| Plutella xylostella is an economically important pest worldwide,which is harmful to cruciferous vegetables.P.xylostella is resistant to insecticides and the resistance develops rapidly.Therefore,it is urgent to establish a new population management strategy against this serious pest.The genetic regulation based on the sex determination shows important application prospect in pest control.It suppresses pest populations after releasing in the field by manipulating key sex-specific infertility or lethal genes of insects.Thus in this paper,we study the role of PxImp and PxPSI in sex determination of P.xylostella,and understand the mechanism of sex determination,by which to establish genetic control targeted to population sex ratio,and provide novel measures for sustainable management.The main results are as follows:1.Cloning and functional analysis of PxImp geneThe PxImp gene contains three non-sex-specific splice variants(PxImp~1、PxImp~2、PxImp~3)and one male-specific splice variant(PxImp~M),and the Open Reading Frame(ORF)length is 1602 bp,1638 bp,1572 bp and 1716 bp,respectively.The expression of PxImp~1,PxImp~2 and PxImp~3 were similar in both sexes from the third instar larvae to the adult stage except that the PxImp~1 was higher in the female than in the male.Among males,the expression of PxImp~M from the fourth instar larvae to the adults was equal and higher than the third instar larvae.When PxImp was down regulated by RNAi in the third instar larvae,the pupation rate and eclosion rate of males decreased significantly,and the pupation and eclosion period of female and male were prolonged more than 1 d.In addition,the expression of key sex determining gene Pxdsx~M in male was decreased,and the size of testis decreased significantly.RNAi down regulated PxImp~M which only reduced the expression of Pxdsx~M,while the expression of PxImp~M decreased after RNAi suppression of Pxy Masc.The results showed that PxImp gene affect the growth and development of larvae,pupae and adults,as well as the development of testis.2.Cloning and functional analysis of PxPSI geneThere was only one splice variant for PxPSI gene.The expression of PxPSI from the first to third larval stage was equal and constant in both sexes,while the expression of PxPSI from the fourth instar larvae to the adult stage increased gradually,and it was higher in the male than the female.RNAi suppression of PxPSI in the third instar larvae led to down regulated expression of Pxdsx~M in male,and the dented surface of testis.What’s more,the clasper was obvious distorted and breakage,and the last segment of abdomen was too long.When PxPSI was knocked out by CRISPR/Cas9,the male pupa has an abnormal black tumor on its abdomen,the male clasper was obviously deformed or even degraded.Additionly,the aedeagus was missing and the genital segment was shortened,which implied that PxPSI gene may play an important role in male sex differentiation of P.xylostella.The above results provide the preliminary evidence for the function of PxImp and PxPSI genes in sex determination of P.xylostella,which lay the foundation for further research of sex-based genetic regulation against P.xylostella population. |
