Proactive resistance management for sustaining the efficacy of RNA interference for pest control.

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.

Negative association between host plant suitability and the fitness cost of resistance to Bacillus thuringiensis (Bacillales: Bacillaceae).

Transgenic crops producing Bacillus thuringiensis (Bt) toxins are commonly used for controlling insect pests. Nearby refuges of non-Bt host plants play a central role in delaying the evolution of resistance to Bt toxins by pests. Pervasive fitness costs associated with resistance, which entail lower fitness of resistant than susceptible individuals in refuges, can increase the ability of refuges to delay resistance. Moreover, these costs are affected  by environmental factors such as host plant suitability, implying that manipulating refuge plant suitability could improve the success of the refuge strategy. Based on results from a previous study of Trichoplusia ni resistant to Bt sprays, it was proposed that low-suitability host plants could magnify costs. To test this hypothesis, we investigated the association between host plant suitability and fitness costs for 80 observations from 30 cases reported in 18 studies of 8 pest species from 5 countries. Consistent with the hypothesis, the association between plant suitability and fitness cost was negative. With plant suitability scaled to range from 0 (low) to 1 (high), the expected cost was 20.7% with a suitability of 1 and the fitness cost increased 2.5% for each 0.1 decrease in suitability. The most common type of resistance to Bt toxins involves mutations affecting a few types of midgut proteins to which Bt toxins bind to kill insects. A better understanding of how such mutations interact with host plant suitability to generate fitness costs could be useful for enhancing the refuge strategy and sustaining the efficacy of Bt crops.

Downregulation of a transcription factor associated with resistance to Bt toxin Vip3Aa in the invasive fall armyworm.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm (Spodoptera frugiperda), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda. Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb, the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Genome-wide analysis reveals distinct global populations of pink bollworm (Pectinophora gossypiella).

The pink bollworm (Pectinophora gossypiella) is one of the world's most destructive pests of cotton. This invasive lepidopteran occurs in nearly all cotton-growing countries. Its presence in the Ord Valley of North West Australia poses a potential threat to the expanding cotton industry there. To assess this threat and better understand population structure of pink bollworm, we analysed genomic data from individuals collected in the field from North West Australia, India, and Pakistan, as well as from four laboratory colonies that originated in the United States. We identified single nucleotide polymorphisms (SNPs) using a reduced-representation, genotyping-by-sequencing technique (DArTseq). The final filtered dataset included 6355 SNPs and 88 individual genomes that clustered into five groups: Australia, India-Pakistan, and three groups from the United States. We also analysed sequences from Genbank for mitochondrial DNA (mtDNA) locus cytochrome c oxidase I (COI) for pink bollworm from six countries. We found low genetic diversity within populations and high differentiation between populations from different continents. The high genetic differentiation between Australia and the other populations and colonies sampled in this study reduces concerns about gene flow to North West Australia, particularly from populations in India and Pakistan that have evolved resistance to transgenic insecticidal cotton. We attribute the observed population structure to pink bollworm's narrow host plant range and limited dispersal between continents.

Refuges of conventional host plants counter dominant resistance of cotton bollworm to transgenic Bt cotton.

Transgenic crops have revolutionized insect pest control, but evolution of resistance by pests threatens their continued success. The primary strategy for combating pest resistance to crops producing insecticidal proteins from Bacillus thuringiensis (Bt) uses refuges of non-Bt host plants to allow survival of susceptible insects. The prevailing paradigm is that refuges delay resistance that is rare and recessively inherited. However, we discovered refuges countered resistance to Bt cotton that was neither rare nor recessive. In a 15-year field study of the cotton bollworm, the frequency of a mutation conferring dominant resistance to Bt cotton surged 100-fold from 2006 to 2016 yet did not rise from 2016 to 2020. Computer simulations indicate the increased refuge percentage from 2016 to 2020 is sufficient to explain the observed halt in the evolution of resistance. The results also demonstrate the efficacy of a Bt crop can be sustained by non-Bt refuges of other crops.

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.

Fitness Costs and Incomplete Resistance Associated with Delayed Evolution of Practical Resistance to Bt Crops.

Insect pests are increasingly evolving practical resistance to insecticidal transgenic crops that produce Bacillus thuringiensis (Bt) proteins. Here, we analyzed data from the literature to evaluate the association between practical resistance to Bt crops and two pest traits: fitness costs and incomplete resistance. Fitness costs are negative effects of resistance alleles on fitness in the absence of Bt toxins. Incomplete resistance entails a lower fitness of resistant individuals on a Bt crop relative to a comparable non-Bt crop. In 66 studies evaluating strains of nine pest species from six countries, costs in resistant strains were lower in cases with practical resistance (14%) than without practical resistance (30%). Costs in F1 progeny from crosses between resistant and susceptible strains did not differ between cases with and without practical resistance. In 24 studies examining seven pest species from four countries, survival on the Bt crop relative to its non-Bt crop counterpart was higher in cases with practical resistance (0.76) than without practical resistance (0.43). Together with previous findings showing that the nonrecessive inheritance of resistance is associated with practical resistance, these results identify a syndrome associated with practical resistance to Bt crops. Further research on this resistance syndrome could help sustain the efficacy of Bt crops.

Molecular Genetic Basis of Lab- and Field-Selected Bt Resistance in Pink Bollworm.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) control some important insect pests. However, evolution of resistance by pests reduces the efficacy of Bt crops. Here we review resistance to Bt cotton in the pink bollworm, Pectinophora gossypiella, one of the world's most damaging pests of cotton. Field outcomes with Bt cotton and pink bollworm during the past quarter century differ markedly among the world's top three cotton-producing countries: practical resistance in India, sustained susceptibility in China, and eradication of this invasive lepidopteran pest from the United States achieved with Bt cotton and other tactics. We compared the molecular genetic basis of pink bollworm resistance between lab-selected strains from the U.S. and China and field-selected populations from India for two Bt proteins (Cry1Ac and Cry2Ab) produced in widely adopted Bt cotton. Both lab- and field-selected resistance are associated with mutations affecting the cadherin protein PgCad1 for Cry1Ac and the ATP-binding cassette transporter protein PgABCA2 for Cry2Ab. The results imply lab selection is useful for identifying genes important in field-evolved resistance to Bt crops, but not necessarily the specific mutations in those genes. The results also suggest that differences in management practices, rather than genetic constraints, caused the strikingly different outcomes among countries.

Global Patterns of Insect Resistance to Transgenic Bt Crops: The First 25 Years.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have improved pest management and reduced reliance on insecticide sprays. However, evolution of practical resistance by some pests has reduced the efficacy of Bt crops. We analyzed global resistance monitoring data for 24 pest species based on the first 25 yr of cultivation of Bt crops including corn, cotton, soybean, and sugarcane. Each of the 73 cases examined represents the response of one pest species in one country to one Bt toxin produced by one or more Bt crops. The cases of practical resistance rose from 3 in 2005 to 26 in 2020. Practical resistance has been documented in some populations of 11 pest species (nine lepidopterans and two coleopterans), collectively affecting nine widely used crystalline (Cry) Bt toxins in seven countries. Conversely, 30 cases reflect no decrease in susceptibility to Bt crops in populations of 16 pest species in 10 countries. The remaining 17 cases provide early warnings of resistance, which entail genetically based decreases in susceptibility without evidence of reduced field efficacy. The early warnings involve four Cry toxins and the Bt vegetative insecticidal protein Vip3Aa. Factors expected to favor sustained susceptibility include abundant refuges of non-Bt host plants, recessive inheritance of resistance, low resistance allele frequency, fitness costs, incomplete resistance, and redundant killing by multi-toxin Bt crops. Also, sufficiently abundant refuges can overcome some unfavorable conditions for other factors. These insights may help to increase 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.

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

Practical resistance to Cry toxins and efficacy of Vip3Aa in Bt cotton against Helicoverpa zea.

BACKGROUND: Crops genetically engineered to make insect-killing proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, the benefits of such transgenic crops are reduced when pests evolve resistance to Bt toxins. We evaluated resistance to Bt toxins and Bt cotton plants using laboratory bioassays and complementary field trials focusing on Helicoverpa zea, one of the most economically important pests of cotton and other crops in the United States. RESULTS: The data from 235 laboratory bioassays demonstrate resistance to Cry1Ac, Cry1Fa, and Cry2Ab occurred in most of the 95 strains of H. zea derived from Arkansas, Louisiana, Mississippi, Tennessee, and Texas during 2016 to 2021. Complementary field data show efficacy decreased for Bt cotton producing Cry1Ac + Cry1Fa or Cry1Ac + Cry2Ab, but not Cry1Ac + Cry1Fa + Vip3Aa. Moreover, analysis of data paired by field site and year shows higher survival in bioassays was generally associated with lower efficacy of Bt cotton. CONCLUSIONS: The results confirm and extend previous evidence showing widespread practical resistance of H. zea in the United States to the Cry toxins produced by Bt cotton and corn, but not to Vip3Aa. Despite deployment in combination with Cry toxins in Bt crops, Vip3Aa effectively acts as a single toxin against H. zea larvae that are highly resistant to Cry toxins. Furthermore, Vip3Aa adoption is increasing and previous work provided an early warning of field-evolved resistance. Thus, rigorous resistance management measures are needed to preserve the efficacy of Vip3Aa against this highly adaptable pest. © 2022 Society of Chemical Industry.

Responses to Bt toxin Vip3Aa by pink bollworm larvae resistant or susceptible to Cry toxins.

BACKGROUND: Transgenic crops that make insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized management of some pests. However, evolution of resistance to Bt toxins by pests diminishes the efficacy of Bt crops. Resistance to crystalline (Cry) Bt toxins has spurred adoption of crops genetically engineered to produce the Bt vegetative insecticidal protein Vip3Aa. Here we used laboratory diet bioassays to evaluate responses to Vip3Aa by pink bollworm (Pectinophora gossypiella), one of the world's most damaging pests of cotton. RESULTS: Against pink bollworm larvae susceptible to Cry toxins, Vip3Aa was less potent than Cry1Ac or Cry2Ab. Conversely, Vip3Aa was more potent than Cry1Ac or Cry2Ab against laboratory strains highly resistant to those Cry toxins. Five Cry-susceptible field populations were less susceptible to Vip3Aa than a Cry-susceptible laboratory strain (APHIS-S). Relative to APHIS-S, significant resistance to Vip3Aa did not occur in strains selected in the laboratory for > 700-fold resistance to Cry1Ac or both Cry1Ac and Cry2Ab. CONCLUSIONS: Resistance to Cry1Ac and Cry2Ab did not cause strong cross-resistance to Vip3Aa in pink bollworm, which is consistent with predictions based on the lack of shared midgut receptors between these toxins and previous results from other lepidopterans. Comparison of the Bt toxin concentration in plants relative to the median lethal concentration (LC50 ) from bioassays may be useful for estimating efficacy. The moderate potency of Vip3Aa against Cry1Ac- and Cry2Ab-resistant and susceptible pink bollworm larvae suggests that Bt cotton producing this toxin together with novel Cry toxins might be useful as one component of integrated pest management. © 2022 Society of Chemical Industry.

Novel genetic basis of resistance to Bt toxin Cry1Ac in Helicoverpa zea.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis have advanced pest management, but their benefits are diminished when pests evolve resistance. Elucidating the genetic basis of pest resistance to Bacillus thuringiensis toxins can improve resistance monitoring, resistance management, and the design of new insecticides. Here, we investigated the genetic basis of resistance to Bacillus thuringiensis toxin Cry1Ac in the lepidopteran Helicoverpa zea, one of the most damaging crop pests in the United States. To facilitate this research, we built the first chromosome-level genome assembly for this species, which has 31 chromosomes containing 375 Mb and 15,482 predicted proteins. Using a genome-wide association study, fine-scale mapping, and RNA-seq, we identified a 250-kb quantitative trait locus on chromosome 13 that was strongly associated with resistance in a strain of Helicoverpa zea that had been selected for resistance in the field and lab. The mutation in this quantitative trait locus contributed to but was not sufficient for resistance, which implies alleles in more than one gene contributed to resistance. This quantitative trait locus contains no genes with a previously reported role in resistance or susceptibility to Bacillus thuringiensis toxins. However, in resistant insects, this quantitative trait locus has a premature stop codon in a kinesin gene, which is a primary candidate as a mutation contributing to resistance. We found no changes in gene sequence or expression consistently associated with resistance for 11 genes previously implicated in lepidopteran resistance to Cry1Ac. Thus, the results reveal a novel and polygenic basis of resistance.

Managing Fall Armyworm in Africa: Can Bt Maize Sustainably Improve Control?

The recent invasion of Africa by fall armyworm, Spodoptera frugiperda, a lepidopteran pest of maize and other crops, has heightened concerns about food security for millions of smallholder farmers. Maize genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) is a potentially useful tool for controlling fall armyworm and other lepidopteran pests of maize in Africa. In the Americas, however, fall armyworm rapidly evolved practical resistance to maize producing one Bt toxin (Cry1Ab or Cry1Fa). Also, aside from South Africa, Bt maize has not been approved for cultivation in Africa, where stakeholders in each nation will make decisions about its deployment. In the context of Africa, we address maize production and use; fall armyworm distribution, host range, and impact; fall armyworm control tactics other than Bt maize; and strategies to make Bt maize more sustainable and accessible to smallholders. We recommend mandated refuges of non-Bt maize or other non-Bt host plants of at least 50% of total maize hectares for single-toxin Bt maize and 20% for Bt maize producing two or more distinct toxins that are each highly effective against fall armyworm. The smallholder practices of planting more than one maize cultivar and intercropping maize with other fall armyworm host plants could facilitate compliance. We also propose creating and providing smallholder farmers access to Bt maize that produces four distinct Bt toxins encoded by linked genes in a single transgene cassette. Using this novel Bt maize as one component of integrated pest management could sustainably improve control of lepidopteran pests including fall armyworm.

Early Warning of Resistance to Bt Toxin Vip3Aa in Helicoverpa zea.

Evolution of resistance by pests can reduce the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Because of the widespread resistance of Helicoverpa zea to crystalline (Cry) Bt toxins in the United States, the vegetative insecticidal protein Vip3Aa is the only Bt toxin produced by Bt corn and cotton that remains effective against some populations of this polyphagous lepidopteran pest. Here we evaluated H. zea resistance to Vip3Aa using diet bioassays to test 42,218 larvae from three lab strains and 71 strains derived from the field during 2016 to 2020 in Arkansas, Louisiana, Mississippi, Tennessee, and Texas. Relative to the least susceptible of the three lab strains tested (BZ), susceptibility to Vip3Aa of the field-derived strains decreased significantly from 2016 to 2020. Relative to another lab strain (TM), 7 of 16 strains derived from the field in 2019 were significantly resistant to Vip3Aa, with up to 13-fold resistance. Susceptibility to Vip3Aa was significantly lower for strains derived from Vip3Aa plants than non-Vip3Aa plants, providing direct evidence of resistance evolving in response to selection by Vip3Aa plants in the field. Together with previously reported data, the results here convey an early warning of field-evolved resistance to Vip3Aa in H. zea that supports calls for urgent action to preserve the efficacy of this toxin.

Evaluating Cross-Resistance to Cry and Vip Toxins in Four Strains of Helicoverpa armigera With Different Genetic Mechanisms of Resistance to Bt Toxin Cry1Ac.

Evolution of resistance by pests has diminished the efficacy of transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt). In China, where transgenic cotton producing Bt toxin Cry1Ac has been planted since 1997, field control failures have not been reported but the frequency of resistance to Cry1Ac has increased in the cotton bollworm, Helicoverpa armigera. This provides incentive to switch to multi-toxin Bt cotton, which is grown in many other countries. Previous work created four laboratory strains of H. armigera with >100-fold resistance to Cry1Ac, with the genetic basis of resistance known in all but the LF256 strain. Here, we analyzed the genetic basis of resistance in Cry1Ac in LF256 and evaluated cross-resistance of all four strains to three toxins produced by widely planted multi-toxin Bt cotton: Cry1Fa, Cry2Ab, and Vip3Aa. DNA sequencing revealed that LF256 lacked the mutations in three genes (HaTSPAN1, HaABCC2, and HaABCC3) that confer resistance to Cry1Ac in two other strains of H. armigera we analyzed. Together with previous results, the data reported here show that each of the four strains examined has a different genetic basis of resistance to Cry1Ac. Significant positive cross-resistance occurred to Cry1Fa in three of the four strains tested but not to Cry2Ab or Vip3Aa in any strain. Thus, Cry2Ab and Vip3Aa are likely to be especially valuable for increasing the efficacy and durability of Bt cotton against H. armigera populations that have some resistance to Cry1Ac.