Insights into the functional properties of a natural free amino acid mix: Effect on growth performance, nutrient metabolism, and immune response in a carnivorous fish, Asian seabass (Lates calcarifer).

Dietary supplements containing a functional feed additive have been shown to be beneficial to fish and shellfish aquaculture. However, the functional properties of aquafeed formulations have rarely been reported in fish. This study aimed to investigate the effects of natural free amino acid mix (FAAM) supplementation as a functional solution on the growth performance and nutrient utilization in a carnivorous fish, Asian seabass (Lates calcarifer). Five isonitrogenous and isolipidic diets were prepared with graded supplementation levels of FAAM at 0 % (control group), 0.25 %, 0.50 %, 0.75 %, and 1.0 %, denoted as FAAM0, FAAM0.25, FAAM0.5, FAAM0.75, and FAAM1.0, respectively. The experimental fish were fed different dietary FAAM supplementations to apparent satiation twice daily for eight weeks. Significant improvements were observed in the growth performance of fish among the five groups (P < 0.05). Fish fed with FAAM0.75 displayed significantly increased activities of lysozyme, myeloperoxidase, catalase, and glutathione peroxidase (P < 0.05). The activities of digestive enzymes, including amylase, protease, and lipase, were enhanced by the supplementation of FAAM in the feed (P < 0.05), especially for the groups that contained more than 0.5 % FAAM in the feed. Furthermore, the morphological profile of the intestinal tract, including the mucosal fold height, width, thickness, and goblet cell, increased in fish fed with FAAM at 1.0 % (P < 0.05). Moreover, FAAM supplementation in diets not only modulated the expression of immune-related genes (glutathione peroxidase (GPx), complement (C)3, C4, and C-reactive protein) in the liver but also positively impacted the growth-ralated genes, including growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I), and IGF-II. In addition, the amounts of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids-especially γ-linolenic acid (C18:3 n6) and α-linolenic acid (C18:3n3)-increased in fish fed with diets containing FAAMs (P < 0.05). Interestingly, the diets supplemented with FAAMs also had a positive effect on the economic indices in terms of revenue-to-cost ratios. These findings provide a scientific basis for the application of FAAMs as a functional solution that can be used in feed formulations for Asian seabass.

Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

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.

Immune response and protective efficacy of Streptococcus agalactiae vaccine coated with chitosan oligosaccharide for different immunization strategy in nile tilapia (Oreochromis niloticus).

In the past decade, the outbreak of Streptococcus agalactiae has caused significant economic losses in tilapia farming. Vaccine immunization methods and strategies have gradually evolved from single-mode to multi-mode overall prevention and control strategies. In this study, an inactivated vaccine of S. agalactiae with a chitosan oligosaccharide (COS) adjuvant was constructed using different administration methods: intraperitoneal injection (Ip), immersion combined with intraperitoneal injection (Im + Ip), immersion combined with oral administration (Im + Or), and oral administration (Or). Safety analysis revealed no adverse effects on tilapia, and the vaccine significantly promoted fish growth and development when administered through Im + Or or Or immunization. Following vaccination, innate immunity parameters including SOD, ACP and CAT activities were all significantly enhanced. Additionally, specific serum IgM antibodies reached their highest level at the 6th week post vaccination. Skin and intestinal mucus IgT antibodies reached peaked at the 6th and 7th week post vaccination, respectively. The relative peak expression values for IL-8, IL-12, MHC-I, MHC-II, IgM, IgT, CD4, CD8, TNFα, IFNγ from Im + Ip group were significantly higher than those in Ip group, Im + Or group and Or group in most cases (p < 0.05). Importantly, the relative protection survival of Im + Ip group was the highest (78.6%), followed by the Ip group (71.4%), the Or group (64.3%) and the Im + Or group (57.1%). In summary, this study encourages further research on multi-channel immunization strategies of other kinds of vaccines in other aquatic economic animals to improve their disease resistance.

Impacts of astaxanthin-enriched Paracoccus carotinifaciens on growth, immune responses, and reproduction performance of broodstock Nile tilapia during winter season.

The growth, immune response, and reproductive performance of broodstock of Nile tilapia (Oreochromis niloticus) under winter stress conditions were investigated the effects of supplementary diets with astaxanthin-enriched Paracoccus carotinifaciens. Throughout an eight-week period in the winter season, male and female tilapia were fed with diets containing different levels of P. carotinifaciens dietary supplementation: 0 g/kg (T1; control), 5 g/kg (T2), 10 g/kg (T3), and 20 g/kg (T4). Subsequently, a four-week mating system was implemented during the winter stress period. The results revealed that there were no significant differences observed in growth, hematological indices, and blood chemical profiles among all treatment groups for both male and female tilapia. However, a significant increase in cholesterol content was noted in both male and female tilapia fed with the T4 diet (p<0.05). The total carotenoid content in the muscle was evaluated, and significantly higher values were found in both male and female tilapia that fed T4 supplementation (p<0.05). Moreover, immunological parameters such as myeloperoxidase and antioxidant parameters in the liver including superoxide dismutase activity and catalase enzyme activity showed significant increases in tilapia fed with the T4 diet. The impact of P. carotinifaciens supplementation on broodstock tilapia indicated a significant increase in spermatozoa concentration in males and increased egg production in females after consumption of the T4 diet (p<0.05). Thus, this study highlighted that the presence of astaxanthin-enriched P. carotinifaciens in the diet of broodstock Nile tilapia can lead to the accumulation of carotenoids in their muscle tissue, improvement in antioxidant status, enhancement of immune function, and potential enhancement of reproductive capabilities, even under overwintering conditions.

Immune response and protective efficacy of mannosylated polyethylenimine (PEI) as an antigen delivery vector, administered with a Streptococcus agalactiae DNA vaccine in Nile tilapia (Oreochromis niloticus).

This study examined the effectiveness of a DNA vaccine for S. agalactiae that was delivered by mannose-based polyethyleneimine (Man-PEI). The results showed that Man-PEI/pcDNA-Sip stimulated a higher serum antibody titer compared to control or other vaccine groups (p < 0.05). Additionally, it induced higher expression of immune-related genes, and increased activities of superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Furthermore, the Man-PEI/pcDNA-Sip group showed an improved relative percent survival (RPS) of 85.71%. These results demonstrate the potential value of Man-PEI as a vaccine delivery vehicle, and suggest that it can be effective in boosting the immune protective rate induced by pcDNA-Sip vaccines.

Effect of dietary gamma aminobutyric acid (GABA) modulated the growth performance, immune and antioxidant capacity, digestive enzymes, intestinal histology and gene expression of Nile tilapia (Oreochromisniloticus).

Dietary GABA plays an important role in a variety of physiological functions in animals, but this has been rarely reported in fish. This study investigated the effects of dietary supplementation with GABA on growth, serum antioxidant indicators, digestive enzyme activities, intestinal morphology, and the gene expression of Nile tilapia. Diets containing three different GABA concentrations, 0 (control), 200 mg/kg (GABA200), and 500 mg/kg (GABA500), were fed to Nile tilapia (6.97 ± 0.34 g) for 56 days. The present study found that increasing dietary GABA content could increase the fish growth performance including final body weight, weight gain, specific growth rate, average daily gain, protein efficiency ratio, and feed efficiency compared to the control diet. Interestingly, the feed conversion ratio was improved by dietary GABA supplementation. The antioxidant enzyme activities against ammonia stress of fish fed the GABA diets were significantly higher than the corresponding control group throughout the 96-h ammonia exposure. Moreover, significant increases in digestive enzyme activities including protease, amylase and lipase were found in fish fed the GABA diets. Intestinal morphology analysis revealed increased heights and widths of intestinal villi as well as thickness of the intestinal muscularis in fish fed the GABA diets compared to the control diet. The supplementation of diets with GABA significantly increased the expression level of immune- and growth-related genes. The above results indicate that dietary GABA can modulate the growth, improve their immune response and antioxidant status, gut health and morphology and gene expression of Nile tilapia. Therefore, GABA is a promising feed additive for Nile tilapia aquaculture.

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Assessment of Bacillus subtilis fermented Caulerpa microphysa byproduct as feed additive on the growth performance, immune regulation and disease resistance of white shrimp (Litopenaeus vannamei).

In this study, the immunomodulatory and antioxidant activity of fermented Caulerpa microphysa byproduct (FCMB) by Bacillus subtilis was evaluated, and its potential as a feed additive for white shrimp (Litopenaeus vannamei) was explored. In vitro experiments showed that the FCMB supernatant contained polysaccharides, polyphenols and flavonoids, and exhibited antioxidant properties as assessed by various antioxidant assays. Additionally, the FCMB supernatant was found to increase the production rate of reactive oxygen species and the activity of phenoloxidase in hemocytes in vitro. Furthermore, the results of the in vivo feeding trial showed that dietary 5 g kg-1 FCMB significantly improved the weight gain and specific growth rate of white shrimp after 56 days of feeding. Although there were no significant differences in total hemocyte count, phagocytosis, superoxide anion production rate, and phenoloxidase activity among the experimental groups, upregulation of immune-related genes was observed, particularly in the hepatopancreas and hemocytes of shrimps fed with 5 g or 50 g FCMB per kg feed, respectively. In the pathogen challenge assay, white shrimp fed with 5 % FCMB exhibited a higher survival rate compared to the control group following Vibrio parahaemolyticus challenge. Therefore, it is concluded that the fermented byproduct of C. microphysa, FCMB, holds potential as a feed additive for enhancing the growth performance and disease resistance against V. parahaemolyticus in white shrimp.

The immune response of fairy shrimp Streptocephalus sirindhornae against bacterial black disease by de novo transcriptome analysis.

To enhance genomic resources and to understand the molecular immune mechanisms underlying the response of fairy shrimp (Streptocephalus sirindhornae) to pathogens, we first performed a comparative gene transcription analysis from Aeromonas hydrophila-immunized shrimp and from a control group through RNA sequencing. Meanwhile, the differentially expressed genes (DEGs) were investigated, and a total of 46,958,894 clean reads were obtained and then assembled into 73,297 unigenes with an average length of 993 bp and an N50 of 1,458 bp. Unigenes were annotated by comparison with the NR/NT/KO/SwissProt/PFAM/GO and KOG databases, and 28,198 unigenes (38.47%) were annotated in at least one database. After a bacterial challenge, 143 and 287 genes were identified as markedly up- or downregulated, respectively, and 345 were associated with 142 pathways, including the classic immune-related apoptosis, toll-like receptor and MAPK signaling pathways. Moreover, ten differently expressed immune-related genes were confirmed by using quantitative real-time PCR. This study characterized a gene expression pattern for normal and Aeromonas hydrophila-immunized S. sirindhornae for the first time and shed new light on its molecular mechanisms, thus enabling the future efforts of disease control programs for this valuable aquaculture species.

Impacts of Aegle marmelos fruit extract as a medicinal herb on growth performance, antioxidant and immune responses, digestive enzymes, and disease resistance against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

An experiment was conducted to investigate the effects of Aegle marmelos fruit (AMF) extract on the growth performance, biochemical parameters, immune response, antioxidative capacity, and digestive enzyme activity of Nile tilapia (Oreochromis niloticus). Fish were fed a diet supplemented with AMF at concentrations of 0 (AMF0; control), 5 (AMF5), 10 (AMF10), 15 (AMF15), or 20 (AMF20) g/kg for 8 weeks. The results show that the final body weight, weight gain, specific growth rate, average daily gain, and feed conversion ratio were significantly higher in fish fed AMF15 and AMF20 compared to those fed the control diet (P < 0.05). Moreover, significant increases in antioxidant enzyme activities and non-specific immune responses were observed in groups fed AMF15 and AMF20. Interestingly, the level of cholesterol decreased with increasing AMF concentrations in the diet. As dietary AMF levels increased, digestive enzyme activities significantly improved. After the feeding trial, fish were injected intraperitoneally with Streptococcus agalactiae, and the 14-day cumulative mortality was calculated. A high survival rate after challenge with S. agalactiae was observed in all groups that received AMF-supplemented feed. Therefore, the present study suggests that supplementing the diet of Nile tilapia with AMF at a concentration of 20 g/kg could encourage their growth, improve their immunity and antioxidant status, and provide strong protection against S. agalactiae.

Optimum dietary sources and levels of selenium improve growth, antioxidant status, and disease resistance: re-evaluation in a farmed fish species, Nile tilapia (Oreochromis niloticus).

The objective of this study was to investigate the effects of sources and levels of selenium (Se) on juvenile Nile tilapia (Oreochromis niloticus). A completely randomized design involving a 2 × 3 factorial arrangement of treatments was used in this study. Organic Se (L-selenomethionine; SeMet) and inorganic Se (sodium selenite; Na2SeO3) were each added to the basal diet at 1, 3 and 5 mg Se/kg. The basal diet, without Se supplementation, was used as a control. There was a total of 7 experimental diets, and each was fed in triplicate to groups of fish with an initial average body weight of 13.5 g for 8 weeks. The results showed that growth performance was significantly affected by dietary sources and levels of Se (P < 0.05). Fish fed diets supplemented with SeMet of 1.0 mg Se/kg resulted in higher growth performance compared to basal diet (P < 0.05), but Na2SeO3 supplementation did not affect growth. The feed conversion ratio was significantly decreased as dietary sources of SeMet (P < 0.05). Interestingly, fish fed diets supplemented with both forms of Se had lower cholesterol levels than those fed the basal diet (P < 0.05). Moreover, dietary sources and levels of Se significantly increased (P < 0.05) the antioxidant enzyme activities such as lysozyme, catalase, myeloperoxidase, superoxide dismutase and glutathione peroxidase. Dietary sources and levels of Se significantly could enhance the Nile tilapia resistance against Streptococcusagalactiae infection (P < 0.05). Overall, it can be concluded that the inclusion level of 1.0 mg Se/kg of organic Se in the diet is suggested to be the optimal level for the growth performance and immune response of Nile tilapia. Therefore, dietary supplementation with Se is useful for improving growth, antioxidant status, immune response, and disease resistance.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

A multi-epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia.

Edwardsiella ictaluri infects several fish species and protection of the all the susceptible fish hosts from the pathogen using a monovalent vaccine is impossible because the species is composed of host-based genotypes that are genetic, serological and antigenic heterogenous. Here, immunoinformatic approach was employed to design a cross-immunogenic chimeric EiCh protein containing multi-epitopes. The chimeric EiCh protein is composed of 11 B-cell epitopes and 7 major histocompatibility complex class II epitopes identified from E. ictaluri immunogenic proteins previously reported. The 49.32 kDa recombinant EiCh protein was expressed in vitro in Escherichia coli BL-21 (DE3) after which inclusion bodies were successfully solubilized and refolded. Ab initio protein modelling revealed secondary and tertiary structures. Secondary structure was confirmed by circular dichroism spectroscopy. Antigenicity of the chimeric EiCh protein was exhibited by strong reactivity with serum from striped catfish and Nile tilapia experimentally infected with E. ictaluri. Furthermore, immunogenicity of the chimeric EiCh protein was investigated in vivo in Nile tilapia juveniles and it was found that the protein could strongly induce production of specific antibodies conferring agglutination activity and partially protected Nile tilapia juveniles with a relative survival percentage (RPS) of 42%. This study explored immunoinformatics as reverse vaccinology approach in vaccine design for aquaculture to manage E. ictaluri infections.

First comprehensive proteome analysis of lysine crotonylation in Streptococcus agalactiae, a pathogen causing meningoencephalitis in teleosts.

BACKGROUD: Streptococcus agalactiae is a common colonizer of the rectovaginal tract and lead to infectious diseases of neonatal and non-pregnant adults, which also causes infectious disease in fish and a zoonotic risk as well. Lysine crotonylation (Kcr) is a kind of histone post-translational modifications discovered in 2011. In yeast and mammals, Kcr function as potential enhancers and promote gene expression. However, lysine crotonylation in S. agalactiae has not been studied yet. METHODS: In this study, the crotonylation profiling of fish pathogen, S. agalactiae was investigated by combining affinity enrichment with LC MS/MS. The Kcr modification of several selected proteins were further validated by Western blotting. RESULTS: In the present study, we conducted the proteome-wide profiling of Kcr in S. agalactiae and identified 241 Kcr sites from 675 screened proteins for the first time. Bioinformatics analysis showed that 164 sequences were matched to a total of six definitively conserved motifs, and many of them were significantly enriched in metabolic processes, cellular process, and single-organism processes. Moreover, four crotonylation modified proteins were predicted as virulence factors or to being part of the quorum sensing system PTMs on bacteria. The data are available via ProteomeXchange with identifier PXD026445. CONCLUSIONS: These data provide a promising starting point for further functional research of crotonylation in bacterial virulence in S. agalactiae.

Insights into the Evolution of the Suppressors of Cytokine Signaling (SOCS) Gene Family in Vertebrates.

The SOCS family are key negative regulators of cytokine and growth factor signaling. Typically, 8-17 SOCS genes are present in vertebrate species with eight known in mammals, classified as type I (SOCS4-7) and type II (CISH and SOCS1-3) SOCS. It was believed that the type II SOCS were expanded through the two rounds of whole genome duplication (1R and 2R WGDs) from a single CISH/SOCS1-3 precursor. Previously, 12 genes were identified in rainbow trout but here we report 15 additional loci are present, and confirm 26 of the genes are expressed, giving rainbow trout the largest SOCS gene repertoire identified to date. The discovery of the additional SOCS genes in trout has led to a novel model of SOCS family evolution, whereby the vertebrate SOCS gene family was derived from CISH/SOCS2, SOCS1/SOCS3, SOCS4/5, SOCS6, and SOCS7 ancestors likely present before the two WGD events. It is also apparent that teleost SOCS2b, SOCS4, and SOCS5b molecules are not true orthologues of mammalian SOCS2, SOCS4, and SOCS5, respectively. The rate of SOCS gene structural changes increased from 2R vertebrates, to 4R rainbow trout, and the genes with structural changes show large differences and low correlation coefficient of expression levels relative to their paralogues, suggesting a role of structural changes in expression and functional diversification. This study has important impacts in the functional prediction and understanding of the SOCS gene family in different vertebrates, and provides a framework for determining how many SOCS genes could be expected in a particular vertebrate species/lineage.

Dissecting the immune pathways stimulated following injection vaccination of rainbow trout (Oncorhynchus mykiss) against enteric redmouth disease (ERM).

Enteric redmouth disease (ERM or yersiniosis) is one of the most important diseases of salmonids and leads to significant economic losses. It is caused by the Gram-negative bacterium Yersinia ruckeri but can be controlled by bacterin vaccination. The first commercial ERM vaccine was licenced in 1976 and is one of the most significant and successful health practices within the aquaculture industry. Although ERM vaccination provides complete protection, knowledge of the host immune response to the vaccine and the molecular mechanisms that underpin the protection elicited is limited. In this report, we analysed the expression in spleen and gills of a large set of genes encoding for cytokines, acute phase proteins (APPs) and antimicrobial peptides (AMPs) in response to ERM vaccination in rainbow trout, Oncorhynchus mykiss. Many immune genes in teleost fish are known to have multiple paralogues that can show differential responses to ERM vaccination, highlighting the necessity to determine whether all of the genes present react in a similar manner. ERM vaccination immediately activated a balanced inflammatory response with correlated expression of both pro- and anti-inflammatory cytokines (eg IL-1ß1-2, TNF-α1-3, IL-6, IL-8 and IL-10A etc.) in the spleen. The increase of pro-inflammatory cytokines may explain the systemic upregulation of APPs (eg serum amyloid A protein and serum amyloid protein P) and AMPs (eg cathelicidins and hepcidin) seen in both spleen and gills. We also observed an upregulation of all the α-chains but only one ß-chain (p40B2) of the IL-12 family cytokines, that suggests specific IL-12 and IL-23 isoforms with distinct functions might be produced in the spleen of vaccinated fish. Notably the expression of Th1 cytokines (IFN-γ1-2) and a Th17 cytokine (IL-17A/F1a) was also up-regulated and correlated with enhanced expression of the IL-12 family α-chains, and the majority of pro- and anti-inflammatory cytokines, APPs and AMPs. These expression profiles may suggest that ERM vaccination activates host innate immunity and expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune response. A late induction of Th2 cytokines (IL-4/13B1-2) was also observed, that may have a homeostatic role and/or involvement in antibody production. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may provide essential information and function as biomarkers in future vaccine development in aquaculture.

Characterisation of arginase paralogues in salmonids and their modulation by immune stimulation/ infection.

In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.

Pathogenicity of Streptococcus iniae causing mass mortalities of yellow catfish (Tachysurus fulvidraco) and its induced host immune response.

The outbreak of mass mortality occurred in Tachysurus fulvidraco farm in Hubei province of China. The pathogenic strain of Streptococcus iniae (termed 2022SI08) was isolated and identified from diseased T. fulvidraco, based on morphological, physiological, and biochemical characteristics, as well as 16S rRNA gene sequence and phylogenetic analysis. Further, the whole genome of isolate S. iniae was sequenced and predicted to contain one single circular chromosome of 1,776,777 bp with a GC content of 37.14%. The genomic sequence analysis showed that 2022SI08 was positive for 204 virulent and 127 antibiotic resistant genes. The experimental challenge demonstrated the high pathogenicity of the retrieved isolate of S. iniae, with a median lethal dosage (LD50) 9.53 × 105 CFU/g. Histopathological examination indicated that the 2022SI08 strain could induce extensive tissue cell degeneration, necrosis, hemorrhage, and inflammation in the skin, gill, fin, spleen, liver, kidney, intestine, eye, and brain. Moreover, the innate immune enzyme activities in serum such as acid phosphatase and alkaline phosphatase were increased significantly at 24 and 48 h post infection (hpi) and then decreased at 168 hpi. The transcriptional profile of immune associated gene in T. fulvidraco following bacterial infection was detected at each point of time, and the results revealed clear transcriptional activation of those genes, which proving their reacting and regulatory role during the response of the host against S. iniae infection. The results revealed that S. iniae was an etiological agent in the mass mortalities of T. fulvidraco and this research will be conducive for increasing our understanding on pathogenesis and host defensive system in S. iniae invasion.

Nutritional profile of the land snail Cyclophorus saturnus, a rich-in-nutrients food item from Thailand.

Cyclophorus saturnus is an edible land snail traditionally harvested for human food, yet little is known about its nutritional value, especially in Thailand. This study aimed to investigate its nutritional potential as an alternative food resource. In the present study, proximate composition, essential mineral content, amino acid, and lipid profiles of the meat were evaluated. Proximate analysis showed that C. saturnus contained 80.04% moisture, 11.88% protein, 6.04% carbohydrate, and 0.93% fat, with 80.01 kcal/100 g fresh matter. For minerals, calcium was the most abundant element in the meat. Its protein contained glutamic and aspartic as the major amino acids, while it was not a good source of tryptophan and methionine but was considered a very rich source of other essential amino acids (amino acid scores greater than 100). Its lipid fraction showed a higher proportion of mono and polyunsaturated fatty acids (MUFA and PUFA, 67.69%) and a lower proportion of saturated fatty acids (SFA) (32.31%). The PUFA/SFA ratio (1.56), hypocholesterolemic/hypercholesterolemic ratio (HH; 5.58), atherogenicity index (AI; 0.48), and thrombogenicity index (TI; 0.20) are considered nutritionally healthy for humans. Overall, this study demonstrates the nutritional potential of C. saturnus to serve as a nutritious part of the human diet and as an alternative ingredient in food systems; therefore, its production and consumption should be more extensively promoted.