The Sexual Attraction And Selection Mechanism During Mating Process Of Bursaphelenchus Xylophilus

Pine wilt disease which caused by pine wood nematode(Bursaphelenchus xylophilus)is a destructive disease of pine trees and has been considered as one of the most dangerous forest disasters in many countries and regions around the world,and the pathogenic nematode has also listed as a first-class hazardous forestry pest in China.In recent years,the spread of the epidemic to the north and the more severe situation has seriously threatening China's forestry and ecological security.Except for the widespread host,vector insects and existing control measures can not meet the demand,the rapid propagation and adaptation mechanism of pine wood nematode is also the key factor for controlling the disease.Therefore,research on pine wood nematode from the view of reproductive biology will provide foundational basics for further study its invasion and spread mechanism,and help to find effective ideas and measures for control the nematode.Mating selection is an important part of sexual mating,mating is a prerequisite for the reproduction of hermaphrodite and rapid reproduction is the basis of population expansion.The study starts from observing the mating choice process of pine wood nematode,and focused on the phenomena of sex attraction in the mating search phase and female mating selection before mating.Accordling,we attempted to explore the internal mechanism under the pnernomena.The main findings are as follows:1)The attraction between females and males were clarified through the two-point chemotaxis assay on agar plates,the attractiveness of females to males is obviously greater than the attractiveness of males to females,and no attraction was observed between the same sexes.Besides,the same effect of females and males secretions on the opposite sex indicated the source of sex attraction.2)LC-MS analysis of the secretions of males and females showed that the main substances of the secretions were ascarosides,the difference between males and females secretions are mainly medium and long chain ascarosides.Among them,there are few chemicals with significant differences before and after mating of female and male.Five ascarosides showed significant difference between virgin and mated males,they are ascr#12,bhas#18,ascr#32,and ibha#24 in virgin males,and ascr#18 in mated males;There are three between virgin and mated females,they are ascr#18 in virgin females,ascr#12 and ibha#18 in mated females.The differences between males and females including ascr#15,bhas#16,bhas#20,ascr#23,icas#12,bhas#34,ibha#20,icas#21 and icas#22.Previous results showed that the secretions of male and female B.xylophilus are attractive to the opposite sex,indicating these long-chain ascarosides may play a role in the bisexual attraction of pine wood nematodes.The high expression of ascr#18 in mated males and virgin females and ascr#12 in virgin males and mated females indicated that they may play a special role in the sex attraction of B.xylophilus.3)Studies have shown that in C.elegans,the acox gene upstream of the ascaroside biosynthesis peroxide ? oxidation pathway is involved in the long-chain ascaroside biosynthesis,while the downstream daf-22 gene control shortens the long-chain ascaroside.Function analysis of Bx-daf-22 in B.xylophilus showed that the RNAi of Bx-daf-22.1,daf-22.2 and Bx-daf-22.3together caused the decreasing of female attraction to males,and the interference of Bx-daf-22.3individually lead to the attractiveness of females to males increased.Since Bx-daf-22 interference can cause a large decrease of short-chain ascarosides,and at the same time cause an increase in upstream long-chain ascarosides,this result indicated that medium-and long-chain ascarosides may the main substance that causes the attractiveness to males.4)In order to further prove the role of medium-long chain ascaroside in the bisexual attraction of pine wood nematode,the RNAi of the Bx-acox gene that controls the medium-long chain ascaroside biosynthesis upstream of the pine wood nematode peroxidase ? oxidation pathway were conducted.It showed that the both interference of Bx-acox1.1 and Bx-acox1.2cause significant decrease of females attractiveness to males,which supplemented the explanation of the role of medium-long chain ascaroside in the female attractiveness of pine wood nematodes to males.5)Micro observation showed after females and males got closer through sex attraction,females would judge and evalue the males through several contacts and finally chose one for mating.When in the face of both virgin and mated males,the female often chose to mate with the former one.The mating preference of females were explored through comparative analysis of the differences in sensory ability,attractiveness to females,and nutritional supplementation of mated and unmated males,results showed that nutritional recovery of unmated males can significantly change female Mating preference.6)Transcriptome analysis of virgin male,mated male without nutritional supplement and nutritionally restored mated males were performed to reveal the reseaon of female mating selection bias.Analysis showed that the differentially expressed genes(DEGs)of virgin and nutritionally restored mated males compared with mated males were mainly related to collagen synthesis and glutathione transferase,cathepsin and other enzymes related to the degradation of exogenous substances.The up regulated DEGs in the two groups were mainly collagen family genes and down regulated DEGs in the two groups were mainly transporters,dehydrogenases,and hydrolases,and other enzymes involved in stimulus response and osmotic regulation signaling pathways.Compared with virgin males,the nutrient recovery males have less DEGs.The up-regulation is mainly glutathione transferase,cellulase and transport proteins and the enzymes that involve in degradation of exogenous substances and self-osmotic adjustment.This may be affected by males' feeding activities during nutritional recovery and adaptability to the external environment.The down regulated DEGs is mainly some collagen genes and cathepsins,which may be associated with the recovery of the male's collagen skeleton structure during its nutirtion supplement.These results indicated the collagen genes and energy metabolism enzyme may related to female selection bias.7)Research on the transcriptome data analysis of virgin and mated females showed that a small amount of collagen genes and peptidases were down regulated after mated,and more than70% of these down regulated genes were also down regulated in mated males.But the down regulated genens in mated males were much more than in mated female,mainly were collagen genes and macromolecular metabolic enzymes.This indicate the energy and body consumption in the mating process of males is greater than females,and it may be the main reason of female mating selection bias.In summary,during the mating process of pine wood nematode,females and males first release chemical substance to get closer,then mating after female selection.Medium and long chain ascarosides may be worked during the sex attraction of females and males;transcriptome analysis results preliminary indicated that most of the collagen genes did not change significantly in mated females,but changed significantly in mated males,and expressed normally in the nutritional recovery males,indicating that these genes play special roles in males.According to previous studies,these collagen genes are relate to the development of nematode male spiculum and radiation sensing systems,and the spiculum and sensing system plays an important role in the positioning and response of males.It speculated that this may cause the mating selction bias of females to males.This study explored the sex attraction between females and males of pine wood nematodes and the selection of females to males before mating,which may provide useful information to further study the reproduction mechanism of the pathogenic nematode.