas.
Del análisis realizado se concluye la existencia de
incrementos en las concesiones en periodos cercanos a
los hitos regulatorios identificados. Desde la región de
Los Lagos hacia el sur, el establecimiento de las nuevas
concesiones exhibe un patrón de uso del territorio que
se caracteriza por patrones de concesiones que se
desplazan de norte a sur y de este a oeste. Los centros
concesionados tendieron a ubicarse primeramente en
lugares cercanos a asentamientos urbanos, para posteriormente expandirse hacia zonas más aisladas. Existe
evidencia estadística que apunta a que los centros se
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Figura 4. Representación de Índices LISA para los años 1991, 2001, 2005 y 2012. Los distritos se clasificaron según su
significancia estadística en: distritos no significantes, distritos significantes de valores altos (HH), distritos significantes de
valores bajos (LL), distritos significantes de valor atípico donde un distrito de valor alto está rodeado principalmente por
distritos de valores bajos (HL) y distritos de valor atípico donde un distrito de valor bajo está rodeado principalmente por
distrito de valores altos (LH).
tienden a concentrar en el espacio. Más aun, cuando se
considera la manera que los centros se concentran en
distintos momentos del tiempo, se observa que los
centros pasaron de concentrarse en torno a Puerto
Montt y la zona este de la isla de Chiloé, a poblar y
agruparse en zonas al sur de la isla de Chiloé.
En principio no se observa un patrón claro respecto
al tamaño de los centros. Sin embargo, después de los
90’ el tamaño promedio del centro se va estabilizando
en torno a las 10 ha. La distancia media entre los centros
disminuyó a través del tiempo, estabilizándose a partir
del año 2005 en torno a 2 km.
Las estimaciones econométricas muestran que
existe una relación inversa entre la distancia de los
centros y el número de centros del distrito en el periodo,
siendo mayor el efecto después de 1991. Este resultado
sugiere que la legislación influyó en cómo se
distribuyeron los centros, propiciando la dispersión
espacial de los centros de cultivo. Este efecto parece ser
más importante entre 1992 y 2005. Las estimaciones
muestran que, en el caso de salmónidos, a mayor
distancia de un distrito respecto de la capital provincial
se incrementa el número de centros. De igual forma, las
concesiones de salmónidos tienden a ubicarse en zonas
donde existen otros centros de estas especies,
concentrándose en zonas geográficas reducidas. Esto
último corrobora lo expuesto por Perlman & JuárezRubio (2010), quienes señalan que existen otros
factores, tales como economías de escala y encadenamientos productivos, que favorece la concentración de
los centros en zonas geográficas reducidas. Entonces,
el modelo corrobora los resultados preliminares
obtenidos a partir del análisis descriptivo, puesto que la
mayor distancia entre los centros genera un menor
número de centros en un distrito dado. Sin embargo,
esto está estrechamente relacionado a que una mayor
distancia y tamaño puede ser alcanzado solo en distritos
con mayor tamaño. Precisamente, estos distritos están
ubicados en zonas más australes, hacia donde debiera
tender a desplazarse la actividad acuícola si se impone
una mayor distancia entre los centros.
La evidencia sugiere entonces que el número de
centros por distrito se reduciría al incrementar la
distancia entre los centros. En este sentido la dirección
que han tomado los cambios regulatorios respecto a
estos dos parámetros (número de centros en una zona
geográfica y distancia entre ellos), podría ser una de las
causas que promovió la expansión de la acuicultura hacia
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Tabla 9. Estimaciones del modelo Poisson panel para el
número de centros de moluscos y centros de salmones por
distrito. *P < 0,1; **P < 0,05; ***P < 0,01.
Variable
Constante
LDmcc
LDmcc # y_92to01
LDmcc # y_02to05
LDmcc # y_06to12
LDmcc# region11
LDmcc# region12
LDmcc#areak2
Lareacenk2
Lareacenk2#y_92to01
Lareacenk2#y_02to05
Lareacenk2#y_06to12
Lareacenk2#region11
Lareacenk2#region12
areak2
areak2#region11
areak2#region12
distkpuert
diskcaprov
ddistptomo
ddistcappr
y_92to01
y_02to05
y_06to12
psalmon
trend
Lsalmonido
region11
region12
Constante Ln(alpha)
Salmónidos
Moluscos
-2.0747***
-0.0155**
-0.0640***
-0.0624***
-0.0512***
0.0084
0.0455***
0.0000009***
-0.2018
-0.4540*
-0.7546**
-1.1497***
5.0681***
-5.6941***
0.0003**
-0.0002
-0.0003*
0.0261***
0.0534***
-0.0230***
-0.0374***
0.3485***
0.4747***
0.5098***
-0.0177
0.0530***
0.0091***
0.0707
-2.7295
0.4448***
-2.6248***
0.0048
-0.2817***
-0.2912***
-0.2872***
0.0046
0.2844***
0.00000008
1.7456***
0.3176
0.9660
3.6688***
11.7434
17.7706
-0.0003
0.0000
0.0002
0.0673***
0.0727***
-0.0627***
-0.0540***
1.4862***
1.8543***
1.5067***
0.0714***
-0.0124***
-5.4439*
-8.3258*
1.1642***
zonas australes, donde es posible emplazar los centros
más alejados unos de otros. La evolución natural de este
poblamiento de zonas australes ha tendido a generarse
en torno a zonas relativamente cercanas a los
asentamientos urbanos.
La actividad acuícola se desarrolla usando espacios
geográficos que son heterogéneos en cuanto a
características ambientales, económicas y sociales.
Considerando esta heterogeneidad en conjunto con el
patrón de desarrollo espacio-temporal de la industria
acuícola en Chile, así como el impacto probable de las
regulaciones que pudieron haber incentivado el
desplazamiento de centros de cultivo hacia las regiones
de Aysén y Magallanes, una implicancia es que el
diseño regulatorio debiera ser diferenciado por zonas
geográficas, lo cual requiere información científica y
tecnológica detallada, previo al otorgamiento de
concesiones así como evaluación frecuente y sostenida
a través del tiempo.
El desplazamiento de la actividad acuícola hacia el
sur de la Patagonia, en conjunto con un incremento en
la dispersión de la actividad productiva en el referido
territorio, podría no ser deseable en términos de las
consecuencias ambientales que genera. No obstante que
el problema trasciende el ámbito de este trabajo, a la luz
de los resultados expuestos, no está claro si una
actividad acuícola desplazada hacia el sur del territorio
y más dispersa, es más o menos deseable que una
actividad concentrada en un espacio más acotado del
territorio. En particular, la mayor dispersión podría ser
una fuente potencial de generación de mayores
externalidades negativas distribuidas espacialmente, y
fuente de conflicto con otros usos del espacio
geográfico, impactos que tienden a atenuarse con la
concentración. En este sentido, las regulaciones futuras
de la acuicultura debieran anticipar eventuales consecuencias no deseadas respecto al comportamiento de la
población regulada, incluyendo, entre otros, las
decisiones de localización.
Finalmente, el patrón detectado respecto a la
expansión de las actividades de la industria acuícola
chilena hacia las regiones de Aysén y Magallanes, tiene
relevancia no solamente asociada a los potenciales
impactos ambientales de la actividad y la necesidad (y
nueva oportunidad), para un mejor diseño regulatorio
que intente conciliar la búsqueda de beneficios
económicos con la protección del medio ambiente, los
impactos sociales involucrados y la competencia por el
uso del espacio geográfico con otras actividades
productivas. El desarrollo de la industria acuícola en la
Patagonia chilena constituye también un desafío en
términos de la necesidad de provisión de infraestructura
apropiada para la actividad y de oferta de trabajo
calificado que pueda satisfacer adecuadamente las
nuevas demandas.
AGRADECIMIENTOS
Los autores agradecen a Leonardo Vargas por su
valioso apoyo en la preparación y manejo de las bases
de datos y la construcción de los mapas para este
artículo. Los autores también agradecen los útiles
comentarios de dos evaluadores anónimos y del Editor
Asociado de Latin American Journal of Aquatic
Research. Finalmente, agradecemos el apoyo financiero
provisto por el Centro Interdisciplinario para la Investigación en Acuicultura (INCAR) a través de CONICYT/
FONDAP/15110027. M. Estay agradece financiamiento parcial para esta investigación a través del
Núcleo Milenio en Economía Ambiental y de Recursos
Naturales Proyecto RS 130001 de la ICM.
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Lat. Am. J. Aquat. Res., 43(4): 718-725, 2015
Stocks de Plagioscion ternetzi de los ríos Paraguay y Paraná
718
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Research Article
Identificación de stocks pesqueros de la corvina de río (Plagioscion ternetzi)
de los ríos Paraguay y Paraná, mediante el análisis morfométrico de sus otolitos
Esteban Avigliano1, Guy Comte1, Juan José Rosso2, Ezequiel Mabragaña2, Paola Della Rosa3
Sebastian Sanchez3, Alejandra Volpedo1, Franco del Rosso4 & Nahuel Federico Schenone1
1
Instituto de Investigaciones en Producción Animal (INPA-CONICET-UBA)
Facultad de Ciencias Veterinarias, Universidad de Buenos Aires
Av. Chorroarín 280 (C1427CWO), Buenos Aires, Argentina
2
Grupo de Biotaxonomía Morfológica y Molecular de Peces (BIMOPE), IIMyC-CONICET
Universidad Nacional de Mar del Plata, Mar del Plata, Argentina
3
Instituto de Ictiología del Nordeste, Facultad de Ciencias Veterinarias
Universidad Nacional del Nordeste, Santiago Cabral 2139 (3400), Corrientes, Argentina
4
Programa Biodiversidad, Áreas Protegidas y Cambio Climático del Ministerio de la Producción y Ambiente
de la Provincia de Formosa, Argentina
Autor corresponsal: Esteban Avigliano ([email protected])
RESUMEN. La identificación de stocks pesqueros es un requisito básico para el manejo y la gestión de la pesca.
El objetivo de este trabajo fue describir por primera vez los otolitos de la corvina de Río P. ternetzi y evaluar la
existencia de diferentes stocks pesqueros entre dos áreas de la Cuenca del Plata, cuenca baja del Río Paraguay
y el Río Paraná medio. Con este fin, cinco índices morfométricos aplicados sobre los otolitos sagittae
(rectangularidad, circularidad AO/LO, SS/SO y PS/PO) fueron comparados entre los sitios de estudio. Los
otolitos sagittae son semicirculares y presentan bordes lisos. El sulcus acusticus es de tipo heterosulcoide y
curvo, con abertura pseudoostio-caudal. Se observaron diferencias significativas para circularidad, rectangularidad, SS/SO y PS/PO (t- test, P < 0,05). El análisis multiparamétrico T2 de Hotteling mostró diferencias
significativas entre los sitios de estudio (P < 0,006) mientras que el análisis canónico discriminante mostró un
alto porcentaje de clasificación de los individuos (>69%). Los resultados obtenidos sugieren que las poblaciones
de esta especie de los ríos Paraguay y Paraná estarían parcialmente separadas, aunque mantendrían flujo de
individuos entre las mismas.
Palabras clave: Plagioscion ternetzi, morfometría, otolito, stocks pesquero, Río Paraná, Río Paraguay.
Identification of fish stocks of river crocker (Plagioscion ternetzi) in Paraná and
Paraguay rivers by using otolith morphometric analysis
ABSTRACT. The identification of fish stocks in basic requirement for fishing management. The objective of
this research was to describe for the first time otoliths river crocker (Plagioscion ternetzi) and to evaluate the
existence of different fish stocks in the Paraguay River lower basin and the middle Paraná River (northeast
Argentinean region and southeast Paraguayan region). For this purpose, five morphometric indexes applied on
sagitta otolith (rectangularity, circularity AO/SO, SS/SO and PS/PO) were compared between the study sites.
The sagittae otoliths are semicircular and with smooth edges. Sulcus acusticusis is heterosulcoid and curved,
with an ostium open widely in the anterior margin of the otolith. Significant differences were observed for
circularity, rectangularity, SS/SO and PS/PO (t-test, P < 0.05). The T2 Hotteling multiparametric analysis
showed significant differences between the study sites (P < 0.006), while the canonical discriminant showed a
high classification percentage of the individuals (>69%). The results indicates that the stocks populations would
be partially separated, with a considerable flow of individuals between these rivers.
Keywords: Plagioscion ternetzi, morphgometry, otolith, fish stocks, Paraná River, Paraguay River.
__________________
Corresponding editor: Guido Plaza
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INTRODUCCIÓN
El género Plagioscion (Gill, 1861) es endémico de las
aguas dulces de América del Sur, donde sus miembros
se distribuyen en los ríos Magdalena (Colombia),
Amazonas (Brasil, Perú y Colombia), Orinoco
(Venezuela), Paraguay y Paraná (Argentina, Brasil,
Paraguay y Uruguay) y diferentes cuencas de las
Guayanas (Casatti, 2003, 2005). Algunas especies
como P. squamosissimus han sido introducidas en el
alto Río Paraná, en el Río São Francisco y diferentes
embalses artificiales del noreste de Brasil (Cassati,
2005).
Los análisis filogenéticos indican que el género
Plagioscion en América del Sur es monofilético (Cooke
et al., 2012). Las relaciones filogenéticas de las
especies del género, el registro fósil, la historia
geomorfológica y los datos de distribución sugieren que
las incursiones hacia los sistemas acuáticos
continentales se habrían producido desde el océano en
el oeste de Venezuela, entre finales del Oligoceno y
Mioceno temprano (Cooke et al., 2012). Estas
incursiones fueron las responsables de la adaptación a
los ambientes dulceacuícolas de las especies de
Plagioscion (Cooke et al., 2012). Actualmente el
género posee cinco especies válidas (Casatti, 2005)
siendo Plagioscion ternetzi (Boulenger, 1895) la que
presenta la distribución más meridional.
Particularmente, P. ternetzi está distribuida en los
ríos Uruguay, Paraguay y Paraná, originalmente hasta
el salto Sete Quedas, hoy sumergido por la represa
Itaipú (Casatti, 2005; Serra et al., 2012). En la cuenca
baja del Río Paraguay y la confluencia con el Río
Paraná, esta especie es de gran importancia tanto para
las pesquerías artesanales como para la pesca deportiva,
siendo además de gran interés cultural para la región.
Los mayores volúmenes de captura se producen en las
cercanías a la Laguna Herradura, un meandro del Río
Paraguay (Formosa, Argentina). En esta área, el
periodo de desove se produce en los meses cálidos, de
enero a marzo. En este periodo la especie prefiere
regiones con poca corriente de agua y ambientes con
meandros (Vera et al., 2005). En contraste, actualmente
las capturas en el Río Paraná son muy escasas y se
realizan en forma no dirigida (accidental) por
pescadores artesanales.
A pesar de la importancia de este recurso, poco se
sabe sobre el estado de la pesquería. No existen
estadísticas sobre los volúmenes de extracción y la
estacionalidad de las capturas. Por otro lado, la
existencia de diferentes poblaciones o stocks pesqueros
y la conectividad entre éstas también es desconocida.
Los estudios sobre estas temáticas están dirigidos casi
exclusivamente a P. squamossissimus (Teixeira et al.,
2002; González et al., 2005).
Los otolitos han sido ampliamente utilizados para la
determinación de stocks pesqueros debido a que la
morfología y morfometría de estas estructuras están
fuertemente influenciadas por el ambiente que
frecuentan los peces y por el tipo de uso del hábitat
(e.g., Longmore et al., 2010; Avigliano et al., 2012,
2014; Cañás et al., 2012; Keating et al., 2014; Vieira et
al., 2014; Avigliano et al., 2015b). Los otolitos de los
peces teleósteos son cuerpos policristalinos compuestos principalmente por carbonato de calcio precipitado
en forma de aragonita, que están alojados en el aparato
vestibular (Campana, 1999). El análisis de la morfometría de los otolitos permite generar una descripción
cuantitativa de la forma y el contorno que puede
compararse estadísticamente (Lestrel, 1997). Entre los
índices morfométricos más utilizados están la
circularidad, rectangularidad, AO/LO y SS/SO (e.g.,
Longmore et al., 2010; Cañás et al., 2012; Tuset et al.,
2013; Avigliano et al., 2015a).
En este contexto, los otolitos sagittae de la corvina
de río P. ternetzi fueron descritos y diferentes índices
morfométricos fueron comparados entre los individuos
que habitan el bajo Río Paraguay y el Río Paraná medio
(noroeste argentino) con el fin de evaluar la existencia
de diferentes stocks pesqueros.
Área de estudio
El área abarcada corresponde a la cuenca baja del Río
Paraguay y a la cuenca media del Río Paraná. Estas
cuencas constituyen un corredor biogeográfico de
características atípicas debido a que tiene sus nacientes
en ambientes del trópico húmedo y su desembocadura
en regiones templadas húmedas (Cabrera & Willink,
1973). El carácter de corredor biogeográfico se
manifiesta en el hecho que todos los bosques en galería
del sistema fluvial Paraguay-Paraná tienen linaje
amazónico (Cabrera & Willink, 1973).
La cuenca baja del Río Paraguay, específicamente
la laguna Herradura y la porción del Río Paraguay
asociada (región oriental de la Provincia de Formosa),
corresponde a la región biogeográfica del Chaco
Húmedo e incluye esteros, selvas en galería, pastizales
y palmeras (Cabrera & Willink, 1973).
El Río Paraná medio y alto (longitud entre 26° y
28°S) se caracteriza por la presencia de selvas y
bosques subtropicales, acompañados por pastizales
dominados por gramíneas megatérmicas (Cabrera &
Willink, 1973).
La ictiofauna de la región de estudio está constituida
por más de 250 especies en el Paraná medio (Menni et
al., 1992) y 143 en el Río Paraguay (Menni, 2004)
pertenecientes a más de 150 géneros y 41 familias
(López et al., 2005). La gran diversidad de ambientes
acuáticos como ríos, esteros, meandros y lagunas
permiten la presencia de especies migradoras,
sedentarias, relacionadas con la vegetación, además de
peces anuales, pulmonados y grandes ictiófagos (López
et al., 2005).
Colecta de muestras
Los sitios de muestreo se indican en la Fig. 1. En el Río
Paraguay y la Laguna Herradura (26°32'13”S,
58°15'38”W) los especímenes se capturaron con
anzuelo a profundidades entre 3 y 9 m durante febrero
de 2012, 2013 y 2014. En el Río Paraná (Sitio 1:
27°18'14”S, 57°51'15”W y Sitio 2: 27°28'36”S,
57°02'48”W) se capturaron con redes de tres mallas de
multifilamento (malla interna de 3x3 cm y externas de
10x10) en octubre 2013 y febrero 2014. Los rangos de
tallas para el Río Paraguay y La Herradura estuvieron
entre 20,2 y 35,0 cm (n = 13) y en el Río Paraná entre
24,3 y 43 cm (n = 41). Los peces fueron traslados al
laboratorio a 4°C donde se registró la longitud total y
se extrajeron los otolitos sagittae. Los otolitos fueron
limpiados con agua destilada y conservados
individualmente en seco, en sobres de papel.
Morfometría de los otolitos
Los otolitos sagittae de P. ternetzi fueron descritos
utilizando la nomenclatura propuesta por Tuset et al.
(2008). Los otolitos derechos se fotografiaron con
microscopio estereoscopio (Leica® EZ4-HD) y se
registraron las siguientes variables morfométricas
sobre las imágenes mediante el procesador de imágenes
Image-ProPlus®4.5: longitud del otolito (LO), ancho
(AO), perímetro del sulcus (PS) y perímetro (PO) en
mm y superficie del otolito (SO) y superficie del sulcus
(SS) en mm2. Posteriormente, se calcularon los
siguientes índices de forma: circularidad (PO2/AO),
rectangularidad (SO/[LO*AO]), AO/LO, SS/SO y
PS/PO. La nomenclatura de los índices utilizados fue
tomada de Tuset et al. (2013) y Volpedo et al. (2008).
La circularidad da información sobre la complejidad
del contorno de los otolitos (Cañás et al., 2012). La
rectangularidad da información sobre la aproximación
a una forma rectangular o cuadrada siendo igual a 1 un
rectángulo o cuadrado perfecto. El índice AO/LO
indica la relación entre el ancho y la longitud del
otolito, siendo igual a 1 para un círculo o rectángulo
perfecto (Avigliano et al., 2014). El índice SS/SO
determina que proporción de la superficie del otolito
está ocupada por el sulcus que a su vez corresponde con
la superficie que ocupa la mácula nerviosa que
transmite al cerebro información relacionada con la
posición relativa del pez en la columna de agua, así
como con la captación auditiva (Volpedo et al., 2008).
La relación entre el perímetro del sulcus y del otolito se
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3
expresa con el índice PS/PO, utilizado por primera vez
para Odontesthes bonariensis (Avigliano et al., 2014).
Análisis estadístico
Las variables circularidad, SS/SO y PS/PO no se
ajustaron a la distribución normal y homogeneidad de
la varianza (Shapiro-Wilk, P < 0,04 y Levene’s, P >
0,05); por este motivo fueron transformadas con la
función log (x+1) para SS/SO y PS/PO y log (x) para
circularidad. Luego de comprobar los supuestos de
normalidad y homogeneidad de varianza, el análisis de
covarianza fue utilizado para corregir el efecto de la
longitud del otolito sobre las variables estudiadas
(ANCOVA, P < 0,01) (Longmore et al., 2010; Kerr &
Campana, 2014). Las constantes utilizadas para la
corrección fueron: circularidad, b = 0,03; rectangularidad, b = -0,001; AO/LO, b = -0,0032; SS/SO, b =
0,0013; PS/PO b = 0,00071.
La prueba t de Student fue utilizada para evaluar la
existencia de diferencias significativas para cada una de
las variables entre los sitios de estudio. La correlación
entre las variables fue testeada utilizando el test de
correlación de Pearson. Esta metodología es aplicada
para evaluar la multicolinealidad entre las variables y
evita su uso redundante, que puede llevar a falsas
conclusiones en el análisis de función discriminante
(Graham, 2003).
Por otro lado, el análisis multivariado de comparaciones de pares T2 de Hotteling fue aplicado para
evaluar las diferencias entre los sitios de estudio
considerando simultáneamente todas las variables
morfométricas.
El análisis discriminante canónico (ADC) fue
realizado utilizando lotes de índices morfométricos
para obtener la matriz de clasificación cruzada y
determinar la capacidad de estas variables para la
identificación del sitio de origen de los peces (e.g.,
Longmore et al., 2010; Kerr & Campana, 2014). Los
coeficientes estandarizados de las funciones discriminantes canónicas (CEFD) se utilizaron para
determinar la contribución de cada variable en la
discriminación de grupos. El programa InfoStat® se
utilizó para todos los análisis estadísticos.
RESULTADOS
Descripción de los otolitos
Los otolitos sagittae de P. ternetzi son semicirculares
con ambos lados globosos. La cara interna o mesial es
cóncava y la externa convexa. El borde dorsal es más
curvo y con mayor profundidad que el ventral. Los
márgenes son lisos, aunque el borde ventral y posterior
puede presentar notables procesos calcáreos (Fig. 2).
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Figura 1. Sitios de muestreo de la corvina de río Plagioscion ternetzi. 1. Río Paraguay, Laguna Herradura, 2. Río Paraná.
Figura 2. Otolito sagitta izquierdo representativo de Plagioscion ternetzi. a) cara interna o mesial, b) cara externa, c) vista
lateral. 1: ostium, 2: cauda, 3: depresión profunda, 4: meseta; A: anterior, P: posterior, D: dorsal, V: ventral, E: cara externa,
I: cara interna. Barra = 3 mm.
El sulcus acusticus es de tipo heterosulcoide, en
posición supramedia y con abertura pseudo ostiocaudal. El sulcus es profundo y claramente delimitado
por una cresta que ocupa casi toda la superficie mesial
(Fig. 2a). La cauda es tubular alargada y curva, su
longitud es aproximadamente el doble que la del
ostium. Hacia su extremo distal se observa una
curvatura hacia la cara ventral adquiriendo una forma
de “U” (Fig. 2a). En el extremo distal la cauda se
estrecha, presenta una fuerte depresión que forma un
canal en el borde ventral y posterior, siguiendo la
morfología del sulcus. Esta depresión se extiende desde
la abertura del ostium hasta los primeros dos tercios de
la cauda. La cara externa del otolito presenta una
meseta con mayor desarrollo hacia el extremo posterior
(Fig. 2b), siendo notable en vista lateral (Fig. 2c).
Morfometría del otolito
Los resultados de las comparaciones de los índices
morfométricos de los otolitos (prueba t de Student) de
los peces provenientes de los diferentes sitios de
estudio se muestran en la Tabla 1. Se observaron
diferencias significativas para todas las variables,
excepto AO/LO. El índice de rectangularidad fue más
elevado para los otolitos de las corvinas capturadas en
Río Paraná, mientras que la circularidad fue significativamente menor en este sitio. Esto indica una tendencia
a la morfometría rectangular. En forma opuesta, la
circularidad fue mayor para los peces del Río Paraguay,
mientras que la rectangularidad fue significativamente
menor, indicando una tendencia a la circularidad de los
otolitos. Por otro lado, los índices PS/PO y SS/SO fueron
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Tabla 1. Media ± desviación estándar de los índices morfométricos por sitio de estudio (transformación log(x) para
circularidad y log(x+1) para SS/SO y PS/PO). Las letras diferentes indican diferencias estadísticamente significativas (t de
Student, P < 0,05).
Circularidad
Rectangularidad
AO/LO
SS/SO
PS/PO
Río Paraguay
Río Paraná
T
P
2,30 ± 0,09
0,74 ± 0,01a
0,63 ± 0,02a
0,16 ± 0,01a
0,32 ± 0,01a
2,23 ± 0,08
0,75 ± 0,01b
0,62 ± 0,03a
0,16 ± 0,01b
0,31 ± 0,01b
-2,9
2,39
-1,0
-2,05
-2,2
0,005
0,020
0,332
0,045
0,029
a
mayores en los otolitos de las corvinas del Río
Paraguay. Se observó correlación entre el AO/LO y la
circularidad (r = -0,38; P = 0,003) y rectangularidad (r
= 0,37; P = 0,01). Por este motivo la variable AO/LO
no fue considerada para los análisis multivariados.
Acorde a la prueba de comparaciones de pares T2 de
Hotteling, se hallaron diferencias significativas entre
los sitios de estudio (P < 0,006). Según los CEFD, los
índices que más contribuyeron a la discriminación
fueron circularidad (CEFD = 0,65) y PS/PO (CEFD =
0,59), seguidos de rectangularidad (CEFD = -0,47), y
SS/SO (CEFD = -0,15). La clasificación cruzada del
ACD (Tabla 2) mostró un alto porcentaje de individuos
bien clasificados para el Río Paraná (69%) y Río
Paraguay (71%).
Tabla 2. Tabla de clasificación cruzada del Análisis
Canónico Discriminante (ACD) de los índices morfométricos. Los números entre paréntesis representan el
porcentaje de clasificación.
Río Paraná
Río Paraguay
Total
Río Paraguay
Río Paraná
Total
4 (31)
29 (71)
33
9 (69)
12 (29)
31
13
41
DISCUSIÓN
Los otolitos sagittae de P. ternetzi fueron descritos por
primera vez en este trabajo. La morfología y
morfometría de los otolitos ha sido ampliamente
utilizada para diferenciar las especies (e.g., Tuset et al.,
2013; Zhuang et al., 2014), describir los patrones
ecomorfológicos (e.g., Volpedo & Echeverría, 2003;
Volpedo & Fuchs, 2010; Jaramillo et al., 2014), como
indicador ambiental (Nelson et al., 1994; Avigliano et
al., 2012), para la determinación de especies fosilizadas
(e.g., Reichenbacher et al., 2007, 2009; Reichenbacher
& Reichard, 2014) y para identificar stocks pesqueros
b
(e.g., Cadrin & Friedland, 1999; Cañás et al., 2012;
Avigliano et al., 2014).
Diferentes autores han utilizado previamente otras
variables morfométricas en especies de sciénidos
marinos. Por ejemplo, Aguilera & Aguilera (2003)
describieron dos especies extintas de sciénidos del
género Plagioscion en sedimentos marinos del
Neogeno en América del Sur, mientras que Monteiro et
al. (2005) estudiaron la variación de forma alométrica
en cinco sciénidos de la costa de Brasil, utilizando
diferentes variables morfométricas de los otolitos. Por
otro lado, Zhang et al. (2014) utilizaron análisis de
forma para diferenciar stocks pesqueros del sciénido
asiático Larimichthys polyactis. A pesar de ello, los
estudios de identificación de poblaciones están
direccionados a especies marinas o estuarinas (Ward et
al., 1994), aún considerando que la existencia de
diferentes grados de diferenciación genética (presencia
de subpoblaciones), generalmente es mayor en especies
de agua dulce en relación a las marinas (Ward et al.,
1994). En este sentido, este es uno de los primeros
trabajos en Sudamérica que emplea la morfometría de
los otolitos para la identificación de stocks en peces de
agua dulce.
Los índices morfométricos más utilizados para la
identificación de stocks son la rectangularidad, la
circularidad, la relación de aspecto y LO/talla del pez
(Tuset et al., 2008; Longmore et al., 2010; Cañás et al.,
2012; Jaramillo et al., 2014), con un menor número de
estudios que utilizan índices en relación al sulcus como
SS/OS (Jaramillo et al., 2014; Zhuang et al., 2014;
Avigliano et al., 2014) o PS/PO (Avigliano et al.,
2014).
En este trabajo, los índices morfométricos que
explicaron la mayor proporción de variabilidad fueron
circularidad, PS/PO y rectangularidad. Los otolitos de
las corvinas capturadas en la cuenca del Río Paraguay
tendieron a la forma circular, mientras que los del Río
Paraná tendieron a la rectangularidad.
Otros investigadores han reportado una relación
positiva entre la salinidad y la tendencia a la circu-
723
6
laridad o elepticidad en especies de diferentes
ambientes como el pejerrey O. bonariensis para el lago
Chasicó (agua salada) (Avigliano et al. 2012, 2015b) y
el Río de la Plata con amplio rango de salinidad
(Avigliano et al. 2014, 2015b). Estas observaciones
concuerdan con los resultados presentados en este
trabajo, debido a que los sistemas lagunares y arroyos
asociados a la región de estudio del Río Paraguay se
caracterizan por poseer salinidad relativamente
elevada, mientras que los afluentes del Río Paraná se
caracterizan por la baja salinidad (Neiff, 2003).
La relación entre el tamaño del sulcus con la
movilidad es bien conocida (Lombarte & Popper, 1994;
Arellano et al., 1995; Tuset et al., 2003; Avigliano et
al., 2014). Por ejemplo, en algunas especies del género
Merluccius, este índice estaría relacionado con el uso
de la columna de agua. Sin embargo, en algunas
especies del género Mullus estaría asociado a
diferencias en el comportamiento de alimentación
(Aguirre & Lombarte, 1999). Avigliano et al. (2014)
han reportado que la superficie del sulcus en relación a
la superficie del otolito tiende a ser mayor en las
poblaciones de O. bonariensis que realizan grandes
migraciones en el estuario del Río de la Plata. En este
trabajo, los índices relacionados con el tamaño del
sulcus fueron mayores para el Río Paraguay.
Presumiblemente, esto estaría relacionado al comportamiento migratorio de la especie en este sitio de estudio.
Las corvinas del Río Paraguay realizarían migraciones
reproductivas hacia sistemas lénticos asociados al
caudal principal (Vera et al., 2005). Además, no existen
barreras artificiales que podrían obstaculizar los
desplazamientos sobre el Río Paraguay. Por otro lado,
sobre el Río Paraná, la migración de la especie podría
estar restringida, debido a la presencia de la represa
Yacyretá aguas arriba de los sitios de muestreo.
Los análisis multivariados mostraron diferencias
significativas entre los sitios de estudio y un porcentaje
de clasificación relativamente alto de los individuos
según su origen. Esto indica que las poblaciones
tenderían a estar parcialmente separadas. Según los
porcentajes de clasificación, es posible que el flujo de
peces entre ambos sitios de estudio sea considerable. En
el futuro, estudios complementarios basados en la
genética de los individuos podrían contribuir para
determinar el grado de conectividad entre los stocks
(Teixeira et al., 2002), descartándose la utilización de
métodos de marcado debido a la alta senilidad de la
especie, que una vez capturada y luego de la devolución
al cuerpo de agua no suele sobrevivir.
La existencia de diferentes poblaciones podría estar
relacionada con la formación y evolución geográfica de
los ríos Paraná y Paraguay. Estos ríos se unieron
durante el Mioceno superior y parte del Plioceno (hace
~4 o 5 millones de años) (Orfeo, 1996; Orfeo &
Stevaux, 2002) y es posible que diferentes poblaciones
hayan existido en el área antes de la formación de la
confluencia. Por otro lado, también es posible que P.
ternetzi se haya distribuido únicamente en uno de estos
ríos, y la población pudo tender a la separación luego
de la formación de la confluencia. Debido a la falta de
registros paleontológicos, no es posible confirmar estas
hipótesis.
Los resultados de este trabajo indican que la
morfometría del otolito, en especial los índices de
circularidad, rectangularidad y AO/LO, podría ser
empleada como un indicador de hábitat en P. ternetzi.
Hay que considerar que el tamaño de muestra obtenida
para el Río Paraná fue relativamente bajo y podría ser
poco representativo. Esto se debe al estado actual de la
pesquería en esta zona de la cuenca que hizo imposible
realizar mayores capturas durante los tres años de
muestreo. Sin embargo, la información presentada traza
una línea de base para el estudio de la biología y
dinámica poblacional de esta especie, aportando
conocimientos para la gestión y el manejo de los
recursos pesqueros en la cuenca baja del Río Paraguay
y en el Río Paraná medio.
AGRADECIMIENTOS
Los autores agradecen al CONICET (PIP 11220120100543CO), ANPCyT (PIP 2010-1372), a la
Universidad de Buenos Aires (UBACYT 20620110
100007) y al Ministerio de la Producción y Ambiente
de la provincia de Formosa por el financiamiento. Los
autores agradecen a los revisores anónimos por los
valiosos comentarios que ayudaron a mejorar la
claridad del manuscrito.
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Lat. Am. J. Aquat. Res., 43(4): 726-738, 2015
Endoparásitos en crustáceos decápodos intermareales
Research Article
Carga parasitaria en crustáceos decápodos de la costa central de Chile:
¿existe alguna asociación con la abundancia de los hospedadores definitivos?
Natalia Leiva1, Mario George-Nascimento2 & Gabriela Muñoz1
1
Facultad de Ciencias del Mar y de Recursos Naturales
Universidad de Valparaíso, P.O. Box 5080, Reñaca, Viña del Mar, Chile
2
Facultad de Ciencias, Universidad Católica de la Santísima Concepción
P.O. Box 297, Concepción, Chile
Corresponding author: Natalia Leiva ([email protected])
RESUMEN. Los crustáceos tienen un rol importante en el ciclo de vida de los parásitos, ya que actúan como
hospedadores intermediarios. No obstante, en Chile y a nivel mundial existen pocos estudios parasitológicos
sobre crustáceos que habitan el intermareal rocoso. En este estudio se registró y comparó la carga parasitaria de
crustáceos decápodos, y se relacionó con la abundancia de sus hospedadores definitivos (peces y aves). Entre
julio y septiembre de 2013 se recolectaron 409 crustáceos, correspondientes a 16 especies, desde el intermareal
rocoso de dos localidades de Chile central (33°S), Las Cruces y Montemar. El 65,5% de la muestra estaba
parasitada, recolectándose 2.410 metacercarias y 18 nemátodos. Algunas metacercarias correspondían a la
familia Opecoelidae, mientras que otras a Microphallidae; los nematodos eran de la familia Cystidicolidae. La
mayor prevalencia y abundancia de Microphallidae se registró en Petrolisthes tuberculosus (42,3%; 4,8 ± 11,08
parásitos/crustáceo) y de Opecoelidae en P. violaceus (96,9%; 13,59 ± 17,50 parásitos/crustáceo), mientras que
Cystidicolidae fue poco prevalente y abundante en ambas localidades. Las infecciones parasíticas fueron
afectadas por la localidad de muestreo, especie y tamaño del hospedador. No se encontró asociación entre la
abundancia de los hospedadores definitivos y las cargas parasitarias que presentaban los crustáceos. La nula
relación entre las cargas parasitarias y la abundancia de hospedadores definitivos puede ocurrir si estos últimos
tienen un amplio espectro trófico o viajan largas distancias, de este modo, la transmisión de los parásitos no
sería afectada directamente por la abundancia de los hospedadores definitivos.
Palabras clave: crustáceos decápodos, hospedadores intermediarios, digeneos, nemátodos, peces intermareales,
aves costeras.
Parasite burden in decapod crustaceans from the central coast of Chile: is there
any association with the relationship with definitive host abundances?
ABSTRACT. Crustaceans play an important role in parasite life cycles, serving as second intermediate hosts.
However, there are scarce parasitological studies in crustaceans from the rocky intertidal habitats, in Chile and
around de world. In this study we aimed to record the parasites in decapod crustaceans, compare their parasitic
loads between localities and relate them with the abundance of the definitive hosts (fishes and birds). Between
July and September 2013, 409 crustacean specimens, corresponding to 16 species, were collected from the rocky
intertidal zone of two localities of central Chile (33°S), Las Cruces and Montemar. Of out the sample, 65.5%
was parasitized; counting 2,410 metacercariae and 18 nematodes. One group of these metacercariae belonged
to the family Opecoelidae; while others corresponded to the family Microphallidae. Nematodes belonged to the
family Cystidicolidae. The highest prevalence and abundance of opecoelids were in P. violaceus (96.9%, 13.59
± 17.50 parasites/crustacean), microphallids were mostly recorded in the crab Petrolisthes tuberculosus (42.3%,
11.08 ± 4.8 parasites/crustacean), while cystidicolids were less prevalent and abundant than digenean at both
localities. Parasite loads was affected by body size, locality and species of crustacean hosts. No association was
found between parasite loads in these intermediate hosts and the abundance of definitive hosts.
__________________
Corresponding editor: Luis Miguel Pardo
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1
727
2
The low relationships between parasite loads and host abundances may be due to several reasons, such as a wide
trophic spectrum and great capacity of movement, which would not contribute to the parasite transmission and
the direct relationship with the definitive host abundances.
Keywords: decapod crustaceans, intermediate hosts, digeneans, nematodes, intertidal fish, coastal birds.
INTRODUCCIÓN
El parasitismo, entendido como la asociación entre dos
organismos (hospedador y parásito), ha sido descrito
como una relación ecológica, donde el parásito depende
completamente de su hospedador quien no solo
representa el hábitat, sino que también tiene los
recursos alimenticios para que el parásito se desarrolle
(Hinojosa-Sáez & González-Acuña, 2005; Rohde,
2005). El parásito puede tener varios estados
ontogenéticos, necesitando a diversos hospedadores
durante su vida, lo que implica que las vías de
transmisión varíen entre un estado y otro (Mouritsen &
Poulin, 2002). La transmisión del parásito es activa
cuando el estado infectante es una larva nadadora, la
cual puede llegar a su hospedador directamente,
mientras que la transmisión es pasiva si la larva parásita
no puede salir por sí misma de un hospedador
intermediario, en este caso, el siguiente hospedador
necesariamente debe depredar sobre el organismo
parasitado (Olsen, 1974; Cribb, 2005a).
Los crustáceos han tenido un rol importante en la
transmisión de parásitos, ya que muchas especies
actúan como primeros o segundos hospedadores
intermediarios de endoparásitos, contribuyendo tanto al
desarrollo del parásito como en su dispersión. Los
digeneos, céstodos, nemátodos y acantocéfalos tienen
al menos un estadio larval que requiere de un crustáceo
(Marcogliese, 1995). En el ambiente marino, los
crustáceos son depredados por un vertebrado (pez, ave
o mamífero) donde el endoparásito se desarrolla hasta
alcanzar el estado adulto (Rohde, 2005). Sin embargo,
a pesar de la importancia que cumplen los crustáceos en
el ciclo de vida de endoparásitos marinos, en Chile son
muy pocos los estudios parasitológicos realizados en
crustáceos decápodos (Muñoz & Olmos, 2008; Oliva et
al., 2008; Saldanha et al., 2009; Zambrano & GeorgeNascimento, 2010), sobre todo en aquellos que habitan
en pozas del intermareal rocoso, pese a la alta
abundancia que tienen en este ambiente.
Las aves y peces son los vertebrados más frecuentes
de la zona intermareal rocosa de Chile central y varios
de sus parásitos son adquiridos mediante la depredación
de presas que habitan en esa misma zona (Muñoz &
Cortés, 2009). Los parásitos que requieren de peces
intermareales para llegar a sus estados adultos, corresponden a nemátodos del orden Spirurida (Guyanemidae
y Cystidicolidae) (Muñoz et al., 2002, 2004; Muñoz &
George-Nascimento, 2007; Muñoz, 2010), y digeneos
del orden Plagiorchiida (Opecoelidae y Lecithasteridae) (Muñoz & Olmos, 2008; Muñoz & Castro,
2012), mientras que en aves costeras se han registrado
digeneos Plagiorchiida (Microphallidae) y acantocéfalos
del orden Polymorphida (Polymorphidae) (HinojosaSáez & González-Acuña, 2005; Muñoz, 2005;
González-Acuña et al., 2009). Por lo tanto, algunos de
estos grupos de parásitos podrían encontrarse en
crustáceos decápodos que habitan la zona intermareal.
Los modelos epidemiológicos suponen que el éxito
de la transmisión de los parásitos a sus hospedadores
intermediarios y definitivos, dependerá de la distribución y/o abundancia local de estos últimos. No
obstante, esta relación puede variar de acuerdo a
múltiples factores, como la movilidad y susceptibilidad
a infecciones del hospedador e historia de vida del
parásito. Particularmente, los endoparásitos necesitan
de uno o más hospedadores intermediarios, y un
hospedador definitivo para completar su ciclo de vida.
Los hospedadores definitivos debido a su gran
movilidad, aseguran la dispersión de los huevos del
parásito, contribuyendo a su propagación (Smith, 2001;
Hansen & Poulin, 2006). Por consiguiente, mientras
mayor sea la abundancia de hospedadores definitivos
en un área, mayor probabilidad habrá para que las
etapas de dispersión del parásito se encuentren con un
hospedador, lo cual se reflejaría en mayores cargas
parasitarias de los hospedadores intermediarios
(Latham & Poulin, 2003; Smith, 2007). Basado en estos
antecedentes, el presente estudio tiene como objetivo
registrar y cuantificar las especies de endoparásitos
presentes en crustáceos decápodos comunes del
intermareal rocoso de Chile central, y asociar sus
prevalencias y abundancias con la abundancia de
hospedadores definitivos (peces y aves). De estas
relaciones se espera encontrar correlaciones directas
entre la carga parasitaria (abundancia y prevalencia) de
los hospedadores intermediarios y las abundancias de
hospedadores intermediarios y definitivos.
Parásitos en crustáceos decápodos
Los crustáceos decápodos fueron recolectados manualmente desde el intermareal rocoso de dos localidades
de Chile central, Las Cruces (33°30’S, 71°37’W) y
Montemar (32°58’S, 71°29’W), abreviados en este
estudio como LC y MO, respectivamente. La recolección de crustáceos se realizó en baja marea, desde
pozas intermareales. Se realizaron tres muestreos en
cada localidad, entre julio y septiembre de 2013,
recolectando al menos 40 ejemplares por muestreo.
Los crustáceos recolectados fueron colocados en
recipientes con agua de mar y llevados al Laboratorio
de Parasitología de la Facultad de Ciencias del Mar y
de Recursos Naturales de la Universidad de Valparaíso.
Posteriormente, el 60% de los ejemplares recolectados
fue guardado en bolsas plásticas y congelado para su
posterior análisis, mientras que el resto de los
especímenes se mantuvo vivo hasta el momento de su
disección, con el fin de obtener parásitos en buenas
condiciones para su identificación. Previo a la
disección, se registró el largo del cefalotórax (LCT) en
aquellos de cuerpo ovalado y deprimido (jaibas y
cangrejos) y la longitud total (LT) en los de cuerpo
alargado y comprimido (camarones y cangrejos
ermitaños), mediante un pie de metro digital (en mm).
La identificación de los crustáceos fue determinada
según los criterios morfológicos indicados en claves
taxonómicas de Viviani (1969), Retamal (1981) y
Zúñiga (2002).
La disección se realizó bajo un microscopio
estereoscópico Leica M80. Previo a la disección, los
crustáceos fueron anestesiados con una solución diluida
de AQUI-S®. Cada individuo fue colocado en una
placa de Petri con agua salina al 8%, caparazón y
apéndices fueron extraídos para luego separar cada
tejido del animal con ayuda de agujas de disección
finas. Luego se procedió a registrar los parásitos
encontrados en distintos tejidos (vísceras y musculatura
del cefalotórax, abdomen y pereiópodos). Todos los
taxa parasitarios fueron contabilizados, fijados en
etanol 70% y guardados en tubos de Eppendorf para su
posterior identificación al nivel taxonómico más bajo
posible.
Para la determinación taxonómica de los digeneos
se consideró su morfología externa y la de sus órganos
internos (tracto digestivo y algunos órganos sexuales),
de acuerdo a las claves taxonómicas de Cribb (2005b)
y Deblock (2008). Para la identificación de nemátodos
se consideró la morfometría en base a la medición de
ciertos caracteres morfológicos como longitud total,
ancho máximo, esófago glandular y muscular, y
posición del anillo nervioso, acorde a la metodología
utilizada por Muñoz et al. (2004). Los nemátodos
larvales fueron comparados con los de ejemplares
adultos machos y hembras de otras especies de
nemátodos en relación a sus medidas relativas (una
determinada medida morfométrica divida por la
longitud corporal), según Martorelli et al. (2000). Para
esto se comparó las medidas relativas mediante pruebas
7283
de Kruskal-Wallis (Zar, 1996) entre nemátodos larvales
de crustáceos decápodos y nemátodos adultos del
intermareal rocoso de Chile (Muñoz et al., 2004;
Muñoz & George-Nascimento, 2007; Muñoz, 2010).
Posterior a la identificación de los parásitos, se
determinó la prevalencia (porcentaje de hospedadores
parasitados de una muestra) y abundancia promedio
(promedio de parásitos por especie de hospedador) de
los taxa parasitarios encontrados en cada crustáceo y
localidad. Se comparó, mediante pruebas de t-Student,
la longitud corporal de los crustáceos por localidad:
LCT en jaibas y cangrejos, y LT en camarones y
cangrejos ermitaños, los que fueron analizados
separadamente. También se realizaron correlaciones de
Spearman entre la abundancia de parásitos y la longitud
corporal de los hospedadores, considerando solo
aquellas especies más abundantes que contenían
parásitos. Además, se consideró correlaciones de la
abundancia promedio y prevalencia de parásitos con
respecto a la LCT promedio de los crustáceos
parasitados entre localidades.
Para asociar la prevalencia de parásitos con la de las
especies de crustáceos según su localidad, se aplicaron
tablas de contingencia (2x2 a 2x5) usando un programa
estadístico online (www.vassarstats.net), para asociar
cada especie de parásito por separado. Según la
naturaleza de los datos se aplicó la corrección de Yates
y probabilidad exacta de Fisher.
Relación entre parásitos y abundancia de hospedadores
La estimación de la abundancia de aves costeras se
realizó el mismo día y lugar de la recolección de los
crustáceos, donde se procedió a identificar y contar in
situ las especies de aves presentes en una sección de la
costa durante una hora en intervalos de 15 min,
realizando cuatro conteos por visita. Para el
reconocimiento de las aves en terreno se consideró las
guías de campo de Araya & Millie (1998) y Jaramillo
(2005).
El registro de la abundancia de peces se realizó en
tres pozas intermareales rocosas de las zonas litorales
escogidas (LC y MO), del mismo sector donde se
realizaron las recolecciones de crustáceos y conteo de
aves. Se realizaron tres muestreos mensuales en LC
(julio-octubre 2013) y otros tres muestreos en MO
(octubre 2013-enero 2014). Para el registro de peces, el
agua contenida en pozas grandes fue aspirada con una
motobomba, lo cual facilitó la extracción de los peces,
mientras que las pozas pequeñas fueron trabajadas con
el agua que contenían. La captura de peces se realizó
mediante el uso de anestésico AQUI-S® y redes de
mano. Posteriormente, los peces se identificaron
taxonómicamente y se contabilizaron.
729
4
Se realizaron disecciones de peces y aves para
determinar la presencia de los grupos parasitarios y
compararlos con los encontrados en los crustáceos
decápodos en las localidades muestreadas. Los peces
extraídos de dos pozas de MO y dos de LC fueron
congelados para su posterior disección (n = 111) para
revisar sus parásitos, mientras que el resto (peces de una
poza de cada localidad) fueron devueltos a su hábitat
natural. Para el registro de parásitos de aves costeras,
solo se trabajó con aquellas encontradas muertas en la
zona intermareal (n = 8). Cada ave recogida fue
disectada para obtener sus parásitos, registrarlos y
contabilizarlos. Del total de peces y aves revisadas, se
calculó la prevalencia y abundancia, solo de los taxa
parasitarios que coincidían con los encontrados en los
crustáceos.
Se calculó la frecuencia numérica y relativa de aves
y peces, obteniendo valores promedios de los tres
muestreos realizados en cada localidad y para cada
grupo de vertebrado (peces y aves). Se aplicaron tablas
de contingencia para asociar la frecuencia de vertebrados con la de grupos de parásitos que suelen tener
según registros bibliográficos (González-Acuña et al.,
2009; Muñoz & Delorme, 2011; Muñoz & Castro 2012;
Muñoz, 2014). La asociación entre abundancia de peces
y aves, abundancia de crustáceos y sus cargas parasitarias se realizó considerando todos los ejemplares
obtenidos. Estas variables fueron asociadas mediante
tablas de contingencia de 2x2 (Zar, 1996).
RESULTADOS
Se recolectó un total de 409 crustáceos correspondientes a 16 especies; 13 en LC y 11 en MO.
Allopetrolisthes angulosus fue la más abundante en LC,
mientras que Petrolisthes violaceus y Petrolisthes
tuberculatus fueron más abundantes en MO, seguidas
de P. tuberculosus y Pagurus edwardsii (Tabla 1).
La LCT de los crustáceos (jaibas y cangrejos)
recolectados en LC varió entre 4,05 y 43,50 mm, siendo
A. angulosus y Taliepus dentatus los que presentaron la
menor y mayor LCT respectivamente, mientras que en
MO la LCT de los crustáceos varió entre 3,25 y 36,52
mm, siendo Petrolisthes laevigatus y Paraxanthus
barbiger los que mostraron la menor y mayor LCT
respectivamente (Tabla 1). El cangrejo ermitaño P.
edwardsii presentó la menor LT en ambas localidades,
mientras que P. edwardsii y el camarón Rhynchocinetes
typus presentaron la mayor LT en MO y en LC
respectivamente (Tabla 1).
Analizando el conjunto de especies de crustáceos, el
promedio de la LCT de jaibas y cangrejos no fue
significativamente diferente entre localidades (LC:
9,37 ± 5,32 mm, MO: 9,45 ± 5,11 mm) (t = 0,59; d.f. =
354; P = 0,577), mientras que el promedio de la LT de
los camarones y cangrejo ermitaños de LC fue
significativamente mayor que los de MO (t = 4,3; d.f.=
51; P < 0,001).
Parásitos en crustáceos decápodos
Se obtuvo un total de 2.428 parásitos, la mayoría de
ellos digeneos en estado de metacercaria (n = 2.410) y
unos pocos nemátodos en fase larvaria (n = 18). Las
metacercarias fueron extraídas principalmente desde la
musculatura del cefalotórax y pereiópodos, y unas
pocas entre las lamelas branquiales; mientras que los
nemátodos fueron encontrados principalmente en la
cavidad celomática y algunos en el intestino. El
porcentaje de prevalencia del parasitismo en el
ensamble de crustáceos fue de 65,5%. De las especies
parasitadas, nueve pertenecían a Anomura, y solo dos a
Brachyura (Tablas 1-2).
De las metacercarias extraídas de los crustáceos, se
determinaron dos morfotipos comunes: una de forma
redondeada y cubierta blanda de color café (morfotipo
I, Fig. 1a), y otra de forma redondeada a ovalada y
cubierta gruesa e incolora (morfotipo II, Figs. 1b-1c).
Las metacercarias se disectaron y caracterizaron
morfológicamente, determinándose que la metacercaria
morfotipo I era de cuerpo alargado y ensanchado a nivel
acetabular, acetábulo protuberante ubicado cerca de la
mitad del cuerpo, dos ciegos intestinales y tegumento
no espinoso; algunos ejemplares de mayor tamaño
presentaban nueve testículos, este último rasgo es
característico del género Helicometrina. Por lo tanto,
este grupo se determinó como Opecoelidae (Fig. 2a).
Las metacercarias morfotipo II eran de cuerpo pequeño
y comprimido, esófago largo, faringe pequeña a la
mitad del esófago, ciegos cortos (hasta la zona media
del cuerpo), desarrollo temprano del sistema genital y
algunos ejemplares tenían saco del cirro. Además,
había ejemplares con o sin espinas en el cuerpo (mitad
o cuerpo entero). Este grupo de metacercarias se
determinó como Microphallidae, compuestos por
varias morfoespecies no determinadas (Figs. 2b-2d).
Un tercer tipo de metacercaria no fue identificada; de
forma esférica embrión pequeño, de similares
características al morfotipo I, pero con esófago largo y
la faringe ubicada a la mitad de éste. Estas
metacercarias fueron encontradas en abundancia de uno
a tres, en seis crustáceos de LC, correspondientes a
Romaleon polyodon, P. edwardsii y R. typus (no
incluidas en la Tabla 2).
Los nemátodos fueron de pequeños tamaños y muy
delgados, con estriaciones cuticulares y presencia de
pseudolabios (Figs. 2e-2h), que son características
propias de Cystidicolidae. Por lo tanto, la morfometría
7305
Tabla 1. Número de especímenes recolectados (n), longitud corporal promedio ( , mm) ± desviación estándar (DE, mm)
(longitud cefalotoráxica para la mayoría de las especies, longitud total para las especies indicadas con asteriscos), y
abundancia relativa (ABU, %) de 16 especies de crustáceos decápodos recolectados en Las Cruces y Montemar.
Las Cruces
n
Anomura
Allopetrolisthes angulosus
Allopetrolisthes punctatus
Pagurus edwardsii
Pagurus gaudichaudii
Petrolisthes granulosus
Petrolisthes laevigatus
Petrolisthes tuberculatus
Petrolisthes tuberculosus
Petrolisthes violaceus
Brachyura
Homalaspis plana
Paraxanthus barbiger
Pisoides edwardsii
Romaleon polyodon
Taliepus dentatus
Caridea
Betaeus emaginatus
Rhynchocinetes typus
Montemar
± DE
ABU (%)
n
49
3
20
8,0 ± 24
10,6 ± 3,0
16,7 ± 5,9*
28,5
1,7
11,6
12
4
23
16
32
8,9 ± 2,7
10,9 ± 5,3
8,2 ± 4,2
7,9 ± 3,8
12,2 ± 4,0
6,9
2,3
13,4
9,3
18,6
9
1
19,2 ± 4,8
36,5 ± --
5,2
0,6
3
21,07 ± 2,8
1,7
113
31
13
1
7,8 ± 2,0
8,5 ± 1,7
14,1 ± 2,2*
20,5 ± --*
47,7
13,1
5,5
0,4
32
10
10,2 ± 3,4
12,4 ± 3,6
13,5
4,2
3
5
5
1
4
29,3 ±5,9
21,1 ±5,5
9,8 ± 1,1
28,5 ± -29,3 ± 10,9
1,27
2,1
2,1
0,4
1,7
1
18
65,08 ±-- *
65,2 ± 14,5*
0,4
7,6
ABU (%)
± DE
Tabla 2. Abundancia promedio (ABU) y prevalencia (PRE, %) de tres grupos de parásitos (Microphallidae, Opecoelidae y
Cystidicolidae), registrados en crustáceos recolectados en Las Cruces y Montemar. Los espacios en blanco indican que la
especie hospedadora no estuvo presente.
Las Cruces
Hospedador
Allopetrolisthes angulosus
Allopetrolisthes punctatus
Homalaspis plana
Paraxanthus barbiger
Petrolisthes granulosus
Petrolisthes laevigatus
Petrolisthes tuberculatus
Petrolisthes tuberculosus
Petrolisthes violaceus
Pisoides edwardsii
Romaleon polyodon
Taliepus dentatus
Opecoelidae
ABU
PRE
1,88
63,7
2,61
80,6
Microphallidae
ABU
PRE
0,95
21,2
0,23
9,7
Montemar
Cystidicolidae
ABU
PRE
0,07
5,31
0
0
0
0
5,4
40
0
0
9,97
13,5
93,8
100,0
3,63
11,7
46,9
80,0
0,06
0,2
6,3
10,0
0,5
25,0
0
0
0
0
de los nemátodos larvales fue comparada con cinco
especies de nemátodos cistidicólidos adultos de peces
del intermareal; tres especies de Similascarophis y dos
de Ascarophis (Muñoz et al., 2004; Muñoz & GeorgeNascimento, 2007). De las siete medidas relativas
utilizadas y comparadas, en todas se detectaron diferencias significativas entre las especies de nemátodos
Opecoelidae
ABU PRE
7,04 87,7
5
100
Microphallidae
ABU
PRE
0,16
6,1
1,33
75,0
Cystidicolidae
ABU
PRE
0,06
4,1
0
0
3,33
3,75
10,87
8,06
13,59
50,0
75,0
69,6
81,3
96,9
0
2,25
0,65
0,56
0
0
50,0
26,1
18,8
0
0,08
0
0,04
0,06
0
8,3
0
4,4
6,3
0
1,33
66,7
0
0
0
0
(prueba de Kruskal-Wallis, H > 38,19; P < 0,001); entre
tres y cuatro de estas variables morfométricas (cavidad
bucal, posición del anillo nervioso, esófago glandular y
ancho corporal), difirieron significativamente entre
Ascarophis draconi, Similascarophis chilensis y
Similascarophis maulensis, mientras que las variables
de los nemátodos larvales no fueron distintas a Ascarophis
731
6
Figura 1. Morfotipos de metacercarias encontradas en
crustáceos decápodos intermareales de Chile central.
a) metacercaria de Opecoelidae, b-c) metacercarias de
Microphallidae.
carvajali (prueba a posteriori, 0,055 < P < 1,0) y
Similascarophis sp. (0,25 < P < 1,0). Además, se
encontró que las características bucales de algunos
ejemplares fueron afines a Ascarophis (pseudolabios
más robustos con una leve proyección hacia el centro
bucal, Fig. 2g) y otros a Similascarophis (pseudolabios
más largos y sin proyección hacia el centro bucal, Fig.
2h), lo que indica la presencia de más de una especie de
nematodo cistidicólido en los crustáceos.
Relación entre parásitos y abundancia de hospedadores
Se encontraron correlaciones entre la abundancia y
prevalencia de parásitos de crustáceos, tanto en LC (rs
= 0,920; P < 0,05) como en MO (rs = 0,891; P < 0,05),
y por cada taxa parasitario (rs = 0,929 en Microphallidae, rs = 0,942 en Opecoelidae y rs = 0,816 en
Cystidicolidae, P < 0,05). En general, la prevalencia de
parásitos se correlacionó positivamente con la LCT de
los hospedadores, aunque solo en LC y para los taxa
parasitarios de Microphallidae y Cystidicolidae, se
obtuvo correlaciones significativas (Fig. 3).
Los crustáceos P. tuberculosus y Allopetrolisthes
punctatus, de LC y MO respectivamente, mostraron la
mayor prevalencia de metacercarias de Microphallidae,
mientras que P. tuberculosus y P. laevigatus presentaron la mayor abundancia de estos digeneos (Tabla 2).
La mayor prevalencia de metacercarias de Opecoelidae
se encontraron en A. punctatus, P. tuberculatus y P.
tuberculosus de ambas localidades (LC y MO), siendo
estas últimas dos especies las que tenían la mayor
abundancia (Tabla 2). Los nemátodos se encontraron en
tres especies de crustáceos (Tabla 2), todos con baja
abundancia, entre 0,05 y 0,12 parásitos/ crustáceo, y
máxima prevalencia de 8,3% en Petrolisthes
granulosus. Las infecciones múltiples ocurrieron en el
18,5% del total de crustáceos. No hubo diferencias en
la prevalencia de co-ocurrencia de parásitos entre
localidades (Microphallidae-Opecoelidae: P exacta de
Fisher = 0,393), (Opecoelidae-Cystidicolidae, P exacta
de Fisher = 1), mientras que la concurrencia de los tres
taxa parasitarios se observó sólo en cuatro especímenes
de crustáceos.
Las cargas parasitarias entre los crustáceos de LC
fueron diferentes a los de MO; la abundancia de
metacercarias de Opecoelidae (prueba de MannWhitney, U = 14.829; P < 0,001) y la de Microphallidae
(U = 17.724; P = 0,024; Fig. 4) fue mayor en LC,
mientras que la abundancia de nemátodos Cystidicolidae no fue diferente entre localidades (U = 0,496; P =
0,619). En cuanto a la prevalencia de parásitos, las
metacercarias de Microphallidae fueron más
prevalentes en LC (χ2 = 8,38; gl = 1, P = 0,038), en
cambio, las metacercarias de Opecoelidae (χ2 = 0,83; gl
= 1; P = 0,361) y nemátodos de Cystidicolidae (χ2 =
0,01; gl = 1; P = 0,874) tuvieron similares prevalencias
entre ambas localidades.
Al comparar las cargas parasitarias de las especies
de crustáceos más abundantes y parasitadas (A. angulosus, A. punctatus, P. tuberculosus y P. tuberculatus)
presentes en LC y MO, se encontró que la prevalencia
de Opecoelidae y Microphallidae (analizadas por separado para cada especie de crustáceo), no mostró
diferencias significativas entre localidades (P > 0,06 en
pruebas de Chi-cuadrado con corrección de Yates o P
exacta de Fisher). La abundancia de metacercarias de
Opecoelidae fue significativamente mayor en A.
angulosus (U = 1.294; P < 0,001), y A. punctatus (U =
20,5; P < 0,013) de MO, mientras que la abundancia de
metacercarias de Microphallidae fue significativamente
mayor en A. angulosus de LC (U = 2.348; P < 0,001).
Otra diferencia importante fue que el crustáceo P.
violaceus solo se encontró en MO con un 96,8% de
732
7
Figura 2. Digeneos y nemátodos encontrados en crustáceos decápodos intermareales de Chile central. a) embrión libre de
una metacercaria de Opecoelidae, b-d) embriones libres de metacercarias de Microphallidae, e) zona apical anterior,
f) estriaciones cuticulares, g-h) región cefálica de nemátodos larvales de Cystidicolidae.
prevalencia y 13,6 ± 17,5 de abundancia promedio
(parásitos/crustáceo). Las abundancias de nemátodos
Cystidicolidae no fueron comparadas dado al bajo
número por especies de crustáceos y por localidad.
La prevalencia y abundancia promedio de cada taxa
parasitario no se correlacionó con la abundancia de
crustáceos (0,098 < rs < 0,8; P > 0,05), excepto la
abundancia de metacercarias de Microphallidae que se
correlacionó negativamente con la abundancia de
crustáceos de MO (rs = -0,90; P < 0,05).
La diversidad de peces intermareales fue mayor en
LC que en MO, con 10 y 4 especies, respectivamente.
En LC, las especies más abundantes fueron Girella
laevifrons, Scartichthys viridis y Helcogrammoides
spp., mientras que en MO fueron G. laevifrons y S.
viridis (Tabla 3). En cuanto a las aves costeras, se
registraron 10 especies en cada localidad, ocho especies
fueron comunes entre ellas (Tabla 3). Considerando los
tres muestreos y cuatro eventos de conteo de aves (de
15 min cada uno) se registraron 81 avistamientos en
total en LC y 241 en MO, lo cual mostró diferencias
significativas entre localidades (χ2 = 42,37; gl = 1; P <
0,001). Las aves con mayor abundancia relativa fueron
Larus dominicanus y Phalacrocorax brasilianus
(Tabla 3), pero con diferencias en frecuencia notorias
entre localidad; 10 avistamientos h-1 para L.
dominicanus en LC, y 28 y 32 avistamientos h-1 para L.
dominicanus y P. brasilianus, respectivamente, en MO.
No se encontró una asociación entre la abundancia
de hospedadores definitivos con la abundancia de
parásitos en crustáceos. La frecuencia de peces en pozas
intermareales fue mayor en LC (χ2 = 5,24; gl = 1; P ˂
0,022), sin embargo, la mayor abundancia y
prevalencia de metacercarias de Opecoelidae, que
maduran en peces, se obtuvo en MO (Fig. 4a). De igual
manera, a pesar que la frecuencia de aves fue significativamente mayor en MO (χ2 = 42,37; gl = 1; P ˂
0,001), la mayor prevalencia y abundancia de
metacercarias de Microphallidae, que maduran en aves,
se observó en crustáceos de LC (Figs. 4b-4c),
encontrándose además un mayor número de crustáceos
en esta localidad (χ2 = 5,08; gl = 1; P ˂ 0,029, Figs.
4b-4c).
8733
Figura 3. Relación entre la prevalencia y la LCT
promedio de distintas especies de crustáceos parasitados
con alguno de los tres taxa parasitarios. a) Opecoelidae,
b) Microphallidae y c) Cystidicolidae, en Las Cruces (LC)
y Montemar (MO). r = coeficiente de correlación de
Pearson.
Sin embargo, no se encontró correlación entre la
abundancia de metacercarias de Microphallidae con la
abundancia de aves, entre los meses de muestreo en
ambas localidades (LC: P = 0,899; MO: P = 0,467).
Figura 4. Variación en la abundancia de hospedadores
(promedio peces y crustáceos por poza, número de aves
observadas por hora) y prevalencia y abundancia promedio
de dos taxa de digeneos en respecto a Las Cruces (LC) y
Montemar (MO).
Tampoco se encontró correlación entre la abundancia
de metacercarias de Opecoelidae y la abundancia de
peces intermareales en la localidad de LC (P = 0,796);
MO no fue considerado ya que los muestreos de peces
fueron realizados en meses distintos a los de crustáceos.
7349
Tabla 3. Promedio de abundancia numérica (AN) ± desviación estándar (DE) y abundancia relativa (AR, %) de peces del
intermareal y aves costeras obtenidas en tres muestreos efectuados en Las Cruces y Montemar.
Especies
Peces intermareales
Girella laevifrons
Auchenionchus spp.
Calliclinus geniguttatus
Scartichthys viridis
Graus nigra
Aphos porosus
Gobiesox marmoratus
Helcogrammoides spp.
Hypsoblennius sordidus
Ophiogobius jenynsi
Myxodes spp.
Aves costeras
Haemotopus palliatus
Larus dominicanus
Numenius phaeopus
Haemotopus ater
Pelecanus thagus
Leucophaeus modestus
Phalacrocorax brasilianus
Muscisaxicola maclovianus
Egretta thula
Aphriza virgata
Lessonia rufa
Cinclodes sp.
Las Cruces
Montemar
AN ± DE
AR
AN ± DE
AR
8,33 ± 14,43
2,67 ± 3,06
0,33 ± 0,58
13,33 ± 12,58
17,9
5,7
0,7
28,6
28 ± 15,13
0,67 ± 1,15
86,6
2,1
0,33 ± 0,58
2,67 ± 4,62
13,67 ± 14,57
0,33 ± 0,58
3,33 ± 5,77
1,67 ± 2,89
0,7
5,7
29,3
0,7
7,1
3,6
2,33 ± 4,04
1,33 ± 2,31
7,2
4,1
4,33 ± 4,04
10,33 ± 3,51
2,67 ± 2,31
0,67 ± 1,15
0,33 ± 0,58
0,67 ± 1,15
0,67 ± 0,58
16,0
38,3
9,9
2,5
1,2
2,5
2,5
3,33 ± 4,16
3,67 ± 4,73
0,33 ± 0,58
12,3
13,6
1,2
0,67 ± 0,58
28,33 ± 3,06
2,33 ± 3,21
2,5 ± 3,54
2,33 ± 3,21
5,33 ± 3,79
32,67 ± 5,13
2,67 ± 2,31
3,67 ± 6,35
0,8
35,3
2,9
2,1
2,9
6,6
40,7
3,3
4,6
0,67 ± 1,15
0,8
De los peces recolectados en la zona intermareal y
que fueron revisados sus parásitos, sólo cinco especies
tenían alguno de los taxa parasitarios encontrados en
crustáceos (Tabla 4). Los digeneos opecoélidos,
estuvieron representados por Helicometrina c.f. nimia,
y se encontraron principalmente en Calliclinus geniguttatus y Auchenionchus microcirrhis, mientras que
Cystidicolidae se encontró en G. nigra y G. laevifrons
(Tabla 4). De las cuatro especies de aves disectadas,
solo la gaviota L. dominicanus tenía digeneos Microphallidae en baja abundancia y prevalencia (Tabla 4).
DISCUSIÓN
Este estudio ha revelado que los crustáceos
decápodos del intermareal de Chile central albergan a
digeneos (Opecoelidae y Microphallidae) y nemátodos
(Cystidicolidae) larvales, lo que indicaría que son
hospedadores intermediarios de estos parásitos. La
abundancia de parásitos en crustáceos estuvo asociada
a cada especie de crustáceo, la LCT y localidad. Sin
embargo, se encontró escasa asociación con la
abundancia de sus hospedadores intermediarios y
definitivos.
Los crustáceos decápodos pueden acumular
parásitos a medida que crecen, es decir a mayor LCT,
ya que tanto las metacercarias como las larvas de
nemátodos no pueden salir del crustáceo hospedador ni
continuar un desarrollo más avanzado y necesariamente
requieren de otra especie de hospedador para completar
su ciclo de vida. La alta abundancia de parásitos en LC,
especialmente de metacercarias, no estuvo relacionada
con la abundancia de hospedadores definitivos, por lo
tanto podría estar relacionada con la composición de
especies de los hospedadores intermediarios primarios,
como por ejemplo gasterópodos (Hechinger &
Lafferty, 2005; Hansen & Poulin, 2006; SantiagoBass & Weis, 2008; Levakin et al., 2013). Es posible
que en LC exista mayor diversidad de gasterópodos y
que a su vez estén más parasitados con digeneos, de este
modo habría larvas cercarias parasitando constantemente a los crustáceos. Sin embargo, las condiciones
del ambiente tienen un rol importante en la transmisión de cercarias, ya que son dependientes de la
735
10
temperatura, luminosidad, salinidad y desecación (e.g.,
Lowenberger & Rau, 1994; Kumar, 1999; Poulin,
2005; Lei & Poulin, 2011), todas condiciones muy
variables en el ambiente intermareal.
En este estudio, no se efectuaron mediciones de
temperatura en las pozas intermareales muestreadas,
pero se tiene datos tomados en la Estación
Meteorológica de Montemar, Bahía de Valparaíso
(http://cienciasdelmar.cl/weather/), a 400 m de la costa
en los primeros 6 m de profundidad. La temperatura en
ese lugar varió de 11,64 a 13,32°C entre julio y
septiembre de 2013, meses en que se realizó los
muestreos de crustáceos. Algunos análisis previos (no
mostrados en este estudio), indicaron que las
prevalencias de metacercarias son similares entre los
meses de muestreo, pero la abundancia de metacercarias de Opecoelidae tienden a aumentar entre julio y
septiembre, mientras que las de Microphallidae tienden
a disminuir. Estas observaciones se podrían asociar al
aumento de temperatura en primavera, que podría
afectar positiva o negativamente la liberación de
cercarias y por lo tanto, afectar la infección en los
crustáceos (Poulin, 2005).
Para determinar la transmisión de parásitos a través
de la dieta, desde los hospedadores intermediarios
(presas) hacia los definitivos (depredadores), es
trascendental que exista un enlace trófico entre ellos.
Esto se determinó entre los grupos de hospedadores
considerados en este estudio, específicamente en
aquellos vertebrados que eran hospedadores definitivos
de los tres taxa parasitarios utilizados. Los peces
Auchenionchus spp. y Gobiesox marmoratus son los
que consumen un mayor porcentaje de crustáceos
decápodos (Muñoz & Ojeda, 1998; Quijada & Cáceres,
2000), y son estos hospedadores los que tienen mayor
abundancia o prevalencia de digeneos Opecoelidae
(particularmente Helicometrina c.f. nimia). Los crustáceos decápodos, P. tuberculosus, P. tuberculatus y P.
violaceus, forman parte de la dieta de estos peces, y
además presentaron las mayores abundancias y
prevalencias de metacercarias de Opecoelidae, lo que
indica que estos crustáceos serían hospedadores
intermediarios para esta familia de digeneos.
En el caso de Microphallidae, es posible que exista
cierta especificidad de las cercarias por sus
hospedadores intermediarios, dada la relación indirecta
entre abundancia de las especies de crustáceos con la
carga parasitaria de estas metacercarias. Los crustáceos
P. tuberculosus, P. tuberculatus, P. laevigatus y P.
barbiger, tuvieron la mayor abundancia y prevalencia
de metacercarias de Microphallidae, lo que indicaría
que actúan como segundos hospedadores intermediarios para esta familia de digéneos. Además, varias
especies de crustáceos son depredadas por aves marinas
comunes de la costa de Chile central como la gaviota
común L. dominicanus, en la cual varias especies de
Petrolisthes conforman parte de su dieta (Bahamondes
& Castilla, 1986). La gaviota común además presenta
digéneos Microphallidae (Maritrema y Microphallus)
en estado adulto (Cremonte & Martorelli, 1998;
González-Acuña et al., 2009), lo cual también fue
corroborado en el presente estudio (Tabla 4).
Igualmente, en la costa de Buenos Aires (Argentina) se
ha registrado digeneos de la familia Microphallidae en
la gaviota cahuil, Larus maculipennis (Etchegoin &
Martorelli, 1997). Sin embargo, en Chile esta gaviota
no presenta registros de digéneos microfálidos, lo que
se debería a que los estudios parasitológicos han sido
realizados exclusivamente en ambientes dulceacuícolas y
estuarinos.
A pesar que estos antecedentes señalan a los
crustáceos decápodos como hospedadores intermediarios
de digeneos y nemátodos, las prevalencias y abundancias
de estos parásitos no estuvieron asociadas a las
abundancias de sus hospedadores definitivos (peces y
aves). Esto demuestra que altas cargas de parásitos en
sus hospedadores intermediarios no implica que estén
en lugares con altas abundancias de hospedadores
definitivos, especialmente al relacionarlos con hospedadores secundarios (e.g., digéneos en crustáceos).
Existen algunos estudios, que han encontrado
relaciones significativas entre la abundancia de
hospedadores definitivos y la prevalencia o abundancia
de parásitos en hospedadores intermediarios (e.g.,
Fredensborg et al., 2006; Zambrano & GeorgeNascimento, 2010), pero esto depende del ciclo de vida
de los parásitos y de su transmisión. Por ejemplo, las
aves que tienen digeneos adultos pueden liberar los
huevos del parásito en las heces, en zonas intermareales
donde se encuentran los hospedadores intermediarios,
es decir gasterópodos que son directamente infectados
con larvas miracidios de digeneos que eclosionan de los
huevos. En este caso, la abundancia de aves se
relacionaría significativamente con la prevalencia de
digeneos (estado de esporoquistos) presentes en los
gasterópodos (Fredensborg et al., 2006). Otros estudios
en cambio, relacionan las abundancias del primer y
segundo hospedador intermediario en relación al nivel
del parasitismo de digeneos (Hansen & Poulin, 2006),
pero solo con uno de los hospedadores suele haber
relaciones positivas, mientras que en otros la abundancia o densidad de los hospedadores no se relaciona con
las cargas parasitarias. Además, los niveles de
parasitismo pueden cambiar en el espacio aun cuando
se trate de pocos metros o muchos kilómetros de
distancia (Smith, 2001). Lo mismo ocurre en el tiempo,
lo que indica que la transmisión de parásitos si bien es
dependiente de la presencia de sus hospedadores, estos
736
11
Tabla 4. Número de especímenes recolectados (n), prevalencia (PRE, %) y abundancia (ABU) promedio de parásitos en
hospedadores muestreados en Las Cruces y Montemar, presente estudio y datos de otras fuentes bibliográficas. * Digeneos
registrados, pero sin datos numéricos.
Las Cruces
Opecoelidae en peces
A. crinitus
A. microcirrhis
A. porosus
C. geniguttatus
G. laevifrons
G. marmoratus
H. chilensis
H. cunninghami
H. sordidus
M. viridis
M. cristatus
O. jenynsi
S. viridisCystidicolidae en peces
G. laevifrons
G. nigra
B. chilensis
Microphallidae en aves
P. thagus
L. dominicanus S. variegata
P. brasilianus
n
PRE
ABU
3
5
1
1
25
8
10
2
1
2
3
10
40
25
0
60
0
100
0
0
10
0
0
0
0
0
0
0
0
8,8
0
2
0
0
0,1
0
0
0
0
0
0
0
Montemar
Otras fuentes
n
PRE
ABU
1
100
5
28
0
0
7
28
4
0
3,6
25
0
0,04
1,8
2
4
1
1
0
25
0
0
0
0,25
0
0
no son el factor principal para el éxito de su transmisión
en la zona intermareal de Chile central, sino que
dependen de una combinación de múltiples factores;
varios de ellos serían factores estocásticos del ambiente
y clima. De hecho, el ambiente intermareal se
caracteriza por ser muy variable, en cuanto a
temperatura, lluvias y oleaje. Más aún, la transmisión
de parásitos larvales desde los hospedadores
intermediarios a los definitivos es pasiva, a través de la
depredación, lo cual también está sujeto al azar.
El espectro trófico del hospedador definitivo
también sería un factor relevante para comprender la
relación entre lo que come y su carga parasitaria. Por
ejemplo, la gaviota L. dominicanus depreda sobre una
amplia diversidad de presas, como moluscos,
crustáceos, peces y otros (Bahamondes & Castilla,
1986), siendo una especie generalista y oportunista
(Yorio & Bertellotti, 2002). Al alimentarse de una
amplia gama de organismos, la depredación de
crustáceos dependerá de la abundancia de estos con
respecto a otros ítems dietarios y de la facilidad de
capturarlos. Por lo tanto, la posibilidad de depredar
sobre una presa parasitada disminuye si hay mayor
variedad de alimento, lo cual disminuiría la transmisión
de sus parásitos a hospedadores definitivos. Conse-
Referencias
n
PRE
ABU
26
141
61,5
53,2
3,6
5,7
7
500
260
826
14,3
0,2
17,7
0,1
0,4
0,01
2,07
0,01
Muñoz & Castro (2012)
Muñoz & Delorme (2011)
Muñoz (2014)
Muñoz & Delorme (2011)
56
40
23
14
1,4
43
30
86
0,02
1
1,1
15
Muñoz-Muga & Muñoz (2010)
Muñoz et al. (2004)
90
*
*
González-Acuña et al. (2009)
cuentemente, si bien L. dominicanus es uno de los
hospedadores definitivos de Microphallidae, la
infección de parásitos no necesariamente se relaciona
con la abundancia de sus hospedadores definitivos.
En suma, las especies de crustáceos decápodos del
intermareal rocoso de Chile central albergan endoparásitos; digeneos de la familia Opecoelidae y Microphallidae, y nemátodos de la familia Cystidicolidae. Sus
hospedadores definitivos son vertebrados, peces y aves,
que habitan comúnmente la zona del intermareal. A
pesar que es lógico pensar que existe una relación entre
las abundancias de sus hospedadores definitivos con la
abundancia de hospedadores intermediarios y sus
cargas parasitarias, tales relaciones no fueron
evidenciadas en este estudio. Es decir, la transmisión de
parásitos está regulada por numerosos factores
abióticos, por lo tanto encontrar los factores determinantes implica enfocarse en las características del
hábitat o cambios del clima. Futuras investigaciones
podrían dirigirse a determinar los ciclos de vida de cada
uno de los taxa parasitarios registrados en el presente
estudio, lo cual resulta esencial para entender la
dinámica de transmisión de parásitos en un ambiente
tan dinámico y variado como es el intermareal rocoso.
737
12
AGRADECIMIENTOS
Se agradece el financiamiento del proyecto FONDECYT
REGULAR 1130304 adjudicado por MG-N.
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Lat. Am. J. Aquat. Res., 43(4): 739-744, 2015
Copepod Acartia tonsa on the feeding of common snook
739
Research Article
Inclusion of copepod Acartia tonsa nauplii in the feeding of
Centropomus undecimalis larvae increases stress resistance
Wanessa de Melo-Costa1,2, Cristina Vaz Avelar de Carvalho2, Gabriel Passini2
Andressa Teles3, Manuela Sozo-Cecchini4 & Vinicius Ronzani-Cerqueira2
1
Fundação Instituto de Pesca do Estado do Rio de Janeiro (FIPERJ)
Guaratiba, Rio de Janeiro-RJ, CEP 23032050, Brasil
2
Universidade Federal de Santa Catarina (UFSC), Laboratório de Piscicultura Marinha
Servidão dos Coroas, s/n, Barra da Lagoa, CEP 88061600, Florianópolis-SC, Brasil
3
Centro de Investigaciones Biológicas del Noroeste S.C., La Paz, Baja California, México
4
Universidade Federal de Santa Catarina, Laboratório de Reprodução e Desenvolvimento Animal Trindade
CEP 88040900, Florianópolis-SC, Brasil
Corresponding author: Wanessa de Melo-Costa ([email protected])
ABSTRACT. This research represents the first result of studies of the common snook Centropomus undecimalis
larvae from broodstock matured in captivity in Brazil. The aim of this study was to evaluate if the inclusion of
Acartia tonsa nauplii improves stress resistance of common snook larvae. The larvae were fed with: rotifers
Brachionus plicatilis (10 to 15 mL-1); A. tonsa nauplii (0.25 to 0.5 mL-1) and rotifers (5 to 7.5 mL-1), and A.
tonsa nauplii (0.12 to 0.25 mL-1). The average percentage of survival of the treatments was 11.9%. At 20 days
of age, larvae were subjected to thermal stress. Subsequently, the stress resistance was evaluated. Common
snook larvae fed B. plicatilis+A. tonsa reached a higher weight and length (7.5 ± 0.00 mg and 9.1 ± 0.23 mm,
respectively) and resisted more heat stress (87.4%) than larvae fed other foods, indicating that the feed mixture is
satisfactory as a starter diet for larvae of common snook. However, more research is needed to confirm these results.
Keywords: crustacean, common snook, larviculture, live feed, marine fish, aquaculture.
La inclusión de nauplios del copépodo Acartia tonsa en la alimentación de larvas
de Centropomus undecimalis aumenta su resistencia al estrés
RESUMEN. Esta investigación constituye el primer resultado de los estudios de larvas de róbalo blanco
Centropomus undecimalis a partir de reproductores maduros mantenidos en cautiverio en Brasil. El objetivo de
este estudio fue evaluar si la inclusión de nauplios de Acartia tonsa mejora la resistencia al estrés de las larvas
de róbalo blanco. Las larvas se alimentaron con rotíferos Brachionus plicatilis (10 a 15 mL-1); nauplios de A.
tonsa (0,25 a 0,5 mL-1) y rotíferos (5 a 7,5 mL-1), y nauplios de A. tonsa (0,12 a 0,25 mL-1). El promedio de
supervivencia de los tratamientos fue 11,9%. A los 20 días de edad, las larvas fueron sometidas a estrés térmico.
Posteriormente, se evaluó la resistencia al estrés. Las larvas de róbalo blanco alimentadas con B. plicatilis+A.
tonsa alcanzaron un mayor peso y longitud (7,5 ± 0,00 mg y 9,1 ± 0,23 mm, respectivamente) y resistieron más
al estrés por calor (87,4%) que las larvas alimentadas con los demás alimentos, lo que indica que la mezcla de
alimentación es satisfactoria como una dieta inicial para larvas de róbalo blanco. Sin embargo, se necesita más
investigación para confirmar estos resultados.
Palabras clave: crustáceos, róbalo blanco, larvicultura, alimento vivo, peces marinos, acuicultura.
INTRODUCTION
Studies on the common snook Centropomus
undecimalis larvae have been conducted primarily in
the United States, Mexico and Brazil, with eggs obtai__________________
Corresponding editor: Mauricio Laterça
ned from wild breeding mature specimens and recently
by Mote Marine Laboratory using fish in captivity
(Yanes-Roca & Main, 2012).
The results of Centropomus spp. larviculture in
Brazil are described for the fat snook, C. parallelus.
740
However, in Brazil, for the common snook, this is
the first result of experiments with larvae obtained from
captive breeding as the only work done previously with
larvae of the common snook was from spawning wild
breeding specimens (Soligo et al., 2011).
One important factor in the production of juvenile
marine fish is the live food supply during incubation,
because they stimulate food intake and secretion of
enzymes, resulting in continued growth and good
survival (Chang et al., 2006).
Marine copepods are sources of protein, lipids
[especially the highly unsaturated fatty acids
eicosapentaenoic acid (EPA) 20:5 n-3 and
docosahexaenoic acid (DHA) 22:6 n-3], carbohydrates,
and enzymes which are essential for the survival,
growth and digestion; metamorphosis of larvae
developing central nervous system, maintaining the
structure and function of the cell membrane and the
development and operation of the vision and stress
tolerance (Sargent et al., 1997; Støttrup, 2000) and
these advantages are important as live food for growing
fish.
Acartia tonsa is one of the most studied species of
copepods. Their nauplii are between 65 and 120 mm
wide and 106 to 250 mm length and can be fully
digested by fish larvae (Schipp et al., 1999). They are
used in farmed fish because they are effective in the
first feeding (Schipp et al., 1999), as when fed with a
mixture of microalgae, these copepods are an excellent
source of highly unsaturated fatty acids in the polar
lipid fraction, which are biologically available to the
larvae and are a source of antioxidants, astaxanthin and
vitamins C and E (Schipp et al., 1999). The ability to
incorporate essential fatty acids for marine fish larvae
through their phytoplankton diet may be the answer to
the success of copepods as live food (Sargent et al.,
1997; Støttrup, 2000). Arachidonic acid (ARA) and
EPA-derived eicosanoids are involved in the
physiological response to stress and, probably, the
optimum ratio of EPA: ARA found in copepods allows
fish larvae to cope better with stressful situations
(McEvoy & Sargent, 1998).
In the larviculture of genus Centropomus spp., the
survival rate is generally low, and a higher incidence of
deaths occurs in the first week because of the difficulty
of adapting to the first food (Yanes-Roca & Main,
2012). Successful larval rearing in the early days is a
key for the production of fish species of commercial
importance (Cara et al., 2005).
Quantifiable indicators of stress have been sought
by farmers to monitor the impact of the conditions and
management in hatcheries (Cara et al., 2005) and
different stress resistance tests are used for this
purpose: the confinement (Arends et al., 1999),
temperature variations and exposure to low levels of
oxygen (Tago et al., 1999), osmotic shock (Van Anholt
et al., 2004), exposure to air (Van Anholt et al., 2004;
Luz & Portella, 2005) used in both the larval fish in
freshwater and saltwater/brackish.
The initial diet of common snook larvae is not well
defined. Several studies have shown that inclusion of
copepods in the initial larval diet of the common snook
and fat snook is positive (Barroso et al., 2013; YanesRoca & Main, 2012); however, it is still necessary to
prove that copepods increase resistance to stress, to
improve the survival and growth when producing
captive specimens. Therefore, the aim of this study was
to evaluate if the thermal stress resistance of C.
undecimalis larvae increases by introducing copepod A.
tonsa nauplii in their food.
This study was conducted at the Laboratório de
Piscicultura Marinha (LAPMAR), Universidade
Federal de Santa Catarina (UFSC), Florianópolis, SC.
The experiment was approved by the Ethics Committee
on the Use of Animals/UFSC (Protocol PP00861).
The common snook broodstock were kept in
circular tanks of 36,000 L in a water recirculation
system. To induce spawning, we chose two males that
had a fluidity of sperm and one female that had oocytes
with an average of 350 µm based on the study of IbarraCastro et al. (2011). The eggs were quantified by a
volumetric method and transferred to fiberglass tanks
with a 100-L useful volume at a density of 15 eggs L-1.
The hatching rate was 98%.
The rotifer B. plicatilis (average size of 120 to 300
µm) was cultured in seawater salinity 35, average
temperature of 26°C and fed once a day with algae
Nannochloropsis oculata (300x104 cells mL-1) and
baker's yeast Saccharomyces cerevisiae (0.8 g 106
rotifers, divided into three parts, which are offered at 9,
13 and 17 h). Before offering larvae, rotifers were
enriched with a commercial emulsion Protein Selco®
Plus, INVE, Belgium (150 g m-3 for 12 h), to improve
the nutritional quality.
Broodstock A. tonsa were obtained from a pond
filled with water of Lagoa da Conceição (FlorianópolisSC) that was filtered through a 100 µm mesh in a
system of air lift (Barroso et al., 2013). Copepods were
isolated, identified and cultured in the laboratory in
fiberglass tanks with 250 L of seawater salinity 35, an
average temperature of 28°C, to obtain the nauplii, with
modified methods (Støttrup et al., 1986). The feeding
of the copepods was conducted with three species of
microalgae in the exponential phase of growth:
Chaetoceros calcitrans, Isochrysis galbana and N.
oculata (500; 400 and 300x104 cells mL-1, respectively), which are microalgae that contain essential fatty
acids for marine fish larvae.
Larviculture took place in the green water system,
to which microalgae N. oculata were added daily in the
tanks while maintaining a density of 500x104 cells mL-1
in fiberglass tanks, circular, with a working volume of
100 L. The renewal of the water of the experimental
units began at 9 days, with 50% until day 13, when they
went to 100% at the end of the experiment.
The larvae were fed from 2-days-old until 19 with
three different diets: 1) rotifers B. plicatilis density 10
to 15 rotifers mL-1; 2) A. tonsa copepod nauplii, density
between 0.25 and 0.5 nauplii mL-1; and 3) rotifers (5 to
7.5 mL-1) + A. tonsa nauplii (0.12 to 0.25 mL-1), half of
the density of each regime. Table 1 contains the
densities and periods in which larvae were offered.
Rotifers were offered to the larvae on the second day
after hatching at the same density used by Ibarra-Castro
et al. (2011).
The number of nauplii and rotifers was measured
once a day, in the morning, to keep the determined
density for each experimental treatment in the hatchery
tanks. With the help of a 100-mL flask, a water sample
was taken from each tank; a sub-sample of 1 mL and
lugol was examined under a microscope to count the
number of organisms.
Temperature (27 ± 1ºC), salinity (34) and dissolved
oxygen (7.3 ± 0.7 mg L-1) remained controlled during
the experimental period and at levels considered
optimal for the species (Yanes-Roca & Main, 2012). A
photoperiod of 10 h light: 14 h dark was maintained.
At the end of the experiment, the wet weight (mg)
of 24 larvae was measured with a precision balance.
The total length (mm), the percentage of larvae with gas
bladder and the notochord flexion of larvae was
measured directly with the aid of a stereomicroscope.
The stress test by heat shock was carried out on 20day-old larvae. Before the test, common snook larvae
741
went through a period of food deprivation for 3 h, in
containers of 5 L of sea water, with the same conditions
of salinity, dissolved oxygen and temperature in which
they were maintained in a 100-L tank hatchery.
Twenty-nine larvae from each treatment in triplicate
were carefully removed from each tank, with the aid of
a 500-mL vessel. The larvae were then placed in a 5-L
vessel (containing the same water in which they were
in before the temperature of 27°C) with a sieve therein
to be transferred to a vessel containing seawater at
37°C. Acute heat shock (27 to 37°C) lasted 10 min.
Then, larvae were carefully returned to their original
containers at 27ºC. Twenty-four hours later the survival
was evaluated for the definition of stress resistance rate
(Re), where (Re) = [(number of live larvae in the
container / total number of larvae in the container)] x
100 (Ako et al., 1994).
Data of wet weight (mg) and length (mm) were
tested for normality (Shapiro-Wilk test) and
homoscedasticity (Levene test) before being analyzed
by ANOVA. Then, when necessary, the means were
compared by Tukey’s test. The data of the rate of
inflation of the gas bladder and flexion notochord were
analyzed by ANOVA.
We used the nonparametric χ2 for comparison of
survival (%) after stress test between treatments. The
first test was with a 3x2 contingency table and notice
significant differences, 2x2 tables were used to find the
differences. All analyses were performed using α =
0.05.
RESULTS
The common snook larval survival reared with different
foods was greater with a mix of rotifers B. plicatilis +
A. tonsa nauplii (13.7%) and showed higher wet
weights (mg) and lengths (mm) than other treatment
(Table 2). All larvae in the study flexed their
notochords (Table 2) between 10 and 12 days of age.
The larvae fed a mixture endured more stress after
heat shock (Table 3).
Table 1. Brachionus plicatilis and Acartia tonsa nauplii densities and periods of feeding Centropomus undecimalis larvae.
Treatment
Live feed
Rotifer
B. plicatilis
Nauplii of copepod
A. tonsa
Mix
Mix
Mix
Mix
B. plicatilis
A. tonsa
B. plicatilis
A. tonsa
Density (mL-1)
Period (days)
10.00
15.00
0.25
0.50
5.00
0.12
7.50
0.25
2º to 10º
11º to 19º
2º to 6º
7º to 19º
2º to 10º
2º to 6º
11º to 19º
7º to 19º
742
Table 2. Survival (S) (%), wet weight (WW) (mg), total length (TL) (mm), inflated gas bladder (IGB) (%) and flexion of
the notochord (FN) (%) in common snook Centropomus undecimalis at 19 days, fed rotifers Brachionus plicatilis (Bp);
Acartia tonsa nauplii (At) and a mixture of them (Bp + At). n = 24. Mean and standard deviation (±SD). Different superscript
letters in the same column indicate significant difference (P < 0.05).
Treatments
S
Bp
At
Bp + At
10.8
11.2
13.7
WW
Table 3. Survival (%) of 20-day-old Centropomus
undecimalis larvae 24 h after heat shock fed Brachionus
plicatilis rotifers, Acartia tonsa nauplii and a mixture of
them (n = 3). Mean and standard deviation (±SD).
Different superscript letters in the same column indicate
significant difference (P < 0.05).
Treatments
Survival
B. plicatilis rotifer
A. tonsa nauplii
Rotifer + A. tonsa nauplii
TL
IGB
FN
1.4 ± 0.00 5.3 ± 0.09 79.17 ± 0.00 100 ± 0.00
4.4b ± 0.00 7.7b ± 0.10 95.83ª ± 0.00 100 ± 0.00
7.5a ± 0.00 9.1a ± 0.23 70.83b ± 0.00 100 ± 0.00
c
4.60c ± 2.31
43.68b ± 5.69
87.36a ± 1.15
DISCUSSION
Barroso et al. (2013), comparing the same types of food
utilized in this study in the newly hatched larvae fat
snook, Centropomus parallelus, found an average
survival rate of 16.0%, with 14-day-old, and claim that
this is a tendency for other marine fish species. This
trend is explained by the fact that in the early days old
larvae of marine fish are very fragile, with low survival,
as the energy demand and protein necessary for
morphological changes such as formation of the mouth,
anus, pigmentation of the eyes, gas bladder, fins, scales
and other organs of the digestive system is very large
(Yúfera & Darias, 2007).
Epinephelus coioides larvae fed rotifers and
copepod nauplii (0.1 nauplii mL-1) increased their
growth and survival (Knuckey et al., 2005), while the
larvae of C. parallelus fed a mixture of rotifers + A.
tonsa nauplii did not differ significantly from larvae fed
rotifers or A. tonsa nauplii, reaching an average of 3.86
mm at 14 days (Barroso et al., 2013). In the present
study, common snook larvae fed mix rotifers + A. tonsa
nauplii showed higher wet weights (mg) and lengths
(mm) than other treatments.
In describing the development of the larvae of C.
undecimalis, reared in the laboratory, Lau & Shafland
(1982) found a total length of 9.5 mm. These authors
fed larvae for the first 12 days of age with natural
zooplankton (mainly copepods nauplii) and rotifers,
and then with newly hatched Artemia sp. In the present
c
ab
study, larvae fed a mixture of rotifers + A. tonsa nauplii
until 19 days old, reached growth levels of larvae also
fed Artemia sp. (Lau & Shafland, 1982). This suggests
an alternative to the use of Artemia for the larval period
studied, since from the point of view of nutrition, a diet
based on rotifers and Artemia can be completely
replaced or supplemented with the use of copepods
compared to the fatty acid profile of the composition of
these animals, which meets the needs of the larval fish
(Sargent et al., 1997).
Gas bladder formation allows to vertically displacing the larvae in the water column while the bending
of the notochord is a prerequisite for the formation of
the fin, which is important in swimming horizontally
(Barroso et al., 2013). Although mixed treatment larvae
had a higher growth, higher gas bladder inflation was
seen in the treatment with nauplii larvae of A. tonsa.
This can be explained because the other treatments had
enriched rotifers, when they are offered to the larvae
take with them the enriching which is mainly composed
of fatty acids which may form a layer on the surface of
the water making it difficult to capture the air larvae at
the time of inflating gas vesicle. Larvae that do not
inflate their gas bladders are less resistant to stresses
such as handling, hypoxia and weaning (Chatain,
1989), but the larvae fed a mixture endured more stress
after heat shock, because stress resistance is also related
to the quality of the food (Luz, 2007) and in this study,
the mixture is described as an option ensures greater
resistance.
Osteological development studies of C. undecimalis
larvae, grown in the laboratory, found that the bending
of the notochord occurred between 10 and 14 days old
and 4.4 mm (Potthof & Tellock, 1993). In the present
study all larvae flexed their notochords between 10 and
12 days of age.
Heat shock at 10°C applied to the common snook
larvae showed significant differences depending on the
type of food (P < 0.05) and resistance to stress after 24
h. The larvae fed nauplii of A. tonsa were more resistant
than larvae fed rotifer only. According to Watanabe et
al. (1983), malnourished fish do not survive in extreme
conditions compared to properly fed fish. In the
intensive hatchery, animal stress is constant (Luz,
2007) and feeding interferes in larval resistance to
stress (Luz, 2007), as verified by Ako et al. (1994),
when it increased the amount of fatty acids in Artemia
to feed the larvae of Mugil cephalus and realized that
they became more resistant to stress responses.
The advantages of copepods relative to other
organisms as feed have been found in several studies.
These results clarify the benefits observed with regard
to resistance to heat stress and common snook larvae
growth. The stress response may represent an important
tool for selecting the best organisms for aquaculture,
especially those raised in intensive systems (Lima et
al., 2006). Furthermore, it has been shown that
organisms, which have induced thermotolerance, also
show increased resistance to other forms of stress
(Spees et al., 2002).
In this study, we conclude that the mix of rotifers
and copepods of Acartia tonsa nauplii provided greater
common snook larval growth and increased resistance
to heat stress. However, more research is needed to
confirm these results.
ACKNOWLEDGEMENTS
This research is part of "Development of systems for
breeding and growth out of common snook
(Centropomus undecimalis) in freshwater and marine
shrimp farms" project, funded by the National Council
Scientific and Technological Development (CNPq) and
the Ministry of Fisheries and Aquaculture (MPA). The
authors thank the Coordination of Improvement of
Higher Education Personnel (CAPES) for the
scholarship granted to the first author, in the Amazon
Blue Program and CNPq for the research fellowship
awarded to Professor Vinicius Cerqueira and the
scholarships to the third and fifth authors. We
acknowledge the collaboration of Professor Mauro de
Melo Júnior to help identify the copepods used in this
research. We also thank the technicians and students
LAPMAR help in the logistics of fish reproduction.
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Lat. Am. J. Aquat. Res., 43(4): 745-754, 2015 Chemical composition of Cryphiops caementarius
745
Research Article
Chemical composition of the freshwater prawn Cryphiops caementarius
(Molina, 1782) (Decapoda: Palaemonidae) in two populations in northern Chile:
reproductive and environmental considerations
Jorge E. Moreno-Reyes1, Carlos A. Méndez-Ruiz1, Gina X. Díaz1
Jaime A. Meruane2 & Pedro H. Toledo2,3
1
Programa de Magister en Acuicultura, Departamento de Acuicultura, Facultad de Ciencias del Mar
Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile
2
Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Católica del Norte
Larrondo 1281, Coquimbo, Chile
3
Centro de Estudios Avanzados en Zonas Áridas, CEAZA, Coquimbo, Chile
Corresponding author: Jorge E. Moreno ([email protected])
ABSTRACT. Reductions of its natural populations have led to recent efforts in small-scale aquaculture of the
freshwater prawn Cryphiops caementarius, either for conservation or commercial purposes. However, the lack
of knowledge about its nutritional requirements has been one of the major obstacles for its successful culture.
Given its importance, this study determines and compares the chemical composition (moisture, ash, crude
protein, total lipids and nitrogen free extract) of whole animals and main storage tissues (gonad, hepatopancreas
and muscle), of C. caementarius adult prawns from two natural populations. Moreover, the relation of this
composition with reproductive and environmental parameters (sex, maturation and habitat) is discussed. The
specimens were collected in Limarí and Choapa rivers (Coquimbo, Chile) during reproductive season, and
divided into six categories according to capture location, gonad maturation stage, and gender. The chemical
composition of whole animals and storage tissues was compared among categories. Significant differences were
observed between tissues, sexes, maturity stages and locations. Regarding tissues, the muscle and the gonads
were rich in protein, whereas the hepatopancreas had high lipid content. According to results, factors such as
sex, habitat and stage of gonad maturation can modify the biochemistry of C. caementarius. Nonetheless, the
main chemical variations were observed in tissues involved in regulatory processes (hepatopancreas and
gonads), and to a lesser extent in structural tissues (muscle). This is the first study known that reports information
about the biochemistry of C. caementarius and its findings may be useful to improve feeding practices in
aquaculture.
Keywords: Cryphiops caementarius, chemical composition, nutritional requirements, protein, lipids, storage
tissues.
Composición química del camarón de río Cryphiops caementarius (Molina, 1782)
(Decapoda: Palaemonidae) en dos poblaciones del norte de Chile: consideraciones
reproductivas y ambientales
RESUMEN. El detrimento de las poblaciones naturales de Cryphiops caementarius ha conducido a un reciente
esfuerzo para implementar actividades de acuicultura a pequeña escala con fines de repoblamiento y
comerciales. Sin embargo, la falta de conocimiento de sus requerimientos nutricionales ha sido uno de los
mayores obstáculos para el éxito de su cultivo. Dada su importancia, este estudio determina y compara la
composición química (humedad, ceniza, proteína cruda, lípidos totales y extractos libres de nitrógeno) de
animales enteros y tejidos (gónada, hepatopáncreas y músculo) de especímenes adultos de C. caementarius
provenientes de dos poblaciones naturales. Los animales fueron capturados en los ríos Limarí y Choapa
(Coquimbo, Chile) durante su estación reproductiva natural y clasificados en seis categorías de acuerdo al sexo,
estado de madurez gonadal y lugar de captura. Se determinaron diferencias significativas entre tejidos, sexos,
estados de madurez y lugares de captura. Con respecto a los tejidos, los valores más altos de proteína se encon-
________________
Corresponding editor: Luis Miguel Pardo
746
traron en el músculo y la gónada, mientras que los de lípidos se encontraron en el hepatopáncreas. De acuerdo
a los resultados obtenidos, factores como el sexo, estado de madurez gonadal y lugar de procedencia de los
animales, pueden modificar la composición química de C. caementarius. No obstante, la principales variaciones
ocurren en tejidos involucrados en procesos regulatorios (gónada y hepatopáncreas) y en menor medida en
tejidos estructurales (músculo). Este es el primer estudio de la composición química de C. caementarius, y sus
resultados podrían ser utilizados para mejorar las prácticas de alimentación en actividades de acuicultura.
Palabras clave: Cryphiops caementarius, composición química, requerimientos nutricionales, proteína, lípidos,
tejidos de almacenamiento.
INTRODUCTION
Cryphiops caementarius (Molina, 1782) commonly
known in Chile as the northern river prawn, is one of
the most important freshwater resources and the only
species of the Palaemonidae family present in Chilean
inland waters (Jara et al., 2006; Meruane et al., 2006).
However, indiscriminate extraction due to its economic
importance and anthropogenic alterations of its habitat,
have reduced its natural populations, putting this
species in danger of extinction in some locations within
its natural distribution range (Jara et al., 2006).
This situation, has encouraged researchers to
investigate the biology and the culture requirements of
the species (Castro, 1966; Bahamonde & Vila, 1971;
Norambuena, 1977; Viacava et al., 1978; Rivera &
Meruane, 1994), with the aim of establish artificial
culture systems that allow to recover its natural
populations. Nevertheless, the difficulty to satisfy its
environmental requirements in captivity due to its
complex life cycle, along with high cannibalism
behavior during mating season and other issues
commonly related with nutritional deficiencies (e.g.,
high mortality rates during ecdysis and low
reproductive performance) has delayed the successful
culture of C. caementarius under controlled conditions.
Since chemical composition analysis is considered
an appropriate way to gather information about the
nutritional requirements in crustaceans, many researchers
have conducted investigations to understand how
different organs store and transfer nutrients to support
physiological events such as growth (HernándezVergara et al., 2003), reproduction (Pillay & Nair,
1973; Castille & Lawrence, 1989; Cavalli et al., 1999;
Palacios et al., 2000; Wen et al., 2001; Rosa & Nunes,
2002; Rodríguez-González et al., 2006) and
maintenance (Rosa & Nunes, 2003; Oliveira et al.,
2007; Vinagre et al., 2007). However, regarding the
biochemistry of crustaceans, it has been stated that
environmental factors such as habitat, food availability,
and seasonality can modify their metabolism (Schirf et
al., 1987; Kucharski & Da Silva, 1991; Oliveira et al.,
2003), and thus their chemical composition (Rosa &
Nunes, 2003).
Because there are no formal studies focused on the
biochemistry of C. caementarius, the objective of the
present work was to determine the chemical
composition of adult male and female prawns from two
natural populations, and to evaluate the influence of
environmental and reproductive factors on the accumulation of nutrients in main storage tissues. As the
knowledge of nutritional requirements in decapods has
been considered crucial to their successful culture in
captivity, this investigation provides basic information
about nutritional requirements of the species, in order
to improve small-scale aquaculture practices as an
alternative way to recovering natural populations.
Study of this species is important because of its social
and economic importance (Meruane et al., 2006), and
its conservation status which is reported as vulnerable
to critically endangered according to Jara et al. (2006)
and vulnerable according to the agreement 6/2014
included into the species conservation status list,
published by the ministry of environment of Chile.
All animals utilized in this research were treated with
proper care, minimizing discomfort and distress. Also,
the number of sampled animals was kept to the
minimum necessary to obtain scientific results,
balancing the gain in knowledge with the long-term
conservation and well-being of the species. The
animals were used with the permission of the Ethic and
Biotechnology Committee of the Universidad Católica
del Norte, Chile.
Biological material
Adults of C. caementarius (cephalotorax length >14.3
mm according to Bahamonde & Vila, 1971) were
extracted from Choapa (31°39′85″S, 71°9′17″W) and
Limarí (30°39′26″S, 71°31′13″W) rivers (Coquimbo,
Chile) between October 2009 and February 2010 (high
reproductive activity), and then carried alive to the
crustaceans laboratory of the Universidad Católica del
Norte in Coquimbo, Chile. Twenty males in stage III
(mature), forty eight females in stage I (immature) and
twenty females in stage IV (advanced maturity) of
Chemical composition of Cryphiops caementarius
gonadic development, along with twenty males in stage
III, fifty females in stage I and twenty females in stage
IV, extracted from Choapa and Limarí rivers, were
utilized for the analysis of chemical composition. All
animals were intermolt hard-shelled and stages of
gonadic maturation were visually identified based on
size, color and gross morphology according to the scale
proposed by Viacava et al. (1978). In the laboratory, the
prawns were divided into six categories according to
their capture location, maturation stage and sex (Table
1). Prawns were then placed into water and kept in to
the fridge (4°C) during 1 h to decrease their metabolism
before being euthanized. Immediately after, some
prawns were conserved intact for the chemical analysis
in whole animals and the others were dissected to
remove the gonads, the hepatopancreas, and the
abdominal muscle. Tissues were individually weighed
and pooled from six to eight individuals when there was
insufficient amount to perform all analyses (e.g.,
immature female gonad). Subsequently, tissue samples
and whole animals were kept in plastic bags covered
with aluminum foil and maintained at -20°C until their
chemical analysis (two weeks maximum).
Chemical composition
Moisture, ash, crude protein and total lipid contents of
gonad, hepatopancreas, abdominal muscle, and whole
animals were determined by triplicate according to the
AOAC procedures (2005). The moisture was obtained
by oven drying at 95°C to constant weight. Ash was
quantified after calcination in muffle furnace at 550°C.
Crude protein was determined using the Kjeldahl
method, with a conversion factor of 6.25. Total lipids
were determined using the Soxhlet method. Nitrogen
free extract (NFE) was calculated with the formula:
NFE = 100 - (crude protein % + total lipids % + ash %)
in accordance with Tacon (1989). The number of
samples used for analysis of whole animals and tissue
was n = 10 except for gonad tissue of LIF and CIF
where the number of samples was n = 5.
Differences in the chemical composition of tissues
analyzed and whole animals between categories (LIF,
LMF, LMM, CIF, CMF and CMM) were tested with a
one-way analysis of variance (one-way ANOVA)
followed by a multiple-comparison test (Holm-sidak)
as needed. Whenever necessary, data were transformed
to satisfy normal distribution and homoscedasticity
requirements. The data reported as percentages were
transformed to arcsine values prior to analysis (Sokal &
Rohlf, 1981). All statistical analyses were tested at the
0.05 level of probability with the software Sigma Stat
3.1 for Windows.
747
RESULTS
The chemical composition of whole animals and main
storage tissues of C. caementarius is presented in Table
2. Significant differences were detected between
tissues, sexes and locations. Regarding whole animals,
females had higher values of crude protein and total
lipids than males, but lower ash contents independently
of the stage of maturation and capture location.
Concerning tissues, the highest values of moisture were
found in abdominal muscle of the six prawn categories
(75.54-77.72%), and gonadal tissue of CIF (74.93%),
LIF (75.08%), LMM (79.58%) and CMM (80.10%).
Independently of capture location, immature females
had higher moisture levels in the gonads
(approximately 30%) than mature females.
In male and female prawns from both rivers, and
independently of the stage of maturation, the muscle
and the gonads were rich in protein, whereas the
hepatopancreas had high lipid content. The highest
values of protein in tissue were found in abdominal
muscle for the six categories ranging from 83.18 to
85.48%. Concerning sexes, muscle of C. caementarius
males had significantly more protein content than
females (Table 2). In relation to location, animals from
Choapa River had generally more protein content in
abdominal muscle than animals from Limarí River.
Independently of location, sex, or stage of
maturation, proteins were the most abundant component in gonads (50.05-74.28%), followed by lipids
(16.73-36.08%) and NFE as minor component (0.8514.10%). In contrast, in the case of the hepatopancreas,
lipids were the most abundant component (63.5372.41%), followed by proteins (16.34-22.02%) and
NFE (3.20-18.07%).
Contrary to the slight sex and location differences
observed in the chemical composition of abdominal
muscle, the variations in the chemical composition of
the gonads and the hepatopancreas showed a
remarkable relation with sex, stage of maturation and
capture location (Table 2). In the case of Limarí River,
mature female prawns showed higher protein and lipid
levels in the gonads than immature female prawns,
whereas in the hepatopancreas, the higher protein and
lipid levels were found in immature females instead of
in mature females. The same results were observed in
prawns from Choapa River, except for the hepatopancreas of mature females, where the lipid levels were
higher than those of immature females. In addition,
regarding mature animals, males from both Limarí and
Choapa rivers had higher protein levels in gonads than
females, but lower lipid levels (Table 2). With regard to
the NFE content, independently of capture location, this
748
Table 1. Categories of prawns according to their capture location, maturation stage and gender.
Categories of prawns
Limarí immature female (LIF)
Limarí mature female (LMF)
Limarí mature male (LMM)
Choapa immature female (CIF)
Choapa mature female (CMF)
Choapa mature male (CMM)
was high in the gonads but low in the hepatopancreas
and muscle of immature females, whereas in mature
females, NFE levels were low in gonadal and muscle
tissues and high in hepatopancreatic tissue.
DISCUSSION
The results presented in this work constitute the first
report of the chemical composition in whole animals
(males and females) and main storage tissues (gonads,
hepatopancreas and muscle) of adult C. caementarius
prawns. According to Dempson et al. (2004), the
proximate body composition (moisture, lipids, protein
and ash) is a good indicator of the nutritional status of
an organism. The greater the protein and lipid content
represents higher the energy density. Despite the
significant differences detected between tissues, sexes
and locations, the high protein levels found during this
study in both whole animals (from 53.58 to 63.32%)
and abdominal muscle tissue (from 83.18 to 85.48%) of
C. caementarius, suggest a good nutritional status of
wild specimens (male and female) from both Limarí
and Choapa rivers and point this species as a
remarkable source of protein for human consumption.
With regard whole animals, moisture levels in male and
female C. caementarius prawns (58-71%) were lower
than the levels reported in Macrobrachium
amazonicum (68.7-78.0%) (Meireles et al., 2013),
whereas the ash contents were higher in C.
caementarius (13.81-28.61%) than in Macrobrachium
vollenhovenii (11.5%) (Ehigiator & Oterai, 2012) but
close to the contents reported in M. amazonicum (21.121.4%) (Meireles et al., 2013). In the case of crude
protein and total lipids, the mean levels in C.
caementarius (53-63% and 15-21% respectively) were
higher than the levels reported in Macrobrachium
jelskii (34-58% and 9-11%) by Ramirez et al. (2010),
but lower than the levels reported for M. rosenbergii
(73.2-78.0% and 5.5-22.4% respectively) by Santos et
al. (2007). Concerning NFE, the levels found in C.
caementarius (0.12-3.55%) were similar to M. jelskii
(0.6-3.4%; Ramirez et al., 2010) and M. vollenhovenii
(2.50%) (Ehigiator & Oterai, 2012).
Cephalothorax
length ± SD (mm)
41.17 ± 4.06
41.86 ± 4.72
59.80 ± 2.49
41.12 ± 4.46
42.21 ± 5.66
61.41 ± 5.50
Wet
weight ± SD (g)
49.55 ± 8.76
52.21 ± 9.08
143.49 ± 20.47
51.65 ± 6.80
55.90 ± 11.12
160.52 ± 23.15
In relation to the differences in the chemical
composition between whole males and females, in
general terms males had higher ash levels than females
but lower protein and lipid levels (Table 2). These
differences in the body composition between sexes
could be associated with reproductive aspects.
According to Rojas et al. (2012) C. caementarius
mature males fight aggressively during mating season
for access to reproductive females, causing superficial
marks and puncture/crack injuries mainly on the
chelipeds. Based on this reproductive behavior, we
suggest that as occurs in other freshwater decapods,
such as Procambarus clarkii, where chelae were more
heavily mineralized than branchiostegites, and Astacus
astacus where statistical differences were detected
between sexual active and inactive animals regarding
the concentration of mineral matter in both chelae and
branchiostegites (Huner & Lindqvist, 1985), mature
males of C. caementarius may increase shell hardness
and thickness by an increase in shell calcification, in
order to reduce possible injuries during intrasexual
combats. As a result of this strategy, the mineral content
in males increases with regard to females. Therefore,
the ash levels rise while the levels of the others nutrients
(protein and lipids mainly) decrease. The presence of a
large number of robust spines in the chelipeds of mature
males (Rojas et al., 2012) in comparison to the
chelipeds of both mature and immature females (per.
obs.), implies an increase in calcium fixation and
supports this suggestion.
Regarding storage tissues, abdominal muscle had
the highest levels of protein and the lowest levels of
NFE. Protein levels in muscle (83-85%) were higher
than others palaemonids such as M. rosenbergii (74%)
(Reddy & Reddy, 2014) and M. carcinus (74-77%)
(Benítez-Mandujano & Ponce-Palafox, 2014), but
similar to commercial crayfishes such as A. astacus
(83.6-84.9%) and P. clarkii (80.7-86.8%) (Huner et al.,
1988). Although proteins can be also accumulated in
the hepatopancreas and the gonads, the high levels
found in abdominal muscle confirm this tissue as the
main protein-storage location in C. caementarius.
Concerning sexes, C. caementarius males had more
749
Table 2. Cryphiops caementarius broodstock, chemical composition in whole animals and tissues, from Limarí and Choapa
rivers. Values are the mean ± standard deviation expressed as percentage dry weight. Means in a row sharing different
superscript letters were significantly different (P < 0.05).The number of samples for whole animals and tissue analysis in
all categories was n = 10, except for gonad tissues of LIF and CIF where the number of samples for all chemical analysis
was n = 5. LIF: Limarí immature female, LMF: Limarí mature female, LMM: Limarí mature male, CIF: Choapa immature
female, CMF: Choapa mature female, CMM: Choapa mature male, NFE: Nitrogen free extract.
Whole animal
Moisture
Ash
Crude protein
Total lipids
NFE
Gonads
Moisture
Ash
Crude protein
Total lipids
NFE
Hepatopancreas
Moisture
Ash
Crude protein
Total lipids
NFE
Muscle
Moisture
Ash
Crude protein
Total lipids
NFE
LIF
LMF
LMM
CIF
CMF
CMM
70.91 ± 0.11e
17.65 ± 0.17c
60.63 ± 0.18d
21.49 ± 0.01e
0.23
65.29 ± 0.15b
19.42 ± 0.05d
61.20 ± 0.19e
19.26 ± 0.15c
0.12
58.06 ± 0.17a
25.91 ± 0.23e
55.32 ± 0.10b
15.22 ± 0.19a
3.55
70.21 ± 0.28d
17.25 ± 0.23b
60.27 ± 0.26c
20.08 ± 0.22d
2.40
66.73 ± 0.21c
13.81 ± 0.18a
63.32 ± 0.16f
21.25 ± 0.25e
1.62
71.58 ± 0.22f
28.61 ± 0.20f
53.58 ± 0.09a
17.52 ± 0.19b
0.29
75.08 ± 0.09c
7.65 ± 0.07c
50.05 ± 0.18ª
28.20 ± 0.27c
14.10
44.42 ± 0.01b
2.79 ± 0.01a
59.23 ± 0.04c
34.79 ± 0.07e
3.19
79.58 ± 0.09d
7.08 ± 0.07b
70.04 ± 0.19e
20.53 ± 0.11b
2.35
74.93 ± 0.12c
7.60 ± 0.05c
51.07 ± 0.71b
28.68 ± 0.02d
12.65
42.86 ± 0.04a
2.92 ± 0.18a
60.15 ± 0.10d
36.08 ± 0.11f
0.85
80.10 ± 0.11e
7.27 ± 0.07b
74.28 ± 0.11f
16.73 ± 0.15a
1.72
48.92 ± 0.04d
2.88 ± 0.03e
22.02 ± 0.11f
71.90 ± 0.23d
3.20
43.35 ± 0.02b
2.06 ± 0.03b
17.49 ± 0.02c
63.67 ± 0.19a
16.78
45.77 ± 0.05c
2.06 ± 0.05b
16.34 ± 0.18b
63.53 ± 0.07a
18.07
49.34 ± 0.17e
2.48 ± 0.06c
20.94 ± 0.07e
66.63 ± 0.29b
9.95
39.19 ± 0.25a
1.60 ± 0.05a
16.11 ± 0.08a
72.41 ± 0.23d
9.88
57.66 ± 0.22f
2.64 ± 0.06d
19.43 ± 0.01d
71.00 ± 0.23c
6.93
76.55 ± 0.18b
5.56 ± 0.08c
83.41 ± 0.14ª
9.03 ± 0.06b
2.00
77.32 ± 0.02bd
5.58 ± 0.03c
83.81 ± 0.12bc
10.32 ± 0.04d
0.29
77.72 ± 0.07d
5.82 ± 0.05d
84.06 ± 0.04b
10.07 ± 0.04cd
0.05
76.68 ± 0.10c
5.43 ± 0.04b
83.55 ± 0.13ac
9.88 ± 0.05c
1.14
75.54 ± 0.10a
5.08 ± 0.04a
83.18 ± 0.07a
11.39 ± 0.05e
0.35
77.67 ± 0.05d
5.81 ± 0.05d
85.48 ± 0.25d
8.50 ± 0.06a
0.21
protein content in abdominal muscle than females
(Table 2). The same result has been reported by Huner
et al. (1988) in A. astacus and P. clarkii. In addition,
concerning locations, animals from Choapa River
(male and female) had generally more protein content
in abdominal muscle in comparison to animals from
Limarí River.
Some studies in crustaceans had reported that
protein levels in whole animals (Santos et al., 2007) and
muscle tissues (Benítez-Mandujano & Ponce-Palafox,
2014) can be influenced by protein levels in diet.
Consequently the higher protein levels found in animals
from Choapa River may be related to a wide variety and
quality of food sources in this river, which is supported
by the larger flora and fauna reported in Choapa River
(SINIA, 2004a) when compared to Limarí River
(SINIA, 2004b). The influence exerted by food (e.g.,
availability, quality and nutritional composition) over
the chemical composition in crustaceans has also been
investigated in natural conditions for Aristeus
antennatus (Crustacea: Penaeidea), Parapenaeus
longirostris (Crustacea: Penaeidea) and Nephrops
norvegicus (Crustacea: Astacidea) by Rosa & Nunes,
(2002, 2003) and in culture conditions for M. jelskii by
Ramírez et al. (2010) and Litopenaeus vannamei by
Ezquerra-Brauer et al. (2003). These studies reported
variations in protein, lipid and carbohydrate contents in
relation to available diet.
In the case of total lipids in muscle, females had
generally a higher content than males, especially in
animals from Choapa River. The higher levels of total
lipids found in abdominal muscle of females in
comparison to males, has also been reported in Cancer
pagurus (Barrento et al., 2010) and M. rosenbergii
(Saravana-Bhavan et al., 2010). In addition C.
caementarius mature females had higher lipid levels in
muscle than immature females. Compared with other
species, C. caementarius had higher lipid levels in
muscle (8.5-9.3%) than M. rosenbergii (3.7-7.3%)
(Cavalli et al., 2001), M. carcinus (5.1%) (BenítezMandujano & Ponce-Palafox, 2014) and Cancer
pagurus (0.7-1.3%) (Barrento et al., 2010). The higher
750
lipid and protein levels found in abdominal muscle of
animals from Choapa River in comparison to animals
from Limarí River, may suggest that animals from
Choapa River have a better physiological condition
than animals from Limarí River. Concerning moisture
and ash contents, C. caementarius males had higher
levels in abdominal muscle than females (Table 2) as
occurs in M. rosenbergii (Saravana-Bhavan et al.,
2010).
Regarding both the gonads and the hepatopancreas,
the high moisture content and low nutrient levels (crude
protein or total lipids or NFE) found in immature
females in comparison to mature females suggest that
during ovarian cycle, these organs, and mostly the
ovary, replace the water inside with nutrients for the
vitellus. The same mechanism has also been reported in
Cherax quadricarinatus, where lipid and protein levels
in the ovary increase during vitellogenesis while
moisture levels decrease (Li et al., 2010), and in
Armases cinereum and Sesarma reticulatum where the
lipid and carbon contents in the ovary increase
throughout ovarian maturation, while water concentration decreases (Hasek & Felder, 2005). The lipid
contents found in the gonads of C. caementarius (1636%) were close to the levels reported in both M.
rosenbergii by Cavalli et al. (2001) (18-55%) and C.
quadricarinatus by Li et al. (2010) (31-37%).
However, in the case of the hepatopancreas the lipid
contents in C. caementarius (63-72%) were higher than
the contents in M. rosenbergii (41-58%) (Cavalli et al.,
2001) but similar to the contents in C. quadricarinatus
(65-77%) (Li et al., 2010). In addition, the protein
levels found in both the gonads (50-74%) and the
hepatopancreas (16-22%) of C. caementarius were
close to the levels reported in C. quadricarinatus
(gonads 65-81% and hepatopancreas 22-26%) (Li et al.,
2010). Concerning moisture, the levels found in the
hepatopancreas (39-57%) and the gonads (42-80%) of
C. caementarius male and female prawns were similar
to the levels reported in C. quadricarinatus
(hepatopancreas 42-53% and gonads 48-63%) (Li et al.,
2010). About ash contents, the levels found in the
gonads (2.70-7.65%) and the hepatopancreas (1.602.88%) of C. caementarius males and females, were
comparable to the levels reported in marine decapods
such as Homarus gammarus (gonad 3.6% and
hepatopancreas 5.2-6.1%) and Homarus americanus
(gonad 4.7% and hepatopancreas 3.6-5.4%) (Barrento
et al., 2009). On the other hand, for all analyzed tissues
(especially in the gonads) high moisture levels were
accompanied by high ash levels (Table 2). The same
result was found by Barrento et al. (2009) in the muscle,
the hepatopancreas and the gonads of marine decapods
of the genus Homarus. The apparent relation between
moisture and ash levels in storage tissues of C.
caementarius may be related to the natural characteristics of Choapa and Limarí rivers (hard waters rich in
minerals) (SINIA, 2004a; SINIA, 2004b) along with
the recognized capacity of crustaceans to accumulate
minerals in shell and soft tissues (Meador et al., 1995;
MacFarlane et al., 2000).
Concerning nutrients, it is known that proteins plays
an important role in morphogenesis and energy supply
in the embryos of decapods (Rosa & Nunes, 2003; Luo
et al., 2004). In the case of C. caementarius, the high
levels found in the gonads of mature males and females
(up to 50%), confirm proteins as the main components
of gametes and also confirm their importance in the
synthesis of egg yolk during ovarian development, as
occurs in C. quadricarinatus (García-Guerrero et al.,
2003) and M. rosenbergii (Revathi et al., 2012). This
condition suggests a high demand for proteins during
gametogenesis by C. caementarius, which is supported
evidence that protein required in several crustacean
broodstocks for maturation and production of eggs is
higher than the level required for growth (Harrison,
1990, 1997).
About total lipids, although these can also
accumulate in the gonad and muscle the high levels
found in the hepatopancreas of males and females in
comparison to the other analyzed tissues, confirm this
organ as the main lipid storage place in C. caementarius
as occurs in other crustaceans (O'Connor & Gilbert,
1968; Herreid & Full, 1988; Kucharski & Da Silva,
1991; Muriana et al., 1993; García et al., 2002).
Furthermore, it is generally established in decapods that
lipids can act as an energy source for physiological
processes such as molting and vitellogenesis, and as the
main source of metabolic energy during embryo
development (García-Guerrero et al., 2003; Yao et al.,
2006; García-Guerrero, 2009). The higher levels of
total lipids found in the gonads of mature females in
comparison to immature females reflect the importance
of lipids as an energy source in the eggs of C.
caementarius, and suggest an elevated lipid
requirement, especially in reproductive females
throughout the mating season. This suggestion is in
agreement with Harrison (1990), who reported higher
lipid requirements for crustacean maturation than for
growth and survival, and reinforced by recent studies
performed on C. quadricarinatus (Li et al., 2010) and
M. rosenbergii (Revathi et al., 2012), where a gradual
accumulation of lipids in the ovary was observed
during vitellogenesis.
In relation to NFE, the low values found in the
gonads of mature animals (male and female) suggest
that these compounds have a secondary role in the
formation of C. caementarius gametes. In contrast, the
high levels observed in the hepatopancreas may suggest
that NFE is a complementary source of energy that
supports, together with lipids, the intense reproductive
behavior documented for this species. Viacava et al.
(1978) reported daily successive mating events in males
because of their polygamous behavior whereas Moreno
et al. (2012) reported in females the capacity to remature and have successive spawning events
throughout the reproductive season. In addition, these
authors also reported in females a molting event
performed prior to spawn, which implies a high energy
demand.
In some crustaceans as in the case of the crayfish
Cherax destructor (Jones & Obst, 2000) and marine
decapods like penaeoideans (Vicent et al., 1988;
Marangos et al., 1989; Bray & Lawrence, 1990;
Palacios et al., 2000) it has been recognized the
capacity to transfer nutrients among tissues to support
the high energy demand associated to gonadal
maturation. In contrast, studies performed in marine
and freshwater species such as Penaeus vannamei
(Palacios et al., 2000; Arcos et al., 2003), M.
rosenbergii (Cavalli et al., 2001) and A. cinereum and
S. reticulatum (Hasek & Felder, 2005), suggest the
possibility of an active mobilization of nutrients from
exogenous sources (instead of the hepatopancreas) to
obtain energy compounds. In addition, Avarre et al.
(2003) also suggested that some yolk nutrients in
Penaeus indicus originate from ingested food either
directly or after storage in the hepatopancreas.
Although the nutrient mobilization among C.
caementarius main storage tissues was not directly
evaluated in this study, the previously mentioned
ability in crustaceans to get nutrients either from
exogenous food or storage tissues, and the differences
observed in this study between mature and immature
animals with regard to the chemical composition of the
gonads and the hepatopancreas (Table 2) led us to
hypothesize that C. caementarius may be able to
quickly assimilate and relocate yolk nutrient extracted
from storage tissues (mainly hepatopancreas) and/or
from exogenous food straight to the ovary, in order to
support gametogenesis. The condition observed in
animals from Limarí River, where immature females
showed higher levels of protein and lipids in the
hepatopancreas than mature females, while mature
females showed higher levels of protein and lipids in
the gonads than immature females, suggest a nutrient
mobilization from the hepatopancreas to the ovary
during gonadal maturation. On the other hand, the fact
that the lipid levels found in the hepatopancreas of
mature females from Choapa River were higher than
the levels found in immature females, suggest
751
mobilization of lipids to the ovary from exogenous
sources instead of hepatopancreas.
This hypothesis must be investigated for males and
females in future experiments to properly understand
how this species obtains and distributes the energy
necessary to support reproductive activity. In summary,
the basic nutritional requierements of C. caementarius
adults prawns include low levels of NFE (5-10%) and
high levels of both proteins (50-60%) and lipids (1020%), specially during the mating season. This
information can be used by local researchers to improve
feeding practices in future activities of reproduction
and culture under controlled conditions whether for
natural population management or commercial
purposes.
In conclusion, the results herein suggest that
reproductive behavior and environmental conditions
can modify the biochemistry of C. caementarius.
Nevertheless, the main changes occurs in tissues
involved in regulatory processes (the hepatopancreas
and the gonads), and to a lesser extent in structural
tissues (muscle).
ACKNOWLEDGEMENTS
We are grateful to Mauricio López Castillo from the
Nutrition Laboratory of the Aquaculture Department,
Universidad Católica del Norte, for his technical
assistance during the chemical analysis. This work was
made possible with funding provided by the DGIP
Research Program (10301260) of the General Direction
of Graduate Research of the Universidad Católica del
Norte, Chile.
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Lat. Am. J. Aquat. Res., 43(4): 755-765, 2015
Risk assessment of Geotrichum spp. for L. vannamei cultures
755
Research Article
Isolation and risk assessment of Geotrichum spp. in the white shrimp
(Litopenaeus vannamei Boone, 1931) from culture ponds
José Luis Ochoa1†, Norma Ochoa-Alvarez1, Maria Antonia Guzmán-Murillo1
Sergio Hernandez2 & Felipe Ascencio1
1
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Instituto Politécnico Nacional Nº195
Col. Playa Palo de Santa Rita, La Paz, BCS, 23096, México
2
Centro Interdisciplinario de Ciencias Marinas (IPN), Instituto Politécnico Nacional s/n
Col. Playa Palo de Santa Rita. La Paz BCS, 23096, México
Corresponding author: Felipe Ascencio ([email protected])
†This study is dedicated in memory of the late Prof. José Luis Ochoa
ABSTRACT. The present study was done in order to identify the fungus invading some of the supralittoral
ponds used for shrimp aquaculture in the CIBNOR facilities in La Paz, Baja California Sur (BCS), México
during the summer season. From the walls and bottoms of the ponds, two strains of Geotrichum spp. were
isolated and morphologically identified. Fungal adhesion towards hemocytes and primary cultures of various
white shrimp (Litopeneaus vannamei) tissues (gill, tegument, and gut) was analyzed to determine infectivity.
Extracellular protease, lipase, and amylase activity were evaluated as virulence factors. Survival of shrimp postlarvae (PL8) exposed to fungal culture supernatant or to their filaments was also investigated. The results showed
that shrimp tegument cells and hemocytes were very susceptible to Geotrichum spp. invasion, and that this
fungus provokes great mortality of post-larvae. Hence, Geotrichum spp. could be considered an opportunistic
pathogen that might represent a serious health risk to shrimp in culture.
Keywords: Geotrichum spp., Fusarium solani, Litopenaeus vannamei, mycotoxins, extracellular enzymes,
aquaculture.
Aislamiento y evaluación de riesgos de Geotrichum spp. en el camarón blanco
(Litopenaeus vannamei Boone, 1931) en estanques de cultivo
RESUMEN. El presente trabajo se realizó con el fin de identificar hongos que invaden algunos de los estanques
supralitorales utilizados para el cultivo del camarón en la instalación del CIBNOR, en La Paz, BCS, México durante
la temporada de verano. De las paredes y el fondo de los estanques se aislaron e identificaron morfológicamente dos
cepas de Geotrichum spp. Se analizó la adhesividad de hongos hacia cultivos primarios de diversos tejidos (hemocitos,
branquias, tegumento, e intestino) de camarón blanco (Litopeneaus vannamei) para determinar la infectividad. La
actividad de lipasas, amilasas, y proteasa extracelular, fueron evaluadas como factores de virulencia. También se
evaluó la supervivencia de post-larvas (PL8) de camarones expuestos a los sobrenadantes del cultivo o filamentos de
hongos. Los resultados muestran que las células de tegumento y hemocitos de camarón son susceptibles a la invasión
por Geotrichum spp. y que este hongo provoca gran mortalidad de post-larvas de camarón. Por lo tanto, Geotrichum
spp. puede ser considerado un patógeno oportunista que podría representar un riesgo grave para la salud de los
camarones en cultivo.
Palabras clave: Geotrichum spp., Fusarium solani, Litopenaeus vannamei, micotoxinas, enzimas extracelulares,
acuicultura.
INTRODUCTION
Shrimp aquaculture is at present an attractive economic
activity of great impact and commercial importance in
Mexico (Gillett, 2008). Unfortunately, disease incidence
__________________
Corresponding editor: Cesar Lodeiros
affects production and commercialization success.
Thus, shrimp-farming success depends on the
application of procedures aimed at preventing and
controlling the presence of pathogenic microorganisms
in the ponds. Fungi are considered opportunistic patho-
756
gens in aquaculture because they usually affect stressed
or immunocompromised animals (Pelczar et al., 2001;
Leslie & Summerell, 2006; Madigan et al., 2009).
Lightner (1996) reported 100% mortalities of shrimp
eggs and larvae exposed to Lagenidium callinectes
while other researchers found that such fungus is also
capable of infecting juvenile and adult shrimps in
culture (Bertke & Aronson, 1992; Nakamura et al.,
1994; Khoa et al., 2004, 2005; Cruz da Silva et al.,
2011). Other fungi belonging to the genera
Haliphtrofos and Sirolpidium provoke diseases in
cultured shrimp larvae (Noga, 1990); Fusarium, on the
other hand, is capable of affecting practically all
developmental stages of shrimp (Bachere et al., 2000;
Bugni & Ireland, 2004). Some toxic strains of
Fusarium have been found responsible for different
epizootic episodes in cultures of Penaeus chinensis
(Chen et al., 1992), P. californiensis (Lightner & Hose,
1984), P. stylirostris (Lightner, 1996), P. japonicus
(Lightner & Hose, 1984; Noga, 1990; Lightner, 1996),
and Litopenaeus vannamei (Cruz da Silva et al., 2011;
Lozano-Olvera et al., 2012).
Our work is the first report referring to the
pathogenicity of Geotrichum strains towards American
white shrimp Litopenaeus vannamei. This yeast like
fungus, found in soil, water, and air worldwide is a
colonizer of the intestinal tract. It may cause
opportunistic infections (geotrichosis) in immunocompromised hosts, which usually acquire it via ingestion
or inhalation (Buchta & Otcenasek, 1998). The
isolation from walls and bottom of a shrimp pond in La
Paz, Baja California Sur (BCS), México was conducted
in order to determine the pathogenicity of isolated
strains of filamentous fungi that implied a potential risk
in shrimp farming success. Pathogenic fungi were
assessed by determining their virulence and adhesive
capacity on cells in primary culture of white shrimp
(Litopenaeus vannamei).
Fungus isolation and characterization
During the preparation of experimental ponds for
cultivation of L. vannamei at Centro de Investigaciones
Biológicas del Noroeste (CIBNOR) in La Paz, BCS,
México, the presence of white spots of microbial
colonies in the walls and bottoms of the ponds were
frequently noticed. The isolation of the corresponding
microorganisms by common microbiological procedures was done (Hyde et al., 2000). The samples,
collected with a sterile scraper and poured in 250 L of
glycerol, were plated in PDA medium and incubated at
22oC for 48 h (Newell, 2001). Purification was done
streaking in various Petri dishes containing the same
culture media and stored at -80°C and -20°C until use
(Hernández-Saavedra, 1990). Microorganism identification was performed by morphological criteria using a
Nikon Optihot-2 microscope (Nikon, Japan) according
to Pitt & Hocking (1997). Distinctive morphological
characteristics for the Geotrichum genus were observed
(Pitt & Hocking, 1997; Kurtzman & Robnett, 1998;
Smith et al., 2000). Identification keys were obtained
from Tortora et al. (2012) and the identification was
done only up to genus.
Growth kinetics determination was done using 125
mL Erlenmeyer flasks containing 25 mL of M-1
medium [glucose 2% (w/v), peptone 1% (w/v), yeast
extract 0.5% (w/v)], and incubated at 25°C with
constant orbital shaking (110 rpm) according to
Hernández-Saavedra (1990). The mycelium was
recovered from the culture by filtration using Whatman
Nº1 paper, washed with distilled water, and afterwards
placed in an oven at 80°C for 24 h to get a constant
weight. The dry weight of the sample was determined
with an analytical balance (Ohaus, AP210S) and
plotted against the time of collection. The analysis of
enzymatic activity, compared with collection strains
obtained from infected shrimp; Fusarium solani
(ATCC 46940), isolated from Penaeus japonicus; and
Fusarium javanicum (CBS 420.76), isolated from
Penaeus californiensis.
Shrimp and primary shrimp tissue culture cells
The white shrimp juveniles (12-14 g) and post-larvae
(PL8) were acquired from two local commercial shrimp
farms (APSA, La Paz, BCS, México and Acuacultores
Marh, La Paz, BCS, México, respectively). Primary
cell cultures of different shrimp tissues (tegument, gill,
intestine, and hemocytes) were prepared by an
enzymatic disaggregation procedure modified from
Fuerst et al. (1991), Jackson et al. (1993), and
Alexopoulos et al. (1996). Hemocyte culture was
prepared from haemolymph obtained by puncture at the
pleopod base of the first abdominal segment near the
genital pore from juvenile shrimp, with a 1-mL syringe
(Hernández et al., 1996). Essentially, aliquots of 100
L of cell suspension (of 2.4x105 cells mL-1) were
placed in 96-wells microplate, mixed with 90 µL
Leibovitz’s L-15 complete medium containing 10%
(v/v) of fetal bovine serum (FBS), (Sigma, Chemical
Co., St Louis, USA) and incubated at 22oC in a CO2
incubator (Shel-Lab, VWR 1810) for 16 h to obtain a
primary culture. Cell counting in 100 µL aliquots of
primary cultures was done with a hematocytometer
using an Optiphot-2 microscope under the contrast
phase mode. Trypan blue staining (Sigma, Chemical
Co., USA), was carried out to estimate cell viability.
In vitro cytotoxicity assay
The in vitro cytotoxic assays were done with 100 µL of
shrimp tissue cell cultures that were first washed with
250 µL of PBS (137 mM NaCl, 0.2 mM KCl, 1.44 mM
Na2HPO4, 0.24 mM KH2PO4; pH 7.2), and mixed with
100 µL of a cytotoxic preparation according to
Varughese et al. (1999). Such preparations consisted of
the supernatant and the fungal extract obtained by
sonication. The culture fluid supernatants of Vibrio
alginolyticus, V. cholera, and V. parahaemolyticus,
which are known to be toxic for shrimps, were used as
positive controls (Aguirre et al., 2003). The plates were
incubated in CO2 atmosphere at 37oC for 2 h. After,
they were washed 3 times with PBS before adding 50
µL of cold methanol and allowed to evaporate under a
hood for 2 min. Cell staining was done with 50 µL
crystal violet in PBS by letting them standstill for 20
min; then the plates were washed three times with PBS,
air dried before adding to each well 200 µL of sodium
duodecyl sulfate (SDS) (1 g/50 mL ethanol), and
incubated for 20 min. Finally, absorbance at 595 nm
was determined in a plate reader (BioRad 3550-UV).
Each toxin preparation was evaluated by triplicate with
each culture.
Adhesion assay
The adhesion capacity of the isolated fungi (mycelium
and spores) to primary cultures of shrimp tissues and
hemocytes was estimated following the procedure
described by Guzmán-Murillo & Ascencio (2001). In
this case, the mycelium was obtained as recommended
by Saha et al. (2008) from M-1 broth culture medium
(pH 4.5) at 22oC and constant orbital shaking (110
rpm). A sample was collected on the 5 th day of
incubation and adjusted to 1.0 optical density
(Beckman DU 640 Spectrophotometer).
The spores were collected from 15 mL assay tubes
containing solid M-1 culture medium (pH 4.5) after
incubation at 22oC for 10-12 days and suspended in 10
mL of an aseptic 0.15 M NaCl solution containing 1%
(w/v) Tween 60. This mixture was carefully transferred
in portions and rotated slowly to a sterile tube. The
recovered spores were counted with a hematocytometer, and their viability was evidenced by staining
with malachite green.
Biotin labeling of fungal mycelium and spores was
done according to Hernández et al. (1996). For this
purpose, 15 mL of mycelium suspension with an optical
density of 1.0 nm and a spore suspension at 1x107
spores-mL-1 in bicarbonate buffer (0.1 M de NaHCO3,
pH 8.0) were centrifuged at 6,000 rpm at 22°C for 5
min. The supernatant was discharged. Cell sediment
was then suspended in 1 mL of bicarbonate buffer and
100 µL of biotin-DMSO (1.3 mg in 1.0 mL). Incubation
757
was carried out at 22°C under darkness with manual
stirring every 30 min for 2-3 h. After this period, 9 mL
of PBS were added, and the mixtures were centrifuged
at 6,000 rpm at 22°C for 20 min. Finally, spores and
mycelium were suspended in 2.5 mL of PBS and stored
under darkness at 4ºC until use. For the adhesion assay,
100 µL aliquots adjusted at 1x104 spores mL-1 were
used. The adhesion assay was done in a 96-wells
microplate containing the primary cell culture of the
various shrimp tissues. The cells were fixed with 100
µL of 2.5% (v/v) glutaraldehyde and rinsed with PBS.
To each well, 100 µL of labeled spore or mycelium
suspension were added and incubated for 0, 30, 60, and
180 min. The plates were washed 3 times with PBS
containing 0.1% (v/v) Tween 20 to eliminate nonadhered cells. Then, 100 µL of streptoavidine-POD (1
µL in 2 mL of PBS) were added to each well, and
incubation was carried out at 37°C for 90 min. Finally,
the wells were washed 3 times with PBS-0.1% (v/v)
Tween 20 and re-suspended in 100 µL of OPD reagent
(2 mg OPD, 12 mL sodium citrate plus 5 mL H2O2).
The plates were incubated under darkness at 22oC for
20 min, and the reaction was stopped by adding 100 µL
at 1 M H2SO4. Absorbance was determined at 490 nm
in a plate reader (BIORAD 3550-UV).
Extracellular enzyme production as virulence
factors
The extracellular production of amylase, lipase,
protease, and chitinase was evaluated in plates
containing M-1 modified medium (glucose 0.4%,
peptone 0.2%, yeast extract 0.5% and agar 4%; all w/v).
The medium in each case was supplemented with the
specific substrate (starch 1% for amylase; 0.5% Tween
80 and 10 mM CaCl2 for lipase; 1% partially
hydrolyzed casein for protease; and 3% colloidal chitin
for chitinase). The plates were inoculated by puncture
with a needle and incubated at room temperature for 4872 h. Amylase production was considered positive by
the appearance of a translucent halo after overlaying 3
mL of fresh lugol (3.3 g Iodine crystals; 6.6. g KI; 1 L
of distilled water) on the gel; lipase production was
revealed by the formation of a precipitate surrounding
the colony; protease and chitinase production was
revealed by the appearance of a halo (Pierce & Leboffe,
2011).
Acute toxicity test on white shrimp post-larvae
(PL8)
Shrimp post-larvae (PL8) survival was evaluated under
two conditions: (a) exposure to mycelium suspension;
and (b) exposure to culture supernatant. In the first case,
the isolated fungi were grown in 25 mL of liquid M-1
medium in 125 mL Erlenmeyer flasks, at 22oC for 3
758
days and under constant orbital shaking (110 rpm) to
reach the logarithmic phase. The cell suspension was
adjusted to different optical densities (0.1, 0.25, 0.5 and
1.0) at 540 nm with a 0.85% NaCl solution. In the
second bioassay, the culture was incubated at 22°C and
constant orbital stirring (110 rpm) for 10 days
(stationary phase).
The cultures were centrifuged at 10,000 rpm
(Beckman J2-HS centrifuge) at 4°C for 10 min, to
obtain both the supernatant and the pellet. To assess
survival of post-larvae (PL8) exposed directly to the
supernatant and mycelium. Before bioassay, the shrimp
post-larvae (PL8) was collected in plastic bags with
seawater for to acclimate at 22°C for 2 h. Specimens
(20) were placed in a 6-well polystyrene plate with flat
bottom (BD Falcon) containing 5 mL sterile seawater.
To each well, 8 mL of the mycelium suspension of
different optical densities and of the culture supernatant
were added. The final volume in each well was adjusted
to 15 mL with sterile seawater, and a coverlid was
applied. The controls were prepared replacing the cell
suspension and supernatant by sterile seawater.
Observations were done with a stereoscope (SP
Southern Precision 1839) at 5x magnification during a
24-h period every 2 h as described Sainz et al. (1998).
All assays were performed in triplicate
All data were normalized using their corresponding
logarithms and ANOVA analysis was performed twoway. Type of cell culture and toxins were considered
assuming absorbance as a dependent variable.
Normalization was done by the Kolmogorov-Smirnov
analysis and homoscedasticity by the Bartlett test.
Whenever significant differences were found, the
Tukey analysis was performed (Zar, 1996).
RESULTS
Fungus identification
The samples collected from CIBNOR´s shrimp ponds
yielded two different yeast-like fungal strains. Both
isolates showed similar morphological features with
white, dry, and dusty colonies (http://www.doctorfungus.org/thefungi/Geotrichum.htm). Hence, the Isolated strains were designed as Geotrichum sp. 1 (Gsp.
1) and Geotrichum sp. 2 (Gsp. 2). Table 1 summarizes
the properties of Gsp. 1 and Gsp. 2. In Gsp. 1 and Gsp.
2 growth kinetics was similar (Fig. 1), which allowed
us to employ similar incubation time for cell biomass
preparation in both cases. Because the start of the
exponential phase was observed very early between the
first and the second day of incubation, and the stationary
Figure 1. Growth kinetics of Geotrichum sp. 1, Geotrichum sp. 2 and Fusarium solani.
phase was reached after 7 days, it was decided to carry
out mycelium collection at day 3, whereas the cell
biomass was collected at day 20. It is important to
mention that by adjusting growth to the equation: y =
K· (1+Ae-bx)-1, an R2 = 0.9926 was found for Gsp. 1 and
an R2 = 0.9949 for Gsp. 2; hence, a difference in growth
rate between the two isolates became apparent, where
Gsp. 1 was faster than Gsp. 2. In consequence, at least
with regards to growth rate, Gsp. 1 and Gsp. 2 showed
different properties, and thus they may correspond to
different strains.
In vitro cytotoxicity of Geotrichum strains on shrimp
tissues
The various shrimp tissue cultures tested showed a
distinct susceptibility to the extract or the supernatant
of each fungal strain (P < 0.001). Hemocytes were more
sensitive than tegument, intestine, and gill cells (Fig. 2).
Interestingly, Gsp. 1 and Gsp. 2 toxicity was higher
than the preparations obtained from V. alginoliticus, V.
parahemoliticus, or V. cholera but lower than the
Fusarium strains. Primary cell cultures of gill and
intestine showed no differences (P > 0.05) with regard
to their susceptibility towards the fungal extracts, and
they were more affected than the tegument tissues. In
general, no differences were observed between fungal
extracts and their corresponding supernatant with
primary shrimp tissue culture gill and intestine cells,
which were also the least affected.
Fungal adhesiveness
Geotrichum sp. 1 and Geotrichum sp. 2 showed a
higher tendency to adhere to hemocytes than to other
shrimp tissues (Fig. 3). However, some differences in
adhesion between spores and filaments were observed.
The spores were always less adhesive than the corres-
759
Table 1. Properties of Geotrichum sp. 1 and Geotrichum sp. 2 isolated from shrimp ponds in Baja California Sur, Mexico.
*Strain used as reference.
Characterístics
Geotrichum sp. 1
Geotrichum sp. 2
Fusarium solani*
Colony diameter
Colony color (Stationary phase)
Mycelium
Spore type
> 45 mm
Black
Cottonish, hyaline
Arthrospore;
cylindrical 3-6 x 6-12 µm
Septed
> 45 mm
Black
Cottonish, hyaline
Arthrospore;
cylindrical 3-6 x 6-12 µm
Septed
60-65 mm
White-cream
Cottonish
Macroconidia (half moon
shape) 3-4 conidias
Septed
Type of hypha
Figure 2. Cytotoxicity of the supernatant (black bars) and sonicated extracts (grey bars) of Geotrichum sp. 1, Geotrichum
sp. 2, Fusarium solani (F45), Fusarium javanicum (F37), Vibrio alginolyticus (Va), Vibrio cholera (Vc), and Vibrio
parahemolyticus (Vp) against primary cell cultures of a) hemocytes, b) gills, c) intestine, and d) tegument of Litopenaeus
vannamei. Each point represents the mean of three experiments; bars indicate SD.
760
Figure 3. Adhesion time-kinetics of Geotrichum sp. 1, Geotrichum sp. 2 spores (top panel), and filaments (bottom panel)
to primary cell cultures of intestine, gills, tegument, and hemocytes of Litopenaeus vannamei. Each point represents the
mean of three experiments; bars indicate SD.
ponding filament stage, indicating some advantage for
the multiple-point attachment that a filament can exert.
Also, a significant increase in adhesion tendency was
observed with elapsed time. No difference in spore
adhesion between the strains was observed, but their
corresponding filament stages showed different
attachment abilities, where Gsp. 2 was more adhesive
than Gsp.1 (P < 0.001; Fig. 3).
Toxicity study on shrimp post-larvae (PL8)
Geotrichum sp. 1 supernatant at a concentration of 0.1
optical density (O.D.) caused 25% post-larvae (PL8)
mortality after 7 h and total loss at 20 h. Lower doses
were innocuous. On the other hand, the culture
supernatant of Geotrichum sp. 2 showed a similar effect
but at earlier times, 25% mortality after 5 h and total
loss at 16 h. It was noted that the toxicity is dosedependent as it is observed that post-larvae mortality
tends to be higher with increasing the optical density at
540 nm of the extract. In the case of Fusarium strains,
the supernatant caused 100% mortalities after only 2 h
(Table 2).
Fungi extracellular enzymatic activity
The strains Geotrichum sp. 1 and Geotrichum sp. 2
produced less amylase than Fusarium reference strains.
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Table 2. Survival percentage of PL8 white shrimp post-larvae exposed to Geotrichum sp. 1, Geotrichum sp. 2, and Fusarium
javanicus CBS, and Fusarium solani ATCC culture media supernatants for a 24-h period. Data correspond to averages
(Standard deviation).
Dilution factor
Geotrichum sp. 1
Geotrichum sp. 2
F. javanicus
F. solani
1
0.5
0.25
0.10
0.01
0.00 (0.00)
3.33 (2.88)
11.66 (7.63)
15.00 (5.00)
90.00 (13.22)
1.66 (2.88)
6.66 (2.88)
61.66 (16.07)
96.66 (2.88)
96.66 (2.88)
0.00 (0.00)
0.00 (0.00)
16.66 (20.81)
65.00 (8.66)
66.66 (14.43)
0.00 (0.00)
3.33 (2.88)
26.66 (7.63)
71.66 (7.63)
50.00 (45.00)
Table 3. Extracellular enzymes of Geotrichum sp. 1, Geotrichum sp. 2, Fusarium javanicum, and Fusarium solani strains.
Data correspond to averages. Standard deviation is indicated in parenthesis. *Hydrolysis halo (mm), **Appearance of
precipitate.
Strain
Geotrichum sp. 1
Geotrichum sp. 2
Fusarium javanicum CBS
Fusarium solani ATCC
Amylase*
Lipase**
Protease*
Chitinase**
1.36 (0.170)
0.27 (0.075)
2.83 (0.170)
1.53 (0.050)
+
+
+
+
0.63 (0.050)
1.83 (0.038)
3.16 (0.340)
-
All strains tested produced lipase. Strains Geotrichum
sp. 2 not produce protease, and none of the tested
strains, produced chitinase (Table 3).
DISCUSSION
As pointed out, from the walls and bottoms of ponds
utilized for shrimp culture at CIBNOR, we isolated 2
Geotrichum strains using M-1 marine culture medium
that favors the growth of marine yeasts and fungi
(Deacon, 2005). The isolated strain, were grown on
PDA medium prepared with distilled water, which
suggests that it may be regarded as facultative marine
fungi strains, and were identified as members of
Geotrichum genus. Their growth characteristics were at
temperatures in the ranges 25-30°C, and both strains
were capable of growing with very low oxygen tension
but not under anaerobic conditions (Pitt & Hocking,
1997).
All pathogenic microorganisms possess some
attributes known as virulence factors by which they can
invade and cause damage to host organisms (Atlas,
1995). Virulence depends to a large extent on two
properties: Invasion capacity and toxin production.
Invasion refers to the capacity of the microorganism to
adhere to host tissues, attack the cells, and proliferate
inside the tissues causing an infection. Toxicity refers
to the ability of the microorganism to produce toxins
capable of altering the normal function of cells or
tissues and/or destroy them. Some toxins are secreted
outside the host and cause severe damage when they
penetrate the body (Atlas, 1995). Based on these facts,
the virulence of Gsp. m1 and Gsp. 2 towards various
shrimp primary cultures and hemocytes was
demonstrated, showing that both have a significant
cytotoxic effect, especially against hemocyte and
tegument cells (Fig. 2). This finding is important
because hemocytes are known to play an important role
in shrimp defense mechanism (Bachere et al., 2000;
Vargas-Albores & Yepiz-Plasencia, 2000), being the
first line of cells that detect invading microorganisms,
and their response or reaction may determine the
susceptibility of the organism towards infection
(Bachere et al., 2000). Adhesion as a virulence factor
has been studied in other pathogens because it is known
that through this mechanism colonization and infection
development is facilitated (Rhem et al., 2000; Krachler
et al., 2011). Other reports have focused on the nature
of the host-pathogen interaction and have identified the
corresponding host receptors to which the pathogens
show affinity (Guzmán-Murillo & Ascencio, 2001;
Wiles et al., 2008). In this case, we observed that the
filaments of both Gsp. 1 and Gsp. 2 strains showed a
significantly higher adhesion capacity (P < 0.001) than
that the spores (Fig. 3).
Such difference may be attributed to the fact that
filaments are developing structures that help the fungus
to attach to different substrates through multiple
contacts, while spores are reproductive structures
aimed to warrant species conservation rather than to
facilitate attachment to a host surface (Tortora et al.,
2012). Alexopoulos et al. (1996), for instance, suggests
762
that fungal spores may require from several hours to
various days to germinate and produce infection under
proper conditions, which is in agreement with our
results. Lozano-Olvera et al. (2012) have reported that
the filaments of Fusarium solani cause death by gill
blockage and the resulting melanization, making the
gill to appear black, causing the death by asphyxia
(Lightner, 1996; Lignot et al., 2000; Nosanchuk et al.,
2002; Pantoja & Lightner, 2008). In the case of the crab
Astacus leptodactyles, Lignot et al. (2000) observed
that F. oxysporum hyphae produced black spots in the
gills after a 36-h exposure, which was attributed to
melanization; on the other hand, Arala-Chavez &
Sequeira (2000) observed an increased hemocyte
proliferation in Penaeus monodon and Drosophila
using various fungal antigens. In our case, exposure of
L. vannamei post-larvae (PL8) to Gsp. 1 and Gsp. 2
filaments produced mortality within the 24 h (Table 2),
and perhaps in this short interval melanization could
not occur (Lignot et al., 2000). Johansson et al. (2000)
reported in shrimp hemolymph, the presence of a
protein which specifically binds β-1,3 glucan in
response to a fungus infection. Apparently, this protein
triggers the shrimp immune system designed to combat
the infection that also agrees with the results of our
experiments in which the filaments reacting with
hemocytes may contribute to accelerate shrimp
response against fungal infection.
Because fungi are unable to produce their own food,
they are prepared to ingest nutrients from the
surrounding medium making use of several
extracellular enzymes that degrade large molecules into
smaller and more assimilated compounds. Hence,
extracellular enzyme production may favor the
infection process. Among the enzymes produced by
fungi for this purpose, lipases and amylases are the
most common (Bugni & Ireland, 2004). In our study,
we observed that both Gsp. 1 and Gsp. 2 strains produce
extracellular lipases, amylases, and proteases (Table 3),
which could promote colonization as it occurs in the
case of G. candidum (Mukkerjee & Kiewitt, 1996).
Nakamura et al. (1994) also confirmed the production
of polygalacturonase as a mechanism of citrus infection
and the development of sour rot in fruits by
Geotrichum.
Finally, we observed that when post-larvae (PL8)
are in contact with the culture supernatant of Gsp. 1 and
Gsp. 2 strains, shrimp started to die after 2 h and up
reaching a total mortality within 20 h (Table 2). This
observation is similar to those made with other
crustacean eggs and post-larvae (PL8) exposed to
filamentous fungi (Noga, 1990; Nakamura et al., 1994).
In particular, Lagenidium, Haliphthoros and Sirolpidium
produce severe mycosis in shrimp protozoa and mysis
with 100% mortality after only 1-2 days. Also
Fusarium strains tested produced 100% mortality after
only a 2-h exposure, which could be attributed to the
excretion of potent toxic secondary metabolites
(Nelson et al., 1995); in the case of Geotrichum,
ammonia excretion could be toxic to shrimp
(O’Donnell, 1996; Aldarf et al., 2002; Bugni & Ireland,
2004). Le Moullac & Haffner (2000) consider that
ammonia is very toxic to aquatic organisms and found
that the number of hemocytes is severely reduced in
shrimp exposed to 3.0 mg L-1 of ammonia. The dose at
which a pathogen may cause damage to a given host is
of extreme importance (Atlas, 1995). In our case,
mortality increased with higher concentrations of the
fungus cells or supernatant (Table 2). In the present
study, it was possible to show that Geotrichum species
may indeed constitute a serious threat in shrimp culture,
and that monitoring and good management practices
are the only strategies that could reduce the risk of
collapse and total culture loss. Isolated fungal strains,
and partially identified as Geotrichum sp. 1 and sp. 2,
produce proteolytic enzymes (Table 3), and these
filamentous fungal strains were cytotoxic for primary
shrimp cell culture, where hemocytes showed greater
susceptibility followed by tegument cells. In
comparison, the adhesion of spores and filaments of
Geotrichum fungi to primary cultured cells was higher
in hemocytes than in any other cell types tested. Shrimp
postlarval PL8 exposed to Geotrichum filaments
showed mortalities in a dose-dependent manner. Based
on our results, we can suggest that isolated strains of
Geotrichum may represent a health risk for white
shrimp culture. It should be noted that Getrichum is a
saprophytic fungus that develops in decaying organic
matter and that its presence can be avoided if proper
measures are taken to minimize the unfavorable
conditions at the bottom and the water column at
pound; thus, having optimal conditions in the cultures,
the chances of that organisms be susceptible to
infection decreases.
It is necessary to mention that micro-flora at ponds
are closely associated with trophic conditions,
ecological factors and physic chemical parameters; so
it has been considered, that the presence of yeast
structures in the digestive tract of organisms in culture
or in aquatic environment can be used as a tool for
monitoring environmental quality to be a valid
instrument for the assessment of eutrophication of the
environment. Thus, the presence of these microorganisms can be used as a bio-marker that allows us to
assess environmental changes; correlating either genus
recovered from various species in different environmental conditions, taking into account the presence of
pollution sources; or, by evaluating the phenotypic
changes in organisms recovered disturbed habitats
(Coelho et al., 2010; Brilhante et al., 2012).
ACKNOWLEDGEMENTS
This work was part of NOA’s M.Sc. thesis. We thank
Drs. Francisco Magallón & Guillermo Portillo,
CIBNOR for introducing us to this problem. We also
acknowledge the assistance and material provided by
Dr. Hector Nolasco to carry out the enzyme test and to
Diana Dorantes for editorial services.
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Lat. Am. J. Aquat. Res., 43(4): 766-775, 2015
Pacific whiteleg shrimp gut microbiota
Research Article
Probiotic modulation of the gut bacterial community of juvenile
Litopenaeus vannamei challenged with Vibrio parahaemolyticus CAIM 170
Irasema E. Luis-Villaseñor1,2, Domenico Voltolina3, Bruno Gomez-Gil4, Felipe Ascencio2
Ángel I. Campa-Córdova2, Juan M. Audelo-Naranjo1 & Olga O. Zamudio-Armenta1
1
Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa (UAS)
Mazatlán, Sinaloa, CP 82000, México
2
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, B.C.S., CP 23096, México
3
Centro de Investigaciones Biológicas del Noroeste, Laboratorio UAS-CIBNOR
4
Centro de Investigación en Alimentación y Desarrollo (CIAD), Mazatlán Unit for Aquaculture
Corresponding author: Ángel I. Campa-Córdova ([email protected])
ABSTRACT. The protective effects of two probiotic mixtures was studied using the fingerprints of the bacterial
community of Litopenaeus vannamei juveniles exposed to probiotics and challenged with Vibrio
parahaemolyticus CAIM 170. Fingerprints were constructed using 16S rRNA gene and the PCR-SSCP (Single
strand conformation polymorphism) technique, and the probiotics used were an experimental Bacillus mixture
(Bacillus tequilensis YC5-2 + B. endophyticus C2-2 and YC3-B) and the commercial probiotic Alibio. The DNA
for PCR-SSCP analyses was extracted directly from the guts of shrimps treated for 20 days with the probiotics
and injected with 2.5×105 CFU g-1 of V. parahaemolyticus one week after suspension of the probiotic treatment.
Untreated shrimps served as positive (injected with V. parahaemolyticus) and negative (not injected) controls
Analysis of the bacterial community carried out after inoculation and 12 and 48 h later confirmed that V.
parahaemolyticus was present in shrimps of the positive control , but not in the negative control or treated with
the probiotic mixtures. A significant difference in the diversity of the bacterial community was observed
between times after infection. The band patterns in 0-12 h were clustered into a different group from that
determined after 48 h, and suggested that during bacterial infection the guts of whiteleg shrimp were dominated
by gamma proteobacteria represented by Vibrio sp. and Photobacterium sp. Our results indicate that the
experimental and the commercial mixtures are suitable to modulate the bacterial community of L. vannamei and
could be used as a probiotic to control vibriosis in juvenile shrimp.
Keywords: Litopenaeus vannamei, Bacillus mix, Vibrio parahaemolyticus, bacterial community, aquaculture.
Modulación por probióticos de la comunidad bacteriana intestinal de juveniles
de Litopenaeus vannamei infectados con Vibrio parahaemolyticus CAIM 170
RESUMEN. Se estudiaron los perfiles de bandeo de la comunidad bacteriana de juveniles de Litopenaeus
vannamei tratados con dos probióticos y expuesto a la bacteria patógena Vibrio parahaemolyticus CAIM 170.
Los perfiles de bandeo se construyeron usando el gen 16S rRNA y la técnica PCR-SSCP (Polimorfismo
conformacional de cadena sencilla) y los probióticos fueron una mezcla experimental de Bacillus (Bacillus
tequilensis YC5-2 y B. endophyticus C2-2 y C3-B) y el probiótico comercial Alibio. El ADN para el análisis
PCR-SSCP se obtuvo de los intestinos de camarones tratados durante 20 días con los probióticos, inyectados
con 2,5×105 UFC g-1 de V. parahaemolyticus una semana después de la suspensión del tratamiento con
probióticos. Camarones no tratados con probióticos sirvieron como control positivo (inyectados con V.
parahaemolyticus) y negativo (no inyectados). El análisis de la comunidad bacteriana durante el reto confirmó
la presencia del patógeno inyectado en el control positivo y su ausencia en el negativo y en los organismos
tratados con probióticos. Durante las 48 h del período experimental se observó una diferencia significativa en la
diversidad de la comunidad bacteriana.
__________________
Corresponding editor: Sandra Bravo
7661
767
2
Los patrones de bandas se agruparon en un grupo a las 0-12 h y en uno diferente después de 48 h y sugirieron
que los intestinos de camarón blanco fueron dominados por gamma proteobacteria representados por Vibrio sp.
y Photobacterium sp. durante la infección bacteriana. Estos resultados indican que las dos mezclas pueden
modular la comunidad bacteriana y pueden ser usadas como probióticos para controlar la vibriosis en camarones
juveniles.
Palabras clave: Litopenaeus vannamei, mezcla de Bacillus, Vibrio parahaemolyticus, comunidad bacteriana,
acuicultura.
INTRODUCTION
The bacterial genus Vibrio is common and widely
distributed in the natural marine environment and in the
microbiota of farmed shrimp ponds (Gopal et al.,
2005), where some of its species may become
opportunistic pathogens and sources of major diseases
when the natural defense mechanisms of cultured
shrimp are suppressed (Lightner, 2005).
Under the common name of vibriosis, these diseases
may cause considerable economic losses, and are
considered among the most serious limiting factors for
the success of marine aquaculture (Lightner, 2005;
Chatterjee & Haldar, 2012). Among the etiological
agents, Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. campbelli, V. alginolyticus and V. penaeicida
have been associated with cultured shrimp diseases
(Ishimaru et al., 1995; Sahul-Hameed et al., 1996;
Jayasree et al., 2006), and among these V. harveyi and
V. alginolyticus are thought to be the most common
causes of disease during larval and postlarval
development (Manefield et al., 2000; Abraham &
Palaniappan, 2004).
The addition of probiotic bacteria to culture systems
has gained attention as a precautionary measure against
pathogens. This addition aims to reduce or eliminate
selected pathogenic species and to improve growth and
survival of the cultured species through the modulation
of the microbial communities of the culture environment (Balcázar et al., 2006; Martínez-Cruz et al.,
2012), because bacteria may affect growth and survival
of aquatic organisms and are a major element in their
well being, since they play distinct roles in the host
organism, which are associated with nutrition, immune
responses and disease resistance (Austin, 2006;
Chaiyapechara et al., 2011; Tuyub-Tzuc et al., 2014).
Culture-independent techniques for population
fingerprinting, such as denaturing gradient gel electrophoresis (DGGE) and single strand conformation
polymorphism (SSCP), are effective tools for a more
complete and rapid assessment of microbial diversity,
especially of complex ecosystems such as intestinal
microbiota (Muyzer & Smalla, 1998; Dohrmann &
Tebbe, 2004; Hassan, 2012).
Previous experiments showed that a Bacillus
mixture which improved survival and development of
Litopenaeus vannamei larvae caused also an increase in
diversity and evenness of the bacterial community of
the larval gut, thus increasing resistance to V.
parahaemolyticus infection (Luis-Villaseñor et al.,
2011, 2013). However, there is no information on the
effect of this or other probiotics on the structure of the
bacterial community of the intestinal tract of juvenile
or adult shrimp challenged with pathogenic bacteria.
This study aimed to evaluate the changes induced by
the same Bacillus mixture on the gut bacterial
community of juvenile Litopenaeus vannamei (Pacific
whiteleg shrimp) infected with V. parahaemolyticus.
Probiotic strains
Cultures of the bacteria Bacillus tequilensis YC5-2, B.
endophyticus C2-2 and B. endophyticus YC3-B were
grown at 37°C for 24 h in 200 mL Erlenmeyer flasks
with 100-mL of TSB medium, and concentrated by
centrifugation at 5000×g for 10 min. Each pellet was
suspended in a sterile saline solution containing 3%
(w/v) NaCl (S-7653, Sigma, St. Louis, MO). The
absorbance was adjusted to an optical density of 1 at
600 nm (approximately 1×109 CFU mL-1), and the
resulting suspensions were added to the shrimp rearing
system at a final concentration of 1×105 CFU mL-1.
Pathogenic bacterium
Strain Vibrio parahaemolyticus CAIM 170, obtained
from the Colección de Microorganismos de Importancia
Acuicola (CIAD, Mazatlan, Mexico, www.ciad.
mx/caim), grown in trypticase soy broth (TS#236950,
Difco, Franklin Lakes, NJ) with 3% (w/v) NaCl, was
centrifuged at 5000×g for 10 min; the pellet was
suspended in 3% (w/v) sterile saline solution. The
bacterial suspension was diluted with filtered sterile
seawater to an optical density of 1.0 (approximate
concentration: 1×109 CFU mL-1), and a 1:10 dilution of
this suspension was used for the challenge experiment.
Probiotic treatment and infection
Juvenile shrimps (mean live weight 8 ± 1 g) were
obtained from a commercial hatchery and acclimated
for five days to laboratory conditions, which did not
change throughout the experiment (5-µm filtered
seawater, 29°C and salinity 36) in a common tank.
After acclimation, 16 groups of 21 shrimps were placed
in 80-L aquaria. Five aquaria (treatment A) were added
daily 1×105 CFU mL-1 of the Bacillus mixture. A
second group of five aquaria (treatment B) received the
dose used by local shrimp farmers (1 mL L-1, with
1×106 CFU mL-1) of a commercial probiotic mixture
(Alibio2135 + AlibioAC + Alibio Bionutre) activated
as recommended by the manufacturer (AliBio S.A. de
C.V., Mexico City). The remaining six aquaria served
as triplicate positive and negative (unchallenged)
controls (treatments C and D, respectively).
Addition of probiotics was suspended after 20 days,
and seven days later all shrimps of treatments A, B and
C were injected into the fifth abdominal section with 20
µL of Vibrio suspension (= 1×108 CFU mL-1), giving
2x106 CFU/shrimp. Shrimps of treatment D were
injected with a sterile saline solution.
Throughout the experiment all treatments were fed
ad libitum a 35% protein commercial diet. Continuous
aeration was maintained in all aquaria, which were
maintained with 50% daily water exchanges.
Sample collection and DNA extraction
One shrimp was randomly selected from each
container immediately after Vibrio injection (time 0) (5
shrimps for each probiotic treatment and 3 shrimps for
positive and negative controls), and sampling was
repeated after 12 and 48 h, in the first case because this
time coincided with the first case of mortality, while the
last was observed 12 h later. Consequently, samples of
live shrimp were taken at times 12 h (onset of mortality)
and 48 h, giving the surviving shrimp 24 to recover
after the last observed death. Immediately after
sampling, the body surface of each shrimp was washed
with sterile seawater, disinfected with 70% ethanol,
dissected with sterile instruments and the entire
intestinal tract was removed, excised with sterile
forceps and scissors, and preserved at -80°C in
individual screw-capped tubes with 1 mL absolute
ethanol.
At the end of the experiment, the chromosomal
DNA was extracted to assay for the diversity of the
intestinal communities, using Wizard genomic DNA
purification kits (Promega, Madison, WI) according to
the manufacturer’s instructions.
Amplification of 16S rRNA
The universal bacterial primers Com1 and Com2ph
were used to amplify a 407 bp fragment corresponding
to positions 519 to 926 (E. coli positions; including
768
3
variable regions 4 and 5 of the 16S gene). The Com1
sequence was 5′-CAGCAGCCGCGGTAATAC and
Com2ph was 3′-CCGTCAATTCCTTTGAGTTT
(Schwieger & Tebbe, 1998). Each PCR was performed
in a total volume of 50 μL in 0.2 mL micro tubes. The
reaction mixtures were contained in 1×PCR buffer with
1.5 mM MgCl2, 0.5 μM of each primer, 200 μM of each
dNTP, and 2.5 U Taq polymerase (GoTaq, Promega).
The total amount of genomic DNA added to the PCR
mixtures was 250 ng. Thermocycling (Peltier Thermal
Cycler, Bio-Rad Laboratories, Hercules, CA) started
with an initial denaturation for 3 min at 94°C, followed
by 30 cycles of 60 s at 94°C, one cycle for of 60 s at
53°C and one of 90 s at 72°C, ending with a final
extension for 5 min at 72°C. The presence of specific
PCR products was confirmed on 1% (w/v) agarose gel.
Single-strand conformation polymorphism
The single-strand removal method (Schwieger &
Tebbe, 1998) was used for profiling bacterial
communities. All PCR products of each replicate were
purified (PCR purification kit, Qiagen, Hilden,
Germany) and diluted in Tris-HCl buffer to a final
volume of 20 μL. Samples were digested for 45 min at
37°C with 1 μL (5 U) of lambda-exonuclease solution
(New England Biolabs, Ipswich, MA), with 3 μL
exonuclease buffer and 6 μL milli-Q H2O, for a total
volume of 30 μL. Digestion was stopped with the first
step of purification with spin columns (MiniElute Kit,
Qiagen), and diluted in 10 μL Tris-HCl buffer.
A 9 μL denaturing loading buffer containing 95%
formamide (v/v), 10 mM NaOH (w/v), 0.25%
bromophenol blue (w/v), and 0.25% xylene cyanole
(w/v) was added before electrophoretic analysis.
Samples were incubated at 95°C for 2 min and
immediately cooled on ice. After 3 min, samples were
loaded onto polyacrylamide gels of 0.625% MDE
(Cambrex, East Rutherford, NJ), and electrophoresis at
260 V at 20°C was carried out for 18 h (DCode
Universal Mutation System, Bio-Rad Laboratories,
Hercules, CA). After electrophoresis was completed,
the gel was stained with AgNO3 (Benbouza et al., 2006)
and scanned using Power Look III (Umax Systems,
Willich, Germany).
Analysis of SSCP profiles
Gel analysis software (Gel Compar II, Applied Maths,
Sint-Martens-Latem, Belgium) was used to calculate
similarities between profiles of bacteria obtained from
the different treatments and times of inoculation, after
image normalization with bacteria markers (B.
licheniformis, Rhizobium trifolii, Flavobacterium
johnsoniae, and R. radiobacter). The calculation of the
similarity matrix was based on Pearson’s correlation
4769
coefficients. The clustering method was the unweighted
pair group method with arithmetic averages (UPGMA).
Elution of bands and DNA sequencing
Bands of interest were cut from the silver-stained
polyacrylamide SSCP gel with a sterile scalpel. The
single-stranded DNA was eluted from the gel by the
crush and soak procedure (Sambrook & Russell, 2001),
resuspended in 12 μL Tris buffer (10 mMTris-HCl, pH
8.0), and amplified via PCR using primers Com1 and
Com2ph under the conditions previously described.
The PCR-amplified products were sequenced by a
commercial firm (Genewiz, South Plainfield, NJ).
The sequences were compared with sequences in
the GenBank database. The BLAST search of the
National Center for Biotechnology Information and the
EzTaxon server database (www.eztaxon.org; Chun et
al., 2007) were used to determine the closest
relationships of the 16s rRNA sequences.
To determine the similarity between treatments, the
data of the metrics obtained from each sample were
exported as a binary matrix (PAST software, palaeoelectronica.org). A PCA was performed from the
correlation matrices generated from a binary matrix,
which was expressed as a value of Pearson’s similarity
coefficient (Fromin et al., 2002). A PCA analysis was
conducted with software Statistica 6.0 (StatSoft, Tulsa,
OK).
RESULTS
Modulation of intestinal microbiota
The dendograms showed a clear modulation of the
intestinal microbiota from 12 h post-infection (onset of
death) to 48 h (organisms recovered), divided into two
clusters. One cluster (48 h) had a similarity value of
40.81% with respect to the second cluster, including
times 0 and 12 h, with a percentage of similarity between
49.64%. Samples Start 1 and Start 2 (samples taken
before probiotic treatment) were within the same cluster
as time 48 h with 54% similarity, indicating a recovery
of the initial microbiota similar to the bioassay (Fig. 1).
Bacterial community of juvenile shrimp infected by
Vibrio parahaemolyticus
The results from the SSCP fingerprint showed that the
taxonomic group Flavobacteria was dominant: α
proteobacteria (mainly Rugeria lacuscaerulensis), γproteobacteria, fusobacteria and Cytophagaceae, represented by Wandonia haliotis Haldis.
V. parahaemolyticus and Vibrio sp. were present
only in the positive control, Cytophaga fermentans was
present only in organisms treated with Alibio while
Photobacterium damselae subs. piscicida was present
in all treatments but not in the positive control. The
individual bands present in the Bacillus mix were
identified as Candidatus bacilloplasma mollicute and
Nautella italica (Table 1). Unidentified bands
(Uncultured bacteria) were also present in the treatment
of the Bacillus mix.
Maribius salinus and Donghicola eburneus(αproteobacteria) were detected only in the Bacillus mix
and the positive control groups (Table 1). Bacteria
species unique to the negative control were
Thioprofundum lithotrophica, Sebaldella termitidis,
Elizabethkingia anophelis, Oceanicola sp., and
Thioclava pacifica. Thalassobium sp. was detected in
both control groups.
PCA Analysis
Principal Component Analysis (PCA) showed that two
of the components explained 91% of the total variance
in the data (CP1 and CP2: 64.8 and 26.2%,
respectively) (Fig. 2), and that their factor loadings
were considered significant at values greater than 0.70.
No significant differences were observed between the
banding profiles at time 0 h and 12 h, but their trends
are separated clearly from that determined after 48 h.
These results coincide with the indications of the
similarity dendrogram.
DISCUSSION
Manipulation of microbiota with probiotics may be a
convenient practice to control or inhibit pathogenic
bacteria in aquaculture, as well as to improve growth
performance and digestive enzymes activities, and
enhance immune responses against pathogens or
physical stress (Balcázar et al., 2006; Pérez et al., 2010;
Zokaeifar et al., 2012).
The Bacillus mix used in this work showed several
effects which may be useful for L. vannamei culture:
after a V. parahaemolyticus challenge which caused
>90% mortality in the control group, juvenile shrimps
treated with this mix had significant higher survival,
different total hemocyte concentrations and a higher
diversity and evenness of their bacterial gut community
than those treated with the commercial product Alibio,
and demonstrated efficient probiotic protection (LuisVillaseñor et al., 2013). However, the underlying
mechanisms for this protection remained unclear.
In this work we showed how the effect of V.
parahaemolyticus CAIM 170 on the bacterial community of juvenile shrimp may be at least partially
avoided in shrimps treated with the Bacillus mix even
770
5
Figure 1. Acrylamide gel-generated via SSCP dendrogram illustrating the relationship (percent similarity) between
bacterial communities in gut of shrimp at time 0 h, 12 h and 48 h: M1-M5 (Bacillus mix), A1-A5 (commercial probiotic),
C1(-)-C3(-): (without probiotics and injected with saline solution) C1(+)-C3(+): (without probiotics and injected with
pathogenic bacteria). Start 1 and 2: initial profiles, before probiotic treatment. The 40-100 scale of the dendrogram shows
percent of similarity of the clusters. The dendrogram was calculated with UPGMA and the Dice coefficient.
after one week after suspension of the treatment.
Although the SSCP distribution profiles displayed little
variation of the number of bands (OTU) at each
sampling period, the greatest variation observed was
their increase 12 h after infection in all treatments with
Vibrio.
The PCA of 0 h and 12 h indicated no evidence of
clustering of individual probiotics groups and no
statistically significant deviation from the baseline
SSCP profile. However, the PCA conducted on the 48
h gut samples showed the probiotic groups clustered
separately from those at the beginning and those at 12
h post-infection. This indicated that the gut microbial
population ecology of the animals at 48 h was
significantly different from that at 0 h and 12 h after
infection, and the separation more evident was that
between probiotic-treated and V. parahaemolyticusinfected shrimps.
6771
Table 1. Closest relative, as determined by Blast search, with similarity (SIM, in %) to the major OTUs from the 16S rRNA
V4 and V5 SSCP gels.
OTU
M1b
M1d
M1c
M2a
M2b
M2a
M3a
M3b
M3c
M3d
M2a
M2c
M2d
M2h
M2i
A1a
A1c
A1d
A4a
A4b
A4c
A1a
A1b
A1c
A1d
A1e
A1f
A1g
A1i
A1j
A3a
A3b
A3c
A4a
A1b
A2a
A2b
A2c
A2d
A3b
A3c
A3d
A3e
SIM (%)
Closest relative
Bacillus mix
h0
Nautella italica
Candidatus bacilloplasma mollicute
Wandonia haliotis Haldis
Maribius salinus
Donghicola eburneus
h 12
84
Uncultured bacterium clone
97
Flavobacteriaceae bacterium
97
97
97
Photobacterium damselae subs. piscicida
h 48
94
Thalassobius gelatinovorus
93
Planktotalea frisia
98
Ruegeria lacuscaerulensis
99
Unidentified alpha proteobacterium
99
Alibio
h0
90
90
Cytophaga fermentans
90
Sebaldella termitidis
90
90
96
Photobacterium damselae subsp. piscicida
h 12
97
96
89
90
97
98
Photobacterium damselae sub sp. Piscicida
89
90
Uncultured bacterium
87
Vibrio furnissii
85
Desulfovibrionaceae bacterium
93
98
90
h 48
92
90
Paracoccus versatus
82
Vibrio sp.
91
100
Donglicola eburneus
90
92
98
98
96
90
90
95
98
Phylogenetic group
α-proteobacteria
Flavobateria
α-proteobacteria
α-proteobacteria
γ- proteobacteria
α-proteobacteria
α-proteobacteria
α-proteobacteria
Flavobacteria
Cytophagaceae
Flavobacteria
Cytophagaceae
γ-proteobacteria
Cytophagaceae
γ-proteobacteria
γ-proteobacteria
Flavobacteria
α-proteobacteria
Flavobacteria
γ-proteobacteria
α-proteobacteria
Flavobacteria
α-proteobacteria
772
7
Continuation
OTU
SIM (%)
C2b
C2c
97
99
C1a
C1b
C2a
C2b
C2c
C2f
C2g
C3a
C3b
C3c
C3f
89
85
91
97
89
96
89
99
93
97
91
C1a
C1b
C1c
C2a
C2b
C2c
C2d
C2e
C3a
C3b
C3c
C3d
92
98
99
95
91
97
92
97
97
92
97
87
C2c
C3b
C3c
99
97
94
C1a
C1b
C1c
C1d
C2a
C2b
C2c
C3a
C3b
C3c
T48
C2a
C2b
C2c
C3a
C3b
C3c
C3d
C3e
C3h
90
97
87
97
93
87
95
87
88
90
91
100
87
98
90
98
98
92
99
Closest relative
Negative control
h0
Photobacterium damselae subsp. piscicida
h 12
Alpha proteobacterium
Sebaldella termitidis
Elizabethkingia anophelis
Thalassobius sp.
Thioprofundum lithotrophica
h 48
Vibrio mediterranei
Ruegeria lacuscaerilensis
Oceanicola sp.
Vibrio sp.
Thalassobius mediterraneus
Thioclava pacifica
Positive control
h0
h 12
Maribius salinus
Thalassobius sp.
Donghicola eburneus
Donghicola sp.
Vibrio parahaemolyticus
Vibrio sp.
Phylogenetic group
γ-proteobacteria
Flavobacteria
Fusobacteria
Flavobacteria
α-proteobacteria
γ-proteobacteria
α-proteobacteria
γ-proteobacteria
α-proteobacteria
α-proteobacteria
γ-proteobacteria
α-proteobacteria
α-proteobacteria
Flavobacteria
α-proteobacteria
α-proteobacteria
α-proteobacteria
α-proteobacteria
α-proteobacteria
Flavobacteria
α-proteobacteria
γ-proteobacteria
γ-proteobacteria
8773
Figure 2. Principal components analysis using the Dice coefficient of single strand conformation SSCP profiles associated
with the intestines of individual L. vannamei inoculated with the treatments and challenged with V. parahaemolyticus for
each time: Time start (▲), 0 h (Δ), 12 h (□), 48 h (×). Each point represents a SSCP profile from one shrimp.
Recent studies on the modulation and stabilization
of gut microbiota by probiotic treatment suggest that
probiotics can exert a positive effect on uncultivable
gut microbiota (Sáenz de Rodrigáñez et al., 2009; Sun
et al., 2012a, 2012b; Yang et al., 2012), which
coincides with the modification of the gut microflora
and the increase in bacterial diversity after probiotic
administration reported in Solea senegalensis by TapiaPaniagua et al. (2010). Several studies indicated that
the culturable intestinal microbial community of
shrimp was mainly composed of Aeromonas,
Plesiomonas, Photobacterium, Pseudoalteromonas,
Pseudomonas and Vibrio species (Moss et al., 2000;
Oxley et al., 2002). Of these, only Photobacterium and
Pseudoalteromonas were detected in this work whereas
Vibrio species were detected only in the positive
control (V. parahaemolyticus, confirming the induced
infection, and Vibrio sp.), and in shrimps treated with
Alibio (Vibrio sp.), possibly because several Vibrio or
Vibrio-related species are common in commercial
probiotic mixtures (Verschuere et al., 2000; Qi et al.,
2009).
In our case, the indigenous intestinal microbiota
tended to be dominated by Wandonia haliotis Haldis,
which may be considered a commensal, because it was
present at all times and in all treatments. The presence
of V. mediterranei in the negative control may be due
to its occurrence in natural microbiota, because this
species is commonly associated to a wide-range of
hosts, with mutual interactions which may range from
mutualism or symbiosis to a pathogenic relation
(Turner et al., 2009; Senderovich et al., 2010).
The fact that shrimps treated with the Bacillus mix
did not show the presence of the pathogen injected may
be taken as an indication of a protective effect of this
probiotic, similar to the effect against V. parahaemolyticus of the indigenous intestinal microbiota
modified with a Bacillus-based probiotic observed by
Wu et al. (2014) in the mud crab S. paramamosain.
Modifications of the intestinal microflora by a probiotic
Bacillus resulting in inhibition of growth of intestinal
Vibrio spp. have been reported also in Penaeus
monodon by Rengpipat et al. (2000) and by Vaseeharan
& Ramasamy (2003), who also noted a positive effect
in the external water environment.
Photobacterium damselae subsp. piscicida was
observed in all our treatments. This microorganism
(formerly Pasteurella piscicida) is a highly pathogenic
bacterium that causes photobacteriosis and does not
show host specificity (Toranzo et al., 1991; Noya et al.,
1995) but, in spite of its generalized presence it did not
show any pathogenic effect, possibly because the
presence of the probiotic Bacillus strains, since these
are known to
modulate shrimp gut bacterial
communities (Luis-Villaseñor et al., 2013), thereby
improving their immune response against pathogenic
bacteria (Zokaeifar et al., 2012, 2014).
The effect of probiotic protection on the structure of
the intestinal bacterial community of shrimp infected
with pathogenic bacteria was unknown, and this work
shows that both probiotic mixtures, Alibio and Bacillus
mix, helped to maintain a natural balance in the
bacterial community of the shrimps intestine,
modulating and increasing diversity and evenness of
bacterial species in shrimps challenged by bacterial
infection.
ACKNOWLEDGMENTS
Supported by SEP-CONACyT Grant 25981. Hector
Acosta of CIBNOR provided technical assistance
during the preparation of the manuscript.
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Lat. Am. J. Aquat. Res., 43(4): 776-780, 2015
Marteilia refringens in the Gulf of California
776
1
Short Communication
Molecular evidence of the protozoan parasite Marteilia refringens in
Crassostrea gigas and Crassostrea corteziensis from the Gulf of California
José Manuel Grijalva-Chon1, Reina Castro-Longoria1, Tania Lizbeth Enríquez-Espinoza1
Alfonso Nivardo Maeda-Martínez2 & Fernando Mendoza-Cano3
1
Departamento de Investigaciones Científicas y Tecnológicas
Universidad de Sonora, Hermosillo, Sonora, México
2
Centro de Investigaciones Biológicas del Noroeste, La Paz, Baja California Sur, México
3
Centro de Investigaciones Biológicas del Noroeste, Laboratorio de Referencia
Análisis y Diagnóstico en Sanidad Acuícola, Hermosillo, Sonora, México
Corresponding author: José Manuel Grijalva-Chon ([email protected])
ABSTRACT. The search for exotic pathogens related to the outbreaks and in surveillance samplings of the
Mexican oyster farms, is a recent activity achieved by academic institutions and state committees for Aquatic
Animal Health, with remarkable results. In samples of Crassostrea gigas collected through December 2009,
January 2010 and November 2010, and of C. corteziensis in September 2011, the protozoan Marteilia refringens
was detected for the first time in the Gulf of California. The carrier oysters were from cultures without abnormal
mortality rates, whereby, the use of histology, in situ hybridization and transmission electron microscopy studies
are necessary to determine if M. refringens has become established in the Gulf of California oyster cultures.
Detection of M. refringens is of great concern to the global oyster farming industry.
Keywords: Marteilia refringens, Crassostrea gigas, Crassostrea corteziensis, Gulf of California.
Evidencia molecular del parásito protozoario Marteilia refringens en
Crassostrea gigas y Crassostrea corteziensis del Golfo de California
RESUMEN. La búsqueda de patógenos exóticos relacionados con brotes de enfermedades y en muestreos de
vigilancia de las granjas ostrícolas de México es una actividad reciente, realizada por instituciones académicas
y comités estatales de sanidad acuícola, con resultados notables. En muestras de Crassostrea gigas colectadas
en diciembre 2009, enero 2010 y noviembre 2010 y de C. corteziensis en septiembre 2011, se detectó por PCR
el protozoario Marteilia refringens por primera vez en el Golfo de California. Los ostiones portadores provenían
de cultivos sin mortalidades anormales, por lo cual, el uso de histología, hibridación in situ y microscopía
electrónica de transmisión son necesarios para determinar si M. refringens se ha establecido en los cultivos de
ostras del Golfo de California. La detección de la presencia de M. refringens es de gran preocupación para la
industria ostrícola.
Palabras clave: Marteilia refringens, Crassostrea gigas, Crassostrea corteziensis, Golfo de California.
The oyster’s culture along the Mexican Pacific coast
began nearly forty years ago, and for almost twenty
years the oyster culture run without major problems,
until massive mortalities were observed since the end
of the 1990’s until 2009. The quest for a pathogen had
shown the evidence of the presence of the ostreid
herpesvirus 1 (OsHV-1) (Vásquez-Yeomans, 2004,
2010; Grijalva-Chon et al., 2013) and the protozoan
Perkinsus marinus (Cáceres-Martínez et al., 2008;
__________________
Enríquez-Espinoza et al., 2010; Escobedo-Fregoso et
al., 2015), which is endemic of the Atlantic coast.
In aquatic cultured species many pathogens are not
specific and infect a wide range of related host species.
In mollusks, several protozoan species seriously
threaten the cultures, and because of the emergence of
exotic diseases of great concern to aquaculture farmers,
countries had implemented strict regulations for trading
live organisms or frozen commodities to avoid its spread.
2777
However, the previous trade of infected broodstock,
spat, or juveniles, before these regulatory rules were in
effect, affected not only the established cultures but
also wild populations.
Marteilia refringens is a protozoan of great concern
to the mollusk aquaculture, mainly in Europe, as it is
responsible for the Aber disease that causes mass
mortalities in Ostrea edulis. This parasite also has the
ability to infect several bivalve species; therefore,
survey studies in areas of mollusk culture are of
worldwide interest. The OIE (2009) listed the
susceptible host species, vectors, and carriers for this
protozoan, but Crassostrea gigas and C. corteziensis
were not included in any category. Thus, the aim of this
study was to investigate the occurrence of M. refringens
in two oyster species cultured in the Gulf of California.
During December 2009 through November 2010,
30 specimens of adult C. gigas (10.35 ± 1.82 cm length)
were monthly collected (n = 360) in La Cruz coastal
lagoon, Sonora, Mexico (2848’87’’N, 11155’03’’W).
The oysters were transported to the Laboratory of
Molecular Ecology at the Sonora University. Tissues of
digestive gland and gills were dissected using sterile
instruments for every oyster and immediately fixed
with 95% ethanol. Additionally, 19 tissue samples of C.
corteziensis cultured during September 2011, from La
Paz, Baja California Sur, Mexico (2408’13’’N,
11025’37’’W) at more than 530 km south of La Cruz,
were included in the current study.
The total genomic DNA from the samples was
isolated with the QIAamp DNA Mini Kit according to
the manufacturer’s instructions (QIAGEN) and PCR
was carried out with Ready-to-Go PCR beads (GE
Healthcare). The nested PCR was performed with
primers and PCR conditions reported by López-Flores
et al. (2004) and López-Flores (2003) that target the
ribosomal DNA intergenic spacer (rDNA IGS). The
first reaction was run with 125 ng DNA and 25 ng of
each primer in a total volume of 12.5 µL using PCRgrade water to amplify a 525 base-pair amplicon. The
primer sequences were MT-1 5’-GCCAAAGACA
CGCCTCTAC-3’ and MT-2 5’-AGCCTTGATCACA
CGCTTT-3’. The PCR conditions were, an initial
denaturalization at 94C for 5 min, 30 cycles of 94C
for 1 min, 60C for 1 min, 72C for 1 min, and a final
step of 72C for 10 min. The nested reaction was made
with Ready-to-Go PCR beads in 12.5 µL of total
volume with 0.5 µL of the first reaction and 0.025 µg
of each primer to amplify a 358 base-pair amplicon.
The nested primers were MT-1B 5’-CGCCACTAC
GACCGTAGCCT-3’ and MT-2B 5’-CGATCGAGTA
AGTGCATGCA-3’, and the PCR conditions were, an
initial denaturalization at 94C for 5 min, 25 cycles of
94C for 30 s, 60C for 30 s, 72C for 30 s, and a final
step of 72C for 10 min. DNA of Ostrea edulis infected
with M. refringens type O and corresponding to
sequence AM292652 of the GenBank was used as
positive control; samples without DNA were included
as negative controls.
Finally, the PCR products were visualized on 2%
agarose gels stained with ethidium bromide. To verify
the identity of the PCR products, only two amplicons
obtained from C. gigas and the two from C. corteziensis
were sequenced in both senses with primers MT-1B
and MT-2B and the chromatograms were revised with
ChromasPro v. 1.5 (Technelysium). The resulting
sequences were analyzed using the basic localalignment search tool (BLAST) of the National Center
for Biotechnology Information (NCBI), USA and a
multiple sequencing alignment was also done using
ClustalX (Thompson et al., 1997) with some M.
refringens sequences reported in GenBank.
In this survey, the majority of the sampled
organisms were diagnosed as negative to the parasite;
however, M. refringens was detected in four different
organisms by nested PCR (1.1%) of the total number of
C. gigas analyzed and two organisms of C. corteziensis
(10.5%). The positive samples of Bahia de Kino,
Sonora, were collected in December 2009 (n = 1),
January 2010 (n = 1) and November 2010 (n = 2). In
accordance with López-Flores et al. (2004), a single
DNA amplicon of 358 base pair (bp) was obtained from
the samples diagnosed as positive (Fig. 1).
Two DNA amplicons from each geographical
region were sequenced and analyzed (GenBank
accession numbers JQ066723-JQ066726). The BLAST
analysis matched 60 M. refringens entries with 94-100%
Figure 1. Nested PCR amplicons (358-bp) of rDNA IGS
agarose gel. M: DNA size marker. Lanes 1-2: amplicons
obtained from Crassostrea gigas tissue. Lanes 3-4:
amplicons obtained from C. corteziensis tissue. Lane 5:
negative control. Lane 6: positive control of Ostrea edulis
infected with Marteilia refringens.
identity and coverage of 100% for most of the entries.
These sequences also matched partially with three
sequences corresponding to a new Marteilia species
(JN820090-JN820092), but with coverage of 60 to 88%
and identities of 80 to 82%. The alignment of sequences
showed that M. refringens from C. gigas has more
substitutions than those from C. corteziensis, when
compared to the European AM292652 sequence (Fig.
2).
Before the first massive mortalities at the end of the
1990s, there was no strict control to prevent the
exchange of farmed oysters from different culture sites,
and there are no official figures regarding the
movement of organisms between farms or geographic
areas. All oyster farmers remember that a batch of
Crassostrea virginica was stocked in the Gulf of
California more than 10 years ago but there are not
official data to support that information. In a recent
study, Escobedo-Fregoso et al. (2015) made a
phylogenetic analysis that suggests the Atlantic coast
origin of the P. marinus from the Mexican Pacific coast
and his would support the version of the translocation
of oysters from the Atlantic to the Pacific, carrying not
only Perkinsus but Marteilia. Furthermore, there is
evidence that C. gigas can carry some primary stages
778
3
of M. refringens without being seriously affected; so C.
gigas is considered as resistant to infection with this
parasite species (OIE, 2009; Berthe, 2004). This would
explain the low prevalence of M. refringens in C. gigas
samples. Nevertheless, a PCR analysis can detect the
presence of a pathogen, but this not necessarily implies
a real infection (Burreson, 2008), and therefore an
extensive study including histology, in situ hybridization or transmission electron microscopy must
prove that C. gigas and C. corteziensis are susceptible
species for M. refringens infections. Another important
aspect of the OIE (2014) is the self-declaration of
freedom from M. refringens for countries or zones and
its repercussion over importations and exportations of
live animals or commodities. Until the C. gigas and C.
corteziensis susceptibility is resolved, the presence of
M. refringens in some locations of the Gulf of
California is of great concern to the oyster culture
industry of the region.
The OIE (2009) recommends the use of primers Pr4
and Pr5 (Le Roux et al., 2001) for detection of M.
refringens, but the primers designed by López-Flores et
al. (2004) were used in this study because of their
higher specificity and sensitivity. The OIE (2009)
mentions that although those primers are more
Figure 2. Nucleotide sequences of the 359-bp PCR product of Marteilia refringens from Crassostrea gigas (JQ066723 and
JQ066724) and C. corteziensis (JQ066725 and JQ066726) and comparison with GenBank sequence AM292652. Dots
represent identical bases to the AM292652 sequence.
4779
sensitive, a thorough study for the evaluation of its
specificity is still necessary; however Carrasco et al.
(2012), found the new M. refringes type C infecting
Cerastoderma edule in Europe for the first time by
using the primers designed by López-Flores et al.
(2004).
The sampled oysters come from cultures without
abnormal mortalities of the same condition that
Grijalva-Chon et al. (2013) describes for oysters with
OsHV-1 in the same location and, fortunately, the
prevalence of M. refringens DNA in the sampled
months is low. All this requires an extensive study that
includes wild mollusk species to determine the genetic
variability of M. refringens in the Gulf of California,
species susceptibility, and possible relationships among
genotypes and host native species. The presence of
OsHV-1, P. marinus and now M. refringens DNA in C.
gigas and the native C. corteziensis clears up doubts, at
least in part, about the possible pathogens involved in
the massive mortality events that threatened cultures
some years ago. Although there may be other pathogens
that may jeopardize the survival of oyster species at
different stages, such as the presence of some Vibrio
bacteria and other protozoan species, the relevance of
this study lies in identifying pathogen species that are
notifiable to the World Organization for Animal Health
(OIE) and which had not been previously reported in
the eastern Pacific.
ACKNOWLEDGEMENTS
Thanks to Tereso Félix-Aispuro and Víctor Lugo from
La Cruz, Sonora, and Manuel Robles from La Paz, Baja
California Sur, for providing organisms and to Dr. Ellis
Glazier for editing the English-language text. Thanks to
Inmaculada López-Flores (Universidad de Granada,
Spain) for providing positive control DNA and to
Josefina Ramos-Paredes (Laboratorio Especializado de
Biología Molecular-SENASICA) for sequencing the
PCR products. Partial funds were provided by Consejo
Nacional de Ciencia y Tecnología through Project CB2009-01-133704.
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of diagnostic tests for aquatic animals. World
Organization for Animal Health-Office International
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Received: 11 September 2014; Accepted: 5 May 2015
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Lat. Am. J. Aquat. Res., 43(4): 781-785, 2015 Invasive Cherax quadricarinatus in Jalisco, Mexico
Short Communication
Wild populations of the invasive Australian red claw crayfish
Cherax quadricarinatus (Crustacea, Decapoda) near the northern
coast of Jalisco, Mexico: a new fishing and profitable resource
Fernando Vega-Villasante1, José J. Ávalos-Aguilar1, Héctor Nolasco-Soria2
Manuel A. Vargas-Ceballos1, José L. Bortolini-Rosales3, Olimpia Chong-Carrillo1
Martín F. Ruiz-Núñez1 & Julio C. Morales-Hernández4
1
Laboratorio de Acuicultura Experimental, Centro de Investigaciones Costeras
Universidad de Guadalajara, Puerto Vallarta, Jalisco, México
2
Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, Baja California Sur, México
3
Departamento de Biología Comparada, Facultad de Ciencias
Universidad Nacional Autónoma de México (UNAM), México City, México
4
Centro de Estudios Meteorológicos de la Costa, Centro Universitario de la Costa
Universidad de Guadalajara, Guadalajara, México
Corresponding author: Héctor Nolasco-Soria ([email protected])
ABSTRACT. The red claw crayfish Cherax quadricarinatus is native to freshwater habitats of northern
Australia and Papua New Guinea. Its high reproductive and adaptive capacity in different environments allows
it to be cultivated, where escaped individuals have established wild populations in countries far from their natural
range. In the late 90’s and beginning of the 21st century, this crayfish was introduced illegally along the coast
of southern Jalisco. Mismanagement led to escape and dispersion. Currently there are wild crayfish in the Cajón
de Peñas Reservoir and surrounding streams in northern Jalisco, Mexico. The aim of this study was to evaluate
the presence of C. quadricarinatus in fisheries in this area of Jalisco and analyze its importance in generating
economic benefits for fishermen, comparing these results with those of the fishery for M. americanum, whose
fishery is traditional. To catch specimens, traps were set for 24 h in the La Sanja Stream and the Cajón de Peñas
Reservoir. The results of the survey showed that C. quadrica rinatus is an important part of the crustacean catch
in this area: 32% of the total catch in the stream corresponded to C. quadricarinatus and the rest to M.
americanum. While 85% of the catch in the dam corresponded to C. quadricarinatus, only 15% referred to M.
americanum. Crayfish fishing in the reservoir is now an important part of the productive activity of local families
dependent on fishing. The ecological consequences of wild crayfish proliferation remain to be studied.
Keywords: Cherax quadricarinatus, crustacean, introduced species, invasion, fishery, Mexico.
Poblaciones silvestres invasoras de langosta australiana de pinzas roja
Cherax quadricarinatus (Crustacea, Decapoda) cerca de la costa norte de
Jalisco, México: un nuevo y rentable recurso pesquero
RESUMEN. La langosta australiana de agua dulce Cherax quadricarinatus es un crustáceo nativo de ríos del
norte de Australia y Papua Nueva Guinea. Su alta capacidad de reproducción y adaptación a diferentes ambientes
le ha permitido establecer poblaciones silvestres en países lejanos a su área de distribución natural. A fines de
los años 90 y comienzo del año 2000, este crustáceo se introdujo ilegalmente a lo largo de la costa del sur de
Jalisco, México. La mala gestión motivó la fuga y dispersión de este crustáceo. Actualmente, se encuentran
ejemplares en el embalse Cajón de Peñas y arroyos circundantes en el norte de Jalisco. El objetivo de este estudio
fue evaluar la presencia de C. quadricarinatus en las capturas realizadas en la zona de Jalisco, y analizar su
importancia en la generación de beneficios económicos para los pescadores, en comparación con la pesquería
de M. americanum, que es una actividad tradicional. Para la captura de los especímenes, las trampas se colocaron
durante 24 h en el arroyo La Sanja y en el embalse Cajón de Peñas. Los resultados mostraron que esta especie
____________________
Corresponding editor: Ingo Wehrtmann
781
782
es parte importante de la captura de crustáceos en esta área. El 32% de la captura total en el arroyo correspondió
a C. quadricarinatus y el resto a M. americanum. Mientras que el 85% de la captura en el embalse, correspondió
a C. quadricarinatus y 15% a M. americanum. La pesca de esta langosta en el embalse es un componente
importante de la actividad productiva para el sostenimiento de las familias locales. Sin embargo, no han sido
aún investigadas las consecuencias ecológicas de su proliferación en los ríos y embalses de la región.
Palabras clave: Cherax quadricarinatus, crustáceo, especie introducida, invasión, pesquería, México.
The Australian red-claw crayfish Cherax quadricarinatus (Von Martens, 1868) is native to freshwater
habitats of northern Australia and Papua New Guinea
(Lawrence & Jones, 2002). The species is currently one
of the most important commercially farmed freshwater
crayfish in the world because it has many advantages,
including omnivorous feeding behavior, fast growth,
and easy cultivation (Bortolini et al., 2007). This
species is cultivated in New Caledonia, Africa, China,
Taiwan, Japan, Malaysia, Israel, Italy, United States,
Mexico, the Caribbean, Puerto Rico, Ecuador, and
Argentina (Ahyong & Yeo, 2007).
The red clay crayfish is a highly invasive species
due to its high reproductive capacity and to its ability to
adapt to many environments, when it can escape from
captivity. Wild populations have been reported from
Jamaica, Mexico, Puerto Rico, Singapore, and South
Africa (Ahyong & Yeo, 2007), Israel (Snovsky & Galil,
2011), and Slovenia (Jaklic & Vrezec, 2011). Taken
into consideration its popularity in aquaculture,
recreational activities, and ability to displace native
species, C. quadricarinatus is of high concern
regarding its potential to distribute globally (Harlioglu
& Harlioglu, 2006). Bortolini et al. (2007) published
the first report of wild populations in Mexico in the
States of Tamaulipas and Morelos, where its cultivation
is well-established. The species has been cultivated also
in the states of Veracruz and Baja California Sur
(INAPESCA, 2012, 2013). Although not officially
cultivated in the State of Jalisco, inhabitants of the
coastal zone mentioned that, starting in the late 90’,
some incipient farms were installed along the southern
coast of Jalisco without official authorization. The
interest in this crayfish by small aquaculture farmers
led to a poorly planned and inadequately managed
transfer of crayfish to other ponds. The poor
management of the ponds favored the escape of C.
quadricarinatus into natural and artificial freshwater
bodies hundreds of miles north of the sites where they
were originally introduced (García de Quevedo, pers.
comm.). According to local fishers who traditionally
exploited the native prawns, particularly Macrobrachium americanum (Bate, 1868), trapping red claw
crayfish was a rare and sporadic event several years
ago. During the past three years, however, catches of C.
quadricarinatus increased progressively (Cajón de
Peña Fisheries Cooperative, pers. comm.). During our
survey, red claw crayfish were caught in fishing traps
in La Sanja, a local stream (20º00’N, 105º27’W) and in
Cajón de Peñas (20º00’N, 105º06’W), a moderately
large reservoir. Both sites are near the northern coast of
Jalisco. Simultaneous capture of the native cauque river
prawn M. americanum was also part of the survey to
compare the relative abundance of the two species. The
aim of this study was to evaluate the presence of C.
quadricarinatus in fisheries in this area of Jalisco and
analyze its importance in generating economic benefits
for fishers, comparing these results with those of the
traditional fishery for M. americanum.
Cherax quadricarinatus and M. americanum were
collected at two sites near the town of Tomatlán,
Jalisco: 1) La Sanja Stream: February 2014; traps were
locally made from 20 L plastic buckets with conical
inlets at the top, 2) Cajón de Peñas Reservoir: April
2014; traps (of cubic shape) were locally made from
PVC pipes and covered with mesh with a conical entry
on one side. Coconut pulp was used as bait in both
locations.
Ten traps were placed at each site in the morning
and removed 24 h later. In the stream and in the
reservoir we performed trapping operations five times
and three times on different days, respectively. The
total number of crayfish per catch was counted and
classified by sex and species. Using a Vernier caliper,
the total length of crayfish and prawns was recorded (in
cm) by measuring the distance from the tip of the
rostrum to the extreme end of the telson. The weight
was recorded (in g) with a digital field scale. Interviews
with fishers from the local fishing cooperative provided
information regarding the costs of fishing and sale
prices of both species during the different fishing
seasons.
In the stream, a total of 105 decapods were captured;
72 individuals of M. americanum (38 males and 34
females; mean weight of 19.2 ± 6.1 g and mean length
of 9.8 ± 2.2 cm) and 33 of C. quadricarinatus (6 males
and 27 females; mean weight of 24.7 ± 6.1 g and mean
length of 9.6 ± 2.5 cm). Of the 34 specimens collected
in the reservoir, five were M. americanum (5 males;
mean weight of 25.4 g ± 8.2 and mean length of 13.4
cm ± 4.5) and 29 C. quadricarinatus (14 males and 15
Invasive Cherax quadricarinatus in Jalisco, Mexico
783
Table 1. Crayfish fishery and its economic value in Cajón de Peñas Reservoir during low and high fishery seasons. *Low
season is from mid-November to June. **High season is from October to early November, about 45 days. Price: US$3.3
kg-1. Total corresponds to 40 fishermen.
Crayfish catch low season*
Fisherman average
Total
Crayfish catch high season**
Fisherman average
Total
Daily catch
(kg)
Monthly catch
(kg)
Total catch
(kg)
Sales profit
per season (US$)
4
160
120
4,800
720
28,800
2,376.00
95,040.00
18
720
540
21,600
810
32,400
2,673.00
106,920.00
Total annual
sales (US$)
201,960.00
Table 2. Prawn fishery and its economic value in Cajón de Peñas Reservoir during low and high fishery seasons. *Low
season is from mid-November to June, **High season is from October to early November, about 45 days. Price: US$13.3
kg-1. Total corresponds to 40 fishermen.
Prawn catch during low season*
Fisherman average
Total
Prawn catch during high season**
Fisherman average
Total
Daily catch
(kg)
Monthly catch
(kg)
Total catch
(kg)
0.5
20
15
600
90
1,800
1,197.00
23,940.00
2.5
100
75
3,000
112.5
4,500
1,496.25
59,850.00
females; mean weight of 29.2 g ± 10.2 and mean length
of 11.7 ± 2.9 cm).
The results confirm that wild populations of red
claw crayfish represent a significant proportion of the
freshwater decapod catch with high commercial value.
There are obvious differences between the two
surveyed sites: large populations of native cauque
prawn are located in drainage channels because these
shrimps require access to downstream estuaries to
complete their reproductive cycles (Bauer, 2013).
These well-established populations may favor the
explosive expansion of the red claw crayfish, because
at the reservoir cauque prawns depend on the annual
arrival of juveniles migrating upstream from the
estuaries. These migrations are difficult or nearly
impossible without a channel to bypass the dam, which
in this case is almost 70 m high (Rodríguez-Uribe et al.,
2014). In contrast, red claw crayfish can maintain stable
populations or expand its territory because the species
does not require migration to brackish water to
complete its reproductive cycle (Ghanawi & Saoud,
2012). This situation may represent a possible threat to
wild populations of cauque prawns in this reservoir,
especially considering that both are benthic species and
prefer places with shelters such as stones, galleries and
Sales profit
per season (US$)
Total anual
sales (US$)
$83,790.00
driftwood (Jones & Ruscoe, 2001; García-Guerrero &
Apun-Molina, 2008).
Although the red claw crayfish is known to be
invasive and a potential disruptor of the wetland
systems when cultured outside their natural range, one
Mexican government agency still includes it as a
species with potential for commercial aquaculture
(INAPESCA, 2013).
Ponce-Palafox et al. (1999) discussed the economic
feasibility of raising red claw crayfish in Mexico, but
also described the risks of accidentally or intentionally
introducing the species into native ecosystems. FAO
(2014) discussed technical and marketing aspects of the
species cultivation, as well as its invasive character, its
global spread for more than two decades, and that the
impacts of dispersion have not been well studied. They
concluded that these impacts are “despicable” without
citing documented cases of significant ecological
consequences, including displacement or competitive
exclusion of native species. In contrast, another federal
government agency from Mexico classified red claw
crayfish as a “high risk species” that affects other
species (CONABIO, 2013).
Fishing and marketing of the crayfish is an
important economic activity at both sampling sites of
784
the present study. At the reservoir, there is an official
fishing cooperative that keeps records of crayfish
catches. The fishing records for crayfish are resumed in
Table 1. During the low season (mid-November
through June), fishers collected 4,800 kg per month
(190.000-240.000 crayfish per month). During the high
fishing season (October and early November; about 45
days), fishers obtained 32,400 kg (about 1,620,000
crayfish). The situation of the native prawn fishery in
this reservoir is resumed in Table 2. During the low
season (same as above) the fishers collected 600 kg per
month (about 30,000 prawns per month). In the peak
season, the fishers obtained 4,500 kg (225,000 prawns
during the season). The total annual sales of the catch
per year was US$280,750 of which 70.7% came from
the sale of red claw crayfish and 29.3% originated from
the sale of native cauque prawns. In these calculations,
the fishing season is nine months because July through
September is an officially closed season for catching
prawns of the genus Macrobrachium. Although the red
claw crayfish is not covered by this prohibition, fishers
are not allowed to place traps under penalty of
prosecution.
In its 2011 report, INAPESCA considered 2010 as
the year with a historical maximum aquaculture
production of 15 ton of red claw crayfish in Mexico,
while trapping of red claw crayfish in the reservoir was
about 66 ton per year. For the stream location, there are
no records available for comparison because these
independent fishers do not report catches. Still, it is
likely that fishing C. quadricarinatus in this stream
provides significant economic benefits to the fishers
because it has a large regional demand and attractive
sale prices. The records of red claw crayfish in Mexican
water bodies should be reviewed and updated by the
government. Jalisco is not officially listed as a state
producing red claw crayfish. Harvesting crayfish in
reservoirs, streams, and irrigation channels is already
an important activity and should be acknowledged and
monitored by the government. Moreover, the
ecological consequences of this invasive expansion
should be thoroughly studied. While red claw crayfish
aquaculture can positively contribute to the local
economy, crayfish farming must be coupled with
measurements to prevent dispersal and damage to the
native ecosystems. This can only be achieved by
concerted efforts in education and sharing scientific
information and public cooperation (Peay, 2009).
ACKNOWLEDGEMENTS
We thank Ira Fogel of CIBNOR for providing
important editorial services. Special thanks to Ingo
Wehrtmann for his kind edition of the manuscript, and
to anonymous referees of LAJAR for improving the
quality of our work. The authors thank the fishermen of
the Cajón de Peñas Fisheries Cooperative (particularly
"Chendo") for the information provided. Also, we
thank Rafael García de Quevedo for the oral
transmission of the origins of this biological invasion in
Jalisco.
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Lat. Am. J. Aquat. Res., 43(4): 786-791, 2015 Digestibility of energetic ingredients by Arapaima gigas
786
1
Short Communication
Apparent digestibility of energetic ingredients by pirarucu
juveniles, Arapaima gigas (Schinz, 1822)
Filipe dos Santos-Cipriano1, Kauana Santos de Lima2, Érica Bevitório-Passinato2
Raildo Mota de Jesus2, Francisco Oliveira de Magalhães Júnior2,
William Cristiane Teles-Tonini6 & Luis Gustavo Tavares-Braga7
1
Universidade Federal de Minas Gerais, Belo Horizonte, MG, CEP 31270901, Brasil
2
Universidade Estadual de Santa Cruz, Ilhéus, BA, CEP 45662900, Brasil
3
Universidade do Estado da Bahia, Xique-Xique, BA, CEP 47400000, Brasil
Corresponding author: Gustavo Braga ([email protected])
ABSTRACT. An understanding of feed ingredient digestibility for the pirarucu is a fundamental step in the
development of feeds that promote proper growth of the specie while in captivity. A digestibility trial was
conducted with four treatments in triplicate (corn starch, corn, rice bran and wheat bran) to evaluate the
digestibility of dry matter, gross energy, crude protein and amino acids by the pirarucu. We used indirect
methodology with the inclusion of chromium oxide at 0.1% in the feeds. In total, 18 juveniles were used, with
an average live weight of 235 ± 36 g. The sampled juveniles were trained to consume the feeds prior to testing.
The corn and cornstarch presented the best apparent digestibility coefficients (ADCs) of dry matter, with 76.37%
and 70.66%, respectively, followed by rice bran (46.23%) and wheat bran (45.13%). The best ADCs of crude
protein were observed in corn (93.44%) and cornstarch (90.94%) compared to rice bran (68.23%) and wheat
bran (68.58%). There was no significant difference in the ADC of gross energy; the values ranged from 47.10%
for corn starch to 40.10% for corn. The corn and corn starch presented the best ADCs for all the amino acids
evaluated, followed by rice bran and wheat bran.
Keywords: Arapaima gigas, carnivorous, feeding, nutritional value, protein, aquaculture.
Digestibilidad aparente de ingredientes energéticos por juveniles de
pirarucu, Arapaima gigas (Schinz, 1822)
RESUMEN. El conocimiento de la digestibilidad de los ingredientes en la alimentación del pirarucu es
primordial para la elaboración de pienso específico, que promueva un crecimiento óptimo. Se realizó un ensayo
de digestibilidad con cuatro ingredientes energéticos, almidón de maíz, harina de maíz, salvado de arroz y
salvado de trigo para la evaluación de las digestibilidades de materia seca, energía bruta, proteína bruta y
aminoácidos. Se utilizaron 18 juveniles con peso de 235 ± 36 g. La harina de maíz y el almidón de maíz presentan
los mejores coeficientes de digestibilidad aparente (CDA) de la materia seca, 76,37% y 70,66% respectivamente,
seguidos por el salvado de arroz (46,23%) y salvado de trigo (45,13%). Los mejores CDA de la proteína bruta
se determinaron para harina de maíz (93,44%) y almidón de maíz (90,94%), en relación al salvado de arroz
(68,23%) y salvado de trigo (68,58%). Para el CDA de la energía bruta no fue registrada diferencia estadística,
variando entre 47,10% para el almidón de maíz y a 40,10%, para la harina de maíz. La harina de maíz y el
almidón de maíz presentaron los mejores CDA de todos los aminoácidos evaluados, seguidos por el salvado de
arroz y salvado de trigo.
Palabras clave: Arapaima gigas, valor nutritivo, alimentación, carnívoro, proteína, acuicultura.
The pirarucu, Arapaima gigas, is a carnivorous fish
endemic to the Amazon basin. The fish offers great
potential for use in aquaculture; its meat is highly
utilized, it provides a good carcass yield, and it has
rapid growth, with the capacity to reach more than 10
kg in one year of cultivation (Imbiriba, 2001).
__________________
Corresponding editor: Jesús Ponce
Knowledge of the digestibility of the ingredients
used in fish feed is of fundamental importance because
it allows the formulation of more efficient feeds, thus
resulting in a better utilization of nutrients, an
optimization of feeding costs and an increase in
productivity and profitability for the producer. Our ob-
2787
jective was to determine the apparent digestibility
coefficient of the dry matter, crude protein, gross
energy and amino acids of the energetic ingredients by
pirarucu juveniles.
The experiment was conducted at the laboratory of
fish nutrition and feeding (AQUANUT) at the State
University of Santa Cruz in October 2012; the
experimental period was 18 days. We used 18 juvenile
pirarucu, with an average live weight of 235 ± 36 g;
specimens were provided by the Canta Galo Farm,
Ibirataia-Ba. Three individuals were housed per tank in
six tanks (310 L) that were later used as feeding tanks.
The tanks were arranged in a closed circulation system
using a water pump (Dancor ®, RJ, Brazil-75 HP) with
biological filters, and constant aeration was provided
by a blower (WEG of 1 HP).
Juveniles were subjected to period of adaptation to
laboratory and routine management conditions for ten
days, during which they received the reference feed
(Table 1) four times a day. During the adjustment
period and the experimental period, daily cleaning was
performed to remove feces and possible scraps of feed.
The reference ration was formulated using the
SUPER CRAC® computational program, which
monitored the crude protein levels as tested by Ituassú
et al. (2005). In total, 1 g kg-1 of chromium oxide
(Cr2O3) was added as an external indicator of the feeds
for the determination of digestibility. For the
manufacture of feed, the ingredients were ground in a
knife type mill, passed through a 0.5 mm sieve and then
homogenized in accordance with the formulation of
each feed. The feeds were processed in a meat grinder
with a reverser using a matrix of 2 mm. Prior to
processing, water (40°C) was added to the mixture.
Feed grains were subsequently dried in a forced
ventilation oven (55°C) for 24 h and disintegrated to an
appropriate size for fish consumption. The test feeds
were formulated using a mixture of 70% of the
reference feed with 30% of the ingredients to be tested
(Table 2). We evaluated the apparent digestibility
coefficients of four energetic ingredients: corn, corn
starch, wheat bran, and rice bran. For each ingredient,
we used three replications.
For each treatment, the fish remained in the tanks
(310 L) during the daytime period where they received
five feedings per day, two in the morning (08:00 and
10:00 h) and three in the afternoon (12:00, 14:00 and
16:00 h). An hour after the last feeding, the fish were
transferred to the digestibility aquariums (200 L) to
perform feces collection. The digestibility aquariums
had a conical shape with a constant aeration system and
were equipped with collectors at the bottom that were
submerged in water and ice during the collection
periods. At 07:00 h the next day, the fish were
transferred to the supply tanks; next, the collectors were
removed and the material was collected. For each feed,
the fish were subjected to a three-day adaptation period
and a subsequent three-day period of feces collection.
The apparent digestibility coefficients of the feed
and the test ingredients were verified using indirect
methods with the use of chromium oxide as the external
indicator. The apparent digestibility coefficients of the
rations (ADCRA) were calculated according to De Silva
(1989). The coefficients of digestibility of the ingredients (ADCI) were calculated using the methodology
employed by Bureau et al. (1999).
After the withdrawal of the collectors containing the
water and feces, the collected material was released
from the water contained in the upper third of the
collector and added to the remaining volume in
disposable aluminum containers for drying in a forced
ventilation oven at 55ºC for 12 h. After drying and
checking for the possible presence of scales, the
samples were identified, stored in plastic containers and
kept in a freezer (-10°C) for subsequent laboratory
analysis of dry matter (DM), mineral matter (MM),
crude protein (CP), gross energy (GE) and the
concentration of chromium. The analysis of crude
protein, crude energy, dry matter and mineral matter
were performed in the laboratory of animal nutrition
and in the laboratory of fish nutrition and feeding at the
State University of Santa Cruz, according to AOAC
(2005) methodology. The analyses of amino acids of
the feeds and feces were performed using ionic
chromatography (Evonik Industries AG). Chromium
concentrations were analyzed at the Electron
Microscopy Centre at the State University of Santa
Cruz in an optical emission spectrometer with
inductively coupled plasma (ICPO-ES), Varian model
710-S series.
The physicochemical variables of the water, pH,
temperature (ºC) and dissolved oxygen (mg L-1) were
monitored daily throughout the trial period using YSI
Professional Plus multi-parameter equipment and
presented the values of 6.8-7.0, 26.8 ± 0.43°C and 7.2
± 1.43 mg L-1, respectively.
Data were subjected to variance analysis and the
differences between the averages were submitted to the
Scott-Knott test at 5% probability using the statistical
program R Core Team (2011).
The apparent digestibility coefficients (ADC) of dry
matter, crude protein and gross energy of the
ingredients evaluated for juvenile pirarucus showed
significant differences (Table 3). The highest apparent
digestibility coefficients of dry matter (ADCDM) were
found for corn and cornstarch. The rice bran and wheat
did not differ between each other, with both shows in
lower ADCDM.
788
3
Digestibility of energetic ingredients by Arapaima gigas
Table 1. Diets composition for pirarucu juveniles.
Ingredients (g kg-1)
Soybean meal
Wheat bran
Corn gluten meal
Corn
Fish meal
Rice bran
Poultry by-product meal
Corn starch
Soybean oil
Mineral and vitamin mix1
Sodium chloride
Cellulose
Chromic oxide III
BHT2
Total
Crude protein
Gross energy (kJ g-1)
Ash
Reference
188.00
140.00
105.00
90.03
370.00
57.67
27.00
8.45
7.00
3.50
2.15
1.00
0.20
1000
432.40
19.63
143.00
Corn starch
130.80
97.41
73.06
62.64
257.44
40.13
318.79
5.88
7.00
3.50
2.15
1.00
0.20
1000
301.81
18.67
123.68
Diet
Rice bran
130.80
97.41
73.06
62.64
257.44
300
40.13
18.79
5.88
7.00
3.50
2.15
1.00
0.20
1000
339.87
20,39
137.94
Corn
130.80
97.41
73.06
362.64
257.44
40.13
18.79
5.88
7.00
3.50
2.15
1.00
0.20
1000
316.15
20.29
117.42
Wheat bran
130.80
397.41
73.06
62.64
257.44
40.13
18.79
5.88
7.00
3.50
2.15
1.00
0.20
1000
342.15
19.68
123.68
Mineral and vitamin mix per kg of product: vitamin A 6000000 UI, vitamin D3 2250000 UI, vitamin
E 75000 mg, vitamin K3 3000 mg, vitamin tiamine (B1) 5000 mg, riboflavin (B2) 10000 mg,
pirodoxine 8000 mg, biotin 2000 mg, ascorbic acid (vitamin C) 192500 mg, niacin 30000 mg, folic
acid 3000 mg, Fe 100000 mg, Cu 600 mg; Mn 60000 mg, Zn 150000 mg, I 4500 mg, Cu 15000 mg,
Co 2000 mg, Se 400 mg2 Butyl-hydroxy-toluene.
1
Similarly, it was observed for the apparent
digestibility coefficient of crude protein (ADCCP), in
which the ingredients that demonstrated the highest
digestibility were corn and cornstarch; the digestibility
did not differ between corn and cornstarch, while rice
bran and wheat bran, showed digestibility below 70%.
The ADC of all amino acids was higher for cornstarch
and corn, the minor values were found for the rice bran
and wheat bran.
There was no significant difference for the apparent
digestibility coefficient of energy (ADCGE) among all
the ingredients. All the ingredients presented low
digestibility; the values ranged between 40.10 and
47.87%.
The best ADCDM were found for corn and
cornstarch. Rice bran and wheat bran showed lower
ADCDM, most likely due to the high ash content
(Table 2) contained in the two ingredients and the
higher levels of phytate (Kumar et al., 2012), which
partially reduces the availability of minerals, in
addition to the higher fiber content present in wheat
bran.
Another factor that likely contributed to the lower
rice bran and wheat bran ADCDM is the large amount
of non-starch polysaccharides in these two ingredients.
According to Conte et al. (2003), this soluble fiber has
the ability to absorb water, making the digested
material more viscous and reducing the activity of
enzymes and nutrient absorption. This result was also
found by Glencross et al. (2012a), who studied the
digestibility of different sources of starch and nonstarch polysaccharides in trout (Oncorhynchus mykiss).
Teixeira et al. (2010) studied the digestibility of
energetic ingredients in Pseudoplatystoma sp. and
found a lower ADCDM for corn (62.30%) and a higher
ADCDM for rice bran (59.67%) than the results of this
study.
In previous studies of carnivorous freshwater fish,
several researchers found lower values for the protein
digestibility of corn, with 51.4% for surubim Pseudoplatystoma reticulatum (Silva et al., 2013), and 64.18%
for painted Pseudoplatystoma corruscans (Gonçalves
& Carneiro, 2003). For the carnivorous marine fish “red
drum”, Sciaenops oceallatus, the ADCCP found by Mc
Googan & Reigh (1996) was lower for corn (81.56%)
and slightly higher for rice bran (77.16%). Wheat bran
and rice bran showed the lowest ADCCP. These results
were similar to those of Gonçalves & Carneiro (2003).
The authors studied the digestibility of ingredients in
the painted Pseudoplatystoma corruscans, in which
they observed higher digestibility with corn compared
to wheat bran and rice bran.
789
4
Table 2. Chemical composition of ingredients for pirarucu juveniles.
Ingredients
Dry matter (g kg-1)
Crude protein (g kg-1)
Gross energy (kJ g-1)
Lipid (g kg-1)
Ash (g kg-1)
Amino acids (%)
Ala
Arg
Asp
Cys
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Val
Corn starch
88.62 ± 0.2
14.35 ± 2.0
17.09 ± 0.6
8.71 ± 0.8
1.60 ± 0.3
Rice bran
91.72 ± 0.4
138.06 ± 5.3
21.34 ± 0.5
217.90 ± 6.6
100.34 ± 1.1
Corn
88.62 ± 0.3
80.75 ± 7.2
16.88 ± 0.4
85.56 ± 1.3
8.65 ± 0.4
Wheat bran
88.56 ± 0.4
177.8 ± 8.2
18.05 ± 0.8
116.11 ± 3.0
50.36 ± 0.6
0.159
0.096
0.210
0.019
0.380
0.136
0.061
0.073
0.213
0.159
0.062
0.088
0.099
0.141
0.101
0.076
0.742
0.822
1.060
0.229
1.500
0.726
0.274
0.432
0.809
0.292
0.308
0.502
0.572
0.515
0.448
0.629
0.532
0.316
0.471
0.159
1.370
0.276
0.244
0.306
0.903
0.085
0.175
0.355
0.619
0.271
0.237
0.396
0.792
1.153
1.143
0.286
2.920
0.900
0.367
0.516
0.993
0.625
0.265
0.615
0.886
0.682
0.521
0.726
Table 3. Apparent digestibility coefficient (ADC) of: dry matter, crude protein and energy, and amino acid of tested
ingredients for pirarucu juveniles. Values followed by the same superscripts within columns do not differ (P > 0.05).
Ingredients (%)
Dry matter
Crude protein
Gross energy
Amino acids
Ala
Arg
Asp
Cys
Glu
Gly
His
Ile
Leu
Lys
Met
Phe
Pro
Ser
Thr
Val
Corn starch
70.66a ± 2.54
90.94a ± 3.50
47.87a ± 5.37
Rice bran
46.23b ± 2.79
68.23b ± 6.27
42.23a ± 2.35
Corn
76.37a ± 0.42
93.44a ± 3.44
40.10a ± 5.42
Wheat bran
45.13b ± 0,80
68.58b ± 2,25
47.37a ± 3.67
89.41a
97.38ª
91.76ª
84.32ª
95.16ª
94.84ª
93.64ª
89.08ª
94.41ª
94.42ª
91.24ª
84.27ª
92.90ª
92.56ª
87.22ª
90.28ª
55.64b
77.20b
55.90b
41.86b
69.80b
70.01b
68.62b
51.95b
61.26b
60.33b
60.92b
39.81b
66.80b
58.80b
44.80b
54.25b
91.95a
96.67a
89.36a
83.10a
96.34a
90.65a
92.18a
89.71a
94.41a
93.42a
89.58a
80.80a
91.12a
89.11a
80.72a
88.54a
57.35b
72.29b
48.50b
50.17b
70.38b
67.48b
66.88b
49.02b
59.41b
60.98b
56.00b
52.76b
72.64b
58.21b
42.50b
51.29b
CV (%)
(P)
5.98
6.39
9.90
0,0010
0,0020
0,1560
9.71
5.95
13.84
15.32
7.60
6.87
7.92
14.68
10.59
10.07
9.15
15.74
6.21
10.22
13.96
12.61
0.0003
0.0005
0.0011
0.0013
0.0008
0.0005
0.0010
0.0014
0.0007
0.0006
0.0003
0.0016
0.0003
0.0006
0.0004
0.0008
Digestibility of energetic ingredients by Arapaima gigas
The diets formulation based on the amount of
available amino acids can result in significant
improvements in performance (Rawles et al., 2006).
The digestibility of amino acids tend to reflect protein
digestibility, however differences may occur in the
ADCAA of some amino acids (Zhang et al., 2015). In
this study, similar to ADCCP, corn and corn starch
showed the highest values for the ADC of all amino
acids. Among the amino acids, lysine is considered the
first limiting to the growth of fish (Abboudi et al.,
2006). Methionine is required in large quantities and
also plays an important role in growth (Bomfim et al.,
2008). The values of the ADC of lysine and methionine
found by Ribeiro et al. (2011) using Nile tilapia are
slightly lower than the present work for corn (80.38 and
80.87%) and slightly higher for wheat bran (79.92 and
79.66%, respectively).
All ingredients showed an ADCGE below 50%. The
test ingredients had high levels of starch; typically,
carnivorous fish species present less activity of amylase
compared to omnivorous species (Hidalgo et al., 1999).
Glencross et al. (2012b) observed a negative
relationship between the higher levels of amylopectin
and digestibility in juveniles of Lates calcarifer.
Similar to the observations of this study, Silva et al.
(2013) and Lundstedt et al. (2004) observed difficulties
in the use of starch as an energy source by the
carnivorous fish P. reticulatum and P. corruscans,
respectively.
Lower ADCGE levels for ingredients of vegetable
origin compared to ingredients of animal origin were
observed for the carnivorous fish Rachycentron
canadum (Zhou et al., 2004), Sebastes schlegeli (Lee,
2002) and P. corruscans (Gonçalves & Carneiro,
2003). Low ADCGE values were also found by
Gonçalves & Carneiro (2003) for wheat bran (53.20%),
rice bran (47.34%), and corn (64.95%) using P.
corruscans (9.80 g). Silva et al. (2013) observed low
ADCGE values for corn (43.24%) and wheat bran
(40.45%) in P. reticulatum (82.40 g). Braga et al.
(2008) evaluated the digestibility of ingredients in
Salminus brasiliensis juveniles (35.51 g) and found that
the ADCGE values were the highest for corn (80.84%)
and wheat bran (77.02%).
In conclusion, pirarucu exhibited a good ability to
utilize protein from corn and cornstarch, although the
species was not able to efficiently digest the energy
contained in any of the tested ingredients.
ACKNOWLEDGMENTS
This project was supported by Coordination for the
Improvement of Higher Education Personnel (CAPES)
and Bahia Research Foundation (FAPESB-TSC0015/
790
5
2012), Brazil. The Cantagalo Farm (Bahia, Brazil)
provided the fish for this project.
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maintenance and for tissue accretion in Atlantic
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Bureau, D.P., A.M. Harris & C.Y. Cho. 1999. Apparent
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Conte, A.J., A.S. Teixeira, E.T. Fialho, N.A. Schoulten &
A.G. Bertechini. 2003. Effect of phytase and xilanase
on the performance and bone characteristics of broiler
chicks fed diets with rice bran. Rev. Bras. Zootec., 32:
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and artificial diets. Asian Fish Soc. Spec., 4: 36-45.
Glencross, B., N. Rutherford & N. Bourne. 2012a. The
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rainbow trout (Oncorhynchus mykiss). Aquaculture,
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M. Anderson, T. Fox-Smith & R. Smullen. 2012b. An
assessment of cereal grains and other starch sources in
diets for barramundi (Latescal carifer) implications
for nutritional and functional qualities of extruded
feeds. Aquacult. Nutr., 18: 388-399.
Gonçalves, E.G. & D.J. Carneiro. 2003. Apparent
digestibility coefficients of protein and energy of some
ingredients used in diets for pintado, Pseudoplatystoma corruscans (Agassiz, 1829). Rev. Bras.
Zootec., 32: 779-786.
Hidalgo, M.C., E. Urea & A. Sanz. 1999. Comparative
study of digestive enzymes in fish with different
nutritional habits. Proteolytic and amylase activities.
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Ituassú, D.R., M. Pereira-Filho, R. Roubach, R.
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Crude protein levels for juvenile pirarucu. Pesqui.
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Kumar, V., A.K. Sinha, H.P.S. Makkar, G. Boeck & K.
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Lee, S.M. 2002. Apparent digestibility coefficients of
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Lundstedt, L.M., J.F.B. Melo & G. Moraes. 2004.
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Pseudoplatystoma coruscans (Teleostei: Siluriformes)
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Received: 5 January 2015; Accepted: 16 June 2015
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J.E.P. Cyrino. 2013. Digestibility of feed ingredients
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D.V. Crepaldi & L.P. Ribeiro. 2010. Apparent
digestibility coefficients of different energetic
ingredients for surubim juveniles. Rev. Bras. Zootec.,
39: 1180-1185.
Zhang, C.X., K.K. Huang, L. Wang, K. Song, L. Zhang &
P. Li. 2015. Apparent digestibility coefficients and
amino acid availability of common protein ingredients
in the diets of bullfrog, Rana (Lithobates) catesbeiana.
Zhou, Q.C., B.P. Tan, K.S. Mai & Y.J. Liu. 2004.
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Lat. Am. J. Aquat. Res., 43(4): 792-797, Anticipation
2015
of Artemia sp. supply in the larviculture of barber goby
Short Communication
Anticipation of Artemia sp. supply in the larviculture of the barber goby
Elacatinus figaro (Gobiidae: Teleostei) influenced growth, metamorphosis
and alkaline protease activity
Maria Fernanda da Silva-Souza1, Juliet Kiyoko Sugai2 & Mônica Yumi Tsuzuki1
Laboratório de Peixes e Ornamentais Marinhos (LAPOM), Departamento de Aquicultura
Centro de Ciências Agrárias, Universidade Federal de Santa Catarina
P.O. Box 476, 88040-970, Florianópolis, Santa Catarina, Brazil
2
Laboratório de Enzimologia Aplicada, Departamento de Bioquímica, Centro de Ciências Biológicas
Universidade Federal de Santa Catarina, P.O. Box 476
88040-900, Florianópolis, Santa Catarina, Brazil
1
Corresponding author: Mônica Yumi Tsuzuki ([email protected])
ABSTRACT. The barber goby Elacatinus figaro is considered endangered due to overexploitation by the
ornamental industry. Farming marine ornamental fishes, especially the threatened ones, can be one of the
measures to minimize the pressure on the natural stocks. Among the priority issues for their production is the
determination of the most appropriate feeding management. The feeding protocol commonly used in the
larviculture of barber goby, when the start of Artemia sp. offer occurred at the 18th DAH (days after hatching)
(treatment T18), was modified, by anticipating brine shrimp supply in 6 days (treatment T12). Alkaline proteases
activity, growth and metamorphosis of larvae were evaluated in both protocols. Juveniles at T12 showed higher
weight (0.04 ± 0.001 g) and lower activity of total alkaline proteases (1.3 ± 0.2 mU mg-1 protein) compared to
T18 (0.02 ± 0.001 g; 2.8 ± 0.4 mU mg-1 protein, respectively). With anticipation of brine shrimp, the
commencing and end of larval transformation was observed earlier (at 24 and 34 DAH, respectively) in
comparison to those with the supply of Artemia sp. at 18 DAH (27 and 41 DAH, respectively). Thus, the Artemia
sp. anticipation was beneficial during the larviculture of the barber goby, considering that larvae reached
metamorphosis earlier.
Keywords: Elacatinus figaro, fish, ornamental, endangered, live food, feeding, aquaculture.
La anticipación del suministro de Artemia sp. en la larvicultura del neón gobi
Elacatinus figaro (Gobiidae: Teleostei) influenció el crecimiento, metamorfosis
y actividad de proteasas alcalinas
RESUMEN. El neón gobi, Elacatinus figaro, se considera en peligro de extinción debido a la sobreexplotación
por la industria ornamental. El cultivo de peces ornamentales marinos, especialmente de las especies
amenazadas, puede ser una de las medidas para minimizar la presión sobre las poblaciones naturales. Entre los
temas prioritarios para su producción es la determinación de la estrategia de alimentación más adecuada. El
protocolo de alimentación de uso común en la larvicultura del neón gobi, cuando se inicia el suministro de
Artemia sp., que ocurre en el 18º DDH (días después de la eclosión) (tratamiento T18), fue modificado mediante
la anticipación de suministro de este microcrustáceo branquiópodo en 6 días (tratamiento T12). La actividad de
las proteasas alcalinas, crecimiento y metamorfosis de las larvas se evaluaron en ambos protocolos. Los juveniles
en T12 mostraron mayor peso (0,04 ± 0,001 g) y la menor actividad del total de proteasas alcalinas (1,3 ± 0,2
mU mg-1 de proteína) en comparación con T18 (0,02 ± 0,001 g; 2,8 ± 0,4 mU mg-1 de proteína, respectivamente).
Con la anticipación del suministro de Artemia sp. se observó que el principio y final de la transformación de las
larvas fue más temprano (a los 24 y 34 DDH, respectivamente), en comparación con aquellos con el
suministro de Artemia sp. en 18 DDH (27 y 41 DDH, respectivamente). Por lo tanto, la anticipación del
______________________
Corresponding editor: Mauricio Laterça
1
792
793
2
suministro de Artemia sp. fue beneficiosa durante el cultivo larval del neón gobi, considerando que la
metamorfosis de las larvas se alcanzó antes.
Palabras clave: Elacatinus figaro, pez, ornamentales, amenazada, alimento vivo, alimentación, acuicultura.
The barber goby, Elacatinus figaro, an endemic marine
ornamental fish from Brazil (Carvalho-Filho, 1999) is
of interest to the aquarium trade because of its small
size, coloration, active behavior and rusticity (Sazima
et al., 2000). It is a cleaner fish (Sazima et al., 1996),
removing ectoparasites, dead tissue, mucus and scales
from the body of other fish and invertebrates (Johnson,
1982; Losey, 1987; DeLoach, 1999). This cleaning
behavior is considered of fundamental importance for
the maintenance of the equilibrium and health of fish in
reef ecosystems (DeLoach, 1999) and in reef
aquariums.
Due to an intensive harvest during the past years to
the aquarium trade (Gasparini et al., 2005), E. figaro
has been included in the list of endangered species, and
its capture and trade is prohibited by the Brazilian
Ministry of Environment (Normative Instruction
Number 5 of 21 May 2004), Ministério do Meio
Ambiente, (Brasil, 2004).
Farming marine ornamental fishes, especially the
threatened ones, can be one of the measures to
minimize the pressure on the natural stocks. Among the
priority issues for the production of these fishes are the
knowledge of the nutritional requirements and the
determination of the most appropriate feeding management (Pezzato, 1997; Avella et al., 2007). Since food
is the source of energy and nutrients for larval growth
and development, the feeding protocol has a strong
influence on the development, digestive and
assimilation potential of nutrients in fish larvae
(Guerreiro et al., 2010). According to preliminary
studies with the barber goby, the early supply of either
an inadequate or an appropriate live food can alter
digestion and food utilization, affecting larval survival
and growth (Côrtes, 2009).
In aquaculture, the identification, quantification,
and evaluation of the changes in the profile of the
activity of digestive enzymes are needed to establish
the most appropriate moment to conduct the dietary
transition and to assist in choosing appropriate
ingredients and developing suitable food strategies in
the larviculture and grow out, based on the digestive
capacity of fish (Kuz’mina, 1996; Galvão et al., 1997;
Fernández et al., 2001; Cara et al., 2002).
In the present study, the feeding protocol commonly
used in the larviculture of this species was modified by
anticipating the Artemia sp. supply in order to evaluate
if this anticipation would affect growth as wet weight,
onset and end of metamorphosis (transformation from
larvae to juvenile) and activity of total alkaline
proteases of juveniles.
The experiment was conducted at the Fish and
Marine Ornamentals Laboratory (LAPOM), Federal
University of Santa Catarina, Brazil. Two couples of
wild Elacatinus figaro breeders, captured from the state
of Espírito Santo/Brazil with permission for activities
with scientific purposes from SISBIO/ICMBio
(Number: 22051-2) were used to obtain natural
spawning.
Breeders were maintained as described by Meirelles
et al. (2009). The photoperiod used was 14 h light and
10 h dark. Breeders were fed to apparent satiation twice
a day (morning and afternoon) with a varied diet
consisting of commercial diets for marine ornamental
fish MarineTetra and TetraVeggie (TETRA, Melle,
Osnabrück, Germany), marine fish weaning diet NRD
(INVE Technologies, Belgium), Artemia sp., enriched
with commercial emulsion of fatty acids (DHA SelcoINVE Technologies, Dendermonde, Belgium), as well
as shellfish, squid, chopped fresh fish and shrimp.
Tanks were daily cleaned to remove uneaten food and
feces.
Every day, the PVC pipe used as substrate for
spawning of each couple was observed, and so the day
before hatching was calculated in order to transfer the
eggs to 40 L hatching/larviculture tanks with the same
physical and chemical water conditions of the parental
tanks.
To verify if the anticipation of the brine shrimp,
Artemia sp., supply in the barber goby larviculture
would affect growth and day of metamorphosis of
larvae, two different feeding protocols (treatments)
(Fig. 1) were performed in triplicate: T18-Standard
feeding protocol, when the start of the brine shrimp
offer occurs at the 18th DAH (days after hatching)
(Côrtes, 2009; Meirelles et al., 2009); T12-Anticipation of the brine shrimp supply in 6 days (12th DAH)
from the standard feeding protocol.
After hatching, larvae were kept at the hatching tank
at a density of 5 larvae L-1, with microalgae
Nannochloropsis oculata (0.5-1.0 x106 cells mL-1 of
water) and rotifers Brachionus sp., until the start of
Artemia sp. supply, either at 12 DAH or 18 DAH.
After hatching, larvae were reared in aquariums at a
density of 5 larvae L-1, with microalgae Nannochloropsis oculata at a concentration of 0.5-1.0x106
cells mL-1 of water, for maintenance of rotifers.
Anticipation of Artemia sp. supply in the larviculture of barber goby
794
3
Figure 1. Dietary protocol of barber goby with Artemia sp. supply at 12 (T12) and 18 DAH (T18).
Rotifers Brachionus sp. (lorica length ranging from
100 to 180 µm) and Artemia sp. nauplii and metanauplii were used as live food. Rotifers were cultured
at a salinity of 25 g L-1, 26ºC, with microalgae
Nannochloropsis oculata (104-105 cells per individual).
Artemia sp. nauplii cysts (INVE Technologies,
Dendermonde, Belgium) were incubated at 29ºC and
salinity of 35 g L-1. Artemia sp. metanauplii were kept
under the same conditions, and after hatching, were
enriched with docosahexaenoic acid (DHA) Selco
(INVE Technologies, Dendermonde, Belgium), 24 h
prior to delivery to the larvae. Dry diet (crude protein
52%; lipid 12%, ash 15%; otohime TDO-A 250 µm;
Reed Mariculture, California, USA) was used for
juveniles.
During the experiment, the water temperature was
maintained at 26 ± 2ºC, salinity 28 ± 3 g L-1, and water
pH at 7.9 ± 0.2. In each experimental unit, dates of
commencement and completion of metamorphosis of
the larvae (larvae settled and pigmented) were
examined.
Ten fish of 41 DAH were collected per experimental
unit (30 fish per treatment) for biometry and quantification of total alkaline proteases activity. This age
was fixed for sampling animals as larvae had already
metamorphosed into juveniles (settled with typical
yellow and black color of the species). In each
sampling, fish were caught and immediately immersed
in iced cold water, where they were killed by thermal
shock. After being dried with paper towel, the biometry
was performed. Finally, they were properly packed in
aluminum foil and stored at -18ºC to determine the
activity of total alkaline proteases. In order to obtain the
homogenates for the enzymatic determination, the tail
and the head were despised and a “pool” of 10 juveniles
of each experimental unit was used for obtaining a
single homogenate.
For the preparation of the enzymatic extract, each
replica of samples was individually homogenized in
iced-cold distilled water in a ratio of 1:5.5 (tissue:
distilled water, w:v), through a van Potter homogenizer
for 2.5 min (5 agitations of 30 s with intervals of about
5 min to cooling). They were then centrifuged at
15,550x g for 15 min at 4ºC. The supernatants were
used for quantifying the activity of alkaline proteases.
The quantification of soluble proteins of the
enzymatic extracts was performed by the method of
Bradford (1976) using bovine serum albumin (BSA)
(Sigma-Aldrich, USA) as standard. The total alkaline
proteases activity of the extracts was measured using
the azocasein (Sigma Chemical Co, St Louis, Missouri,
USA) hydrolysis, method described by García-Carreño
et al. (1979).The enzymatic activity was expressed as
specific activity (U mg-1 protein), i.e., units ([∆Abs366nm
(Test-Control) min-1 mL-1]) per milli-gram of protein.
Data were subjected to analysis of variance
(ANOVA) with a significance level of 5% (software
Statistica 7.0). When present, the statistical differences
were measured using the Tukey test. The results were
presented as mean ± standard deviation (SD).
Survival rates did not differ among treatments
(mean of 13.5%). Growth of juvenile (41 DAH) barber
goby differed between the two treatments (P < 0.05), as
795
4
larvae with anticipated supply of Artemia sp. (T12)
showed higher weight (0.04 ± 0.001 g) compared to
those that started consuming this live food at the 18th
DAH (T18) (0.02 ± 0.001 g). This higher growth at T12
also affected the start of metamorphosis (Fig. 2).
Juveniles of 41 DAH fed Artemia sp. at the 12th DAH,
showed a lower activity of total alkaline proteases (1.3
± 0.2 mU mg-1 protein) compared to the activity found
in fish when Artemia sp. was offered at T18 (2.8 ± 0.4
mU mg-1 protein) (P < 0.05).
Assuming that larval biomass with anticipated
supply of Artemia sp. doubled, compared with the
traditional protocol, the decline in specific activity of
alkaline proteases is not related to a decrease in enzyme
synthesis, but might be a result of an increase in tissue
protein due to larval growth as cited by ZaboninoInfante & Cahu (2001) and Jimenez-Martinez et al.
(2012).
The day when Artemia sp. was offered to barber
goby larvae, significantly affected metamorphosis. In
larvae with anticipation of Artemia sp. (T12), the
commencing and end of transformation was observed
earlier (at 24 and 34 DAH, respectively) in comparison
to those at T18 (27 and 41 DAH, respectively) (Fig. 2).
Thus, the Artemia sp. anticipation proposed in this
study was beneficial, once barber goby larvae reached
metamorphosis earlier than the commonly used feeding
protocol for E. figaro, and even in comparison with
similar species. In the cultivation of Elacatinus
oceanopsis, when brine shrimp was added in the
larviculture at 15 DAH, fish entered metamorphosis
between days 30 and 40 post-hatching (Olivotto et al.,
2005). Considering that inert diet is supplied when
larvae reached metamorphosis, early metamorphosis of
fish would diminish the need of live food.
The success in the hatchery production of marine
fish is largely dependent on the availability of suitable
live food for feeding fish larvae. Live food organisms
contain all the nutrients such as essential proteins,
lipids, carbohydrates, vitamins, minerals, amino acids
and fatty acid and hence are commonly known as
“living capsules of nutrition” (Das et al., 2012).
The live food contribution to the nutrition, digestion
and assimilation process of the barber goby might be
considered when defining a feeding protocol for the
species. For example, the enrichment techniques
commonly used in Artemia sp. nauplii increased the
availability of highly unsaturated fatty acids to marine
fish larvae, such as docosahexaenoic acid (DHA) and
eicosapentaenoic acid (EPA), essential components in
Figure 2. Start and end day of the metamorphosis (mean
± SD, n = 10) of the barber goby in larviculture with
beginning of Artemia sp. supply at 12 (T12) and 18 DAH
(T18). Different letters in the same parameter evaluated
indicate significant differences (P < 0.05).
the diet (Han et al., 2000). Barroso (2010) demonstrated that Artemia sp. has greater amounts of incorporated fatty acids when compared to rotifers, in the
larviculture of the fat snook Centropomus parallelus,
using the same feeding protocol of this study. Possibly,
the greater availability of fatty acids in brine shrimp
caused an increment in growth in barber goby larvae in
which Artemia sp. supply was anticipated. The supply
of live food with administration of these fatty acids has
also been shown to increase the growth and development in ornamental species such as in yellowtail
damselfish Chrysiptera parasema (Olivotto et al.,
2003), in the goby Elacatinus evelynae (Olivotto et al.,
2005) and in clownfish Amphiprion ocellaris (Avella et
al., 2007).
Furthermore, because of its greater size when
compared with rotifers, Artemia sp. motility by the
digestive tract may cause mechanical stimuli by
increasing the peristaltic movements, which triggers the
larval digestive processes (Tandler & Kolkovski,
1991).
In the protocols for marine fish larviculture, the
beginning of the use of Artemia sp. nauplii (450-700
µm) is indicated when the larvae are able to consume
food larger than rotifers (80-340 µm) (Côrtes &
Tsuzuki, 2012). It is important that the introduction of
larger food during fish developing is supplied at the
right time, because there is a moment when the use of
rotifers is not most beneficial for the larvae, i.e., when
the energy spent by larvae to capture food is not
compensated by the energy contained in it.
Consequently, in the larviculture of barber goby, the
anticipation in the supply of brine shrimp caused higher
larval growth and an advance in their development and,
as a consequence, their earlier metamorphosis to
juvenile.
Anticipation of Artemia sp. supply in the larviculture of barber goby
ACKNOWLEDGEMENTS
The authors thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for Master's
scholarship to the first author and CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico) for grant to M.Y. Tsuzuki.
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Lat. Am. J. Aquat. Res., 43(4): 798-806, 2015 Electrophoretic protein profiles of copepod fed diatoms
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1
Short Communication
Electrophoretic protein profiles of mid-sized copepod Calanoides patagoniensis
steadily fed bloom-forming diatoms
Victor M. Aguilera1,2, Rubén Escribano2 & José Martínez-Oyanedel3
1
Instituto de Ciencias Naturales Alexander von Humboldt, Universidad de Antofagasta
P.O. Box 170, Antofagasta, Chile
2
Millenium Institute of Oceanography, Universidad de Concepción, Concepción, Chile
3
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas
Universidad de Concepción, Concepción, Chile
Corresponding author: Victor M. Aguilera ([email protected])
ABSTRACT. Recent field and experimental evidence collected in the southern upwelling region off
Concepción (36°5’S, 73°3’W) showed an abrupt reduction (<72 h) in the egg production rates (EPR) of
copepods when they were fed steadily and solely with the local bloom-forming diatom Thalassiosira rotula.
Because diatoms were biochemically similar to dinoflagellate Prorocentrum minimum, a diet which supported
higher reproductive outcomes, the fecundity reduction observed in copepod females fed with the diatom may
have obeyed to post-ingestive processes, giving rise to resources reallocation. This hypothesis was tested by
comparing feeding (clearance and ingestion rates), reproduction (EPR and hatching success) and the structure
of protein profiles (i.e., number and intensity of electrophoretic bands) of copepods (adults and eggs) incubated
during 96 h with the two food conditions. The structure of protein profiles included molecular sizes that were
calculated from the relative mobility of protein standards against the logarithm of their molecular sizes. After
assessing the experimental conditions, feeding decreased over time for those females fed with T. rotula, while
reproduction was higher in females fed with P. minimum. Electrophoretic profiles resulted similar mostly at a
banding region of 100 to 89-kDa, while they showed partial differences around the region of 56-kDa band,
especially in those females fed and eggs produced with T. rotula. Due to reproductive volume was impacted
while larvae viability, a physiological processes with specific and high nutritional requirements, was
independent on food type; post-ingestive processes, such as expression of stress-related proteins deviating
resources to metabolic processes others than reproduction, are discussed under framework of nutritional-toxic
mechanisms mediating copepod-diatoms relationships in productive upwelling areas.
Keywords: diatoms, blooms, food, copepods, reproduction, protein profiles.
Perfiles electroforéticos de proteínas del copépodo de talla media
Calanoides patagoniensis alimentado sostenidamente con diatomeas
formadoras de florecimientos
RESUMEN. Evidencia experimental y de campo recolectada en la región austral de surgencia frente a
Concepción (36°5’S, 73°3’W), mostró una abrupta (<72 h) reducción en la tasa de producción de huevos (EPR)
de copépodos cuando fueron alimentados sostenida y exclusivamente con cepas locales de la diatomea
formadora de florecimientos masivos Thalassiosira rotula. En vista que las diatomeas fueron bioquímicamente
similares al dinoflagelado Prorocentrum minimum, dieta que permitió mejores resultados reproductivos, la
reducción en la fecundidad en hembras de copépodos alimentadas con diatomea pudo obedecer a procesos postingestivos, dando lugar a una redistribución de recursos nutricionales. Se evaluó esta hipótesis mediante la
comparación de la alimentación (tasas de aclaramiento y de ingestión), reproducción (TPH y eclosión de huevos)
y estructura de perfiles de proteínas (i.e., número e intensidad de bandas electroforéticas) de copépodos (adultos
y huevos) incubados durante 96 h en ambas condiciones de alimento. La estructura de los perfiles de proteínas
incluyó los tamaños moleculares obtenidos desde la movilidad relativa de los estándares de proteínas contra el
logaritmo de su peso molecular. Luego de evaluar las condiciones experimentales, la alimentación de hembras
alimentadas
__________________
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2
con T. rotula disminuyó en el tiempo, mientras que la reproducción fue mayor en hembras alimentadas con P.
minimum. Los perfiles electroforéticos resultaron mayormente similares en la región de la banda de 100 a 89kDa, mientras que estos mostraron diferencias parciales en la región de la banda de 56-57-kDa, especialmente
en aquellas hembras alimentadas y huevos producidos con T. rotula. Dado que el volumen reproductivo fue
impactado mientras que la viabilidad de las larvas (proceso fisiológico con específicos y altos requerimientos
nutricionales), fue independiente del tipo de alimento; procesos post-ingestivos, tales como la expresión de
proteínas de estrés desviando recursos hacia otros procesos metabólicos distintos de la reproducción, se discuten
en el marco de los mecanismos nutricionales-tóxicos mediando las relaciones copépodos-diatomeas en sistemas
productivos de surgencia.
Palabras clave: diatomeas, florecimientos masivos, alimento, copépodos, reproducción, perfiles de proteínas.
Inter-specific relationship between primary producers
and their consumers in the ocean involve multiple and
diverse mechanisms that from the trophodynamic
viewpoint ultimately modulate how much photosynthetic carbon is available for higher trophic levels. A
specific issue of this relationship concerns the
“goodness” of food for marine grazers represented by
diatom blooms, which are highly prevalent biological
features in the most productive ocean ecosystems
(Irigoien et al., 2002). In terms of food for copepods,
main diatom grazers, such conditions are determined by
the size-spectra, cell concentration, and biochemical
properties of species forming the blooms (Jones &
Flynn, 2005; Flynn, 2008; Koski et al., 2008). On the
matter, diversity and nutritional value associated to
these microalgae aggregations can be greatly decreased
by allelophatic mechanisms during the establishment
and prevalence of the bloom (Legrand et al., 2001;
Flynn, 2008).
Chemical interactions among algae during blooms
may in turn modify diversity and prey size-structure
available at the time for grazers (Legrand et al., 2003).
Since size-distribution of food particles may restrict the
efficient detection and capture of prey by the copepods,
diatom blooms may thus compromise the achievement
of the food ration, especially for those mid and largesized species with higher food requirements (Price &
Paffenhöfer, 1984).
Functionally, the high cell concentrations observed
during diatom blooms (Scholin et al., 2000) may induce
high ingestion rates and, hence, low gut passage time
and incomplete digestion of the ingested cells
(Besiktepe & Dam, 2002). Both, passage time and
partial digestion modulate assimilation efficiency and
growth of copepods (Dutz et al., 2008). Ultimately,
bloom forming diatoms as many others microalgae
(Turner, 2014) are able to produce an array of
biologically-active metabolites, many of which have
been attributed as a form of grazing deterrent (Turner,
2014 and references therein). Thus, some chainforming diatoms, such as the species Thalassiosira
rotula, have been found capable to alternate from just
physical to more complex and compensatory chemical
defense mechanisms against grazers (Miralto et al.,
1999; Hamm et al., 2003; Fontana et al., 2007).
Therefore, when copepods were fed with different
strains of T. rotula their egg production dropped, their
embryos failed to develop, or hatched into malformed
nauplii that die soon after birth (Ianora & Miralto,
2010).
Calanoides patagoniensis (Copepoda, Calanoidea)
is a mid-size copepod species (2.5-2.7 mm length) that
co-exists with T. rotula in the productive southern
upwelling regions of the Humboldt Current System,
where this diatom is one of the most common and
dominant phytoplankton species (Anabalón et al.,
2007; Vargas et al., 2007). In these ecosystems, this
diatoms species was associated with reproductive
failures in other large-sized co-existing copepod
species, Calanus chilensis, expressed as low egg
production rates, low egg hatching, and high percentage
of larvae abnormality (Poulet et al., 2007).
More recently and studying reproductive traits of C.
patagoniensis upon local T. rotula strains, winter
flagellate assemblages, and Prorocentrum minimum;
Aguilera & Escribano (2013) found that although of
copepod egg viability was unaffected by food
treatments, reproductive activity in the form of egg
production rates resulted 30% lower after sustained (3
days) ingestion of T. rotula . Interestingly, both diets
had similar and relatively low C:N ratios (T. rotulaC:N
ratio = 4.3; P. minimumC:N ratio = 3.3). That is to say, both
diets provided relatively high nitrogen compounds and
thus, metabolic process with high proteins demand,
such as reproduction, should not be limited (Checkley,
1980). Whether tested diets were similar in providing C
and N for copepod females, post-ingestive processes,
such as the reorganization of nutritional compounds,
could lead to changes in copepod egg production rates.
We tested this possibility through the comparison of
feeding and reproductive traits as well as electrophoresis gel profiles of copepod females steadily fed (96 h)
with both food treatments and their spawned eggs.
Electrophoretic protein profiles of copepod fed diatoms
Copepods were collected between spring of 2007
and summer of 2008 at the upper 20 m of a shallow
nearshore station (ca. 5 km from the shoreline) in the
upwelling area off Concepción, Chile, in southern
Pacific Ocean (36°5’S, 73°3’W). Samples were
collected through vertical hauls of a WP-2 net with a
200-µm mesh size, and equipped with a non-filtering 1
L cod-end. Immediately after sampling, the cod-end
contents were transferred into a 60 L thermo box and
transported to a laboratory at the Marine Biology
Station of Dichato. Within 2 h of capture, fertilized and
unda-maged females of C. patagoniensis were carefully
sorted out using a dissecting microscope Leica Leitz
MZ6. Mature and reproductive copepod females were
selected and gently transferred into 0.2 µm filtered sea
water using the following criteria: 1) fully integrated
antenna, 2) presence and pigmentation of gonadal
segment, 3) visual recognition of oocytes in
vitellogenesis phase II (Yehezkel et al., 2000). After
sorting, females were acclimated by 24 h in filtered sea
water without food before to starting the experiments
(for more details please see Aguilera & Escribano,
2013). Food media to feed spring cohorts of copepods
consisted in a T. rotula culture collected from the study
area during the spring of 2007, when diatom blooms
dominate the phytoplankton structure and biomass
(Vargas et al., 2006, 2007).
The most abundant diatom T. rotula was then
successfully isolated and cultured into 0.2 µm filtered
sea water enriched with K-medium at 12°C with a
12:12 light: dark cycle (Guillard & Ryther, 1962; Keller
et al., 1987). Additionnally, it was supplied a culture of
the dinoflagellate P. minimum as food for copepod
cohorts obtained during summer 2008: this alga has
proved to be a suitable food resource that has widely
been used on feeding and reproduction experiments
with marine copepods (Paffenhöfer et al., 2005). Both
microalga cultures were supplied during their
exponential growth phase to ensure their nutritional
quality as food for copepods (Diekmann et al., 2009).
Linear dimensions of algae (length and width) were
measured under the microscope to later determine
volume and equivalent mean spherical diameter.
Carbon and nitrogen content were measured in algae
filtered onto precombusted filters using a Thermo
Finningan EA FLASH 1112 elemental analyzer.
Four experimental series were performed with both
food treatments, each one consisting on 96-h individual
incubations with daily food renewing and daily
monitoring of clearance (CR), ingestion (IR), egg
production (EPR) and hatching success (H) in 30
mature copepod females. Animals for experiments
were individually and gently pipetted into 300 mL acidwashed crystallizing dishes (300 mL glass capsules
800
3
with concave walls and flat floor) and incubated in a
temperature-controlled chamber (13 ± 1°C). The uses
of dishes allow a better individual monitoring of
simultaneous copepod responses, such egg production,
and fecal pellets production. Whereas turbulent
environment that eventually could impair fecal pellets
is only subjected to the aquatic perturbations derived
from copepod swimming, more dense eggs and fecal
pellets are deposited in the flat floor or gently in the
concave walls of experimental dishes without major
impairments. Furthermore, ad libitum food supply
(>100 g C L-1) based on fast growing cell supplied
during their exponential growth phase should promote
large-sized and dense pellets (Butler & Dam, 1994).
Estimations of CR and IR, measured as cell removal,
considered a food concentration of 194 ± 52 µg C L-1
(T. rotula) and 175 ± 41 µg C L-1 (P. minimum).
Clearance or filtration rate is the volume of water
cleared of food particles by a consumer per unit time,
whereas IR is the amount of food particles ingested by
the consumer per unit time (Båmstedt et al., 2000). Six
control dishes with no animals and six dishes
containing single adult females were incubated by 8 h
and mixed periodically to avoid cell sedimentation in
the case of diatoms. After sieving through 80 µm the
content of experimental dishes (to separate eggs 151 ±
6 m diameter, and fecal pellets >150 m length),
water volumes of all dishes were filtered directly onto
0.7 mm precombusted (450ºC) glass-fiber filters and
then were analyzed for elemental compounds as above.
Thus, IR was expressed in carbon units (µg C f-1 d-1)
following standard method (Frost, 1972). Food media
during reproductive experiments was daily renewed
maintaining a similar food concentration as in feeding
estimation experiments. In case of T. rotula, food media
was periodically and gently mixed to minimize cells
settling; in turn, eggs produced over 24 h by single
females during the incubations were quantified to
obtain daily averages of egg production rates (EPR).
From these batches produced daily with both diets,
random groups of 30 eggs were allowed to hatch after
60 h incubation in 3-5 mL of filtered sea water to
estimate hatching success (H). The rest of the daily
EPR was cleaned with filtered sea water and then were
carefully concentrated into cryovials and kept at -80°C
until electrophoretic analysis. When each 96 h
experimental series ended, females were gently
cumulated, cleaned and kept separated from egg
samples at -80°C until electrophoretic analysis.
Furthermore, a sample of copepod males collected
throughout the study from field samples was also
included to compare electrophoretic protein profile, due
we did not control food intake by copepods in the field.
4801
Total soluble proteins were extracted by mechanical
disruption of samples (copepods and eggs) in 0.5 mL of
extracting buffer (Tris 100 mM (pH 7.5), NaCl 100
mM, EDTA 5 mM, PMSF 1 mM) (Tartarotti & Torres,
2009). Samples were sonicated during 3 cycles of 10 s
followed by 10 s rest in a vibracell sonics sonicator at
50% gain. Afterwards, the samples were centrifuged at
15000 g for 15 min at 4°C and the supernatant was
recovered. The protein concentration was determined
using the Bradford method (Bradford, 1976) and
Biorad reagents according to the manufacturer
instructions. Bovine serum albumin was utilized as
standard. Approximately 10 µg of proteins were mixed
with the appropriate volume of 4X Laemmli sample
buffer, heated, and charged into a 12% SDSpolyacrylamide gel. The electrophoresis was run at 100
mA until the tracking dye reached the gel bottom. The
gel was stained with Comassie blue in a mixture
Ethanol, water, acetic acid in the proportion of 4:6:1.
The gels were distained in the same mixture without the
colorant. The molecular sizes were calculated from a
calibration curve constructed from the relative mobility
of the proteins standard against the logarithm of their
molecular sizes.
The effect of food offer (T. rotula and P. minimum)
and incubation length (h) was assessed on daily
averages of CR, IR, EPR, and H through a two-way
ANOVA test. Mean averages included into the analysis
were computed by compiling daily averages observed
during the four experimental series performed with
each food treatment. The potential association between
food C and N contents and copepod responses (IR,
EPR, and H), as well as between IR and reproduction
(EPR and H), was addressed by means of simple
regression and Spearman correlation tests depending on
the degree of deviation from normality. Due to some
eggs accounted to determine daily average of EPR were
destined later to estimate H, the eggs quantity finally
available to develop electrophoretic analysis was
rounded around 300 eggs. Therefore, the reported
concentration of soluble proteins to elaborate protein
profiles with females and eggs was expressed in terms
of µg f-1 L-1 and µg egg-1 L-1, respectively. Statistical
analyses were performed using the software STAT
version 7.0.
Feeding activity in terms of CR ranged between 3240 (T. rotula) and between 30-45 mL f -1 d-1 (P.
minimum), while IR fluctuated between 10-14 (T.
rotula) and 7-11 µg C f-1 d-1 (P. minimum). These
variations in copepod feeding responses are shown in
Figs. 1a-1b, while their statistical comparisons appear
in Table 1. After assessing the two feeding conditions,
CR and IR decreased over time for those females fed
with T. rotula, while CR and IR increased for those fed
with P. minimum. For reproductive traits the analysis
revealed that EPR (egg f-1 d-1) ranged between 27 ± 6
(T. rotula) and 31 ± 4 (P. minimum), which tended to
decreased over time for those females fed with T.
rotula. Although EPR decreased after 48 h with P.
minimum, it recovered to their original levels after 72
h, and remained high until the end of the experiments
(Fig. 1c). The interaction between incubation length
and food type resulted in smaller brood sizes that
decreased fecundity about 40% in those females fed
with T. rotula, after 72 h of incubation. Other hand, H
was relatively high (>90%) with both food treatments
(Fig. 1d), although H was statistically lower with T.
rotula (92 ± 4%). Spearman correlation analysis of
pooled elemental composition data of food types
showed significant but antagonistic correlations
between the N and C:N ratio of diet and copepod IR,
and while the first one positive (n = 16, R = 0.4, P-value
< 0.05) the latter was negative (n = 16, R = -0.5, P-value
< 0.05). Likewise, EPR varied correlated and
significantly with CR (N = 16, R = 0.5, P-value < 0.05).
Concentration of soluble proteins ranged from 2.72
to 6.29 µg f-1 L-1 in adults and from 0.26 to 0.42 µg egg-1
L-1 in eggs (Table 2). Between 6 and 10 electrophoretic
bands were retained in SDS-polyacrylamide electrophoresis gel elaborated with females and egg
preparations, respectively (Fig. 2). Proteins derived
from female preparations have molecular weights
varying between 56 and 219-kDa, whereas these ranged
between 56 and 170-kDa in eggs-derived samples. In
general terms female’s electro-phoretic profiles fed
both food treatments resulted quite similar although
band at 56-kDa was more intense in those females fed
with T. rotula (Fig. 2, S2), while the structure of
electrophoretic profiles of eggs spawned by females fed
T. rotula showed greater number of electrophoretic
bands than eggs spawned by females fed P. minimum.
These bands corresponded to proteins retained at 73
and 56-kDa at 56-kDa electrophoretic bands which
were more intense in those eggs spawned by females
fed with T. rotula (Fig. 2, S4).
This experimental exercise lies on the assumption
that as mid-sized copepod, C. patagoniensis could face
difficulties to diversify their diet under a massive
diatoms bloom. In this sense, some authors have
proposed that multi-algal consortiums would allow
copepods to avoid poorly diverse food resources in the
field; such that even during blooms, unicellular or short
chains of individual diatom cells are dispersed, mixed
and often consumed together with other taxa (Flynn &
Irigoien, 2009). Moreover, as copepods have the ability
to eat different food particles including a variety of
planktonic groups in their daily ration, they may
enhance the probability of obtaining a nutritionally com-
802
5
Figure 1. Simultaneous effect of food type (Thalassiosira rotula and Prorocentrum minimum) and feeding time on:
a) ingestion rates, b) clearance rates, c) egg production rates, and d) hatching success of Calanoides patagoniensis during
consecutive incubation experiments. Scatter plot as well as vertical bars denote daily means ± SD.
Table 1. Statistical results of two-way ANOVA analysis conducted to establish the effect of food treatment [T. rotula (T.r.)
and P. minimum (P.m.)] and feeding time in copepod responses during consecutive incubation experiments. Copepod
responses were: clearance rate (CR), ingestion rate (IR), egg production rate (EPR), and hatching success (H). Effect of
incubation time is denoted as the trend that each response acquired over feeding time (equal, increase or decrease).
df: degrees of freedom.
Variable
IR
CR
EPR
H
Factor
Diet
Time
Interaction
Diet
Time
Interaction
Diet
Time
Interaction
Diet
Time
Interaction
ANOVA
P.m. < T.r.
equal
P.m. > T.r.
increase
P.m. > T.r.
decrease
P.m. > T.r.
increase
-
plete ration in variable and nutritionally diluted
environments (Kleppel, 1993). Certainly, this usually
does occur in the ocean but elevated concentrations and
spatial coverage of diatom blooms (Tiselius &
Kuylenstierna, 1996; Miralto et al., 2003; Vidoudez et
al., 2011) give them the character of mesoscale events
that deserve special considerations. Firstly, diatom
blooms are beyond a diluted environment in terms of
food particles, and copepods tend to readily migrate and
aggregate at localized diatom patches (Bainbridge,
F-value
34
0.5
18
7
6
35
21
19
13
19
4
8
df
1,16
3,16
3,16
1,16
3,16
3,16
1,16
3,16
3,16
1,16
3,16
3,16
P-value
0.0001
> 0.05
0.0001
0.02
0.005
0.0001
0.0003
0.0001
0.0001
0.001
0.02
0.001
1953; Tiselius, 1992; Atkinson & Shreeve, 1995;
Bochdansky & Bollens, 2004). Further, bloom-forming
diatoms are capable to reduce phytoplankton diversity
through nutrients depletion, physical constraints, and
allelophatic mechanisms (Price & Paffenhöfer, 1984;
Legrand et al., 2001; Turner, 2014). Thus, highly dense
diatom blooms may induce a shortcut in the food
diversity and field prey for mid and large-sized
copepods, which could be more efficient feeding on
large and highly abundant diatoms than small and diluted
6803
Table 2. Details of electrophoretic banding (B) of produced with females and eggs of C. patagoniensis fed and produced
after sustained feeding with T. rotula and P. minimum. This information primarily comprises protein complexes of high
molecular weight, while molecular sizes of male bands were provided only as a reference. Sample size denotes number of
females, eggs and males required for electrophoretic preparations.
Sample
codes
Type of
sample
Sample
size
Food type
(µg mL-1)
Total soluble
proteins
Band codes
(kDa)
Proteins molecular
weights
S1
S2
S3
Females
Females
Males
72
65
25
P. minimum
T. rotula
--
195.66
295.75
157.30
S4
S5
Eggs
Eggs
300
300
P. minimum
T. rotula
79.12
126.92
B1-B2-B3-B4-B5-B6
B1-B2-B3-B4-B5-B6-B7
B1-B2-B3-B4-B5-B6-B7-B8B9-B10
B1-B2-B3-B4-B5-B6-B7-B8
B1-B2-B3-B4-B5-B6
213-183-150-133-113-62.9
219-186-150-137-114-73-56
229-225-167-158-139-129-10365-54-47
170-161-145-111-93-79-69-57
167-145-111-97-73-56
Figure 2. SDS-poly acrylamide gel electrophoresis
profiles of copepods samples containing between 79 and
up to 290 µg mL-1 of the total soluble proteins. Lanes: ST
(protein standards with molecular sizes shown in kDa), S1
(females fed P. minimum), S2 (females fed T. rotula), S3
(males after-samplings preserved), S4 (eggs produced on
P. minimum) and S5 (eggs produced on T. rotula). Black
arrows highlight some specific electrophoretic bands: 167
and 56-kDa in S2, 113-kDa in S4, and 56-kDa in S5.
flagellates that possibly co-occur with the diatoms
bloom.
We recently showed the egg production of C.
patagoniensis steadily fed T. rotula decreased
significantly after 72 h, besides these egg production
rates were negatively associated with the IR and
assimilation efficiency (AE) of T. rotula (Aguilera &
Escribano, 2013); it suggests us sustained ingestion and
assimilation of T. rotula could cause the drop of
copepod gross growth efficiency (i.e., carbon
ingestion/egg mass production). Such kind of postingestive processes have been observed, for instance,
under sustained stimulus of toxic compounds in the diet
(Kozlowsky-Suzuki et al., 2003); whereas other
possible explanation considers the food quality that T.
rotula represent for copepods. Current nutritional
assessment was unfortunately limited since we only
quantified and compared C and N contributions of both
food treatments. This comparison revealed T. rotula
reported the highest contribution of both elements
(Aguilera & Escribano, 2013). Furthermore, previous
feeding and reproductive studies developed in the study
area indicate that diatoms (including T. rotula) were an
adequate food resource to sustain secondary production
(Vargas et al., 2006) as well as reproductive
performance of small-sized copepods (Aguilera et al.,
2011). Besides, both food treatments were supplied
during their exponential growth phase to ensure their
cellular goodness and thus, nutritional quality.
Conversely to EPR, H (offspring viability) was
relatively high (>90%) and unaffected by food type and
incubation time, a reproductive outcome that has been
previously documented in copepod females fed on
several bloom-forming diatoms (Ianora & Miralto,
2010). Because larvae viability, a physiological process
highly-demanding of specific nutritional resources, was
not affected by the food treatments, it seems unlikely
that a nutritional deficit may have caused the
reproductive decline observed in those females fed on
T. rotula.
Previous assumptions could be better understood by
considering results of the comparison of protein
profiles elaborated with females fed on- and eggs
spawned with both food types. Protein profiles of
copepod females resulted mostly similar in terms of
structure, although electrophoretic bands in the range of
60 till 200-kDa were more intensely expressed in
females fed T. rotula (Fig. 2, S2). More dissimilar
structures of protein profiles were observed in
electrophoretic gels prepared with egg samples (Fig. 2,
S4-S5). Thus, proteins in the retained in the band close
to 103.9-kDa were only observed in egg spawned by
females fed with P. minimum, while those in band of
56-kDa only observed in preparations derived from T.
rotula. Several proteins with molecular weights of 86,
177, and 196-kDa circulate through the hemolymph
and are transported to the growing oocytes during the
second phase of crustacean vitellogenesis (Yehezkel et
al., 2000; Warrier & Subramoniam, 2002), providing a
source of proteins, lipids, and carbohydrates to
developing embryos (Wallace et al., 1967; Adiyodi &
Subramoniam, 1983; Shafir et al., 1992). Due the
electrophoretic bands that retained proteins in the band
of 80-200 kDa were similar in profiles of females fed
both food treatments, nutritional complexes such those
mentioned above should have been available as
demonstrated by the high and food-independent larvae
viability.
Among microalgae species, the diatom T. rotula is
considered capable of producing active metabolites
with negative effects on his predators (Fontana et al.,
2007; Ribalet et al., 2009, Caldwell, 2010). Through a
specific metabolic pathway involving the oxidation of
fatty acids, the local specie of T. rotula seems to be able
to affect the physiology of the large-sized copepod C.
chilensis, finally inducing their reproductive collapse
(Poulet et al., 2007). Such that, healthy females may
experience reproductive impairments under sustained
conditions of food containing or producing toxic
compounds (Turner, 2014). We observed C.
patagoniensis had a moderate AE on T. rotula (AE =
45%), inversely correlated with EPR and interpreted as
AE was not entirely assigned to reproductive efforts
(Aguilera & Escribano, 2013). Recent molecular
evidence showed up- and down-regulation of stressrelated proteins expression in Calanus helgolandicus
after it was fed for 48 h on the oxylipin-producing
diatom Skeletonema marinoi (Lauritano et al., 2012).
Regulation of gene expression was associated to the
ability or inability to activate stress/detoxification
proteins, such as the cytochrome P450 enzyme
(CYP1A, 56-57-kDa) to cope with the toxic diet. In the
current study we found that not only 56-kDa protein
band was far more intense in copepods fed T. rotula,
but it was also only present in eggs spawned by females
fed diatoms. This may suggest that both stages could
have activated stress/detoxification mechanisms to
cope potentially detrimental compounds derived from
eat diatoms. Interestingly, the majority of the eggs
succeed to hatch despite the decline on egg production.
8047
Chemical co-evolution between plant defenses and
animal offenses has been proposed to explain some
traits of the diatom-copepod relationship (Lauritano et
al., 2012); and both species, T. rotula and C.
patagoniensis, co-exists and strongly interacts in this
productive area during the upwelling period. The
expression of non-essential proteins such that stressrelated ones may represent new metabolic demands,
that undermine other expensive processes like
reproduction (Kurihara et al., 2004), and growth
(Chinnery & Williams, 2004). This possibility deserves
to be further evaluated given the ecological and
functional relevance of diatoms blooms in these highly
productive marine ecosystems.
ACKNOWLEDGEMENTS
Authors are very grateful to the staff of Marine Biology
Station of Dichato: Jose Marileo, Gisela Letelier,
Claudia Pérez, Katty Donoso, Marcelo Fuentes, and
Jose Caamaño; they also thank Javier Seiter for his
cooperation in electrophoretic preparations. Partial
support to Dr. V. Aguilera-Ramos was provided by
Programa Bicentenario of Chile (PBCT) of CONICYT
Grant Rue-02 and FONDECYT 1080037. Funding
during the final stage of this study was provided by
grants from the Chilean Scientific and Technologic
Commission through FONDECYT project N°11130495,
and Millennium Scientific Initiative Grant IC120019.
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