The identification, evolutionary analysis, and immune roles of Rab family members in red swamp crayfish, Procambarus clarkii.

The Rab GTPase constitutes the largest family of small GTPases that regulate intracellular trafficking. Different eukaryotes possess varying numbers of Rab paralogs. However, limited knowledge exists regarding the evolutionary pattern of Rab family in most major eukaryotic supergroups. This study cloned 24 Rab genes from transcriptome data of Procambarus clarkii haemocytes. The multiple sequence alignment and phylogenetic tree analysis revealed a relatively high degree of conservation for PcRab. Furthermore, PcRab exhibited similarities in motif composition with all members showing presence of G, PM, RabF, and RabSF motifs. The tertiary structure indicated that PcRab proteins mainly consisted of α-helices and ß-strands, and most PcRab proteins shared similar tertiary structures, and it was indicated that they have similar protein characteristics. Protein-protein interaction prediction identified a total of 20 interacting proteins involved in vesicle trafficking, phagocytosis, and signal transduction with 193 interactions. Expression analysis showed wide expression patterns for PcRab in P. clarkii organs. Upon infection by white spot syndrome virus and Aeromonas veronii, significant induction was observed for PcRab gene expression levels, indicating their involvement in pathogen response mechanisms. The present study represents the pioneering effort in comprehensively identifying and cloning the Rab family genes in crustacean, followed by a systematic investigation into their evolutionary patterns and immune response upon pathogen infection. The results provided valuable insights for further investigation into the molecular mechanism underlying the response of P. clarkii to pathogen infection.

The molecular characterization of Rab11 and its immune roles in red swamp crayfish (Procambarus clarkii).

The Rab proteins primarily regulate vesicular transport between membrane-bound organelles and are important for innate immune. However, there is currently a lack of studies on crustaceans regarding Rab proteins, particularly core Rabs. We identified a Rab11 gene from Procambarus clarkii (PcRab11) and evaluated its potential involvement in immune response. The results showed PcRab11 was 1789 bp long, with an open reading frame of 645 bp encoding 211 amino acids and an estimated molecular weight of 23.8 kDa. Sequence analysis revealed its remarkable evolutionary conservation. The PcRab11 was widely expressed in various tissues, with highest levels in hepatopancreas, and localized within the cell cytoplasm. Upon infection with white spot syndrome virus (WSSV) or Aeromonas veronii, the expression of PcRab11 in immune organs was significantly induced. Furthermore, silencing PcRab11 reduced phagocytosis-related genes expression and haemocytes' phagocytic activity to FITC-labeled A. veronii, as well as decreased mortality and death time in WSSV or A. veronii infected P. clarkii. Additionally, the potential protein interaction between PcRab11 and 14-3-3ε was identified in haemocytes. Overall, our findings provided evidence for the involvement of Rab11 in P. clarkii's immune response, establishing a foundation to explore the immune role of core Rab proteins in crustaceans' innate immune system.

Single-cell RNA-seq revealed heterogeneous responses and functional differentiation of hemocytes against white spot syndrome virus infection in Litopenaeus vannamei.

Shrimp hemocytes are the vital immune cells participating in innate immune response to defend against viruses. However, the lack of specific molecular markers for shrimp hemocyte hindered the insightful understanding of their functional clusters and differential roles in combating microbial infections. In this study, we used single-cell RNA sequencing to map the transcriptomic landscape of hemocytes from the white spot syndrome virus (WSSV)-infected Litopenaeus vannamei and conjointly analyzed with our previous published single-cell RNA sequencing technology data from the healthy hemocytes. A total of 16 transcriptionally distinct cell clusters were identified, which occupied different proportions in healthy and WSSV-infected hemocytes and exerted differential roles in antiviral immune response. Following mapping of the sequencing data to the WSSV genome, we found that all types of hemocytes could be invaded by WSSV virions, especially the cluster 8, which showed the highest transcriptional levels of WSSV genes and exhibited a cell type-specific antiviral response to the viral infection. Further evaluation of the cell clusters revealed the delicate dynamic balance between hemocyte immune response and viral infestation. Unsupervised pseudo-time analysis of hemocytes showed that the hemocytes in immune-resting state could be significantly activated upon WSSV infection and then functionally differentiated to different hemocyte subsets. Collectively, our results revealed the differential responses of shrimp hemocytes and the process of immune-functional differentiation post-WSSV infection, providing essential resource for the systematic insight into the synergistic immune response mechanism against viral infection among hemocyte subtypes. IMPORTANCE: Current knowledge of shrimp hemocyte classification mainly comes from morphology, which hinder in-depth characterization of cell lineage development, functional differentiation, and different immune response of hemocyte types during pathogenic infections. Here, single-cell RNA sequencing was used for mapping hemocytes during white spot syndrome virus (WSSV) infection in Litopenaeus vannamei, identifying 16 cell clusters and evaluating their potential antiviral functional characteristics. We have described the dynamic balance between viral infestation and hemocyte immunity. And the functional differentiation of hemocytes under WSSV stimulation was further characterized. Our results provided a comprehensive transcriptional landscape and revealed the heterogeneous immune response in shrimp hemocytes during WSSV infection.

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

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

SpSR-B2 functions as a potential pattern recognition receptor involved in antiviral and antibacterial immune responses of mud crab Scylla paramamosain.

Although class B scavenger receptors (SR-Bs) in mammals are multifunctional molecules, the functions of SR-Bs in invertebrates remain largely unknown. In this study, we characterized an SR-B homolog, namely SpSR-B2, from Scylla paramamosain. SpSR-B2 shared high similarity with mammalian SR-Bs, and exhibited specific binding activity to ac-LDL, indicating that it may be a new member of SR-B class in invertebrates. SpSR-B2 was upregulated after challenge with white spot syndrome virus (WSSV) or bacteria. Binding assays showed that SpSR-B2 specifically interacted with WSSV envelope protein VP24. Besides, SpSR-B2 could bind to all tested bacterial cells and agglutinate these bacteria. SpSR-B2 also exhibited a strong binding activity to LPS but weak binding activities to other tested polysaccharides. These findings indicated that SpSR-B2 was a potential recognition molecule for viral protein VP24 and bacterial LPS. Knockdown of SpSR-B2 resulted in dramatically decreased expressions of certain antimicrobial peptides (AMPs), and overexpression of SpSR-B2 led to the increased expression of the AMP of SpALF2, suggesting that SpSR-B2 could regulate the expression of AMPs. Taken together, this study revealed that SpSR-B2 functioned as a potential pattern recognition receptor participating in antiviral and antibacterial immunity, and provided new insights into the immune functions of invertebrate SR-Bs.

Evaluation of the relationship between the 14-3-3ε protein and LvRab11 in the shrimp Litopenaeus vannamei during WSSV infection.

The 14-3-3 proteins interact with a wide variety of cellular proteins for many diverse functions in biological processes. In this study, a yeast two-hybrid assay revealed that two 14-3-3ε isoforms (14-3-3ES and 14-3-3EL) interacted with Rab11 in the white shrimp Litopenaeus vannamei (LvRab11). The interaction of 14-3-3ε and LvRab11 was confirmed by a GST pull-down assay. The LvRab11 open reading frame was 645 bp long, encoding a protein of 214 amino acids. Possible complexes of 14-3-3ε isoforms and LvRab11 were elucidated by in silico analysis, in which LvRab11 showed a better binding energy score with 14-3-3EL than with 14-3-3ES. In shrimp challenged with the white spot syndrome virus (WSSV), the mRNA expression levels of LvRab11 and 14-3-3ε were significantly upregulated at 48 h after challenge. To determine whether LvRab11 and binding between 14-3-3ε and LvRab11 are active against WSSV infection, an in vivo neutralization assay and RNA interference were performed. The results of in vivo neutralization showed that LvRab11 and complexes of 14-3-3ε/LvRab11 delayed mortality in shrimp challenged with WSSV. Interestingly, in the RNAi experiments, the silencing effect of LvRab11 in WSSV-infected shrimp resulted in decreased ie-1 mRNA expression and WSSV copy number. Whereas suppression of complex 14-3-3ε/LvRab11 increased WSSV replication. This study has suggested two functions of LvRab11 in shrimp innate immunity; (1) at the early stage of WSSV infection, LvRab11 might play an important role in WSSV infection processes and (2) at the late stage of infection, the 14-3-3ε/LvRab11 interaction acquires functions that are involved in immune response against WSSV invasion.

Effects of dietary quercetin on the innate immune response and resistance to white spot syndrome virus in Procambarusclarkii.

In recent years, the use of natural products with immune-stimulating and antimicrobial properties has attracted increasing attention in aquaculture researches. In our study, the effect of diet supplemented with quercetin, a flavonoid commonly found in some types of plants substance on the innate immune response and disease resistance in crayfish (Procambarus clarkii) against white spot syndrome virus (WSSV) is reported. It was found that dietary 40 mg/kg quercetin significantly reduced the mortality of crayfish and WSSV copy number after WSSV challenge. Dietary quercetin increased catalase (CAT), and lysozyme (LZM) activity in crayfish. Dietary quercetin increased the expression of NF-κB, anti-lipopolysaccharide factor (ALF) and toll-like receptor (TLR) genes in crayfish. The apoptosis rate of hemocyte was increased by quercetin supplement in crayfish. Our results suggest that dietary quercetin may affect the innate immunity of crayfish and protect crayfish from WSSV infection.

Detection of white spot syndrome virus in seafood samples using a magnetosome-based impedimetric biosensor.

White spot syndrome virus (WSSV) is a significant threat to the aquaculture sector, causing mortality among crabs and shrimps. Currently available diagnostic tests for WSSV are not rapid or cost-effective, and a new detection method is therefore needed. This study demonstrates the development of a biosensor by functionalization of magnetosomes with VP28-specific antibodies to detect WSSV in seafood. The magnetosomes (1 and 2 mg/ml) were conjugated with VP28 antibody (0.025-10 ng/µl), as confirmed by spectroscopy. The magnetosome-antibody conjugate was used to detect the VP28 antigen. The binding of antigen to the magnetosome-antibody complex resulted in a change in absorbance. The magnetosome-antibody-antigen complex was then concentrated and brought near a screen-printed carbon electrode by applying an external magnetic field, and the antigen concentration was determined using impedance measurements. The VP28 antigen (0.025 ng/µl) bound more efficiently to the magnetosome-VP28 antibody complex (0.025 ng/µl) than to the VP28 antibody (0.1 ng/µl) alone. The same assay was repeated to detect the VP28 antigen (0.01 ng/µl) in WSSV-infected seafood samples using the magnetosome-VP28 antibody complex (0.025 ng/µl). The WSSV in the seafood sample was also drawn toward the electrode due to the action of magnetosomes controlled by the external magnetic field and detected using impedance measurement. The presence of WSSV in seafood samples was verified by Western blot and RT-PCR. Cross-reactivity assays with other viruses confirmed the specificity of the magnetosome-based biosensor. The results indicate that the use of the magnetosome-based biosensor is a sensitive, specific, and rapid way to detect WSSV in seafood samples.

An invertebrate gene encoding a Mab21-containing protein involves in antiviral response through regulating the STING pathway.

The cGAS-STING pathway plays essential roles in detecting cytosolic dsDNA and initiating antiviral and antibacterial responses in vertebrates. However, knowledge about its function in antiviral response of invertebrates is very limited. In the present study, a gene encoding a Mab21-containing protein, a cGAS homologue, was identified from a decapod crustacean Litopenaeus vannamei and designated as LvMab21cp. LvMab21cp was mainly distributed in intestine and hepatopancreas, showing similar expression profile with other genes in the cGAS-STING pathway, such as LvSTING and LvIRF. The expression levels of LvMab21cp, LvSTING and LvIRF were up-regulated in intestine and hepatopancreas of shrimp after white spot syndrome virus (WSSV) infection. Knockdown of LvMab21cp by dsRNA-mediated RNA interference could decrease the expression levels of its putative downstream genes, including LvSTING, LvIRF, LvVago4 and LvVago5, and enhance the in vivo propagation of WSSV in shrimp. Overexpression of LvMab21cp and LvSTING in HEK 293T cells activated the expression of mammalian IFNs upon simulation with interferon stimulatory DNA (ISD). These data suggest that LvMab21cp was a cGAS homologue, a member of the shrimp cGAS-STING pathway, and play an important role during WSSV infection. To our knowledge, this is the first report to show the role of the cGAS-STING pathway in the antiviral response of invertebrates, which will provide new insights into the innate immunity of invertebrates.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

The identification of a nuclear factor Akirin with regulating the expression of antimicrobial peptides in red swamp crayfish (Procambarus clarkii).

Akirin is a highly conserved nuclear factor among different species. It is closely related to skeletal muscle development, innate immune response, and tumorigenesis in a variety of animals. In invertebrates, Akirin is mainly involved in gene transcription and NF-κB dependent natural immune response. In the present study, a nuclear factor Akirin was identified from Procambarus clarkii. The Akirin protein of crayfish consists of 204 amino acids and is conserved among its family members, especially the nuclear localization signal peptide motif (KRRR). PcAkirin was highly expressed in stomach, intestines, and hepatopancreas. After A. hydrophila challenge, the transcription level of Akirin significantly increased in hemocyte and hepatopancreas. In addition, the recombinant Akirin protein was produced successfully and helpful to resist WSSV infection by increasing the expression level of some immune related genes. On the contrary, after interfering with Akirin gene by dsRNA, the crayfish increased the sensitivity to A. hydrophila and WSSV infections. The results are more obvious in the accumulated mortality of P. clarkii infected with A. hydrophila and WSSV. All these results suggested that Akirin played a significant role in innate immune responses and protected it from WSSV and bacterial infection in crayfish.

The T cell factor, pangolin, from Litopenaeus vannamei play a positive role in the immune responses against white spot syndrome virus infection.

As a downstream interactor of ß-catenin, Pangolin which is the homologous protein of the T cell factor/lymphoid enhancer factor (TCF/LEF) in vertebrates is less understood in the research field of immunity. In this study, two isoforms of Litopenaeus vannamei Pangolin (LvPangolin1 and LvPangolin2) were identified. Phylogenetic tree analysis revealed that all of the Pangolin proteins from invertebrates were represented the same lineage. The mRNA expression profiles of the LvPangolin1 and LvPangolin2 genes differed across different tissues. The expression of LvPangolin1 and the amount of LvPangolin1and LvPangolin2 combined (LvPangolinComb) were significantly increased in the haemocyte, intestine and gill but reduced in the hepatopancreas after white spot syndrome virus (WSSV) challenge. The inhibition of LvPangolin1 but not LvPangolinComb significantly reduced the survival rates of L. vannamei after WSSV infection, while significantly higher WSSV viral loads in both LvPangolin1-inhibited and LvPangolinComb-inhibited L. vannamei were observed. Knockdown of LvPangolin by RNAi could distinctly decrease the expression of antimicrobial peptide (AMP) genes and their related transcription factors. All of these results indicate that LvPangolin plays a positive role in the response to WSSV infection and that this may be mediated through regulating the immune signalling pathways which control the expression of AMPs with antiviral abilities.

In Litopenaeus vannamei, the cuticular chitin-binding proteins LvDD9A and LvDD9B retard AHPND pathogenesis but facilitate WSSV infection.

Acute hepatopancreatic necrosis disease (AHPND) is a serious bacterial disease caused by V. parahaemolyticus strains which contain a virulent plasmid that encodes a binary pore-forming Pir toxin. Typically, these AHPND-causing bacteria first colonize in the shrimp stomach and then later cross to the hepatopancreas. To do this, they must pass through structural barriers which include the pliant cuticular lining of the stomach lumen. A previous transcriptomic study of shrimp challenged with the virulent 5HP strain of V. parahaemolyticus found significant upregulation of a contig associated with the cuticular proteins LvDD9A and LvDD9B. Here, we confirmed that the mRNA levels of these two genes were significantly upregulated not only in 5HP-infected shrimp, but also in the stomach of shrimp challenged with the white spot syndrome virus (WSSV). Using dsRNA-mediated gene silencing, we found that AHPND-causing bacteria migrated to the hepatopancreas within 3 h of AHPND infection in LvDD9A/B-silenced shrimp. Shrimp shell hardness of LvDD9A/B-silenced shrimp was also significantly decreased. Conversely, we found that silencing of LvDD9A/B significantly inhibited both WSSV gene expression and genome replication. Taken together, our data suggests that LvDD9A and LvDD9B are involved in both AHPND and WSSV infection. However, in AHPND, these cuticular proteins help to prevent bacterial migration from the stomach to the hepatopancreas, whereas in WSSV infection, they facilitate viral gene expression and genome replication.

CqPP2A inhibits white spot syndrome virus infection by up-regulating antimicrobial substances expression in red claw crayfish Cherax quadricarinatus.

Protein phosphatase 2A (PP2A) is an important serine/threonine phosphatase, a highly conserved enzyme widely expressed in eukaryotic cells, which accounts for a majority of the serine/threonine phosphatase activity in cells implicated in regulation of immune signaling pathways and antiviral response. However, most of studies about PP2A have been conducted in mammals but few in crustaceans. In this study, two subunits of PP2A (named as CqPP2Ab and CqPP2Ac) were characterized to be involved in white spot syndrome virus (WSSV) infection in the haematopoietic tissue (Hpt) cells from red claw crayfish Cherax quadricarinatus. The open reading frame (ORF) of CqPP2Ab was 1341 bp encoding 446 amino acids with seven WD40 domains, and the ORF of CqPP2Ac was 930 bp encoding 309 amino acids with a PP2Ac domain. Tissue distribution analysis showed that the mRNA transcript of CqPP2Ab and CqPP2Ac were both widely expressed in all the tested tissues with the highest expression in hemocyte, followed by high expression in Hpt. The gene expressions of CqPP2Ab and CqPP2Ac were both significantly down-regulated at 6 h post WSSV infection (6 hpi) in Hpt cells. Importantly, the expression of viral immediate early gene IE1 and late viral gene envelope protein VP28 were both significantly increased post WSSV infection after gene silencing of CqPP2Ab or CqPP2Ac in Hpt cells, suggesting that CqPP2Ab and CqPP2Ac could inhibit WSSV infection in Hpt cells, probably by increasing the antimicrobial substances expression in consideration to the significantly reduced expression of anti-lipopolysaccharide factor, crustin, and lysozyme after gene silencing of CqPP2Ab or CqPP2Ac, respectively. These findings provide a new light on the mechanism of WSSV infection and the antiviral response in crustaceans.

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

The Toll family of receptors are a group of conserved pattern recognition receptors (PRRs) essentially controlling the initiation of innate immune responses. The white spot syndrome virus (WSSV) and Vibrio parahaemolyticus are major pathogens of aquaculture shrimp. Previous study has suggested that expression of the Toll2 receptor in Pacific white shrimp Penaeus vannamei was up-regulated by white spot syndrome virus (WSSV) infection but did not significantly changed upon infection with the bacterial pathogen Vibrio parahaemolyticus. The current study intends to investigate the role of P. vannamei Toll2 in antibacterial and antiviral immunity. We demonstrated that compared with the control, the Toll2-silenced shrimp was more susceptible to V. parahaemolyticus infection, suggesting that Toll2 may play a positive role in antibacterial immunity. However, silencing of Toll2 significantly enhanced survivorship of shrimp infected with WSSV and reduced the viral load in shrimp tissues. The expression of WSSV structural protein VP28 was also inhibited in Toll2-silenced shrimp. Histologic pathology analysis further showed that the WSSV infection was attenuated in stomach tissues from Toll2-silenced shrimp. These suggested that Toll2 could promote WSSV infection in shrimp. In Toll2-silenced shrimp, expression of antimicrobial peptides ALFs and PENs was significantly changed, which may contribute to the role of Toll2 in antibacterial immunity and WSSV infection.

Innate immune responses against viral pathogens in Macrobrachium.

Some members of genus Macrobrachium are important economically prawns and valuable objects for studying the innate immune defense mechanism of crustaceans. Studies have focused on immune responses against bacterial and fungal infections and have expanded to include antiviral immunity over the past two decades. Similar to all living organisms, prawns are exposed to viruses, including white spot syndrome virus, Macrobrachium rosenbergii nodavirus, and Decapod iridescent virus 1 and develop effective defense mechanisms. Here, we review current understanding of the antiviral host defense in two species of Macrobrachium. The main antiviral defense of Macrobrachium is the activation of intracellular signaling cascades, leading to the activation of cellular responses (apoptosis) and humoral responses (immune-related signaling pathways, antimicrobial and antiviral peptides, lectins, and prophenoloxidase-activating system).

Leucine-rich repeat-containing G-protein-coupled receptor 2 (LGR2) can regulate PO activity and AMP genes expression in Macrobrachium nipponense.

Leucine-rich repeat-containing G-protein-coupled receptors (LGRs) form a subfamily of the large superfamily of G-protein-coupled receptors. LGRs can be divided into three groups. LGR2 from Drosophila melanogaster is involved in cuticle tanning (melanization and sclerotization). In this study, one LGR2 (MnLGR2) was identified from Macrobrachium nipponense. MnLGR2 has an open reading frame of 4515 bp encoding a protein with 1504 amino acids. MnLGR2 is comprised of a 7-transmembrane domain, 12 leucine-rich repeats, and 5 low-complexity regions. The highest expression level of MnLGR2 was observed in gills. The expression levels of MnLGR2 in gills and stomach could be regulated by bacterial challenge. Knockdown of MnLGR2 upregulated the expression of anti-microbial peptide (AMP) genes. Further study indicated that inhibition of AMP expression by MnLGR2 was through inhibition of relish-mediated AMP expression. In addition to the negative regulation of AMP expression, MnLGR2 participated in positive regulation of phenol oxidase (PO) activity and expression of proPO activating pathway-related genes (proPO-activating factor and proPO-activating enzymes). Therefore, MnLGR2 plays an important role in prawn innate immunity.

Identification of a dorsal transcription factor (MnDorsal) from Macrobrachium nipponense and its role in innate immunity.

Rel/nuclear factor (NF)-κB family of transcription factors paly vital roles in innate immunity response to bacterial and viral infection. Here, we cloned and identified a dorsal homologue (named as MnDorsal) from Macrobrachium nipponense. The full-length cDNA of MnDorsal is 2573 bp with a 1986 bp open reading frame that encodes 661 amino acids. Predicted MnDorsal protein contained a RHD (Rel homology domain), an IPT (Iglike, plexins, and transcriptions factors) domain, and two low complexity regions. Phylogenetic analysis showed that MnDorsal has a closer genetic distance with dorsal homologues from invertebrates. MnDorsal was widely expressed in a variety of tissues, including hemocytes, heart, hepatopancreas, gills, stomach, and intestine. Expression patterns analysis showed that the transcriptional level of MnDorsal in the gills was evidently up-regulated after Staphylococcus aureus, Vibrio parahaemolyticus, white spot syndrome virus, or polyinosinic-polycytidylic acid challenge, suggesting that MnDorsal participates in the immune defenses against pathogens and stimulant challenges. Additionally, the dsRNA-mediated RNA interference analysis showed that knockdown of MnDorsal can significantly inhibit the expression of anti-lipopolysaccharide factor (ALF) and crustin. Further studies revealed that the up-regulated expression of ALFs (MnALF2, MnALF3, and MnALF4) and crustins (MnCrustin3 and MnCrustin4) caused by S. aureus infection were obviously decreased after silencing MnDorsal. These findings suggest that MnDorsal positively regulate the expression of antibacterial peptides (AMPs) during S. aureus infection. Our study will promote to better understand the role of Toll-Dorsal-AMPs pathway in innate immunity response to gram-positive bacterial infection in crustacean.

Shedding the Light on Litopenaeus vannamei Differential Muscle and Hepatopancreas Immune Responses in White Spot Syndrome Virus (WSSV) Exposure.

White Spot Syndrome Virus (WSSV) is one of the main threats to farming Litopenaeus vannamei, the most important crustacean commercialized in aquaculture worldwide. Here, we performed RNA-seq analyses in hepatopancreas and muscle from WSSV-negative (healthy) and WSSV-positive (unhealthy) L. vannamei, previously exposed to the virus, to obtain new insights about the molecular basis of resistance to WSSV. We detected 71% of our reads mapped against the recently described L. vannamei genome. This is the first report mapping RNA-seq transcripts from shrimps exposed to WSSV against the species reference genome. Differentially expressed gene (DEG) analyses were performed for four independent comparisons, and 13,338 DEGs were identified. When the redundancies and isoforms were disregarded, we observed 8351 and 6514 DEGs, respectively. Interestingly, after crossing the data, we detected a common set of DEGs for hepatopancreas and healthy shrimps, as well as another one for muscle and unhealthy shrimps. Our findings indicate that genes related to apoptosis, melanization, and the Imd pathway are likely to be involved in response to WSSV, offering knowledge about WSSV defense in shrimps exposed to the virus but not infected. These data present potential to be applied in further genetic studies in penaeids and other farmed shrimp species.

Antigenic properties of VP15 from white spot syndrome virus in kuruma shrimp Marsupenaeus japonicus.

White spot syndrome virus (WSSV) is known as one of the most lethal pathogenic viruses in shrimp causing massive damage to shrimp aquaculture industries. To date, no effective treatment or prevention has been found. In this study, five recombinant viral proteins VP15, VP19, VP24, VP26, and VP28 were expressed and purified in E. coli, which were employed as candidates against WSSV in Kuruma shrimp Marsupenaeus japonicus. In vivo antiviral assay in this study newly revealed that VP15 of major nucleocapsid protein, being known as a DNA-binding protein provided the substantial protection against the viral infection when pre-injected into shrimps. Furthermore, we also verified the immunogenic effects of purified VP15 and VP19 proteins produced in a silkworm-bacmid expression system. Taken together, our study identified VP15 as an effective candidate against WSSV infection in the Kuruma shrimp. It is interesting to uncover why and how VP15 is involved in the immune memory in shrimp in the future study.