Juan Luis Jurat-Fuentes

Helicoverpa zea Boddie (corn earworm or bollworm) is a primary target of transgenic crops expressing insecticidal Cry and Vip3Aa proteins from Bacillus thuringiensis (Bt). Widespread resistance to Cry toxins has increased reliance on Vip3Aa. We evaluated Vip3Aa susceptibility in 71 field populations of H. zea collected across the southeastern USA from 2018 through 2025 using diet-overlay bioassays of F₁ progeny. Vip3Aa1 was used in 2018 and Vip3Aa39 from 2019 through 2025. Median lethal concentrations (LC₅₀) were estimated and resistance ratios calculated relative to two susceptible laboratory colonies, a field-collected susceptible and the commercially available Benzon colony. Resistance ratios depended strongly on the susceptible reference used. Three populations evaluated in 2018 had resistance ratios ≥ 10 relative to the field-collected susceptible colony, whereas all Benzon-referenced resistance ratios were 50 values showed no temporal trend from 2019 through 2025, when all populations were evaluated with Vip3Aa39. These results provide no evidence of a sustained regional decline in Vip3Aa susceptibility in this region, but they highlight the importance of stable susceptible benchmarks, protein quality control, and complementary approaches for long-term resistance monitoring.

For almost three decades, crystal (Cry) insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used as plant-incorporated protectants (PIPs) in transgenic corn, resulting in areawide suppression of the European corn borer (Ostrinia nubilalis). Control of this once-considered most damaging pest of corn in North America is now threatened by the evolution of practical resistance to PIPs in Bt corn. Practical resistance to the Cry1F protein has been detected in eastern Canada, and cases of resistance to the other available Bt PIPs (Cry1Ab, Cry1A.105, and Cry2Ab) are starting to emerge in Canada and the USA. A proactive insect resistance management (IRM) plan greatly benefits from an effective resistance monitoring program. Accordingly, the development and implementation of high-throughput DNA-based resistance monitoring could bring substantial benefits to IRM for Bt corn. In this study, we report the development and testing of a highly multiplexed (HiPlex) targeted sequencing panel amplifying the ABC transporter subfamily C2 gene in O. nubilalis (OnABCC2), a proposed Cry1F receptor altered in resistant O. nubilalis. Using this molecular diagnostic tool, we identified polymorphisms associated with practical O. nubilalis resistance to Cry1F and examined their frequency in O. nubilalis populations collected in four Canadian provinces. The results identify disruptive mutations in OnABCC2 as closely associated with the Cry1F resistance phenotype and support the use of HiPlex targeted sequencing in monitoring for O. nubilalis resistance to Bt PIPs.

Pyramided crops producing Cry and Vip3Aa toxins from Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) play a central role in managing lepidopteran pests in the United States, in part because the major corn and cotton pest Helicoverpa zea Boddie (Lepidoptera: Noctuidea) has evolved practical resistance to all lepidopteran-active Cry toxins produced in transgenic crops. Although several factors affect the evolution of resistance to pyramided crops, the implications of synergy between Cry and Vip3Aa toxins for managing resistance have rarely been considered. Here, we analyzed data from the literature and new data from diet-overlay bioassays of 2 related strains of H. zea susceptible (GA-RS) or resistant (GA-R13) to Cry1Ac and Vip3Aa to evaluate interactions between Cry and Vip3 toxins. Our analysis of 258 cases evaluating interactions between Cry and Vip3 toxins from 16 studies based on artificial-diet bioassays of 18 species of lepidopteran pests shows that synergy occurs more frequently (42.7%) than independent (34.7%) or antagonistic (22.6%) interactions. Our analysis of data from 4 studies of 4 species of lepidopteran pests shows that synergy between Cry1Ab and Vip3Aa typically occurs for pyramided Cry1Ab + Vip3Aa corn. The level of synergy between Cry1Ac and Vip3Aa in diet bioassays was similar for GA-RS and GA-R13, demonstrating that the evolution of resistance to these toxins did not affect synergy. We discuss how considering synergy between Cry and Vip3Aa toxins could improve the monitoring of Vip3Aa resistance in H. zea with sentinel plots and the development of more realistic models to forecast the evolution of resistance to pyramided Bt crops.

The fall armyworm (Spodoptera frugiperda; Lepidoptera: Noctuidae) is a major pest threatening global agriculture. Insecticidal gene silencing by RNA interference (RNAi) induced by a double-stranded RNA (dsRNA) has emerged as a promising strategy for sustainable pest management. However, RNAi efficacy against S. frugiperda is constrained by physiological barriers, including rapid degradation by gut nucleases and reduced cytosolic availability upon dsRNA uptake. Paperclip dsRNA (pcRNA) is a structurally optimized dsRNA that exhibits increased stability and clathrin-independent uptake in mosquito cells. In this study, we evaluated the stability, silencing efficacy, and insecticidal activity of pcRNA in S. frugiperda larvae, compared to conventional linear dsRNA. Ex vivo assays with larval gut fluids and leaf disk bioassays with S. frugiperda larvae showed that, for the same target gene, pcRNA has greater stability and induced significantly higher mortality than dsRNA, hairpin RNA (hpRNA), and small interfering RNA (siRNA). For the same target gene, the pcRNA induced strong, specific gene silencing and significantly higher mortality in diet bioassays compared to negative control treatments (GFP-pcRNA and water) and linear dsRNA. Notably, pcRNAs targeting the membrane septate junction protein (MESH) gene and a multiplexed pcRNA mix targeting chitin synthase 2 (CHS2), acetylcholinesterase 1 (ACE1), and juvenile hormone esterase (JHE) caused more than 50% mortality in diet-based bioassays. Our findings underscore pcRNA multiplexing as a promising platform to increase silencing efficiency and mortality for RNAi-based management of S. frugiperda and highlight the importance of RNA structure and gene target combinations for RNAi efficiency in this pest.

Damage to the lepidopteran larval midgut by insecticidal Cry proteins from Bacillus thuringiensis (Bt) induces molecular signals that activate intestinal stem cell (ISC) proliferation and differentiation, enabling epithelial repair. We previously reported that exposing Chloridea (Heliothis) virescens (tobacco budworm) larvae to the Cry1Ac Bt protein increases production of midgut α-arylphorin, a hexamerin protein. Subsequent reports support a central role for α-arylphorin as a potent mitogen in the regenerative response to Cry intoxication. Here, we examined the effects of feeding purified α-arylphorin to C. virescens larvae on ISC activity and larval susceptibility to Cry1Ac. Purified α-arylphorin stimulated proliferation of primary C. virescens ISC cultures in vitro. Histological analyses revealed that oral ingestion of purified α-arylphorin elicited a hormetic response in C. virescens midgut ISCs. Feeding on lower α-arylphorin concentrations induced dose-dependent ISC proliferation, resulting in a pseudostratified midgut epithelium and reduced C. virescens larval susceptibility to Cry1Ac. In contrast, increasing α-arylphorin concentration failed to stimulate ISC proliferation or reduce susceptibility to Cry1Ac. At the highest α-arylphorin concentrations tested, reduced larval susceptibility occurred in the absence of ISC proliferation. Binding assays suggest that α-arylphorin may interact with Cry1Ac in the midgut lumen and hinder toxin binding to epithelial receptors, explaining reduced larval susceptibility when ISC proliferation is not observed. Together, these findings support a model in which local α-arylphorin titers determine whether protection from Cry1Ac intoxication occurs through ISC proliferation or via α-arylphorin acting as a putative decoy toxin-binding factor.

In Brazilian eucalyptus farms, the brown looper caterpillar (Thyrinteina arnobia) causes severe defoliation and up to 40% reduction in biomass production. In controlling T. arnobia, transgenic eucalyptus event 1521K059 was developed to produce the Cry1Ab, Cry1Bb, and Cry2Aa pesticidal proteins from the bacterium Bacillus thuringiensis. We performed bioassays with leaf material from eucalyptus events and the purified individual pesticidal proteins to establish susceptibility in T. arnobia larvae, and competition binding assays with the three Cry proteins and midgut brush border membrane vesicles of T. arnobia. The results from bioassays support high toxicity against T. arnobia for all three Cry proteins and that eucalyptus event 1521K059 is a high-dose event for T. arnobia. Lack of competition between the three Cry proteins supports that event 1521K059 is a pyramided trait. These observations support the high activity of eucalyptus event 1521K059 against T. arnobia larvae and a low risk of resistance evolution.

Corn genetically engineered to produce insecticidal proteins from Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (ie Bt corn) has toxicity against specific insect pests. However, field-evolved resistance to Bt crops has emerged in several species, including the European corn borer (ECB, Ostrinia nubilalis Hübner). The first case of practical resistance in ECB to Bt corn producing the Cry1F insecticidal protein was documented in 2018 in Nova Scotia, Canada. Six mutations in an ABC transporter gene (OnABCC2) were associated with this resistance; five of these caused nonsynonymous amino acid changes and one causes a two base pair deletion (TA12076-) resulting in a frameshift and premature stop codon. We genotyped 364 ECB individuals collected between 2022 and 2023 across the USA at these six loci and complemented the analysis with targeted OnABCC2 exome sequencing and ECB strain association analysis. Allele frequencies significantly varied across markers and locations. The TA12076- allele was detected in 2023 at a low frequency (0.01) across all sites. Targeted OnABCC2 exome sequencing identified 130 alleles, 41 of them which cause nonsynonymous changes, but no additional indels. Strain genotyping matched previously known ECB strain distributions but showed no association with Bt resistance Our data indicated that the TA12076- allele occurred at low frequency in U.S. populations, consistent with the absence of reported Cry1F field failures, as resistance is recessive. This indel was detected in heterozygous individuals from 3 populations (New Hampshire, Ohio, and Iowa). These observations underscore the importance of efforts monitoring for Bt resistance evolution and spread in ECB populations.