Ecotoxicological Effects Of Cd²⁺、Hg²⁺ On Meretrix Lamarckii Under The Ocean Acidification Conditions

Since the concept of "ocean acidification" has been described in the 《Nature》 in 2003, it aroused wide attention of scholars and has become one of the important research contents in the field of international marine science research. Ocean acidification not only directly changed the p H value, but also caused a series of chemical equilibrium fluctuations, which affected the physiological, growth, reproduction, metabolism and survival of most marine organisms, and could eventually lead to irreversible changes in marine ecosystems. At present, about ocean acidification research mainly concentrated in calcifying organisms, such as corals, echinoderms, crustaceans, shellfish and foraminifera, coccoliths etc. Most studies just only consider the impact of a single factor on life activities, however, research of effect of ocean acidification combination with other environmental stress factors（heavy metal pollution） on origanisms are weak. It is not conducive to a comprehensive understanding of the possible dangers of ocean acidification on marine organisms and ecosystems. Ocean acidification is essential to further research. In this paper, M. lamarckii is selected as the research object. In order to artificially simulating future ocean acidification（p H7.80, 7.60.7.40）, we used high purity CO2 and air mixed gas to produce acidic seawater. The researchs of ecotoxicological effects of Cd²⁺ and Hg²⁺ on M. lamarckii under different seawater acidification conditions were performed. The main research results are as follows:1、 Effect of ocean acidification on the acute toxicity of Cd²⁺ and Hg²⁺ to juvenile M. lamarckiiThe results showed that the survival rate of juvenile M. lamarckii was not significantly affected by ocean acidification solely. However, compared to that of control, ocean acidification significantly increased the toxicity of Cd²⁺ and Hg²⁺ to juvenile M. lamarckii. More acidified experimental seawater applied, more toxic the cadmium and mercury were shown. Both Cd²⁺ and Hg²⁺ showed the greatest toxicity at the most acidified experimental trials（p H at 7.4）, with 96 h LC50 concentrations at 4.068 and 10.332mg/L for Cd²⁺ and Hg²⁺, respectively. The 96 h LC50 concentrations of other experimental trials were successively increased as the p H value of experimental seawater increased, which were 4.728, 5.947, 6.458mg/L and 10.595, 11.169, 12.027mg/L for Cd²⁺ and Hg²⁺ at p H7.6, 7.8 and 8.2, respectively. Compared to that of control, the toxicity of Cd²⁺ and Hg²⁺ on juvenile M. lamarckii increased about 1.4 and 1.2 times respectively at lower p H. The results of present study will enrich the study contents of ocean acidification and heavy metal contamination and contribute to the restoration of M. lamarckii population and environmental protection.2、 Effect of ocean acidification on the antioxidant activity of Cd²⁺ and Hg²⁺ to M.lamarckiiWhether dealed with a single or double factor effect, the effect of SOD, CAT and GPx activity on M. lamarckii showed rising first and then reducing according to the time extension. At the same time, we also found that the sensitivity of the antioxidant enzymes was enhanced under the ocean acidification condition. With the increase of the degree of acidification, the antioxidant enzyme activity of the acid group increased and decreased rapidly. The antioxidant enzyme activity of p H7.40 group rised first and was also inhibited first during the period of experiment. The amplitude variations of the enzyme activity of superoxide dismutase（SOD） was lower than that of the CAT and GPX during the experimental treatment. We conjected that M. lamarckii produced a large amounts of H2O2, which needed CAT and GPX to clear them when subjected environmental stresses. Under similar conditions, M. lamarckii’s gill expressed more sensitive than visceral mass to oxidative damage, but lower degree of injure.3、 Cloning and expression analysis of metallothionein gene in M. lamarckiiMetallothionein（MT） is a family of cysteine-rich, low molecular weight, and heavy-metal binding proteins. MT plays important roles in essential metals homeostasis, non-essential metals detoxification and antioxidation. In this study, the RACE technique is used to obtain full-length c DNA sequences of the M. lamarckii Metallothionein gene（Ml-MT） and a bioinformatics analysis was performed to obtain the molecular properties of Ml-MT. The results showed that the Ml-MT c DNA sequencing length was 636 bp, containing 65 bp of 5’Untranslated Region（UTR） and 340 bp of 3’UTR in addition to 213 bp of the open reading frame（ORF）, which can then encode 76 amino acids. Of this, cysteine accounts for 27% of the amino acids and there are 16 MT specific Cys-Xn-Cys structures, where the molecular weight and theoretical isolectric point were predicted to be 7.704 k Da and 7.138 respectively. The MT amino acid sequence alignment comparative analysis indicated that the M. lamarckii and M. lusoria were similar at more than 88%, with M. meretrix shared a common ancestry of 87%. The quantification real-time PCR results of Ml-MT m RNA in 5 tissues of M. lamarckii were uniformly detected. These expressions had tissue specificity where visceral mass had the highest levels, followed by the Gill filaments, adductor muscle, mantle and foot.4、 Effect of ocean acidification on expression of metallothionein gene of Cd²⁺ and Hg²⁺ to M. lamarckiiWhether dealed with a single or double effect factor, the expression of Ml-MT m RNA increased with different levels during the experiment. This is a detailed description in the following: Ml-MT m RNA appeared a short "rise-fall" due to the stress response by ocean acidification solely in the 6h-12 h, similar to the "parabola" changes. Compared with the control group, the changes of the acid group was close to the control group during 12-96 h. Under the environment of Hg²⁺ participation, the expression of Ml-MT m RNA exhibited only a single round of "high-low" wave; However, with the environment of Cd²⁺ participation Ml-MT m RNA presented "high-low-high-low", which were Similar with two wheeled ladder type rise throughout the all experiment time. At the same time, we also found that the sensitivity of the Ml-MT m RNA was enhanced under the ocean acidification condition. With the increase of the degree of acidification, the Ml-MT m RNA of the acid group increased and decreased rapidly. p H7.40 group is the first to rise and is also inhibited firstly with the extension of time, So we speculate that ocean acidification can enhance Ml-MT m RNA expression.