Exploring the cobia (Rachycentron canadum) genome: unveiling putative male heterogametic regions and identification of sex-specific markers.

BACKGROUND: Cobia (Rachycentron canadum) is the only member of the Rachycentridae family and exhibits considerable sexual dimorphism in growth rate. Sex determination in teleosts has been a long-standing basic biological question, and the molecular mechanisms of sex determination/differentiation in cobia are completely unknown. RESULTS: Here, we reported 2 high-quality, chromosome-level annotated male and female cobia genomes with assembly sizes of 586.51 Mb (contig/scaffold N50: 86.0 kb/24.3 Mb) and 583.88 Mb (79.9 kb/22.5 Mb), respectively. Synteny inference among perciform genomes revealed that cobia and the remora Echeneis naucrates were sister groups. Further, whole-genome resequencing of 31 males and 60 females, genome-wide association study, and sequencing depth analysis identified 3 short male-specific regions within a 10.7-kb continuous genomic region on male chromosome 18, which hinted at an undifferentiated sex chromosome system with a putative XX/XY mode of sex determination in cobia. Importantly, the only 2 genes within/between the male-specific regions, epoxide hydrolase 1 (ephx1, renamed cephx1y) and transcription factor 24 (tcf24, renamed ctcf24y), showed testis-specific/biased gene expression, whereas their counterparts cephx1x and ctf24x, located in female chromosome 18, were similarly expressed in both sexes. In addition, male-specific PCR targeting the cephx1y gene revealed that this genomic feature is conserved in cobia populations from Panama, Brazil, Australia, and Japan. CONCLUSION: The first comprehensive genomic survey presented here is a valuable resource for future studies on cobia population structure and dynamics, conservation, and evolutionary history. Furthermore, it establishes evidence of putative male heterogametic regions with 2 genes playing a potential role in the sex determination of the species, and it provides further support for the rapid evolution of sex-determining mechanisms in teleost fish.

Establishment of a cobia (Rachycentron canadum) gill cell line: A valuable tool for immune response studies.

Cobia (Rachycentron canadum), a commercially important marine fish, has been used to develop a novel gill cell line, designated CG, for the first time. The CG cell line was cultured in Leibovitz's-15 medium with 5% fetal bovine serum (FBS) and successfully sub-cultured more than 110 passages. It underwent verification through sequencing of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Optimal growth rate was achieved when the CG cell line was cultured in a medium supplemented with 5% FBS, 1% Penicillin-Streptomycin (P/S), and 5 parts per thousand (ppt) of coral sea salt water, maintained at a temperature of 27 °C. The addition of 5 ppt of salt in the growth medium suggests that this cell line could be a viable in vitro tool for marine ecosystem toxicological studies or for culturing marine parasitic microorganisms. The CG cell line was also successfully transfected using the pTurbo-GFP plasmids, showing an 18% efficiency, with observable GFP expression. Furthermore, the cell line has been effectively cryopreserved. Gene expression analysis indicated that the CG cell line exhibits responsive regulation of immune gene expression when exposured to various stimulants, highlighting its potential as an in vitro platform for immune response studies. This makes it suitable for exploring dynamic immune signaling pathways and host-pathogen interactions, thereby offering valuable insights for therapeutic development.

Genome-wide characterization of microsatellites in cobia Rachycentron canadum (Linnaeus, 1766): Survey and analysis of their abundance and diversity.

The cobia Rachycentron canadum, mainly distributed in the warm waters of tropical and subtropical regions around the world, remains a fish of considerable economic importance. Detailed diversity and the number of microsatellite sequences in the cobia genome are still unintelligible. The primary aim of this work was to identify and quantify the miscellaneous SSR sequences in the cobia genome. More than 280,000 sequences were sequenced and screened using next-generation sequencing technology and microsatellite identification. Perfect mononucleotide repeats, dinucleotide microsatellites, and trinucleotide microsatellites contain (A)10 /(T)10 , (AC)6 /(TG)6 , and (AAT)5-32 as the largest number of motifs in each type of microsatellite, respectively. The tetranucleotide and pentanucleotide microsatellites (TTM and PTM) consist of the largest number of motifs of both (ATCT)5-32 and (TCAT)5-31 in TTMs, and (CTCTC)5-9 in PTMs, whereas the hexanucleotide microsatellites are rarely observed in the cobia genome. All c. 38000 sequences of composite microsatellites are extremely diverse, including compound (11.71%), interrupted compound (71.77%), complex (0.45%), and interrupted complex (16.07%). In this study, we developed a convenient and useful recording system for writing down and categorizing diverse composite microsatellite types. This system will provide great support for exploring repeat origins, evolutionary mechanisms, and the application of polymorphic microsatellites.

Feed Regime Slightly Modifies the Bacterial but Not the Fungal Communities in the Intestinal Mucosal Microbiota of Cobia Fish (Rachycentron canadum).

The bacterial community of the intestinal microbiota influences many host functions, and similar effects have been recently reported for the fungal community (mycobiota). Cobia is a tropical fish that has been studied for its potential in marine aquaculture. However, the study of its bacterial community has been underreported and the mycobiota has not been investigated. We analyzed the gut bacterial and fungal profile present in the intestinal mucosa of reared adult cobias fed two diets (frozen fish pieces (FFPs) and formulated feed (FF)) for 4 months by sequencing the 16S rRNA (V3-V4) and internal transcribed spacer-2 (ITS2) regions using Illumina NovaSeq 6000. No significant differences in the alpha diversity of the bacterial community were observed, which was dominated by the phyla Proteobacteria (~96%) and Firmicutes (~1%). Cobia fed FF showed higher abundance of 10 genera, mainly UCG-002 (Family Oscillospiraceae) and Faecalibacterium, compared to cobia fed FFPs, which showed higher abundance of 7 genera, mainly Methylobacterium-Methylorubrum and Cutibacterium. The inferred bacterial functions were related to metabolism, environmental information processing and cellular processes; and no differences were found between diets. In mycobiota, no differences were observed in the diversity and composition of cobia fed the two diets. The mycobiota was dominated by the phyla Ascomycota (~88%) and Basidiomycota (~11%). This is the first study to describe the gut bacterial and fungal communities in cobia reared under captive conditions and fed on different diets and to identify the genus Ascobulus as a new member of the core fish mycobiota.

Comparative gonad transcriptome analysis in cobia (Rachycentron canadum).

Background: Cobia (Rachycentron canadum) is a species of fish with high commercial potential particularly due to fast growth rates. The evidence of sexual size dimorphism favoring females indicate potential benefits in having a monosex culture. However, the involvement of genetic factors responsible for sexual development and gonadal maintenance that produces phenotypic sex in cobia is largely unknown. Methods: In the present study, we performed transcriptome sequencing of cobia to identify sex-biased significantly differentially expressed genes (DEGs) in testes and ovaries. The reliability of the gonad transcriptome data was validated by qPCR analysis of eight selected significantly differential expressed sex-related candidate genes. Results: This comparative gonad transcriptomic analysis revealed that 7,120 and 4,628 DEGs are up-regulated in testes or ovaries, respectively. Further functional annotation analyses identified 76 important candidate genes involved in sex determination cascades or sex differentiation, including 42 known testis-biased DEGs (dmrt1, amh and sox9 etc.), and 34 known ovary-biased DEGs (foxl2, sox3 and cyp19a etc.). Moreover, eleven significantly enriched pathways functionally related to sex determination and sex differentiation were identified, including Wnt signaling pathway, oocyte meiosis, the TGF-beta signaling pathway and MAPK signaling pathway. Conclusion: This work represents the first comparative gonad transcriptome study in cobia. The putative sex-associated DEGs and pathways provide an important molecular basis for further investigation of cobia's sex determination, gonadal development as well as potential control breeding of monosex female populations for a possible aquaculture setting.

Selection of Autochthonous Yeasts Isolated from the Intestinal Tracts of Cobia Fish (Rachycentron canadum) with Probiotic Potential.

Some yeast strains have been proposed as probiotics to improve the health of cultured fish. Cobia is a tropical benthopelagic fish species with potential for marine aquaculture; however, one of the main limitations to its large-scale production is the high mortality of fish larvae. In this study, we evaluated the probiotic potential of autochthonous yeasts from the intestines of cobia. Thirty-nine yeast isolates were recovered from the intestinal mucosa of 37 adult healthy cobia by culture methods. Yeasts were identified by sequencing of the ITS and D1/D2 regions of the 28S rRNA gene and typed by RAPD-PCR using the M13 primer. Yeast strains with unique RAPD patterns were characterized in terms of their cell biomass production ability; anti-Vibrio, enzymatic, and hemolytic activity; biofilm production; hydrophobicity; autoaggregation; polyamine production; safety; and protection of cobia larvae against saline stress. Candida haemuloni C27 and Debaryomyces hansenii C10 and C28 were selected as potential probiotics. They did not affect the survival of larvae and showed biomass production >1 g L-1, hydrophobicity >41.47%, hemolytic activity γ, and activity in more than 8 hydrolytic enzymes. The results suggest that the selected yeast strains could be considered as potential probiotic candidates and should be evaluated in cobia larvae.

Monitoring of Harmful Algal Bloom (HAB) of Noctiluca scintillans (Macartney) along the Gulf of Mannar, India using in-situ and satellite observations and its impact on wild and maricultured finfishes.

In the Gulf of Mannar, Noctiluca scintillans blooms have been observed three times in September 2019, September and October 2020, and October 2021. It was determined and measured how the bloom period affects ichthyo-diversity. Noctiluca cell density varied slightly from year to year, ranging from1.8433 × 103 cells/L to 1.3824 x 106cells/L. In surface and sea bottom waters, high ammonia levels and low dissolved oxygen levels were noted. During the bloom period a significant increase in chlorophyll concentration was found. The amount of chlorophyll in GOM was extremely high, according to remote sensing photos made using MODIS-Aqua 4 km data. Acute hypoxia caused the death of wild fish near coral reefs and also in fish reared in sea cages. The decay of the bloom resulted in significant ammonia production, a dramatic drop in the amount of dissolved oxygen in the water, and ultimately stress, shock, and mass mortality of fishes.

Microbiome composition and autochthonous probiotics from contrasting probiosis/dysbiosis states in cobia (Rachycentron canadum) fish epitheliocystis.

Microbiome components and bacterial isolates related to healthy and epitheliocystis states in aquaculture cycles of cobia fish were studied. We detected well-defined 16S rRNA amplicon gene sequence variants showing differential abundance in healthy or diseased cycles. Isolation trials were performed, and experimental tests were used to determine probiotic potential of the bacterial strains obtained from water, tissues or live food used in this aquaculture model. The taxonomic affiliation of these strains was cross-compared against microbiome components, finding that some of them had close or identical affiliation to the abundant types found in healthy cycles. Strains belonging to the groups already identified as predominant by culture-independent means were screened as potential probiotics based on desirable activities such as antagonism and antibiosis against marine pathogenic bacteria, quorum quenching, bile acid resistance, antibiotic sensitivity and enzymatic activities for improved nutrient digestion. We have also found that in the tracking of microbiome composition across different developmental stages of cobia, healthy cycles exhibited a consistent high relative abundance of a Mesobacillus sp., while in the diseased cycle the emergence of a Vibrio sp. was observed. Our study suggests that epithelocystis in cobia is associated with a displacement of a symbiotic microbiome community linked to the increase frequency of Vibrio species.

Comparative evaluation of fish larval preservation methods on microbiome profiles to aid in metagenomics research.

Applications of microbiome research through metagenomics promise to generate microbiome manipulation strategies for improved larval survival in aquaculture. However, existing lacunae on the effects of sample preservation methods in metagenome profiles hinder the successful application of this technique. In this context, four preservation methods were scrutinized to identify reliable methods for fish larval microbiome research. The results showed that a total of ten metagenomics metrics, including DNA yield, taxonomic and functional microbiome profiles, and diversity measures, were significantly (P < 0.05) influenced by the preservation method. Activity ranking based on the performance and reproducibility showed that three methods, namely immediate direct freezing, room temperature preservation in absolute ethanol, and preservation at - 20 °C in lysis, storage, and transportation buffer, could be recommended for larval microbiome research. Furthermore, as there was an apparent deviation of the microbiome profiles of ethanol preserved samples at room temperature, the other methods are preferred. Detailed analysis showed that this deviation was due to the bias towards Vibrionales and Rhodobacterales. The microbial taxa responsible for the dissimilarity across different methods were identified. Altogether, the paper sheds light on the preservation protocols of fish larval microbiome research for the first time. The results can help in cross-comparison of future and past larval microbiome studies. Furthermore, this is the first report on the activity ranking of preservation methods based on metagenomics metrics. Apart from methodological perspectives, the paper provides for the first time certain insights into larval microbial profiles of Rachycentron canadum, a potential marine aquaculture species. KEY POINTS: • First report on effects of preservation methods on fish larval microbiome profiles. • First report on activity ranking of preservation methods based on metagenomics metrics. • Storage methods influenced DNA yield, taxonomic and functional microbiome profiles.

Transcriptomic Profiling and Microsatellite Identification in Cobia (Rachycentron canadum), Using High-Throughput RNA Sequencing.

Cobia (Rachycentron canadum) is a marine teleost species with great productive potential worldwide. However, the genomic information currently available for this species in public databases is limited. Such lack of information hinders gene expression assessments that might bring forward novel insights into the physiology, ecology, evolution, and genetics of this potential aquaculture species. In this study, we report the first de novo transcriptome assembly of R. canadum liver, improving the availability of novel gene sequences for this species. Illumina sequencing of liver transcripts generated 1,761,965,794 raw reads, which were filtered into 1,652,319,304 high-quality reads. De novo assembly resulted in 101,789 unigenes and 163,096 isoforms, with an average length of 950.61 and 1,617.34 nt, respectively. Moreover, we found that 126,013 of these transcripts bear potentially coding sequences, and 125,993 of these elements (77.3%) correspond to functionally annotated genes found in six different databases. We also identified 701 putative ncRNA and 35,414 putative lncRNA. Interestingly, homologues for 410 of these putative lncRNAs have already been observed in previous analyses with Danio rerio, Lates calcarifer, Seriola lalandi dorsalis, Seriola dumerili, or Echeneis naucrates. Finally, we identified 7894 microsatellites related to cobia's putative lncRNAs. Thus, the information derived from the transcriptome assembly described herein will likely assist future nutrigenomics and breeding programs involving this important fish farming species.

Enhanced oxygen unloading in two marine percomorph teleosts.

Teleost fishes are diverse and successful, comprising almost half of all extant vertebrate species. It has been suggested that their success as a group is related, in part, to their unique O2 transport system, which includes pH-sensitive hemoglobin, a red blood cell ß-adrenergic Na+/H+ exchanger (RBC ß-NHE) that protects red blood cell pH, and plasma accessible carbonic anhydrase which is absent at the gills but present in some tissues, that short-circuits the ß-NHE to enhance O2 unloading during periods of stress. However, direct support for this has only been examined in a few species of salmonids. Here, we expand the knowledge of this system to two warm-water, highly active marine percomorph fish, cobia (Rachycentron canadum) and mahi-mahi (Coryphaena hippurus). We show evidence for RBC ß-NHE activity in both species, and characterize the Hb-O2 transport system in one of those species, cobia. We found significant RBC swelling following ß-adrenergic stimulation in both species, providing evidence for the presence of a rapid, active RBC ß-NHE in both cobia and mahi-mahi, with a time-course similar to that of salmonids. We generated oxygen equilibrium curves (OECs) for cobia blood and determined the P50, Hill, and Bohr coefficients, and used these data to model the potential for enhanced O2 unloading. We determined that there was potential for up to a 61% increase in O2 unloading associated with RBC ß-NHE short-circuiting, assuming a - 0.2 ∆pHa-v in the blood. Thus, despite phylogenetic and life history differences between cobia and the salmonids, we found few differences between their Hb-O2 transport systems, suggesting conservation of this physiological trait across diverse teleost taxa.

Effects of three medicinal herbs Bidens pilosa, Lonicera japonica, and Cyathula officinalis on growth and non-specific immune responses of cobia (Rachycentron canadum).

This study investigates the effects of three medicinal herbal extracts, namely Bidens pilosa (BPE), Lonicera japonica (LJE), and Cyathula officinalis (COE), on nonspecific immune parameters of cobia (Rachycentron canadum) in vitro and in vivo. During in vitro tests, BPE treatment increased reactive oxygen species (ROS) production in a dose-dependent manner in primary head kidney leukocytes. Similarly, ROS production rates were enhanced by LJE (50 and 100 mg/ml) and COE (100 mg/ml). This suggests that these three herbal extracts possess immunostimulating properties. We then conducted two feeding trials to examine the effects of these three herbal extracts on growth and innate immune parameters of cobia, and sought an optimal dietary supplementation proportion required for activating the non-specific immune responses. In the first trial, we supplemented the diet with 1, 5, or 10% of the individual extracts. After a ten-week feeding trial, no negative impacts on weight gain, feed conversion rate, and survival rate were observed in fish offered experimental diets. Further, ROS production, phagocytic capacity of the head kidney leukocytes, and serum lysozyme activity were enhanced by differing degrees in fish fed the herbal extracts compared to fish in the control group. A similar albumin/globulin ratio was seen between each experimental group and the control group regardless of the type and dose of herbal extract used, indicating these medicinal herbal extracts are safe for cobia. We then performed a 30-day feeding trial with lower extract concentrations (1, 3, and 5% of the diet) to identify dose responses in cobia at various time points so that we could establish a cost-effective manner of administering the three extracts for cobia. All BPE fed fish had higher ROS production compared to the control group, while phagocytosis rate and index were simultaneously raised in only the BPE30 group (3% BPE). Immune parameters such as ROS production, phagocytic rate, and serum lysozyme activity were triggered when fish received 30 g LJE per kg of feed. However, ROS production only increased in the LJE10 group (1% LJE) on day 30 and was not enhanced in the LJE50 group (5% LJE). Additionally, although the phagocytic rate and phagocytic index were induced in the LJE50 group, serum lysozyme activity was not elevated in this group (LJE50) at any time point examined. ROS production was greatly improved in all COE fed groups, but only the COE30 group (3% COE) showed prolonged enhanced phagocytic rate over the 30-day feeding trial.

In the face of climate change and exhaustive exercise: the physiological response of an important recreational fish species.

Cobia (Rachycentron canadum) support recreational fisheries along the US mid- and south-Atlantic states and have been recently subjected to increased fishing effort, primarily during their spawning season in coastal habitats where increasing temperatures and expanding hypoxic zones are occurring due to climate change. We therefore undertook a study to quantify the physiological abilities of cobia to withstand increases in temperature and hypoxia, including their ability to recover from exhaustive exercise. Respirometry was conducted on cobia from Chesapeake Bay to determine aerobic scope, critical oxygen saturation, ventilation volume and the time to recover from exhaustive exercise under temperature and oxygen conditions projected to be more common in inshore areas by the middle and end of this century. Cobia physiologically tolerated predicted mid- and end-of-century temperatures (28-32°C) and oxygen concentrations as low as 1.7-2.4 mg l-1. Our results indicated cobia can withstand environmental fluctuations that occur in coastal habitats and the broad environmental conditions their prey items can tolerate. However, at these high temperatures, some cobia did suffer post-exercise mortality. It appears cobia will be able to withstand near-future climate impacts in coastal habitats like Chesapeake Bay, but as conditions worsen, catch-and-release fishing may result in higher mortality than under present conditions.

α-Enolase as a novel vaccine candidate against Streptococcus dysgalactiae infection in cobia (Rachycentron canadum L.).

Streptococcus dysgalactiae is an important pathogenic bacterium that has caused economic loss for the cobia industry in Taiwan, ROC. This study presents a highly effective subunit vaccine composed of a moonlight protein, α-enolase, for the prevention of S. dysgalactiae infection. First, α-enolase was cloned, transformed, and expressed in E. coli for production of recombinant protein. Then, the protective efficacies of α-enolase recombinant protein were evaluated in combination with either a pro-inflammatory cytokine, TNF-α, or an oil adjuvant, ISA 763 AVG. The results showed that the combination of α-enolase and ISA 763 AVG was highly protective (RPS = 88.89%), while a negative effect was found in the group immunised with α-enolase adjuvanted with TNF-α (RPS = 22.22%). A further study was conducted with double dose of ISA 763 AVG, which led to an increased RPS value of 97.37%. Moreover, immunised cobia exhibited significantly greater lysozyme activity, antibody responses, and expression of certain immune-related genes post-challenge. Altogether, our results demonstrated that a combination of α-enolase recombinant protein with ISA 763 AVG adjuvant is a promising vaccine that can be employed for protection of cobia against S. dysgalactiae infection.

Differential expression of immune-related genes in head kidney and spleen of cobia (Rachycentron canadum) having Streptococcus dysgalactiae infection.

Streptococcus dysgalactiae is a gram-positive bacterium and a harmful aquaculture pathogen. To investigate the immune response against S. dysgalactiae, we performed transcriptome analysis of the head kidney and spleen of cobia (Rachycentron canadum) using RNA-seq. Total RNA was extracted from the head kidney and spleen of cobia, 1 and 2 days after treatment with S. dysgalactiae or control PBS. After RNA purification and cDNA library generation, sequencing was performed using the Illumina HiSeq™ 4000 platform. The filtering and de novo assembling transcripts were annotated using several databases. To identify differentially expressed genes (DEGs) between the S. dysgalactiae and PBS groups, the mapped values of fragments per kilobase of transcripts per million fragments were calculated. After de novo assembly, a total of 106,984 transcripts were detected, with an N50 of 3020 bp. These transcripts were annotated and categorised into a total of 7608 genes based on the KEGG pathway database. DEGs (2-fold difference) were calculated by comparing the S. dysgalactiae and PBS control group gene expression levels at each time point. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were significantly enriched in the immune-related pathways - "cytokine-cytokine receptor interaction", "complement and coagulation cascades", and "hematopoietic cell linage". In this study, immune-related genes responding to S. dysgalactiae were detected, and several immune system pathways were categorized. We identified the IL17C-related pathway for inducing the expression of pro-inflammatory cytokine genes (IL-1ß, IL-6, and IFNγ). Additionally, neutrophil-related genes (CSF3, CD121, and CD114) were induced in the spleen after S. dysgalactiae infection. It was suggested that these pathways contribute to immune responses against S. dysgalactiae infection. The data revealed in this study may offer improved strategies against S. dysgalactiae infection in cobia.

Preparation and Identification of Novel Antihypertensive Peptides from the In Vitro Gastrointestinal Digestion of Marine Cobia Skin Hydrolysates.

This research focuses on cobia skin hydrolysates and their antihypertensive effects via the inhibitory activities of angiotensin I-converting enzyme (ACE). Marine fish Cobia (Rachycentron canadum) skin was hydrolysed for 5 h using Protamex and Protease N to obtain the cobia skin protein hydrolysates PX-5 and PN-5, respectively. The soluble protein and peptide contents of the PX-5 were 612 and 270 mg/g, respectively, and for the PN-5, 531 and 400 mg/g, respectively. The IC50 of PX-5 and PN-5 on ACE was 0.221 and 0.291 mg/mL, respectively. Increasing the IC50 from 0.221 to 0.044 mg/mL by simulated gastrointestinal digestion (PX-5G) reduced the ACE-inhibitory capacity of PX-5. Using gel filtration chromatography, the PX-5G was fractioned into eight fractions. The molecular weight of the fifth fraction from PX-5G was between 630 and 450 Da, and the highest inhibitory efficiency ratio on ACE was 1552.4%/mg/mL. We identified four peptide sequences: Trp-Ala-Ala, Ala-Trp-Trp, Ile-Trp-Trp, and Trp-Leu, with IC50 values for ACE of 118.50, 9.40, 0.51, and 26.80 µM, respectively. At a dose of 600 mg PX-5 powder/kg body weight, in spontaneously hypertensive rats PX-5's antihypertensive effect significantly reduced systolic and diastolic blood pressure by 21.9 and 15.5 mm Hg, respectively, after 4 h of oral gavage.

The role of dietary methionine concentrations on growth, metabolism and N-retention in cobia (Rachycentron canadum) at elevated water temperatures.

This study determined impacts of dietary methionine concentrations at two temperatures on growth, feeding efficiency and N-metabolites in juvenile cobia. Methionine concentrations of the experimental diets were deficient (M9; 9 g/kg), sufficient (M12; 12 g/kg) and surplus (M16, 16 g/kg). Water temperature was normal (30°C) or elevated (34°C). Twenty cobia in triplicate tanks were fed the experimental diets for 6 weeks. Both methionine and temperature affected cobia's growth and feeding efficiency. Cobia fed M9 performed lower than the fish fed M12 and M16 diets. Additionally, cobia reared at 34°C performed poorer than at 30°C, probably due to lower voluntary feed intake in the fish reared at 34°C. Protein efficiency ratio and protein productive value in cobia fed M9 diet were less than M12 or M16 diets. This was confirmed with the improved retentions of indispensable amino acids (AAs). No interactions between methionine and temperature were observed in growth and protein accretion. At 30°C, CF improved, while HSI and VSI declined upon methionine supplementation levels. Of which an interaction between temperature and methionine was present. Plasma, muscle and liver free AA and N-metabolites were affected by methionine and temperature. Furthermore, temperature affected cobia's lipid class composition, resulting in increased phospholipids and cholesterol at 34°C.

Total Bioavailable Organic Selenium in Fishmeal-Based Diet Influences Growth and Physiology of Juvenile Cobia Rachycentron canadum (Linnaeus, 1766).

The study examined the effects of supplemental organic selenium (Se) extracted from selenoyeast on the growth performance, glutathione peroxidase (GPx) activity, biochemical status and liver histochemistry of juvenile cobia Rachycentron canadum. Six experimental diets were prepared supplemented with Se with total concentration of 1.52 (Se-1.52), 1.93 (Se-1.93), 2.29 (Se-2.29), 2.71 (Se-2.71) and 3.14 (Se-3.14) mg/kg of total Se in the diets and a fishmeal-based control diet without Se supplementation containing 1.15 (Se-1.15) mg/kg of Se was used as control. Experimental diets were fed to the fish of six treatment groups in triplicate twice daily for 8 weeks. Juvenile cobia fed dietary Se of 1.93, 2.29 and 2.71 mg/kg showed increased final body weight (FBW), specific growth rate (SGR) and feed intake (FI) than the fish fed the control diet. Se accumulations in the muscle and liver tissue displayed a positive linear relationship with dietary Se levels. Se deficiency was apparent in fish fed the control diet and displayed reduced growth and feed efficiency. Red blood cell (RBC) counts were significantly (P < 0.05) higher in cobia fed dietary Se between 1.52 to 2.71 mg/kg than the fish fed 3.14-mg/kg Se diet. Glutathione peroxidase activity significantly (P < 0.05) declined in the group fed with control diet compared to fish fed Se-supplemental diet. Juvenile cobia fed the highest Se level (Se-3.14 mg/kg) showed toxic effects in the liver, including histopathological lesions in the liver. Based on the results obtained for FBW, SGR, tissue Se retention and haematological parameters, we conclude that optimal dietary Se requirement for juvenile cobia fed commercial diets is 2.32 mg/kg.

Identification of protective protein antigens for vaccination against Streptococcus dysgalactiae in cobia (Rachycentron canadum).

Streptococcus dysgalactiae is considered a causative agent of severe infection and economic loss for the cobia industry in Taiwan. In this study, protective antigens of this pathogenic bacterium were identified and screened in cobia (Rachycentron canadum). Outer surface proteins (OMPs) of this pathogen were extracted using mutanolysin digestion. Immunogenic targets were detected by western blot and then subjected to peptide sequencing using NanoLC-MS/MS. Two surface proteins, namely phosphoenolpyruvate protein phosphotransferase (PtsA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), showed strong reactions with cobia antisera against S. dysgalactiae. Recombinant proteins were produced in Escherichia coli cells and their protective efficacies were investigated in cobia. Fish immunised with recombinant proteins, rPtsA + ISA (ISA 763 AVG) and rGAPDH + ISA, elicited higher levels of specific antibody responses against the recombinant proteins and had high levels of lysozyme activity. Notably, vaccinated fish were protected from lethal challenge with relative percentage of survival (RPS) values for rPtsA + ISA and rGAPDH + ISA groups being 91.67% and 83.33%, while 0% RPS value was found in both ISA injected and control groups. The results presented in the study demonstrate that the GAPDH and PtsA are promising vaccine candidates for preventing S. dysgalactiae disease in cobia.

Isolation and characterization of pathogenic Vibrio alginolyticus from sea cage cultured cobia (Rachycentron canadum (Linnaeus 1766)) in India.

Mass mortalities of cobia, Rachycentron canadum, sub-adults occurred during August 2013 in cage culture in the Gulf of Mannar, Mandapam Tamil Nadu, India. The epizootic of disease was started with typical classical clinical signs followed by acute mortality. Grossly, severe haemorrhage and congestion were observed in the gastric mucosa. The abdomen was distended with peritoneal fluid. The heart revealed haemopericardium and fibrinous pericardium. Histologically, the gastric mucosa showed severe erosion and necrosis. Haemorrhagic pericarditis and an increased size of the melano macrophage centre (MMC) in the tail kidney were other histopathological changes. Vibrio sp. was isolated from the gastric lesions and heart blood swab of moribund fishes and it was found to be virulent to the cobia fingerlings. After the challenge, the same bacterium could be re-isolated from moribund fingerlings. The 16S ribosomal RNA of the isolate was amplified and blast analysis of the sequence confirmed that the pathogen was Vibrio alginolyticus. The confirmation was also correlated with its cultural, biochemical and pathomorphological changes. This is the second report and the first incidence of epizootics with severe pathological lesions in cultured cobia in India. The study throws light on the pathology of vibriosis. By practising cage farm management measures, occurrences of infection may be prevented. SIGNIFICANCE AND IMPACT OF THE STUDY: The epizootics of vibriosis caused serious economic losses to farmers. Natural blooms of the pathogen can be prevented by sea cage management measures such as, changing the inner net of the cages, changing the location of the cages to relatively clean water (about 50 m apart) from the affected site and providing shade over the cages while the water temperature rises. Supplementation of the feed with immunostimulants and mineral mixture may be practised to improve the immune response against infection. Early diagnosis and sea cage management measures may prevent occurrences of the infection.