Resistance Of Helicoverpa Armigera Research Articles

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.

Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.

Up-Regulated Serpin Gene Involved in Cry1ac Resistance in Helicoverpa Armigera

Up-Regulated Serpin Gene Involved in Cry1ac Resistance in Helicoverpa Armigera

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.

Identification of the 2-tridecanone cis-acting element in the promoter of cytochrome P450 CYP6B7 in Helicoverpa armigera.

The expression level of cytochrome P450 genes in insects can be induced by plant allelochemicals, which is important for insects to adapt to host plants. Cytochrome P450 CYP6B7 has been reported to be involved in pyrethroid insecticide resistance in Helicoverpa armigera, and its transcription level was induced by some inducers. Currently, the regulatory mechanism of the induced expression of CYP6B7 remains unknown, although it is very important for understanding the detoxification mechanism to allelochemicals in host plants. The objective of the present study was to investigate the cis-acting element in the promoter of CYP6B7 mediating the inducible up-regulation of CYP6B7 in H. armigera by 2-tridecanone. The promoter region of CYP6B7 was cloned by genome walking technique and analyzed by transient transfection assay. Progressive 5' deletion of the promoter region of CYP6B7 revealed that the relative luciferase activity of construct -320/+232 could be significantly induced by 2-tridecanone. Further stepwise deletion between -320 and -238 bp found that construct -292/+232 could also be significantly induced by 2-tridecanone, but the adjacent construct -256/+232 could not, suggesting the essential role of the sequence between -292 and -257 bp for 2-tridecanone induction. Nucleotide mutations between -292 and -281 bp had no influence on the induction effect by 2-tridecanone, but nucleotide mutations between -280 and -257 bp significantly decreased the induction effect. These results demonstrated that the cis-acting element for 2-tridecanone induction was between -280 and -257 bp in the promoter of CYP6B7.

Heterologous expression of Helicoverpa armigera cytochrome P450 CYP6B7 in Pichia pastoris and interactions of CYP6B7 with insecticides.

Previous studies indicated that constitutive over-expression of cytochrome P450 CYP6B7 was involved in fenvalerate resistance in Helicoverpa armigera. In this study, the CYP6B7 gene from H. armigera (namely HaCYP6B7), was heterologously expressed in Pichia pastoris GS115. A vector pPICZA-HaCYP6B7 was constructed and transformed into P. pastoris GS115, the transformant of pPICZA-HaCYP6B7-GS115 was then cultured and induced by 1% (v/v) methanol and the heterologous expression of HaCYP6B7 protein in P. pastoris was confirmed by SDS-PAGE and western blot. Microsomes containing the expressed HaCYP6B7 showed activities against model substrate p-nitroanisole and 7-ethoxycoumarin, with p-nitroanisole O-demethylation (PNOD) and 7-ethoxycoumarin O-deethylation (ECOD) activities of 15.66- and 4.75-fold of the control, respectively. Moreover, it showed degradation activities against the insecticides bifenthrin, fenvalerate and chlorpyrifos, with clearance activities of 6.88-, 1.49- and 2.27-fold of the control, respectively. The interactions of HaCYP6B7 with insecticides were further confirmed by molecular docking in silico with binding scores of 5.450, 5.295 and 2.197 between putative HaCYP6B7 protein and bifenthrin, fenvalerate and chlorpyrifos, respectively. The results of present study provided more direct and important evidence on the role of HaCYP6B7 conferring pyrethroid resistance in H. armigera. © 2017 Society of Chemical Industry.

Planting window requirements for Bt cotton in Australia: do they limit the exposure ofHelicoverpaspp. (Lepidoptera: Noctuidae) to Bt toxins?

The threat of Bt resistance in Helicoverpa armigera (Hubner 1808) and H. punctigera (Wallengren 1860) (Heliothinae) is a major impediment to the sustainability of transgenic (Bt) cotton crops in Australia. A resistance management plan has, therefore, been instigated that in part requires farmers to sow their Bt cotton within specified dates to limit the number of generations of Helicoverpa spp. that are exposed to Bt toxins during the growing season. This study evaluated this strategy by utilising industry-wide databases of planting dates and other cropping/pest management events throughout 11 growing seasons. The analysis included model predictions of when Helicoverpa is likely to enter winter (pupal) diapause and emerge as moths in spring, and the long-term pheromone trapping records of moths. In New South Wales and southern Queensland (Qld), most first-generation moths were caught at the same time, or before, cotton was planted (mostly in October). This generation was, therefore, unlikely to have been exposed significantly to Bt toxins as feeding larvae in spring, but some individuals (especially H. armigera) may have been exposed to Bt prior to pupating in the previous autumn. Three subsequent generations, which could have been exposed to Bt during summer–autumn, were recognisable in trap catches before moths became very rare in early April. By then, the majority of Helicoverpa had entered winter diapause as pupae and cotton harvest was well underway. We conclude that the current planting window (pre-mid-November in these regions) achieves little in limiting the exposure of Helicoverpa to Bt. However, insufficient is known of the population dynamics of Helicoverpa in central Qld to be as definitive about the need for planting windows there. Importantly, Helicoverpa are active all year round in central Qld and cotton can be grown over a longer period, thus increasing the risks of greater exposure to Bt.

Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera

The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids.

Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera.

Toxins from the bacterium Bacillus thuringiensis (Bt) are used widely for insect control in sprays and transgenic plants, but their efficacy is reduced when pests evolve resistance. Previous work showed that mutations in a gene encoding the transporter protein ABCC2 are linked with resistance to Bt toxins Cry1Ab, Cry1Ac or both in four species of Lepidoptera. Here we compared the ABCC2 gene of Helicoverpa armigera (HaABCC2) between susceptible strains and a laboratory-selected strain with >1,000-fold resistance to Cry1Ac relative its susceptible parent strain. We discovered a 73-base pair (bp) insertion in the cDNA of the resistant strain that generates a premature stop codon expected to yield a truncated ABCC2 protein. Sequencing of genomic DNA revealed that this insertion is an intron that is not spliced out because of a 6-bp deletion at its splicing site. Analysis of progeny from crosses revealed tight genetic linkage between HaABCC2 and resistance to Cry1Ac. These results provide the first evidence that mis-splicing of a gene encoding an ABCC2 protein confers resistance to a Bt toxin.

Up-regulated death-associated LIM-only protein contributes to fitness costs of Bacillus thuringiensis Cry1Ac resistance in Helicoverpa armigera

The evolution of Bacillus thuringiensis (Bt) resistance provides a useful pathway for the study of fitness trade-offs associated with stress adaptation. In a previous study, we used cDNA-amplification fragment length polymorphism (cDNA-ALFP) analysis to identify differentially expressed genes (DEGs) in Cry1Ac-susceptible (96S) and -resistant (Bt-R) Helicoverpa armigera larvae. Among these DEGs, a death-associated LIM-only protein (HaDALP) was identified. In the current study, the full-length cDNA encoding HaDALP was cloned using rapid amplification of cDNA ends according to an expressed sequence tag derived from the previous cDNA-ALFP analysis. HaDALP expression patterns indicated that this gene was differentially expressed in tissues and stages and was highest in the midgut and epidermis of the 5th instar larvae. It is up-regulated in Cry1Ac-resistant H. armigera larvae. Fitness parameters, such as larval and pupal weight, pupal duration, and survival rate, which are the most sensitive indicators, were evaluated after HaDALP knockdown or feeding of the HaDALP protein in vivo. Our findings suggested that up-regulation of HaDALP might be related to Cry1Ac resistance and an adaption to Bt toxins.

Heterologous Expression and Biochemical Characterisation of Fourteen Esterases from Helicoverpa armigera

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.

Proteomic and molecular analyses of esterases associated with monocrotophos resistance in Helicoverpa armigera

We have obtained a Helicoverpa armigera strain (MonoR) showing high level resistance to monocrotophos by 15 generations of selection on a weakly resistant field-caught strain. Bioassays using various synergists show a major role for esterases in the resistance, and little if any role for cytochrome P450s or glutathione S-transferases. Biochemical data also show elevated levels of esterase activity in the resistant line. There were also minor differences among our strains in the kinetics of acetylcholinesterase activity and in its susceptibility to monocrotophos inhibition, and sequence comparisons revealed one amino acid substitution in AChE1 but none in AChE2 in the MonoR strain. However the AChE1 substitution does not match any others linked to resistance in this or other species and would be located well away from the enzyme active site, so it may not be causally involved with resistance. Native PAGE shows several individual esterase isozymes are more intensely staining in the resistant line and native Western analysis with an antibody against Clade 1 esterases show that several of these belong to Clade 1 and that their greater staining intensity in MonoR is due to greater amounts of the respective enzymes. Proteomic analysis of gel slices also matches the overexpressed bands with up to six Clade 1 enzymes, with some involvement also from esterases in three other Clades. Two of the isozymes that are overexpressed in MonoR are also overexpressed in a fenvalerate resistant line and proteomic analysis of that line bears out previous proteomics of another fenvalerate resistant line which also implicates Clade 1 isozymes.

Reduced fitness associated with spinosad resistance in Helicoverpa armigera

The fitness cost of spinosad resistance was investigated in the cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Laboratory experiments were conducted to compare relative fitness of H. armigera between the spinosad-susceptible and -resistant strains. During the experiments, the average development periods of the resistant strain were lengthened by 4–5 days, reflected in a prolongation of egg, larval and pupal periods. Furthermore, pupal survival, pupal weight, the mean life span of emerged adults, eggs laid and hatched decreased greatly in the resistant strain in comparison with the susceptible strain. Other life-cycle parameters such as larval survival, larval wet weights, prepupal periods, pupation ratio, and sex ratio did not change significantly. As a result, both net replacement rate (R 0) and intrinsic rate of increase (r m) were reduced for the resistant strain. Our results clearly indicated that relative fitness of resistant individuals was reduced in the absence of spinosad. Rational measures including pesticide rotations should be expected to delay development of resistance to spinosad in H. armigera field populations from China.

The effects of diet on the detection of resistance to Cry1Ac toxin in Australian Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

The effects of raw or heat-denatured soybean flour in an artificial diet on the detection of Cry1Ac resistance in Helicoverpa armigera were examined. Resistant neonate larvae reared on denatured soybean flour diet showed resistance factors of 7980 and 16,901 at the LC 50 and LC 99.9 levels, respectively. By comparison, resistance could not be detected in neonate larvae reared on raw flour diet. Third instar larvae reared on denatured flour diet showed resistance factors of 322 and 21,190 at the LC 50 and LC 99.9 levels. Resistance was not detected in third instar larvae reared on raw flour diet. There was 68% survival of resistant neonate larvae on Bollgard II cotton leaf feeding assays, compared to 100% mortality in a susceptible strain. We conclude that detection of CRY1Ac resistance in H. armigera from Australia can be masked, if an artificial diet gives chronic exposure to potent, protease inhibitors present in raw soy flour.

Variation in Resistance to Pyrethroids in <I>Helicoverpa armigera</I> From Benin Republic, West Africa

Pyrethroid resistance in Helicoverpa armigera (Hübner) field populations was investigated in Benin over several years by using third- and fourth-instar larval topicalbioassays. H. armigera was resistant to pyrethroids tested as cypermethrin, deltamethrin, bifenthrin, and fenvalerate. Pretreatment with piperonyl butoxide significantly decreased the LD50 value of cypermethrin and deltamethrin, and the resistance suppression by this synergist effect was observed. No significant decrease in the LD50 value was obtained when S,S,S-tributyl phosphoro-trithioate was applied before deltamethrin. In the field, cypermethrin's LD50 value varied, and the highest LD50 values were observed during the rainy season, the cotton, Gossypium hirsutum L., crop period, when pyrethroids are extensively used. In the dry season when there was no cotton cultivation, the lowest LD50 values were obtained. However, reversion was never total in the field; resistance did not revert to the level we observed in the susceptible strain. In the laboratory, when field populations were reared in insecticide-free conditions, resistance decreased and total reversion was observed. Results are discussed with regard to insecticide resistance fitness cost and resistance management strategies.

Investigation of the role of glutathione S-transferase isozymes in pyrethroid resistance of Helicoverpa armigera in Turkey

Investigation of the role of glutathione S-transferase isozymes in pyrethroid resistance of Helicoverpa armigera in Turkey

Functional expression of Helicoverpa armigera CYP9A12 and CYP9A14 in Saccharomyces cerevisiae

Elevated oxidative detoxification is a major mechanism responsible for pyrethroid resistance in Helicoverpa armigera from Asia. Constitutive overexpression of CYP9A12 and CYP9A14 was associated with pyrethroid resistance in the YGF strain of H. armigera. CYP9A12 and CYP9A14 were functionally expressed in the W(R) strain of yeast ( Saccharomyces cerevisiae) transformed with a plasmid shuttle vector pYES2. The cell lysates prepared from yeast transformed with CYP9A12 and CYP9A14, respectively, exhibited considerable O-demethylation activities against two model substrates p-nitroanisole (0.59 and 0.42 nmol p-nitrophenol min −1 mg protein −1) and methoxyresorufin (2.98 and 5.41 pmol resorufin min −1 mg protein −1), and clearance activity against the pyrethroid esfenvalerate (8.18 and 4.29 pmol esfenvalerate min −1 mg protein −1). These results provide important evidence on the role of CYP9A12 and CYP9A14 in conferring pyrethroid resistance in H. armigera, and also demonstrate that the yeast expression system can provide necessary redox environment for insect P450s to metabolize xenobiotics.

Tracking pyrethroid resistance in the polyphagous bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae), in the shifting landscape of a cotton-growing area

In cotton-growing areas of Central Africa, timing of host crops and pest management practices in annual rainfed cropping systems result in a shifting mosaic of habitats that influence the dynamics and resistance of Helicoverpa armigera (Hübner) populations on spatial scales, both within and across seasons. From 2002 to 2006, regional and local resistance was monitored among cotton fields and among the major host plants of the bollworm. From 2002, pyrethroid resistance increased within and across cotton-growing seasons to reach a worrying situation at the end of the 2005 growing season. Cotton crops played a fundamental role in the increase in seasonal resistance, even if the intensive use of insecticides on local tomato crops strongly concentrated resistance alleles in residual populations throughout the off-season. Due to the relative stability of resistance in H. armigera populations despite a long off-season, we believe that after the dispersal of the moths southwards at the end of the growing season, reverse migration mainly accounts for the reconstitution of populations at the onset of the following growing season. In addition, local resistance monitoring in 2005 and 2006 showed that it was possible to control the increase in resistance by temporarily stopping the use of pyrethroids during the period of peak infestation of cotton by H. armigera. On the other hand, the similar resistance frequency of populations sampled from sprayed and unsprayed synchronous hosts confirmed the absence of reproductive isolation between adults. As a result, diversity in cropping systems should be encouraged by planting alternative host plants to provide a mosaic of habitats, which in return would provide insecticide-free refuges. The implications for insecticide resistance management in annual cropping systems are discussed.

Changes of inheritance mode and fitness in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) along with its resistance evolution to Cry1Ac toxin

The changes of inheritance mode and fitness of resistance in Helicoverpa armigera (Hübner) along with its resistance evolution to Cry1Ac toxin were evaluated in the laboratory. The resistance levels reached 170.0-, 209.6- and 2893.3-fold, on selection of the field population in the 16th (BtR-F 16), 34th (BtR-F 34) and 87th (BtR-F 87) generation with artificial diet containing Cry1Ac toxin, respectively. As the resistance levels increased, more larvae feeding on the Bt cotton expressing Cry1Ac toxin survived. Most larvae of BtR-F 87 could develop to the 5th instar and about 3% individuals reached the adult stage. The inheritance of Cry1Ac resistance trait at three resistant levels was autosomal and incompletely recessive, but the degree of dominance decreased as the resistance increased. The resistance was primarily monogenic in BtR-F 16 strain, but polygenic as resistance increased. The relative fitness of H. armigera, measured as a ratio of R 0 (the net replacement rate) of resistant strain divided by R 0 of the susceptible strain, decreased with an increase of the resistance levels, with ratios of 0.79, 0.64 and 0.59 in their respective BtR-F 16, BtR-F 34 and BtR-F 87 strains.

Note: Insecticide resistance inHelicoverpa armigera from cotton-growing areas in Turkey

Insecticide resistance inHelicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most important constraints on cotton production in Turkey. We investigated the susceptibility ofH. armigera strains collected from cotton fields in the Adana, Hatay and Antalya provinces to insecticides which are in wide use. LD50 values for tralomethrin, lambda-cyhalothrin, endosulfan, profenofos and methomyl were determined by topical bioassay. At the LD50 levels, resistance ratios for tralomethrin were 24.7-, 19.7- and 15.7-fold in the Adana, Hatay, and Antalya strains, respectively; and for lambda-cyhalothrin were 41-, 20-and 40-fold, respectively. Resistance ratios ranged from 1.2- to 2.1-fold in all field strains for endosulfan, profenofos and methomyl, with no significant resistance. These results suggest the presence of resistance to tralomethrin and lambda-cyhalothrin inH. armigera strains collected from cotton fields, but no resistance to endosulfan, profenofos or methomyl could be observed.