The Complete Mitochondrial Genome Sequence and Characterization of Single-Nucleotide Polymorphisms in the Control Region of the Asian Seabass (Lates calcarifer)

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.     

Use of INFIC data base for amino acid requirement studies

The International Network of Feed Information Centers (INFIC) database contains amino acid profiles of over 15000 feedstuffs prepared by various techniques by the countries of the world. Preliminary filtering of appropriate feedstuffs which might be used in fish feed formulations has generated a data base of over 100 commonly used ingredients. Many of these have digestibility coefficients for protein and some have amino acid digestibility coefficients listed. The data base is faulted by the variety of techniques used to measure amino acid profiles, and by many ingredients which have not had digestibility coefficients for the protein component determined. This data base is now being re-examined by the National Research Council to generate more exact values for the indispensable and dispensable amino acids present in feedstuffs. It is anticipated that the base will be further improved by cooperating laboratories specifically interested in amino acid profiles of various fish feed formulas. Regardless of these deficiencies, the existing INFIC data base and the scheduled improved one should provide fish feed formulae, chemical amino acid profiles, for least cost and for research diet functions. These data will form a first proximation for satisfying dietary requirements for various fish species used in nutritional requirement studies.     

Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata)

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the ‘MedFish’ SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4–99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.     

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.     

Dietary protein source affects lipid metabolism in the European seabass (Dicentrarchus labrax)

The study was undertaken to evaluate the effects of dietary protein sources on lipogenesis and fat deposition in a marine teleost, the European seabass (Dicentrarchus labrax). Four isonitrogenous (crude protein (CP, Nx6.25), 44% DM) and isoenergetic (22-23 kJ/g DM) diets were formulated to contain one of the following as the major protein source: fish meal (FM), one of two soy protein concentrates (SPC) and corn gluten meal (CGM). Apparent digestibility coefficients of the diets and raw ingredients, as well as soluble nitrogen (ammonia and urea) and phosphorus excretion were measured. Growth rates of seabass fed plant protein-based diets were significantly lower than those fed fish meal based diet. The protein utilisation was strongly correlated to the dietary essential amino acids index. Measurements of N excretion (ammonia and urea nitrogen) confirmed these data. Daily fat gain at the whole body level ranged between 1.1 to 1.7 g/kg BW, with the highest values being recorded in fish fed the fish meal based diet. Levels of plasma triglycerides and cholesterol were lower in fish fed soy protein diets than in those fed the diet solely based on fish meal. Soy protein rich diets decreased the activities of selected hepatic lipogenic enzymes (glucose 6-phosphate dehydrogenase, malic enzyme, ATP-citrate lysase, acetylcoenzyme A carboxylase and fatty acid synthetase). Highest lipogenic enzyme activities where found in fish fed the fish meal diet, except for fatty acid synthetase which was increased in seabass fed the corn-gluten meal based diets. Overall data suggest that dietary protein sources affects fat deposition and the lipogenic potential in European seabass.     

Complement consumption by Photobacterium damselae subsp. piscicida in seabream, red porgy and seabass normal and immune serum. Effect of the capsule on the bactericidal effect

A virulent strain of Photobacterium damselae subsp. piscicida (Pdp) was grown without (C form) or with (C+ form) glucose supplementation, the latter to enhance capsule formation. Both forms were resistant to killing by normal serum of seabream, red porgy and seabass. However, the C form was killed by immune serum of all three fish species while the C+ form was killed only by seabream and red porgy sera and to a lesser extent than the C form. Both C and C+ forms consumed complement in normal serum and this consumption was enhanced by precoating the bacteria in specific fish antibody. Complement consumption was greatest in seabass serum, especially with antibody-coated C+ form yet in this case the bacteria were not killed. The killing of the C form in immune serum of all three fish species was completely inhibited by EGTA/Mg(2+), indicating that the mechanism of complement activation leading to killing of the bacteria was by the classical pathway. The results suggest that immune serum killing by the classical complement pathway may provide some degree of protection against pasteurellosis, but enhanced expression of the capsule by Pdp in vivo may restrict complement-mediated killing, especially in immunised seabass.     

Multicentric infiltrative lipoma in a farmed Mediterranean seabass Dicentrarchus labrax: a pathological and biochemical case study

A tumour diagnosed as multicentric infiltrative lipoma affecting a single farmed seabass is described. The fish had 3 masses on the lateral side of its back, deforming the skin surface. The masses showed a tendency to invade the underlying musculature. Histologically, the neoplasm consisted of differentiated adipocytes. Biochemically, the neoplastic tissue showed lower values of monounsaturated fatty acids and higher values of polyunsaturated fatty acids compared with adjacent normal muscular tissue, particularly of the n3 series, such as eicosapentanoic (C20:5n3) and docosahexanoic (C22:6n3) acids. Data obtained suggest a metabolic disturbance in the lipid component of the muscular tissue metabolic pathway, which could be the starting point to promote lipoma formation. This is the first report of lipoma in Mediterranean seabass.     

A note on the use of plasma urea level to validate the arginine requirement assessed by growth data in seabass (Dicentrarchus labrax)

Duplicate groups of 60 European seabass (mean weight 2.1 g) were fed for 9 weeks with 7 isonitrogenous (46% crude protein, CP) and isolipidic (12% ether extract) semipurified diets obtained by adding graded levels of L-arginine to a basal diet. By analysis of dose-weight gain relationship, the dietary arginine requirement was found to be 3.9 g/100 g CP (N'6.25). A similar value (3.8 g/100 g CP) was obtained when the 5 h (peak) post-prandial plasma urea response relative to that of fish fed the basal diet were regressed against the dietary arginine level, suggesting the possible use of this parameter to validate the arginine requirement estimated from growth data.     

Welfare status of cultured seabass (Dicentrarchus labrax L.) and seabream (Sparus aurata L.) assessed by blood parameters and tissue characteristics

Seabass (SBS) and seabream (SBM) juveniles were reared with the goal of obtaining three different final densities (SINT = 40 kg m^?3; INT = 20 kg m^?3; SEM = 0.2 kg m^?3) to ascertain the effects thereof on the welfare of the fish. Significant blood metabolites and hepatic glycogen were determined every 3 months and at harvest. Trials lasted 18 months in seabass and 17 months in seabream. At the end of experiment the main biometric productive parameters and quality of body composition were also recorded. Regarding intensively reared seabass (SINT‐SBS, INT‐SBS), the plasma triglycerids, total cholesterol and transaminases (AST, ALT) were always significantly higher than in semi‐intensively maintained fish (SEM‐SBS). At the final sampling in the SINT‐SBS batch, the total protein and glucose were also markedly increased. Conversely, at harvest the liver glycogen content decreased in SINT‐SBS (34 ± 8 mg g^?1 liver) with respect to INT‐SBS (57 ± 12 mg g^?1 liver) and SEM‐SBS (63 ± 11 mg g^?1 liver). No differences among groups were observed for creatine kinase (CK) and lactate dehydrogenase (LDH). With regard to seabream, SINT‐SBM and INT‐SBM constantly showed plasma triglycerids and total cholesterol as being significantly higher when compared to SEM‐SBM. In the final two blood samplings, SINT‐SBM exhibited the most elevated values for LDH. At harvest, AST, ALT, total protein and glucose markedly increased in SINT‐SBM, whereas liver glycogen content was reduced (22.5 ± 9 mg g^?1 liver), more than in INT‐SBM (70 ± 16 mg g^?1 liver) and SEM‐SBM (75 ± 20 mg g^?1 liver). In both seabass and seabream, body composition was very similar in the different stocking densities, except for total cholesterol. Total n‐3 polyunsaturated fatty acid (PUFA) in seabream was significantly different from fish of the semi‐intensive groups; however, nutritional values and fatty acid profiles were equally good. The intermediate final density of seabass and seabream at 20 kg m^?3 seemed to give the best results in terms of their well being when compared to fish reared at 40 kg m^?3. The absence of differences in blood metabolites and hepatic glycogen levels between the intensive batch and the semi‐intensive groups until harvest was a reference to the positive status of the fish. A density of 20 kg m^?3 can be considered acceptable for farm strategy planning for raising healthy on‐growing seabass and seabream juveniles.     

Morphological differences between wild and farmed Mediterranean fish

Gilthead seabream (Sparus aurata L.) and European seabass (Dicentrarchus labrax L.) are important commercial marine fish species both for aquaculture and fisheries in the Mediterranean. It is known that farmed individuals escape from farm facilities, but the extent of escape events is not easy to report and estimate because of the difficulty to distinguish between wild and farmed individuals. In this study, significant differences provided through morphometry evidence that the cranial and body regions of seabream and seabass are different regarding their farm or wild origin at different scales. Morphological variations have been shown to be a valuable tool for describing changes in shape features. Therefore, the biomass contribution of escapees to local habitats could be determined by identifying escaped individuals from fisheries landings as a first step to assess the potential negative effects of fish farm escapees on the environment, and their influence on wild stocks and local fisheries.     

Lipidomics focusing on serum polar lipids reveals species dependent stress resistance of fish under tropical storm

The serum polar lipid metabolic changes for two common cage-cultured fishes, yellow coraker Pseudosciaena crocea and Japanese seabass Lateolabrax japonicus, after tropical storm attack have been studied by ultra-performance liquid chromatography—quadrupole-time of flight mass spectrometry (UPLC-qTOF-MS). The full scan mass spectrometry combined with principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated that yellow croaker underwent significant chemico-physiological changes during the recovery process, whereas Japanese seabass did not show such noticeable time-dependent consistent metabolites change patterns. Further identification of the metabolite biomarkers showed the increase of phosphatidylcholine with high unsaturated fatty acid and lysophospholipids, and the decrease of phosphatidylcholine with saturated fatty acids and plasmologens, which indicated the need of energy supplement and successive stressful inflammation. The increase of taurocholic acid and decrease of cortol could be regarded as the physiological alleviation measure during the recovery period. This is the first metabolomic study to tackle the fish physiological response for the complex environmental changes, and demonstrated that lipidomics is an effective analytical tool for predicting the stress resistance of fish to ultra uncontrolled environmental stress.     

Immunocytochemical localization of piscidin in mast cells of infected seabass gill

Annual losses of ～5–10% of the juvenile stock of European seabass, Dicentrarchus labrax (L.) in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans. Immunocytochemical, light and ultrastructural studies were carried out on seabass naturally parasitized with this monogenean. The site of the worm's attachment was marked by the common presence of haemorrhages and white mucoid exudate. In histological sections, infected gills showed hyperplasia, as well as proliferation of mucous cells and rodlet cells. Disruption and fusion of the secondary lamellae were common in all infected seabass, with several specimens also showing marked inflammation and erosion of the primary and secondary lamellar epithelium. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of mast cells that were positive. Mast cells were both within and outside the blood vessels of the primary and secondary lamellae, and often made intimate contact with vascular endothelial cells. Mast cells were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Our data provide evidence showing the presence of piscidin 3 in the cytoplasmic granules of an important group of fish inflammatory cells, the mast cells resident in seabass gill tissue. There was no significant difference in the number of HAGR-positive mast cells between infected and uninfected fish (ANOVA, p > 0.05). However, mast cells in parasitized gills usually showed much stronger immunostaining intensity compared to those in unparasitized gills. These data are the first to document a response of piscidins or any other antimicrobial peptide of fish to parasite infection and suggest that mast cells may play a role in certain inflammatory responses without a detectable increase in their numbers.     

QTL Mapping for Resistance to Iridovirus in Asian Seabass Using Genotyping-by-Sequencing

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81 cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5 days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.     

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.     

Pterocladia (Rhodophyta) and Ulva (Chlorophyta) as feed supplements for European seabass, Dicentrarchus labrax L., fry

The aim of this study was to evaluate the suitability of two marine macroalgae, Ulva lactuca (Chlorophyta) and Pterocladia capillacea (Rhodophyta), meals as a supplement to enhance the nutritional value of formulated feeds for European seabass Dicentrarchus labrax fry. Seven isoproteic (50 % crude protein), isocaloric (500 Kcal/100 g gross energy) diets containing four levels (0 or control, 5, 10, and 15 %) of either Ulva meal (UM) or Pterocladia meal (PM) were tested. Each diet was fed to triplicate groups of D. labrax fry (initial body weight, 0.23?±?0.02 g for Ulva- and 0.14?±?0.01 g for Pterocladia-fed fish), to apparent visual satiety for 8 weeks. The results indicated that feeding seabass at 5 % UM or PM level (U₅ and P₅ diets) produced the best growth, feed utilization, nutrient retention, and survival rates among all the dietary groups. Feeding fish with a 5 % PM-added diet has also improved stress response after a 5-min air exposure test, prior to the termination of the feeding trial. These findings suggest that both Pterocladia and Ulva meals could be potentially used as an additional feed component (at 5 %) for enhancement of seabass D. labrax fry performance, nutrients composition, and stress resistance especially when subjected to transportation from hatchery to weaning ponds/tanks.