Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca2+-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a T-terminal. untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7-and 4.9-fold at 6 h after injury and 8 h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury heating and the immune response in A. irradians. (c) 2008 Elsevier Ltd. All rights reserved.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development. (C) 2010 Elsevier Ltd. All rights reserved.

CfLGBP, a pattern recognition receptor in Chlamys farreri involved in the immune response against various bacteria

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coil, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of the immune effects of CpG motifs that protect Japanese flounder (Paralichthys olivaceus) against bacterial infection

CpG-containing oligodeoxynucleotides (ODNs) are known to be immunostimulatory in vertebrate systems and can activate both innate and adaptive immune responses. In this report, we described the selection, identification, and analysis of CpG motifs with immunoprotective effects in Japanese flounder. Sixteen CpG ODNs were synthesized and examined for the ability to inhibit bacterial dissemination in Japanese flounder blood. Four ODNs with the strongest inhibitory effects were selected and mixed to form ODNs 4M. In addition, a plasmid, pCN6, was constructed that contains the sequences of the four selected ODNs. When administered into Japanese flounder via intraperitoneal injection, both ODNs 4M and pCN6 could, in dose and time dependent manners, afford short-term protection against the infections of two different bacterial pathogens. Immunological analyses showed that ODNs 4M and, especially, pCN6 activated head kidney macrophages and enhanced serum bactericidal activity via probably the alternative pathway of complement activation. When used as a DNA vaccine to immunize Japanese flounder, pCN6 conferred apparent protections (42.9% and 52.6%, respectively, in terms of relative percent survival) against the challenges of two different fish pathogens at 4-week post-vaccination. Transcriptional analysis showed that vaccination with pCN6 upregulated the expression of the genes encoding NKEF, MHC II alpha, IL-1 beta, Mx, and MHC I alpha. These results demonstrate that ODNs 4M and pCN6 are immunostimulatory in Japanese flounder and can induce short- and long-term nonspecific protections against bacterial infections. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and analysis of a Sciaenops ocellatus ISG15 homologue that is involved in host immune defense against bacterial infection

ISG15 is an interferon-stimulated gene that encodes a ubiquitin-like protein. ISG15 homologues have been identified in a number of fish species, some of which are known to be regulated at expression level by virus infection and lipopolysacchande (LPS) treatment However, the relationship between ISG15 and live bacterial infection has not been investigated in piscine models. In this study, an ISG15 homologue, SoISG15, was identified from red drum Scraeriops ocellaws and analyzed at expression and functional levels The open reading frame ofSolSG15 is 477 base pairs (bp) and mtronless, with a 5'-untranslated region (UTR) of 91 bp and a 3'-UTR of 415 bp The deduced amino acid sequence of S0ISG15 shares 60-67% overall identities with the ISG15 of several fish species. S0ISG15 possesses two conserved ubiquinn-like domains and the canonical ubiquitin conjugation motif, LRGG, at the C-terminus. Expressional analysis showed that constitutive expression of SolSG15 was highest in blood and lowest in kidney Experimental challenges with LPS and bacterial pathogens induced significant S0ISG15 expression in the kidney but not in the liver Similar differential induction was also observed at cellular level with primary hepatocytes and head kidney (HK) lymphocytes. Poly(' C), however, effected drastic induction of S0ISG15 expression in kidney and liver at both tissue and cellular levels. Immunoblot analysis showed that S0ISG15 was secreted by cultured HK lymphocytes into the extracellular milieu. Recombinant S0ISG15 expressed in and purified from Eschenclua colt was able to enhance the respiratory burst activity, acid phosphatase activity, and bactericidal activity of HK macrophages. Taken together, the results of this study indicated that SoISG 15 possesses apparent immunological property and is likely to be involved in host immune defense against bacterial infection. (C)2010 Elsevier Ltd All rights reserved.

Peptidoglycan recognition protein of Chlamys farreri (CfPGRP-S1) mediates immune defenses against bacterial infection

Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in detecting and eliminating the invading bacteria. Several PGRPs have been identified from mollusk, but their functions and the underlined mechanism are still unclear. In the present study, the mRNA expression profiles, location, and possible functions of PGRP-S1 from Zhikong scallop Chlamys farreri (CfPG RP-St) were analyzed. The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation (P < 0.01), while moderately after the stimulations of LPS (P < 0.01) and beta-glucan (P < 0.05). The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bind beta-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn2+. These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders. (C) 2010 Elsevier Ltd. All rights reserved.

Physiological and immune responses of zhikong scallop Chlamys farreri to the acute viral necrobiotic virus infection

The Zhikong Scallop, Chlamys farreri, is one of the most Important bivalve mollusks cultured in northern China However, mass mortality of the cultured C farreri has posed a serious threat to the maricultural Industry in recent years. Acute Viral Necrobiotic Virus (AVNV) is believed as an important etiological agent causing the scallop mass mortalities To understand the mechanism behind the AVNV associated scallop disease and mortality, we assessed the physiological and immune responses of C farreri to the virus infection using oxygen consumption rate, ammonium-nitrogen excretion rate, hemocyte copper, zinc superoxide dismutase gene expression, and plasma superoxide dismutase activity and alkaline phosphatase activity as indicators Scallops challenged by AVNV at 25 C developed typical disease signs 2 days after virus injection Before the disease manifested, scallop oxygen consumption and NH4+-N excretion rates rose and then fell back. Real-time PCR revealed that the hemocyte cytosol Cu, Zn SOD gene expression was upregulated followed by recovery The plasma SOD activity, however, augmented consistently following virus injection Moreover, plasma AKP activity first lowered and then elevated gradually to the highest level at 24 h post virus injection Scallops challenged by AVNV at 17 degrees C neither developed notable disease nor showed obvious responses that could be associated with the virus infection. While the results suggested a correlation between the elevated seawater temperature and the AVNV infection associated C farreri mortalities, they also indicated that the viral infection provoked multiple physiological and immune responses in the host scallops (C) 2010 Elsevier Ltd All rights reserved

栉孔扇贝免疫致敏（immune priming)现象及其机制的初步研究

栉孔扇贝是我国重要的海水养殖品种，自1997年以来陆续爆发的大规模死亡，不仅造成了巨大的经济损失，而且直接威胁到现有产业的生存和发展。本研究通过比较扇贝在连续刺激条件下的不同免疫反应，推测栉孔扇贝中有无免疫致敏和记忆现象，观察短期免疫刺激对扇贝再次遇到相同病原物刺激时死亡率的影响，探讨栉孔扇贝的免疫致敏作用，并对其机制进行初步探讨，以期更好地了解扇贝的免疫防御机制，为扇贝病害防治提供参考。 利用荧光实时定量PCR技术检测了扇贝受到鳗弧菌连续等量刺激后两种重要病原识别受体（PRR）基因的表达。结果显示，在受到连续两次刺激后CfPGRP-S1、LGBP基因的表达量均有不同程度的增加。其中CfPGRP-S1基因在受到第二次刺激后表达量增加的幅度高于第一次，且基因表达显著性增加的时间比第一次提前；LGBP基因在第二次刺激后，表达量显著性增加的时间也比第一次提前。同时，对同批处理的扇贝进行重要免疫指标的检测分析发现两次刺激后吞噬率变化不显著，而第二次刺激后吞噬指数的最大峰值高于第一次，并且最大峰值出现的时间比第一次提前；血淋巴中的抗菌活力和肝胰腺中SOD、MDA含量在两次刺激前后的变化不显著。在脊椎动物中，识别能力和抗菌能力的提高是免疫记忆的两个重要特点，栉孔扇贝在第二次免疫刺激后PRR识别能力的提高和吞噬作用的增强，说明扇贝中可能存在类似脊椎动物免疫记忆的免疫致敏作用。对扇贝进行两次鳗弧菌浸泡刺激，第一次为短期浸泡刺激，第二次为长期浸泡刺激。结果发现，第一次浸泡过的扇贝在遭受相同病原第二次刺激时，在一定时间段内其死亡率比未受第一次刺激的扇贝极显著降低，表明第一次的短期病原刺激增强了扇贝对第二次刺激的抵抗能力。同时比较第二次刺激过程中扇贝免疫指标的变化发现经过短期刺激扇贝的吞噬作用、ACP和AKP活性比未受过刺激的扇贝显著增强，而其他指标包括POL、SOD则差异不显著，说明增强的吞噬作用、ACP和AKP活性与扇贝的死亡率下降有关。研究结果表明栉孔扇贝中存在免疫致敏（immune priming）现象，短期刺激可以提高扇贝的免疫反应能力，增强扇贝机体对相同病原物的抵抗能力。

Growth, survival and immune activity of scallops, Chlamys farreri Jones et Preston, compared between suspended and bottom culture in Haizhou Bay, China

We examined the growth, survival and immune response of the scallop, Chlamys farreri, during a 1-year period in deep water of Haizhou Bay. Scallops were cultured using two methods: (1) in lantern nets at a 5 m depth and (2) in a bottom culture system (sleeves) on the seabed at about a 25 m depth. Shell heights, meat dry weight and immune activities in the haemolymph (superoxide dismutase and myeloperoxidase) were measured bimonthly or quarterly from July 2007 to June 2008. Survival was measured at the end of the study and environmental parameters in the experimental layers were monitored during the experiment. The growth and immune activities of scallops were lower when the water temperature was high, which was consistent with the main mortality occurring in summer. The growth and immunity of scallops were higher in the suspended culture than in the bottom culture during the experiment, with the exception of shell growth during the last study period. Survival of scallops in the suspended culture (54.6 +/- 12.3%) was significantly lower than that in the bottom culture (86.8 +/- 3.5%) at the end of this study. We conclude from our results that the high mortality of C. farreri can be prevented by culturing them in a bottom culture system before November of the first year, and then transferring them to a suspended culture to improve scallop production.

Modulatory effects of ammonia-N on the immune system of Penaeus japonicus to virulence of white spot syndrome virus

To study response to white spot syndrome virus (WSSV) under ammonia stress, Penaeus japonicus were exposed to 5 mg l(-1) ammonia-N and challenged orally with WSSV (NW). Controls consisted of an ammonia-N-exposed control group (N), a WSSV-challenged positive control group (W), and an untreated control group (control). Immune parameters measured were total haemocyte count (THC), haemocyte phagocytosis, plasma protein content and haemolymph enzymatic activities for prophenoloxidase (proPO), alkaline phosphatase (ALP), and nitric oxide synthase (NOS). THC and plasma protein had downward trends with time in all treatment groups (NW, N, and W) in contrast to the untreated control group (control). The percentage phagocytosis, NOS activity, and ALP and proPO activity of W and NW decreased initially then increased from 6 to 78 h (except for NOS and ALP, from 6 to 54 h) before declining thereafter until the end of the experiment. Compared with untreated controls (control), there was a downward trend for all measured parameters in the treatment groups (N, NW, and W), but the degree was W > NW > N. WSSV was detected at 78 h postchallenge in both W and NW. In conclusion, 5 mg l(-1) ammonia-N reduced the immunocompetence of P japonicus and may have decreased the virulence of WSSV (C) 2004 Elsevier B.V. All rights reserved.

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.

Immune responses of the scallop Chlamys farreri after air exposure to different temperatures

The present study examined the influence of air exposure at different temperatures: a common perturbation associated with aquaculture handling practices, on immune responses in zhikong scallop Chlamys farreri. Scallops were exposed to air for 2 h, 6 h, 12 h and 24 h at 5 degrees C, 17 degrees C and 25 degrees C respectively. Thereafter, a recovery period of 24 h at 17 degrees C was applied. Haemocyte mortality, phagocytosis and reactive oxygen species (ROS) production of haemocytes, acid phosphatase (ACP) and superoxide dismutase (SOD) activity in haemocyte lysates were chosen as immumomarkers of anoxic stress. The results showed that an increase of haemocyte mortality and a decrease of phagocytosis and ACP activity were observed after 2 h of air exposure for all temperatures tested. Moreover, a significant increase of ROS production occurred following 2 h of air exposure at 25 degrees C and 24 h of air exposure at 17 degrees C. Significant differences were also observed in haemocyte mortality, percentage of phagocytic cells and ACP and SOD activity depending on the temperature of air exposure. Finally, after 24 h of recovery at 17 degrees C, percentage of phagocytic haemocytes and ACP activity did not return to initial values. ROS production was significantly higher than before the recovery period and initial values for scallops subjected to air exposure at 5 degrees C. In our study, scallops showed a relative low anoxia tolerance under a high temperature. All the scallops air exposed to 25 degrees C died after the 6 h sampling. In conclusion, air exposure associated to aquaculture practices was demonstrated to strongly affect functional immune activities of scallop haemocytes, and high temperature air exposure caused reduced survival of scallops. (c) 2007 Elsevier B.V. All rights reserved.

Effects of acute temperature or salinity stress on the immune response in sea cucumber, Apostichopus japonicus

Invertebrates are increasingly raised in mariculture, where it is important to monitor immune function and to minimize stresses that could suppress immunity. The activities of phagocytosis, superoxide dismutase (SOD), catalase (CAT), myeloperoxiclase (MPO), and lysozyme (LSZ) were measured to evaluate the immune capacities of the sea cucumber, Apostichopus japonicus, to acute temperature changes (from 12 degrees C to 0 degrees C, 8 degrees C, 16 degrees C, 24 degrees C, and 32 degrees C for 72 h) and salinity changes (from 30 parts per thousand to 20 parts per thousand, 25 parts per thousand, and 35 parts per thousand for 72 h) in the laboratory. Phagocytosis was significantly affected by temperature increases in 3 h, and by salinity (25 parts per thousand and 35 parts per thousand) changes in 1 h. SOD activities decreased significantly in 0.5 h to 6 h samples at 24 degrees C. At 32 degrees C, SOD activities decreased significantly in 0.5 h and 1 h exposures, and obviously increased for 12 h exposure. CAT activities decreased significantly at 24 degrees C for 0.5 h exposure, and increased significantly at 32 degrees C in 3 h to 12 h exposures. Activities of MPO increased significantly at 0 degrees C in 0.5 h to 6 In exposures and at 8 degrees C for 1 h. By contrast, activities of MPO decreased significantly in 24 degrees C and 32 degrees C treatments. In elevated-temperature treatments, activities of LSZ increased significantly except at 32 degrees C for 6 h to 12 h exposures. SOD activity was significantly affected by salinity change. CAT activity decreased significantly after only 1 h exposure to salinity of 20 parts per thousand.. Activities of MPO and LSZ showed that A. japonicus tolerates limited salinity stress. High-temperature stress had a much greater effect on the immune capacities of A. japonicus than did low-temperature and salinity stresses. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.

Immune condition of Apostichopus japonicus during aestivation

Aestivation is an indispensable state in the life history of sea cucumbers, Apostichopus japonicus. The immune characteristics of the coelomic fluid of A. japonicus, were investigated during aestivation. Samples were collected between July and November 2006 from a coastal pond located off the Yellow Sea in Jiaonan, Shandong Province, China. The total coelomocytes counts (TCC), total superoxide dismutase (T-SOD), catalase (CAT), myeloperoxidase (MPO), and lysozyme (IZM) in the coelomic fluid were measured. The activities of catecholamines, [adrenaline (AD), noradrenaline (NA), and dopamine (DOP)] were estimated. TCC decreased from July to September, indicating weakness of the cellular immune activity at that time. Activities of SOD, CAT, MPO, and LZM changed significantly from July to October. Catecholamines AD and NA in coelomic fluid were significantly higher on August 21 and November 27. There was no significant variation in DOP during the sampling period. Thus, immune characters in coelomic fluid of A. japonicus changed significantly during aestivation. Water temperature was significantly and negatively correlated with TCC, and salinity was significantly and positively correlated with AD and NA. The mechanism of aestivation in A. japonicus is complex and might not be attributed only to environmental changes, such as temperature and salinity, as shown in previous studies. (C) 2008 Elsevier B.V. All rights reserved.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.