Effects Of Cu²⁺ On Growth, Reproduction And Expression Of Chemosensory Gene In Daphnia Carinata

In this paper, Daphnia carinata were taken as the research object. Methods and techniques of molecular biology, toxicology, biochemistry and so on were used. Impacts of copper ion on growth and reproduction of Daphnia carinata were researched as well as mRNA expression of Chemosensory gene by controlling the external environmental condition. The main results were as following:1. Impacts of copper ion on growth, reproduction and antioxidase activities of Daphnia carinataIn order to research the impacts of water polluted by copper ion on Daphnia carinatd’s growth and reproduction and antioxidase activity, the culture experiment lasted 21 days with five different concentration groups (0μg·L-1,2μg·L-1,4μg·L-1,8 μg·L-1 and 16 μg·L-1). According to the obtained results, different concentrations of copper ion would not change Daphnia carinatd’s reproductive method directly. The intrinsic rate of increase, fecundity and net reproductive rate showed a decrease trend after the primal increase due to the increased copper ion concentration and the top values appeared at 4 μg·L-1. However, the time of producting the first generation and mean generation time were prolonged and the results of high concentration group (16 μg·L-1) were much more obvious (P< 0.05). Meanwhile, the lifetime decreased and high concentration group (16 μg·L-1) lived for about 10 days. The body of Daphnia carinata at 4 μg·L-1 was longest, while the body at 16 μg·L-1 was shortest. Besides, copper ion had no obvious impacts on Daphnia carinatd’s reproductive interval.Both SOD and CAT activities had a decrease trend after the primal increase because of copper ion and the peak value emerged at 4 μg·L-1. However, the activities of POD showed an increase trend all the time.2. DacaCSP2 and DacaCSP3 full-length cloning and bioinformatics analysiscDNA sequences of two CSP genes were cloned from Daphnia carinata, named as DacaCSP2 (GenBank accession no:KM624608) and DacaCSP3 (GenBank accession no:KM624609) respectively. The full-length of DacaCSP2 was 632 bp and DacaCSP3 was 935 bp. The ORF coded protein of both DacaCSP2 and DacaCSP3 possessed OS-D superfamily domain, four cysteine sites,2Fe-2S ferredoxin-type iron-sulfur binding domain, anaphylatoxin domain and so on. The sequences were rather conservative through the analysis of amino acids coded by DacaCSP2 and DacaCSP3 of Daphnia carinata, and Daphnia pulex had the highest similarity (83%). Analysis of its biological origin showed that Daphnia carinata had the highest phylogenetic relationship with Daphnia pulex.3. DacaCSP2 and DacaCSP3 expression analysisReal-time PCR analysis showed that whether DacaCSP2 or DacaCSP3 mRNA expression levels were different in the different Daphnia carinata, obviously. For male, the mRNA expression levels were the lowest, for the sexual female, the mRNA expression levels were highest. According to the research results, it could be presumed that DacaCSP2 and DacaCSP3 probably played a role in the adjustment and control at the Daphnia carinata’s growth stage.4. DacaCSP2 and DacaCSP3 spatial orientation analysisIn situ hybridization indicated that mRNA of DacaCSP2 and DacaCSP3 were mostly expressed in ovaries, summer eggs and the second antennas of sexual female and also expressed in thoractic limbs and rectum. As for parthenogenetic female, mRNA of DacaCSP2 and DacaCSP3 were mostly expressed in ovaries and the second antennas and also expressed in thoractic limbs and rectum. The above results showed that DacaCSP2 and DacaCSP3 perhaps had an impacts on different reproductive methods of Daphnia carinata.5. Impacts of copper ion on the expression of DacaCSP2 and DacaCSP3Then the difference of DacaCSP2 and DacaCSP3 mRNA expression levels were analyzed after treating Daphnia carinata with different concentrations of copper ion. The results showed that the expression levels of both DacaCSP2 and DacaCSP3 mRNA increased at first and then decreased as the concentration of copper ion increasing. When the concentration was 4μg·L-1, the expression levels of DacaCSP2 and DacaCSP3 mRNA were the highest.According to the results, CSPs would begin to play a role when Daphnia carinata were stimulated by adverse environment, which indicated that CSPs plays an important function in the stress reaction, not only in order to prevent itself hurting, but also provide more opportunities for the survival of their offspring. Therefore, it was presumed that DacaCSP2 and DacaCSP3 genes may play a certain role in growth and reproduction of Daphnia carinata.