Dissecting Phosphine Resistance: Worldwide populations of pest insects—such as the lesser grain borer, Rhyzopertha dominica , and the rust-red flour beetle, Tribolium castaneum —have become highly resistant to the fumigant phosphine, providing a potential threat to global food security. The nematode, Caenorhabditis elegans is vulnerable to phosphine, but phosphine-resistant strains are known. Schlipalius et al. (p. 807 ) show that mutations in the delta-1-pyrroline-5-carboxylate dehydrogenase and dihydrolipoamide dehydrogenase ( dld-1 ) genes both give rise to phosphine resistance in C. elegans . Phosphine resistance mutants in R. dominica , and T. castaneum also map to the dld-1 gene, which codes for a core metabolic enzyme. These mutants are, however, hypersensitive to arsenic, mimics of which might thus synergize with phosphine.

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2×) resistant strain. This gene was also found in the strongly resistant (431×) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12–20× resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431×) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.

Next-generation sequencing methods enable identification of the genetic basis of traits in species that have no prior genomic information available. The combination of next generation sequencing, variant analysis and linkage is a powerful way of identifying candidate genes for a trait of interest. Here we used a comparative transcriptomics (RNAseq) and genetic linkage analysis approach to identify the rph1 gene. rph1 variants are responsible for resistance to the fumigant phosphine (PH₃) that is used to control insect pests of stored grain. In each of the four major species of pest insect of grain we have investigated, there are two major resistance genes, rph1 and rph2, which interact synergistically to produce strongly phosphine-resistant insects. Using RNAseq and genetic linkage analyses we identified candidate resistance (rph1) genes in phosphine resistant strains of three species, Rhyzopertha dominica (129 candidates), Sitophilus oryzae (206 candidates) and Crypotolestes ferrugineus (645 candidates). We then compared these candidate genes to 17 candidate resistance genes previously mapped in Tribolium castaneum and found only one orthologous gene, a cytochrome b5 fatty acid desaturase (Cyt-b5-r), to be associated with the rph1 locus in all four species. This gene had either missense amino acid substitutions and/or insertion/deletions/frameshift variants in each of 18 phosphine resistant strains that were not observed in the susceptible strains of the four species. We propose a model of phosphine action and resistance in which phosphine induces lipid peroxidation through reactive oxygen species (ROS) generated by dihydrolipoamide dehydrogenase (DLD), whereas disruption of Cyt-b5-r in resistant insects decreases the polyunsaturated fatty acid content of membranes, thereby limiting the potential for lipid peroxidation.