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.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity.

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-ß. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs' expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

Activation and characterization of G protein-coupled receptors for CHHs in the mud crab, Scylla paramamosain.

Crustacean hyperglycemic hormone (CHH) superfamily peptides constitute a group of neurohormones, including the crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), which reportedly play an essential role in regulating various biological activities by binding to their receptors in crustaceans. Although bioinformatics analyses have identified G protein-coupled receptors (GPCRs) as potential CHH receptors, no validation through binding experiments has been carried out. This study employed a eukaryotic expression system, HEK293T cell transient transfection, and ligand-receptor interaction tests to identify the GPCRs of CHHs in the mud crab Scylla paramamosain. We found that four GPCRs (Sp-GPCR-A34-A37) were activated by their corresponding CHHs (Sp-CHH1-v1, Sp-MIH, Sp-VIH) in a dose-dependent manner. Of these, Sp-GPCR-A34 was exclusively activated by Sp-VIH; Sp-GPCR-A35 was activated by Sp-CHH1-v1 and Sp-VIH, respectively; Sp-GPCR-A36 was activated by Sp-CHH1-v1 and Sp-MIH; Sp-GPCR-A37 was exclusively activated by Sp-MIH. The half-maximal effective concentration (EC50) values for all CHHs/GPCRs pairs (both Ca2+ and cAMP signaling) were in the nanomolar range. Overall, our study provided hitherto undocumented evidence of the presence of G protein-coupled receptors of CHH in crustaceans, providing the foothold for further studies on the signaling pathways of CHHs and their corresponding GPCRs.

Effects of the tumor necrosis factor on hemocyte proliferation and bacterial infection in Chinese mitten crab (Eriocheir sinensis).

Tumor necrosis factor (TNF) is an important cytokine that can regulate a variety of cellular responses by binding tumor necrosis factor receptor (TNFR). We studied whether the TNF of Eriocheir sinensis can regulate hemocyte proliferation. The results showed that the EsTNF and EsTNFR were constitutively expressed in all tested tissues, including the heart, hepatopancreas, muscles, gills, stomachs, intestines, and hemocytes. We found that low levels of EsTNF and EsTNFR transcripts were present in hemocytes. The gene expression levels were significantly increased in the hemocytes after being stimulated by Staphylococcus aureus or Vibrio parahaemolyticus. We also found some genes related to cell proliferation were expressed at a higher level in pulsing rTNF-stimulated hemocytes compared with the control group. We also knocked down the EsTNFR gene with RNAi technology. The results showed that the expression level of these genes related to cell proliferation was significantly down-regulated compared with the control group when the TNF does not bind TNFR. We used Edu technology to repeat the above experiments and the results were similar. Compared with the control group, the hemocytes stimulated by rTNF showed more significant proliferation, and the proliferation rate was significantly down-regulated after knocking down the EsTNFR gene. Therefore, we indicate that TNF binding TNFR can affect the proliferation of E. sinensis hemocytes, which might be manifested by affecting the expression of some proliferation-related genes.

Integrated application of transcriptomics and metabolomics provides insights into acute hepatopancreatic necrosis disease resistance of Pacific white shrimp Litopenaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) has caused a huge economic loss to shrimp aquaculture. Vibrio parahaemolyticus (VpAHPND) is regarded as a major causative agent of AHPND in the Pacific white shrimp Litopenaeus vannamei. However, knowledge about how shrimp resist to AHPND is very limited. In order to learn the molecular mechanisms underlying AHPND resistance of shrimp, comparison between disease-resistant family and susceptible family of L. vannamei were performed at transcriptional and metabolic levels. Integrated analysis of transcriptomics and metabolomics on hepatopancreas of shrimp, the target tissue of VpAHPND, showed that significant differences existed between resistant family and susceptible family of shrimp. The susceptible family showed higher level of glycolysis, serine-glycine metabolism, purine and pyrimidine metabolism, but lower level of betaine-homocysteine metabolism in the hepatopancreas in comparison with the resistant family without VpAHPND infection. Curiously, VpAHPND infection induced up-regulation of glycolysis, serine-glycine metabolism, purine metabolism, pyrimidine metabolism, and pentose phosphate pathway, and down-regulation of betaine-homocysteine metabolism in resistant family. In addition, arachidonic acid metabolism and some immune pathways, like NF-κB and cAMP pathways, were up-regulated in the resistant family after VpAHPND infection. In contrast, amino acid catabolism boosted via PEPCK-mediated TCA cycle flux was activated in the susceptible family after VpAHPND infection. These differences in transcriptome and metabolome between resistant family and susceptible family might contribute to the resistance of shrimp to bacteria. IMPORTANCE Vibrio parahaemolyticus (VpAHPND) is a major aquatic pathogen causing acute hepatopancreatic necrosis disease (AHPND) and leads to a huge economic loss to shrimp aquaculture. Despite the recent development of controlling culture environment, disease resistant broodstock breeding is still a sustainable approach for aquatic disease control. Metabolic changes occurred during VpAHPND infection, but knowledge about the metabolism in resistance to AHPND is very limited. Integrated analysis of transcriptome and metabolome revealed the basal metabolic differences exhibited between disease-resistant and susceptible shrimp. Amino acid catabolism might contribute to the pathogenesis of VpAHPND and arachidonic acid metabolism might be responsible for the resistance phenotype. This study will help to enlighten the metabolic and molecular mechanisms underlying shrimp resistance to AHPND. Also, the key genes and metabolites of amino acid and arachidonic acid pathway identified in this study will be applied for disease resistance improvement in the shrimp culture industry.

Identification of a Double-ß-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

ß-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian ß-defensin 11 (AvBD11) is unique, with two ß-defensin motifs with a broad range of antimicrobial activities. However, a double-sized ß-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-ß-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to ß-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD in vivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-ß-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

Deubiquitinase cylindromatosis (CYLD) regulates antibacterial immunity and apoptosis in Chinese mitten crab (Eriocheir sinensis).

Ubiquitination and deubiquitination of target proteins is an important mechanism for cells to rapidly respond to changes in the external environment. The deubiquitinase, cylindromatosis (CYLD), is a tumor suppressor protein. CYLD from Drosophila melanogaster participates in the antimicrobial immune response. In vertebrates, CYLD also regulates bacterial-induced apoptosis. However, whether CYLD can regulate the bacterial-induced innate immune response in crustaceans is unknown. In the present study, we reported the identification and cloning of CYLD in Chinese mitten crab, Eriocheir sinensis. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that EsCYLD was widely expressed in all the examined tissues and was upregulated in the hemolymph after Vibrio parahaemolyticus challenge. Knockdown of EsCYLD in hemocytes promoted the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation and increased the expression of corresponding antimicrobial peptides. In vivo, silencing of EsCYLD promoted the removal of bacteria from the crabs and enhanced their survival. In addition, interfering with EsCYLD expression inhibited apoptosis of crab hemocytes caused by V. parahaemolyticus stimulation. In summary, our findings revealed that EsCYLD negatively regulates the nuclear translocation of Relish to affect the expression of corresponding antimicrobial peptides and regulates the apoptosis of crab hemocytes, thus indirectly participating in the innate immunity of E. sinensis.

The hematopoietic cytokine Astakine play a vital role in hemocyte proliferation and innate immunity in Scylla paramamosain.

Astakine may induce hematopoietic response in crustaceans, as it is necessary for hemocyte proliferation. In this study, we produced the recombinant Scylla paramamosain Astakine (rspAstakine) and assessed its immunomodulatory function. We analyzed its amino acid sequences and generated a three-dimensional model, then ligand binding sites and enzyme commission of spAstakine were predicted. The rspAstakine was identified at 21.3 kDa by Western blot and liquid chromatography-mass spectrometry. The results showed that rspAstakine induced proliferation of hemocytes in mud crab in vivo and in vitro. The expression of immune-related genes was up-regulated after rspAstakine treatment, similarly to the immunity-related parameters, activities of superoxide dismutase, phenoloxidase, lysozyme, and peroxidase. Additionally, the intracellular content of reactive oxygen species was higher in the rspAstakine treatment group than PBS group. The rspAstakine also enhanced the rate of phagocytosis, while reduced the apoptosis rate of hemocytes after Vibrio alginolyticus infection. The mortalities of the V. alginolyticus only group and rspAstakine + V. alginolyticus group were 83.3 % and 58.3 %, respectively, which illustrated that rspAstakine plays a protective role against V. alginolyticus infection in S. paramamosain. Our results demonstrate the potential of Astakine to enhance the proliferation and immunomodulatory function of hemocytes in crustaceans.

Eight TRIM32 isoforms from oriental river prawn Macrobrachium nipponense are involved in innate immunity during white spot syndrome virus infection.

Tripartite motif (TRIM) proteins comprise a large family of RING-type ubiquitin E3 ligases that regulate important biological processes. In this study, full-length MnTRIM32 cDNA was obtained from oriental river prawn Macrobrachium nipponense, and eight MnTRIM32 isoforms generated by alternative splicing were identified. The open reading frames of the eight MnTRIM32 isoforms were predicted to be separately composed of 402, 346, 347, 346, 414, 358, 359, and 358 amino acid residues. Protein structural analysis revealed that all MnTRIM32 isoforms contained a RING domain and a coiled coil region. MnTRIM32 was ubiquitously expressed in all tissues tested, with the highest expression in the hepatopancreas. The mRNA levels of MnTRIM32 in the gills, stomach, and intestine of prawns were found to undergo time-dependent enhancement following white spot syndrome virus (WSSV) stimulation. Double-stranded RNA interference studies revealed that MnTRIM32 silencing significantly downregulated the expression levels of interferon (IFN) regulatory factor MnIRF, IFN-like factor MnVago4, and tumor necrosis factor MnTNF. Furthermore, knockdown of MnTRIM32 in WSSV-challenged prawns increased the expression of VP28 and the number of WSSV copies, suggesting that MnTRIM32 plays a positive role in limiting WSSV infection. These findings provided strong evidence for the important role of MnTRIM32 in the antiviral innate immunity of M. nipponense.

Single-cell RNA sequencing analysis of shrimp immune cells identifies macrophage-like phagocytes.

Despite the importance of innate immunity in invertebrates, the diversity and function of innate immune cells in invertebrates are largely unknown. Using single-cell RNA-seq, we identified prohemocytes, monocytic hemocytes, and granulocytes as the three major cell-types in the white shrimp hemolymph. Our results identified a novel macrophage-like subset called monocytic hemocytes 2 (MH2) defined by the expression of certain marker genes, including Nlrp3 and Casp1. This subtype of shrimp hemocytes is phagocytic and expresses markers that indicate some conservation with mammalian macrophages. Combined, our work resolves the heterogenicity of hemocytes in a very economically important aquatic species and identifies a novel innate immune cell subset that is likely a critical player in the immune responses of shrimp to threatening infectious diseases affecting this industry.

A Deacetylase CqSIRT1 Promotes WSSV Infection by Binding to Viral Envelope Proteins in Cherax quadricarinatus.

Sirtuin 1 (SIRT1), a member of the class III lysine deacetylases, exhibits powerful functional diversity in physiological processes and disease occurrences. However, the potential molecular mechanism underlying the role of SIRT1 during viral infection in crustaceans is poorly understood. Herein, SIRT1 was functionally characterized from the red claw crayfish Cherax quadricarinatus, which possesses typically conserved deacetylase domains and strong evolutionary relationships across various species. Moreover, gene knockdown of CqSIRT1 in crayfish haematopoietic tissue (Hpt) cell culture inhibited white spot syndrome virus (WSSV) late envelope gene vp28 transcription. In contrast, enhancement of deacetylase activity using a pharmacological activator promoted the replication of WSSV. Mechanically, CqSIRT1 was co-localized with viral envelope protein VP28 in the nuclei of Hpt cells and directly bound to VP28 with protein pulldown and co-immunoprecipitation assays. Furthermore, CqSIRT1 also interacted with another two viral envelope proteins, VP24 and VP26. To the best of our knowledge, this is the first report that WSSV structural proteins are linked to lysine deacetylases, providing a better understanding of the role of CqSIRT1 during WSSV infection and novel insights into the basic mechanism underlying the function of lysine deacetylases in crustaceans.

Potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and VP(AHPND) resistance in Pacific white shrimp (Litopenaeus vannamei, Boone, 1931).

The potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and disease resistance against Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) in Pacific white shrimp, were intensively investigated. Three effective strains of Bacillus amyloliquefaciens (A), Bacillus pumilus (P) and Bacillus subtilis (S) were mixed in pairs at a ratio of 5 × 108:5 × 108 CFU/kg diet and coated with the prebiotic chitosan (C) at a concentration of 20 mL/kg diet. Five different feed treatments were used to feed experimental shrimp for 5 weeks: control (control, no synbiotics), chitosan (coat, C) and the synbiotic treatments PAC, PSC and ASC. At week 5, the final length, final weight gain, weight gain, length, average daily gain, specific growth rate and feed conversion ratio, measured as growth parameters, were significantly upregulated in the PSC and ASC groups compared with the control and coat groups (P < 0.05). This result was consistent with the expression analysis of two growth-related genes (Rap-2a and GF-II) in the hepatopancreas and intestines of treated shrimp, as determined using qRT-PCR. The prebiotic chitosan and synbiotics PAC, PSC and ASC strongly induced significant differences in the expression of the Rap-2a and GF-II genes in the target organs compared with the expression in the control group at various time points (P < 0.05). Additionally, application of the synbiotic treatments also significantly enhanced the hepatopancreas characteristics and epithelial and intestinal wall thicknesses of the shrimp compared with the control. Interestingly, all the synbiotic treatments elevated phagocytic activity significantly at weeks 3 and 5 compared with that in the other groups. qRT-PCR analysis of immune-related genes also indicated that the prebiotic group and all synbiotic groups showed strong expression of anti-lipopolysaccharide (ALF) and prophenoloxidase (proPO) genes in the intestine. Finally, the synbiotic groups PAC, PSC and ASC exhibited stronger VPAHPND resistance at 120 h after exposure than the chitosan coat and control groups, with survival rates of 41.7 ± 11.55, 41.7 ± 0.00, 52.8 ± 5.77, 30.6 ± 15.28 and 22.2 ± 5.77%, respectively (P < 0.05). Based on the obtained information, all synbiotics were recommended for improved growth and immune responses, while ASC was the best for disease resistance against VPAHPND in Pacific white shrimp.

SpBNIP3 regulates apoptosis and autophagy in mud crab (Scylla paramamosain) during white spot syndrome virus infection.

BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), which is a pro-apoptotic protein in the BCL-2 family involves a variety of cell signaling pathways, including mitochondrial dysfunction, mitochondrial autophagy, and apoptosis in vertebrates. However, the role of BNIP3 in the regulation of apoptosis and/or autophagy in crustaceans suffering virus infection is still limited. In this study, the mud crab (Scylla paramamosain) BNIP3 (SpBNIP3) was identified and studied to elucidate its association with the white spot syndrome virus (WSSV) infection. SpBNIP3 was widely expressed in all tested tissues and significantly down-regulated in the hemocytes of mud crab after WSSV infection. Knockdown of SpBNIP3 using RNA interference increased the apoptosis rate and Caspase 3 activity but decreased the mitochondrial membrane potential and autophagy levels, as well as viral copy number in mud crabs infected with WSSV. Additionally, the relationship between the viral infection and the autophagy of hemocytes was observed. The level of autophagy was reduced upon WSSV infection, and the activation of autophagy enriched the viral copy number. Taken together, the results of this study provide a new finding on the mechanism that SpBNIP3 may participate in the WSSV infection through the regulation of apoptosis and autophagy processes in mud crabs.

AKT regulates hemocyte proliferation via glucose metabolism in Eriocheir sinensis.

Threonine-serine protein kinase (AKT) plays an important role in the regulation of essential biological processes such as cell metabolism and cell proliferation in several organisms. Eriocheir sinensis is a widely distributed crab that is exposed to complex and diverse biological environments and various diseases. We aimed to elucidate the immune function of AKT and the underlying mechanism in E. sinensis. In this study, we performed bioinformatics analysis of E. sinensis AKT (EsAkt) and found that the AKT protein was highly conserved across species. qRT-PCR showed high AKT expression in the liver and muscle tissues, and low expression in hemocytes. After stimulation with gram-positive Staphylococcus aureus or gram-negative Vibrio parahaemolyticus, E. sinensis AKT (EsAkt) was significantly up-regulated in hemocytes. Further, knockout of the EsAkt gene weakened cell glucose metabolism and inhibited cell proliferation. Taken together, these results suggest that EsAkt plays a key role in regulating hemocyte glucose metabolism and cell proliferation in Eriocheir sinensis.

Protein profiling of hemolymph in Haemaphysalis flava ticks.

BACKGROUND: Tick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava. METHODS: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ). RESULTS: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-ß, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-ß. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ. CONCLUSIONS: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.

SpBAG3 assisted WSSV infection in mud crab (Scylla paramamosain) by inhibiting apoptosis.

The function of B-cell lymphoma-2 (Bcl-2) family proteins can be divided into two categories: anti-apoptotic and pro-apoptotic. As an anti-apoptotic protein, Bcl2-associated athanogene 3 (BAG3) plays a key role in regulating apoptosis, development, cell movement, and autophagy, and mediating the adaptability of cells to stimulation. However, SpBAG3 has not been reported in mud crab (Scylla paramamosain), and the regulatory effect of SpBAG3 on apoptosis in mud crab and its function in antiviral immunity is still unknown. In this study, SpBAG3 was found, and characterized, which encoded a total of 175 amino acid (molecular mass 19.3 kDa), including a specific conserved domain of the BAG family. SpBAG3 was significantly down-regulated at 0-48 h post-infection with WSSV in vivo. The antiviral effect of SpBAG3 was investigated using RNA interference. The results indicated that SpBAG3 might be involved in assisting the replication of WSSV in the host. SpBAG3 could change the mitochondrial membrane potential (△ψm), and affect cell apoptosis through mitochondrial apoptotic pathways. Therefore, the results of this study suggested that SpBAG3 could assist WSSV infection by inhibiting the apoptosis of the hemocytes in mud crab.

Genomic Identification and Functional Analysis of JHAMTs in the Pond Wolf Spider, Pardosa pseudoannulata.

Juvenile hormone (JH) plays a critical role in many physiological activities of Arthropoda. Juvenile hormone acid methyltransferase (JHAMT) is involved in the last steps of JH biosynthesis as an important rate-limiting enzyme. In recent studies, an increasing number of JHAMTs were identified in arthropods, but no JHAMT was reported in spiders. Herein, eight JHAMTs were identified in the pond wolf spider, Pardosa pseudoannulata, all containing the well conserved S-adenosyl-L-methionine binding motif. JHAMT-1 and the other seven JHAMTs were located at chromosome 13 and chromosome 1, respectively. Multiple alignment and phylogenetic analysis showed that JHAMT-1 was grouped together with insect JHAMTs independently and shared high similarities with insect JHAMTs compared to the other seven JHAMTs. In addition, JHAMT-1, JHAMT-2, and JHAMT-3 were highly expressed in the abdomen of spiderlings and could respond to the stimulation of exogenous farnesoic acid. Meanwhile, knockdown of these three JHAMTs caused the overweight and accelerated molting of spiderlings. These results demonstrated the cooperation of multi-JHAMTs in spider development and provided a new evolutionary perspective of the expansion of JHAMT in Arachnida.

miR-2 contributes to WSSV infection by targeting Caspase 2 in mud crab (Scylla paramamosain).

Caspase 2 is widely studied for its function in the regulation of apoptosis in mammals. Despite the fundamental role of apoptosis during the anti-viral immune response, the relationship between Caspase 2 and virus infection has not been extensively explored in invertebrates. Also, whether or not miRNAs involve this process remains unclear. To address this issue, the miRNA-mediated regulation of Caspase 2 in mud crab (Scylla paramamosain) (Sp-Caspase 2) was characterized in this study. Sp-Caspase 2 contains an open reading frame (ORF) of 969 bp encoding 322 deduced amino acids and possesses a conserved CASc domain. The results suggested that Sp-Caspase 2 could suppress white spot syndrome virus infection via apoptosis induction. The further data showed that Sp-Caspase 2 was directly targeted by miR-2 in mud crab. Silencing or overexpression of miR-2 could affect apoptosis and WSSV replication through the regulation of Sp-Caspase 2 expression. Taken together, these results demonstrated the crucial role of the miR-2-Caspase 2 pathway in the innate immunity of mud crabs and revealed a novel mechanism in the anti-viral immune response in marine invertebrates.

IgE Epitopes of the House Dust Mite Allergen Der p 7 Are Mainly Discontinuous and Conformational.

Background: Several studies indicate that Der p 7 is an important and clinically relevant allergen of Dermatophagoides pteronyssinus which should be included in vaccines for treatment of house dust mite (HDM) allergy. Aim of this study was to characterize the IgE epitopes of Der p 7. Methods: Recombinant Der p 7 was expressed and purified, analyzed for fold by circular dichroism and tested for its allergenic activity by basophil activation. Seven overlapping, surface-exposed peptides (P1-P7) with a length of 27 to 37 amino acids, which spanned the Der p 7 sequence, were synthesized and tested for IgE reactivity and allergenic activity by basophil activation assay. Carrier-bound peptides were studied for their ability to induce allergen-specific IgG antibodies in rabbits. Peptide-specific antibodies were used to inhibit allergic patients` IgE binding to Der p 7 by ELISA for mapping of IgE epitopes. Results: rDer p 7 showed high allergenic activity comparable with Der p 5, Der p 21, and Der p 23. None of the seven tested peptides showed any IgE reactivity or allergenic activity when tested with HDM- allergic patients indicating lack of sequential IgE epitopes on Der p 7. IgE inhibition experiments using anti-peptide specific IgGs and molecular modeling enabled us to identify discontinuous, conformational IgE epitopes of Der p 7. Conclusion and Clinical Relevance: IgE epitopes of Der p 7 belong to the conformational and discontinuous type whereas sequential Der p 7 peptides lack IgE reactivity. It should thus be possible to construct hypoallergenic vaccines for Der p 7 based on carrier-bound allergen peptides.