Identification and characterization of a TLR4 homologue in Eriocheir sinensis based on structure analysis.

Toll-like receptor 4 (TLR4) plays an essential role in the activation of innate immunity by recognizing diverse pathogenic components of bacteria. Six Tolls were found in Eriocheir sinensis but have not yet been identified as mammalian TLR4 homolog. For this purpose, we predicted three-dimensional (3D) structures of EsTolls (EsToll1-6) with AlphaFold2. 3D structure of LRRs and TIR most had high accuracy (pLDDT >70). By structure analysis, 3D structures of EsToll6 had a high overlap with HsTLR4. Moreover, we also predicted potential 11 hydrogen bonds and 3 salt bridges in the 3D structure of EsToll6-EsML1 complex. 18 hydrogen bonds and 7 salt bridges were predicted in EsToll6-EsML2 complex. Co-immunoprecipitation assay showed that EsToll6 could interact with EsML1 and EsML2, respectively. Importantly, TAK242 (a mammalian TLR4-specific inhibitor) could inhibit the generation of ROS stimulated by lipopolysaccharides (LPS) in EsToll6-EsML2-overexpression Hela cells. Collectively, these results implied that EsToll6 was a mammalian TLR4 homolog and provided a new insight for researching mammalian homologs in invertebrates.

The interaction between 20-hydroxyecdysone and AMPK through PI3K activation in Chinese mitten crab, Eriocheir sinensis.

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKß and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKß induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis, which appeared to be helpful for a better understanding in molting regulation.

Identification of toll-like receptor family and the immune function of new Sptlr-6 gene of Scylla paramamosain.

As a crucial member of pattern-recognition receptors (PRRs), the Tolls/Toll-like receptors (TLRs) gene family has been proven to be involved in innate immunity in crustaceans. In this study, nine members of TLR gene family were identified from the mud crab (Scylla paramamosain) transcriptome, and the structure and phylogeny of different SpTLRs were analyzed. It was found that different SpTLRs possessed three conserved structures in the TIR domain. Meanwhile, the expression patterns of different Sptlr genes in examined tissues detected by qRT-PCR had wide differences. Compared with other Sptlr genes, Sptlr-6 gene was significantly highly expressed in the hepatopancreas and less expressed in other tissues. Therefore, the function of Sptlr-6 was further investigated. The expression of the Sptlr-6 gene was up-regulated by Poly I: C, PGN stimulation and Vibrio parahaemolyticus infection. In addition, the silencing of Sptlr-6 in hepatopancreas mediated by RNAi technology resulted in the significant decrease of several conserved genes involved in innate immunity in mud crab after V. parahaemolyticus infection, including relish, myd88, dorsal, anti-lipopolysaccharide factor (ALF), anti-lipopolysaccharide factor 2 (ALF-2) and glycine-rich antimicrobial peptide (glyamp). This study provided new knowledge for the role of the Sptlr-6 gene in defense against V. parahaemolyticus infection in S. paramamosain.

Metabolomics and microbiome co-analysis reveals altered innate immune responses in Charybdis japonica following Aeromonas hydrophila infection.

A comprehensive bioinformatics analysis was conducted to elucidate the innate immune response of Charybdis japonica following exposure to Aeromonas hydrophila. This study integrated metabolomics, 16S rRNA sequencing, and enzymatic activity data to dissect the immune mechanisms activated in response to infection. Infection with A. hydrophila resulted in an increased abundance of beneficial intestinal genera such as Photobacterium spp., Rhodobacter spp., Polaribacter spp., Psychrilyobacter spp., and Mesoflavibacter spp. These probiotics appear to suppress A. hydrophila colonization by competitively dominating the intestinal microbiota. Key metabolic pathways affected included fatty acid biosynthesis, galactose metabolism, and nitrogen metabolism, highlighting their role in the crab's intestinal response. Enzymatic analysis revealed a decrease in activities of hexokinase, phosphofructokinase, and pyruvate kinase, which are essential for energy homeostasis and ATP production necessary for stress responses. Additionally, reductions were observed in the activities of acetyl-CoA carboxylase and fatty acid synthase. Gene expression analysis showed downregulation in Peroxiredoxin 1 (PRDX1), Peroxiredoxin 2 (PRDX2), glutathione-S-transferase (GST), catalase (CAT), and glutathione (GSH), with concurrent increases in malondialdehyde (MDA) levels, indicating severe oxidative stress. This study provides insights into the molecular strategies employed by marine crabs to counteract bacterial invasions in their natural habitat.

Rhodobacter sphaeroides supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against Shewanella putrefaciens infection via intestinal flora and metabolism regulation.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection.

In vitro immune analysis of serum from the hemolymph of the anomuran crab Albunea symmysta (Linnaeus, 1758) displayed diversified reactions.

The present investigation aims to substantiate that serum from the hemolymph of anomuran crab Albunea symmysta encompasses multiple immunological reactions in in vitro condition. The serum highly agglutinated human O erythrocytes in the presence of Ba2+. Distinct and unique sugar binding capacity of serum towards laminarin, N-acetyl sugars and higher binding specificity towards a glycoprotein, fetuin was inferred. In vitro enhancement of melanin synthesis due to enhanced oxidation of 3, 4-dihydroxy-dl-phenylalanine (dl-DOPA) by preincubation of nonself molecules with serum phenoloxidase (PO) was documented. Similarly, dl-DOPA oxidation by serum PO was reduced when preincubated with chemical inhibitors and copper chelators. Further, the crab serum lysed the vertebrate erythrocytes with maximum hemolysis against chicken and it unveiled dependency on divalent cation, serum concentration, ionic strength, pH, temperature and time interval. Occurrence of maximum hemolysis at a concentration of 30 µl, pH 8.0, temperature 37 °C and time interval of 60 min in the presence of Ba2+ were documented. Interestingly, serum hemolysis was reduced by different osmoprotectants suggesting a colloid-osmotic mechanism involving in hemolysis. It was observed that A. symmysta serum had antimicrobial activity against Gram-positive Staphylococcus aureus and fungal pathogen Candida albicans. The serum showed higher glycan content, potent lysozyme and free radical scavenging activity suggesting the existence of potential immune molecules of therapeutic use. These results clearly demonstrated the diversified immunogenicity of A. symmysta serum confirming a highly conserved non-specific immunity of crustaceans.

Glutaredoxin 2 in the mud crab Scylla paramamosain: Identification and functional characterization under hypoxia and pathogen challenge.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that plays a key role in antioxidant defense. In this study, a novel Grx2 gene (SpGrx2) was identified from the mud crab Scylla paramamosain, which consists of a 196 bp 5' untranslated region, a 357 bp open reading frame, and a 964 bp 3' untranslated region. The putative SpGrx2 protein has a typical single Grx domain with the active center sequence C-P-Y-C. The expression analysis revealed that the SpGrx2 mRNA was most abundant in the gill, followed by the stomach and hemocytes. Both mud crab dicistrovirus-1 and Vibrioparahaemolyticus infection as well as hypoxia could differentially induce the expression of SpGrx2. Furthermore, silencing SpGrx2 in vivo affected the expression of a series of antioxidant-related genes after hypoxia treatment. Additionally, SpGrx2 overexpression significantly increased the total antioxidant capacity of Drosophila Schneider 2 cells after hypoxia, resulting in a reduction of reactive oxygen species and malondialdehyde content. The subcellular localization results indicated that SpGrx2 was localized in both the cytoplasm and the nucleus of Drosophila Schneider 2 cells. These results indicate that SpGrx2 plays a crucial role as an antioxidant enzyme in the defense system of mud crabs against hypoxia and pathogen challenge.

New insight of variable lymphocyte receptor-likes in anti-bacteria activity from Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is a vital freshwater aquaculture species in China, however, is also facing various crab disease threats. In the present study, we identify three novel variable lymphocyte receptor-like (VLR-like) genes-VLR-like1, VLR-like3 and VLR-like4-from E. sinensis, which play vital roles in adaptive immune system of agnathan vertebrates. The bacterial challenge, bacterial binding and antibacterial-activity experiments were applied to study immune functions of VLR-likes, and the transcriptomic data from previous E. sinensis bacterial challenge experiments were analyzed to speculate the possible signaling pathway. VLR-like1 and VLR-like4 can respond to Staphylococcus aureus challenge and inhibit S. aureus specifically. VLR-like1 and VLR-like4 possess broad-spectrum bacteria-binding ability whereas VLR-like3 do not. VLR-likes in E. sinensis could associate with the Toll-like receptor (TLR) signaling pathway. The above results suggest that VLR-likes defend against bacteria invasion though exerting anti-bacteria activity, and probably connect with the TLR signaling pathway. Furthermore, studying the immune functions of these VLR-likes will provide a new insight into the disease control strategy of crustacean culture.

Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus.

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Biochemical, metabolic, and immune responses of mud crab (Scylla paramamosain) after mud crab reovirus infection.

Mud crab reovirus (MCRV) is a serious pathogen that leads to large economic losses in the mud crab farming. However, the molecular mechanism of the immune response after MCRV infection is unclear. In the present study, physiological, transcriptomic, and metabolomic responses after MCRV infection were investigated. The results showed that MCRV infection could increase lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase activities. MCRV infection decreased antioxidant enzyme activity levels, induced oxidative stress, and caused severe histological damage. Transcriptome analysis identified 416 differentially expressed genes, including 354 up-regulated and 62 down-regulated genes. The detoxification, immune response, and metabolic processes-related genes were found. The results showed that two key pathways including phagocytosis and apoptosis played important roles in response to MCRV infection. The combination of transcriptomic and metabolomic analyses showed that related metabolic pathways, such as glycolysis, citrate cycle, lipid, and amino acid metabolism were also significantly disrupted. Moreover, the biosynthesis of unsaturated fatty acids was activated in response to MCRV infection. This study provided a novel insight into the understanding of cellular mechanisms in crustaceans against viral invasion.

Hematodinium sp. infection does not drive collateral disease contraction in a crustacean host.

Host, pathogen, and environment are determinants of the disease triangle, the latter being a key driver of disease outcomes and persistence within a community. The dinoflagellate genus Hematodinium is detrimental to crustaceans globally - considered to suppress the innate defences of hosts, making them more susceptible to co-infections. Evidence supporting immune suppression is largely anecdotal and sourced from diffuse accounts of compromised decapods. We used a population of shore crabs (Carcinus maenas), where Hematodinium sp. is endemic, to determine the extent of collateral infections across two distinct environments (open-water, semi-closed dock). Using a multi-resource approach (PCR, histology, haematology, population genetics, eDNA), we identified 162 Hematodinium-positive crabs and size/sex-matched these to 162 Hematodinium-free crabs out of 1191 analysed. Crabs were interrogated for known additional disease-causing agents; haplosporidians, microsporidians, mikrocytids, Vibrio spp., fungi, Sacculina, trematodes, and haemolymph bacterial loads. We found no significant differences in occurrence, severity, or composition of collateral infections between Hematodinium-positive and Hematodinium-free crabs at either site, but crucially, we recorded site-restricted blends of pathogens. We found no gross signs of host cell immune reactivity towards Hematodinium in the presence or absence of other pathogens. We contend Hematodinium sp. is not the proximal driver of co-infections in shore crabs, which suggests an evolutionary drive towards latency in this environmentally plastic host.

Identification of arginine kinase as an allergen of brown crab, Callinectes bellicosus, and in silico analysis of IgE-binding epitopes.

In recent years there has been an increase in the prevalence of allergic reactions to contact with/or consumption of crustaceans by immune responses mediated by IgE antibodies. Arginine kinase (AK) is considered one of the main allergens present in marine invertebrates. Currently, the allergenic potential of the brown crab (Callinectes bellicosus), which is a crustacean of great economic importance, has not been studied. Therefore, the aim of this work was to identify C. bellicosus AK as an allergen and to predict IgE-binding epitopes through immunobioinformatic analysis. AK was purified by precipitation with ammonium sulfate and ion- exchange chromatography. AK allergenicity was evaluated by IgE reactivity against sera from crustacean-allergic and non-allergic patients in both native and denaturing conditions. Additionally, a homology model was built based on the deduced amino acid sequence. A single band (~40 kDa) was found in SDS-PAGE, which was identified as an AK by mass spectrometry. AK showed immunoreactivity against crab-allergenic sera in both native and denaturing conditions with 70% and 80% positive reactions, respectively. Additionally, a 1073 bp ORF was obtained which codes for a deduced sequence of 357 amino acids corresponding to AK with > 90% identity with other AKs. Structural homology model of AK showed two main domains with conserved / folding of phospho-guanidine kinases. BediPred and Discotope were used for epitope prediction analysis, which suggests eight possible linear epitopes and seven conformational epitopes, respectively; and shows to be similar to other crustaceans AKs. C. bellicosus AK was identified as an allergenic protein by IgE reactivity and immunobioinformatic analysis indicates that both linear and conformational epitopes could be located in the surface of C. bellicosus AK structure.

Identification and function analysis of an immune deficiency homolog in swimming crab, Portunus trituberculatus.

The immune deficiency (IMD) pathway is involved in both antiviral and antibacterial immune responses in Drosophila. IMD protein is the key adaptor to link the extracellular signal and the intracellular reaction to initiate the signal transduction in IMD pathway. In present study, the cDNA of the IMD (Pt-IMD) was identified from a marine crab, Portunus trituberculatus. The Pt-IMD is predicted to encode 170 amino acids with a death domain. Real-Time quantitative PCR analysis showed that Pt-IMD was constitutively expressed in hemocytes, intestine, gill, heart, muscle and hepatopancreas in normal crab. Moreover, the transcript of Pt-IMD in large-granule hemocytes is approximately 6-fold higher than semi-granular cells and agranular cells. Intracellular localization showed Pt-IMD was distributed mainly in the cytoplasm when it was over-expressed in Drosophila Schneider 2 (S2) cell. Functionally, over-expression of Pt-IMD could activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. Furthermore, Pt-IMD expression was also knock-down by RNAi to determine the function of Pt-IMD on regulation of the expression of different antimicrobial peptides (AMPs) in crab. In the primary cultured hemocytes challenged with or without Vibrio alginolyticus, after Pt-IMD was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), ALF3, crustin1, crustin3, arasin2, hyastatin1and hyastatin3 have been significantly inhibited in normal cell or bacterial infected cell, while the expression of lysozyme was normal in non-infected cells and was significantly induced in bacterial infected cells, which compared to the non-specific dsRNA treated cells.

SpBAG3 assisted WSSV infection in mud crab (Scylla paramamosain) by inhibiting apoptosis.

The function of B-cell lymphoma-2 (Bcl-2) family proteins can be divided into two categories: anti-apoptotic and pro-apoptotic. As an anti-apoptotic protein, Bcl2-associated athanogene 3 (BAG3) plays a key role in regulating apoptosis, development, cell movement, and autophagy, and mediating the adaptability of cells to stimulation. However, SpBAG3 has not been reported in mud crab (Scylla paramamosain), and the regulatory effect of SpBAG3 on apoptosis in mud crab and its function in antiviral immunity is still unknown. In this study, SpBAG3 was found, and characterized, which encoded a total of 175 amino acid (molecular mass 19.3 kDa), including a specific conserved domain of the BAG family. SpBAG3 was significantly down-regulated at 0-48 h post-infection with WSSV in vivo. The antiviral effect of SpBAG3 was investigated using RNA interference. The results indicated that SpBAG3 might be involved in assisting the replication of WSSV in the host. SpBAG3 could change the mitochondrial membrane potential (△ψm), and affect cell apoptosis through mitochondrial apoptotic pathways. Therefore, the results of this study suggested that SpBAG3 could assist WSSV infection by inhibiting the apoptosis of the hemocytes in mud crab.

Immunomodulatory role of crustacean cardioactive peptide in the mud crab Scylla paramamosain.

Crustacean cardioactive peptide (CCAP) is a pleiotropic neuropeptide, but its immunomodulatory role is not clear. Herein, the mud crab Scylla paramamosain provides a primitive model to study crosstalk between the neuroendocrine and immune systems. In this study, in situ hybridization showed that Sp-CCAP positive signal localized in multiple cells in the nervous tissue, while its conjugate receptor (Sp-CCAPR) positive signal mainly localized in the semigranular cells of hemocytes. The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide (LPS) stimulation, but the Sp-CCAP mRNA expression level was up-regulated firstly and then down-regulated after the stimulation of polyriboinosinic polyribocytidylic acid [Poly (I:C)]. After the injection of Sp-CCAP synthesis peptide, the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group. Simultaneously, the mRNA expression of phagocytosis related gene (Sp-Rab5), nuclear transcription factor NF-κB homologues (Sp-Relish), C-type lectin (Sp-CTL-B), prophenoloxidase (Sp-proPO), pro-inflammatory cytokines factor (Sp-TNFSF, Sp-IL16) and antimicrobial peptides (Sp-ALF1 and Sp-ALF5) in the hemocytes were also significantly up-regulated at different time points after the injection of Sp-CCAP synthetic peptide, but Sp-TNFSF, Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h. In addition, RNA interference of Sp-CCAP suppressed the phagocytic activity of hemocytes and inhibited the mRNA expression of Sp-Rab5, Sp-Relish, Sp-CTL-B, Sp-TNFSF, Sp-IL16 and Sp-ALF5 in the hemocytes, and ultimately weakened the ability of hemolymph bacteria clearance of mud crab. Taken together, these results revealed that CCAP induced innate immune and increased the anti-infection ability in the mud crab.

The Elevated Expressions of Anti-lipopolysaccharide Factors After Priming Stimulation Confer Lastingly Humoral Protection in Crab Eriocheir sinensis.

Evidence of immune memory in invertebrates (immune priming) has accumulated in various organisms, and both cellular and humoral immune reactions are speculated to be involved in immune priming. However, there is a lack of understanding of the molecular mechanisms involved. In the present study, the protective effect of primed haemolymph was further validated by the increased survival rate of naïve crabs receiving a transfusion of primed haemolymph. By proteomic analysis, there were 474 proteins identified from the primed haemolymph, and most of them were functionally annotated in transport and metabolism classes. A total of 70 proteins were found to be differentially expressed in haemolymph at 12 hours and 7 days after priming stimulation with Aeromonas hydrophila, among which anti-lipopolysaccharide factor 1 (EsALF-1) and 3 (EsALF-3) were identified as the most significant (p < 0.05). After being challenged with A. hydrophila, EsALF-1 and EsALF-3 were highly expressed at both mRNA (in haemocytes) and protein (in haemolymph) levels compared with blank crabs, and the mRNA expressions of components in the EsTLR1-EsMyd88-EsPelle-EsALF pathway also increased significantly (p < 0.05). The EsALF-3 and EsMyd88 were even significantly higher expressed in response to the second A. hydrophila challenge, but their expressions all decreased (p < 0.05) when EsTLR1 was knocked down by RNAi. After the naïve crabs received an injection with the recombinant protein of EsALF-1 (rEsALF-1) or EsALF-3 (rEsALF-3), their survival rate increased significantly (p < 0.05) upon A. hydrophila stimulation. In contrast, the survival rate of the primed crabs reduced significantly (p < 0.05) after they received an injection with the antibody of EsALF-1 or EsALF-3. The enhanced expressions of EsALF-1 and EsALF-3 after A. hydrophilap riming stimulation could sustain for four weeks. All the results suggested that the EsTLR1-mediated productions of EsALF-1 and EsALF-3 in haemolymph played an indispensable role in the month-long humoral immune protection induced by A. hydrophila, which provides solid evidence of immune priming in crabs and a valuable reference for further understanding immune memory in invertebrates.

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

SpTIA-1 suppresses WSSV infection by promoting apoptosis in mud crab (Scylla paramamosain).

TIA-1 (T cell restricted intracellular antigen-1) is a kind of RNA-binding protein which serves as the downstream of CED-9 (a BCL2 homolog) and induces apoptosis under stress conditions. So far, the function of apoptosis mediated by TIA-1 has been extensively studied in higher animals, and apoptosis happens to be related to biological immune defense. However, the involvement of TIA-1 in the study of immune function during viral infection has not been clearly studied, especially in marine invertebrates. In the study, SpTIA-1 in mud crab (Scylla paramamosain) was specifically identified. The Open Reading Frame (ORF) of SpTIA-1 was consisted of 1116 nucleotide bases and encoded 372 amino acids. Besides, the results showed that the expression of SpTIA-1 was obviously up-regulated during WSSV (White Spot Syndrome Virus) infection in hemocytes of mud crab. Furthermore, through RNAi approach, we found that SpTIA-1 could activate Caspase-3 signaling and increase ROS levels to reduce mitochondrial membrane potential, resulting in the increase of apoptosis rate in hemocytes, which eventually suppressed WSSV multiplication in mud crab. The current study therefore improves the knowledge of antiviral immunity in mud crab and provides new insights into the innate immunity of marine crustaceans.

Identification and characterization of Crassostrea angulata arginine kinase, a novel allergen that causes cross-reactivity among shellfish.

Oyster is a common food that causes allergy. However, little information is available about its allergens and cross-reactivity. In this study, arginine kinase (AK) was identified as a novel allergen in Crassostrea angulata. The primary sequence of AK was cloned which encoded 350 amino acids, and recombinant AK (rAK) was obtained. The immunodot results, secondary structure and digestive stability showed that native AK and rAK had similar IgG/IgE-binding activity and physicochemical properties. Serological analysis of 14 oyster-sensitive individuals demonstrated that AK exhibited cross-reactivity among oysters, shrimps, and crabs. Furthermore, nine epitopes in oyster AK were verified using inhibition dot blots and inhibition enzyme linked immunosorbent assay, six of which were similar to the epitopes of shrimp/crab AK. The most conserved epitopes were P5 (121-133) and P6 (133-146), which may be responsible for the cross-reactivity caused by AK. These findings will provide a deeper understanding of oyster allergens and cross-reactivity among shellfish.