Morphologic, cytometric, quantitative transcriptomic and functional characterisation provide insights into the haemocyte immune responses of Pacific abalone (Haliotis discus hannai).

In recent years, the abalone aquaculture industry has been threatened by the bacterial pathogens. The immune responses mechanisms underlying the phagocytosis of haemocytes remain unclear in Haliotis discus hannai. It is necessary to investigate the immune mechanism in response to these bacterial pathogens challenges. In this study, the phagocytic activities of haemocytes in H. discus hannai were examined by flow cytometry combined with electron microscopy and transcriptomic analyses. The results of Vibrio parahaemolyticus, Vibrio alginolyticus and Staphylococcus aureu challenge using electron microscopy showed a process during phagosome formation in haemocytes. The phagocytic rate (PP) of S. aureus was higher than the other five foreign particles, which was about 63%. The PP of Vibrio harveyi was about 43%, the PP peak of V. alginolyticus in haemocyte was 63.7% at 1.5 h. After V. parahaemolyticus and V. alginolyticus challenge, acid phosphatase, alkaline phosphatase, total superoxide dismutase, lysozyme, total antioxidant capacity, catalase, nitric oxide synthase and glutathione peroxidase activities in haemocytes were measured at different times, differentially expressed genes (DEGs) were identified by quantitative transcriptomic analysis. The identified DEGs after V. parahaemolyticus challenge included haemagglutinin/amebocyte aggregation factor-like, supervillin-like isoform X4, calmodulin-like and kyphoscoliosis peptidase-like; the identified DEGs after V. alginolyticus challenge included interleukin-6 receptor subunit beta-like, protein turtle homolog B-like, rho GTPase-activating protein 6-like isoform X2, leukocyte surface antigen CD53-like, calponin-1-like, calmodulin-like, troponin C, troponin I-like isoform X4, troponin T-like isoform X18, tumor necrosis factor ligand superfamily member 10-like, rho-related protein racA-like and haemagglutinin/amebocyte aggregation factor-like. Some immune-related KEGG pathways were significantly up-regulated or down-regulated after challenge, including thyroid hormone synthesis, Th17 cell differentiation signalling pathway, focal adhesion, melanogenesis, leukocyte transendothelial migration, inflammatory mediator regulation of TRP channels, ras signalling pathway, rap1 signalling pathway. This study is the first step towards understanding the H. discus hannai immune system by adapting several tools to gastropods and providing a first detailed morpho-functional study of their haemocytes.

Two NF-κB subunits are associated with antimicrobial immunity in Hyriopsis cumingii.

The NF-κB pathway activated by bacteria and viruses produces a series of antimicrobial peptides that participate in the innate immune response. In this study, two NF-κB subunits were cloned and identified from Hyriopsis cumingii (named Hcp65 and Hcp105) using RT-PCR and RACE. The predicted Hcp65 protein possessed a N-terminal Rel homology domain (RHD) and an Ig-like/plexins/transcription factors domain (IPT); the Hcp105 contained an RHD, an IPT domain, 6 ankyrin (ANK) domain and a death domain. Quantitative reverse transcription PCR (qRT-PCR) showed that Hcp65 and Hcp105 were constitutively expressed in the detected tissues, and were significantly up-regulated in hemocytes, hepatopancreas and gill of mussels challenged with lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic-polycytidylic acid (poly I: C). The dsRNA-mediated silencing of Hcp65 and Hcp105 caused significant reduction of immune genes such as lysozyme (HcLyso), theromacin (Hcther), whey acid protein (HcWAP), LPS-binding protein/bactericidal permeability protein (HcLBP/BPI) 1 and 2. In addition, subcellular localization experiments showed that Hcp65 and Hcp105 proteins were expressed in both the nucleus and cytoplasm of HEK-293T cells, and Hcp50 proteins (mature peptide of Hcp105) were mainly localized in the nucleus. The recombinant Hcp65 and Hcp50 protein could form homodimer and heterodimer and bind κB site in vitro. These results provide useful information for understanding the role of NF-κB in mollusks.

The role of caspase 3 in the mud crab (Scylla paramamosain) after Vibrio parahaemolyticus infection.

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection.

Functional characterization of a putative tumor necrosis factor superfamily member 10 in blood clam (Tegillarca granosa).

Tumor necrosis factor superfamily member 10 (TNFSF10), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Apo-2L, is one of the important members of the TNF superfamily. It is well demonstrated that TNFSF10 preferentially induces a variety of tumor cell apoptosis, and therefore exerts an important role in tumor immune surveillance. However, the function of TNFSF10 in pathogen defense is poorly understood, especially in invertebrates. The blood clam (Tegillarca granosa), an important commercial marine bivalve, plays an important ecological role in the marine ecosystem. The identification of immune genes will provide new perspective for disease control in the blood clam (T. granosa) farming. To better understand the biological function of TNFSF10 protein, the full-length cDNA of TNFSF10 homologous gene of T. granosa (TgTNFSF10) was cloned and identified for the first time, which was found to contain 1239 base pairs and encode 254 amino acids with a molecular weight of 29.5 kDa and a conserved TNF domain in the C-terminal. Quantitative RT-PCR analysis showed that TgTNFSF10 gene was constitutively expressed in all tested tissues, with the highest expression in hemocytes. LPS, Vibrio alginolyticus and Vibrio parahaemolyticus stimulations dramatically increased the expression of TgTNFSF10 in T. granosa (11.47-fold, 3.71-fold and 8.29-fold compared with the control respectively). In vitro experiments showed that recombinant TgTNFSF10 protein strongly inhibited the proliferation of HepG2 cells. Further confocal microscopy and flow cytometry analysis showed that obvious apoptosis occurred in TgTNFSF10-treated hemocytes and HepG2 cells. To sum up, our study demonstrated that TgTNFSF10 had strong apoptosis-inducing activity, which may participate in the innate immune response of T. granosa to pathogen invasion.

Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

SpBark Suppresses Bacterial Infection by Mediating Hemocyte Phagocytosis in an Invertebrate Model, Scylla paramamosain.

Scavenger receptors are cell surface membrane-bound receptors that typically bind multiple ligands and promote the removal of endogenous proteins and pathogens. In this study, we characterized a novel scavenger receptor-like protein, namely, SpBark. SpBark was upregulated in hemocytes after challenges with bacteria, suggesting that it might be involved in antibacterial defense. SpBark is a type I transmembrane protein with four extracellular domains, including three scavenger receptor cysteine-rich domains (SRCRDs) and a C-type lectin domain (CTLD). Western blot assay showed that SpBark CTLD possessed a much stronger binding activity to tested microbes than the three SRCRDs. It also exhibited apparent binding activities to lipopolysaccharide (LPS) and acetylated low-density lipoprotein (ac-LDL), whereas the other SRCRDs showed much lower or no binding activities to these components. Agglutination activities were observed in the presence of Ca2+ by incubating microorganisms with SpBark CTLD instead of SRCRDs. These results suggested that SpBark CTLD was the major binding site for ac-LDL and LPS. Coating Vibrio parahemolyticus with SpBark CTLD promoted bacterial clearance in vivo. This finding indicated that SpBark might participate in the immune defenses against Gram-negative bacteria through a certain mechanism. The promotion of bacterial clearance by SpBark was further determined using SpBark-silenced crabs injected with V. parahemolyticus. SpBark knockdown by injection of SpBark dsRNA remarkably suppressed the clearance of bacteria in hemolymph. Meanwhile, it also severely restrained the phagocytosis of bacteria. This finding suggested that SpBark could modulate the phagocytosis of bacteria, and the promotion of bacterial clearance by SpBark was closely related to SpBark-mediated phagocytosis activity. The likely mechanism of bacterial clearance mediated by SpBark was as follows: SpBark acted as a pattern recognition receptor, which could sense and bind to LPS on the surface of invading bacteria with its CTLD in hemolymph. The binding to LPS made the bacteria adhere to the surface of hemocytes. This process would facilitate phagocytosis of the bacteria, resulting in their removal. This study provided new insights into the hemocyte phagocytosis mechanisms of invertebrates and the multiple biological functions of Bark proteins.

Antimicrobial properties and phenoloxidase activation of the lectin isolated from kadal shrimp (Metapenaeus dobsoni).

The present study reveals purification and characterization of the lectin from the haemolymph of Metapenaeus dobsoni. The Md-Lec was purified by affinity chromatography with mannose coupled sepharose CL-4B column and it exhibits single band with a molecular weight of 68 kDa in SDS-PAGE. Furthermore, the molecular mass was confirmed by MALDI-TOF and functional groups present were analysed by FTIR. The surface morphology of purified Md-Lec displays the homogeneous nature of protein. The X-ray diffraction (XRD) analysis expresses three peaks at 10.7716̊, 21.6258̊ and 31.7523̊which indicate the crystalline nature of the protein and the retention time of 3.068 min evident from HPLC reveals the purity of the sample. Functional analysis of purified Md-Lec exhibits yeast agglutination activity against Saccharomyces cerevisiae and has the ability to agglutinate the human erythrocytes, which was observed by light microscopy. It also exhibited phenoloxidase activation, encapsulation and phagocytic activities. In addition, purified Md-Lec showed the broad spectrum of bacterial agglutination activity against Gram negative Vibrio parahaemolyticus and Aeromonas hydrophila, important fish pathogens. Antiviral potential and anticancer activity of purified Md-Lec against CyHV-2 virus and MDA-MB-231 breast cancer cell lines were also evaluated in this study.

Molecular characterization, expression and antimicrobial activities of a c-type lysozyme from the mud crab, Scylla paramamosain.

Lysozyme is an important immune protein involved in the first line of defense for crustaceans. In the present study, a c-type lysozyme gene (SpLyzC) was cloned and characterized from the mud crab, Scylla paramamosain. The full-length cDNA was 849 bp with an open reading frame of 669 bp, and encoded a polypeptide of 223 amino acids with a calculated molecular mass of 23.7 kDa and an isoelectric point of 8.90. SpLyzC shared conserved active sites with c-type lysozymes from other species, detected in all tested tissues and had higher expression levels in hepatopancreas and gill tissues. The expression of SpLyzC was up-regulated in hepatopancreas and gill after infection with Vibrio parahaemolyticus and Staphylococcus aureus. The density of bacteria in the hemolymph and the mortality of crabs increased following infection with V. parahaemolyticus after SpLyzC expression was silenced by injecting double-strand RNA of SpLyzC. The recombinant protein of the S. paramamosain c-type lysozyme (rSpLyzC) exhibited antibacterial activities against Micrococcus lysodeikticus, S. aureus, Vibrio harveyi and V. parahaemolyticus. These results indicate that SpLyzC could help eliminate bacteria in S. paramamosain and may play an important role in resistance to bacterial infection.

Differential Recognition of Vibrio parahaemolyticus OmpU by Toll-Like Receptors in Monocytes and Macrophages for the Induction of Proinflammatory Responses.

Vibrio parahaemolyticus is a human pathogen, and it is a major cause of severe gastroenteritis in coastal areas. OmpU is one of the major outer membrane porins of V. parahaemolyticus Host-immunomodulatory effects of V. parahaemolyticus OmpU (VpOmpU) have not been elucidated yet. In this study, in an effort towards characterizing the effect of VpOmpU on innate immune responses of the host, we observed that VpOmpU is recognized by the Toll-like receptor 1/2 (TLR1/2) heterodimer in THP-1 monocytes but by both TLR1/2 and TLR2/6 heterodimers in RAW 264.7 macrophages. To the best of our knowledge, this is the first report of a natural pathogen-associated molecular pattern (PAMP) recognized by both TLR1/2 and TLR2/6 heterodimers; so far, mainly the synthetic ligand Pam2CSK4 has been known to be recognized by both the TLR1/2 and TLR2/6 heterodimers. We also have shown that VpOmpU can activate monocytes and macrophages, leading to the generation of proinflammatory responses as indicated by tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and NO production in macrophages and TNF-α and IL-6 production in monocytes. VpOmpU-mediated proinflammatory responses involve MyD88-IRAK-1 leading to the activation of mitogen-activated protein (MAP) kinases (p38 and Jun N-terminal protein kinase [JNK]) and transcription factors NF-κB and AP-1. Further, we have shown that for the activation of macrophages leading to the proinflammatory responses, the TLR2/6 heterodimer is preferred over the TLR1/2 heterodimer. We have also shown that MAP kinase activation is TLR2 mediated.

Two host gut-derived lactic acid bacteria activate the proPO system and increase resistance to an AHPND-causing strain of Vibrio parahaemolyticus in the shrimp Litopenaeus vannamei.

Lactic acid bacteria (LAB) are group of beneficial bacteria that have been proposed as relevant probiotics with immunomodulatory functions. In this study, we initially isolated and identified host-derived LAB from the gut of the Pacific white shrimp Litopenaeus vannamei. Analysis of the bacterial 16S rRNA gene sequence revealed two candidate LAB, the Lactobacillus plantarum strain SGLAB01 and the Lactococcus lactis strain SGLAB02, which exhibited 99% identity to the L. plantarum strain LB1-2 and the L. lactis strain R-53658, which were isolated from bee gut, respectively. The two LAB displayed antimicrobial activities against gram-positive and gram-negative bacteria, including the virulent acute hepatopancreatic necrosis disease (AHPND)-causing strain of Vibrio parahaemolyticus (VPAHPND). Viable colony count and SEM analysis showed that the two candidate LAB, administered via oral route as feed supplement, could reside and adhere in the shrimp gut. Double-stranded RNA-mediated gene silencing of LvproPO1 and LvproPO2 revealed a significant role of two LvproPOs in the proPO system as well as in the immune response against VPAHPND infection in L. vannamei shrimp. The effect of LAB supplementation on modulation of the shrimp proPO system was investigated in vivo, and the results showed that administration of the two candidate LAB significantly increased hemolymph PO activity, the relative mRNA expression of LvproPO1 and LvproPO2, and resistance to VPAHPND infection. These findings suggest that administration of L. plantarum and L. lactis could modulate the immune system and increase shrimp resistance to VPAHPND infection presumably via upregulation of the two LvproPO transcripts.

Molecular cloning and functional characterization of a homolog of the transcriptional regulator CSL in Litopenaeus vannamei.

The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag-1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1-DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation-related genes (LvHey2 and LvAstakine), and attenuated the expression of immune-related genes L. vannamei hypoxia inducible factor alpha (LvHIF-α), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens.

Novel C-type lectin from razor clam Sinonovacula constricta agglutinates bacteria and erythrocytes in a Ca2+-dependent manner.

Among its other physiological roles, C-type lectins functioned as pattern recognition receptors (PRR) in innate immunity received much attention. In the present study, a novel C-type lectin was identified and characterized from the invertebrate razor clam Sinonovacula constrict and designated as ScCTL. The complete cDNA sequence of ScCTL was 828 bp in length and coded a secreted polypeptide of 158 amino acids with a typical CRD domain. Multiple sequence alignments combined with phylogenetic analysis both collectively confirmed that ScCTL was a novel member belong to lectin family. Spatial expression distribution analysis revealed that ScCTL was extensively expressed in all of the examined tissues, and the highest expression was detected in the hepatopancreas. After 1 × 107 CFU/mL Vibrio parahaemolyticus challenge by immersion infection, the ScCTL transcript in hepatopancreas and gill were markedly upregulated and arrived the maximum levels at 24 or 12 h after challenge, respectively. Recombinant ScCTL could agglutinate not only all tested bacteria but sheep and mouse erythrocyte in the presence of Ca2+. All of our studies suggested that ScCTL performed important roles in protecting cells from pathogenic infection in S. constrict.

Identification and characterization of a laccase from Litopenaeus vannamei involved in anti-bacterial host defense.

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defences of various invertebrates. Whether or not laccase exists in shrimp and its function is still poorly understood. In this study, a laccase (LvLac) was cloned and identified from Litopenaeus vannamei for the first time. The full length of LvLac is 3406 bp, including a 2034 bp open reading frame (ORF) coding for a putative protein of 677 amino acids with a signal peptide of 33 aa. LvLac contains three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine, respectively. Phylogenetic analysis revealed that LvLac was close to insects laccase 1 family. LvLac expression was most abundant in heart and the crude LvLac protein could catalyze the oxidation of hydroquinone. Real-time PCR showed that LvLac expression was responsive to Vibrio parahaemolyticus, Micrococcus lysodeikticus and white spot syndrome virus (WSSV) infection. Knockdown of LvLac enhanced the sensitivity of shrimps to V. parahaemolyticus and M. lysodeikticus challenge, suggesting that LvLac may play a positive role against bacterial pathogens.

Identification and functional analysis of a TEP gene from a crustacean reveals its transcriptional regulation mediated by NF-κB and JNK pathways and its broad protective roles against multiple pathogens.

Thioester-containing proteins (TEPs) are present in a wide range of species from deuterostomes to protostomes and are thought to be involved in innate immunity. In the current study, a TEP gene homologous to insect TEPs (iTEP) from the crustacean Litopenaeus vannamei, named LvTEP1, is cloned and functionally characterized. The open reading frame (ORF) of LvTEP1 is 4383 bp in length, encoding a polypeptide of 1460 amino acids with a calculated molecular weight of 161.1 kDa LvTEP1, which is most similar to other TEPs from insects, contains some conserved sequence features, including a N-terminal signal peptide, a canonical thioester (TE) motif, and a C-terminal distinctive cysteine signature. LvTEP1 is expressed in most immune-related tissues, such as intestine, epithelium, and hemocytes, and the mRNA level of LvTEP1 is upregulated in hemocytes after bacterial and viral challenges, indicating its involvement in the shrimp innate immune response. An expression assay in Drosophila S2 cells shows LvTEP1 to be a full-length secretory protein, and processed forms are present in the supernatant. Of note, only the processed form of LvTEP1 protein can bind to both the gram-negative bacterium Vibrio parahaemolyticus and the gram-positive bacterium Staphylococcus aureus in vitro, and its abundance can be induced after bacterial treatment. Moreover, knockdown of LvTEP1 renders shrimps more susceptible to both V. parahaemolyticus and S. aureus, as well as white spot syndrome virus (WSSV) infection, suggesting its essential defensive role against these invading microbes. We also observe that the expression of LvTEP1 is regulated in a manner dependent on both NF-κB and AP-1 transcription factors in naive shrimps and in vitro, suggesting that LvTEP1 could be poised in the body cavity prior to infection and thus play an important role in basal immunity. Taken together, our findings provide some in vitro and in vivo evidence for the involvement of LvTEP1 in shrimp innate immunity and provide some insight into its expression regulation mediated by multiple transcription factors or signaling pathways.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. The full-length 2492 bp TRAF6 (Sp-TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N-terminal RING-type zinc finger, two TRAF-type zinc fingers and a conserved C-terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp-TRAf6 has highest identity of 88% with Pt-TRAF6 from Portunus trituberculatus, while the similarity of Sp-TRAF6 with other crustacean sequences was 54-55%. RT-PCR analysis indicated that Sp-TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp-TRAF6 transcripts were significantly up-regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp-TRAF6. The qRT-PCR results showed that silencing Sp-TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute-phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp-TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain.

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs).

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1ß and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Report of leucine-rich repeats (LRRs) from Scylla serrata: Ontogeny, molecular cloning, characterization and expression analysis following ligand stimulation, and upon bacterial and viral infections.

Leucine-rich repeat (LRR) proteins are present in all living organisms, and their participation in signal transduction and defense mechanisms has been elucidated in humans and mosquitoes. LRRs possibly involve in protein-protein interactions also and show differential expression pattern upon challenge with pathogens. In the present study, a new LRR gene was identified in mud crab, Scylla serrata. LRR gene mRNA levels in different developmental stages and various tissues of S. serrata were analysed. Further, the response of the gene against different ligands, Gram-negative bacterium, and white spot syndrome virus (WSSV) was investigated in vitro and in vivo. Full-length cDNA sequence of S. serrata LRR (SsLRR) was found to be 2290 nucleotide long with an open reading frame of 1893bp. SsLRR encodes for a protein containing 630 deduced amino acids with 17 conserved LRR domains and exhibits significant similarity with crustacean LRRs so that these could be clustered into a branch in the phylogenetic tree. SsLRR mRNA transcripts were detected in all the developmental stages (egg, Zoea1-5, megalopa and crab instar), haemocytes and various tissues such as, stomach, gill, muscle, hepatopancreas, hematopoietic organ, heart, epithelial layer and testis by reverse-transcriptase PCR. SsLRR transcripts in cultured haemocytes showed a 2-fold increase in expression at 1.5 and 12h upon Poly I:C induction. WSSV challenge resulted in significant early up-regulation at 3h in-vitro and late up-regulation at 72h in-vivo. Peptidoglycan (PGN)-induction resulted in marginal up-regulation of SsLRR at timepoints, 6, 12 and 24h (fold change below 1.5) and no significant change in the expression at early timepoints. LPS-stimulation, on the other hand, showed either down-regulation or normal level of expression at all timepoints. However, a delayed 5-fold up-regulation was observed in vivo against Vibrio parahaemolyticus infection at 72hpi. The constitutive expression of the LRR gene in all the early life-stages, and its response to various ligands and to viral challenge suggest the possible role of the LRR in immune defense in mud crab. The result provides additional information which would help in future studies in understanding the innate immune pathways in crustaceans.

Differentially expressed transcripts in stomach of Penaeus monodon in response to AHPND infection.

Acute Hepatopancreatic Necrosis Disease (AHPND) is an emerging disease in aquacultured shrimp caused by a pathogenic strain of Vibrio parahaemolyticus. As with several pathogenic bacteria, colonization of the stomach appeared to be the initial step of the infection for AHPND-causing Vibrio. To understand the immune responses in the stomach of black tiger shrimp (Penaeus monodon), differentially expressed transcripts (DETs) in the stomach during V. parahaemolyticus strain 3HP (VP3HP) infection was examined using Ion Torrent sequencing. From the total 42,998 contigs obtained, 1585 contigs representing 1513 unigenes were significantly differentially expressed with 1122 and 391 unigenes up- and down-regulated, respectively. Among the DETs, there were 141 immune-related unigenes in 10 functional categories: antimicrobial peptide, signal transduction pathway, proPO system, oxidative stress, proteinases/proteinase inhibitors, apoptotic tumor-related protein, pathogen recognition immune regulator, blood clotting system, adhesive protein and heat shock protein. Expression profiles of 20 of 22 genes inferred from RNA sequencing were confirmed with the results from qRT-PCR. Additionally, a novel isoform of anti-lipopolysaccharide factor, PmALF7 whose transcript was induced in the stomach after challenge with VP3HP was discovered. This study provided a fundamental information on the molecular response in the shrimp stomach during the AHPND infection that would be beneficial for future research.

B-cell activating CpG ODN 1668 enhance the immune response of Pacific red snapper (Lutjanus peru) exposed to Vibrio parahaemolitycus.

B-class CpG ODN 1668 is known to possess clear immunostimulatory properties. In this study, we investigated the potential ability of CpG ODN 1668 to enhance the immune response of Pacific red snapper exposed to Vibrio parahaemolyticus. Four different treatments were evaluated in Pacific red snapper: (1) stimulatory CpG ODN 1668, (2) stimulatory CpG ODN 1668 and V. parahaemolyticus, (3) exposure only to V. parahaemolyticus and (4) PBS. Samples were taken at 24, 72, 168 and 240 h of stimulation/infection. The results show that intraperitoneal injection of CpG-ODN 1668 enhanced the anti-protease, superoxide dismutase and catalase activities in serum. CpG ODN 1668 upregulated TLR9 and IgM gene expression in head-kidney, intestine and skin, with higher expression in head-kidney. A higher correlation was observed between TLR9 and IgM in head-kidney and intestine. Finally, no histopathological damages were observed in fish stimulated with CpG ODN 1668. In contrast, melanomacrophages-like structures were present in higher numbers in infected fish. Taken together, these results indicate that CpG ODN 1668 activates innate immune response and upregulate the TLR9 and IgM-mediated immune response. These results may be exploited for the control of Vibriosis in farmed Pacific red snapper.

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) stimulates prophenoloxidase activating system in Chinese mitten crab (Eriocheir sinensis).

Melanization mediated by prophenoloxidase (proPO) activating system play an essential role in killing invading microorganisms in invertebrates. Lipopolysaccharide and ß-1, 3-glucan binding protein (LGBP) as a pattern recognition protein have been demonstrated to active the proPO cascade in insect and shrimp. In this study, we investigated the role of LGBP in prophenoloxidase cascade-induced melanization in Chinese mitten crab (Eriocheir sinensis). By RT-PCR analysis, EsLGBP was detected in all tested tissues, and showed highest expression in hemocytes, gill, intestine and brain. The expression of EsLGBP was up-regulated in the hemocytes following injections of LPS and ß-1, 3-glucan. The recombinant EsLGBP protein (rEsLGBP) was produced via prokaryotic expression system and affinity chromatography. By western blotting, rEsLGBP was discovered to exhibit the ability to bind to all tested microorganisms, including Gram-negative bacteria, Gram-positive bacteria and yeast (Pichia pastoris). Meanwhile we found rEsLGBP has a high binding activity towards microbial immune elicitors such as LPS and ß-1, 3-glucan whereas no binding activity is detected with peptidoglycan. Moreover, the effects of RNAi-mediated blockade of EsLGBP were investigated on bacterial counts in the hemolymph and cumulative mortality rate of crabs infected with Vibrio parahaemolyticus in vivo. Further experiments demonstrate that rEsLGBP can trigger the whole hemolymph dependent melanization and stimulate to proPO cascade in vitro. Taken together, these results provide experimental evidence for role of LGBP in innate immunity, especially in the activation of prophenoloxidase activating system.