Molecular evidence of insecticide resistance in Helicoverpa armigera and expression profiles of genes involved in metabolic resistance

Abstract

Cotton bollworm, Helicoverpa armigera (Hübner) is a polyphagous pest which has been reported from a wide array of plants and has cosmopolitan distribution across Asia, Australia, Africa, Europe and South America. It is one of the most widespread pest species of economically important crops. To prevent the damage caused by H. armigera, a variety of methods are being used, including the use of chemical and biological pesticides. Chemical pesticides are the first choice for H. armigera control and 30% of all the pesticides worldwide are used against this pest. The insecticides used for its control include organophosphates, carbamates and pyrethroids but extensive and unattended use of chemical pesticides has resulted in high levels of resistance in H. armigera. In Pakistan, H. armigera has developed high level of resistance against pyrethroid insecticides mainly cypermethrin but the resistance mechanism and the underlying genes have remained unidentified. The current study was designed to identify and investigate the novel genes and mechanisms involved in metabolic insecticide resistance in H. armigera with particular focus on a pyrethroid (cypermethrin) and to find the genes regulating the over-expression of cytochrome P450 in the resistant insects. Helicoverpa armigera were collected from cotton/chickpea fields in Faisalabad (FSD), Pakistan and tested for resistance against cypermethrin. It was revealed that FSD has evolved 6.9-fold resistance compared to Australian cypermethrin susceptible strain TWBS (Toowoomba). To determine the resistance mechanism of the FSD strain, larvae were treated with piperonyl butoxide (PBO), a known P450 and carboxylesterase inhibitor, prior to cypermethrin treatment and the observed cypermethrin resistance was a metabolic resistance. To find out the genes involved in metabolic resistance, amplified fragment length polymorphism (AFLP) was used. The AFLP based linkage map of FSD strain revealed that a single linkage group consisting of 13 AFLPs showed a strong correlation with resistance, indicating the involvement of a single gene. Thus a previously reported metabolic resistance gene CYP337B3 was considered as a possible candidate gene for cypermethrin resistance in FSD strain and it was further confirmed by PCR analysis which showed the presence of CYP337B3 gene in toxicity bioassays survived larvae and absence in killed larvae. Survivorship of Abstract XVII the cypermethrin toxicity bioassay was most strongly correlated with the CYP337B3 genotype. To evaluate the metabolic capability of CYP337B3 gene to catabolise cypermethrin, CYP337B3 was isolated from FSD strain, ligated in heterologous expression vector and transformed into insect cell line (HA2302). It was found that heterologous CYP337B3 enzyme is able to degrade cypermethrin in in vitro assays using high performance liquid chromatography (HPLC). To evaluate the possible role of other P450 genes in the cypermethrin resistance of the FSD strain, the expression patterns of 59 P450 genes were analysed in FSD strain, Australian fenvalerate resistant TWBR strain and susceptible strain (TWBS) using quantitative PCR (qPCR). There was a higher expression (>5-fold) of three genes (CYP340G1, CYP340H1 and CYP341B2) in FSD. Expression levels all P450 genes in TWBR were similar to TWBS only with the exception of CYP321B1 significant down-regulation. Moreover, expression profile of these genes was also examined in backcross progeny possessing CYP337B3 to study whether the upregulated P450 genes (CYP340G1, CYP340H1 and CYP341B2) in FSD were genetically linked to the cypermethrin resistance. However, it was revealed that these genes were not overexpressed in backcross progeny. Genetic diversity and presence or absence of resistance, in different populations of H. armigera from Pakistan, were determined by using DNA barcoding (COI gene) and CYP337B genes family, respectively. It was found that pyrethroid resistance gene, CYP337B3 was present in all the populations (Northern, Central, East- Southern) from Pakistan. Moreover, no significant population structure of H. armigera was observed in Pakistan, indicating that this migratory pest is not restricted to specific geographic region. The study concludes that H. armigera in Pakistan has evolved CYP337B3 mediated metabolic resistance against pyrethroids and CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles