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Copepods are small crustaceans that live in microorganism-rich aquatic environments and provide a key supply of live food for fish and shellfish larviculture. To better understand the host-pathogen interaction between the copepod and Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP AHPND ), the comparative transcriptome and microbiome analyses were conducted in copepod Apocyclops royi-TH following VP AHPND infection. Transcriptome analysis identified a total of 836 differentially expressed genes, with 275 upregulated and 561 downregulated genes. Subsequent analysis showed that a total of 37 differentially expressed genes were associated with the innate immune system, including 16 upregulated genes related to Toll-like receptor signaling pathway, antimicrobial peptides, and stress response genes, and 21 downregulated genes associated with immunological modulators, signaling molecules, and apoptosis-related proteins. Analysis of the copepod microbiome following VP AHPND infection showed that the microbes changed significantly after bacterial infection, with a reduced alpha diversity accompanied by the increased level of Proteobacteria and decreased levels of Bdellovibrionota, Bacteroidota, and Verrucomicrobiota. The population of Vibrio genera were increased significantly, while several other genera, including Denitromonas, Nitrosomonas, Blastopirellula, Fusibacter, Alteromonas, KI89A_clade, and Ruegeria, were decreased significantly after infection. These findings suggest that VP AHPND infection has a significant impact on the immune defense and the composition of the copepod microbiota., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ubiquitination is a critical posttranslational modification that regulates host immune responses to pathogens. In this study, we investigated the ubiquitination of hemocyanin (PvHMC [Penaeus vannamei hemocyanin]) mediated by the mitochondrial E3 ubiquitin ligase (PvMulan) in shrimp Penaeus vannamei. We characterized distinct ubiquitination patterns of PvHMC in response to different pathogen challenges, both in vitro and in vivo. Specifically, we found that Vibrio parahaemolyticus infection led to an increase in PvMulan, which resulted in K48-linked ubiquitination and subsequent proteasomal degradation of PvHMC. In contrast, PvMulan primarily enhanced the SUMOylation of PvHMC, bolstering its immune functions against white spot syndrome virus challenges. Inhibition of PvMulan-mediated PvHMC ubiquitination significantly affected the proliferation of V. parahaemolyticus and the survival rate of infected shrimps. This study sheds light on the role of hemocyanin ubiquitination in immune regulation, illustrating its dual function in response to distinct pathogens., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Litopenaeus vannamei is a widely distributed euryhaline aquatic animal, affected by low salinity, which can impact its disease resistance and immunity. However, there is a limited understanding of the adaptation mechanisms of L. vannamei with different genetic backgrounds to low salinity. Therefore, the present study aimed to compare the immunity characteristics and transcriptomics of L. vannamei low salt-tolerant (FG I/J) and low salt-sensitive (control) families. Also, the disease resistance and immune parameters (including [THC], hemolymph cell viability, lysozyme activity [LZM], phenoloxidase content [PO], interleukin-6 [IL-6], and tumor necrosis factor-alpha [TNF-α]) of the FG I/J and control families of L. vannamei under low salinity (5‰) and ambient salinity (24‰) were examined. Additionally, hepatopancreas transcriptomics of the FG I/J and control families were analyzed at a salinity of 5‰. The results showed that the FG I/J family had higher disease resistance to Vibrio parahaemolyticus and stronger immunological capacity than the control family. Transcriptomic analysis showed significantly enriched energy metabolism and immune regulation pathways. Therefore, we speculated that energy metabolism provides sufficient energy for immunological modulation in the FG I/J family to deal with long-term low-salt stress and achieve high growth and survival rates., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The pathogen recognition system involves receptors and genes that play a crucial role in activating innate immune response in brown-marbled grouper (Epinephelus fuscoguttatus) as a control agent against various infections including vibriosis. Here, we report the molecular cloning of partial open reading frames, sequences characterization, and expression profiles of Pattern Recognition Receptors (PRRs) in brown-marbled grouper. The PRRs, namely pglyrp5, tlr5, ctlD, and ctlE in brown-marbled grouper, possess conserved domains and showed shared evolutionary relationships with other fishes, humans, mammals, birds, reptilians, amphibians, and insects. In infection experiments, up to 50% mortality was found in brown-marbled grouper fingerlings infected with Vibrio alginolyticus compared to 27% mortality infected Vibrio parahaemolyticus and 100% survival of control groups. It is also demonstrated that all four PRRs had higher expression in samples infected with V. alginolyticus compared to V. parahaemolyticus. This PRRs gene expression analysis revealed that all four PRRs expressed rapidly at 4-h post-inoculation even though the Vibrio count was only detected earliest at 12-h post-inoculation in samples. The highest expression recorded was from V. alginolyticus inoculated fish spleen with up to 73-fold change for pglyrp5 gene, followed by 14 to 38-fold expression for the same treatment in spleen, head kidney, and blood samples for other PRRs, namely tlr5, ctlD, and ctlE genes. Meanwhile less than a 10% increase in expression of all four genes was detected in spleen, head kidney, and blood samples inoculated with V. parahaemolyticus. These findings indicated that pglyrp5, tlr5, ctlD, and ctlE play important roles in the early immune response to vibriosis infected, brown-marbled grouper fingerlings., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai . It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus , Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ma, Wu, Zhang, Zhang, Jiang, Shen, Wu, Chen and Di.)

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks., (Copyright © 2021. Published by Elsevier Ltd.)

The indirect enzyme-linked immunosorbent assay (ELISA) is the gold standard method for monoclonal antibody (McAb) detection and plays a unique role in the preparation of bacterial antibodies. To solve the laborious issues associated with indirect ELISA, a novel bacterial coloration immunofluorescence strip (BCIFS) for antibody detection using colored bacteria instead of a labeled antibody as the antigen and tracer simultaneously and goat anti-mouse IgG as the test line was developed. The affinity range survey of BCIFS indicated that hybridoma cell cultures of E. coli O157:H7 (D3, E7) and Vibrio parahemolyticus (H7, C9) were detected, which complied with the results of indirect ELISA. Compared with the traditional indirect ELISA, the BCIFS sensitivity for E7 cell cultures, ascites, and purified antibodies was at least 4-fold more sensitive, and the BCIFS cross-reactivity for E7 cell cultures was almost consistent with that of indirect ELISA. In addition, the BCIFS isotypes for E. coli O157:H7 cell cultures and Vibrio parahemolyticus were IgG2a and IgG1, respectively, which were identical to the indirect ELISA. Furthermore, the BCIFS method was confirmed by McAb preparation, effective antibody use, and targeted antibody-secreted hybridoma preparation and screening, which showed excellent performance and substitution of the indirect ELISA method. Combined with methylcellulose semisolid medium, BCIFS offers a novel, easy to operate, rapid preparation method for antigen-specific hybridomas. This is the first report using BCIFS instead of indirect ELISA for bacterial antibody detection and application in different samples, which demonstrates a rapid and powerful tool for antibody engineering., (Copyright © 2021. Published by Elsevier B.V.)

Vibrio parahaemolyticus is an important marine pathogen that cause inflammation even death in teleost. It has brought huge economic losses to aquaculture and serious threats to the sustainable development of marine fisheries. Here, we isolated the DNA, RNA, and total flagellin from V. parahaemolyticus, and obtained the primary spleen and head kidney cells (including leukocytes) from Tetraodon nigroviridis. V. parahaemolyticus DNA, RNA, and total flagellin were used to treat the T. nigroviridis primary cells described above. The results show that the nitric oxide (NO) production and respiratory burst response were significantly induced after stimulation with V. parahaemolyticus total flagellin in T. nigroviridis head kidney and spleen cells. And total flagellin could promote the gene expression and protein production of IL-1β in T. nigroviridis leukocytes. T. nigroviridis TLR5M (TnTLR5M) and TLR5S (TnTLR5S) ORF sequences were obtained as the main recognition receptor for flagellin. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect the expression of pattern recognition receptor TnTLR5M and TnTLR5S, the important signal molecule of inflammatory system TnMyD88 and TnTRAF6, and inflammatory cytokines TnIL-1β and TnIFN-γ2. The results show that there were a significant upregulation after challenge with V. parahaemolyticus total flagellin. We further demonstrated that the total flagellin of V. parahaemolyticus could activate the luciferase activity of the NF-κB reporter gene mediated by TnTLR5M. Overall, our results suggest that V. parahaemolyticus total flagellin activated the NO production, respiratory burst response, and inflammatory cytokines expressions, such as TnIL-1β and TnIFN-γ2, through the TnTLR5M-NF-κB signaling pathway in T. nigroviridis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor superfamily member 10 (TNFSF10), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Apo-2L, is one of the important members of the TNF superfamily. It is well demonstrated that TNFSF10 preferentially induces a variety of tumor cell apoptosis, and therefore exerts an important role in tumor immune surveillance. However, the function of TNFSF10 in pathogen defense is poorly understood, especially in invertebrates. The blood clam (Tegillarca granosa), an important commercial marine bivalve, plays an important ecological role in the marine ecosystem. The identification of immune genes will provide new perspective for disease control in the blood clam (T. granosa) farming. To better understand the biological function of TNFSF10 protein, the full-length cDNA of TNFSF10 homologous gene of T. granosa (TgTNFSF10) was cloned and identified for the first time, which was found to contain 1239 base pairs and encode 254 amino acids with a molecular weight of 29.5 kDa and a conserved TNF domain in the C-terminal. Quantitative RT-PCR analysis showed that TgTNFSF10 gene was constitutively expressed in all tested tissues, with the highest expression in hemocytes. LPS, Vibrio alginolyticus and Vibrio parahaemolyticus stimulations dramatically increased the expression of TgTNFSF10 in T. granosa (11.47-fold, 3.71-fold and 8.29-fold compared with the control respectively). In vitro experiments showed that recombinant TgTNFSF10 protein strongly inhibited the proliferation of HepG2 cells. Further confocal microscopy and flow cytometry analysis showed that obvious apoptosis occurred in TgTNFSF10-treated hemocytes and HepG2 cells. To sum up, our study demonstrated that TgTNFSF10 had strong apoptosis-inducing activity, which may participate in the innate immune response of T. granosa to pathogen invasion., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora., (Copyright © 2021. Published by Elsevier B.V.)

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3-6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3-6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3-6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5-6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection., Competing Interests: APC provided support in the form of salary to JC. The specific roles of this author are articulated in the ‘author contributions’ section. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Recently, l-amino acid oxidases (LAAOs) have been identified in several fish species as first-line defense molecules against bacterial infection. Here, we report the cloning and characterization of a fish LAAO gene, EcLAAO2, from orange-spotted grouper (Epinephelus coioides). The full-length cDNA is 3030 bp, with an ORF encoding a protein of 511 amino acids. EcLAAO2 is mainly expressed in the fin, gill, and intestine. Its expression is upregulated in several immune organs after challenge with lipopolysaccharide (LPS) and poly (I:C). The recombinant EcLAAO2 protein (rEcLAAO2), expressed and purified from a baculovirus expression system, was determined to be a glycosylated dimer. According to a hydrogen peroxide-production assay, the recombinant protein was identified as having LAAO enzyme activity with substrate preference for L-Phe and L-Trp, but not L-Lys as other known fish LAAOs. rEcLAAO2 could effectively inhibit the growth of Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus subtilis while exhibiting less effective inhibition of the growth of Escherichia coli. Finally, protein models based on sequence homology were constructed to predict the three-dimensional structure of EcLAAO2 as well as to explain the difference in substrate specificity between EcLAAO2 and other reported fish LAAOs. In conclusion, this study identifies EcLAAO2 as a novel fish LAAO with a substrate preference distinct from other known fish LAAOs and reveals that it may function against invading pathogens., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD 50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Dorsal is a Rel/NF-κB transcription factor, which forms a key part of the Toll pathway. Lysozyme is a ubiquitous enzyme that degrades bacterial cell walls. In this study, a Dorsal homolog was cloned and characterized from triangle sail mussel Hyriopsis cumingii, namely, HcDorsal. Dorsal consisted of 3041 bp, including a 1938 bp open reading frame encoding a 645 amino acid protein. The deduced HcDorsal protein contained a Rel homology domain and an Ig-like, plexin, transcription factor domain. Analysis of expression patterns showed that HcDorsal was highly expressed in the hepatopancreas of H. cumingii. The expression level of HcDorsal continuously increased after Vibrio parahaemolyticus stimulation. When HcDorsal was knocked down by siRNA interference, two phage lysozyme genes (HcLyso1 and HcLyso2) obtained by horizontal gene transfer were significantly downregulated in hemocytes of mussels. Furthermore, knockdown of HcLyso1 and HcLyso2 could weaken V. parahaemolyticus clearance ability. Recombinant HcLyso1 and HcLyso2 proteins accelerated the bacterial clearance in vivo in mussels and evidently inhibited the growth of V. parahaemolyticus. These results suggested that HcDorsal could be activated after V. parahaemolyticus stimulation and then modulate the immune response through the transcriptional regulation of HcLyso1 and HcLyso2, thereby playing a protective role in mussels., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

B-type allatostatins (AST-B) are neuropeptides that have important physiological roles in arthropods, they have also been identified in a number of crustacean species. Recent research on neuroendocrine-immune (NEI) regulatory system in invertebrates has exploded, it reveals that the NEI network plays an indispensable role in optimizing the immune response and maintaining homeostasis. Herein, mud crab Scylla paramamosain provides a primitive and ancient model to study crosstalk between the neuroendocrine and immune systems. In this study, qRT-PCR analysis showed that the nervous system was the main production site for Sp-AST-B mRNA in S. paramamosain, while its receptor gene (Sp-AST-BR) mRNA could be detected in all the analyzed tissues including hemocytes. This reveals that AST-B might act as a pleiotropic neuropeptide. In situ hybridization further confirmed that granular cells of hemocyte subpopulations express Sp-AST-BR. Time-course analysis revealed that bacteria-analog LPS or virus-analog Poly (I:C) challenge significantly induced Sp-AST-B expression in the thoracic ganglion, and the expression of Sp-AST-BR in hemocytes were also positively changed. Furthermore, mud crabs treated with a synthetic AST-B peptide significantly increased the mRNA levels of AST-BR, nuclear factor-κB (NF-κB) transcription factor (Dorsal and Relish), pro-inflammatory cytokine (IL-16) and immune-effector molecules, and also dramatically enhanced the nitric oxide (NO) production and phagocytic activity in hemocytes. Meanwhile dsRNA-mediated knockdown of Sp-AST-B remarkably suppressed the NO concentrations, phagocytic activity and the expression of immune related genes, resulting in markedly impaired ability of crabs to inhibit bacterial proliferation in vivo. Combined, these data demonstrate that AST-B induced innate immune in the mud crab., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Acute hepatopancreatic necrosis disease (AHPND) is a lethal disease in marine shrimp that has caused large-scale mortalities in shrimp aquaculture in Asia and the Americas. The etiologic agent is a pathogenic Vibrio sp. carrying binary toxin genes, pir A and pir B in plasmid DNA. Developing AHPND tolerant shrimp lines is one of the prophylactic approaches to combat this disease. A selected genetic line of Penaeus vannamei was found to be tolerant to AHPND during screening for disease resistance. The mRNA expression of twelve immune and metabolic genes known to be involved in bacterial pathogenesis were measured by quantitative RT-PCR in two populations of shrimp, namely P1 that showed susceptibility to AHPND, and P2 that showed tolerance to AHPND. Among these genes, the mRNA expression of chymotrypsin A (ChyA) and serine protease (SP), genes that are involved in metabolism, and crustin-P (CRSTP) and prophenol oxidase activation system 2 (PPAE2), genes involved in bacterial pathogenesis in shrimp, showed differential expression between the two populations. The differential expression of these genes shed light on the mechanism of tolerance against AHPND and these genes can potentially serve as candidate markers for tolerance/susceptibility to AHPND in P. vannamei . This is the first report of a comparison of the mRNA expression profiles of AHPND tolerant and susceptible lines of P. vannamei ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mai, Caro, Cruz-Flores, White and Dhar.)

This experimental studies investigated the protective efficiencies and the potential immune mechanisms of vibrio monovalent and polyvalent autogenous formalin-inactivated whole-cell bacterins (FKC) in Gilthead sea bream (Sparus aurata) cultured in Egypt. Two months post-vaccination, the relative percentage survival (RPS) was estimated after challenge with the vaccine's homologues pathogenic strains. The survival values were 100% and 83.3% in groups immunized with monovalent V. alginolyticus or V. parahaemolyticus FKC bacterins, respectively. On the other hand, survival values were 91.75% and 75% in fish groups subjected to polyvalent (V. parahaemolyticus O11: K40 & V. alginolyticus) and (V. parahaemolyticus O3: K6 & V. alginolyticus) FKC bacterins, respectively. Overall, the tested vaccine preparations were significantly increased (P < 0.05) the agglutination antibody titer, phagocytic activity, respiratory burst activity, when compared to the non-immunized control group. The current results conclude that, autogenous Vibrio vaccines provoked a promising protection against vibriosis in Gilthead sea bream cultured in Egypt, it was superior in monovalent FKC V. alginolyticus vaccine and polyvalent FKC of V. parahaemolyticus O11: K40 with V. alginolyticus vaccine that could be useful means of prevention and control of vibriosis., (Copyright © 2020. Published by Elsevier Ltd.)

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca 2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aquatic animals are frequently threated by bacterial pathogens. The most economic and efficient protection against bacterial infection are through vaccine immunization. The various serotypes of the pathogens, such as Vibrios, hurdle the development of the vaccines, especially polyvalent vaccines. Here, we demonstrate that recombinant bacterial ghost is a good candidate for multivalent vaccine. By expressing PhiX174 gene E alone or co-expressing the gene E with two genes encoding outer membrane proteins (VP1667 and VP2369) in V. parahaemolyticus, we generated the recombinant V. parahaemolyticus ghosts VPG and rVPGs respectively. Fish immunized with either VPG or rVPG showed increased survival against the infection by either V. parahaemolyticus or V. alginolyticus, with a better protective effect by immunization with rVPG. Our furthermore studies show that rVPG stimulates stronger innate immune responses by increasing the expression of tnfα, il1β, il6, il8 and il10 as well as that of c3b, lyz, and tlr5, the key players linking the innate and adaptive immune responses upon microbial stimulation. In summary, VPG and rVPG can protect zebrafish against the infection from at least two Vibrio species, suggesting its potential value for further aquaculture vaccines development., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis after the consumption of contaminated food, wound infection, and seizures. Antibiotic therapy is the main method for controlling Vibrio infections, which inevitably leads to drug resistance. Therefore, a vaccine is urgently needed to avoid this problem. Outer membrane proteins (OMPs) play a pivotal role in the interaction between the host immune system and bacteria. VP1243 is an OMP of V. parahaemolyticus, and it possessed immunogenicity in our previous study. The present study found that VP1243 was widely distributed, highly conserved and possessed similar surface epitopes among the major Vibrio species. The protein stimulated a strong antibody response and induced cross-reactive immune responses in V. parahaemolyticus, V. alginolyticus and V. vulnificus. Notably, it provided 100% immune protection against lethal challenges by the three Vibrio species in mice immunized with VP1243. Efficient clearance of cells of the three Vibrio bacterial species was observed in immunized mice. These findings provide solid evidence for VP1243 as a promising candidate for the development of a versatile vaccine to protect against Vibrio infections., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections., Competing Interests: The authors have declared that no competing interests exist.

When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan-gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp ( p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased ( p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei .

Leucine-rich repeat (LRR) is a vital structure in some pattern recognition receptors such as TLRs, NLRs and newly reported LRR-containing proteins. Apart from some limited reported LRR-containing proteins, most of LRR proteins, especially immune-related proteins, remain uncharacterized functionally. In the present study, a transmembrane protein containing several LRR motifs, designated as LvLRRm, was identified from the shrimp Litopenaeus vannamei. LvLRRm contained a long signal peptide, one LRRNT region, 12 LRR motifs, one LRRCT region and a transmembrane region. The transcripts of LvLRRm were widely distributed in all tested tissues of shrimp and they were responsive to Vibrio parahaemolyticus infection in several immune-related tissues including Oka, intestine, gill and hemocytes. Knockdown of LvLRRm by dsRNA interference led to a decreased survival rate of shrimp infected by Vibrio parahaemolyticus and an increased in vivo Vibrio propagation. Meanwhile, knockdown of LvLRRm also down-regulated the expression levels of genes involved in antibacterial immune signaling pathways, including the transcription factors LvDorsal and LvRelish, and several antimicrobial peptides. These data suggested that LvLRRm played important roles in shrimp against Vibrio infection, which was probably functioning through activation of antibacterial immune signaling pathways. The present study provided new evidence to elucidate the immune function of LRR-containing proteins in invertebrates., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nanogold enhanced surface plasmon resonance (SPR), colloidal gold immunochromatographic test strips (ICTS), and polymerase chain reaction (PCR), combined with immunomagnetic separation (IMS) were established in this study for the rapid detection of Vibrio parahaemolyticus (VP). The sensitivities of SPR, ICTS, and PCR was determined to be 10 1 , 10 3 , and 10 3  CFU/mL for VP, respectively. After separation and enrichment by IMS, the sensitivities of SPR, ICTS, and PCR were 10 0 , 10 1 , and 10 2  CFU/mL for VP, respectively, which were improved by 10-, 100-, and 10-fold compared to the direct detection by SPR, ICTS, and PCR, respectively. When the VP-polluted water samples were directly assessed by SPR, ICTS, and PCR, the results were negative. By contrast, after separation and enrichment for 45 min by IMS, the results were all positive. The IMS-SPR, IMS-ICTS, and IMS-PCR detection methods were able to yield results in approximately 1.5 h, 55 min, and 3.5 h, respectively. These combined detection methods have advantages in being high-throughput and easy to operate without the need for sophisticated equipment or specialized skills. These methods might aid in the development of SPR, ICTS, and PCR technologies for simultaneously examining multiple food-borne pathogens in food products.

Vibrio parahaemolyticus (V. parahaemolyticus) is a well-known food-borne human pathogen that can cause a variety of clinical manifestations after the consumption of raw or undercooked seafoods. The crucial roles of Vibrio OmpU in bacterial pathogenesis have been found in recent studies. In the present study, we screened for single domain antibody fragment (sdAb) candidates that bind to V. parahaemolyticus OmpU by using a sdAb phage display library and isolated several positive phage clones. The UAb28, which was one of the positive clones, was shown high enrichment and affinity. The CDRs of UAb28 are speculated to perform the OmpU binding function by molecular docking. The capable of recognizing OmpU was verified by binding and inhibition assays. The UAb28 might be useful in future studies to develop the potential sdAb-based immunotherapeutics against V. parahaemolyticus infection., Competing Interests: Declaration of competing interest There are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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&#95;NT domain, thirteen LRR domains and one LRR&#95;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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The myeloid differentiation protein 2 (MD2)-related lipid-recognition (ML) proteins display diverse biological functions in host immunity and lipid metabolism by interacting with different lipids. Human MD2, an indispensable accessory protein in TLR4 signaling pathway, specifically recognizes lipopolysaccharides (LPS), thereby leading to the activation of TLR4 signaling pathway to produce many effectors that participate in inflammatory and immuneresponses against Gram-negative bacteria. Toll and immune deficiency (IMD) pathways are first characterized in Drosophila and are reportedly present in crustaceans, but the recognition and activation mechanism of these signaling pathways in crustaceans remains unclear. In the present study, a novel ML protein was characterized in mud crab (Scylla paramamosain) and designated as SpMD2. The complete SpMD2 cDNA sequence is 1114 bp long with a 465 bp open reading frame; it encodes a protein that contains 154 amino acids (aa). In the deduced protein, a signal peptide (1-21 aa residues) and a ML domain (43-151 aa residues) were predicted. SpMD2 shared a similar three-dimensional structure and a close evolutionary relationship with human MD2. SpMD2 was highly expressed in gills, hemocytes, intestine, and hepatopancreas and was upregulated in gills and hemocytes after challenges with bacteria, thereby suggesting its involvement in antibacterial defense. Western blot assay showed that SpMD2 possesses strong binding activities to different bacteria and two fungi. ELISA demonstrated that SpMD2 exhibits binding abilities to LPS, lipid A, peptidoglycan (PGN), and lipoteichoic acid (LTA). Its binding ability to LPS and lipid A were stronger than to PGN or LTA, implying that SpMD2 was an important LPS-binding protein in mud crab. Bacterial clearance assay revealed that the pre-incubation of Vibrio parahemolyticus with SpMD2 facilitates bacterial clearance in vivo and that knockdown of SpMD2 dramatically suppresses the bacterial clearance and decreases the expression of several antimicrobial peptides (AMPs). Furthermore, SpMD2 overexpression could enhance the promoter activity of SpALF2. These results revealed that SpMD2 affects bacterial clearance by regulating AMPs. Thus, by binding to LPS and by regulating AMPs, SpMD2 may function as a potential receptor, which is involved in the recognition and activation of a certain immune signaling pathway against Gram-negative bacteria. This study provides new insights into the diverse functions of ML proteins and into the antibacterial mechanisms of crustaceans., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

While Vibrio parahaemolyticus (VP AHPND ) has been identified as the cause of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, mechanisms of host response remain unknown. Understanding these processes is important to improve farming practices because this understanding will help to develop methods to enhance shrimp immunity. Pre-treatment of shrimp with 5-minute chronic non-lethal heat stress (NLHS) for 7 days was found to significantly increase Litopenaeus vannamei survival against VP AHPND infection. To elucidate the mechanism involved, mRNA and miRNA expression profiles from the hemocyte of L. vannamei challenged with VP AHPND after NLHS with corresponding control conditions were determined by RNA-Seq. A total of 2,664 mRNAs and 41 miRNAs were differentially expressed after the NLHS treatment and VP AHPND challenge. A miRNA-mRNA regulatory network of differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) was subsequently constructed and the interactions of DEMs in regulating the NLHS-induced immune-related pathways were identified. Transcriptomic data revealed that miRNA and mRNA interactions contribute to the modulation of NLHS-induced immune responses, such as the prophenoloxidase-activating system, hemocyte homeostasis, and antimicrobial peptide production, and these responses enhance VP AHPND resistance in L. vannamei.

Due to increased temperatures and aquaculture density, thermal and hypoxia stresses have become serious problems for the aquaculture of abalone Haliotis diversicolor. Stresses lead to immunosuppression, which can cause severe negative impacts on aquaculture farms. To study the mechanism of immunosuppression after hypoxia stress and bacterial challenge, transcriptomes of H. diversicolor hemocytes involved in immunity were profiled. A total of 307,395,572 clean reads were generated and assembled into 99,774 unigenes. KEGG analysis indicated that 225 unigenes with immunologic function were mapped into immune-related pathways. Expression of 41 unigenes measured by quantitative real-time PCR (qRT-PCR) showed consistent results with that of transcriptome analysis. When exposure challenge of Vibrio parahaemolyticus, it is indicated that the PI3K-AKT, MAPK, NF-κB and P53 signal pathways were involved in the hypoxia-induced immunosuppression of H. diversicolor. Furthermore, when the AKT gene (HdAKT) was inhibited by double-stranded RNA (dsRNA), expression levels of HdAKT was lower than the blank and control group in hemocytes at 4 h, 12 h and 24 h (p < 0.05).

Kruppel-like factors (KLFs) belong to a family of zinc finger-containing transcription factors that are widely present in eukaryotes. In the present study, a novel KLF from the giant river prawn Macrobrachium rosenbergii (designated as MrKLF) was successfully cloned and characterized. The full-length cDNA of MrKLF was 1799 bp with an open reading frame of 1332 bp that encodes a putative protein of 444 amino acids, including three conserved ZnF&#95;C2H2 domains at the C-terminus. Multiple alignment analysis showed that MrKLF and other crustacean KLFs shared high similarity. Quantitative real-time PCR analysis revealed that MrKLF mRNA was found in different tissues of prawns and detected in the gills, hepatopancreas, and intestines. After the challenge with Vibrio parahaemolyticus and Aeromonas hydrophila, different expression patterns of MrKLF in the gills, intestines, and hepatopancreas were observed. RNA interference analysis indicated that MrKLF was involved in regulating the expression of four antimicrobial peptides, namely, Crustin (Crus) 2, Crus8, anti-lipopolysaccharide factor (ALF) 1, and ALF3. These results help promote research on M. rosenbergii innate immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Two lipopolysaccharides (LPS) and β-1, 3-glucan binding protein (LGBP), designated as PcLGBP isoform1 and PcLGBP isoform2, respectively, were identified from Procambarus clarkii in this study. The full-length cDNA of PcLGBP isoform1 was 1308 bp containing an open reading frame (ORF) of 1113 bp encoding a protein of 370 amino acids. The full-length cDNA of PcLGBP isoform2 was 1440 bp containing an ORF of 1245 bp encoding a protein of 414 amino acids. Predicted PcLGBP isoform1 and PcLGBP isoform 2 proteins contained a signal peptide, a glycoside hydrolase domain, and a low-complexity region. The difference between the two LGBP isoforms was that PcLGBP isoform2 had 44 more amino acids behind the signal peptide than the PcLGBP isoform1. The PcLGBP isoform1 and PcLGBP isoform2 transcripts mainly expressed in the hepatopancreas in female and male crayfish. Moreover, the expression levels of the two genes in the hepatopancreas were higher in male than that in female crayfish. Upon being challenged with Vibrio parahaemolyticus or LPS, the expression levels of PcLGBP isoform1 and PcLGBP isoform2 in the hepatopancreas of female and male crayfish were most significantly up-regulated at different time points. The transcripts of anti-lipopolysaccharide factors (ALF5, ALF6, ALF8, and ALF9) and crustins (CRU1, CRU2, CRU3, and CRU4) were evidently down-regulated in the hepatopancreas of V. parahaemolyticus-challenged total PcLGBP (including PcLGBP isoform1 and PcLGBP isoform2)-silenced male crayfish. In addition, the phenoloxidase (PO) activity in the hepatopancreas of male crayfish was evidently higher than that of female crayfish. PcLGBP knock down could significantly decrease the PO activity in the hepatopancreas lysate (HLS) in male crayfish. The PO activity of male crayfish HLS was significantly increased when incubated with a mixture of recombinant LGBP protein and LPS or β-1, 3 glucan. We conclude that LGBP isoforms from P. clarkii function as a pattern recognition protein for recognizing and binding LPS and β-1, 3 glucan, and thus regulate the synthesis of antimicrobial peptides and activate the prophenoloxidase system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Our previous work showed that using Lactobacillus pentosus HC-2 as a probiotic could improve the growth performance, immune response, gut bacterial diversity and disease resistance of Litopenaeus vannamei. However, the probiotic mechanism had not been fully characterized. In the present study, histology and proteomic analysis were performed to explore the influence of HC-2 surface protein on its probiotic effects on L. vannamei after feeding either the intact surface proteins, the probiotic treated with lithium chloride (LiCl) to remove noncovalently bound surface proteins or no probiotic for four weeks. Histological observation found that feeding with normal HC-2 obviously improved the intestinal histology and enhanced the protective effect against pathogen damage, but feeding with LiCl-treated HC-2 did not improve the intestinal environment. A total of over 2764 peptides and 1118 uniproteins were identified from the L. vannamei midgut; 211 proteins were significantly differentially expressed in the normal HC-2 group compared with the control group; 510 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the control group, and 458 proteins were significantly differentially expressed in the LiCl-treated HC-2 group compared with the normal HC-2 group. GO/KEGG enrichment analysis of the significantly different proteins demonstrated that feeding normal HC-2 mainly induced immune response, metabolic, cell adhesion and cell-cell signaling-related protein upregulation, which contributed to bacterial adhesion and colonization in the midgut to improve the shrimp immune system and growth, but these proteins were suppressed after the shrimp were fed bacteria deprived of surface proteins. Taken together, these results indicate that the surface proteins were indispensable for HC-2 to execute probiotic effects in the shrimp midgut., (Copyright © 2019. Published by Elsevier Ltd.)

Vibrio parahaemolyticus is a major food-borne pathogen. V. parahaemolyticus infections are associated with various serotypes; to date, 71 K-serogroups of V. parahaemolyticus have been determined based on capsular polysaccharide (CPS) diversity. In this study, the capsular polysaccharide gene clusters (CPSgcs) of 55 K-serogroups were identified by whole-genome sequencing and analysis. These CPSgcs exhibit a high level of genetic diversity. A microsphere-based suspension array (MSA) was established for the detection and identification of 55 V. parahaemolyticus K-serogroups based on CPSgc-specific genes. To evaluate our array, a double-blind test with 120 clinical isolates was carried out. In addition, an in silico K-serotyping system was established based on V. parahaemolyticus CPSgc-specific genes. This system was then used to examine 845 publicly available V. parahaemolyticus genomes; the results demonstrated that 813 isolates belong to one of 43 K-serogroups. Taken together, these results demonstrate that the molecular system developed in this study is suitable for rapid serotyping of V. parahaemolyticus isolates from environmental and clinical samples. In addition, the system could be applied to epidemiological investigations of this important food-borne pathogen., (Copyright © 2019 Elsevier B.V. All rights reserved.)

White spot syndrome, which is caused by white spot syndrome virus (WSSV), is a highly contagious disease of penaeid shrimp. However, there is currently incomplete understanding of the infection mechanism and pathogenesis of WSSV. In this study, a novel gene of a previously uncharacterized WSSV response protein (LvWRP) in Litopenaeus vannamei was identified and characterized. The LvWRP gene has an open reading frame (ORF) of 879 bp encoding a putative protein of 292 amino acids. Sequence analysis revealed that LvWRP shared 24.9% identity with an uncharacterized protein of Penaeus monodon nudivirus. Real-time qPCR analysis showed that LvWRP was ubiquitously expressed in shrimp tissues, with transcript levels induced in hemocytes upon immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and WSSV. In addition, RNA interference-mediated knockdown of LvWRP followed by WSSV challenge revealed significant decrease in the transcript levels of WSSV IE1 and VP28 genes coupled with a reduction in WSSV copies in shrimp hemocytes. Moreover, depletion of LvWRP followed by WSSV challenge significantly increased the transcript levels of Vago4 and Vago5 as well as increased the phosphorylation of STAT, while hemocytes apoptosis in terms of caspase 3/7 activity was decreased. These results suggest that LvWRP is important for WSSV replication in shrimp, and therefore one of the vital host factors in WSSV infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection., (Copyright © 2019. Published by Elsevier Ltd.)

TAK1-binding proteins (TABs) are important immune protein involved in various intracellular signalling pathways. Here, TAB1-3 (lcTAB1-3) were characterized from Larimichthys crocea. The predicted 1524 bp coding sequence of lcTAB1 encoded a 507-residue protein, while lcTAB2 (2271 bp) and lcTAB3 (1836 bp) encoded 756 and 611 residue proteins, respectively. Their sequence shared conserved domain structures and functional sites with their orthologs from other species. The expression of lcTAB1-3 were detected in all tested tissues, which were upregulated in spleen, liver and kidney following Vibrio parahemolyticus infection. Immunofluorescence staining revealed that lcTAB1 were localized in cytoplasm, while lcTAB2 and lcTAB3 were in the endsome. Moreover, the NF-κB protein level was obviously upregulated after the co-overexpression of lcTAK1 and lcTABs, higher than that after the overexpression of lcTAK1 or lcTABs alone. Co-immunoprecipitation proved the direct interaction of lcTAB1/lcTAB2/lcTAB3 and lcTAK1. These findings indicated the roles of lcTABs in immune response of Larimichthys crocea., (Copyright © 2019. Published by Elsevier Ltd.)

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, Sp Bark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. Sp Bark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that Sp Bark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca 2+ by incubating microorganisms with Sp Bark CTLD instead of SRCRDs. These results suggested that Sp Bark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with Sp Bark CTLD promoted bacterial clearance in vivo . This finding indicated that Sp Bark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by Sp Bark was further determined using SpBark -silenced crabs injected with V. parahemolyticus . SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by Sp Bark was closely related to Sp Bark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by Sp Bark was as follows: Sp Bark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Hemocyanin is primarily a respiratory copper-containing glycoprotein present in the hemolymph of mollusks and arthropods. Recently, hemocyanin has attracted huge research interest due to its multifunctionality and polymorphism. Most previous immune-related studies on shrimp hemocyanin have focused on the C-terminal. Moreover, we previously reported that the C-terminal domain of Litopenaeus vannamei hemocyanin possesses single nucleotide polymorphisms (SNPs), but little is known about the molecular diversity of the N-terminal domain. In the current study, diversity within the N-terminal domain of L. vannamei hemocyanin (LvHMC-N) was explored using bioinformatics and molecular biology techniques as well as immune challenge. Twenty-five LvHMC-N variants were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing, with multiple sequence alignment showing that the 25 variants shared 87%-99 % sequence homology with LvHMC (AJ250830.1). In different shrimp individuals and different shrimp tissues (i.e., hemocytes, stomach, muscle and hepatopancreas), the LvHMC-N variants were expressed differently. Pathogen challenge could modulate the molecular diversity of LvHMC-N, as three LvHMC-Nr variants (LvHMC-Nr1, LvHMC-Nr2 and LvHMC-Nr3) were identified by sequencing following Vibrio parahaemolyticus challenge. Most importantly, recombinant proteins of these three variants (rLvHMC-Nr1, rLvHMC-Nr2 and rLvHMC- Nr3) had relatively high in vitro agglutinative activities against V. parahaemolyticus, Vibrio alginolyticus and Streptoccocus iniae. Our present data indicates that the N-terminus of L. vannamei hemocyanin also possess molecular diversity, which seems to be associated with immune resistance to pathogenic infections., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix-leucine zipper protein that plays a key role in cell proliferation, survival and immune defense through the direct transcriptional control of downstream genes. We have found that MITF participates in the immune response to Vibrio parahaemolyticus infection in the clam Meretrix petechialis . In this study, we focused on how MITF functions in immunity. First, PO, CTSK , and BCL-2 were identified as the target genes of MpMITF in the clam by RNAi. EMSAs showed direct binding between the MpMITF protein and the E-box of the MpPO, MpCTSK , and MpBCL-2 promoters. Yeast one-hybrid assays also suggested that MpMITF could activate the expression of these three downstream genes. These results demonstrated that the transcriptional expression of MpPO, MpCTSK , and MpBCL-2 is directly regulated by MpMITF. Second, we analyzed the roles of MpPO, MpCTSK , and MpBCL-2 in clam immunity. The mRNA expression of MpPO, MpCTSK , and MpBCL-2 increased significantly after V. parahaemolyticus challenge, which implied that these genes might take part in the immune defense against V. parahaemolyticus challenge in clams. The purified recombinant proteins, MpPO and MpCTSK, inhibited the growth of V. parahaemolyticus . Additionally, the apoptosis rate of clam haemocytes rose significantly when the activity of MpBCL-2 was suppressed. These results revealed that MpPO, MpCTSK , and MpBCL-2 are involved in the immune defense against V. parahaemolyticus . This study supports the idea that the MpMITF pathway plays a key role in immune defense through the direct regulation of the downstream genes MpPO, MpCTSK , and MpBCL-2 in the clam, M. petechialis .

Live attenuated bacteria is a promising candidate vector for the delivery of vaccines in clinic trials. In the field of aquaculture industry, live vector vaccine also could provide long-term and effective protection against fish bacterial diseases. In our previous work, we demonstrated attenuated Listeria monocytogenes (Lm) had the potential to be an aquaculture vaccine vector in cellular level and zebrafish model. To further investigate the potential application of attenuated Lm in aquaculture vaccines, the outer membrane protein K (OmpK) from Vibrio parahaemolyticus (V. parahaemolyticus), as a conservative protective antigen, was fused to a new antigen-delivery system, and introduced into double-gene attenuated Lm strain (EGDe-ΔactA/inlB, Lmdd) to get live-vector vaccine strain Lmdd-OmpK. The strain Lmdd-OmpK showed the stable secrete efficacy of OmpK and was tested the cross-protective immunity against Vibrio species. After intraperitoneal administration in zebrafish, Lmdd and Lmdd-OmpK strain both improved the survival rates of zebrafish infected by V. parahaemolyticus, Vibrio alginolyticus (V. alginolyticus) and Vibrio anguillarum (V. anguillarum), respectively. In summary, attenuated Lm is able to protect zebrafish against Vibrio species challenge, illustrating its potential value for further aquaculture vaccines development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Superoxide dismutases (SODs) are important antioxidant enzymes that occur in virtually all oxygen-respiring organisms, and copper/zinc SOD (Cu/ZnSOD) is one of the most important SODs. In the present study, Macrobrachium rosenbergii Cu/Zn-SOD was expressed in a yeast eukaryotic system. The open reading frame (ORF) of MrCu/ZnSOD was cloned into the plasmid vector pHAC181, and the recombinant plasmid was integrated into the downstream region of the GAL1 promoter in Saccharomyces cerevisiae strain GAL1-ScRCH1 via homologous recombination. The resulting recombinant MrCu/ZnSOD consisted of a 3 × HA-tag at its C-terminal. Via western blot, the molecular weight of the recombinant MrCu/ZnSOD was estimated at about 30 kDa. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this recombinant MrCu/ZnSOD ranged from 0.556 to 0.840 μM, and from 0.967 to 2.015 μM, respectively. The recombinant MrCu/ZnSOD protein was able to agglutinate four Gram-negative bacterial strains, as well as two of three Gram-positive strains (except Staphylococcus aureus). This demonstrated that the recombinant protein possessed some antimicrobial activity against certain Gram-positive and Gram-negative bacteria. M. rosenbergii were fed with the recombinant yeast strain MrCu/ZnSOD for 4 weeks and then challenged with the most common crustacean pathogen, Vibrio parahaemolyticus. This group of prawns presented lower mortality, higher enzymatic activity, and higher expression of the mRNA of immune-related genes than that in the control groups. Taken together, these results suggest that MrCu/ZnSOD is an antioxidant enzyme and antimicrobial peptide involved in the crustacean innate immune system and offers protection to the host against pathogenic bacteria., (Copyright © 2019. Published by Elsevier Ltd.)

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The p38 mitogen-activated protein kinases (MAPKs) are evolutionally conserved from yeasts to mammals, and are involved in the regulation of cells response to various extracellular stimuli. In this study, the p38 MAPK gene (designated as Spp38) of mud crab (Scylla paramamosain) was identified and studied. Spp38 contained the conserved Thr-Gly-Tyr (TGY) motif and a Ala-Thr-Arg-Trp (ATRW) substrate-binding site. Spp38 transcript was ubiquitously expressed in all tissues examined, with the highest expression found in muscle and hepatopancras. Quantitative real-time PCR revealed that Spp38 was upregulated in hemocytes and hepatopancras after infection with Vibrio parahemolyticus and Lipopolysaccharides (LPS). Reporter gene assays indicated that Spp38 activated the expression of anti-lipopolysaccharides (SpALF1 - SpALF6) in S. paramamosian. RNA interference (RNAi)-mediated knockdown of Spp38 or inhibition of Spp38 by SB203580 decreased the expression levels of SpALF1-6 and dual oxidase (SpDuox1 and SpDuox2) in S. paramamosian, which consequently reduced reactive oxygen species (ROS) production thereby significantly increasing the bacterial count in the hemolymph of mud crabs. Similarly, there was a significant reduction in bacterial clearance ability of hemolymph after Spp38 knockdown followed by V. parahemolyticus infection. Taken together, the current data indicated that Spp38 could play a vital role in maintaining the homeostasis of hemolymph microbiota in S. paramamosain., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

As a major intracellular iron storage protein, ferritin plays important roles in iron homeostasis and innate immunity. In this study, two novel ferritin subunits from noble scallop Chlamys nobilis (CnFer1 and CnFer2) were identified and analyzed. The open reading frame of CnFer1 and CnFer2 was 522 and 519bp long, encoding 173 and 172 amino acids, respectively. Both ferritins contained a putative iron-binding region signature (IBRS). Analysis of putative conserved domains showed the two CnFer genes contained three key domains of ferritin subunits, a ferroxidase diiron center (E25, Y32, E59, E60, H63, E105, and Q139), an iron ion channel (H116, D129, E132) and a ferrihydrite nucleation center (D58, E59, and E62) that present in M type subunits. A putative iron response element (IRE) was observed at both CnFer genes in the 5' UTR. Phylogenetic analysis result suggested that the two genes are cytoplasmic ferritins and have the closest evolution relationship with ferritins from Mizuhopecten yessoensis. The two ferritin genes were wildly expressed in examined tissues and the highest level was found in gill. After V. parahaemolyticus challenged, both CnFer genes were significantly up-regulated suggesting that they are important proteins involved in host immune defense. Moreover, under bacterial challenge, the expression levels of both two genes in Golden scallops (rich in carotenoids) were significantly higher than that in Brown scallops (less in carotenoids) which suggesting that carotenoids enhance the immunity in scallops to defense against the bacterial stress., (Copyright © 2019. Published by Elsevier Ltd.)

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam 2 CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated., (Copyright © 2019 American Society for Microbiology.)