Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae)

Miwa Uchibori-Asano,Yoshitaka Suetsugu,Tetsuya Kobayashi,Chiharu Ishizuka,Yoshiaki Nakagawa,Gaku Akiduki,Takaaki Daimon,Tetsuro Shinoda,Kakeru Yokoi,Saki Minami,Akihito Ozawa,Akiya Jouraku,Toru Uchiyama

doi:10.1038/s41598-019-40863-5

Abstract

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai.

Highlights

Diacylhydrazine-type insect growth regulators (DAH-IGRs) are non-steroidal ecdysteroid agonists and tebufenozide, methoxyfenozide, chromafenozide, and halofenozide are representatives of commercially available DAH-IGRs1
A high correlation was observed in the sum of four CYP9A genes (r = −0.7845, p = 0.0015). These results suggested that high expression of CYP9A family genes in the LG12 region is involved in the tebufenozide-resistance in A. honmai
One is the decreased sensitivity to tebufenozide caused by an amino acid substitution in the target molecule ecdysone receptor gene (EcR), and the other is increased detoxification activity caused by the overexpression of multiple CYP9A and CCE genes

Summary

Introduction

Diacylhydrazine-type insect growth regulators (DAH-IGRs) are non-steroidal ecdysteroid agonists and tebufenozide, methoxyfenozide, chromafenozide, and halofenozide are representatives of commercially available DAH-IGRs1. The former three DAH-IGRs are specific to lepidopteran pests, while halofenozide is effective to both lepidopteran and coleopteran pests. Www.nature.com/scientificreports feeding and head-capsule slippage at unnatural timing, and lead to lethal moulting[4]. Because of such unique actions, DAH-IGRs are effective against insect pests that have already developed high resistance to conventional insecticides targeting the nervous system, such as organophosphates, carbamates, neonicotinoids, organochlorines, and pyrethroids. The molecular and biochemical mechanisms of tebufenozide-resistance in A. honmai remain largely unknown

Methods

Results

Conclusion