Functional Study Of Natalisin And Its Receptor SIA32 In The Regulation Of Reproductive Behavior Of Spodoptera Litura

Spodoptera litura(Lepidoptera:Noctuidae)is a worldwide polyphagous pest,which can harm more than 380 plant species from 109 families,including vegetables,fruit trees,flowers,lawns,cotton,corn and tobacco.In China,this insect is an important pest of economic crops such as grain,cotton,tobacco and vegetables.Insect reproductive behavior is the basis of sexual selection and evolution,and is also important for pest control and the utilization of beneficial insects.Neuropeptides are a special class of important information substances,which is mainly secreted by the brain and other neurosecretory cells.Neuropeptides regulate the development,physiology and behavior of animals.Tachykinins(TKs)are a special class of neuropeptides found in vertebrates.The representative members of TKs are TKs ???,DTK 1-6 and Natalisin(NTL).NTL has been found in a number of insects such as fruit flies,red flour beetle and silkworms.Previous studies have found that NTL may be related to mating behavior in Drosophila,while in the red flour beetle it may function in regulating of egg laying.However,whether NTL has similar functions in silkworms or other moth species remains to be determined.In the present study,therefore,we cloned the full-length mRNA sequences of NTL and its receptor NTLR(SlA32)by using RNA-seq and RACE technology.And then the relative expression levels of NTL and SlA32 in different tissues and different developmental stages were detected by real-time fluorescence quantitative technique.Finally,we carried out the functional analysis of NTL and SlA32 by using bacterial-mediated RNA interference and physiological and behavioral assay.The main findings of this study are as follows:(1)The full-length mRNA sequence of NTL is 1593 bp.The full mRNA sequence contained a putative ORF of 1317 nucleotides encoding a 438-amino acid protein,flanked by a 5-UTR of 95 bp and a 3-UTR of 181 bp.The full-length SlA32 mRNA is 1618 bp,and its 5,and 3'UTR are 62 bp and 233 bp.Its ORF is 1323 bp,encoding a protein of 440 amino acids.Homology analysis showed that the proteins encoded by the NTL and SlA32 of S.litura showed high homology with lepidopteran insects but low homology with species from other insect'orders.Predictive analysis by transmembrane domain indicated that S1A32 is a typical G protein coupled receptor(GPCR).(2)Real-time fluorescence quantitative analysis showed that the expression patterns of NTL and S1A32 were similar at different developmental stages,they were lower in the egg and larval stage,increased in the pupa stage,and reached the maximum in the adult stage.This expression pattern is related to the sexual maturity reproductive behavior of this moth.However,during adult stage,the expression of SlA32 was delayed compared with NTL;NTL showed the highest expression in 0-day-old males and 1-day-old females,while SIA32 showed the highest expression in 1-day-old males and 2-day-old females.This result indicates that NTL and SlA32 are closely related,and NTL expression may contribute to the expression of SIA32.The expression of NTL and SIA32 in different tissues of S.litura also showed a certain regularity;the expression in the head was higher than that of the chest and reproductive system,and there was a significant decreasing trend from the head to the chest and then to the reproductive system.The expression level in male heads is higher than that of in female heads.The expression differences between sexes and tissues suggest that NTL and SlA32 may have different functions in different tissues of males and females.(3)The bacterial-mediated RNA interference system of NTL and SIA32 was successfully constructed,and achieved a high interference efficiency(more than 60%).Knocking down the expression of NTL and SIA32 significantly reduced male courtship and female calling behavior,ultimately resulting in a lower mating percentage.The mating percentages ofcontrol females(feeding sterile water or E.coli expressing EGFP dsRNA)were 100%and 92%,and the males in the control group were 85%and 77%.However,the males and females in the experimental group(feeding E.coli expressing NTL dsRNA or SIA32 dsRNA)had a lower mating percentage(23-46%).Knocking down the expression of female NTL and SIA32 significantly reduced the fecundity of females.The normal females mating with NTL and SlA32 RNAi males laid fewer eggs after mating than that of controls.These results indicate that ANTL and its receptor SIA32 play important roles both in female and male reproductive process,and exhibit significant functional and mechanical differences between males and females.In conclusion,in the present study,we cloned the full-length mRNA sequence of NTL and its receptor S1A32 for the first time,and confirmed their important roles in the reproductive process of S.litura.It is also the first time to study the function of NTL and SlA32 in Lepidoptera,which provides useful information for the in-depth exploration of reproductive mechanism in these insects.In this study we also constructed a bacterial-mediated RNA interference system for the first time in S.litura.The bacterial-mediated RNA interference technology is a low-cost,high-efficiency technique for gene silencing and functional analysis,which can also be used for pest control.