(Genypterus Chilensis) Cultured in a Chilean Farm

162, Bull. Eur. Ass. Fish Pathol., 35(5) 2015

ȱȱ ȱȱ¢ȱȱ ȬȱȱȱȱȱǻGenypterus chilensisǼȱȱȱȱȱ

A. Levican1,2,# and R. Avendaño-Herrera1,2,3*

1Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile; 2Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile; #Current address: Facultad de Medicina, Universidad Andrés Bello, Viña del Mar, Chile; 3Centro de Investigación Marina de Quintay, CIMARQ, Quintay, Chile

Abstract The red conger eel (Genypterus chilensis, Ǽȱȱȱȱȱȱęȱ ȱǯȱ ǰȱȱȱȱȱȱ¢ȱȱȱȱ ȱȱ ȱȱȱǯȱȱȱ¢ȱ ȱȱȱ¢ȱȱ£ȱŘŗȱȱ ȱ ȱȱȱ¢ȱȱ ȱȬȱȱȱȱȱ¢ǯȱ¢ȱ ȱȱȱȱȱŗŜȱȱȱȱȱȱ¢ȱ ȱȱȱ Vibrio, ȱȱ¢ȱȱ ȱȱȱȱęȱȱȱV. anguillarum, V. ordalii and V. tapetisǰȱȱ ȱȱȱȱȱ¢ȱȱȱȱV. toranzoniae. Despite this, other genera such as Pseudoalteromonas, Psychrobacter, Alteromomas, Marinobacter, Planococcus and Pseudomonasȱ ȱȱęǯȱȱȱȱ ȱȱǰȱ ǰȱ ȱȱȱ¢ȱȱ¢ȱ ȱȱȱȱȱȱȱ- ȱȱȱęȱȱȱȱȱǯ

Introduction Red conger eel (Genypterus chilensis, Guichenot) ȱȱȱęȱ¢ȱȱȱȱȱ ȱȱǰȱ¢ȱęȱȱȱ increasing prices and growing unmet demand ȱ¢ȱĴȱȱȱȱȱȱ estimated at 3,048 tons per year. Considering ȱ ȱȱȱęȱȱȱ this, new aquaculture programs have concen- ȱȱȱȱǻĴǱȦȦ ǯęǯ ȱȱȱ¢ȱȱȱȱ org/summary/Genypterus-chilensis.html). This ȱȱȱȱȱǻ Ȭȱȱǯǰȱ ęȱȱȱȱȱȱȱȱȱ 2011). Vega et al. (2012) obtained high survival ȱ¢ȱȱȱȱȱ£ȱ ȱȱȱȱȱȱȱȱȱ by high gastronomic demand and seasonal an incubation system with a closed water circuit exploitation, which is mainly carried out by and there have also been reported advances ȱęȱȱǻȱȱǯǰȱŘŖŗŘǼǯȱ on growing and conditioning brood stock and ǰȱȱȱȱŗŘȱ¢ȱȱȱȱȱ post-larvae (Maldonado et al., 2012).

* Corresponding author’s email: [email protected] or [email protected] Bull. Eur. Ass. Fish Pathol., 35(5) 2015, 163

ȱȱǰȱ ȱȱŘŖŗŖȱȱ Phenotypic characterization of the isolates ¢ȱŘŖŗŗǰȱȱȱ¢ȱȱȱȱȱ The isolates were phenotypically characterized post-larvae occurred and it was associated with as described by MacFaddin (2003) using the ȱǰȱȱȱȱȱ ȱǱȱ ȱǰȱcytochrome ȱȱę¢ȱȱǻȱ oxidaseǰȱ¢ǰȱ¡£ȱȱȱ ȱǯǰȱŘŖŗŘǼǯȱȱȱȱȱȱ ȱǰȱȱȱ¢ȱ¡¢- ęȱȱȱȱȱȱ lase, amylase, gelatinase and lipase production. ȱȱȱȱǰȱȱȱȱȱ- Growth on TSA-1 under aerobic conditions at tion on the bacteria associated with its diseased ěȱȱǻŚǰȱŗśǰȱŗŞǰȱŘŘȱȱřŝķǼȱ larvae, this study aimed to characterize those ȱȱȱȱ ȱěȱȱ ȱȱ ȱȱ¢ȱȱȱȱ concentrations (0, 2, 4, 6, 8 and 10%) was also conger eel early stage larvae reared in a Chilean tested. The haemolytic capacity was tested on aquaculture nursery. TSA supplemented with 5% sheep blood agar (BA, Biomerieux) incubated under aerobic con- Materials and methods ȱȱŗŞķȱȱśȱǯȱȱ¢ȱȱȱ Sampling ȱȱĚȱǻřŖȱΐǼǰȱĚȱǻřŖȱ Post-larvae between 134 and 175 days post- ΐǼǰȱ¡ȱȱǻŘŖȱΐǼǰȱ¢¢ȱǻŗśȱ hatching were produced at the Centro de Inves- ΐǼǰȱĚ¡ȱǻśȱΐǼȱ¡¢¢ȱǻřŖȱΐǼȱ tigación Marina Quintay (CIMARQ), Valparaiso, ȱȱȱǻŗŖȱΐȱȱŗśŖȱΐǼǰȱ ȱ ǰȱȱȱȱ¢ȱȱȱ carried out ȱȮ ȱȱǻ¡Ǽȱ maintained in the same centre. Samples consist- supplemented with 1% NaCl as recommended ȱȱśȱ ȱǻȱȱ¢ȱ Ǽȱ by the Clinical and Laboratory Standards Insti- post-larvae were washed with sterile saline tute in guideline M42-A (CLSI, 2006) ȱ ȱ (0.9% NaCl) and then directly inoculated onto 2 organisms (obligate halophilic strains). tryptone soy agar (Oxoid) supplemented with 1% (w/v) sodium chloride (TSA-1) and thio- ȱęȱ¢ȱȱȱȱ- ȬȬȬȱǻǰȱ¡Ǽǯȱȱ ȱȱ ȱĴȱȱȱȱ ȱ ȱȱȱŚŞȱȱȱśȱȱȱŗŞǚȱ ȱęȱȱȱȱV. an- ȱȱǯȱȱȱȱȱ guillarum (serotypes O1, O2 and O3) and V. ȱ ȱȱ£ȱȱȮŞŖȱķȱȱ ordalii (serotype O2) as described by Toranzo Criobille tubes (AES Laboratory). et al. (1987). These Vibrio species were selected because they are among the main pathogens ¢ȱ¢ȱȱȬȱȱ ȱȱ¡¢ȱ reported in Chile. saturation and water temperature data were collected and their possible correlation with Genetic characterization ¢ȱ ȱ¢ȱȱȱȱ ȱ ȱȱ ȱ¡ȱȱȱȱ Version 20 (IBM, USA) using the Pearson cor- ȱ ȱȱęȱ¡ȱǻȬ relation and T tests. Ǽȱ ȱȱȼȱǯȱ Colonies were genotyped by Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) 164, Bull. Eur. Ass. Fish Pathol., 35(5) 2015 using the primer pair ERIC-1R and ERIC-2 and ȱȱȱ¢ȱȱ ȱȱȱ conditions as reported by Versalovic et al. (1991). ȱȱȱȱȱȱ- PCR products were analyzed on a 2% agarose sumptively belonging to Vibrio spp. as they gel and stained with 1/10,000 GelRedTM Nucleic were Gram-negative rods, oxidase positive Acid Gel Stain (Biotium, CA). The GeneRulerTM ȱȱ ȱȱȱ- ŗŖŖȱȱȱȱȱǻȱęǰȱ ȱȱȱȱ ȱȱȱȱ USA) was used as a molecular mass marker and vibriostatic agent (Table 1). On the other hand, gels were photographed using the UV transil- ŝŘǯŘƖȱȱȱȱ ȱȱ¢ǰȱ luminator Gel Doc XR+ (Bio Rad, USA). whereas only 47.6% and 28.6% showed amylase and gelatinase activity, respectively. Despite the ȱŗŜȱȱȱ ȱęȱȱȱ hydrolytic enzymes tested are common among universal primer pair pA and pH as described ęȱǰȱ¢ȱȱȱȱ ȱȱ by Edwards et al. (1989). The expected 1,500 capacity ȱȱȱȱȱȱ bp amplicon obtained was then sequenced by (Austin et al., 2005). However, whether these Macrogen (Seoul, Korea). The resulting 16S isolates possess a higher pathogenic capacity rRNA sequences were analysed using the Basic remains to be assessed. Regarding the antibiotic ȱȱȱȱǻǰȱĴǱȦȦ ¢ǰȱȱȱȱȱȱ ȱ blast.ncbi.nlm.nih.gov/). Then, the sequences ȱȱȱȱ ȱȱȱȱȱ ȱȱ¢ȱȱȱȱȱȱȱ ǻȱŗǼǯȱȱȱȱȱȱȱȱ same genus were downloaded and they all ȱȱȱȱȱěȱȱ ȱȱȱ ȱ ȱȱśȱ ȱȱȱǯ (Tamura et al., 2011). Genetic distances were obtained using Kimura’s two-parameter model ȱȱ¢ȱȱȱŗŜȱȱȱ (Kimura, 1980) and clustered with the neigh- sequence allocated the isolates predominantly bour joining algorithm. within the genus Vibrio, ȱ¢ȱAltero- monas, Pseudoalteromonas and Psychrobacter. The Results and discussion ¡ȱȱȱȱȱ ȱȱ ȱ¢ȱȱ ȱşŚƖȱǻƽȱřŜśǼȱȱȱȱ ȱ ȱȱęȱ¢ȱȱȱ ȱȱȱ ȼȱ¢ȱǻƽȱřŞŞǼǯȱ ȱȱ¢ȱȱȱȱ¢ȱȱȱȱ ȱȱȱȱŗŚǯŖȱķȱȱŗŝǯřȱ ȱȱ ȱȱȱȱȱȱȱ ķȱȱ¡¢ȱǰȱȱşŜƖȱȱŗŖśƖǰȱ ǻȱŘǼǯȱȱȱȱȱȱȱVibrio but ȱęȱȱ ȱȱ ǯǰȱȱȱȱȱȱȱȱŗŖŖȱ parameters and daily mortality was observed species that are widely distributed in aquatic (data not shown). Twenty-one predominant ǰȱȱȱȱȱ ȱ colonies (i.e. those colonies that presented the ȱȱȱęǰȱȱȱ same morphology and were the most abundant crustaceans, in commensal or pathogenic rela- ȱȱǼȱ ȱȱȱȱȱ ȱǻȱȱǯǰȱŘŖŗŚǼǯȱ ȱǰȱvibriosis ȱȬȱȱȱȱ¢ȱ ȱȱȱȱȱȱȱȱęȱ £ȱȱ¢ȱȱěȱ and other aquaculture-reared organisms and antibiotics are shown in Table 1. ȱ ¢ȱȱȱȱȱȱ- Bull. Eur. Ass. Fish Pathol., 35(5) 2015, 165

Table 1.ȱ¢ȱȱȱ¢ȱȱěȱȱǻǼȱȱȱȱȱ ȱ ȱGenypterus chilensis post larvae. +, Positive and -, negative.

aȱǱȱ¡ǲȱǱȱǲȱb Vibriostatic agent

ȱ¢ȱ ȱǻĴȱȱ ȱȱ ȱęȱȱVibrio tapetis Haldar, 2012). ǰȱȱ¢ȱ (Isolate 20), which is widely recognized as the tree was constructed including the sequences ȱȱȱ ȱȱȱȱcauses ȱȱşȱȱȱȱȱ¢ȱȱȱ massive mortality in cultured Manila clams this genus (data not shown). Isolates 2 and 3 (Venerupis philippinarum) (Borrego et al., 1996). were very close to the commonly aquaculture However, V. tapetis has been isolated during pathogenic species V. anguillarum and V. ordalii ȱȱȱęȱȱȱȱ (Chaterjee and Haldar, 2012). However, serol- halibut (Hipoglossus hipoglossus) (Reid et al., ¢ȱȱǻ£ȱȱǯǰȱŗşŞŝǼȱȱȱȱ 2003), corkwing wrasse (Symphodus melops) gave negative results indicating that they could (Jensen et al., 2003) and wedge sole (Dicologo- ȱȱȱ¢ȱěȱȱȱ glossa cuneataǼȱǻ£ȱȱǯǰȱŘŖŗŗǼǯȱȱȱ tested. These serotypes include those previously ǰȱ¢ȱȱȱȱȱ ȱ ȱ ȱV. ȱȱȱǲȱǰȱȱ tapetis shown to be pathogenic under laboratory ȱȱȱȱęȱ conditions (Jensen et al., 2003). ȱȱȱȱȱǯ 166, Bull. Eur. Ass. Fish Pathol., 35(5) 2015

35 400

Daily mortality Cumulative mortality 350 30

300 25

250 20

200

15 150

10 100 Daily mortality cases) (n Cumulave mortality cases) (n 5 50

0 0 134 136 138 140 142 144 146 148 150 152 154 156 158 160 162 164 166 168 170 172 174

Days post hatching

Figure 1.ȱ¢ȱȱȱ¢ȱȱȱȱȱȬȱǻȱƽřŜśǼȱ ȱ¢ȱŗřŚȱȱ ŗŝśȱȬǯȱȱȱȱȱȱȱȱȬȱ ȱřŞŞǯȱ

ȱȱȱŜȱǰȱęȱȱȱǻŞǰȱ ǻȱŘǼǯȱȱȱȱȱȱȱ ǯŗŝǰȱǯŗŞǰȱǯŗşȱȱŘřǼȱ ȱęȱȱVibrio ȱȱȱȱȱ ȱȱȱ- toranzoniae. Moreover, those isolates showed a sal relation with aquatic organisms (Leon and ȱ ȬȱĴȱǻȱȱ Ǽȱ Tapia, 1999; Costa-Ramos and Rowley, 2004; ȱȱǯŗŝǰȱǯŗŞȱȱǯŗşȱ ȱęȱ Figueras and Novoa, 2013). Even though the as V. toranzoniae by means a polyphasic taxo- ȱȱȱȱ¢ȱcould be due nomic approach in a recent study by Lasa et to bȱǰȱit is not clear whether ǯȱǻŘŖŗśǼǯȱ ȱȱ¢ǰȱȱ¢ȱȱȱ the recovered species acted as commensal or ȱȱǻŗŞǼȱ ȱȱęȱ as opportunistic pathogens, because there is in turbot (Scophthalmus maximus) because it pro- ȱȱȱȱȱȱ ȱ¢ȱȱ¢ȱȱȱȱ in healthy red conger eel larvae. In this sense, (Lasa et al., 2015). In addition, the taxonomic ȱȱ ȱȱ¢ȱȱ ȱȱȱŘŘȱȱȱȱȱ these bacteria to healthy specimens need to be because it clustered close to several species, i.e. conducted. Nevertheless, studies on the com- V. atlanticus, V. tasmaniensis, V. kanaloae, V. lentus ȱȱȱȱȱȱ and V. cyclotrophicus (data not shown). its susceptibility to therapies in culture systems are also necessary because this is a ¢ȱȱȱ Other bacterial genera that predominated were ȱȱęȱȱǯ Alteromonas, Pseudoalteromonas and Psychrobacter Bull. Eur. Ass. Fish Pathol., 35(5) 2015, 167

Figure 2. Neighbour joining tree based on 16S rRNA sequences (1397 bp) showing the phylogenetic position ȱȱŘŗȱȱȱȱ ȱȱȱȱȬȱȱȱ¢ȱȱȱȱȱȱ ȱȱ¢ȱȱȱȱȱǻin bold). Bootstrap values (>70%) based on 1000 replications are shown ȱȱȱȱȱǯȱǰȱŘȱȱȱŗŖŖȱǯȱ

Acknowledgments ȱȱȱ¢ȱ ȱȱȱȱ 3140296 and also by Grant CONICYT/ by Postdoctoral Grant FONDECYT number FONDAP/15110027 ȱȱàȱ- 168, Bull. Eur. Ass. Fish Pathol., 35(5) 2015

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