Physiological Response Of Ark Shell Scapharca Broughtonii To Cu²⁺ And Ocean Acidification Stress

Global climate change and human activities lead to exposure of coastal ecosystems to multiple environmental stresses.Heavy metal pollution has been a focal point of coastal aquaculture and food security for a long time,while the acidification of coastal waters is a direct reflection of climate change and turns into a hot topic in the filed of marine ecology.The key physiological activities of marine shellfish could be significantly affected by stresses of heavy metals and ocean acidification.Thus,it is meaningful to explore the detrimental effects of heavy metals and acidification on physiology variation of coastal marine shellfish and its responding mechanism.Nowadays,only few studies were reported about the physiological influence of heavy metals on coastal shellfish metabolism.Few researches studied the immunological and trancrioptional influence of Cu2 + pollution and acidification stresses to marine shellfish.In this research,we focused on the ark shell(Scapharca broughtonii)and studied the effects of Cu2 + and acidification stresst on metablosim,histopathological structure and immune response of ark shell.Besides,the regulationg mechanisms of ark shell to these stresses were investigaged through analyzing the immunological and trancriotional responses.The main results are as follows:1.Effect of Cu2+ stress on physiology biochemistry and histopathological structure of ark shellThe stressful effect of Cu2+ following exposure duration 96 hours on physiology metabolism,histopathological structure and enzyme activity of ark shell was studied following the methods of biological toxicity test.The incubating concentrations of Cu2+ were set at(0.01,0.05,0.10,0.50,1.00)mg/L and a control group was set with no Cu2+ stress.Results showed that both incubating concentration and exposure duration had significant influence on physiological indices of the ark shell,including oxygen consumption rates(OR),ammonia excretion rates(NR)and ratios of O to N(O:N).The OR,NR and O:N all decreased sharply after initial exposure to the Cu2+ stress.The minimum value for OR,NR and O:N were found to be(0.005±0.001)mg/(g·h),(0.5±0.05)?mol/(g·h),(0.7±0.1)with the 1.00 mg/L treatment at the end of the experiment,which equaled 1%,29%,3% to control groups,respectively.Generally,apparently reduced metabolic rate of ark shell individuals exposed to Cu2+ stress were observed within 72 h.After 72 h,each group with different Cu2+ concentration behaves differently.After being incubated at 0.01 m g/L for 96 h,t he ark shells seemed to acclimted to Cu2+ stress.Metabolic rates of the animals were restored to the control level,while O:N was no di fferent with the control groups and no de tectable histopathologic damage was found.The physiological metabolism and histopathological structure of individuals under concentrations that higher than 0.05 m g/L Cu2+ were significantly affected after 96 h,w hile most of the O:N in all the experimental groups were below 9 a nd histopathologic damage such as gills damage and tissues messy structure were found.The ACP and ALP activities of individuals exposed to 0.10 mg/L Cu2+ increased in gill and decreased in hepatopancreas,while GPX and GST increased in both tissues.Our results demonstrated that incubating concentration(?0.05 mg/L)after 96 h exposure significantly affect physiological metabolism and histopathological structure of cockle.ACP,ALP,GPX and GST activities of individuals incubated at 0.10 mg/L were significantly affected.2.Immune response of ark shell to Cu2 + and ocean acidificationThe stressful effect of different treatment following exposure duration 96 hours on enzyme activity in gill,mantle and hepatopancreas of ark shell was studied following the methods of biological toxicity test.There were five groups: blank group(A),control group(C),ocean acidification group(L,p H= 7.7),Cu2+stress group(H,0.1mg/L),Cu2+stress+ocean acidification group(A,0.1mg/L and p H=7.7).6 parallels were setup in each group.Results showed that Cu2+ significantly accumulated in the gill,mantle and hepatopancreas of ark shell in group H and A.The highest concentration were in found in the gill,f ollowed by hepatopancreas.However,the acidification had no obvi ous effect on biological accumulation.The total protein in all groups were not significantly affected.The activity of ACP,GST and GPX were elevated in all tissues of group H and A,comparing to the inhibited activity of ALP.Also,SOD and CAT were inhibited in the gill and mantle,CAT activity were increased in hepatopancreas.Moreover,the concentration of MDA in gills where were highest and hepatopancreas were increased.The acidification had no obvious effect on enzyme activity in gill,mantle and hepatopancreas.These results indicated that Cu2+ mainly accumulates in the gill and hepatopancreas of ark shell and the acidification has no obvious effect on immune response.The activity of ACP,ALP,SOD,CAT,GPX,GST and MDA in ark shell were sensitive to Cu2+ stress,which could be used as a b iomarker for Cu2+ stress.The acidification treatment had limited effect on these biomarkers and the Cu2+ stress under the acidification did not cause extra reaction in the group.3.Transcription analysis of ark shell in response to Cu2 + and ocean acidificationThe transcriptome sequencing analysis of the different group was conducted to investigate the transcriptome response of ark shell to Cu2+ and ocean acidification.A control group(C),a Cu2+stress group(H,0.1mg/L)and a Cu2+stress + ocean acidification group(A,0.1mg/L and p H=7.7)were setop.There were 3 parallels in each group.Results showed that 9 sample of ark shell obtained 43.90?59.47×106 clean reads and 360647 unigenes.81.17% of unigenes was successfully annotated in the seven databases,which were mapped to 55 sub-categories,26 categories and 32 m etabolic pathways through GO analysis,KOG analysis and KEGG analysis.Results show that compared with group C,the total number of differentially expressed genes of ark shell in group H was 4182 and 7489 in group A.Numbers of up genes were 2690 and 4219,respectively,and Numbers of down genes were 2122 and 3270,respectively.Compared with group H,there were 3,972 genes differentially expressed in group A.1780 and 2192 genes were up-regulated and down-regulated,respectively.In GO enrichment analysis,the up-regulated genes in the H group were mainly related to the process of DNA replication and synthesis.The up-regulated genes in group A were related to the process of DNA replication and DNA repair.Compared with group H,the up-regulated genes in group A were mainly related to the process of ribosomal processes.In KEGG enrichment analysis,the genes differentially expressed in group H also were related to the DNA replication.The genes differentially expressed in group A also were related to DNA replication and mismatch repair pathways.What's more,difference between group A and group H gene enrichment to the ribosome metabolic pathways.This study showed that Cu2+ and seawater acidification stress could cause DNA damage in the tissue of ark shell,which results in the difference expression of DNA replication and mismatch repair genes.Besides,the toxicity of Cu2+ under acidification to ark shell was exacerbated,which induced extra gene expression variation.