Molecular characterization of phosphine (fumigant) resistance in grain insect pests using the rust red flour beetle, Tribolium castaneum (Herbst) as a model organism

Rajeswaran Jagadeesan (2010). Molecular characterization of phosphine (fumigant) resistance in grain insect pests using the rust red flour beetle, Tribolium castaneum (Herbst) as a model organism PhD Thesis, School of Biological Sciences, The University of Queensland.

       
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Author Rajeswaran Jagadeesan
Thesis Title Molecular characterization of phosphine (fumigant) resistance in grain insect pests using the rust red flour beetle, Tribolium castaneum (Herbst) as a model organism
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2010-08
Thesis type PhD Thesis
Total pages 184
Total colour pages 6
Total black and white pages 178
Subjects 06 Biological Sciences
Abstract/Summary Insect resistance to pesticides is an issue of increasing global importance and a vital one for our future food security. The problem of phosphine resistance is particularly acute in tropical and sub-tropical countries like Australia, where it is used to protect 80% of the AU$8 billion annual grain harvest from insect pests of storage. The major reliance of the grains industries in these countries on phosphine are because alternative control measures are generally either expensive, leave residues on the grain or otherwise reduce the quality of the product. When markets increasingly demand insect-free and residue-free products, the resistance problem threatens the export competitiveness of the nation’s grain industry, as similar resistance problems do not exist in other wheat exporting countries, especially those in temperate regions where pest population growth is inhibited over the winter months. In the present study we used the genetic model and the Tenebrionid grain pest, rust red flour beetle, Tribolium castaneum, as a model organism to understand the genetic and molecular basis of evolution of resistance to phosphine in grain insect pests. The genetic dissection of weakly (~3.2×) and strongly (~431×) resistant strains of T. castaneum revealed that a single major gene (named tc_rph1) is responsible for weak resistance to phosphine whereas two major genes (tc_rph1 and tc_rph2) confer strong resistance, one of which is shared with the weakly resistant strain. Neither weak nor strong resistance genes were sex linked but both were inherited as an incompletely recessive autosomal trait. Genetic interaction and complementation analyses between crosses of weak and strongly resistant strains revealed the synergistic interaction of the tc_rph1 and tc_rph2 loci. The genetic confirmation that two genes conferred high-level resistance to phosphine in T. castaneum prompted me to focus on identifying these genes within the T. castaneum genome. Next-generation sequencing (Illumina- GAII) methods allowed us to resequence the whole genome of selected and unselected F4 and F19 T. castaneum populations of a single pair intercross, Susceptible X Strong-Resistant, that segregated for genes of interest in a whole-genome bulk segregant-type analysis. Subsequent differential detection of SNP homozygosity between selected and unselected datasets identified two regions, on Chr8 and Chr9 that are responsible for strong resistance. The linkage of these regions to resistance loci was confirmed by conversion of identified SNPS to polymorphic CAPS markers and fine scale mapping revealed the closest markers for the two resistance loci, tc_rph1 and tc_rph2 on Chr8 and Chr9,respectively, are approximately 0.08 cm (~30 kb) and 0.37cm (~140 kb) away from their respective most likely candidate genes, Na+/K+ ATPase and dihydrolipoamide dehydrogenase (DLD). Genotyping analysis of F4 selected resistant individuals, using the CAPS markers tcc8-597m and tccU7-138.2k, tightly linked to tc_rph1 and tc_rph2 respectively, confirmed the strongly synergistic interaction of tc_rph1 and tc_rph2 resistance alleles. These markers were also used for genotypic fitness analysis by determining the change in allelic frequency of tc_rph1 and tc_rph2 resistance loci over nineteen generations in a cross segregating for strong resistance alleles without phosphine exposure. The results showed a significant decrease in homozygous resistant tc_rph2 allelic frequency in unselected beetles at multiple generations and revealed the existence of a significant fitness cost associated with that resistance allele only, clearly demonstrating that strong resistance to phosphine in T. castaneum carries a significant fitness disadvantage, which may have significant impact on designing resistance management strategies. Phosphine also induces some significant changes in gene expression at high concentrations in strong resistant T. castaneum. A set of genes associated with amino acid metabolism, redox signalling and defence response were found differentially expressed after phosphine exposure. These genes may provide information as to some of the effects of phosphine toxicity and how organisms can react to such a chemical challenge. Interestingly, none of our candidate genes were found to significantly change expression, suggesting that resistance is possibly conferred by point mutations within those genes rather than an alteration of expression.
Keyword Insecticide resistance, phosphine, stored grain insects, genetics, genomics
Additional Notes Colour pages:91; 95; 96; 101; 110; 114

 
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Created: Tue, 29 Mar 2011, 11:27:18 EST by Rajeswaran Jagadeesan on behalf of Library - Information Access Service