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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., (© 2023. The Author(s).)

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., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

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., (© 2022. The Author(s).)

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Evolution of pest resistance threatens the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Field populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton, have evolved practical resistance to transgenic cotton producing Bt toxin Cry2Ab in India, but not in the United States. Previous results show that recessive mutations disrupting an autosomal ATP-binding cassette gene (PgABCA2) are associated with pink bollworm resistance to Cry2Ab in field-selected populations from India and in one lab-selected strain from the United States (Bt4-R2). Here we discovered that an independently derived, lab-selected Cry2Ab-resistant pink bollworm strain from the United States (BX-R) also harbors mutations that disrupt PgABCA2. Premature stop codons introduced by mis-splicing of PgABCA2 pre-mRNA were prevalent in field-selected larvae from India and in both lab-selected strains. The most common mutation in field-selected larvae from India was also detected in both lab-selected strains. Results from interstrain crosses indicate BX-R has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. We conclude that recessive mutations disrupting PgABCA2 are the primary, but not the only, mechanism of resistance to Cry2Ab in pink bollworm.

Nitrogen (N) plays a key role in plants because it is a major component of RuBisCO and chlorophyll. Hence, N is central to both the dark and light reactions of photosynthesis. Genotypic variation in canopy greenness provides insights into the variation of N and chlorophyll concentration, photosynthesis rates, and N 2 fixation in legumes. The objective of this study was to identify significant loci associated with the intensity of greenness of the soybean [Glycine max (L.) Merr.] canopy as determined by the Dark Green Color Index (DGCI). A panel of 200 maturity group IV accessions was phenotyped for canopy greenness using DGCI in three environments. Association mapping identified 45 SNPs that were significantly (P ≤ 0.0003) associated with DGCI in three environments, and 16 significant SNPs associated with DGCI averaged across all environments. These SNPs likely tagged 43 putative loci. Out of these 45 SNPs, eight were present in more than one environment. Among the identified loci, 21 were located in regions previously reported for N traits and ureide concentration. Putative loci that were coincident with previously reported genomic regions may be important resources for pyramiding favorable alleles for improved N and chlorophyll concentrations, photosynthesis rates, and N 2 fixation in soybean.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are cultivated extensively, but rapid evolution of resistance by pests reduces their efficacy. We report a 3,370-bp insertion in a cadherin gene associated with resistance to Bt toxin Cry1Ac in the pink bollworm (Pectinophora gossypiella), a devastating global cotton pest. We found the allele (r15) harboring this insertion in a field population from China. The insertion is a miniature inverted repeat transposable element (MITE) that contains two additional transposons and produces two mis-spliced transcript variants (r15A and r15B). A strain homozygous for r15 had 290-fold resistance to Cry1Ac, little or no cross-resistance to Cry2Ab, and completed its life cycle on Bt cotton producing Cry1Ac. Inheritance of resistance was recessive and tightly linked with r15. For transformed insect cells, susceptibility to Cry1Ac was greater for cells producing the wild-type cadherin than for cells producing the r15 mutant proteins. Recombinant cadherin protein occurred on the cell surface in cells transformed with the wild-type or r15A sequences, but not in cells transformed with the r15B sequence. The similar resistance of pink bollworm to Cry1Ac in laboratory- and field-selected insects from China, India and the U.S. provides a basis for developing international resistance management practices.

Foraging exposes organisms to rewarding and aversive events, providing a selective advantage for maximizing the former while minimizing the latter. Honey bees (Apis mellifera) associate environmental stimuli with appetitive or aversive experiences, forming preferences for scents, locations, and visual cues. Preference formation is influenced by inter-individual variation in sensitivity to rewarding and aversive stimuli, which can be modulated by pharmacological manipulation of biogenic amines. We propose that foraging experiences act on biogenic amine pathways to induce enduring changes to stimulus responsiveness. To simulate varied foraging conditions, freely-moving bees were housed in cages where feeders offered combinations of sucrose solution, floral scents, and aversive electric shock. Transient effects were excluded by providing bees with neutral conditions for three days prior to all subsequent assays. Sucrose responsiveness was reduced in bees that had foraged for scented rather than unscented sucrose under benign conditions. This was not the case under aversive foraging conditions, suggesting an adaptive tuning process which maximizes preference for high quality, non-aversive floral sites. Foraging conditions also influenced antennal lobe octopamine and serotonin, neuromodulators involved in stimulus responsiveness and foraging site evaluation. Our results suggest that individuals' foraging experiences durably modify neurochemistry and shape future foraging behaviour.

Bt cotton was genetically modified to produce insecticidal proteins targeting Lepidopteran pests and is therefore only minimally affected by caterpillar damage. This could lead to reduced levels of inherent, systemically inducible defensive compounds in Bt cotton which might benefit other important cotton herbivores such as plant bugs. We studied the effects of plant defense induction on the performance of the plant bug Lygus hesperus by caging nymphs on different food sources (bolls/squares) of Bt and non-Bt cotton which were either undamaged, damaged by Bt tolerant caterpillars, or treated with jasmonic acid (JA). Terpenoid induction patterns of JA-treated and L. hesperus-damaged plants were characterized for different plant structures and artificial diet assays using purified terpenoids (gossypol/heliocide H1/4) were conducted. Nymphs were negatively affected if kept on plants damaged by caterpillars or sprayed with JA. Performance of nymphs was increased if they fed on squares and by the Bt-trait which had a positive effect on boll quality as food. In general, JA-sprayed plants (but not L. hesperus infested plants) showed increased levels of terpenoids in the plant structures analyzed, which was especially pronounced in Bt cotton. Nymphs were not negatively affected by terpenoids in artificial diet assays indicating that other inducible cotton responses are responsible for the found negative effects on L. hesperus. Overall, genetically engineered plant defenses can benefit plant bugs by releasing them from plant-mediated indirect competition with lepidopterans which might contribute to increasing numbers of hemipterans in Bt cotton.

Socially exchanged fluids are a direct means by which an organism can influence conspecifics. It was recently shown that when workers of the carpenter ant Camponotus floridanus feed larval offspring via trophallaxis, they transfer Juvenile Hormone III (JH), a key developmental regulator, as well as paralogs of JH esterase (JHE), an enzyme that catalyzes the hydrolysis of JH. Here we combine proteomic, phylogenetic and selection analyses to investigate the evolution of this esterase subfamily. We show that Camponotus JHE-like proteins have undergone multiple duplications, experienced positive selection, and changed tissue localization to become abundantly and selectively present in trophallactic fluid. The Camponotus trophallactic esterases have maintained their catalytic triads and contain a number of positively-selected amino acid changes distributed throughout the protein, which possibly reflect an adaptation to the highly acidic trophallactic fluid of formicine ants. To determine whether these esterases might regulate larval development, we fed workers with a JHE-specific pharmacological inhibitor to introduce it into the trophallactic network. This inhibitor increased the likelihood of pupation of the larvae reared by these workers, similar to the influence of food supplementation with JH. Together, these findings suggest that JHE-like proteins have evolved a new role in the inter-individual regulation of larval development in the Camponotus genus.

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.

Drought is the No. 1 factor that limits agricultural production in the world, thus, making crops more drought tolerant is a major goal in agriculture. Many genes with functions in abiotic stress tolerance were identified, and overexpression of these genes confers increased drought tolerance in transgenic plants. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis is one of them. Interestingly, when IPT-transgenic cotton was field-tested at two different sites, Texas and Arizona, different results were obtained. To explain this phenomenon, reduced irrigation experiments with different timing in applying water deficit stress were conducted. It was found that the timing of water deficit stress is critical for IPT-transgenic cotton to display its yield advantage over control plants (i.e. wild-type and segregated non-transgenic plants). If water deficit stress occurs before flowering (vegetative phase), IPT-transgenic cotton would outperform control plants; however, if water deficit stress occurs at or after flowering (reproductive phase), there would not be a yield difference between IPT-transgenic and control cotton plants. This result suggests that an early induction of IPT expression (before first flowering) is needed in order to realize the benefits of IPT-expression in transgenic plants that face water-deficit stress later in development.

Cabbage looper, Trichoplusia ni (Hübner) is an important lepidopteran pest on many vegetable and greenhouse crops, and some field crops. Although there are no commercial transgenic Bt vegetable or greenhouse crops, T. ni is a target of Bollgard II cotton, which produces Cry1Ac and Cry2Ab. We expand on previous work that examined the effect of Bt crops on parasitoids using Bt-resistant lepidopteran populations as hosts. Cry1Ac/Cry2Ab-resistant T. ni larvae were used to eliminate host quality effects and to evaluate the direct effects of Bt cotton on the parasitoids Copidosoma floridanum (Ashmead) and Cotesia marginiventris (Cresson). These tri-trophic studies confirm that Bt cotton had no significant impact on development, success of parasitism, survival and adult longevity of C. marginiventris when using Bt-resistant T. ni fed on Bt cotton. Similarly, this Bt cotton had no significant impact on the development, mummy weight and the number of progeny produced by C. floridanum. Our studies verified that lyophilized Bt crop tissue maintained its insecticidal bioactivity when incorporated into an artificial diet, demonstrating that hosts and parasitoids were exposed to active Cry proteins. The egg-larval parasitoid C. floridanum, or similar species that consume their entire host, should be considered useful surrogates in risk assessment of Bt crops to non-target arthropods.

Transcriptional coordination is a vital process contributing to metabolic homeostasis. As one of the key nodes in the metabolic network, the forkhead transcription factor FOXO has been shown to interact with diverse transcription co-factors and integrate signals from multiple pathways to control metabolism, oxidative stress response, and cell cycle. Recently, insulin/FOXO signaling has been implicated in the regulation of insect development via the interaction with insect hormones, such as ecdysone and juvenile hormone. In this study, we identified an interaction between Drosophila FOXO (dFOXO) and the zinc finger transcription factor Kruppel homolog 1 (Kr-h1), one of the key players in juvenile hormone signaling. We found that Kr-h1 mutants show delayed larval development and altered lipid metabolism, in particular induced lipolysis upon starvation. Notably, Kr-h1 physically and genetically interacts with dFOXO in vitro and in vivo to regulate the transcriptional activation of insulin receptor (InR) and adipose lipase brummer (bmm). The transcriptional co-regulation by Kr-h1 and dFOXO may represent a broad mechanism by which Kruppel-like factors integrate with insulin signaling to maintain metabolic homeostasis and coordinate organism growth.

In insect societies, both queens and workers produce chemicals that reliably signal caste membership and reproductive status. The mechanisms that help to maintain the honesty of such queen and fertility signals, however, remain poorly studied. Here we test if queen signal honesty could be based on the shared endocrine control of queen fertility and the production of specific signals. In support of this "hormonal pleiotropy" hypothesis, we find that in the common wasp, application of methoprene (a juveline hormone analogue) caused workers to acquire a queen-like cuticular hydrocarbon profile, resulting in the overproduction of known queen pheromones as well as some compounds typically linked to worker fertility. By contrast, administration of precocene-I (a JH inhibitor) had a tendency to have the opposite effect. Furthermore, a clear gonadotropic effect of JH in queens was suggested by the fact that circulating levels of JH were ca. 2 orders of magnitude higher in queens than those in workers and virgin, non-egg-laying queens, even if methoprene or precocene treatment did not affect the ovary development of workers. Overall, these results suggest that queen signal honesty in this system is maintained by queen fertility and queen signal production being under shared endocrine control.

Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India., Competing Interests: This is a cooperative investigation between USDA ARS, the University of Arizona, and DuPont-Pioneer with J.A.F., B.E.T. and Y.C. receiving partial funding from DuPont-Pioneer to support this work (agreement #58-3K95-4-1666). J.A.F. is coauthor of a patent "Cadherin Receptor Peptide for Potentiating Bt Biopesticides" (patent numbers: US20090175974A1, US8354371, WO2009067487A2, WO2009067487A3). B.E.T. is a coauthor of a patent on modified Bt toxins, "Suppression of Resistance in Insects to Bacillus thuringiensis Cry Toxins, Using Toxins that do not Require the Cadherin Receptor" (patent numbers: CA2690188A1, CN101730712A, EP2184293A2, EP2184293A4, EP2184293B1, WO2008150150A2, WO2008150150A3). Bayer CropScience, Dow AgroSciences, Monsanto, and Syngenta did not provide funding to support this work, but may be affected financially by publication of this paper and have funded other work by some of the authors.

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.