Field Populations Of Helicoverpa Armigera Research Articles

Absence of known knockdown resistance mutations but fixation of CYP337B3 was detected in field populations of Helicoverpa armigera across China

The cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest that infests many important crops. Pyrethroids targeting the voltage-gated sodium channel (VGSC) have been long used in the control of the cotton bollworm. Two amino acid substitutions (D1561V and E1565G) in H. armigera VGSC (HaVGSC) and the presence of a chimeric P450 gene (CYP337B3) have been documented to be associated with pyrethroid resistance. To understand the current occurrence of kdr mutations and the CYP337B3 gene in Chinese H. armigera populations, high-throughput amplicon sequencing was adopted to detect potential nucleotide variations in three fragments of the VGSC gene that cover 10 reported knockdown resistance (kdr) sites in insects, and gene-specific PCR was performed to examine the presence of CYP337B3 gene in H. armigera samples collected across China. The nucleotide variation analysis revealed a wealth of nucleotide variations in not only exons but also introns in the VGSC gene in Chinese H. armigera populations. However, neither previously reported kdr-conferring amino acid replacements nor other non-synonymous mutations were observed in a total of 1439 examined individuals. Population genetic analysis suggested that the H. armigera population in Nanchang, Jiangxi Province (JNC) had a moderate genetic differentiation from other populations, while no significant divergence was observed in other populations in northern and northwestern China. The CYP337B3 was present in all the examined individuals, indicating that CYP337B3 is extensively fixed in H. armigera populations across China. These results support that point mutations in VGSC are not a major factor involved in the current pyrethroid resistance in H. armigera. Instead, CYP337B3 plays a prevalent role in the development of resistance to pyrethroids in H. armigera.

Population Genomics of Nonrecessive Resistance to Bt Toxin Cry1Ac in Helicoverpa armigera From Northern China.

Transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have provided control of some key pests since 1996. However, the evolution of resistance by pests reduces the benefits of Bt crops. Resistance to Bt crops that is not recessively inherited is especially challenging to manage. Here we analyzed nonrecessive resistance to Bt toxin Cry1Ac in eight field populations of Helicoverpa armigera sampled in 2018 from northern China, where this global pest has been exposed to Cry1Ac in Bt cotton since 1997. Bioassays revealed 7.5% of field-derived larvae were resistant to Cry1Ac of which 87% had at least one allele conferring nonrecessive resistance. To analyze this nonrecessive resistance, we developed and applied a variant of a genomic mapping approach called quantitative trait locus (QTL)-seq. This analysis identified a region on chromosome 10 associated with nonrecessive resistance to Cry1Ac in all 21 backcross families derived from field-collected moths. Individual sequencing revealed that all 21 field-collected resistant grandparents of the backcross families had a previously identified dominant point mutation in the tetraspanin gene HaTSPAN1 that occurs in the region of chromosome 10 identified by QTL-seq. QTL-seq also revealed a region on chromosome 26 associated with nonrecessive resistance in at most 14% of the backcross families. Overall, the results imply the point mutation in HaTSPAN1 is the primary genetic basis of nonrecessive resistance to Cry1Ac in field populations of H. armigera from northern China. Moreover, because nonrecessive resistance is predominant, tracking the frequency of this point mutation could facilitate resistance monitoring in the region.

Cry1Ac resistance allele frequency in field populations of Helicoverpa armigera (Hübner) collected in Telangana and Andhra Pradesh, India

Bt cotton expressing Cry1Ac insecticidal protein was introduced in India in 2002. Within a span of 12 years, Bt-cotton constituted more than 90% of cropping area covering 11.6 mHa. This large scale cultivation favours resistance evolution in target pests. One of the major factors that influences rate of resistance is frequency of resistance alleles. Using F2 screen methodology and a discriminating dose, we estimated the level of resistance to Cry1Ac toxin in field populations of Helicoverpa armigera (Hübner). We report an increase in Cry1Ac resistance allele frequency in field populations of H. armigera collected in Telangana and Andhra Pradesh states of India. Collective allele frequency was 0.085 (95% CI) for 2013 and 0.035 (95% CI) for 2014, which was 65 times and 29 times higher than the initial frequencies estimated during 2004. To prevent further increase in Bt resistance frequency in H. armigera, it is necessary to integrate other resistance management tactics that preserves pest susceptibility to Bt cotton.

Baseline Susceptibility of Field Populations of Helicoverpa armigera to Bacillus thuringiensis Vip3Aa Toxin and Lack of Cross-Resistance between Vip3Aa and Cry Toxins.

The cotton bollworm Helicoverpa armigera (Hübner) is one of the most damaging cotton pests worldwide. In China, control of this pest has been dependent on transgenic cotton producing a single Bacillus thuringiensis (Bt) protein Cry1Ac since 1997. A small, but significant, increase in H. armigera resistance to Cry1Ac was detected in field populations from Northern China. Since Vip3Aa has a different structure and mode of action than Cry proteins, Bt cotton pyramids containing Vip3Aa are considered as ideal successors of Cry1Ac cotton in China. In this study, baseline susceptibility of H. armigera to Vip3Aa was evaluated in geographic field populations collected in 2014 from major cotton-producing areas of China. The LC50 values of 12 field populations ranged from 0.053 to 1.311 μg/cm2, representing a 25-fold range of natural variation among populations. It is also demonstrated that four laboratory strains of H. armigera with high levels of resistance to Cry1Ac or Cry2Ab have no cross-resistance to Vip3Aa protein. The baseline susceptibility data established here will serve as a comparative reference for detection of field-evolved resistance to Vip3Aa in H. armigera after future deployment of Bt cotton pyramids in China.

Multiple Resistances Against Formulated Organophosphates, Pyrethroids, and Newer-Chemistry Insecticides in Populations of Helicoverpa armigera (Lepidoptera: Noctuidae) from Pakistan.

Field populations of Helicoverpa armigera Hübner from 15 localities across the Punjab, Pakistan, were assessed by the leaf dip method for resistance against formulated organophosphates, pyrethroids, and newer insecticide groups. Resistance levels in H. armigera have been incrementally increasing for organophosphate and pyrethroid insecticides after decades of use in Pakistan. Resistance ratios (RRs) documented for organophosphates were 24- to 116-fold for profenofos and 22- to 87-fold for chlorpyrifos. For pyrethroids, RRs were 3- to 69-fold for cypermethrin and 3- to 27-fold for deltamethrin. Resistance levels against newer chemistries were 2- to 24-fold for chlorfenapyr, 1- to 22-fold for spinosad, 1- to 20-fold for indoxacarb, 1- to 18-fold for abamectin, and 1- to 16-fold for emamectin benzoate. Resistant populations of H. armigera were mainly in the southern part of the Punjab, Pakistan. The most resistant populations were collected from Pakpattan, Multan, and Muzzafargarh. Of the nine insecticides tested, LC50 and LC90 values were lower for newer insecticide groups; resistance levels were moderate to very high against organophosphates, very low to high against pyrethroids, and very low to low against the newer-chemistry insecticides. These findings suggest that the newer-chemistry insecticides with different modes of action could be included in insecticide rotations or replace the older insecticides. Supplementing the use of synthetic insecticides with safer alternatives could help to successfully lower the farmer's reliance on insecticides and the incidence of resistance due to repeated use of insecticides against major insect pests.

DNA-based screening for an intracellular cadherin mutation conferring non-recessive Cry1Ac resistance in field populations of Helicoverpa armigera

A number of cadherin mutants conferring resistance to Bt toxin Cry1Ac have been reported in three major lepidopteran pests, including Helicoverpa armigera. Unlike most of the cadherin mutants conferring recessive resistance, an allele (r15) with a 55aa deletion in the intracellular domain of cadherin (HaCad) was previously identified to cause non-recessive resistance to Cry1Ac in H. armigera. In the present study, a DNA-based PCR method was developed to screen the r15 allele from field populations of H. armigera collected from the main cotton planting areas of China in 2011 and 2012. Three heterozygous r15 alleles were detected from 562 moths collected from northern China (with intensive Bt cotton planting), and r15 allele frequency was estimated to be 0.0027. However, no r15 allele was detected from 314 moths collected from Xinjiang (with limited Bt cotton use). Although all the r15 alleles have the same deletion in the cDNA sequence, at least four different indels causing loss of exon 32 have been detected in the genomic DNA sequences flanking exon 32 of HaCad. Multiple origins of the r15 alleles illustrate parallel genotypic adaption of H. armigera to the selection pressure of Bt cotton.

Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

BackgroundEvolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.Methodology/Principal FindingsBioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.Conclusion/SignificanceThis is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.

Variation in the time-mortality responses of laboratory and field populations ofHelicoverpa armigerainfected with nucleopolyhedrovirus

The virulence of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) to South African laboratory and field populations of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) was evaluated. After determining the volume imbibed by each population, bioassays were performed using the droplet feeding method and gradient-purified HearNPV occlusion bodies. Evaluation of dose- and time-mortality data showed that the median lethal doses (LD 50 s) did not differ significantly between the populations but that the median lethal time (LT 50 ) of the laboratory population was significantly shorter than the LT 50 s of the field populations. The study shows the importance of considering both the dose- and time-mortality responses when comparing the response variability of populations to a nucleopolyhedrovirus infection.

Effects of spinosad on Helicoverpa armigera (Lepidoptera: Noctuidae) from China: tolerance status, synergism and enzymatic responses

Spinosad is increasingly used in pest management programmes, and resistance to it has been detected in recent years. However, there is no report on the susceptibilities of field populations of Helicoverpa armigera (Hübner) from China. Furthermore, the impact of spinosad on metabolic enzymes in this pest remains unknown. Four populations of H. armigera from different locations in China displayed less than 6.5-fold difference in LC(50) to spinosad, the highest being in the Xinjiang population, followed by Xiajin, Taian and Hubei populations, while there was no significant difference at LC(99) level among the four populations. The toxicity of spinosad could be synergised by piperonyl butoxide (PBO) and triphenylphosphate (TPP), but not by diethyl maleate (DEM). Spinosad exposure for 48 h significantly increased the activities of p-nitroanisole O-demethylase (ODM), while no significant changes in glutathione-S-transferase (GST) and carboxyl esterase (CarE) were observed. Field populations of H. armigera from China displayed marginally different susceptibilities to spinosad and had a relatively low LC(50). Cytochrome P450 monooxygenase might be involved in the metabolism of, and hence resistance to, spinosad in this pest in China.

Evaluation of resistance to different insecticides in field strains of Helicoverpa armigera (Lepidoptera: Noctuidae) in Punjab, Pakistan

AbstractSusceptibility of field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) from Punjab, Pakistan to selected insecticides was evaluated using the leaf dip method. The resistance factors varied between populations. The general trends were low to moderate resistance to endosulfan; very low to low resistance to chlorpyrifos and profenofos; very low resistance to spinosad, indoxacarb and thiodicarb; moderate to high resistance to bifenthrin; high to very high resistance to cypermethrin; and very high resistance to lambda‐cyhalothrin. Integrated pest management strategies aimed at reducing pesticide application, such as rotating the conventional insecticides having low resistance with newly developed synthetic insecticides and conserving natural enemies, are recommended.

Resistance Allele Frequency to Bt Cotton in Field Populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in China

Resistance evolution in target insects to Bacillus thurningiensis (Bt) cotton, Gossypium hirsutum L., is a main threat to Bt cotton technology. An increasing trend of population density of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) has been observed since 2001 in Qiuxian County (Hebei, China), where Bt cotton has been planted dominantly since 1998. This region was selected in 2006 and 2007 for estimating frequency of gene alleles conferring resistance to Bt cotton by screening the F1 progeny from single-pair cross between field-collected male and laboratory female of the Bt-resistant strain of H. armigera (F1 screen). F1 offspring from each single-pair line were screened for resistance alleles based on larval growth, development, and survival on Bt cotton leaves for 5 d. Two-year results indicated that ≈20% of field-collected males carried resistance alleles. The conservative estimate of the resistance allele frequency was 0.094 (95% CI, 0.044–0.145) for 2006 and 0.107 (95% CI, 0.055–0.159) for 2007. This is the first report of resistance allele frequency increase to such a high level in the field in China. Long-term adoption of Bt sprays, dominant planting of single-toxin–producing Bt cotton, and lack of conventional cotton refuge system might accelerate the resistance evolution in the region.

Potentiation between pyrethroid and organophosphate insecticides in resistant field populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan

The combined action of pyrethroids plus organophosphates was assessed on putatively resistant field populations of Helicoverpa armigera from Pakistan by using a leaf-dip bioassay. Ethion showed a good potentiation with bifenthrin, lambdacyhalothrin, cyfluthrin, betacyfluthrin, fenpropathrin, esfenvalerate, fluvalenate and tralomethrin. Profenofos was potentiating with bifenthrin but additive with lambdacyhalothrin. Methyl parathion also exhibited potentiation with bifenthrin. Contrarily, quinalphos produced an antagonism with bifenthrin. Chlorpyrifos potentiated lambdacyhalothrin in one population but had an additive effect in the other. A strong potentiation of pyrethroids by ethion in some populations indicates that esteratic detoxification is a key mechanism involved in imparting resistance to pyrethroids in Pakistani H. armigera.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera: Noctuidae) and of its relationship to H. zea

BackgroundHelicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.ResultsWe obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.ConclusionOur mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.

Changes in Cry1Ac Bt Transgenic Cotton in Response to Two Environmental Factors: Temperature and Insect Damage

The efficacy of Cry1Ac Bacillus thuringiensis (Bt) cotton plants against field populations of Helicoverpa armigera (Hübner) has been inconsistent over the growing season. Any reduction in efficacy (where efficacy is the capacity of the plant to affect the survival of the insect) increases the opportunities for H. armigera to evolve resistance to Bt toxin. Changes in efficacy could be due to changes at the level of gene expression and/or in the physiological makeup of the plant and may be induced by environmental conditions. Two environmental factors, temperature and insect damage, were investigated. Temperature was found to affect efficacy, whether plants were grown at different temperatures continuously or were exposed to a change in temperature for a short period. Damage caused by chewing insects (H. armigera larvae) produced a dramatic increase in the efficacy of presquare Bt cotton. In contrast, damage by sucking insects (aphids) did not induce changes in efficacy. Changes in efficacy seemed to be mediated through modification of the physiological background of the plant rather than changes in the level of Cry1Ac expression or in the concentration of the Bt toxin. The impact of the non-Bt responses of plants on strains of H. armigera should be evaluated. It is possible that by enhancing existing defensive mechanisms of plants, the rate of evolution of resistance to Bt toxins could be retarded by increasing the plants overall toxicity through the additive effects of the toxins and plant defenses.

Potentiation/antagonism of deltamethrin and cypermethrins with organophosphate insecticides in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

The joint action of pyrethroids deltamethrin and cypermethrins in combination with organophosphates ethion, profenofos, chlorpyrifos, quinalphos, and triazophos was studied on putatively resistant field populations of Helicoverpa armigera from Pakistan by using a leaf-dip method. Ethion produced a good potentiation with deltamethrin, cypermethrin, alphacypermethrin, and zetacypermethrin, whereas profenofos, chlorpyrifos, quinalphos, and triazophos exhibited an antagonism with deltamethrin as well as cypermethrins. Implications of using mixtures for counteracting insecticide resistance are discussed.

Susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to new chemistries in Pakistan

Toxicities of new chemistries viz. fipronil, chlorfenapyr, indoxacarb, spinosad, abamectin and emamectin benzoate, having novel modes of action, were evaluated using an IRAC leaf-dip method against Pakistani field populations of Helicoverpa armigera, which were highly resistant to conventional chemistries. Whereas the majority of populations exhibited susceptibility close to the baselines, there were, nevertheless, signs of resistance development to the new chemistries as demonstrated by a low level of tolerance in many populations. This may be due to a cross-resistance from the resistance mechanisms, particularly metabolic, already selected against older chemistries. If used judiciously and in rotation, the new chemistries can restore the profitability of crop production by counteracting and preventing insecticide resistance in H. armigera and other pests in the future.

Patterns of resistance to organophosphate insecticides in field populations ofHelicoverpa armigera in Pakistan

The pattern of organophosphate resistance in field populations of Helicoverpa armigera (Hübner) was monitored in Pakistan from 1994 through 1997 using an IRAC leaf-dip method. Generally, moderate to high resistance was found to an orthophosphorate, monocrotophos and to a dithiophosphorate, ethion. Resistance to the commonly used thiophosphorates, chlorpyrifos and profenofos, was very low during 1994 and 1995 but showed an upward trend during 1996 and 1997. Resistance factors to the other thiophosphorates such as quinalphos, parathion-methyl, methamidophos and triazophos remained very low. Baseline lethal concentrations for ethion, methamidophos and triazophos were at least four to five times higher than those for the other organophosphates tested, suggesting their low intrinsic efficacy against H armigera. © 1999 Society of Chemical Industry

Patterns of resistance to organophosphate insecticides in field populations ofHelicoverpa armigerain Pakistan

The pattern of organophosphate resistance in field populations of Helicoverpa armigera (Hübner) was monitored in Pakistan from 1994 through 1997 using an IRAC leaf-dip method. Generally, moderate to high resistance was found to an orthophosphorate, monocrotophos and to a dithiophosphorate, ethion. Resistance to the commonly used thiophosphorates, chlorpyrifos and profenofos, was very low during 1994 and 1995 but showed an upward trend during 1996 and 1997. Resistance factors to the other thiophosphorates such as quinalphos, parathion-methyl, methamidophos and triazophos remained very low. Baseline lethal concentrations for ethion, methamidophos and triazophos were at least four to five times higher than those for the other organophosphates tested, suggesting their low intrinsic efficacy against H armigera. © 1999 Society of Chemical Industry

Pyrethroid resistance of Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan

AbstractThe status of pyrethroid resistance in some field populations of Helicoverpa armigera (Hübner) from Pakistan was determined using an IRAC leaf-dip method. Resistance factors varied between populations, and the general trend was for a moderate to high resistance to cypermethrin and cyfluthrin; a low to moderate resistance to deltamethrin and alpha-cypermethrin; and a comparatively low resistance to bifenthrin, lambda-cyhalothrin and zeta-cypermethrin. Depending on which physiological mechanisms are shown to be present in Pakistani field strains, the latter group of pyrethroids may serve as useful tools in the management of insecticide resistance.

Section 8. Pyrethroid resistance: field resistance mechanisms

SummaryThe dual insecticide ± synergist discriminating dose technique proved highly successful in determining the relative importance of pyrethroid resistance mechanisms present in field populations of Helicoverpa armigera, without the problems of alternative techniques. Synergist pre-treatment proved unnecessary therefore allowing use of the more convenient and labour efficient discriminating dose pre-mix.Oxidative metabolic detoxification, probably via a polysubstrate monooxygenase system, was the major pyrethroid resistance mechanism in both larval and adult H. armigera. Residual piperonyl butoxide insensitive resistance (presumably nerve insensitivity, possibly acting in combination with the penetration resistance factor) was also present but at a low level. This latter resistance mechanism was expressed in larvae but moths appeared to express only weak phenotypic resistance. The predominant pyrethroid resistance mechanism employed by insects is discussed in relation to their feeding habit. The tenet that nectar feeding adult Lepidoptera are unable to express metabolic pyrethroid resistance is challenged.Unrestrained pre-strategy pyrethroid selection pressure on sequential generations resulted in selection for elevated levels of kdr type nerve insensitivity and possibly even super kdr. Restriction of pyrethroid selection pressure to one generation per season favoured selection of the oxidative over the nerve insensitivity resistance mechanism. Two possibly complementary explanations are put forward for this; differential genetic dominance (semidominant oxidative mechanism versus recessive nerve insensitivity) and/or selection in more than one life stage (moths and larvae for the oxidative mechanism versus predominantly larvae only for the nerve insensitivity mechanism). It is suggested that insecticide resistance management strategies should be designed to avoid selection of elevated levels of the intractable nerve insensitivity resistance mechanism whereas low levels of this mechanism (normal kdr) are not considered difficult to manage. The demonstration that the strategy has favoured selection of the more amenable oxidative resistance mechanism invites the opportunity to develop possible chemical countermeasures.