Preliminary Study On The Mechanism Of Autolysis In The Perinereis Aibuhitensis

Perinereis aibuhitensis not only has high nutritional value,but also can be used as an environmental pollution monitoring organism,and has become an important economic and popular breeding species in China’s coastal and Southeast Asian regions.However,in a previous study,we found that Perinereis aibuhitensis was prone to autolysis during transportation and preservation,similar to that seen in sea cucumbers(Stichopus japonicus),and this phenomenon has not been reported yet.Therefore,in this paper,we analyzed sandworms at different times and temperatures,and preliminarily investigated the autolysis mechanism of the sandworms by high-throughput transcriptome sequencing,tissue structural changes and enzyme activity and gene expression.Firstly,we found out the different genes of autolysis at two time points(0 h and 72 h)under the same temperature(20)by second generation transcriptome sequencing,and then we investigated the mechanism of autolysis by the tissue structure,related enzyme activity and relative expression of related genes at different stages of autolysis(0 h,24 h,48 h,72 h,96 h and 120 h).Secondly,the effects of temperature on autolysis were investigated by observing and measuring the tissue structure,related enzyme activities and relative expression of related genes at the same time points(72 h)under different temperatures(10,15,20 and 25).The results of the study were as follows:1.Tissue structure,related biochemical indicators and genetic changes in different stages of autolysis in the Perinereis aibuhitensisWe found that the autolysis of Perinereis aibuhitensis began at 24 h.The most significant effects of autolysis on muscle fibers and body structure were observed at 72 h and beyond.CAT activity decreased significantly during the autolysis process(P<0.05);MDA content showed a trend of decreasing and then increasing,and reached the minimum value at 72 h.GSH,GST and GPx all showed a trend of decreasing and then increasing with different time periods of autolysis,and GSH content was significantly lower than other groups at 24 h,48 h and 72 h(P<0.05);The level of GST at 48 h was significantly lower than those of the other groups(P<0.05),while GPx reached the minimum value at 24 h(P<0.05).MMP-9 always maintained a higher level at 24 h and afterwards,and the highest level was at 48 h.By secondgeneration transcriptome sequencing we filtered genes with significant expression differences at different stages of autolysis and identified collagen、col6a6、gsto1、gpx3、cela2b、mua-3、bcl-xl、traf6-b、stpg1、casp3、calpa、hyal2、mmp9、hsp70b2、agrn、kazal、hsp23、prdx5、cstb、kpi and fp genes were used as autolysis-related genes for RT-q PCR.The results showed that both gpx3 and gsto1 genes showed a trend of increasing,then decreasing,and prdx5 gene showed a trend of increasing,then decreasing with time,and the expression was significantly higher than other groups at 96 h(P<0.05).hyal2,hsp70b2,hsp23,collagen,and mua-3 were not significantly different with time(P>0.05).The relative expression of agrn and col6a6 genes showed a trend of increasing,then decreasing,then increasing over time.the relative expression of kpi,kazal,cela2 b,and casp3 genes did not differ significantly between groups over time(P>0.05).the relative expression of fp and calpa genes showed a trend of increasing,then decreasing,then increasing over time,and the relative expression of cstb and mmp9 genes showed an increasing trend over time.The relative expression of traf6-b showed an increasing trend over time,and the relative expression of bcl-xl and stpg1 showed an increasing,then decreasing,then increasing trend over time.2.Tissue structure,related biochemical indicators and genetic changes of the Perinereis aibuhitensis at different temperaturesWe found that the internal tissues and myofibrils of the Perinereis aibuhitensis remained relatively intact and regularly arranged at 10°C.As the temperature increased,the myofibrils of the Perinereis aibuhitensis would dissolve and break.The CAT activity in the sandworms first increased and then decreased,and was significantly higher than the other groups at 20°C(P<0.05);MDA and GSH both showed a trend of increasing,then decreasing,and then increasing.GST first decreased and then increased with increasing temperature,and was significantly lower than the other groups at 20°C(P<0.05).The content of GPx and MMP 9in sandworms increased significantly with the increase of temperature(P<0.05).Immunerelated genes gpx3,gsto1 and prdx5 showed a trend of decreasing then increasing.hyal2,hsp70b2 and mua-3 were not significantly different at each temperature treatment(P>0.05).the relative expression of hsp23 was significantly higher than other groups at 10°C(P<0.05),the relative expression of collagen was lowest at 15°C and highest at 20°C,and agrn was significantly higher than other groups at 20°C(P<0.05).The relative expression of kpi,calpa,mmp9 and casp3 showed no significant difference between the groups(P>0.05).The relative expression of cstb gene was significantly higher than other groups at 10(P<0.05),and the relative expression of cela2 b gene was significantly higher than other groups at 20(P<0.05).traf6-b and stpg1 showed no significant difference in relative expression at each temperature gradient(P>0.05).the relative expression of bcl-xl gene increased and then decreased with temperature.The relative expression of bcl-xl gene showed a trend of increasing and then decreasing with temperature.In summary,the hyal2,agrn,mua-3,kazal,cela2 b,fp,mmp9,and traf6-b genes are probably the main factors that damage the tissue structure of the organism during autolysis,while gpx3,gsto1,prdx5,kpi,cstb,bcl-xl genes play an important role in alleviating oxidative stress,inhibiting the activity of autolysis-related enzymes and regulating apoptosis in the body.The optimal transport time may be within 24 h～48 h and the optimal storage temperature may be between 15°C～20°C.