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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 www.scielo.cl/imar.htm

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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

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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).

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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).

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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.

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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

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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

Feeding ecology of marine fishes in a shallow coastal lagoon

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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

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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.

Feeding ecology of marine fishes in a shallow coastal lagoon

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

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were captured under the permit for collection awarded by the National Commission of Aquaculture and Fisheries number DGOPA/04031/310510.1940 REFERENCES Amundsen, P.A., H.M. Gabler & F.J. Staldvik. 1996. A new approach to graphical analysis of feeding strategy from stomach contents data-modification of the Costello (1990) method. J. Fish. Biol., 48: 607-614. Arceo-Carranza, D. 2002. Comparación trófica de la familia Belonidae en el Sistema Lagunar de Alvarado, Veracruz, México. Tesis de Licenciatura, Universidad Nacional Autónoma de México, México D.F., 64 pp. Arceo-Carranza, D. & M.E. Vega-Cendejas. 2009. Spatial and temporal characterization of fish assemblages in a tropical coastal system influenced by freshwater inputs: northwestern Yucatan Peninsula. Rev. Biol. Trop., 57: 89-103. Arceo-Carranza, D., J. Franco-López, G.L. Waggy & R. Chavez-López. 2004. Trophic comparison of two species of needlefish (Belonidae) in the Alvarado Lagoonal System, Veracruz, México. Gulf Caribb. Res., 16: 81-88. Bacha, M. & R. Amara. 2009. Spatial, temporal and ontogenetic variation in diet of anchovy (Engraulis encrasicolus) on the Algerian coast (SW Mediterranean). Estuar. Coast. Shelf Sci., 85: 257-264. Baker, R. & M. Sheaves. 2005. Redefining the piscivore assemblage of shallow estuarine nursery habitats. Mar. Ecol. Prog. Ser., 291: 197-213. Blaber, J.S.M. 1997. Fish and fisheries of tropical estuaries. Chapman & Hall, London, 367 pp. Blay, J. Jr, W.K. Awittor & D. Agbeko. 2006. Seasonal variation in food preference and feeding ecology of two juvenile marine fishes, Pseudotolithus senegalensis (Sciaenidae) and Brachydeuterus auritus (Haemulidae) off Cape coast, Ghana. WAJAE, 9: 1-6. Carr, W. & C.A. Adams. 1973. Food habits of juvenile marine fishes occupying seagrass beds in the estuarine zone near Crystal River, Florida. Trans. Am. Fish. Soc., 102: 511-540. Castro-Aguirre, J.L., H.S. Espinoza-Pérez & J.J. Schmitter-Soto. 1999. Ictiofauna estuarina lagunar y vicaria de México. Colección Textos Politécnicos, Serie Biotecnologías, Editorial Limusa, México, 711 pp. Clarke, K.R. & R.M. Warwick. 2001. Change in marine communities: an approach to statistical analysis and interpretation. Natural Environment Research Council, Plymouth Marine Laboratory, Plymouth, 176 pp. Clench, H. 1979. How to make regional list of butterflies: some thoughts. J. Lepid. Soc., 33: 216-31.

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Hixon, M.A. 1980. Competitive interactions between California reef fishes of the genus Embiotoca. Ecology, 61(4): 918-931. Hixon, M.A. & M.H. Carr. 1997. Synergistic predation, density dependence, and population regulation in marine fish. Science, 277: 946-949. Hyslop, E.J. 1980. Stomach contents analysis-a review of methods and their application. J. Fish Biol., 17: 411429. Jansen, P.A., H. Slettvold, A.G. Finstad & A. Langeland. 2002. Niche segregation between Arctic char (Salvelinus alpinus) and brown trout (Salmo trutta): an experimental study of mechanisms. Can. J. Fish. Aquat. Sci., 59: 6-11. Jiménez-Valverde, A. & J. Hortal. 2003. Las curvas de acumulación de especies y la necesidad de evaluar la calidad de los inventarios biológicos. Rev. Ibérica Aracnol., 8: 151-161. Krebs, J.C. 1999. Ecological methodology. Addison Welsey, Menlo Park CA, 620 pp. Laegdsgaard, P. & C.R. Johnson. 1995. Mangrove habitats as nurseries: unique assemblages of juvenile fish in subtropical mangroves in eastern Australia. Mar. Ecol. Prog. Ser., 126: 67-81. Laegdsgaard, P. & C.R. Johnson. 2001. Why do juvenile fish utilize mangrove habitats? J. Exp. Mar. Biol. Ecol., 257: 229-253. Link, J.S. & L.P. Garrison. 2002. Changes in piscivory associated with fishing induced changes to the finfish community on Georges Bank. Fish Res., 55: 71-86. Nagelkerken, I. & G. Van der Velde. 2002. Do nonestuarine mangroves harbor higher densities of juvenile fish than adjacent shallow-water and coral reef habitats in Curaçao (Netherlands Antilles)? Mar. Ecol. Prog. Ser., 245: 191-204. Nagelkerken, I., M. Dorenbosch, W.C.E.P. Verberk, E. Cocheret de la Morinière & G. Van Der Velde. 2000. Day-night shifts of fishes between shallow-water biotopes of a Caribbean Bay, with emphasis on the nocturnal feeding of Haemulidae and Lutjanidae. Mar. Ecol. Prog. Ser., 194: 55-64. Pinkas, L., M.S. Oliphant & I.L.K. Iverson. 1971. Food habits of albacore, bluefin tuna, and bonito in California waters. Calif. Fish Game. Fish. Bull., 152: 1-105. Platell, M.E., G.A. Sarre & I.C. Potter. 1997. The diets of two co-occurring marine teleosts, Parequula melbournensis and Pseudocaranx wrighti, and their relationships to body size and mouth morphology, and the season and location of capture. Env. Biol. Fish, 49: 361-367.

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Ramírez-Luna, V., A.F. Navia & E.A. Rubio. 2008. Food habits and feeding ecology of estuarine fish assemblage of northern Pacific coast of Ecuador. PANAMJAS, 3(3): 361-372. Root, R.B. 1967. The niche exploitation pattern of the blue-gray gnatcatcher. Ecol. Monogr., 37: 317-350. Ross, S.T. 1986. Resource partitioning in fish assemblages: a review of field studies. Copeia, 1986(2): 352-388. Ruiz, G.M., A.H. Hines & M.H. Posey. 1993. Shallow water as a refuge habitat for fish and crustaceans in non-vegetated estuaries: an example from Chesapeake Bay. Mar. Ecol. Prog. Ser., 99: 1-16. Shoener, T.W. 1971. Theory of feeding strategies. Annu. Rev. Ecol. Syst., 2: 369-404. Silvano, R.A.M. 2001. Feeding habits and interspecific feeding associations of Caranx latus (Carangidae) in a subtropical reef. Env. Biol. Fish., 60(4): 465-470.

Received: 26 September 2014; Accepted: 2 February 2015

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Tse, P., T.H.M. Nip & C.K. Wong. 2008. Nursery function of mangrove: a comparison with mudflat in terms of fish species composition and fish diet. Estuar. Coast. Shelf Sci., 80: 235-242. Tzeek-Tuz, J.G. 2013. Biología de la reproducción de Strongylura notata y Sphoeroides testudineus, de la laguna “La Carbonera” en Sisal, Yucatán. Tesis de Maestría, Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, México D.F., 125 pp. Verweij, M.C., I. Nagelkerken, S. Wartenbergh, I.R. Pen & G. Van Der Velde. 2006. Caribbean mangroves and seagrass beds as daytime feeding habitats for juvenile French grunts, Haemulon flavolineatum. Mar Biol., 149: 1291-1299. Wallace, Jr., R.K. 1981. An assessment of diet-overlap indexes. Trans. Am. Fish. Soc., 110: 72-76. Yáñez-Arancibia, A. & R.S. Nugent. 1977. El papel ecológico de los peces en estuarios y lagunas costeras. An. Inst. Cienc. Mar Limnol., 4(1): 107-114.

Lat. Am. J. Aquat. Res., 43(4): 632-640, 2015 DOI: 10.3856/vol43-issue4-fulltext-2

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. MATERIALS AND METHODS 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

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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).

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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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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

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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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Slater, C.H., C.B. Schreck & D.F. Amend. 1995. GnRHa injection accelerates final maturation and ovulation/ spermiation of sockeye salmon (Oncorhynchus nerka) in both fresh and salt water. Aquaculture, 130: 279285. Sylvia, G. 1995. Global markets and products of hake. In: J. Alheit & T.J.Pitcher (eds.). Hake. Springer Netherlands, pp. 415-435. Tamaru, C.S., C.D. Kelley, C.-S. Lee, K. Aida, I. Hanyu & F. Goetz. 1991. Steroid profiles during maturation and induced spawning of the striped mullet, Mugil cephalus L. Aquaculture, 95: 149-168. Thomas, P. & J. Trant. 1989. Evidence that 17α, 20β,21 trihydroxy-4- pregnen-3-one is a maturation-inducing steroid in spotted seatrout. Fish Physiol. Biochem., 7: 185-191. Tingley, G.A., L.V. Purchase, M.V. Bravington & S.J. Holden. 1995. Biology and fisheries of hakes (M. hubbsi and M. australis) around the Falkland Islands. In: J. Alheit & T.J. Pitcher (eds.). Hake. Springer, Netherlands, pp. 269-303. Trant, J.M. & P. Thomas. 1989. Isolation of a novel maturation-inducing steroid produced in vitro by ovaries of Atlantic croaker. Gen. Comp. Endocrinol., 75: 397-404. Ünal, G., H. Karakişi & M. Elp. 2005. Ovarian follicle ultrastructure and changes in levels of ovarian steroids during oogenesis in Chalcalburnus tarichi (Palla, 1811). Turk J. Vet. Anim. Sci., 29: 645-653.

Received: 12 February 2014; Accepted: 10 March 2015

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Vermeirssen, E.N.L.M., R.J. Shields, C. Mazorra de Quero & A.P. Scott. 2000. Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus). Fish Physiol. Biochem., 22: 77-87. Vermeirssen, E.N.L.M., A.P. Scott, C.C. Mylonas & Y. Zohar. 1998. Gonadotrophin releasing hormone agonist stimulates milt fluidity and plasma concentrations of 17,20-dihydroxylated and 5reduced, 3-hydroxylated C21 steroids in male plaice (Pleuronectes platessa). Gen. Comp. Endocrinol., 112: 163-177. Yamauchi, K., H. Kagawa, M. Ban, N. Kasahara & Y. Nagahama. 1984. Changes in plasma estradiol-17ß and 17a,20ß- dihydroxy-4-pregnen-3-one levels during final oocyte maturation of the masu salmon Oncorhynchus masou. Bull. Jap. Soc. Sci. Fish, 50: 2137. Zohar, Y. & C.C. Mylonas. 2001. Endocrine manipulations of spawning in cultured fish: from hormones to genes. Aquaculture, 197: 99-136. Zohar, Y., G. Pagelson & M. Tosky. 1988. Daily changes in reproductive hormone levels in the female gilthead seabream Sparus aurata at the spawning period. In: Y. Zohar & B. Breton (eds.). Reproduction in fish: basic and applied aspects in endocrinology and genetics. Institut National de la Recherche Agronomique, Paris, pp. 119-125.

Lat. Am. J. Aquat. Res., 43(4): 641-650, 2015 DOI: 10.3856/vol43-issue4-fulltext-3

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

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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. MATERIALS AND METHODS 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-

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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).

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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:

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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). Statistical analysis 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

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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).

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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.

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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.

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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.

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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. REFERENCES Alarcón, F.J., F.L. García-Carreño & M.A. Navarrete del Toro. 2001. Effect of plant protease inhibitors on digestive proteases in fish species, Lutjanus argentiventris and L. novemfasciatus. Fish Physiol. Biochem., 4: 179-189. Alarcón, F.J., F.J. Moyano & M. Díaz. 2002. Evaluation of different protein sources for aquafeeds by an optimized pH-stat system. J. Sci. Food. Agr., 82: 1-8. Alarcón, F.J., M. Diaz, F.J. Moyano & E. Abellan. 1998. Characterization of functional properties in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex). Fish. Physiol. Biochem., 19: 257-267. Allen, G.R. 1995. Lutjanidae. Pargos. In: W. Fisher, K. Krup, W. Scheider, C. Sommer, K.E. Carpenter, V.H. Niem (eds.). Guía FAO para la identificación de especies para los fines de la pesca. Pacífico CentroOriental. Volumen III Vertebrados, Parte 2. FAO, Roma, pp.1231-1244. Alvarez-Lajonchère, L., M.I. Abdo de la Parra, L.E. Rodríguez-Ibarra, G. Velasco-Blanco, A.C. PuelloCruz, B. González-Rodríguez, A. Ibarra-Soto & L. Ibarra-Castro. 2012. The scale-up of spotted rose snapper, Lutjanus guttatus, larval rearing at Mazatlán, Mexico. J. World Aquacult. Soc., 43(3): 411-442. Bassompierre, M., T.H. Ostenfeld, E. McLean & K. Rungruangsak-Torrissen. 1998. In vitro protein digestion and growth of Atlantic salmon with different trypsin isozymes. Aquacult. Int., 6: 47-56. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann. Biochem., 72: 248-254. Buddington, R.K. & J.M. Diamond. 1986. Aristotle revisited: the function of pyloric caeca in fish. Proc. Nat. Acad. Sci., 83: 8012-8014. Chakrabarti, R., R.M. Rathore, P. Mittal & S. Kumar. 2006. Functional changes in digestive enzymes and characterization of proteases of silver carp and bighead

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Mazatlán, Mexico. J. World Aquacult. Soc., 42: 564574. Khantaphant, S. & S. Benjakul. 2008. Comparative study on the proteases from fish pyloric caeca and the use for production of gelatin hydrolysate with antioxidative activity. Comp. Biochem. Physiol. B, 151: 410-419. Khantaphant, S. & S. Benjakul. 2010. Purification and characterization of trypsin from the pyloric caeca of brownstripe red snapper (Lutjanus vitta). Food Chem., 120: 658-664. Kishimura, H., K. Hayashi & S. Ando. 2006. Characteristics of carboxypeptidase B from pyloric caeca of starfish Asterina pectinifera. Food Chem., 95: 264269. Kirschke, H. & A.J. Barret. 1987. Chemistry of lysosomal proteases In: H. Glaumann & F. Ballard (eds.). Lysosomes: their role in protein breakdown. Academic Press, London, pp.193-218. Klomklao, S. 2008. Digestive proteinases from marine organisms and their applications. Songklanakarin J. Sci. Technol., 30(1): 37-46. Klomklao, S., H. Kishimur, M. Yabe & S. Benjakul. 2007. Purification and characterization of two pepsins from the stomach of pectoral rattail (Coryphaenoides pectoralis). Comp. Biochem. Physiol. B, 147(4): 682689. Kristjansson, M.M., S. Cudmundsdottir, J.W. Fox & J.B. Bjarnason. 1995. Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua). Comp. Biochem. Physiol. B, 110: 707-717. Kuz᾽mina, V.V. 1996. Influence of age on digestive enzyme activity in some freshwater teleosts. Aquaculture, 148: 25-37. Matus de la Parra, A., A. Rosas, J.P. Lazo & M.T. Viana. 2007. Partial characterization of the digestive enzymes of Pacific bluefin tuna Thunnus orientalis under culture conditions. Fish Physiol. Biochem., 33: 223231. Moguel-Hernández, I., R. Peña, H. Nolasco-Soria, S. Dumas & I. Zavala-Leal. 2013. Development of digestive enzyme activity in spotted rose snapper, Lutjanus guttatus (Staeindacher, 1969) larvae. Fish Physiol. Biochem., 40(3): 839-848.

Received: 24 October 2014; Accepted: 16 April 2015

Pérez-Jiménez, A., G. Cardenete, A.E. Morales, A. García-Alcázar, E. Abellán & M.C. Hidalgo. 2009. Digestive enzymatic profile of Dentex dentex and response to different dietary formulations. Comp. Biochem. Physiol. A, 154: 157-164. Rathore, R.M., S. Kumar & R. Chakrabarti. 2005. Digestive enzyme patterns and evaluation of protease classes in Catlacatla (Family: Cyprinidae) during early developmental stages. Comp. Biochem. Physiol. B, 142: 98-106. Sarath, G., R.S. De la Motte & F.W. Wagner. 1989. Protease assay methods. In: R. Beynon & J. Bond (eds.). Proteolytic enzymes: a practical approach. IRL, Oxford, pp. 25-56. Silva-Carrillo, Y., C. Hernández, R.W. Hardy, B. González-Rodríguez & S. Castillo-Vargasmachuca. 2012. The effect of substituting fish meal with soybean meal on growth, feed efficiency, body composition and blood chemistry in juvenile spotted rose snapper Lutjanus guttatus (Steindachner, 1869). Aquaculture, 364-365: 180-185. Torrissen, K.R. 1987. Genetic variation of trypsin-like isozymes correlated to fish size at Atlantic salmon (Salmo salar). Aquaculture, 62: 1-10. Torrissen, K.R. & K.D. Shearer. 1992. Protein digestion, growth and food conversion in Atlantic salmon and Arctic charr with different trypsin-like isozyme patterns. J. Fish. Biol., 41: 409-415. Unajak, S., P. Meesawat, A. Paemanee, N. Areechon, A. Engkagul, U. Kovitvadhi, S. Kovitvadhi, K. Rungruangsak-Torrissen & K. Choowongkomon. 2012. Characterization of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L). Food Chem., 134(3): 1533-1541. Walter, H.E. 1984. Proteinases: methods with haemoglobin, casein and azocoll as substrates. In: H.U Bergmeyer (ed.). Methods of enzymatic analysis. Verlag Chemie, Weinheim, pp. 270-277. Yasumaru, F. & D. Lemos. 2014. Species specific in vitro protein digestion (pH-stat) for fish: method development and application for juvenile rainbow trout (Oncorhynchus mykiss), cobia (Rachycentron canadum), and Nile tilapia (Oreochromis niloticus). Aquaculture, 426-427: 74-84. Zeef, A.H. & C. Dennison. 1988. A novel cathepsin from the mussel (Perna perna Linne). Comp Biochem. Physiol. B, 90: 204-210.

Lat. Am. J. Aquat. Res., 43(4): 651-661,Risk 2015 assessment of shrimp trawl fishery in the Gulf of California DOI: 10.3856/vol43-issue4-fulltext-4

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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

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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. MATERIALS AND METHODS 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

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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:

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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

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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.

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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%.

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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

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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

Risk assessment of shrimp trawl fishery in the Gulf of California

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. REFERENCES Almendarez-Hernández, L.C. 2008. El potencial de certificación de la pesquería de arrastre de camarón del Golfo de California. Tesis de Maestría. Instituto Politécnico Nacional (CICIMAR), México, 117 pp.

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Almendarez-Hernández, L.C. 2013. Caracterización y comportamiento económico de las embarcaciones camaroneras de alta mar del litoral del Pacífico mexicano como unidades de producción. Tesis de Doctorado, Instituto Politécnico Nacional (CICIMAR), México, 109 pp. Baca-Urbina, G. 2010. Evaluación de Proyectos. McGraw Hill, México, 318 pp. Banco Mundial. 2013. Datos. Estados Unidos de América: Grupo del Banco Mundial. Datos de libre acceso del Banco Mundial: acceso abierto y gratuito a datos sobre desarrollo de los países en todo el mundo. Grupo del Banco Mundial [http://datos.bancomundial.org/]. Reviewed: 10 February 2013. Banco de México (BANXICO). 2013. Inflación. Banco de México. Índice Nacional de Precios al Consumidor (INPC) e Índice Nacional de Precios al Productor (INPP). Banco de México. [http://www.bne.es/cata. htm]. Reviewed: 16 February 2013. Castro-Ortiz, J.L. & D. Lluch-Belda. 2008. Impacts of interanual environmental variation on the shrimp fishery off the Gulf of California. CalCOFI Rep., 49: 183-190. Comisión Nacional de Acuacultura y Pesca (CONA PESCA). 2011. Anuario Estadístico de Acuacultura y Pesca 2011. Comisión Nacional de Acuacultura y Pesca (CONAPESCA), Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA), México, 305 pp. Comisión Nacional de Acuacultura y Pesca (CONA PESCA). 2012. (online): Anuario 2012. Mazatlán, Sinaloa, México: Comisión Nacional de Acuacultura y Pesca. "Anuario 2012". Anuario Estadístico de Acuacultura y Pesca. [http://www.conapesca.sagarpa. gob.-mx/wb/cona/anuario_2012_zip]. Reviewed: 20 March 2014. Csirke, J. 1989. Introducción a la dinámica de poblaciones de peces. FAO, Roma, Doc. Téc. Pesca, 192: 82 pp. Diario Oficial de la Federación (DOF). 2012. Actualización de la Carta Nacional Pesquera. Diario Oficial de la Federación. México, 24 agosto 2012. Fideicomisos Instituidos en Relación con la Agricultura (FIRA). 2009. Situación actual y perspectiva del camarón en México. Fideicomisos Instituidos en Relación con la Agricultura. Boletín informativo, Nueva Época, Núm. 3. México. García-Caudillo, J.M. & J.V. Gómez-Palafox. 2005. La pesca industrial de camarón en el Golfo de California: situación económico-financiera e impactos socioambientales. Conservación Internacional-Región Golfo de California. Guaymas, Sonora, México, 104 pp.

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Hastie, T.J. & R.J. Tibshirani. 1990. Generalized additive models. Chapman & Hall, London, 353 pp. Instituto Nacional de la Pesca (INP). 2000. (CD-ROM). La pesquería de camarón del Pacífico. Sustentabilidad y pesca responsable en México, evaluación y manejo 1999-2000. Instituto Nacional de la Pesca, México. Instituto Nacional de la Pesca (INP). 2006. Plan de manejo para la pesquería de camarón en el litoral del Océano Pacífico. Instituto Nacional de la Pesca, México, 76 pp. Lluch-Belda, D. 1974. La pesquería de camarón de alta mar en el noroeste: un análisis biológico/pesquero. Secretaría de Industria y Comercio, Subsecretaria de Pesca, Instituto Nacional de Pesca, Serie Informativa (INP/SI: 116), México, 76 pp. Lluch-Cota, D.B., C.A. Salinas-Zavala, P. del MonteLuna & D. Lluch-Belda. 1995. El Niño y la pesca en el noroeste de México. Oceanografía, 4(8): 19-41. Lluch-Cota, D.B., S. Hernández-Vázquez, E.F. BalartPáez, L.F. Beltrán-Mórales, P. Del Monte- Luna, A. González-Becerril, S.E. Lluch-Cota, A.F. Navarrete del Proó, G. Ponce-Díaz, C.A. Salinas-Zavala, J. López-Martínez & S. Ortega-García. 2006. Desarrollo sustentable de la pesca en México: orientaciones estratégicas. Centro de Investigaciones Biológicas del Noroeste/ Comisión de Medio Ambiente, Recursos Naturales y Pesca del Senado de la República, México, 436 pp. López-Martínez, J. 2000. Dinámica de la pesquería de camarón café (Penaeus californiensis) en el litoral sonorense y su relación con algunos parámetros océano-atmosféricos. Tesis de Doctorado, Instituto Politécnico Nacional (CICIMAR), México, 160 pp. Medina-Neri, H. 1982. México en la pesca 1939-1976. HMN, México, 384 pp. Murase, H., H. Nagashima, S. Yonezaki, R. Matsukura & T. Kitakado. 2009. Application of a generalized additive model (GAM) to reveal relationships between environmental factors and distributions of pelagic fish and krill: a case study in Sendai Bay, Japan. ICES J. Mar. Sci., 66: 1417-1424. National Ocean and Atmospheric Administration (NOAA). 2013. [online]: Pacific Fisheries Environmental Laboratory. [United States of America]: National Ocean and Atmospheric Administration. "Upwelling Indices" Indices. [http://www.pfeg.noaa. gov/products/pfel/modeled/indices/upwelling/-upwelling.html]. Reviewed: 20 November 2014. Quimbar, J.R. 2004. Análisis de redimensionamiento de la flota camaronera de alta mar del Pacífico mexicano. Secretaría de Pesca y Acuacultura, Gobierno del Estado de Sonora, México, 97 pp.

R. Core Team. 2013. R: A language and environment for statistical computing, reference index version 3.0.1. R Foundation for Statistical Computing, Vienna. Ramírez-Rodríguez, M. & L.C. Almendarez-Hernández. 2013. Subsidies in the jumbo squid fishery in the Gulf of California, Mexico. J. Mar. Pol., 40: 117-123. Richardson, J.W. & H.P. Mapp Jr. 1976. Use of probabilistic cash flows in analyzing investment under conditions of risk and uncertainty. Southern J. Agricult. Econ., 8(2): 19-24. Richardson, J.W. & J.L. Outlaw. 2008. User´s guide and documentation for MexSim©. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA), Universidad Autónoma de Chapingo, México, 36 pp. Richardson, J.W., S.L. Klose & A.W. Gray. 2000. An applied procedure for estimating and simulating multivariate empirical (MVE) probability distributions in farm-level risk assessment and policy analysis. J. Agricult. App. Econ., 32: 299-315. Richardson, J.W., K. Schumann & P. Feldman. 2004. SIMETAR, Simulation for Excel to analyze risk. Agricultural and Food Policy Center, Department of Agricultural Economics, Texas Agricultural Experimental Station, Texas A&M University, Texas, 53 pp. Richardson, J.W., K. Schumann & P. Fieldman. 2008. SIMETAR, Simulation & econometrics to analyze risk. College Station, Texas A&M University, Texas, 96 pp. Rodríguez, de la Cruz, M.C. 1981. Aspectos pesqueros del camarón de alta mar en el Pacífico Mexicano. Ciencia Pesquera, INP, Depto. Pesca. México, 1(2): 1-19. Rodríguez de la Cruz, M.C. 2000. Reclutamiento, cambios de la abundancia y composición de los recursos camaroneros de la parte central del Golfo de California. Mexicoa, 2(1): 23-32. Rodríguez, de la Cruz, M.C. & F.J. Juárez-Rosales. 1976. El camarón del noroeste de México. Secretaría de Pesca-Instituto Nacional de la Pesca, Programa Camarón del Pacifico, México, 36 pp. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA). 2010. Reporte de unidades representativas de producción acuícola y pesquera. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación, Universidad Autónoma de Chapingo, México, 77 pp. Shapiro, D. & P. Switzer. 1989. Extracting time trends from multiple monitoring sites. Department of Statistics, Stanford University, Tech. Rep., 132 pp. Solow, A.R. 1994. Detecting change in the composition of a multispecies community. Biometrics, 50: 56-565.

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Soto, L.R. 1969. Mecanismo hidrobiológico del sistema lagunar Huizache-Caimanero y su influencia sobre la producción camaronera. Tesis Profesional, Universidad Autónoma de Baja California (UABC), México, 75 pp. Wood, S. 2006. Generalized additive models: an introduction with R. Texts in Statistical Sciences, Chapman and Hall/CRC, Boca Raton, 410 pp.

Received: 15 August 2014; Accepted: 6 May 2015

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Lat. Am. J. Aquat. Res., 43(4): 662-674, 2015 DOI: 10.3856/vol43-issue4-fulltext-5

Periphyton and planktonic bacteria in shallow lakes

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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-

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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. MATERIALS AND METHODS 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

Periphyton and planktonic bacteria in shallow lakes

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

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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

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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

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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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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

Periphyton and planktonic bacteria in shallow lakes

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

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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

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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

Periphyton and planktonic bacteria in shallow lakes

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

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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.

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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

Periphyton and planktonic bacteria in shallow lakes

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). REFERENCES Allende, L., G. Tell, H. Zagarese, A. Torremorell, G. Pérez, J. Bustingorry, R. Escaray & I. Izaguirre. 2009. Phytoplankton and primary production in clearvegetated, inorganic-turbid, and algal-turbid shallow lakes from the pampa plain (Argentina). Hydrobiologia, 624: 45-60. Alonso-Sáez, L. & J. Gasol. 2007. Seasonal variations in the contributions of different bacterial groups to the uptake of low-molecular-weight compounds in north-

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western Mediterranean coastal waters. Appl. Environ. Microbiol., 73: 3528-3535. Amann, R.I. & B.M. Fuchs. 2008. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat. Rev. Microbiol., 6: 339-348. Amann, R.I., L. Krumholz & D.A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J. Bacteriol., 172: 762-770. Amann, R.I., W. Ludwig & K.-H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev., 59: 143-169. American Public Health Association (APHA). 2005. Standard methods for the examination of water and wastewaters. Washington DC, 1217 pp. Araya, R., K. Tani, T. Takagi, N. Yamaguchi & M. Nasu. 2003. Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis. FEMS Microbiol. Ecol., 43: 111-119. Bertoni, R., C. Callieri, E. Balseiro & B. Modenutti. 2008. Susceptibility of bacterioplankton to nutrient enrichment of oligotrophic and ultraoligotrophic lake waters. J. Limnol., 67: 120-127. Brinson, M.M. 2004. Niveles extremos de variación de patrones y procesos en humedales. In: A.I. Malvárez & R. Bó (eds.). Documentos del Curso-Taller: bases ecológicas para la clasificación e inventario de humedales en Argentina. Buenos Aires, pp. 19-24. Busscher, H.J. & H.C. Van der Mei. 2000. Initial microbial adhesion events: mechanisms and implications. In: D.G. Allinson, P. Gilbert, H.M. Lappin-Scott & M. Wilson (eds.). Community structure and co-operation in biofilms. Society for General Microbiology, University Press, Cambridge, pp. 25-36. Daims, H., A. Brühl, R. Amann, K.-H. Schleifer & M. Wagner. 1999. The domain-specific probe EUB 338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol., 22: 434-444. De Figueiredo, D.R., M.J. Pereira & A. Correia. 2010. Seasonal modulation of bacterioplankton community at a temperate eutrophic shallow lake. World J. Microbiol. Biot., 26: 1067-1077. Glöckner, F.O., B.M. Fuchs & R. Amann. 1999. Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization. Appl. Environ. Microbiol., 65: 3721-3726. Glöckner, F.O., E. Zaichickov, N. Belkova, L. Dessinova, J. Pernthaler & R. Amann. 2000. Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an

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and six other Adirondack lakes. Limnol. Oceanogr., 43: 368-374. Newton, R.J., S.E. Jones, A. Eiler, K.D. McMahon & S. Bertilsson. 2011. A guide to the natural history of freshwater lake bacteria. Microbiol. Mol. Biol. Rev., 75: 14-49 Pérez, G.L., A. Torremorell, J. Bustingorry, R. Escaray, P. Pérez, M. Diéguez & H. Zagarese. 2010. Optical characteristics of shallow lakes from the Pampa and Patagonia regions of Argentina. Limnologica, 40: 3039. Pernthaler, A., J. Pernthaler & R. Amann. 2004. Sensitive multi-color fluorescence in situ hybridization for the identification of enviromental microorganisms. Molec. Microb. Ecol. Manage., 2: 771-726. Pernthaler, J., F.O. Glöckner, W. Schönhuber & R. Amann. 2001. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. Method. Microbiol., 30: 207-226. Pernthaler, A., C.M. Preston, J. Pernthaler, E.F. DeLong & R. Amann. 2002. Comparison of fluorescently labeled oligonucleotide and polynucleotide probes for the detection of pelagic marine bacteria and archaea. Appl. Environ. Microbiol., 68: 661-667. Pohlon, E., J. Marxsen & K. Küsel. 2010. Pioneering bacterial and algal communities and potential extracellular enzyme activities of stream biofilms. FEMS Microbiol. Ecol., 71: 364-373. Posch, T., B. Mindl, K. Horňák, J. Jezbera, M.M. Salcher, B. Sattler, B. Sonntag, J. Vrba & K. Šimek. 2007. Biomass reallocation within freshwater bacterioplankton induced by manipulating phosphorus availability and grazing. Aquat. Microb. Ecol., 49: 223-232. Quirós, R., A.M. Renella, M.B. Boveri, J. Rosso & A. Sosnovsky. 2002. Factores que afectan la estructura y el funcionamiento de las lagunas pampeanas. Ecol. Aust., 12: 175-185. Quirós, R., M.B. Boveri, C.A. Petrachi, A.M. Renella, J.J. Rosso, A. Sosnovsky & H.T. Von Bernard. 2006. Los efectos de la agriculturización del humedal pampeano sobre la eutrofización de sus lagunas. In: J.G.T. Tundisi, C. Matsumura-Tundisi & G. Sidagis (eds.). Eutrofização na América do Sul: causas, conseqüências e tecnologias de gerenciamento e controle, São Carlo, pp. 1-16. Salcher, M.M., J. Pernthaler & T. Posch. 2011. Seasonal bloom dynamics and ecophysiology of the freshwater sister clade of SAR11 bacteria “that rule the waves” (LD12). ISME J., 5: 1242-1252. Salcher, M.M. 2014. Same same but different: ecological niche partitioning of planktonic freshwater prokaryotes. J. Limnol., 73: 74-87.

Periphyton and planktonic bacteria in shallow lakes

Sánchez, M.L., H. Pizarro, G. Tell & I. Izaguirre. 2010. Relative importance of periphyton and phytoplankton in turbid and clear vegetated shallow lakes from the Pampa Plain (Argentina): a comparative experimental study. Hydrobiologia, 646: 271-280. Sánchez, M.L., G.L. Pérez, I. Izaguirre & H. Pizarro. 2013. Influence of underwater light climate on periphyton and phytoplankton communities in shallow lakes from the Pampa plain (Argentina) with contrasting steady states. J. Limnol., 72: 62-78. Scheffer, M. 2009. Critical transitions in nature and society. Princeton University, Oxford, 384 pp. Scheffer, M. 1998. Ecology of shallow lakes. Chapman & Hall, London, 357 pp. Scheffer, M., S.H. Hosper, M.-L. Meijer, B. Moss & E. Jeppesen. 1993. Alternative equilibria in shallow lakes. TREE, 8: 275-279. Sekar, R., A. Pernthaler, J. Pernthaler, F. Warnecke, T. Posch & R. Amann. 2003. An improved protocol for the quantification of freshwater actinobacteria by fluorescence in situ hybridization. Appl. Environ. Microbiol., 69: 2928-2935. Šimek, K., K. Hornák, J. Jezbera, J. Nedoma, J. Vrba, V. Straskrábová, M. Macek, J.R. Dolan & M.W. Hahn. 2006. Maximum growth rates and possible life strategies of different bacterioplankton groups in relation to phosphorus availability in a freshwater reservoir. Environ. Microbiol., 8: 1613-1624. Silvoso, J., I. Izaguirre & L. Allende. 2010. Picoplankton structure in clear and turbid eutrophic shallow lakes: a seasonal study. Limnologica, 41: 181-190. Stoodley, P., L. Hall-Stoodley, J.D. Boyle, F. Jørgensen & H.M. Lappin-Scott. 2000. Environmental and genetic factors influencing biofilm structure. In: D.G. Allinson, P. Gilbert, H.M. Lappin-Scott & M. Wilson (eds.). Community structure and co-operation in biofilms. Society for General Microbiology, University Press, Cambridge, pp. 53-64.

Received: 10 June 2014; Accepted: 10 May 2015

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Lat. Am. J. Aquat. Res., 43(4): 675-683, 2015 DOI: 10.3856/vol43-issue4-fulltext-6

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

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Latin American Journal of Aquatic Research 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. MATERIALS AND METHODS 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

Sea turtle stranding on Bahia, Brazil

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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.).

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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 80 cm

70 cm

21-75 cm

>75 cm

11-46 cm

>46 cm

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. Statistical analysis 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.

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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

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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

Chemical composition of Cryphiops caementarius

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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

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Latin American Journal of Aquatic Research

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

Chemical composition of Cryphiops caementarius

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

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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. REFERENCES Association of Official Analytical Chemists (AOAC). 2005. In: G.W. Latimer & W. Horwitz (eds.). AOAC, Gaithersburg, MD. Arcos, F.G., A.M. Ibarra, E. Palacios, C. VazquezBoucard & I.S. Racotta. 2003. Feasible predictive criteria for reproductive performance of white shrimp Litopenaeus vannamei: egg quality and female physiological condition. Aquaculture, 228: 335-349. Avarre, J.C., D. Saulnier, Y. Labreuche, D. Ansquer, A. Tietz & E. Lubzens. 2003. Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida. J. Invertebr. Pathol., 82: 23-33. Bahamonde, N. & I. Vila. 1971. Sinopsis sobre la biología del camarón de río del norte. Rev. Biol. Pesq. Chile, 5: 3-6.

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Santos, F.L., V.B. Azeredo & A.S. Martins. 2007. Effect of supplying food complemented with linseed on the chemical composition of Malaysian shrimp (Macrobrachium rosenbergii). Cienc. Tecnol. Alimentos, 27(4): 851-855. Saravana-Bhavan, P., S. Radhakrishnan, C. Seenivasan, R. Shanti, R. Poongodi & S. Kannan. 2010. Proximate composition and profiles of amino acids and fatty acids in the muscle of adult males and females of commercially viable prawn species Macrobrachium rosenbergii collected from natural culture environments. Int. J. Biol., 2(2): 107-119. Schirf, V.R., L.S. Turner, C. Hanapel, P. De La Cruz & P.F. Dehn. 1987. Nutritional status and energy metabolism of crayfish (Procambarus clarkii, Girardi) muscle and hepatopancreas. Comp. Biochem. Physiol. A, 88: 383-386. Sistema Nacional de Información Ambiental (SINIA). 2004a. Diagnóstico y clasificación de los cursos y cuerpos de agua según objetivos de calidad: cuenca del río Choapa. Ministerio de Obras Públicas, Santiago, 131 pp. Sistema Nacional de Información Ambiental (SINIA). 2004b. Diagnóstico y clasificación de los cursos y cuerpos de agua según objetivos de calidad: cuenca del río Limarí. Ministerio de Obras Públicas, Santiago, 137 pp.

Received: 4 September 2014; Accepted: 23 June 2015

Sokal, R.R. & F.J. Rohlf. 1981. Biometry: the principles and practice of statistics of biological research. W.H. Freeman & Co., San Francisco, 859 pp. Tacon, A.G.J. 1989. Nutrición y alimentación de peces y camarones cultivados. Manual de Capacitación. Organización de las Naciones Unidas para la Agricultura y la Alimentación. FAO, Roma, 592 pp. Viacava, M., R. Aitken & J. Llanos. 1978. Estudio del camarón en el Perú. Bol. Inst. Mar del Perú, 3: 165232. Vicent, M., L. Ramos & M. Oliva. 1988. Variations qualitatives et quantitatives des pigments caroténoides dans l´ovaire, et l´hépatopancréas de Penaeus schmitti au cours de la maturation ovarienne. Arch. Int. Physiol. Biochim., 96: 155-164. Vinagre, A.S., A.P.N. Amaral, F.P. Ribarcki, E.F. Silveira & E. Périco. 2007. Seasonal variation of energy metabolism in ghost crab Ocypode quadrata at Siriú Beach (Brazil). Comp. Biochem. Physiol. A, 146: 514519. Wen, X., L. Chen, C. Ai, Z. Zhou & H. Jiang. 2001. Variation in lipid composition of Chinese mittenhanded crab, Eriocheir sinensis during ovarian maturation. Comp. Biochem. Physiol. B, 130: 95-104. Yao, J.J., Z. Yun-long, Q. Wang, Z. Zhong-liang, H. XianCheng & D.A. Xiao-Wei. 2006. Biochemical compositions and digestive enzyme activities during the embryonic development of prawn Macrobrachium rosenbergii. Aquaculture, 253: 573-582.

Lat. Am. J. Aquat. Res., 43(4): 755-765, 2015 Risk assessment of Geotrichum spp. for L. vannamei cultures DOI: 10.3856/vol43-issue4-fulltext-14

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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-

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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). MATERIALS AND METHODS 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.

Risk assessment of Geotrichum spp. for L. vannamei cultures

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

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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

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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 Statistical analysis 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-

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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.

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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

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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

Risk assessment of Geotrichum spp. for L. vannamei cultures

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. REFERENCES Aguirre, G.G., R. Vázquez-Juárez & F. Ascencio. 2003. Efecto de diferentes especies de Vibrio sobre la sobrevivencia y desarrollo larval del camarón blanco de acuacultura (L. vannamei). VI Congreso Ecuatoriano de Acuacultura. V Congreso Latinoamericano de Acuicultura, pp. 94-103.

Aldarf, M., A. Amrane & Y. Prigent. 2002 Reconstruction of the biomass history from carbon and nitrogen substrate consumption, ammonia release and proton transfer during solid cultures of Geotrichum candidum and Penicillium camembertii. Appl. Microbiol. Biotechnol., 58: 823-829. Alexopoulos, C.J., C.W. Mims & M. Blackwell. 1996. Introductory mycology. John Wiley & Sons, New York, 869 pp. Arala-Chavez, M. & T. Sequeira. 2000 Is there any kind of adaptative immunity in invertebrates? Aquaculture, 191: 247-258. Atlas, R.M. 1995. Microorganisms in our world. Mosby Year Book, St. Louis, pp. 5-13. Bachere, E., D. Destoumieux & P. Bulet. 2000. Penaeidins, antimicrobial peptides of shrimp: a comparison with other effectors of innate immunity. Aquaculture, 191: 71-88. Bertke, C.C. & J.M. Aronson. 1992. Hyphal wall chemistry of Lagenidium callinectes and L. chthamalophilum. Bot. Mar., 35: 147-152. Brilhante, R.S.N., D.S.C.M. Catelo-Branco, G.P.S. Duarte, M.A.N. Paiva, C.E.C. Teixeira, J.P.O. Zeferino, A.J. Monteiro, R.A. Cordeiro, J.J. Sidrim & M.F.G. Rocha. 2012. Yeast microbiota of raptor: a possible tool for environmental monoitoring. Environ. Microbiol. Rep., 4: 189-193. Buchta, V. & M. Otcenasek. 1998. Geotrichum candidum, an opportunistic agent of mycotic diseases. Mycoses, 31: 363-370.

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Bugni, T. & C. Ireland. 2004. Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep., 21: 143-163. Chen, B.O., Y. Wu & J. Yang. 1992. Studies on pathogenicity of a species Fusarium in the cultured adult prawn (Penaeus chinensis Donghai). Mar. Sci., 10: 7-15. Coelho, M.A., J.M.F. Almeida, I.M. Martins, A.J. Silva & J.P. Sampaio. 2010. The dynamics of the yeast community of the Tangs River Estuary: testing the hypothesis of the multiple origins of the estuarine yeast. Antonie van Leeuwenhoek, 98: 331-342. Cruz da Silva, L.R., O.C. de Souza, M.J. dos Santos Fernandes, D.M. Massa Lima, R.R. Rodrigues Coelho & C.M. Souza-Motta. 2011. Culturable fungal diversity of shrimp Litopenaues vannamei Boone from breeding farms in Brazil. Braz. J. Microbiol., 42: 4956. Deacon, J. 2005 Environmental conditions for growth, and tolerance of extremes, in fungal biology, Blackwell Publishing, Malden. doi: 10.1002/9781118685068.ch8 Fuerst, J.A., S.K. Sambhi, J.L. Paynter, J.A. Hawkins & J.G. Atherton. 1991. Isolation of a bacterium resembling Pirellula species from primary tissue culture of the giant tiger prawn (Penaeus monodon). Appl. Environ. Microbiol., 57: 3127-3134. Gillett, R.D. 2008. Global study of shrimp fisheries. FAO Doc. Tech. Pêches, 475: 331 pp. Guzmán-Murillo, M.A. & F. Ascencio. 2001. Enzymelinked, biotin-streptavidin bacterial-adhesion assay for Helicobacter pylori lectin-like Interactions with cultured cells. J. Microbiol. Biotechnol., 11: 35-39. Hernández, L.J., T. Gollas-Galvan & F. Vargas-Albores. 1996. Activation of the prophenoloxidase system of the brown shrimp (Penaeus californiensis Holmes). Comp. Biochem. Phys. C, 113: 61-66. Hernández-Saavedra, N.Y. 1990. Aislamiento y caracterización de levaduras marinas aisladas de la costa occidental de Baja California Sur, México. Bachelor Thesis, Universidad Nacional Autónoma de México, Ciudad de México, 92 pp. Hyde, K., V. Sarma & E. Jones. 2000. Morphology and taxonomy of higher marine fungi. In: K.D. Hyde & S.B. Pointing (eds.). Marine mycology. A practical approach. Fungal Diversity Press, Hong Kong, pp. 172-204. Jackson, R.J., K. Fuhihashi, J. Xu-Amano, H. Kiyono, C.O. Elson & J.R. McGhee. 1993. Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as adjuvant. Infect. Immunity, 61(10): 4272-4279.

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Johansson, M.W., P. Keyser, K. Sritunyalucksana & K. Soderhall. 2000. Crustacean haemocytes and haematopoiesis. Aquaculture, 191: 45-52. Khoa, L.V., K. Hatai & T. Aoki. 2004. Fusarium incarnatum isolated from black tiger shrimp, Penaeus monodon Fabricius, with black gill disease cultured in Vietnam. J. Fish. Dis., 27: 507-515. Khoa, L.V., K. Hatai, A. Yuasa & K. Sawada. 2005. Morphology and molecular phylogeny of Fusarium solani isolated from kuruma prawn Penaeus japonicus with black gills. Fish. Pathol., 40: 103-109. Krachler, A.M., H. Ham & K. Orth. 2011. Outer membrane adhesion factor multivalent adhesion molecule 7 initiates host cell binding during infection by Gram-negative pathogens. www.pnas.org/cgi/doi/ 10.1073/pnas.1102360108. Kurtzman, C.P. & C.J. Robnett. 1998. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antoine van Leeuwenhoek, 73: 331-371. Le Moullac, G. & P. Haffner. 2000. Environmental factors affecting immune responses in Crustacea. Aquaculture, 191: 121-132. Leslie, J. & B. Summerell. 2006. The Fusarium laboratory manual. Blackwell Publishing, New Jersey, 338 pp. Lightner, D.V. 1996. A handbook of shrimp pathology and diagnostic procedures for disease of cultured penaeid shrimp. Baton Rouge, Louisiana, World Aquaculture Society, 304 pp. Lightner, D.V. & J.E. Hose. 1984. Observations on the pathogenesis of the imperfect fungus, Fusarium solani, in the California brown shrimp Penaeus californensis. J. Invertebr. Pathol., 44: 292-303. Lignot, J.H., C. Spanings-Pierrot & G. Chamantier. 2000. Osmorregulatory capacity as a tool in monitoring the physiological condition and the effect of stress in crustaceans. Aquaculture, 191: 209-246. Lozano-Olvera, R., F.I. Marrujo-López & S.M. AbadRosales. 2012. Necrosis cuticular en camarón Litopenaeus vannamei asociada a Fusarium sp. Redvet, 13(7): 1-7. Madigan, M.T., J.M. Martinko, P.V. Dunlap & D.P. Clark. 2009. Biología de los microorganismos. Pearson Educación, Madrid, pp. 592-601. Mukkerjee, K.D. & I. Kiewitt. 1996. Enrichment of verylong-chain mono-unsaturated fatty acids by lipasecatalyzed hydrolysis and transesterification. Appl. Microbiol. Biotechnol., 44: 557-562. Nakamura, K., S. Wada, K. Hatai & T. Sugimoto. 1994. Lagenidium myophilum infection in the coonstripe shrimp, Pandalus hypsinotus. Mycoscience, 35: 99104.

Nelson, P.E., M.C. Dignan & E.J. Anaissie. 1995. Taxonomi, biology and clinical aspects of Fusarium species. Clin. Microbiol. Rev., 7(4): 479-504. Newell, S.Y. 2001. Fungal biomass and productivity. In: J. Paul (ed.). Methods in microbiology. Marine microbiology. Academic Press, New York, pp. 357366. Noga, E.J. 1990. A synopsis of mycotic diseases of marine fish and invertebrates. In: F.O. Perkins & T. Cheng (eds.). Patology in marine science. Academic Press, New York, pp. 143-160. Nosanchuk, J.D., B.L. Gómez, S. Diez, S. Youngchim, P. Aisen, R.M. Zancope-Oliviera, A. Restrepo, A. Casadevall & A.J. Hamilton. 2002. Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection. Abs. Gen. Meeting Am. Soc. Microbiol., 102: 204-205. O’Donnell, K. 1996. Progress towards a phylogenetic classification of Fusarium. Sydowia, 48(1): 57-70. Pantoja, C.R. & D.V. Lightner. 2008. Enfermedades causadas por hongos. In: V. Morales & J. Cuellar (eds.). Guía técnica-patología e inmunología de camarones peneidos. Programa CYTED Red II Vannamei, Panamá, pp. 174-186. Pelczar, M.J., E.C.S. Chan & N.R. Krieg. 2001. Microbiology. Tata McGraw Hill Publishing Company, New Delhi, 524 pp. Pierce, B.E. & M.J. Leboffe. 2011. Exercises for the mirobiology laboratory. Morton Publishing Company, Denver, pp. 79-123. Pitt, J.L. & A.D. Hocking. 1997. Fungi and food spoilage. Blackie Academic & Professional, London, 39 pp. Rhem, M.N., E.M. Lech, J.M. Patti, D. McDevitt, M. Höök, D.B. Jones, &. K.R. Wilhelmus. 2000. The collagen-binding adhesin is a keratitis virulence factor in Staphylococcus aureus. Infect. Immunity, 68(6): 3776-3779. Saha, A., P. Mandal, S. Dasgupta & D. Saha. 2008. Influence of culture media and environmental factors on mycelial growth and sporulation of Lasiodiplodia theobromae (Pat.) Griffon and Maubl. J. Environ. Biol., 29(3): 407-410. Sainz, J.C., A.N. Maeda-Martínez & F. Ascencio-Valle. 1998. Experimental vibriosis induction with Vibrio alginolyticus of larvae of the catarina scallop (Argopecten ventricosus: circularis) (Sowerby II, 1842). Microbial. Ecol., 35: 188-192. Smith, M., G. Poot & A. de Cock. 2000. Re-examination of some species of the genus Geotrichum. Antonie van Leeuwenhoek, 77: 71-81. Tortora, G.J., R.F. Berdell & C.L. Case. 2012. Microbiology: an introduction. Benjamin Cumming Publishing, San Francisco, pp. 122-320.

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Vargas-Albores, F. & G. Yepiz-Plasencia. 2000. Beta glucan binding protein and its role in shrimp immune response. Aquaculture, 191: 13-21. Varughese, M., A.V. Teixeira, S. Liu & H. Leppla. 1999. Identification of a receptor-binding region within domain 4 of the protective antigen component of anthrax toxin. Infect. Immunity, 67: 1860-1865.

Received: 22 October 2013; Accepted: 10 July 2015

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Wiles, T.J., R.R. Kulesus & M.A. Mulvey. 2008. Origins and virulence mechanisms of uropathogenic Escherichia coli. Exp. Mol. Pathol., 85(1): 11-19. Zar, J.H. 1996. Bio-statistical analysis. Prentice Hall, New Jersey, 662 pp.

Lat. Am. J. Aquat. Res., 43(4): 766-775, 2015 DOI: 10.3856/vol43-issue4-fulltext-15

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 Mazatlán, Sinaloa, CP 82000, México 4 Centro de Investigación en Alimentación y Desarrollo (CIAD), Mazatlán Unit for Aquaculture Mazatlán, Sinaloa, CP 82000, México 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

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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. MATERIALS AND METHODS 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

Pacific whiteleg shrimp gut microbiota

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

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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

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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. Statistical analysis 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

Pacific whiteleg shrimp gut microbiota

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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.

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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 Flavobacteriaceae bacterium 97 Flavobacteriaceae bacterium 97 Photobacterium damselae subs. piscicida h 48 94 Thalassobius gelatinovorus 93 Planktotalea frisia 98 Ruegeria lacuscaerulensis 99 Unidentified alpha proteobacterium 99 Uncultured bacterium clone Alibio h0 90 Wandonia haliotis Haldis 90 Cytophaga fermentans 90 Sebaldella termitidis 90 Wandonia haliotis Haldis 90 Cytophaga fermentans 96 Photobacterium damselae subsp. piscicida h 12 97 Flavobacteriaceae bacterium 96 Flavobacteriaceae bacterium 89 Uncultured bacterium clone 90 Cytophaga fermentans 97 Flavobacteriaceae bacterium 98 Photobacterium damselae sub sp. Piscicida 89 Uncultured bacterium clone 90 Uncultured bacterium 87 Vibrio furnissii 85 Desulfovibrionaceae bacterium 93 Wandonia haliotis Haldis 98 Ruegeria lacuscaerulensis 90 Wandonia haliotis Haldis h 48 92 Flavobacteriaceae bacterium 90 Paracoccus versatus 82 Vibrio sp. 91 Uncultured bacterium clone 100 Donglicola eburneus 90 Wandonia haliotis Haldis 92 Flavobacteriaceae bacterium 98 Ruegeria lacuscaerulensis 98 Uncultured bacterium clone 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

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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 Flavobacteriaceae bacterium Photobacterium damselae subsp. piscicida h 12 Flavobacteriaceae bacterium Alpha proteobacterium Wandonia haliotis Haldis Flavobacteriaceae bacterium Sebaldella termitidis Uncultured bacterium clone Elizabethkingia anophelis Thalassobius sp. Thioprofundum lithotrophica Ruegeria lacuscaerulensis Uncultured bacterium clone h 48 Vibrio mediterranei Ruegeria lacuscaerilensis Uncultured bacterium clone Oceanicola sp. Flavobacteriaceae bacterium Ruegeria lacuscaerulensis Vibrio sp. Uncultured bacterium clone Thalassobius mediterraneus Thioclava pacifica Ruegeria lacuscaerulensis Uncultured bacterium clone Positive control h0 Uncultured bacterium clone Uncultured bacterium clone Flavobacteriaceae bacterium h 12 Wandonia haliotis Haldis Ruegeria lacuscaerulensis Uncultured bacterium clone Maribius salinus Uncultured bacterium clone Uncultured bacterium clone Thalassobius sp. Uncultured bacterium clone Uncultured bacterium clone Donghicola eburneus Uncultured bacterium clone Donghicola sp. Uncultured bacterium clone Uncultured bacterium clone Wandonia haliotis Haldis Ruegeria lacuscaerulensis Vibrio parahaemolyticus Vibrio sp. Uncultured bacterium clone

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

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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

Pacific whiteleg shrimp gut microbiota

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. REFERENCES Abraham, J.T. & R. Palaniappan. 2004. Distribution of luminous bacteria in semi-intensive penaeid shrimp hatcheries of Tamil Nadu, India. Aquaculture, 232: 8190. Austin, B. 2006. The bacterial microflora of fish, revised. Sci. World J., 6: 931-945. Balcázar, J.L., I. de Blas, I. Ruiz-Zarzuela, D. Cunningham, D. Vendrell & J.L. Múzquiz. 2006. The role of probiotics in aquaculture. Vet. Microbiol., 114: 173-186. Benbouza, H., J.M. Jacquemin, J.P. Baudoin & G. Mergeai. 2006. Optimization of a reliable, fast, cheap and sensitive silver staining method to detect SSR markers in polyacrylamide gels. Biotechnol. Agron. Soc. Environ., 10: 77-81. Chaiyapechara, S., W. Rungrassamee & I. Suriyachay. 2011. Bacterial community associated with the intestinal tract of P. monodon in commercial farms. Microbiol. Ecol., 63: 938-953. Chatterjee, S. & S. Haldar. 2012. Vibrio related diseases in aquaculture and development of rapid and accurate identification methods. J. Mar. Sci. Res. Dev., S1:002. doi:10.4172/2155-9910.S1-002. Chun, J., J.H. Lee, Y. Jung, M. Kim, S. Kim, B.K. Kim & Y.W. Lim. 2007. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int. J. Syst. Evol. Microbiol., 57: 2259-2261. Dohrmann, A.B. & C.C. Tebbe. 2004. Microbial community analysis by PCR-single-strand conformation polymerphism (PCR-SSCP). In: G.A. Kowalchuk, F.J. de Bruijn, I.M. Head, A.J. Van der Zijpp & J.D. Van Elsas (eds.). Molecular microbial ecology manual. Springer, New York, pp. 809-838. Fromin, N., J. Hamelin, S. Tarnawski, D. Roesti, K. Jourdain-Miserez, N. Forestier, F. Gillet, M. Aragno & P. Rossi. 2002. Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environ. Microbiol., 4: 634-643.

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Gopal, S., S.K. Otta, S. Kumar, I. Karunasagar, M. Nishibuchi & I. Karunasagar. 2005. The occurrence of Vibrio species in tropical shrimp culture environments; implications for food safety. Int. J. Food Microbiol., 102: 151-159. Hassan, H.A. 2012. 16S rRNA gene-based bacterial community in polychlorinated biphenyls (PCBs) contaminated site using PCR-single-strand conformation polymorphism (SSCP). Life Sci. J., 9: 935-939. Ishimaru, K., M. Akagawa-Matsushita & K. Muroga. 1995. Vibrio penaeicida sp. nov., a pathogen of kuruma prawns (Penaeus japonicus). Int. J. Syst. Bacteriol., 45: 134-138. Jayasree, L., P. Janakiram & R. Madhavi. 2006. Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). J. World Aquacult. Soc., 37: 523-532. Lightner, D.V. 2005. Biosecurity in shrimp farming: pathogen exclusion through use of SPF stock and routine surveillance. J. World Aquacult. Soc., 36: 229248. Luis-Villaseñor, I.E., M.E. Macías-Rodríguez, B. GómezGil, F. Ascencio-Valle & A.I. Campa-Córdova. 2011. Beneficial effects of four bacillus strains on the larval cultivation of Litopenaeus vannamei. Aquaculture, 321: 136-144. Luis-Villaseñor, I.E., T. Castellanos-Cervantes, B. Gomez-Gil, A.E. Carrillo-García, A.I. CampaCórdova & F. Ascencio. 2013. Probiotics in the intestinal tract of juvenile whiteleg shrimp Litopenaeus vannamei: modulation of the bacterial community. World J. Microbiol. Biotechnol., 29: 257265. Manefield, M., L. Harris, S.A. Rice, R. De Nys & S. Kjelleberg. 2000. Inhibition of luminescence and virulence in the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists. Appl. Environ. Microbiol., 66: 20792084. Martínez-Cruz, P., A.L. Ibáñez, O.A. Monroy-Hermosillo & H.C. Ramírez-Saad. 2012. Use of probiotics in aquaculture. ISRN Microbiol., 2012: Article ID 916845. doi: 10.5402/2012/916845. Moss, S.M., B.R. LeaMaster & J.N. Sweeney. 2000. Relative abundance and species composition of gramnegative, aerobic bacteria associated with the gut of juvenile white shrimp Litopenaeus vannamei reared in oligotrophic well water and eutrophic pond water. J. World Aquacult. Soc., 31: 255-263. Muyzer, G. & K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek, 73: 127-141.

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Noya, M., B. Magarinos & J. Lamas. 1995. Interactions between peritoneal exudate cells (PECs) of gilthead seabream (Sparus aurata) and Pasteurella piscicida. A morphological study. Aquaculture, 131: 11-21. Oxley, A.P.A., W. Shipton, L. Owens & D. McKay. 2002. Bacterial flora from the gut of the wild and cultured banana prawn, Penaeus merguiensis. J. Appl. Microbiol., 93: 214-223. Pérez, T., J.L. Balcázar, I. Ruiz-Zarzuela, N. Halaihel, D. Vendrell, I. de Blas & J.L. Muzquiz. 2010. Hostmicrobiota interactions within the fish intestinal ecosystem. Mucosal. Immunol., 3: 355-360. Qi, Z., X.H. Zhang, N. Boon & P. Bossier. 2009. Probiotics in aquaculture of China-Current state, problems and prospect. Aquaculture, 290: 15-21. Rengpipat, S., S. Rukpratanporn, S. Piyatiratitivorakul & P. Menasaveta. 2000. Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11). Aquaculture, 191: 271-288. Sáenz de Rodrigáñez, M.A., P. Díaz-Rosales, M. Chabrillón, H. Smidt, S. Arijo, J.M. León-Rubio, F.J. Alarcón, M.C. Balebona, M.A. Moriñigo, J.B. Cara & F.J. Moyano. 2009. Effect of dietary administration of probiotics on growth and intestine functionality of juvenile senegalese sole (Solea senegalensis, Kaup 1858). Aquacult. Nutr., 15: 177-185. Sahul-Hameed, A.S., P.V. Rao, J.J. Farmer, W. HickmanBrenner & G.R. Fanning. 1996. Characteristics and pathogenicity of a Vibrio cambelli-like bacterium affecting hatchery-reared Penaeus indicus (Milne Edwards, 1837) larvae. Aquacult. Res., 27: 853-863. Sambrook, J. & D.W. Russell. 2001. Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, New York, 2231 pp. Schwieger, F. & C.C. Tebbe. 1998. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Appl. Environ. Microbiol., 64: 4870-4876. Senderovich, Y., I. Izhaki & M. Halpern. 2010. Fish as reservoirs and vectors of Vibrio cholerae. PLoS ONE, 5(1), e8607. doi:10.1371/journal.pone.0008607. Sun, Y.Z., H.L. Yang, R.L. Ma & S.W. Zhai. 2012a. Does dietary administration of Lactococcus lactis modulate the gut microbiota of grouper, Epinephelus coioides. J. World Aquacult. Soc., 43: 198-207. Sun, Y.Z., H.L. Yang, R.L. Ma, K.P. Huang & J.D. Ye. 2012b. Culture-independent characterization of the autochthonous gut microbiota of grouper Epinephelus coioides following the administration of probiotic Enterococcus faecium. Aquacult. Int., 20(4), 791-801. doi:10.1007/s10499-012-9503-y.

Received: 5 February 2015; Accepted: 4 August 2015

Tapia-Paniagua, S.T., M. Chabrillón, P. Díaz-Rosales, I.G. de la Banda, C. Lobo, M.C. Balebona & M.A. Moriñigo. 2010. Intestinal microbiota diversity of the flat fish Solea senegalensis (Kaup, 1858) following probiotic administration. Microbiol. Ecol., 60: 310319. Toranzo, A.E., S. Barreiro, J.F. Casal, A. Figueras, B. Magariños & J.L. Barja, 1991. Pasteurellosis in cultured gilthead seabream (Sparus aurata): first report in Spain. Aquaculture, 99: 1-15. Turner, J.W., B. Good, D. Cole & E.K. Lipp. 2009. Plankton composition and environmental factors contribute to Vibrio seasonality. ISME J., 3: 10821092. Tuyub-Tzuc, J.T., D. Rendíz-Escalante, R. Rojas-Herrera, G. Gaxiola-Cortés & M.L. Arena-Ortíz. 2014. Microbiota from Litopenaeus vannamei: digestive tract microbial community of Pacific white shrimp (Litopenaeus vannamei). SpringerPlus, 3: 280. doi: 10.1186/2193-1801-3-280. Vaseeharan, B. & P. Ramasamy. 2003. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Lett. Appl. Microbiol., 36: 83-87. Verschuere, L., G. Rombaut, P. Sorgeloos & W. Verstraete. 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mol. Biol. Rev., 64: 655-671. Wu, H.J., L.B.Sun, C.B. Li, Z.Z. Li, Z. Zhang, X.B. Wen Z. Hu, Y.L. Zhang, S.K. Li. 2014. Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain). Fish Shellfish Immunol., 41: 156-162. Yang, H.L., Y.Z. Sun, R.L. Ma & J.D. Ye. 2012. PCRDGGE analysis of the autochthonous gut microbiota of grouper Epinephelus coioides following probiotic Bacillus clausii administration. Aquacult. Res., 43: 489-497. Zokaeifar, H., N. Babaei, C.R. Saad, M.S. Kamarudin, K. Sijam & J.L. Balcazar. 2014. Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol., 36: 68-74. Zokaeifar, H., J.L. Balcázar, C.R. Saad, M.S. Kamarudin, K. Sijam, A. Arshad & N. Nejat. 2012. Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol., 33: 683-689.

Lat. Am. J. Aquat. Res., 43(4): 776-780, 2015 DOI: 10.3856/vol43-issue4-fulltext-16

Marteilia refringens in the Gulf of California

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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; __________________ Corresponding editor: Sergio Palma

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.

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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.

Marteilia refringens in the Gulf of California

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

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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.

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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. REFERENCES Berthe, F.C.J., F. Le Roux, R.D. Adlard & A. Figueras. 2004. Marteiliosis in molluscs: a review. Aquat. Living Resour., 17: 433-448. Burreson, E.M. 2008. Misuse of PCR assay for diagnosis of mollusc protistan infections. Dis. Aquat. Organ., 80: 81-83. Cáceres-Martínez, J., R. Vásquez-Yeomans, G. PadillaLardizábal & M.R. Portilla. 2008. Perkinsus marinus

in pleasure oyster Crassostrea corteziensis from Nayarit, Pacific coast of Mexico. J. Invertebr. Pathol., 99: 66-73. Carrasco, N., K.B. Andree, B. Lacuesta, A. Roque, C. Rodgers & M.D. Furones. 2012. Molecular characterization of the Marteilia parasite infecting the common edible cockle Cerastoderma edule in the Spanish Mediterranean coast. A new Marteilia species affecting bivalves in Europe? Aquaculture, 324-325: 20-26. Enríquez-Espinoza, T.L., J.M. Grijalva-Chon, R. CastroLongoria & J. Ramos-Paredes. 2010. Perkinsus marinus in Crassostrea gigas in the Gulf of California. Dis. Aquat. Organ., 89: 269-273. Escobedo-Fregoso, C., I. Arzul, N. Carrasco, J.N. Gutiérrez-Rivera, R. Llera-Herrera & R. Vázquez. 2015. Polymorphism at the ITS and NTS loci of Perkinsus marinus isolated from cultivated oyster Crassostrea corteziensis in Nayarit, Mexico and phylogenetic relationships to P. marinus along the Atlantic coast. Transbound. Emerg. Dis., 62: 137-147. Grijalva-Chon, J.M., R. Castro-Longoria, J. RamosParedes, T.L. Enríquez-Espinoza & F. Mendoza-Cano. 2013. Detection of a new OsHV-1 DNA strain in the healthy Pacific oyster, Crassostrea gigas, Thunberg, from the Gulf of California. J. Fish Dis., 36(11): 965968. Le Roux, F., G. Lorenzo, P. Peyret, C. Audemard, A. Figueras, C. Vivares, M. Gouy & F. Berthe. 2001. Molecular evidence for the existence of two species of Marteilia in Europe. J. Eukariot. Microbiol., 48: 449454. López-Flores, I. 2003 Estudio molecular integrado de las ostras y de su parásito Marteilia refringens mediante el análisis del ADN repetido. PhD. Thesis, Universidad de Granada, Granada, 252 pp. López-Flores, I., R. de la Herrán, M.A. Garrido-Ramos, J.I. Navas, C. Ruiz-Rejón & M. Ruiz-Rejón. 2004. The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence. Parasitology, 129: 411-419. Office International des Epizooties (OIE). 2009. Manual of diagnostic tests for aquatic animals. World Organization for Animal Health-Office International des Epizooties, Paris, 532 pp. Office International des Epizooties (OIE). 2014. Aquatic animal health code. World Organization for Animal Health-Office International des Epizooties, Paris, 300 pp. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin & D.G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment

Marteilia refringens in the Gulf of California

aided by quality analysis tools. Nucleic Acids Res., 25: 4876-4882. Vásquez-Yeomans, R., J. Cáceres-Martínez & A.F. Huerta. 2004. Herpes-like virus associated with eroded gills of the Pacific oyster Crassostrea gigas in Mexico. J. Shellfish Res., 23: 417-419.

Received: 11 September 2014; Accepted: 5 May 2015

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Vásquez-Yeomans, R., M. García-Ortega & J. CáceresMartínez. 2010. Gill erosion and herpesvirus in Crassostrea gigas cultured in Baja California, Mexico. Dis. Aquat. Organ., 89: 137-144.

Lat. Am. J. Aquat. Res., 43(4): 781-785, 2015 Invasive Cherax quadricarinatus in Jalisco, Mexico DOI: 10.3856/vol43-issue4-fulltext-17

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

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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

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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

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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. REFERENCES Ahyong, S.T. & D.C.J. Yeo. 2007. Feral populations of the Australian red-claw crayfish (Cherax quadricarinatus Von Martens) in water supply catchments of Singapore. Biol. Invas., 9: 943-946. Bauer, R.T. 2013. Amphidromy in shrimps: a life cycle between rivers and the sea. Lat. Am. J. Aquat. Res., 41: 633-650. Bortolini, J.L., F. Alvarez & G. Rodríguez-Almaraz. 2007. On the presence of the Australian redclaw crayfish, Cherax quadricarinatus, in Mexico. Biol. Invas., 9: 615-620. Comisión Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO). 2013. Anexo 1. Listado de especies a considerar para la convocatoria para elaborar diagnósticos sobre el estado de las invasiones biológicas de especies exóticas en algunos ecosistemas de México, ubicados dentro de áreas prioritarias para la conservación, 2013. [http://www.conabio.gob.mx/ institucion/proyectos/doctos/Anexo1_Especies_2013 pdf]. Reviewed: 10 February 2014. Food and Agriculture Organization of the United Nations (FAO). 2014. Cherax quadricarinatus (Von Martens, 1868). Cultured Aquatic Species Information Programme. Cherax quadricarinatus. In: FAO Fisheries and Aquaculture Department Rome. [http://www.fao. org/fishery/culturedspecies/Cherax_quadricarinatus/]. Reviewed: 10 May 2014. García-Guerrero, M.U. & J.A. Apun-Molina. 2008. Density and shelter influence the adaptation of wild juvenile cauque prawns Macrobrachium americanum to culture conditions. North Am. J. Aquacult., 70: 343346. Ghanawi, J. & I.P. Saoud. 2012. Molting, reproductive biology, and hatchery management of redclaw crayfish Cherax quadricarinatus (Von Martens, 1868). Aquaculture, 358: 183-195. Harlioglu, M.M. & A.G. Harlioglu. 2006. Threat of nonnative crayfish introductions into Turkey: global lessons. Rev. Fish. Biol. Fish., 16: 171-181. Instituto Nacional de Pesca (INAPESCA). 2011. Carta Nacional Acuícola, Diario Oficial de la Federación, 31 de enero de 2011. Instituto Nacional de la Pesca, Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Ciudad de México.

Invasive Cherax quadricarinatus in Jalisco, Mexico

[http://www.inapesca.gob.mx/portal/documentos/publ icaciones/2011/SAGARPA_CNA.pdf] Reviewed: 10 December 2014. Instituto Nacional de Pesca (INAPESCA). 2012. Carta Nacional Acuícola, Diario Oficial de la Federación, 6 de junio de 2012. Instituto Nacional de la Pesca, Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Ciudad de México. [http://www.inapesca.gob.mx/portal/documentos/publ icaciones/2011/SAGARPA_CNA.pdf] Reviewed: 10 December 2014. Instituto Nacional de Pesca (INAPESCA). 2013. Actualización de la Carta Nacional Acuícola, Diario Oficial de la Federación, 9 de septiembre de 2013. Instituto Nacional de la Pesca, Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. Ciudad de México. [http://www. inapesca.gob.mx/portal/documentos/publicaciones/20 11/SAGARPA_CNA.pdf] Reviewed: 10 December 2014. Jaklic, M. & A. Vrezec. 2011. The first tropical alien crayfish species in European waters: the redclaw Cherax quadricarinatus (Von Martens, 1868) (Decapoda: Parastacidae). Crustaceana, 84: 651-665. Jones, C.M. & I.M. Ruscoe. 2001. Assessment of five shelter types in the production of redclaw crayfish Cherax quadricarinatus (Decapoda: Parastacidae) under earthen pond conditions. J. World Aquacult. Soc., 32: 41-52.

Received: 4 November 2014; Accepted: 27 May 2015

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Lawrence, C. & C. Jones. 2002. Cherax. In: D.M. Holdich (ed.). Biology of freshwater crayfish. Blackwell Science, Oxford, pp. 645-666. Peay, S. 2009. Invasive non-indigenous crayfish species in Europe: recommendations on managing them. Knowl. Manage. Aquat. Ecosyst., 3: 294-395. Ponce-Palafox, J.T., J.L. Arredondo-Figueroa & X. Romero. 1999 Análisis del cultivo comercial de la langosta de agua dulce (Cherax quadricarinatus) y su posible impacto en América Latina. Contactos, 31: 5461. Rodríguez-Uribe, M.C., F. Vega-Villasante, M. GuzmánArroyo & L.D. Espinosa. 2014. Efectos de una barrera antrópica sobre la migración río arriba del langostino anfídromo Macrobrachium tenellum (Smith, 1871) (Decapoda: Palaemonidae) en la costa del Pacífico mexicano. Gayana, 78: 10-20. Snovsky, G. & B.S. Galil. 2011. The Australian redclaw crayfish Cherax quadricarinatus (Von Martens, 1868) (Crustacea: Decapoda: Parastacidae) in the Sea of Galilee, Israel. Aquat. Invas., 6(Suppl.1): 29-31.

Lat. Am. J. Aquat. Res., 43(4): 786-791, 2015 Digestibility of energetic ingredients by Arapaima gigas DOI: 10.3856/vol43-issue4-fulltext-18

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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-

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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.

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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.

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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/

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2012), Brazil. The Cantagalo Farm (Bahia, Brazil) provided the fish for this project. REFERENCES Abboudi, T., M. Mambrini, W. Ooghe, Y. Larondelle & X. Rollin. 2006. Protein and lysine requirements for maintenance and for tissue accretion in Atlantic salmon (Salmo salar) fry. Aquaculture, 261: 369-383. Association of Official Analytical Chemists (AOAC). 2005. Official methods of analysis of AOAC, Maryland, 1234 pp. Bomfim, M.A.D., E.A.T. Lanna, J.L. Donzele, A.S. Ferreira, F.B. Ribeiro & S.S. Takishita. 2008. Methionine plus cystine requirement based on ideal protein concept, in diets for Nile tilapia fingerlings. Rev. Bras. Zootec., 37: 783-790. Braga, L.G.T., R. Borghesi & J.E.P. Cyrino. 2008. Apparent digestibility of ingredients in diets for Salminus brasiliensis. Pesqui. Agropec. Bras., 43: 271-274. Bureau, D.P., A.M. Harris & C.Y. Cho. 1999. Apparent digestibility of rendered animal protein ingredients for rainbow trout (Oncorhynchus mykiss). Aquaculture, 180: 345-358. 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: 1147-1156. De Silva, S.S. 1989. Digestibility evaluations of natural and artificial diets. Asian Fish Soc. Spec., 4: 36-45. Glencross, B., N. Rutherford & N. Bourne. 2012a. The influence of various starch and non-starch polysaccharides on the digestibility of diets fed to rainbow trout (Oncorhynchus mykiss). Aquaculture, 356: 141-146. Glencross, B., D. Blyth, S. Tabrett, N. Bourne, S. Irvin, 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. Aquaculture, 170: 267-283.

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Imbiriba, E.P. 2001. Production potential of pirarucu, Arapaima gigas, in captivity. Acta Amaz., 31: 299316. Ituassú, D.R., M. Pereira-Filho, R. Roubach, R. Crescêncio, B.A.S. Cavero & A.L. Gandra. 2005. Crude protein levels for juvenile pirarucu. Pesqui. Agropec. Bras., 40: 255-259. Kumar, V., A.K. Sinha, H.P.S. Makkar, G. Boeck & K. Becker. 2012. Phytate and phytase in fish nutrition. J. Anim. Physiol. Anim. Nutr., 96(3): 335-364. Lee, S.M. 2002. Apparent digestibility coefficients of various feed ingredients for juvenile and grower rockfish (Sebastes schlegeli). Aquaculture, 207: 7995. Lundstedt, L.M., J.F.B. Melo & G. Moraes. 2004. Digestive enzymes and metabolic profile of Pseudoplatystoma coruscans (Teleostei: Siluriformes) in response to diet composition. Biochem. Mol. Biol., 137: 331-339. McGoogan, B.B. & R.C. Reigh. 1996. Apparent digestibility of selected ingredients in red drum (Sciaenops ocellatus) diets. Aquaculture, 141: 233244. Rawles, S.D., M. Riche, T.G. Gaylord, J. Webb, D.W. Freeman & M. Davis. 2006. Evaluation of poultry byproduct meal in commercial diets for hybrid striped bass (Morone chrysops ♀ × M. saxatilis ♂) in recirculated tank production. Aquaculture, 259: 377389.

Received: 5 January 2015; Accepted: 16 June 2015

Ribeiro, F.B., E.A.T. Lanna, M.A.D. Bomfim, J.L. Donzele, M. Quadros & P.S.L. Cunha. 2011. True and apparent digestibility of protein and amino acids of feed in Nile tilapia. Rev. Bras. Zootec., 40: 939-946. Silva, T.S.C., G.V. Moro, T.B.A. Silva, J.K. Dairiki & J.E.P. Cyrino. 2013. Digestibility of feed ingredients for the striped surubim Pseudoplatystoma reticulatum. Aquacult. Nutr., 19: 491-498. Teixeira, E.A., E.O.S. Saliba, A.C.C. Euler, P.M.C. Faria, 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. Aquaculture, 437: 38-45. Zhou, Q.C., B.P. Tan, K.S. Mai & Y.J. Liu. 2004. Apparent digestibility of selected feed ingredients for juvenile cobia Rachycentron canadum. Aquaculture, 241: 441-451.

Lat. Am. J. Aquat. Res., 43(4): 792-797, Anticipation 2015 of Artemia sp. supply in the larviculture of barber goby DOI: 10.3856/vol43-issue4-fulltext-19

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

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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.

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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

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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. REFERENCES Avella, M.A., I. Olivotto, G. Gioacchini, F. Maradonna & O. Carnevali. 2007. The role of fatty acids enrichments in the larviculture of false percula clownfish Amphiprion ocellaris. Aquaculture, 273: 87-95. Barroso, M.V. 2010. Utilização do copépode Acartia tonsa nas diferentes fases de desenvolvimento da larva do robalo-peva Centropomus parallelus. Ph.D. Thesis, Universidade Federal de Santa Catarina, Santa Catarina, 82 pp. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Brasil, 2004. [http://www.icmbio.gov.br/cepsul/legislacao/instrucao-normativa/342-2004.html]. Reviewed: 19 August 2015. Cara, J.B., F.J. Moyano, S. Cárdenas, M. Fernández-Díaz & M. Yúfera. 2002. Assessment of digestive enzyme activities during larval development of white bream. J. Fish Biol., 63: 48-58. Carvalho-Filho, A. 1999. Peixes: costa brasileira. Editora Melro Ltda., São Paulo, 320 pp. Côrtes, G.F. 2009. Produção e utilização de diferentes fontes de alimento vivo na fase inicial de larvicultura do neon gobi (Elacatinus figaro). M.Sc. Dissertation, Universidade Federal de Santa Catarina, Santa Catarina, 46 pp. Côrtes, G.F. & M.Y. Tsuzuki. 2012. Effect of different live food on survival and growth of first feeding barber goby, Elacatinus figaro (Sazima, Moura & Rosa 1997) larvae. Aquacult. Res., 43(6): 831-834. Das, P., S.C. Mandal, S.K. Bhagabati, M.S. Akhtar & S.K. Singh. 2012. Important live food organisms and their role in aquaculture. In: S. Munilkumar (ed.). Frontiers in Aquaculture. Chapter: Important live food organisms and their role in aquaculture. Narendra Publishing House, New Delhi, pp. 69-86. DeLoach, N. 1999. Reef fish behavior: Florida, Caribbean, Bahamas. New World Publications, Jacksonville, 359 pp. Fernández, I., F.J. Moyano, M. Díaz & T. Martínez. 2001. Characterization of α-amylase activity in five species

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of Mediterranean sparid fishes (Sparidae, Teleostei). J. Exp. Mar. Biol. Ecol., 262: 1-12. Galvão, M.S.N., N. Yamanaka, N. Fenerich-Verani & C.M.M. Pimentel. 1997. Estudos preliminares sobre enzimas digestivas proteolíticas da tainha Mugil platanus Gunther, 1880 (Osteichthyes, Mugilidae) durante as fases larval e juvenil. Bol. Inst. Pesca, 24: 101-110. Garcia-Carreño, F.L., M.A. Navarrete del Toro, J.M. Ezquerra. 1997. Digestive shrimp proteases for evaluation of protein digestibility in vitro. I: effect of protease inhibition in protein ingredients. J. Mar. Biotechnol., 5: 36-40. Gasparini, J.L., S.R. Floeter, C.E.L. Ferreira & I. Sazima. 2005. Marine ornamental trade in Brazil. Biodivers. Conserv., 14: 2883-2899. Guerreiro, I., M. Vareilles, P. Pousão-Ferreira, V. Rodrigues, M.T. Dinis & L. Ribeiro. 2010. Effect of age-at-weaning on digestive capacity of white seabream (Diplodus sargus). Aquaculture, 300: 194205. Han, K., I. Geurden & P. Sorgeloos. 2000. Enrichment strategies for Artemia sp. using emulsions providing different levels of n-3 highly unsaturated fatty acids. Aquaculture, 183: 335-347. Jimenez-Martinez, L.D., C.A. Alvarez-González, D. Tovar-Ramírez, G. Gaxiola, A. Sanchez-Zamora, F.J. Moyano, F.J. Alarcón, G. Márquez-Couturier, E. Gisbert, W.M. Contreras-Sánchez, N. Perales-García, L. Arias-Rodríguez, J.R. Indy, S. Páramo-Delgadillo & I.G. Palomino-Albarrán. 2012. Digestive enzyme activities during early ontogeny in common snook (Centropomus undecimalis). Fish. Phys. Biochem., 38: 441-454. Johnson, W.S. 1982. A record of cleaning symbiosis involving Gobiosoma sp. and a large Caribbean octopus. Copeia, 1982(3): 712-714. Kuz’mina, V.V. 1996. Influence of age on digestive enzyme activity in some freshwater teleosts. Aquaculture, 148: 25-37. Losey, G.S.J. 1987. Cleaning symbiosis. Symbiosis, 4: 229-258. Meirelles, M.E., M.Y. Tsuzuki, F.F. Ribeiro, R. Medeiros & I. Diniz. 2009. Reproduction, early development and larviculture of the barber goby, Elacatinus figaro (Sazima, Moura & Rosa 1997). Aquacult. Res., 41: 1118. Olivotto, I., M. Cardinali, L. Barbaresi, M. Maradonna & O. Carnevalli. 2003. Coral reef fish breeding: the secrets of each species. Aquaculture, 224: 69-78. Olivotto, I., A. Zenobi, A. Rollo, B. Migliarini, M. Avella & O. Carnevali. 2005. Breeding, rearing and feeding

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studies in the cleaner goby Gobiosoma evelynae. Aquaculture, 250: 175-182. Pezzato, L.E. 1997. O estabelecimento das exigências nutricionais das espécies de peixes cultivadas. Simpósio sobre manejo e nutrição de peixes. CBNA, Piracicaba, pp. 45-62. Sazima, I., R.L. Moura & R.S. Rosa. 1996. Elacatinus figaro sp. n. (Perciformes: Gobiidae), a new cleaner goby from the coast of Brazil. J. Ichthyol. Aquat. Biol., 2: 33-38. Sazima, I., C. Sazima, R.B. Francini-Filho & R.L. Moura. 2000. Daily cleaning activity diversity of clients of the barber goby, Elacatinus figaro, on rocky reefs in southeastern Brazil. Environ. Biol. Fish., 59: 69-77.

Received: 14 February 2015; Accepted: 8 July 2015

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Lat. Am. J. Aquat. Res., 43(4): 798-806, 2015 Electrophoretic protein profiles of copepod fed diatoms DOI: 10.3856/vol43-issue4-fulltext-20

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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 (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.

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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-

Electrophoretic protein profiles of copepod fed diatoms

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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

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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

Electrophoretic protein profiles of copepod fed diatoms

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.

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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. REFERENCES Anabalón, V., C. Morales, R. Escribano & M. Varas. 2007. The contribution of nano-and micro-planktonic assemblages in the surface layer (0-30 m) under different hydrographic conditions in the upwelling area off Concepción, central Chile. Prog. Oceanogr., 75: 396-414. Adiyodi, K.G. & T. Subramoniam. 1983. Oogenesis, oviposition and oosorption, Arthropoda-Crustacea. In: K.G. Adiyodi & R.G. Adiyodi (eds.). Reproductive biology of invertebrates. Wiley, New York, pp. 443495. Aguilera, V., K. Donoso & R. Escribano. 2011. Reproductive performance of small-sized dominant copepods with a highly variable food resource in the coastal upwelling system off the Chilean Humboldt Current. Mar. Biol. Res., 7: 1-15. Aguilera, V. & R. Escribano. 2013. Experimental studies on the feeding and reproduction of Calanoides patagoniensis (Copepoda, Calanoida) in a southern upwelling ecosystem of the Humboldt Current. Env. Mar. Res., 91: 23-26.

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Received: 10 October 2014; Accepted: 4 August 2015

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