# Monitoring the molecular mechanisms of insecticide resistance in Spodoptera frugiperda populations from Thailand

Marlen Saladini Di Rovetino, Bettina Lueke, Kamonporn Masawang, Narisara Piyasaengthong, Maroot Kaewwongse, Saksit Nobsathian, Thierry Fricaux, Kiwoong Nam, Emmanuelle d'Alençon, Vasakorn Bullangpoti, et al.

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## ► To cite this version:

Marlen Saladini Di Rovetino, Bettina Lueke, Kamonporn Masawang, Narisara Piyasaengthong, Maroot Kaewwongse, et al. Monitoring the molecular mechanisms of insecticide resistance in Spodoptera frugiperda populations from Thailand. Pesticide Biochemistry and Physiology, 2025, 214, pp.106599. 10.1016/j.pestbp.2025.106599. hal-05200267

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# Monitoring the molecular mechanisms of insecticide resistance in *Spodoptera frugiperda* populations from Thailand

Marlen Saladini di Rovetino<sup>a</sup>, Bettina Lueke<sup>b</sup>, Kamonporn Masawang<sup>c</sup>, Narisara Piyasaengthong<sup>c</sup>, Maroot Kaewwongse<sup>c,d</sup>, Saksit Nobsathian<sup>c,e</sup>, Thierry Fricaux<sup>a</sup>, Kiwoong Nam<sup>f</sup>, Emmanuelle d'Alençon<sup>f</sup>, Vasakorn Bullangpoti<sup>c</sup>, Ralf Nauen<sup>b,*</sup>, Gaëlle Le Goff<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Fall armyworm

Resistance

Acetylcholinesterase

Voltage-gated sodium channel

CYP9A

## ABSTRACT

*Spodoptera frugiperda* is a major crop pest that invaded Thailand in 2018 which cause significant damage, particularly to maize. In recent years, a loss of efficacy of certain insecticides has been observed, suggesting the emergence of resistance. The aim of our study was to investigate the molecular mechanisms of resistance in *S. frugiperda* populations collected in several regions in Thailand. Analysis of the presence of mutations in insecticide targets revealed three mutations (M944L, F1005C and I1011M) in the voltage-dependent sodium channel, and the F290V mutation in acetylcholinesterase (AChE). These mutations can confer resistance to pyrethroids, organophosphates and carbamates, respectively. The impact of the F290V as well as two other AChE mutations have been functionally validated by inhibition studies with recombinantly expressed AChE-1 variants. The potential involvement of metabolic resistance was also investigated by monitoring the expression level of several cytochrome P450 genes. Most of the CYP9A subfamily P450s were overexpressed, regardless of the geographical origin of the populations, a worrying situation given the demonstrated ability of these P450s to metabolize insecticides belonging to different chemical classes including pyrethroids. Transcription factors such as CncC and Maf were also overexpressed in these field populations. All these results should be taken into consideration in order to propose effective and sustainable control of this pest in Thailand. Our results also demonstrated the need to develop monitoring strategies that take into account the two main resistance mechanisms, target modification and metabolic detoxification.

## 1. Introduction

Fall armyworm (FAW), *Spodoptera frugiperda* (Lepidoptera: Noctuidae), is a major crop pest feeding on more than 350 host plants (Montezano et al., 2018), including plants of agronomic importance such as corn, rice and sorghum. Its invasive nature and expansion in recent years make it a major concern for food security in many parts of the world. While its presence in continental Europe has only been recently shown (FAO, 2024), it has been detected for several years in Asia, particular in Thailand, since December 2018 (IPPC, 2018). In this

country, FAW quickly proved to be a major pest of maize cultivation, with yield losses estimated at 25 % to 40 % (Plant Protection Research and Development Office, 2021). At the same time, an increase in maize production costs has been observed, linked to the costs associated with combating FAW and purchasing pesticides (Plant Protection Research and Development Office, 2021). Thailand is dependent on imports for its pesticide market, and the most commonly imported insecticides belong to the organophosphate, carbamate and pyrethroid classes (Laohaudomchok et al., 2021). In the management of FAW, the insecticides recommended by the Department of Agriculture in May 2024

<footnote><sup>a</sup> Université Cote d'Azur, INRAE, CNRS, ISA, Sophia Antipolis, France</footnote>

<footnote><sup>b</sup> Bayer AG, Crop Science Division, R&D, Monheim, Germany</footnote>

<footnote><sup>c</sup> Animal Toxicology Specialty Research Unit, Department of Zoology, Kasetsart University, Bangkok, Thailand</footnote>

<footnote><sup>d</sup> Division of Physiology, School of Medical Sciences, University of Phayao, Phayao, Thailand</footnote>

<footnote><sup>e</sup> Nakhonsawan Campus Mahidol University, Nakhonsawan, Thailand</footnote>

<footnote><sup>f</sup> DGIMI, INRAE, Univ Montpellier, Montpellier, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: ralf.nauen@bayer.com (R. Nauen), gaëlle.le-goff@inrae.fr (G. Le Goff).</footnote>
have been emamectin benzoate, spinetoram, cyantraniliprole, chlorfenapyr, indoxacarb, chlorantraniliprole, flubendiamide and methoxyfenozide. However, in other regions FAW has already evolved resistance to some of those insecticides mentioned above (Garlet et al., 2021; Thiesen et al., 2025; Zeng et al., 2025) and is ranked in the top 15 of the world's most resistant pest species (Sparks et al., 2020).

The two main mechanisms by which insects become resistant to insecticides are a modification in the molecular target of the insecticide and detoxification by converting the molecule into a less toxic compound (Feyereisen, 1995). Mutations in the targets of different chemical classes of insecticides have been identified in FAW. Mutations in acetylcholinesterase (AChE) such as the A201S, G227A and F290V, account for resistance to organophosphates and carbamates (Carvalho et al., 2013). Mutations in the voltage-dependent sodium channel (VGSC), such as T929I, L932F and L1014F, have been associated with resistance to pyrethroids (Carvalho et al., 2013), while resistance to diamides has been linked to mutations, in the ryanodine receptor (RyR), specifically I4790M (Boaventura et al., 2020a), and more recently I4790K in field-collected Brazilian FAW populations (Okuma et al., 2022). Mutations A309V or A309T in the glutamate-gated chloride channel (GluCl) account for resistance to avermectin (Cai et al., 2024). Metabolic insecticide resistance often involves the overexpression of detoxification enzymes such as cytochrome P450 monooxygenases (P450), choline/carboxylesterases (CCE), glutathione S-transferases (GST) and UDP-glycosyltransferases (UGT) (Amezian et al., 2021a; Hilliou et al., 2021). Several of these enzymes have been shown to be overexpressed in FAW field collected populations resistant to insecticides. For example, the CYP9As which are overexpressed in pyrethroid-resistant populations in the native range of FAW on the American continent (Boaventura et al., 2021; Gimenez et al., 2020). The overexpression of detoxification enzymes can have multiple origins, such as a variation in the number of genes (duplication and amplification) or modifications in cis or trans regulatory elements that affect expression (Feyereisen et al., 2015; Nauen et al., 2022). Gimenez et al. (2020) have shown that the overexpression of CYP9As in a FAW population from Puerto Rico is due to a duplication of the entire CYP9A cluster. Other examples involve trans-regulation with transcription factors and nuclear receptors. Some of these may be constitutively overexpressed in resistant populations, this is the case for Cap'n' Collar C (CncC), a transcription factor of the basic leucine zipper superfamily in another close *Spodoptera* species, *S. exigua* resistant to chlorpyrifos, cypermethrin and deltamethrin (Hu et al., 2021).

Data on FAW resistance in Thailand are scarce, however Thirawut et al. (2023) have shown increasing levels of resistance between 2019 and 2022–2023 to emamectin benzoate, indoxacarb and chlorfenapyr, but the mechanisms of resistance were not yet investigated. In addition, being an invasive species, it is likely that the invading populations are already resistant (Yainna et al., 2021). The aim of our study was to determine the molecular mechanisms of resistance in FAW populations collected in the main maize-growing areas of Thailand. The presence of target-site mutations conferring resistance to AChE-inhibiting insecticides and to pyrethroids was specifically monitored. In addition, the involvement of detoxification mechanisms was investigated by measuring the expression levels of candidate genes such as CYP9As, along with nuclear receptors and transcription factors that may regulate their expression. Furthermore, we recombinantly expressed FAW AChE carrying different reported mutations to investigate their impact on OP and carbamate efficacy at the molecular level.

## 2. Materials and methods

### 2.1. Insect collection and rearing

Larvae of FAW were collected in corn fields in five locations of Thailand, Phayao - Top North (PYO), Nakhon Ratchasima - East (NRS), Saraburi - Center (PCPCPD), Nakhon Sawan - Center (NSN) and

Kanchanaburi - West (KRI) (Fig. 1 and Table S1). Most of the fields have been treated with chemical insecticides, except for Saraburi, where biological control was used (Table S1). Larvae were kept either in 70 % ethanol or in RNAlater (Invitrogen™). The susceptible reference strain corresponds to a strain that has been maintained in the laboratory for over 20 years. The individuals were initially collected in Guadeloupe in 2001, and this strain was used to obtain the reference genome (Gouin et al., 2017).

### 2.2. DNA extraction

Genomic DNA was extracted from individual larvae. The larva was ground with mortar and pestle in liquid nitrogen, then the powder was added to 500 µL lysis buffer (100 mM Tris HCl (pH 8.0), 1.5 M NaCl, 2 % cetyltrimethylammonium bromide and 0.1 % β-mercaptoethanol) and heated for 1 h at 65 °C. Subsequently, chloroform isoamyl alcohol (24:1) was added and mixed for 10 min. The tubes were centrifuged at 10 min at 10,000 x g and the aqueous phase was collected. An equal volume of ice-cold isopropanol was added, mixed for 5 min and then centrifuged at 10,000 xg for 10 min. The pellet was washed with 70 % ethanol, centrifuged for 5 min at 10,000 x g. Then the supernatant was discarded, and the pellet dried for a few minutes. Finally, the pellet was dissolved in 40 µL of nuclease-free water and stored at -20 °C.

### 2.3. Target-site mutation detection by sanger sequencing

Primers were designed to amplify the region of the following mutations A201S, G227A (AChE); L1014F, L932F, T929I (VGSC); G4946E (RyR); A309V/T and G315E (GluCl). Sequences deposited at the National Center for Biotechnology Information (NCBI) were used as a reference to design the primers, i.e. VGSC (GenBank KC435025) and Ace-1 (GenBank KC435023), RyR (GenBank MK226188) and GluCl (GenBank XP_035452745.1). PCR amplifications for Sanger sequencing were performed in 50 µL containing 10 µL 5× Q5 Reaction Buffer (New

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Map showing the geographic origin of five fall armyworm populations collected in Thailand (more details about collection sites in Table S1).</figcaption></figure>
England Biolabs, Ipswich, MA, USA), 1 µL 10 mM dNTPs, 2.5 µL 10 µM forward and reverse primers (Table S2), 1 µL 50 ng of gDNA, 0.5 µL Q5® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and nuclease-free water. The conditions of PCR amplification were 98 °C for 30 s, followed by 35 cycles of 98 °C for 10 s, the respective annealing temperature according to table S2 for 20 s and 72 °C for 45 s, and a final elongation step at 72 °C for 2 min. PCR products were purified with GenElute™ PCR Clean-Up Kit (Sigma-Aldrich, St Louis, MO, USA) then sent for Sanger sequencing (GENEWIZ, Germany). The sequences were analysed with BioEdit 7.2.5 (Tom Hall, Ibis Biosciences, Carlsbad, CA, USA).

## 2.4. Fluorescence based allelic discrimination assays

To detect the target site mutations F290V (AChE) and I4790M (RyR) fluorescence-based allelic discrimination assays were carried out according to Boaventura et al. (2020b). For a final volume of 10 µL, 5 µL SsoAdvanced™ Universal Probes Supermix (Bio-Rad, Hercules, CA, USA), 0.7 µL 10 µM of forward and reverse primers, 0.4 µL 5 µM of probes (Table S3), 1 µL 50 ng of gDNA and nuclease-free water, two technical replicates were done for each sample and at least ten individuals per location were tested. The amplification was done in CFX Opus 96 (Bio-Rad, Hercules, CA, USA) under the following conditions: 95 °C for 5 min and 40 cycles at 95 °C for 15 s and 56 °C for 30 s. The results were analysed by using Bio-Rad qPCR analysis software CFX Maestro 1.0 (Bio-Rad, Hercules, CA, USA).

## 2.5. RNA extractions, cDNA synthesis and RT-qPCR

According to the manufacturer's instructions, the RNA was extracted using TRI Reagent (Sigma-Aldrich, France) from midguts of fifth-instar larvae of *S. frugiperda*. Each biological replicate was composed by five midguts for each field populations and three biological replicates were performed. Subsequently, 1 µg of the total RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad, France) following the manufacturer's guidelines.

Quantitative real-time qPCR reactions were done on CFX Opus 96 (Bio-Rad, Hercules, CA, USA) using Takyon™ No ROX SYBR 2× MasterMix blue dTTP (Eurogentec, Belgium). Each reaction was performed in duplicate, and PCR conditions were as follows: 3 min at 95 °C, 40 cycles of 10 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C for cytochrome P450 genes and three reference genes, ribosomal protein L4 (GSSPFG00028146001-RA from BIPAA https://bipaa.genouest.org/s/p/spodopteraFrugiperda_corn/), ribosomal protein L10 (GenBank MN044628) and ribosomal protein L17 (GenBank MN044629). For the transcription factors, CncC (GenBank OL781184), cAMP-response element binding protein (CREB) (GSSPFG00013352001-RA from BIPAA) and hormone receptor-like in 96 (HR96) (GenBank JX680322) the conditions were 40 cycles of 5 s at 95 °C and 30 s at 60 °C and 20 s at 72 °C, except for muscle aponeurosis fibromatosis (Maf) (GenBank OL781185) 10 min at 95 °C, 40 cycles of 5 s at 95 °C and 30 s at 60 °C. A melting curve was performed at the end of each reaction. Primers were designed by Primer3 V4.1.0 (Untergasser et al., 2012), the required information (sequences, amplicon size and efficiency) on the primers is available in Table S4. SATQPCR tool (https://satqpcr.sophia.inrae.fr/cgi/home.cgi) was used to calculate the relative expression of the mean of three independent biological replicates and setting the control condition corresponding to the laboratory susceptible strain to 1 (Rancurel et al., 2019). All biological replicates were compared using a pair-wise *t*-test with a pooled standard error (satqpcr).

## 2.6. Functional expression of *S. frugiperda* AChE and site-directed mutagenesis

The gene sequence of *S. frugiperda* AChE-1 wildtype was retrieved from GenBank (SUB15330148). The respective expression plasmid was

created using GeneArt server (ThermoFisher, Waltham, MA, USA), and pFastBac1 with BamHI and HindIII restriction sites was chosen. The plasmid was transformed into MaxEfficiencyDH10 (ThermoFisher, Waltham, MA, USA) competent cells according to manufacturer's instructions. The sequence was codon optimized for final expression in Sf9 cells. A pFastBac1 vector containing no insert served as a control. For the recombinant expression of AChE1 we followed the baculovirus expression protocol previously published (Manjon et al., 2018). In brief: MaxEfficiencyDH10 (ThermoFisher, Waltham, MA, USA) competent *E. coli* cells containing a baculovirus shuttle vector (bacmid) were transformed according to manufacturer's instructions. The final bacmid was extracted using Large Construct Kit (Qiagen, Hilden, Germany) following standard protocols. Subsequently Sf9 cells (Gibco™, kept in Sf-900 III-SFM (1×) cell culture medium, containing 10 µg/mL gentamycin) were virus transfected and the virus titer was determined according to Rapid Titer Kit (Takara Bio, San Jose, CA, USA). Sf9 cells were kept at 27 °C and 120 rpm in Sf-900 III-SFM (1×) Gibco, cell culture medium, containing 10 µg/mL gentamycin. Cells were diluted to a concentration of 2 × 10⁶/mL and incubated with the respective amount of virus (MOI 1) for 72 h at 27 °C and 120 rpm. After harvesting, cells were resuspended in 10 mL homogenization buffer / g wet cell weight (0.1 M K2HPO4, 0.5 % Triton X-100, pH 7.6). A Dounce Tissue Grinder was used for grinding the cells followed by a 30 min solubilization on ice and a centrifugation step at 4 °C and 10,000 x g. The resulting supernatant was used as enzyme source. Protein amount was determined using PierceTM BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer instructions.

The introduction of the mutations A201S, G227A, and F290V (Torpedo californica AChE numbering, PDB 1EA5) into wildtype *S. frugiperda* AChE-1 was done by site-directed mutagenesis (Fig. S1). Generated pFastBac vector (as described above) containing wildtype AChE-1 was processed using a Q5-site-directed mutagenesis kit following manufacturer instructions (New England Biolabs, Frankfurt, Germany). Specific primers were generated using NEBaseChanger (https://nebasechanger.neb.com/). Twenty-five ng of total plasmid DNA and each primer with a final concentration of 0.5 µM in 25 µL reactions were used. PCR conditions were as follows: 98 °C for 30 s, followed by 25 cycles of 98 °C for 10 s, 66 °C for 30 s and 72 °C for 3 min and 10 s. Final extension step was at 72 °C for 2 min. A kinase, ligase and DpnI treatment containing 1 µL PCR product, 5 µL KLD Reaction buffer, 10× KLD Enzyme Mix (New England Biolabs, Frankfurt, Germany) and 3 µL nuclease-free water was added and incubated at room temperature for 5 min. Afterwards 5 µL of KLD mix was added to 5-alpha-competent *E. coli* cells for transformation (New England Biolabs) following manufacturer's instructions. Cells were diluted 10- and 40-times and incubated overnight on LB-Agar plates (MP Biomedicals, 113002201-CF, capsules, concentration: 40 g/L) containing carbenicillin (100 µg/mL, CAS: 4800-94-6). Mini- (3 mL) and Midi (25 mL) preps in LB medium (MP biomedicals, 113002011-CF, capsules, concentration: 25 g/L) containing 100 µg/mL carbenicillin were generated and plasmids were isolated using Qiafilter Plasmid Midi Kit following manufacturer's instructions (Qiagen, Hilden, Germany). DNA concentrations were photometrically determined using 260/280 ratio (NanoQuant Infinite 200, Tecan, Switzerland) and normalized to 100 ng/µL. To confirm successful mutagenesis samples were sent for sequencing (TubeSeq Service, Eurofins, Ebersberg, Germany). Plasmids containing substituted nucleotides were further processed as described above.

## 2.7. AChE activity measurement, kinetic parameters and inhibition studies

Activity of AChE in the supernatant of homogenized Sf9 cells was measured according to Ellman et al. (1961) by the hydrolysis of the substrate analogue acetylthiocholine (ATChl) as described earlier (Devonshire and Moores, 1984) with some minor changes. Buffered solution of ATChl and dithionitrobenzoic acid (DTNB) in 0.1 M K2HPO4,
0.1 % Tween 20 (w/w) was added (50 µL) to the wells of a clear 384-well plate (Greiner bio-one, F-bottom, PS) containing 25 µL enzyme solution, giving a final concentration of 0.2 mM ATChI and 0.5 mM DTNB in a final volume of 75 µL. AChE activity was measured continuously at 23 °C in kinetic mode at 5 min intervals at 405 nm in a microplate reader (Tecan, Spark), and replicated four times. Determination of Michaelis-Menten parameters of AChE variants such as K<sub>M</sub> and Vmax were determined with the same assay format at 23 °C using at least 11 ATChI concentrations (ranging from 3.9 to 4000 µM) replicated four times. Kinetic parameters were calculated by using GraphPad Prism 9.0 (GraphPad Software, Boston MA, USA).

The effect of inhibitors chlorpyrifos-oxon, thiodicarb and methomyl (all obtained internally at Bayer Crop Science at a purity of >95 %) on AChE activity was determined in the absence of substrate. A stock solution of inhibitors was prepared in dimethyl sulfoxide (DMSO) and diluted with buffer, and the final DMSO concentration in each well was 0.6 %. At least seven concentrations (1:5 dilutions) in four replicates were tested for each variant in a clear 384-well plate. Twenty µL of the diluted supernatant were pre-incubated for 30 min at 20 °C ± 1 °C with 5 µL of inhibitor solution. Enzyme solution in buffer served as the positive control (full reaction rate), while buffer containing 18.2 µM chlorpyrifos-oxon served as the negative control (no reaction rate) due to complete inhibition. Afterwards 50 µL of substrate and DTNB solution (0.2 mM or 0.05 mM ATChI and 0.5 mM DTNB final concentration) were added to the plate followed by a 50 min incubation at 20 °C ± 1 °C. Each reaction was replicated four times, and the absorbance was measured at 405 nm in a microplate reader (Tecan, Spark). Enzyme inhibition was expressed as the mean percentage of activity remaining at different inhibitor concentrations and IC<sub>50</sub> values were calculated by non-linear regression using GraphPad Prism 9.0 (GraphPad Software, Boston MA, USA).

## 3. Results

### 3.1. Target site mutations

The presence of target site mutations that are known to confer resistance to different classes of insecticides was determined by using two techniques, TaqMan and PCR amplification followed by Sanger sequencing (Table 1). The VGSC mutations T929I, L932F and L1014F, which are known to confer pyrethroid resistance in FAW, have not been detected in any Thai populations, regardless of their geographical origin. However, sequencing analysis has revealed the presence of three other mutations M944L, F1005C and I1011M (numbering according to the Musca domestica sequence and corresponding to M979L, F1040C and I1046M in FAW sequence SFRUCORN610000020360-PA from BIPAA https://bipaa.genouest.org/sp/spodoptera_frugiperda_corn/). Ten percent of Kanchanaburi (KRI) larvae analysed and 30 % of Phayao (PYO) larvae were heterozygous for M944L. The situation is different for the other two mutations, F1005C and I1011M, which were detected in the heterozygous state in all the Thai populations analysed. For F1005C, the percentages of heterozygous individuals ranged from 70 % to 90 %, with 90 % being found in three populations, KRI, Saraburi (PCPCPD) and Nakhon Ratchasima (NRS) while lower percentages were obtained for Nakhon Sawan (NSN) and PYO. In the case of the I1011M mutation, approximately 60 % of the genotyped larvae were heterozygous for the NSN, PYO and PCPCPD populations. Higher percentages, up to 90 %, were found for NRS and KRI. Concerning the AChE, which is the target of organophosphates and carbamates, the mutations A201S and G227A (numbering according to Torpedo californica AChE) were absent in all the samples analysed, while the mutation F290V was found frequently in all locations (Table 1). Twenty percent to 50 % of the FAW larvae collected were heterozygotes for F209V and 10 % homozygotes resistant in NSN, PCPCPD. For KRI, 80 % of samples were homozygous for the V290 resistance allele and none of the individuals tested carried the F290 susceptible allele. No known mutations have been detected in the

<caption>**Table 1**</caption>

Target-site mutation frequencies in major insecticide targets. Several field populations were analysed from different locations in Thailand. Abbreviations: Number of insects (N), Homozygous susceptible (SS), heterozygotes (RS) and homozygous resistant (RR).

<table><thead><tr><th>Target</th><th>Mutation</th><th>Population</th><th>N</th><th>SS (%)</th><th>RS (%)</th><th>RR (%)</th></tr></thead><tbody><tr><td rowspan="10"></td><td rowspan="5">T929I</td><td>KRI</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>100</td><td>0</td><td>0</td></tr><tr><td rowspan="5">L932F</td><td>KRI</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>100</td><td>0</td><td>0</td></tr><tr><td rowspan="5">M944L</td><td>KRI</td><td>10</td><td>90</td><td>10</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>70</td><td>30</td><td>0</td></tr><tr><td rowspan="5">F1005C</td><td>KRI</td><td>10</td><td>10</td><td>90</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>30</td><td>70</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>10</td><td>90</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>10</td><td>90</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>16,7</td><td>83,3</td><td>0</td></tr><tr><td rowspan="5">I1011M</td><td>KRI</td><td>10</td><td>10</td><td>90</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>40</td><td>60</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>40</td><td>60</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>20</td><td>80</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>36,7</td><td>63,3</td><td>0</td></tr><tr><td rowspan="5">L1014F</td><td>KRI</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>100</td><td>0</td><td>0</td></tr><tr><td rowspan="5">A201S</td><td>KRI</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NSN</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PCPCPD</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>NRS</td><td>10</td><td>100</td><td>0</td><td>0</td></tr><tr><td>PYO</td><td>30</td><td>100</td><td>0</td><td>0</td></tr><tr><td rowspan="5">G227A</td><td>KRI</td><td>10</td><td>0</td><td>20</td><td>80</td></tr><tr><td>NSN</td><td>10</td><td>70</td><td>20</td><td>10</td></tr><tr><td>PCPCPD</td><td>10</td><td>40</td><td>50</td><td>10</td></tr><tr><td>NRS</td><td>10</td><td>40</td><td>50</td><td>10</td></tr><tr><td>PYO</td><td>30</td><td>53,3</td><td>46,7</td><td>0</td></tr><tr><td>Acetylcholinesterase (AChE)</td><td>F290V</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

target of diamides (Table S5), the RyR (I4790M and G4946E, numbering according to Plutella xylostella), nor in the glutamate-gated chloride channel (A309V/T and G314D numbering according to P. xylostella), the target of emamectin benzoate (Table S5).

### 3.2. AChE characterization and inhibition studies

We recombinantly expressed wildtype FAW AChE (Ace-1) in Sf9-cells and Michaelis-Menten kinetics confirmed its functional catalytic capacity to hydrolyse the substrate acetylthiocholine exhibiting a K<sub>M</sub>-value of 115 ± 6.16 µM. Site-directed mutagenesis inserting the known mutations A201S, G227A and F290V revealed AChE variants with slightly different Michaelis-Menten parameters, but still with the capacity to hydrolyse acetylthiocholine iodide (Table S6). Inhibition studies with three common AChE inhibitor insecticides revealed low, 3-4-fold, insensitivity levels for chlorpyrifos-oxon, thiodicarb and methomyl in the F290V variant when compared to wildtype AChE (Table 2). We also tested AChE variants with commonly reported mutations A201S and G227A and the combination of A201S and F290V, despite their absence in the FAW samples from Thailand genotyped in
<table><caption>**Table 2**
Acetylcholinesterase (AChE) inhibition studies. Inhibition of recombinantly expressed wildtype and mutant AChE (Ace-1) variants of Spodoptera frugiperda by chlorpyrifos-oxon (CPO), thiodicarb (TDC) and methomyl (MET). Data are mean values ± SD (n = 4).</caption><thead><tr><th rowspan="2">AChE variant</th><th>CPO</th><th>TDC</th><th>MET</th></tr><tr><th>IC<sub>50</sub> [nM]</th><th>IC<sub>50</sub> [µM]</th><th>IC<sub>50</sub> [µM]</th></tr></thead><tbody><tr><td rowspan="3">Wildtype</td><td>5.32 ±</td><td>1.43 ±</td><td>0.356 ±</td></tr><tr><td>0.208</td><td>0.0946</td><td>0.0121</td></tr><tr><td>83.6 ±</td><td>28.1 ±</td><td>29.0 ±</td></tr><tr><td rowspan="2">A201S</td><td>3.26</td><td>0.937</td><td>0.890</td></tr><tr><td>6.00 ±</td><td>2.62 ±</td><td>0.852 ±</td></tr><tr><td rowspan="2">G227A</td><td>0.225</td><td>0.135</td><td>0.0314</td></tr><tr><td>16.2 ±</td><td>4.05 ±</td><td>1.53 ±</td></tr><tr><td>F290V</td><td>0.519</td><td>0.168</td><td>0.0407</td></tr><tr><td>A201S + F290V</td><td>219 ±</td><td>35.8 ±</td><td></td></tr><tr><td></td><td>6.16</td><td>1.11</td><td>65.5 ± 1.46</td></tr></tbody></table>

this study. The mutation A201S exhibited the strongest impact on the inhibitory potential of chlorpyrifos-oxon, thiodicarb and methomyl resulting in insensitivity ratios 15.7-, 19.7- and 81.5-fold, respectively when compared to wildtype AChE (Table 2).

### 3.3. Cytochrome P450 expression levels

To explore the role of P450s in resistance, the expression levels of several P450s were monitored by RT-qPCR in Thai field populations and compared to the susceptible reference strain (FAW-S) (Fig. 2). The genes were selected based on some of our previous studies showing their impact on insecticide resistance (Amezian et al., 2022; Giraudo et al., 2015). The CYP9A subfamily genes are organised in a cluster, among which six genes (CYP9A26, CYP9A29, CYP9A30, CYP9A31, CYP9A58 and CYP9A59) were overexpressed in all the field populations tested. It should be noted that all the CYP9As studied were overexpressed in the NSN population with CYP9A26 overexpression level reaching more than 300-fold. In contrast, low levels of expression were obtained for CYP9A28 and CYP9A75 in the PYO, NRS and PCPCPD populations. Some P450s were repressed in most populations, such as Cyp4L12, while others show contrasting expression depending on the population.

Cyp4M15 was repressed in KRI and NSN, whereas it was over-expressed in NRS.

### 3.4. Expression levels of selected transcription factors and nuclear receptors

Based on previous studies several transcription factors and nuclear receptors have been identified and shown to be involved in the regulation of detoxification gene expression in pest insects including FAW (Amezian et al., 2021b). Their expression levels were investigated to see if it correlates with the observed overexpression of certain P450s in Thai field populations (Fig. 3). CncC and Maf were overexpressed by more than a factor of 2 in certain localities such as KRI. However, the situation was surprising for the NRS locality, where CncC was repressed and Maf overexpressed, even though these two transcription factors are known to function together. For CREB and HR96, the only significant difference observed was a repression for insects collected at NRS and PCPCPD.

## 4. Discussion

The aim of our molecular study was to characterise potential causes of the recently reported lack in efficacy of several insecticides against FAW in Thailand (Thirawut et al., 2023). We explored the status of common target-site mutations and the expression level of P450s known to confer resistance to different chemical classes of insecticides, in order to support appropriate recommendation of insecticide application schemes for controlling this major pest. If the resistance is based on a modification of the target, it will be preferable to use a pesticide with a different mode of action, whereas if the resistance is due to an increase in detoxification, the recommendations will be less obvious because of the cross-resistance to several chemical classes provided by some of the detoxification enzymes. Decisions will then have to be based on bioassay results. In the present study, we were able to track populations from 5 localities in Thailand, corresponding to the main maize-growing regions.

Our work has highlighted the presence of three mutations (M944L, I1005C and I1011M) in the VGSC, different from those previously identified in FAW (T929I, L932F and L1014F) (Carvalho et al., 2013). Further work is needed to assess the potential of these mutations to

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Expression levels of P450s in FAW populations collected in the field in Thailand and compared with a susceptible reference strain by using RT-qPCR. Gene expression was normalized using the expression of the ribosomal protein L4, L10 and L17 reference genes and shown as fold-change relative to the expression of FAW-S. Data are mean values ± SEM. A pairwise t-test was performed to test for statistically significant differences in expression levels in field populations compared to the susceptible reference strain (* P < 0.05, ** P < 0.01, *** P < 0.001).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Expression levels of transcription factors in FAW populations collected in the field in Thailand and compared with a susceptible reference strain by using RT-qPCR. Gene expression was normalized using the expression of the ribosomal protein L4, L10 and L17 reference genes and shown as fold-change relative to the expression of FAW_S. Data are mean values ± SEM. A pairwise *t*-test was performed to test for statistically significant differences in expression levels in field populations compared to the susceptible reference strain (* *P* < 0.05, ** *P* < 0.01, *** *P* < 0.001).</figcaption></figure>

confer pyrethroid resistance in FAW, including toxicological tests. However, one of these mutations (I1011M) has already been identified in *Aedes aegypti* and was shown to confer resistance to pyrethroids (Brengues et al., 2003). I1011M was demonstrated by expression in Xenopus oocytes to be involved in reduced sensitivity to type I but not type II pyrethroids (Du et al., 2013). It is localised in a receptor site and is essential for insecticide binding (Du et al., 2013). This mutation (I1011M) is detected in the FAW populations of the five localities with a percentage of heterozygotes ranging from 60 to 90 % and the absence of RR homozygotes. In this case, the status of the allele will affect the phenotype: if it is dominant, the phenotype will be resistant, whereas it will be susceptible if the allele is recessive. Interestingly, we found this I1011M mutation in association with the F1005C mutation in 47 of the 62 individuals that carried either mutation. It is not uncommon for the concomitant presence of several mutations in the VGSC to reduce sensitivity to pyrethroids to a greater extent than the presence of a single mutation. Indeed, this has been demonstrated in several insects and for several mutation combinations (Dong et al., 2014), as in the case of the VGSC in *Drosophila melanogaster*, where the combination T929I + L1014F confers a 30,000-fold reduction in sensitivity to deltamethrin compared with the T929I mutation alone (Vais et al., 2003). Whether this is also the case with the combination of mutations F1005C + I1011M in FAW is a hypothesis that remains to be demonstrated. The third mutation we detected was M944L, and not yet recorded elsewhere, but located between two mutated amino acids (G943A and Q945R) identified in pyrethroid resistant populations of human and salmon lice (Fallang et al., 2005; Kristensen, 2005). However, its potential role in pyrethroid resistance remains to be investigated.

Of the three AChE mutations known to confer varying levels of resistance to organophosphates and carbamate A201S, G227A and F290V, only F290V was detected in Thai FAW populations, and its frequency varied across locations. This mutation is in the acyl pocket and close to the residues of the catalytic site. It has been shown in the *Culex pipiens* mosquito to interfere with insecticide binding while having little effect on the endogenous activity of the enzyme (Alout et al., 2007). The presence of the F290V mutation in Thai populations is in line with the data available on FAW populations collected throughout the world. This mutation appears to be widespread and is detected on all 5 continents (Boaventura et al., 2020b; Tay et al., 2022; Yainna et al., 2021). Enzyme kinetics conducted in the present study using recombinantly expressed FAW AChE carrying the F290V mutation revealed only low levels of OP and carbamate resistance against those insecticides tested here, i.e. chlorpyrifos-oxon, thiodicarb and methomyl. Insensitivity factors of

3-4-fold are unlikely to compromise recommended label rates of these insecticides. However, it remains to be demonstrated that OPs and carbamates work against FAW under applied conditions, at least based on the absence of target-site mutations other than F290V. Conversely, the G227A mutation is only present in certain regions of the world, and is absent from Thai populations, in line with its low detection rate in Asia (Tay et al., 2022), however, we have shown that this mutation in functionally expressed AChE has almost no impact on the inhibition potential of the AChE inhibitors tested. The strongest impact on OP and carbamate-mediated AChE inhibition was demonstrated by the A201S mutation, which was absent in all Thai samples, but shown to be present in FAW samples collected in other regions such as Kenya, Indonesia and Brazil (Boaventura et al., 2020b). The impact is significant and likely to compromise field rates of both OPs and carbamates.

Several molecules from the diamide insecticide family are recom-
mended for control of FAW in Thailand and based on the absence of
known RyR mutations (I4790M and G4946E) they are important tools to
keep FAW under economic damage thresholds in various crops. While
no known resistance mutations were found in RyR, the efficacy of di-
amides may also be influenced by metabolic resistance, which warrants
further study. The G4946E mutation was first identified in *P. xylostella*
(Troczka et al., 2012) and has since been identified in several lepidop-
teran pests (Richardson et al., 2020), but to our knowledge has not been
detected in FAW field samples. Sequence analysis shows that the glycine
at position 4946 is encoded by GGC in FAW and that the change to
glutamic acid (GAA or GAG) requires the mutation of two bases (fig. S2),
which is a very rare event (Feyereisen et al., 2015). The I4790M mu-
tation has already been found in Asia, where isoleucine has been
substituted by two other amino acids (V and T) in Chinese populations
(Cai et al., 2024).

Emamectin benzoate is the most commonly used insecticide against FAW in Thailand, and although a decrease in sensitivity has been observed (Thirawut et al., 2023), no mutations have been detected in its molecular target, the GluCl. On the other hand, the presence of mutations in GluCl has been identified in Chinese populations of FAW with the A309V or A309T mutation and the G315D mutation at low frequencies (Cai et al., 2024). These mutations were initially identified in *P. xylostella* for A309V (Wang et al., 2016), and in *Tetranychus urticae* for G323D in Tu_GluCl1 and G326E in Tu_GluCl3 (both equivalent to position G315 in PxGluCl) (Dermauw et al., 2012; Kwon et al., 2010) and shown to play a role in resistance by reducing sensitivity to abamectin when the channel is expressed in Xenopus oocytes (Mermans et al., 2017; Wang et al., 2017). However, resistance to emamectin benzoate is
more commonly reported to be dependent on detoxification mechanisms and in particular dependent on P450s in *S. exigua* and *S. litura* (Shi et al., 2025; Teng et al., 2022; Zuo et al., 2021).

We explored the potential involvement of oxidative metabolic detoxification mechanisms by monitoring the expression level of certain candidate P450s for which data have been reported in relation to their inducibility by xenobiotics (Amezian et al., 2021a; Giraudo et al., 2015) or their involvement in insecticide resistance (Boaventura et al., 2021; Gimenez et al., 2020). Indeed, CYP9A subfamily members have been shown to be overexpressed in field-collected resistant strains of both *S. frugiperda* and *S. exigua* (Boaventura et al., 2021; Gimenez et al., 2020; Han et al., 2024). Our data revealed significant expression levels for certain CYP9A subfamily members. In all the populations we studied, CYP9As were overexpressed compared to the susceptible strain. In the NSN population, all CYP9As were overexpressed whereas in the PYO population, only some of them were. The expression levels reached when compared to the susceptible strain (> 100-fold) are close to those reported by Boaventura et al. (2021) in the Brazilian population Sf-Des resistant to insecticides such as deltamethrin and chlorpyrifos. Indeed, CYP9As have been associated with resistance to pyrethroids, diamides and avermectins (Boaventura et al., 2021; Han et al., 2024; Zuo et al., 2021). Between 11 and 15 genes encode for CYP9As in the genomes of *Spodoptera* depending on species (*exigua*, *frugiperda*, *littoralis* and *litura*) and the majority of these genes are organised in a cluster (Cheng et al., 2017; Gouin et al., 2017; Hilliou et al., 2021; Zuo et al., 2021). Deletion of the entire cluster using the CRISPR/Cas9 technique resulted in greater sensitivity to esfenvalerate and abamectin in *S. frugiperda* (42- and 15-fold, respectively) and *S. exigua* (22- and 5-fold, respectively) and also to indoxacarb, but only in *S. frugiperda* (Shi et al., 2023). Heterologous expression of CYP9As has demonstrated their ability to metabolize certain insecticides and substrate specificity. Despite high homology within the CYP9A cluster, each enzyme has its own metabolic capabilities. Of the three insecticides (esfenvalerate, abamectin and indoxacarb) tested by Shi et al., only CYP9A32 is able to metabolize all three, while CYP9A31 only indoxacarb and CYP9A60 none. (Shi et al., 2023). Whether the NSN population, which strongly overexpresses CYP9A31 and CYP9A32, is resistant to indoxacarb remains to be demonstrated. In contrast to CYP9As, certain P450s are repressed in field populations compared to the susceptible reference strain. These include CYP4L13 in the PYO and PCPCPD populations. This P450 was shown to be overexpressed in field strains of FAW in China (Guo et al., 2024). Of the 42 P450s for which the authors monitored expression levels, it was the most over-expressed (approx. 30-fold). When its expression was reduced by RNAi, insects became more sensitive to chlorantraniliprole.

One of the possible causes of P450 overexpression is the involvement of transcription factors which may themselves be overexpressed. In recent years, several transcription factors and nuclear receptors have been identified to play an essential role in controlling the expression of detoxification genes and thus driving the extent of metabolic resistance (Amezian et al., 2021b). Our results showed an overexpression of CncC and Maf in some of the populations, while CREB and HR96 have an expression level comparable to the susceptible strain and are repressed as in NRS and PCPCPD. The overexpression of CncC and Maf is a potential indicator for the upregulation of P450s as observed in our study. In *S. exigua*, it has been shown that the constitutive overexpression of CncC and Maf is involved in the overexpression of CYP321A8, a P450 responsible for resistance to chlorpyrifos and cypermethrin (Hu et al., 2021), while in *S. litura* it is the control of CYP307A1 expression which is involved in resistance to bistrifluron (Gong et al., 2022). The population-dependent expression profile of CncC correlates with the level of expression of CYP9A75 (the only CYP9A outside the cluster), and is consistent with our previous work which showed that overexpression of CncC in Sf9 cells induced overexpression of CYP9A75 (Amezian et al., 2023). There are other possible causes of P450 overexpression, including modifications to the promoter region of genes. A well-known example is the insertion of a transposable element into the CYP6G1

promoter in *Drosophila melanogaster*. This insertion leads to the over-expression of CYP6G1, which has been shown to be capable of metabolising DDT and neonicotinoids [for review (Le Goff and Hilliou, 2017)]. Gene duplication or amplification is also a possible cause of over-expression, and in the case of CYP9A, duplication of the entire cluster has been demonstrated in a population of pyrethroid-resistant FAWs (Gimenez et al., 2020). Further analysis is required to understand the impact of the expression profiles obtained in Thai populations on metabolic insecticide resistance.

In conclusion, to our knowledge, our study is the first to investigate field-collected Thai FAW populations for the presence of molecular markers linked to insecticide resistance and possibly explaining the decrease in susceptibility observed for certain insecticides against FAW in Thailand (Thirawut et al., 2023). Genotyping diagnostics revealed the presence of mutations in VGSC and AChE, which are likely to confer resistance to pyrethroids and carbamates/OPs, respectively. While monitoring the expression of detoxification genes showed that over-expression of CYP9As was widespread in the populations studied. This situation is worrying for the integrated management of FAW in Thailand, as these genes can confer cross-resistance to insecticides from different chemical classes (avermectins, diamides, pyrethroids and oxadiazines). In addition, the concomitant presence of mutations in the targets and an increase in detoxification can have a synergistic effect. In *S. exigua*, *Zuo et al.* (2025) demonstrated the contribution of each mechanism in nearly isogenic lines. The presence of the L1014F mutation in VGSC results in a resistance factor of 6.2, while the over-expression of CYP9A9 confers a factor 79. The combination of the two factors resulted in a resistance level of 631 (Zuo et al., 2025). Our results highlight the need to take into account the role of metabolic resistance in the implementation of resistance monitoring in FAW. Most studies are limited to investigate the presence of mutations in insecticide targets rather than building a holistic view by including markers of metabolic resistance such as CYP9A subfamily members. There is a real need to set up appropriate tools for monitoring insecticide resistance evolution in FAW, an invasive pest organism that is spreading across the globe and endangering sustainable crop yields.

### CRediT authorship contribution statement

**Marlen Saladini di Rovetino:** Writing – review & editing, Writing – original draft, Visualization, Resources, Investigation, Formal analysis.
**Bettina Lueke:** Writing – review & editing, Resources, Investigation, Formal analysis.
**Kamonporn Masawang:** Writing – review & editing, Resources, Investigation.
**Narisara Piyasaengthong:** Writing – review & editing, Resources, Investigation.
**Maroot Kaewwongse:** Writing – review & editing, Resources, Investigation.
**Saksit Nobsathian:** Writing – review & editing, Resources, Investigation.
**Thierry Fricaux:** Writing – review & editing, Resources, Investigation.
**Kiwoong Nam:** Writing – review & editing, Resources, Investigation.
**Emmanuelle d'Alençon:** Writing – review & editing, Resources.
**Vasakorn Bullangpoti:** Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.
**Ralf Nauen:** Writing – review & editing, Writing – original draft, Supervision, Investigation, Conceptualization.
**Gaëlle Le Goff:** Conceptualization, Resources, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.

### Funding

MSdR was supported by a PhD scholarship funded by 50 % INRAE-SPE and 50 % Bayer AG. This research was financially supported by the "PHC SIAM" program (project number: 48219WC), implemented by the French Ministry for Europe and Foreign Affairs, the French Ministry of Higher Education and Research and the Thai Ministry of Higher Education, Science, Research and Innovation.
Declaration of competing interest

BL and RN are employed by Bayer AG, a manufacturer of in-
secticides. All other authors declare no competing interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.pestbp.2025.106599.

Data availability

Data will be made available on request.

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