D3252: Outpacing the beetles: Transgenic technology for combatting phosphine resistance

Tribolium castaneum infest grain stores globally, compromising food security and farmer’s profits. For decades, agriculturalists’ best defence has been fumigation with phosphine. Unfortunately, resistance to phosphine has been recorded widely. Two metabolic enzymes (Cytochrome-b5 fatty acid desaturase (Cyt-b5-r) and Dihydrolipoamide dehydrogenase (DLD)) are the major contributors to phosphine resistance. Termed resistance to phosphine 1 (rph1) and resistance to phosphine 2 (rph2) respectively, the genes encoding these enzymes act synergistically to produce strong phosphine resistance. In both cases, mutations decreasing or eliminating enzyme function cause phosphine resistance. No rph1 knock-out mutations have been observed in T. castaneum.
We report our efforts to understand the molecular mechanisms underpinning phosphine resistance, and their impacts on fitness. To unravel how rph1 contributes to phosphine resistance, we used CRISPR-Cas9 on a strain of T. castaneum, inducing a null mutation in this gene. If homozygous null individuals are viable, we’ll investigate their phosphine resistance phenotype and fitness. We are also using CRISPR-Cas9 to knock a strep-tagged Wild Type copy of rph1 into an x-linked eye colour locus termed Cardinal within a phosphine resistant strain. This will then determine if complementation at the rph1 locus is possible, and pulldown Cyt-b5-r protein and identify its molecular partners. We’re sampling wild T. castaneum from across Victoria, Australia, which are likely to bear rph1 mutations. To assess potential fitness costs within rph1 mutants available to us, we’re building a flight-mill that records flight parameters.