Functional Study Of Prothoracicotropic Hormone In The Regulation Of Reproductive Behavior Of Spodoptera Litura Females

Spodoptera litura(Lepidoptera:Noctuidae)is one of the most destructive pests of many agricultural crops,which lies all around the world.Because of its polyphagy and fertility,it is easy to break out and cause great losses to agriculture and forestry.Therefore,we need to find more ways to prevent the outbreak of Spodoptera litura.Insect hormones are signalling molecules essential for the correct regulation of many different physiological,behavioural and developmental events in insects.The study of insect hormones can not only understand the signal pathway of growth,development and reproduction,and further increasing our understanding of the life,we also can screen genes in this process as an alternative methods for pest control.Prothoracicotropic Hormone(PTTH)is a brain neuropeptide which can consists of stimulating the secretion of ecdysone(the moulting hormone)by the prothoracic glands(PGs)in larval,then it can cause the development of target tissue.Its release and blocking can be used as a switch to stop or start larval pupa diapause.It has been reported in Lepidoptera,Diptera and Hymenoptera.The transcriptional data showed that there was a significant difference in the expression of PTTH mRNA between the vrigin moth and non-vrigin moth.Meanwhile,there was a high expression of PTTH mRNA in the brain of adult Lepidoptera,indicating that PTTH may be regulate the reproductive behavior of Spodoptera litura.However,the reproductive function of PTTH in Spodoptera litura is still unkown.For these reasons,we amplified the PTTH gene of Spodoptera litura and analyzed the expression of PTTH in different tissues during the whole life by fluorescence quantitative method.Then we study the function of PTTH gene by bacterial-mediated RNAi.The PTTH mRNA sequence contained a putative ORF of 681 nucleotides encoding 227 amino acids.The similarity with Spodoptera exigua and Bombyx mori is 84.5%and 58.7%respectively.The phylogenetic tree based on Spodoptera litura PTTH is consistent with the traditional taxonomic classification.The overall homology of all Lepidoptera insects was 50.39%.The similarity of PTTH amino acids between Spodoptera litura and other insects order is very low,only 12%-18%.But they share some common conservative structures,such as the presence of seven cysteines.The results of real-time fluorescence quantitative analysis showed that PTTH was expressed in the whole life of Spodoptera litura,and the expression in the egg period was the highest.The expression of the fourth and fifth instar larvae was lower,but the expression increased from the last instar larva to the pupal stage which suggest that the function of PTTH is related to insect molting metamorphosis,but the high expression of PTTH in egg stage and low instar larvae should re-evaluate the function of PTTH.We detected the expression of PTTH mRNA in different tissues.The results of relative expression levels showed the highest experssion is in ovary,higher than head.which suggests that PTTH participates in or regulates insect reproductive behavior in adult stage.The expression of PTTH mRNA was higher in wings than that of reproductive tract,chest and other tissues.The expression in intestinal tissue was the lowest.In this study,we constructed a bacterial-mediated RNAi system with PTTH as the target gene.IPTG was used to induced the E.coli to produce dsRNA.We fed the larva E.coli expressing PTTH dsRNA to interfere the expression of PTTH gene,and the interference efficiency reached 75%.There was no significant change in weight between pupa stage and adult stage after feeding,but the time of pupation and eclosion was earlier.In the four artificial nights,the mating rate of Spodoptera litura decreased significantly after the interference of PTTH gene.And in the group-female which was interfered,it affected the behavior of the paired male moth.The proportion of the male moth that occured courting reached 100%,and significantly increased the courting times in the first artificial night,and the calling rate and calling times of the disturbed female moth also decreased.The average life both the experimental group and the control group was about 7 days,and interference with PTTH gene did not affect the life of Spodoptera litura.At the same time,through non-parametric test analysis,the average number of eggs per female in the experimental group and the control group did not change significantly,and the hatching rates of the experimental group and the control group were more than 98%,indicating that the PTTH gene did not affect the hatching rate of Spodoptera litura offspring.In this study,we found that PTTH played an important role in the reproductive process first time through the way of RNAi and phenotypic observation,which deepened the understanding of the functional diversity of the brain neuropeptide,and it can provid technical support and molecular targets for the exploration of new methods of pest control.