Analysis Of Fatty Acid Composition And Dynamic Changes Of Lipid Droplets Of Bursaphelenchus Xylophilus Under Different Influencing Factors

Bursaphelenchus xylophilus is the direct pathogenic factor of Pine Wilt Disease and an important invasive species,which has caused serious ecological damage and economic losses to many countries and regions in East Asia.J2 larva feeds to form lipid droplets which larger with continuous feeding.J_Ⅲlarva has super-large lipid droplets and strong stress resistance and a long life span.It depends on the fat stored in lipid droplets to survive and spread.Therefore,clarifying the dynamic changes of fatty acid components and lipid droplets during the growth and development of B.xylophilus is of great significance to reveal the spreading mechanism.In this paper,four treatments,including low temperature,exogenous addition of fatty acids,interference of transcription factor daf-12 gene and inhibition of TOR signal pathway,were used to analyze the changes in fatty acid composition,content and dynamic changes of lipid droplets of B.xylophilus in different developmental stages.The main results are as follows:1.In different development stages of B.xylophilus,the fatty acids are the same and the relative contents are similar.The content of polyunsaturated fatty acids is the highest in each stage,accounting for 59.8%,55.68%,63.38%and 62.08%of the total fatty acids in this stage,respectively.And the content of polyunsaturated fatty acids changes with the development progress of B.xylophilus.In J2 stage,the fatty acid with the highest content is arachidonic acid,accounting for 27.15%.In J3,J4 and adult stage,the fatty acid with the highest content is linoleic acid,accounting for 27.00%,36.02%and 36.43%of each stage respectively.At low temperature,the fusion of lipid droplets can be promoted,the relative content of saturated fatty acids is kept at about 31%,and about 7.5%of monounsaturated fatty acids are dehydrogenated into polyunsaturated fatty acids,and the unsaturated degree of fatty acids increases.2.Exogenous addition of oleic acid can make B.xylophilus produce super-large lipid droplets with a diameter of more than 10μm,which can increase the palmitic acid content and decrease the linoleic acid content.The addition of exogenous palmitic acid and linoleic acid induced the lipid droplets to nearly double,and mainly changed the relative contents of palmitic acid,stearic acid,oleic acid and linoleic acid.The daf-12 gene and SCD gene of B.xylophilus were up-regulated by exogenous fatty acids.3.The transcription factor daf-12 gene,which is closely related to fatty acid metabolism and the formation of spreading larvae,is expressed in all developmental stages of B.xylophilus,and the expression level of daf-12 gene increases at first and then decreases in the reproductive period,with a wide range of expression sites.The daf-12 gene and SCD gene are closely related to the low temperature response process,and they can both make up-regulated transcription responses to low temperature signals in different degrees.The life of daf-12(RNAi)is shortened at room temperature,but it does not change at low temperature.The lipid droplets of all stages increased significantly after being RNAi,and the diameter of lipid droplets increased from 4μm to 8μm.The fatty acid components,polyunsaturated fatty acids and total fatty acids of J2,J3 and adults increased to different degrees after RNAi,but the total fatty acid content in J4 stage remained unchanged and the polyunsaturated fatty acids decreased.4.Treating B.xylophilus with TOR signal pathway inhibitor rapamycin can promote fat accumulation and increase lipid droplets.GC-MS showed that rapamycin increased the polyunsaturated fatty acid composition.The transformation of reproductive larvae of B.xylophilus into dispersive larvae is the key link to realize the spread and epidemic of B.xylophilus.The above research results show that low temperature,exogenous fatty acid,daf-12 gene and rapamycin can promote fat storage,lipid droplets enlargement and fusion,and play an important role in the formation and spread of dispersive larvae.The results provide theoretical support for an in-depth analysis of the formation mechanism of dispersive larvae,and provide research ideas for developing and designing prevention and control measures to specifically block the formation of dispersive larvae.