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Crops engineered to produce insecticidal crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) have revolutionised pest control in agriculture. However field-level resistance to Bt has developed in some targets. Utilising novel vegetative insecticidal proteins (Vips), also derived from Bt but genetically distinct from Cry toxins, is a possible solution that biotechnical companies intend to employ. Using data collected over two seasons we determined that, before deployment of Vip-expressing plants in Australia, resistance alleles exist in key targets as polymorphisms at frequencies of 0.027 (n = 273 lines, 95% CI = 0.019-0.038) in H. armigera and 0.008 (n = 248 lines, 0.004-0.015) in H. punctigera. These frequencies are above mutation rates normally encountered. Homozygous resistant neonates survived doses of Vip3A higher than those estimated in field-grown plants. Fortunately the resistance is largely, if not completely, recessive and does not confer resistance to the Bt toxins Cry1Ac or Cry2Ab already deployed in cotton crops. These later characteristics are favourable for resistance management; however the robustness of Vip3A inclusive varieties will depend on resistance frequencies to the Cry toxins when it is released (anticipated 2016) and the efficacy of Vip3A throughout the season. It is appropriate to pre-emptively screen key targets of Bt crops elsewhere, especially those such as H. zea in the USA, which is not only closely related to H. armigera but also will be exposed to Vip in several varieties of cotton and corn.

The highly polyphagous Old World cotton bollworm Helicoverpa armigera is a quarantine agricultural pest for the American continents. Historically H. armigera is thought to have colonised the American continents around 1.5 to 2 million years ago, leading to the current H. zea populations on the American continents. The relatively recent species divergence history is evident in mating compatibility between H. zea and H. armigera under laboratory conditions. Despite periodic interceptions of H. armigera into North America, this pest species is not believed to have successfully established significant populations on either continent. In this study, we provide molecular evidence via mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and cytochrome b (Cyt b) partial gene sequences for the successful recent incursion of H. armigera into the New World, with individuals being detected at two sites (Primavera do Leste, Pedra Preta) within the State of Mato Grosso in Brazil. The mtDNA COI and Cyt b haplotypes detected in the Brazilian H. armigera individuals are common throughout the Old World, thus precluding identification of the founder populations. Combining the two partial mtDNA gene sequences showed that at least two matrilines are present in Brazil, while the inclusion of three nuclear DNA Exon-Primed Intron-Crossing (EPIC) markers identified a further two possible matrilines in our samples. The economic, biosecurity, resistance management, ecological and evolutionary implications of this incursion are discussed in relation to the current agricultural practices in the Americas.

Cooperative management of pest susceptibility to transgenic Bacillus thuringiensis (Bt) crops is pursued worldwide in a variety of forms and to varying degrees of success depending on context. We examine this context using a comparative socioecological analysis of resistance management in Australia, Brazil, India, and the United States. We find that a shared understanding of resistance risks among government regulators, growers, and other actors is critical for effective governance. Furthermore, monitoring of grower compliance with resistance management requirements, surveillance of resistance, and mechanisms to support rapid implementation of remedial actions are essential to achieve desirable outcomes. Mandated resistance management measures, strong coordination between actors, and direct linkages between the group that appraises resistance risks and growers also appear to enhance prospects for effective governance. Our analysis highlights factors that could improve current governance systems and inform other initiatives to conserve susceptibility as a contribution to the cause of public good.

The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.

Combinations of dissimilar insecticidal proteins (\"pyramids\") within transgenic plants are predicted to delay the evolution of pest resistance for significantly longer than crops expressing a single transgene. Field-evolved resistance to Bacillus thuringiensis (Bt) transgenic crops has been reported for first generation, single-toxin varieties and the Cry1 class of proteins. Our five year data set shows a significant exponential increase in the frequency of alleles conferring Cry2Ab resistance in Australian field populations of Helicoverpa punctigera since the adoption of a second generation, two-toxin Bt cotton expressing this insecticidal protein. Furthermore, the frequency of cry2Ab resistance alleles in populations from cropping areas is 8-fold higher than that found for populations from non-cropping regions. This report of field evolved resistance to a protein in a dual-toxin Bt-crop has precisely fulfilled the intended function of monitoring for resistance; namely, to provide an early warning of increases in frequencies that may lead to potential failures of the transgenic technology. Furthermore, it demonstrates that pyramids are not 'bullet proof' and that rapid evolution to Bt toxins in the Cry2 class is possible.

The global reliance on Bacillus thuringiensis (Bt) proteins for controlling lepidopteran pests in cotton, corn, and soybean crops underscores the critical need to understand resistance mechanisms. Vip3Aa, one of the most widely deployed and currently effective Bt proteins in genetically modified crops, plays a pivotal role in pest management. This study investigates the molecular basis of Vip3Aa resistance in Australian Helicoverpa armigera through genetic crosses, and integrated genomic and transcriptomic analyses. We identified a previously uncharacterized gene, LOC110373801 (designated HaVipR1 ), as potentially important in Vip3Aa resistance in two field-derived resistant lines. Functional validation using CRISPR/Cas9 knockout in susceptible lines confirmed the gene’s role in conferring high-level resistance to Vip3Aa. Despite extensive laboratory selection of Vip3Aa-resistant colonies in Lepidoptera, the biochemical mechanisms underlying resistance have remained elusive. Our research identifies HaVipR1 as a potential contributor to resistance, adding to our understanding of how insects may develop resistance to this important Bt protein. The identification of HaVipR1 contributes to our understanding of potential resistance mechanisms and may inform future resistance management strategies. Future work should explore the biochemical pathways influenced by HaVipR1 and assess its interactions with other resistance mechanisms. The approach utilized here underscores the value of field-derived resistant lines for understanding resistance in agricultural pests and highlights the need for targeted approaches to manage resistance sustainably.

I examined the long-term consequences of a trade-off between predation risk and resource acquisition for the garden skink (Lampropholis guichenoti) by rearing hatchlings to maturity in outdoor enclosures covered with snake predator scent (mimicking high predator densities) or control scent (mimicking low predator densities). Open areas provided optimal foraging and basking sites but were covered with scent, whereas retreat sites provided suboptimal foraging and basking opportunities and were not scented. During the initial six months of the experiment, lizards reared in enclosures covered with scent from a natural predator became active later in the day, showed reduced mobility, and selected \"safer\" substrate microhabitats than did lizards raised in enclosures covered with control scent. These behavioral shifts reduced opportunities to forage and bask for lizards in the predator-scented enclosures. During the study, however, lizards from predator-scented enclosures became gradually less responsive to snake chemical cues, and after one year there were no differences in the activity patterns and substrate microhabitat use of lizards in both treatments. This pattern of behavior is paralleled by variation in growth rates of lizards. Throughout the study, lizards exposed to predator scent were lighter and shorter than were lizards exposed to control scent. However, this result reflects differential rates of growth by lizards only during the first six months of the experiment. Nevertheless, lizards that grew slowly early in life, as a consequence of predator avoidance, attained smaller body sizes at maturity and produced lighter clutch masses and offspring. Thus, predator avoidance tactics employed early in a lizard's life can impose long-term fitness costs.

The cotton bollworm, Helicoverpa armigera (Hübner) is one of the most serious insect pest species to evolve resistance against many insecticides from different chemical classes. This species has evolved resistance to the pyrethroid insecticides across its native range and is becoming a truly global pest after establishing in South America and having been recently recorded in North America. A chimeric cytochrome P450 gene, CYP337B3, has been identified as a resistance mechanism for resistance to fenvalerate and cypermethrin. Here we show that this resistance mechanism is common around the world with at least eight different alleles. It is present in South America and has probably introgressed into its closely related native sibling species, Helicoverpa zea. The different alleles of CYP337B3 are likely to have arisen independently in different geographic locations from selection on existing diversity. The alleles found in Brazil are those most commonly found in Asia, suggesting a potential origin for the incursion of H. armigera into the Americas.

Since 2004–2005 cotton expressing Cry1Ac and Cry2Ab insecticidal proteins from the bacterium Bacillus thuringiensis has been commercially available in Australia to manage the target pests Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren). In both target species, the frequency of alleles conferring resistance to Cry2Ab is unexpectedly high in field populations. A significant challenge for managing these pests would occur if resistance to Cry2Ab toxins inadvertently selected for resistance to other insecticides used to control them. Dose-response bioassays were performed to measure the toxicity of currently registered insecticide sprays on isogenic strains of Cry2Ab-resistant and Cry2Ab-susceptible H. armigera and H. punctigera. Within-species comparisons of Cry2Ab-resistant and Cry2Ab-susceptible strains of H. armigera and H. punctigera indicate no cross-resistance with pyrethroid insecticides. Additionally, Cry2Ab-resistant strains were not cross-resistant to the following selective insecticides: indoxacarb, chlorantraniliprole, and avermectins. In both H. armigera and H. punctigera, Cry2Ab-resistant colonies exhibited a small, but significant, degree of enhanced susceptibility in response to chlorpyrifos and methomyl. We report higher tolerance to conventional insecticides in H. armigera compared with H. punctigera. Our results indicate that there is no significant interplay between Cry2Ab resistance frequencies in H. armigera and H. punctigera and frequencies of resistance to a range of insecticide sprays currently registered for cotton. Therefore, we conclude that any increases in frequencies of the common Cry2Ab resistance phenotypes identified in Australian populations of Helicoverpa spp. are unlikely to increase resistance risk for the indoxacarb, chlorantraniliprole, or avermectin classes of insecticide.

Transgenic crops that express insecticide genes from Bacillus thuringiensis (Bt) are used worldwide against moth and beetle pests. Because these engineered plants can kill over 95% of susceptible larvae, they can rapidly select for resistance. Here, we use a model for a pyramid two-toxin Bt crop to explore the consequences of spatio-temporal variation in the area of Bt crop and non-Bt refuge habitat. We show that variability over time in the proportion of suitable non-Bt breeding habitat, Q, or in the total area of Bt and suitable non-Bt habitat, K, can increase the overall rate of resistance evolution by causing short-term surges of intense selection. These surges can be exacerbated when temporal variation in Q and/or K cause high larval densities in refuges that increase density-dependent mortality; this will give resistant larvae in Bt fields a relative advantage over susceptible larvae that largely depend on refuges. We address the effects of spatio-temporal variation in a management setting for two bollworm pests of cotton, Helicoverpa armigera and H. punctigera, and field data on landscape crop distributions from Australia. Even a small proportion of Bt fields available to egg-laying females when refuges are sparse may result in high exposure to Bt for just a single generation per year and cause a surge in selection. Therefore, rapid resistance evolution can occur when Bt crops are rare rather than common in the landscape. These results highlight the need to understand spatio-temporal fluctuations in the landscape composition of Bt crops and non-Bt habitats in order to design effective resistance management strategies.