Differential metabolism of neonicotinoids by brown planthopper, Nilaparvata lugens, CYP6ER1 variants
Introduction
Nilaparvata lugens Stål severely damages rice plants by sucking up and transmitting viruses, including rice ragged stunt virus and rice grassy stunt virus. Imidacloprid, which was introduced in 1991, was very effective against planthoppers and was used heavily (Bass et al., 2015). Then, imidacloprid-resistant N. lugens appeared in Asian countries, including Vietnam, China, and Japan (Matsumura et al., 2008). Since 2005, there have been outbreaks of N. lugens in East Asian countries (Nakao, 2017). Imidacloprid, which acts as a nicotinic acetylcholine receptor (nAChR) competitive modulator, belongs to Insecticide Resistance Action Committee (IRAC) group 4A. IRAC 4A includes seven neonicotinoids: imidacloprid, nitenpyram, acetamiprid, thiamethoxam, thiacloprid, clothianidin, and dinotefuran. Structure of dinotefuran is different from those of neonicotinoids, because dinotefuran has a tetrahydrofuran structure instead of pyridine or thiazole ring. According to chemoinformatic analysis of chemical similarity relations (Tanimoto indices), dinotefuran was clearly separated from other neonicotinoids (Nauen et al., 2015). According to the Arthropod Pesticide Resistance Database (APRD) (Michigan State University, 2019), there are many cases of resistance to neonicotinoids. It is interesting to investigate whether the different structure of dinotefuran causes the lack of cross-resistance.
CYP6ER1 overexpression in N. lugens has been associated with imidacloprid resistance in five countries in South and East Asia (Garrood et al., 2016). RNA interference of CYP6ER1 indicated that CYP6ER1 expression was sufficient to confer imidacloprid resistance (Liang et al., 2017). Comparison of a reference sequence (CYP6ER1vL) (Fig. S1) from lab-susceptible N. lugens with the nucleotide sequences from imidacloprid-resistant N. lugens in Asia revealed seven amino acid sequence variants: CYP6ER1vA (Fig. S1), CYP6ER1vB, CYP6ER1vC, CYP6ER1vD1, CYP6ER1vD2, CYP6ER1vE, and CYP6ER1vF (Zimmer et al., 2018). CYP6ER1vA was the main variant expressed in N. lugens from Thailand, Vietnam, and Indonesia, whereas CYP6ER1vB was expressed mainly in N. lugens from India (Zimmer et al., 2018). Recombinantly expressed CYP6ER1vA and CYP6ER1vB metabolized imidacloprid, but other CYP6ER1 variants did not (Zimmer et al., 2018). The findings of susceptibility studies employing transgenic Drosophila over-expressing CYP6ER1 variants revealed that T318S + A375del + A376G in CYP6ER1vA and T318S + P377del in CYP6ER1vB contribute greatly to imidacloprid resistance. CYP6ER1 homology models indicated these mutations give rise to the imidacloprid-binding cavity's hydrophobic interface (Zimmer et al., 2018).
We demonstrate lack of evidence of dinotefuran metabolism by CYP6ER1 variants carrying A375del + A376G (del3) (Fig. S1) and T318S + A375del + A376G (T318Sdel3) (Fig. S1) mutations. In fact, transgenic Drosophila over-expressing CYP6ER1vA exhibited 288.7-fold resistance to imidacloprid but only 3.6-fold resistance to dinotefuran.
Moreover, we investigated the metabolism of other neonicotinoids, as well as sulfoxaflor and flupyradifurone, by CYP6ER1 variants carrying the del3 mutations, either with or without the T318S mutation.
Section snippets
Chemicals
Dinotefuran, sulfoxaflor, and flupyradifurone, with >99% purity, were synthesized at Mitsui Chemicals Agro, Inc. (Chiba, Japan). Other neonicotinoids (imidacloprid, acetamiprid, clothianidin, nitenpyram, thiacloprid, and thiamethoxam), with >98% purity, were purchased from the FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) as analytical standards.
Expression of CYP6ER1 using baculovirus/Sf9 expression system
Sequences of CYP6ER1-del3 variant (GenBank accession number JF928994) (Fig. S1) and Drosophila melanogaster NADPH cytochrome P450 reductase
Expression of CYP6ER1 variants and Drosophila melanogaster NADPH cytochrome P450 reductase (CPR)
Mutations of CYP6ER1-del3 and CYP6ER1-T318Sdel3 were suggested to confer imidacloprid resistance (Zimmer et al., 2018). SDS-PAGE analysis revealed that CYP6ER1 variants carrying CYP6ER1-del3 and CYP6ER1-T318Sdel3 mutations with CPR were expressed successfully in Sf9 cells using baculovirus (Fig. 1). The reduced carbon monoxide difference spectrum of CPY6ER1-del3, and CPY6ER1-T318Sdel3 proteins in microsomes, exhibited distinct peaks at 450 nm (Fig. 2), indicating functional expression of these
Discussion
The emergence of imidacloprid-resistant N. lugens is a grave problem, which is made worse by the apparent cross-resistance to other neonicotinoids. Recent research has shown that mutations in CYP6ER1, T318Sdel3, and T318S + P377del contribute significantly to resistance to imidacloprid (Zimmer et al., 2018). However, whether these mutations confer cross-resistance to other neonicotinoids is not known.
In this study, we confirmed the metabolism of imidacloprid by CYP6ER1-del3 and
Acknowledgments
The authors thank Ms. Yoshimi Umezu for helpful assistance, and Vincent L. Salgado for helpful discussions and for critically reading the manuscript.
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