Effects of selenoprotein extracts from Cardamine hupingshanensis on growth, selenium metabolism, antioxidant capacity, immunity and intestinal health in largemouth bass Micropterus salmoides.

This study aimed to assess the impact of dietary selenoprotein extracts from Cardamine hupingshanensis (SePCH) on the growth, hematological parameters, selenium metabolism, immune responses, antioxidant capacities, inflammatory reactions and intestinal barrier functions in juvenile largemouth bass (Micropterus salmoides). The base diet was supplemented with four different concentrations of SePCH: 0.00, 0.30, 0.60 and 1.20 g/Kg (actual selenium contents: 0.37, 0.59, 0.84 and 1.30 mg/kg). These concentrations were used to formulate four isonitrogenous and isoenergetic diets for juvenile largemouth bass during a 60-day culture period. Adequate dietary SePCH (0.60 and 1.20 g/Kg) significantly increased weight gain and daily growth rate compared to the control groups (0.00 g/Kg). Furthermore, 0.60 and 1.20 g/Kg SePCH significantly enhanced amounts of white blood cells, red blood cells, platelets, lymphocytes and monocytes, and levels of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin in the hemocytes. In addition, 0.60 and 1.20 g/Kg SePCH increased the mRNA expression levels of selenocysteine lyase, selenophosphate synthase 1, 15 kDa selenoprotein, selenoprotein T2, selenoprotein H, selenoprotein P and selenoprotein K in the fish liver and intestine compared to the controls. Adequate SePCH not only significantly elevated the activities of antioxidant enzymes (Total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), the levels of total antioxidant capacity and glutathione, while increased mRNA transcription levels of NF-E2-related factor 2, Cu/Zn-superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase. However, adequate SePCH significantly decreased levels of malondialdehyde and H2O2 and the mRNA expression levels of kelch-like ECH-associated protein 1a and kelch-like ECH-associated protein 1b in the fish liver and intestine compared to the controls. Meanwhile, adequate SePCH markedly enhanced the levels of immune factors (alkaline phosphatase, acid phosphatase, lysozyme, complement component 3, complement component 4 and immunoglobulin M) and innate immune-related genes (lysozyme, hepcidin, liver-expressed antimicrobial peptide 2, complement component 3 and complement component 4) in the fish liver and intestine compared to the controls. Adequate SePCH reduced the levels of pro-inflammatory cytokines (tumour necrosis factor-α, interleukin 8, interleukin 1ß and interferon γ), while increasing transforming growth factor ß1 levels at both transcriptional and protein levels in the liver and intestine. The mRNA expression levels of mitogen-activated protein kinase 13 (MAPK 13), MAPK14 and nuclear factor kappa B p65 were significantly reduced in the liver and intestine of fish fed with 0.60 and 1.20 g/Kg SePCH compared to the controls. Histological sections also demonstrated that 0.60 and 1.20 g/Kg SePCH significantly increased intestinal villus height and villus width compared to the controls. Furthermore, the mRNA expression levels of tight junction proteins (zonula occludens-1, zonula occludens-3, Claudin-1, Claudin-3, Claudin-5, Claudin-11, Claudin-23 and Claudin-34) and Mucin-17 were significantly upregulated in the intestinal epithelial cells of 0.60 and 1.20 g/Kg SePCH groups compared to the controls. In conclusion, these results found that 0.60 and 1.20 g/Kg dietary SePCH can not only improve growth, hematological parameters, selenium metabolism, antioxidant capacities, enhance immune responses and intestinal functions, but also alleviate inflammatory responses. This information can serve as a useful reference for formulating feeds for largemouth bass.

Dietary antarctic krill improves antioxidant capacity, immunity and reduces lipid accumulation, insights from physiological and transcriptomic analysis of Plectropomus leopardus.

BACKGROUND: Due to its enormous biomass, Antarctic krill (Euphausia superba) plays a crucial role in the Antarctic Ocean ecosystem. In recent years, Antarctic krill has found extensive application in aquaculture, emerging as a sustainable source of aquafeed with ideal nutritional profiles. However, a comprehensive study focused on the detailed effects of dietary Antarctic krill on aquaculture animals, especially farmed marine fishes, is yet to be demonstrated. RESULTS: In this study, a comparative experiment was performed using juvenile P. leopardus, fed with diets supplemented with Antarctic krill (the krill group) or without Antarctic krill (the control group). Histological observation revealed that dietary Antarctic krill could reduce lipid accumulation in the liver while the intestine exhibited no obvious changes. Enzyme activity measurements demonstrated that dietary Antarctic krill had an inhibitory effect on oxidative stress in both the intestine and the liver. By comparative transcriptome analysis, a total of 1,597 and 1,161 differentially expressed genes (DEGs) were identified in the intestine and liver, respectively. Functional analysis of the DEGs showed multiple enriched terms significantly related to cholesterol metabolism, antioxidants, and immunity. Furthermore, the expression profiles of representative DEGs, such as dhcr7, apoa4, sc5d, and scarf1, were validated by qRT-PCR and fluorescence in situ hybridization. Finally, a comparative transcriptome analysis was performed to demonstrate the biased effects of dietary Antarctic krill and astaxanthin on the liver of P. leopardus. CONCLUSIONS: Our study demonstrated that dietary Antarctic krill could reduce lipid accumulation in the liver of P. leopardus, enhance antioxidant capacities in both the intestine and liver, and exhibit molecular-level improvements in lipid metabolism, immunity, and antioxidants. It will contribute to understanding the protective effects of Antarctic krill in P. leopardus and provide insights into aquaculture nutritional strategies.

The effects of four paralogous piscidin antimicrobial peptides on the chemotaxis, macrophage respiratory burst, phagocytosis and expression of immune-related genes in orange-spotted grouper (Epinephelus coicodes).

Antimicrobial peptides (AMPs) are an essential part of the vertebrate innate immune system. Piscidins are a family of AMPs specific in fish. In our previous investigation, we identified four paralogous genes of piscidins in the orange-spotted grouper (Epinephelus coicodes), which exhibited distinct activities against bacteria, fungi, and parasitic ciliated protozoa. Piscidins demonstrated their capability to modulate the expression of diverse immune-related genes; however, their precise immunoregulatory functions remain largely unexplored. In this study, we examined the immunomodulatory properties of putative mature peptides derived from four E. coicodes piscidins (ecPis1S, ecPis2S, ecPis3S, and ecPis4S) in head kidney leukocytes (HKLs) or monocytes/macrophages (MO/MΦ)-like cells isolated from E. coicodes. Our data demonstrate that E. coicodes piscidins exhibit immunomodulatory activities supported by multiple lines of evidence. Firstly, all four piscidins displayed chemotactic activities towards HKLs, with the most potent chemotactic activity observed in ecPis2S. Secondly, stimulation with E. coicodes piscidins enhanced respiratory burst and phagocytic activity in MO/MФ-like cells, with ecPis3S showing the highest efficacy in increasing phagocytosis of MO/MΦ-like cells. Thirdly, mRNA expression levels of chemokine receptors, Toll-like receptors, T cell receptors, and proinflammatory cytokines were modulated to varying extents by the four piscidins in E. coicodes HKLs. Overall, our findings indicate that the immunological activities of these four paralogous piscidins from E. coicodes are exhibited in a paralog-specific and concentration-dependent manner, highlighting their distinct and versatile immunomodulatory properties. This study makes a significant contribution to the field of fish AMPs immunology by elucidating the novel mechanisms through which members of the piscidin family exert their immunomodulatory effects. Moreover, it provides valuable insights for further exploration of fish immunomodulating agents.

Sea perch (Lateolabrax japonicus) UBC9 augments RGNNV infection by hindering RLRs-interferon response.

Small ubiquitin-like modifier (SUMO) is a reversible post-translational modification that regulates various biological processes in eukaryotes. Ubiquitin-conjugating enzyme 9 (UBC9) is the sole E2-conjugating enzyme responsible for SUMOylation and plays an important role in essential cellular functions. Here, we cloned the UBC9 gene from sea perch (Lateolabrax japonicus) (LjUBC9) and investigated its role in regulating the IFN response during red-spotted grouper nervous necrosis virus (RGNNV) infection. The LjUBC9 gene consisted of 477 base pairs and encoded a polypeptide of 158 amino acids with an active site cysteine residue and a UBCc domain. Phylogenetic analysis showed that LjUBC9 shared the closest evolutionary relationship with UBC9 from Paralichthys olivaceus. Tissue expression profile analysis demonstrated that LjUBC9 was significantly increased in multiple tissues of sea perch following RGNNV infection. Further experiments showed that overexpression of LjUBC9 significantly increased the mRNA and protein levels of RGNNV capsid protein in LJB cells infected with RGNNV, nevertheless knockdown of LjUBC9 had the opposite effect, suggesting that LjUBC9 exerted a pro-viral effect during RGNNV infection. More importantly, we found that the 93rd cysteine is crucial for its pro-viral effect. Additionally, dual luciferase assays revealed that LjUBC9 prominently attenuated the promoter activities of sea perch type Ⅰ interferon (IFN) in RGNNV-infected cells, and overexpression of LjUBC9 markedly suppressed the transcription of key genes associated with RLRs-IFN pathway. In summary, these findings elucidate that LjUBC9 impairs the RLRs-IFN response, resulting in enhanced RGNNV infection.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Evolutionary history and functional characterization of duplicated G protein-coupled estrogen receptors in European sea bass.

Across vertebrates, the numerous estrogenic functions are mainly mediated by nuclear and membrane receptors, including the G protein-coupled estrogen receptor (GPER) that has been mostly associated with rapid non-genomic responses. Although Gper-mediated signalling has been characterized in only few fish species, Gpers in fish appear to present more mechanistic functionalities as those of mammals due to additional gene duplicates. In this study, we ran a thorough investigation of the fish Gper evolutionary history in light of available genomes, we carried out the functional characterization of the two gper gene duplicates of European sea bass (Dicentrarchus labrax) using luciferase reporter gene transactivation assays, validated it with natural and synthetic estrogen agonists/antagonists and applied it to other chemicals of aquaculture and ecotoxicological interest. Phylogenetic and synteny analyses of fish gper1 and gper1-like genes suggest their duplication may have not resulted from the teleost-specific whole genome duplication. We confirmed that both sbsGper isoforms activate the cAMP signalling pathway and respond differentially to distinct estrogenic compounds. Therefore, as observed for nuclear estrogen receptors, both sbsGpers duplicates retain estrogenic activity although they differ in their specificity and potency (Gper1 being more potent and more specific than Gper1-like), suggesting a more conserved role for Gper1 than for Gper1-like. In addition, Gpers were able to respond to estrogenic environmental pollutants known to interfere with estrogen signalling, such as the phytoestrogen genistein and the anti-depressant fluoxetine, a point that can be taken into account in aquatic environment pollution screenings and chemical risk assessment, complementing previous assays for sea bass nuclear estrogen receptors.

Integrated analysis of microbiome and host transcriptome reveals the damage/protective mechanism of corn oil and olive oil on the gut health of grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu).

As digestive and immune organs of animals, the gut was frequently used to evaluate the health status of aquatic animals. In previous oil source alternatives study, corn oil (CO) had been found to induce gut inflammation, while olive oil (OO) had been found to be effective in protecting intestinal health. Three diets with different oil sources (fish oil, CO, OO) were formulated for an 8-week culture experiment, and it was proposed to combine 16S sequencing and transcriptome sequencing analysis to preliminarily elucidate the damage/protection mechanism of CO and OO on the gut health of grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu). We found that CO indeed damaged to gut health and destroyed the gut structure, while OO had a positive outcome in protecting the gut structure, promoting digestibility and relieving enteritis. Photobacterium, Romboutsia and Epulopiscium were significantly enriched in OO group and Staphylococcus were significantly enriched in CO group. Transcriptome sequencing further revealed CO could activated Complement and coagulation cascades, Staphylococcus aureus infection, Systemic lupus erythematosus, and Tuberculosis pathways; conversely, OO activated B-cell signaling receptors, promoted B-cell proliferation and apoptosis, and thus activated B-cell signaling pathways to enhance immunity, whereas OO can regulate IL17 signaling pathway and TNF signaling pathway to inhibit NF-κB signaling pathway to reduce pro-inflammatory response. By integrating the microbiome and transcriptome, further identified all differential microorganisms were directly and significantly correlated with differential genes, and Clostridium_sensu_stricto_1, Romboutsia, Staphylococcus might as the core regulates the expression of differential gene in the organism. These results reveal that different oil sources alter gut gene expression mainly by modulating the composition and abundance of gut microbiota, further regulating the health status of the gut. Gut microbiota could be used as biomarkers to provide reference and solutions for the mitigation of inflammation in aquatic animals.

G protein-coupled bile acid receptor 1 reduced hepatic immune response and inhibited NFκB, PI3K/AKT, and PKC/P38 MAPK signaling pathway in hybrid grouper.

The mammalian G protein-coupled bile acid receptor 1 (TGR5) is involved in the inflammatory response. However, the functions of TGR5 in the immune response of fish remain unclear. In this study, the full-length sequence of tgr5 from hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) was cloned, and the function of TGR5 in the immune response was explored. The results showed that the ORF of tgr5 gene in hybrid grouper was 1029 bp and encoded 342 amino acids. Activation of TGR5 by INT-777 significantly decreased the activities and mRNA expression of TNFα and IL1ß, whereas inhibition of TGR5 by SBI-115 showed the opposite effect. SBI-115 treatment significantly increased the expression of phosphorylated inhibitor κB α (p-IKBα) protein. After the INT-777 treatment, the concentration of protein kinase C (PKC) and expression of the p38 mitogen-activated protein kinases (p38a), p38b and p38c, were significantly decreased in vivo. INT-777 agonist significantly decreased the expression of phosphorylated phosphoinositide 3-kinase (p-PI3K) protein and the ratio of phosphorylated and nonphosphorylated serine/threonine-protein kinase (p-AKT/AKT). In conclusion, activation of hepatic TGR5 inhibited the PKC/P38 MAPK, PI3K/AKT, NFκB signaling pathway and improved hepatic immune responses of hybrid grouper in vivo and in vitro.

Recent studies have shown that mammalian G protein-coupled bile acid receptor 1 (TGR5) is involved in inflammatory response. However, the functions of TGR5 in immune response of hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂) remain unclear. In this study, the full-length sequence of tgr5 from hybrid grouper was cloned and characterized for the first time, and the functions of TGR5 in the immune response was explored by activating/inhibiting hepatic TGR5 in vivo and in vitro. These results showed that activation of hepatic TGR5 inhibited PKC/P38 MAPK and PI3K/AKT signaling, attenuated the NFκB pathway, and improved the hepatic immune responses of hybrid grouper in vivo and in vitro. The inhibition of TGR5 had the opposite effects. Understanding the functions of hepatic TGR5 may help to develop management strategies to reduce the liver inflammation in fish or other animals.

Genome-wide association study (GWAS) analysis of black color trait in the leopard coral grouper (Plectropomus leopardus) using whole genome resequencing.

The leopard coral grouper (Plectropomus leopardus) is a coral reef fish species that exhibits rapid and diverse color variation. However, the presence of melanoma and the high proportion of individuals displaying black color in artificial breeding have led to reduced economic and ornamental value. To pinpoint single nucleotide polymorphisms (SNPs) and potential genes linked to the black pigmentation characteristic in this particular species, This study gathered a cohort of 360 specimens from diverse origins and conducted a comprehensive genome-wide association analysis (GWAS) employing whole-genome resequencing. As a result, 57 SNPs related to the black skin trait were identified, and a grand total of 158 genes were annotated within 50 kb of these SNPs. Subsequently, GWAS was applied to three populations (LED, QHH, and QHL), and the corresponding results were compared with the analysis results of the total population. The results of the four GWAS models showed significant enrichment in Rap1 signaling pathway, melanin biosynthesis, metabolic pathways, tyrosine metabolism, cAMP signaling pathway, AMPK signaling pathway, PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, HIF-1 signaling pathway, Ras signaling pathway, MAPK signaling pathway, etc. (p < 0.05), which were mainly associated with eleven genes (POL4, MET, E2F2, COMT, ZBED1, TYRP2, FOXP2, THIKA, LORF2, MYH16 and SOX2). Significant differences (p < 0.05) were observed in the expression of all 11 genes in the dorsal skin tissue, in 10 genes except COMT in the ventral skin tissue, and in all 11 genes in the caudal fin tissue. These findings imply that the control of body color in the P. leopardus is the result of the joint action of multiple genes and signaling pathways. These findings will contribute to a more profound comprehension of the genetic attributes that underlie the development of black skin in the vibrant P. leopardus, thus furnishing a theoretical foundation for genetic enhancement.

The role of salinity on genome-wide DNA methylation dynamics in European sea bass gills.

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change.

Methyltransferase-like 3 suppresses red spotted grouper nervous necrosis virus and viral hemorrhagic septicemia virus infection by enhancing type I interferon responses in sea perch.

Methylation at the N6 position of adenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, tightly associating with regulation of viral life circles and immune responses. Here, a methyltransferase-like 3 homolog gene from sea perch (Lateolabrax japonicus), designated LjMETTL3, was cloned and characterized, and its negative role in fish virus pathogenesis was uncovered. The cDNA of LjMETTL3 encoded a 601-amino acid protein with a MT-A70 domain, which shared the closest genetic relationship with Echeneis naucrates METTL3. Spatial expression analysis revealed that LjMETTL3 was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. LjMETTL3 expression was significantly upregulated at 12 and 24 h post RGNNV and VHSV infection in vitro. In addition, ectopic expression of LjMETTL3 inhibited RGNNV and VHSV infection in LJB cells at 12 and 24 h post infection, whereas knockdown of LjMETTL3 led to opposite effects. Furthermore, we found that LjMETTL3 may participate in boosting the type I interferon responses by interacting with TANK-binding kinase. Taken together, these results disclosed the antiviral role of fish METTL3 against RGNNV and VHSV and provided evidence for understanding the potential mechanisms of fish METTL3 in antiviral innate immunity.

Effects of tributyrin and alanyl-glutamine dipeptide on intestinal health of largemouth bass (Micropterus salmoides) fed with high soybean meal diet.

To investigate the effects of dietary tributyrin (TB) and alanyl-glutamine (AGn) on the intestinal health of largemouth bass (Micropterus salmoides) fed with high-level soybean meal (SM) diet, six isonitrogenous (41.36%) and isolipidic (10.25%) diets were formulated and fed to largemouth bass (initial body weight 25.5 ± 0.5g) for 8 weeks. The two control diets contained 34.8% peanut meal (PM) and 41.3% SM, while the other four experimental diets supplemented TB at 0.1% (TB0.1), 0.2% (TB0.2) and AGn at 1% (AGn1), 2% (AGn2) in SM, respectively. The results showed that there were no significant differences in weight gain, survival rate, and hepatosomatic index among all groups (P>0.05), while feed coefficient rate in AGn1, AGn2 and TB0.2 groups was significantly lower than that in SM group (P< 0.05). Compared with the PM group, the intestinal inflammation of largemouth bass in SM group were obvious, accompanied by the damage of intestinal structure, the decrease of digestive enzyme activity, and the up-regulation of proinflammatory cytokines. Compared with the SM group, the activities of intestinal trypsin, lipase and foregut amylase in TB and AGn groups increased significantly (P<0.05), and the gene expression levels of acetyl-CoA carboxylase (ACC), caspase-3, caspase-8, caspase-9, tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß) were down-regulated, while the gene expression levels of target of rapamycin (TOR) and eIF4E-binding protein (4E-BP) were up-regulated in all experimental groups (P<0.05). It can be concluded that supplementation of 1%-2% AGn and 0.1%-0.2% TB can alleviate enteritis caused by high-level soybean meal, and the recommend level is 2% AGn and 0.2% TB.

A novel scavenger receptor (EcSRECII) as a lipopolysaccharide recognition molecule involved in regulating NF-κB activation through extracellular EGF-like cysteine-rich repeat domains with lysosomes in Epinephelus coioides.

Scavenger receptors (SRs), as multifunctional pattern recognition receptors, play an important role in innate immunity in mammals, however, their function in fish is limited. Herein, scavenger receptor F2 in Epinephelus coioides (EcSRECII) induced an innate immune response to LPS in GS cells. EcSRECII markedly enhanced LPS-induced NF-κB and IFN-ß signaling pathways, whereas knockdown of EcSRECII significantly inhibited LPS-induced NF-κB and IFN-ß promoter activation. Interestingly, only retain of epidermal growth factor (EGF)/EGF-like domain in EcSRECII resulted in a punctate cytoplasmic distribution, while the C-terminal domain exhibited a distinct cytoskeletal cytoplasmic distribution. Moreover, devoid of this EGF/EGF-like domain fragment more sharply impaired its ability to activate EcSRECII-induced NF-κB activation than deletion of the C-terminal domain region, but both domains significantly induced IFN-ß promoter activation. Full-length EcSRECII and the deletion mutant of C-terminal domain could partly colocalize with lysosomes by LPS derived from V. parahaemolyticus (V.p. LPS) in GS cells, but there was no similar distribution in the deletion mutant of EGF/EGF-like domain. This finding firstly suggested that the N-terminal EGF/EGF-like domain was necessary for the NF-κB signaling pathway to trigger resistance to vibrio infection and its functional exertion may be associated with lysosomes, thus providing insights into the regulation of resistance to vibrio infection in teleosts.

Influences of chronic copper exposure on intestinal histology, antioxidative and immune status, and transcriptomic response in freshwater grouper (Acrossocheilus fasciatus).

Copper (Cu) contamination is commonly found in both natural water environments and fish farms, and it can cause severe damage to different fish organs, but Cu-induced intestinal damage has been rarely studied. This study subjected three groups of freshwater grouper (Acrossocheilus fasciatus) (initial weight: 1.56 ± 0.10 g) to 0 mg/L, 0.01 mg/L, and 0.04 mg/L Cu2+ for 30 days, named Con, Cu0.01, and Cu0.04 groups, respectively. The histological observation indicated that the Cu0.04 group caused a significant decrease in villus length, lamina propria width, and muscular thickness compared to the Con group (P < 0.05). Additionally, the Cu0.04 group significantly increased intestinal superoxide dismutase (SOD), glutathione peroxidase (GPx), lysozyme (LZM) activities, as well as malondialdehyde (MDA) content than the Con group (P < 0.05). Meanwhile, the Cu0.01 and Cu0.04 groups showed significantly increased immunoglobulin M (IgM), complement 3 (C3), and glutathione (GSH) contents than the Con group (P < 0.05). Transcriptomic analysis revealed a total of 101 differentially expressed genes (DEGs), including 47 up-regulated and 54 down-regulated DEGs, were identified between the Cu0.04 and Con groups. Notably, the DEGs were mainly related to intestinal structure construction, immune functions, apoptosis, and resistance to DNA damage and pathogen infection. The findings suggest that chronic Cu exposure caused intestinal histological alterations, activated the antioxidative and immune systems, and induced systematic adaptation to cope with the physical barrier injury, DNA damage, and potential pathogen growth.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

Molecular characterization, structural and expression analysis of twelve CXC chemokines and eight CXC chemokine receptors in largemouth bass (Micropterus salmoides).

The chemokine-receptor system plays important roles in the leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. In the present study, the sequence features, structures and expression patterns of twelve CXC chemokine ligands (CXCL8a.1, CXCL8a.2, CXCL8b.1, CXCL8b.2, CXCL12a, CXCL12b, CXCL13.1, CXCL13.2, CXCL14, CXCL18a, CXCL18b and CXCL19) and eight CXC chemokine receptors (CXCR1, CXCR2, CXCR3.1, CXCR3.2, CXCR3.3, CXCR4a, CXCR4b and CXCR5) of largemouth bass (Micropterus salmoides) were analyzed. All the CXCLs and CXCRs of largemouth bass shared high sequence identities with their teleost counterparts and possessed conserved motifs and structures of CXCLs and CXCRs family. Realtime qPCR revealed that these CXCLs and CXCRs were ubiquitously expressed in all examined tissues, with high expression levels in the immune-related tissues (spleen, head kidney, and gill). Following lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (polyI:C) stimulations, most of these CXCLs and CXCRs were significantly up-regulated in spleen. In addition, the potential interacted molecules of these CXCLs and CXCRs were analyzed by protein-protein interaction network analysis. To the best of our knowledge, this is the first study that in detail analyzes the CXCLs and CXCRs of largemouth bass. Our results provide valuable basis for study the function and mechanism of chemokine-receptor system in largemouth bass.

Molecular characterization, immune expression, and functional delineation of peroxiredoxin 1 in Epinephelus akaara.

Peroxiredoxin 1 is a member of the typical 2-Cys peroxiredoxin family, which serves diverse functions in gene expression, immune and inflammatory responses, and tumor progression. In this study, we aimed to analyze the structural, functional, and immunomodulatory properties of peroxiredoxin 1 from Epinephelus akaara (EaPrx1). The open reading frame of EaPrx1 is 597 base pairs in length, encoding 198 amino acids, with a molecular weight of approximately 22 kDa. The in silico analysis revealed that EaPrx1 shares a conserved thioredoxin fold and signature motifs that are critical for its catalytic activity and oligomerization. Further, EaPrx1 is closely related to Epinephelus lanceolatus Prx1 and clustered in the Fishes group of the vertebrate clade, revealing that EaPrx1 was conserved throughout evolution. In terms of tissue distribution, a high level of EaPrx1 expression was observed in the spleen, brain, and blood tissues. Likewise, in immune challenge experiments, significant transcriptional modulations of EaPrx1 upon lipopolysaccharide, polyinosinic:polycytidylic acid, and nervous necrosis virus injections were noted at different time points, indicating the immunological role of EaPrx1 against pathogenic infections. In the functional analysis, rEaPrx1 exhibited substantial DNA protection, insulin disulfide reduction, and tissue repair activities, which were concentration-dependent. EaPrx1/pcDNA™ 3.1 (+)-transfected fathead minnow cells revealed high cell viability upon arsenic toxicity, indicating the heavy metal detoxification activity of EaPrx1. Taken together, the transcriptional and functional studies imply critical roles of EaPrx1 in innate immunity, redox regulation, apoptosis, and tissue-repair processes in E. akaara.

The role of TNF-α in the phagocytosis of largemouth bass (Micropterus salmoides) leukocytes.

Phagocytosis is an important innate immune process in which immune cells recognize, ingest and eliminate pathogens. Largemouth bass (Micropterus salmoides) has become an important economic farmed fish in many regions, while few studies has focused on phagocytosis of its leucocytes. In present study, largemouth bass peripheral blood leucocytes were separated using Percoll gradient to establish the phagocytic function. Flow cytometric analysis showed that largemouth bass leukocytes exhibited the phagocytic capacity to fluoresbrite microspheres and Aeromonas hydrophila, where higher phagocytic capacity to A. hydrophila were observed in granulocytes/monocytes than that of lymphocytes. The leukocytes engulfing fluoresbrite microspheres and A. hydrophila were also observed by fluorescence microscopy. Besides, manygenes associated with phagocytosis and TNF-α in leukocytes were up-regulated following A. hydrophila stimulation. Subsequently, the largemouth bass TNF-α was recombinantly expressed to investigate its role in regulating phagocytosis. The results showed that TNF-α in largemouth bass could significantly enhance the phagocytic ability of granulocytes/monocytes to A. hydrophila, but not lymphocytes. Moreover, we also found that TNF-α could not only significantly increase the ROS activity of granulocytes/monocytes, but also had the function of inducing its apoptosis. These results demonstrated that granulocytes/monocytes play more important role in phagocytosis, meanwhile, TNF-α has the function of enhancing the phagocytic ability of granulocytes/monocytes in largemouth bass.

Involvement of MMP-9 in collagen degradation of sea bass (Lateolabrax japonicus): Cloning, expression, and characterization.

Disintegration of intramuscular connective tissue is responsible for postmortem tenderization of fish muscles during chilled storage. Matrix metalloproteinase-9 (MMP-9) was reported to be involved in this process, whereas the mechanism has not been revealed. In the present study, purified type I and V collagens from the connective tissues of sea bass (Lateolabrax japonicus) muscles were first prepared. These two kinds of collagens comprise three polypeptide chains (α), forming a typical triple-helical domain as determined by circular dichroism. The complete coding region of MMP-9 containing an open reading frame of 2070 bp encoding 689 amino acid residues was then cloned. The recombinant MMP-9 catalytic domain (rcMMP-9) was expressed in Escherichia coli and exhibited high hydrolyzing activity toward gelatin. Besides, rcMMP-9 was effective in degrading type V collagen rather than type I collagen at 4°C. The enzymatic activity of rcMMP-9 was highly pH-dependent, and its enzymatic activity under neutral and basic conditions was higher than that under acidic conditions. Metal ion Ca2+ was necessary for the maintenance of rcMMP-9 activity, whereas Zn2+ inhibited its activity. Our present study indicated that MMP-9 is responsible for the disintegration of intramuscular connective tissues by cleaving type V collagen during postmortem tenderization of fish muscle. PRACTICAL APPLICATION: Elucidation the involvement of MMP-9 in collagen degradation will deliver a reference for the prevention of muscular protein decomposition during chilled storage of fish fillets.

Detoxification effects of ascorbic acid on the oxidative stress, neurotoxicity, and metallothionein (MT) gene expression in juvenile rockfish, Sebastes schlegelii by the dietary chromium exposure.

Juvenile rockfish Sebastes schlegelii (mean length 10.8 ± 1.4 cm, and mean weight 31.7 ± 3.6 g) were exposed for 4 weeks with the different levels of dietary chromium (Cr6+) at 0, 120 and 240 mg/L and ascorbic acids (AsA) at 100, 200 and 400 mg/L. Superoxide dismutase (SOD) activity, glutathione S-transferase (GST) activity, and glutathione (GSH) level of liver and gill were evaluated as antioxidant response indicators for the 4 weeks exposure. The SOD and GST activity of liver and gill were substantially increased by the high concentrations of dietary Cr exposure, whereas a significant decrease was observed in the GSH levels of liver and gill. Metallothionein (MT) gene in liver was significant stimulated in the response to the dietary Cr exposure. In neurotoxicity, AChE activity was considerably inhibited in brain and muscle tissues by dietary Cr exposure. The high levels of AsA supplementation were highly effective to attenuate the alterations in the antioxidant responses, MT gene expression, and AChE activity by the dietary Cr exposure.