Toxicological and molecular profiling of insecticide resistance in a Brazilian strain of fall armyworm resistant to Bt Cry1 proteins.

BACKGROUND: Spodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and to Bacillus thuringiensis (Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross-resistance in FAW by investigating the toxicological profile of a Bt-resistant Brazilian strain (Sf_Des) in comparison to a Bt-susceptible strain (Sf_Bra). RESULTS: Laboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross-resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited - depending on the substrate - up to 19-fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA-Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed that CYP9A-like and CYP6B39 are significantly upregulated (>200-fold) in Sf_Des in comparison to Sf_Bra strain. No target-site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation-reduction and metabolic processes. CONCLUSION: Our results indicate that multiple/cross-resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Molecular characterization of Cry1F resistance in fall armyworm, Spodoptera frugiperda from Brazil.

Fall armyworm, Spodoptera frugiperda (J.E. Smith) is a major lepidopteran pest of maize in Brazil and its control particularly relies on the use of genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins such as Cry1F. However, control failures compromising the efficacy of this technology have been reported in many regions in Brazil, but the mechanism of Cry1F resistance in Brazilian fall armyworm populations remained elusive. Here we investigated the molecular mechanism of Cry1F resistance in two field-collected strains of S. frugiperda from Brazil exhibiting high levels of Cry1F resistance. We first rigorously evaluated several candidate reference genes for normalization of gene expression data across strains, larval instars and gut tissues, and identified ribosomal proteins L10, L17 and RPS3A to be most suitable. We then investigated the expression pattern of ten potential Bt toxin receptors/enzymes in both neonates and 2nd instar gut tissue of Cry1F resistant fall armyworm strains compared to a susceptible strain. Next we sequenced the ATP-dependent Binding Cassette subfamily C2 gene (ABCC2) and identified three mutated sites present in ABCC2 of both Cry1F resistant strains: two of them, a GY deletion (positions 788-789) and a P799 K/R amino acid substitution, located in a conserved region of ABCC2 extracellular loop 4 (EC4) and another amino acid substitution, G1088D, but in a less conserved region. We further characterized the role of the novel mutations present in EC4 by functionally expressing both wild type and mutated ABCC2 transporters in insect cell lines, and confirmed a critical role of both sites for Cry1F binding by cell viability assays. Finally, we assessed the frequency of the mutant alleles by pooled population sequencing and pyrosequencing in 40 fall armyworm populations collected from maize fields in different regions in Brazil. We found that the GY deletion being present at high frequency. However we also observed many rare alleles which disrupt residues between sites 783-799, and their diversity and abundance in field collected populations lends further support to the importance of the EC4 domain for Cry1F toxicity.