Genetics - Theses

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Subject: Insecticide resistance ×

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    Molecular and genetic analysis of Bt and spinosad resistance in diamondback moth, Plutella xylostella
    Baxter, Simon Wade. (University of Melbourne, 2005)
    Diamondback moth, Plutella xylostella, is a destructive global pest of nutritional and economically important cruciferous plants. Over the last 50 years, field strains have developed resistance to all major classes of insecticides used against them, including Bacillus thuringiensis (Bt) toxins and spinosyns. Molecular and genetic analysis of Bt resistance Diamondback moth is the only insect to develop open field resistance to Bt toxins. Many other insect species have been selected for Bt resistance under laboratory conditions, and in three lepidopteran pests, resistance has been genetically associated with mutations in a CrylA-binding midgut cadherin protein. Here, diamondback moth strains SCI and NO-QA were analysed. Both are resistant to CrylAa, CrylAb, CrylAc, CryIF and CrylJ however SCI was also resistant to Cry1C. AFLP linkage mapping was used to identify twenty-nine of the expected 31 linkage groups in SCI. CrylA resistance was significantly associated with a single linkage group (LG22 ?2=15.6, dfi=1, P<0.0001) and Cry 1C resistance was associated with two others (?2 =6.3 and 4.8, df=1). This data confirms different genetic mechanisms are responsible for CrylAc and CrylC resistance in this strain. Genes for lepidopteran Bt binding proteins were identified and mapped in SCI, including the 12-domain midgut cadherin, aminopeptidase N, alkaline phosphatase, glycosyl transferase, and P252-like genes, however none mapped to CrylAc or CrylC resistance associated linkage groups. Molecular analysis and interstrain complementation tests for allelism show mutations in the same genes are responsible for CrylAc resistance in SCl and the second resistant strain, NO-QA, Comparative genomics identified seven ribosomal genes that mapped to the CrylAc resistance linkage. Where possible, these single copy anchor loci were used to construct linkage maps for NO-QA and SCI. In NO-QA, 9 AFLP markers clustered around the Cry 1 Ac resistance locus were identified using bulked segregant analysis. Sequencing and inverse PCR were carried out on two of these AFLP markers and a PCR based resistance detection assay designed from one of these markers. Molecular and genetic analysis of spinosad resistance Spinosad is a novel class of insecticides that primarily targets nACh Receptors. The mechanism for field evolved spinosad resistance in diamondback moth strain Pearl-Sel (18,600 fold resistant) is hypothesised to be a single, recessive, autosomal gene. AFLP linkage mapping identified 28 linkage groups including one autosome responsible for spinosad resistance (?2=21.1, df=1, P<0.0001). Degenerate PCR identified five nAChR genes, two of which mapped to the resistance linkage group (nAChR a7-1 and a7-2). A linkage map of the resistance linkage group positioned nAChR ?7-1 more than 30 Haldane centimorgans (cM) and nAChR ?7-2 only 4.2 cM from the theoretical resistance locus, SpR- 1.
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    Genomics of insecticide resistance and xenobiotic metabolism in helicoverpa armigera
    Wee, Choon Wei. (University of Melbourne, 2005)
    Helicoverpa armigera is a polyphagous lepidopteran that causes severe agricultural losses globally and it has a great capacity to develop resistance to insecticides. Previous research has shown that metabolic based resistance is the major cause of pyrethroid resistance in Australia and both cytochrome P450 monooxygenases and carboxylesterases have been implicated. AN02, a field strain of H. armigera exhibits a 50 fold resistance to fenvalerate in the larval stage. Resistance can be almost completely synergised by a P450 inhibitor, piperonyl butoxide, suggesting that resistance is metabolic based and likely to be mediated by P450 enzymes. The research presented in this thesis utilised two techniques, cDNA-AFLP and cDNA microarrays to identify differentially expressed genes between fenvalerate resistant and susceptible backcross progeny from an AN02 heterozygous resistant female and susceptible male. A novel cytochrome P450, CYP337B1 was found to be constitutively overexpressed in resistant individuals, and was mapped to within 1 cM of the resistance locus RFen1. Two other P450s which have previously been identified to be overexpressed in fenvalerate resistant H. armigera in Australia, CYP4G8 and CYP6B7 were also overexpressed in AN02, as were two glutathione S-transferases and one carboxylesterase. H. armigera larvae were also challenged with different xenobiotics; piperonyl butoxide, phenobarbital, esfenvalerate and jasmonic acid. Induction was studied in three separate tissues, integument, fatbody and midgut. Suites of inducible genes were identified, which may play a role in detoxifying these compounds. Notably, the three cytochrome P450s identified as constitutively upregulated in AN02 (CYP337B1, CYP4G8 and CYP6B7) were among the genes induced by esfenvalerate.