Identification And Functional Analysis Of Halloween Genes In Plutella Xylostella

Ecdysteroids play fundamental roles in controlling insect development and reproduction,and they are involved in a series of physiological processes,including Vg biosynthesis and transport,germ cell differentiation,oogenesis,ovarian development and so on.The sequential hydroxylation of steroid precursors into the active ecdysteroid 20E(20-hydroxyecdysone)is mediated by a group of enzymes encoded by genes in the Halloween family,but some of these genes are species-specific.Therefore,understanding the molecular characteristics and functions of Halloween genes is biologically important for illuminating hormonal regulation mechanisms in the development and reproduction of pests and for controlling population growth of and damage by pests.Although Plutella xylostella is a worldwide pest of many economically important cruciferous crops,there are currently no effective strategies to control this prolific and rapidly reproducing insect in China.To gain a better understanding of the hormonal regulation mechanism of development and reproduction in P.xylostella,we propose to carry out studies using 20E synthase genes with the reproduction hormone as a core.The study includes three components:(1)identifying the number,molecular characteristics and phylogenetic relationship of Halloween genes;(2)measuring the stage-and tissue-specific expression profiles of Halloween genes;and(3)uncovering the functions of Halloween genes in the 20E synthesis,development and reproduction of P.xylostella.The main results were as follows:Six Halloween genes,including disembodied(dib),shadow(sad),shade(shd),spook(spo),nonmolting glossy/shroud(sro),and neverland(nvd),were identified in the P.xylostella genome;contained ORFs of 1431,1231,1552,1620,918,and 1128 bp,respectively;and encoded 476,305,470,539,305,and 375 aa,respectively.The converted amino acid sequences were compared with those of other insects and showed that spo,dib,sad and shd belonged to the P450 superfamily,while nvd and sro were contained in the dieske and SDR(short-chain dehydrogenase reductase)superfamily,respectively.The phylogenetic analysis of predicted amino acid sequences with Halloween orthologues showed they were all highly conserved with other homologous genes,especially in Lepidoptera.The expression profiles showed that Halloween genes were expressed at each developmental stage,suggesting their important functions in the physiological activity of P.xylostella.Among the genes,the highest expression of dib,sad and shd was in the adult stage,and nvd and spo were highly expressed in the egg and pupa stage,respectively.However,there was a relatively lower transcript level of sro in each developmental stage.Tissue-specific expression profiles showed that five Halloween genes(nvd,sro,spo,dib and sad)were highly expressed specifically in the prothorax,which is the major site of ecdysone production,but shd was expressed predominantly in the fat body to convert ecdysone to 20-hydroxyecdysone.RNAi-based knockdown of sad expression,which is involved in the last step of ecdysone biosynthesis,was conducted to explore the functions of Halloween genes.Injection of dssad into fourth-instar larvae within 12 and 24 hours significantly inhibited sad expression and the 20E titer of P.xylostella and resulted in a longer developmental duration and lower pupation of fourth-instar larvae.However,with injection of dssad into female pupae,sad expression was suppressed significantly within 6 and 12 hours,but the 20E titer was only significantly reduced within 12 hours;the 20E titer also caused shorter ovarioles,vitelline depletion in the oocyte,and fewer mature eggs.The number of eggs laid by females was significantly reduced after 3 days,suggesting the important roles of 20E in P.xylostella reproduction.These results showed that Halloween genes play a critical role in the biosynthesis of ecdysteroids and are involved in the development and reproduction of P.xylostella.This study will establish the theoretical foundations on which it will be possible to use hormonal regulation mechanisms of development and reproduction of P.xylostella to screen potential genes to better control P.xylostella populations under various environmental conditions.