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.

Down Syndrome Cell Adhesion Molecule Triggers Membrane-to-Nucleus Signaling-Regulated Hemocyte Proliferation against Bacterial Infection in Invertebrates.

Down syndrome cell adhesion molecule (Dscam) generates tens of thousands of isoforms by alternative splicing, thereby providing crucial functions during immune responses. In this study, a novel Dscam signaling pathway was investigated in crab, which remains poorly characterized in invertebrates. Bacterial infection induced the cytoplasmic cleavage of Dscam intracellular domains (ICDs) by γ-secretase, and then the released ICDs carrying specific alternatively spliced exons could directly interact with IPO5 to facilitate nuclear translocation. Nuclear imported ICDs thus promoted hemocyte proliferation and protect the host from bacterial infection. Protein-interaction studies revealed that the ectodomain of Dscam bound to a disintegrin and metalloprotease domain 10 (ADAM10) rather than ADAM17. Inhibition or overexpression of ADAM10 impaired or accelerated Dscam shedding activity post-bacterial stimulation, respectively. Moreover, the shedding signal then mediated Dscam with an intact cytoplasmic domain to promote the cleavage of ICDs by γ-secretase. Furthermore, the transcription of ADAM10 was regulated by Dscam-induced canonical signaling, but not nuclear imported ICDs, to serve as a feedback regulation between two different Dscam pathways. Thus, membrane-to-nuclear signaling of Dscam regulated hemocyte proliferation in response to bacterial infection.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

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.

miR-2 contributes to WSSV infection by targeting Caspase 2 in mud crab (Scylla paramamosain).

Caspase 2 is widely studied for its function in the regulation of apoptosis in mammals. Despite the fundamental role of apoptosis during the anti-viral immune response, the relationship between Caspase 2 and virus infection has not been extensively explored in invertebrates. Also, whether or not miRNAs involve this process remains unclear. To address this issue, the miRNA-mediated regulation of Caspase 2 in mud crab (Scylla paramamosain) (Sp-Caspase 2) was characterized in this study. Sp-Caspase 2 contains an open reading frame (ORF) of 969 bp encoding 322 deduced amino acids and possesses a conserved CASc domain. The results suggested that Sp-Caspase 2 could suppress white spot syndrome virus infection via apoptosis induction. The further data showed that Sp-Caspase 2 was directly targeted by miR-2 in mud crab. Silencing or overexpression of miR-2 could affect apoptosis and WSSV replication through the regulation of Sp-Caspase 2 expression. Taken together, these results demonstrated the crucial role of the miR-2-Caspase 2 pathway in the innate immunity of mud crabs and revealed a novel mechanism in the anti-viral immune response in marine invertebrates.

A novel JNK induces innate immune response by activating the expression of antimicrobial peptides in Chinese mitten crab Eriocheir sinensis.

c-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinases (MAPKs) that participates in the regulation of various physiological and pathological processes. In this study, we identified a novel JNK (EsJNK) and determined the cDNA sequence of its isoform (EsJNK-a) from the Chinese mitten crab Eriocheir sinensis. The open reading frame (ORF) of EsJNK was predicted to encode 421 peptides with a serine/threonine protein kinase, a catalytic (S_TKc) domain, and a low complexity region. The ORF of EsJNK-a was 1380 bp encoding a protein with 459 amino acids, which was 38 amino acids more than that of EsJNK. The predicted tertiary structure of EsJNK was conserved and contained 15 α-helices and 10 ß-sheets. Phylogenetic tree analysis revealed that EsJNK was clustered with the JNK homologs of other crustaceans. Quantitative real-time PCR assays showed that EsJNK was expressed in all the tissues examined, but it was relatively higher in hemocytes, muscles, and intestines. The expression of EsJNK mRNA in the hemocytes was upregulated by lipopolysaccharides and peptidoglycans, as well as by Staphylococcus aureus or Vibrio parahaemolyticus challenge. Functionally, after silencing EsJNK by siRNA in crabs, the expression levels of two antimicrobial peptides (AMPs), namely, anti-lipopolysaccharide factor and crustin, were significantly inhibited. The purified recombinant EsJNK protein with His-tag accelerated the elimination of the aforementioned bacteria in vivo. However, knockdown of EsJNK had an opposite effect. These findings suggested that EsJNK might be involved in the antibacterial immune defense of crabs by regulating the transcription of AMPs.

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea.

BACKGROUND: Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity. METHODS AND RESULTS: The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients. CONCLUSIONS: This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

Exosomal miR-224 contributes to hemolymph microbiota homeostasis during bacterial infection in crustacean.

It is well known that exosomes could serve as anti-microbial immune factors in animals. However, despite growing evidences have shown that the homeostasis of the hemolymph microbiota was vital for immune regulation in crustaceans, the relationship between exosomes and hemolymph microbiota homeostasis during pathogenic bacteria infection has not been addressed. Here, we reported that exosomes released from Vibrio parahaemolyticus-infected mud crabs (Scylla paramamosain) could help to maintain the homeostasis of hemolymph microbiota and have a protective effect on the mortality of the host during the infection process. We further confirmed that miR-224 was densely packaged in these exosomes, resulting in the suppression of HSP70 and disruption of the HSP70-TRAF6 complex, then the released TRAF6 further interacted with Ecsit to regulate the production of mitochondrial ROS (mROS) and the expression of Anti-lipopolysaccharide factors (ALFs) in recipient hemocytes, which eventually affected hemolymph microbiota homeostasis in response to the pathogenic bacteria infection in mud crab. To the best of our knowledge, this is the first document that reports the role of exosome in the hemolymph microbiota homeostasis modulation during pathogen infection, which reveals the crosstalk between exosomal miRNAs and innate immune response in crustaceans.