Characterization and function analysis of cathepsin C in Marsupenaeusjaponicus.

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.

Identification and characterization of four Bacillus species from the intestine of hybrid grouper (Epinephelus fuscoguttatus♀ × E. lanceolatus♂), their antagonistic role on common pathogenic bacteria, and effects on intestinal health.

As an alternative to the criticized antibiotics, probiotics have been adopted for their eco-friendly nature and ability to enhance host growth and immunity. Nevertheless, reports suggest ineffectiveness in commercially available probiotics since most are from non-fish sources; thus, this study was envisaged to isolate and characterize new Bacillus spp. from the gut of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂) which could serve as potential probiotics. The isolation and characterization were performed based on their morphological and biochemical properties, and 16S rRNA sequencing homology analysis. A subsequent 30-day in vivo biosafety feeding trial was conducted to ascertain isolates' non-pathogenicity, as well as their effects on fish growth, and intestinal mucosal microvilli via scanning electron microscopy (SEM) analysis. Four Bacillus spp. strains, namely, B. velezensis strain PGSAK01 (accession number OQ726606), B. stercoris strain PGSAK05 (accession number OQ726607), B. velezensis strain PGSAK17 (accession number OQ726601), and B. subtilis strain PGSAK19 (accession number OQ726605), were identified and characterized in the current study. The strains showed promising probiotic properties such higher adhesion capability, higher thermotolerance, displaying higher survivability to 0.5 % bile, lower pH tolerance, γ-haemolytic activity, and multispecies characteristics. Among the 24 antibiotics tested, while all isolates showed susceptibility to 21, the PGSAK01 strain showed resistance to furazolidone antibiotics. None of the isolates showed possession of i) virulence factor genes encoding enterotoxigenic (hblA, hblC, hblD, nheA, nheB, and entFM) and emetic (cereulide synthetase gene, ces) genes, and ii) streptomycin resistance gene (vat c), ampicillin-resistant genes (mecA and bla), and vancomycin-resistant gene (van B). Nevertheless, the PGSAK01 and PGSAK17 strains showed possession of tek K, cat, and ant(4')-Ia (adenylyltransferase) (except the PGSAK01) resistant genes. All isolates displayed better antimicrobial effects against pathogenic bacteria Streptococcus agalactiae, S. iniae, Vibrio harveyi, and V. alginolyticus. The in vivo biosafety trial involved hybrid grouper fish being grouped into five (average weight 32 ± 0.94 g), namely, the group fed the basal diet void of isolate's supplementation (control), and the remaining four groups fed the basal diet with 1 × 108 CFU/g diet of individual strain PGSAK01, PGSAK05, PGSAK17, and PGSAK19 supplementation. At the end of the study, a significantly higher WGR, K (except the PGSAK01 group), VSI; lysozyme (except PGSAK01 group), total antioxidant activity, alkaline phosphatase, superoxide dismutase enzyme activities; highly dense intestinal mucosal villi (based on the scanning electron microscopy analysis); and significantly lower malondialdehyde levels were witnessed in the isolated treated groups compared to the control, supporting the results obtained in the auto-aggregation and cell-surface hydrophobicity test. This work's results have provided thought-provoking targets; thus, studies involving extensive genome sequencing and functional annotation analysis will be explored to offer unfathomable insights into their mechanisms of action and potential health benefits, further establishing the four Bacillus strains' (PGSAK01, PGSAK05, PGSAK17, and PGSAK19) potential role in probiotic fields and functional foods.

Involvement of Nrf2 in the immune regulation of Litopenaeus vannamei against Vibrio harveyi infection.

NF-E2-related factor-like-2 (Nrf2) is a transcription factor that belongs to the Cap'n'Collar transcription factor family and plays a role in regulating inflammation, autophagy, metabolism, proteostasis, and cancer prevention. However, its influence on Vibrio spp infection in L. vannamei remains uncertain. In this study, the effects of Nrf2 on the immune response in Vibrio spp infection was determined by RT-PCR and histopathological analysis. The results showed that RNAi of Nrf2 significantly decreased the expression of antioxidant-related genes (CAT, SOD and GST; p < 0.05), and significantly up-regulated inflammation-related genes (IMD, pro-PO, P38, Toll, Hsp70, NFκB and RAB6A; p < 0.05) and the apoptosis gene (caspase3). Under the infection of V. harveyi, histopathological analysis showed that after RNAi of Nrf2, the hepatopancreas of shrimp has an abnormal arrangement of hepatic tubules and vacuolization of hepatocyte; The basement membrane is peeled off and the epithelial cells are massively necrotic. Compared with the RNAi of Nrf2 group, the tissue damage in the SFN group was much lessened, and there were fewer apoptosis signals in the TUNEL assay. In conclusion, this experiment indicated that Nrf2 is involved in the regulation of inflammatory response, oxidative stress,and apoptosis induced by V. harveyi in L. vannamei.

A novel C-type lectin for Litopenaeus vannamei involved in the innate immune response against Vibrio infection.

C-type lectins (CTLs), as a member of pattern recognition receptors, play a vital role in the innate immune response of invertebrates to eliminate micro-invaders. In this study, a novel CTL of Litopenaeus vannamei, namely, LvCTL7, was successfully cloned, with an open reading frame of 501 bp and a capability to encode 166 amino acids. Blast analysis showed that the amino acid sequence similarity between LvCTL7 and MjCTL7 (Marsupenaeus japonicus) was 57.14%. LvCTL7 was mainly expressed in hepatopancreas, muscle, gill and eyestalk. Vibrio harveyi can significantly affect LvCTL7 expression level in hepatopancreases, gills, intestines and muscles (p < 0.05). LvCTL7 recombinant protein can bind to Gram-positive bacteria (Bacillus subtilis) and Gram-negative bacteria (Vibrio parahaemolyticus and V. harveyi). It can cause the agglutination of V. alginolyticus and V. harveyi, but it had no effect on Streptococcus agalactiae and B. subtilis. The expression levels of SOD, CAT, HSP 70, Toll 2, IMD and ALF genes in the challenge group added with LvCTL7 protein were more stable than those in the direct challenge group (p < 0.05). Moreover, knockdown of LvCTL7 by double-stranded RNA interference downregulated the expression levels of genes (ALF, IMD and LvCTL5) that protect against bacterial infection (p < 0.05). These results indicated that LvCTL7 had microbial agglutination and immunoregulatory activity, and it was involved in the innate immune response against Vibrio infection in L. vannamei.

Heme Oxygenase-1 Is Involved in the Repair of Oxidative Damage Induced by Oxidized Fish Oil in Litopenaeus vannamei by Sulforaphane.

Heme oxygenase-1 (HO-1), which could be highly induced under the stimulation of oxidative stress, functions in reducing the damage caused by oxidative stress, and sulforaphane (SFN) is an antioxidant. This study aims to investigate whether HO-1 is involved in the repair of oxidative damage induced by oxidized fish oil (OFO) in Litopenaeus vannamei by sulforaphane (SFN). The oxidative stress model of L. vannamei was established by feeding OFO feed (OFO accounts for 6%), and they were divided into the following four groups: control group (injected with dsRNA-EGFP and fed with common feed), dsRNA-HO-1 group (dsRNA-HO-1, common feed), dsRNA-HO-1 + SFN group (dsRNA-HO-1, supplement 50 mg kg-1 SFN feed), and SFN group (dsRNA-EGFP, supplement 50 mg kg-1 SFN feed). The results showed that the expression level of HO-1 in the dsRNA-HO-1 + SFN group was significantly increased compared with the dsRNA-HO-1 group (p < 0.05). The activities of SOD in muscle and GPX in hepatopancreas and serum of the dsRNA-HO-1 group were significantly lower than those of the control group, and MDA content in the dsRNA-HO-1 group was the highest among the four groups. However, SFN treatment increased the activities of GPX and SOD in hepatopancreas, muscle, and serum and significantly reduced the content of MDA (p < 0.05). SFN activated HO-1, upregulated the expression of antioxidant-related genes (CAT, SOD, GST, GPX, Trx, HIF-1α, Nrf2, prx 2, Hsp 70), and autophagy genes (ATG 3, ATG 5), and stabilized the expression of apoptosis genes (caspase 2, caspase 3) in the hepatopancreas (p < 0.05). In addition, knocking down HO-1 aggravated the vacuolation of hepatopancreas and increased the apoptosis of hepatopancreas, while the supplement of SFN could repair the vacuolation of hepatopancreas and reduce the apoptosis signal. In summary, HO-1 is involved in the repair of the oxidative damage induced by OFO in L. vannamei by SFN.

Immune-enhancing effects of Astragalus polysaccharides and Ganoderma lucidum polysaccharides on Vibrio harveyi flgJ DNA vaccine in grouper.

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1ß, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

Effect of sub-lethal ammonia and nitrite stress on autophagy and apoptosis in hepatopancreas of Pacific whiteleg shrimp Litopenaeusvannamei.

Oxidative stress caused by ammonia and nitrite, affect the health and growth of aquaculture animals, results in oxidative damages. However, the toxic mechanism and pathogenesis of ammonia and nitrite to aquatic invertebrates are not completely clear. The present study was conducted to investigate the effects of sub-lethal ammonia and nitrite on autophagy and apoptosis in hepatopancreas of Pacific whiteleg shrimp Litopenaeus vannamei. Shrimps were exposed to sub-lethal ammonia (20 mg/L) and nitrite (20 mg/L) for 72 h, respectively. Hepatopancreas was collected for investigating the autophagy and apoptosis under stress conditions. The results showed that ammonia stress could induce up-regulated of autophagy (ATG3, ATG4, ATG10 and ATG12) and apoptosis (Caspase3 and P53) genes transcription. Nitrite stress could also induce up-regulated of autophagy (ATG3, ATG4, ATG5 and ATG10) and apoptosis (Caspase3) genes transcription. The expression of the autophagy related genes increased at first and then decreased with increasing exposure time. The atrophy, lysis, vacuolation of cell and other tissue damages in hepatopancreas were observed after 72h exposure to ammonia and nitrite. The results indicated that ammonia and nitrite stress could induce autophagy and apoptosis, and results in oxidative damage.

Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus).

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

The role of dctP gene in regulating colonization, adhesion and pathogenicity of Vibrio alginolyticus strain HY9901.

Vibriosis caused by Vibrio alginolyticus has severely affected the development of mariculture industry in recent decades. DctP, a tripartite ATP-independent periplasmic transporter solute-binding subunit, is thought to be one of the virulence factors in Vibrio. In this study, the results displayed no difference in morphological characteristics and growth between ΔdctP (dctP mutant strain) and WT (wild-type strain). Nevertheless, the ability of swarming motility, biofilm formation, ECPase formation, cell adhesion and colonized ability of ΔdctP significantly decreased compared to those of WT. The LD50 of ΔdctP significantly increased by 40-fold compared to that of WT. The transcriptome analysis demonstrated the deletion mutation of dctP could regulate the expression levels of 22 genes related to colonization, adhesion and pathogenicity in V. alginolyticus. The analysis of qRT-PCR showed the transcriptome data were reliable. These results reveal the effect of attenuated function of DctP on colonization, adherence and pathogenicity by controlling the expression of related gene.

Mass mortalities associated with viral nervous necrosis in Murray cod in China.

In December 2019, a mass mortality among cultured Murray cod (Maccullochella peelii peelii) fry occurred on a freshwater farm located at Foshan city of Guangdong province, China. The cumulative mortality was up to 45% within 15 days. The diseased fish showed clinical signs, including abnormal swimming behaviour, loss of appetite and dark body colouration before mass mortality. Samples of brain and retina tissues were collected from affected fish and subjected to reverse transcriptase polymerase chain reaction detection and virus isolation in cell culture. Approximately 430 bp product was detected from the brain and retina tissues and culture supernatant of betanodavirus-infected SSN-1 cells. The typical cytopathic effect of betanodavirus infection, which is characterized by vacuolation, was observed in SSN-1 cells at three days after inoculating with the tissue filtrate of diseased Murry cod fry, and the TCID50 of the infected SSN-1 cell supernatant was 107.8 . Histopathological examinations revealed vacuolation and necrosis in the brain and retina of naturally and experimentally infected Murray cod fry. Electron microscopic observation also showed the aggregation of numerous spherical, non-enveloped viral particles measuring 22-28 nm in diameter in the cytoplasm of betanodavirus-infected SSN-1 cells. Sequence and phylogenetic analysis based on RdRp and Cp genes further indicated that the betanodavirus isolated from Murray cod belonged to the RGNNV genotype. Much higher mortality was obtained in challenged Murray cod fry compared with the controls through immersion challenge. This study is the first report of the natural infection of betanodavirus in freshwater fish in China.

Construction and evaluation of Vibrio alginolyticus ΔclpP mutant, as a safe live attenuated vibriosis vaccine.

Vibrio alginolyticus is a common and serious pathogen threatening the progress of coastal aquaculture. ClpP protease has been proved to be closely associated with biofilm formation, stress tolerance, autolysis and virulence in several pathogens. Hence, targeting ClpP may be a potentially viable, attractive option for the preparation of vaccine in preventing vibriosis. In this study, an in-frame deleted mutant strain (ΔclpP) was constructed by allelic exchange mutagenesis to investigate physiological role of clpP in pathogenicity of V. alginolyticus and evaluate its potential as a live attenuated vaccine. The results exhibited that ΔclpP showed no differences in external morphology, growth, swarming motility and ECPase activity. However, ΔclpP represented an increment in biofilm formation, and a decrement in adherence to CIK cells. In addition, virulence of ΔclpP was examined in pearl gentian grouper and was found to be seriously attenuated. ΔclpP induced high antibody titers and provided a valid protection with a relative percent survival value of 83.8% without histopathologic abnormality. Our results indicated ΔclpP showed a great potential to be a live attenuated vaccine.

Immune effect of Vibrio harveyi formalin-killed cells vaccine combined with chitosan oligosaccharide and astragalus polysaccharides in ♀Epinephelus fuscoguttatus×♂Epinephelus lanceolatus.

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 × â™‚E. lanceolatus. The results indicated the vaccine triggered a remarkably higher expression levels of IL-1ß, 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.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

Protective effects of ß-glucan as adjuvant combined inactivated Vibrio harveyi vaccine in pearl gentian grouper.

Vaccination is one of the strategies for preventing Vibrio harveyi infection in marine-cultured animals. In this study, we prepared a formalin-killed cells of V. harveyi ZJ0603 vaccine (FKC) combined with ß-glucan to immune pearl gentian grouper. The results indicated that the expression levels of IgM, TNF-α, MHC-Iα, IL-1ß and IL-16 significantly increased in the spleen of the vaccinated fish. Antibody titers, activities of lysozyme and superoxide dismutase were significantly prompted in blood of the vaccinated fish. After 35 d post-vaccination, all fish were challenged intraperitoneally by virulent V. harveyi, and the relative percentage of survival (RPS) of FKC+ß-glucan, FKC, ß-glucan and PBS were 68 ± 5.7%, 55 ± 8.5%, 42 ± 7.5% and 32 ± 6.9%, respectively. These results demonstrated that ß-glucan could be as a potential adjuvant of FKC and provide good protective effect against V. harveyi infection in the pearl gentian grouper culture.

Protection against Vibrio alginolyticus in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatu) immunized with an acfA-deletion live attenuated vaccine.

Vibrio alginolyticus is well-known as an opportunistic Gram-negative pathogen, which endangers the development of global aquaculture as well as human health. In this study, a ΔacfA mutant strain and complementation of the ΔacfA mutant (C-acfA) were constructed. The ΔacfA mutant was tested in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) to observe the changes in virulence and evaluate its potential as an attenuated live vaccine. The results showed that the ΔacfA mutant caused a high antibody titer and a significant reduction in the ability to colonize the intestine of pearl gentian grouper. Grouper vaccinated with ΔacfA mutant were more tolerant of the infection by virulent V. alginolyticus HY9901 without inducing clinical symptoms and obvious pathological changes. The relative percent survival value of pearl gentian grouper vaccinated with ΔacfA mutant intraperitoneal injection reached 81.1% after challenging with V. alginolyticus HY9901. The specific antibody titers immunized with ΔacfA was significantly higher than that in the PBS group. The antibody titer of ΔacfA group displayed the tendency of rising up from the first to fourth week and declining from fifth to eighth week and reached the peak at the fourth week. In the meanwhile, the expression level of genes associated with immunity, including IL-1ß, TNF-α, IL-16, IgM, CD8α and MHC-Iα, was up-regulated after vaccination, indicating that the ΔacfA can induce effective and durable immune response in pearl gentian grouper and it may be an effective attenuated live vaccine candidate for the prevention of infections by V. alginolyticus.

Identification and functional characterization of a c-type lysozyme from Fenneropenaeus penicillatus.

Lysozyme is an important defense molecule of the innate immune system and possess high antimicrobial activities. In this study, a full-length c-type lysozyme cDNA (Fplysc) was cloned and characterized from Fenneropenaeus penicillatus. The cDNA contains an open reading frame of 477 bp encoding 158 amino acids, with 53-94% identity with those of other crustaceans. The recombinant Fplysc had antibacterial activities against Gram-positive bacteria (Streptococcus agalactiae and Micrococcus luteus) and Gram-negative bacteria (Vibrio alginolyticus and Escherichia coli), and showed antiviral activity against WSSV and IHHNV. The qRT-PCR analysis showed that Fplysc expression levels were most abundant in hemocytes and less in eyestalk. The expression levels of Fplysc were significantly upregulated in gill, intestine and hemocytes when challenged with WSSV and V. alginolyticus. Fplysc-silencling suppressed Fplysc expression in cephalothoraxes and increased mortality caused by WSSV and V. alginolyticus, and exogenous rFplysc led to a significant decrease of shrimp mortality by injecting rFplysc into Fplysc silenced shrimp, suggesting Fplysc is the important molecule in shrimp antimicrobial and antiviral response. In conclusion, the results provide some insights into the function of Fplysc in shrimp against bacterial and viral infection.

Superoxide dismutase B (sodB), an important virulence factor of Vibrio alginolyticus, contributes to antioxidative stress and its potential application for live attenuated vaccine.

Vibrio alginolyticus is an opportunistic and halophilic Gram-negative pathogen in limiting the development of aquatic industry and affecting human health. SODs are oxidative enzymes that play a critical role in oxidative defense. In this study, an in-frame deleted mutant strain (ΔsodB) was constructed by allelic exchange mutagenesis to investigate physiological role of sodB in pathogenicity of V. alginolyticus. The results exhibited that ΔsodB showed no differences in growth compared with wild-type strain HY9901 (WT), but led to increasing in biofilm formation, ECPase activity and sensitivity to hydrogen peroxide, decreasing in swarming motility, adherence to CIK cells, SOD activity and virulence. In addition, ΔsodB induced a high antibody titer and provided a valid protection with a relative percent survival value of 86.5% without inducing clinical symptoms after challenging with WT. These results suggest that sodB is important for normal physiological function, oxidation resistance and virulence in V. alginolyticus, and ΔsodB may be considered as an effective live attenuated vaccine against V. alginolyticus.

Characterization and function analysis of interleukin-1 receptor-associated kinase-1 (IRAK-1) from Fenneropenaeus penicillatus.

The interleukin-1 receptor-associated kinase-1 (IRAK-1) is an important adapter protein which links downstream of MyD88, and involved in the complex composed of MyD88 and TRAF6 to activate TLRs signaling pathway. In this study, an IRAK-1 homolog (FpIRAK-1) was cloned from the red tail shrimp Fenneropenaeus penicillatus. The ORF of FpIRAK-1 consisted of 2874 bp encoding a protein of 957 amino acids which contains a death domain (DD) and a catalytic domain of serine/threonine kinases (STKc). Homology analysis revealed that the predicted amino acid sequence of FpIRAK-1 shared 71% similarities with IRAK-1 of Litopenaeus vannamei. Real-time RT-PCR indicated that FpIRAK-1 was constitutively expressed in various tissues of F. penicillatus. The expression level of FpIRAK-1 mRNA was significantly up-regulated and then decreased gradually after white spot syndrome virus (WSSV) and Vibrio alginolyticus challenge. Gene knockdown of FpIRAK-1 enhanced the sensitivity of shrimps to WSSV and V. alginolyticus challenge, suggesting FpIRAK-1 could play a positive role against bacterial and viral pathogens. In conclusion, the results of this study provide some insights into the function of FpIRAK-1 in activating Toll signaling pathway and the host defense against invading pathogens.

Immunogenicity and efficacy of DNA vaccine encoding antigenic AcfA via addition of the molecular adjuvant Myd88 against Vibrio alginolyticus in Epinephelus coioides.

DNA vaccines had been widely used against microbial infection in animals. The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been increasingly studied in recent years. MyD88 is one of the adapter molecules to activate the signaling cascades and produces inflammatory mediators, and its immunological role and adjuvant potential which had been proved in mammals were rarely reported in fish species. In this study, plasmid pcMyD88 was constructed and the capacity of MyD88 as molecular adjuvant was explored by co-injecting with a DNA vaccine encoding AcfA against Vibrio alginolyticus infection in orange spotted grouper. The results suggested that it needed at least 7 days to transported DNA vaccine pcacfA or molecular adjuvant pcMyD88 from the injected muscle to kidney and spleens and stimulate host's immune system for later protection. The co-injection of pcMyD88 with DNA vaccine pcacfA could increase significantly specific antibody levels and the expression levels of the immune-related genes including MHCIα, MHCIIα, CD4, CD8α, IL-1ß and TNFα. Furthermore, pcMyD88 enhanced the immunoprotection of pcacfA against V. alginolyticus infection, with the significantly higher RPS of 83.3% in pcMyD88 + pcacfA group compared with that of pcacfA alone (73.3%) at challenging test of 10 weeks post vaccination. Together, these results clearly demonstrate that MyD88 is an effective adjuvant for the DNA vaccine pcacfA in orange spotted grouper.