Impact of dietary administration of Arthrospira platensis free-lipid biomass on growth performance, body composition, redox status, immune responses, and some related genes of pacific whiteleg shrimp, Litopenaeus vannamei.

The current study aimed to assess the influence of dietary inclusion of cyanobacterium Arthrospira platensis NIOF17/003 as a dry material and as a free-lipid biomass (FL) on the growth performance, body composition, redox status, immune responses, and gene expression of whiteleg shrimp, Litopenaeus vannamei postlarvae. L. vannamei were fed five different supplemented diets; the first group was fed on an un-supplemented diet as a negative control group (C-N), the second group was fed on a commercial diet supplemented with 2% of A. platensis complete biomass as a positive control group (C-P20), whereas, the three remaining groups were fed on a commercial diet supplemented with graded amounts of FL at 1%, 2%, and 3% (FL10, FL20, and FL30, respectively). The obtained results indicated that the diet containing 1% FL significantly increased the growth performance, efficiency of consumed feed, and survival percentage of L. vannamei compared to both C-N and C-P20 groups. As for the carcass analysis, diets containing A. platensis or its FL at higher levels significantly increased the protein, lipid, and ash content compared to the C-N group. Moreover, the shrimp group fed on C-P20 and FL10 gave significantly stimulated higher digestive enzyme activities compared with C-N. The shrimp fed C-P20 or FL exhibited higher innate immune responses and promoted their redox status profile. Also, the shrimp fed a low FL levels significantly upregulated the expression of both the peroxiredoxin (Prx) and prophenoloxidase (PPO1) genes than those receiving C-N. The current results recommended that dietary supplementation with 1% FL is the most effective treatment in promoting the performance and immunity of whiteleg shrimp.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis.

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight ß-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Nucleic Acid Sensing by STING Induces an IFN-like Antiviral Response in a Marine Invertebrate.

The cytosolic detection of pathogen-derived nucleic acids has evolved as an essential strategy for host innate immune defense in mammals. One crucial component in this process is the stimulator of IFN genes (STING), which acts as a vital signaling adaptor, connecting the cytosolic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) to the downstream type I IFN signaling pathway. However, this process remains elusive in invertebrates. In this study, we present evidence demonstrating that STING, an ortholog found in a marine invertebrate (shrimp) called Litopenaeus vannamei, can directly detect DNA and initiate an IFN-like antiviral response. Unlike its homologs in other eukaryotic organisms, which exclusively function as sensors for cyclic dinucleotides, shrimp STING has the ability to bind to both double-stranded DNA and cyclic dinucleotides, including 2'3'-cGAMP. In vivo, shrimp STING can directly sense DNA nucleic acids from an infected virus, accelerate IFN regulatory factor dimerization and nuclear translocation, induce the expression of an IFN functional analog protein (Vago4), and finally establish an antiviral state. Taken together, our findings unveil a novel double-stranded DNA-STING-IKKε-IRF-Vago antiviral axis in an arthropod, providing valuable insights into the functional origins of DNA-sensing pathways in evolution.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

BACKGROUND: Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. METHODS: Epithelial activation and/or barrier effects upon exposure to 2-50 µg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 µg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. RESULTS: Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. CONCLUSIONS: Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Integrated analysis of intestinal microbiota and transcriptome reveals that a coordinated interaction of the endocrine, immune system and gut microbiota response to heat stress in Litopenaeus vannamei.

Due to the ongoing global warming, the risk of heatwaves in the oceans is continuously increasing while our understanding of the physiological response of Litopenaeus vannamei under extreme temperature conditions remains limited. Therefore, this study aimed to evaluate the physiological responses of L. vannamei under heat stress. Our results indicated that as temperature rose, the structure of intestinal and hepatopancreatic tissues was damaged sequentially. Activity of immune-related enzymes (acid phosphatase/alkaline phosphatase) initially increased before decreased, while antioxidant enzymes (superoxide dismutase and glutathione-S transferase) activity and malondialdehyde content increased with rising temperature. In addition, the total antioxidant capacity decreased with rising temperature. With the rising temperature, there was a significant increase in the expression of caspase-3, heat shock protein 70, lipopolysaccharide-induced tumor necrosis factor-α, transcriptional enhanced associate domain and yorkie in intestinal and hepatopancreatic tissues. Following heat stress, the number of potentially beneficial bacteria (Rhodobacteraceae and Gemmonbacter) increased which maintain balance and promote vitamin synthesis. Intestinal transcriptome analysis revealed 852 differentially expressed genes in the heat stress group compared with the control group. KEGG functional annotation results showed that the endocrine system was the most abundant in Organismal systems followed by the immune system. These results indicated that heat stress leads to tissue damage in shrimp, however the shrimp may respond to stress through a coordinated interaction strategy of the endocrine system, immune system and gut microbiota. This study revealed the response mechanism of L. vannamei to acute heat stress and potentially provided a theoretical foundation for future research on shrimp environmental adaptations.

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.

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.

Shrimp Parvovirus Circular DNA Fragments Arise From Both Endogenous Viral Elements and the Infecting Virus.

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

Probiotic, Lactobacillus pentosus BD6 boost the growth and health status of white shrimp, Litopenaeus vannamei via oral administration.

This study aims to assess and determine the oral-administration of probiotic, Lactobacillus pentosus BD6 on growth performance, immunity and disease resistance of white shrimp, Litopenaeus vannamei. Lac. pentosus BD6 effectively inhibited the growth of aquatic pathogens, which was used in the test. Shrimp were fed with the control diet (without probiotic supplement) for 60 days and the probiotic-containing diets at 107, 108, 109, and 1010 cfu kg-1, respectively. Shrimp fed with the diet containing probiotic at the doses of 109-10 cfu kg-1 showed significant increase in growth performance as well as feed efficiency than that of the control. After a challenge test with Vibrio alginolyticus, shrimp fed with a probiotic diet at a dose of 1010 cfu kg-1 showed a significantly lower mortality as compared to the control and that of shrimp fed the diet containing probiotic at the levels up to 107-8 cfu kg-1. In addition, a therapeutic potential of Lac. pentosus BD6 was discovered because the cumulative mortalities of shrimp fed with probiotic and pathogen V. parahaemolyticus simultaneously were significantly lower when compared to control shrimp. Probiotic in diet at a dose of 109-10 cfu kg-1 significantly increased PO activity of shrimp, while shrimp receiving probiotic at the doses of 108-10 cfu kg-1 showed significant increase in lysozyme activity and phagocytic activity. Shrimp fed with the diet containing probiotic at the level of 1010 cfu kg-1 also indicated higher gene expression of prophenoloxidase (proPO) I, but not proPO II, lipopolysaccharide and ß-1,3-glucan-binding protein and penaeidin 4. Analysis of the bacterial microbiota of the shrimp intestine revealed that oral administration of probiotic increased the relative abundance of beneficial bacteria and reduced the abundance of harmful pathogenic bacteria in the gut flora of shrimp. Despite no statistically significant difference, an analysis of microbial diversity recorded higher species richness, Shannon-Weaver diversity index and evenness in the probiotic group, compared to the control group. It was concluded that Lac. pentosus BD6 has great antibacterial ability against a wide range of pathogens and has therapeutic potential to reduce the mortality of shrimp infected with V. parahaemolyticus. Additionally, dietary Lac. pentosus BD6 at the level of 1010 cfu kg-1 was recommended to improve growth performance, immunity and disease resistance of shrimp against V. alginolyticus.

Identification and function of an Arasin-like peptide from Litopenaeus vannamei.

Antimicrobial peptides (AMPs) play an important role in the host defense system of shrimps. In this study, an Arasin-like peptide, named as LvArasin-like, was identified from the hemocytes of the pacific white shrimp, Litopenaeus vannamei. The complete open reading frame (ORF) of LvArasin-like was 213 bp, encoding 70 amino acid residues with a predicted molecular mass of 5.68 kDa and a theoretical isoelectric point (pI) of 6.73. The predicted peptide consisted of a signal peptide, an N-terminal Pro/Arg-rich domain, and a C-terminal cysteine-rich domain. LvArasin-like expression was most abundant in the gills and was up-regulated in hemocytes after LPS or Poly I:C injection as well as challenges by Vibrio parahaemolyticus or Staphylococcus aureus infection. In the heterologous expression system, LvArasin-like protein (rLvArasin-like) was recombinantly expressed in the forms of a dimer or both a monomer and dimer. The rLvArasin-like could directly bind to gram-positive and gram-negative bacteria and exhibited broad-spectrum antimicrobial activity towards them, with 50 % of minimal inhibitory concentrations (MIC50) of 6.25-50 µM. Moreover, dsRNA-mediated knockdown of LvArasin-like enhanced the susceptibility of shrimp to V. parahaemolyticus. In addition, the transcriptional level of LvArasin-like was downregulated when silencing of the transcription factors LvDorsal and LvRelish using RNAi in vivo. All of these results suggest that LvArasin-like is involved in host defense against bacterial infection. Therefore, it is a potential therapeutic agent for disease control in shrimp aquaculture.

Therapeutic effect and immune mechanism of chitosan-gentamicin conjugate on Pacific white shrimp (Litopenaeus vannamei) infected with Vibrio parahaemolyticus.

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.

Characterization of Litopenaeus vannamei secreted protein acidic and rich in cysteine -like in WSSV infection.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular and non-structural glycoprotein. In shrimp, a significant function of SPARC in WSSV infection remains unclear. In this study, the full-length cDNA sequence of a secreted protein acidic and rich in cysteine -like was cloned from shrimp Litopenaeus vannamei (named as LvSPARC-L). LvSPARC-L contained an open reading frame of 1002 bp, encoding 333 amino acids. Bioinformatics analysis showed that LvSPARC-L contained a SPARC Ca2+-binding region in the C-terminus, a Kazal-type serine protease inhibitor domain and a BUD22 domain. Tissue distribution assay indicated that LvSPARC-L generally expressed in all tissues selected with a higher expression in hemocyte, stomach and pleoplod. In hepatopancreas and intestine, the relative expression of LvSPARC-L was significantly up-regulated following the WSSV challenge. Besides, the relative expression of viral immediately early gene IE1 and a late gene VP28 was significantly increased in the LvSPARCL-silenced shrimp. Furthermore, the relative expression of LvP53 and LvCaspase3 was extremely decreased in the stomach of dsLvSPARC-L treated shrimp, while that of LvP38 was not affected significantly. All data together suggest that LvSPARC-L might play an antiviral role by regulating apoptosis.

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

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

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon.

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

LvCSN5 is involved in WSSV infection via interaction with wsv006.

As an extremely virulent pathogen, white spot syndrome virus (WSSV) greatly threatens shrimp aquaculture worldwide. The interaction between virus and host is important for viral infection. In the present study, a yeast two-hybrid (Y2H) library was constructed to clarify the functions of wsv006, and the interaction between wsv006 and shrimp Litopenaeus vannamei (L. vannamei) was analyzed. Furthermore, we explored the role of the wsv006-interacting molecule L. vannamei COP9 constitutive photomorphogenic-like protein subunit 5 (LvCSN5) in WSSV infection. Y2H assay showed that wsv006 interacted with LvCSN5, and co-immunoprecipitation (Co-IP) assay confirmed such interaction. Multiple alignments of amino acid sequences with other species revealed that the LvCSN5 had high identity with Penaeusmonodon CSN5 (PmCSN5). LvCSN5 was mainly expressed in intestine, eye and hepatopancreas. In addition, the relative expression of LvCSN5 was significantly up-regulated both in intestine and hepatopancreas following the WSSV challenge. Besides, the relative expressions of IE1 and VP28, as well as the viral copy numbers were significantly increased in the LvCSN5-silenced shrimp. Our findings suggested that LvCSN5 was involved in WSSV infection by interacting with wsv006.

F1 ATP synthase ß subunit is a putative receptor involved in white spot syndrome virus infection in shrimp by binding with viral envelope proteins VP51B and VP150.

White spot syndrome virus (WSSV) is highly virulent toward shrimp, and F1 ATP synthase ß subunit (ATPsyn-ß) has been suggested to be involved in WSSV infection. Therefore, in this study, interactions between Penaeus monodon ATPsyn-ß (PmATPsyn-ß) and WSSV structural proteins were characterized. Based on the results of yeast two-hybrid, co-immunoprecipitation, and protein pull-down assays, WSSV VP51B and VP150 were identified as being able to interact with PmATPsyn-ß. Membrane topology assay results indicated that VP51B and VP150 are envelope proteins with large portions exposed outside the WSSV virion. Cellular localization assay results demonstrated that VP51B and VP150 co-localize with PmATPsyn-ß on the membranes of transfected cells. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA results demonstrated that VP51B and VP150 bound to PmATPsyn-ß in a dose-dependent manner, which could be competitively inhibited by the addition of WSSV virions. In vivo neutralization assay results further showed that both recombinant VP51B and VP150 could delay mortality in shrimp challenged with WSSV.

A Litopenaeus vannamei TRIM32 gene is involved in oxidative stress response and innate immunity.

Tripartite motif (TRIM) family proteins are named by the presence of tripartite motifs in their amino terminal domains. Apart from the amino terminal, their carboxyl terminal contain variable domains which mediate diverse functions of the TRIM proteins. It had been found that TRIM proteins played important roles in distinct biological processes, such as innate immunity, anti-tumor immunity, cell cycle regulation and so on. In the present study, we cloned a TRIM32 (LvTRIM32) gene from Litopenaeus vannamei. LvTRIM32 was highly expressed in hemocytes, gills and epidermis, and subcellular localization analysis indicated that it was widely distributed in S2 cells. In vitro ubiquitination assays indicated that LvTRIM32 had E3 ubiquitin ligase activity. Results of real-time RT-PCR assay showed that LvTRIM32 was induced in shrimp hemocytes upon oxidative stress. It was also proved that the promoter activity of LvTRIM32 was enhanced by NF-E2-related factor, and knocked-down expression of LvTRIM32 depressed the expression of malic enzyme and epoxide hydrolase. Downregulated LvTRIM32 suppressed the cumulative mortality of shrimp under oxidative stress. Moreover, it was found that LvTRIM32 could be induced in shrimp hemocytes upon immunostimulation, and downregulated LvTRIM32 increased the cumulative mortality of shrimp infected with white spot syndrome virus (WSSV) or Vibrio alginolyticus. Collecting results suggested that LvTRIM32 was a member of shrimp antioxidant stress system, and it was also involved in WSSV- or V. alginolyticus-infection resistance.

Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense.

Activating transcription factor 2 (ATF2), a member of the bZIP transcription factor family, is involved in multiple physiological and developmental processes, yet its role in the innate immunity remains unclear. In this study, two isoforms (named as MnATF2a and MnATF2b) of ATF2 gene were identified in Macrobrachium nipponense and were produced by exon skipping. The full length of MnATF2a is 2328 bp with an open reading frame of 2079 bp that encode 692 amino acids. MnATF2a has 237 bp nucleotides more than MnATF2b and the extra 237 bp is a complete exon. MnATF2a and MnATF2b proteins contain the same conserved and typical bZIP domain at the C-terminus. MnATF2a has 79 amino acids more than MnATF2b. MnATF2a and MnATF2b are widely distributed in a variety of immune tissues. After Vibrio parahaemolyticus and Staphylococcus aureus infection, the expression levels of MnATF2a and MnATF2b were significant up-regulated in the gills and stomach at 12 h. RNA interference analysis showed that knockdown of the total MnATF2 gene significantly inhibits the transcription of tumor necrosis factor (TNF) and promotes the expression of crustins (including Cru3, Cru4, and Cru7). Further study showed that knockdown of MnTNF evidently increase the expression of Cru3, Cru4, and Cru7. Our research indicates that ATF2 negatively regulate the expression of AMPs by regulating the transcription of TNF in M. nipponense. This study provides valuable information about the function of ATF2 family in the innate immunity in crustacean.

Effects of biogenic amines on the immune response and immunoregulation mechanism in hemocytes of Litopenaeus vannamei in vitro.

The effects of three biogenic amines (DA, NE and 5-HT) on the immune signaling pathway and immune response of hemocytes in shrimp were investigated through in vitro experiments. The results showed that the G protein effectors (AC, PLC), the second messengers (cAMP, DAG), Calmodulin (CaM) and protein kinases (PKA, PKC) of DA and NE groups shared a similar trend in which all intracellular signaling factors increased significantly and reached the maximum at 3 h. The concentrations of AC, cAMP and PKA in 5-HT groups decreased significantly compared with the control group, while the concentrations of PLC, CaM, DAG and PKC in 5-HT groups increased markedly. The immune parameters such as total hemocyte count (THC), cell viability, antibacterial activity and bacteriolytic activity, as well as prophenoloxidase (proPO) activity in three biogenic amines groups decreased significantly, while the phenoloxidase (PO) activity increased significantly. The phagocytic activity in DA and NE groups decreased significantly, while that in 5-HT groups increased markedly and reached the highest level at 1 h. Among these three biogenic amines, DA showed the strongest effect on the immune activity of the hemocytes, whereas 5-HT had the least effect. In addition, we speculated that DA and NE might regulate phagocytosis by activating intracellular AC-cAMP-PKA pathway while 5-HT might inhibit intracellular AC-cAMP-PKA pathway. Moreover, the activation of proPO system might be related to PLC-DAG-PKC and PLC-CaM pathway.

TNF-Receptor-Associated Factor 3 in Litopenaeus vannamei Restricts White Spot Syndrome Virus Infection Through the IRF-Vago Antiviral Pathway.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.