Effects of Environmental Cadmium on Cadmium Accumulation,Oxidative Response,and Microelements Regulation in the Liver and Kidney of Hexagrammos otakii

The effects of cadmium(Cd)on metal accumulation,microelements contents,and antioxidant responses in Hexagram-mos otakii were studied.The fish were exposed to 0.2,2.5,or 10μg L?1 Cd for 12 or 24 days.Then,the concentration of Cd and mi-croelements(Ca,Fe,Zn,and Se)were determined in the liver and kidney.Moreover,the activities of antioxidant enzymes including superoxide dismutase(SOD),catalase(CAT),and glutathione-S-transferase(GST),and the content of malondialdehyde(MDA)in the liver and kidney were also measured.A continuous accumulation of Cd was observed throughout the experimental period.Cd accumulation was higher in liver than that in the kidney,while Ca and Fe contents were lower in liver than those in the kidney.Cd exposure resulted in a decrease of Ca and Fe concentrations in the kidney,while there was no effect on the liver.Zn and Se remained unaffected with exposure to Cd.Cd exposure induced severe oxidative stress in H.otakii,as indicated by significant induction of the activities of SOD,CAT,and GST,and a simultaneous increase of MDA content.These data show that antioxidant enzymes and mi-croelements contents can be used as potential biomarkers to monitor environmental health in fish.     

The influence of cadmium on the antioxidant enzyme activities in polychaete Perinereis aibuhitensis Grube (Annelida: Polychaeta)

The infaunal polychaete Perinereis aibuhitensis Grube, distributed widely along Asian coasts and estuaries, is considered a useful animal model in ecotoxicological tests and a promising candidate in biomonitoring programs. This paper deals with the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidases (GSH-Px) in infaunal polychaete P. aibuhitensis exposed to a series of sublethal water-bound cadmium (Cd) concentrations (0, 0.34, 1.72, 3.44, 6.89, and 17.22 mg L⁻¹) under a short-term exposure (1–8 d). The results indicate that the SOD and GSH-Px activities in P. aibuhitensis are stimulated first and then renewed to the original level. The CAT activity of worms decreases at an earlier exposure time but increases to the control values at a later exposure time. Our study suggests that Cd can interfere with the antioxidant defense system of P. aibuhitensis. However, the changes in antioxidant enzyme activities for this species do not show the best promise as biomarkers in Cd biomonitoring of estuarine and coastal zones because weak or non-dose-effect relationships between the antioxidant enzymes activities and Cd levels are found.     

Effect of benzo[a]pyrene on detoxification and the activity of antioxidant enzymes of marine microalgae

The objective of this study was to examine the effect of benzo[a]pyrene(Ba P) on the detoxification and antioxidant systems of two microalgae,Isochrysis zhanjiangensis and Platymonas subcordiformis.In our study,these two algae were exposed to Ba P for 4 days at three different concentrations including 0.5 μg~(L-1)(low),3 μg~(L-1)(mid) and 18 μg~(L-1)(high).The activity of detoxification enzymes,ethoxyresorufin O-deethylase(EROD) and glutathione S-transferase(GST) increased in P.subcordiformis in all Ba P-treated groups.In I.zhanjiangensis,the activity of these two enzymes increased at the beginning of exposure,and then decreased in the groups treated with mid-and high Ba P.The activity of antioxidant enzyme superoxide dismutase(SOD) increased in I.zhanjiangensis in all Ba P-treated groups,and then decreased in high Ba P-treated group,while no significant change was observed in P.subcordiformis.The activity of antioxidant enzyme catalase(CAT) increased in I.zhanjiangensis and P.subcordiformis in all Ba Ptreated groups.The content of malondialdehyde(MDA) in Isochrysis zhanjiangensis increased first,and then decreased in high Ba P-treated group,while no change occurred in P.subcordiformis.These results demonstrated that Ba P significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae.The metabolic related enzymes(EROD,GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of Ba P in marine water.     

Immunotoxicity Effect of Benzo[α]Pyrene on Scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 ugL-1) on scallop (Chla-mys farreri) immune system were studied. The results showed that B[a]P had significant toxic effects on the haemocyte counts, neu-tral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolvtic activity in all B[α]P treatment groups as well as antibacterial activity, in groups of 0.5, 1, 2.5 and 7.5 ugL-1 B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentra-tion-response to B[α]P. Bacteriolytic activity in 0.1ugL-1 B[α]P treatment group and antibacterial activity in 0.1 ugL-1 and 0.5 ugL-1 B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 ugL-1 B[α]P treatment group (P<0.05). Thus, B[α]P has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immu-nomodulation in aquatic organisms.     

Immunotoxicity effect of benzo[α]pyrene on scallop Chlamys farreri

The toxic effects of benzo[α]pyrene (B[α]P) at different concentrations (0.1, 0.5, 1, 2.5 and 7.5 μgL⁻¹) on scallop (Chlamy farreri) immune system were studied. The results showed that B[α]P had significant toxic effects on the haemocyte counts, neutral red uptake, phagocytosis, bacteriolytic and antibacterial activity (P<0.05), while the seawater control and acetone control had no significant differences. The haemocyte counts, neutral red uptake, phagocytosis and bacteriolytic activity in all B[α]P treatment groups as well as antibacterial activity in groups of 0.5, 1, 2.5 and 7.5 μgL⁻¹ B[α]P decreased significantly (P<0.05). Some of these indices tended to be stable on the sixth day and others on the ninth day, and the indices showed clear time- and concentration-response to B[α]P. Bacteriolytic activity in 0.1μgL⁻¹ B[α]P treatment group and antibacterial activity in 0.1 μgL⁻¹ and 0.5 μgL- B[α]P treatment groups increased at the beginning of exposure and reached their peaks on day 1 and day 6, respectively. Following that, both activities decreased gradually and became stable after day 9. When all the indices reached stability, they were significantly lower than those in control group (P<0.05), except for antibacterial activity in 0.1 μgL⁻¹ B[α]P treatment group (P>0.05). Thus, B[α] has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms.     

Expression profiles of two small heat shock proteins and antioxidant enzyme activity in Mytilus galloprovincialis exposed to cadmium at environmentally relevant concentrations

Small heat shock proteins encompass a widespread but diverse class of proteins,which play key roles in protecting organisms from various stressors.In the present study,the full-length cDNAs of two small heat shock proteins(MgsHSP22 and MgsHSP24.1)were cloned from Mytilus galloprovincialis,which encoded peptides of 181 and 247 amino acids,respectively.Both MgsHSP22 and MgsHSP24.1 were detected in all tissues examined by real-time PCR,with the highest expression being observed in muscle and gonad tissues.The real-time PCR results revealed that Cd signifi cantly inhibited MgsHSP22 expression at 24 h and MgsHSP24.1 at 24 and 48 h under 5 μg/L Cd 2 + exposure.MgsHSP24.1 expression was also signifi cantly inhibited after 50 μg/L Cd 2+ exposure for 48 h.With regard to antioxidant enzymes,increased GPx and CAT activity were detected under Cd 2+ stress(5 and 50 μg/L),while no signifi cant difference in SOD activity was observed throughout the experiment.Overall,both MgsHsps and antioxidant enzymes revealed their potential as Cd stress biomarkers in M.galloprovincialis.     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)     

Antioxidant response of ridgetail white prawn <Emphasis Type="Italic">Exopalaemon carinicauda</Emphasis> to harmful dinoflagellate <Emphasis Type="Italic">Prorocentrum minimum</Emphasis> exposure and its histological change

The dinoflagellate Prorocentrum minimum, one of the most widespread red tide causing species, affects marine aquaculture and ecosystems worldwide. In this study, ridgetail white prawn Exopalaemon carinicauda were exposed to P. minimum cells (5 × 10⁴ cells mL^?1) to investigate its harmful effects on the shrimp. Antioxidant activities and histological changes were used as indicators of health status of the shrimp. In 72 hours, the mortality of E. carinicauda was not affected, but its antioxidant response and histology were statistically different from those of control. Elevated superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and depressed catalase (CAT) activity were observed in gill; while increased SOD, glutathione S-transferase (GST), CAT activities and modulated GPX activity were observed in hepatopancreas. Thus, antioxidant activities in gill and hepatopancreas seem to respond differentially to harmful alga exposure. Increased malondialdehyde (MDA) content in early a few hours indicates the damage of the antioxidant defense system. Although MDA content recovered to a low level thereafter, a series of histological abnormalities including accumulation or infiltration of hemocytes, tissue lesions and necrosis were discovered in gill and hepatopancreas. Exposure to P. minimum induced sublethal effects on E. carinicauda, including temporary oxidative damage and histological injury.     

Chitosan oligosaccharide-Ca complex accelerates the depuration of cadmium from Chlamys ferrari

This study investigated the effect of a chitosan oligosaccharide-Ca complex (COS-Ca) on the depuration of cadmium (Cd) from Chlamys ferrari. After exposure to 0.5 mg L-1 CdCl2 for 3 or 7 d, the scallops were treated by COS-Ca prior to determina-tion of Cd, calcium (Ca) and zinc (Zn) contents, Cd distribution in organs, malondialdehyde (MDA) content and antioxidant variables. Results showed that COS-Ca reduced Cd content in the viscera of the scallops, with highest Cd depuration rate (47%) observed on day 3. The COS-Ca concentration substantially affected Cd depuration, and the exposure to 8.75 mg L-1 COS-Ca led to significantly higher Cd depuration rate compared with those of lower COS-Ca concentrations (1.75, 3.5, 5.25, and 7.00 mg L-1). Distribution analysis of Cd in scallop organs indicated that COS-Ca significantly reduced Cd content in the kidney throughout the 5-d experiment, as well as in the gill during the early stage of Cd depuration. In addition, COS-Ca treatment decreased glutathione peroxidase (GSH-Px) activity and MDA content while increasing superoxide dismutase (SOD) and catalase (CAT) activities on different days. Our work suggested COS-Ca complex treatment as an effective method for acceleration of Cd depuration from Cd-contaminated bivalves.     

Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

Tissue-specific bioaccumulation and oxidative stress responses in juvenile Japanese flounder (Paralichthys olivaceus) exposed to mercury

To understand mercury (Hg) toxicity in marine fish, we measured Hg accumulation in juvenile Japanese flounder (Paralichthys olivaceus) and assessed the effects on growth and antioxidant responses. After Hg exposure (control, 5, 40, and 160 μg/L Hg) for 28 d, fish growth was significantly reduced. The accumulation of Hg in fish was dose-dependent and tissue-specific, with the maximum accumulation in kidney and liver, followed by gills, bone, and muscle. Different antioxidants responded differently to Hg exposure to cope with the induction of lipid peroxidation (LPO), which was also tissue-specific and dosedependent. As Hg concentration increased, superoxide dismutase (SOD) and catalase (CAT) activities increased significantly, whereas glutathione S-transferase (GST) activity and glutathione (GSH) levels decreased significantly in the gills. SOD and glutathione peroxidase (GPx) activities and the GSH level increased significantly in the liver. SOD activity and GSH levels increased significantly, but CAT activity decreased significantly with an increase in Hg concentration in the kidney. LPO was induced significantly by elevated Hg in the gills and kidney but was least affected in the liver. Therefore, oxidative stress biomarkers in gills were more sensitive than those in the liver and kidney to Hg exposure. Thus, the gills have potential as bioindicators for evaluating Hg toxicity in juvenile flounder.     

The genotoxic effects of benzo[a]pyrene and methamidophos on black porgy evaluated by comet assay

In this study, two common pollutants （benzo[a]pyrene and methamidophos） in marine environment were tested by comet assay for their inducement of in vivo genotoxic effect to the blood cells of black porgy （ Acanthopagrus schlegeli）. The fish was exposed to 2 μg/L of benzo[a]pyrene （BaP） and methamidophos, and their mixture. The assay was performed on whole blood at 2 h, 5 h, 24 h and 96 h exposure intervals. A significant increase in DNA damage was observed in each treatment with the pollutants. Additive effect of BaP and methamidophos was also found in the experiment. However, the decrease ratios of DNA damage for 5 h and 96 h exposure interals compared with 2 h and 24 h exposure ones, respectively, were noticed. This phenomenon may be explained by the function of repairing process via enzyme cytochrome P450 in the animal. Evidence of the genotoxicity of organophosphorus pesticides （OPs） and polynuclear aromatic hydrocarbons （PAHs） on marine fish are discussed in this paper.     

Cloning and sequence analysis of a full-length cDNA of SmPPlcb encoding turbot protein phosphatase 1 beta catalytic subunit

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 （PP1） is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase l（PPlcb）, was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPPlcb, by the rapid amplification of cDNA ends （RACE） technique. The cDNA sequence of SmPPlcb we obtained contains a 984 bp open reading frame （ORF）, flanked by a complete 39 bp 5＇ untranslated region and 462 bp 3＇ untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B （PP2B）. And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPPlcb is extremely conserved in both amino acid and nucleotide acid levels compared with the PPlcb of other vertebrates and invertebrates, and its Kozak motif contained in the 5＇UTR around ATG start codon is GXXAXXGXXATGG, which is different from mammalian in two positions A6 and G3, indicating the possibility of different initiation of translation in turbot, and also the 3＇UTR of SmPPlcb is highly diverse in the sequence similarity and length compared with other animals, especially zebraf＇lsh. The cloning and sequencing of SmPPlcb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.     

BAC end sequencing of Pacific white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 paired- ends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.     

Cloning and expression analysis of a ubiquitin gene (Ub L40 ) in the haemocytes of Crassostrea hongkongensis under bacterial challenge

Ubiquitin, a highly conserved stress-related protein, is assigned multiple functions, such as DNA processing, protein degradation, and ribosome synthesis. The Crassostrea hongkongensis ubiquitin gene (designated ChUbL40) was cloned by a combination of suppressive subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The full-length cDNA of ChUbL40 is 496 bp in length, consisting of a 5’ untranslated region (UTR) of 34 bp, a 3’-UTR of 75 bp and an open reading frame of 387 bp encoding a ubiquitin fusion protein of 128 amino acids. Analysis of the amino acid sequence of ChUbL40 reveals that UbL40 is highly conservative during evolution. The expression patterns of ChUbL40 gene in various tissues were examined by real-time PCR. The expression level of ChUbL40 in haemocytes is down-regulated at 4 h and gradually returned to its original level from 6 h to 24 h after Vibrio alginolyticus challenge. Our results suggest that ChUbL40 is ubiquitously expressed and plays an important role in immune defense against bacterial challenge.     

哈维氏弧菌GST核酸疫苗制备及其对斜带石斑鱼的免疫保护作用

通过同源引物从致病性哈维氏弧菌(Vibrio harveyi)ZJ0603基因组中克隆GST的开放阅读框(ORF),构建真核表达质粒pcDNA-GST。大量抽提重组质粒后,于背鳍基部肌肉注射重组质粒免疫斜带石斑鱼(Epinephelus coioides),分析重组质粒的免疫效果。通过核酸水平检测重组质粒在鱼体肝、肌肉、头肾和脾脏组织的分布;用ELISA法检测鱼体血清的抗体水平,用Western-blot检测目的蛋白的表达情况。结果表明:该序列全长615 bp;免疫7 d后,鱼体中均有质粒分布;斜带石斑鱼血清中产生抗GST的高效抗体(1∶4 096);相应的目的蛋白也在鱼体中成功表达。攻毒后,疫苗免疫保护率达80%,表明GST可作为防治哈维氏弧菌病有效候选抗原。     

Effects of benzo(a)pyrene exposure on the antioxidant enzyme activity of scallop <Emphasis Type="Italic">Chlamys farreri</Emphasis>

Scallop Chlamys farreri was exposed to different concentrations of benzo(a)pyrene (BaP) (0.5 μg/L, 1.0 μg/L, 10.0 μg/L and 50.0 μg/L) for 30 days in seawater. The 7-ethoxyresorufin O-deethylase (EROD) activity was significantly induced, and increased with the increasing BaP concentration. The glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx) activities increased in short time at low concentration of BaP, and was significantly depressed at high concentrations. Scallop gill was more sensitive to BaP than the digestive gland, and the digestive gland was the main tissue to deal with oxyradicals. The contents of malondialdehyde (MDA) increased with the exposure time and there was a positive correlation (concentration-effect) between the MDA content and the concentration of BaP. The biomarkers determined in this experiment had important roles in detoxification, and showed great potential as biomarkers for oxidative stress. Controlled laboratory experiments designed to simulate field exposure scenarios are particularly useful in ascertaining biomarkers suitable for use with complex contaminant mixtures in the marine environment. Supported by the Technology Development Program of Shandong (No. 2008GG1005010) and the Program of Introducing Talents of Discipline of Universities (111 Project, No. B08049)