Estimated frequency of nonrecessive Bt resistance genes in bollworm,Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in eastern North Carolina

In summer 2000, adult female bollworm moths, Helicoverpa zea (Boddie), were collected from light-traps at four locations near the Tidewater Research Station, Plymouth, NC. Female moths were allowed to lay eggs, and at hatch, 72 larvae from each female were screened for growth rate on normal artificial diet and on diets containing 5.0 microg of either Cry1Ac or Cry2Aa Bt toxin per milliliter of diet. The growth rate bioassays were performed to isolate nonrecessive Bt resistance genes present in field populations of bollworm. We found one individual out of 583 screened that appeared to carry a major gene for resistance to Cry1Ac. Assuming four alleles per individual, the gene frequency is 1/2332 or 0.0003. Other females appeared to have minor genes for Cry1Ac resistance or major genes with lower levels of dominance. We also found one individual out of 646 screened that appeared to carry a major gene for resistance to Cry2Aa. The gene frequency for Cry2Aa resistance was estimated at 1/2584 or 0.00039. Again, other females seemed to carry additional minor resistance genes. Along with other results that indicate partially dominant inheritance of Cry1Ac resistance in bollworm, these allele frequency estimates are important for determining the rate of resistance evolution in H. zea to specific Bt toxins.     

Cannibalism of Helicoverpa zea (Lepidoptera: Noctuidae) from Bacillus thuringiensis (Bt) transgenic corn versus non-Bt corn

Because of the importance of cannibalism in population regulation of Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in corn, Zea mays L., it is useful to understand the interactions between Bacillus thuringiensis (Bt) transgenic corn and cannibalism. To determine the effects of Bt corn on cannibalism in H. zea, pairs of the same or different instars were taken from Bt or non-Bt corn and placed on artificial diet in proximity. Cannibalism occurred in 91% of pairs and was approximately 7% greater for pairs of larvae reared from Bt transgenic corn (95%) than from non-Bt corn (88%). Also, first instar by first instar pairs had a lower rate of cannibalism than other pairs. Time until cannibalism was not different for larvae from Bt corn versus non-Bt corn. Pupation rate of cannibals and surviving victims was not different for pairs from Bt corn versus non-Bt corn. Finally, cannibalism increased pupation rate of cannibals from both Bt and non-Bt corn by approximately 23 and 12%, respectively, although the increases were not significant. Thus, negative effects of Bt on larvae were compensated by increased cannibalism in comparison with larvae reared on non-Bt corn, which increased larval survival to levels comparable with larvae reared on non-Bt plants.     

Interaction of Bacillus thuringiensis toxins with larval midgut binding sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the "stacked" gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of (125)I-labeled Cry1Ab protein ((125)I-Cry1Ab) and (125)I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either (125)I-Cry1Ab or (125)I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with (125)I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. (125)I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, (125)I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Cold hardiness of Helicoverpa zea (Lepidoptera: Noctuidae) pupae

An insect's cold hardiness affects its potential to overwinter and outbreak in different geographic regions. In this study, we characterized the response of Helicoverpa zea (Boddie) pupae to low temperatures by using controlled laboratory measurements of supercooling point (SCP), lower lethal temperature (LT(50)), and lower lethal time (LLTime). The impact of diapause, acclimation, and sex on the cold hardiness of the pupae also were evaluated. Sex did not significantly affect the SCP, LT(50), or LLTime. However, the mean SCP of diapausing pupae (-19.3°C) was significantly lower than nondiapausing pupae (-16.4°C). Acclimation of nondiapausing pupae to constant temperatures from 10 to 20°C before supercooling also produced a significantly lower SCP than nondiapausing pupae held at 25°C. The LT(50)s of nondiapausing and diapausing were not significantly different, but confirmed that H. zea pupae are chill-intolerant because these lethal temperatures are warmer than the corresponding mean SCPs. Diapausing pupae survived longer than nondiapausing pupae at the same, constant, cold temperatures, a finding consistent with the SCP results. Both of these results suggest enhanced cold hardiness in diapausing pupae. When laboratory results were compared with field temperatures and observed distributions of H. zea in the contiguous United States, the laboratory results corroborated what is currently perceived to be the northern overwintering limit of H. zea; approximately the 40(th) parallel. Moreover, our research showed that areas north of this limit are lethal to overwintering pupae not because of low temperature extremes, but rather the length of time spent at near-zero temperatures.     

Efficacy of insecticides of different chemistries against Helicoverpa zea (Lepidoptera: Noctuidae) in transgenic Bacillus thuringiensis and conventional cotton

Six insecticides of different chemistries were evaluated against the cotton bollworm, Helicoverpa zea (Boddie), in non-B.t. (Deltapine 'DP 5415', Deltapine 'DP 5415RR') and transgenic Bacillus thuringiensis (Berliner) (B.t.) (Deltapine 'NuCOTN 33B', Deltapine 'DP 458 B/RR') cotton. In 1998, treatments consisted of three rates each of a pyrethroid (lambda-cyhalothrin), spinosyn (spinosad), carbamate (thiodicarb), pyrrole (chlorfenapyr), oxadiazine (indoxacarb), and avermectin (emamectin benzoate) in a nonirrigated field. In 1999, treatments consisted of three rates each of lambda-cyhalothrin, spinosad, thiodicarb, and indoxacarb in an irrigated and a nonirrigated (dryland) field. The highest rate of each insecticide corresponded to normal grower-use rates. Spinosad and thiodicarb controlled H. zea in non-B.t. cotton, whereas other materials were less effective. Even though H. zea is becoming increasingly resistant to pyrethroid insecticides, lambda-cyhalothrin was highly effective in dryland B. thuringiensis cotton. Spinosad and thiodicarb were equally effective. Data indicated that reduced rates of lambda-cyhalothrin, spinosad, and thiodicarb could be used for control of H. zea in dryland B.t. cotton systems. However, reduced rates of these insecticides in a heavily irrigated B.t. cotton system did not provide adequate control.     

Cross-resistance to alpha-cypermethrin after xanthotoxin ingestion in Helicoverpa zea (Lepidoptera: Noctuidae)

Cytochrome P450 monooxygenases (P450) are membrane-bound hemoproteins that play important roles in conferring protection against both naturally occurring phytochemicals and synthetic organic insecticides. Despite the potential for common modes of detoxification, cross-resistance between phytochemicals and synthetic organic insecticides has rarely been documented. In this study, we examined the responses of a susceptible strain of corn earworm, Helicoverpa zea (Boddie), a polyphagous noctuid, to exposure by an allelochemical infrequently encountered in its host plants and by an insecticide widely used for control purposes. Within a single generation, survivors of xanthotoxin exposure displayed higher levels of tolerance to alpha-cypermethrin than did unexposed control larvae. The F1 offspring of xanthotoxin-exposed survivors also displayed higher alpha-cypermethrin tolerance than did offspring of unexposed control larvae, suggesting that increased alpha-cypermethrin tolerance after xanthotoxin exposure represents, at least in part, heritable resistance. Administration of piperonyl butoxide, a P450 synergist, demonstrated that resistance to both xanthotoxin and alpha-cypermethrin is P450-mediated. Alpha-cypermethrin-exposed survivors, however, failed to show superior growth on xanthotoxin diets. Assays with control larvae, larvae induced by both xanthotoxin and alpha-cypermethrin, and survivors of LD50 doses of both compounds indicated that H. zea midgut P450s are capable of metabolizing both xanthotoxin and alpha-cypermethrin. Metabolism of each compound is significantly inhibited by the presence of the other compound, suggesting that at least one form of P450 in H. zea midguts degrades both compounds and may constitute the biochemical basis for possible cross-resistance. Compared with control larvae, xanthotoxin- and alpha-cypermethrin-induced larvae displayed 2- to 4-fold higher P450-mediated metabolism of both compounds. However, xanthotoxin- and alpha-cypermethrin-exposed survivors exhibited much higher (2.5- to 11-fold) metabolism of both compounds than did the induced larvae. The metabolism results, like the bioassay results, are consistent with the interpretation that increased alpha-cypermethrin tolerance after xanthotoxin exposure is attributable mainly to heritable resistance.     

Toxicity of Bacillus thuringiensis Cry proteins to Helicoverpa armigera (Lepidoptera: Noctuidae) in South Africa

The susceptibility of one of the most important pests in southern Africa, Helicoverpa armigera (Lepidoptera: Noctuidae), to Bacillus thuringiensis Cry proteins was evaluated by bioassay. Cry proteins were produced in Escherichia coli BL21 cells that were transformed with plasmids containing one of six cry genes. The toxicity of each Cry protein to H. armigera larvae was determined by the diet contamination method for second instar larvae and the droplet feeding method for neonate larvae. For each of the proteins, dose-mortality and dose-growth inhibition responses were analyzed and the median lethal dose (LD(50)) and median inhibitory dose (ID(50)) determined. Second instar larvae were consistently less susceptible to the evaluated Cry proteins than neonate larvae. The relative toxicity of Cry proteins ranked differently between neonate larvae and second instar larvae. On the basis of the LD(50) and ID(50) values, Cry1Ab, Cry1Ac, and Cry2Aa were the most toxic of the evaluated proteins to H. armigera larvae. The study provides an initial benchmark of the toxicity of individual Cry proteins to H. armigera in South Africa.     

Field efficacy of sweet corn hybrids expressing a Bacillus thuringiensis toxin for management of Ostrinia nubilalis (Lepidoptera: Crambidae) and Helicoverpa zea (Lepidoptera: Noctuidae)

Field studies were done in 1995-1996 to assess the efficacy of three sweet corn hybrids that express the Bacillus thuringiensis (Bt) toxin, CrylAb, against two lepidopteran pests, Ostrinia nubilalis (Hubner) and Helicoverpa zea (Boddie). The Bt hybrids tested were developed by Novartis Seeds, using the event BT-11, which expresses Bt toxin in green tissue as well as reproductive tissues including the tassel, silk, and kernel. Bt hybrids were compared with a standard non-Bt control or the non-Bt isoline for each hybrid; none of the hybrids were treated with insecticides during the study. Hybrid efficacy was based on larval control of each pest, as well as plant or ear damage associated with each pest. In both years, control of O. nubilalis larvae in primary ears of all Bt hybrids was 99-100% compared with the appropriate non-Bt check. Plant damage was also significantly reduced in all Bt hybrids. In 1996, control of H. zea in Bt hybrids ranged from 85 to 88% when compared with the appropriate non-Bt control. In 1996, a University of Minnesota experimental non-Bt hybrid (MN2 x MN3) performed as well as the Bt hybrids for control of O. nubilalis. Also, in 1996, two additional University of Minnesota experimental non-Bt hybrids (A684su X MN94 and MN2 X MN3) performed as well as Bt hybrids for percent marketable ears (ears with no damage or larvae). In addition, compared with the non-Bt hybrids, percent marketable ears were significantly higher for all Bt hybrids and in most cases ranged from 98 to 100%. By comparison, percent marketable ears for the non-Bt hybrids averaged 45.5 and 37.4% in 1995 and 1996, respectively. Results from the 2-yr study strongly suggest that Bt sweet corn hybrids will provide high levels of larval control for growers in both fresh and processing markets. Specifically, Bt sweet corn hybrids, in the absence of conventional insecticide use, provided excellent control of O. nubilalis, and very good control of H. zea. However, depending on location of specific production regions, and the associated insect pests of sweet corn in each area, some insecticide applications may still be necessary.     

Resistance monitoring of Helicoverpa armigera (Lepidoptera Noctuidae) to Bacillus thuringiensis insecticidal protein in China

Sensitivities of Helicoverpa armigera (Hübner) field populations to Bacillus thuringiensis (Bt) insecticidal protein CrylAc were monitored during 1998-2000 in China. A total of 41 strains was sampled, and most of them were collected from Bt cotton planting regions. The range of IC50 values (concentration producing 50% inhibition of larval development to third instar) among different populations in 1998,1999, and 2000 were 0.020-0.105 microg/ml, 0.016-0.099 microg/ml, and 0.016-0.080 microg/ml, respectively. Diagnostic concentration studies (IC99) showed that the percentage of individuals reaching third instar ranged from 0 to 4.35%, with only eight of the 41 tested populations showing values above 0%. Also interesting was a trend over successive years in which fewer populations contained individuals that survived the diagnostic concentration by reaching third instar. Considering these data, it was determined that the field populations sampled during the 3-yr study were susceptible to CrylAc protein, and that movement toward resistance among H. armigera populations was not apparent.     

Acute, sublethal and combination effects of azadirachtin and Bacillus thuringiensis toxins on Helicoverpa armigera (Lepidoptera: Noctuidae) larvae

The efficacy of neem (1500 ppm azadirachtin (AI)), Delfin WG, a biological insecticide based on selected strain of Bacillus thuringiensis Berliner (Bt) subspecies kurstaki, and Cry1Ac protein, either individually or in combination, were examined against first to fourth instar Helicoverpa armigera (Hübner) larvae. Using an oral administration method, various growth inhibitory concentrations (EC) and lethal concentrations (LC) were determined for each bioagent. Combinations of sublethal concentrations of Bt spray formulation with azadirachtin at EC50 or EC95 levels not only enhanced the toxicity, but also reduced the duration of action when used in a mixture. The LC20 and LC50 values for Cry1Ac toxin were 0.06 and 0.22 microg ml-1, respectively. Bt-azadirachtin combinations of LC50+EC20 and LC50+EC50 result in 100% mortality. The mortality also was significant in LC20+EC20 and LC20+EC50 mixtures. These studies imply that the combined action is not synergistic but complimentary, with azadirachtin particularly facilitating the action of Bt. The Bt spray-azadirachtin combination is more economical than combinations that involve isolating the toxic protein, as the Bt spray formulations can be combined in a spray mixture with neem. These combinations may be useful for controlling bollworm populations that have acquired resistance to Bt as they may not survive the effect of mixture. Azadirachtin may be useful as a means of reducing the endotoxin concentrations in a mixture, to promote increased economic savings and further reduce the probability of resistance development to either insect control agent.     

Detection and evolution of resistance to the pyrethroid cypermethrin in Helicoverpa zea (Lepidoptera: Noctuidae) populations in Texas

The bollworm, Helicoverpa zea (Boddie), is a key pest of cotton in Texas. Bollworm populations are widely controlled with pyrethroid insecticides in cotton and exposed to pyrethroids in other major crops such as grain sorghum, corn, and soybeans. A statewide program that evaluated cypermethrin resistance in male bollworm populations using an adult vial test was conducted from 2003 to 2006 in the major cotton production regions of Texas. Estimated parameters from the most susceptible field population currently available (Burleson County, September 2005) were used to calculate resistance ratios and their statistical significance. Populations from several counties had statistically significant (P < or = 0.05) resistance ratios for the LC(50), indicating that bollworm-resistant populations are widespread in Texas. The highest resistance ratios for the LC(50) were observed for populations in Burleson County in 2000 and 2003, Nueces County in 2004, and Williamson and Uvalde Counties in 2005. These findings explain the observed pyrethroid control failures in various counties in Texas. Based on the assumption that resistance is caused by a single gene, the Hardy-Weinberg equilibrium formula was used for estimation of frequencies for the putative resistant allele (q) using 3 and 10 microg/vial as discriminatory dosages for susceptible and heterozygote resistant insects, respectively. The influence of migration on local levels of resistance was estimated by analysis of wind trajectories, which partially clarifies the rapid evolution of resistance to cypermethrin in bollworm populations. This approach could be used in evaluating resistance evolution in other migratory pests.     

Toxicity of several delta-endotoxins of Bacillus thuringiensis against Helicoverpa armigera (Lepidoptera: Noctuidae) from Spain

Toxicity and larval growth inhibition of 11 insecticidal proteins of Bacillus thuringiensis were evaluated against neonate larvae of Helicoverpa armigera, a major pest of important crops in Spain and other countries, by a whole-diet contamination method. The most active toxins were Cry1Ac4 and Cry2Aa1, with LC50 values of 3.5 and 6.3 microg/ml, respectively. At the concentrations tested, Cry1Ac4, Cry2Aa1, Cry9Ca, Cry1Fa1, Cry1Ab3, Cry2Ab2, Cry1Da, and Cry1Ja1, produced a significant growth inhibition, whereas Cry1Aa3, Cry1Ca2, and Cry1Ea had no effect.     

Field Evolved Resistance in Helicoverpa armigera (Lepidoptera: Noctuidae) to Bacillus thuringiensis Toxin Cry1Ac in Pakistan

Helicoverpa armigera (Hübner) is one of the most destructive pests of several field and vegetable crops, with indiscriminate use of insecticides contributing to multiple instances of resistance. In the present study we assessed whether H. armigera had developed resistance to Bt cotton and compared the results with several conventional insecticides. Furthermore, the genetics of resistance was also investigated to determine the inheritance to Cry1Ac resistance. To investigate the development of resistance to Bt cotton, and selected foliar insecticides, H. armigera populations were sampled in 2010 and 2011 in several cotton production regions in Pakistan. The resistance ratios (RR) for Cry1Ac, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin and deltamethrin were 580-fold, 320-, 1110-, 1950-, 200-, 380, 690, and 40-fold, respectively, compared with the laboratory susceptible (Lab-PK) population. Selection of the field collected population with Cry1Ac in 2010 for five generations increased RR to 5440-fold. The selection also increased RR for deltamethrin, chlorpyrifos, profenofos, cypermethrin, spinosad, indoxacarb, abamectin to 125-folds, 650-, 2840-, 9830-, 370-, 3090-, 1330-fold. The estimated LC_50s for reciprocal crosses were 105 µg/ml (Cry1Ac-SEL female × Lab-PK male) and 81 g µg/ml (Lab-PK female × Cry1Ac-SEL male) suggesting that the resistance to Cry1Ac was autosomal; the degree of dominance (D_LC) was 0.60 and 0.57 respectively. Mixing of enzyme inhibitors significantly decreased resistance to Cry1Ac suggesting that the resistance to Cry1Ac and other insecticides tested in the present study was primarily metabolic. Resistance to Cry1Ac was probably due to a single but unstable factor suggesting that crop rotation with non-Bt cotton or other crops could reduce the selection pressure for H. armigera and improve the sustainability of Bt cotton.     

Species diagnosis and Bacillus thuringiensis resistance monitoring of Heliothis virescens and Helicoverpa zea (Lepidoptera: Noctuidae) field strains from the southern United States using feeding disruption bioassays

Validation of a feeding disruption bioassay for the detection of resistance to Bacillus thuringiensis toxin and species identification is reported using field strains of Heliothis virescens and Helicoverpa zea collected from the southern United States in 1998. Feeding disruption is measured by a lack of fecal production from larvae exposed to a diagnostic concentration of CryIAc in a blue indicator diet. The bioassay provided rapid (24 h) diagnosis of the species composition of larvae tested and also monitored for the presence of resistance in H. virescens. An additional diagnostic concentration was established for monitoring resistance in H. zea. A probit model was used to compare the fecal production responses of insect strains over a range of CryIAc doses. Probability calculations, derived from our assay results, are also presented to aid in the interpretation of future results from field trials. Integration of the feeding disruption bioassay into integrated pest management programs is discussed.     

Interaction of Bacillus thuringiensis Toxins with Larval Midgut Binding Sites of Helicoverpa armigera (Lepidoptera: Noctuidae)

In 1996, Bt-cotton (cotton expressing a Bacillus thuringiensis toxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either new B. thuringiensis genes or with a combination of cry genes. However, one requirement for the “stacked” gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of ¹²⁵I-labeled Cry1Ab protein (¹²⁵I-Cry1Ab) and ¹²⁵I-Cry1Ac to brush border membrane vesicles (BBMV) of Helicoverpa armigera was analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either ¹²⁵I-Cry1Ab or ¹²⁵I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from four H. armigera populations were also tested with ¹²⁵I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined. ¹²⁵I-Cry1Ac binding was strongly inhibited by N-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast, ¹²⁵I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.     

Citrus pollen retention by adult Helicoverpa zea (Lepidoptera: Noctuidae) after exposure to citrus blooms

In previous migration studies, the presence of citrus pollen on adult Helicoverpa zea (Boddie), in conjunction with synoptic weather systems and 72-h backtrack trajectories, were used to identify source zones of migrants. However, data are lacking regarding the retention of citrus pollen for 72-h by H. zea adults. We exposed laboratory-reared and feral H. zea adults to citrus blooms for a 12-h period in laboratory and field studies and examined insects for the external presence of citrus pollen (i.e., marking) at 12-h intervals through 72 h. Citrus pollen marking was higher for females than males at the time of removal from citrus blooms. Fifteen to 100% of H. zea adults were marked with citrus pollen at 72 h after removal from citrus blooms. Pollen loads ranged from rare (< or = 10 pollen grains) to moderate (101 to 500 grains) during 1995; only rare and light (11 to 100 grains) pollen loads were detected during 1996. Citrus pollen marking of H. zea adults through 72 h after removal from citrus blooms has not been previously confirmed. These data provide evidence of H. zea using available blooming citrus groves in the absence of a host crop and will impact current perspectives regarding H. zea and host plant interactions and timing of pest management tactics.     

Comparative production of Helicoverpa zea (Lepidoptera: Noctuidae) from transgenic cotton expressing either one or two Bacillus thuringiensis proteins with and without insecticide oversprays

Transgenic cotton, Gossypium hirsutum (L.), expressing either one or two Bacillus thuringiensis ssp. kurstaki Berliner (Bt) proteins was compared with the conventional sister line in field experiments with regard to production of bollworm, Helicoverpa zea (Boddie), and bolls damaged by bollworm. The relative numbers of bollworms that developed on Bollgard (Monsanto Co., St. Louis, MO), Bollgard II (Monsanto Co.), and conventional cotton were estimated under nontreated conditions in 2000 and both insecticide-treated and nontreated conditions in 2001-2002 in North Carolina tests. Averaged across seven field studies under nontreated conditions, Bollgard cotton generated statistically similar numbers of large (L4-L5) bollworm larvae compared with the conventional variety; however, Bollgard cotton produced significantly fewer damaged bolls and bollworm adults than the conventional variety. Production of large larvae, damaged bolls, and adults was decreased dramatically by Bollgard II cotton as compared with Bollgard and conventional varieties. When comparing insecticide-treated and nontreated cotton genotypes, both Bt cotton sustained less boll damage than the conventional variety averaged across insecticide regimes; furthermore, Bollgard II cotton had fewer damaged bolls than the Bollgard variety. When averaged across cotton genotypes, pyrethroid oversprays reduced the numbers of damaged bolls compared with the nontreated cotton. Insecticide-treated Bollgard cotton, along with insecticide-treated and nontreated Bollgard II cotton reduced production of bollworm larvae, pupae, and adults. However, the addition of pyrethroid oversprays to Bollgard II cotton seemed to be the best resistance management strategy available for bollworm because no bollworms were capable of completing development under these conditions.     

Resistance to Bacillus thuringiensis toxin Cry2Ab in a strain of Helicoverpa armigera (Lepidoptera: Noctuidae) in Australia

Transgenic cotton, Gossypium hirsutum L., expressing the crylAc and cry2Ab genes from Bacillus thuringiensis (Bt) Berliner variety kurstaki in a pyramid (Bollgard II) was widely planted for the first time in Australia during the 2004-2005 growing season. Before the first commercial Bollgard II crops, limited amounts of cotton expressing only the crylAc gene (Ingard) was grown for seven seasons. No field failures due to resistance to CrylAc toxin were observed during that period and a monitoring program indicated that the frequency of genes conferring high level resistance to the CrylAc toxin were rare in the major pest of cotton, Helicoverpa armigera (Htibner) (Lepidoptera: Noctuidae). Before the deployment of Bollgard II, an allele conferring resistance to Cry2Ab toxin was detected in field-collected H. armigera. We established a colony (designated SP15) consisting of homozygous resistant individuals and examined their characteristics through comparison with individuals from a Bt-susceptible laboratory colony (GR). Through specific crosses and bioassays, we established that the resistance present in SP15 was due to a single autosomal gene. The resistance was recessive. Homozygotes were highly resistant to Cry2Ab toxin, so much so, that we were unable to induce significant mortality at the maximum concentration of toxin available. Homozygotes also were unaffected when fed leaves of a cotton variety expressing the cry2Ab gene. Although cross-resistant to Cry2Aa toxin, SP15 was susceptible to CrylAc and to the Bt product DiPel.     

Toxicity of Bacillus thuringiensis insecticidal proteins for Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae), major pests of cotton

The susceptibilities of the major pests of cotton in Australia, Helicoverpa armigera and Helicoverpa punctigera, to some insecticidal proteins from Bacillus thuringiensis were tested by bioassay. A commercial formulation, DiPel, and individual purified insecticidal proteins were tested. H. armigera was consistently more tolerant to B. thuringiensis insecticidal proteins than was H. punctigera, although both were susceptible to only a limited range of these proteins. Only Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab, and Vip3A killed H. armigera at dosages that could be considered acceptable. There was no significant difference in the toxicities of Cry1Fa and Cry1Ac for H. punctigera but Cry1Fa had little toxicity for H. armigera. The five instars of H. armigera did not differ significantly in their susceptibility to DiPel on the basis of LC(50). However, there were significant differences in the susceptibility to Cry1Ac and Cry2Aa of three strains of H. armigera. Bioassays conducted with Cry1Ac and Cry2Aa showed that there was a small but significant negative interaction between these delta-endotoxins.     

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

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.