The Fitness Cost Of The Insecticide-resistant Diamondback Moth And Its Association With The JHEH Gene

Given the existence of biological fitness costs in the insecticide-resistantdiamondback moth (DBM), Plutella xylostella, comparisons between resistant (RS)and susceptible (SS) DBM strains under conditions without insecticide stress wereperformed to study the developmental period, weight, and survival rate of larvae, andthe oviposition quantity, hatching rates and survival period of male and female adults.High performance liquid chromatography (HPLC) was used to measure the amount ofjuvenile hormone (JH) present during larval development. One member of the DBMjuvenile hormone epoxide hydrolase (JHEH) gene family was cloned using the RACEtechnique, and its expression during larval development was determined usingreal-time PCR. The RNAi technique was employed to silence JHEH to determine itsrole in the development of DBM larvae. The main results are as follows.1. Fitness costs prevail in insecticide-resistant DBM strainsThe resistance ratio of the chlorpyrifos-resistant strain is573.56times that of thesusceptible strain and4.03times that observed in the fipronil-resistant strain. Theduration of larval developmental for RS DBMs was6.4days, which was faster thanthe7.5days observed for SS DBMs. Larval body weight for RS DBMs increasedmarkedly faster than in SS DBMs, but the final weight showed a distinctdisadvantage. The larval survival rate and the oviposition rate of adults did not showany significant differences between RS and SS DBMs. The egg-hatching rate of theRS moths (91%) also showed a distinct disadvantage when compared with the SSmoths (97%). RS female moths survived for6.6days compared with7.3days forfemale SS moths, showing a distinct disadvantage. However, male survival was10.7days for RS moths compared with9.0days for SS moths, showing a significantadvantage.2. The dynamic changes in the JH content relate to developmental differencesThe JH titers of both SS and RS moths increased similarly with the age of themoth. For the same instar, the JH titer in the single RS larva was more than that of the SS larva, though some significant differences were observed over thedevelopmental period. The JH titer per unit of body weight for both RS and SSmoths was reduced with aging. As a whole, for the same time of the same instar, theJH titer per unit of body weight for RS moths was more than that of the SS moths,except for the time points of96h,132h and144h after hatching.3. Cloning of a DBM JHEH gene whose expression correlated with the JH titerA1,777bp full-length cDNA of the JHEH gene was cloned; the clone containeda predicted open reading frame of1,644bp encoding a547amino acid protein, a40bp5'-untranslated region (UTR), a64bp3'-UTR, and a19bp poly (A) tail. Thepredicted molecular weight and isoelectric point were61.2kD and6.34, respectively.Because the JHEH protein was more hydrophilic than hydrophobic, the JHEH proteinshould be soluble. A typical epoxide hydrolase domain and epoxy hydrolaseN-terminal domain between amino acids98and207of the JHEH protein were found,indicating that the gene belongs to the epoxy hydrolase protein superfamily. Aphylogenetic tree was constructed using the neighbor-joining method, which indicatedthat P. xylostella was closely related to Bombyx mori, more distantly related to thehymenopteran insects, and most distantly related to Trichoplusia ni and Spodopteraexigua of the lepidopteran insects.The mRNA levels of the JHEH gene were measured using real-time PCR. Theresults showed that changes in the relative expression levels of the JHEH gene aregenerally consistent in both strains. The expression levels of the JHEH gene in the RRmoths were lower than in the SS moths, except for the time point of120h afterhatching. The changes in the expression of the DBM JHEH gene in both strainsconformed to changes in the per unit body weight of the JH titer, indicating that t heJHEH gene may play an important role in the degradation of JH.4. Functional characterization of the DBM JHEH gene using RNAiWhen fed with dsRNA of0.6583μg/μL, significant differences were observed insurvival rate between the treatment and control after feeded48h and72h. The expression of the JHEH gene did not show any significant differences when the larvaewere fed JHEH-dsRNA compared with the control, indicating that dsRNA mayinterfere with the expression of JHEH mRNA. However, because the larvaldevelopmental period and expression of the JHEH gene of the treatment did not showsignificant differences compared with the control, further studies are required forunderstanding the role of JHEH in the development of DBMs.