Analysis Of Lipid Droplet Dynamics And Fatty Acid Composition Changes In Different Developmental Stages Of Bursaphelenchus Xylophilus

Pine wilt disease is a devastating epidemic disease of pine caused by pine wood nematode,Bursaphelenchus xylophilus.Once a pine is infected with Bursaphelenchus xylophilus,it can die within two months at the earliest.The transformation and development of B.xylophilus reproductive larvae into dispersive larvae is the key link in the realization of disease transmission and epidemic.At this stage,the dispersive third stage larvae continue to feed and accumulate fat into the lipid droplets in the body,while the dispersive fourth stage larvae in the vector insects do not feed,relying on the fat stored in the lipid droplets to survive,and rely on lipolysis.The energy drives it to overflow from the long beetle and infect healthy pine.Therefore,the dynamic changes of lipid droplets and the changes of fatty acid composition and content during the growth and development of pine wood nematode are closely related to its infection and transmission cycle.Based on the previous observations,we systematically studied the dynamic changes of pine wood nematode lipid droplets and changes in fatty acid composition and content.In this study,a modified Bellman’s device was used to collect adult larvae and two types of reproductive and dispersive larvae of different stages in B.xylophilus.With reference to the related technology in the model organism Caenorhabditis elegans,4 types of lipid droplets staining methods were used.Comparison of lipid droplets staining methods and selection of the best method for observing lipid droplet dynamics;clarify the differences in triglyceride content of pine wood nematodes of different stages by ultraviolet spectrophotometer;use gas chromatography-mass spectrometry to identify the fatty acid types and content at different stages.in B.xylophilus The research results can not only provide references for the formation mechanism of dispersive larvae in B.xylophilus,but also lay a foundation for further revealing the spread and transmission mechanism,and provide new ideas for exploring specific pine wilt disease prevention and control measures.The main findings of this paper are as follows:1.Using Sudan Black B,Nile Red,and Oil Red O dyes to quantify the fat of pine wood nematode,considering the simplicity of the dyeing method,the length of time,the observation effect of dyeing and the comparison of pixel intensity after Image J software image processing,the pixel intensity of Lipid droplets stained by Sudan Black is 200×10¹⁷μm²,41.64×10¹⁷μm ² stained by Nile Red,52.12×10¹⁷μm² stained by Oil Red O,and 83.85×10¹⁷μm ² stained by Post Oil Red O.Lipid droplets stained by Sudan black had the highest average pixel intensity,and lipid droplets stained by Nile red had the lowest average pixel intensity.The post-oil red O staining method is the best way to reflect the fat storage in B.xylophilus.The size of the lipid droplet is a manifestation of the fat content.In the process of transformation and development,the lipid droplets of the spreading larva of B.xylophilus has a phenomenon that several lipid droplets transforms into a super lipid droplet.2.Ultraviolet spectrophotometer results show that lipid droplets are the sites where neutral lipids such as triglycerides are stored in cells.Among them,the average triglyceride content of J4 larvae was53umol/L,the average triglyceride content of J3 larvae was 45.89 umol/L,and the average triglyceride content of adults was 46.92 umol/L.The average triglyceride content of the larvae during the proliferation stage was 61.76 umol/L,the average triglyceride content of the larvae during the spreading stage was45.89 umol/L,and the average triglyceride content of the nematodes during the breeding stage was 49.18umol/L.There were significant differences in triglyceride content between larvae and adults of B.xylophilus breeding and diffusion types（p<0.05）.It indicates that there are certain differences in the triglyceride content of B.xylophilus at different developmental stages,which may be related to the different host environment and the body fat composition of the different developmental stages.3.The results of gas chromatography-mass spectrometry showed that the fatty acid composition of larvae and adults of B.xylophilus reproductive and dispersive larvae covered 23 species of C11～C22,mainly 16,18 and 20 carbon fatty acids.and the relative content of saturated fatty acids of J2,J3,J4 and adults were（35.04±0.62）%,（25.95±0.58）%,（41.42±0.67）%,（32.08±0.62）%;relative content of monounsaturated fatty acids are（0.80±0.01）%,（7.04±0.05）%,（18.88±0.03）%,（13.56±0.02）%;the relative content of polyunsaturated fatty acids are（2.03±0.05）%,（5.26±0.05）%,（53.8±0.27）%,（17.77±0.21）%.The relative contents of saturated fatty acids in the dispersal third-stage and the dispersal fourth-stage stages were（40.48±0.40）%,（25.65±0.65）%,the relative contents of monounsaturated fatty acids were（3.65±0.07）%,（4.24±0.06）%,and the relative contents of polyunsaturated fatty acids were respectively It is（15.04±0.15）%,（0.76±0.13）%.The relative fatty acid content of larvae and adults of B.xylophilus breeding and dispersal types were significantly different（p<0.05）.The composition of each fatty acid is similar,but there are significant differences in relative content（p<0.05）.It shows that the fatty acid composition of B.xylophilus at different developmental stages has certain differences in species and relative content,which may be related to the different host environment and the special physiological structure of the worm at different developmental stages.The above research results clarify the dynamic changes of lipid droplets in different developmental stages of B.xylophilus,as well as the analysis of fat composition and content.It is speculated that lipid droplets changes may play an important role in the transformation and development of diffuse larvae,host transfer,and energy consumption.