A Negative Regulatory Feedback Loop within the JAK-STAT Pathway Mediated by the Protein Tyrosine Phosphatase DUSP14 in Shrimp.

The JAK-STAT pathway is a central communication node for various biological processes. Its activation is characterized by phosphorylation and nuclear translocation of the transcription factor STAT. The regulatory balance of JAK-STAT signaling is important for maintenance of immune homeostasis. Protein tyrosine phosphatases (PTPs) induce dephosphorylation of tyrosine residues in intracellular proteins and generally function as negative regulators in cell signaling. However, the roles of PTPs in JAK-STAT signaling, especially in invertebrates, remain largely unknown. Pacific white shrimp Penaeus vannamei is currently an important model for studying invertebrate immunity. This study identified a novel member of the dual-specificity phosphatase (DUSP) subclass of the PTP superfamily in P. vannamei, named PvDUSP14. By interacting with and dephosphorylating STAT, PvDUSP14 inhibits the excessive activation of the JAK-STAT pathway, and silencing of PvDUSP14 significantly enhances humoral and cellular immunity in shrimp. The promoter of PvDUSP14 contains a STAT-binding motif and can be directly activated by STAT, suggesting that PvDUSP14 is a regulatory target gene of the JAK-STAT pathway and mediates a negative feedback regulatory loop. This feedback loop plays a role in maintaining homeostasis of JAK-STAT signaling and is involved in antibacterial and antiviral immune responses in shrimp. Therefore, the current study revealed a novel inhibitory mechanism of JAK-STAT signaling, which is of significance for studying the regulatory mechanisms of immune homeostasis in invertebrates.

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.

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.

Genetic structural analysis of different breeds and geographical groups of Fenneropenaeus chinensis reveals population diversity.

Fenneropenaeus chinensis is a commercially important shrimp species cultured in China. This study investigated eight F. chinensis populations in China, including four geographical populations, three commercial breeds, and one wild population captured from the Yellow Sea. Population stratification analysis revealed that the Hebei geographical population and commercial breeding "Huanghai No. 4" were relatively independent and stable, reflecting a relatively closed breeding environment, whereas gene introgression was present between other populations. Selective signature analysis detected artificial selection for vision, growth, and disease resistance in the Hebei population. Neuronal development-related genes were detected to be under selection in the Changyi and Rizhao populations. Fertility of the Rizhao population was also investigated. Additionally, genes in the glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathway were involved in the high pH tolerance of the "Huanghai No. 4" population. This study provided support for the genetic mechanism of parsing economic traits and the development of molecular breeding technologies.

Genome-wide QTL and eQTL mapping reveal genes associated with growth rate trait of the Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Growth rate is a crucial economic trait for farmed animals, but the genetic regulation of this trait is largely unknown in non-model organisms such as shrimp. RESULTS: In this study, we performed genome-wide phenotypic quantitative trait loci (QTL) and expression quantitative trait loci (eQTL) mapping analyses to identify genes affecting the growth rate of Pacific white shrimp (Litopenaeus vannamei), which is the most commercially-farmed crustacean worldwide. We used RNA-sequencing of 268 individuals in a mapping population, and subsequently validated our findings through gene silencing and shrimp growth experiments. We constructed a high-density genetic linkage map comprising 5533 markers spanning 44 linkage groups, with a total distance of 6205.75 cM and an average marker interval of 1.12 cM. Our analyses identified 11 QTLs significantly correlated with growth rate, and 117,525 eQTLs. By integrating QTL and eQTL data, we identified a gene (metalloreductase STEAP4) highly associated with shrimp growth rate. RNA interference (RNAi) analysis and growth experiments confirmed that STEAP4 was significantly correlated with growth rate in L. vannamei. CONCLUSIONS: Our results indicate that the comprehensive analysis of QTL and eQTL can effectively identify genes involved in complex animal traits. This is important for marker-assisted selection (MAS) of animals. Our work contributes to the development of shrimp breeding and available genetic resources.

A high-density linkage map and sex-determination loci in Pacific white shrimp (Litopenaeus vannamei).

BACKGROUND: Expansion of genomic resources for the Pacific white shrimp (Litopenaeus vannamei), such as the construction of dense genetic linkage maps, is crucial for the application of genomic tools in order to improve economically relevant traits. Sexual dimorphism exists in Pacific white shrimp, and the mapping of the sex-determination region in this species may help in future reproductive applications. We have constructed male, female, and sex-averaged high-density genetic maps using a 50 K single-nucleotide polymorphism (SNP) array, followed by a genome-wide association study (GWAS) to identify genomic regions associated with sex in white shrimp. RESULTS: The genetic map yielded 15,256 SNPs assigned to 44 linkage groups (LG). The lengths of the male, female, and sex-averaged maps were 5,741.36, 5,461.20 and 5,525.26 cM, respectively. LG18 was found to be the largest for both sexes, whereas LG44 was the shortest for males and LG31 for females. A sex-determining region was found in LG31 with 21 statistically significant SNPs. The most important SNP was previously identified as a sex-linked marker and was able to identify 99% of the males and 88% of the females. Although other significant markers had a lower ability to determine sex, putative genes were intercepted or close to them. The oplophorus-luciferin 2-monooxygenase, serine/arginine repetitive matrix protein and spermine oxidase genes were identified as candidates with possible participation in important processes of sexual differentiation in shrimp. CONCLUSIONS: Our results provide novel genomic resources for shrimp, including a high-density linkage map and new insights into the sex-determining region in L. vannamei, which may be usefulfor future genetics and reproduction applications.

Identification of structural variation related to spawn capability of Penaeus vannamei.

BACKGROUND: The genetic basis underlying spawning abilities in the Pacific white shrimp, Penaeus vannamei, remains largely unexplored. To investigate genetic variations potentially related to reproductive performance, a systematic bioinformatic analysis was conducted to identify structural variations (SVs) with different polymorphic spectra in P. vannamei with high fertility (HF) and low fertility (LF). RESULTS: A total of 2,323 and 1,859 SV events were identified exclusively in the HF and LF groups, respectively. These SVs were mapped to 277 genes in the HF group and 231 genes in the LF group. Gene Ontology (GO) enrichment analysis based on SNPs (single nucleotide polymorphism) and SVs revealed several neural-related processes, suggesting the importance of neural regulation in reproduction. Notably, we identified a set of promising genes, including Cttn, Spast, Ppp4c, Spire1, Lhcgr, and Ftz-f1, which may enhance fertility in shrimp. CONCLUSION: In conclusion, this study is the first to establish a link between SVs and reproductive traits in P. vannamei. The promising genes discovered have the potential to serve as crucial markers for enhancing reproductive traits through targeted genotyping.

Genomic mining of Vibrio parahaemolyticus highlights prevalence of antimicrobial resistance genes and new genetic markers associated with AHPND and tdh + /trh + genotypes.

BACKGROUND: Acute Hepatopancreatic Necrosis Disease (AHPND) causes significant mortality in shrimp aquaculture. The infection is primarily instigated by Vibrio parahaemolyticus (Vp) strains carrying a plasmid encoding the binary toxin PirAB. Yet, comprehension of supplementary virulence factors associated with this relatively recent disease remains limited. Furthermore, the same holds for gastroenteritis in humans caused by other Vp genotypes. Additionally, given the prevalent use of antibiotics to combat bacterial infections, it becomes imperative to illuminate the presence of antimicrobial resistance genes within these bacteria. RESULTS: A subsampled number of 1,036 Vp genomes was screened for the presence of antimicrobial resistance genes, revealing an average prevalence of 5 ± 2 (SD) genes. Additional phenotypic antimicrobial susceptibility testing of three Vp strains (M0904, TW01, and PV1) sequenced in this study demonstrated resistance to ampicillin by all tested strains. Additionally, Vp M0904 showed multidrug resistance (against ampicillin, tetracycline, and trimethoprim-sulfamethoxazole). With a focus on AHPND, a screening of all Vibrio spp. for the presence of pirA and/or pirB indicates an estimated prevalence of 0.6%, including four V. campbellii, four V. owensii, and a Vibrio sp. next to Vp. Their pirAB-encoding plasmids exhibited a highly conserved backbone, with variations primarily in the region of the Tn3 family transposase. Furthermore, an assessment of the subsampled Vp genomes for the presence of known virulence factors showed a correlation between the presence of the Type 3 Secretion System 2 and tdh, while the presence of the Type 6 Secretion System 1 was clade dependent. Furthermore, a genome-wide association study (GWAS) unveiled (new) genes associated with pirA, pirB, tdh, and trh genotypes. Notable associations with the pirAB genotype included outer membrane proteins, immunoglobulin-like domain containing proteins, and toxin-antitoxin systems. For the tdh + /trh + genotypes (containing tdh, trh, or both genes), associations were found with T3SS2 genes, urease-related genes and nickel-transport system genes, and genes involved in a 'minimal' type I-F CRISPR mechanism. CONCLUSIONS: This study highlights the prevalence of antimicrobial resistance and virulence genes in Vp, identifying novel genetic markers associated with AHPND and tdh + /trh + genotypes. These findings contribute valuable insights into the genomic basis of these genotypes, with implications for shrimp aquaculture and food safety.

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.

Comparative transcriptome analysis of hepatopancreas reveals the potential mechanism of shrimp resistant to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus carrying a pathogenic plasmid (VPAHPND) is one of the main causative agents of acute hepatopancreatic necrosis disease (AHPND) in shrimp aquaculture. Knowledge about the mechanism of shrimp resistant to VPAHPND is very helpful for developing efficient strategy for breeding AHPND resistant shrimp. In order to learn the mechanism of shrimp resistant to AHPND, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp from different families with different resistance to VPAHPND. Through comparative analysis on the hepatopancreas of shrimp from VPAHPND resistant family and susceptible family, we found that differentially expressed genes (DEGs) were mainly involved in immune and metabolic processes. Most of the immune-related genes among DEGs were highly expressed in the hepatopancreas of shrimp from resistant family, involved in recognition of pathogen-associated molecular patterns, phagocytosis and elimination of pathogens, maintenance of reactive oxygen species homeostasis and other immune processes etc. However, most metabolic-related genes were highly expressed in the hepatopancreas of shrimp from susceptible family, involved in metabolism of lipid, vitamin, cofactors, glucose, carbohydrate and serine. Interestingly, when we analyzed the expression of above DEGs in the shrimp after VPAHPND infection, we found that the most of identified immune-related genes remained at high expression levels in the hepatopancreas of shrimp from the VPAHPND resistant family, and most of the identified metabolic-related genes were still at high expression levels in the hepatopancreas of shrimp from the VPAHPND susceptible family. The data suggested that the differential expression of these immune-related and metabolic-related genes in hepatopancreas might contribute to the resistance variations of shrimp to VPAHPND. These results provided valuable information for understanding the resistant mechanism of shrimp to VPAHPND.

Shrimp SIRT4 promotes white spot syndrome virus replication.

In WSSV pathogenesis, the molecular mechanisms and the key host factors that regulate the viral replication and morphogenesis remain unclear. However, like most viruses, WSSV is known to induce metabolic reprogramming in several metabolic pathways including the host glutamine metabolism, and several recent reports have suggested that the sirtuins SIRT3, SIRT4, and SIRT5, which belong to a family of NAD+-dependent deacetylases, play an important role in this regulation. Here we focus on characterizing LvSIRT4 from Litopenaeus vannamei and investigate its role in regulating glutamine dehydrogenase (GDH), an important enzyme that promotes glutaminolysis and viral replication. We found that LvSIRT4 silencing led to significant decreases in both WSSV gene expression and the number of viral genome copies. Conversely, overexpression of LvSIRT4 led to significant increases in the expression of WSSV genes and the WSSV genome copy number. Immunostaining in Sf9 insect cells confirmed the presence of LvSIRT4 in the mitochondria and the co-localization of LvSIRT4 and LvGDH in the same cellular locations. In vivo gene silencing of LvSIRT4 significantly reduced the gene expression of LvGDH whereas LvSIRT4 overexpression had no effect. However, neither silencing nor overexpression had any effect on the protein expression levels of LvGDH. Lastly, although GDH activity in uninfected shrimp was unchanged, the GDH enzyme activity in WSSV-infected shrimp was significantly increased after both LvSIRT4 silencing and overexpression. This suggests that although there may be no direct regulation, LvSIRT4 might still be able to indirectly regulate LvGDH via the mediation of one or more WSSV proteins that have yet to be identified.

Effects of sand substrate removal on the intestinal antioxidant and metabolism in Marsupenaeus japonicus.

M. japonicus is an important specie for factory farming, and factory farming requires an environment with sand at the bottom of the pond. However, the physiological responses as well as survival in the process of factory farming without laying sand are currently unknown. In the present study, we explored the effect of sand substrate removal on the intestinal histomorphology, antioxidant enzyme activity, and metabolic profile of M. japonicus. Our results indicate a gradual increase in the mortality rate of kuruma shrimp in ponds lacking sand substrate. The intestinal mucosa exhibited necrosis and the presence of vacuoles, with their number gradually increasing over time. The intestinal villi showed significant erosion, accompanied by a decrease in intestinal superoxide dismutase (SOD) activity and catalase (CAT) activity, and consistent with an upregulation in the expression of apoptosis-related genes such as caspase-3, indicating an adaptive response to the adverse environmental conditions. Additionally, the metabolomic analysis revealed that most significantly differential metabolites were linked to amino acid and lipid metabolism. These findings enhance our understanding of the sand substrate removal on the intestinal health of kuruma shrimp, which provides a basis for the factory farming.

Comparative transcriptomic analysis of hepatopancreas reveals that more genes are involved in the exposure response of Vibrio parahaemolyticus PirAvp compared to PirBvp.

Vibrio parahaemolyticus (VP-AHPND) is regarded as one of the main pathogens that caused acute hepatopancreatic necrosis disease (AHPND) in the Pacific white shrimp Litopenaeus vannamei. PirAvp and PirBvp toxin proteins are the main pathogenic proteins of AHPND in shrimp. Knowledge about the mechanism of shrimp response to PirAvp or PirBvp toxin is very helpful for developing new prevention and control strategy of AHPND in shrimp. In this study, the pathological sections showed that after 4 h treatment, significant pathological changes were observed in the PirBvp treated group, and no obvious pathological changes was found in PirAvp treated group. In order to learn the mechanism of shrimp response to PirAvp and PirBvp, comparative transcriptome was applied to analyze the different expressions of genes in the hepatopancreas of shrimp after treatment with PirAvp or PirBvp. A total of 9978 differentially expressed genes (DEGs) were identified between PirAvp or PirBvp-treated and PBS control shrimp, including 6616 DEGs in the PirAvp treated group and 3362 DEGs in the PirBvp treated group. There were 2263 DEGs that were commonly expressed, 4353 DEGs were only expressed in PirAvp VS PBS group and 1099 DEGs were uniquely expressed in PirBvp VS PBS group. Among these DEGs, the anti-apoptosis related pathways and immune response related genes significantly expressed in the commonly expressed DEGs of PirAvp VS PBS group and PirBvp VS PBS group, and small GTPase-mediated signaling and DNA metabolic process might relate to the host special reaction towards PirAvp and PirBvp exposure. The data suggested that the differential expression of these immune and metabolic-related genes in hepatopancreas might contribute to the pathogenicity variations of shrimp to VP-AHPND. The identified genes in this study will be useful for clarifying the response mechanism of shrimp toward different toxins of VP-AHPND and will further provide molecular basis for understanding the pathogenic mechanism of VP-AHPND.

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.

In silico identification and functional study of long non-coding RNA involved in acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus infection in white shrimp, Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) caused by toxin-producing Vibrio parahaemolyticus (VpAHPND) has severely affected shrimp production. Long non-coding RNA (lncRNA), a regulatory non-coding RNA, which can play important function in shrimp disease responses. This study aimed to identify and investigate the role of lncRNA involved in VpAHPND infection in Pacific white shrimp, Litopenaeus vannamei. From a total of 368,736 de novo assembled transcripts, 67,559 were identified as putative lncRNAs, and only 72 putative lncRNAs showed differential expression between VpAHPND-infected and normal shrimp. The six candidate lncRNAs were validated for their expression profiles during VpAHPND infection and tissue distribution using RT-qPCR. The role of lnc2088 in response to VpAHPND infection was investigated through RNA interference. The result indicated that the suppression of lnc2088 expression led to an increase in shrimp mortality after VpAHPND infection. To explore the set of genes involved in lnc2088 knockdown, RNA sequencing was performed. A total of 275 differentially expressed transcripts were identified in the hepatopancreas of lnc2088 knockdown shrimp. The expression profiles of five candidate metabolic and immune-related genes were validated in lnc2088 knockdown and VpAHPND-infected shrimp. The result showed that the expression of ChiNAG was significantly increased, while that of NCBP1, WIPF2, and NFKB1 was significantly downregulated in ds2088-injected shrimp. Additionally, the expression of NFKB1, NCBP1 and WIPF2 was significantly increased, whereas that of ChiNAG and CUL5 were significantly decreased after infection with VpAHPND. Our work identified putative lncRNA profiles in L. vannamei in response to VpAHPND infection and investigated the role of lncRNA in shrimp immunity.

White feces syndrome in shrimp: Comprehensive understanding of immune system responses.

White feces syndrome (WFS) is a multifactorial disease that affects global shrimp production. The diagnostic approach to identify WFS involves traditional and molecular scientific methods by examining histopathology, bioassays, PCR (polymerase chain reaction), and calorimetric estimation. The pathogenesis of WFS is closely associated with Vibrio spp., intestinal microbiota (IM) dysbiosis, and Enterocytozoon hepatopenaei (EHP). It also has caused over 10-15 % loss in the aquaculture industry and is also known to cause retardation, lethargy and slowly leading to high mortality in shrimp farms. Therefore, it is necessary to understand the molecular mechanisms processed under the association of IM dysbiosis, Vibrio spp., and EHP to analyze the impact of disease on the innate immune system of shrimp. However, only very few reviews have described the molecular pathways involved in WFS. Hence, this review aims to elucidate an in-depth analysis of molecular pathways involved in the innate immune system of shrimp and their response to pathogens. The analysis and understanding of the impact of shrimp's innate immune system on WFS would help in developing treatments to prevent the spread of disease, thereby improving the economic condition of shrimp farms worldwide.

Identification of COP9 signalosome (CSN) subunits and antiviral function analysis of CSN5 in shrimp.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Study on the mechanism of miR-7562 regulating ATG5 and ATG12 genes in Penaeus monodon under Vibrio harveyi infection.

MicroRNAs (miRNAs) play a fundamental role in the post-transcriptional regulation of genes and are pivotal in modulating immune responses in marine species, particularly during pathogen assaults. This study focused on the function of miR-7562 and its regulatory effects on autophagy against Vibrio harveyi infection in the black tiger shrimp (Penaeus monodon), an economically important aquatic species. We successfully cloned and characterized two essential autophagy-related genes (ATGs) from P. monodon, PmATG5 and PmATG12, and then identified the miRNAs potentially involved in co-regulating these genes, which were notably miR-7562, miR-8485, and miR-278. Subsequent bacterial challenge experiments and dual-luciferase reporter assays identified miR-7562 as the principal regulator of both genes, particularly by targeting the 3'UTR of each gene. By manipulating the in vivo levels of miR-7562 using mimics and antagomirs, we found significant differences in the expression of PmATG5 and PmATG12, which corresponded to alterations in autophagic activity. Notably, miR-7562 overexpression resulted in the downregulation of PmATG5 and PmATG12, leading to a subdued autophagic response. Conversely, miR-7562 knockdown elevated the expression levels of these genes, thereby enhancing autophagic activity. Our findings further revealed that during V. harveyi infection, miR-7562 continued to influence the autophagic pathway by specifically targeting the ATG5-ATG12 complex. This research not only sheds light on the miRNA-dependent mechanisms governing autophagic immunity in shrimp but also proposes miR-7562 as a promising target for therapeutic strategies intended to strengthen disease resistance within the crustacean aquaculture industry.

Ago4-piRNA complex is a key component of genomic immune system against transposon expression in Penaeus monodon.

Argonaute proteins are key constituents of small RNA-guided regulatory pathways. In crustaceans, members of the AGO subfamily of Argonaute proteins that play vital roles in immune defense are well studied, while proteins of the PIWI subfamily are less established. PmAgo4 of the black tiger shrimp, Penaeus monodon, though phylogenetically clustered with the AGO subfamily, has distinctive roles of the PIWI subfamily in safeguarding the genome from transposon invasion and controlling germ cell development. This study explored a molecular mechanism by which PmAgo4 regulates transposon expression in the shrimp germline. PmAgo4-associated small RNAs were co-immunoprecipitated from shrimp testis lysate using a PmAgo4-specific polyclonal antibody. RNA-seq revealed a majority of 26-27 nt long small RNAs in the PmAgo4-IP fraction suggesting that PmAgo4 is predominantly associated with piRNAs. Mapping of these piRNAs on nucleotide sequences of two gypsy and a mariner-like transposons of P. monodon suggested that most piRNAs were originated from the antisense strand of transposons. Suppression of PmAgo4 expression by a specific dsRNA elevated the expression levels of the three transposons while decreasing the levels of transposon-related piRNAs. Taken together, these results imply that PmAgo4 exerts its suppressive function on transposons by controlling the biogenesis of transposon-related piRNAs and thus, provides a defense mechanism against transposon invasion in shrimp germline cells.