Copper (Cu) is an essential element in the metabolic process of humans and animals, but it can cause toxicity at high concentrations of exposure. Bioflocs has been proved to have antioxidant, immune-enhancing and anti-inflammatory properties. Here, the purpose of this study was to evaluate potential mechanisms and protective effects of bioflocs and Cu exposure on inflammatory response, oxidative stress and immune-related genes and protein expression in Rhynchocypris lagowski Dybowski. 360 healthy R. lagowski were irregularly distributed among 12 tanks (3 tanks per group, 30 fish per tank). The experiment was divided into two parts: the feeding experiment was carried out in the first eight weeks, followed by acute copper exposure for 96 h. Then we selected the stressed fish for experimental analysis. The results provided evidences that bioflocs protected the R. lagowski by inhibiting the accumulation of copper, the activity of immune enzymes and the expression of NF-κB signaling pathway related genes and proteins, and the activity of antioxidant enzymes and the expression of Nrf2 signaling pathway related genes. Overall, these findings suggest that bioflocs could regulate the activation of Nrf2 and protect acute copper exposure induced inflammatory response by inhibiting the NF-κB signaling pathway in R. lagowski.

Chinese mitten crab Eriocheir sinensis is probably the most important freshwater cultured crab in China. A tiny minority of brownish-orange individuals have been discovered in the long period of artificial breeding history of E. sinensiss. Those mutants are usually accompanied with slow growth rate, low molting frequency and poor survival rate, which may be the results of growth defects and immunodeficiency. To better understand the relationship between body color determination and the immune system as well as the related genes expression in E. sinensiss, we performed the whole-body transcriptome analysis in different color of first stage zoea (ZI) larvae using next-generation sequencing (NGS) technology. We randomly assembled 175.40 and 177.52million clean reads from the wild and mutant ZIs, respectively. Finally, we identified 7153 differentially expressed genes (DEGs) (p < 0.05), with 5194 up-regulated and 1959 down-regulated. A total of 13 KEGG pathways related to immune system were detected among 248 pathways. Except the first whole-body RNA sequencing of color-specific transcriptomes for E. sinensis, this study will offer a better understanding of the underlying molecular mechanisms of interaction between color determination and the immune system.

The present study was conducted to investigate the effects of CoQ10 dietary supplementation on growth performance, feed utilization, blood profile, immune response, and oxidative status of Nile tilapia (12.4 ± 0.11 g, initial body weight). Five experimental diets were formulated containing CoQ10 at levels of 0, 10, 20, 30, 40 mg kg−1 diet (D1, D2, D3, D4, and D5, respectively). The results of a 56-days feeding trial showed that, significantly higher weight gain % (WG %), specific growth rate (SGR), feed intake (FI), and feed efficiency ratio (FER) were recorded in fish groups fed diets supplemented with different levels of CoQ10 than fish fed the control diet, while survival rate (SR%), condition factor (CF), hepatosomatic index (HSI) and viscerasomatic index (VSI) showed no obvious differences (P > 0.05) among all experimental groups. The highest activities of digestive enzymes (protease, amylase, and lipase) were recorded in D3, D4, and D5 groups. Moreover, blood status of all experimental fish was within normal rates and significant alterations were only in the case of glucose, cortisol, total cholesterol (T-Chol), triglycerides, and total protein (TP), where fish fed on D3, D4 and D5 diets exhibited lower values of glucose, cortisol, T-Chol, and triglycerides and higher values of TP. Furthermore, the lowest values of immune response [lysozyme, bactericidal, respiratory burst (NBT), and alternative complement pathway activities (ACP)], antioxidant capacity and oxidative related genes expressions [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX)] resulted from feeding on the basal diet (D1) compared to CoQ10 diets, especially with its high levels {≥20 mg kg−1 diet (D3, D4, and D5)} in most cases. In conclusion, our results suggest that the use of ≥20 mg CoQ10 kg-1 diet improves the growth and health being of Nile tilapia.

A relatively large repertoire of type I interferon (IFN) genes is apparent in rainbow trout/Atlantic salmon, that includes six different IFN subgroups (IFNa-IFNf) belonging to the three known type I IFN groups (1–3) in bony fish. Whether this is true for other salmonids, and how the various type I subgroups evolved in teleost fish was studied using the extensive genomic resources available for fish. This confirmed that salmonids, at least the Salmoninae, indeed have a complex (in terms of IFN subgroups present) and large (number of genes) IFN repertoire relative to other teleost fish. This is in part a consequence of the salmonid 4R WGD that duplicated the growth hormone (GH) locus in which type I IFNs are generally located. Divergence of the IFN genes at the two GH loci was apparent but was not seen in common carp, a species that also underwent an independent 4R WGD. However, expansion of IFN gene number can be found at the CD79b locus of some perciform fish (both freshwater and marine), with expansion of the IFNd gene repertoire. Curiously the primordial gene order of GH-IFNc-IFNb-IFNa-IFNe is largely retained in many teleost lineages and likely reflects the tandem duplications that are taking place to increase IFN gene number. With respect to the evolution of the IFN subgroups, a complex acquisition and/or loss has occurred in different teleost lineages, with complete loss of IFN genes at the GH or CD79b locus in some species, and reduction to a single IFN subgroup in others. It becomes clear that there are many variations to be discovered regarding the mechanisms by which fish elicit protective (antiviral) immune responses.

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.

Interleukin 11 (IL-11) is a secretory cytokine with pleotropic properties, including anti-inflammatory and anti-apoptotic functions. This study aimed to functionally characterize a teleostean IL-11a ortholog from redlip mullet (LhIL-11a) through bioinformatic analysis, transcriptional expression profiling and protein function assays. The deduced LhIL-11a protein sequence is 200 amino acids long, with a predicted molecular weight of 23.168 kDa. Multiple sequence alignment indicates that LhIL-11a has a typical four-bundle architecture of α-helixes as observed in other IL-11s. The identity–similarity matrix show a higher identity between LhIL-11a and other fish IL-11a sequences. Phylogenetic analysis demonstrated that LhIL-11a falls within a clade including other fish counterparts. In the tissue distribution analysis, the highest constitutive expression of LhIL-11a mRNA was observed in the mullet gastrointestinal tract and brain tissues. Following the challenges with LPS, poly I:C and Lactococcus garvie, the transcription levels of LhIL-11a were significantly upregulated in both PBCs and liver. In the biological functional assay, recombinant LhIL-11a protein showed strong activities of suppressing pro-inflammatory cytokines and apoptotic gene expression in mullet kidney cells and reducing LPS stimulated NO production in murine macrophage cells. Overall, the findings in this study provide the experimental clues to understanding the functional roles of fish IL-11a in inflammation and apoptosis regulation during host defense against invading microbial pathogens.

Cathepsin K belongs to the family of cysteine cathepsins. It is well known that the cysteine cathepsins participate in various physiological processes and host immune defense in mammals. However, in teleost fish, the function of cathepsin K is very limited. In the present study, a cathepsin K homologue (SsCTSK) from the teleost black rockfish (Sebastes schlegelii) was identified and examined at expression and functional levels. In silico analysis showed that three domains, including signal peptide, cathepsin propeptide inhibitor I29 domain, and functional domain Pept_C1, are existed in SsCTSK. SsCTSK also possesses a peptidase domain with three catalytically essential residues (Cys25, His162 and Asn183). Phylogenetic profiling indicated that SsCTSK was evolutionally close to the cathepsin K of other teleost fish. Expression of SsCTSK occurred in multiple tissues and was induced by bacterial infection. Purified recombinant SsCTSK (rSsCTSK) exhibited apparent maximal peptidase activity at 45 °C, and its enzymatic activity was remarkably declined in the presence of the cathepsin inhibitor E−64. Moreover, rSsCTSK possesses the ability to bind with PAMPs and bacteria. Finally, knockdown of SsCTSK expression facilitated bacterial invasion in black rockfish. Collectively, these results indicated that SsCTSK functions as a cysteine protease and may serves as a target for pathogen manipulation of host defense system.

Columnaris disease, induced by Flavobacterium columnare, seriously affects the health of freshwater fish species and damages the mucosal tissues, such as the fins, skin, and gills. Teleosts represent the first bony vertebrate to contain both innate and adaptive immune responses against pathogens. So far, three immunoglobulin isotypes (IgM, IgD, and IgT/IgZ) have been identified in teleost fish, and IgT in mucosal tissues of teleost fish was reported to perform a similar function to IgA in mammals during parasitic infection. However, very limited information is known about the function of IgT in gill mucosal tissues during bacterial infection. In the present study, rainbow trout (Oncorhynchus mykiss) was infected with F. columnare (Fc) via immersion. After Fc infection, the gill structure of rainbow trout showed serious hyperplasia symptoms on the secondary lamellae at 12 h post infection (hpi). Moreover, the mRNA expression levels of NOS2 and cathelicidin-1 were significantly upregulated immediately at 12 hpi and showed high expression throughout the experiment. IgT and IgM showed much higher mRNA expression levels at 28 days post infection (dpi) and 75 dpi, while IgD only showed high mRNA expression levels at 28 dpi. Importantly, the accumulation of IgT+ B cells and strong bacteria-specific IgT responses were detected in the gill lamellae of both infected fish (28 dpi) and survivor fish (75 dpi). Overall, our results suggest that IgT and IgT+ B cells play a central role in the adaptive immune responses of fish gill mucosa against bacterial infection.

We investigated differential gene expression in Tilapia infected with the Tilapia Lake virus (TiLV).We used high-throughput sequencing to identify mRNAs and miRNAs involved in TiLV infection progression We identified 25,359 differentially expressed genes that included 863 new genes. We identified 1770, 4142 and 4947 differently expressed genes comparing non-infected controls with 24 and 120 h infections and between the infected groups, respectively. These genes were enriched to 291 GO terms and 62 KEGG pathways and included immune system progress and virion genes. High-throughput miRNA sequencing identified 316 conserved miRNAs, 525 known miRNAs and 592 novel miRNAs. Furthermore, 138, 198 and 153 differently expressed miRNAs were found between the 3 groups listed above, respectively. Target prediction revealed numerous genes including erythropoietin isoform X2, double-stranded RNA-specific adenosine deaminase isoform X1, bone morphogenetic protein 4 and tapasin-related protein that are involved in immune responsiveness. Moreover, these target genes overlapped with differentially expressed mRNAs obtained from RNA-seq. These target genes were significantly enriched to GO terms and KEGG pathways including immune system progress, virion and Wnt signaling pathways. Expression patterns of differentially expressed mRNA and miRNAs were validated in 20 mRNA and 19 miRNAs by qRT-PCR. We also were able to construct a miRNA-mRNA target network that can further understand the molecular mechanisms on the pathogenesis of TiLV and guide future research in developing effective agents and strategies to combat TiLV infections in Tilapia.

Bacterial enteritis is an important deadly threat to farmed seahorses. However, its pathogenesis is obscure because of the paucity of reproducible experimental intestinal inflammation models. Herein, a strain of Edwardsiella tarda YT1 from farmed seahorse Hippocampus erectus was isolated and identified by morphological, phylogenetic, and biochemical analysis, and confirmed as a pathogen of enteritis for the first time by challenge experiment. Two E. tarda concentrations (1 × 105 and 1 × 107 colony forming units [cfu] ml−1) were confirmed suitable for an enteritis model by intraperitoneal injection. To develop and evaluate the experimental model, we challenged seahorses with E. tarda and found that (1) the infection inhibited body length increase, significantly decreased body weight (P < 0.05), and induced typical pathological features including anorexia, anal inflammation, and intestinal fluid retention; (2) 19 external (weight, height, anal inflammation, feeding status, and intestinal fluid retention), histological (goblet and inflammatory cell numbers and thickening of lamina propria and muscularis mucosae), and molecular (hepcidin, liver-expressed antimicrobial peptide, lysozyme, piscidin, interleukin [IL]-1β, IL-1β receptor, IL-2, IL-10, interferon1, tumor necrosis factor [TNF]-α, and toll-like receptor 5 [TLR5]) indicators were suitable for model evaluation, as they could sensitively respond and varied similarly throughout the experiment, indicating the high sensitivity of seahorses against pathogen invasion; (3) TLR5 may play an essential role in triggering host immune responses during E. tarda-induced chronic enteritis, and (4) the evaluating system could reflect the pattern and intensity of disease progression. Thus, we developed an experimental model and an evaluating system of bacterial enteritis in farmed seahorses, helping us to reveal the pathogenesis of bacterial enteritis, identify potential therapeutic drugs, and search suitable genetic markers for seahorse molecular breeding.

Interleukin (IL)-21 is a pleiotropic cytokine and plays a vital role in immunity. In the current study, we examined the immune function of Japanese flounder Paralichthys olivaceus IL-21 (PoIL-21). PoIL-21 shares moderate (25.17%–46.25%) sequence identities with other teleost IL-21. PoIL-21 expression occurred in multiple tissues, especially intestine, and was regulated by bacterial infection in a time dependent manner. PoIL-21 was secreted by peripheral blood leukocytes (PBL) upon LPS stimulation. Recombinant PoIL-21 (rPoIL-21) bound to a wide range of Gram-negative and Gram-positive bacteria and inhibited the growth of the fish bacterial pathogen Streptococcus iniae. rPoIL-21 also interacted with PBL, resulting in enhanced cell proliferation, ROS production, and expression of IL-1β, TNF-α, CD8β, T-bet, PoIL-21, PoIL-21 receptor, and STAT. Consequently, the presence of rPoIL-21 significantly reduced bacterial infection in PBL. In vivo study showed that rPoIL-21 upregulated the expression of inflammatory cytokines and PoIL-21. Taken together, these results indicate that PoIL-21 is an inducible, secreted cytokine with a broad range of binding capacities and plays an important role in the regulation of anti-bacterial immunity.

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H2O2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20∼Val140, Ser169∼Tyr245, Thr258∼Pro390, Phe414∼Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1β, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

The present study was designed to investigate the dietary effects of ginger extract (Zingiber officinale) on common carp (Cyprinus carpio). Three hundred and sixty fish weighing 10.9 ± 0.17g were randomly divided into four experimental treatments in triplicates. Four experimental diets were designed containing different inclusion levels of ginger extract as 0% (control), 0.1%, 0.2%, and 0.4% named T1, T2, T3, and T4, respectively, and fish were fed 3% of body weight daily for 60 days. Feed conversion ratio, weight gain, and specific growth rate parameters were measured higher in the groups fed with ginger extract compared to the control, especially in T3 (P < 0.05). Also, fish treated with ginger extract showed lower carcass moisture and lipid, but higher protein contents compared to the control. The results showed significant increase in the number of erythrocytes and leucocytes, as well as the level of hematocrit and hemoglobin in fish fed with supplemented diets. In addition, significant enhancement was observed in cases of serum parameters, including total protein, albumin, globulin, lysozyme, superoxide dismutase, catalase, and total immunoglobins T3 and T4 compared to the control. The lowest serum cortisol level was noticed in T3. Furthermore, the T3 group showed the highest activities of skin mucus lysozyme, alkaline phosphatase, and protease (P < 0.05). In conclusion, the present results demonstrated that Zingiber officinale extract at a 0.2% level can effectively improve the growth and health status of Common Carp (Cyprinus carpio).

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation.

Microbial exopolysaccharides (EPS) from Lactococcus have been found to have an important role in the probiotic activity of this bacterium; however, the immunomodulatory and antioxidant activities have not been fully explored in aquaculture. In the present study, we investigated EPS-2 from Lactococcus lactis Z-2, isolated from healthy common carp, for its immunomodulatory and antioxidant effects and disease resistance against Aeromonas hydrophila in Cyprinus carpio L. We found that the molecular weight of EPS-2 was 18.65 KDa. The monosaccharide composition of this polymer was rhamnose, xylose, mannose, glucose, and galactose at a molar percentage of 13.3%, 14.1%, 18.5%, 27.4%, and 26.7%, respectively. EPS-2 treatment could modulate the immune responses in vitro and in vivo. In vitro tests showed that EPS-2 could significantly enhance the proliferation and phagocytosis activities (P < 0.05) as well as induce the production of nitic oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and anti-inflammatory cytokines (IL-10, TGF-β) (P < 0.05) in head kidney cells. When the fish were gavaged with three different concentrations of EPS-2 (250, 500, 1000 μg/mL) for 7 days and infected with A. hydrophila, different expression patterns of the NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in the serum and of antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas were observed. Before infection with A. hydrophila, EPS-2 supplementation significantly up-regulated the NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), LZM and AKP activities, and levels of antioxidant molecules compared to those in the negative (G1) group (P < 0.05), whereas levels of NO and pro-inflammatory cytokines and LZM and AKP activities were significantly lower than those in the positive (G2) group after infection (P < 0.05). However, whether infected or not, the expression levels of anti-inflammatory cytokines (IL-10, TGF-β) were significantly increased in the EPS-2 treatment groups (P < 0.05). These results indicate that EPS-2 has immunomodulatory and antioxidant effects on common carp, both in vitro and/or in vivo, and can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), are crucial sensors with a conserved structure in cytoplasm, inducing the production of cytokines, chemokines and host restriction factors which mediate a variety of intracellular activities to interfere with distinct PAMPs (pathogen-associated molecular patterns) for eliminating pathogens in innate immune system. Although RLR genes have been investigated in most vertebrates and some invertebrates, the systematic identification and characterization of RLR genes have not been reported in scallops. In this study, four RLR genes (PY-10413.4, PY-10413.5, PY-443.7 and PY-443.8, designated PyRLRs) were identified in Yesso scallop (Patinopecten yessoensis) through whole-genome scanning through in silico analysis, including two pairs of tandem duplicate genes located on the same scaffold (PY-10413.4 and PY-10413.5, PY-443.7 and PY-443.8, respectively). Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of these genes. The expression profiles of PyRLRs were determined in all developmental stages, in healthy adult tissues, and in mantles that simulated ocean acidification (OA) exposure (pH = 6.5 and 7.5) at different time points (3, 6, 12 and 24 h). Spatiotemporal expression patterns suggested the functional roles of PyRLRs in all stages of development and growth of the scallop. Regulation expressions revealed PY-10413.4 and PY-10413.5 with one or two CARD(s) (caspase activation and recruitment domain) were up-regulated expressed at most time points, whereas PY-443.8 and PY-10413.4 without CARD were significantly down-regulated at each time points, suggesting functional differentiations in the two pairs of PyRLRs based on the structural differences in response to OA. Collectively, this study demonstrated gene duplication of RLR family genes and provide primary analysis for versatile roles in the response of the bivalve innate immune system to OA challenge.

The protective role of β-glucan (BG) on liver function, histopathology, immune and antioxidant related gene expressions in Nile tilapia exposed to subacute deltamethrin (DLM) was investigated for 30 days. Fish (28.18 ± 1.34 g) of the 1st and 2nd groups fed the control diet, while the 3rd and 4th groups fed BG at 0.5 g/kg and the 2nd and 4th groups were exposed to DLM (15 μg/L) in rearing water. DLM-treated fish displayed a considerable increase in blood biochemical parameters (creatinine, urea and bilirubin) as well as hepatic enzymes (ALP, AST and ALT) (P < 0.05). Blood total protein, globulin, albumin, WBCs, RBCs, Hb, phagocytic index, phagocytic and lysozyme activities were significantly decreased in fish subjected to DLM (P < 0.05). Fish fed BG showed significantly the lowest cortisol and glucose levels, while fish exposed to DLM without feeding BG showed the highest cortisol and glucose levels (P < 0.05) after 15 and 30 days. Additionally, DLM toxicity caused downregulation in antioxidant (CAT and GPx) and immune (IL-1β and IL-8) related gene expressions, while and IFN-γ, HSP70 and CASP3 were upregulated. The histopathological examination of Nile tilapia exposed to DLM revealed damage in gills, intestine, spleen and liver which confirmed the toxic effects. Conversely, BG presented protective effects and restored the above-mentioned parameters when fish exposed to DLM and fed BG. Thus, BG supplementation exhibited defensive effects against DLM toxicity in Nile tilapia through improving blood biochemical responses, immune, and antioxidant related gene expressions as well as histopathological effects.

Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles - arginine, ornithine and citrulline - perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24 h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.

Inactivated vaccines are often applied with adjuvants in commercial fish farming. Although some mineral or non-mineral oil adjuvants show efficient improvement with inactivated vaccines, but sometimes bring side effects such as tissue adhesion and granulomatous lesion at the injection site. CpG ODN is a novel type of soluble adjuvant which has been proved to possess excellent advantages in fish vaccine development. In this study, we designed a tandem sequence of CpG ODN synthesized in plasmid pcDNA 3.1, and an inactivated Vibrio anguillarum vaccine developed in our previous work was chosen for determining the efficiency of the CpG-riched plasmids (pCpG) as an adjuvant. Results showed that pCpG we designed can offer higher immunoprotection with the vaccine. Interestingly, even below the minimum immune dosage of the vaccine, a high RPS of 84% was observed once the vaccine was administrated with the pCpG. Serum specific antibody titer, superoxide dismutase and total protein were enhanced and some immune genes related to both innate and adaptive immune response were upregulated, implying an effective auxiliary function of the pCpG. Totally, our study suggested that the pCpG is a potential and available adjuvant for turbot vaccine development.

The present study assessed the potential benefits of myrcene administration to suppress negative effects of copper exposure on immune-, antioxidant-, tight junction-, stress- and osmoregulatory-related gene expressions in common carp (Cyprinus carpio) gill. Fish were fed with diets containing 0% (control), 0.5% (M0.5) and 1% (M1) myrcene for 6 weeks, and then, exposed to 0.25 mg/L copper for further two weeks. The fish gill samples were taken for gene expression assays after six and eight weeks. The results showed that there were interaction effects of myrcene levels and copper exposure on superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione-s-transferase (gst), glutathione reductase (gr), heat shock protein-70 (hsp70), interleukin 1-beta (il1b), interleukin 10 (il10), tumor necrosis factor-alpha (tnfa), occludin (occl), caludin 3 (cld3), caludin 7 (cld7), and Na+-K+-ATPase (nka) genes expressions. Overall, the M0.5 treatment had significantly lower antioxidant genes expression, and higher hsp70, cytokines, tight-junction proteins, and nka genes expression, compared to the control treatment, before copper exposure. Copper exposure significantly down-regulated most of the tested genes (except il10), however, the M0.5 treatment had significantly higher antioxidant (except gpx), hsp70, cld7, and nka gene expression compared to the control treatment. The M1 treatment showed fluctuated antioxidant gene expressions, down-regulated gene expression of the pro-inflammatory cytokines, and occl, and up-regulation of cld3 gene expressions, before copper exposure. After copper exposure, this treatment had significantly higher gr and cat expression compared to the control; moreover, there was a marked up-regulation in il10 gene expression in this treatment, which was the highest value among all treatment combinations. In conclusion, copper exposure significantly down-regulates antioxidant-, inflammatory-, and tight junction-related along with hsp70 and nka genes expression in common carp gills. Pre-treatment with 0.5% myrcene is beneficial to suppress such negative effects, probably due to its antioxidant properties. However, myrcene administration must be done with caution, as higher levels may interfere with antioxidant and immune defenses.

The histone deacetylase, sirtuin 6 (SIRT6), plays an essential role in the regulation of oxidative stress, mitochondrial function and inflammation in mammals. However, the specific role of SIRT6 in invertebrate immunity has not been reported. Here, we characterized for the first time, a sirtuin 6 homolog in Litopenaeus vannamei (LvSIRT6), with full-length cDNA of 2919 bp and 1536 bp open reading frame (ORF) encoding a putative protein of 511 amino acids, which contains a typical SIR2 domain. Sequence and phylogenetic analysis revealed that LvSIRT6 shares a close evolutionary relationship with SIRT6 from invertebrates. Real-time quantitative PCR analysis of LvSIRT6 transcripts revealed that they were ubiquitously expressed in shrimp and induced in hepatopancreas and hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting the involvement of LvSIRT6 in shrimp immune response. Moreover, knockdown of LvSIRT6 decreased mitochondrial membrane potential and increased total ROS level in hemocytes, especially upon V. parahaemolyticus challenge. Depletion of LvSIRT6 also increased hemocytes apoptosis in terms of decreased expression of pro-survival LvBcl-2, but increased expression of pro-apoptotic LvBax and LvCytochrome C, coupled with high LvCaspase3/7 activity. Shrimp were rendered more susceptible to V. parahaemolyticus infection upon LvSIRT6 knockdown. Taken together, our present data suggest that LvSIRT6 plays an important role in shrimp immune response by modulating hemocytes ROS production and apoptosis during pathogen challenge.

Procambarus clarkii is one of the most important aquatic invertebrates in China and has high commercial value. However, aquaculture has suffered great economic loss due to outbreaks of infectious diseases in P. clarkii. To identify red swamp crayfish related genes involved in the response to bacterial infection, we analysed immune-related genes following lipopolysaccharide (LPS) stimulation by quantitative proteomics. The proteome of the hepatopancreas of P. clarkii challenged with LPS and phosphate-buffered saline was analysed to evaluate the immune response. Based on liquid chromatography coupled with tandem mass spectrometry, 16 upregulated and 29 downregulated proteins were identified. A Gene Ontology analysis demonstrated 5 biological process, 11 cellular component, and 6 molecular function subcategories. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the identified proteins were mainly involved in metabolism, phagosome, and ribosome. Real-time quantitative reverse transcription-PCR revealed that eight immune-related genes were upregulated after LPS stimulation compared to the control. Taken together, the data enhance our understanding of the immune response of crayfish to LPS.

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a β-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, β-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that β-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, β-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in β-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of β-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Anti-lipopolysaccharide factors (ALFs), as main host-defense molecules of crustaceans, form a unique family of antimicrobial peptides (AMPs). MnALF4 is one isoform of ALFs isolated from the freshwater prawn Macrobrachium nipponense. In the present study, MnALF4 gene was successfully expressed in the yeast Pichia pastoris and the recombinant MnALF4 protein exhibited efficient and broad-spectrum antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria in vitro. When prawns were injected with rMnALF4 before bacterial challenge with E. coli, the recombinant protein effectively promote the elimination of bacteria by the host. It manifested that rMnALF4 could effectively kill the invading bacteria in vivo. Treatment with rMnALF4 led to remarkable changes in bacterial morphology, such as spheroidization, oversized bacteria, and cell lysis. In addition, rMnALF4 showed weak hemolysis activity to the rabbit red blood cells. Our work suggests that MnALF4 plays an important role in Macrobrachium immunity and is worthy of further investigation as a potential antibacterial agent with high efficacy against bacterial infection and low toxicity to host cells.

Astakine is a crucial factor in the proliferation and differentiation of hematopoietic stem cells and is directly involved in hematopoiesis in crustaceans. To assess the role of Astakine in the innate immune system of Scylla paramamosain, the immune responses in healthy and Astakine-inhibited S. paramamosain were investigated in the present study. The RNA transcripts of Astakine were widely distributed in all examined tissues, with significantly higher levels of expression in hemocytes of both healthy and challenged S. paramamosain with Vibrio alginolyticus and WSSV. When Astakine was knocked down by RNA interference technology, immune-related genes, including Janus kinase, prophenoloxidase, hemocyanin, β-actin, myosin II essential light chain-like protein, signal transducer and activator of transcription, Relish, and C-type-lectin, were significantly down-regulated in hemocytes. The levels of phenoloxidaseactivity (PO), total hemocyte counts (THC) and hemocyte proliferation decreased significantly in hemocytes of Astakine-dsRNA treated S. paramamosain. After being challenged with V. alginolyticus and WSSV, the THC decreased significantly and the levels of hemocyte apoptosis increased significantly in Astakine-dsRNA treated S. paramamosain in comparison with those in infected groups without Astakine-dsRNA treatment. After being challenged with WSSV, the WSSV copies were significantly lower in Astakine-dsRNA treated groups than those in the WSSV infection group, which suggested that knockdown of Astakine was not conductive to WSSV replication and this might be associated with the decreasing THC. The results of survival analysis showed that the survival rate of V. alginolyticus or WSSV infected S. paramamosain decreased significantly following Astakine knockdown. These results suggested that RNA interference of Astakine might weaken the resistance of S. paramamosain to V. alginolyticus or WSSV infection. The weaken resistivity after knockdown Astakine might be related to the changes of important immune-related gene expression, THC, PO activity, proliferation and apoptosis of hemocytes.

Toll is essential in innate immune system which is important for defense against bacterial, fungal and viral infections in invertebrates. Our previous study showed that cadmium (Cd) could change the expression pattern of ShToll3 in the epithelium (gills and midgut from the freshwater crab Sinopotamon henanense) infected by Aeromonas hydrophila. To investigate the diverse innate immune roles of crustacean homolog Tolls, in this study, we cloned Shtoll1 from S. henanense. The full-length cDNA of Shtoll1 was 4746 bp, with an ORF of 3033 bp encoding a putative protein of 111 amino acids, a 5′-untranslated region of 255 bp and a 3′-untranslated region of 1713 bp. Phylogenetic analysis showed that ShToll1 was clustered into the group of DmToll1, DmToll 4 and DmToll 5. In addition, the tissue distribution results showed that Shtoll1 was expressed widely in different tissues, with the highest expression in heamocytes. Besides, Shtoll1 expressions were upregulated in heamocytes and hepatopancreas after A. hydrophila infection. At the same time, the increase of Shtoll1 expressions were examined in heamocytes in response to Cd exposure and A. hydrophila infection in combination. Through western blotting and immunohistochemical analysis, the ShToll1 expressions in heamocytes were increased in response to A. hydrophila and Cd independently as well as in combination. Moreover, the mRNA level of three antimicrobial peptides (AMPs) alf5, alf6, and c-lys, which possibly responded to Cd and A. hydrophila stimulation through Shtoll1, were analyzed. Thus, we conclude that Cd expand the susceptibility of ShToll1 to A. hydrophila infection in heamocytes. This suggest that ShToll1 may contribute to the innate immune defense of S. henanense against A. hydrophila and Cd in heamocytes.

The ectoparasite, Lepeophtheirus salmonis (Kroyer 1837), is effective at avoiding elimination from its host, Atlantic salmon, Salmo salar L., by inhibiting the recruitment of immune cells to the site of attachment. In other ectoparasitic arthropods, numerous factors have been identified that bind or neutralize chemokines preventing their interaction with receptors on the surfaces of immune cells. To determine if L. salmonis is utilizing a similar mechanism of immune modulation, the chemotactic activity of peripheral blood leukocytes (PBL) to leukotriene B4 (LTB4) and the secreted/excreted products (SEPs) of the sea louse were investigated in vitro. The results showed that incubation of LTB4 with SEPs reduced leukocyte migration compared to LTB4 immune stimulation alone. Data suggests that one of the mechanisms L. salmonis may be using to regulate immune cell recruitment in Atlantic salmon is by inhibiting or neutralizing the activity of chemokines.

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F1F0-ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (MjATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). MjATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant MjATPIF1 protein injection. Further study found that MjATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that MjATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation.

L-type lectins (LTLs) belong to the lectin family and are characterized by a conserved structural motif in their carbohydrate recognition domain. LTLs are homologous to leguminous lectins. In this study, we identified and functionally characterized an LTL from kuruma shrimp Marsupenaeus japonicus. We designated this LTL as MjLTL2. MjLTL2 contains a signal peptide, a Lectin_leg domain, a coiled coil, and transmembrane domain. MjLTL2 is distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestine; higher expression levels are seen in hemocytes and the hepatopancreas than in other tissues. MjLTL2 was upregulated following challenge of shrimp with Vibrio anguillarum and white spot syndrome virus (WSSV). MjLTL2 can agglutinate several bacteria without Ca2+. In addition, MjLTL2 could bind to several Gram-positive and -negative bacteria by binding to their lipopolysaccharide and peptidoglycan. However, MjLTL2 could not enhance the clearance of V. anguillarum in vivo. In the presence of WSSV infection, MjLTL2 knockdown by RNA interference resulted in a 7-day lower cumulative mortality of M. japonicus. Moreover, less VP19, VP24, VP26, and VP28 mRNAs were extracted from the hemocytes of MjLTL2 knockdown shrimp than from the control. These results suggest that MjLTL2 is involved in immune responses in shrimp.

Effect of bio-augmentation of Bacillus spp in biofloc on growth, survival and immunity in Indian white shrimp Penaeus indicus was evaluated. Nine Bacillus strains were isolated and screened individually as well as in the form of a consortia. To maintain a C:N ratio of 12:1 a blend of carbohydrate sources was used. Bio-augmentation with bacterial consortium and Virgibacillus sp. produced improved growth and immunity. Shrimp survival ranged from 80 to 95% among treatments. Production was higher (35%) in the biofloc tanks with an average body weight (ABW) of 10.89 ± 1.2 g. On evaluating the immune responses, it was found that trypsin significantly (P < 0.05) enhanced Prophenoloxidase (PO) activity in Lysinibacillus, Bacillus cereus, Bacillus licheniformis and Bacillus subtilis bio-augmented groups. Laminarin induced PO activity was observed in groups supplemented with Oceanobacillus sp., Bacillus sp.and Bacillus megaterium. The lysozyme (LZ) activity was significantly (P < 0.05) higher in B. cereus and Microbial Consortia (MC), while other treatments were less effective. Total hemocyte count (THC) significantly (P < 0.05) increased in all treatment groups compared to the control. Hyaline hemocyte (HH) count was significantly (P < 0.05) higher in the control group (14.43%). Semi granular hemocytes (SGH) was higher in groups treated with Lysinibacillus, Bacillus sp., B. licheniformis and B. subtilis. The granular hemocyte (GH) count was significantly (P < 0.05) higher in Virgibacillus sp., B. cereus, B.megaterium and Oceanobacillus sp. The biofloc alone (BF), treated and augmented with B. megaterium significantly (P < 0.05) increased phagocytic activity. Highly significant phagocytic index (PI) was observed in bio-augmented groups, BF and MC. The relative expression levels of immune genes were found to be significantly up-regulated in shrimps grown in bio-augmented groups. Enhanced immunological parameters implies that bio-augmentation of biofloc with Bacillus spp. improved immunity in shrimps. Hence, bio-augmentation of probiotics in biofloc may be useful in improving culture conditions to produce P. indicus.

Previous studies have indicated that white spot syndrome virus (WSSV) infection induces apoptosis in many shrimp organs. However, the mechanism by which WSSV causes host apoptosis remains largely unknown. In this study, we demonstrated the function of wsv152, the first mitochondrial protein identified as encoded by WSSV. Glutathione S-transferase pulldown and co-immunoprecipitation analysis revealed that wsv152 interacts with the shrimp mitochondrial protein cytochrome c oxidase 5a (COX5a), a subunit of the COX complex. We also found that wsv152 expression significantly increased the rate of apoptosis, suggesting a role of wsv152 in WSSV-induced apoptosis in shrimp. Knockdown of wsv152 in vivo led to downregulation of several apoptosis-related shrimp genes, including cytochrome c, apoptosis-inducing factor and caspase-3. Suppression of wsv152 also resulted in significant reductions in the number of WSSV genome copies in tissues and in the mortality of WSSV-infected shrimp. Together, these results suggest that wsv152 targets host COX5a and is associated with the expression profiles of apoptosis-related shrimp genes. Wsv152 is likely also involved in WSSV-induced apoptosis, thereby facilitating virus infection and playing a complex role in WSSV pathogenesis.

Cryptocaryon irritans infection could cause huge economic losses to the marine fish industry. Larimichthys crocea, a special economic species in China, suffered from the threat of serious infection, and L. crocea could enhance the level of piscidin 5-like to defense against the infection. This study set out to observe the main histopathological changes of some key tissues caused by infection, and determineed how an ectoparasite affected the expression of piscidin-5 like in its hosts. Pathological changes and immune response were assessed using histological and in situ hybridization (ISH) technologies. The infection induced inflammation occurring, especially in the gill where epithelium cells swell, hyperplasia, necrosis shedding adjacent to the parasites attachment sites. Infected hepatic cells grown big vacuoles in the cytoplasm. The boundary between red pulp and white pulp turned indistinct, splenic corpuscle lost the normal structure, the number and size of melano-macrophage centers increased apparently in the infected spleen. The whole structure of head kidney became loose. Immunostaining with RNA probes against piscidin 5-like showed subpopulations of mast cells (MCs) were positive. Piscidin 5-like-positive MCs existed mainly in the head kidney where they distributed around melano-macrophage center, followed in the gill located at different positions they also distributed in the margin of spleen, and randomly and sparsely existed in the liver. After being infected by C. irritans, the gill arch arose positive MCs groups, and they also migrated to spleen, while the positive staining deepen in other detected tissues. Therefore, organism enhanced the expression level through improving expression ability of positive MCs, or increasing the number of positive MCs.

In this study, rainbow trout (Onchorhynchys mykiss) weighing 27.75 ± 0.34 g were orally subjected to eight experimental diets each in three replicates containing varying amounts of chitosan and nano-chitosan (0.05, 0.5 and 5 g kg−1) loaded in clinoptilolite (14.28 g kg−1) for 70 days; and the growth and immune responses were evaluated. Results showed that growth parameters in fish fed diets chit + clin2, chit + clin3, nchit + clin1, nchit + clin2 and nchit + clin3 were significantly higher than in fish fed the control diet. All feeds, except chit + clin3, and nchit + clin3, significantly increased the total protein level. Feeds containing chit + clin2, nchit + clin1, and nchit + clin2 significantly elevated serum lysozyme activity compared with the control group. All treatments, except chit + clin3, and nchit + clin3 exhibited higher serum immunoglobulin (Ig) level than control one. In contrast, diet nchit + clin1 significantly unregulated the expression of Ig M gene in fish head-kidney compared to other groups. Additionally, all feeds, except clinoptilolite, and nchit + clin3, significantly improved the serum complement activity. Diets chit + clin2, nchit + clin1, and nchit + clin2 also significantly elevated antibacterial activity against Yersinia ruckeri compared with the control diet. Expression of inducible nitric oxide synthase (iNOS) gene in fish fed diets clinoptilolite, chit + clin1, chit + clin3, nchit + clin1, nchit + clin2, and nchit + clin3 was significantly higher than the control diet. All diets, except clinoptilolite, increased IL-1β gene expression compared to the control group. Present results suggest that diets supplemented with nchit + clin, especially at 0.05 g kg−1 nano-chitosan inclusion, could improve growth performance and immune parameters of rainbow trout.

Quantification real-time PCR (qRT-PCR) is a common method in analysis of gene expression, but the stable reference genes for the normalization analysis have not been appreciated before identifying expression pattern of genes in teleost fishes. In this study, we selected eight candidate reference genes (18S, Actin, EF-1α, 40S, B2M, TUBA, UBCE and GAPDH) basing on transcriptome analysis and the traditional housekeeping genes, and analyzed the stability of the reference genes in spleen, head kidney and head kidney leukocytes (HKL) after pathogen challenge in Schizothorax prenanti (S. prenanti). Three common programs (geNorm, NormFinder and Bestkeeper) were used to evaluate the stability of the candidate reference genes. Two reference genes, Actin and EF-1α presented higher stability, while 18S and GAPDH were the lower stable genes, both in in vitro and in vivo. An important immune gene, toll-like receptor 22a (TLR22a), was selected to validate the stability of the proposed reference genes (Actin and EF-1α) across different experiment treatments. The results reveal that Actin and EF-1α are quite suitable reference genes for the normalization analysis. Otherwise, using the most stable gene Actin to validate the reliable of transcriptome data showed the high correlation between the fold change of transcriptome data and qRT-PCR data. In conclusion, our study not only acquired the suitable reference gene for the qRT-PCR assay under specific experiment condition, but also provided a comprehensive method to evaluate and validate the reference gene based on transcriptome analysis in teleost fishes.

Vibrio harveyi is the pathogen causing vibriosis in marine-cultured animals, leading to massive deaths in farmed grouper around the world. It is urgent to develop an effective vaccine to prevent vibriosis. In the previous study, we developed a V. harveyi formalin-killed cells vaccine (FKC), and sought an effective adjuvant for enhancing the immune efficacy of vaccine. In this study, we aimed to evaluate the immune responses and protective effect of FKC combined with chitosan oligosaccharide (COS) or Astragalus polysaccharides (APS) in the pearl gentian grouper♀Epinephelus fuscoguttatus × ♂E. lanceolatus. The results indicated the vaccine triggered a remarkably higher expression levels of IL-1β, IL-16, TNF-α, MHC-Iα and IgM in the kidney and spleen of groupers post-vaccination. Antibody titers, lysozyme, catalase, superoxide dismutase and total protein were significantly elevated in the vaccinated fish compared with those in the control. The experimental groupers were challenged intraperitoneally by V. harveyi at 35 d post-vaccination, and the relative percentage of survival (RPS) of group FKC + COS, FKC + APS, COS, APS and FKC were 80%, 72%, 52%, 47% and 55%, respectively. These results demonstrated COS and APS was the potential adjuvants for FKC against V. harveyi in aquaculture.

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 109 cfu kg diet−1. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analyses. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

The present study addressed the possible effects of fishwort (Houttuynia cordata) powder (FWP) on Nile tilapia's skin mucus parameter, serum immune response, and growth performance. Three hundred twenty tilapia fingerlings (average weight of 39.06 ± 0.16 g) were divided into four treatments and fed four levels of FWP; 0, 5, 10, and 20 g kg−1 for 72 days. Completed randomised design of the four replications was applied and revealed that fish fed FWP significantly improved skin mucus lysozyme activity (SMLA). The highest value (P < 0.05) was recorded in fish fed 10 g kg−1 FWP. However, no significant difference in SMLA was observed by feeding the fish 5 and 20 g kg−1 FWP. Significant (P < 0.05) enhanced skin mucus peroxidase activity (SMPA) was observed in fish fed 10 g kg−1 FWP, but no significant difference in SMPA was detected between FWP supplemented diets (5 and 20 g kg−1 FWP) and the control group. Regarding serum immunity, dietary administration of FWP showed significantly (P < 0.05) improved serum lysozyme, peroxidase, alternative complement (ACH50), and phagocytosis vs. the control. The highest values of serum immunity (P < 0.05) were recorded in fish fed 10 g kg−1 FWP. However, no significance in respiratory burst activity was observed. Similarly, no significant difference in growth performance, feed conversion ratio, and survival rate was observed in fish fed FWP compared to the control. In summary, diets supplemented with FWP (10 g kg−1) increased the serum and mucosal immunity; however, no FWP supplementations had effects on Nile tilapia growth and survival rate.

Proteins in the tumor necrosis factor receptor (TNFR) superfamily play significant roles in many physiological and pathological events, such as inflammation, apoptosis, autoimmunity, and organogenesis. Here, two TNFR gene homologs (PmTNFR1 and PmTNFR5) were identified in the pearl oyster Pinctada fucata martensii. The predicted PmTNFR1 and PmTNFR5 protein sequences were 406 and 533 amino acids long, respectively, and both possessed motifs characteristic of the TNFR family, including a TNFR homology domain (CRD), a transmembrane domain (TM), and death domains. However, the predicted amino acid sequences of PmTNFR1 and PmTNFR5 had low identity (∼16–23%) with sequences of vertebrate TNFR family proteins. Furthermore, PmTNFR5 had a death domain at the C-terminal, indicating that this protein may be a novel member of the TNFR superfamily. Constitutive PmTNFR1 and PmTNFR5 mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the hepatopancreas and gill. The gene expression levels of PmTNFR1 and PmTNFR5, as well as those of downstream signaling molecules related to the NF-κB pathway (RIP, TRAF2, TRAF3, IKK, and NF-κB), were quantified in the gill after LPS challenge and in the hemocytes after nucleus insertion surgery using real-time PCR (qRT-PCR). We found that all genes were significantly upregulated at 6 h and 12 h post-injection, as well as at 15 d post-insertion. We used RNAi to inhibit the expression of the PmTNFR1 and PmTNFR5 genes. We then quantified the expression levels of PmTNFR1 and PmTNFR5, as well as downstream genes, using qRT-PCR. We found that RNAi inhibition of PmTNFR1 and PmTNFR5 downregulated the downstream genes (RIP, TRAF2, TRAF3, IKK, and NF-κB). Therefore, our results suggested that PmTNFR1 and PmTNFR5 mediate the NF-κB signaling pathway, and are closely related to immune defense, particularly allograft immunity, in the pearl oyster P. fucata martensii.

Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 3 was 1562 bp and included a 175 bp 5′-untranslated region (UTR), a 141 bp 3′-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, ChCas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of ChCas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis.

Granulocyte colony-stimulating factor (GCSF) is a growth factor that drives the proliferation and differentiation of granulocytes and monocytes/macrophages. Currently, two copies of GCSF, named GCSFa and GCSFb, have been identified in teleost fish, but data on the functions and signal pathways of these fish GCSFs are still limited. In the present study, a GCSFa homologue (LcGCSFa) was identified from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LcGCSFa is 636 bp long and encodes a protein of 211 amino acids (aa), with a 19-aa signal peptide and a typical IL-6 domain, conserved in fish GCSF sequences. The phylogenetic analysis showed that LcGCSFa clustered with other fish GCSFa homologues. LcGCSFa was constitutively expressed in all tissues tested and significantly up-regulated in head kidney and spleen by Vibrio alginolyticus or poly(I:C). LcGCSFa transcripts were also detected in primary head kidney leucocytes (PKL), primary head kidney macrophages (PKM), and primary head kidney granulocytes (PKG), and significantly up-regulated in PKL and PKG by LPS or poly(I:C). These data indicated that LcGCSFa may be involved in the immune responses induced by bacterium and virus. The recombinant LcGCSFa protein (rLcGCSFa) produced in Pichia pastoris promoted the proliferation of PKL both in vivo and in vitro. Furthermore, rLcGCSFa significantly increased both transcription and phosphorylation levels of the signal transducers and activators of transcription (STAT) proteins (LcSTAT3 and LcSTAT5) in PKL, which are required for the GCSF-dependent proliferation. These results showed that LcGCSFa may promote the proliferation of PKL via the activation of LcSTAT3 and LcSTAT5, suggesting a conserved role across vertebrate GCSFs.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

Mannose-binding lectin (MBL) is a crucial pattern recognition receptor in the host innate immune system. Previously, we reported the biological function of Ctenopharyngodon idella MBL (CiMBL) in initiating the lectin pathway of the complement system. In the present study, we further explored its biological function including the agglutinating ability, binding capacity and protective role in vitro and in vivo. After Aeromonas hydrophila infection, western blot analysis revealed that the CiMBL were fluctuated and expressed in the serum and major immune-related tissues. The result of quantitative PCR (qPCR) showed that the recombinant CiMBL (rCiMBL) significantly inhibited the mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in liver, spleen and hepatic cells. Due to rCiMBL bound to d-mannose, d-galactose, d-glucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS), peptidoglycan (PGN) and Agar in the presence of Ca2+, herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca2+-dependent manner. More importantly, rCiMBL enhanced the survival rate of grass carp following bacterial infection. Overall, the results provide an evidence that CiMBL can protect grass carp against A. hydrophila infection in aquaculture.

Troponin C (TnC) is one member of the EF-hand superfamily. In many species, this gene had been identified and related functions had been elucidated. The TnC gene was still blank in the Scylla paramamosain. We obtained the TnC gene for the first time in the S. paramamosain. And we systematically analyzed the possible role of this gene in the innate immunity of S. paramamosain while infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The full-length 1427 bp sequence of TnC contains a 453 bp open reading frame (ORF) for encoding a 151 amino acid protein. Detection of tissue specificity of gene expression showed that the TnC was primarily expressed in muscle tissue. The expression of TnC was successfully inhibited by RNA interference technology, and several immune genes were affected. The activity of phenoloxidase and superoxide dismutase increased, and the total hemocytes counts increased after RNAi of TnC. It was found that after infection with V. alginolyticus and WSSV, the expression of TnC in hemocytes decreased. Infected with V. alginolyticus and WSSV, the cumulative mortality and apoptotic rate of hemocytes increased after silencing the TnC gene. Our results indicate that TnC takes participate in the innate immunity of S. paramamosain and may plays a different role in the antiviral and antibacterial immune response.

Tyramine (TA), a biogenic monoamine, plays various important physiological roles including immunological regulation in invertebrates. In this study, the effects of TA on the regulation of immune resistance, carbohydrate metabolism and biogenic monoamine, as well as its signaling pathway in Macrobrachium rosenbergii were determined. Results showed that total haemocyte count, hyaline cells, semigranular cells, and phenoloxidase activity per 50 μL of haemolymph and per granulocyte (the sum of semigranular and granular cells) at 0.5 h as well as phagocytic activity and clearance efficiency to Lactococcus garvieae at 1 h of prawn injected with TA at 1 nmol prawn−1 significantly increased, but the significantly decreased plasma lysozyme activity, phagocytic activity, clearance efficiency, and haemolymph glucose and dopamine were observed in prawn injected with TA at 10 nmol prawn−1 for 0.5 h. Respiratory bursts and haemolymph lactate in two TA-injection treatments at 0.5 h and 0.5–1 h, respectively, were significantly higher than those of the saline control, and in addition, TA depressed dopamine release in a dose-dependent manner after 0.5 h of TA injection. All the examined parameters returned to control levels after prawn injected with TA for 2 h. The inhibited effect of TA (at 10 nmol prawn−1 injection) on the phagocytic activity and clearance efficiency to pathogens was blocked by prazosin (an α1 adrenoceptors antagonist). For prawn received TA for 1 h then challenged with Lactococcus garvieae at 2 × 105 colony-forming units prawn−1, the survival ratio of TA 1 nmol prawn−1-injected prawn significantly increased by 20%, compared to the saline-challenged control or TA 10 nmol prawn−1-injected prawn after 144 h of challenge. These results suggested that the level of dopamine release suppression regulated by TA resulted in the immunoenhancing or immunosuppressive effects in prawn, and the signaling pathways of TA in mediating immune function were through octopamine (OA)/TA receptors.

Two in vitro trials were conducted to identify a peptide with antioxidant activity and immunoenhancement from cottonseed meal protein hydrolysate (CPH) for fish. Primary hepatocytes of Megalobrama amblycephala were treated with CPH. In experiment 1, CPH significantly increased aspartate aminotransferase (GOT), alanine aminotransferase (GPT), total superoxide dismutase (t-SOD), catalase (CAT), and lysozyme activities, as well as up-regulated SOD, CAT, antimicrobial peptides 1 (Leap 1) and Leap 2 mRNA levels (p < 0.05). However, CPH significantly down-regulated the expression of NADPH oxidase-2 (NOX2), Kelch-like-ECH-associated protein 1 (Keap1), NF-E2-related factor 2 (Nrf2) and BTB and CNC homolog 1 (Bach1) mRNA (p < 0.05) in fish hepatocytes. Experiment 2 showed that the molecular mass of CPH was distributed mainly in the 700–1024 Da range. Peptide 1 (P1) and P2 significantly decreased GOT and GPT activities in conditioned medium (p < 0.05); however, P4 and P6 did not affect GOT and GPT activities (p > 0.05). Furthermore, P4 significantly increased hepatocyte GOT, GPT, t-SOD, CAT levels and lysozyme activities (p < 0.05), up-regulated SOD, CAT, Leap1 and Leap2 mRNA expression levels, and down-regulated the expression of Nrf2 and NOX2 mRNA (p < 0.05) in fish hepatocytes. The above results indicated that CPH and P4 enhanced hepatocyte metabolism, as well as improved antioxidant capacities and innate immunity of blunt snout bream hepatocytes.

Apart from mitigating endoplasmic reticulum (ER) stress, vast studies have demonstrated the crucial role of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α) - spliced X-box binding protein 1 (XBP1s) signaling pathway in inflammatory response in mammals. In addition, palmitic acid (PA)-induced inflammation has been verified in large yellow croaker (Larimichthys crocea). However, whether the IRE1α-XBP1s signaling pathway is involved in inflammatory response caused by PA remains poorly studied in fish. The present study was aimed at elucidating the role of the IRE1α-XBP1s signaling pathway in inflammatory response induced by PA in primary hepatocytes from large yellow croaker. In the present study, the full-length cDNA of ire1α and xbp1s were cloned and comprised 3793 bp and 1789 bp with an open reading frame of 3279 bp and 1170 bp, encoding 1093 and 390 amino acids, respectively. IRE1α protein possessed a protein kinase and endoribonuclease domain and XBP1s protein possessed a basic-leucine zipper domain. The IRE1α protein and XBP1s protein located to the ER membrane and nucleus respectively. The ire1α and xbp1s were widely transcribed in various tissues with the higher level in intestine, liver, adipose and head kidney. The ER stress-inducing agent tunicamycin (Tm) and PA treatment significantly activated the IRE1α-XBP1s signaling pathway and increased the pro-inflammatory genes expression including tumor necrosis factor α (tnfα), interleukin 6 (il-6) and interleukin 1β (il-1β) (P < 0.05). When KIRA6, the IRE1α kinase inhibitor, was used to block the IRE1α-XBP1s signaling pathway, the Tm and PA-induced pro-inflammatory genes expression was significantly suppressed (P < 0.05). These data indicated that the IRE1α-XBP1s signaling pathway was involved in the PA-induced inflammatory response in large yellow croaker.

Ghrelin is a peptide hormone secreted by gastrointestinal tract which regulates multiple physiological processes such as appetite, metabolism, growth and gonad development in fish. In the present study, the effects of ghrelin on hybrid tilapia infected with Aeromonas hydrophila are elucidated. Juvenile hybrid tilapia fish (20.0 ± 5.0 g) were intraperitoneally injected with 0, 0.1, 1.0, or 10.0 ng/g ghrelin/body weight synthetic ghrelin alone or in combination with A. hydrophila (0.5 × 106 CFU). At 10 days post treatment, the survival rate in the group that received 1.0 ng/g ghrelin/body weight ghrelin in combination with A. hydrophila was higher (66.66%) than that of the Ah group (13.33%) that received A. hydrophila alone. In tilapia that received ghrelin injections, reactive oxygen species (ROS) levels tended to increase at 5 h, while injection of 10.0 ng/g ghrelin/body weight ghrelin resulted in a significant decrease in ROS levels at 10 h. No changes in serum immune or antioxidant-related indicators were observed in fish injected with A. hydrophila compared to controls. However, ghrelin injection decreased Albumin (ALB), glutathione peroxidase (GSH-Px), lysozyme (LZM) and superoxide dismutase (SOD). Histological analysis showed that ghrelin injection alleviated the pathological changes in liver and spleen caused by A. hydrophila infection. Overall, the expression of HSP70, IL-1β, and TGF-β in the liver tended to upregulate compared to the control. In the kidney, HSP70, IL-1β and TGF-β levels were increased, and TNF-α expression levels were decreased compared to the control. The HSP70 level in the spleen was decreased, and IL-1β, TGF-β, and TNF-α were expressed at significantly higher levels in the spleen in the tilapia that received ghrelin injections. Taken together, our results indicate that injection with 1.0 ng/g ghrelin/body weight ghrelin may effectively protect juvenile hybrid tilapia against A. hydrophila infection by improving hematological indicators, maintaining normal histology and regulating cytokine gene expression.

Fish mucosal organs are directly exposed to aquatic environment, which is suited to the colonization and growth of microorganisms, and thus are considered to play an important role in maintaining homeostasis and preventing entry of invading pathogens. Researches on fish mucosal immunity have shown that mucosal organs in fish such as gill, skin, intestine, olfactory organs harbour lymphoid cells including T and B cells as well as dendritic-like cells. The findings on immune responses following direct administration with antigens into the mucosal organs will help to shed light on developing fish mucosal vaccines. The present review highlighted vaccine deliveries via mucosal organs, especially focusing on methods other than typical mucosal vaccine platforms, such as oral and immersion vaccines. In addition, we proposed a hypothesis that mucosal tissues are important sites for generating cell-mediated immunity following vaccination with extracellular antigens.

Mass mortality of juvenile hybrid (Haliotis discus hannai ♀× H. fulgens ♂, DF) and adult H. discus hannai (DD) occurs in south China during the summer. This study showed that the juvenile DF and adult DD exhibited significantly lower survival rates than juvenile DD and adult DF under 72 h pathogenic bacteria (Vibrio harveyi) challenge at different temperatures (20 °C and 28 °C). Phenoloxidase (PO) and superoxide dismutase (SOD) activities were significantly higher in juvenile DD compared to juvenile DF, whereas that in adult abalone was the opposite. Juvenile DD and adult DF also exhibited advantages in terms of immune-related gene expression (TRAF, TLR, MIF, Lys, Spi, Cat, TNF, and SOD) compared to juvenile DF and adult DD. The data reveals immunocompetence differences in DD and DF at the juvenile and adult stages.

Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.

Transcription factor activator protein 1 (AP1) plays an irreplaceable role in the response to a variety of external stimulants, such as cellar stress, bacterial and viral infections, and inflammatory cytokines. In this study, we identified a novel AP1 gene from Macrobrachium nipponense and named it MnAP1, which has a full length of 1747 bp contains an 882 bp open reading frame, and encodes a protein with 293 amino acids. The MnAP1 protein contains Pfam and bZIP domains. MnAP1 is widely distributed in hemocytes, heart, hepatopancreas, gill, stomach, and intestinal tissues. The expression levels of MnAP1 in the gills and stomach were significantly upregulated after Vibrio parahaemolyticus and Staphylococcus aureus attacks. We studied the relationship between MnAP1 and the transcripts of antimicrobial peptides (AMPs) in gills through RNA interference. Interestingly, the regulatory effects of MnAP1 on the expression of different AMPs were different. We found that the expression levels of crustins, including Cru1, Cru3, and Cru4 in the gills were evidently decreased, whereas the synthesis of Cru5 and anti-lipopolysaccharide factors (ALF3 and ALF4) were obviously increased. We further explored the effect of MnAP1 on the expression of transcription factor relish from M. nipponense. The result showed that the knockdown of MnAP1 can remarkably upregulate the expression of MnRelish. Relish as a member of the nuclear factor κB family that regulates the expression of AMPs in the innate immunity of crustacean. Hence, we also detected the expression levels of Cru5, ALF3, and ALF4 in the gills of MnRelish-silenced prawns. The Data showed that the expression levels of these three AMPs were evidently reduced after MnRelish silencing. Our results indicated that MnAP1 plays a positive role in regulating the expression of AMPs, promotes the JNK/AP1 signaling pathway, and exerts a negative regulatory effect on the synthesis of AMPs by inhibiting the transcription of NF-κB factor in the innate immunity of M. nipponense.

Coelomic fluid contains a population of coelomocytes, enzymes, nutrients and kinds of molecules that could be essential for Apostichopus japonicus live. The coelom and polian vesicle are the main tissues that hold the most coelomic fluid in the animal, but whether there exists any immunological difference of the coelomic fluid from the two tissues remains unknown. In this study, we first extracted the coelomic fluid both from the coelom and polian vesicle, and compared their non-specific immune factors. It was found that the ACP and AKP activities in the polian vesicle were significantly higher than those in the coelom, but it was contrary for the SOD and CAT. Meanwhile, the expression levels of several immune-related genes including AjC3-2, AjMKK3/6, AjTLR3 and AjToll in the polian vesicle were significantly lower than those in the coelom. Besides, the early changes of non-specific immune factors were further monitored after eviscerated. During 7 days post evisceration, the immunoenzymes activities of ACP, AKP, SOD and CAT were decreased first and then recovered gradually in the coelomic fluid from the coelom. In the polian vesicle, the ACP and AKP activities showed a similar trend with the coelom, while the SOD and CAT activities showed a transitory increase during 2 h post evisceration (hpe) to 12 hpe. Moreover, the expression profiles of the immune-related genes in the coelom reached the peak at 3 days post evisceration (dpe), while their expression levels in the polian vesicle reached the peak at 7 dpe. All the results suggested that the immunocompetence of coelomic fluid differed in the coelom and polian vesicle, and thus may exert their respective immunological functions. It was likely that the respond speed in the coelom would be faster than that in the polian vesicle after evisceration. Our data will provide a basis for better understanding of the immune defense mechanism of A. japonicus.

In Chile, the salmon and trout farmed fishing industries have rapidly grown during the last years, becoming one of the most important economic sources for the country. However, infectious diseases caused by bacteria, virus, mycoses and parasites, result in losses of up to 700 million dollars per year for the Chilean aquaculture production with the consequent increase of antibiotic and antiparasitic usage. After 30 years of its first appearance, the main salmon health problem is still the salmonid rickettsial septicaemia (SRS), which together with other disease outbreaks, reveal that vaccines do not provide acceptable levels of long-lasting immune protection in the field. On the other hand, due to the large dependence of the industry on salmonids production, the Chilean government promoted the Aquaculture diversification program by 2009, which includes new species such as Merluccius australis, Cilus gilberti and Genypterus chilensis, however, specific research regarding the immune system and vaccine development are issues that still need to be addressed and must be considered as important as the farm production technologies for new fish species. Based on the experience acquired from the salmonid fish farming, should be mandatory an effort to study the immune system of the new species to develop knowledge for vaccination approaches, aiming to protect these aquaculture species before diseases outbreaks may occur. This review focuses on the current status of the Chilean aquaculture industry, the challenges related to emerging and re-emerging microbial pathogens on salmonid fish farming, and the resulting needs in the development of immune protection by rational designed vaccines. We also discussed about what we have learn from 25 years of salmonid researches and what can be applied to the new Chilean farmed species on immunology and vaccinology.

Calpains (CAPNs) belong to the papain superfamily of cysteine proteases, and they are calcium-dependent cytoplasmic cysteine proteases that regulate a variety of physiological processes. We obtained the sequence of CAPN3 from an NGS-based analysis of Pagrus major (PmCAPN3) and confirmed the conserved molecular biological properties in the predicted amino acid sequence. The amino acid sequence and predicted domains of CAPN3 were found to be highly conserved in all of the examined species, and one catalytic domain and four calcium binding sites were identified. In healthy P. major, the PmCAPN3 mRNA was most abundantly expressed in the muscle and skin, and ubiquitously expressed in the other tissues used in the experiment. After artificial infections with fish pathogens, significant changes in its expression levels were found in immune-related tissues, most of showed upregulation. In particular, the highest level of expression was found in the liver, a tissue associated with protease activity. Taken together, these results suggest a physiological activity for PmCAPN3 in P. major and reveal functional possibilities that have not yet been reported in the immune system.

Scavenger receptors play a central role in defending against infectious diseases in mammals. However, the function of SRECII remains unknown in teleost fish. In this study, type F scavenger receptor expressed by endothelial cells-II (SRECII) cDNA sequence was first identified from Epinephelus coioides, named EcSRECII, which contained an N-terminal signal peptide, eight EGF/EGF-like cysteine-rich motifs and a C-terminal low-complexity region. The gene location maps revealed that EcSRECII has the conservation of synteny among selected species. Subcellular localization showed that EcSRECII was mainly located in the cytoplasm in HEK293T cells and GS cells. In healthy E. coioides, EcSRECII mRNA was highly expressed in spleen, skin, gill, thymus and head kidney. The relative EcSRECII mRNA expression after V. parahaemolyticus infection was significantly up-regulated at 12 h in spleen, head kidney and thymus, but downregulated at 1 d in skin and reduced at 3 d and 1 w in spleen. Furthermore, overexpression of EcSRECII activated NF-κB and IFN-β signaling pathway in vitro. Taken together, these results indicated that EcSRECII could be as the potential pathogen recognition receptor for involving in bacterial infection by regulating innate immunity responses in E. coioides.