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.

The Hippo-Yki Signaling Pathway Positively Regulates Immune Response against Vibrio Infection in Shrimp.

In the Hippo pathway, activation of Hippo and Warts (Wts) kinases results in the phosphorylation of Yorkie (Yki), to prevent its nuclear translocation. Shrimp aquaculture is threatened by Vibrio genus bacteria. In this study, we examine the role of the Hippo pathway in immune defense against Vibrio parahaemolyticus in Pacific white shrimp Penaeus vannamei. We show that V. parahaemolyticus infection promotes the expression of Yki and facilitates the dephosphorylation and nuclear translocation of Yki, indicating the inhibition of Hippo signaling upon bacterial infection. There is a complex regulatory relationship between the Hippo pathway components Hippo, Wts, and Yki and the immune-related transcription factors Dorsal, Relish, and STAT. Silencing of Hippo and Wts weakened hemocyte phagocytosis, while the silencing of Yki enhanced it, suggesting a positive regulation of shrimp cellular immunity by Hippo signaling activation. In vivo silencing of Hippo and Wts decreased the survival rates of V. parahaemolyticus-infected shrimp and elevated the bacterial content in tissues, while the silencing of Yki showed the opposite results. This suggests that the activation of Hippo signaling and the inhibition of Yki enhance antibacterial immunity in shrimp.

Determination of the efficacy of using a serine protease gene as a DNA vaccine to protect against Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Serine proteases are proteolytic enzymes that exhibit biological roles in many biological systems. Previously, a Vibrio parahaemolyticus serine protease was reported to be a virulence factor. Here, the serine protease gene of V. parahaemolyticus was investigated as a DNA vaccine against V. parahaemolyticus in Litopenaeus vannamei. The serine protease gene was mutated to replace the conserved residues His82, Asp131 and Ser231 with Gly, Asp and Pro, respectively. Then, a pcDNA3.1 vector to express mutVpSP (mutant serine protease) was constructed for in vitro and in vivo DNA vaccine investigation. In vivo mutVpSP transcriptional analysis revealed expression in various immunized white shrimp tissues, such as hemocytes, hepatopancreas, stomach, intestine, gills, and muscle. The efficiency of prevention of V. parahaemolyticus infection was investigated in vaccinated shrimp, and the lowest cumulative mortality percentage was 30%, while the control shrimp had a 60% cumulative mortality rate. The immune system was stimulated in shrimp vaccinated with the DNA vaccine. The mRNA expression of the shrimp immune-responsive genes phenoloxidase, peroxinectin and C-type lectin was significantly upregulated. Additionally, the humoral and cellular immune responses, including the PO, phagocytic, and encapsulation activities and nodule formation, were elevated. These results suggested that the serine protease could be a V. parahaemolyticus virulence determinant and that this DNA vaccine could be applied as an effective vaccine candidate for control of acute hepatopancreatic necrosis disease syndrome (AHPND) in shrimp.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

The ECSIT Mediated Toll3-Dorsal-ALFs Pathway Inhibits Bacterial Amplification in Kuruma Shrimp.

The Toll signaling pathway plays an important role in animal innate immunity. However, its activation and signal transmission greatly differ across species and need to be investigated. Shrimp farming is a worldwide economic activity affected by bacterial disease from the 1990s, which promoted research on shrimp immunity. In this study, we first proved that, among the three identified Toll receptors in Marsupenaeus japonicus kuruma shrimp, Toll 3 plays a pivotal role in initiating the antibacterial response in vivo, especially upon anti-Staphylococcus aureus infection. Further research showed that this result was due to the activation of the Dorsal transcription factor, which induced the expression of two anti-lipopolysaccharide factors (Alfs). Moreover, the evolutionarily conserved signaling intermediate in Toll pathways, ECSIT, was proved to be needed for signal transmission from Toll 3 to Dorsal and the expression of anti-lipopolysaccharide factors. Finally, the mortality assay showed that a Toll3-ECSIT-Dorsal-Alf axis was functional in the anti-S.aureus immunity of M. japonicus shrimp. The results provide new insights into the function and signal transduction of the Toll pathway in aquatic species and offer basic knowledge for shrimp disease control and genetic breeding.

Shrimp Plasma MANF Works as an Invertebrate Anti-Inflammatory Factor via a Conserved Receptor Tyrosine Phosphatase.

For a long time, how anti-inflammatory factors evolved was largely unknown. In this study, we chose a marine invertebrate, Litopenaeus vannamei, as a model and identified that shrimp mesencephalic astrocyte-derived neurotrophic factor (MANF) was an LPS-induced plasma protein, which exerted its anti-inflammatory roles on shrimp hemocytes by suppressing ERK phosphorylation and Dorsal expression. In addition, we demonstrated that shrimp MANF could be associated with a receptor protein tyrosine phosphatase (RPTP) to mediate negative regulation of ERK activation and Dorsal expression. More interestingly, shrimp RPTP-S overexpression in 293T cells could switch shrimp and human MANF-mediated ERK pathway activation to inhibition. In general, our results indicate that this conserved RPTP is the key component for extracellular MANF-mediated ERK pathway inhibition, which gives a possible explanation about why this neurotropic factor could both protect neuron cells from apoptosis and inhibit immune cell M1 activation in various species.

Dietary recombinant human lysozyme improves the growth, intestinal health, immunity and disease resistance of Pacific white shrimp Litopenaeus vannamei.

The present study was conducted to investigate the effects of dietary recombinant human lysozyme (RHL) on the growth, immune response, anti-oxidative activity, intestinal morphology, intestinal microflora and disease resistance of shrimp Litopenaeus vannamei. Shrimps with an initial body weight of 2.36 ± 0.02 g were fed diets supplemented with 0 (control group, R0), 0.0025% (R1), 0.005% (R2), 0.01% (R3), 0.02% (R4) and 0.04% (R5) of RHL, respectively. After a 10-week feeding trial, the final body weight, survival rate, weight gain ratio and protein efficiency rate of the shrimps in dietary RHL supplemented groups were significantly higher than that in the control group, while feed conversion ratio was significantly lower (P < 0.05). The total haemocyte count, total anti-oxidative capacity, respiratory burst, activities of phagocytosis, nitric oxide synthase, phenol oxidase and lysozyme in serum were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Meanwhile, the intestinal pile height and wall thickness were significantly higher in dietary RHL supplemented groups than those in the control group (P < 0.05). Dietary RHL significantly improved the expressions of immune-related genes in gill, such as lipopolysaccharide-ß-glucan binding protein, Toll, immune deficiency, heat shock protein 70 and Crustin (P < 0.05). The abundance of proteobacteria and bacteroidetes in intestine was higher, while the abundance of firmicutes and cyanobacteria was lower than those in the control group at the phylum level. In addition, dietary RHL supplementation significantly improved the protective ability of shrimp against V. parahaemolyticus infection (P < 0.05). Based on the broken-line model analysis for weight gain ratio after the feeding trial, the optimal level of dietary RHL supplementation for shrimp was estimated to be 0.006375%.

The potential role of eyestalk in the immunity of Litopenaeus vannamei to Vibrio infection.

The X-organ-sinus gland complex (XO-SG) in the eyestalk is an important neuroendocrine regulatory organ of crustaceans such as Litopenaeus vannamei, a prominent aquaculture species. The current study found significant changes in the enzyme activities of ALP, ACP, and T-SOD of hepatopancreatic in response to Vibrio parahaemolyticus exposure following eyestalk ablation, indicating that they were all involved in the immunological regulation of shrimps against V. parahaemolyticus infection. A total of 52,656 unigenes were obtained after RNA-Seq, with an average length of 1036 bp and an N50 of 1847 bp. Subsequently, 1899 eyestalk positive regulation genes (EPRGs), 745 eyestalk negative regulation genes (ENRGs), and 2077 non-eyestalk regulatory genes (NEGs) were identified. KEGG analysis of EPRGs revealed that eyestalk ablation might activate the neuroendocrine-immune (NEI) system. The RNA-Seq data were validated using quantitative real-time PCR (qRT-PCR). The findings suggested that eyestalk ablation might affect the expression of genes involved in the prophenoloxidase-activating system, the TLR signaling pathway, and numerous other immune-related genes in L. vannamei. All of these findings revealed that the eyestalk might have a role in the immune response of L. vannamei. The genes and pathways discovered in this study will help to elucidate the molecular mechanisms of hemocytes' immune response to V. parahaemolyticus following eyestalk ablation in shrimp, as well as provide the framework for building crustacean immunity theory.

Shoc2 recognizes bacterial flagellin and mediates antibacterial Erk/Stat signaling in an invertebrate.

Flagellin is a key bacterial virulence factor that can stimulate molecular immune signaling in both animals and plants. The detailed mechanisms of recognizing flagellin and mounting an efficient immune response have been uncovered in vertebrates; however, whether invertebrates can discriminate flagellin remains largely unknown. In the present study, the homolog of human SHOC2 leucine rich repeat scaffold protein in kuruma shrimp (Marsupenaeus japonicus), designated MjShoc2, was found to interact with Vibrio anguillarum flagellin A (FlaA) using yeast two-hybrid and pull-down assays. MjShoc2 plays a role in antibacterial response by mediating the FlaA-induced expression of certain antibacterial effectors, including lectin and antimicrobial peptide. FlaA challenge, via MjShoc2, led to phosphorylation of extracellular regulated kinase (Erk), and the subsequent activation of signal transducer and activator of transcription (Stat), ultimately inducing the expression of effectors. Therefore, by establishing the FlaA/MjShoc2/Erk/Stat signaling axis, this study revealed a new antibacterial strategy in shrimp, and provides insights into the flagellin sensing mechanism in invertebrates.

Evaluation of immune stimulatory products for whiteleg shrimp (Penaeus vannamei) by a metabolomics approach.

The use of probiotics, prebiotics and dietary fiber has become a common practice in shrimp aquaculture as alternatives to antibiotic treatment. However, not much is known about the metabolic mechanisms underlying the effects of probiotics and immunostimulant used in shrimp aquaculture. In this study, a gas chromatography-mass spectrometry (GC-MS) based metabolomics approach was used to characterize metabolite profiles of haemolymph and gills of whiteleg shrimp (Penaeus vannamei) exposed to four treatments (cellulose fiber, probiotics with Vibrio alginolyticus, a combination of cellulose fiber and V. alginolyticus and a control treatment). The cellulose fiber was administrated as a feed additive (100 mg⋅Kg-1 feed), while the probiotics was applied in the water (105 UFC⋅mL-1 culture water). The results showed significant differences in haemolymph metabolite profiles of immune stimulated treatments compared to the control and among treatments. The combination of cellulose fiber and probiotics resulted in greater differences in metabolic profiles, suggesting a better immune stimulation with this approach. The changes in haemolymph metabolome of treated shrimp reflected several biochemical pathway modifications, including changes in amino acid and fatty acid metabolism, disturbances in energy metabolism and antimicrobial activity and stress responses. For gill tissues, significant differences were only found in lactic acid between the probiotic group and the control. Among the altered metabolites, the increases of itaconic acid in haemolymph, and lactic acid in both haemolymph and gill tissues of immune-stimulated suggest the potential use of these metabolites as biomarkers for health assessment in aquaculture.

Effects of Lactobacillus pentosus combined with Arthrospira platensis on the growth performance, immune response, and intestinal microbiota of Litopenaeus vannamei.

Litopenaeus vannamei is one of the most productive shrimp species in the world. However, shrimp farming is suffering from adverse environmental conditions and disease outbreaks. Typically, Lactobacillus pentosus and Arthrospira platensis are used as substitutes for some antibiotics. In the present study, we assessed the effects of dietary supplements along with living bacteria or cell-free extracts of L. pentosus combined with A. platensis on the growth performance, immune response, intestinal microbiota, and disease resistance of L. vannamei against Vibrio alginolyticus. Shrimp fed L. pentosus live bacteria combined with A. platensis showed the best growth performance and lowest feed conversion rate. The supplementation diet with L. pentosus live bacteria and A. platensis could significantly enhance the trypsin activity in shrimp after the feeding trial. Given the lowest feed conversion rate in shrimp fed L. pentosus live bacteria combined with A. platensis, we reasonably speculated that the decrease in feed conversion rate may be related to the increase in trypsin activity. In addition, dietary cell-free extracts of L. pentosus combined with A. platensis enhanced the expression of immune-related genes after the feeding trial or challenge test. Moreover, results of the bacterial challenge test indicated that the shrimp fed cell-free extracts of L. pentosus combined with A. platensis diet resulted in the highest survival rate, which suggested that cell-free extracts of L. pentosus and A. platensis could improve the disease resistance against V. alginolyticus by up-regulating the expressions of immune-related genes. Dietary L.pentosus or A. platensis, or their combination, reduced the abundance of harmful bacteria, including Proteobacteria in shrimp intestine, which suggested that L. pentosus and A. platensis could improve the growth performance and health of shrimp by regulating the structure of the intestinal microbiota. The findings of this study demonstrated that L. pentosus live bacteria and A. platensis exerted synergistic effects on the growth performance and digestion in shrimp, while cell-free extracts of L. pentosus and A. platensis showed synergistic effects on the immune response and disease resistance of shrimp against V. alginolyticus.

Transcriptome profiling reveals the novel immunometabolism-related genes against WSSV infection from Fenneropenaeus merguiensis.

The white spot syndrome virus (WSSV) has been considered a serious threat to shrimp aquaculture. Besides, the activation of cell metabolism as an immune reaction to the virus is now recognized as a piece of the pivotal puzzle of the antiviral responses. Hence, this study explores the relationship between metabolic gene expression and antiviral responses in shrimp using transcriptome analysis. The RNA-seq libraries of Fenneropenaeus merguensis hemocytes after WSSV challenge at early (6 hpi) and late (24 hpi) stages of infection were analyzed to identify differentially expressed genes (DEGs) that the WSSV subverted the expression. One-hundred-thirty-three DEGs that were expressed in response to WSSV infection at both stages were identified. Based on the GO annotation, they were related to innate immunity and metabolic pathway. The expression correlation between "full term" (NGS) and qRT-PCR of 16 representative DEGs is shown. Noticeably, the expression profiles of all the selected metabolic genes involved in glucose metabolism, lipid metabolism, amino acid metabolism, and nucleotide metabolism showed a specific correlation between NGS and qRT-PCR upon WSSV infection. Of these, we further characterized the function related to the WSSV response of glutamine: fructose-6-phosphate aminotransferase (FmGFAT), the rate-limiting enzyme of the hexosamine biosynthesis pathway, which was found to be up-regulated at the late stage of WSSV infection. Suppression of FmGFAT by RNA interference resulted in postponing the death of WSSV-infected shrimp and reduction of viral copy number. These results suggested that the FmGFAT is linked between metabolic change and WSSV responses in shrimp, where the virus-induced metabolic rewiring hijack biological compounds and/or energy sources to benefit the viral replication process.

Shrimp Vago5 activates an innate immune defense upon bacterial infection.

Acute hepatopancreatic necrosis disease, AHPND, caused by a specific Vibrio parahaemolyticus (VPAHPND) strain, results in a great loss of global shrimp production. This study performed suppression subtractive hybridization (SSH) to identify differentially expressed genes from white shrimp Penaeus vannamei hemocyte upon VPAHPND infection. Among the immune-related genes identified, Vago5, kunitz, secretory leukocyte proteinase inhibitor, and profilin are the most abundant genes classified as the up-regulated genes in the SSH library. The qRT-PCR results show that only Vago5 was highly up-regulated at 3 and 6 h post-VPAHPND challenge, whereas kunitz, secretory leukocyte proteinase inhibitor, and profilin were highly up-regulated at 48 h post-VPAHPND challenge. As an early VPAHPND infection-responsive gene, Vago5 was further functional characterized by RNA interference. Knockdown of Vago5 gene resulted in the significantly rapid increase of shrimp mortality and the number of bacteria in the stomach and hepatopancreas upon VPAHPND infection. Moreover, downstream genes of Toll, IMD, and JAK/STAT pathways and phenoloxidase system were analyzed for the expression in the VPAHPND-infected shrimp hemocyte after dsVago5 treatment. Vago5 gene knockdown resulted in a significant decrease in transcript levels of PEN4, TNF, and PO2 genes as well as PO activity in the hemolymph, suggesting that Vago5 might modulate antibacterial infection through activation of the genes in the NF-κB mediated pathways, JAK/STAT pathway, and phenoloxidase system.

Process optimization for fermented siwu decoction by multi-index-response surface method and exploration of the effects of fermented siwu decoction on the growth, immune response and resistance to Vibrio harveyi of Pacific white shrimp (Litopenaeus vannamei).

The purpose of this study was to explore the optimal fermentation technology of Chinese herbal medicine formula-Siwu Decoction and the effects of fermented Siwu Decoction (FSW) on the growth performance, immune response, intestinal microflora and anti microbial ability of Litopenaeus vannamei. Response to surface methodology (RSM) was used to optimize the fermentation process of Siwu Decoction. The optimal fermentation conditions were obtained as follows: inoculation amount of mixed strains was 4.5%, fermentation time was 36 h, and the ratio of material to liquid was 20%. A total of 1260 shrimps were selected and divided into seven groups, three in parallel in each group. The dietary level of each group was as follows: Control （No additions）, USW1 (0.2% unfermented herbal medicine), USW2 (0.5% unfermented herbal medicine), USW3 (0.8% unfermented herbal medicine), FSW1 (0.2% fermented herbal medicine), FSW2 (0.5% fermented herbal medicine), FSW3 (0.8% fermented herbal medicine). The immune response and antioxidant defense ability of hemocytes and intestine were measured at 21 and 42 days of feeding and the intestinal flora and growth performance were measured at 42 days of feeding, after that, a 7-day challenge test against Vibrio harveyi was conducted. The results showed that fermented Siwu Decoction significantly improved the growth performance and body composition of Litopenaeus vannamei; significantly increased the total number of hemocytes, phagocytic activity, antibacterial activity and bacteriolytic activity of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei; the addition of fermented Siwu Decoction significantly increased the gene expression level of hemocytes and intestinal tract of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei. The abundance of Bacillus increased, while the abundance of Vibrio decreased. After Vibrio harveyi challenge, the cumulative mortality of FSW group was significantly lower than that of control group. Fermented Siwu Decoction may be a potential physiological enhancer in aquaculture, and can be widely used in aquaculture.

Phenylalanine hydroxylase (PAH) plays a positive role during WSSV and Vibrio parahaemolyticus infection in Litopenaeus vannamei.

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei, with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l-DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l-DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Nitric Oxide Synthase Regulates Gut Microbiota Homeostasis by ERK-NF-κB Pathway in Shrimp.

The gut microbiota is a complex group of microorganisms that is not only closely related to intestinal immunity but also affects the whole immune system of the body. Antimicrobial peptides and reactive oxygen species participate in the regulation of gut microbiota homeostasis in invertebrates. However, it is unclear whether nitric oxide, as a key mediator of immunity that plays important roles in antipathogen activity and immune regulation, participates in the regulation of gut microbiota homeostasis. In this study, we identified a nitric oxide synthase responsible for NO production in the shrimp Marsupenaeus japonicus. The expression of Nos and the NO concentration in the gastrointestinal tract were increased significantly in shrimp orally infected with Vibrio anguillarum. After RNA interference of Nos or treatment with an inhibitor of NOS, L-NMMA, NO production decreased and the gut bacterial load increased significantly in shrimp. Treatment with the NO donor, sodium nitroprusside, increased the NO level and reduced the bacterial load significantly in the shrimp gastrointestinal tract. Mechanistically, V. anguillarum infection increased NO level via upregulation of NOS and induced phosphorylation of ERK. The activated ERK phosphorylated the NF-κB-like transcription factor, dorsal, and caused nuclear translocation of dorsal to increase expression of antimicrobial peptides (AMPs) responsible for bacterial clearance. In summary, as a signaling molecule, NOS-produced NO regulates intestinal microbiota homeostasis by promoting AMP expression against infected pathogens via the ERK-dorsal pathway in shrimp.

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.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei.

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC's diversity and multiple immune activities.

Effects of Dietary Mannan Oligosaccharides on Non-Specific Immunity, Intestinal Health, and Antibiotic Resistance Genes in Pacific White Shrimp Litopenaeus vannamei.

This study was conducted to comprehensively investigate the beneficial effects of a mannan oligosaccharide product (hereinafter called MOS) on Litopenaeus vannamei and optimum level of MOS. Five isonitrogenous and isolipid diets were formulated by adding 0%, 0.02%, 0.04%, 0.08%, and 0.16% MOS in the basal diet. Each diet was randomly fed to one group with four replicates of shrimp in an 8-week feeding trial. The results showed that dietary MOS improved the growth performance and the ability of digestion of shrimp. Dietary MOS significantly increased the activity of total superoxide dismutase, catalase, and glutathione peroxidase and decreased the content of malondialdehyde in plasma of shrimp. Dietary MOS significantly increased the activity of alkaline phosphatase and lysozyme in plasma and the hemocyte counts. Dietary MOS significantly upregulated the expression of Toll, lysozyme, anti-lipopolysaccharide factor, Crustin, and heat shock protein 70 in the hepatopancreas. And dietary MOS significantly upregulated the expression of intestinal mucin-2, mucin-5B, and mucin-19, while it decreased the expression of intestinal mucin-1 and macrophage migration inhibitory factor. Dietary MOS improved the bacterial diversity; increased the abundance of Lactobacillus, Bifidobacterium, Blautia, and Pseudoalteromonas; and decreased the abundance of Vibrio in the intestine. Shrimp fed MOS diets showed lower mortality after being challenged by Vibrio parahaemolyticus. Notably, this study found a decrease in antibiotic resistance genes and mobile genetic elements after MOS supplementation for the first time. The present results showed that diet with MOS supplementation enhanced the organismal antioxidant capacity and immunity, improved intestinal immunity, optimized intestinal microecology, mitigated the degree of antibiotic resistance, and increased the resistance to V. parahaemolyticus in L. vannamei, especially when supplemented at 0.08% and 0.16%.