The expression of peptidoglycan recognition protein-S1 gene in the scallop Chlamys farreri was enhanced after a second challenge by Listonella anguillarum

A more rapid and powerful response against repeated exposure of same pathogen in vertebrates is usually considered as the reflection of immunological memory, but it is not well understood in invertebrates. In the present Study, the temporal expression profiles of Chlamys farreri peptidoglycan recognition protein-S1 (CfPGRP-S1) gene after two challenges of Listonella anguillarum were examined to evaluate priming response in scallops. The up-regulation of CFPGRP-S1 mRNA occurred 3 h earlier, and the expression level was significant higher (P < 0.05), after the second challenge than that after the first challenge. The preliminary results Provided new insights into invertebrate immunological memory, and they also would be helpful to develop strategies for disease control. (C) 2008 Elsevier Inc. All rights reserved.

cDNA cloning and gene expression pattern following bacterial challenge of peroxinectin in Chinese shrimp Fenneropenaeus chinensis

Peroxinectin, a cell-adhesive hemoperoxidase that binds superoxide dismutase and mediates blood cells adhesion and migration in invertebrate, is believed to play an important role in cellular immune reaction. In this study, we reported a new peroxinectin gene homologue from Chinese shrimp Fenneropenaeus chinensis. Based on expressed sequence tags (ESTs) of haemocyte cDNA library, we cloned a 2,611 bps full-length cDNA of peroxinectin gene homologue encoded 801 amino acids. Motif scanning of the predicted polypeptide revealed a peroxidase domain and an integrin binding motif (Lys-Gly-Asp, KGD). Peroxinectin gene expressed constitutively in haemocyte as determined by quantitative real-time RT-PCR, the expression level varied following bacterial challenge. These findings suggested that peroxinectin expression is susceptible to exterior stimulus and maintains at a high expression level during bacterial infection.

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.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids. with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned ANT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 mu g L-1 and 200 mu g L-1 CdCl2. The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment. (C) 2008 Elsevier Inc. All rights reserved.

A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972 bp, encoding 757 amino acids with a calculated molecular mass of 84.96 kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value = 1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge. (c) 2006 Elsevier Ltd. All rights reserved.

A Dorsal homolog (FcDorsal) in the Chinese shrimp Fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge

Rel/NF kappa B is a family of transcription factors. In the present study, a Rel/NF kappa B family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627 bp, revealed a 1071 bp open reading frame encoding 357 aa. The predicted molecular weight (MW)of the deduced amino acid sequence of FcDorsal was 39.78 kDa, and its theoretical pl was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NF kappa B member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Immune condition of Chlamys farreri in response to acute temperature challenge

The effects of acute temperature challenge on some immune parameters of haemocyte in Zhikong scallop, Chlamys farreri, recognised as a temperature sensitive bivalve species, were evaluated over a short period of time. Scallops were suddenly transferred from 17 degrees C to 11 degrees C, 23 degrees C and 28 degrees C for a period of 72 h. Total haemocyte count (THC), percentage of phagocytic haemocytes, reactive oxygen species (ROS) production, acid phosphatase (ACP) and superoxide dismutase (SOD) activities (in both haemocyte lysate and cell-free haemolymph) were chosen as biomarkers of temperature stress. Results demonstrated that the percentage of phagocytic haemocytes and ACP activity in cell-free haemolymph of scallops challenged at 28 degrees C for 72 h significantly decreased. By contrast, reactive oxygen species production by haemocytes increased when compared to the initial values. It is concluded that haemocyte activities of C. farreri appear to be compromised when scallops were transferred from 17 degrees C to 28 degrees C. Meanwhile, no obvious negative effect of acute temperature stress was detected on haemocyte activities of C. farreri challenged at 11 degrees C, which highlighted the high tolerance of scallops to acute decrease of seawater temperatures. (C) 2007 Elsevier B.V. All rights reserved.