Effects of replacing fishmeal with soybean meal on the immune and antioxidant capacity, and intestinal metabolic functions of red swamp crayfish Procambarus clarkii.

Excess utilization of plant protein sources in animal feed has been found to adversely affect the antioxidant properties and immunity of animals. While the role of gut microbes in plant protein-induced inflammation has been identified in various models, the specific mechanisms regulating gut microbes in crustaceans remain unclear. Accordingly, this study was designed to investigate the effects of replacing fishmeal with soybean meal (SM) on the hepatopancreas antioxidant and immune capacities, and gut microbial functions of crayfish, as well as the potential microbial regulatory mechanisms. 750 crayfish (4.00 g) were randomly divided into five groups: SS0, SS25, SS50, SS75, and SS100, and fed diets with different levels of soybean meal substituted for fishmeal for six weeks. High SM supplementation proved detrimental to maintaining hepatopancreas health, as indicated by an increase in hemolymph MDA content, GPT, and GOT activities, the observed rupture of hepatopancreas cell basement membranes, along with the decreased number of hepatopancreatic F cells. Moreover, crayfish subjected to high SM diets experienced obvious inflammation in hepatopancreas, together with up-regulated mRNA expression levels of nfkb, alf, and tlr (p<0.05), whereas the lzm mRNA expression level exhibited the highest value in the SS25 group. Furthermore, hepatopancreas antioxidant properties highly attenuated by the level of dietary SM substitution levels, as evidenced by the observed increase in MDA content (p<0.05), decrease in GSH content (p<0.05), and inhabitation of SOD, CAT, GPx, and GST activities (p<0.05), along with down-regulated hepatopancreas cat, gpx, gst, and mmnsod mRNA expression levels via inhibiting nrf2/keap1 pathway. Functional genes contributing to metabolism identified that high SM diets feeding significantly activated lipopolysaccharide biosynthesis, revealing gut dysfunction acted as the cause of inflammation. The global microbial co-occurrence network further indicated that the microbes contributing more to serum indicators and immunity were in module eigengene 17 (ME17). A structural equation model revealed that the genes related to alf directly drove the serum enzyme activities through microbes in ME17, with OTU399 and OTU533 identified as major biomarkers and classified into Proteobacteria that secrete endotoxins. To conclude, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the innate immune system of crayfish.

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.

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 effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

iTRAQ-based proteomic analysis of the hepatopancreas from Litopenaeus vannamei after trans-vp28 gene Synechocystis sp. PCC6803 immunization.

Litopenaeus vannamei (Pacific white shrimp) is one of the most commercially important varieties of shrimp cultivated in the world. Shrimp farming is a high-risk, capital-intensive industry that is susceptible to periodic outbreaks of diseases caused by viral and bacterial pathogens. Thus, there is a need to develop economically viable methods of disease control. The hepatopancreas of crustaceans are known to have an important role in their innate immune response. In this study, we have explored the immune response of the hepatopancreas from L. vannamei fed with trans-vp28 gene Synechocystis sp. PCC6803 using iTRAQ-based proteomics. A total of 214 differentially expressed proteins (DEPs) were identified, of which 143 were up-regulated and 71 were down-regulated. These proteins have diverse roles in the cell cytoskeleton and cell phagocytosis, antioxidant defense process and the response of immune related proteins. Among these proteins, the immunity associated with the functional annotation of L. vannamei was further analysed. In addition, 4 DEPs (act1, N/A, H and C7M84_013542) were analysed using parallel reaction monitoring (PRM). This is the first report of proteomics in the hepatopancreas of L. vannamei immunized with trans-vp28 gene Synechocystis sp. PCC6803.

The red swamp crayfish, Procambarus clarkii cathepsin C, participates in the innate immune response to the viral and bacterial pathogens.

The cathepsin C, a lysosomal cysteine protease, involves the modulation of immune and inflammatory responses in living organisms. However, the knowledge on cathepsin C in red swamp crayfish (Procambarus clarkii), a freshwater crustacean with economic values, remained unclear. In the present study, we provide identification and molecular characterization of cathepsin C from P. clarkii. (Hereafter Pc-cathepsin C). The Pc-cathepsin C cDNA contained a 1356 bp open reading frame that encoded a protein of 451 amino acid residues. The deduced amino acid sequence comprised of cathepsin C exclusion domain and pept_C1 domain, and also catalytic residues (Cys248, His395 and Asn417). Analysis of the transcriptional patterns of the Pc-cathepsin C gene revealed that it was broadly distributed in various tissues of P. clarkii, and it was more abundant in the hepatopancreas and gut. Following a challenge with viral and bacterial pathogen-associated molecular patterns, the expression of Pc-cathepsin C was strongly enhanced at different time points. The knockdown of Pc-cathepsin C, altered the expression of immune-responsive genes, suggesting its immunoregulatory role in P. clarkii. This study has identified and provided the immunoregulatory function of Pc-cathepsin C, which will contribute to further investigation of the molecular mechanism of cathepsin C in crustaceans.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

The response of phenoloxidase to cadmium-disturbed hepatopancreatic immune-related molecules in freshwater crayfish Procambarus clarkii.

There has been extensive research on local and systemic oxidative stress and immunosuppression in cadmium exposed crustaceans, but the underlying mechanisms remain to be elucidated. Because of multiple functions of epithelial cells, such as storing and detoxifying heavy metals, producing and secreting immune-related molecules (i.e. hemocyanin, NF-κB and CBS/H2S et al.), hepatopancreas may play an important role in immune system. In the present study, as an indication of systemic and local immune status in crayfish Procambarus clarkii, the relationship between PO activities in haemolymph and levels of CBS/H2S/NF-κBp65 in hepatopancreas was evaluated following a 96 h exposure to sub-lethal Cd2+ concentrations (1/40, 1/8 and 1/4 of the 96 h LC50). The results indicated that there was a significant increase in ROS contents accompanied by markedly decreased THC and PO levels (P < 0.01) in a dose- and time- dependent manner. The evolutionarily conserved CBS and NF-κB p65 showed obvious difference (P < 0.01) (including cellular distribution and expression level) between the healthy and pathological conditions based on IHC analysis. Even small change of endogenous H2S content may be closely related to NF-κB p65 level and PO activity (P < 0.01). There was significantly negative correlation (P < 0.05) between PO activity and expression levels of CBS and NF-κB p65. Obviously, crayfish innate immunity was a highly complex network of various cells, molecules, and signaling pathways which operate, at least partly, through small signaling molecules such as H2S. ROS-mediated CBS/H2S/NF-κB pathway probably allowed hepatopancreas to inhibit PO activity under cadmium stress.

Transcriptome analysis of Procambarus clarkii infected with infectious hypodermal and haematopoietic necrosis virus.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) is a major viral pathogen in cultured penaeid shrimp. IHHNV has many hosts, mainly including crustaceans. It has recently been reported that Procambarus clarkii can be infected by IHHNV. In the present study, we studied the hepatopancreas of P. clarkii by transcriptome high-throughput sequencing to analyze the response of P. clarkii to IHHNV infection. After de novo assembly, there were 400,340,760 clean reads. A total of 237 differentially expressed genes (DEGs) were obtained, including 77 significantly up-regulated unigenes and 160 significantly down-regulated ones. The expression levels of 12 immune-related DEGs were validated by qRT-PCR, substantiating the reliability of RNA-Seq results. The enrichment analysis of DEGs showed that the immune-related pathways were closely related to apoptosis and phagocytosis. Moreover, a large number of pathways related to metabolic function were down-regulated, suggesting that IHHNV infection might affect the growth of P. clarkii.

Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge.

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

Evolutionary and functional analysis of Cyclina sinensis c-Jun AP-1 gene in response to LPS stimulation.

The transcription factor activator protein-1 (AP-1) plays an essential and critical role in the regulation of numerous downstream genes involved in various physiological and chemical responses. In this study, we identified a full-length cDNA of the c-Jun AP-1 gene (termed Csc-Jun) from the transcriptome library in Cyclina sinensis. The cDNA contains an 825-bp open reading frame that encodes a 274-amino acid protein sequence, including a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature that shares 90% identity to that of Ruditapes philippinarum. Furthermore, a phylogenetic analysis using MrBayes and PhyML software (with Bayesian and maximum likelihood approaches, respectively) revealed that the c-Jun AP-1 family genes might be involved in adapting to various environments in different invertebrates. We implemented the PAML software with the maximum likelihood method to further select and verify the positive selection sites (PSSs) in the Mollusca c-Jun AP-1 genes, and we detected four PSSs located in the Jun transcription factor domain. In addition, a spatial expression analysis showed that the Csc-Jun cDNA transcript was ubiquitously expressed in all of the tested tissues and was strongly expressed in the hepatopancreas and weakly expressed in the tissues of the hemocytes, gill filaments, mantle and adductor muscle. Quantitative real-time PCR showed that the expression profiles of Csc-Jun were significantly upregulated at different times in all of the tested tissues when challenged with lipopolysaccharide (LPS). Furthermore, knockdown of Csc-Jun by RNA interference resulted in a higher mortality of C. sinensis following LPS exposure. Finally, we explored the function of the TLR13-MyD88 signaling pathway in the innate immunity of C. sinensis by RNA interference and immune challenges. The results revealed that the mRNA expression levels of Csc-Jun were all decreased (P < 0.01) in normal and stimulated C. sinensis hemocytes. These data collectively indicated that the c-Jun AP-1 gene might play vital roles in innate immunity and provide new evidence for the evolutionary patterns of innate immune genes in Mollusca.

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.

Expression of Cathepsin F in response to bacterial challenges in Yesso scallop Patinopecten yessoensis.

Cathepsin F is a unique papain cysteine proteinase with highly conserved structures: catalytic triad and a cystatin domain contained in the elongated N-terminal pro-region. It has been reported that cathepsin F is associated with the establishment of innate immune in several vertebrate including fish in aquaculture, but not known in bivalves. In this study, we firstly identified and characterized cathepsin F in the Yesso scallop (Patinopecten yessoensis). The protein structural and phylogenetic analyses were then conducted to determine its identity and evolutionary position. We've also investigated the expression levels of cathepsin F gene at different embryonic developmental stages, in healthy adult tissues and especially in the hemocytes and hepatopancreas after Gram-positive (Micrococcus luteus) and negative (Vibrio anguillarum) challenges using quantitative real-time PCR (qPCR). Cathepsin F was significantly up-regulated 3 h after infection of V. anguillarum in hemocytes, suggesting its participation in immune response. Our findings have provided strong evidence that cathepsin F may be a good target for enhancing the immune activity in Yesso scallop.

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.

Chicken-type lysozyme functions in the antibacterial immunity in red swamp crayfish, Procambarus clarkii.

Lysozymes possess antibacterial activities, making them crucial defense proteins in innate immunity. In this study, a chicken-type (c-type) lysozyme (designated PcLyzc) was cloned and characterized from red swamp crayfish Procambarus clarkii. The full-length cDNA had an open reading frame of 435 base pairs encoding a polypeptide of 144 amino acid residues. Multiple alignments and phylogenetic analysis revealed that PcLyzc shared high similarity to the other known invertebrate c-type lysozymes. PcLyzc transcripts were steadily expressed in a wide range of tissues in healthy crayfish, and were prominently up-regulated in the hepatopancreas and gills after Vibrio anguillarum or Aeromonas hydrophila challenge. Recombinant PcLyzc showed inhibitory activity in vitro against both Gram-positive bacteria, including Staphylococcus aureus, Micrococcus luteus and Bacillus thuringiensis, and Gram-negative bacteria, including A. hydrophila, V. anguillarum and Escherichia coli. By overexpressing PcLyzc through introducing exogenous recombinant protein, or silencing PcLyzc expression through injecting double strand RNA, it was found that PcLyzc could help eliminate the invading bacteria in crayfish hemolymph and could protect crayfish from death, possibly by promoting the hemocytic phagocytosis. These results indicated that PcLyzc played a role in the antibacterial immunity of crustaceans, and laid a foundation of developing new therapeutic agents in aquaculture.

A novel C1q-domain-containing protein from razor clam Sinonovacula constricta mediates G-bacterial agglutination as a pattern recognition receptor.

Complement component 1q (C1q) with a characteristic C1q globular domain is an important pattern recognition molecule in the classical complement systems and plays a major role in the crosslinking between innate immunity and specific immunity in vertebrates. In this study, a homologous gene encoding typically C1q domains was obtained from the razor clam Sinonovacula constricta (designated ScC1qDC) by rapid amplification of the cDNA end. The full-length cDNA of ScC1qDC was 1225 bp in length with a 5'UTR of 258 bp, a 3'UTR of 223 bp, and an open reading frame of 744 bp encoding a polypeptide of 247 amino acids containing a typical C1q globular domain. The mRNA transcripts of ScC1qDC were constitutively transcribed in all examined tissues with higher expression in the hepatopancreas. Time-course expression analysis indicated that ScC1qDC was significantly up-regulated both in hepatopancreas and gills after Vibrio parahaemolyticus challenge. The recombinant ScC1qDC (rScC1qDC) displayed high binding activities to various pathogen-associated molecular patterns, including LPS, PGN, and MAN. Recombinant ScC1qDC showed no agglutinating activity to Gram-positive bacterium of Micrococcus luteus but showed obvious activities towards all the three examined Gram-negative bacteria. All our results indicated that ScC1qDC might be served as a pattern recognition receptor and promoted Gram-negative bacteria agglutination during the pathogen challenge.

Transcriptome profiling of red swamp crayfish (Procambarus clarkii) hepatopancreas in response to lipopolysaccharide (LPS) infection.

The RNA-sequencing followed by de novo assembly generated 61,912 unigene sequences of P. clarkii hepatopancreas. Comparison of gene expression between LPS challenged and PBS control samples revealed 2552 differentially expressed genes (DEGs). Of these sequences, 1162 DEGs were differentially up-regulated and 1360 DEGs differentially down-regulated. The DEGs were then annotated against gene ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Some immune-related pathways such as PPAR signaling pathway, lysosome, Chemical carcinogenesis, Peroxisome were predicted by canonical pathways analysis. The reliability of transcriptome data was validated by quantitative real time polymerase chain reaction (qRT-PCR) for the selected genes. The data presented here shed light into antibacterial immune responses of crayfish. In addition, these results suggest that transcriptomic data provides valuable sequence resource for immune-related gene identification and helps to understand P. clarkii immune functions.

A role of tumor susceptibility gene 101 (TSG101) in innate immune response of crayfish Procambarus clarkii.

Tumor susceptibility gene 101 (TSG101) is a multi-functional gene involved in cell growth and proliferation in vertebrates. However, its role in the innate immune response of crustaceans remains unclear. Here, a TSG101 gene was identified in crayfish Procambarus clarkii with an open reading frame of 1320 bp that encoded a predicted 48.3-kDa protein highly homologous to those in other invertebrates. TSG101 mRNA was highly expressed in stomach and hepatopancreas, and its expression was induced significantly in different tissues (hemocytes, gills and intestine) by lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C) with various expression patterns. Recombinant TSG101 protein was expressed in Escherichia coli, and a possible protein-protein interaction between TSG101 and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was explored by far-western blotting. RNA interference of TSG101 affected the gene expression of members of the Toll pathway. These results suggest that TSG101 is involved in the innate immune responses of P. clarkii.

The fragmentation mechanism and immune-protective effect of CfTEP in the scallop Chlamys farreri.

Thioester-containing proteins (TEPs), characterized by a unique intrachain ß-cysteinyl-γ-glutamyl thioester bond, form an ancient and diverse family of secreted proteins that play central roles in the innate immune response. But the existence form and immune protection mechanism of TEP in invertebrates still remain unclear, especially in the mollusks. The fragmentation and the immune-protective effect of thioester bond in CfTEP, a previously identified thioester-containing protein in scallop Chlamys farreri, were characterized in the present study. During the early embryonic development of scallop, the mRNA transcript of CfTEP could be detected in all the stages, and its expression levels in D-larvae, veliger larvae and eye-spot larvae were significantly higher than that in eggs. The CfTEP protein was also detected in peripheral of D-larvae, veliger larvae and eye-spot larva by immunofluorescence. In the adult scallop, the CfTEP protein was mainly distributed in the hepatopancreas, gill, kidney, gonad, and mantle. The expression of CfTEP mRNA in the hemocytes of adult scallop was significantly up-regulated when the scallops were stimulated by LPS, PGN or ß-glucan. Two bands (100 and 55 kDa) were detected using anti-CfTEP-R1 (spanned the C-terminal portion of the thioester, A2M-comp and A2M-recep domain, 942-1472), and a single band (46 kDa) was detected by using anti-CfTEP-R2 (the N-terminal portion of the following A2M-N-2 domain, 452-496) in the serum of scallop at 12 h after LPS stimulation. When the thioester bond of CfTEP protein was inactivated by injecting methylamine, the survival rate of scallop was significantly decreased after challenged by Vibrio angulillarum. All these results suggested that CfTEP protein existed as fragments similar to vertebrate C3, and played central roles in the immune response against pathogen in the innate immunity of scallops.

Characterization and expression analysis of serpinB3, the first clade B serine protease inhibitor in Pacific white shrimp, Litopenaeus vannamei.

Currently, about nine serpin clades (A-I) were preferentially observed in higher animals and clustered on the basis of function. Of these, eight clades contain extracellular proteins, while clade B contains predominantly intracellular proteins. In the present study, the first clade B serpin (named LvserpinB3) was identified from the Pacific white shrimp Litopenaeus vannamei. LvserpinB3 encoded a 412-amino acid protein with a 19-amino acid signal peptide and a serpin domain. Moreover, a transmembrane helix (TMHs) was predicted to be located on the N-terminal of LvserpinB3. Alignment with the cDNA sequence indicated that the genomic LvserpinB3 gene contains 2 exons and 1 intron. The P1-P1' scissile bond of the core feature reactive center loop (RCL) represented for Arginine-Isoleucine (RI), which was in accordance with PmserpinB3, Msserpin-4, -5 and -7. The highest mRNA expression level of LvserpinB3 was detected in hepatopancreas. A significant decrease of LvserpinB3 was detected in hepatopancreas at 6 h post Vibrio anguillarum injection, and later on, the expression of LvserpinB3 was remarkably elevated at 24 h post bacterial challenge. Suppression of LvserpinB3 in vivo by double-stranded RNA (dsRNA) mediated RNA interference (RNAi) led to a significant increase in the transcripts of LvSP1 (Serine protease 1), LvPPAE2 (Prophenoloxidase-activating Enzyme 2) and cumulative mortality. Furthermore, rLvserpinB3 protein was expressed and purified in vitro for the prophenoloxidase inhibition assay. The rLvserpinB3 protein can strongly impede the extent of proPO cascade. All above imply that LvserpinB3 might be an inhibitor for prophenoloxidase-activating system.