Frequency and diversity of indoxacarb resistance in Australian Helicoverpa armigera (Lepidoptera: Noctuidae).

Annual indoxacarb resistance in Helicoverpa armigera (Hübner) populations collected from various crops in Australia was monitored between 2013 and 2023. Resistance frequency determined by F2 screening using a predetermined discriminating dose of indoxacarb, was lowest in the 2013-2014 and 2015-2016 seasons at 0.0164 and 0.0246, respectively. Resistance then increased significantly to a ten-year high of 0.0869 in 2018-2019 but declined to 0.0557 in 2019-2020 during a severe drought, remaining relatively stable thereafter to 2023. Indoxacarb resistance was first detected in H. armigera collected from maize in the Gwydir valley, New South Wales, in 2013 (strain GY7-39). In 2017, a second indoxacarb resistant H. armigera strain (UN1U3-10) was isolated from a population collected in chickpeas in the Liverpool Plains, New South Wales. Indoxacarb resistance of this strain was characterized to evaluate its potential to compromise the ongoing effectiveness of insecticide resistance management strategies in Australian farming systems. Survival at the discriminating dose of indoxacarb in UN1U3-10 was 28.9, 52.6, 86.7, and 92.9% in the F2, F3, F4, and F5, respectively. Following introgression with a susceptible strain and reselection with the discriminating dose of indoxacarb, the resistance ratio of UN1U3-10 was approximately 800-fold. Resistance was autosomal, incompletely dominant and conferred by more than 1 locus. While indoxacarb resistance in UN1U3-10 did not confer to emamectin benzoate or spinetoram and there was no evidence of major cross-resistance to the Bt toxins Cry1A, Cry2A or Vip3A, there was 5-fold reduced sensitivity to chlorantraniliprole. Indoxacarb resistance was suppressed by approximately 10-fold by PBO with no synergism by TPP or DEM, suggesting the involvement of cytochrome P450 enzymes. A stability analysis indicated a fitness cost may be associated with the genes that confer resistance in the UN1U3-10 strain. The potential risk for diverse indoxacarb resistance in the Australian H. armigera population is discussed.

Insecticide resistance in Australian Spodoptera frugiperda (J.E. Smith) and development of testing procedures for resistance surveillance.

Spodoptera frugiperda (J.E. Smith) is a highly invasive noctuid pest first reported in northern Australia during early 2020. To document current status of resistance in S. frugiperda in Australia, insecticide toxicity was tested in field populations collected during the first year of establishment, between March 2020 and March 2021. Dose-response was measured by larval bioassay in 11 populations of S. frugiperda and a susceptible laboratory strain of Helicoverpa armigera. Emamectin benzoate was the most efficacious insecticide (LC50 0.023µg/ml) followed by chlorantraniliprole (LC50 0.055µg/ml), spinetoram (LC50 0.098µg/ml), spinosad (LC50 0.526µg/ml), and methoxyfenozide (1.413µg/ml). Indoxacarb was the least toxic selective insecticide on S. frugiperda (LC50 3.789µg/ml). Emamectin benzoate, chlorantraniliprole and methoxyfenozide were 2- to 7-fold less toxic on S. frugiperda compared with H. armigera while spinosyns were equally toxic on both species. Indoxacarb was 28-fold less toxic on S. frugiperda compared with H. armigera. There was decreased sensitivity to Group 1 insecticides and synthetic pyrethroids in S. frugiperda compared with H. armigera: toxicity was reduced up to 11-fold for methomyl, 56 to 199-fold for cyhalothrin, and 44 to 132-fold for alpha cypermethrin. Synergism bioassays with metabolic inhibitors suggest involvement of mixed function oxidase in pyrethroid resistance. Recommended diagnostic doses for emamectin benzoate, chlorantraniliprole, spinetoram, spinosad, methoxyfenozide and indoxacarb are 0.19, 1.0, 0.75, 6, 12 and 48µg/µl, respectively.

Relative fitness and stability of resistance in a near-isogenic strain of indoxacarb resistant Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUND: A strain of Helicoverpa armigera with 171-fold resistance to indoxacarb was introgressed with a susceptible strain by serial backcrossing and reselection with indoxacarb resulting in the creation of the near-isogenic GY7-39BC4 strain. Fitness was compared on artificial diet under diapause and non-diapause conditions in resistant, susceptible and F1 progeny from a reciprocal backcross of the two strains using life history trait analyses. Selection experiments were used to determine stability of resistance. RESULTS: There were no significant differences between strains in survival, female fertility or realized fecundity. A comparison of the intrinsic rate of population increase showed similar relative fitness between strains. Lower male fertility and male longevity in the resistant strain and one of the F1 strains compared with the susceptible strain suggests small non-recessive costs may be associated with male reproductive capacity in individuals with indoxacarb resistance alleles. However, there was no significant decline in resistance in the GY7-39 strain when reared in the absence of insecticide for five generations. Following an artificially induced diapause, survival was reduced by 52% and pupal weights were significantly lower in the resistant strain compared with the susceptible strain. CONCLUSIONS: These results suggest indoxacarb resistance does not confer a major fitness cost under standard laboratory conditions. However, a survival cost associated with overwintering highlights the imperative for adoption of management strategies in northern regions of Australia where a winter diapause does not occur. © 2020 Society of Chemical Industry.

Linkage mapping an indoxacarb resistance locus in Helicoverpa armigera (Lepidoptera: Noctuidae) by genotype-by-sequencing.

BACKGROUND: A major challenge to sustainable agricultural pest control is the rapid evolution of insecticide resistance. This is caused by mechanisms that reduce insecticide efficacy. Understanding the genetic mechanisms of resistance is essential for DNA-based monitoring of resistance in field populations. One such insecticide is indoxacarb, an important selective control option for Helicoverpa armigera in a range of crops including grain, horticulture and cotton. Recently, a strain of H. armigera (GY7-39) resistant to indoxacarb (198-fold) was isolated from field-collected moth. RESULTS: To identify the indoxacarb resistance locus, GY7-39 was backcrossed for six generations to susceptible strain New GR. In each generation, only resistant males were used to cross back to New GR. Genotype-by-sequencing was carried out on 95 H. armigera samples. In total, 13 203 tags with 8697 unique locations on the H. armigera genome were obtained. The indoxacarb resistance locus in strain GY7-39 was mapped to a 2.6 Mbp region on chromosome 16. In this region, two closely linked loci (IndoR1 and IndoR2) were found to be associated with indoxacarb resistant GY7-39. CONCLUSIONS: We mapped indoxacarb resistance in GY7-39 to two closely linked loci IndoR1 and IndoR2 in a narrowed 2.6 Mbp region of H. armigera chromosome 16. The results provide essential background data for future genetic investigations including fine mapping of the indoxacarb resistance gene and the eventual development of an effective DNA-based diagnostic to support resistance management. © 2019 Society of Chemical Industry.

Baseline Susceptibility of Helicoverpa punctigera (Lepidoptera: Noctuidae) to Indoxacarb, Emamectin Benzoate, and Chlorantraniliprole.

Susceptibility in Helicoverpa punctigera (Wallengren) to emamectin benzoate, chlorantraniliprole, and indoxacarb was established from feeding assays on insecticide-incorporated artificial diet in the laboratory. The variation in dose responses was examined in H. punctigera field populations collected in eastern Australia between September 2013 and January 2016 and compared with a laboratory strain. Chlorantraniliprole was the most toxic insecticide with an average LC50 of 3.7 µg of insecticide per liter of diet (n = 12 field strains). The average LC50 for emamectin benzoate was 5.6 µg of insecticide per liter of diet (n = 11 field strains), whereas indoxacarb had the lowest toxicity with an average LC50 of 172 µg of insecticide per liter of diet (n = 14 field strains). Variation in susceptibility between field strains was low at 1.9-, 2.4-, and 2-fold for chlorantraniliprole, emamectin benzoate, and indoxacarb, respectively. Narrow ranges of intra-specific tolerance, high slopes, and goodness-of-fit to a probit binomial model suggested feeding bioassays using insecticide-incorporated diet were a more effective laboratory method for measuring dose responses of these insecticides in H. punctigera than traditional topical bioassays. We propose discriminating concentrations of 0.032, 0.026, and 4 µg of insecticide/ml of diet for chlorantraniliprole, emamectin benzoate, and indoxacarb, respectively, to monitor insecticide resistance in H. punctigera. Although the potential for H. punctigera to develop insecticide resistance is considered low based on historical records, recent changes in population dynamics of this species in eastern Australia may have increased the risk of resistance development.

Multiple recombination events between two cytochrome P450 loci contribute to global pyrethroid resistance in Helicoverpa armigera.

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.

Genetics, cross-resistance and synergism of indoxacarb resistance in Helicoverpa armigera (Lepidoptera: Noctuidae).

BACKGROUND: The cotton bollworm Helicoverpa armigera is a global pest of field and horticultural crops and has developed resistance to insecticides from many chemical classes. Indoxacarb is an important option for selective control of H. armigera in a range of crops that play host to this species. A strain of H. armigera resistant to indoxacarb (designated GY7-39) was detected from the field by F2 screening and characterised by comparison with a near-isogenic indoxacarb-susceptible laboratory strain to determine inheritance, cross-resistance profile and synergism of indoxacarb resistance. RESULTS: The level of indoxacarb resistance in the GY7-39 strain was 139-198-fold compared with the susceptible strain. Genetic analysis showed that resistance was autosomal, incompletely dominant and conferred by one or a few closely linked loci. Indoxacarb resistance in the GY7-39 strain did not confer cross-resistance to chlorantraniliprole. The GY7-39 strain was more susceptible to emamectin benzoate, fenvalerate, Cry1Ac and Cry2Ab compared with the susceptible strain. Indoxacarb resistance was synergised by the metabolic inhibitor PBO. CONCLUSIONS: Rapid selection of indoxacarb resistance in the GY7-39 strain indicates the potential risk of resistance development to indoxacarb in field populations of H. armigera. Lack of cross-resistance indicates that resistance could be managed effectively by the use of rotational strategies that incorporate transgenic technologies. Synergism studies indicate the potential involvement of metabolic detoxification enzymes as the mechanism of resistance to indoxacarb. © 2016 Society of Chemical Industry.

Baseline Susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to Indoxacarb, Emamectin Benzoate, and Chlorantraniliprole in Australia.

Baseline susceptibility of Helicoverpa armigera (Hübner) to emamectin benzoate, chlorantraniliprole, and indoxacarb was determined in feeding assays on insecticide-incorporated artificial diet in the laboratory. The intraspecific variation of H. armigera was established from field populations collected between September 2012 and March 2013, primarily from commercial farms across eastern Australia. Emamectin benzoate had the highest toxicity with a median lethal concentration (LC50) of 0.01 µg/ml diet (n=20 strains). The LC50 for chlorantraniliprole was 0.03 µg/ml diet (n=21 strains), while indoxacarb had the lowest relative toxicity with an average LC50 of 0.3 µg/ml diet (n=22 strains). Variation in susceptibility amongst field strains was 2.3-fold for emamectin benzoate and 2.9-fold for chlorantraniliprole and indoxacarb. Discriminating concentrations of 0.2, 1, and 12 µg of insecticide per milliliter of diet for emamectin benzoate, chlorantraniliprole, and indoxacarb, respectively, were calculated from toxicological data from field H. armigera strains as a first step in resistance management of these classes of insecticide in Australia. The low intraspecific tolerance, high slope values, and goodness-of-fit to a probit binomial model obtained in this study suggest that a feeding assay using diet incorporated insecticide is an effective laboratory method for measuring the dose-responses of these classes of insecticides in H. armigera.

Toxicity and Cross-Resistance of Insecticides to Cry2Ab-Resistant and Cry2Ab-Susceptible Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae).

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.

Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins.

Intra-specific variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia to the Cry1Ac and Cry2Ab delta-endotoxins from Bacillus thuringiensis (Berliner) (Bt) was determined to establish a baseline for monitoring changes that might occur with the use of Bt cotton. Strains of H. armigera and H. punctigera were established from populations collected primarily from commercial farms throughout the Australian cotton belts. Strains were evaluated for susceptibility using two bioassay methods (surface treatment and diet incorporation) by measuring the dose response for mortality (LC50) and growth inhibition (IC50). The variation in LC50 among H. armigera (n=17 strains) and H. punctigera (n=12 strains) in response to Cry1Ac was 4.6- and 3.2-fold, respectively. The variation in LC50 among H. armigera (n=19 strains) and H. punctigera (n=12 strains) to Cry2Ab was 6.6- and 3.5-fold, respectively. The range of Cry1Ac induced growth inhibition from the 3rd to 4th instar in H. armigera (n=15 strains) was 3.6-fold and in H. punctigera (n=13 strains) was 2.6-fold, while the range of Cry2Ab induced growth inhibition from neonate to 3rd instar in H. armigera (n=13 strains) was 4.3-fold and in H. punctigera (n=12 strains) was 6.1-fold. Variation in susceptibility was also evaluated for two age classes (neonates and 3rd instars) in laboratory strains of H. armigera and H. punctigera. Neonates of H. punctigera had the same or higher sensitivity to Bt than 3rd instars. Neonates of H. armigera were more sensitive to Cry2Ab than 3rd instars, while being less sensitive to Cry1Ac than 3rd instars. Differences in the two methods of bioassay used affected relative sensitivity of species to Bt toxins, highlighting the need to standardize bioassay protocols.

Fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac.

The performance of Helicoverpa armigera (Hübner) on 15-wk-old cotton plants was compared for a susceptible strain, a near-isogenic laboratory-selected strain, and F1 progeny of the two strains. Glasshouse experiments were conducted to test the three insect types on conventional plants and transgenic plants that produced the Bacillus thuringiensis (Bt) toxin Cry1Ac. At the time of testing (15 wk), the Cry1Ac concentration in cotton leaves was 75% lower than at 4 wk. On these plants, < 10% of susceptible larvae reached the fifth instar, and none survived to pupation. In contrast, survival to adulthood on Cry1Ac cotton was 62% for resistant larvae and 39% for F1 larvae. These results show that inheritance of resistance to 15-wk-old Cry1Ac cotton is partially dominant, in contrast to results previously obtained on 4-wk-old Cry1Ac cotton. Growth and survival of resistant insects were similar on Cry1Ac cotton and on non-Bt cotton, but F1 insects developed more slowly on Cry1Ac cotton than on non-Bt cotton. Survival was lower and development was slower for resistant larvae than for susceptible and F1 larvae on non-Bt cotton. These results show recessive fitness costs are associated with resistance to Cry1Ac.

Relative fitness of Cry1A-resistant and -susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton.

Glasshouse and laboratory experiments were conducted to evaluate the relative fitness of Cry1A-susceptible and laboratory-selected resistant strains of Helicoverpa armigera (Hübner). Life history parameters of H. armigera larvae feeding on young cotton plants showed a significant developmental delay of up to 7 d for the resistant strain compared with the susceptible strain on non-Bacillus thuringiensis (Bt) cotton. This fitness cost was not evident on artificial diet. There was no developmental delay in the F1 hybrid progeny from the reciprocal backcross of the resistant and susceptible strains, indicating that the fitness cost is recessive. In two cohorts tested, survival to pupation of resistant larvae on Bt cotton expressing Cry1Ac was 54 and 51% lower than on non-Bt cotton, whereas all susceptible and F1 larvae tested on Cry1Ac cotton were killed. Mortality of susceptible larvae occurred in the first or second instar, whereas the F1 larvae were able to develop to later instars before dying, demonstrating that resistance is incompletely recessive. The intrinsic rate of increase was reduced by >50% in the resistant strain on Cry1Ac cotton compared with the susceptible strain on non-Bt cotton. There was a significant reduction in the survival of postdiapausal adults from the resistant strain and the F1 strains, indicating that there is a nonrecessive overwintering cost associated with Cry1A resistance in H. armigera.

Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

Three laboratory strains of Helicoverpa armigera (Hübner) were established by mating of field-collected insects with an existing insecticide-susceptible laboratory strain. These strains were cultured on artificial diet containing the Cry1Ac protoxin of Bacillus thuringiensis using three different protocols. When no response to selection was detected after 7-11 generations of selection, the three strains were combined by controlled mating to preserve genetic diversity. The composite strain (BX) was selected on the basis of growth rate on artificial diet containing Cry1Ac crystals. Resistance to Cry1Ac was first detected after 16 generations of continuous selection. The resistance ratio (RR) peaked approximately 300-fold at generation 21, after which it declined to oscillate between 57- and 111-fold. First-instar H. armigera from generation 25 (RR = 63) were able to complete their larval development on transgenic cotton expressing Cry1Ac and produce fertile adults. There appeared to be a fitness cost associated with resistance on cotton and on artificial diet. The BX strain was not resistant to the commercial Bt spray formulations DiPel and XenTari, which contain multiple insecticidal crystal proteins, but was resistant to the MVP formulation, which only contains Cry1Ac. The strain was also resistant to Cry1Ab but not to Cry2Aa or Cry2Ab. Toxin binding assays showed that the resistant insects lacked the high affinity binding site that was detected in early generations of the strain. Genetic analysis confirmed that resistance in the BX strain of H. armigera is incompletely recessive.