The Biological Effects Of Ocean Acidification On Copper Toxicity Of Thalassiosira Pseudonana

The increasing atmosphere carbon dioxide?CO₂?induced ocean acidification?OA?changes the seawater carbonate chemistry and hence the bio-toxicity of copper,a heavy metal ion in ocean and a widespread threat to marine organisms.Diatoms contribute approximately 40%of the marine primary productivity,functionally sequestering carbon and bio-accumulating copper and critical foundation of marine food web.However,there are few comprehensive studies on how ocean acidification affects the copper metabolism of diatoms and the safety of seafood related to diatom.Here,we put Thalassiosira pseudonana CCMP1335 as the research object,and from multi-perspectice of physiological ecology,ecological toxicology,molecular biology,deeply analyze copper metabolic regulation mechanism and its ecological effect of T.pseudonana.The main research contents contain:?1?Determining the copper toxicity effect on T.pseudonana.Clear understanding the influence of copper exposure on the specific growth rate and the median lethal effect?EC50,96 h?and the toxicity threshold of copper;the effects of copper within the toxicity threshold on the photosynthetic characteristics,net photosynthetic efficiency of PSII and respiratory efficiency were analyzed.?2?Analyzing the absorption and accumulation of copper in T.pseudonana.Studies on copper absorption kinetics were carried out to obtain the semi-saturation constant?Km?and maximum absorption efficiency?Vmax?in T.pseudonana.HPLC-ICP-MS and other methods were used to determine the content of copper in the tissue cells of T.pseudonana.?3?Biological response of copper metabolism in T.pseudonana to ocean acidification.We simulate the atmospheric CO₂concentration ranges?400?atm,1000?atm,2000?atm?between now and the next 300years,and set up different acidification intensity to determine the influence of ocean acidification on the biological characteristics?growth,photosynthesis,respiration,copper metabolism?in T.pseudonana.?4?Comparative analysis of copper metabolism of T.pseudonana at different spatial and temporal scales.Different time scales of the copper metabolism of T.pseudonana were comparative analysis:the short-term stress experiment of ocean acidification and the long-term adaptability experiment of ocean acidification were set to compare and analyze the effects of short-term stress acidification and long-term adaptability acidification on the copper metabolism response of T.pseudonana.Comparative analysis of the copper metabolism of T.pseudonana at different spatial scales:optimizing the culture system,improving the in-situ experiment platform in natural sea area,ocean acidification studies were carried out in the laboratory and in the in-situ sea areas respectively,and the effects of ocean acidification on the copper metabolism response of T.pseudonana were compared and analyzed in different spatial scales.?5?Predict and evaluate of copper enrichment kinetics and potential ecological effects.Enrichment kinetics of copper:the transfer experiment of copper in the food chain from T.pseudonana to Argopecten irradians was carried out,and the enrichment kinetics equation of copper was constructed to clarify the enrichment rule and potential ecotoxicological effects of copper in typical food chain.Predicting and evaluating potential ecological effects:build and optimize dynamic response model of T.pseudonana under the condition of ocean acidification and build prediction figure of dynamic track and dynamics curves copper metabolism and accumulation,which combine with future climate change under different carbon dioxide path data.?6?Molecular regulation mechanism of copper metabolism of T.pseudonana under ocean acidification.Transcriptional data of T.pseudonana under different acidification conditions were obtained,and the differences in transcriptional expression of key genes in the copper metabolic pathway were confirmed by bioinformatics to complete the classification and functional prediction of key genes in the copper metabolic pathway and analyze evolution and adaptation mechanisms.Their functions were verified by fluorescence quantitative PCR and other methods.We found that:?1?The lethal concentration of Cu in 96h was 20?M.?2?Copper toxicity can significantly inhibit the growth and photosynthesis of T.pseudonana and enhance respiration.?3?The increasing pCO₂ had no significant effect on respiration,but promoted the growth and photosynthesis,reduced the accumulation of copper in T.pseudonana,which reduced the toxic effect of copper on T.pseudonana.Besides,the absorption of copper in T.pseudonana has circadian rhythm of more days than nights.?4?Both short-term acidizing stress and long-term acidizing adaptation can promote the growth and reduce the accumulation of copper in T.pseudonana.However,under long-term acidification conditions,algae have an evolutionary adaptability to the increase of carbon dioxide,which is irreversible.Situ experiments in natural sea area showed that under natural conditions,rising carbon dioxide promoted the growth and also decreased the accumulation of copper in T.pseudonana,which was basically consistent with the trend of indoor experiments.Feeding experiments in sea area indicated that copper could be transferred from T.pseudonana to its predator of Argopecten irradians through the food chain,and the decline copper concentration of T.pseudonana led to the decrease of copper accumulation in Argopecten irradians under ocean acidification.?5?Transcriptome results further showed that copper overdose could induce the detoxification reaction of T.pseudonana and further produce optimized copper detoxification strategy under the condition of further ocean acidification,including up-regulated the expression of glutathione?GSH?,phytochelatins?PCs?and efflux transporter P1B-ATPases?CTP?,and down-regulated the expression of synthesis of cytochrome c oxidase 1?SCO1?and cytochrome c oxidase copper chaperone?COX17?.In summary,our results show that increasing CO₂ reduces copper accumulation and alleviates copper toxicity in T.pseudonana.This may be attributed to optimizing the detoxification strategy of T.pseudonana,This may be due to increased carbon dioxide providing more energy for growth in the algae,optimizing the detoxification strategy of T.pseudonana,including down-regulated the expression of copper chaperone?SCO1?and cytochrome c oxidase copper chaperone?COX17?as well as up-regulated the expression of efflux transporter P1B-type ATP ases?CTP?.Therefore,we predict that ongoing ocean acidification will reduce the accumulation of copper in diatoms,but may also reduce the bioremediation ability of heavy metal pollution.Changes in copper metabolism will affect the production of marine primary productivity and the global biogeochemical cycle of copper.This study will provide important scientific basis and clues for comprehensively analyzing the copper biogeochemical cycle of marine diatom,provide basic data for clarifying the response mode of microalgae to heavy metal pollution under the condition of global marine environmental change,and provide theoretical and technical support for the ecological risk assessment and restoration of heavy metal pollution.