Analysis Of Ruditapes Philippinarum Responses To Acute Ocean Acidification Based On The MRNA And MiRNA Levels

Ocean,as the earth’s natural "carbon sink",has absorbed as much as 50%of totally anthropogenic CO₂ emitted to the atmosphere.The phenomenon that too much CO₂ absorption usually leads to the decrease of seawater pH,was known as "Ocean Acidification（OA）".Ocean acidification has broken the original chemical equilibrium state of calcium carbonate on the surface of the ocean,so that OA has an effect on the ability of biocalcification and physiobiochemical characteristics for the ocean.To alleviate the anthropogenic CO₂ emissions in the atmosphere,a technology,called "Carbon Capture and Storage（CCS）",is being put into practice internationally,but it is not yet mature.There still exist many risks,such as leakage of CO₂ in the process of transportation,which will lead to local and acute seawater acidification,and make seawater pH sharply decline in during a short period of time.Perhaps,this phenomenon will have potential impacts on marine biota in different organizational levels of all forms of biological,physical and evolution.The present study selected the bivalves Ruditapes philippinarum as the research object.Based on the above research background and the habitat characteristics of the species,we designed the experimental stress conditions.Through the methods of histological observation,traditional physiological and biochemical tests and the analysis of the transcriptome level,we will systematically explored the influence and changes on organizational structure,physiological and biochemical metabolism for the main tissues.Moreover,the key metabolism network and molecular signal transduction pathway of the gill tissue will be researched on the transcriptome levels.On the one hand,the results of the study will provide a reference for the researches of the bivalves and other marine creatures stressed by acute acidification;on the other hand,the transcriptome sequencing（mRNA and miRNA）data of the gill tissue will also enrich the transcriptome information of marine shellfish.1.The physiological and biochemical responses of R.philippinorum to acute ocean acidification（1）The main tissues,including gill,mantle,hepatopancreas and pipes were detected by the method of histological observation.The results showed that these tissues stressed by acidification（14d）had different degree of damage,particularly in the extreme-acidified group,as follows:the gill filaments were much looser and the gill cilium clutter;the acinar cavity of hepatopancreas was significantly enlarged;the outer epidermis of the mantle was detached,and the gland cells of inner epidermal were highly concentrated;the mucus cells in outer epidermal of the water pipe were increased and closely aligned.（2）The change trend of physiological and biochemical metabolism on the main tissues,（gill,mantle and hepatopancreas）were observed.The results showd that there may be a stress phenomenon for the three tissues after the first day.The low pH could have a certain inhibitory effect on the CAT activity and antioxidant capacity of the gill.The AMS activity of hepatopancreas was not significantly affected by acidification,and the activity of PK,HK and SDH decreased with the prolonged acidification stress.The change trend of total protein content in the mantle was different from those of gill and hepatopancreas,and the negative impact on PO in the extreme-acidified group was more obvious.In conclusion,as the acidification degree deepens and the processing time increases,the acidification stress will cause the cellular structure damage,so as to break the original stable state of metabolism and immunity.In addition,R.philippinarum responded rapidly to the stress of the extreme-acidified group,but prolonged high-acidified group also produced the same negative effects.2.The transcriptome effects on the gill tissue stressed by 96h acute ocean acidification（1）mRNA-seq analysis:After de novo assembly,a total of 106,831 unigenes were obtained from all the samples.And 105,627（98.87%）unigenes were annotated after sequence alignment,including MC（101,605（95.11%））,MT1（1100,733（94.29%））,MT2（99,955（93.56%））,respectively.Moreover,analysis of the genes expressed differentially among MCVS-MT1,MC-VS-MT2 and MT1-VS-MT2,showed that the number of differentially expressed genes（DEGs）in MC-VS-MT2 was the top.Compared to MC,a total of 916 DEGs in the two acidified group（MT1 and MT2）.The up-regulated DEGs were enriched to Ras signaling pathway,Apoptosis and NF-kappa B signaling pathway,et al.The down-regulated DEGs were enriched to Base excision repair,Phagosome and ECM-receptor interaction,et al.Additionally,different levels of ocean acidification affected the expression of some significant genes related to immune or gill structures,such as:elenium-dependent glutathione peroxidase,sialic acidbinding lectin,Toll-like receptor 4 and Collagen alpha-2 isoforms,et al.（2）miRNA-seq analysis:After the miRNA-seq analysis,a total of 272 known-miRNA and 146 novel-miRNA were obtained from all the samples（SC,ST1 and ST2）.There were 16 differentially expressed miRNAs（DEMs）（5 novel-miRNA and 11 known-miRNA）obtained from the statistical analysis to three groups（SC-VS-ST1,SC-VS-ST2 and ST1-VSST2）.The target genes of DEMs from acidified groups（ST1 and ST2）were annotated to Endocytosis,Lysosome,VEGF signaling pathway,Calcium signaling pathway,Citrate cycle and RNA transport.And the target genes of DEMs from SC-VS-ST1 was not enriched to immune related pathways.（3）Association analysis:After the correlation analysis of DEGs and DEMs,we obtained 16 DEMs with correlation（indicated that they have common mRNA or common miRNA,the common target genes of miR-200 and miR-236-3p were the top）,and 8 DEMs without correlation（indicated that one DEM was related to multi-mRNA,the target genes of novelm0002-3p were the top）.To sum up,by the integrated analysis of histological observation,physiological and biochemical indicator detection and transcriptome（mRNA-seq,miRNA-seq）of the overall analysis,this experiments revealed the effects on the main tissues of R.philippinarum exposed to high-and extreme-acidified groups,especially showed how the gill tissue response to acute acidification stimuli in molecular levels.The results of the experiment provided some scientific reference value for the study of shellfish and other marine organisms exposed to ocean acidification.