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.     

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.     

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 microg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection.     

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.     

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.     

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.     

Osteological development of the feeding apparatus in early stage larvae of the seabass,Lates calcarifer

The osteological development of the feeding apparatus was examined in early stage larvae of laboratory-reared seabass,Lates calcarifer. At initial mouth openings 40 hours after hatching, the larvae were equipped with the fundamental elements forming the oral cavity, such as the trabecular roof, the lower branchial and hyoid arches forming the floor, the quadrate and symplectic-hyomandibular cartilages making up the sides, and the maxilla and Meckel's cartilage bordering the jaws. The cleithrum appeared almost simultaneously. The mechanics of creating a negative pressure in the oral cavity, which results in a “sucking” mode of feeding, were elucidated from these elements. During a period from 50–60 to 100–110 hours after initial mouth opening (HAMO), new elements such as the premaxilla and jaw teeth appeared, and the ossification of existing elements started. The new elements apparently enabled the larvae to “grasp” food organisms, in addition to the already existing and increasing sucking ability, from 100–110 HAMO.     

Effect of feeding frequency as a rearing system on biological performance,survival, body chemical composition and economic efficiency of Asian seabass Lates calcarifer (Bloch, 1790) reared under controlled environmental conditions

A 70-day rearing trial was done to determine the optimal frequency of feeding on growth performance (GP), feed conversion rate (FCR), cannibalism, survival rate (SR), body chemical composition and economic efficiency of the Asian sea bass. This study tested four different treatments of feeding frequencies (FF), once (T1), twice (T2), three times (T3), and four times (T4) per day. An average initial weight of Asian sea bass fry was 0.2 g (SD = ±0.12) were stocked 10 individuals per m³ (9.14 m × 1.82 m × 1.22 m, L × W × H; water depth 0.61 m) with two replicates per treatment (4 × 2 = 8). Fry were fed a mixture of larval commercial feed and shrimp with a pellet diet containing (46% CP). Initially, the feeding rate of 8% biomass per day was further adjusted according to fish biomass on a weekly basis. Results showed that, the FF significantly affected (p < 0.05) on growth indictors and survival rate (SR). Specifically fry fed three times a day (T3) had the best FBW, FL, SGR, ADWG and FCR followed by T4 and T2 while fry fed one time a day was the lowest in these parameters. Also, VSI, HSI and CF (k) significantly differed among the treatments. The fish whole body content of protein, moisture and ash did not significantly (p < 0.05) be affected by feeding frequency, but lipid content differed and both T3, T4 were the highest. It could be concluded that, increasing FF up to three times a day had a positive effect on weight gain, survival rate and feed utilization of Lates calcarifer. The second degree polynomial regression indicates that fed three times a day is optimum for best growth performance and survival for Asian sea bass.     

Variable growth rates of the tropical estuarine fish barramundi Lates calcarifer (Bloch) under different freshwater flow conditions

Relationships between freshwater flows and growth rates of the opportunistic predatory finfish barramundi Lates calcarifer in a dry tropical estuary were examined using data from a long-term tag-recapture programme. Lagged effects were not investigated. After accounting for length at release, time at liberty and seasonal variation ( e.g. winter, spring, summer and autumn), growth rates were significantly and positively related to fresh water flowing to the estuary. Effects were present at relatively low levels of freshwater flow ( i.e. 2·15 m³ s⁻¹, the 5th percentile of the mean flow rate experienced by fish in the study during time at liberty). The analysis, although correlative, provides quantitative evidence to support the hypothesis that freshwater flows are important in driving the productivity of estuaries and can improve growth of species high in the trophic chain.     

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.     

Structure and development of free neuromasts in barramundi, Lates calcarifer (Block)

This study was conducted to clarify the development of free neuromasts with growth of the barramundi, Lates calcarifer. A pair of free neuromasts was observed behind the unpigmented eyes in newly hatched eleutheroembryos with a mean total length of 1.93 mm, and two-hour-old eleuthero-embryos could respond to an approaching pipette. At 2 days after hatching, the egg yolk sac was mostly consumed, the eyes were pigmented, and the larvae commenced feeding on rotifers. Free neuromasts increased in number with growth and commenced developing into canal neuromasts in barramundi 15 days old with a mean total length of 8.07 mm. The average length of the major axis of the trunk free neuromasts attained approximately 12.9-15.5 microm, and the number of sensory cells was 15.4-17.5 at 15-20 days old. Developed cupulae of free neuromasts were observed in 1-day-old eleutheroembryos. The direction of maximum sensitivity of free neuromasts, determined from the polarity of the sensory cells, coincided with the minor axis of the lozenge-shaped outline of the apical surface of the free neuromasts. The polarity of trunk neuromasts was usually oriented along the antero-posterior axis of the fish body, but a few had a dorso-ventral direction. On the head, free neuromasts were oriented on lines tangential to concentric circles around the eye.     

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.