New genomic resources for an invasive mite pest expand the known diversity of pesticide resistance mechanisms and guide development of KASP diagnostic markers

The redlegged earth mite, Halotydeus destructor, is an invasive pest of grain and pasture crops in Australia. Australian populations are increasingly developing resistance to broad-spectrum pesticides. Here, we assembled a chromosome-level genome for H. destructor, comprising nine chromosomes and totalling approximately 46 Mb. We also generated a genomic dataset from 190 populations collected across thousands of kilometres of its invasive Australian range and across four years (2018, 2021, 2022, and 2023), and used this dataset to develop KASP (Kompetitive allele-specific PCR) markers for identifying resistance mutations. We combined pooled whole-genome sequencing with resistance testing for organophosphate pesticides, chlorpyrifos and omethoate, and the pyrethroid pesticide bifenthrin. Target-site resistance to chlorpyrifos was strongly associated with the common ace G119S mutation. A second mutation, ace A201S, was also common, although its association with chlorpyrifos or omethoate resistance was less clear. Target-site resistance to bifenthrin was strongly associated with the common para L1024F mutation. We also detected several rare target-site mutations that likely contribute to resistance in specific populations, including F331Y in ace, and M918T, M918V, L925I, F1020S, and F1538I in para. Copy number variation in ace showed mixed associations with resistance, potentially reflecting the co-occurrence of variable gene copies and target-site mutations segregating within and among populations. Finally, we observed putative compositional and structural differences in genomic regions containing carboxylesterases with high ace-like homology, a group of genes that might be involved in organophosphate metabolism. This study advances the genomic and molecular resources available for H. destructor, and represents the most comprehensive investigation to date of resistance mechanisms and their spatiotemporal distribution in this invasive pest. Our data showed that pesticide resistance in H. destructor likely involves many genes. Nevertheless, target-site mutations are likely to remain key markers for rapid resistance diagnostics, for which our KASP markers will provide valuable tools.