Non-recessive Resistance Research Articles

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105 + Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic cropsproducing insecticidal proteins fromBacillus thuringiensis Berliner (Bt,Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.)rapidly evolved in some FAW populations, pyramided Bt maizehybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.

Global perspectives on field-evolved resistance to transgenic Bt crops: a special collection.

Crops genetically engineered to produce insect-killing proteins from Bacillus thuringiensis (Bt) have revolutionized management of some major pests, but their efficacy is reduced when pests evolve resistance. Practical resistance, which is field-evolved resistance that reduces the efficacy of Bt crops and has practical implications for pest management, has been reported in 26 cases in seven countries involving 11 pest species. This special collection includes six original papers that present a global perspective on field-evolved resistance to Bt crops. One is a synthetic review providing a comprehensive global summary of the status of the resistance or susceptibility to Bt crops of 24 pest species in 12 countries. Another evaluates the inheritance and fitness costs of resistance of Diabrotica virgifera virgifera to Gpp34/Tpp35Ab (formerly called Cry34/35Ab). Two papers describe and demonstrate advances in techniques for monitoring field-evolved resistance. One uses a modified F2 screen for resistance to Cry1Ac and Cry2Ab in Helicoverpa zea in the United States. The other uses genomics to analyze nonrecessive resistance to Cry1Ac in Helicoverpa armigera in China. Two papers provide multi-year monitoring data for resistance to Bt corn in Spain and Canada, respectively. The monitoring data from Spain evaluate responses to Cry1Ab of the corn borers Sesamia nonagrioides and Ostrinia nubilalis, whereas the data from Canada track responses of O. nubilalis to Cry1Ab, Cry1Fa, Cry1A.105, and Cry2Ab. We hope the new methods, results, and conclusions reported here will spur additional research and help to enhance the sustainability of current and future transgenic insecticidal crops.

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.

Inheritance of Resistance to Cry1A.105 in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae).

Simple SummaryThe corn earworm, Helicoverpa zea (Boddie), is a target pest of Bacillus thuringiensis (Bt) cotton and maize in North America. This pest has evolved resistance to both Bt crops in the United States, which is a significant threat to their sustainable use for H. zea control. Cry1A.105 is a Bt toxin commonly used in commercial transgenic maize hybrids to control above-ground caterpillar pests, such as H. zea. The aim of this study is to describe the inheritance of Cry1A.105 resistance in H. zea. Genetic-cross studies revealed that the resistance to Cry1A.105 is controlled by a single, autosomal, nonrecessive gene. Data generated from the study will be useful in resistance risk assessment and refining Bt resistance mitigation programs for H. zea control.Cry1A.105 is a bioengineered Bacillus thuringiensis (Bt) insecticidal protein consisting of three domains derived from Cry1Ac, Cry1Ab, and Cry1F. It is one of the two pyramided Bt toxins expressed in the MON 89034 event, a commonly planted Bt maize trait in the Americas. Recent studies have documented that field resistance of the corn earworm, Helicoverpa zea (Boddie), to the Cry1A.105 toxin in maize plants has become widespread in the United States. To investigate the inheritance of resistance to Cry1A.105 in H. zea, two independent tests, each with various genetic crosses among susceptible and Cry1A.105-resistant populations, were performed. The responses of these susceptible, resistant, F1, F2, and backcrossed insect populations to Cry1A.105 were assayed using a diet overlay method. The bioassays showed that the resistance to Cry1A.105 in H. zea was inherited as a single, autosomal, nonrecessive gene. The nonrecessive nature of the resistance could be an important factor contributing to the widespread resistance of maize hybrids containing Cry1A.105 in the United States. The results indicate that resistance management strategies for Bt crops need to be refined to ensure that they are effective in delaying resistance evolution for nonrecessive resistance (nonhigh dose).

Multiple and non-recessive resistance to Bt proteins in a Cry2Ab2-resistant population of Helicoverpa zea

Transgenic corn and cotton expressing Cry and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) have been planted for managing lepidopteran and coleopteran insect pests for more than two decades in the U.S. Evolution of insect resistance to these Bt proteins is a major threat for the continued efficacy of Bt technology. Understanding patterns of cross-resistance and dominance of resistance in target insects is essential for resistance management and pest control. The bollworm, also called corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt crops in the U.S. In this study, we evaluated the responses of Cry2Ab2-resistant (RR), susceptible (SS), and heterozygous (RS) strains of H. zea to the Cry1Ac, Cry1A.105, Cry2Ab2 and Vip3Aa39 proteins using diet-overlay bioassays. The Cry2Ab2-resistant H. zea also possessed high levels of resistance to Cry1Ac and Cry1A.105, but were very susceptible to Vip3Aa. The resistance to Cry1 and Cry2 proteins in RR H. zea varied from incompletely recessive to completely dominant depending on the protein concentration. These data suggest that the RR H. zea possess multiple non-recessive resistance to Cry1/Cry2 proteins. These results have significant implications for understanding field-evolved resistance of H. zea to Cry1 and Cry2 proteins in U.S. Bt corn and cotton fields and should contribute to developing effective management strategies for control of H. zea.

Combining refuges with transgenic insect releases for the management of an insect pest with non-recessive resistance to Bt crops in agricultural landscapes

Reinforcing the high-dose/refuge strategy with releases of transgenic insects has been suggested as a method for simultaneously managing agricultural pest populations and resistance to transgenic crops. Theoretical and empirical studies have shown that these approaches can work when deployed against closed populations and the assumptions of the HDR strategy are met. However, field-evolved resistance is often linked to non-recessive resistance or refuge non-compliance, and pest management regimes are likely to take place at the landscape-level. It is therefore important to understand how effective such strategies are when resistance is non-recessive, and how they could be employed in agricultural landscapes. We developed a spatially-explicit model to investigate the efficacy of strategies combining refuges with transgenic insect releases to manage a pest with non-recessive resistance in agricultural landscapes. We compared two release strategies, area-wide releases and localised releases targeted at population hotspots, and analysed the effects of refuge and release parameters on population and resistance dynamics. Area-wide releases reliably achieved landscape-level pest eradication. Localised releases also eradicated the pest when low release thresholds were combined with high release ratios, and maintained the pest at low densities when insufficient to achieve extinction. Reinforcing refuges with localised releases also greatly enhanced the probability of resistance extinction. However, when resistance remained in the population, localised releases prevented resistance from reaching fixation rather than greatly delaying or reversing resistance evolution. Our work indicates that combining refuges with simple release policies is effective for landscape-level pest suppression when the HDR assumptions are violated, but more nuanced release strategies may be required to enhance the benefits to resistance management.

Transcriptome and differentially expressed genes of Busseola fusca (Lepidoptera: Noctuidae) larvae challenged with Cry1Ab toxin

Busseola fusca (Fuller) (Lepidoptera: Noctuidae), a major insect pest of maize in sub-Saharan Africa, has developed high levels of non-recessive resistance to Cry1Ab toxin expressed in genetically modified Bt maize. Multiple resistance mechanisms to various Cry toxins have been identified in Lepidoptera, but no study has yet been done to determine the mechanism of Cry1Ab resistance in B. fusca. Therefore, the larval transcriptome of B. fusca was sequenced, de novo assembled and characterized. Differential expression analysis was performed to compare gene expression profiles of Cry toxin challenged and unchallenged neonate larvae to assess the molecular basis of the defence mechanism employed by this insect. Several genes associated with Cry toxin resistance in other lepidopteran pests were detected in B. fusca. Results suggest that differential expression of metabolic and immune-related genes might explain Cry1Ab toxin defence in this pest (supplemental file). Transcript expression profiles of neonates demonstrated that 33.59% and 60.31% of the 131 differentially expressed genes were upregulated and downregulated in the toxin-challenged neonate larvae, respectively. Transcripts were grouped into two subclusters according to the similarity of their expression patterns. Transcripts in subcluster 1 were moderately upregulated in the toxin-challenged neonate larvae, and, conversely, downregulated in the unchallenged neonate larvae. The solute carrier organic anion transporter, which is involved in insecticide detoxification, was upregulated in the toxin-challenged neonate larvae. Conversely, most of the transcripts in subcluster 2 were moderately downregulated in the toxin-challenged neonate larvae, and upregulated for neonates feeding on non-challenged maize. Four unidentified transcripts were extremely down-regulated in the toxin-challenged neonate larvae, and upregulated in the unchallenged neonate larvae. Further studies are recommended to establish if there is a direct correlation between these differentially expressed genes and the observed resistance. Elucidation of such defence mechanisms is crucial for developing insect resistance management strategies to ensure sustainable use of genetically modified maize in Africa. Nevertheless, this is the first study on gene expression profiles of B. fusca strains challenged with Cry toxin. The transcriptome characterized in this study provides a significant resource base for future studies on B. fusca and contributes to understanding some of the gene regulation and signalling networks involved in the defence of B. fusca against Cry1Ab toxin.

Inheritance and fitness costs of Cry1A.105 resistance in two strains of Spodoptera frugiperda (J.E. Smith)

Cry1A.105 is a Bacillus thuringiensis (Bt) protein expressed in many Bt corn hybrids for controlling moth pests including the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith). The objective of this study was to compare the inheritance and fitness costs of two Cry1A.105-resistant FAW strains (RR32 and RR67) from Florida, U.S. To assess inheritance of the resistance, larval mortality of one susceptible strain (SS), RR32, RR67, and eight FAW strains generated from various genetic crosses was assayed on Cry1A.105-treated diet and Cry1A.105 corn leaf tissues. In the fitness cost study, survival, growth, development, and fecundity of SS, RR32, RR67, and two F1 heterozygous-resistant FAW strains were evaluated on non-Bt corn leaf tissue. The Cry1A.105 resistance in both RR32 and RR67 was likely inherited as a single autosomal gene with no fitness costs. The dominance levels differed between the two strains and varied from incompletely recessive to incompletely dominant, depending on Cry1A.105 concentrations and assay materials. The results of this study indicate a relatively high risk of resistance development to single-gene Cry1A.105 corn in FAW. Together with published data, the results also show that non-recessive resistance and lack of fitness costs in resistance to Bt crops are not uncommon, which provides a cautionary evidence against the assumption that resistance is recessive in an resistance management plan for Bt crops.

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.

Dominant Inheritance of Field-Evolved Resistance to Bt Corn in Busseola fusca

Transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been adopted worldwide, notably in developing countries. In spite of their success in controlling target pests while allowing a substantial reduction of insecticide use, the sustainable control of these pest populations is threatened by the evolution of resistance. The implementation of the “high dose/refuge” strategy for managing insect resistance in transgenic crops aims at delaying the evolution of resistance to Bt crops in pest populations by promoting survival of susceptible insects. However, a crucial condition for the “high dose/refuge” strategy to be efficient is that the inheritance of resistance should be functionally recessive. Busseola fusca developed high levels of resistance to the Bt toxin Cry 1Ab expressed in Bt corn in South Africa. To test whether the inheritance of B . fusca resistance to the Bt toxin could be considered recessive we performed controlled crosses with this pest and evaluated its survival on Bt and non-Bt corn. Results show that resistance of B . fusca to Bt corn is dominant, which refutes the hypothesis of recessive inheritance. Survival on Bt corn was not lower than on non-Bt corn for both resistant larvae and the F1 progeny from resistant × susceptible parents. Hence, resistance management strategies of B . fusca to Bt corn must address non-recessive resistance.

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.

Diverse genetic basis of field-evolved resistance to Bt cotton in cotton bollworm from China

Evolution of pest resistance reduces the efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays or in transgenic crops. Although several pests have evolved resistance to Bt crops in the field, information about the genetic basis of field-evolved resistance to Bt crops has been limited. In particular, laboratory-selected resistance to Bt toxin Cry1Ac based on recessive mutations in a gene encoding a toxin-binding cadherin protein has been identified in three major cotton pests, but previous work has not determined if such mutations are associated with field-selected resistance to Bt cotton. Here we show that the most common resistance alleles in field populations of cotton bollworm, Helicoverpa armigera, selected with Bt cotton in northern China, had recessive cadherin mutations, including the deletion mutation identified via laboratory selection. However, unlike all previously studied cadherin resistance alleles, one field-selected cadherin resistance allele conferred nonrecessive resistance. We also detected nonrecessive resistance that was not genetically linked with the cadherin locus. In field-selected populations, recessive cadherin alleles accounted for 75-84% of resistance alleles detected. However, most resistance alleles occurred in heterozygotes and 59-94% of resistant individuals carried at least one nonrecessive resistance allele. The results suggest that resistance management strategies must account for diverse resistance alleles in field-selected populations, including nonrecessive alleles.