Characterization,expression and function analysis of DAX1 gene of scallop (Chlamys farreri jones and preston 1904) during its gametogenesis

DAX1, a member of nuclear receptor superfamily, has a function in the sex determination and gonadal differentiation of several vertebrate species. However, little information about DAX1 of invertebrates is available. Here we cloned a homolog of scallop (Chlamys farreri Jones and Preston 1904) dax1, Cf-dax1, and determined its expression characteristics at mRNA and protein levels. The cDNA sequence of Cf-dax1 was 2093 bp in length, including 1404 bp open reading frame (ORF) encoding 467 amino acids. Unlike those of vertebrates, no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1. Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes. Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues, with the highest expression level found in adductor muscle, moderate level in mantle, gill and testis, and low level in kidney, ovary and hepatopancreas. The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher (P<0.05) in testis than in ovary at the same stage, showing a sex-dimorphic expression pattern. Furthermore, immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary, implying that DAX1 may involve in gametogenesis of bivalves.     

Genotoxic potential of copper oxide nanoparticles in the bivalve mollusk <Emphasis Type="Italic">Mytilus trossulus</Emphasis>

Copper oxide nanoparticles (CuO-NPs) are among the most widely used metal oxide nanoparticles, which increases the chance of their being released into the marine environment. As the applications of these particles have increased in recent years, their potential impact on the health of marine biota has also increased. However, the toxicological effects of these NPs in the marine environment are poorly known. In the present study, the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures. Genotoxicity was assessed by the single cell gel electrophoresis (comet) assay in mussel gill and digestive gland cells. The results showed that copper in both forms (CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues. The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland. In contrast to these results, it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills. A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels. Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only. It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells, while the gill cells were more sensitive to the genotoxic effects of CuO-NPs. These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.     

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)     

Cloning of the cDNA encoding adenosine 5′-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5’-UTR of 78 bp, a 3’-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.     

Isolation and characterization of an enoyl-acyl carrier protein reductase gene from microalga Isochrysis galbana

In most bacteria,plants and algae,fatty acid biosynthesis is catalyzed by a group of freely dissociable proteins known as the type II fatty acid synthase(FAS II) system.In the FAS II system,enoylacyl carrier protein reductase(ENR) acts as a determinant for completing the cycles of fatty acid elongation.In this study,the cDNA sequence of ENR,designated as IgENR,was isolated from the microalga Isochrysis galbana CCMM5001.RACE(rapid amplification of cDNA ends) was used to isolate the full-length cDNA of IgENR(1 503 bp),which contains an open reading frame(ORF) of 1 044 bp and encodes a protein of 347 amino acids.The genomic DNA sequence of IgENR is interrupted by four introns.The putative amino acid sequence is homologous to the ENRs of seed plants and algae,and they contain common coenzymebinding sites and active site motifs.Under different stress conditions,real-time quantitative polymerase chain reaction(RT-qPCR) showed the expression of IgENR was upregulated by high temperature(35℃),and downregulated by depleted nitrogen(0 mol/L).To clarify the mechanism of lipids accumulating lipids,other genes involved in lipids accumulation should be studied.     

Genotoxic Potential of Copper Oxide Nanoparticles in the Bivalve Mollusk Mytilus trossulus

Copper oxide nanoparticles(CuO-NPs) are among the most widely used metal oxide nanoparticles,which increases the chance of their being released into the marine environment.As the applications of these particles have increased in recent years,their potential impact on the health of marine biota has also increased.However,the toxicological effects of these NPs in the marine environment are poorly known.In the present study,the DNA damaging potential of CuO-NPs in the marine eastern mussel Mytilus trossulus was evaluated and compared to that of dissolved copper exposures.Genotoxicity was assessed by the single cell gel electrophoresis(comet) assay in mussel gill and digestive gland cells.The results showed that copper in both forms(CuO-NPs and dissolved copper) was accumulated to different extents in mussel tissues.The mussel exposed to the dissolved copper attained higher concentrations of copper in the gills than in the digestive gland.In contrast to these results,it was found that CuO-NPs could induce much higher copper accumulation in the digestive gland than in the gills.A clear and statistically significant increase in DNA damage was found in both tissues of the Cu-exposed group compared to the control mussels.Our results indicated that the CuO-NP exposure produced remarkable effects and increased DNA damage significantly in mussel gill cells only.It should be noted that the digestive gland cells were prone to accumulation following CuO-NPs when compared to the gill cells,while the gill cells were more sensitive to the genotoxic effects of CuO-NPs.These results also suggested the need for a complete risk assessment of engineered particles before its arrival in the consumer market.     

Temporal variation in biodeposit organic content and sinking velocity in long-line shellfish culture

We measured the organic content and sinking velocities of biodeposits from two scallop species (Chlamys farreri, Patinopecten yessoensis) and abalone (Haliotis discus hannai) that were cultured on suspended long-lines. Measurements were conducted every two months from April 2010 to February 2011. The shellfish were divided into three size groups (small, middle, and big sizes). At each sample point, we assessed biodeposit organic content, average sinking velocity, the frequency distribution of sinking velocities, and the correlation between organic content and sinking velocity. The organic content of biodeposits varied significantly among months (P<0.05) and the pattern of change varied among species. Sinking velocities varied significantly, ranging from <0.5 cm/s to >1.9 cm/s. The sinking velocities of biodeposits from C. farreri and P. yessoensis were 0.5–1.5 cm/s and from H. discus hannai were <0.7 cm/s. The organic content was significantly negatively correlated to the sinking velocity of biodeposits in C. farreri (P<0.001) and P. yessoensis (P<0.05).     

合浦珠母贝丝氨酸蛋白酶抑制因子基因pfser1克隆与表达

【目的】克隆合浦珠母贝(Pinctada fucata)丝氨酸蛋白酶抑制因子pfser1基因,探讨该基因的组织表达及其在天然免疫过程中的作用,以及与生物矿化过程的关系。【方法】通过RACE技术获得pfser1基因的全长,通过生物信息学分析其序列结构特征,利用实时荧光定量PCR方法检测pfser1基因在不同组织中的表达,检测健康合浦珠母贝在被大肠杆菌(Escherichia coli)MG1655刺激后和在贝壳损伤修复实验中pfser1基因表达量的变化。【结果】合浦珠母贝pfser1基因cDNA全长为1240 bp,包含1035 bp的开放阅读框(ORF),编码344个氨基酸,氨基酸序列的功能结构域含有丝氨酸蛋白酶抑制因子Serpin家族保守结构域。pfser1基因在合浦珠母贝各个组织中均有表达,在外套膜边缘膜中表达量最高;大肠杆菌MG1655刺激后,该基因表达量显著升高;在贝壳损伤修复过程中,pfser1基因表达先升高后受到抑制。【结论】pfser1基因所表达的蛋白参与了合浦珠母贝的天然免疫应答过程,并与生物矿化过程有一定关系。     

Biomarker responses in the bivalve Chlamys farreri to the water-soluble fraction of crude oil

To investigate the effect of the water soluble fraction of crude oil(WSF) on marine bivalves, the scallop C hlamys farreri was exposed to three WSF concentrations(0.18 mg/L, 0.32 mg/L, and 0.51 mg/L, respectively) in seawater. Petroleum hydrocarbon contents in scallops and a suite of enzymes [7-Ethoxyresorufin-O-deethylase(EROD), aryl hydrocarbon hydroxylase(AHH), glutathione S-transferase(GST), and glutathione peroxidase(GPx)] in gills and digestive glands were monitored over 10 days. The results revealed that WSF affected the activity of the four enzymes in the gills and digestive glands. EROD activity in the gills was significantly induced in most individuals of the three test groups, while in the digestive gland it was significantly induced in the low-concentration group within 4 days but was inhibited in the middle- and high-concentration groups on days 1, 4, and 10. AHH activity in the gills of all treatment groups was significantly induced on day 1. In the digestive gland, AHH activity was induced in most individuals from the treatment groups. In all treatment groups, GST activity was significantly inhibited from days 2 to 10 in the gills and was induced after day 4 in the digestive gland. GPx activity in the gills was significantly inhibited throughout the exposure period in all treatment groups. There was no overall significant difference in GPx activity in the digestive gland between the control and treatment groups. Our results also revealed that petroleum hydrocarbon concentrations in the tissues increased linearly with exposure time. EROD activity in the digestive gland and GST and GPx activity in the gill tissue were negatively correlated with petroleum hydrocarbon body burden. These enzymes play important roles in detoxification and can act as potential biomarkers for monitoring petroleum hydrocarbon contaminants in the marine environment.