Demographics and Genetic Variability of the New World Bollworm (Helicoverpa zea) and the Old World Bollworm (Helicoverpa armigera) in Brazil

Helicoverpa armigera is one of the primary agricultural pests in the Old World, whereas H. zea is predominant in the New World. However, H. armigera was first documented in Brazil in 2013. Therefore, the geographical distribution, range of hosts, invasion source, and dispersal routes for H. armigera are poorly understood or unknown in Brazil. In this study, we used a phylogeographic analysis of natural H. armigera and H. zea populations to (1) assess the occurrence of both species on different hosts; (2) infer the demographic parameters and genetic structure; (3) determine the potential invasion and dispersal routes for H. armigera within the Brazilian territory; and (4) infer the geographical origin of H. armigera. We analyzed partial sequence data from the cytochrome c oxidase subunit I (COI) gene. We determined that H. armigera individuals were most prevalent on dicotyledonous hosts and that H. zea were most prevalent on maize crops, based on the samples collected between May 2012 and April 2013. The populations of both species showed signs of demographic expansion, and no genetic structure. The high genetic diversity and wide distribution of H. armigera in mid-2012 are consistent with an invasion period prior to the first reports of this species in the literature and/or multiple invasion events within the Brazilian territory. It was not possible to infer the invasion and dispersal routes of H. armigera with this dataset. However, joint analyses using sequences from the Old World indicated the presence of Chinese, Indian, and European lineages within the Brazilian populations of H. armigera. These results suggest that sustainable management plans for the control of H. armigera will be challenging considering the high genetic diversity, polyphagous feeding habits, and great potential mobility of this pest on numerous hosts, which favor the adaptation of this insect to diverse environments and control strategies.     

Genetic diversity and demographic history of the Old World Bollworm,Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), in Ethiopia inferred from mitochondrial gene sequences

The Old World bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a globally distributed agricultural and horticultural insect pest. Despite the economic importance of this insect in Ethiopia, its genetic diversity and demographic history are poorly understood. We examined the nucleotide variation of the mitochondrial cytochrome c oxidase subunit I (COI) gene fragment of 74 H. armigera individuals from six collection sites in Ethiopia. We recorded 15 COI haplotypes in H. armigera, ten globally shared and five exclusive to Ethiopia (HaET15, HaET14, HaET10, HaET7, and HaET4). Haplotype HaET1 was the most widely geographically distributed and frequent (71.62%). Analysis of molecular variance (AMOVA) revealed a high and significant level of variation within H. armigera populations (θ_ST  = −0.0135). Negative values of the neutrality test and nonsignificant index of mismatch distribution supported the demographic expansion of H. armigera populations in Ethiopia; furthermore, this was also supported by the nonsignificant values of the sum of squared deviations (SSD) and raggedness index (r). The high genetic variation and population expansion of H. armigera have immense implications for devising locally adapted management strategies in area‐wide integrated pest management IPM programs. However, a comprehensive study of H. armigera genetic diversity and population structure using various molecular markers is needed for future confirmation.     

Mitochondrial DNA analysis of field populations of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) and of its relationship to <Emphasis Type="Italic">H. zea</Emphasis>

Background  

Helicoverpa 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.     

Population structure and gene flow in the global pest,Helicoverpa armigera

Helicoverpa armigera is a major agricultural pest that is distributed across Europe, Asia, Africa and Australasia. This species is hypothesized to have spread to the Americas 1.5 million years ago, founding a population that is at present, a distinct species, Helicoverpa zea. In 2013, H. armigera was confirmed to have re‐entered South America via Brazil and subsequently spread. The source of the recent incursion is unknown and population structure in H. armigera is poorly resolved, but a basic understanding would highlight potential biosecurity failures and determine the recent evolutionary history of region‐specific lineages. Here, we integrate several end points derived from high‐throughput sequencing to assess gene flow in H. armigera and H. zea from populations across six continents. We first assemble mitochondrial genomes to demonstrate the phylogenetic relationship of H. armigera with other Heliothine species and the lack of distinction between populations. We subsequently use de novo genotyping‐by‐sequencing and whole‐genome sequences aligned to bacterial artificial chromosomes, to assess levels of admixture. Primarily, we find that Brazilian H. armigera are derived from diverse source populations, with strong signals of gene flow from European populations, as well as prevalent signals of Asian and African ancestry. We also demonstrate a potential field‐caught hybrid between H. armigera and H. zea, and are able to provide genomic support for the presence of the H. armigera conferta subspecies in Australasia. While structure among the bulk of populations remains unresolved, we present distinctions that are pertinent to future investigations as well as to the biosecurity threat posed by H. armigera.     

Population genetics and recent colonization history of the invasive drosophilid Zaprionus indianus in Mexico and Central America

Zaprionus indianus, also known as the African fig fly, is an invasive pest of a variety of commercial and native fruit. The species was first reported in Brazil in 1999, but has established itself in much of the New World within the last 10–15 years. We used nucleotide sequences from a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene to examine haplotype relationships, population structure, and infer the colonization history of Z. indianus in Mexico and Panama. Construction of a haplotype network showed that six COI haplotypes, obtained from flies collected at six localities in Mexico and one in Panama, clustered into three distinct clades. Clade composition was generally consistent in flies from Panama to northwestern Mexico, and analysis of molecular variance indicated no significant structure among populations. Three of the six haplotypes from Mexico and Panama were identical to previously reported haplotypes from Brazil. None of the six haplotypes, however, were shared with previously reported haplotypes from potential source populations in the Old World. The results of our genetic analysis suggest that the invasion of Z. indianus into Central America and Mexico most probably includes a northward migration of individuals from Brazil, with the possibility of at least one additional introduction of Z. indianus to the New World. Additional sequence data from potential source populations in the Old World will be required to confidently determine the number of introductions of Z. indianus into the New World, and to identify the geographic source.     

A Multiplex Real-Time PCR Assay to Diagnose and Separate Helicoverpa armigera and H. zea (Lepidoptera: Noctuidae) in the New World

The Old World bollworm, Helicoverpa armigera (Hübner), and the corn earworm, H. zea (Boddie), are two of the most important agricultural pests in the world. Diagnosing these two species is difficult—adults can only be separated with a complex dissection, and larvae cannot be identified to species using morphology, necessitating the use of geographic origin for identification in most instances. With the discovery of H. armigera in the New World, identification of immature Helicoverpa based on origin is no longer possible because H. zea also occurs in all of the geographic regions where H. armigera has been discovered. DNA barcoding and restriction fragment length polymorphism (RFLP) analyses have been reported in publications to distinguish these species, but these methods both require post-PCR processing (i.e., DNA sequencing or restriction digestion) to complete. We report the first real-time PCR assay to distinguish these pests based on two hydrolysis probes that bind to a segment of the internal transcribed spacer region 2 (ITS2) amplified using a single primer pair. One probe targets H. armigera, the second probe targets H. zea, and a third probe that targets a conserved segment of 18S rDNA is used as a control of DNA quality. The assay can be completed in 50 minutes when using isolated DNA and is successfully tested on larvae intercepted at ports of entry and adults captured during domestic surveys. We demonstrate that the assay can be run in triplex with no negative effects on sensitivity, can be run using alternative real-time PCR reagents and instruments, and does not cross react with other New World Heliothinae.     

Morphological Characterization of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Hübner) (Lepidoptera: Noctuidae: Heliothinae)

The cotton bollworm Helicoverpa armigera (Hübner) is a widespread lepidopteran pest found in various crops worldwide. This highly polyphagous species, commonly found both in the Old and New World, has caused significant economic damage as an invasive agricultural pest in Brazil since 2013. The goal of the present study is to provide a detailed morphological assessment of adults and immature stages of H. armigera, as this species is often confused with H. zea (Boddie), a congeneric species that is native to the New World. The biology data were acquired during four full life cycles, and observations on general behavior, nocturnal habits of larvae and adults, and sensitivity of larvae to humidity were recorded. Larval chaetotaxy differs between the first and the remaining instars, which bear L2 on the meso- and metathorax and L3 on A3 through A6, along with conspicuous chalazae and longitudinal bands. Important morphological characters of this species include the following: eggs with four micropylar openings, lined with 12 cells arranged in the shape of a rosette; pupa adecticous and obtect, with prominent spiracles; adults with the distal antennomere striate. Adults exhibit sexual dimorphism in the number of setae on the frenulum and spines on the prothoracic leg. Illustrations of the critical morphological features of this species are provided.     

DNA barcoding and the differentiation between North American and West European Phormia regina (Diptera,?Calliphoridae,Chrysomyinae)

Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4–0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.     

Mitochondrial DNA genomes of five major Helicoverpa pest species from the Old and New Worlds (Lepidoptera: Noctuidae)

Five species of noctuid moths, Helicoverpa armigera, H. punctigera, H. assulta, H. zea, and H. gelotopoeon, are major agricultural pests inhabiting various and often overlapping global distributions. Visual identification of these species requires a great deal of expertise and misidentification can have repercussions for pest management and agricultural biosecurity. Here, we report on the complete mitochondrial genomes of H. assulta assulta and H. assulta afra, H. gelotopoeon, H. punctigera, H. zea, and H. armigera armigera and H. armigera conferta’ assembled from high‐throughput sequencing data. This study significantly increases the mitogenome resources for these five agricultural pests with sequences assembled from across different continents, including an H. armigera individual collected from an invasive population in Brazil. We infer the phylogenetic relationships of these five Helicoverpa species based on the 13 mitochondrial DNA protein‐coding genes (PCG's) and show that two publicly available mitogenomes of H. assulta ( KP015198 and KR149448 ) have been misidentified or incorrectly assembled. We further consolidate existing PCR‐RFLP methods to cover all five Helicoverpa pest species, providing an updated method that will contribute to species differentiation and to future monitoring efforts of Helicoverpa pest species across different continents. We discuss the value of Helicoverpa mitogenomes to assist with species identification in view of the context of the rapid spread of H. armigera in the New World. With this work, we provide the molecular resources necessary for future studies of the evolutionary history and ecology of these species.     

Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zea for larvae of Heliothis armigera and Heliothis punctigera

Laboratory bioassays indicated that the potency of a nuclear polyhedrosis virus from Heliothis zea derived from a commercial American formulation was similar to that of a naturally occurring nuclear polyhedrosis virus from H. punctigera in Australia. Both viruses exhibited high virulence for neonate larvae of H. armigera and H. punctigera, the major pest species in this genus in Australia. Hence evaluation of the virus in Australia can proceed employing virus from either H. punctigera or H. zea.     

Invasion origin,rapid population expansion,and the lack of genetic structure of cotton bollworm (Helicoverpa armigera) in the Americas

In 2013, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was officially declared as present in Brazil and, after two years, the species was detected in the Caribbean and North America. Information on genetic features and accurate distribution of pests is the basis for agricultural protection policies. Furthermore, such knowledge is imperative to develop control strategies, understand the geographical range, and genetic patterns of this species in the Americas. Here, we carried out the widest sampling of H. armigera in the South American continent and Puerto Rico, after we estimated the diversity, demographic parameters, and genetic structure. The Internal Transcribed Spacer 1 (ITS1) nuclear marker was used to investigate the presence of putative hybrids between H. armigera and H. zea, and they were observed at a frequency of 1.5%. An ABC analysis, based in COI gene fragment, suggested Europe as the origin of South America specimens of H. armigeraand following a movement northward through the Caribbean. Three mtDNA genes and three nDNA markers revealed high genetic diversity distributed without the defined population structure of H. armigera in South America. Most of the genetic variation is within populations with a multidirectional expansion of H. armigera among morphoclimatic regions. High genetic diversity, rapid population expansion, and hybridization have implications for pest management since they suggest that adaptive alleles are spread through wide areas in South America that favor rapid local adaptation of H. armigera to new and disturbed environments (e.g., in agricultural areas).     

Population Genetic Structure of the Cotton Bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India as Inferred from EPIC-PCR DNA Markers

Helicoverpa armigera is an important pest of cotton and other agricultural crops in the Old World. Its wide host range, high mobility and fecundity, and the ability to adapt and develop resistance against all common groups of insecticides used for its management have exacerbated its pest status. An understanding of the population genetic structure in H. armigera under Indian agricultural conditions will help ascertain gene flow patterns across different agricultural zones. This study inferred the population genetic structure of Indian H. armigera using five Exon-Primed Intron-Crossing (EPIC)-PCR markers. Nested alternative EPIC markers detected moderate null allele frequencies (4.3% to 9.4%) in loci used to infer population genetic structure but the apparently genome-wide heterozygote deficit suggests in-breeding or a Wahlund effect rather than a null allele effect. Population genetic analysis of the 26 populations suggested significant genetic differentiation within India but especially in cotton-feeding populations in the 2006–07 cropping season. In contrast, overall pair-wise F _ST estimates from populations feeding on food crops indicated no significant population substructure irrespective of cropping seasons. A Baysian cluster analysis was used to assign the genetic make-up of individuals to likely membership of population clusters. Some evidence was found for four major clusters with individuals in two populations from cotton in one year (from two populations in northern India) showing especially high homogeneity. Taken as a whole, this study found evidence of population substructure at host crop, temporal and spatial levels in Indian H. armigera, without, however, a clear biological rationale for these structures being evident.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Molecular markers to discriminate among four pest species of Helicoverpa (Lepidoptera: Noctuidae)

The four significant pest species in the Helicoverpa genus (H. armigera, H. assulta, H. punctigera and H. zea) are morphologically similar and can only be reliably distinguished through dissection of adult genitalia. Two partial regions of the mitochondrial DNA (mtDNA), the cytochrome oxidase subunit I (COI) and the cytochrome b (Cyt b) genes were amplified by PCR and digested with restriction endonucleases. The restriction patterns, generated by the endonucleases BstZ17I and HphI, demonstrated reliable differentiation of the four Helicoverpa pest species. This technique is fast, reliable and effective at distinguishing specimens irrespective of their life stages and offers support to conventional taxonomic differentiation based on morphological characters.     

A host-plant specialist, Helicoverpa assulta, is more tolerant to capsaicin from Capsicum annuum than other noctuid species

Plant secondary compounds not only play an important role in plant defense, but have been a driving force for host adaptation by herbivores. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), an alkaloid found in the fruit of Capsicum spp. (Solanaceae), is responsible for the pungency of hot pepper fruits and is unique to the genus. The oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae), is a specialist herbivore feeding on solanaceous plants including Capsicum annuum, and is one of a very few insect herbivores worldwide capable of feeding on hot pepper fruits. To determine whether this is due in part to an increased physiological tolerance of capsaicin, we compared H. assulta with another specialist on Solanaceae, Heliothis subflexa, and four generalist species, Spodoptera frugiperda, Heliothis virescens, Helicoverpa armigera, and Helicoverpa zea, all belonging to the family Noctuidae. When larvae were fed capsaicin-spiked artificial diet for the entire larval period, larval mortality increased in H. subflexa and H. zea but decreased in H. assulta. Larval growth decreased on the capsaicin-spiked diet in four of the species, was unaffected in H. armigera and increased in H. assulta. Food consumption and utilization experiments showed that capsaicin decreased relative consumption rate (RCR), relative growth rate (RGR) and approximate digestibility (AD) in H. zea, and increased AD and the efficiency of conversion of ingested food (ECI) in H. armigera; whereas it did not significantly change any of these nutritional indices in H. assulta. The acute toxicity of capsaicin measured by injection into early fifth instar larvae was less in H. assulta than in H. armigera and H. zea. Injection of high concentrations produced abdominal paralysis and self-cannibalism. Injection of sub-lethal doses of capsaicin resulted in reduced pupal weights in H. armigera and H. zea, but not in H. assulta. The results indicate that H. assulta is more tolerant to capsaicin than the other insects tested, suggesting that this has facilitated expansion of its host range within Solanaceae to Capsicum after introduction of the latter to the Old World about 500 years ago. The increased larval survival and growth due to chronic dietary exposure to capsaicin suggests further adaptation of H. assulta to that compound, the mechanisms of which remain to be investigated.     

Resistance to insecticides in Heliothine Lepidoptera: a global view

The status of resistance to organophosphate, carbamate, cyclodiene and pyrethroid insecticides in the heliothine Lepidoptera is reviewed. In particular, resistance in the tobacco budworm, Heliothis virescens, and the corn earworm, Helicoverpa zea, from the New World, and the cotton bollworm, Helicoverpa armigera, from the Old World, are considered in detail. Particular emphasis has been placed on resistance to the most widely used of these insecticide groups, the pyrethroids. In each case, the incidence and current status of resistance are considered before a detailed view of the mechanisms of resistance is given. Controversial issues regarding the nature of mechanisms of resistance to pyrethroid insecticides are discussed. The implications for resistance management are considered.     

Old World and New World Cupressus pollen: morphological and cytological remarks

The aim of this work was to collect new information about pollen morphology and pollen wall structure comparing Cupressus species from the Old World and New World. Cupressus is a nonmonophyletic genus that includes species that appear to be divided into Old World and New World clades. Observations in this study on cypress pollen indicate that grain size and composition of intine β-glucans are different between the considered Old World and New World species. Different from all the other American cypress species, pollen of C. macrocarpa reacted to dying in a similar manner to Old World species. Rehydrated pollen grains collected from 20 Asian, Afro-Mediterranean and American cypress species were measured under a light microscope. The size of the pollen grains and the percentage of intine in relation to the pollen grain diameter were significantly different between Old World and New World species. Pollen wall composition was tested after addition of different dyes to the hydration solution, and subsequent observations were carried out by light and fluorescence microscopy. Lugol and calcofluor staining showed differences in composition of the middle and inner intine layers between New World and Old World species.     

Susceptibility of larvae ofHeliothis zea,H. virescens,andH. armigera [lep.: Noctuidae] to 3 baculoviruses

The pattern of virulence (based on inclusion bodies) for 3 baculoviruses ofHeliothis, i.e. a unicapsid, nuclear polyhedrosis virus (HzSNPV); a multicapsid, nuclear polyhedrosis virus (HaMNPV); and a granulosis virus (HaGIV) was the same (HzSNPV>HaMNPV>HaGIV) for 3 species ofHeliothis. Based on numbers of nucleocapsids, however, the HaGIV was ca 2X more virulent than the HaMNPV for larvae ofH. virescens, (F.), and the HaMNPV was about 6X more virulent than the HaGIV for larvae ofH. armigera (Hübner). The fastest rate of larval mortality was obtained with HzSNPV. Although the mortality rate for HaGIV was faster than that of HaMNPV forH. virescens andH. armigera, it was slower than that of HaMNPV for larvae ofH. zea (Boddie). The pattern of susceptibility ofHeliothis species to HzSNPV and HaMNPV wasH. zea>H. virescens>H. armigera. Differences in susceptibility of the least susceptible species (H. armigera) and the most susceptible species (H. zea) to HzSNPV was ca. 1.6 X. Larvae ofH. zea, however, were ca. 4 to 6 X more susceptible to HaMNPV than were larvae ofH. virescens orH. armigera. A different pattern of susceptibility was recorded for HaGIV when larvae were challenged with HzSNPV and HaMNPV. Larvae ofH. virescens were ca. 20 and 35 X more susceptible to HaGIV than were larvae ofH. zea andH. armigera, respectively.     

The Potential Distribution of Invading Helicoverpa armigera in North America: Is It Just a Matter of Time?

Helicoverpa armigera has recently invaded South and Central America, and appears to be spreading rapidly. We update a previously developed potential distribution model to highlight the global invasion threat, with emphasis on the risks to the United States. The continued range expansion of H. armigera in Central America is likely to change the invasion threat it poses to North America qualitatively, making natural dispersal from either the Caribbean islands or Mexico feasible. To characterise the threat posed by H. armigera, we collated the value of the major host crops in the United States growing within its modelled potential range, including that area where it could expand its range during favourable seasons. We found that the annual value of crops that would be exposed to H. armigera totalled approximately US$78 billion p.a., with US$843 million p.a. worth growing in climates that are optimal for the pest. Elsewhere, H. armigera has developed broad-spectrum pesticide resistance; meaning that if it invades the United States, protecting these crops from significant production impacts could be challenging. It may be cost-effective to undertake pre-emptive biosecurity activities such as slowing the spread of H. armigera throughout the Americas, improving the system for detecting H. armigera, and methods for rapid identification, especially distinguishing between H. armigera, H. zea and potential H. armigera x H. zea hybrids. Developing biological control programs, especially using inundative techniques with entomopathogens and parasitoids could slow the spread of H. armigera, and reduce selective pressure for pesticide resistance. The rapid spread of H. armigera through South America into Central America suggests that its spread into North America is a matter of time. The likely natural dispersal routes preclude aggressive incursion responses, emphasizing the value of preparatory communication with agricultural producers in areas suitable for invasion by H. armigera.     

Mutated Cadherin Alleles from a Field Population of Helicoverpa armigera Confer Resistance to Bacillus thuringiensis Toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r₁) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r₂ and r₃) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r₂ and r₃ alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.