| Since the Industrial Revolution,massive amount of anthropogenic carbon dioxide(CO2)generated has elevated the atmospheric CO2 concentration.About one fourth to one third of the anthropogenic CO2 has been absorbed by the ocean which leading to reductions in both oceanic pH and carbonate ions concentrations,a process known as'ocean acidification(OA)'.The release of gametes into natural seawater environment for external fertilization is an ancestral mating strategy commonly employed by marine invertebrates.It has been shown that OA generally has a negative impact on the fertilization success of broadcast spawning marine organisms.Compared to open ocean,coastal environment is more susceptible to the elevated atmospheric CO2 concentrations and these organisms inhabited here are more susceptible to OA.To date,the effects of OA on the fertilization success of marine intertidal shellfish and the underneath mechanism are yet to be explored.The blood clam,Tegillarca granosa,is a traditional aquaculture bivalve species in China and is a typical representative of broadcast spawning invertebrates.Therefore,the effects of OA on the fertilization success of T.granosa were studied in this study.Results obtained in this study showed that the fertilization capability of both gametes were significantly reduced in either HCl-or CO2-acidified seawater.Moreover,the sperm velocity decreased significantly in seawater acidified by CO2 aeration,while no significant difference was observed between HCl addition groups and the control.A series of consecutive events including sperm-egg collisions,gamete fusion,and triggering Ca2+ oscillations,are essential for successful fertilization of broadcast spawning invertebrates.In order to examine the impact mechanism of the OA induced reductions in fertilization success,the effects of OA on the three important aspects were studied in this research.The study on gamete collisions found that the sperm velocity of T.granosa was observed in the pCO2-acidified trials and would thus lead to a reduced the probability of gamete collisions.According to the fertilization kinetics model,the pCO2-induced declining sperm velocity observed in this study was predicted to result in about 7.2%and 20.8%drop in fertilization success at pH 7.8 and pH 7.4 of that of the control,respectively.Since the adenosine triphosphate(ATP)level of sperm is closely related to their motility,the ATP content and activities of key enzymes during ATP synthesis under different OA scenarios were analyzed in this research.Results obtained indicated that the decreased ATP content and inhibited ATP synthesis are the main reason for the reduced sperm velocity.The study on gamete fusion found that probability of gamete fusion per collision decreased sharply with an increase in pCO2 levels,suggesting OA hampers sperm and eggs fusions.According to the fertilization kinetics model,gamete fusion probability per sperm-egg collision of T.granosa was markedly decreased to approximately 69.4%and 46.8%of the control for the experimental groups at pH 7.8 and 7.4,respectively.Proteomic analysis in the egg membrane of T.granosa under different pH levels found that the expression levels many proteins have been altered after OA treatment,including these protein involved in the regulation of fertilization related cell functions and gamete fusion process.Immunofluorescence and real-time PCR in the study suggested that the the decreased probability of gamete fusions in pCO2 acidified trials could be due to the disturbed function of gamete recognition and binding process.The study on egg activation found that intracellular Ca2+ fluorescence intensities of eggs decreased with the decrease of experimental pH,suggesting OA hamper the increase of intracellular Ca2+ during the fertilization events.Since a series of intracellular Ca2+ oscillations are an integral part of egg activation and a prerequisite for subsequent fertilization events,results obtained in this research revealed that the pCO2 induced reduction in fertilization success may be partially due to the depression of Ca2+ oscillations detected.In addition,real-time PCR suggested that the down-regulated expressions of Ca2+ transport related proteins under OA are responsible for the disturbed intracellular Ca2+ signaling.In conclusion,the results obtained in the present study suggest that OA could narrow the window for successful fertilization of the marine bivalve,T.granosa,through a comprehensive mechanism that hampers sperm-egg collisions,gamete fusion,and generation of Ca2+ oscillations. |
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