Occurrence of Bothriocephalus acheilognathi in cyprinid fish from three lakes in the flood plain of the Yangtze River, China

Cyprinid fish, Hemiculter leucisculus, Cultrichthys erythropterus and Culter dabryi, were sampled from Liangzi, Honghu and Tangxun lakes in the flood plain of the Yangtze River. The cestode Bothriocephalus acheilognathi Yamaguti, 1934 was found in the 3 lakes, but C. erythropterus sampled from Liangzi lake was found uninfected due probably to the small sample size. Findings of the cestode in the 3 lakes represent the first record of the parasite in the flood plain of the Yangtze River, indicating that B. acheilognathi may be distributed much wider in China than previously recognized.

A detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription polymerase chain reaction

A rapid, sensitive and highly specific detection method for grass carp hemorrhagic virus (GCHV) based on a reverse transcription-polymerase chain reaction (RT-PCR) has been developed. Two pairs of PCR primers were synthesized according to the cloned cDNA sequences of the GCHV-861 strain. For each primer combination, only one specific major product was obtained when amplification was performed by using the genomic dsRNA of GCHV-861 strain. The lengths of their expected products were 320 and 223 bp, respectively. No products were obtained when nucleic acids other than GCHV-861 genomic RNA were used as RT-PCR templates. To assess the sensitivity of the method, dilutions of purified GCHV-861 dsRNA total genome (0.01 pg up to 1000 pg) were amplified and quantities of as little as 0.1 pg of purified dsRNA were detectable when the amplification product was analyzed by 1.5% agarose gel electrophoresis. This technique could detect GCHV-861 not only in infected cell culture fluids, but also in infected grass carp Ctenopharyngodon idellus and rare minnow Gobiocypris rarus with or without hemorrhagic symptoms. The results show that the RT-PCR amplification method is useful for the direct detection of GCHV.

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG). in concentrations of 5-30% for 10, 30, or 60 min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30 min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30 min exposure, the hatching rate of the embryos immersed in PG was 93.3 +/- 7.0% (mean +/- S.D.), however. in DMSO. EG, Gly. and MeOH, it was 82.7 +/- 10.4, 22.0 +/- 5.7, 0.0 +/- 0.0, and 0.0 +/- 0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P < 0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used. (C) 2008 Elsevier Inc. All rights reserved.

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Three members of the tetraspanin/TM4SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM4SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM4SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression

Clip domain serine protease (cSP), characterized by conserved clip domains, is a new serine protease family identified mainly in arthropod, and plays important roles in development and immunity. In the present study, the full-length cDNA of a cSP (designated EscSP) was cloned from Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1380 bp EscSP cDNA contained a 1152 bp open reading frame (ORF) encoding a putative cSP of 383 amino acids, a 5'-untranslated region (UTR) of 54 bp, and a 3'-UTR of 174 bp. Multiple sequence alignment presented twelve conserved cysteine residues and a canonical catalytic triad (His(185), Asp(235) and Ser(332)) critical for the fundamental structure and function of EscSP. Two types of cSP domains, the clip domain and tryp_spc domain, were identified in the deduced amino acids sequence of EscSP. The conservation characteristics and similarities with previously known cSPs indicated that EscSP was a member of the large cSP family. The mRNA expression of EscSP in different tissues and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EscSP mRNA transcripts could be detected in all examined tissues, and were higher expressed in muscle than that in hepatopancreas. gill, gonad, haemocytes and heart. The EscSP mRNA expression in haemocytes was up-regulated after L anguillarum challenge and peaked at 2 h (4.96 fold, P < 0.05) and 12 h (9.90 fold, P < 0.05). Its expression pattern was similar to prophenoloxidase (EsproPO), one of the components of crab proPO system found in our previous report. These results implied that EscSP was involved in the processes of host-pathogen interaction probably as one of the proPO system members. (C) 2009 Elsevier Ltd. All rights reserved.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes; (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans. (C) 2009 Elsevier Ltd. All rights reserved.

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares similar to 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia colt, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular beta-agarase. E. coli DH5 alpha harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5 alpha/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5 alpha/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5 alpha/pTAET21, is an effective vaccine candidate against E. tarda infection. (C) 2009 Elsevier Ltd. All rights reserved.

First molluscan TNFR homologue in Zhikong scallop: Molecular characterization and expression analysis

Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge

Arthropod defence responses (e.g. prophenoloxidase (proPO) activation and Toll pathway initiation) are mediated by serine proteinase cascades and regulated by serpins in haemolymph. A serpin (Fc-serpin) cDNA was cloned from the haemocytes of Fenneropenaeus chinensis by rapid amplification of cDNA ends (RACE) PCR and haemocyte cDNA library screening. The full-length cDNA consists of 1734 bp, encoding 411 amino acids with a calculated molecular mass of 46.55 kDa and a theoretical isoelectric point of 7.70. Fc-serpin contains a typical serpin-like homologue (serine proteinase inhibitors domain). The deduced protein contains a putative signal peptide of 19 amino acids and the serpin's signature sequence ((FHCNRPFLFLI389)-F-379). Fc-serpin showed some identity with Pacifastacus leniusculus serpin (42%) and Manduca sexta serpin-6 (34%). The reactive centre loop (RCL) sequences of Fc-serpin, P leniusculus serpin, M. sexta serpin-6 and Bombyx mori serpin-2 are highly similar. An Arg at the PI position of the reactive site indicates that Fc-serpin may have inhibitory activity against prophenoloxidase activating proteinase (PAP) and clotting enzyme. Transcripts of Fc-serpin mRNA were mainly detected in haemocytes and the lymphoid organ by RT-PCR. The variation of the mRNA transcription level in haemocytes followed by artificial infection with bacteria OF white spot syndrome virus (WSSV) was quantified by SYBR Green real-time PCR analysis. Expression profiles of Fc-serpin greatly fluctuated after challenge. This work represents the first report Of a serpin in penaeid shrimp. The data provide clues that Fc-serpin might play potential roles in the innate immunity of shrimp. (C) 2008 Elsevier Ltd. All rights reserved.

A thioredoxin with antioxidant activity identified from Eriocheir sinensis

Thioredoxin, with a redox-active disulfide/dithiol in the active site, is the major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. In the present study, the cDNA encoding thioredoxin-1 (designated EsTrx1) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsTrx1 was of 641 bp, containing a 51 untranslated region (UTR) of 17 bp, a 3' UTR of 306 bp with a poly (A) tail, and an open reading frame (ORF) of 318 bp encoding a polypeptide of 105 amino acids. The high similarity of EsTrx1 with Trx1s from other animals indicated that EsTrx1 should be a new member of the Trx1 sub-family. Quantitative real-time PCR analysis revealed the presence of EsTrx1 transcripts in gill, gonad, hepato-pancreas, muscle, heart and haemocytes. The expression of EsTrx1 mRNA in haemocytes was up-regulated after Listonella anguillarum challenge, reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to elucidate its biological functions, EsTrx1 was recombined and expressed in E. coli BL21 (DE3). The rEsTrx1 was demonstrated to possess the expected redox activity in enzymatic analysis, and to be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could function as an important antioxidant in a physiological context, and perhaps is involved in the responses to bacterial challenge. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5' untranslated region (UTR) of 69 bp, a 3' UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12 h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.