Genome mapping coupled with CRISPR gene editing reveals a P450 gene confers avermectin resistance in the beet armyworm.

The evolution of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm, Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15-16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance.

Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States.

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world's most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest's population size. In Arizona, the program started in 2006 and decreased the pest's estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera.

The insecticidal crystalline (Cry) and vegetative insecticidal (Vip) proteins derived from Bacillus thuringiensis (Bt) are used globally to manage insect pests, including the cotton bollworm, Helicoverpa armigera, one of the world's most damaging agricultural pests. Cry proteins bind to the ATP-binding cassette transporter C2 (ABCC2) receptor on the membrane surface of larval midgut cells, resulting in Cry toxin pores, and ultimately leading to cell swelling and/or lysis. Insect aquaporin (AQP) proteins within the membranes of larval midgut cells are proposed to allow the rapid influx of water into enterocytes following the osmotic imbalance triggered by the formation of Cry toxin pores. Here, we examined the involvement of H. armigera AQPs in Cry1Ac-induced osmotic cell swelling. We identified and characterized eight H. armigera AQPs and demonstrated that five are functional water channel proteins. Three of these (HaDrip1, HaPrip, and HaEglp1) were found to be expressed in the larval midgut. Xenopus laevis oocytes co-expressing the known Cry1Ac receptor HaABCC2 and each of the three HaAQPs displayed abnormal morphology and were lysed following exposure to Cry1Ac, suggesting a rapid influx of water was induced after Cry1Ac pore formation. In contrast, oocytes producing either HaABCC2 or HaAQP alone failed to swell or lyse after treatment with Cry1Ac, implying that both Cry1Ac pore formation and HaAQP function are needed for osmotic cell swelling. However, CRISPR/Cas9-mediated knockout of any one of the three HaAQP genes failed to cause significant changes in susceptibility to the Bt toxins Cry1Ac, Cry2Ab, or Vip3Aa. Our findings suggest that the multiple HaAQPs produced in larval midgut cells compensate for each other in allowing for the rapid influx of water in H. armigera midgut cells following Cry toxin pore formation, and that mutations affecting a single HaAQP are unlikely to confer resistance to Bt proteins.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin.

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

High frequency of ryanodine receptor and cytochrome P450 CYP9A186 mutations in insecticide-resistant field populations of Spodoptera exigua from China.

The beet armyworm, Spodoptera exigua is a global agricultural pest that is polyphagous, highly dispersive, and often difficult to control due to resistance to many insecticides. Previous studies showed that a target site mutation in the S. exigua ryanodine receptor (SeRyR) corresponding to I4743M contributes approximately 20-fold resistance to chlorantraniliprole, whereas a mutation in the cytochrome P450 enzyme CYP9A186 corresponding to F116V confers 200-fold to emamectin benzoate through enhanced metabolic detoxification. Here, high frequencies of mutations were found among six China S. exigua field populations collected from 2016 to 2019 resulting in SeRyR I4743M and CYP9A186 F116V substitutions, with some populations having high levels of resistance to chlorantraniliprole and emamectin benzoate, respectively. Whereas we found a significant correlation between emamectin benzoate resistance level and the allele frequency of CYP9A186 F116V, no significant correlation was found between chlorantraniliprole resistance level and SeRyR I4743M allele frequency in the six field populations. These results suggest that CYP9A186 F116V is a major resistance mechanism for emamectin benzoate in the tested field populations, whereas it is likely that resistance mechanisms other than SeRyR I4743M are responsible for resistance to chlorantraniliprole in the six China field populations. Because of the growing resistance to these two insecticides by S. exigua in China, the use of insecticidal compounds with different modes of action and/or other integrated pest management strategies are needed to further delay the evolution of insecticide resistance and effectively manage S. exigua in China.

A long non-coding RNA regulates cadherin transcription and susceptibility to Bt toxin Cry1Ac in pink bollworm, Pectinophora gossypiella.

Extensive planting of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has spurred increasingly rapid evolution of resistance in pests. In the pink bollworm, Pectinophora gossypiella, a devastating global pest, resistance to Bt toxin Cry1Ac produced by transgenic cotton is linked with mutations in a gene (PgCad1) encoding a cadherin protein that binds Cry1Ac in the larval midgut. We previously reported a long non-coding RNA (lncRNA) in intron 20 of cadherin alleles associated with both resistance and susceptibility to Cry1Ac. Here we tested the hypothesis that reducing expression of this lncRNA decreases transcription of PgCad1 and susceptibility to Cry1Ac. Quantitative RT-PCR showed that feeding susceptible neonates small interfering RNAs (siRNAs) targeting this lncRNA but not PgCad1 decreased the abundance of transcripts of both the lncRNA and PgCad1. Moreover, neonates fed the siRNAs had lower susceptibility to Cry1Ac. The results imply that the lncRNA increases transcription of PgCad1 and susceptibility of pink bollworm to Cry1Ac. The results suggest that disruption of lncRNA expression could be a novel mechanism of pest resistance to Bt toxins.

Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua.

Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to "pyramid" two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.

Doublesex is essential for masculinization but not feminization in Lygus hesperus.

In most holometabolous insects, sex differentiation occurs via a hierarchical cascade of transcription factors, with doublesex (dsx) regulating genes that control sex-specific traits. Although less is known in hemimetabolous insects, early evidence suggests that substantial differences exist from more evolutionarily advanced insects. Here, we identified and characterized dsx in Lygus hesperus (western tarnished plant bug), a hemipteran pest of many agricultural crops in western North America. The full-length transcript for L. hesperus dsx (Lhdsx) and several variants encode proteins with conserved DNA binding and oligomerization domains. Transcript profiling revealed that Lhdsx is ubiquitously expressed, likely undergoes alternative pre-mRNA splicing, and, unlike several model insects, is sex-biased rather than sex-specific. Embryonic RNA interference (RNAi) of Lhdsx only impacted sex development in adult males, which lacked both internal reproductive organs and external genitalia. No discernible impacts on adult female development or reproductivity were observed. RNAi knockdown of Lhdsx in nymphs likewise only affected adult males, which lacked the characteristic dimorphic coloration but had dramatically elevated vitellogenin transcripts. Gene knockout of Lhdsx by CRISPR/Cas9 editing yielded only females in G0 and strongly biased heterozygous G1 offspring to females with the few surviving males showing severely impaired genital development. These results indicate that L. hesperus male development requires Lhdsx, whereas female development proceeds via a basal pathway that functions independently of dsx. A fundamental understanding of sex differentiation in L. hesperus could be important for future gene-based management strategies of this important agricultural pest.

ß-tubulin functions in spermatogenesis in Lygus hesperus Knight.

Lygus hesperus Knight is an important insect pest of crops across western North America, with field management heavily reliant on the use of chemical insecticides. Because of the evolution of resistance to these insecticides, effective and environmentally benign pest management strategies are needed. Traditional sterile insect technique (SIT) has been successfully employed to manage or eradicate some insect pests but involves introducing irradiated insects with random mutations into field populations. New genetically-driven SIT techniques are a safer alternative, causing fixed mutations that manipulate individual genes in target pests to produce sterile individuals for release. Here, we identified seven ß-tubulin coding genes from L. hesperus and show that Lhßtub2 is critical in male sperm production and fertility. Lhßtub2 is expressed primarily in the male testes and targeting of this gene by RNA interference or gene editing leads to male sterility.

An initial event in the insect innate immune response: structural and biological studies of interactions between ß-1,3-glucan and the N-terminal domain of ß-1,3-glucan recognition protein.

In response to invading microorganisms, insect ß-1,3-glucan recognition protein (ßGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of ßGRP (N-ßGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-ßGRP with laminarihexaose, a glucose hexamer containing ß-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing ß-1,3 and ß-1,6 links that activates the proPO pathway, to N-ßGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-ßGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-ßGRP-laminarin complex in solution differs from the one in which a single N-ßGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-ßGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-ßGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-ßGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of ß-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the ßGRP-ß-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway.

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.

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 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.

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.

Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton.

The pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is a major global pest of cotton. Current management practices include chemical insecticides, cultural strategies, sterile insect releases, and transgenic cotton producing crystalline (Cry) protein toxins of the bacterium Bacillus thuringiensis (Bt). These strategies have contributed to the eradication of P. gossypiella from the cotton-growing areas of the United States and northern Mexico. However, this pest has evolved resistance to Bt cotton in Asia, where it remains a critical pest, and the benefits of using transgenic Bt crops have been lost. A complete annotated reference genome is needed to improve global Bt resistance management of the pink bollworm. We generated the first chromosome-level genome assembly for pink bollworm from a Bt-susceptible laboratory strain (APHIS-S) using PacBio continuous long reads for contig generation, Illumina Hi-C for scaffolding, and Illumina whole-genome re-sequencing for error correction. The pseudo-haploid assembly consists of 29 autosomes and the Z sex chromosome. The assembly exceeds the minimum Earth BioGenome Project quality standards, has a low error rate, is highly contiguous at both the contig and scaffold levels (L/N50 of 18/8.26 MB and 14/16.44 MB, respectively), and is complete, with 98.6% of lepidopteran single-copy orthologs represented without duplication. The genome was annotated with 50% repeat content and 14,107 protein-coding genes, further assigned to 41,666 functional annotations. This assembly represents the first publicly available complete annotated genome of pink bollworm and will serve as the foundation for advancing molecular genetics of this important pest species.

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.

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug.

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

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.

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.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.