A standard panel of microsatellites for Asian seabass (Lates calcarifer)

Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 ± 0.71 (range: 6–19), and the expected heterozygosity averaged 0.76 ± 0.02 (range: 0.63–1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM‐T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3–5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 × 10 and 20 × 20) demonstrated a high power of these markers for revealing parent‐sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.     

Cloning and characterization of the calreticulin gene in Asian seabass (Lates calcarifer)

Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. We cloned and characterized the CRT gene in an important marine food fish species Asian seabass (Lates calcarifer). The full-length DNA of the CRT gene was 2194 bp, including a complete open reading frame encoding 420 amino acid residues, a 113 bp 5'-untranslated region and an 818 bp 3'-untranslated region. The CRT gene contained nine exons and eight introns covering a total of 2772 bp genomic DNA from the start to stop codon. Ten single nucleotide polymorphisms (SNPs) were detected in introns and an exon in six individuals collected from five different locations. The CRT gene was assigned to linkage group 4 of the linkage map of Asian seabass. Quantitative real-time PCR revealed that the CRT gene was highly expressed in liver at the age of 1, 3 and 7 months under normal conditions, whereas its expression in liver reduced sharply after 0.5 to 2 h cold challenge at 16°C, and then increased slowly. A preliminary association analysis showed a significant (P < 0.001) association between the SNP6 in the CRT gene and the mortality after cold challenge at 16°C. Our results suggest that the CRT gene is associated with cold tolerance of Asian seabass and further investigation will be necessary to illustrate the underlying mechanisms.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Significant associations of polymorphisms in the prolactin gene with growth traits in Asian seabass (Lates calcarifer)

Prolactin (encoded by PRL) is a multifunctional hormone involved in osmoregulation, reproduction, growth, development, immunomodulation, endocrine and metabolic regulation. We cloned the full-length cDNA of Asian seabass PRL, searched for polymorphism in the DNA sequence, and conducted association analyses. Twelve SNPs and one 4-bp deletion were identified in PRL. The SNP c.264+127C>G was used for linkage mapping, and this gene was mapped to linkage group 11. The c.264+980_983delTTGT, c.264+127C>G, c.264+138T>G, c.264+269T>C and c.330C>G polymorphisms were genotyped in 521 individuals with growth trait records. Association analyses between single markers and growth traits revealed that the c.264+269T>C SNP was significantly associated with body weight (BW), total length (TL), standard length (SL) and Fulton's condition factor (KTL and KSL), while the other four were not. Analysis of haplotypes showed that there were 10 haplotypes and 22 haplotype combinations in the population. The differences of BW, TL, KTL and KSL among different haplotype combinations were significant.     

Characterization of two parvalbumin genes and their association with growth traits in Asian seabass (Lates calcarifer)

Parvalbumins are extremely abundant in fish muscle and play an important role in muscle relaxation. In this study, two parvalbumin genes (PVALB1 and PVALB2) were cloned from Asian seabass (Lates calcarifer). The cDNAs for PVALB1 and PVALB2 were 840 and 667 bp respectively. Both genes consisted of five exons and four introns, encoded 109 amino acids, and were of beta lineage. Using real-time polymerase chain reaction, expression of PVALB1 was detected in all 10 tissues tested, with expression in brain, kidney, muscle and small intestine being 15- to 322-fold higher than in the other tissues. Expression of PVALB2 was detected only in muscle, brain and intestine, and was up to 10-fold lower than PVALB1 expression. A (CT)(17) microsatellite in the 3'-untranslated region of PVALB1 and three single nucleotide polymorphisms (SNPs) in the third intron of PVALB2 were identified. The microsatellite in PVALB1 was significantly associated with body weight and body length at 90 days post-hatch (P < 0.01), whereas the SNPs in PVALVB2 were not associated with these traits.     

Azadirachta indica (neem) leaf dietary effects on the immunity response and disease resistance of Asian seabass,Lates calcarifer challenged with Vibrio harveyi

The present study was aimed to address the possible evaluation of Azadirachta indica (neem) leaf-supplemented diets on innate immune response in Asian seabass, Lates calcarifer fingerlings against Vibrio harveyi infection. Fish were fed for two weeks diets containing six graded levels of neem leaf at 0 g, 1 g, 2 g, 3 g, 4 g and 5 g per kg feed. Fish fed neem leaf-supplemented diet displayed significant differences (p < 0.05) in weight gain, specific growth rate (SGR) and feed conversion ratio (FCR) compared to the control group fed without neem leaf-supplemented diet. Various innate immune parameters were examined pre-challenge and post-challenge. Fish was injected intraperitoneally with a lethal dose of V. harveyi containing 10⁸ cells mL^?1. Supplementation of neem leaf diet significantly increased phagocytic activity, superoxide anion production, serum lysozyme, serum bactericidal activity, serum anti-protease activity throughout the experimental period when compared with the control group. Dietary doses of neem leaf diet significantly influenced the immune parameters, haematological parameters and blood biochemical indices of treated fish. The results suggested that fish fed neem leaf-supplemented diet improved the immune system and increased survival rate in L. calcarifer fingerlings against V. harveyi infection.     

Viral nerve necrosis in hatchery-produced fry of Asian seabass Lates calcarifer: sequential microscopic analysis of histopathology

We studied the natural progression of viral nerve necrosis (VNN) in larvae of Asian seabass Lates calcarifer Bloch from 0 to 40 days post-hatch (dph). The hatchlings were reared in the vicinity of a confirmed nodavirus-affected older batch. Using light and electron microscopy (EM), we made a sequential analysis of histopathological manifestations in nerve tissue and other organs. There were no changes from the day of hatching until 4 dph. Larvae at 4 dph had viral particles in the intramuscular spaces underlying the skin, but the nerve cells of the brain were normal. The first signs of necrosis of the brain cells were observed at 6 dph. EM observations revealed characteristic membrane-bound viral particles measuring 30 nm in the cytoplasm of nerve cells of the brain, spinal cord and retina. Histological samples of fry examined when group mortalities reached 20 to 35% revealed highly vacuolated brains, empty nerve cell cytoplasm and viral particles in the intercellular spaces. Viral particles occurred extensively in the intramuscular spaces and the epidermal layers. These observations were corroborated by positive immunostaining of the virus-rich intramuscular spaces. EM studies also revealed progressive necrotic changes in the cells harboring the virus. Results emphasize the need to maintain hygiene in the hatchery environment and to develop strategies for prevention of disease spread among cohabiting seabass and other susceptible fish larvae. Intramuscular localization of the nodavirus in both preclinical healthy-looking and post-clinical moribund larvae suggests that virus neutralization strategies during larval development could be effective in controlling VNN-associated mortalities.     

Parasite fauna of seabass (Lates calcarifer) under mariculture conditions in Lampung Bay, Indonesia

Fish parasites have been repeatedly reported to be a major threat to the developing industry of finfish mariculture in Indonesia, due to severe parasite and disease outbreaks. The aim of this study was to identify the metazoan parasite fauna and trichodinid ciliates that infect Lates calcarifer in a representative mariculture farm in Indonesia. Examined were 105 L. calcarifer (seabass) for the metazoan parasite fauna and trichodinid ciliates. Thirty‐five specimens each from the net cages of the National Sea Farming Development Centre (Balai Budidaya Laut, BBL) in Lampung Bay, South Sumatra, Indonesia were investigated in three consecutive seasons (two dry and one rainy season from 2002 to 2003). Nineteen parasite species were identified; all fish specimens were infected with two to 10 parasite species, demonstrating a species‐rich parasite fauna. Protozoans (1 species), myxozoans (1), digeneans (3), monogeneans (5), cestodes (3), nematodes (5) and acanthocephalans (1) were found, including 11 new host records in cultured L. calcarifer from Indonesia. Larval and adult parasite stages were isolated, demonstrating that this fish species, although kept inside the net cages, still functions as an intermediate and final host for marine fish parasites. During all seasons, the six detected monoxenous (single host life cycle) parasite species showed a higher prevalence than the 13 heteroxenous (multiple hosts) species. Most abundant were the fish pathogenic monogeneans Pseudorhabdosynochus epinepheli, Pseudorhabdosynochus lantauensis, Benedenia epinepheli and Neobenedenia melleni with a high prevalence. Most heteroxenous parasites (Digenea, Cestoda, Nematoda and Acanthocephala) occurred with a low prevalence below 26%, caused by the specific culture conditions. Diversity of the heteroxenous parasites was higher in the dry seasons than in the rainy season. Though some seasonality could be observed for the fish pathogenic monogeneans, severe disease outbreaks of these ectoparasites cannot be excluded in either the dry or rainy season.     

Methodology in the study of seabass (Lates calcarifer Bloch): nutritional requirements in Singapore

This paper describes the effort of the Marine Aquaculture Section of the Primary Production Department in Singapore and the Institut Francais de Recherche pour l'Exploitation de la Mer in developing adequate facilities for fish nutritional requirement studies in Singapore, and in introducing appropriate nutritional methodology so that results are reliable and accurate. One of the first achievements was the demonstration of homogeneous fish (tropical seabass, Lates calcarifer Bloch) growth response in a newly established tank system before any nutritional experiments were initiated. The application of statistical procedures that take into consideration the power of a test and number of replications required has allowed for more accurate interpretation of results. This can be seen in the results of the first nutritional requirement experiment on the optimal dietary protein level of the seabass at constant energy.     

DNA barcoding reveals a likely second species of Asian sea bass (barramundi) (Lates calcarifer)

DNA barcoding – the sequencing of a c. 650 base pair region of the mitochondrial cytochrome c oxidase I gene – strongly suggests that barramundi ( Lates calcarifer ) from Australia and from Myanmar are different species (Kimura 2 parameter distance of c. 9·5%). Cytochrome b sequence data support this conclusion (distance c. 11·3%). Further examination, both genetic and morphological, of L. calcarifer throughout its range is recommended.     

Cymothoa indica (Crustacea, Isopoda, Cymothoidae) parasitizes the cultured larvae of the Asian seabass Lates calcarifer under laboratory conditions

Parasitic disease in fishes is one of the most important factors limiting aquaculture production and its economic viability. Cymothoa indica, a cymothoid isopod, is reported here for the first time parasitizing cultured larvae of the Asian seabass Lates calcarifer in India. Fourteen-day-old L. calcarifer larvae of mean weight 8.73 +/- 0.03 mg were fed with wild zooplankton in the laboratory. On Day 14 of rearing, larvae were found parasitized by cymothoids. Infected larvae reached a mean (+/- SE) weight of 98.86 +/- 0.30 mg, while uninfected specimens weighed 117 +/- 0.43 mg at the end of the experiment (Day 21). C. indica occurred in the branchial and anterodorsal regions of infected fish, where resultant skin lesions were red, hemorrhagic, without scales and with abundant secreted mucus. The cumulative mortality over the 3 wk period was 16.54 %. These parasites are transferred to the host via the zooplankton used as food; this could easily be overcome, either by filtering wild zooplankton to remove the infectious swimming larvae of C. indica or by using cultured copepods.     

Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer)

Two separate experiments were conducted to determine the dietary requirements of juvenile Asian sea bass Lates calcarifer Bloch for lysine and arginine. Fish (average initial weight: lysine experiment, 13.12 ± 0.12 g; arginine experiment, 2.56 ± 0.13 g) were given amino acid test diets for 12 weeks containing fish meal, zein, squid meal, and crystalline amino acids. Each set of isonitrogenous and isocaloric test diets contained graded levels of L ‐lysine or L ‐arginine. The feeding rate in the lysine experiment was at 4–2.5% of the body weight day^?1, while in the arginine experiment it was at 10–4% of the body weight day^?1. The fish (20 per tank, lysine experiment; 15 per tank, arginine experiment) were reared in 500‐L fibreglass tanks with continuous flowthrough sea water at 27 °C and salinity of 31 ppt in the lysine experiment and at 29 °C and salinity of 29 ppt in the arginine experiment. The experiments were in a completely randomized design with two replicates per treatment. Survival was high in fish given adequate lysine or arginine. Mean percentage weight gains were significantly different in fish fed varying levels of lysine or arginine. Fish fed high levels of L ‐arginine suffered high mortalities. No significant differences were obtained in the feed efficiency ratios (FER, g gain g^?1 feed) of fish fed graded lysine, although the values tended to increase as the dietary lysine level was increased up to the requirement level. In contrast, in the arginine experiment, significant differences in FER of fish among treatments were obtained; the highest FER was observed in fish fed the diet containing an optimum arginine level. On the basis of the growth response, survival, and FER, the lysine and arginine requirements of juvenile Asian sea bass were estimated to be 20.6 g kg^?1 dry diet (4.5% protein) and 18.2 g kg^?1 dry diet (3.8% protein), respectively. These data will be useful in the further refinement of practical diet formulations for the Asian sea bass.     

An inexpensive tag for short-term studies in milkfish (Cbanos chanos Forsskal) and in seabass (Lates calcarifer Bloch)

An opercular tag for marking adult milkfish ( Chanos chanos Forsskal) and seabass ( Lates calcarifer Bloch) is described. High tag retention and relatively low mortality rates were observed in adult fish handled two to ten times during 14-to 60-day tests. The features and advantages of the tag for marking large-sized fish in short-term studies are discussed.

Zusammenfassung

Eine preiswerte Markierung für Kurzzeitstudien des Milchfisches (Chanos chanos Forsskal) und der Centropomidae (Lates calcarifer Bloch)
Eine Kiemendeckel-Markierung für adulte Milchfische (Chanos chanos Forsskal) und Centropomidae (Lates calcacifer Bloch) wird beschrieben. Sie zeichnet sich durch gute Haltbarkeit aus und verursacht relativ geringe Mortalität bei adulten Fischen, die in Versuchen von 14 bis 60 Tage Dauer 2-bis 10mal untersucht wurden. Die Eigenschaften und Vorteile dieser Markierung für große Fische in Kurzzeitstudien werden diskutiert.

Résumé

Un marquage économique pour des études de courte durée du chanidé (Chanos chanos Forsskal) et du centropomidé (Lates calcarifer Bloch)
Un marquage d'opercule pour les chanidés (Chanos chanos Forsskal) et les centropomidés (Lates calcarifer Bloch) adultes est décrit. Une bonne conservation et une mortalité relativement basse ont été observées chez des poissons adultes examinés 2 à 10 fois pendant des expériences d'une durée de 14 à 60 jours. Les caractéristiques et les avantages du marquage de poissons de grande taille pendant des expériences d'une courte durée sont discutés.     

Tryptophan requirement of juvenile Asian sea bass Lates calcarifer

The dietary requirement of tryptophan for juvenile Asian sea bass (Lates calcarifer Bloch) was studied. The juveniles (mean initial weight, 5.30 ± 0.06 g) were given semi‐purified test diets containing fish meal, gelatin, squid meal, and crystalline amino acids, for 12 weeks. Each set of isonitrogenous and isocaloric test diets contained graded levels of tryptophan. Fish (15 per tank) were reared in 250‐L fiberglass tanks provided with continuous flow‐through sea water at 26°C and salinity of 28 p.p.t. Fish were fed twice daily at a feeding rate of 8% of the body weight day^?1 for the first 4 weeks and at 3.5–2.5% of the body weight day^?1 from 5 to 12 weeks. The experiment was in a completely randomized design with two replicates per treatment. Mean percentage weight gains and feed efficiency ratios were significantly different in fish fed varying tryptophan levels. Survival was 100% in all treatments. On the basis of break‐point analysis of the growth response, the dietary tryptophan requirement of juvenile Asian sea bass is 0.41% of the dietary protein. This information will be useful in further refinement of practical feed formulations for the Asian sea bass.