Overexpression of a P450 gene (CYP6CW1) in buprofezin-resistant Laodelphax striatellus (Fallén)

Abstract

Cytochrome P450 monooxygenase (P450)-mediated detoxification is an important mechanism involved in the resistance to neurotoxic pesticides. However, the molecular basis of the mechanism of P450s, as associated with the resistance to growth regulator insecticides (IGRs) remains largely unknown. In this study, a resistance strain (YN-BPF) of Laodelphax striatellus was developed, with 59.9-fold resistance to buprofezin, through 42 generations of discontinuous selections of the susceptible strain (YN) with buprofezin (an IGR). A synergistic study in vivo and a biochemical study in vitro indicated that an enhanced detoxification mediated by P450s to some extent contributes to the buprofezin resistance in the YN-BPF strain. A total of 38 cDNA sequences encoding tentative unique P450 genes were identified in an L. striatellus transcriptome database, and the mRNA expression level of these genes was examined in the YN and YN-BPF strains using quantitative real-time PCR (qPCR). A single P450 gene, CYP6CW1, was highly overexpressed (22.78-fold) in the YN-BPF strain compared with the YN strain. Based on the analysis of insects with similar genetic backgrounds, our results provided evidence for the role of CYP6CW1 in the resistance of L. striatellus to buprofezin.

Introduction

The small brown planthopper, Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), a notorious agricultural pest, causes serious damage to rice due to transmission of rice stripe virus and rice black streaked dwarf virus [1]. Buprofezin is an insect growth regulator insecticide (IGR) that is mainly used in the control of homopteran pests, such as L. striatellus. Buprofezin is active against larval developmental stages, causing cuticular lesions that result from the disruption of chitin synthesis. Because this insecticide has generally been considered to have a good efficacy against the target pests while being harmless to beneficial insects, it has been used widely in integrated pest management (IPM) programs [2], [3]. However, the extensive applications of buprofezin inevitably results in the development of resistance in natural populations of L. striatellus [4]. Thus, to better control this important agricultural pest, it is important to reveal the underlying resistance mechanism of this commonly used insecticide.

Cytochrome P450 monooxygenases (P450s) can be involved in the detoxification and bioactivation of insecticides [5]. Moreover, the transcriptional overexpression of P450 genes is often thought to enhance the metabolism of insecticides and appears to be a common phenomenon in the evolution of resistance development in insects. In fact, as a reductionist’s means of understanding resistance mechanisms in insects, overexpression has become a dominant criterion in identifying insecticide resistance associated with the P450 genes. The increased metabolism of neurotoxic insecticides in correlation with the overexpression of P450 genes has been well demonstrated in many insect species, including the house fly Musca domestica (CYP6D1), fruit fly Drosophila melanogaster (CYP6G1), mosquito Culex pipiens pallens (CYP6F1), brown planthopper Nilaparvata lugens (CYP6ER1), mosquito Culex quinquefasciatus (CYP9M10) and whitefly Bemisia tabaci (CYP6CW1) [6], [7], [8], [9], [10], [11]. Although the molecular basis of the mechanism of the P450-associated resistance of IGRs remained largely unknown when compared with neurotoxic insecticides, a few studies have suggested that P450s are involved in the catabolism of IGRs [12], [13]. In particular, Karatolos et al. (2012) found that the overexpression of CYP4G61 was associated with pyriproxyfen (a juvenile hormone analogue) resistance [14]. However, there has been no evidence for the correlation between P450 and buprofezin resistance in L. striatellus reported thus far.

In this study, a significantly buprofezin-resistant strain of L. striatellus was found in our laboratory. Compared with the susceptible (YN) strain, the buprofezin-selected (YN-BPF) strain developed a 59.9-fold resistance to buprofezin. The establishment of this buprofezin-resistant strain in addition to a transcriptome database of L. striatellus provided an excellent platform and opportunity to systematically identify P450 genes and assess their roles in buprofezin resistance in L. striatellus. Here, we report the following: (1) a synergistic study in vivo and a biochemical analysis in vitro to evaluate the role of P450s in buprofezin resistance in the YN-BPF strain and (2) the expression patterns of 38 P450 genes in an effort to identify candidate genes associated with the buprofezin-resistant phenotype.