A Negative Regulatory Feedback Loop within the JAK-STAT Pathway Mediated by the Protein Tyrosine Phosphatase DUSP14 in Shrimp.

The JAK-STAT pathway is a central communication node for various biological processes. Its activation is characterized by phosphorylation and nuclear translocation of the transcription factor STAT. The regulatory balance of JAK-STAT signaling is important for maintenance of immune homeostasis. Protein tyrosine phosphatases (PTPs) induce dephosphorylation of tyrosine residues in intracellular proteins and generally function as negative regulators in cell signaling. However, the roles of PTPs in JAK-STAT signaling, especially in invertebrates, remain largely unknown. Pacific white shrimp Penaeus vannamei is currently an important model for studying invertebrate immunity. This study identified a novel member of the dual-specificity phosphatase (DUSP) subclass of the PTP superfamily in P. vannamei, named PvDUSP14. By interacting with and dephosphorylating STAT, PvDUSP14 inhibits the excessive activation of the JAK-STAT pathway, and silencing of PvDUSP14 significantly enhances humoral and cellular immunity in shrimp. The promoter of PvDUSP14 contains a STAT-binding motif and can be directly activated by STAT, suggesting that PvDUSP14 is a regulatory target gene of the JAK-STAT pathway and mediates a negative feedback regulatory loop. This feedback loop plays a role in maintaining homeostasis of JAK-STAT signaling and is involved in antibacterial and antiviral immune responses in shrimp. Therefore, the current study revealed a novel inhibitory mechanism of JAK-STAT signaling, which is of significance for studying the regulatory mechanisms of immune homeostasis in invertebrates.

Oral delivery of bacteria expressing wsv108 gene-specific dsRNA protects shrimp from white spot syndrome virus (WSSV) infection.

Double-stranded RNA (dsRNA) can specifically inhibit gene expression by RNA interference and has important application potential in animal disease control. White spot syndrome virus (WSSV) is one of the most harmful pathogens in shrimp aquaculture, causing huge economic losses every year. In this study, we investigated the function of the WSSV-encoded wsv108 protein. We demonstrated that wsv108 could promote apoptosis by interacting with heat shock protein 70 (HSP70) and enhancing the expression of multiple apoptosis-related genes. Silencing of wsv108 gene by injection with specific dsRNA prepared by in vitro transcription significantly increased the survival rate of WSSV-infected shrimp and reduced the viral load in tissues, suggesting that wsv108 is important for WSSV pathogenicity. Based on this, we expressed the wsv108 specific dsRNA in engineered Escherichia coli. Oral feeding of this bacterium could inhibit the expression of wsv108, increase the survival rate of WSSV-infected shrimp, and decrease the viral load of WSSV in tissues. Therefore, this study developed a new method for treatment of WSSV disease by oral administration of bacterially expressed dsRNA against a novel therapeutic target molecule, which could be a potential candidate strategy for WSSV control in aquaculture.

Identifying phenotype-associated subpopulations through LP_SGL.

Single-cell RNA sequencing (scRNA-seq) enables the resolution of cellular heterogeneity in diseases and facilitates the identification of novel cell types and subtypes. However, the grouping effects caused by cell-cell interactions are often overlooked in the development of tools for identifying subpopulations. We proposed LP_SGL which incorporates cell group structure to identify phenotype-associated subpopulations by integrating scRNA-seq, bulk expression and bulk phenotype data. Cell groups from scRNA-seq data were obtained by the Leiden algorithm, which facilitates the identification of subpopulations and improves model robustness. LP_SGL identified a higher percentage of cancer cells, T cells and tumor-associated cells than Scissor and scAB on lung adenocarcinoma diagnosis, melanoma drug response and liver cancer survival datasets, respectively. Biological analysis on three original datasets and four independent external validation sets demonstrated that the signaling genes of this cell subset can predict cancer, immunotherapy and survival.

Basigin binds bacteria and activates Dorsal signaling to promote antibacterial defense in Penaeus vannamei.

The NF-κB pathway plays an important role in immune regulation. Basigin, an immunoglobulin superfamily membrane protein, is involved in the activation of NF-κB. However, its role in NF-κB signaling in response to pathogen infection remains unclear. In this study, we identified the Basigin gene from Pacific white shrimp, Penaeus vannamei, a representative species for studying the innate immune system of invertebrates. Basigin promoted the degradation of the IκB homolog Cactus, facilitated the nuclear translocation of the NF-κB family member Dorsal, and positively regulated the expression of Dorsal pathway downstream antimicrobial peptide genes. Interestingly, recombinant Basigin protein could bind a variety of Gram-positive and Gram-negative bacteria. Silencing of Basigin inhibited the Dorsal signaling activated by V. parahaemolyticus infection and significantly decreased the survival rate of V. parahaemolyticus-infected shrimp. The expression levels of the antimicrobial peptides ALF1 and ALF2 were downregulated, and the phagocytosis of hemocytes was attenuated in Basigin-silenced shrimp. Similar results were observed in shrimp treated with a recombinant extracellular region of the Basigin protein that was able to compete with endogenous Basigin. Therefore, to the best of our knowledge, this study is the first to demonstrate the function of Basigin as a pathogen recognition receptor that activates NF-κB signaling for antibacterial immunity in shrimp.

The Hippo-Yki pathway downstream transcription factor Scalloped negatively regulates immune defense against Vibrio parahaemolyticus infection in shrimp.

The Hippo-Yki signaling pathway plays a crucial role in numerous biological processes. Previous studies have demonstrated the significance of signal transduction components of the Hippo pathway in the immune response of shrimp. In this study, the downstream transcription factor of Hippo signaling, Scalloped, was analyzed in the context of Vibrio parahaemolyticus infection in Pacific white shrimp, Penaeus vannamei. Upon bacterial and fungal infections, the expression of Scalloped was upregulated in hemocytes. Scalloped was found to localize in the nucleus and interact with the Hippo pathway downstream transcriptional co-activator Yki. With the assistance of Yki, Scalloped activated the promoter of Cactus, a cytoplasmic inhibitor of the NF-κB pathway, leading to the inhibition of the nuclear translocation of the NF-κB family member Dorsal in shrimp. By inhibiting the Dorsal pathway, Scalloped reduced the expression of immune functional proteins and negatively regulated the immune response against bacterial infection in shrimp. RNAi-mediated silencing of Scalloped significantly enhanced the survival rate of V. parahaemolyticus-infected shrimp and reduced the bacterial load in tissues. These findings demonstrate the potential of Scalloped as a therapeutic target for vibriosis in crustaceans and contribute to our understanding of the shrimp's antibacterial defense and the functional roles of Hippo signaling in animal immunity.

A novel chitinase Chi6 with immunosuppressive activity promotes white spot syndrome virus (WSSV) infection in Penaeus vannamei.

Chitinases, a group of glycosylase hydrolases that can hydrolyze chitin, are involved in immune regulation in animals. White spot syndrome virus (WSSV) causes huge losses to crustacean aquaculture every year. We identified a novel chitinase Chi6 from Pacific white shrimp Penaeus vannamei, which contains a catalytic domain but no chitin-binding domain. The Chi6 expression was regulated by multiple immune signaling pathways and increased after immune stimulations. Silencing of Chi6 by RNAi in vivo did not affect Vibrio parahaemolyticus infection, but significantly increased the survival rate of WSSV-infected shrimp. The expression of multiple WSSV immediate early and structural genes was also decreased upon Chi6 silencing. The recombinant Chi6 protein showed no effect on bacterial growth but could attenuate shrimp hemocyte phagocytosis. The mRNA levels of several key elements and downstream genes of the MAPK and Dorsal pathways in Chi6-silenced shrimp were significantly up-regulated, suggesting an inhibitory effect of Chi6 on humoral immune response. Moreover, Chi6 enhanced the regulatory effect of Dorsal on the expression of WSSV ie1 gene. Therefore, Chi6 promotes WSSV infection through immunosuppression and regulation of WSSV gene expression. Targeting Chi6 could be a potential strategy for controlling WSSV disease in shrimp farming.

White Spot Syndrome Virus (WSSV) Inhibits Hippo Signaling and Activates Yki To Promote Its Infection in Penaeus vannamei.

White spot syndrome virus (WSSV) is a serious threat to shrimp aquaculture, especially Pacific white shrimp, Penaeus vannamei, the most farmed shrimp in the world. Activation of the Hippo-Yki signaling pathway, characterized by the intracellular Hippo-Wts kinase cascade reactions and the phosphorylation and cytoplasmic retention of Yki, is widely involved in various life activities. The current work established the fundamental structure and signal transduction profile of the Hippo-Yki pathway in P. vannamei and further investigated its role in viral infection. We demonstrated that WSSV promoted the dephosphorylation and nuclear translocation of Yki, suggesting that Hippo signaling is impaired and Yki is activated after WSSV infection in shrimp. In vivo, Yki gene silencing suppressed WSSV infection, while Hippo and Wts silencing promoted it, indicating a positive role of Hippo signaling in antiviral response. Further analyses showed that Yki suppressed Dorsal pathway activation and inhibited hemocyte apoptosis in WSSV-infected shrimp, while Hippo and Wts showed opposite effects, which contributed to the role of Hippo signaling in WSSV infection. Therefore, the current study suggests that WSSV annexes Yki to favor its infection in shrimp by inhibiting Hippo signaling. IMPORTANCE White spot syndrome virus (WSSV) is one of the most harmful viral pathogens to shrimp. The pathological mechanism of WSSV infection remains unclear to date. The Hippo-Yki signaling pathway is important for various biological processes and is extensively involved in mammalian immunity, but little is known about its role in infectious diseases in invertebrates. Based on revealing the fundamental structure of the shrimp Hippo pathway, this study investigated its implication in the pathogenesis of WSSV disease. We demonstrated that WSSV enhanced Yki activation by inhibiting Hippo signaling in shrimp. The activated Yki promoted WSSV infection by inhibiting hemocyte apoptosis and suppressing the activation of Dorsal, an NF-κB family member in shrimp that is critical for regulating antiviral response. Therefore, this study suggests that WSSV can hijack the Hippo-Yki signaling pathway to favor its infection in shrimp.

The Hippo-Yki Signaling Pathway Positively Regulates Immune Response against Vibrio Infection in Shrimp.

In the Hippo pathway, activation of Hippo and Warts (Wts) kinases results in the phosphorylation of Yorkie (Yki), to prevent its nuclear translocation. Shrimp aquaculture is threatened by Vibrio genus bacteria. In this study, we examine the role of the Hippo pathway in immune defense against Vibrio parahaemolyticus in Pacific white shrimp Penaeus vannamei. We show that V. parahaemolyticus infection promotes the expression of Yki and facilitates the dephosphorylation and nuclear translocation of Yki, indicating the inhibition of Hippo signaling upon bacterial infection. There is a complex regulatory relationship between the Hippo pathway components Hippo, Wts, and Yki and the immune-related transcription factors Dorsal, Relish, and STAT. Silencing of Hippo and Wts weakened hemocyte phagocytosis, while the silencing of Yki enhanced it, suggesting a positive regulation of shrimp cellular immunity by Hippo signaling activation. In vivo silencing of Hippo and Wts decreased the survival rates of V. parahaemolyticus-infected shrimp and elevated the bacterial content in tissues, while the silencing of Yki showed the opposite results. This suggests that the activation of Hippo signaling and the inhibition of Yki enhance antibacterial immunity in shrimp.

The Non-Receptor Protein Tyrosine Phosphatase PTPN6 Mediates a Positive Regulatory Approach From the Interferon Regulatory Factor to the JAK/STAT Pathway in Litopenaeus vannamei.

SH2-domain-containing protein tyrosine phosphatases (PTPs), belonging to the class I PTP superfamily, are responsible for the dephosphorylation on the phosphorylated tyrosine residues in some proteins that are involved in multiple biological processes in eukaryotes. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway transduce signaling responding to interferons and initiate cellular antiviral responses. The activity of the JAK/STAT pathway is generally orchestrated by the de-/phosphorylation of the tyrosine and serine residues of JAKs and STATs, in which the dephosphorylation processes are mainly controlled by PTPs. In the present study, an SH2-domian-contianing PTP, temporally named as LvPTPN6, was identified in Litopenaeus vannamei. LvPTPN6 shares high similarity with PTPN6s from other organisms and was phylogenetically categorized into the clade of arthropods that differs from those of fishes and mammals. LvPTPN6 was constitutively expressed in all detected tissues, located mainly in the cytoplasm, and differentially induced in hemocyte and gill after the challenge of stimulants, indicating its complicated regulatory roles in shrimp immune responses. Intriguingly, the expression of LvPTPN6 was regulated by interferon regulatory factor (IRF), which could directly bind to the LvPTPN6 promoter. Surprisingly, unlike other PTPN6s, LvPTPN6 could promote the dimerization of STAT and facilitate its nuclear localization, which further elevated the expression of STAT-targeting immune effector genes and enhanced the antiviral immunity of shrimp. Therefore, this study suggests a PTPN6-mediated regulatory approach from IRF to the JAK/STAT signaling pathway in shrimp, which provides new insights into the regulatory roles of PTPs in the JAK/STAT signaling pathway and contributes to the further understanding of the mechanisms of antiviral immunity in invertebrates.

A kelch motif-containing protein KLHDC2 regulates immune responses against Vibrio parahaemolyticus and white spot syndrome virus in Penaeus vannamei.

The kelch motif-containing proteins are widely present in organisms and known to be involved in various biological processes, but their roles in immunity remain unclear. In this study, a kelch motif-containing protein KLHDC2 was identified from Pacific white shrimp Penaeus vannamei and its immune function was investigated. The klhdc2 gene was widely expressed in shrimp tissues and its protein product was mainly present in the nucleus. Expression of klhdc2 was regulated by shrimp NF-κB family members Dorsal and Relish, and changed after immune stimulation. KLHDC2 could enhance the immune defense against Vibrio parahaemolyticus in shrimp but inhibit that against white spot syndrome virus (WSSV). Further analyses showed that KLHDC2 did not affect the phagocytosis of hemocytes but regulated the expression of a series of immune effector genes. KLHDC2 has a complex regulatory relationship with Dorsal and Relish, which may partly contribute to its positive role in antibacterial response by regulating humoral immunity. Moreover, the regulatory effect of KLHDC2 on WSSV ie1 gene contributed to its negative effect on antiviral response. Therefore, the current study enrichs the knowledge on the Kelch family and helps to learn more about the regulatory mechanism of shrimp immunity.

Wnt5b plays a negative role in antibacterial response in Pacific white shrimp Penaeus vannamei.

The Wnt family genes are essentially implicated in development and growth in animals. Accumulating clues have pointed to the importance of Wnts in invertebrate immunity, but the underlying mechanisms are still unclear to date. The Wnt5b has been known to promote white spot syndrome virus (WSSV) infection in shrimp but its role in antibacterial response remains unclear. In the current study, we focused on the involvement of Wnt5b in Vibrio parahaemolyticus infection in Pacific white shrimp Penaeus vannamei. We demonstrated that the expression of Wnt5b was regulated by the IMD-Relish and JAK-STAT pathways but not the Dorsal pathway and was suppressed upon bacterial infection. Although Wnt5b did not affect the cellular immunity in shrimp, it was involved in regulation of humoral immunity. Silencing of Wnt5b in vivo significantly increased expression of several antimicrobial peptides but decreased that of many immune functional proteins including C-type lectins and lysozymes. Treatment with recombinant Wnt5b protein increased the susceptibility of shrimp to V. parahaemolyticus infection, while silencing of Wnt5b in vivo showed an opposite result. These suggested that Wnt5b plays a negative role in antibacterial response in shrimp. Together with previous reports, the current study shows that Wnt5b functions as an inhibitor for shrimp immunity, which is a potential target for improving immune responses against infection.

miR-10c Facilitates White Spot Syndrome Virus Infection by Targeting Toll3 in Litopenaeus vannemei.

Toll-like receptors (TLRs) are canonical cell membrane receptors functioning to recognize pathogens and transduce signals to activate immune responses. It has been known that Toll3 in Pacific white shrimp Litopenaeus vannamei (LvToll3) plays a critical role in antiviral immunity by inducing the transcription of interferon regulatory factor (IRF), which mediates a signaling axis that is similar to the interferon system of vertebrates. However, the regulatory mechanism of the Toll3-IRF signaling is still unclear. In this study, a novel microRNA (miRNA) of miR-10 family, temporarily named as miR-10c, was identified from L. vannamei. miR-10c may play a nonnegligible regulatory role in shrimp immune responses since it was constitutively expressed in all detected tissues and transcriptionally induced by immune stimulation. Functional analysis validated that miR-10c could target LvToll3 to inhibit its expression, through which miR-10c blocked the nuclear translocation of IRF and facilitated white spot syndrome virus (WSSV) infection. To our knowledge, the present study revealed the first report of a Toll targeted by miRNA in crustaceans and provided a solid evidence base for supporting the role of LvToll3 in antiviral defense by activating IRF signaling in L. vannamei. Identification of the miR-10c/Toll3/IRF regulatory axis in shrimp provides new insights into the participation of miRNA in the regulation of immune responses and contributes to in-depth understanding of the mechanisms of Toll-induced immune responses in L. vannamei.

The LARK protein is involved in antiviral and antibacterial responses in shrimp by regulating humoral immunity.

The LARK proteins containing a C2HC-type zinc finger motif and two RNA recognition motifs are conserved across vertebrates and invertebrates. Previous studies have suggested that invertebrate LARKs and their mammalian counterparts, the RBM4 proteins, regulate gene expression by affecting RNA stability and post-transcriptional processing, participating in multiple life processes. In the current study, the LARK gene from Pacific white shrimp Litopenaeus vannamei was identified and functionally explored in the context of immunity. The LARK protein was mainly present in the nucleus of its expression vector-transfected S2 cells, and the LARK mRNA was detectable in all the tested shrimp tissues. Expression of LARK in gill was up-regulated by immune stimulation with various pathogens. In vivo experiments demonstrated that LARK played positive roles in both antiviral and antibacterial responses and silencing of LARK could make shrimp more susceptible to infection with Vibrio parahaemolyticus and white spot syndrome virus (WSSV). Although silencing of LARK did not affect the phagocytic activity of hemocytes, it regulated expression of many components of the NF-κB and JAK-STAT pathways and a series of immune function proteins. These suggested that LARK could be mainly involved in regulation of humoral immunity. The current study could help reveal the roles of LARK/RBM4 in immunity and further explore the regulatory mechanisms of shrimp immunity.

Toll receptor 2 (Toll2) positively regulates antibacterial immunity but promotes white spot syndrome virus (WSSV) infection in shrimp.

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.

A Novel Forkhead Box Protein P (FoxP) From Litopenaeus vannamei Plays a Positive Role in Immune Response.

The forkhead box protein P (FoxP) family members have been known to be important for regulation of immune responses in vertebrates, but their roles in invertebrate immunity remain unclear. In this study, a novel FoxP gene (LvFoxP) was identified from Pacific white shrimp Litopenaeus vannamei and functionally studied in the context of immune response. Possessing a conserved FoxP coiled-coil domain and a forkhead domain, LvFoxP shared homology to vertebrate FoxP family members, in particular FoxP1. Expression of LvFoxP was detectable in all the examined tissues and could be up-regulated by immune challenge in gill and hemocytes. The LvFoxP protein was present in both the cytoplasm and nucleus of hemocytes and could be nuclear-translocated upon immune stimulation. Silencing of LvFoxP increased the susceptibility of shrimp to infections by Vibrio parahaemolyticus and white spot syndrome virus (WSSV) and down-regulated the expression of multiple components of NF-κB and JAK-STAT pathways and almost all the examined immune effector genes. Moreover, the phagocytic activity of hemocytes from LvFoxP-silenced shrimp against V. parahaemolyticus was decreased. These suggested that LvFoxP could play a positive role in immune response. The current study may provide novel insights into the immunity of invertebrates and the functional evolution of the FoxP family.

A double chitin catalytic domain-containing chitinase targeted by c-Jun is involved in immune responses in shrimp.

Chitinases are a group of chitin-degrading enzymes widely distributed in organisms. Chitinases containing two chitin catalytic domains have been widely found in arthropods but their functions remain unclear. In this study, a member of these chitinases from Litopenaeus vannamei (dChi) was identified and functionally studied in the context of immunity. The promoter of dChi contained activator protein 1 (AP-1) binding sites and could be regulated by c-Jun. The recombinant dChi protein showed no bacteriostatic activity in vitro but knockdown of dChi in vivo increased the mortality of shrimp and the bacterial load in tissues after Vibrio parahaemolyticus infection, suggesting that dChi could play a positive role in antibacterial responses. However, silencing of dChi expression significantly decreased the mortality of WSSV-infected shrimp and down-regulated the viral load in tissues, indicating that dChi could facilitate WSSV infection. We further demonstrated that dChi was involved in regulation of the bacterial phagocytosis of hemocytes and expression of a series of immune related transcription factors and antimicrobial peptides. These indicated that the roles of dChi in antibacterial responses and anti-WSSV responses in vivo could result from its regulatory effects on the immune system. Taken together, the current study suggests that double chitin catalytic domain-containing chitinases could be important players in immune regulation in crustaceans.

A MicroRNA-1-Mediated Inhibition of the NF-κB Pathway by the JAK-STAT Pathway in the Invertebrate Litopenaeus vannamei.

The JAK-STAT and NF-κB pathways are conserved cellular signaling cascades orchestrating a variety of biological processes. The regulatory interactions between these two pathways have been well studied in vertebrates but less concerned in invertebrates, hindering further understanding of immune signaling evolution. The Pacific white shrimp Litopenaeus vannamei is now an important model for studying invertebrate immunity and cellular signaling mechanisms. In this study, the microRNA-1 (miR-1) molecule from L. vannamei was identified, and its mature and precursor sequences were analyzed. The miR-1 promoter contained a STAT binding site and its transcriptional activity could be regulated by the JAK-STAT pathway. The target gene of miR-1 was identified as MyD88, the upstream component of the Dorsal (the NF-κB homolog) pathway. By suppressing the expression of MyD88, miR-1 attenuated activation of the Dorsal pathway. With miR-1 as the mediator, STAT also exerted a negative regulatory effect on the Dorsal pathway. Moreover, miR-1 was involved in regulation of the expression of a set of immune effector genes and the phagocytic activity of hemocytes and had an inhibitory or excitatory effect on antibacterial or antiviral responses, respectively. Taken together, the current study revealed a microRNA-mediated inhibition of the NF-κB pathway by the JAK-STAT pathway in an invertebrate, which could contribute to immune homeostasis and shaping immune responses.

A shrimp gene encoding a single WAP domain (SWD)-containing protein regulated by JAK-STAT and NF-κB pathways.

Regulation of immune responses in animals is largely governed by the JAK-STAT and NF-κB pathways, which are conserved across vertebrates and invertebrates. At present, the relationship between these two pathways in invertebrates remains unclear. In the current study, a novel antimicrobial peptide termed LvSWD5 belonging to the Crustin family was identified from Pacific white shrimp Litopenaeus vannamei. The mature LvSWD5 peptide containing a single WAP domain (SWD) could directly bind bacteria and fungi and inhibit the growth of both Gram-positive and -negative bacteria in vitro. The LvSWD5 promoter was predicted to contain binding sites for STAT and NF-κB and could be regulted by the JAK-STAT and Relish pathways. The expression of LvSWD5 was up-regulated during bacterial, viral and fungal infections and silencing of LvSWD5 in vivo affected the expression of a series of immune related genes and decreased the phagocytic activity of hemocytes against V. Parahaemolyticus. Moreover, the susceptibility of shrimp to V. parahaemolyticus and white spot syndrome virus (WSSV) was significantly increased after silencing of LvSWD5, indicating that LvSWD5 could be involved in antibacterial and antiviral responses. These suggested that the JAK-STAT and NF-κB pathways could converge at the promoter level of a common target gene to regulate the immunity in shrimp.

A novel C-type lectin with microbiostatic and immune regulatory functions from Litopenaeus vannamei.

C-type lectins (CTLs) are a group of lectins with at least one carbohydrate recognition domain (CRD), the binding of which to carbohydrates requires the presence of calcium ions. CTLs generally function as pattern recognition receptors (PRRs), essentially participating in innate immunity. In the current study, a novel CTL termed LvCTL5 was identified from Pacific white shrimp Litopenaeus vannamei, which shared sequence identities with other crustacean CTLs. LvCTL5 was highly expressed in hepatopancreas and could be activated by infection with bacteria, virus and fungi. The recombinant LvCTL5 protein purified from E. coli showed microbiostatic and agglutination activities against bacteria and fungi in vitro. Silencing of LvCTL5 in vivo could significantly affect expression of a series of immune effector genes and down-regulate the phagocytic activity of hemocytes. Compared with controls, the LvCTL5-silenced shrimp were highly susceptible to Vibrio parahaemolyticus and white spot syndrome virus (WSSV) infections. These suggest that LvCTL5 has microbiostatic and immune regulatory activities and is implicated in antiviral and antibacterial responses.

A JAK-STAT pathway target gene encoding a single WAP domain (SWD)-containing protein from Litopenaeus vannamei.

In shrimp, the JAK-STAT pathway is essentially implicated in both antiviral and antibacterial responses. However, few regulatory target genes of the JAK-STAT pathway in shrimp have been reported so far. In this study, a novel single WAP domain-containing peptide (LvSWD4) was identified from Pacific white shrimp Litopenaeus vannamei. The promoter of LvSWD4 was predicted to harbor multiple STAT-binding DNA motifs. Over-expression of the JAK-STAT pathway components STAT, JAK and Domeless in vitro significantly enhanced the transcriptional activity of the LvSWD4 promoter, and in vivo silencing of STAT and the the JAK-STAT pathway upstream regulator IRF down-regulated the expression of LvSWD4, suggesting that LvSWD4 could be a target gene of the JAK-STAT pathway. The expression of LvSWD4 was significantly increased after infection with Gram-negative and positive bacteria, fungi and virus, and silencing of LvSWD4 increased the susceptibility of shrimp to V. parahaemolyticus and WSSV infections. In vitro experiments also demonstrated that the recombinant LvSWD4 protein had significant inhibitory activities against Gram negative bacteria V. parahaemolyticus and E. coli and Gram positive bacteria S. aureus and B. subtilis. Furthermore, silencing of LvSWD4 in vivo significantly affected expression of various immune functional genes and attenuated the phagocytic activity of hemocytes. These suggested that as a target gene of STAT, LvSWD4 was essentially implicated in shrimp immunity, which could constitute part of the mechanism underlying the immune function of the shrimp JAK-STAT pathway.