Immune signaling of Litopenaeus vannamei c-type lysozyme and its role during microsporidian Enterocytozoon hepatopenaei (EHP) infection.

The microsporidian Enterocytozoon hepatopenaei (EHP) is a fungi-related, spore-forming parasite. EHP infection causes growth retardation and size variation in shrimp, resulting in severe economic losses. Studies on shrimp immune response have shown that several antimicrobial peptides (AMPs) were upregulated upon EHP infection. Among those highly upregulated AMPs is c-type lysozyme (LvLyz-c). However, the immune signaling pathway responsible for LvLyz-c production in shrimp as well as its function against the EHP infection are still poorly understood. Here, we characterized major shrimp immune signaling pathways and found that Toll and JAK/STAT pathways were up-regulated upon EHP infection. Knocking down of a Domeless (DOME) receptor in the JAK/STAT pathways resulted in a significant reduction of the LvLyz-c and the elevation of EHP copy number. We further elucidated the function of LvLyz-c by heterologously expressing a recombinant LvLyz-c (rLvLyz-c) in an Escherichia coli. rLvLyz-c exhibited antibacterial activity against several bacteria such as Bacillus subtilis and Vibrio parahaemolyticus. Interestingly, we found an antifungal activity of rLvLyz-c against Candida albican, which led us to further investigate the effects of rLvLyz-c on EHP spores. Incubation of the EHP spores with rLvLyz-c followed by a chitin staining showed that the signals were dramatically decreased in a dose-dependent manner, suggesting that rLvLyz-c possibly digest a chitin coat on the EHP spores. Transmission electron microscopy analysis revealed that an endospore layer, which is composed mainly of chitin, was digested by rLvLyz-c. Lastly, we observed that EHP spores that were treated with rLvLyz-c showed a significant reduction of the spore germination rate. We hypothesize that thinning of the endospore of EHP would result in altered permeability, hence affecting spore germination. This work provides insights into shrimp immune signaling pathways responsible for LvLyz-c production and its anti-EHP property. This knowledge will serve as important foundations for developing EHP control strategies.

Litopenaeus vannamei heat shock protein 90 (LvHSP90) interacts with white spot syndrome virus protein, WSSV322, to modulate hemocyte apoptosis during viral infection.

As cellular chaperones, heat shock protein can facilitate viral infection in different steps of infection process. Previously, we have shown that the suppression of Litopenaeus vannamei (Lv)HSP90 not only results in a decline of white spot syndrome virus (WSSV) infection but also induces apoptosis in shrimp hemocyte cells. However, the mechanism underlying how LvHSP90 involved in WSSV infection remains largely unknown. In this study, a yeast two-hybrid assay and co-immunoprecipitation revealed that LvHSP90 interacts with the viral protein WSSV322 which function as an anti-apoptosis protein. Recombinant protein (r) LvHSP90 and rWSSV322 inhibited cycloheximide-induced hemocyte cell apoptosis in vitro. Co-silencing of LvHSP90 and WSSV322 in WSSV-infected shrimp led to a decrease in expression level of viral replication marker genes (VP28, ie-1) and WSSV copy number, while caspase 3/7 activity was noticeably induced. The number of apoptotic cells, confirmed by Hoechst 33342 staining assay and annexin V/PI staining, was significantly higher in LvHSP90 and WSSV322 co-silenced-shrimp than the control groups. Moreover, the co-silencing of LvHSP90 and WSSV322 triggered apoptosis by the mitochondrial pathway, resulting in the upregulation of pro-apoptotic protein expression (bax) and the downregulation of anti-apoptotic protein expression (bcl, Akt). This process also involved the release of cytochrome c (CytC) from the mitochondria and a decrease in mitochondrial membrane potential (MMP). These findings suggest that LvHSP90 interacts with WSSV322 to facilitate viral replication by inhibiting host apoptosis during WSSV infection.

Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon.

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Antiviral properties of Penaeus monodon cyclophilin A in response to white spot syndrome virus infection in the black tiger shrimp.

Cyclophilin A (CypA) or peptidylprolyl isomerase A, plays an important role in protein folding, trafficking, environmental stress, cell signaling and apoptosis etc. In shrimp, the mRNA expression level of PmCypA was stimulated by LPS. In this study, all three types of shrimp hemocytes: hyaline cell, granulocyte and semi-granulocyte expressed the PmCypA protein. The mRNA expression level of PmCypA was found to be up-regulate to four-fold in white spot syndrome virus (WSSV) infected hemocytes at 48 h. Interestingly, PmCypA protein was only detected extracellularly in shrimp plasma at 24 h post WSSV infection. To find out the function of extracellular PmCypA, the recombinant PmCypA (rPmCypA) was produced and administrated in shrimp primary hemocyte cell culture to observe the antiviral properties. In rPmCypA-administrated hemocyte cell culture, the mRNA transcripts of WSSV intermediate early gene, ie1 and early gene, wsv477 were significantly decreased but not that of late gene, vp28. To explore the antiviral mechanism of PmCypA, the expression of PmCypA in shrimp hemocytes was silenced and the expression of immune-related genes were investigated. Surprisingly, the suppression of PmCypA affected other gene expression, decreasing of penaeidin, PmHHAP and PmCaspase and increasing of C-type lectin. Our results suggested that the PmCypA might plays important role in anti-WSSV via apoptosis pathway. Further studies of PmCypA underlying antiviral mechanism are underway to show its biological function in shrimp immunity.

Heat Shock Protein 70 Is a Damage-Associated Molecular Pattern That by Binding to Lipopolysaccharide and ß-1,3-Glucan-Binding Protein Activates the Prophenoloxidase System in Shrimp.

Recent studies have initiated a paradigm shift in understanding heat shock protein 70 (HSP70) functions in the shrimp immune system. However, the mechanism by which Litopenaeus vannamei (Lv)HSP70 modulates the innate immune response remains unclear. This study shows that LvHSP70 binds to the pattern recognition receptor LPS and ß-1,3-glucan-binding protein (LvLGBP), and subsequently leads to the activation of the prophenoloxidase system. Injection of shrimp with rLvHSP70 significantly (p < 0.05) upregulated the gene and protein expression of the key pattern recognition receptor LvLGBP. A coimmunoprecipitation and ELISA-based binding assay strongly confirmed the binding of LvHSP70 to LvLGBP at polysaccharide recognition motifs (PLS motifs) with a Kd of 4.44 µM and its competitive binding with LPS (IC50) is 8.036 µM. Conversely, LPS efficiently competed with LvHSP70 for binding to LvLGBP in a concentration-dependent manner with an IC50 of 7.662 µM, indicating that both are ligands of LvLGBP and likely bind at the same site. Binding of LvHSP70 to LvLGBP highly activated phenoloxidase activity in shrimp hemocyte lysate supernatants. Gene silencing of LvLGBP impaired the activation of phenoloxidase activity in shrimp by rLvHSP70, indicating that LvHSP70-LvLGBP interaction was essential for stimulating the immune cascade. Taken together, these results demonstrated that LvHSP70 is a ligand of LvLGBP similar to LPS and acts as a damage-associated molecular pattern to modulate the shrimp immune system via the prophenoloxidase system, eventually leading to the production of melanin and toxic reactive intermediates against invading pathogens.

Role of hemocytin from Litopenaeus vannamei in immune response against microsporidian, Enterocytozoon hepatopenaei.

Hemocytin, a multidomain hemostasis-related protein, is a homologous protein of hemolectin in Drosophila melanogaster and von Willebrand factor (vWF) in humans. The vWF type D (VWD) domain in hemocytin is thought to be a major mediator of hemocyte aggregation and the prophenoloxidase (proPO) activation system. Here, we report for the first time the role of hemocytin from Litopenaeus vannamei (LvHCT) against Enterocytozoon hepatopenaei (EHP), the pathogenic microsporidian causing hepatopancreatic microsporidiosis in Pacific white shrimp (L. vannamei). The LvHCT gene contains 58,366 base pairs consisting of 84 exons encoding for 4267 amino acids. Multiple sequence alignment and phylogenetic analysis revealed that LvHCT was clustered with crustacean hemocytins. Gene expression analysis by quantitative real-time RT-PCR showed that LvHCT in hemocytes was significantly upregulated at 9 and 11 days post-EHP cohabitation, which was consistent with EHP copy numbers in the infected shrimp. To further investigate the biological function of LvHCT in EHP infection, a recombinant protein containing an LvHCT-specific VWD domain (rLvVWD) was expressed in Escherichia coli. In vitro agglutination assays showed that rLvVWD was functionally representative of LvHCT and induced aggregation of pathogens, including Gram-negative and -positive bacteria, fungi, and EHP spore. LvHCT suppression resulted in higher EHP copy numbers and proliferation due to the lack of hemocytin-mediated EHP spore aggregation in LvHCT-silenced shrimp. Moreover, immune-related genes in the proPO-activating cascade and Toll, IMD and JAK/STAT signaling pathways were upregulated to eliminate the over-controlled EHP in LvHCT-silenced shrimp. Furthermore, the impaired phenoloxidase activity due to LvLGBP suppression was recovered after rLvVWD injection, suggesting that LvHCT may be directly involved in phenoloxidase activation. In conclusion, a novel LvHCT is involved in shrimp immunity against EHP via EHP spore aggregation and possible activation of the proPO-activating cascade.

Molecular characterization of turtle-like protein in whiteleg shrimp (Litopenaeus vannamei) and its role in Enterocytozoon hepatopenaei infection.

Enterocytozoon hepatopenaei (EHP) is a microsporidian parasite that infects shrimp hepatopancreas, causing growth retardation and disease susceptibility. Knowledge of the host-pathogen molecular mechanisms is essential to understanding the microsporidian pathogenesis. Turtle-like protein (TLP) is part of the immunoglobulin superfamily of proteins, which is widely distributed in the animal kingdom. TLP has multiple functions, such as cell surface receptors and cell adhesion molecules. The spore wall proteins (SWPs) of microsporidia are involved in the infection mechanisms. Some SWPs are responsible for spore adherence, which is part of the activation and host cell invasion processes. Previous studies showed that TLP from silkworms (Bombyx mori) interacted with SWP26, contributing to the infectivity of Nosema bombycis to its host. In this study, we identified and characterized for the first time, the Litopenaeus vannamei TLP gene (LvTLP), which encodes an 827-aa protein (92.4 kDa) composed of five immunoglobulin domains, two fibronectin type III domains, and a transmembrane region. The LvTLP transcript was expressed in all tested tissues and upregulated in the hepatopancreas at 1 and 7 days post-cohabitation (dpc) and at 9 dpc in hemocytes. To identify the LvTLP binding counterpart, recombinant (r)LvTLP and recombinant (r)EhSWP1 were produced in Escherichia coli. Coimmunoprecipitation and enzyme-linked immunosorbent assays demonstrated that rLvTLP interacted with rEhSWP with high affinity (KD = 1.20 × 10-7 M). In EHP-infected hepatopancreases, LvTLP was clustered and co-localized with some of the developing EHP plasmodia. Furthermore, LvTLP gene silencing reduced the EHP copy numbers compared with those of the control group, suggesting the critical role of LvTLP in EHP infection. These results provide insight into the molecular mechanisms of the host-pathogen interactions during EHP infection.

Identification of Potential Druggable Targets and Structure-Based Virtual Screening for Drug-like Molecules against the Shrimp Pathogen Enterocytozoon hepatopenaei.

Enterocytozoon hepatopenaei (EHP) causes slow growth syndrome in shrimp, resulting in huge economic losses for the global shrimp industry. Despite worldwide reports, there are no effective therapeutics for controlling EHP infections. In this study, five potential druggable targets of EHP, namely, aquaporin (AQP), cytidine triphosphate (CTP) synthase, thymidine kinase (TK), methionine aminopeptidase2 (MetAP2), and dihydrofolate reductase (DHFR), were identified via functional classification of the whole EHP proteome. The three-dimensional structures of the proteins were constructed using the artificial-intelligence-based program AlphaFold 2. Following the prediction of druggable sites, the ZINC15 and ChEMBL databases were screened against targets using docking-based virtual screening. Molecules with affinity scores ≥ 7.5 and numbers of interactions ≥ 9 were initially selected and subsequently enriched based on their ADMET properties and electrostatic complementarities. Five compounds were finally selected against each target based on their complex stabilities and binding energies. The compounds CHEMBL3703838, CHEMBL2132563, and CHEMBL133039 were selected against AQP; CHEMBL1091856, CHEMBL1162979, and CHEMBL525202 against CTP synthase; CHEMBL4078273, CHEMBL1683320, and CHEMBL3674540 against TK; CHEMBL340488, CHEMBL1966988, and ZINC000828645375 against DHFR; and CHEMBL3913373, ZINC000016682972, and CHEMBL3142997 against MetAP2.The compounds exhibited high stabilities and low binding free energies, indicating their abilities to suppress EHP infections; however, further validation is necessary for determining their efficacy.

Heat shock protein 90 of Pacific white shrimp (Litopenaeus vannamei) is possibly involved in promoting white spot syndrome virus infection.

Viruses cause up to 60% of disease-associated losses in shrimp aquaculture, and the white spot syndrome virus (WSSV) is a major viral pathogen in shrimp. Heat shock proteins (HSPs) are host chaperones that help promote many viral infections. We investigated the involvement of Litopenaeus vannamei (Lv) HSP90 in WSSV infections. Expression of LvHSP90 at the transcript and protein levels were upregulated after WSSV infection. Silencing LvHSP90 resulted in the increased cumulative mortality rate and the reduction of circulating hemocytes. The inhibition of LvHSP90 also induced the expression of apoptosis-related genes which indicated the induction of apoptotic pathway and might lead to shrimp death. However, lower the number of WSSV-infected cells and viral copy numbers were detected in the LvHSP90-silenced shrimp compared with those of the controls, corresponding with significantly decreased expressions of viral genes, including the immediate-early genes WSV083 and WSV249 and viral DNA polymerase. Conversely, injecting shrimp with WSSV that had been co-incubated with a recombinant LvHSP90 (rLvHSP90) promoted WSSV infection as evidenced by an increased cumulative mortality rate and viral copy numbers at 40-48 h post infection (hpi). Subcellular localization of LvHSP90 in WSSV-infected hemocytes at 3, 6 and 12 hpi demonstrated increased expression and translocation of LvHSP90 into the nucleus where WSSV DNA can replicate. Thus, LvHSP90 might be involved in the WSSV pathogenesis by promoting WSSV replication.

Stimulator of interferon gene (STING) and interferon regulatory factor (IRF) are crucial for shrimp antiviral defense against WSSV infection.

The cytosolic DNA-sensing immune response is essential for recognizing and establishing an effective host immune response to pathogens. However, the importance of the cytosolic signalling molecules responsible for facilitating an appropriate immune response following infection with a DNA virus in shrimps remains unknown. Here, we report the discovery of the Penaeus monodon stimulator of interferon gene (PmSTING) and interferon regulatory factor (PmIRF) genes and their important roles in the host defense against viral infection. High expression levels of PmSTING transcripts were detected in the midgut, hepatopancreas, and hindgut, with lower levels in foregut, while PmIRF was highly expressed in the hindgut, foregut, and hepatopancreas of P. monodon. The mRNA expression level of both PmSTING and PmIRF was up-regulated in the foregut in response to white spot syndrome virus (WSSV; dsDNA virus) infection. RNA-interference-mediated gene silencing of PmSTING and PmIRF rendered shrimps to be more susceptible to WSSV infection; suppression of PmIRF decreased the mRNA transcript level of PmSTING; and silencing of the cytosolic sensor PmDDX41 suppressed both PmSTING and PmIRF gene transcript levels. Thus, PmSTING and PmIRF are likely to be important for the antiviral innate response against the dsDNA WSSV pathogen and may mediate the antiviral immune defenses via PmDDX41/PmSTING/PmIRF signaling cascade in P. monodon.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon.

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Genome organization and definition of the Penaeus monodon viral responsive protein 15 (PmVRP15) promoter.

The viral responsive protein 15 from the black tiger shrimp Penaeus monodon (PmVRP15) is a highly responsive gene upon white spot syndrome virus (WSSV) challenge. It is identified from hemocyte and important for WSSV trafficking and assembly. However, the knowledge of PmVRP15 gene regulation is limited. In the present study, the genome organization and 5'upstream promoter sequences of PmVRP15 gene were investigated. The PmVRP15 gene was found to contain 4 exons interrupted by 3 introns and the start codon was located in the exon 2. The transcription start site and TATA box were also determined from the 5' upstream sequence. By using the narrow down experiment, the 5' upstream promoter active region was determined to be at the nucleotide positions -525 to +612. Mutagenesis of the putative transcription factor (TF) binding sites revealed that the binding site of interferon regulatory factor (IRF) (-495/-479) was a repressor-binding site whereas those of the octamer transcription factor 1 (Oct-1) (-275/-268) and the nuclear factor of activated T-cells transcription factor (NFAT) (-228/-223) were activator-binding sites. This is the first report on the transcription factors that might play essential roles in modulating the PmVRP15 gene expression. Nevertheless, the underlying regulation mechanism of PmVRP15 gene expression needs further investigation.

Correction: Caspase-1-Like Regulation of the proPO-System and Role of ppA and Caspase-1-Like Cleaved Peptides from proPO in Innate Immunity.

[This corrects the article DOI: 10.1371/journal.ppat.1004059.].

Differentially expressed genes in hemocytes of Litopenaeus vannamei challenged with Vibrio parahaemolyticus AHPND (VPAHPND) and VPAHPND toxin.

While toxin-harboring Vibrio parahaemolyticus has been previously established as the causative agent of early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp, information on the mechanistic processes that happen in the host during infection is still lacking. Here, we examined the expression responses of the shrimp hemocyte transcriptome to V. parahaemolyticus AHPND (VPAHPND) by RNA sequencing (RNA-seq). Using libraries (SRA accession number SRP137285) prepared from shrimp hemocytes under experimental conditions, a reference library was de novo assembled for gene expression analysis of VPAHPND-challenged samples at 0, 3/6, and 48 h post infection (hpi). Using the library from 0-hpi as the control, 359 transcripts were found to be differentially expressed in the 3/6-hpi library, while 429 were differentially expressed in the 48-hpi library. The expression patterns reported in the RNA-seq of 9 representative genes such as anti-lipopolysaccharide factor (LvALF), crustin p (CRU), serpin 3 (SER), C-type lectin 3 (CTL), clottable protein 2 (CLO), mitogen-activated protein kinase kinase 4 (MKK4), P38 mitogen-activated protein kinase (P38), protein kinase A regulatory subunit 1 (PKA) and DNAJ homolog subfamily C member 1-like (DNJ) were validated by qRT-PCR. The expression of these genes was also analyzed in shrimp that were injected with the partially purified VPAHPND toxin. A VPAHPND toxin-responsive gene, LvALF was identified, and its function was characterized by RNA interference. LvALF knockdown resulted in significantly rapid increase of shrimp mortality caused by toxin injection. Protein-protein interaction analysis by molecular docking suggested that LvALF possibly neutralizes VPAHPND toxin through its LPS-binding domain. The data generated in this study provide preliminary insights into the differences in the immune response of shrimp to the bacterial and toxic aspect of VPAHPND as a disease.

WSSV-responsive gene expression under the influence of PmVRP15 suppression.

The viral responsive protein 15 from black tiger shrimp Penaeus monodon (PmVRP15), is highly up-regulated and produced in the hemocytes of shrimp with white spot syndrome virus (WSSV) infection. To investigate the differential expression of genes from P. monodon hemocytes that are involved in WSSV infection under the influence of PmVRP15 expression, suppression subtractive hybridization (SSH) of PmVRP15-silenced shrimp infected with WSSV was performed. The 189 cDNA clones of the forward library were generated by subtracting the cDNAs from WSSV-infected and PmVRP15 knockdown shrimp with cDNAs from WSSV-infected and GFP knockdown shrimp. For the opposite subtraction, the 176 cDNA clones in the reverse library was an alternative set of genes in WSSV-infected shrimp hemocytes in the presence of PmVRP15 expression. The abundant genes in forward SSH library had a defense/homeostasis of 26%, energy/metabolism of 23% and in the reverse SSH library a hypothetical protein with unknown function was found (30%). The differential expressed immune-related genes from each library were selected for expression analysis using qRT-PCR. All selected genes from the forward library showed high up-regulation in the WSSV-challenged PmVRP15 knockdown group as expected. Interestingly, PmHHAP, a hemocyte homeostasis associated protein, and granulin-like protein, a conserved growth factor, are extremely up-regulated in the absence of PmVRP15 expression in WSSV-infected shrimp. Only transcript level of transglutaminase II, that functions in regulating hematopoietic tissue differentiation and inhibits mature hemocyte production in shrimp, was obviously down-regulated as observed from SSH results. Taken together, our results suggest that PmVRP15 might have a function relevant to hemocyte homeostasis during WSSV infection.

Silencing of a Kazal-type serine proteinase inhibitor SPIPm2 from Penaeus monodon affects YHV susceptibility and hemocyte homeostasis.

In shrimp, the Kazal-type serine proteinase inhibitors (KPIs) are involved in host innate immune defense system against pathogenic microorganisms. A five-Kazal-domain SPIPm2 is the most abundant KPIs in the black tiger shrimp Penaeus monodon and up-regulated in response to yellow head virus (YHV) infection. In this study, the role of SPIPm2 in YHV infection was investigated. The expression of SPIPm2 in hemocytes, gill and heart from 48-h YHV-infected shrimp was increased. The expression of SPIPm2 in hemocytes was significantly increased after 12 h of infection and gradually increased higher afterwards. Silencing of SPIPm2 by dsRNA interference resulted in the increased expression of different apoptosis-related genes, the increased expression of transcriptional factors of antimicrobial synthesis pathways, the reduction of circulating hemocytes in the shrimp hemolymph, and the increased susceptibility of the silenced shrimp to YHV infection. The activities of caspase-3 and caspase-7 in the hemocytes of SPIPm2-silenced shrimp was also increased by 5.32-fold as compared with those of the control shrimp. The results suggested that the SPIPm2 was involved in the hemocyte homeostasis.

Shrimp hemocyte homeostasis-associated protein (PmHHAP) interacts with WSSV134 to control apoptosis in white spot syndrome virus infection.

Hemocyte homeostasis-associated protein (PmHHAP) was first identified as a viral-responsive gene, due to a high upregulation in transcription following white spot syndrome virus (WSSV) infection. Functional studies using RNA interference have suggested that PmHHAP is involved in hemocyte homeostasis by controlling apoptosis during WSSV infection. In this study, the role of PmHHAP in host-viral interactions was further investigated. Yeast two-hybrid assay and co-immunoprecipitation revealed that PmHHAP binds to an anti-apoptosis protein, WSSV134. The viral protein WSSV134 is a late protein of WSSV, expressed 24 h post infection (hpi). Gene silencing of WSSV134 in WSSV-infected shrimp resulted in a reduction of the expression level of the viral replication marker genes VP28, wsv477, and ie-1, which suggests that WSSV134 is likely involved in viral propagation. However, co-silencing of PmHHAP and WSSV134 counteracted the effects on WSSV infection, which implies the importance of the host-pathogen interaction between PmHHAP and WSSV134 in WSSV infection. In addition, caspase 3/7 activity was noticeably induced in the PmHHAP and WSSV134 co-silenced shrimp upon WSSV infection. Moreover, PmHHAP and WSSV134 inhibited caspase-induced activation of PmCasp in vitro in a non-competitive manner. Taken together, these results suggest that PmHHAP and WSSV134 play a role in the host-pathogen interaction and work concordantly to control apoptosis in WSSV infection.

Antimicrobial activity of a serine proteinase inhibitor SPIPm5 from the black tiger shrimp Penaeus monodon.

A two-domain Kazal-type serine proteinase inhibitor, SPIPm5, from Penaeus monodon was studied. Its transcript was expressed in all tissues tested including the hemocytes, stomach, gill, lymphoid organ, muscle, intestine and heart albeit less in hepatopancreas and eyestalk. The expression of SPIPm5 gene was also up-regulated by heat stress, white spot syndrome virus (WSSV) infection and yellow head virus (YHV) infection. Injection of recombinant rSPIPm5 protein into normal shrimp to mimic heat stress condition did not have or had little stimulating effect on the expression of other immune genes: crustinPm1, penaeidin3, penaeidin5, Hsp70, SPIPm2 and SPIPm5. Like some other proteinase inhibitors, the rSPIPm5 could inhibit the hemolymph proPO activity. In survival experiments, the rSPIPm5 could prolong the life of WSSV-infected shrimp similar to the effect of heat stress. The rSPIPm5 also helped the YHV-, Vibrio harveyi- and V. parahaemolyticus-infected shrimp survive longer. The increased endurance against microbial infection was due to the inhibitory effects presumably activated by rSPIPm5 on viral replication and bacterial growth but not the expression of antimicrobial peptides. Therefore, the SPIPm5 plays an important role in shrimp innate immunity against the viral and bacterial infection.

Anti-melanization mechanism of the white spot syndrome viral protein, WSSV453, via interaction with shrimp proPO-activating enzyme, PmproPPAE2.

Inhibition of the host melanization reaction, activated by the prophenoloxidase activating (proPO) system, is one of the crucial evasion strategies of pathogens. Recently, the shrimp pathogen, white spot syndrome virus (WSSV), was found to inhibit melanization in the shrimp Penaeus monodon. The viral protein WSSV453 was previously shown to interact with PO-activating enzyme 2 (PmPPAE2) and reported to be involved in suppressing the shrimp melanization response after WSSV infection. Here, we characterized how WSSV453 inhibits melanization. WSSV453 is a non-structural viral protein, which was first detected in shrimp haemocytes at 6 hours post-infection (hpi) by WSSV and in shrimp plasma at 24 hpi. We produced recombinant proteins for three components of the P. monodon proPO system: PmproPPAE2, PmproPO1 and PmproPO2. Functional assays showed that active PmPPAE2 processed PmproPO1 and 2 to produce functional PO. Incubation of WSSV453 with PmproPPAE2 dose-dependently reduced PmPPAE2 activity toward PmPO1 or PmPO2. In contrast, WSSV453 had no effect on activated PmPPAE2. The addition of active PmPPAE2 to WSSV-infected shrimp plasma at day 2 post-infection also rescued PO activity. Taken together, these results indicate that the anti-melanization activity of WSSV is due to WSSV453, which interacts with PmproPPAE2 and interferes with its activation to active PmPPAE2.

HSP70 and HSP90 are involved in shrimp Penaeus vannamei tolerance to AHPND-causing strain of Vibrio parahaemolyticus after non-lethal heat shock.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus carrying toxin-producing plasmid, has led to severe mortalities in farmed penaeid shrimp throughout Asia. Previous studies reported that a non-lethal heat shock (NLHS) could enhance disease tolerance in aquatic animals. Here, we investigate whether the NLHS could enhance the survival of shrimp Penaeusvannamei upon challenge with an AHPND-causing strain of V. Parahaemolyticus (VPAHPND). Two NLHS conditions, acute and chronic NLHSs, were used. The former abruptly exposed the juveniles shrimp from 28 °C to 38 °C for 30 min only once whereas the latter exposed the shrimp to 38 °C for 5 min every day for 7 days. The treated shrimp were, then, challenged with VPAHPND at day 3, day 7, and day 30 during the recovery time after the treatment. The results showed that the shrimp exposed to either acute or chronic NLHS had higher survival rate (>50%) than that of the non-heated shrimp control (20%) when they were challenged with VPAHPND at day 3 recovery time. However, only those exposed to chronic NLHS showed the VPAHPND protection at day 7 and day 30 recovery times. Furthermore, the qRT-PCR analysis revealed that the expression of heat shock proteins, LvHSP70, LvHSP90 as well as other immune-related genes, LvproPO and LvCrustin, were induced upon exposure of shrimp to chronic NLHS. Interestingly, gene silencing of LvHSP70 and LvHSP90 eliminated the VPAHPND tolerance in the chronic NLHS shrimp and had decreasing PO activity suggesting that these LvHSPs played crucial roles in bacterial defense in shrimp. All together, we show for the first time that the NLHS enhance the shrimp tolerance to VPAHPND infection and this is likely mediated by the induction of LvHSP70, LvHSP90 and subsequent activation of the proPO system.