Effect Of Cladocera Life History Parameters And Gene Expression Of Antioxidant Enzymes In Cyanobacteria 华衍生 Stress Factor

One of the most troublesome symptoms of eutrophication is the proliferation of toxic, food-web altering cyanobacterial harmful blooms, or CyanoHABs. CyanoHABs are expanding geographically and now representing a serious threat to the water quality and function of freshwater ecosystem. The accumulation and breakdown of huge CyanoABs biomasses often cause organism mortality due to hypoxia and toxins (e.g. microcystins, ammonia and nitrite). The main aim of this study is to illustrate the effects of stressful factors during CyanoHABs breakdown stage on life-history parameters and antioxidant systems in cladoceran. We selected Daphnia similoides and Daphnia similis (ubiquitous species in Lake Taihu), as well as Daphnia magna (classic ecotoxicological test organism) to conduct a series of single-factor and combined-factor experiments. Results are shown below:1. The effect of different dissolved oxygen levels on life-history parameters of Daphnia similioides was investigated. Results showed that survival time, number of moult, number of broods, offspring at each brood, and total offspring per female decreased significantly with decreasing DO levels. A two-parameter single rectangular hyperbola model was suitable for fitting the variation of following four traits:mean survival day, number of moult, number of broods and number of total offspring per female. According to the models, the EC50s for survival, moult, number of broods, and total offspring per female indicated that reproduction is more sensitive to low DO. We can conclude that reduction in DO levels have adverse effects to D. similoides as low DO could contribute to enhanced mortality, decreased moult, reduced growth, delayed maturation, and lower reproduction.2. The sub-lethal effects of ammonia and hypoxia on life-history parameters of Daphnia similis was investigated. Experiments lasted for14d and we recorded the life-history traits such as survival, moult, maturation, and fecundity. Results showed that hypoxia significantly decreased survival time and the number of moults of D. similis, whereas ammonia had no effect on them. Elevated ammonia significantly delayed development to maturity in tested animals and decreased their body sizes at maturity. Both ammonia and hypoxia were significantly detrimental to the number of broods, the number of offspring per female, and the number of total offspring per female, and significantly synergistic interactions were detected. Our data clearly demonstrate that elevated ammonia and hypoxia derived from cyanobacterial blooms synergistically affect the cladoceran D. similis.3. Here, we cloned and sequenced the full-length cDNA of Mn-SOD from Daphina magna (Dm-Mn-SOD) and then analyzed the gene expression pattern under stimulation of microcystin-LR (MC-LR) and nitrite. The full-length Dm-Mn-SOD sequence was consisted of955bp nucleotides, encoding215amino acids, showing well-conserved domains that were required for metal binding and several common characteristics. Its theoretical molecular mass was24594.24kDa, pI7.10. The deduced amino acid sequence of Dm-Mn-SOD showed that it shared high identity with other Mn-SOD in Dromia personata (75%), Cancer pagurus (75%) and Carcinus maenas (75%). The phylogenetic analysis indicated that Dm-Mn-SOD was highly homologous to other species. We investigated the reponses of Dm-Mn-SOD gene to MC-LR and nitrite via real-time PCR. The results showed that both MC-LR and nitrite induced Dm-Mn-SOD transcript levels, indicating Dm-Mn-SOD could play an outstanding role in eliminating reactive oxygen species in mitochondria. In addition, this study also provided a potential a useful biomarker for the risk assessment of environmental pollutants.4. In the present study, we cloned the Cu/Zn-SOD cDNA from the model cladoceran Daphnia magna, analyzed its catalytic properties, and investigated mRNA expression patterns after the exposure to known oxidative stressors. The full-length Cu/Zn-SOD of D. magna (Dm-Cu/Zn-SOD) sequence was consisted of537bp nucleotides, encoding178amino acids, showing well-conserved domains that were required for metal binding and several common characteristics. The deduced amino acid sequence of Dm-Cu/Zn-SOD showed that it shared high identity with Daphnia pulex (88%), Alvinella pompejana (56%) and Cristaria plicata (56%), respectively. The phylogenetic analysis indicated that Dm-Cu/Zn-SOD was highly homologous to D. pulex. The variation of Dm-Cu/Zn-SOD mRNA expression was quantified by real-time PCR, and the results indicated that the expression was up-regulated after48-h exposure to unionized ammonia and dissolved oxygen. This study shows that the Dm-Cu/Zn-SOD mRNA could be successfully employed as a biomarker of oxidative stress, which is a common mode of toxicity for many other aquatic hazardous materials.5. The accumulation and breakdown of huge CyanoHABs biomasses often cause hypoxia in lakes. Acute changes of dissolved oxygen (DO) have adverse effects on metabolic responses and survival of Daphnia. To reveal the responses of Daphnia magna in different life stages to hypoxia, four age groups of D. magna (1,4,7, and14d) were exposed to different DO levels (2.0,4.0,8.0mg L’^in48-h acute bioassay. Results from acute bioassays were used to estimate the survival rate for every age group. Meantime, gene expressions of hemoglobin (DHB), a-esterase (EST), catalase (CAT), heat-shock protein70(HSP70) were assessed. Results showed as below:(1) no significant mortality occurred in three age groups (1,4,7); whereas the significant changes was observed in14-d group indicating more sensitive to hypoxia than others group.(2) D. magna DHB transcript levels at both ages increased at4mg L-1DO followed by decreased transcript level at the lowest DO, suggesting that hypoxia induced DHB expression, while excessive-low DO inhibited the expression in turn.(3)1-d group’s EST transcript levels increased with decreasing DO level;4-d and7-d groups’ levels increased at first and then fell down subsequently along with decreasing DO. EST is thought to be related to the digestion of lipids and related to energy generation. Induced EST transcript levels when D. magna exposed to hypoxia suggested that organisms need elevate energy synthesis efficiency to relieve hypoxia.(4) the same pattern was seen in both CAT and HSP70, which has substantial potentials in repairing damaged cell:gene transcripts increased and then fell down in1-d and7-d groups; but only decreased in4-d and14-d groups along with reduced DO levels. The responses of CAT and HSP70suggested that hypoxia induced the two expression, while excessive-low DO inhibited the expression in turn.