Mechanisms Of Juvenile Hormone Action In Helicoverpa Armigera And The Regulation Of Fitness Cost In Bt Resistance

The cotton bollworm, Helicoverpa armigera, which belongs to Lepidoptera Noctuidae, distributes broadly in the word and harms amount of crops including cotton, cereal, corn and wheat. As one of the major agricultural pests in China, the cotton bollworm often brings great lost in the economy. Transgenic crops that produce Bacillus thuringiensis(Bt) Cry toxins have become an important tool of insect pest management, however, the control efficacy of Bt crops will be diminished if pest evolves resistance to Bt toxins. To date, several studies have reported insect pests including Helioverpa zea and Pectinophora gossypiella to have evolved resistance to Bt crops in the field, and in China, it was found that the median LC50 of H. armigera grown annually, so the resistance of H. armigera to Bt has been the key factor for sustainable utilization of Bt cotton. The resistance of insect pests to Bt is often accompanied with the fitness cost which can delay or prevent the development of resistance, and the fitness component of fecundity was the most important factor linked to the population. The study of fitness costs associated with insect resistance to Bt insecticidal proteins is important for understanding resistance evolution and evaluating resistance management practices used to mitigate resistance to transgenic crops. In our study, we analyzed the fitness cost on the reproduction in the Bt resistant strains selected from three different regions. The new genes involved in the juvenile hormone(JH) regulation were identified with the method of RNA-sequence, the expression pattern of these genes during development period were tested by qRT-PCR and the affection on the development or reproduction were verficated by RNA interference. The main results are summarized as follows:We compared the life history traits of three independent sets of Helicoverpa armigera adults, of each there were a susceptible population and a Cry1Ac-resistant population derived by selection from it. It was found that resistant individuals exhibited fewer progeny, less fecundity, lower egg hatching rate and longer adult longevity than susceptible strains. For instance, the fecundity in 96 S,LFS and XJS was 817.15±27.32, 712.35±23.16 and 833.96±29.43, receptively, which was higher than that in resistant strains(565.60±16.40-666.74±28.12); the hatchability in susceptible strains was 55.19-69.51%, which was higher than that in resistant strains. Interestingly, the juvenile hormone(JH) level, appeared higher in resistant strains relative to susceptible strains. In the resistant strains of LF120 and BtR, the higher JH appeared in the previtellogenic stage, while there were no differences among susceptible and resistant strains during the vitellogenin stage. In the XJ series, the JH level was always high during the development stage.By the de nove sequencing of the transcriptome of cotton bollworm at the different stages, a total of 177539 unigens were produced. We identified 21 new genes involved in the synthesis and metabolism of JH and tested the JH titer during the developmental time. We found the JH appeared high level after ecdysiasts. The results of expression level during the development stages showed that FPPS4 played dominate role in the JH synthesis and JHEH2 was the main factor in the metabolism of JH.In the present study, laboratory observations were made on the development of H.armigera after treated with different concentration of Methoprene by injection method, the result showed that 2nd and 5th instar larval were susceptible to Methoprene, while there was no affection of Methoprene on the development of 3rd instar larval. And it showed that the prolongation of larval occurred with 0.3d-0.8d when the concentration under 2.5 μg/μl, while the higher concentration harms to the development of cotton bollworm in 2nd instar larval, such as the mortality rate was 60% and 95% with the concentration of 2.5 μg/μl and 5.0 μg/μl. For the 3rd larval instar, the larval developmental duration prolonged 2d when treated with Methoprene under the concentration of 5.0 μg/μl, and the survival rate were not affected by the application of Methoprene. It was also found that the mortality rate was 80% of 5th larval instar with 5.0 μg/μl Methoprene. By the de nove sequencing of the transcriptome of cotton bollworm treated with Methoprene and control, a total of 50481 unigenes were produced. Through the different expressed genes analysis, 4416 unigenes were found to express differently between them, among which 583 unigens were up regulated after treated with Methoprene, while the other 3833 were down-regulated. In the Methoprene treated individuals the genes related to metabolism of JH and 20 E were up-regulated, while the expression of some cytochrome P450 s genes and JHAMT were down-regulated in the Methoprene treated groups.Based on the results of RNA-sequence database, the CDS of Met, Kr-h1 and FTZ-F1 were cloned. The expression level showed that these genes expressed during the whole developmental stage with the peak at the last instar larval, and the expression level of Met in females was significantly higher than that in males. Analysis the function of these genes using the method of RNA interference, it showed that knockdown of Met and Kr-h1 caused shorter pupation time about 48 hours with changing the expression of genes associated with 20 E, and injection of Met and Kr-h1 dsRNAs reduced half of the number of laid eggs and hatchability. Knockdown of FTZ-F1 caused larval lethality and abnormal phenotype, and silencing this gene affected the expression of JHAMT which was associated with JH biosynthesis.