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.

Identification of mapk genes, and their expression profiles in response to low salinity stress, in cobia (Rachycentron canadum).

Mitogen-activated protein kinases (MAPKs) are a class of protein kinases that regulate various physiological processes, and play a crucial role in maintaining the osmotic equilibrium of fish. The objective of this study was to identify and characterize the mapk family genes in cobia (Rachycentron canadum) and examine their expression profiles under different low salinity stress regimes (acute: from 30‰ to 10‰ in 1 h, sub-chronic: from 30‰ to 10‰ over 4 d). A total of 12 cobia mapk genes (Rcmapks) were identified and cloned, including six erk subfamily genes (Rcmapk1/3/4/6/7/15), three jnk subfamily genes (Rcmapk8/9/10) and three p38 mapk subfamily genes (Rcmapk 11/13/14). Domain analysis indicated that the RcMAPKs possessed the typical domains including S_TKc and PKc_like domain. Phylogenetic analysis revealed that the Rcmapks were most closely related to those of the turbot (Scophthalmus maximus). The tissue distribution of mapk genes in adult cobia and the expression patterns of Rcmapks under different low salinity stress regimes were investigated using quantitative real-time PCR (qRT-PCR). The results revealed that Rcmapk3/9/10/11/13/14 exhibited a relatively broad expression distribution across 14 different tissues. For all these genes the highest expression level was in the brain, except for Rcmapk14 (highly expressed in the stomach, gill, and skin). The genes Rcmapk1/6/15 showed significantly higher expression in the testis. Under acute low salinity stress, expression of Rcmapk1/3/6/7/9/11/13/14 was significantly altered in the gill, intestine, and trunk kidney, however, the aforementioned genes exhibited very different expression patterns among the three tissues. In the gill, most of the genes from the erk (Rcmapk3/6/7) and p38 mapk subfamily (Rcmapk11/13/14) were significantly up-regulated at almost all the time points (P < 0.05); Similarly, the expression of Rcmapk3/9/11/13/14 genes were significantly increased in the trunk kidney; while in the intestine, most of the altered genes (Rcmapk6/7/9/11/13/14) were significantly down-regulated at 1 h. Following the sub-chronic low salinity stress, expression of Rcmapk1/3/6/7/9/11/13/14 genes were significantly altered in all three tissues. These findings provide important reference data for elucidating the roles of cobia mapk family genes in response to low salinity stress.

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.

A novel chemerin receptor 1 (Chemerin1) takes part in the immune response of cobia (Rachycentron canadum).

Chemerin receptor 1 (Chemerin1) plays a critical role in innate and adaptive immune systems. In this study, a cobia (Rachycentron canadum) Chemerin1 was identified, and its molecular characterization and expression patterns were analyzed. Multiple sequence alignment revealed that the RcChemerin1 possessed a typical dynein regulatory complex (DRC) motif. There was also a potential N-glycosylation site in the extracellular regions of the N-terminus and intracellular loops (ICL) 1 region. Phylogenetic analysis demonstrated that the RcChemerin1 was clustered together with homologous proteins from other fish species. RcChemerin1 was constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, which suggests that the RcChemerin1 plays different roles in un-stimulated tissues. RcChemerin1 expression showed up-regulation in the head kidney after Vibrio harveyi challenge. Up-regulation in the head kidney and spleen was also observed after polyinosinic-polycytidylic acid (poly I: C) challenge, which suggests that RcChemerin1 may play vital roles during bacterial and viral infection. The differential responses of immune organs to bacteria and poly I: C imply the differences in defense mechanisms against viruses and bacteria.

First report on genetic characterization, cell-surface properties and pathogenicity of Lactococcus garvieae, emerging pathogen isolated from cage-cultured cobia (Rachycentron canadum).

The diseased cage-cultured cobia (Rachycentron canadum) displayed clinical signs, haemorrhagic eyes, dorsal darkness and gross pathological lesions, enlargement of spleen and liver. Haemorrhages were found in brain, heart and liver with cumulative mortality rates ranging from 20% to 50%. Extensive congestion in the heart, liver, spleen, kidney and brain was observed histopathologically. Epicarditis and meningitis were also revealed in diseased cobia. All isolates recovered from the organs (liver, spleen, head kidney, posterior kidney, brain and muscle) of cobia were found to be gram-positive, non-motile, ovoid cocci, short-chain-forming (diplococci) and α-haemolytic. The API 32 strep system together with the polymerase chain reaction assay for species-specific primers (pLG1 and pLG2) and the internal transcribed spacer (ITS) region (G1 and L1 primers) confirmed all four selected isolates as Lactococcus garvieae. Partial 16S rDNA nucleotide sequence (~1,100 bp) of one representative L. garvieae isolate AOD109191 (GenBank accession number, MW328528.1) shared 99.9% identities with the 16S rDNA nucleotide sequence of L. garvieae (GenBank accession numbers: MT604790.1). Transmission electron microscopy (TEM) evaluation of one representative L. garvieae isolate (AOD109191) and the results of multiplex PCR did not reveal the presence of the capsular gene cluster (CGC), thus categorizing the isolate as the KG+ phenotype. Capsule staining and TEM observations confirmed the presence of a hyaluronic acid-like capsule, a possible virulence factor in KG+ phenotype L. garvieae isolates. The pathogenic potential of the representative isolate (AOD109191) was assessed through intraperitoneal injection challenges in cobia. The gross lesions and histopathological changes found in experimentally infected cobia were similar to those seen in naturally infected fish. This is the first report that confirms L. garvieae-induced 'warm water lactococcsis' can cause outbreaks of diseases in cage-cultured cobia.

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.

Identification and expression analysis of cobia (Rachycentron canadum) liver-related miRNAs under hypoxia stress.

At present, due to the influence of global warming, seasonal change, diurnal variation, and eutrophication of the water body, hypoxia has become one of the major factors limiting the stable development of cobia (Rachycentron canadum) culture. In this study, the miRNAs involved in hypoxia stress were screened, and the target genes of miRNAs were annotated and analyzed. The results showed that a total of 184 conservative microRNA (miRNA) and 121 newly predicted miRNA were obtained by sequencing the liver of control (C) and hypoxic (dissolved oxygen, DO (2.64 ± 0.25) mg/L; 3 h) (S) groups. The pathways involved in energy metabolism included starch and sucrose metabolism (ko00500), glycosaminoglycan degradation (ko00531), and galactose metabolism (ko00052). The results indicate that the body maintains physiological activities by regulating some important pathways at the transcriptional level under hypoxia stress, such as the conversion of aerobic metabolism and anaerobic metabolism, the reduction of energy consumption, and the promotion of red blood cell proliferation to maintain the homeostasis of the body.

Hepatic antioxidant activity, immunomodulation, and pro-anti-inflammatory cytokines manipulation of κ-carrageenan (κ-CGN) in cobia, Rachycentron canadum against Lactococcus garvieae.

The effects of dietary k-Carrageenan (k-CGN) at 10, 20, and 30 g kg-1 on growth rate, hemato-biochemical indices, innate-adaptive parameters and modification of pro- and/or anti-inflammatory cytokines and chemokines pathway in cobia, Rachycentron canadum against Lactococcus garvieae is reported. The weight gain (WG) increased substantially (P < 0.05) in all k-CGN treated groups; the specific growth rate (SGR) was significant in healthy uninfected normal (HuN) and L. garvieae challenged (LaC) groups fed with 20 g kg-1k-CGN diet on 45 and 60 days. The white blood cell (WBC) counts, total protein (TP) level, total anti-oxidant (T-AOC), catalase (CAT), and glutathione (GSH) activities increased significantly when fed with 20 g and 30 g kg-1k-CG diets on 45th and 60th day. The immunological parameters such as phagocytic (PC) index and the activity of phagocytic (PC), respiratory burst (RB), superoxide dismutase (SOD), alternate complement pathway (ACH50), and lysozyme (LZM) were significantly enhanced with all k-CG diets in 45 and 60 days of treatment. No cumulative mortality (CM) in HuN group fed by control or any k-CGN diets. CM was 5% in LaC group fed with 20 g kg-1k-CGN diet whereas in LaC groups fed with 10 g and 30 g kg-1k-CGN diets the CM was 10%. The interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNFα) pro-inflammatory cytokines mRNA transcripts were one-fold high (P < 0.05) in both HuN and LaC group fed all k-CGN enriched diets on 45 and 60 days. Similarly, IL-18 and TLR2 mRNA was one-fold high expression in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 or 60 days. Interferon gamma (IFNγ) and interferon regulatory factor 3/7 (IRF3/IRF7) mRNA transcripts did not change with any diet. IL-6, IL-10, and IL-11 mRNA were one-fold high expressions in both groups fed the 20 g and 30 g kg-1k-CGN enriched diets on 45 and 60 days. However, the expression of CC1, CC3, and CCR9 pro-inflammatory chemokines mRNA did not vary with any control or k-CGN enriched diets. The results indicate that diet enriched with k-CGN at 20 g kg-1 significantly influences the growth, antioxidant and innate-adaptive immune performance, and pro-anti-inflammatory cytokines and chemokines regulation in cobia against L. garvieae.

Cloning of Mn-SOD gene and its mRNA expression difference and antioxidant enzyme activities under hypoxia stress of cobia Rachycentron canadum.

BACKGROUND: Environmental hypoxia affects the survival and development of organisms. It is also an important environmental factor that leads to oxidative damage. Hypoxia is a condition in which tissues are deprived of oxygen; reoxygenation is the phenomenon in which hypoxic tissues are exposed to oxygen. Hypoxia-reoxygenation is vital in pathogenesis, where the production of reactive oxygen species and antioxidant disparity significantly contribute to disease progression, and it is one of the most common physiological stressors in the aquaculture industry. METHODS AND RESULTS: In this study, the full length of complementary DNA (cDNA) of the manganese superoxide dismutase (Mn-SOD) gene of healthy cobia Rachycentron canadum was analysed using rapid amplification of cDNA ends. The real-time quantitative Polymerase Chain Reaction was used to measure the expression levels of Mn-SOD mRNAs in various tissues (heart, muscle, brain, liver, kidney, gill, intestine, and spleen). The 2-ΔΔCT method was used to performed the expression analysis. The experimental data were analysed using SPSS ver. 19.0 ( https://spss.software.informer.com/19.0/ ). P < 0.05 and P < 0.01 were set as significant differences. The values were articulated as mean ± standard deviation. The Mn-SOD gene cDNA sequence was 1209 bp long, including a 684 bp open reading frame, 42 bp 5'UTR and 483 bp 3'UTR, encoding 227 amino acids. Under hypoxia-reoxygen stress, the expression of Mn-SOD in brain tissue was significantly lower than in the control group after 8 h of reoxygenation and higher than the control group after 24 h. Hypoxia and subsequent reoxygenation triggered a disturbance in antioxidant homeostasis, displayed in the modification of GPx expression/activity in the liver: GPx was improved. CONCLUSIONS: These results provide valuable information on the role of Mn-SOD regulation in oxidative stress caused by hypoxia.

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.

RNA-seq analysis reveals divergent adaptive response to hyper- and hypo-salinity in cobia, Rachycentron canadum.

Salinity is an important abiotic stress that affects metabolic and physiological activities, breed, development, and growth of marine fish. Studies have shown that cobia (Rachycentron canadum), a euryhaline marine teleost fish, possesses the ability of rapid and effective hyper/hypo iono- and osmoregulation. However, genomic studies on this species are lacking and it has not been studied at the transcriptome level to identify the genes responsible for salinity regulation, which affects the understanding of the fundamental mechanism underlying adaptation to fluctuations in salinity. To describe the molecular response of cobia to different salinity levels, we used RNA-seq analysis to identify genes and biological processes involved in response to salinity changes. In the present study, 395,080,114 clean reads were generated and then assembled into 65,318 unigenes with an N50 size of 2758 bp. There were 20,671 significantly differentially expressed genes (DEGs) including 8805 genes adapted to hypo-salinity and 11,866 genes adapted to hyper-salinity. These DEGs were highly represented in steroid biosynthesis, unsaturated fatty acid metabolism, glutathione metabolism, energy metabolism, osmoregulation, and immune response. The candidate genes identified in cobia provide valuable information for studying the molecular mechanism of salinity adaptation in marine fish. Furthermore, the transcriptomic sequencing data acts not only as an important resource for the identification of novel genes but also for further investigations regarding cobia biology.

Effects of dietary lipid-to-carbohydrate ratio on growth and carbohydrate metabolism in juvenile cobia (Rachycentron canadum).

This experiment was conducted to investigate the effects of dietary lipid-to-carbohydrate ratio on growth and carbohydrate metabolism in juvenile cobia (Rachycentron canadum). Six isonitrogenous diets were prepared to vary in lipid-to-carbohydrate ratio (g/g) as follows: D1, 2.26; D2, 1.31; D3, 0.78; D4, 0.47; D5, 0.34; and D6, 0.23. Cobias were fed to satiety for 8 weeks. The weight gain and protein efficiency ratio in D1 group were significantly lower than those in other groups (P < 0.05), accompanied by a lower level of feed conversion ratio (P < 0.05). Protein retention efficiency in D4 and D6 and whole body protein in D4 and D5 were significantly higher than those in D1 group (P < 0.05). Survival rate in D4 group was the highest among all groups and was significantly higher than that in D1, D2 and D5 (P < 0.05). In terms of serum triglyceride, D1 and D2 were significantly higher than D6 (P < 0.05). Hepatosomatic index in D3 and D4 was significantly lower than that in D1 (P < 0.05). Fructose-1,6-diphosphatase in D4 was significantly higher than that in D1 and D3 (P < 0.05). Phosphofructokinase in D3 and D4 and malic enzyme in D4 and D5 were significantly higher than those in other groups (P < 0.05). Results indicate that cobia utilizes carbohydrates as energy source more efficiently than it utilizes lipids. The optimal lipid-to-carbohydrate ratio in juvenile cobia diets is 0.47.

Molecular characterization of cobia (Rachycentron canadum) CD4 homologues revealed the first evidence of soluble CD4 in fish.

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia.

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.

Efeito do processamento de defumação na qualidade de bijupirá (Rachycentron canadum): atributos bacteriológicos, químicos e sensoriais / Effect of smoking-method on the keeping quality of cobia (Rachycentron canadum): bacteriological, chemical and sensory attributes

O objetivo do presente trabalho foi desenvolver a carne de bijupirá defumada, assim como avaliar o rendimento, a qualidade bacteriológica, a composição centesimal e a aceitabilidade do produto. Análises microbiológicas de pesquisa de Salmonella sp. e contagens de Staphylococcus aureus, coliformes totais e Escherichia coli foram realizadas. Foi determinada a composição centesimal e realizado o teste de aceitação do produto. O rendimento médio da carne após a salga foi de 83,41%, com base no peso do charuto. A análise microbiológica da carne defumada apresentou-se positiva em apenas uma amostra para Staphylococcus aureus e negativa para as demais bactérias. O produto possui boa qualidade nutricional e alcançou 97% de aceitação para o aspecto global, atingindo média de 6,26 (± 0,99). A carne de bijupirá defumada obteve condições higiênico-sanitárias satisfatórias, boa qualidade nutricional, além de alto índice de aceitação sensorial, destacando-se os atributos textura e sabor. Evidenciou-se, assim, que esse tipo de processo pode ser realizado em escala artesanal ou industrial.(AU)

The objective of the study was to develop the smoked cobia meat and to evaluate the yield, the bacteriological quality, the centesimal composition, and the acceptability of the product. Microbiological analysis of Salmonella sp. and Staphylococcus aureus, total coliforms and Escherichia coli counts were performed. The centesimal composition was determined and the acceptance test was performed. The average yield of the product after salting was 83.41% based on the fish roll weight. The microbiological analysis of the smoked meat showed positive in only one sample for Staphylococcus aureus and negative for the other bacteria. The product has a good nutritional quality and was accepted obtaining 97% for the overall aspect, average of 6.26 (± 0.99). The smoked cobia meat obtained satisfactory hygienic-sanitary conditions, good nutritional quality, besides a high index of sensorial acceptance emphasizing the texture and flavor attributes. Smoked process can be easily carried out on an artisanal or industrial scale.(AU)

Nutritional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase at the enzymatic and molecular levels in cobia Rachycentron canadum.

Despite being a carnivorous fish species, cobia (Rachycentron canadum) can utilize high levels of dietary carbohydrate (up to 360 g kg-1). By contrast, rainbow trout (also carnivorous) cannot, due to the absence of molecular induction of glycolytic enzyme and inhibition of gluconeogenic enzyme gene expressions such as pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK). We hypothesized that this phenomenon is species-specific and will not be observed in cobia. Our results show that, at the molecular level, the mRNA abundance of the important glycolytic (PK) and gluconeogenic (PEPCK) enzymes in cobia liver are regulated by dietary carbohydrate-to-lipid (CHO:L) ratios and nutritional status (fed, unfed, and refed). Significantly upregulated hepatic PK and depressed PEPCK gene expressions were observed when the fish were fed with an increasing CHO/L-ratio diet or were refed. However, in contrast to gene expression, there was no significant effect of dietary CHO/L ratios on PK enzyme activity. The decrease in PEPCK activity was significantly found between low CHO/L ratio and high CHO/L ratio diets, whereas the moderate CHO/L ratio group showed intermediate values. But PEPCK activity appeared to be independent of nutritional status. These results suggest that nutritional regulation is obvious, at least at the molecular level, in the key hepatic enzymes (PK and PEPCK) of the glucose metabolism pathway, in response to different dietary CHO/L ratios and to the transition from being starved to fed. Determining whether other key enzymes involved in hepatic glucose metabolism contribute to glucose tolerance in cobia is necessary for further investigation of this phenomenon at the enzymatic and molecular levels.

Molecular cloning of IL-6, IL-10, IL-11, IFN-ɤ and modulation of pro- and anti-inflammatory cytokines in cobia (Rachycentron canadum) after Photobacterium damselae subsp. piscicida infection.

Photobacterium damselae subsp. piscicida (P. damselae subsp. piscicida) is the agent of Photobacteriosis, a serious fish disease that produces an acute infection and high mortality in farmed cobia. It has been proved that regulation of pro- and anti-inflammatory cytokines play a central role in initiation of proper inflammatory responses against bacterial infection. Here we have analyzed the expression of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8 and IFN-ɤ) and anti-inflammatory cytokines (IL-10 and IL-11) in spleen and head kidney during acute P. damselae subsp. piscicida infection of cobia. Our data revealed that cytokines tested showed distinct patterns of expression. While TNF-α and IL-8 showed a decay pattern of expression, IL-1ß response was quite late. Moreover, P. damselae subsp. piscicida infection induced the simultaneous expressions of pro-inflammatory (IL-6, IFN-ɤ) and anti-inflammatory (IL-10, IL-11) cytokines. Together these results indicate the innate immunity of cobia is actively suppressed by P. damselae subsp. piscicida.

Effect of increased rearing temperature on digestive function in cobia early juvenile.

The present study is focused to elucidate the main characteristics of the digestive function of this carnivorous fast-growing fish living at high temperatures. With this aim, we have examined the effects of an increased temperature from 30 to 34 °C on the daily pattern of gastrointestinal pH, enzymatic proteolytic digestive activity and the feed transit time in early juveniles of cobia (Rachycentron canadum), a species living in tropical and subtropical waters with an increasing aquaculture production. Fish were fed two meals a day. Gastric luminal pH was permanently acidic (mean pH values: 2.76-4.74) while the intestinal pH increased from neutral/slightly acidic to slightly alkaline when the digesta was present, with an increasing alkalinity from proximal to distal intestine (mean pH values: 6.05 to 7.69). The temperature did not affect the gastric pH but a slightly higher acidity was induced in the intestine at 34 °C. Pepsin activity showed a daily rhythm at 30 °C with maximum in the middle of the light period, while at 34 °C some hourly changes coinciding with feed adding without a clear daily trend during the 24-h period were observed. The trypsin activity exhibited a daily rhythm at both temperatures with an increase after morning feeding to reach a maximum several hours later. Average pepsin activity during the daily cycle was slightly higher at 34 °C (6.1 and 7.3 U mg-1 BW at 30 and 34 °C respectively), but values were significantly different only at 8 and 24 h after the morning meal. Similarly, the trypsin activity was significantly affected by the temperature only at 8 and 16 h after the morning meal, but daily activity averages were similar (1.20 and 1.29 U g-1 BW at 30 and 34 °C respectively). The partial transit rates of the first meal in the stomach for each period inter-samplings were higher during the first 4-h period and decreased progressively along the rest of the 24-h cycle at both temperatures, but no significant differences were detected at 30 °C. In addition, the transit was notably faster at 34 °C particularly during the first 8 h after feeding, with rates between 100 and 65% of total volume displaced (intake or released) during each 4-h period. In the intestine the transit rate was relatively constant and similar at both temperatures during 12 h after feeding. Then the rates remained very low during the following 12 h. Residence time of the first meal was longer at 30 than at 34 °C, particularly in the stomach (12 h:02 min vs 4 h:54 min respectively). In the intestine the difference was not so large (8 h:18 min vs 6 h:24 min respectively). In a parallel study under same conditions, cobia reared at 30 °C grew faster and showed a more favorable feed conversion ratio than those at elevated temperature (34 °C). The present results indicate that at 34 °C, a subtle increase of proteolytic activity cannot compensate for the faster gut transit rate. Therefore, 30 °C is more appropriate temperature for the early on-growing of cobia because at higher temperatures the digestion efficiency decrease being one of the causes for a lower growth.