PHB2 inhibits WSSV replication by promoting the nuclear translocation of STAT.

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses and cell proliferation. However, the function of the PHBs in immune regulation has largely not been determined. In the present study, we identified PHB2 in the red swamp crayfish Procambarus clarkii. PHB2 was found to be widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge. PHB2 significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. Here, we observed that PHB2 promotes the nuclear translocation of STAT by binding to STAT. After blocking PHB2 or STAT with antibodies or interfering with PHB2 or STAT, the expression levels of the antiviral genes ß-thymosin (PcThy-4) and crustin2 (Cru2) decreased. The gene sequence of PHB2 was analyzed and found to contain a nuclear introgression sequence (NIS). After in vivo injection of PHB2 with deletion of NIS (rΔNIS-PHB2), the nuclear translocation of STAT did not change significantly compared to that in the control group. These results suggest that PHB2 promoted the nuclear translocation of STAT through NIS and mediated the expression of antiviral proteins to inhibit WSSV infection.

Insect cells of Spodoptera frugiperda support WSSV gene replication but not progeny virion assembly.

The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.

Characterization and function analysis of cathepsin C in Marsupenaeusjaponicus.

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.

KCMF1-like suppresses white spot syndrome virus infection by promoting apoptosis in mud crab (Scylla paramamosain).

Potassium channel modulatory factor 1 (KCMF1), an E3 ubiquitin ligase, plays a vital role in renal tubulogenesis, preeclampsia, and tumor development in mammals. Nevertheless, the function of KCMF1 in invertebrates remains to be investigated. Here, we identified KCMF1-like from Scylla paramamosian, encoding 242 amino acids with two zinc finger domains at the N-terminal. Real-time quantitative PCR analysis revealed that KCMF1-like was expressed in all tested tissues, including hemocytes, brain, mid-intestine, subcuticular epidermis, gills, muscle, heart, and stomach, with higher levels in muscle and mid-intestine. KCMF1-like was up-regulated in the hemocytes of mud crabs challenged with white spot syndrome virus (WSSV). RNA interference (RNAi) was performed to investigate the impact of KCMF1-like on the proliferation of WSSV in mud crabs. Knock-down of KCMF1-like resulted in an increase of the WSSV copy number and an impairment of the hemocytes apoptosis rate in vivo. In addition, KCMF1-like could also affect the mitochondrial membrane potential. Collectively, these results revealed that KCMF1-like might play a crucial role in the defense against virus infection in mud crab. This study contributes a novel insight into the role of KCMF1-like in the antiviral immune defense mechanism in crustaceans.

Effects of dietary supplementation of Anabaena sp. PCC7120 expressing VP28 protein on survival and histopathology after WSSV infection in Macrobrachium nipponense.

Shrimp are especially susceptible to the White Spot Syndrome Virus (WSSV). Oral administration of the WSSV envelop protein VP28 is a promising approach to protect shrimp against WSSV. In this study, Macrobrachium nipponense (M. nipponense) were fed for 7 days with food supplemented with Anabaena sp. PCC 7120 (Ana7120) expressing VP28 and then challenged with WSSV. The survival rates of M. nipponense in three groups, including control, WSSV-challenged, and VP28-vaccinated, were subsequently determined. We also determined the WSSV content of different tissues and the tissue morphology in the absence of and after viral challenge. The survival rate of the positive control group (no vaccination and challenge, 10%) and empty vector group (fed with Ana7120 pRL-489 algae and challenged, 13.3%) was much lower than the survival rate of M. nipponense in wild type group (fed with Ana7120 and challenged, 18.9%), immunity group 1 (fed with 3.33% Ana7120 pRL-489-vp28 and challenged, 45.6%) or immunity group 2 (fed with 6.66% Ana7120 pRL-489-vp28 and challenged, 62.2%). RT-qPCR showed that WSSV content of the gill, hepatopancreas and muscle of immunity groups 1 and 2 were substantially lower than the positive control. Microscopic examination revealed that WSSV-challenged positive control exhibited large number of cell rupture, necrosis, nuclear exfoliation in gills and hepatopancreatic tissues. The gill and hepatopancreas of immunity group 1 showed partial symptoms of infection, yet the tissue was visibly healthier than that of the positive control group. No symptoms were visible in the gills and hepatopancreatic tissue of immunity group 2. The results demonstrate that the probability of M. nipponense infected by WSSV can be diminished by oral administration of cyanobacteria-expressed VP28. Such an approach could improve the disease resistance and delay the death of M. nipponense in the commercial production of this shrimp.

SpTNF regulates apoptosis and antimicrobial peptide synthesis in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

Tumor necrosis factor (TNF) is an inflammatory cytokine that is important in cell survival, proliferation, differentiation, and death. However, the functions of TNF in the innate immune responses of invertebrates have been less studied. In this study, SpTNF was cloned and characterized from mud crab (Scylla paramamosain) for the first time. SpTNF contains an open reading frame of 354 bp encoding 117 deduced amino acids, with a conserved C-terminal TNF homology domain (THD) domain. RNAi knockdown of SpTNF reduced hemocyte apoptosis and antimicrobial peptide (AMP) synthesis. Expression of SpTNF was initially down-regulated but subsequently up-regulated after 48 h in hemocytes of mud crabs after WSSV infection. Results of RNAi knockdown and overexpression showed that SpTNF inhibits the WSSV infection through activating apoptosis, NF-κB pathway, and AMP synthesis. Furthermore, the lipopolysaccharide-induced TNF-α factor (SpLITAF) can regulate the expression of SpTNF, induction of apoptosis, and activation of the NF-κB pathway and AMP synthesis. The expression and nuclear translocation of SpLITAF were found to be regulated by WSSV infection. Knocking down of SpLITAF increased the WSSV copy number and expression of VP28 gene. Taken together, these results proved the protective function of SpTNF, which is regulated by SpLITAF, in the immune response of mud crabs against WSSV through the regulation of apoptosis and activation of AMP synthesis.

The first description of the blue swimming crab (Portunus pelagicus) transcriptome and immunological defense mechanism in response to white spot syndrome virus (WSSV).

In the global shellfish farming industry, white spot syndrome virus (WSSV) is a major cause of mortality and a significant factor in economic losses. However, information on molecular immune responses to WSSV in blue swimming crabs (Portunus pelagicus) has never been reported. First, viral loads were measured in the gills, hepatopancreas, intestines, subcuticular epithelium and hemocytes of blue swimming crabs (50 ± 10 g) (n = 4) after WSSV induction at 0, 24, 48 and 96 h post injection (hpi). A significant increase in WSSV particles was observed in gills at 48 and 96 hpi, as supported by histopathology. To further investigate the acute immune response to WSSV, total RNA from the same gill tissues at 0, 24, and 96 hpi was used to construct 16 high-quality RNA-seq cDNA libraries. In summary, 162,740 unigenes were discovered in these transcriptomic libraries analyzed with the GO, KO, KOG, NR, NT, PFAM and SwissProt databases. Intensive sequence analysis against control crabs using three major categories of gene oncology (GO) of DEGs, biological processes (BPs), molecular functions (MFs), and cellular components (CCs), indicated that induction of WSSV in blue swimming crabs strongly affected the immune responses of the target animals significantly during the early stages of infection from 24 to 96 hpi. Furthermore, KEGG identified approximately twenty biological pathways of gene expression that were both downregulated and upregulated. Interestingly, at 24 and 96 hpi, several immune-related genes involved in virus defense in the blue swimming crab, particularly crustin 2, chitinase, anti-lipopolysaccharide, proteinase inhibitor, and lysozyme, were highly expressed during the WSSV early infection stages. At the same time, viral mRNA transcripts, including WSV289, WSV343, WSV306, deoxyuridine 5' triphosphate nucleohydrolase, RING finger containing E3 ubiquitin-protein ligase WSV403 and WSV404, were recorded in the top twenty upregulated genes. Moreover, some immune-responsive genes related to growth development, such as chitinase, tubulin alpha and beta chains, trypsin, and the cathepsin family, were also differentially expressed during these periods. Expression validation of 20 upregulated and 11 downregulated immune-related genes using qRT‒PCR showed similar patterns with transcriptome information. Overall, the data showed that during WSSV infection, a number of immune-, metabolism-, and growth-related pathways were activated, and several of the pathways involved differed depending on the stage of virus invasion. These findings could effectively help us better understand the impact of WSSV on the physiology of blue swimming crabs and serve as a valuable reference for future research on the immune system and disease control in this target species.

Fucosyltransferase 2 is involved in immune-related functions in Penaeus vannamei by modulating antimicrobial peptides' expression.

In mammals fucosyltransferase 2 (FUT2) plays an important regulatory role in inflammation, bacterial or viral infection, and tumor metastasis. However, the specific role of FUT2 in invertebrate immunity has not been reported. Here, the FUT2 homolog of Penaeus vannamei (designated as PvFUT2) was cloned and found to have a full-length cDNA of 1104 bp with an open reading frame (ORF) encoding 316 amino acids. PvFUT2 is constitutively expressed in all shrimp tissues tested with the highest found in intestines. Moreover, PvFUT2 was induced in the main immune organs (hemocytes and hepatopancreas) of shrimp by Gram-positive (Vibrio parahaemolyticus), Gram-negative (Streptococcus iniae) bacteria and virus (White Spot Syndrome Virus), indicating the involvement of PvFUT2 in shrimp antimicrobial response. Intriguingly, PvFUT2 knockdown with or without pathogen challenge reduced the expression of Pvß-catenin and antimicrobial peptides genes, particularly anti lipopolysaccharide factor and lysozyme. Further analysis revealed that the knockdown of PvFUT2 increased Vibrio abundance in hemolymph and resulted in an increase in shrimp cumulative mortality rate. Thus, during pathogen challenge, the expression of PvFUT2 is induced to regulate ß-catenin and subsequently antimicrobial peptides expression to augment shrimp antimicrobial immune response.

Eight TRIM32 isoforms from oriental river prawn Macrobrachium nipponense are involved in innate immunity during white spot syndrome virus infection.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. In this study, full-length MnTRIM32 cDNA was obtained from oriental river prawn Macrobrachium nipponense, and eight MnTRIM32 isoforms generated by alternative splicing were identified. The open reading frames of the eight MnTRIM32 isoforms were predicted to be separately composed of 402, 346, 347, 346, 414, 358, 359, and 358 amino acid residues. Protein structural analysis revealed that all MnTRIM32 isoforms contained a RING domain and a coiled coil region. MnTRIM32 was ubiquitously expressed in all tissues tested, with the highest expression in the hepatopancreas. The mRNA levels of MnTRIM32 in the gills, stomach, and intestine of prawns were found to undergo time-dependent enhancement following white spot syndrome virus (WSSV) stimulation. Double-stranded RNA interference studies revealed that MnTRIM32 silencing significantly downregulated the expression levels of interferon (IFN) regulatory factor MnIRF, IFN-like factor MnVago4, and tumor necrosis factor MnTNF. Furthermore, knockdown of MnTRIM32 in WSSV-challenged prawns increased the expression of VP28 and the number of WSSV copies, suggesting that MnTRIM32 plays a positive role in limiting WSSV infection. These findings provided strong evidence for the important role of MnTRIM32 in the antiviral innate immunity of M. nipponense.

Molecular characterization of an LRR-only protein gene in Pacific white shrimp Litopenaeus vannamei: Sequence feature, expression pattern, and protein activity.

Leucine-rich repeat (LRR)-only proteins have been proved to be involved in the innate immune responses as they could mediate protein-protein or protein-ligand interactions. In the present study, a novel LRR-only protein (LvLRRop-1) was identified and characterized from Pacific white shrimp Litopenaeus vannamei. The complete cDNA sequence of LvLRRop-1 contains an open reading frame (ORF) of 1488 bp, which encoded a polypeptide of 495 amino acids with a predicted molecular mass of 55.67 kDa and a calculated theoretical isoelectric point of 6.435. There are five LRR motifs, six LRR_TYP motifs in the protein sequence of LvLRRop-1 with consensus signature sequences of LxxLxxLxLxxNxL. The LvLRRop-1 mRNA transcripts could be detected in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, especially highest in hemocytes and hepatopancreas. The mRNA transcripts of LvLRRop-1 increased within the first 6 h in hemocytes and hepatopancreas after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenges. The recombinant LvLRRop-1 could bind four typical pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polycytidine-polycytidylic acid (poly IC), in a dose-dependent manner, and inhibit the growth of bacteria Micrococcus luteus. These data indicated that LvLRRop-1 could play a pivotal role in the innate immune response of shrimps as a kind of pattern recognition receptor (PRR).

Gain of function studies on predicted host receptors for white spot virus.

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from -8.46 to -11.82 cal mol-1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Identification of a Yorkie homolog from Litopenaeus vannamei as a negative regulator in anti-WSSV immune response.

Hippo signaling pathway is a serine threonine kinase cascade that is evolutionary conserved with well-established roles in organ size control, development, tumorigenesis and immunity. As its core molecule, Yorkie also plays an important role against pathogen. In this study, we cloned and characterized a Yorkie homolog from Litopenaeus vannamei, designed as LvYKI, which has a 1650 bp open reading frame. It has the characterized domains of Yokie family, and displayed to be close to the insects and crustacean. Quantitative Real-time PCR showed that LvYKI had different regulatory mechanisms in different tissues. The transcriptional level of Lvyki was down-regulated in gill, while up-regulated in hepatopancreas post white spot syndrome virus (WSSV) infection. Moreover, the expression and phosphorylation of LvYKI was reduced upon WSSV infection, which indicated that LvYKI was involved in WSSV infection. Furthermore, RNAi was performed to evaluate the role of LvYKI in shrimp immune responses. Knocking down of Lvyki resulted in inhibition of the transcription of WSSV gene ie1 and vp28, and delayed mortality of shrimp post WSSV infection. Meanwhile, the apoptosis of hemocyte was increased as well. All results suggested that shrimp can promote apoptosis to resist WSSV infection mediated by down-regulation of LvYKI. In addition, it was found that LvYKI could interact with Lvß-catenin, which cross-linked the Wnt and Hippo signaling pathway in innate immunity. Conclusively, our study provided clues that LvYKI plays an important role in the interaction between shrimp and virus. It will promote our understanding of the molecular mechanism in innate immunity.

Immunostimulation of shrimp through oral administration of silkworm pupae expressing VP15 against WSSV.

White spot syndrome virus (WSSV) is one of the most concerning pathogens in penaeid shrimp and can cause severe loss in shrimp aquaculture worldwide. Among the WSSV structural proteins, VP15, a DNA-binding protein located in the WSSV nucleocapsid, is an antiviral protein candidate to protect kuruma shrimp (Marsupenaeus japonicus) from WSSV infection. We identified that the truncated VP15, VP15(26-57), is responsible for the protective effect against the WSSV. This study attempts to develop an immunizing agent against WSSV using silkworm pupa as a delivery vector through oral administration. The VP15, VP15(26-57), and SR11 peptide derived from VP15(26-57) were expressed in silkworm pupae. Oral administration of feed mixed with the powdered pupae that expressed VP15-derived constructs enhanced the survivability of kuruma shrimp with an overall relative percent survival (RPS) higher than 70%. There is no death for the group receiving pupa/VP15(26-57), and the RPS is 100%. In addition, we also investigated the relative mRNA expression levels of immune-related genes by qPCR at different time points. Our results indicate that the oral administration of pupa/VP15-derived products could provide a high protective effect against WSSV and be a practical approach for controlling WSSV in aquaculture.

SpBNIP3 regulates apoptosis and autophagy in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), which is a pro-apoptotic protein in the BCL-2 family involves a variety of cell signaling pathways, including mitochondrial dysfunction, mitochondrial autophagy, and apoptosis in vertebrates. However, the role of BNIP3 in the regulation of apoptosis and/or autophagy in crustaceans suffering virus infection is still limited. In this study, the mud crab (Scylla paramamosain) BNIP3 (SpBNIP3) was identified and studied to elucidate its association with the white spot syndrome virus (WSSV) infection. SpBNIP3 was widely expressed in all tested tissues and significantly down-regulated in the hemocytes of mud crab after WSSV infection. Knockdown of SpBNIP3 using RNA interference increased the apoptosis rate and Caspase 3 activity but decreased the mitochondrial membrane potential and autophagy levels, as well as viral copy number in mud crabs infected with WSSV. Additionally, the relationship between the viral infection and the autophagy of hemocytes was observed. The level of autophagy was reduced upon WSSV infection, and the activation of autophagy enriched the viral copy number. Taken together, the results of this study provide a new finding on the mechanism that SpBNIP3 may participate in the WSSV infection through the regulation of apoptosis and autophagy processes in mud crabs.

The MIP-T3 from shrimp Litopenaeus vannamei restricts white spot syndrome virus infection via regulating NF-κB activation.

In vertebrate, MIP-T3 (microtubule-interacting protein associated with TRAF3) functions as a regulator of innate immune response that involves many cellular processes. However, the immune response regulated by shrimp (an arthropod) MIP-T3 remains unrevealed. In the present study, a MIP-T3 homolog from shrimp Litopenaeus vannamei (named as LvMIP-T3) was cloned and identified. LvMIP-T3 had a 2076 bp open reading frame (ORF), encoding a polypeptide of 691 amino acids that contained a classic coiled-coil domain in the C-terminal that showed a high degree of conservation to other homologs. LvMIP-T3 could interact with LvTRAF6, a member of the canonical NF-κB pathway, but not LvTRAF3, which implies that LvMIP-T3 is able to regulate NF-κB activity via its interaction with LvTRAF6. In addition, LvMIP-T3 was substantially inducted in response to white spot syndrome virus (WSSV) challenge, and we demonstrated that LvMIP-T3 facilitated the expression of NF-κB-mediated several Penaeidins (antimicrobial peptides, AMPs) to oppose infection. Taken together, we identified a MIP-T3 homolog from shrimp L. vannamei that played a positive role in the TRAF6/NF-κB/AMPs axis mediated defense response, which will contribute to better understand the regulator relationship among members of the canonical NF-κB pathway in shrimp, and provides some insights into disease resistance breeding.

Molecular characterization and expression of the autophagy-related gene Atg14 in WSSV-infected Procambarus clarkii.

Atg14 (autophagy-related gene 14), also known as Atg14L or Barkor (Beclin-1 associated autophagy-related key regulator), plays an important role in a variety of biological processes including immunity, development, tumor inhibition, longevity, and protection against some cardiac and neurodegenerative diseases. However, very few studies have characterized Atg14 expression in invertebrates, particularly crustaceans. Here, a novel Atg14 gene from Procambarus clarkii (named PcAtg14) was characterized via RACE technology. Bioinformatics analysis showed that the total length of the PcAtg14 gene sequence was 2,880 bp, and it was predicted to encode 488 amino acids. The results of homology comparison showed that PcAtg14 exhibited the highest homology with crustacean the American lobster (Homarus americanus). Quantitative real-time PCR expression analysis showed that PcAtg14 was expressed in all tissues of P. clarkii, with the hepatopancreas having the highest expression and the eyestalk exhibiting the lowest expression. Upon white spot syndrome virus (WSSV) infection, the relative expression of PcAtg14 in the hepatopancreas, muscle, hemocyte, gill, heart and epidermis were significantly up-regulated at different time periods. After PcAtg14 gene silencing via RNA interference (RNAi), the proliferation of WSSV in P. clarkii was significantly inhibited, which coincided with a significant increase in P. clarkii mortality and an increase in the expression of autophagy-related genes (ATGs). Transmission electron microscopy analysis demonstrated an increase in the number of autophagosomes in the hepatopancreas of the PcAtg14 gene silencing group compared to the control group after WSSV infection. Collectively, these results indicated that PcAtg14 suppressed autophagy by reduce the fusion of autophagosomes and lysosomes, thereby promoting WSSV replication in P. clarkii. The findings here therefore provide novel insights into the immune mechanisms through which P. clarkii responds to WSSV infection.

Transcriptome profiles of red swamp crayfish Procambarus clarkii hematopoietic tissue in response to WSSV challenge.

The crayfish Procambarus clarkii could achieve a high cumulative mortality after WSSV infections. To better understand the immune response to WSSV in hematopoietic tissue, the present study investigated the immunological response of P. clarkii and analyzed the expression of some hematopoietic cytokines. After assembly, there was an average of 47,712,411 clean reads were obtained in control and treatment groups. A total of 35,945 unigenes were discovered with N50 length of 1554 bp. Under functional classification, enrichment, and pathway analysis using different database, there were about 257 differentially expressed genes (DEGs) identified, of which 139 were up-regulated and 118 were down-regulated. The GO function analysis of these DEGs were mostly participated in activation of immune response, complement activation, complement binding, negative regulation of humoral immune response and secretory granule membrane. Under KEGG analysis, these DEGs were involved in ECM-receptor interaction, HIF-1 signaling pathway, Glycolysis/Gluconeogenesis, Thyroid hormone signaling pathway and Glucagon signaling pathway. The real-time quantitative PCR (RT-qPCR) analysis of 9 selected genes confirmed the reliability of RNA-Seq results. The present research provide for the first time the transcriptomic profile of P. clarkii hematopoietic tissue in response to WSSV infection and reveals the astakines may play important roles in antiviral immune response. The results of the present study will further enrich the theoretical basis of the crayfish immune system and provide new ideas for disease prevention and control.

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.

KLF13 induces apoptotic cell clearance in Penaeus vannamei as an essential part of shrimp innate immune response to pathogens.

Although, in mammals, the Krüppel-like transcription factor 13 (KLF13) plays an essential role in cell proliferation, survival, differentiation, apoptosis, tumorigenesis, immune regulation, and inflammation, its role in penaeid shrimp is unclear. In the current study, we characterized a KLF13 homolog in Penaeus vannamei (PvKLF13), with full-length cDNA of 1677 bp and 1068 bp open reading frame (ORF) encoding a putative protein of 355 amino acids, which contains three ZnF_C2H2 domains. Sequence and phylogenetic analysis revealed that PvKLF13 shares a close evolutionary relationship with KLF13 from invertebrates. Transcript levels of PvKLF13 were ubiquitously expressed in shrimp and induced in hemocytes upon challenge with Vibrio parahaemolyticus, Streptococcus iniae, and white spot syndrome virus (WSSV), suggesting the involvement of PvKLF13 in shrimp immune response to pathogens. Besides, knockdown of PvKLF13 decreased hemocytes apoptosis in terms of increased expression of pro-survival PvBcl-2, but decreased expression of pro-apoptotic PvBax and PvCytochrome C, coupled with high PvCaspase3/7 activity, especially upon V. parahaemolyticus challenge. The findings here indicate the involvement of PvKLF13 in apoptotic cell clearance as an essential part of shrimp innate immune response to pathogens.

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.