Shrimp Innate Immunity Research Articles

Shrimp Intestinal Microbiota Homeostasis: Dynamic Interplay Between the Microbiota and Host Immunity

ABSTRACTThe shrimp intestine harbors a microbiota that has pivotal roles for host's physiology. Imbalance of shrimp intestinal microbiota has been shown closely related to the occurrence of diseases. The morphological and biological features of the shrimp intestine are considered suboptimal for stable microbial colonization, making the intestinal microbiota composition highly susceptible to the impact of environmental changes or stressors, and particularly unstable. Therefore, the relative unsteadiness of the microbiota composition represents a continuous threat to host survival. Shrimp intestinal microbiota homeostasis is achieved through a dynamic interplay between the microbiota and the host's innate immunity. The shrimp intestine possesses effective innate immune mechanisms that can suppress the uncontrolled proliferation of microbiota components, and simultaneously protect the microbiota from elimination. The mechanism(s) by which the microbial components and the intestinal innate immunity interact with each other to achieve homeostasis represents an interesting interplay between host and microbiota. This review summarizes the current knowledge about intestinal microbiota colonization in shrimp, as well as the intricate mechanisms employed by the intestinal immune system to regulate this microbiota. Moreover, the potential intervention strategies to promote and protect shrimp intestinal homeostasis by modulating the microbiota are also discussed. Thus, this review seeks to comprehensively analyze the current information and contribute to a deeper understanding of the complex interplay between the shrimp intestinal microbiota and innate immunity in maintaining shrimp intestinal homeostasis and overall health. This enhanced understanding may potentially open new avenues for aquaculture management and disease mitigation strategies, ultimately benefiting the shrimp farming industry.

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 β-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.

Characterization of a novel Type-I Crustin (carcininPm2) from black tiger shrimp Penaeus monodon

Carcinins are type-I crustins from crustaceans and play an important role in innate immune system. In this study, type-I crustins, carcininPm1 and carcininPm2, from the hemocytes of Penaeus monodon were identified. Comparison of their amino acid sequences and the phylogenetic tree revealed that they were closely related to the other crustacean carcinin proteins, but were clustered into different groups of the carcinin proteins. The full-length amino acids of carcininPm1 and carcininPm2 were 92 and 111 residues, respectively. CarcininPm1 and carcininPm2 were expressed mainly in hemocytes and intestine compared to the other tissues. The expression of carcininPm1 and carcininPm2 were dramatically increased in early time of bacterial challenged shrimp hemocytes. In contrast, the carcininPm1 and carcininPm2 were expressed in response to late state of YHV-infected shrimp hemocytes where the copy number of virus was high. The recombinant carcininPm2 (rcarcininPm2) but not its WAP domain (rcarcininPm2_WAP) exhibited antimicrobial activity against Vibrio harveyi and Vibrio parahaemolyticus AHPND but not other bacteria tested. The rcarcininPm2 was able to prolong the survival rate of VH-treated post larval shrimp from about 102 h to 156 h. These studies indicated that the carcininPm2 possessed the potential and challenges as antibacterial in innate immunity of shrimp.

Identification of an Ortholog of MALT1 from Shrimp That Induces NF-κB-Mediated Antiviral Immunity.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) serves as a pivotal mediator for NF-κB activation in response to a wide spectrum of transmembrane receptor stimuli. In the present study, a homolog of MALT1, named LvMALT1, is cloned from the Pacific white shrimp (Litopenaeus vannamei) and its potential function in shrimp innate immunity is explored. The open reading frame of LvMALT1 is 2364 bp that encodes 787 amino acids. The predicted LvMALT1 protein structure comprises a death domain, three immunoglobulin domains, and a caspase-like domain, exhibiting remarkable similarity to other homologs. LvMALT1 is a cytoplasmic-localized protein and could interact with LvTRAF6. Overexpression of LvMALT1 induces the activation of promoter elements governing the expression of several key antimicrobial peptides (AMPs), including penaeidins (PENs) and crustins (CRUs). Conversely, silencing of LvMALT1 leads to a reduction in the phosphorylation levels of Dorsal and Relish, along with a concomitant decline in the in vivo expression levels of multiple AMPs. Furthermore, LvMALT1 is prominently upregulated in response to a challenge by the white spot syndrome virus (WSSV), facilitating the NF-κB-mediated expression of AMPs as a defense against viral infection. Taken together, we identified a MALT1 homolog from the shrimp L. vannamei, which plays a positive role in the TRAF6/NF-κB/AMPs axis-mediated innate immunity.

The transcriptome of Litopenaeus vannamei in zoea larvae and adults infected by Vibrio parahaemolyticus

IntroductionThe rapid development of the Litopenaeus vannamei industry largely depends on the acquisition of high-quality larvae. Shrimp larval development is a unique metamorphic process that results in mortality due to disease vulnerability. Currently, studies on innate immunity in shrimp are mainly focused on the adult stage, but little has been done on innate immunity in larvae.MethodsIn this study, utilizing a high-throughput Illumina sequencing approach, we compare the transcriptome of L. vannamei Zoea larvae and adults infected by Vibrio parahaemolyticus.ResultsA total of 487,887,650 high quality reads were obtained, assembled, and aggregated into 23,802 genes, among which 3315 were identified as differentially expressed genes. Then GO term enrichment analysis, KEGG pathway enrichment analysis and functional gene analysis were performed. We found that pathways that are involved in the innate immune response, such as mTOR signaling pathway, MAPK signaling pathway, and Notch signaling pathway, were more enriched in the Zoea stage compared to the adult stage, suggesting that innate immunity against Vibrio vulnificus may exist during the Zoea larvae stage.Discussionwere more enriched in the Zoea stage compared to the adult stage, suggesting that innate immunity against Vibrio vulnificus may exist during the Zoea larvae stage. These findings will provide useful information for the study of innate immunity in shrimp larvae.

Investigating host-gut microbial relationship in Penaeus monodon upon exposure to Vibrio harveyi

To understand the host-gut microbial relationship, we used multidisciplinary platforms, metagenome, transcriptome, and metabolome analyses, to determine shrimp and intestinal microbial interactions upon 0, 6, 12, 24, and 48 h exposure to 107 CFU/mL of Vibrio harveyi, a shrimp pathogen. The bacterial communities in the intestine of shrimp were disrupted during exposure to V. harveyi. The abundance of Vibrio ASVs in the Harveyi and Vulnificus clades was significantly increased after exposure to the pathogen (6, 12, and 24 h) and decreased later after 48 h. Conversely, Pseudoalteromonas was found in lower abundance (6, 12, and 24 h) but significantly increased after 48 h of the bacterial challenge. Gene expression analysis revealed that genes belonging to several immune-related pathways, including the Toll pathway, the immune deficiency (IMD) pathway, and pattern recognition proteins (PRPs), were significantly upregulated. Early responses in the first 6 h after exposure were the genes involved in phagocytosis, pattern recognition proteins (PRPs), and signal transduction (spätzle and ankyrin). Late responses (12–48 h) were genes related to proteinases and proteinase inhibitors (PPIs), antimicrobial peptides (AMPs), and oxidative stress. Genes related to lipid metabolisms such as fatty acid metabolism and choline metabolism were also upregulated. Metabolomics analysis also showed an increase in phospholipids, including phosphocholine groups, in the intestine of shrimp exposed to the pathogen. Taken together, the gene expression and metabolomics analyses suggest the importance of lipid metabolism in the defense mechanism against bacterial invasion. Moreover, our metabolomic analysis showed a decrease in tryptophan and indole-3-acrylic acid, metabolites related to intestinal immune homeostasis, after bacterial infection. Our observations suggest that pathogenic Vibrio disrupted biological processes in the shrimp intestine, resulting in a decrease in indole-3-acrylic acid and its derivatives which in turn compromised intestinal immunity. In addition, shrimp responded to the bacterial invasion by activating metabolites related to eicosanoid and phospholipid biosynthesis. Our findings on the interactions between shrimp and intestinal microbiota will be an indispensable gateway to systems biology to better understand the function of shrimp gut microbiota and its influence on shrimp innate immunity.

Taxifolin Inhibits WSSV Infection and Transmission by Increasing the Innate Immune Response in Litopenaeus vannamei.

An outbreak of white spot syndrome virus (WSSV) can hit shrimp culture with a devastating blow, and there are no suitable measures to prevent infection with the virus. In this study, the activity of active molecules from Chinese herbs against WSSV was evaluated and screened. Taxifolin had the highest rate (84%) of inhibition of the WSSV infection. The viral infectivity and genome copy number were reduced by 41% when WSSV virion was pretreated with taxifolin prior to shrimp infection. A continuous exchange of taxifolin significantly reduced the mortality of shrimp infected with WSSV. Due to the WSSV virion infectivity being affected by taxifolin, the horizontal transmission of the virus was blocked with an inhibition rate of up to 30%, which would further reduce the cost of a viral outbreak. Additionally, the viral genome copy number was also reduced by up to 63% in shrimp preincubated in taxifolin for 8 h. There may be a connection to the enhancement of innate immunity in shrimp that resulted in a 15% reduction in mortality for taxifolin-fed shrimp after the WSSV challenge. After dietary supplementation with taxifolin, the resistance of larvae to WSSV was improved, indicating that taxifolin may be a potential immunostimulant for shrimp to prevent WSD. Therefore, the results indicate that taxifolin has application potential for blocking a WSSV outbreak and reducing the loss of shrimp culture.

Effects of dietary supplementation of chamomile oil on Indian shrimp (Penaeus indicus) performance, antioxidant, innate immunity, and resistance to Vibrio parahaemolyticus infection

The stimulatory effects of dietary chamomile (Matricaria chamomilla) oil (ChO) on Indian shrimp (Penaeus indicus)’s growth performance, biochemical parameters, antioxidants, innate immunity, and resistance to Vibrio parahaemolyticus infection were investigated. Shrimp (4.0–4.5 g) were fed with diets enriched with 0.0 (control), 1.0, 3.0, and 5.0 g ChO/kg feed three times a day to apparent satiety for 56 days. Intramuscularly, V. parahaemolyticus were injected after the feeding trial, and clinical symptoms and mortalities were observed for 96 h. In ChO-fed animals, growth-stimulating effects were found on final weight, weight gain, specific growth rate, and feed intake with an optimum level of 3 g/kg diet. Dietary ChO did not significantly affect feed conversion ratio, protein efficienct ratio, protein productive value, and energy utlilization. Serum protein (total protein, albumin, and globulin) profile, as well as antioxidant and immunity variables, significantly higher in ChO-fed animals particularly at the 3.0 and 5.0 g/kg feed treatments. Conversily, significant reductions in levels of aspartate and alanine aminotransferase, creatinine, urea, malondialdehyde, and nitric oxide were detected in animals fed 3.0 or 5.0 g ChO/kg feed. Vibrio spp. showed low abundance in the gills and intestine of ChO-fed Indian shrimp compared to the control one; particularly in the treatments of 3.0 and 5.0 g/kg feed. The control group died completely after the bacterial challenge; however, ChO-fed animals were more resistant, with relative survival rates of 80.0% and 86.7% in the 3.0 and 5.0 g ChO/kg feed treatments, respectively, with no significant difference between them. Therefore, diets containing 3.0 g ChO/kg feed were found to be optimum for Indian shrimp, boosting its growth, antioxidant capacity, and innate immunity. Furthermore, ChO-enriched diets effectively increased its resistance to infection by V. parahaemolyticus.

NLRP3-like protein negatively regulates the expression of antimicrobial peptides in Penaeus vannamei hemocyates

NACHT, LRR and PYD domains-containing protein 3 (NLRP3) protein is the core actor involved in inflammasome formation, and plays a pivotal role in the innate immune response. However, whether NLRP3 participates in the regulation of innate immunity in invertebrates is still unknown. In the present study, we characterized a NLRP3 ortholog in Penaeus vannamei (designated PvNLRP3-like) with 2514 bp length of open reading frame (ORF) encoding a putative protein of 837 amino acids. Sequence analysis revealed that PvNLRP3-like contained only NACHT domain and shared closely homology with other invertebrates, but the topological structure of NACHT domain of PvNLRP3-like is similar with that in human NLRP3. PvNLRP3-like was ubiquitously expressed in tissues and induced in hemocytes by Vibrio parahaemolyticus, Streptococcus iniae and White Spot Syndrome Virus (WSSV) challenge, suggesting that PvNLRP3-like participated in the immune responses to pathogens. Furthermore, silencing of PvNLRP3-like followed by V. parahaemolyticus stimulation negatively regulated the transcripts of antimicrobial peptides (AMPs) including Lysozyme (LYZ) 3, Crustin (CRU) 2, Anti-lipopolysaccharide factor (ALF) 2/3 and Penaeidins (PEN) 3/4. This study enriches our current knowledge on shrimp innate immunity, and provides novel perspective to understand the immune regulation role of PvNLRP3-like.

Evaluation of the relationship between the 14-3-3\u03b5 protein and LvRab11 in the shrimp Litopenaeus vannamei during WSSV infection

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

A novel C-type lectin LvCTL 4.2 has antibacterial activity but facilitates WSSV infection in shrimp (L. vannamei)

Glycan-binding protein C-type lectin (CTL), one of the pattern recognition receptors (PRRs), binds to carbohydrates on the surface of pathogens and elicits antimicrobial responses in shrimp innate immunity. The objective was to identify and characterize a novel C-type lectin LvCTL 4.2 in Litopenaeus vannamei. The LvCTL 4.2 protein consisted of a signal peptide at the N terminal and a carbohydrate-recognition domain (CRD) with a mutated mannose-binding (Glu-Pro-Ala; EPA) motif at the C terminal, and thereby has a putative secreted mannose-binding C-type lectin architecture. LvCTL 4.2 was highly expressed in nervous tissue and stomach. Infection with white spot syndrome virus (WSSV) induced expression of LvCTL 4.2 in shrimp stomach at 12 h post infection. Conversely, there was no obvious upregulation in expression of LvCTL 4.2 in stomach or hepatopancreas of shrimp with AHPND (acute hepatopancreas necrosis disease). Pathogen binding assays confirmed recombinant LvCTL 4.2 protein (rLvCTL 4.2) had significant binding ability with the WSSV virion, Gram-negative, and Gram-positive bacteria. Moreover, rLvCTL 4.2 had strong growth inhibition of Vibrio parahaemolyticus. Silencing LvCTL 4.2 suppressed WSSV replication, whereas pretreatment of WSSV with rLvCTL 4.2 facilitated viral replication in vivo. In conclusion, LvCTL 4.2 acted as a PRR that inhibited AHPND-causing bacteria, but facilitated WSSV pathogenesis.

Phosphorylation of Shrimp Tcf by a Viral Protein Kinase WSV083 Suppresses Its Antiviral Effect.

Nuclear DNA-binding TCF proteins, which act as the main downstream effectors of Wnt signaling, are essential for the regulation of cell fate and innate immunity. However, their role during viral infection in shrimp remains unknown. Herein, we demonstrated that Litopenaeus vannamei TCF (LvTcf) acts independently of Lvβ-catenin to promote interferon-like protein LvVago1 production, thus mounting the response to WSSV infection. Further, we observed that WSV083, a WSSV serine/threonine protein kinase, bound to LvTcf and phosphorylated it. Phosphorylated LvTcf was then recognized and degraded via the ubiquitin-proteasome pathway. Moreover, mass spectrometry analyses indicated that the T39 and T104 residues of LvTcf were target sites phosphorylated by WSV083. Point mutation analyses suggested that additional sites of LvTcf may undergo phosphorylation via WSV083. Taken together, the current work provides valuable insights into host immunity and viral pathogenesis. LvTcf is not only a modulator of shrimp innate immunity but is also an important target for WSSV immune evasion. Thus, the current findings will help improve disease control in shrimps.

The Notch receptor-ligand Delta is involved in the immune response of Penaeus vannamei

In the Notch signaling pathway in vertebrates and invertebrates, the ligand Delta plays crucial roles in cell proliferation, differentiation, and immunity. Although the Notch signaling pathway has recently been implicated in the immune defense of Penaeus vannamei, the association of Delta with this immune response remains unclear. Here, we cloned and characterized the Delta homolog in P. vannamei (designated as PvDelta). PvDelta has a 2493 bp open reading frame (ORF) encoding a putative protein of 830 amino acids. Bioinformatics analysis revealed that PvDelta contains an N-terminal signal peptide, a conserved Notch ligand (MNNL) domain, a Delta/Serrate/Lag-2 segment, 9 epidermal growth factors segments, a transmembrane domain, and shares high homology with other Delta family members. Transcripts of PvDelta were detected in all shrimp tissues tested and were induced by Vibrio parahaemolyticus, white spot syndrome virus (WSSV), and lipopolysaccharide (LPS), indicating its involvement in shrimp immune response. Moreover, after PvDelta knockdown followed by LPS stimulation, the expression of Notch signaling pathway genes (i.e., PvNotch, PvCSL, and PvHey) was downregulated. Finally, shrimp depleted of PvDelta showed a lower survival rate in response to V. parahaemolyticus challenge. In sum, our data reveal that PvDelta is involved in the innate immunity of shrimp by positively modulating the Notch signaling pathway.

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.

Metagenomics in bioflocs and their effects on gut microbiome and immune responses in Pacific white shrimp

Biofloc systems generate and accumulate microbial aggregates known as bioflocs. The presence of bioflocs has been shown to change gut bacterial diversity and stimulate innate immunity in shrimp. The microbial niche of bioflocs may therefore have the potential to drive shifts in the shrimp gut microbiota associated with stimulation of innate immunity. We performed shotgun metagenomic analysis and 16S rRNA-based amplicon sequencing to characterize complex bacterial members in bioflocs and the shrimp digestive tract, respectively. Moreover, we determined whether biofloc-grown shrimp with discrete gut microbiomes had an elevation in local immune-related gene expression and systemic immune activities. Our findings demonstrated that the bacterial community in bioflocs changed dynamically during Pacific white shrimp cultivation. Metagenomic analysis revealed that Vibrio comprised 90% of the biofloc population, while Pseualteromonas, Photobacterium, Shewanella, Alteromonas, Bacillus, Lactobacillus, Acinetobacter, Clostridium, Marinifilum, and Pseudomonas were also detected. In the digestive tract, biofloc-grown shrimp maintained the presence of commensal bacteria including Vibrio, Photobacterium, Shewanella, Granulosicoccus, and Ruegeria similar to control shrimp. However, Vibrio and Photobacterium were significantly enriched and declined, respectively, in biofloc-grown shrimp. The presence of bioflocs upregulated immune-related genes encoding serine proteinase and prophenoloxidase in digestive organs which are routinely exposed to gut microbiota. Biofloc-grown shrimp also demonstrated a significant increase in systemic immune status. As a result, the survival rate of biofloc-grown shrimp was substantially higher than that of the control shrimp. Our findings suggested that the high relative abundance of vibrios in bioflocs enriched the number of vibrios in the digestive tract of biofloc-grown shrimp. This shift in gut microbiota composition may be partially responsible for local upregulation of immune-related gene expression in digestive organs and systemic promotion of immune status in circulating hemolymph.

A novel and convenient bacterial platform for production of encapsidated double‐stranded RNA targeting white spot syndrome virus in shrimp

White spot syndrome virus (WSSV) is a highly infectious virus that can cause 100% mortality in farmed shrimp in the matter of days, leading to significant economic losses to shrimp aquaculture worldwide. To mitigate the adverse impact of WSSV, effective antiviral strategies are necessary. Currently, double‐stranded RNA (dsRNA), which can silence viral gene expression through an RNA interference (RNAi) pathway, is among the most promising therapeutic tools for WSSV, but its farm‐scale utilization is held back by high costs and the lack of delivery approach. Recently, virus‐like particles (VLPs) have been applied as nanocarriers to deliver dsRNA into shrimp tissues, resulting in prolonged half‐life of the encapsidated dsRNA and stimulation of shrimp innate immunity. However, recombinant VLPs and dsRNA are traditionally expressed in two different E. coli strains: a protease‐deficient BL21(DE3) strain and an RNase III‐deficient HT115(DE3) strain, respectively. The ability to produce both VLPs and dsRNA simultaneously in a cell will reduce the costs and steps associated with the production, but attempts at VLP‐dsRNA coexpression in each of these strains resulted in degradation of either VLP or dsRNA due to the presence of proteases or RNaseIII, respectively. Here, to streamline VLP‐dsRNA production and allow simultaneous expression of VLP and dsRNA in the same cell, P1 transduction was employed to generate a novel E. coli strain which is both protease‐ and RNase III‐deficient. The newly engineered E. coli strain is able to express both MrNV‐VLP and VP28 dsRNA efficiently. Therefore, this new expression platform will pave the way for development of effective and low‐cost production of VLP‐encapsulated dsRNA for protection against viral infection in farmed shrimp.Support or Funding InformationThe authors would like to thank the following institutions for supporting research and scholarship to graduate student: Thailand Graduate Institute of Science and Technology (TGIST), National Science and Technology Development Agency (NSTDA) and Mahidol University.

RNA interference of Hsp70 in Artemia franciscana nauplii and its effect on morphology, growth, survival and immune response

Brine shrimp Artemia franciscana release either encysted embryos via oviparous reproduction or motile nauplii via ovoviviparous reproduction. Artemia cysts contain an abundant amount of ArHsp21, ArHsp22, artemin, and p26, small heat shock proteins which regulate diapause and stress tolerance of this branchiopod crustacean. However, not much is known of the role of molecular chaperone, Hsp70 in this animal. In this study, the immunostimulatory role of Hsp70 in A. franciscana was investigated, work which included examining the function of this protein in embryo development, growth, survival and stress tolerance. Knockdown of Hsp70 was successfully performed by RNA interference (RNAi), with specific Hsp70 dsRNA construct delivered to female broods by microinjection to knockdown the protein in nauplii. The loss of Hsp70 neither affected the growth nor morphology as Artemia nauplii hatched normally and thereafter grew to adult within 28 days, observations similar to the control animals, the latter injected with GFP dsRNA. The survival of nauplii lacking Hsp70 appeared lower upon 28 days culture but the differences were insignificant when comparisons were made with the controls (P > .05). On other aspect, knockdown of Hsp70 reduced approximately 31% survival upon Vibrio campbellii challenge, indicating that Hsp70 assisted in the tolerance of Artemia nauplii during pathogen infection, perhaps through immune stimulation. Initial findings revealed that Hsp70 knockdown did not affect the expression of prophenoloxidase (proPO), a crucial immune protein of the shrimp innate immunity. By examining the morphology, growth, viability and molecular changes upon Hsp70 elimination, this study contributes to our understanding of the important roles of Hsp70 in A. franciscana, an important live food species used in aquaculture.

Comprehensive expression analysis of the beta integrin from Penaeus monodon indicating its participation in innate immunity and ammonia nitrogen stress response

The aim of the present study was to investigate the function of the beta integrin (PmItgb) in Penaeus monodon. The 3011 bp cDNA sequence of PmItgb was cloned from P. monodon using rapid amplification of cDNA ends (RACE) PCR. Phylogenetic tree analyses indicated that the amino acid sequence of PmItgb should be merged into Fenneropenaeus chinensis (93%). Quantitative real-time PCR (q RT-PCR) revealed that PmItgb mRNA was highly expressed in the hemocytes. In addition, with regard to developmental stages, PmItgb showed significantly higher expression in oosperm, nauplius IV, zoea I and III, and post larval stages than that in other development stages. PmItgb expression in the shrimp epidermis was higher in the postmolt (B) stage, and lower in other molting stages. We also found that Vibrio harveyi and V. anguillarum challenge enhanced PmItgb expression in the hepatopancreas and gills. When PmItgb was inhibited, innate immunity-related genes such as ALF, crustin 1, crustin 7, penaeidin 3, and penaeidin 5 were significantly down-regulated. Furthermore, we demonstrated that PmItgb knock-down by specific dsRNA reduced bacterial clearance. In high ammonia nitrogen concentrations, PmItgb was significantly up-regulated in the hepatopancreas and gills. After PmItgb was silenced, the rate of mortality owing to high ammonia nitrogen concentrations decreased; the expression of related anti-apoptotic genes was up-regulated, and that of the apoptotic genes was slightly down-regulated. These results suggested that PmItgb may be involved in shrimp innate immunity and mediate apoptosis of hepatopancreatic cells induced by high ammonia nitrogen environments.

Litopenaeus vannamei CK2 is involved in shrimp innate immunity by modulating hemocytes apoptosis

Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase with multiple cellular functions in vertebrates including apoptosis, differentiation, proliferation, survival, tumorigenesis, signal transduction, immune regulation and inflammation. In the current study, the catalytic and regulatory subunit homologs of Litopenaeus vannamei protein kinase CK2 (LvCK2α and LvCK2β) were cloned and characterized. LvCK2α has a full-length cDNA sequence of 1764 bp with a 1053 bp open reading frame (ORF) encoding a putative protein of 351 amino acids, which contains a typical serine/threonine kinase domain. On the other hand, LvCK2β has a 1394 bp full-length cDNA with an ORF of 663 bp encoding a protein with 221 amino acids, which contains a Casein kinase II regulatory subunit domain. Sequence and phylogenetic analysis revealed that LvCK2 was evolutionary related with the CK2 of invertebrates. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that LvCK2α and LvCK2β transcripts were widely expressed in all shrimp tissues tested, and were both induced in hemocytes and hepatopancreas upon challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), suggesting their involvement in shrimp immune response. Moreover, RNA interference (RNAi) of LvCK2α resulted in increased hemocytes apoptosis, shown by high caspase 3/7 activity, increased number of apoptotic cells, coupled with an elevation in transcript levels of pro-apoptotic LvCaspase3 and LvCytochrome C, and a reduction in mRNA levels of pro-survival LvBcl2, LvIAP1, and LvIAP2. In addition, LvCK2α knockdown followed by V. parahaemolyticus challenge resulted in higher cumulative mortality of shrimp. Taken together, our current findings suggest that LvCK2 modulates shrimp hemocytes apoptosis as part of the innate immune response to pathogens.

Heat shock protein 70 from Litopenaeus vannamei (LvHSP70) is involved in the innate immune response against white spot syndrome virus (WSSV) infection

White spot syndrome (WSS) caused by white spot syndrome virus (WSSV) is a severe infectious disease in shrimp aquaculture. To find effective therapeutics to control WSSV, it is indispensable to understand the innate immune responses of shrimp to WSSV infection. Previous report demonstrated that the Litopenaeus vannamei heat shock protein 70 (LvHSP70) could induce shrimp innate immunity against bacterial infection. Herein, we further investigate the role of LvHSP70 in anti-WSSV infection. The temporal expression of LvHSP70 was significantly upregulated 2.5- and 1.5-fold at 6 and 24 h post systemic WSSV infection suggesting that the LvHSP70 was a WSSV responsive gene. The recombinant protein of LvHSP70 (rLvHSP70) was produced in an Escherichia coli system and its effect in protection against WSSV infection was investigated. Intramuscularly injection of juvenile shrimp with 1 nmol of rLvHSP70 could significantly prolong 50% mortality of WSSV-infected shrimp from 3 days to 5 days as compared to the control group injected with bovine serum albumin (BSA). Consistently, the injection of rLvHSP70 resulted in 24-fold, 20-fold and 100-fold decrease in the viral copy number after 6, 12 and 24 h post injection, respectively, compared to the control shrimp injected with BSA. Interestingly, it was found that the rLvHSP70 enhanced the expression of the key gene in the prophenoloxidase (proPO) activating system, LvproPO, but reduced the expression of Lvcaspase2 and LvIAP in WSSV-infected shrimp. These results suggested that the LvHSP70 is an important molecule involved in antiviral defense in shrimp presumably via modulating the proPO system and apoptosis.