Protein inhibitor of activated STAT (PIAS) proteins are activation-suppressing proteins for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. The inhibitory mechanism of PIAS proteins in the Janus kinase (JAK)/STAT signaling pathway has been well studied in mammals and Drosophila. However, the roles of PIAS in crustaceans are unclear. In the present study, we identified PIAS in kuruma shrimp Marsupenaeus japonicus and found that its relative expression could be induced by Vibrio anguillarum stimulation. To explore the function of PIAS in shrimp infected with V. anguillarum, we performed an RNA interference assay. After knockdown of PIAS expression in shrimp subjected to V. anguillarum infection, bacterial clearance was enhanced and the survival rate increased compared with those in the control shrimp (dsGFP injection). Simultaneously, the expression levels of antimicrobial peptides (AMPs), including anti-lipopolysaccharide factor (ALF) A1, C1, C2, and CruI-1, increased. Further study revealed that knockdown of PIAS also enhanced STAT phosphorylation and translocation. Pulldown assay indicated that PIAS interacts with activated STAT in shrimp. In conclusion, PIAS negatively regulates JAK/STAT signaling by inhibiting the phosphorylation and translocation of STAT through the interaction between PIAS and STAT, which leads to the reduction of AMP expression in shrimp. Our results revealed a new mechanism of PIAS-mediated gene regulation of the STAT signal pathway.

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus , was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein ( Mj pIgR for short). Mj pIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein levels. Knockdown of MjpIgR , and blocking Mj pIgR with its antibody inhibited WSSV infection in shrimp and overexpression of Mj pIgR facilitated the invasion of WSSV. Further analyses indicated that Mj pIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of Mj pIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin ( Mj CaM). Mj pIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of Mj pIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that Mj pIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that Mj pIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway.

The Toll signaling pathway plays an important role in the innate immunity of Drosophila melanogaster and mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense against Staphylococcus aureus by regulating expression of antimicrobial peptides in shrimp. We then found that ␤-arrestins negatively regulate Toll signaling in two different ways. ␤-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of ␤-arrestin⅐Cactus⅐ Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. ␤-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser 276 that impairs Dorsal transcriptional activity. Our study suggests that ␤-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

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The Ras GTPase superfamily, including more than 100 members, plays a vital role in a number of cellular processes, such as cytoskeleton recombination, gene expression, and signaling pathway regulation. Some members of the superfamily participate in innate immunity in animals. However, there have been few studies of RhoA on this aspect. In the present study, we identified a RhoA GTPase in the shrimp Marsupenaeus japonicus and named it MjRhoA. Expression of MjRhoA was significantly upregulated in hemocytes and heart of shrimp challenged with Vibrio anguillarum. Overexpression of MjRhoA in shrimp caused the total bacterial number to decrease significantly and knockdown of MjRhoA increased the bacterial number obviously, with a consequent decline in shrimp survival. These results confirmed the antibacterial function of MjRhoA in shrimp. Further study showed that rate of phagocytosis of hemocytes was decreased in MjRhoA-knockdown shrimp. Interestingly, we observed that MjRhoA was translocated onto the hemocyte membrane at 1 h post V. anguillarum challenge. The expression levels of the β-integrin-mediated phagocytosis markers ROCK2 and Arp2/3 declined significantly after knockdown of MjRhoA. These results suggested that the antibacterial function of MjRhoA was related to β-integrin-mediated phagocytosis in shrimp. Our previous study identified that a C-type lectin, hFcLec4, initiated β-integrin mediated phagocytosis after bacterial infection. Thus, knockdown of hFcLec4 and β-integrin was performed. The results showed that the translocation of MjRhoA from the cytoplasm to membrane was inhibited and the expression level of MjRhoA was decreased, suggesting that MjRhoA participated in hFcLec4-integrin mediated phagocytosis. Therefore, our study identified a new hFcLec4-integrin-RhoA dependent phagocytosis against bacterial infection in shrimp.

Impaired phosphatase activity leads to the persistent activation of signal transducers and activators of transcription (Stat). In mammals, Stat family members are often phosphorylated or dephosphorylated by the same enzymes. To date, only one Stat similar to mammalian Stat5a/b has been found in crustaceans and there have been few studies in Stat signal regulation in crustaceans. Here, we report that β-arrestin1 interacts with TC45 (45-kDa form of T cell protein tyrosine phosphatase) in the nucleus to attenuate Stat signaling by promoting dephosphorylation of Stat. Initially, we showed that Stat translocates into the nucleus to induce antimicrobial peptide (AMP) expression after bacterial infection. βArr1 enters the nucleus of hemocytes and recruits TC45 to form the βarr1-TC45-Stat complex, which dephosphorylates Stat efficiently. The interaction of TC45 with Stat decreased and Stat phosphorylation increased in βarr1-silenced shrimp (Marsupenaeus japonicus) after challenge with Vibrio anguillarum. βArr1 directly interacts with Stat in nucleus and accelerates Stat dephosphorylation by recruiting TC45 after V. anguillarum challenge. Further study showed that βarr1 and TC45 also affect AMP expression, which is regulated by Stat. Therefore, βarr1 and TC45 are involved in the anti-V. anguillarum immune response by regulating Stat activity negatively to decrease AMP expression in shrimp.

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.