Effects Of Ocean Acidification And Warming On Resistance Of Mussels Mytilus Coruscus To Vibrio Alginolyticus And Its Potential Mechanisms

Global warming is caused by the large amount of greenhouse gas CO₂emitted by human activities,which leads to the warming of the oceans,and the absorption of excess CO₂in the atmosphere by the oceans leads to ocean acidification.At present,the change of the ocean physical and chemical properties is still in progress,which has a great impact on the marine organisms,and is likely to change the original biological relationship,thus affecting the continuation of the biological community.Marine shellfish and Vibrio have a close relationship.Most Vibrio bacteria are opportunistic pathogens for shellfish.Changes in the environment,especially when tilted in the direction of Vibrio,can lead to increased risk of Vibriosis in shellfish.The risk of mussel infection or resistance to Vibrio would change due to the different temperature and p H tolerance ranges of mussels and Vibrio.To find the change,we studied the effects of acidification and warming on the Vibrio resistance of mussels with thick shells,and analyzed the causes of the final results from the perspectives of host and bacteria,respectively.The effects of acidification and warming on Vibrio alginolyticus virulence were studied first.Vibrio was cultured in four conditions,two p H（environment 8.1,acidification 7.7）×two temperatures（average environment temperature 20℃,maximum temperature 30℃）.The 14-hour growth curve was measured.Subculture was performed every 12 h,and samples were taken to detect virulence gene expression,including fur,tlh,and trh.The results showed that Vibrio grew faster at30℃,and the growth rate was slightly higher at p H 7.7 than that at p H 8.1.The influence of temperature was far greater than that of acidification.The relative expression levels of virulence genes fur and tlh increased with culture time,and the relative expression levels of all three genes increased with temperature.Acidification had little effect on virulence genes fur and tlh,but significantly increased the relative expression levels of trh at common temperature.In general,temperature accelerated the growth of Vibrio alginolyticus,and also increased the expression of virulence genes.The influence of p H on growth and virulence genes was far less than that of temperature.Then the effects of acidification and warming on hemolymph antioxidant,immune-related enzymes and metabolome of mussels were then studied.Mussels were exposed to four physicochemical conditions,a combination of two p H（environment p H 8.1,acidification 7.7）and two temperatures（environment average20℃,maximum 30℃）.At the end of exposure,hemolymph was extracted for the detection of SOD,GSH,MDA,ACP,AKP,LZM and metabolomics.Experimental results showed that the antioxidant index SOD,GSH,MDA had two consistent rules,that acidification in high temperature group significantly increased their content and high temperature in low p H group significantly increased the content of them.In addition,acidification and heating increased the content of GSH significantly.The interaction between acidification and warming was observed in all three indicators.The immune enzymes ACP,AKP and LZM did not show regular changes.ACP was only significantly different between the two groups treated with high temperature,AKP was only significantly different between the two groups treated with common temperature,and LZM was only significantly different between the two groups treated with normal p H.There was no interaction between acidification and warming in the three indexes.There were significant differences in hemolymph metabolites of mussels in the four groups.Temperature affected the differential expression of 22metabolites,and no metabolite differences caused by p H were observed.These metabolites are mainly involved in amino acid-related pathways,including Amino Acids&Derivatives,Lysine degradation,Monobactam biosynthesis,Phenylalanine metabolism,Folate Biosynthesis and Arginine and proline metabolism.Finally,the effects of acidification and warming on resistance of mussels to Vibrio were studied.The hepatopancreas of mussels exposed to acidification and warming for two months were collected to detect bacterial load every 24 h after infected by Vibrio alginolyticus,and the hemolymph of mussels with thick shells was collected to detect antioxidant,immune enzymes and metabolome after 5 days of challenge.The experimental results showed that the bacterial load in hepatopancreas increased with the time of exposure to Vibrio,and the rate was significantly increased on the fourth and fifth days.In addition,the amount of bacteria in the high-temperature exposed group was significantly higher than that in the low-temperature group,and that in the acidified group was significantly higher than that in the normal p H group.SOD was significantly different only in low p H group and was induced by high temperature.The contents of GSH and MDA showed the same trend,that is,acidification significantly increased their levels in the high temperature group,and high temperature significantly increased their levels in the high temperature group.Interaction between p H and temperature was observed in GSH and MDA,but not in SOD.ACP and AKP showed the same pattern,acidification significantly reduced their levels in the high-temperature group,and high temperature significantly increased their levels in the normal p H group.Lysozyme showed no difference between the four groups.There is p H and temperature interaction in ACP and AKP,but not in lysozyme.Results can be seen that the four groups from the metabolism of mussels haemolymph metabolites have significant differences.7 kinds of different metabolites were caused by temperature alone.10were caused by p H alone.The five metabolites affected by both p H and temperature decreased with the increase of temperature at low p H,increased with the increase of temperature at normal p H,increased with the decrease of p H at common temperature,and decreased with the decrease of p H at high temperature.In terms of pathway,amino acid metabolism is still the main factor involved.Correlation analysis was conducted between physiological parameters and differential metabolites.The results showed that the changes of bacterial load were positively correlated with SOD,GSH,MDA and LZM,but not with metabolites.The metabolites n-Ribosylhistidine and L-beta-Aspartyl-L-Glycine were negatively correlated with SOD,GSH and MDA.（2R,2’）-isobuteine was negatively correlated with SOD,GSH and MDA,but positively correlated with AKP.Gly-gly-gly-gly and Tetrahydropentoxyline were negatively correlated with MDA and positively correlated with ACP and AKP.In conclusion,amino acid metabolites affect the antioxidant and immune capacity of mussel hemolymph,thus increasing the risk of Vibrio infection to mussels in acidified and warming environments.In this study,the risk of Vibrio infection in Marine shellfish increases with ocean warming and acidification.The increased risk was caused not only by the increased pathogenicity of Vibrio but also partly by the reduced resistance of mussels to Vibrio.The decrease of Vibrio resistance of mussels was directly related to the activity of antioxidant system and immune enzyme,and the activity of antioxidant system and immune enzyme were highly related to hemolymph metabolites.Metabolites affect the energy metabolism pathway and antioxidant GSH metabolism pathway by affecting the metabolism of amino acids.