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Genome mapping coupled with CRISPR gene editing reveals a P450 gene confers avermectin resistance in the beet armyworm

['Yayun Zuo', 'The Key Laboratory Of Plant Immunity', 'College Of Plant Protection', 'Nanjing Agricultural University', 'Nanjing', 'Institute Of Pesticide Science', 'Northwest A F University', 'Yangling', 'Shaanxi', 'Yu Shi']

Date: 2021-09

The evolution 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.

Insecticide resistance is a global constraint to agricultural production, and rapid identification of resistance genes is critical for better monitoring and management of resistant insect pests. Identification of metabolic resistance genes has always been a challenging task due to the high diversity of insect detoxification enzyme genes. 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 avermectin susceptibility. 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 avermectin. 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 field monitoring of such mutations, for making improved decisions on appropriate use of effective chemistries, as well for improvements in the design of future compounds that target S. exigua.

The beet armyworm, Spodoptera exigua, is an important global pest of many crops [ 6 , 19 ] and because of repeated overuse of EB, it has evolved high levels of insecticide resistance in the field in some parts of Asia, including Pakistan and China [ 6 , 19 ]. Bulk segregant analysis (BSA) has recently been used to rapidly identify genomic regions associated with unique phenotypes in numerous different species [ 20 ]. To better understand the molecular genetic basis of EB resistance, we first produced a chromosome-level de novo genome assembly from a EB-susceptible reference beet armyworm laboratory strain from China. We then used BSA to identify loci containing major resistance genes and used CRISPR/Cas9 gene-editing to map resistance to the CYP9A186 gene. Furthermore, the natural substitution (F116V) in the substrate binding site 1 (SRS1) region of CYP9A186 was functionally verified to confer resistance to both avermections, EB and abamectin. Hence, the combined use gene editing with BSA provided a rapid and highly efficient means to identify metabolic resistance genes responsible for insecticide resistance. Such results are critical for the monitoring of field populations of the beet armyworm for avermectin resistance and for the future use and/or design of new chemistries to manage resistant populations.

At least two major mechanisms are known to cause avermectin resistance in arthropods: target-site insensitivity and metabolic enzyme-based resistance [ 8 ]. Target site mutations in the glutamate-gated chloride channels are associated with resistance to avermectins in Drosophila melanogaster [ 9 ], Plutella xylostella [ 10 , 11 ] and Tetranychus urticae [ 5 , 12 ]. Enhanced oxidative metabolism of abamectin has also been reported as a resistance mechanism in Leptinotarsa decemlineata [ 13 ], Bemisia tabaci [ 14 ], P. xylostella [ 15 ] and T. urticae [ 16 ]. Enhanced avermectin metabolism due to increased expression of cytochrome P450 was shown in T. urticae [ 17 ] and in Pediculus humanus humanus [ 3 ]. Other detoxification mechanisms, including sequestration and/or the metabolic action of esterases, carboxylesterases, and/or glutathione S-transferases (GSTs) may also play a role in avermectin resistance in some arthropods [ 16 , 18 ]. However, the underlying molecular mechanisms of resistance to EB and abamectin remain largely unknown in lepidopteran pests.

Avermectins are a group of 16-membered macrocyclic lactones possessing potent anthelmintic and insecticidal activities [ 1 ]. Emamectin benzoate (EB) [(4″R)-4″-deoxy-4″-(methylamino) avermectin B1 benzoate] is a macrocyclic lactone semi-synthetic derivative of the avermectin family and has much higher potency than abamectin against a number of lepidopterous pests [ 2 ]. Similar to abamectin and ivermectin, the mode of action of EB in invertebrates is through the overstimulation of glutamate-gated chloride channels (GluCls) [ 1 – 3 ]. With extensive field application of avermectins, the evolution of resistance in mites, insects, and parasitic worms has become an increasingly serious problem in agriculture [ 4 – 7 ].

These distinct hydroxy- and O-desmethyl-metabolites were only found in CYP9A186-F116V samples containing NADPH ( S3A and S3D Fig ), as the Ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) multireaction monitoring (MRM) signals corresponding to these two metabolites were not above the noise signal for the CYP9A186wt reaction samples ( S3B and S3E Fig ). These results confirm that unlike the CYP9A186-F116V samples, CYP9A186wt does not metabolize EB or abamectin under our experimental conditions.

EB, abamectin and their multiple metabolites showed excellent separation and linearity under our experimental conditions ( Figs 5 and S3 ). Recovery rates for both EB and abamectin were nearly 100% for the non-insertion control samples. Our in vitro assays showed that EB and abamectin were both metabolized by CYP9A186-F116V microsomal fractions at higher rates than by CYP9A186wt microsomes ( Fig 5A ). Whereas CYP9A186-F116V microsomes metabolized EB and abamectin at 1.03 ± 0.14 pmol/min/pmol P450 and 0.91 ± 0.09 pmol/min/pmol P450, respectively, the metabolic activity of both insecticides by CYP9A186wt microsomal fractions was negligible (0.11 ± 0.02 pmol/min/pmol P450 for EB and 0.12 ± 0.04 pmol/min/pmol P450 for abamectin). Indeed, specific activities of CYP9A186wt were not significantly higher than the limits of detection (LOD) for EB or abamectin (0.10 ± 0.01 pmol/min/pmol P450 and 0.13 ± 0.02 pmol/min/pmol P450, respectively) ( Fig 5A ). We observed no decrease of either EB or abamectin with microsomal protein fractions from cells infected with “non-insertion” control virus.

Both wild-type (CYP9A186wt) and mutant (CYP9A186-F116V) CYP9A186 enzymes were expressed in Trichoplusia ni High Five (Hi5) cells using a baculovirus system. Reduced CO-difference spectra of CYP9A186wt and CYP9A186-F116V in purified recombinant microsomes showed a maximum peak near 450 nm ( S2 Fig ), indicating successful expression of functional enzymes within the Hi5 cells. In addition, both recombinant P450s showed O-debenzylation activities to 7-benzyloxy-4-trifluoromethyl coumarin (BFCOD), again indicating that the recombinant enzymes had correct folding and were active. Microsomes with CYP9A186wt and CYP9A186-F116V had BFCOD specific activities of 0.083 ± 0.001 and 0.057 ± 0.003 pmol/min/pmol P450, respectively (mean values ± SEM, n = 4). Non-insertion control Hi5 microsomes showed no BFCOD activity.

( A ) Alignment of the deduced CYP9A186 amino acid sequences from WH-EB and WH-S with a translated human cytochrome P450 (e.g., CYP3A4). The F116V substitution is shown with a red arrow. The predicted locations of alpha helical secondary structures are marked based on the template of the CYP3A4 protein structure (1TQN) using ESPript 3.0. ( B ) Representative chromatograms from direct sequencing of CYP9A186 PCR products from WH-EB and WH-S showing the T346G mutation. ( C ) qRT-PCR analysis of CYP9A107, CYP9A27, CYP9A11, CYP9A186 and CYP9A98 in midgut and fat body from WH-S and WH-EB. Data shown represent means ± SE derived from four biological replicates. Asterisks indicate significant differences between the strains (Student’s t-test, p<0.05).

To determine if one of the P450 genes in the defined region associated with avermectin resistance differed between susceptible and resistant strains, we PCR-amplified, cloned and sequenced the full-length cDNAs corresponding to the open reading frames (ORF) of CYP9A107, CYP9A27, CYP9A11, CYP9A186 and CYP9A98 from WH-S and WH-EB, respectively. We found a single T346G mutation resulting in the F116V substitution within the SRS1 region of CYP9A186 (GenBank accession no. MN179472) in WH-EB and not WH-S ( Fig 4A ). DNA based genotyping of 20 larvae from each strain further revealed that WH-EB is homozygous for Val (GTT) at codon 116, whereas WH-S is homozygous for Phe (TTT) ( Fig 4B ). No other consistent mutations were found in the other four genes. Additionally, we also observed a 10-fold increase in CYP9A186 transcript abundance (i.e., overexpression) in fat body tissue from WH-EB compared with WH-S ( Fig 4C ). None of the other four P450 candidate genes showed higher transcription in WH-EB compared to WH-S ( Fig 4C ). These data therefore show that a naturally-occurring point mutation and/or overexpression of CYP9A186 is associated with avermectin resistance in WH-EB strain and provides additional supports for its involvement in metabolic insecticide resistance in the beet armyworm.

To map resistance in the WH-EB strain, we created four homozygous knockout strains (dA40-A98, dA40-A107, dA107-A98, and dA186) and observed their corresponding resistance phenotypes ( Fig 3 ). The first knockout of the entire CYP9A40 to CYP9A98 gene cluster (ΔCYP9A98-CYP9A40) resulted in a strain (e.g., dA40-A98) with complete restoration of susceptibility to EB ( Table 3 ), indicating that at least one resistance gene is located in this region. Subsequent CRISPR/Cas9 knockout of the chromosomal fragment corresponding to CYP9A40 to CYP9A107 (ΔCYP9A107-CYP9A40) resulted in a strain (dA40-A107) that remained fully resistant to both insecticides ( Table 3 ). In contrast, susceptibility was fully restored in the dA107-A98 strain where the chromosome fragment from CYP9A107 to CYP9A98 (ΔCYP9A107-CYP9A98) was deleted ( Table 3 ), further narrowing the resistance gene to this region. Finally, we used CRISPR/Cas9 to generate a strain (dA186) homozygous for a 4-bp deletion in CYP9A186 (ΔCYP9A186) that results in the introduction of a premature stop codon and the loss of downstream active sites within the cytochrome P450 enzyme ( S1 Fig ). Bioassays show that susceptibility to abamectin and EB was fully restored in the dA186 strain ( Table 3 and Fig 3 ), confirming that resistance maps to the CYP9A186 gene.

( A ) Single-pair crosses between JZ-S and WH-EB virgin adults produced families of hybrid F 1 offspring. A male F 1 was backcrossed to a female JZ-S to produce the backcross (BC) family. Progeny from the BC family were exposed to a diagnostic concentration of EB of which 120 survivors and their F 1 parents were analyzed by next-generation sequencing (NGS). ( B ) BSA mapping revealed a 1 Mb genomic region in chromosome 17 highly correlated with EB resistance in WH-EB. The mean frequency deviation values obtained from a sliding window analysis (1 Mb window with 100 Kb step) were plotted across all 31 chromosomes of the genome. The red dashed line corresponds to the top 1% threshold of the SNP-index likely containing changes linked with EB resistance. ( C ) Expanded map of chromosome 17 (from 15 Mb to 16 Mb) that includes the CYP9A gene cluster (15.71 Mb to 15.81Mb).

Tissue samples (120 moth legs) from EB-resistant individuals (survivors) derived from a backcrossed family generated by crossing a resistant WH-EB female with a susceptible JZ-S male ( Fig 2A ) were pooled and used for bulked sequence analysis (BSA). In doing so, we identified a 1 Mb genomic region in chromosome 17 (from 15–16 Mb) that was significantly biased with resistance ( Fig 2B ). A cluster (chromosomal locations: 15.71 Mb to 15.81Mb) of CYP genes belonging to the CYP9A family is located in this region of chromosome 17 ( Fig 2C ), whose functions are frequently associated with xenobiotic detoxification [ 22 – 25 ]. Detailed analysis of the CYP9A gene cluster from the genome of S. exigua identified ten CYP9A genes, which were named CYP9A40, CYP9A187, CYP9A30, CYP9A9a and CYP9A9b (duplication), CYP9A107, CYP9A27, CYP9A11, CYP9A186 and CYP9A98 according to the International P450 Nomenclature Committee ( Fig 2C ). We therefore hypothesize that the dominant EB resistance in WH-EB is associated with one or more P450 genes in the CYP9A gene cluster of WH-EB.

Using de novo prediction and library alignments to detect repetitive DNA, we found that 33% (147.97 Mb) of the beet armyworm genome corresponds to putative transposable elements (TEs). This is consistent with the reported repeat content (32%) published for S. litura [ 22 ]. Several families of conserved TEs were found in the beet armyworm genome, including long interspersed nuclear elements (LINEs, 15%), rolling-circle transposition (RC, 4%), long terminal repeat elements (LTR, 3%), short interspersed nuclear elements (SINE, 3%), and DNA transposons (2%) ( S3 Table ). Simple repeats also occupied approximately 1% of the genome ( S3 Table ). Consistent with other lepidopterans [ 21 ], the beet armyworm chromosome W is enriched with DNA transposons and LTR retrotransposons when compared with the other 30 automosomes and the Z chromosome ( Fig 1 ).

To comprehensively annotate genes in our assembled beet armyworm genome, we integrated ab initio, transcriptome- and protein homology-based strategies to predict 17,727 protein-coding genes (e.g., gene models) with an overall mean length of 9,605 bp ( Table 2 ). BUSCO assessment of the gene models positively identified 1,601 of the 1,658 reference genes (96.6%). Of these genes, only 45 (2.7%) were duplicated, 12 (0.7%) were fragmented, and 45 (2.7%) were missing, indicating the assemble genome is well-represented by its protein-coding genes. Comparison of the genome with RNA-seq data indicated a total mean exon and intron count of 6.4 and 5.2 per gene, with a mean length of 335 bp and 1,435 bp, respectively ( Table 2 ). Among the predicted genes, 82% had transcriptome support and 96% had matches within the UniProt database. InterProScan identified protein domains for 13,186 (74%) genes, GO terms for 8,227 (47%) genes and Reactome pathways for 2,731 (15%) genes, respectively. EggNOG predicted GO terms for 6,706 genes, KEGG orthology (KO) matches for 7,643 genes, KEGG pathway matches for 4,493 genes, EC matches for 2,675 genes, and COG matches for 12,987 genes ( Table 2 ).

To facilitate genetic mapping of resistance, we first sequenced the beet armyworm genome using both Illumina and PacBio whole genome sequencing (WGS). We generated 3,919,515 PacBio subreads yielding 38.98 Gb (87X coverage) and 56.32 Gb of Hi-C sequencing data (126X coverage), respectively. Estimated genome size ranged from 408.58 Mb to 448.90 Mb with a low heterozygosity for the sequenced susceptible WH-S strain ( S1 Table ). The assembled genome consisted of 667 contigs spanning 446.80 Mb with a scaffold/contig N50 length of 14.36/3.47 Mb ( Table 1 ); among them, 367 contigs were anchored into 32 pseudo-chromosomes ( Fig 1 ), accounting for greater than 96% (429.74 Mb) of the genome. Consistent with lepidopteran females being heterogametic for sex chromosomes [ 21 ], 30 autosomes plus two sex chromosomes (Z and W) were identified from the sequenced female pupa. When compared with the insect_odb9 reference dataset consisting of 1,658 functional genes, BUSCO completeness analyses showed our final genome assembly was 97.9% complete, with only 0.2% fragmented and 1.9% missing BUSCO genes ( S2 Table ). Reflecting the high degree of completeness and accuracy of the genome assembly, we mapped 97.62% Illumina short reads and 93.78% of the PacBio long reads to our final assembly with very low redundancy (2.1% duplicated BUSCOs) ( Table 2 ). Among four Noctuidae pest species ( Table 1 ), assemblies of S. exigua and S. frugiperda have the highest genome contiguity (contig N50 > 1 Mb, contig number < 1,000) and the lowest gap content (< 0.1%).

Discussion

The results reported here show the molecular genetic and biochemical basis of resistance to EB and abamectin in a laboratory-selected strain of beet armyworm from China. We first produced a chromosome-level de novo genome assembly and used BSA to identify the CYP9A gene cluster as the major locus associated with EB resistance. We then coupled CRISPR/Cas9 gene-editing to knockout regions within this gene cluster and mapped resistance to the CYP9A186 gene. Heterologous expression of recombinant CYP9A186 tested with in vitro metabolism assays confirmed that a natural mutation (T346G), resulting in the F116V substitution located in the substrate binding site 1 (SRS1) region of CYP9A186, causes resistance to both EB and abamectin. Hence, the combined use of CRISPR/Cas9 gene editing with BSA provided a rapid and highly efficient means to identify metabolic resistance genes responsible for avermectin insecticide resistance in the beet armyworm.

The evolution of pesticide resistance is an increasingly intractable problem affecting crop production worldwide [26]. To combat resistance, there is an urgent need for the rapid identification of not only markers for resistance, but also genes that cause pesticide resistance. Numerous studies point to target-site resistance and metabolic resistance as the two primary mechanisms by which insects evolve resistance to insecticides [27,28]. Target-site resistance involves alterations (e.g., mutations) in the insecticide target protein that reduce its sensitivity to insecticides [29]. For many insecticides, the primary insecticide targets are known, and identification of mutations can be directly assessed. In contrast, metabolic resistance can involve numerous different pathways consisting of unrelated gene products, making their identification difficult due to the high diversity of detoxification enzyme genes in insect pests [30,31].

One efficient way to overcome the above problem is to map resistance genes within finite regions of insect genomes by BSA [32]. To do this, a high-quality genome map is an absolute prerequisite for precise genetic localization. Hence, we generated a chromosome-level assembly of the beet armyworm genome by single-molecule real-time PacBio and Hi-C sequencing. Even with a precise genomic map, BSA can often be difficult to identify specific and functional genes responsible for the observed phenotype. Improvements in BSA will likely come as sequencing technologies and methods are further developed to increase their accuracy and shorten the confidence interval of genetic mapping, especially in regions of chromosomes dense with gene clusters and/or with low recombination rates. In addition to increased sequencing resolution, methods to improve how genetic loci identified by BSA are linked to resistance phenotypes via functional genomics are needed. Recently, the CRISPR/Cas9 genome editing system was shown to be effective in several lepidopterans, including S. exigua [33–35], providing a powerful functional genomics tool for the exploration of the complex biology of insecticide resistance. Here, we show for the first time that BSA used in conjunction with CRISPR/Cas9 gene editing allows for the specific and rapid identification of a gene responsible for metabolic insecticide resistance. Such information is important for tracking resistant beet armyworm individuals in the field and for making better informed decisions about the appropriate use of avermectin and related insecticides to delay the further spread of resistance. Furthermore, the identification of specific mutations involved in the structure and detoxification function of CYP9A186 may enable the rational design of new and more potent chemistries that target pest insects.

A P450 gene, CYP392A16, is highly over-expressed in the abamectin resistant Mar-ab strain of T. urticae, and this P450 can metabolize abamectin in vitro [17]. However, P450 point mutations that increase the metabolism of avermectins have not been previously reported. For cytochrome P450s, six substrate recognition sites (SRSs) are key to the acquisition of novel functions [36]. Site-directed mutagenesis studies previously showed that SRS1, SRS4, SRS5, and SRS6 possess amino acid residues that form the catalytic site, while SRS2 and SRS3 participate in the formation of the substrate access channel [37]. Conservative amino acid substitutions in these SRS regions can alter P450 activities and/or substrate profiles, and some substitutions in non-SRS regions on the proximal surface of a P450 can also improve kinetic properties due to alterations in interactions with the P450 redox partners [37]. Here, we show that the single amino acid substitution F116V in SRS1 of CYP9A186 is a gain-of-function mutation that enables enhanced metabolism of EB and abamectin. Although this Phe to Val substitution appears to be relatively minor, the replacement of the bulky phenyl aromatic ring for the isopropyl moiety apparently has a major affect on the CYP9A186 active site. Indeed, CYP9A186 belongs to the CYP3 clan of cytochrome P450s, which contains other well-characterized P450s, including the human CYP3A4 whose three-dimensional structure has been solved [38]. Alignment of CYP9A186 with CYP3A4 (Fig 4) shows that F116 in CYP9A186 likely corresponds with S119 of CYP3A4 within the B’-C loop. This residue likely protrudes into the active site, where it is in close proximity to the heme and opposite the C-terminal loop, allowing for direct interaction with substrate [38,39]. We speculate that the F116V substitution could reduce structural hindrance within the active site and allow for acceptance of a broader suite of substrates hydroxylated by the P450.

Changes in P450 transcription as well as structural changes appear to alter insecticide susceptibility and must be evaluated on a case-by-case basis. Recently, CYP321A8 was shown to contribute resistance to pyrethroid and organophosphate insecticides in S. exigua through overexpression regulated by cis- and trans-regulatory elements [40]. In D. melanogaster, transgenic overexpression of CYP6G2 leads to resistance to diazinon [41], whereas changes in transcript abundance was not involved for the closely related CYP6G1. It was rationalized that CYP6G2 has multiple hydrophobic substitutions that increased the size of its catalytic site along with one positive-to-polar substitution that eliminated a potentially critical charge in SRS2 [37,42]. Similarly, structural changes in a P450 active site are important in Anopheles gambiae, where CYP6Z1 metabolizes DDT but CYP6Z2 does not [43,44]. Overlays of the predicted CYP6Z1 and CYP6Z2 structures indicated that R210 (SRS2), I298 and E302 (SRS4) of the CYP6Z2 protrude sufficiently enough into the CYP6Z2 catalytic site that it could no longer dock large hydrophobic substrates such as DDT [37]. In Anopheles funestus, alleles of CYP6P9b that confer high pyrethroid resistance are characterized by three point mutations, as well as constitutive overexpression [45]. More recently, Zimmer et al. [46] reported several point mutations were associated with gene duplications for CYP6ER1 gene variants of imidacloprid resistant Nilaparvata lugens strains. When two SRS5 mutations (A375del and A376G) were tested in transgenic Drosophila, imidacloprid resistance increased 20-fold. A similar level of resistance was obtained with the T318S (in SRS4) substitution. When this T318S substitution was combined with both A375del and A376P, this combination exhibited the highest resistance of all transgenic lines (35-fold). Such examples indicate that variation in both SRS regions and non-SRS regions of CYPs contribute to differences in metabolic capability and that gain-of-function mutations are common for several classes of insecticides.

In vivo and in vitro metabolism studies in vertebrates have identified three major oxidative metabolites of abamectin: 24-hydroxymethyl (24-OH), 26-hydroxymethyl (26-OH), and 3’-O-desmethyl abamectin [47]. In our study, CYP9A186-F116V also catalyzes the hydroxylation and O-demethylation of EB and abamectin, most likely also leading to the (24’ or 26’) hydroxy-metabolites and 3’-O-desmethyl-metabolites. In contrast, CYP392A16 from T. urticae did not O-demethylate abamectin, but rather catalyzed the hydroxylation of the insecticide to produce metabolites that were less toxic to T. urticae than the parental compound [17].

Alleles with fitness costs are known to have a decreased ability to survive and reproduce in the absence of the insecticide [48]. Previous research reported that the WH-EB strain of S. exigua developed 1,110-fold resistance to EB and that two or a few genes contributed to resistance [49]. Subsequently, the WH-EB strain was reared without further selection with EB. This strain now maintains a relatively stable resistance level (~300 fold) to EB. It is therefore likely that one or a few minor genes that contributed to resistance in the WH-EB strain were lost due to fitness costs, and only the mutated CYP9A186 with little or no associated fitness costs, was retained.

Here, our combined approach of coupling gene editing with BSA allowed for the rapid identification of CYP9A186 as the primary gene involved in metabolic resistance to both EB and abamectin in a strain of the beet armyworm from China. The accuracy and speed at which we identified, mapped, and validated the functional role of CYP9A186 suggests that such methods are highly adoptable for the rapid identification of resistance in many arthropod pest/insecticide systems. We can further use this information to proactively track beet armyworm avermectin resistance in field populations and make better informed decisions on the use of appropriate chemistries to further delay the evolution of insecticide resistance. Our findings also further enhance the understanding of avermectin resistance evolution and suggests that gain-of-function mutations in the SRS regions of P450 are important for the acquisition of novel detoxification functions. As such, future work to monitor CYP9A186 for mutations in the field as well the design of specific compounds to target/inhibit CYP9A186 could be important for resistance management of S. exigua.

[END]

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