Joanne C. Daly

Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.

Genetic variation in Heliothis in Australia: species identification and gene flow in the two pest species H. armigera (Hübner) and H. punctigera Wallengren (Lepidoptera: Noctuidae).

Genetic (electrophoretic) variation was examined in the two pest species of Heliothis found in Australia, H. armigera (Hubner) and H. punctigera Wallengren. They could be differentiated by seven loci which enabled identification of individual eggs and small larvae not normally distinguishable by their morphology. The traditional criteria for distinguishing the larger larvae of the two species were shown to be unreliable. The genetic distance between the two species was 0·34 ± 0·02. The percentage of loci polymorphic in both species, 32%, and the mean heterozygosities, 11·3% for H. armigera and 10·8% for H. punctigera , are lower than those reported in the American species, H. virescens (F.) and H. zea (Boddie). Populations throughout Australia were differentiated from each other, but there did not appear to be a marked geographic pattern to the variation. Genetic distances between populations of H. armigera were low ( Heliothis is large and that significant gene flow probably occurs between widely separated regions.

Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae).

Abstract In Australia, transgenic cotton plants expressing the cry1Ac gene from Bacillus thuringiensis Berliner variety kurstaki are less toxic to first-instar Helicoverpa armigera (Hübner) after the plant is producing fruit. We developed two bioassay methods (leaf mush, leaf disk) to test if the physiological state of the plants explained changes in toxicity and a third method (diet incorporation) was developed to quantify the toxicity of Bt leaves when mixed in chickpea diet. Cry1Ac protein was less toxic to H. armigera larvae when the protein was mixed with leaves from fruiting versus presquare conventional cotton. Differences in LC50 varied from 2.4- to 726-fold, depending on the source of toxin and conventional plant material. These results suggest that plant-toxin interactions in fruiting cotton are reducing the toxicity of the Cry1Ac protein. The possible role of tannins in these changes is discussed.

Inheritance of metabolic resistance to the synthetic pyrethroids in Australian Helicoverpa armigera (Lepidoptera: Noctuidae)

Two strains of Helicoverpa armigera (Hubner), resistant to synthetic pyrethroids, were isolated from field populations collected in the irrigated cotton growing areas of northern New South Wales: an isofemale strain, Mfo, and a composite field strain, Field. A series of crosses were undertaken between the Mfo and susceptible strains to determine the genetic basis of the resistance. Dose-responses were obtained by bioassay using topical application of fenvalerate in acetone. Considerable variation in tolerance was observed in both resistant and susceptible strains. In the Mfo strain, the major shift in resistance phenotype (33-fold resistance) was due to a single major gene with incomplete dominance. The resistance phenotype was not diluted by 23 generations of backcrossing. The major resistance phenotype could be eliminated almost completely by the addition of piperonyl butoxide which affects oxidative breakdown of the pyrethroids by mixed function oxidase (mfo) enzymes. Similar results were observed in the Field strain. Monitoring for resistance in field populations will be imprecise because a true discriminating dose does not exist for susceptible and heterozygous resistant individuals.

Ecology and resistance management for bacillus thuringiensis transgenic plants

Transgenic plants expressing the endotoxin proteins of Bacillus thuringiensis (Bt) will be released for commercial production during this decade. While these plants have a great potential to reduce our dependence on pesticides for insect control, their success will be short‐lived if insects rapidly develop resistance to the Bt toxins or if chemical use is needed to control other pests in the same production systems. Ecological studies can play a vital role in the design of strategies that avoid these problems by, for example, identifying key factors in insect life histories that influence the rate at which resistance evolves, validating management models that often make simple assumptions about the population biology of insects, identifying critical insect‐plant interactions and determining whether pest control by transgenic plants is affected when the plants are stressed. These points can be illustrated from studies of non‐transgenic ecosystems in the areas of host plant resistance, insecticide resistanc...

Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae).

Abstract Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein “lipophorin” and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.

Sex-linked inheritance of endosulphan resistance in Helicoverpa armigera

Endosulphan resistance is common in Australian populations of Helicoverpa armigera (Hübner), but resistance frequencies have not increased above 10–40% of the population, despite widespread use of this insecticide. We examined the inheritance of endosulphan resistance as part of a study of its evolution. Two field-resistant strains were selected in the laboratory. Results from a series of F1s and backcrosses in both larvae and adults indicated that resistance was sex-linked, with almost complete dosage compensation in the hemizygous females. In males, resistance was partially dominant in fourth instar larvae but semi-recessive in adults. Estimates of resistance factors depended on the life stage and instar tested and on the bioassay method used. In larvae, resistance factors were highest in neonates (14-fold) and then declined to twofold in fifth instar larvae. Our results indicate that the genetic basis of resistance should enhance the rate of evolution in field populations compared with an autosomal locus.