Over-expression of multiple cytochrome P450 genes in fenvalerate-resistant field strains of Helicoverpa armigera from north of China

https://doi.org/10.1016/j.pestbp.2016.01.003Get rights and content

Abstract

Pyrethroid resistance was one of the main reasons for control failure of cotton bollworm Helicoverpa armigera (Hübner) in China. The promotion of Bt crops decreased the application of chemical insecticides in controlling H. armigera. However, the cotton bollworm still kept high levels of resistance to fenvalerate. In this study, the resistance levels of 8 field-collected strains of H. armigera from north of China to 4 insecticides, as well as the expression levels of related P450 genes were investigated. The results of bioassay indicated that the resistance levels to fenvalerate in the field strains varied from 5.4- to 114.7-fold, while the resistance levels to lambda-cyhalothrin, phoxim and methomyl were low, which were ranged from 1.5- to 5.2-, 0.2- to 1.6-, and 2.9- to 8.3- fold, respectively, compared to a susceptible strain. Synergistic experiment showed that PBO was the most effective synergist in increasing the sensitivity of H. armigera to fenvalerate, suggesting that P450 enzymes were involved in the pyrethroid resistance in the field strains. The results of quantitative RT-PCR indicated that eight P450 genes (CYP332A1, CYP4L11, CYP4L5, CYP4M6, CYP4M7, CYP6B7, CYP9A12, CYP9A14) were all significantly overexpressed in Hejian1 and Xiajin1 strains of H. armigera collected in 2013, and CYP4L5 was significantly overexpressed in all the 6 field strains collected in 2014. CYP332A1, CYP6B7 and CYP9A12 had very high overexpression levels in all the field strains, indicating their important roles in fenvalerate resistance. The results suggested that multiple P450 genes were involved in the high-level fenvalerate-resistance in different field strains of H. armigera collected from north of China.

Introduction

The cotton bollworm, Helicoverpa armigera (Hübner), is an important polyphagous agricultural pest with a wide range of distribution in Asia, Africa, Australia and Europe. H. armigera had developed resistance to almost all kinds of insecticides, including pyrethroids, organophosphates, carbamates, organochlorines, spinosad and Bt toxin in different areas and countries [1], [2], [3], [4]. In China, H. armigera was a major pest on cotton before the commercialization of transgenic Bacillus thuringiensis (Bt) crops [5]; pyrethroids were introduced to control this insect pest in the early 1980s; resistance of H. armigera to pyrethroids was first reported in the late 1980s and heavily broke out in 1992, which led to more than ¥5 billion economic losses [6].

Increasing metabolism mediated by detoxification enzymes, such as esterases, GSTs, cytochrome P450s, have been considered to be responsible for insecticide resistance [7], [8], [9]. Among the 3 detoxification enzymes, cytochrome P450s was reported to be the major detoxification enzyme to pyrethroids and responsible for high level resistance to different pyrethroid insecticides [10], [11], [12].

Cytochrome P450s is encoded by a superfamily genes and capable of metabolizing endogenous and exogenous substance in virtually all organisms, and contribute to the adaptability of animals to a wide range of host plants and insecticides resistance of pest [10], [13], [14], [15]. In H. armigera, multiple cytochrome P450 genes have been reported to be overexpressed and related to insecticide resistance, such as CYP6B7, CYP9A12, CYP9A14, CYP337B3 [3], [16], [17], [18], [19], [20], [21], [22], [23], [24]. However, the expression levels of specific genes are different in different strains and the contribution of a CYP gene varied among different strains, e.g. CYP337B3, which was arisen from unequal crossing-over of CYP337B1 and CYP337B2, was reported to be involved in fenvalerate resistance of H. armigera in an Australia strain and cypermethrin resistance in a Pakistan strain, and could metabolize fenvalerate to 4′-hydroxyfenvalerate [3] and cypermethrin to 4′-hydroxycypermethrin [24], respectively. While in several field-derived resistant populations in China, fenvalerate resistance was validated to be not correlated with CYP337B3 [25], suggesting the complexity of resistance mediated by cytochrome P450 in H. armigera. Such phenomenon also occurred in other insect species. In Drosophila melanogaster, DDT resistance resulted from the over-transcription of a single P450 gene CYP6G1, which can metabolize a wide range of insecticides, including imidacloprid and lufenuron [26]. Overexpression of CYP12A4 was also reported conferring lufenuron resistance in another field population of D. melanogaster [27]. CYP6G1 and CYP12D1 were found both over-expressed in a field strain of D. melanogaster selected by DDT. When CYP6G1 was knocked down, CYP6A8 was selected instead [28]. In housefly, over-expression of CYP6D1 was the major reason for permethrin resistance in a permethrin-selected strain LPR [29], while CYP6D3v2, CYP6A24, and CYP12A1 were reported to be overexpressed in some other permethrin-resistant strains of housefly [30], [31], [32].

High levels of fenvalerate resistance in H. armigera had been reported after 15 years of cotton planting in China [33].This can be explained by the inevitable application of insecticides for the control of mirid bugs and late-season cotton bollworms. Investigation on the resistance levels of H. armigera to commonly used insecticides and the resistance mechanisms could provide information for the effective control of pyrethroid resistance of H. armigera in field. In the present study, we detected the resistance levels of 8 field-collected strains of H. armigera to 4 insecticides, investigated the expression levels of eleven cytochrome P450 genes in order to understand the roles of different P450 genes in pyrethroids resistance in field strains of H. armigera from north of China.

Section snippets

Insects

HDS strain of H. armigera, was first collected from Handan, Hebei province in 1988, maintained in laboratory on artificial diet without insecticide exposure and served as a susceptible strain. Field strains of H. armigera were collected from six locations (see Table 1) from north of China in 2013 and 2014. Most H. armigera were collected from corns except for that of Weifang strain, which was collected from non-Bt cotton. The larvae were all reared on artificial diet at 27 ± 1 °C, 70 ± 10% humidity

Insecticide resistance

The results of bioassay (Table 2, Table 3) showed that the field strains of H. armigera collected in 2014, including Hejian2, Xiajin2, Weifang, Beijing and Anyang, showed quite high levels of resistance to fenvalerate, with resistance ratio (RR) of 55.8-, 96.1-, 68.9-, 114.7- and 66.3-fold respectively, comparing with the susceptible HDS strain. Since PBO, DEF and DEM are well known inhibitors of cytochrome P450s, esterases and GSTs, respectively, a synergist experiment was performed to detect

Discussion

In China, organophosphates and carbamates were introduced to control the cotton bollworm in the late 1970s, while pyrethroids were introduced in the early 1980s due to its high efficiency and low toxicity. With the occurrence of severe resistance problems of pyrethroids in the 1990s, mixture of organophosphates and pyrethroids were used to control the cotton bollworm. Previous resistance monitoring data showed that the resistance levels of H. armigera to fenvalerate were high in the population

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant No. 30971943).

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