This project resolved two long-standing anomalies regarding Cry2Ab resistance in H. armigera. The first was the observation that frequently a greater proportion of survivors (assumed to be homozygous resistant insects) were observed in F2 tests than the expected 1/16. The second was that F1 tests yield a greater frequency of resistance than F2 tests. The latter anomaly has created concern as some observers question which test, or indeed do either test, provide reliable data. We have found that both anomalies stem from a common cause, namely that the offspring from single pairs of field-collected insects mate infrequently in the laboratory. As a result some F2 tests yield a distorted ratio of resistant and susceptible insects. Also when few females mate, the informative mating type (heterozygote x heterozygote) may not occur and a false negative F2 test is generated. This problem is not shared by F1 tests as only one round of mating is required (field insect x laboratory homozygous resistant) and if that mating does not occur, viable offspring are not generated and no result can be scored. Thus F1 tests provide a reliable measure of the frequency of resistance in the population.

While not part of the objectives of the project, studies on Vip3A resistance were also initiated. This proved fortuitous as Monsanto has announced an intention to add the vip3a gene to their Bollgard II variety to produce Bollgard III. Our work has permitted the determination of preliminary estimates of the frequency of Vip3A resistance in both species. For H. armigera 2.5% of all alleles are of the resistant form and thus resistance is exceptionally high. Preliminary characterisation of Vip3A resistance isolates of both H. armigera and H. punctigera is underway. Further work to detect and then test through complementation tests additional isolates is required. Other forms of resistance may be present that have quite different characteristics to the ones already examined and therefore pose a different level of threat. Further work is also required to determine if fitness costs are associated with Vip3A resistance before an appropriate RMP can be developed for Bollgard III that causes minimal constraints on growers while protecting the longevity of Bt technology.When contemplating a RMP for Bollgard III, one key point merits emphasis. The resistance status of Cry2Ab toxin is presently of concern and if the susceptibility of H. armigera populations is lost through the evolution of resistance before Bollgard III is widely deployed, much of the longevity of the new variety will be lost. The existing frequency of Vip3A resistance will ensure that susceptibility to the new toxin will be quickly lost. Then, despite Bollgard III being a three toxin product, it will be rendered the equivalent of a single toxin (Cry1Ac) product, perhaps indistinguishable in efficacy from the early transgenic variety Ingard. Then as functionally a single gene product, a reversion to a more restrictive RMP would be necessary to provide even limited longevity of the susceptibility of H. armigera to Cry1Ac. Recently, the Bt monitoring program has identified for the first time several instances of Cry1Ac resistance in H. punctigera, so that species would also need to be watched. The possibility that alternative technology companies with a different array of toxins may enter the Australian market is unlikely to offer a practical solution, as all varieties of transgenic cotton already commercialised, or nearing commercialisation, express one or more of the Cry1, Cry2 or Vip classes of toxins derived from Bacillus thuringiensis. Cross resistance occurs between different Cry1’s for other Lepidoptera (Ferré and Van Rie 2002) and is evident among different Cry2’s in H. armigera (Mahon et al. 2007a, Caccia et al. 2010). As our isolations of Vip resistance are the first for any species, cross resistance among Vip toxins is yet to be tested, but are also likely. Thus from theauthors perspective, in the longer term, if the Cry2Ab resistance situation deteriorates further, the industry should go to whatever lengths necessary to protect the susceptibility of Cry2Ab until Bollgard III becomes available. This might cause pain in the short term, but models of the evolution of resistance incorporating 10 years of data gathered on resistance to the three Bt toxins suggest that the payoff to growers through greatly increased longevity of the Bt technology would eclipse the temporary pain.