Effects Of High Salinity Stress On The Survival,Physiological And Biochemical,and Transcriptome In Razor Clam Sinonovacula Constricta

Salinity is an important environmental factor that affects the osmoregulation,immune defense,energy metabolism,and survival of aquatic organisms.Different from open seas with relatively stable salinity levels,intertidal zones,estuaries and land-based seawater ponds are characterized by varying salinity levels.Having a strong ability to regulate osmotic pressure is essential for the survival of shellfish living in these areas.The razor clam,Sinonovacula constricta,is a marine bivalve and naturally distributes in the estuary and intertidal region where salinity is usually lower than that of normal seawater.Since it can bring better ecological benefits and higher economic income,integrated culture in pond has recently become a popular mode for the razor clam cultivation in northern China.However,water conditions in pond can change greatly due to external environmental factors including drought,high temperature,etc.Although the razor clam can withstand salinity stress to some extent,the acute and dramatic increase in salinity often leads to large-scale mortality of individuals.Thus,in this study,we investigated the survival,enzyme activity,gill tissue structure and free amino acids of razor clams under acute high salt stress,as well as the construction of a whole transcriptome network of gill tissues,in order to provide varieties for studying the mechanism of osmotic pressure regulation in razor clams and breeding new high salt tolerant species.The specific results are shown as follows.1.In order to investigate the effects of high salt stress on the survival and enzyme activity of S.constricta juvenile with shell length of about 2 cm,the individuals were randomly divided into six groups with different salinities of 20(S₂₀),25(S₂₅),30(S₃₀),35(S₃₅),40(S₄₀),and 45 ppt(S₄₅).The mortalities of S.constricta juvenile in each group were calculated and the half lethal salinity(LC₅₀)at different stress times were analyzed by two methods.And also,the activities of Na⁺-K⁺-ATPase（NKA）,acid phosphatase（ACP）,and alkaline phosphatase（AKP）of those individuals from groups S₂₀,S₃₀ and S₄₀ were respectively analyzed.The results showed that the mortality rates of groups S₂₀,S₂₅,S₃₀,S₃₅,S₄₀ and S₄₅ at 120 h were 0,（2.17±0.85）%,（9.50±0.82）%,（30.67±3.70）%,（73.50±7.08）%,and（94.67±3.06）%,respectively,and all the individuals of group S₄₅ died at 168 h.The LC₅₀ of high salinity at 72 h,96 h,120 h,144 h and 168h analyze using linear regression method were 46.03 ppt,39.85 ppt,35.7ppt,34.57 ppt,and 33.69 ppt respectively,and the LC₅₀ obtained by probit analysis method were 44.31 ppt,40.74 ppt,36.96 ppt,35.67 ppt and 34.47 ppt respectively.As the salinity increased and the treatment lasted longer,the activities of NKA,AKP and ACP first increased,then decreased and then stabilized.The three enzymes showed similar change trend with different effect time.Salinity above 30 ppt might cause great damage to the body with reducing the osmotic pressure regulation and immune defense function.2.Wild adult clams S.constricta with a shell length of 5.14±0.38 cm were obtained.The gill samples of each group were obtained at 0 h,6 h,12 h,24 h,48 h,72h,96 h,and 120 h in S₂₀,S₃₀ and S₄₀.Using a microscope to analysis,we measured the gill filament length（GFL）,gill filament width（GFW）and gill thickness（GT）of S.constricta.Furthermore,paraffin section observation revealed that the gill of razor clam shrunk quickly under high-salinity and dissolved at 48 h under salinity of 40 ppt,but not under 30 ppt.The minimum GFL,GFW,and GT of S₃₀ accounted for 83.04%,76.64%,and 58.55%of those of the control group,and those of S₄₀ accounted for70.96%,59.53%,and 57.88%of those of the control group.These data further corroborated our results.3.Gills from groups S₂₀,S₃₀,and S₄₀ at 48 h of treatment were selected for free amino acids（FAA）content analysis.Besides,the contents of total FAAs（TFAAs）in the gill tissues were 61.25±1.58,89.36±4.18,and 111.98±1.46 mg·g^-1 at salinity of 20 ppt,30 ppt,and 40 ppt,respectively,indicating increasing TFAA contents with salinity.In addition,several amino acid species including taurine,alanine,proline,glycine and glutamic showed significantly changed contents in response to salinity variation,suggesting these amino acids might play an important role in razor clam’s adaptation to salinity stress.Taurine was the most abundant amino acid among all detected FAAs.Alanine was also the most abundant amino acid among all detected FAAs.Proline was the highest rising multiplier.Notably,research has shown that the contents of delicious amino acids（DAAs）in aquatic animals in higher salinity water are higher than those in lower salinity water,and the increased DAA contents contribute to the improvement of flavor.4.Gills from groups S₂₀,S₃₀,and S₄₀ at 24 h of treatment were selected for building whole transcriptome regulatory network.The results showed that a total of 83,262lnc RNAs,52,422 m RNAs,2,890 circ RNAs and 498 mi RNAs were identified in the nine samples,while a large number of differentially expressed transcripts were screened in three comparison groups.The differentially expressed m RNA and lnc RNA target genes were mainly functionally enriched for analysis involved in osmoregulation such as amino acid metabolism and membrane transport,further analysis of the annotated information revealed that amino acid synthesis and membrane transport are the main factors involved in the adaptation of constricting razor clams to increased salinity,while signal transduction and lipid metabolism plays a secondary role.Based on these differentially expressed genes,and lnc RNA/circ RNA-mi RNA-m RNA regulatory networks were constructed and a portion was screened for mapping.Functional enrichment analysis revealed that the differentially expressed m RNAs in the network were mainly involved in amino acid synthesis and metabolism as well as membrane composition pathways,these include significant upregulation of tyrosine aminotransferase（TAT）,which can improve osmotic stress tolerance,and significant downregulation of G protein inhibitors,which can reduce K+channel degradation.Further,the q RT-PCR experiment of one up-regulated gene and one down-regulated gene in ce RNA networks verified the up and down-regulated characteristics of the two genes,and determined their expression characteristics that increased with the stress time,which laid the foundation for the full verification of our subsequent network.Then,the expression characteristics of rate-limiting enzyme-cysteine sulfinic acid decarboxylase（CASD）in taurine synthesis pathway and rate-limiting enzyme-△¹-Pyrroline-5-carboxylate synthase（P5CS）in proline synthesis pathway were studied.Combined with the analysis of free amino acid content data,it was found that proline would be synthesized rapidly in the early stage of stress,but taurine synthesis was relatively delayed.These findings may help to understand how nc RNAs regulate osmotic pressure.Combining all these results,it was referred that razor clams may adapt to moderately high-salinity of 30 ppt in a short time through osmotic adjustment.Although a small proportion of individuals could survive under the salinity of 35 ppt,the death of most individuals still suggested it can hardly be accepted in aquacultural production.Notably,living under a salinity of 40 ppt for more than 48 h could cause great damage to razor clams.Therefore,the seawater salinity is recommended to be lower than 30 ppt for S.constricta aquaculture.Further,the free amino acids mainly involved in hypertonicity regulation should be alanine,taurine and proline.At the same time,nc RNA should also be involved in osmolarity regulation.