Screening for insecticide resistance in Australian field populations of Bemisia tabaci (Hemiptera: Aleyrodidae) using bioassays and DNA sequencing.

BACKGROUND: Species within the Bemisia tabaci cryptic species complex can cause significant crop damage. We used high-throughput amplicon sequencing to identify the species composition and resistance allele genotypes in field populations from cotton fields in Australia. For selected populations, the resistance phenotype was determined in bioassays and compared with sequencing data. RESULTS: A metabarcoding approach was used to analyse the species composition in 144 field populations collected between 2013 and 2021. Two mixed AUS I and MEAM1 populations were detected, whereas the remaining 142 populations consisted of MEAM1 only. High-throughput sequencing of organophosphate and pyrethroid resistance gene amplicons showed that the organophosphate resistance allele F331W was fixed (> 99%) in all MEAM1 populations, whereas the pyrethroid resistance allele L925I in the voltage-gated sodium channel gene was detected at varying frequencies [1.0%-7.0% (43 populations); 27.7% and 42.1% (two populations); 95%-97.5% (three populations)]. Neither organophosphate nor pyrethroid resistance alleles were detected in the AUS I populations. Pyrethroid bioassays of 85 MEAM1 field-derived populations detected no resistance in 51 populations, whereas 32 populations showed low frequency resistance, and 2 populations were highly resistant. CONCLUSIONS: We demonstrate that high-throughput sequencing and bioassays are complementary approaches. The detection of target site mutations and the phenotypic provides a comprehensive analysis of the low-level resistance to pyrethroids that is present in Australian cotton farms. By contrast, a limited survey of whitefly populations from horticulture found evidence of high-level resistance against pyrethroids. Furthermore, we found that the F331W allele (linked to organophosphate resistance) is ubiquitous in Australian MEAM1. © 2022 Commonwealth of Australia. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci.

Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.