Killing of xenogenous and virally infected homogenous target cells by shrimp lymphocyte-like haemocytes.

Haemocytes play a crucial role in the invertebrate's immune system. In our lab, five subpopulations of shrimp haemocytes were identified in the past: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. In the latter two subpopulations, their characteristics such as having small cytoplasmic rims and not adhering to plastic cell-culture plates are very similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity like natural killer cells, with the ability to recognize and kill target cells. In our study, K562 cells and Sf9 cells were used as xenogenous target cells to detect the cytotoxic activity of the shrimp non-adherent lymphocyte-like haemocytes. Non-adherent haemocytes were collected and mixed with K562 cells and Sf9 cells at a 5:1 ratio and the binding activity was examined under a microscope. The binding rate of non-adherent haemocytes to K562 cells and Sf9 cells reached 6.6 % and 2.4 % after 240 min of culture, respectively. Then, the killing activity of non-adherent haemocytes was detected by an EMA staining (fluorescence microscopy), which showed 3.75 % dead K562 cells and 1.025 % dead Sf9 cells, and by Sytox® blue staining (flow cytometry), which showed 4.97 % of dead K562 cells. Next, a killing assay was developed to visualize the killing activity of shrimp non-adherent haemocytes. Non-adherent haemocytes were pre-labeled in blue (CellTracker blue) and K562/Sf9 cells in green (CFSE); dead cells were differentially stained red with ethidium bromide. The cytotoxic activity increased and reached a level of 2.59 % in K562 cells and 0.925 % in Sf9 cells at 120 min after co-culture. Furthermore, in the co-cultures of non-adherent haemocytes with K562 cells and Sf9 cells, upregulation of the gene and protein expression of the cytotoxic molecules torso-like protein and granzyme B was observed by RT-qPCR at 240 min and western blotting at 180 min. Additionally, non-adherent haemocytes were co-cultured with WSSV-inoculated shrimp ovary and lymphoid organ cells to detect the cytotoxicity to homogenous target cells. The binding activity started at 60 min in both the ovary and lymphoid organ cultures and reached at 240 min 50.62 % and 40.7 %, respectively. The killing activity was detected by EMA staining and the percentage of dead ovary and lymphoid organ cells increased respectively from 10.84 % to 6.89 % at 0 min to 13.09 % and 8.37 % at 240 min. In conclusion, we demonstrated the existence of cytotoxic activity of shrimp lymphocyte-like haemocytes against xenogenous cells from mammals and insects and against WSSV-infected homogenous shrimp cells.

Identification of a FOXO gene and its roles in anti-WSSV infection through regulation of Dicers and Argos in Macrobrachium nipponense.

Forkhead box O (FOXO) proteins are a subgroup of the forkhead family of transcription factors that play important roles in the immune response. In this study, we cloned and identified a FOXO gene named MnFOXO from Macrobrachium nipponense. The full-length cDNA of MnFOXO is 2086 bp and contains a 1302 bp open reading frame, which encodes 433 amino acids. MnFOXO consists of five low-complexity regions and a conserved DNA-binding domain (forkhead domain). Evolutionary analyses indicate that MnFOXO proteins cluster with FOXO proteins from crustaceans. Tissue distribution analysis showed that MnFOXO was expressed in all detected tissues, with relatively higher expression levels in the intestine, eyestalks, stomach, and hemocytes than in the hepatopancreas, gills, and heart. The expression levels of MnFOXO in the hepatopancreas and intestine were significantly up-regulated in M. nipponense infected with white spot syndrome virus (WSSV) at 24 and 48 h. Furthermore, knockdown of MnFOXO increased the expression of WSSV envelope protein VP28 during WSSV infection. Further studies showed that knockdown of the MnFOXO gene in M. nipponense inhibited the synthesis of Dicers (MnDicer1, MnDicer2) and Argonautes (MnArgo1, MnArgo2) during WSSV invasion. These findings suggest that MnFOXO positively regulates the expression of Dicers and Argos, and inhibits the expression of VP28. This study provides new evidence for understanding the role of FOXO in antiviral innate immunity in crustaceans.

Screening high-efficiency promoter to construct trans-vp28 gene Anabaena sp. PCC7120 against white spot syndrome virus of Litopenaeus vannamei.

This study aimed to select high-quality promoters to construct trans-vp28 gene Anabaena sp. PCC7120 and feed Litopenaeus vannamei to assess the effect of L.vannamei against white spot syndrome virus (WSSV). Transgenic algae were created using five plasmids containing PrbcL, Pcpc560, Ptrc, Ptac, and PpsbA. According to the gene expression efficiency and the growth index of transgenic algae, Pcpc560 was determined to be the most efficient promoter. Shrimps were continuously fed trans-vp28 gene Anabaena sp. PCC7120 for one week and then challenged with WSSV. After the challenge, the transgenic algae group (vp28-7120 group) was continuously immunized [continuous immunization for 0 days (vp28-7120-0d); continuous immunization for 2 days (vp28-7120-2d); continuous immunization for 4 days (vp28-7120-4d)]. After seven days, the daily survival rate of each experimental group was continuously tracked. Following the viral challenge, the hepatopancreas samples were assayed for their levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thioredoxin peroxidase (TPX), acid phosphatase (ACP), and alkaline phosphatase (AKP) at varying time intervals. In comparison to the positive control group (challenge and no vaccination) and the wild-type group (challenge, fed wild-type Anabaena sp. PCC7120), the vp28-7120 group (challenge, fed trans-vp28 gene Anabaena sp. PCC7120) exhibited a remarkable increase in survival rates, reaching 50 % (vp28-7120-0d), 76.67 % (vp28-7120-2d), and 80 % (vp28-7120-4d). Furthermore, the vp28-7120 group consistently displayed significantly higher activities of SOD, CAT, GSH-Px, ACP, and AKP, while exhibiting notably lower TPX activity, when compared to the control group. These results indicate that the Pcpc560 promoter effectively elevated the expression level of the exogenous vp28 gene and spurred the growth of the trans-vp28 gene Anabaena sp. PCC7120. Consequently, trans-vp28 gene Anabaena sp. PCC7120 significantly bolstered the immunity of L.vannamei. Therefore, utilizing the Pcpc560 promoter to develop trans-vp28 gene Anabaena sp. PCC7120 based oral vaccine is highly beneficial for industrial-scale cultivation, advancing its commercialization prospects.

Newly developed mRNA vaccines induce immune responses in Litopenaeus vannamei shrimps during primary vaccination.

White spot syndrome virus (WSSV) causes highly destructive infection in crustacean aquaculture, often resulting in 100% mortality within a week. However, there is lack of studies addressing the safety issues of WSSV vaccines in shrimps. In this study, WSSV VP28 mRNA vaccines were developed using codon deoptimization approach. These vaccines were administered to Litopenaeus vannamei shrimps at various dosages to access their safety and the shrimps' immune responses using quantification PCR (qPCR). The findings of this study indicate that the expression level of codon deoptimized VP28 mRNA vaccines are lower compared to the wild type VP28 vaccines, as observed through a comparison of bioinformatic predictions and experimental results. Additionally, the total haemocyte count (THC) in shrimps injected with codon deoptimized VP28 vaccine was higher than those injected with wild type VP28 vaccines. Furthermore, the expression of immune-related genes differed between codon deoptimized and wild type VP28 vaccines. In summary, the results suggest that 0.01 µg codon deoptimized VP28-D1 mRNA vaccine is the most promising WSSV mRNA vaccine, displaying low pathogenicity and expression in shrimps. To the best of our knowledge, this research represents the first attempt to attenuate WSSV using codon deoptimization method and development of a potential mRNA vaccine for shrimp purpose. The study addresses an important gap in shrimp vaccine research, offering potential solutions for WSSV control in shrimps.

Screening for White Spot Syndrome Virus (WSSV) in pink shrimp (Penaeus paulensis) from Rocha Lagoon, Uruguay.

Penaeus paulensis (pink shrimp) is an important resource for small-scale fisheries in the brackish coastal lagoons of Uruguay. No viral diseases have been detected in shrimp populations in the Uruguayan territory. The presence of viral pathogens, such as White Spot Syndrome Virus (WSSV) and Infectious Hypodermal Haematopoietic Necrosis Virus (IHHNV) in wild shrimp populations has been previously reported in Brazil and Argentina. We investigated the presence of WSSV in wild populations of penaeid shrimp from Rocha Lagoon, Uruguay. We sampled 70 specimens of juvenile P. paulensis and assessed the presence of these viral pathogens using nested PCR and histology. Gill tissue from the 70 samples was divided into 14 pools of 5 individuals for DNA extraction and PCR analysis. We also retested each pooled sample individually. The nested PCR procedure described in the WOAH aquatic animal manual was used. A subset of 20 individual specimens were also processed using standard histological techniques. The results showed that WSSV was not detected in the pooled or individually tested samples. We found no evidence of the presence of the viral genome or gill lesions in the samples analysed. This indicates that the fishery is still likely to be free of WSSV infection. The procedures and information generated can be used as a baseline study for future implementation of surveillance programmes in the country.

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.

Estimation of Genetic Parameters for Growth and WSSV Resistance Traits in Litopenaeus vannamei.

The current study aimed to provide a precise assessment of the genetic parameters associated with growth and white spot syndrome virus (WSSV) resistance traits in Pacific white shrimp (Litopenaeus vannamei). This was achieved through a controlled WSSV challenge assay and the analysis of phenotypic values of five traits: body weight (BW), overall length (OL), body length (BL), tail length (TL), and survival hour post-infection (HPI). The analysis included test data from a total of 1017 individuals belonging to 20 families, of which 293 individuals underwent whole-genome resequencing, resulting in 18,137,179 high-quality SNP loci being obtained. Three methods, including pedigree-based best linear unbiased prediction (pBLUP), genomic best linear unbiased prediction (GBLUP), and single-step genomic BLUP (ssGBLUP) were utilized. Compared to the pBLUP model, the heritability of growth-related traits obtained from GBLUP and ssGBLUP was lower, whereas the heritability of WSSV resistance was higher. Both the GBLUP and ssGBLUP models significantly enhanced prediction accuracy. Specifically, the GBLUP model improved the prediction accuracy of BW, OL, BL, TL, and HPI by 4.77%, 21.93%, 19.73%, 19.34%, and 63.44%, respectively. Similarly, the ssGBLUP model improved prediction accuracy by 10.07%, 25.44%, 25.72%, 19.34%, and 122.58%, respectively. The WSSV resistance trait demonstrated the most substantial enhancement using both genomic prediction models, followed by body size traits (e.g., OL, BL, and TL), with BW showing the least improvement. Furthermore, the choice of models minimally impacted the assessment of genetic and phenotypic correlations. Genetic correlations among growth traits ranged from 0.767 to 0.999 across models, indicating high levels of positive correlations. Genetic correlations between growth and WSSV resistance traits ranged from (-0.198) to (-0.019), indicating low levels of negative correlations. This study assured significant advantages of the GBLUP and ssGBLUP models over the pBLUP model in the genetic parameter estimation of growth and WSSV resistance in L. vannamei, providing a foundation for further breeding programs.

Genome-wide identification of ATG genes and their expression profiles under biotic and abiotic stresses in Fenneropenaeus chinensis.

BACKGROUND: Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. Extensive research has shown that autophagy plays a pivotal role in both viral infection and replication processes. Despite the increasing research dedicated to autophagy, investigations into shrimp autophagy are relatively scarce. RESULTS: Based on three different methods, a total of 20 members of the ATGs were identified from F. chinensis, all of which contained an autophagy domain. These genes were divided into 18 subfamilies based on their different C-terminal domains, and were found to be located on 16 chromosomes. Quantitative real-time PCR (qRT-PCR) results showed that ATG genes were extensively distributed in all the tested tissues, with the highest expression levels were detected in muscle and eyestalk. To clarify the comprehensive roles of ATG genes upon biotic and abiotic stresses, we examined their expression patterns. The expression levels of multiple ATGs showed an initial increase followed by a decrease, with the highest expression levels observed at 6 h and/or 24 h after WSSV injection. The expression levels of three genes (ATG1, ATG3, and ATG4B) gradually increased until 60 h after injection. Under low-salt conditions, 12 ATG genes were significantly induced, and their transcription abundance peaked at 96 h after treatment. CONCLUSIONS: These results suggested that ATG genes may have significant roles in responding to various environmental stressors. Overall, this study provides a thorough characterization and expression analysis of ATG genes in F. chinensis, laying a strong foundation for further functional studies and promising potential in innate immunity.

Infection and intracellular transport of white spot syndrome virus require the ESCRT machinery in shrimp.

The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.

Comparative Transcriptomic Analysis of WSSV-Challenged Penaeus vannamei with Variable Resistance Levels.

The Pacific white shrimp, Penaeus vannamei, is highly susceptible to white spot syndrome virus (WSSV). Our study explored the transcriptomic responses of P. vannamei from resistant and susceptible families, uncovering distinct expression patterns after WSSV infection. The analysis revealed a higher number of differentially expressed genes (DEGs) in the susceptible family following WSSV infection compared to the resistant family, when both were evaluated against their respective control groups, indicating that the host resistance of the family line influences the transcriptome. The results also showed that subsequent to an identical duration following WSSV infection, there were more DEGs in P. vannamei with a high viral load than in those with a low viral load. To identify common transcriptomic responses, we profiled DEGs across families at 96 and 228 h post-infection (hpi). The analysis yielded 64 up-regulated and 37 down-regulated DEGs at 96 hpi, with 33 up-regulated and 34 down-regulated DEGs at 228 hpi, showcasing the dynamics of the transcriptomic response over time. Real-time RT-PCR assays confirmed significant DEG expression changes post-infection. Our results offer new insights into shrimp's molecular defense mechanisms against WSSV.

WSSV early protein WSSV004 enhances viral replication by suppressing LDH activity.

White spot syndrome virus (WSSV) is known to upregulate glycolysis to supply biomolecules and energy for the virus's replication. At the viral genome replication stage, lactate dehydrogenase (LDH), a glycolytic enzyme, shows increased activity without any increase in expression. In the present study, yeast 2-hybrid screening was used to identify WSSV proteins that interacted with LvLDH isoform 1 and 2, and these included the WSSV early protein WSSV004. The interaction between WSSV004 and LvLDH1/2 was confirmed by co-immunoprecipitation. Immunofluorescence showed that WSSV004 co-localized with LvLDH1/2 in the cytoplasm. dsRNA silencing experiments showed that WSSV004 was crucial for WSSV replication. However, although WSSV004 silencing led to the suppression of total LvLDH gene expression during the viral late stage, there was nevertheless a significant increase in LvLDH activity at this time. We also used affinity purification-mass spectrometry to identify cellular proteins that interact with WSSV004, and found a total of 108 host proteins and 3 WSSV proteins with which it potentially interacts. Bioinformatics analysis revealed that WSSV004 and its interacting proteins might be responsible for various biological pathways during infection, including vesicular transport machinery and RNA-related functions. Collectively, our study suggests that WSSV004 serves as a multifunctional modulator to facilitate WSSV replication.

Molecular Characterization and Expression Analysis of the C-Type Lectin Domain Family 4 Member F in Litopenaeus vannamei against White Spot Syndrome Virus.

White spot disease (WSD) outbreaks pose a significant threat to the Pacific white shrimp (Litopenaeus vannamei) farming industry. The causative agent is the white spot syndrome virus (WSSV). There are no effective treatments for WSD so far. Therefore, understanding the resistance mechanisms of L. vannamei against the WSSV is crucial. C-type lectins (CTLs) are important pattern recognition receptors (PRRs) that promote agglutination, phagocytosis, encapsulation, bacteriostasis, and antiviral infections. This study cloned the C-type lectin domain family 4 member F (LvCLEC4F) from L. vannamei. LvCLEC4F contains a 492 bp open reading frame (ORF) encoding a protein of 163 amino acids, including a carbohydrate recognition domain (CRD). Following a challenge with the WSSV, the expression profile of LvCLEC4F was significantly altered. Using RNA interference (RNAi) technology, it was found that LvCLEC4F promotes WSSV replication and affects the expression levels of genes related to the regulation of apoptosis, signaling and cellular stress response, and immune defense. Meanwhile, the hemolymph agglutination phenomenon in vivo was weakened when LvCLEC4F was knocked down. These results indicated that LvCLEC4F may play an important role in the interaction between L. vannamei and WSSV.

Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

MicroRNA sequencing analysis reveals immune responses in hepatopancreas of Fenneropenaeus penicillatus under white spot syndrome virus infection.

White Spot Disease is one of the most harmful diseases of the red tail shrimp, which can cause devastating economic losses due to the highest mortality up to 100% within a few days. MicroRNAs (miRNAs) are large class of small noncoding RNAs with the ability to post-transcriptionally repress the translation of target mRNAs. MiRNAs are considered to have a significant role in the innate immune response of crustaceans, particularly in relation to antiviral defense mechanisms. Numerous crustacean miRNAs have been verified to be required in host immune defense against viral infection, however, till present, the miRNAs functions of F. penicillatus defense WSSV infection have not been studied yet. Here in this study, for the first time, miRNAs involved in the F. penicillatus immune defense against WSSV infection were identified using high-throughput sequencing platform. A total of 432 miRNAs were obtained including 402 conserved miRNAs and 30 novel predicted miRNAs. Comparative analysis between the WSSV-challenged group and the control group revealed differential expression of 159 microRNAs in response to WSSV infection. Among these, 48 were up-regulated and 111 were down-regulated. Ten candidate MicroRNAs associated with immune activities were randomly selected for qRT-PCR analysis, which confirming the expression profiling observed in the MicroRNA sequencing data. As a result, most differentially expressed miRNAs were down-regulated lead to increase the expression of various target genes that mediated immune reaction defense WSSV infection, including genes related to signal transduction, Complement and coagulation cascade, Phagocytosis, and Apoptosis. Furthermore, the genes expression of the key members in Toll and Imd signaling pathways and apoptotic signaling were mediated by microRNAs to activate host immune responses including apoptosis against WSSV infection. These results will help to understand molecular defense mechanism against WSSV infection in F. penicillatus and to develop an effective WSSV defensive strategy in shrimp farming.

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.

Transcriptomics reveals different response mechanisms of Litopenaeus vannamei hemocytes to injection of Vibrio parahaemolyticus and WSSV.

As the most important cultural crustacean species worldwide, studies about Pacific white shrimp (Litopenaeus vannamei) have received more attention. It has been well-documented that various pathogens could infect L. vannamei, resulting in huge economic losses. The studies about the responding mechanism of L. vannamei to sole pathogens such as Vibrio parahaemolyticus and white spot virus (WSSV) have been extensively reported, while the studies about the differently responding mechanisms remain unclear. In the present study, we identified the differently expressed genes (DEGs) of L. vannamei hemocytes post V. parahaemolyticus and WSSV infection with RNA-seq technology and compared the DEGs between the two groups. The results showed 2672 DEGs post the V. parahaemolyticus challenge (1079 up-regulated and 1593 down-regulated genes), while 1146 DEGs post the WSSV challenge (1067 up-regulated and 513 down-regulated genes). In addition, we screened the genes that simultaneously respond to WSSV and V. parahaemolyticus (434), solely respond to WSSV (1146), and V. parahaemolyticus challenge (2238), respectively. Six DEGs involved in innate immunity were quantified to validate the RNA-seq results, and the results confirmed the high consistency of both methods. Furthermore, we found plenty of innate immunity-related genes that responded to V. parahaemolyticus and WSSV infection, including pattern recognition receptors (PRRs), the proPO activating system, antimicrobial peptides (AMPs), and other immunity-related proteins. The results revealed that they were differently expressed after different pathogen challenges, demonstrating the complex and specific recognition systems involved in defending against the invasion of different pathogens in the environment. The present study improved our understanding of the molecular response of hemocytes of L. vannamei to V. parahaemolyticus and WSSV stimulation.

Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus.

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these âˆ¼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

Immune-stealth VP28-conjugated heparin nanoparticles for enhanced and reversible anticoagulation.

Heparins are a family of sulfated linear negatively charged polysaccharides that have been widely used for their anticoagulant, antithrombotic, antitumor, anti-inflammatory, and antiviral properties. Additionally, it has been used for acute cerebral infarction relief as well as other pharmacological actions. However, heparin's self-aggregated macrocomplex may reduce blood circulation time and induce life-threatening thrombocytopenia (HIT) complicating the use of heparins. Nonetheless, the conjugation of heparin to immuno-stealth biomolecules may overcome these obstacles. An immunostealth recombinant viral capsid protein (VP28) was expressed and conjugated with heparin to form a novel nanoparticle (VP28-heparin). VP28-heparin was characterized and tested to determine its immunogenicity, anticoagulation properties, effects on total platelet count, and risk of inducing HIT in animal models. The synthesized VP28-heparin trimeric nanoparticle was non-immunogenic, possessed an average hydrodynamic size (8.81 ± 0.58 nm) optimal for the evasion renal filtration and reticuloendothelial system uptake (hence prolonging circulating half-life). Additionally, VP28-heparin did not induce mouse death or reduce blood platelet count when administered at a high dosein vivo(hence reducing HIT risks). The VP28-heparin nanoparticle also exhibited superior anticoagulation properties (2.2× higher prothrombin time) and comparable activated partial thromboplastin time, but longer anticoagulation period when compared to unfractionated heparin. The anticoagulative effects of the VP28-heparin can also be reversed using protamine sulfate. Thus, VP28-heparin may be an effective and safe heparin derivative for therapeutic use.

Smart nano generation of transgenic algae expressing white spot syndrome virus in shrimps for inner ear-oral infection treatments using the spotted hyena optimizer (SHO)-Long short-term memory algorithm.

Nanotechnology offers a promising avenue to amplify the effectiveness and precision of using transgenic algae in managing WSSV in shrimp by possibly crafting nano-carriers for targeted therapeutic agent delivery or modifying algae cells at a molecular level. Leveraging the capabilities of nano-scale interventions, this study could explore innovative means to manipulate cellular processes, control biological interactions, and enhance treatment efficacy while minimizing undesirable impacts in aquatic environments. The White Spot Syndrome Virus (WSSV) is a double-stranded DNA virus with a tail and rod form that belongs to theNimaviridaefamily. There is no workable way to manage this illness at the moment. This research proposes a new model based on the Long Short-Term Memory (LSTM) and Spotted Hyena Optimizer (SHO) method to control the inner ear-oral infection, utilizing transgenic algae (Chlamydomonas reinhardtii). It is pretty tricky to modify the weight matrix in LSTM. The output will be more accurate if the weight of the neurons is exact. Histological examinations and nested polymerase chain reaction (PCR) testing were performed on the challenged shrimp every 4 h to assess the degree of white spot disease. The SHO-LSTM has shown the highest accuracy and Roc value (98.12% and 0.93, respectively) and the lowest error values (MSE = 0.182 and MAE = 0.48). The hybrid optimized model improves the overall inner ear-oral linked neurological diseases detection ratio. Additionally, with the slightest technical complexity, it effectively controls the forecast factors required to anticipate the ENT. Algal cells were found to be particularly well-suited for inner ear-oral infections, and shrimps fed a transgenic line had the best survival ratio in WSSV infection studies, with 87% of the shrimp surviving. This shows that using this line would effectively stop the spread of WSSV in shrimp populations.

Integrase-associated niche differentiation of endogenous large DNA viruses in crustaceans.

IMPORTANCE: Crustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.