Insecticide resistance in a major pest, Spodoptera frugiperda

President of the jury :                       

• Janice DE ALMEIDA-ENGLER, Directrice de Recherche Institut Sophia Agrobiotech, France  

Rapporteurs :                     

• Fabrice CHANDRE, Directeur de Recherche IRD Université de Montpellier, France
• Valérie RAYMOND, Professeur, Université de Angers, France                          

Examiners :

• Chris BASS, Professeur, University of Exeter, Royaume-Uni     

Thesis Directors :

• Ralf NAUEN, Distinguished Science Fellow, Bayer AG, Allemagne
• Gaëlle LE GOFF, Directrice de Recherche Institut Sophia Agrobiotech, France       

Abstract :

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is considered one of the world’s most destructive insect pests. It is a highly polyphagous agricultural pest capable of feeding on several economically important crops, including maize, rice, and sorghum. Native to the Americas, FAW has rapidly spread across five continents since 2016 and has recently reached Europe. To mitigate the severe agronomic and economic damages caused by this pest, FAW management has relied heavily on insecticide applications. This strong selection pressure has driven the evolution of resistance to numerous insecticide classes; according to the Arthropod Pesticide Resistance Database, FAW has evolved resistance to at least 47 insecticides. This adaptation relies on mechanisms that enable FAW to cope with xenobiotics, including plant secondary metabolites and synthetic insecticides. Among these, two main mechanisms drive insecticide resistance: mutations in insecticide target sites and enhanced metabolic detoxification mediated by overexpressed enzymes. Understanding the mechanisms responsible for the resistance phenotypes observed in field populations is essential for maintaining effective control strategies against this pest. In this context, the aim of this thesis was to investigate the molecular basis of insecticide resistance in FAW, integrating population monitoring with functional analyses of key target and detoxification genes.

The first part of the thesis focused on monitoring molecular mechanisms of insecticide resistance in FAW field populations from Thailand. Resistance-associated mutations in four insecticide target-site genes were investigated, revealing three mutations (M944L, F1005C and I1011M) in the voltage-gated sodium channel associated with pyrethroid resistance, and the F290V mutation in acetylcholinesterase, linked to resistance to organophosphates/carbamates, at varying frequencies across the analysed populations. As resistant populations often exhibit enhanced detoxification mediated by gene overexpression, the expression levels of cytochrome P450 monooxygenase (P450) genes were also analysed, with particular focus on the CYP9A subfamily known to be involved in insecticide resistance. The analysis reveals a significant up-regulation of these genes in the populations studied. In addition, to investigate the causes of this overexpression, the expression of several transcription factors (CncC, Maf, CREB, and HR96) potentially involved in CYP9A regulation was examined. While this monitoring study provided important insights, it also highlighted the urgent need for rapid and sensitive tools to detect resistance-associated mutations. To address this gap, a droplet digital PCR (ddPCR) based method was developed, enabling pooled sample analysis and the detection of seven target site mutations associated with resistance to organophosphates/carbamates, pyrethroids, diamides, and avermectins for the first time in FAW.

The final part of the thesis aimed to characterize and validate the role of the CYP9A subfamily in FAW insecticide resistance using Sf9 cells. Attention was given to the individual contribution of CYP9A30, CYP9A31, CYP9A32, and CYP9A75, as these genes are strongly induced by xenobiotics and overexpressed in resistant populations. Stable Sf9 cell lines either overexpressing or knocked out by CRISPR/Cas9 for these genes were generated and used to assess changes in cell viability following exposure to several insecticides, including methoprene, chlorpyrifos, chlorpyrifos-oxon, emamectin benzoate and abamectin. In addition, recombinant expression and inhibitor kinetic assays were performed to further validate the functional roles of CYP9A enzymes in insecticide detoxification.
Overall, these findings integrate applied and fundamental approaches to advance the understanding of insecticide resistance in FAW and to support sustainable resistance management strategies.

Keywords :

Spodoptera frugiperda, resistance, cytochromes P450, target site mutation.