Genetic mapping and pharmacological analysis of phosphine toxicity and resistance pathways in Caenorhabditis elegans

The fumigant phosphine is critically important for the protection of stored commodities frominsect and rodent pests. Phasing out the use of ozone-depleting substances in accordance with theUnited Nations Montreal Protocol has left phosphine as the only economically viable fumigant forroutine use. The heavy reliance on phosphine for many years has resulted in highly-resistant pestinsects, a situation that threatens the safe and economical, long-term use of phosphine. Thenematode Caenorhabditis elegans has been used as a model organism for the study of phosphinetoxicity. The creation of phosphine-resistant C. elegans mutants (pre) has additionally allowed thisorganism to be used to model phosphine resistance.Phosphine gas is highly toxic to aerobic organisms, making it a highly effective fumigant, butalso a severe occupational health and safety risk. To address the safety concerns of working withphosphine in a university research environment, a custom-designed chamber was created for thesafe containment of the phosphine gas generated from aluminium phosphide tablets. In animprovement on previous generation systems, this chamber can be completely sealed to controlthe escape of phosphine, even if there is a complete failure of the laboratory ventilation system.The development of this novel chamber, has allowed fumigation facilities to be established atthe University of Queensland. This has allowed the phosphine resistance phenotype of the pre-7mutant of C. elegans to be characterised and has facilitated the identification of multiplephosphine synergists. The LC50 value for 24-hour phosphine exposure at 20°C is 732ppm for thewild-type N2 strain. Under the same conditions, the resistant mutant pre-7 has an LC50 value of4269ppm - a resistance factor of 5.8× greater than wild-type.Based on the proposed mode of action of phosphine as a mitochondrial toxin and initiator ofoxidative stress, a range of compounds were tested for their ability to synergistically enhance thetoxicity of phosphine. This resulted in mitochondrial uncouplers being identified as extremelypotent synergists with phosphine, capable of causing 100% mortality when used in combination.This was despite the fact that the concentrations of phosphine and uncouplers used wereindividually nonlethal. In addition to being highly potent synergists in the wild-type strain,mitochondrial uncouplers have also been shown to be effective against genetic phosphineresistance in two different pre mutant strains. In the case of pre-7, exposure to uncouplerappeared to completely negate the genetic resistance to phosphine toxicity.The reported capacity of phosphine to induce oxidative stress suggested an alternative class ofpotential phosphine synergists. As such, diethyl maleate, a compound that depletes the cellularantioxidant glutathione, was tested for a synergistic interaction with phosphine. While it waspossible to cause mortality in C. elegans with individually sub-lethal concentrations of phosphineand the glutathione depletor diethyl maleate, the synergism was not as strong as that previouslyseen with mitochondrial uncouplers. In contrast to the 100% mortality resulting from combinedtreatments of nonlethal phosphine and uncoupler concentrations; the corresponding treatmentusing dimethyl disulphide caused mortality to a level of around 30%.A potential alternative fumigant to phosphine, dimethyl disulphide (DMDS), was alsoinvestigated to determine its comparative toxicity to phosphine. Both DMDS and phosphine inhibitthe mitochondrial respiratory enzyme, cytochrome c oxidase, though the subsequent toxicitypathways of the two compounds are thought to be quite different. Under identical treatmentconditions DMDS was found to be almost 600 times more toxic to C. elegans than phosphine atthe LC50 level. No significant cross-resistance to DMDS was displayed by the phosphine-resistantmutant pre-7, as a concentration of 3ppm of DMDS for 24 hours at 20°C caused equal mortality inwild-type and pre-7.Using both classical and molecular techniques, a genetic study was also carried out on the pre-7mutant. The factor responsible for the phosphine resistance of pre-7 was determined to be due toa completely recessive autosomal genetic element. This factor was mapped to a 96 kb region ofthe C. elegans chromosome II. Subsequent gene knockdown and gene expression analysis of thisregion identified two plausible candidates from the 17 predicted genes at this locus, but the generesponsible for the resistance phenotype was not unambiguously identified.As a result of this work, the nematode C. elegans has been established as the premiere modelorganism with which to study phosphine toxicity and resistance. Synergist discovery has become astraightforward procedure that may result in new grain protection strategies to alleviate theeconomic and food security problems resulting from phosphine resistance. The work presented inthis thesis presents C. elegans as an excellent model for testing of prevailing theories regarding themode of phosphine toxicity. Furthermore, the genetic characterisation of the phosphine-resistantpre-7 mutant is the most advanced genetic analysis of phosphine resistance ever conducted

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