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Insect ryanodine receptors (RyRs) are the targets of diamide insecticides. Two point mutations G4946E and I4790M (numbering according to Plutella xylostella, PxRyR) in the transmembrane domain of the insect RyRs associated with diamide resistance have so far been identified in three lepidopteran pests, P. xylostella, Tuta absoluta and Chilo suppressalis. In this study, we identified one of the known RyR target site resistance mutations (I4790M) in a field-collected population of Spodoptera exigua. The field-collected WF population of S. exigua exhibited 154 fold resistance to chlorantraniliprole when compared with the susceptible WH-S strain. Sequencing the transmembrane domains of S. exigua RyR (SeRyR) revealed that the resistant WF strain was homozygous for the I4743M mutation (corresponding to I4790M in PxRyR), whereas the G4900E allele (corresponding to G4946E of PxRyR) was not detected. The 4743M allele was introgressed into the susceptible WH-S strain by crossing WF with WH-S, followed by three rounds of backcrossing with WH-S. The introgressed strain 4743M was homozygous for the mutant 4743M allele and shared about 94% of its genetic background with that of the recipient WH-S strain. Compared with WH-S, the near-isogenic 4743M strain showed moderate levels of resistance to chlorantraniliprole (21 fold), cyantraniliprole (25 fold) and flubendiamide (22 fold), suggesting that the I4743M mutation confers medium levels of resistance to all three diamides. Genetic analysis showed diamide resistance in the 4743M strain was inherited as an autosomal and recessive trait. Results from this study have direct implications for the design of appropriate resistance monitoring and management practices to sustainably control S. exigua.

The evo of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm, Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance.

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P-glycoprotein [P-gp or the ATP-binding cassette transporter B1 (ABCB1)] is an important participant in multidrug resistance of cancer cells, yet the precise function of this arthropod transporter is unknown. The aim of this study was to determine the importance of P-gp for susceptibility to insecticides in the beet armyworm (Spodoptera exigua) using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) geneediting technology. We cloned an open reading frame (ORF) encoding the S. exigua P-gp protein (SeP-gp) predicted to display structural characteristics common to P-gp and other insect ABCB1 transporters. A knockout line with a frame shift deletion of four nucleotides in the SeP-gp ORF was established using the CRISPR/Cas9 gene-editing system to test its potential role in determining susceptibility to chemical insecticides or insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Results from comparative bioassays demonstrate that knockout of SeP-gp significantly increases susceptibility of S. exigua by around threefold to abamectin and emamectin benzoate (EB), but not to spinosad, chlorfenapyr, betacypermethrin, carbosulfan indoxacarb, chlorpyrifos, phoxim, diafenthiuron, chlorfluazuron, chlorantraniliprole or two Bt toxins (Cry1Ca and Cry1Fa). Our data support an important role for SeP-gp in susceptibility of S. exigua to abamectin and EB and imply that overexpression of SeP-gp may contribute to abamectin and EB resistance in S. exigua.

NADPH–cytochrome P450 reductase (CPR) plays an important role in the cytochrome P450 (CYP)-mediated metabolism of endogenous and exogenous substrates. CPR has been found to be associated with insecticide metabolism and resistance in many insects. However, information regarding CPR in the bird cherry-oat aphid, Rhopalosiphum padi, is unavailable. In the current study, a full-length cDNA (2,476 bp) of CPR (RpCPR) encoding 681 amino acids was cloned from R. padi. Nucleotide sequence and deduced amino acid sequence analysis showed that RpCPR exhibits characteristics of classical CPRs and shares high identities with those of other insects, especially with the pea aphid, Acyrthosiphon pisum. The mRNA of RpCPR was expressed at all developmental stages, with the highest expression level found in the second instar and the lowest in adult. Expression levels of RpCPR in isoprocarb-resistant and imidacloprid-resistant strains were 3.74- and 3.53-fold higher, respectively, than that of a susceptible strain. RpCPR expression could also be induced by low concentrations (LC30) of isoprocarb and imidacloprid. Moreover, we sequenced the open reading frame (ORF) of RpCPR from 167 field samples collected in 11 geographical populations. Three hundred and thirty-four SNPs were detected, of which, 65 were found in more than two individuals. One hundred and ninety-four missense mutations were present in the amino acid sequence, of which, the P484S mutant had an allele frequency of 35.1%. The present results suggest that RpCPR may play an important role in the P450-mediated insecticide resistance of R. padi to isoprocarb and imidacloprid and possibly other insecticides. Meanwhile, RpCPRmaintains high genetic diversity in natural individuals, which provides the possibility of studying potential correlations between variants and certain special physiological characters.

The voltage-gated sodium channel (VGSC) is the target of sodium-channel-blocking insecticides. Traditionally, animals were thought to have only one VGSC gene comprising a α-subunit with four homologous domains (DI–DIV). The present study showed that Rhopalosiphum padi, an economically important crop pest, owned a unique heterodimeric VGSC (H1 and H2 subunits) encoded by two genes (Rpvgsc1 and Rpvgsc2), which is unusual in insects and other animals. The open reading frame (ORF) of Rpvgsc1 consisted 1150 amino acids, and the ORF of Rpvgsc2 had 957 amino acids. Rpvgsc1 showed 64.1% amino acid identity to DI–DII of Drosophila melanogaster VGSC and Rpvgsc2 showed 64.0% amino acid identity to DIII–DIV of D. melanogaster VGSC. A M918L mutation previously reported in pyrethroids-resistant strains of other insects was found in the IIS4-S6 region of R. padi field sample. The two R. padi VGSC genes were expressed at all developmental stages and showed similar expression patterns after treatment with beta-cypermethrin. Knockdown of Rpvgsc1 or Rpvgsc2 caused significant reduction in mortality rate of R. padi after exposure to beta-cypermethrin. These findings suggest that the two R. padi VGSC genes are both functional genes.