Identification and characterization of a Reeler domain containing protein in Procambarus clarkii provides new insights into antibacterial immunity in crustacean.

Crayfish, as an invertebrate, relies only on the innate immune system to resist external pathogens. In this study, a molecule containing a single Reeler domain was identified from red swamp crayfish Procambarus clarkii (named as PcReeler). Tissue distribution analysis showed that PcReeler was highly expressed in gills and its expression was induced by bacterial stimulation. Inhibiting the expression of PcReeler by RNA interference led to a significant increase in the bacterial abundance in the gills of crayfish, and a significant increase in the crayfish mortality. Silencing of PcReeler influenced the stability of the microbiota in the gills revealed by 16S rDNA high-throughput sequencing. Recombinant PcReeler showed the ability to bind microbial polysaccharide and bacteria and to inhibit the formation of bacterial biofilms. These results provided direct evidence for the involvement of PcReeler in the antibacterial immune mechanism of P. clarkii.

Subcellular Translocation of Yorkie through the PRP4K-CRM1 Axis Regulates Antimicrobial Peptides Transcription and Defense against Bacterial Infection in Crab.

The Hippo signaling pathway plays important roles in innate immunity. In the current study, we found that bacterial infection did not influence mRNA and protein levels of yorkie (Yki), which is an important terminal molecule of the Hippo signaling pathway. However, bacterial infection promoted the translocation of Yki from the nucleus to the cytoplasm in Chinese mitten crab (Eriocheir sinensis), thus attenuating Yki-suppressed transcription of antimicrobial peptides through Cactus. Chromosome region maintenance 1 (CRM1)-silenced crab hemocytes significantly suppressed Yki translocation from the nucleus to the cytoplasm upon bacterial infection, resulting in significantly increased expression of Cactus, decreased expression of antimicrobial peptides, and higher bacterial susceptibility, which demonstrated the regulatory role of CRM1 in subcellular localization of Yki. However, RNA interference of Scalloped (Sd) exhibited no effect on the subcellular localization of Yki and its regulation of Cactus/antimicrobial peptides. Moreover, we elucidated that both CRM1 and Sd could interact with Yki and that the PRP4K-mediated phosphorylation of a conserved serine amino acid residue in the nuclear export signal of Yki is essential for interaction between Yki and CRM1; however, the phosphorylation did not affect the binding of Yki with Sd. We also found that bacterial infection significantly promoted the expression of PRP4K in hemocytes; RNA interference of PRP4K and phosphatase inhibitor suppressed Yki translocation from the nucleus to the cytoplasm, promoting Cactus expression and inhibiting antimicrobial peptide expression. Thus, subcellular localization of Yki regulates antibacterial infection through both PRP4K and CRM1 in crabs.

The role of ficolin as a pattern recognition receptor in antibacterial immunity in Eriocheir sinensis.

Ficolin, a member of the fibrinogen-related proteins family (FREPs), functions as a pattern recognition receptor (PRR) in vertebrates and in invertebrates as a novel lectin. In this study, we discovered the Ficolin homolog of Chinese mitten crab (Eriocheir sinensis), which we named EsFicolin. The obtained sequence showed that it has a highly conserved C-terminal fibrinogen-related domain (FReD) and a coiled-coil structure for trimer formation. EsFicolin was up-regulated in hemocytes after being stimulated by bacteria. Recombinant EsFicolin protein binds to gram-negative and gram-positive bacteria and agglutinates bacteria through pathogen-associated molecular patterns. In-depth study found that recombinant EsFicolin could effectively remove bacteria and showed direct antibacterial activity. EsFicolin could also promote the phagocytosis of hemocytes to enhance bacterial clearance. These findings suggest that EsFicolin plays an important role in the crab antibacterial immune response.

Effect of polystyrene nanoplastics on cell apoptosis, glucose metabolism, and antibacterial immunity of Eriocheir sinensis.

The adverse effects of plastic waste and nanoplastics on the water environment have become a focus of global attention in recent years. In the present study, using adult Chinese mitten crabs (Eriocheir sinensis) as an animal model, the bioaccumulation and the in vivo and in vitro toxicity of polystyrene nanoplastics (PS NPs), alone or in combination with the bacteria, were investigated. This study aimed to investigate the effects of PS NPs on apoptosis and glucose metabolism in Chinese mitten crabs, and whether PS NPs could synergistically affect the antibacterial immunity of crabs. We observed that NPs were endocytosed by hemocytes, which are immune cells in crustaceans and are involved in innate immunity. The RNA sequencing data showed that after hemocytes endocytosed NPs, apoptosis and glucose metabolism-related gene expression was significantly induced, resulting in abnormal cell apoptosis and a glucose metabolism disorder. In addition, exposure to NPs resulted in changes in the antimicrobial immunity of crabs, including changes in antimicrobial peptide expression, survival, and bacterial clearance. In summary, NPs could be endocytosed by crab hemocytes, which adversely affected the cell apoptosis, glucose metabolism, and antibacterial immunity of Eriocheir sinensis. This study revealed the effects of NPs on crab immunity and lays the foundation for further exploration of the synergistic effect of NPs and bacteria.

A Novel Ig Domain-Containing C-Type Lectin Triggers the Intestine-Hemocyte Axis to Regulate Antibacterial Immunity in Crab.

The C-type lectin family with the signature C-type lectin-like domain promotes antibacterial host defense within the animal kingdom. We examined the role of Chinese mitten crab, Eriocheir sinensis (H. Milne-Edwards) (Decapoda: Grapsidae) Ig domain-containing C-type lectin (EsIgLectin), a novel and poorly understood member of the C-type lectin family. EsIgLectin was expressed primarily by both hemocytes (E sinensis) and intestines, with significantly induced mRNA expression on intestinal or hemolymph bacterial infections. As a soluble protein, both its C-type lectin-like domain and the Ig domain were required for bacterial binding, bacterial agglutination, bacterial growth inhibition, and in vivo bacterial clearance. Polymeric EsIgLectin could be constructed via the disulfide bond in the Ig domain, significantly enhancing EsIgLectin antibacterial activity. EsIgLectin promoted bacterial phagocytosis in an Ig domain-dependent manner in hemocytes, while it controlled microbial homeostasis and protected against bacteria-induced inflammation in the intestine. Protein interaction studies revealed that the EsIgLectin Ig domain bound to the first Ig domain of the polymeric Ig receptor, which was essential for EsIgLectin-induced bacterial phagocytosis. The temporal sequence of cell interactions during intestinal inflammation is only beginning to be understood. In this article, we show that hemocyte-derived EsIgLectin entered the intestinal wall at the later phase of intestinal inflammation. Moreover, EsIgLectin protected the host against intestinal and hemolymph infections in a polymeric Ig receptor-dependent manner. Therefore, the EsIgLectin promoted bacterial clearance and protected against inflammatory disease through an independent or synergistic effect of hemocytes and intestines in invertebrates.

FADD regulates antibacterial immune responses via the immune deficiency signaling pathway in the Chinese mitten crab.

In invertebrates, innate immune responses are the only defense against invading pathogens. The immune deficiency (IMD) signaling pathway protects invertebrates from bacterial infection by secreting antimicrobial peptides (AMPs). Fas-associated protein with death domain (FADD) activates AMPs and triggers apoptosis. However, FADD's function in crustaceans is unclear. Herein, the full-length FADD cDNA (EsFADD) was cloned from the Chinese mitten crab, Eriocheir sinensis. Vibrio parahaemolyticus infection upregulated EsFADD expression markedly. Knockdown of EsFADD in hemocytes suppressed the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation, which in turn reduced the expression of several AMPs. In vivo, silencing of EsFADD rendered crabs susceptible to bacterial infection and impaired their bacterial clearance. The results suggest that EsFADD is indispensable in IMD signal transduction in E. sinensis. In contrast to Drosophila, EsFADD barely promoted apoptosis. Our findings revealed the evolutionary conservation of FADD in crustaceans and provided insights into IMD signaling in invertebrates.

Immunological functional differentiation of two transmembrane variants of Dscam in Chinese mitten crab.

Down syndrome cell adhesion molecule (Dscam), also called hypervariable Dscam (Dscam-hv), is an important player in arthropod alternative splicing that connects neurons and immune regulation, acting as a pathogen-specific recognition molecule. Dscam-hv has two forms: transmembrane (TM) Dscam (mDscam) and soluble Dscam (sDscam). Herein, we investigated two transmembrane variants of mDscam resulting from alternative splicing of the transmembrane domain, focusing on differences in their immune regulation. We characterized the Dscam[TM1] and Dscam[TM2] genes of Chinese mitten crab (Eriocheir sinensis) through bioinformatics analysis. Both genes are expressed in the gill, intestine, and other immune tissues. Following gram-positive and gram-negative bacteria stimulation, EsDscam[TM1] and EsDscam[TM2] mRNA expression levels increased significantly in hemocytes. Sequencing showed that EsDscam[TM1] was more abundant in hemocytes than EsDscam[TM2]. Additionally, the two subtypes differ in their regulation of antimicrobial peptides, the proportion of exon 33 carried, and bacterial phagocytosis.

A novel ML protein functions as a pattern recognition protein in antibacterial responses in Eriocheir sinensis.

The myeloid differentiation factor 2 (MD-2)-related lipid recognition (ML) domain is present in MD-2, MD-1, GM2-activator protein (GM2A) and Niemann-Pick disease type C2 (NPC2). ML proteins function in antibacterial signal transduction and lipid metabolism in vertebrates, but the mechanism in invertebrates is unknown. In this study, we found that ML proteins were involved in bacterial resistance in Chinese mitten crab (Eriocheir sinensis). One member, EsML3, a soluble, bacterial-induced pattern recognition protein was upregulated in hemocytes following bacterial challenge. Recombinant EsML3 bound to Gram-negative bacteria (Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by interaction with peptidoglycan, lipopolysaccharide. EsML3 showed no direct bacteriostatic or bacteriocidal activity. Pre-incubating bacteria with rEsML3 significantly promoted in vivo bacterial clearance. EsML3 also promoted phagocytic activity and plays a role against bacterial infection. In summary, EsML3 mediates cellular immune responses by recognising invasive microorganisms, promoting bacterial clearance and phagocytosis against bacterial infection in crab.

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.

Vitellogenin receptor expression in ovaries controls innate immunity in the Chinese mitten crab (Eriocheir sinensis) by regulating vitellogenin accumulation in the hemolymph.

The vitellogenin receptor (Vgr), which is specific for vitellogenin (Vtg), recognises and transports Vtg into the ovaries. Accumulating evidence suggests that Vtg also performs an immune defence function and plays critical roles in innate immunity in oviparous animals. However, whether Vgr is involved in innate immunity in the Chinese mitten crab (Eriocheir sinensis) is unknown. In this study, we obtained a 3009 nucleotide partial cDNA of the E. sinensis vitellogenin receptor gene (Es-vgr) encoding an open reading frame of 1003 amino acid residues. Bioinformatics analysis showed that the domains of Es-vgr were conserved during evolution. Quantitative real-time PCR and western blotting revealed that the highest Es-vgr expression levels occurred in the ovary, and expression was specific. Comparison of the expression levels of Es-vgr and the Vtg gene (Es-vtg1) at different ovary developmental stages suggested that there may be some regulatory relationship between them. Bacterial challenge induced high-level expression of antimicrobial peptide genes and reduced Es-vgr expression in ovaries, resulting in massive accumulation of Vtg in the hemolymph. The survival rate of crabs increased significantly after injection with recombinant Es-vtg1 protein following bacterial infection. Collectively, these results demonstrate that Es-vgr plays critical roles in antimicrobial function by regulating the accumulation of Vtg in the hemolymph.

Bacteria-induced IMD-Relish-AMPs pathway activation in Chinese mitten crab.

The innate immune response is an important line of defense against invading pathogens in invertebrates. Signaling pathways, including the IMD pathway, play critical roles in the production of antimicrobial peptides (AMPs), which induce the transcription of immune effectors that protect against bacterial invasion. In the present study, the cDNA of IMD from Eriocheir sinensis was cloned (designated EsIMD) and shown to be significantly upregulated following Gram-positive and Gram-negative bacterial infection. In vivo and in vitro studies collectively suggested that both the Gram-negative bacterium Vibrio parahemolyticus and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis elicit the translocation of Relish. Moreover, EsIMD positively regulated EsRelish translocation from the cytoplasm to the nucleus following stimulation with both Gram-positive and Gram-negative bacteria. EsRelish knockdown in hemocytes significantly suppressed AMPs' expression. Furthermore, both Lys-type and DAP-type peptidoglycan-containing bacteria activated the IMD pathway and elicited antibacterial responses in crab. Conclusively, these findings demonstrate that both Gram-positive and Gram-negative bacteria activate IMD signaling, via a mechanism that is distinct with that by which Gram-negative bacteria activate IMD signaling in Drosophila. These findings might pave the way for a better understanding of the innate immune system and the fundamental network of the IMD signaling pathway in crustacean.

Rab7 controls innate immunity by regulating phagocytosis and antimicrobial peptide expression in Chinese mitten crab.

The Rab family is the most significant subfamily of small GTP-binding proteins. These proteins have widespread intracellular localization and play an important role in many biological processes. Rab7 plays a crucial role in the innate immune system of crustaceans. In the present study, we cloned and characterized Rab7 from Chinese mitten crab (Eriocheir sinensis), designated EsRab7. The full-length of the EsRab7 cDNA sequence is 1,257 bp and contains a 618-bp open reading frame encoding a 205-amino acid polypeptide. Bioinformatics analysis showed that the Rab7 protein was highly conserved during evolution. Quantitative real-time PCR showed the highest tissue expression in muscle, followed by hepatopancreas. EsRab7 was significantly upregulated in hemocytes after stimulation by Gram-positive Staphylococcus aureus or Gram-negative Vibrio parahaemolyticus. Further studies showed that EsRab7 knockdown during bacterial stimulation resulted in decreased bacterial phagocytosis. In addition, EsRab7 regulated the expression of antimicrobial peptides via the Toll signaling pathway. Collectively, these results demonstrate that EsRab7 plays critical roles in antimicrobial function in the Chinese mitten crab.

EsGPCR89 regulates cerebral antimicrobial peptides through hemocytes in Eriocheir sinensis.

G protein-coupled receptors (GPCRs) are important transmembrane receptors that participate in diverse physiological processes including metabolism, cell growth and immune processes by transmitting extracellular signals to intracellular effectors. In this study, a gene belonging to the GPCR family was cloned from Eriocheir sinensis and named EsGPCR89. The full-length gene includes an open reading frame (ORF) of 465 amino acid residues, and bioinformatic analysis confirmed the high conservation between species. EsGPCR89 was detected in various tissues of E. sinensis, and was up-regulated in brain following Staphylococcus aureus infection. Expression levels of cerebral antimicrobial peptides (AMPs) were also up-regulated following bacterial challenge, reflecting their function in cerebral immunity. Additionally, EsGPCR89 silencing in hemocytes by RNA interference, down-regulated AMPs in brain after S. aureus infection. Moreover, through Immunisation assay and Polyacrylamide gel electrophoresis (SDS-PAGE) experiments, we could infer that bacterially infected hemocytes released effectors under the regulation of EsGPCR89, thereby activating transcription of cerebral AMPs. These results demonstrate that EsGPCR89 plays important roles in cerebral antimicrobial function via hemocytes.

Deleted in azoospermia-associated protein 2 regulates innate immunity by stimulating Hippo signaling in crab.

The Hippo-signaling pathway plays a critical role in both normal animal physiology and pathogenesis. Because pharmacological interventions targeting this pathway have diverse clinical implications, a better understanding of its regulation in various conditions and organisms is crucial. Here, we identified deleted in azoospermia-associated protein 2 (DAZAP2) in the Chinese mitten crab (Eriocheir sinensis), designated EsDAZAP2, as a Hippo-regulatory protein highly similar to proteins in various species of insects, fish, and mammals. We found that a bacterial infection significantly induces EsDAZAP2 expression, and an EsDAZAP2 knockdown both suppresses antimicrobial peptide (AMP) expression in vitro and results in increased viable bacterial counts and mortality in vivo, suggesting that EsDAZAP2 plays a critical role in innate immunity. Using yeast two-hybrid screening and co-immunoprecipitation assays, we found that EsDAZAP2 regulates the Toll pathway rather than the immune deficiency and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. Our findings also demonstrate that EsDAZAP2 binds to the Hippo protein, Salvador (Sav). Moreover, by examining the regulation of Dorsal, a transcription factor that regulates AMP expression in E. sinensis, we provide experimental evidence indicating that EsDAZAP2 promotes Hippo pathway activation in innate immunity, with EsDAZAP2 and Hippo binding to different Sav domains. To the best of our knowledge, this is the first report of a DAZAP2-regulated Hippo-signaling pathway operating in animal innate immunity.

Ecdysone inducible gene E75 from black tiger shrimp Penaeus monodon: Characterization and elucidation of its role in molting.

Molting is controlled by ecdysteroids, which are synthesized and secreted by the Y-organ in crustaceans. Ecdysone inducible gene, E75, is an early-response gene in the 20-hydroxyecdysone (20E) signaling pathway, with crucial roles in arthropod development. Complementary DNA (cDNA) encoding Penaeus monodon E75 (PmE75) was cloned using RT-PCR and RACE. PmE75 cDNA was 3526 bp long and encoded a 799-amino acid protein. Tissue distribution analysis showed that PmE75 was expressed ubiquitously in selected tissues, and was relatively abundant in the epidermis, muscle, and hepatopancreas. Developmental expression revealed that PmE75 was expressed throughout its life cycle. Silencing PmE75 significantly decreased PmE75 expression. Shrimps injected with PBS and dsGFP started molting on Day 7 and had almost completed molting on Day 9, whereas dsPmE75-injected shrimp presented no signs of molting. These results suggested that PmE75 might be involved in molting. In situ hybridization results support this hypothesis. To explore the role of 20E and eyestalks in the regulation of molting in P. monodon, exogenous 20E injection and eyestalk ablation (ESA) were performed, and showed that 20E can induce the transcription and expression of PmE75 in the hepatopancreas, epidermis, and muscle, which were significantly elevated after ESA. These results provide further insights into our understanding of molting.

Characterization and expression analysis of a chitinase gene (PmChi-5) from black tiger shrimp (Penaeus monodon) under pathogens infection and ambient ammonia-N stress.

Chitinases are crucial enzymes for crustaceans. Previous researches had already revealed that chitinases play important roles in digestion, molting and defense against viruses. In the present study, a chitinase cDNA was identified from black tiger shrimp (Penaeus monodon) and designated as PmChi-5. The full-length PmChi-5 cDNA was 2860 bp in size, containing an open reading frame (ORF) of 1731 bp that encoded a protein of 576 amino acids with a deduced molecular weight of 64.8 kDa. Expression of the PmChi-5 mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill and hepatopancreas. PmChi-5 was expressed throughout the whole larvae stages, and the highest level at Mysis3 stage, which indicated that PmChi-5 may be involved in larval metamorphosis. After challenged with Streptococcus agalactiae and Vibrio harveyi, the transcripts of PmChi-5 were found to be up-regulated significantly both in hepatopancreas and gill. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-5 transcripts were significantly changed in hepatopancreas and gill. The results showed that PmChi-5 may be involved in molting, larval metamorphosis, the immune defenses to pathogens infection and ammonia-N stress.

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

Chitinase is a multi-gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase-4 gene (PmChi-4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi-4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi-4 protein was 67.7 kDa and shared 61%-88% identity with the type of Chi-4s from other crustaceans. Quantitative real-time (qRT-PCR) analysis indicated that PmChi-4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi-4 was expressed throughout the whole larvae stages, and the highest level of PmChi-4 transcripts was detected at Mysis3 stage, which indicated that PmChi-4 may be involved in larval metamorphosis. In order to know whether PmChi-4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi-4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi-4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi-4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi-4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon.