White Spot Syndrome Virus Copy Number Research Articles

C-type lectins containing an immunoglobulin domain have an anti-WSSV function in Procambarus clarkii

Crayfish, as an invertebrate, cannot produce specific antibodies to defend against viruses. However, they have their own survival strategy to protect themselves from virus infection. In this study, two C-type lectins (CTLs) containing one immunoglobulin (IG) domain (PcIgLec2 and PcIgLec3) were characterized from Procambarus clarkii. PcIgLec2 and PcIgLec3 were widely expressed in various tissues, and their expression levels in the intestine were upregulated by white spot syndrome virus (WSSV). Knockdown of PcIgLec2 or PcIgLec3 resulted in a significant increase in VP28 expression and WSSV copy number, and injection of recombinant PcIgLec2 (rPcIgLec2) or recombinant PcIgLec3 (rPcIgLec3) protein could reduce VP28 expression and WSSV copy number. Further investigation indicated that the IG domain of PcIgLec2 or PcIgLec3 inhibited WSSV replication, whereas their carbohydrate recognition domain (CRD) had no substantial effect on WSSV replication. WSSV binding assays showed that rPcIgLec2, rPcIgLec3, rPcCRD2, and rPcCRD3 had the ability to bind to WSSV. However, their IG domain could not bind to WSSV. In conclusion, the two CTLs with an IG domain participated in antiviral innate immunity. Their CRD was responsible for WSSV binding, and their IG domain played negative roles in WSSV replication.

Participation of shrimp pva-miR-166 in hemocyte homeostasis by modulating apoptosis-related gene PvProsaposin during white spot syndrome virus infection.

Tiny controllers referred to as microRNAs (miRNAs) impede the expression of genes to modulate biological processes. In invertebrates, particularly in shrimp as a model organism, it has been demonstrated that miRNAs play a crucial role in modulating innate immune responses against viral infection. By analyzing small RNAs, we identified 60 differentially expressed miRNAs (DEMs) in Penaues vannamei hemocytes following infection with white spot syndrome virus (WSSV). We predicted the target genes of WSSV-responsive miRNAs, shedding light on their participation in diverse biological pathways. We are particularly intrigued by pva-miR-166, which is the most notably elevated miRNA among 60 DEMs. At 24 h post-infection (hpi), the negative correlation between the expression of pva-miR-166 and its target gene, PvProsaposin, was evident and their interaction was confirmed by a reduction in luciferase activity in vitro. Suppression of PvProsaposin in unchallenged shrimp led to decreased survival rates, reduced total hemocyte count (THC), and increased caspase 3/7 activity, suggesting its significant role in maintaining hemocyte homeostasis. In WSSV-infected shrimp, a lower number of hemocytes corresponded to a lower WSSV load, but higher shrimp mortality was observed when PvProsaposin was suppressed. Conformingly, the introduction of the pva-miR-166 mimic to WSSV-infected shrimp resulted in decreased levels of PvProsaposin transcripts, a significant loss of THC, and an increase in the hemocyte apoptosis. Taken together, we propose that pva-miR-166 modulates hemocyte homeostasis during WSSV infection by suppressing the PvProsaposin, an anti-apoptotic gene. PvProsaposin inhibition disrupts hemocyte homeostasis, rendering the shrimp's inability to withstand WSSV invasion.IMPORTANCEGene regulation by microRNAs (miRNAs) has been reported during viral infection. Furthermore, hemocytes serve a dual role, not only producing various immune-related molecules to combat viral infections but also acting as a viral replication site. Maintaining hemocyte homeostasis is pivotal for the shrimp's survival during infection. The upregulated miRNA pva-miR-166 could repress PvProsaposin expression in shrimp hemocytes infected with WSSV. The significance of PvProsaposin in maintaining hemocyte homeostasis via apoptosis led to reduced survival rate, decreased total hemocyte numbers, and elevated caspase 3/7 activity in PvProsaposin-silenced shrimp. Additionally, the inhibitory ability of pva-miR-166-mimic and dsRNA-PvProsaposin on the expression of PvProsaposin also lowered the THC, increases the hemocyte apoptosis, resulting in a lower WSSV copy number. Ultimately, the dysregulation of the anti-apoptotic gene PvProsaposin by pva-miR-166 during WSSV infection disrupts hemocyte homeostasis, leading to an immunocompromised state in shrimp, rendering them incapable of surviving WSSV invasion.

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.

API5-Hsp20 axis regulate apoptosis and viral infection in mud crab (Scylla paramamosain).

Apoptosis Inhibitor 5 (API5) is a widely concerned nuclear protein with diverse functions in organisms, so far, study of API5 is still quite limited in lower animals, and its role in viral immune response has not been addressed. Here, we explored the function of API5 in mud crab (Scylla paramamosain) during White Spot Syndrome Virus (WSSV) infection. The interacting protein Hsp20 of API5 was screened by pull-down assay, and API5 and hsp20 were knocked down by RNAi interference. The results showed that API5 was upregulated along with virus infection, silencing of API5 led to increased WSSV copy numbers and apoptotic rate of hemocytes, highlighting its significance in the immune response. Moreover, we discovered a novel interaction between API5 and Heat Shock Protein 20 (Hsp20), and then revealed that Hsp20 could promote cell apoptosis of hemocytes and reduce viral copy numbers by suppressing API5. The current study therefore improves the knowledge of API5-Hsp20 axis and provides novel insights into intricate mechanisms governing the antiviral response in marine crustaceans.

Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein–protein docking modelling suggests the helveticin-J homolog may be able to block VP28–PmRab7 interactions and interrupt WSSV infection.

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.

The MEF2 homolog of Penaeus vannamei is essential for maintaining the WSSV latent infection

White spot syndrome virus (WSSV) is a lethal shrimp pathogen that has a latent infection cycle. The latent virus can easily turn into an acute infection when the culture environment changes, leading to widespread shrimp mortality. However, the mechanism of WSSV latent infection is poorly understood. Bioinformatic analysis revealed that the promoters of WSSV latency-related genes (i.e., wsv151, wsv366, wsv403, and wsv427) contained putative myocyte enhancer factor 2 (MEF2) binding sites. This suggested that the transcription factor MEF2 may be involved in WSSV latent infection. To further investigate this, a MEF2 homolog (PvMEF2) was cloned from Penaeus vannamei and its role in WSSV latent infection was explored. The results showed that knockdown of PvMEF2 led to an increase in the copy number of WSSV, indicating reactivation of WSSV from a latent infection. It was further demonstrated that suppression of PvMEF2 significantly decreased expression of the viral latency-related genes in WSSV-latent shrimp, while overexpression of PvMEF2 in Drosophila S2 cells activated the promoter activity of the viral latency-related gene. Additionally, we demonstrated that silencing of PvMEF2 was able to upregulate the expression of pro-apoptosis genes, thereby promoting cell apoptosis during latent infection. Collectively, the present data suggest that PvMEF2 could promote the expression of virus latency-related genes and enhance cell survival to maintain WSSV latent infection. This finding would contribute to a better understanding of the maintenance mechanism of WSSV latent infection.

Effects of PmDOME and PmSTAT knockdown on white spot syndrome virus infection in Penaeus monodon

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway plays an important role in antiviral immunity. This research reports the full-length DOME receptor gene in Penaeus monodon (PmDOME) and examines the effects of PmDOME and PmSTAT silencing on immune-related gene expressions in shrimp hemocytes during white spot syndrome virus (WSSV) infection. PmDOME and PmSTAT were up-regulated in shrimp hemocytes upon WSSV infection. Suppression of PmDOME and PmSTAT showed significant impacts on the expression levels of ProPO2 (melanization), Vago5 (interferon-like protein) and several antimicrobial peptides, including ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7. Silencing of PmDOME and PmSTAT reduced WSSV copy numbers and delayed the cumulative mortality caused by WSSV. We postulated that suppression of the JAK/STAT signaling pathway may activate the proPO, IFN-like antiviral cytokine and AMP production, resulting in a delay of WSSV-related mortality.

DnaJC16, the molecular chaperone, is implicated in hemocyte apoptosis and facilitates of WSSV infection in shrimp.

Chaperone proteins, including heat shock proteins (HSPs) and DnaJ proteins, are highly conserved and well known for their quick responses to environmental stresses and pathogen infections, especially viruses. However, how DnaJ, an HSP family member, in Penaeus vannamei responds to viral invasion has not been reported. In this research, the novel DnaJ homolog subfamily C member 16-like, or DnaJC16, was characterized in P. vannamei. It contains the DnaJ and thioredoxin domains. Phylogenetic tree analysis demonstrated the conservation of DnaJC16 among penaeid shrimp, where PvDnaJC16 was found to be closely related to DnaJC16 from Fenneropenaeus chinensis and Marsupenaeus japonicus. The transcripts of PvDnaJC16 were expressed in all the tissues tested, and the highest expression was in the lymphoid organs. As hemocytes are major immune tissue, we found significant upregulation of PvDnaJC16 in shrimp hemocytes after white spot syndrome virus (WSSV) infection. Furthermore, the suppression of PvDnaJC16 expression by RNA interference in WSSV-infected shrimp showed a decrease in replication and WSSV copy number. Interestingly, a dramatically high cumulative survival rate following the WSSV challenge (over 60%) was observed in PvDnaJC16-silenced shrimp. Meanwhile, the total hemocyte number was significantly increased in PvDnaJC16 knockdown. In addition, the expression of caspase-3 was reduced, as was the caspase-3/7 activity in PvDnaJC16 silencing. Additionally, the percentage of late apoptotic hemocytes diminished after PvDnaJC16 reduction, whereas the percentage of hemocyte viability increased. Our data reflect the fact that the upregulation of PvDnaJC16 expression upon WSSV infection enhances hemocyte apoptosis, which can accelerate viral spreading in shrimp.

Enhance the biocontrol efficiency of Bacillus velezensis Bs916 for white spot syndrome virus in crayfish by overproduction of cyclic lipopeptide locillomycin

White spot disease, caused by white spot syndrome virus (WSSV), is a serious crustacean disease in China, and currently, there is no specific treatment for it. In this study, the addition of locillomycin could help to improve the cumulative survival rate of WSSV-infected crayfish. Then further analysis showed that Spo0A and SpoIIID could negatively regulate the production of locillomycin in Bacillus velezensis Bs916, and the disruption of them caused a significant increase in the production of locillomycin, which was 8.9 times higher than that of wild-type. Subsequently, we investigated the protective effects of B. velezensis Bs916 and ΔSpo0A + SpoIIID as probiotics, and the results showed that addition of their cultures to the basal control diet significantly reduced the mortality of WSSV-challenged crayfish. Further results indicated that dietary B. velezensis Bs916 or ΔSpo0A + SpoIIID supplement reduced the copy number of WSSV and improved the immune activities of acid phosphatase (ACP), alkaline phosphatase (AKP), nitric oxide synthase (NOS) and lysozyme (LZM) in crayfish. Moreover, compared with B. velezensis Bs916, all above performances were further enhanced when ΔSpo0A + SpoIIID was provided, so we speculated that overproduction of locillomycin might contribute to the enhancement of biocontrol efficiency of B. velezensis Bs916 for WSSV in crayfish. In summary, this study provides some insight into the therapeutic or prophylactic intervention with B. velezensis Bs916 or its metabolic product, locillomycin, to protect crayfish against WSSV infection, and also provides a theoretical basis for the construction of high yielding locillomycin strains.

Effect of dietary Pediococcus pentosaceus MR001 on intestinal bacterial diversity and white spot syndrome virus protection in Pacific white shrimp

This study investigated the effect of Pediococcus pentosaceus MR001 on white spot disease (WSD) resistance and the intestinal microbiota of Litopenaeus vannamei. In a two-way white spot syndrome virus (WSSV) infection, probiotic supplementation significantly increased the survival of L. vannamei (p < 0.05) when compared to the control group. This result coincided with the upregulation of immunity (hemocyanin, prophenoloxidase, superoxide dismutase, LvToll, and STAT) and the reduced WSSV copy number in shrimp given P. pentosaceus MR001 treatment. The probiotic feeding group also significantly enhanced the expression of intestinal digestion (amylase, trypsin, chymotrypsin, fatty acid binding protein, and fatty acid synthase) -related genes. Moreover, 16S rRNA gene analysis was performed using Illumina sequencing technology to investigate the bacterial composition in the intestine of shrimp during probiotic administration. The results showed that P. pentosaceus MR001 could considerably increase the abundance of potential probiotics and reduce the abundance of potential pathogenic bacteria in the shrimp intestine. Additionally, the metagenomic prediction showed that gut microbiota in shrimp with and without probiotic feedings are related to the diverse pathways. In the probiotic feeding group, the microbiota was mostly related to metabolism, especially the degradation pathway. Our findings suggest that MR001 can improve immune responses and alter the gut microbiota, which may help in preventing WSD in L. vannamei.

PmKuSPI is regulated by pmo-miR-bantam and contributes to hemocyte homeostasis and viral propagation in shrimp

The Kunitz-type serine protease inhibitor (KuSPI) is a low molecular weight protein that plays a role in modulating a range of biological processes. In Penaeus monodon, the PmKuSPI gene has been found to be highly expressed in the white spot syndrome virus (WSSV)-infected shrimp and is predicted to be regulated by a conserved microRNA, pmo-miR-bantam. We reported that, despite being upregulated at the transcriptional level, the PmKuSPI protein was also upregulated after WSSV infection. Silencing the PmKuSPI gene in healthy shrimp had no effect on phenoloxidase activity or apoptosis but resulted in a delay in the mortality of WSSV-infected shrimp as well as a reduction in the total hemocyte number and WSSV copies. According to an in vitro luciferase reporter assay, the pmo-miR-bantam bound to the 3′UTR of the PmKuSPI gene as predicted. In accordance with the loss of function studies using dsRNA-mediated RNA interference, the administration of the pmo-miR-bantam mimic into WSSV-infected shrimp lowered the expression of the PmKuSPI transcript and the PmKuSPI protein, as well as the WSSV copy number. According to these results, the protease inhibitor PmKuSPI is posttranscriptionally controlled by pmo-miR-bantam and plays a role in hemocyte homeostasis, which in turn affects shrimp susceptibility to WSSV infection.

Molecular characterization of two newly identified glycine rich crustin genes (Mn-Gly-Cru3, Mn-Gly-Cru4) and their function in Macrobrachium nipponense innate immunity

Crustin, as an important antibacterial peptide in crustacean, plays a role in the defense against the invasion of foreign pathogens in the innate immune system. In this study, two glycine rich crustin genes (named as Mn-Gly-Cru3 and Mn-Gly-Cru4) were cloned in Macrobrachium nipponense. The full-length cDNA of Mn-Gly-Cru3 and Mn-Gly-Cru4 were 740 and 781 bp with open reading frames of 525 and 576 bp that encode 174 and 191 amino acids, respectively. The genome structure of Mn-Gly-Cru3 and Mn-Gly-Cru4 shows that both contain two exons and one intron. Both Mn-Gly-Cru3 and Mn-Gly-Cru4 protein contains a signal peptide, a cysteine-rich region, a glycine-rich region, and a whey acidic protein domain. Evolutionary analysis showed that Mn-Gly-Cru1, Mn-Gly-Cru2, Mn-Gly-Cru3 and Mn-Gly-Cru4 were clustered together. Mn-Gly-Cru3 and Mn-Gly-Cru4 were widely distributed in multiple tissues. After white spot syndrome virus (WSSV), Staphylococcus aureus or Vibrio parahaemolyticus challenge, the expression levels of Mn-Gly-Cru3 and Mn-Gly-Cru4 in the hemocytes, gills, and stomach were significantly changed. Mn-Gly-Cru3 and Mn-Gly-Cru4 knockdown remarkably increased the copy number of WSSV and the number of S. aureus and V. parahaemolyticus colonies in vivo. Purified rMn-Gly-Cru3 and rMn-Gly-Cru4 proteins could bind to a variety of bacteria and they also had binding activity toward LPS or PGN. Furthermore, rMn-Gly-Cru4 had a stronger binding activity to polysaccharides than rMn-Gly-Cru3. Our study indicates that Mn-Gly-Cru3 and Mn-Gly-Cru4 in M. nipponense are involved in the innate immune defense against bacteria or viruses.

A Novel Hemocyte-Specific Small Protein Participates in White Spot Syndrome Virus Infection via Binding to Viral Envelope Protein.

Hemocytes are essential components of the immune system against invading pathogens in shrimp. Many uncharacterized transcripts exist in hemocytes but the knowledge of them is very limited. In the present study, we identified a novel small protein from the uncharacterized transcripts in hemocytes of Litopenaeus vannamei. This transcript was specifically expressed in hemocytes and encoded a novel secretory protein, which was designated as hemocyte-specific small protein (LvHSSP). The expression level of LvHSSP was significantly up-regulated in the hemocytes of shrimp infected with white spot syndrome virus (WSSV). After knockdown of LvHSSP by RNA interference, the WSSV copy number in shrimp decreased significantly. Conversely, WSSV copy number increased in shrimp when they were infected by WSSV after incubation with recombinant LvHSSP protein. These results suggested that LvHSSP might promote viral infection in shrimp. Immunocytochemical assay showed that the recombinant LvHSSP protein was located on the membrane of hemocytes. Co-IP results showed that LvHSSP could interact with VP26, the main envelope protein of WSSV, suggesting that LvHSSP might mediate WSSV adhesion and entry into host cells by binding to viral envelope protein. Meanwhile, the total hemocyte counts were significantly decreased after LvHSSP knockdown while increased after supplementing with recombinant LvHSSP protein, supporting the idea of hemocytes as the carrier for systemic dissemination of WSSV. This study reported a novel small protein in hemocytes, which modulated the viral infection in shrimp. Our results will enrich the knowledge of invertebrate innate immunity and provide a new field in the study of hemocyte function.

Shrimp pmo-miR-750 regulates the expression of sarcoplasmic calcium-binding protein facilitating virus infection in Penaeus monodon

MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and play crucial roles in antiviral responses. Penaeus monodon miR-750 (pmo-miR-750) was found to be strongly up-regulated in the late phase of white spot syndrome virus (WSSV) infection, but its function remains uncharacterized. Herein, the targets that were translationally down-regulated in the shrimp stomach following a pmo-miR-750 mimic injection were identified using two-dimensional gel electrophoresis. Sarcoplasmic calcium-binding protein (Scp) and actin1 (Act1) were revealed to be down-regulated protein spots. The genuine binding of pmo-miR-750 mimic to Scp but not Act1 mRNA was validated in vitro. In addition, a negative correlation between the Scp transcript and pmo-miR-750 expression level in WSSV-infected P. monodon stomach implies that pmo-miR-750 regulates Scp expression in vivo. When injected into WSSV-infected shrimp, the pmo-miR-750 mimic suppressed Scp expression but significantly increased the WSSV copy number. Consistent with the miRNA mimic-mediated Scp suppression, the loss of function assay of Scp in WSSV-challenged shrimp by RNA interference revealed a decreased survival rate with a dramatic increase in viral copy number. Besides that, apoptosis was activated in the hemocytes of the Scp knockdown shrimp upon WSSV infection. Collectively, our findings reveal that up-regulated pmo-miR-750 suppresses Scp expression at both the transcript and protein levels in the late stage of WSSV infection, which contributes to modulating apoptosis and eventually enabling viral propagation.

Effects of Janus kinase (JAK) on hemocyte proliferation and disease resistance in Scylla paramamosain

Janus kinase (JAK), a nonreceptor tyrosine kinase, is the JAK-signal transducer and activator of transcription (STAT) signaling pathway. In this study, RNA interference technology was used to knockdown the expression of JAK to explore the immune function of JAK in Scylla paramamosain. JAK expression was relatively high in the intestine and relatively low in the heart and hemocytes of S. paramamosain. JAK was highly expressed in all examined tissues of S. paramamosain after infection with white spot syndrome virus (WSSV) or Vibrio alginolyticus. The expression levels of Astakine, STAT, CAP, TLR, proPO, and CTL were downregulated, while MCM7, Myosin and Relish expression was upregulated in hemocytes at 24 h after injection of JAK-dsRNA. Astakine expression was significantly downregulated within 72 h after injection of JAK-dsRNA. The total hemocyte count and hemocyte proliferation were significantly decreased after treatment with Astakine-dsRNA or JAK-dsRNA. The results showed that JAK may promote hemocyte proliferation, probably by regulating Astakine expression. The activities of phenoloxidase, superoxide dismutase, and lysozyme were decreased significantly, while production of reactive oxygen species was increased significantly in hemocytes after treatment with JAK-dsRNA. After JAK knockdown, the phagocytosis rate of WSSV or V. alginolyticus in hemocytes was increased. After WSSV challenge, the WSSV copy number in the JAK-dsRNA-treated group was higher than that in the WSSV group. After V. alginolyticus challenge, the apoptosis rate of hemocytes in the JAK-dsRNA group was higher than that in the V. alginolyticus group. The mortality rate of WSSV or V. alginolyticus-infected crabs after JAK knockdown was higher than that of the infection group. These results suggest that JAK can regulate the expression of some immune-related genes, such as Astakine, to influence the innate immune response of S. paramamosain against pathogens (WSSV or V. alginolyticus), mostly by regulating enzyme activities, hemocyte phagocytosis and apoptosis.

Identification and functional characterization of two Bcl-2 family proteins in swimming crab, Portunus trituberculatus

Bcl-2 family proteins are regulatory factors of the mitochondrial apoptosis pathway that are vital to immune defenses and many other biological processes. However, the effects of Bcl-2 family proteins on apoptosis and immunity in crustaceans is poorly studied. In the study, two novel genes including Bax gene (PtBax) and Bcl-2 (PtBcl-2) from Portunus trituberculatus were identified and characterized. The deduced amino acid sequences showed that PtBax and PtBcl-2 possess domains of typical Bcl-2-related proteins. Both genes were widely expressed in all tested tissues, with highest expression levels in the hepatopancreas. Fluorescence in situ hybridization revealed that PtBax and PtBcl-2 were mainly expressed in the cytoplasm, where they play an important role in pathogen infection. Both PtBax and PtBcl-2 were significantly upregulated in the hepatopancreas at both mRNA and protein levels following challenge with Vibrio parahaemolyticus or white spot syndrome virus (WSSV). Moreover, double-stranded RNA-mediated silencing of both genes altered apoptosis levels in the hepatopancreas and WSSV copy number in muscles. Interactions between PtBax and PtBcl-2 were analyzed at transcriptional and protein levels. Yeast two-hybrid system analysis showed that PtBcl-2 could interact with PtBax directly. These results suggest that PtBax and PtBcl-2 may participate in pathogen infection by regulating apoptosis.

Anti-lipopolysaccharide factors regulated by Stat, Dorsal, and Relish are involved in anti-WSSV innate immune defense in Macrobrachium nipponense

Anti-lipopolysaccharide factors (ALF) is an important antimicrobial peptide and critical effector molecule with a broad spectrum of antimicrobial activities in crustaceans. In addition to the previously reported five ALFs (MnALF1-5), another three ALFs [MnALF1, which is different from MnALF1 (ALF02818) that has been reported; MnALF6; and MnALF7] and an isoform of MnALF4 (MnALF4-isoform2) were newly identified from Macrobrachium nipponense in this study. MnALF6 has 134 amino acids and one single nucleotide polymorphism (SNP) in MnALF6 resulted in the change of 107th amino acid from E to D. Intron 1 retention produced longer transcript of MnALF6. The full length of MnALF7 has 691 bp with a 363 bp ORF encoding 120 amino acid protein. Three SNPs in MnALF2 resulted in the conversion of amino acids at positions 70, 73, and 91 from T70I73P91 to K70L73S91. The deletion of 13 bp in MnALF4 resulted in early termination of ORF, resulting in MnALF4-isoform2 with only 98 amino acids. The gDNAs of MnALF1, MnALF2, MnALF5, and MnALF6 contain three exons and two introns, while those of MnALF3 and MnALF7 contain three exons, one known intron, and one unknown intron. The MnALF1–7 in M. nipponense were widely distributed in multiple tissues. After white spot syndrome virus (WSSV) stimulation, the expression levels of MnALF1–7 changed. Knockdown of MnALF1–7 could evidently increase the expression of the envelope protein VP28 and the copy number of WSSV during viral infection. Further studies found that silencing of three transcription factors (Stat, Dorsal, and Relish) in M. nipponense significantly inhibit the synthesis of MnALF1–7 during the process of WSSV challenge. This study adds to the knowledge about the roles of ALFs in the innate immune responses to WSSV infection in M. nipponense.

WSV056 Inhibits Shrimp Nitric Oxide Synthase Activity by Downregulating Litopenaeus vannamei Sepiapterin Reductase to Promote White Spot Syndrome Virus Replication.

Sepiapterin reductase (Spr) plays an essential role in the biosynthesis of tetrahydrobiopterin (BH4), a key cofactor of multiple enzymes involved in various physiological and immune processes. Suppression of Spr could result in BH4 deficiency-caused diseases in human and murine models. However, information on the biological function of Spr in invertebrates is limited. In this study, two Sprs (CG12116 and Sptr) from Drosophila melanogaster were found to be downregulated in transgenic flies overexpressing white spot syndrome virus (WSSV) immediate-early protein WSV056. CG12116 and Sptr exerted an inhibitory effect on the replication of the Drosophila C virus. A Litopenaeus vannamei Spr (LvSpr) exhibiting similarity of 64.1–67.5% and 57.3–62.2% to that of invertebrate and vertebrate Sprs, respectively, were cloned. L. vannamei challenged with WSSV revealed a significant decrease in LvSpr transcription and Spr activity in hemocytes. In addition, the BH4 co-factored nitric oxide synthase (Nos) activity in shrimp hemocytes was reduced in WSSV-infected and LvSpr knockdown shrimp, suggesting WSSV probably inhibits the LvNos activity through LvSpr downregulation to limit the production of nitric oxide (NO). Knockdown of LvSpr and LvNos caused the reduction in NO level in hemocytes and the increase of viral copy numbers in WSSV-infected shrimp. Supplementation of NO donor DETA/NO or double gene knockdown of WSV056 + LvSpr and WSV056 + LvNos recovered the NO production, whereas the WSSV copy numbers were decreased. Altogether, the findings demonstrated that LvSpr and LvNos could potentially inhibit WSSV. In turn, the virus has evolved to attenuate NO production via LvSpr suppression by WSV056, allowing evasion of host antiviral response to ensure efficient replication.

Natural ingredient paeoniflorin could be a lead compound against white spot syndrome virus infection in Litopenaeus vannamei.

White spot syndrome virus (WSSV) is an important pathogen causing high mortality in the shrimp industry in aquaculture, yet there is no treatment available to date. In order to find a treatment against WSSV infection, this study examined the anti-WSSV activity of eight natural compounds using shrimp larvae as a model. Among the eight compounds, paeoniflorin showed the most obvious anti-WSSV effect, with a maximum protection efficiency of WSSV-infected shrimp >60% at 100μM. Furthermore, pretreatment and post-treatment experiments revealed that paeoniflorin could prevent and treat WSSV infection in shrimp. The antiviral activity of paeoniflorin in aquaculture water decreased rapidly with time, and the results showed that the stable anti-WSSV activity of paeoniflorin could only remain in water for 1day. Thus, the dosing pattern of continuous medication changes was evaluated. Obviously, in the model of continuous change of paeoniflorin, WSSV copy numbers in the virus-treated shrimp group still progressively increased, while the virus content in WSSVpaeoniflorin -treated group continued to decrease. Interestingly, paeoniflorin inhibited horizontal transmission of WSSV to a certain extent. Notably, paeoniflorin significantly increased the expression of antimicrobial peptides of shrimp to resist WSSV. In conclusion, paeoniflorin has the potential to protect shrimp against WSSV.