The second major research project has been focused on the different genetic resistance options adopted by different insect pest species. Ultimately we seek to understand why one genetic mechanism is used in one species and not in another. To date we have concentrated on pyrethroid and organophosphorus chemical resistance with a focus on lab-based research. While wanting to continue this work, we wish to expand this project to include the identification of field resistance mechanisms to chemical insecticides in L. cuprina, H. armigera and D. melanogaster. This will help us to understand the evolution of resistance in the field and will also provide molecular monitoring tools useful in the effective management of field resistance.

A strain of L. cuprina isolated at Tara in Queensland has previously been shown to be resistant to several different insecticides. Resistance to the insect growth regulator insecticides cyromazine and diflubenzuron have been genetically mapped. Both of these resistances map to more than one locus. The Cyp genes 6A27, 6A28 and 12A7 are up-regulated in the Tara strain. Zhenzhong Chen (Postdoctoral Fellow) has shown that Cyp6a27 and Cyp6a28 are tightly linked on the chromosome 4 and may explain resistances that have mapped to this chromosome. These genes are being overexpressed in vivo in D. melanogaster by Alex Blasetti to test for an association between overexpression and resistance.

A field strain of H. armigera, AN02, exhibits a 50 fold resistance to the pyrethroid fenvalerate in larvae. 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. Jeffrey Wee used two independent but complementary techniques, cDNA-AFLP and cDNA microarrays, to identify genes that are differentially expressed between fenvalerate resistant and susceptible backcross progeny from an ANO2 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 over-expressed in fenvalerate resistant H. armigera in Australia, Cyp4G8 and Cyp6B7 were also over-expressed in AN02, as were two glutathione S-transferases and one carboxylesterase. The Cyp337B1gene is being expressed in D. melanogaster to test its capacity to confer pyrethroid resistance.

In D. melanogaster the overexpression of Cyp6g1 is sufficient to cause resistance to many different insecticides. However this is not the only resistance mechanism we have found in field populations of this species. Michael Bogwitz conclusively showed that in the NB16 strain that lufenuron resistance is the result of the upregulation of the cytochrome P450 Cyp12a4. In the WC2 strain, another unknown factor on chromosome III is able to confer lufenuron resistance, using a mechanism that is also independent of Cyp6g1. Emily Remnant has found a region around map position 68 on chromosome III important for this resistance. Microarray analysis of the WC2 strain indicates that the resistance present on chromosome III is unlikely to be due to upregulation of a detoxification enzyme, and that a potentially novel mechanism of lufenuron resistance exists. Fine scale genetic mapping is currently underway to identify the gene responsible and characterise the resistance mechanism.

Spatial and longitudinal variation of pyrethroid resistance in diamondback moth

When strong selection acts on populations, phenotypes reflect effects of selection and gene flow. Nancy Endersby (postgraduate student) has investigated the relative impact of these factors by assessing resistance to synthetic pyrethroids in an 11-year study of diamondback moth, Plutella xylostella, from southern Australia. She estimated resistance levels in populations from weeds, canola, forage crops and vegetables. Differences in resistance among local populations sampled repeatedly were stable over several years. Levels were lowest in samples from weeds and highest in vegetables. Resistance in canola samples increased over time as insecticide use increased. There was no evidence that selection in one area influenced resistance in adjacent areas. Microsatellite variation among 13 populations was low (0.25%), compared with an AMOVA estimate of 13.8% for the resistance trait. Results suggest that local selection rather than gene flow of resistance alleles dictated variation in resistance across populations. Therefore, regional resistance management strategies may not limit resistance evolution.

Resistance to pyrethroids in head lice

Stephen Barker and Anna Murrell (Postdoctoral Fellow) discovered that the T929I-L932F allele is present in head lice, Pediculus capitis, in Brisbane and Townsville, Australia, and in Argentina. This allele confers kdr-like resistance to synthetic pyrethroids in head lice, P. capitis. T929I-L932F was discovered in P. capitis in the US and UK in 2000, in Japan in 2003, and in Europe in 2005.

Genetics of Bt Resistance in Helicoverpa armigera: Resistance to Cry2Ab.

The genetically modified cotton, Bollguard II, expresses two different toxin genes derived from bacteria - Cry1Ac and Cry2Ab.  Expression of Cry1Ac (but NOT Cry2Ab) declines over the course of the growing season, resulting in selection for Cry2Ab resistance later in the season.  Cry2Ab has been found in field populations of H. armigera, albeit at low frequencies.  Phoebe Heard and David Heckel are working to map Cry2Ab resistance in H. armigera using AFLPs using DNA from the progeny of crosses produced by Rod Mahon (CSIRO Entomology).  A resistant gene has been mapped to a specific linkage group. More detailed mapping of this gene will allow DNA diagnostic tools to be developed for the monitoring of resistance in field populations of H. armigera. This will be an important tool in maintaining susceptibility in the field.