BACKGROUND Diet‐overlay bioassays suggest that Helicoverpa zea (Lepidoptera: Noctuidae) field populations have developed resistance to some of the Bt insecticidal proteins that are constituents of the pyramids expressed in the second and third generation Bt cotton technologies. Unfortunately, these bioassays are not always a reliable indicator for how a seemingly resistant population will perform in an actual cotton field, and thus, leaf tissue bioassays have been suggested as a method to better assess field performance. However, bollworm larvae typically prefer to feed on floral tissue rather than leaf tissue, and an alternative cotton structure type may be more ideal for use in plant tissue‐based bioassays. A series of diet‐overlay bioassays using Bt proteins and Bt cotton plant tissue were conducted with laboratory susceptible (Bz‐SS) and resistant (Cry‐RR, resistant to Cry1Ac and Cry2Ab) H. zea strains to determine if plant tissue overlays could detect resistance and which cotton plant structure type would be most ideal for use in bioassays. RESULTS Results suggest that diet overlays using lyophilized plant tissue were able to detect resistance. Lyophilized tissue from white flowers was most ideal for use in bioassays, whereas tissue from non‐Bt bolls and leaves affected larval health and behavior, confounding assay results. CONCLUSION Overlays using white flower tissue could potentially be used to supplement Bt protein overlays and provide an improved assessment of larval performance on Bt cotton technologies. © 2021 Society of Chemical Industry.

Transgenic corn and cotton expressing Cry and Vip insecticidal proteins from the bacterium,Bacillus thuringiensis(Bt), have been a valuable tool for the management of lepidopteran pests. In 2019, a Vip3Aa-resistant strain ofHelicoverpa zea(CEW-Vip-RR) was isolated from F2screens of field populations in Texas. Characterizing the resistance mechanism in this strain is important for predicting the sustained efficacy of current commercial Bt traits and guiding the development of future transgenic traits. Resistance to insecticidal proteins in Bt traits is commonly associated with reduced toxin binding, with the exception of Vip3Aa resistance being associated to altered proteolytic processing in the insect host gut. Therefore, Vip3Aa protoxin processing was tested by incubation with midgut fluids from CEW-Vip-RR relative to a susceptible strain (CEW-SS). Finding no significant processing differences, alterations in Vip3Aa binding were tested by comparing binding of radiolabeled and biotinylated Vip3Aa toxin to midgut brush border membrane vesicles (BBMV) from CEW-Vip-RR and CEW-SS larvae. Specific Vip3Aa binding to CEW-Vip-RR BBMV in these experiments was consistently reduced when compared with CEW-SS BBMV. These results support that an altered Vip3Aa-receptor is associated with resistance in CEW-Vip-RR. Understanding this resistance mechanism could have important implications for resistance management decisions considering widespread Cry1 and Cry2 resistance inH. zeapopulations.

Foliar-applied insecticide treatments may be necessary to manage thrips in cotton (Gossypium hirsutum L.) under severe infestations or when at-planting insecticide seed treatments do not provide satisfactory protection. The most common foliar-applied insecticide is acephate. Field observations in Tennessee suggest that the performance of acephate has declined. Thus, the first objective was to perform leaf-dip bioassays to assess if tobacco thrips, Frankliniella fusca (Hinds) (Thysanoptera: Thripidae), in cotton production regions have evolved resistance to foliar-applied insecticides. A second objective was to assess the performance of commonly applied foliar insecticides for managing thrips in standardized field trials in Arkansas, Tennessee, Mississippi, and Texas. For both objectives, several insecticides were evaluated including acephate, dicrotophos, dimethoate, lambda-cyhalothrin, imidacloprid, and spinetoram. Field trials and bioassays were completed from 2018 to 2021. Dose-response bioassays with acephate were performed on tobacco thrips field populations and a susceptible laboratory population. Bioassay results suggest that tobacco thrips have developed resistance to acephate and other organophosphate insecticides; however, this resistance seems to be most severe in Arkansas, Tennessee, and the Delta region of Mississippi. Resistance to other classes of insecticides were perhaps even more evident in these bioassays. The performance of these insecticides in field trials was variable, with tobacco thrips only showing consistent signs of resistance to lambda-cyhalothrin. However, it is evident that many populations of tobacco thrips are resistant to multiple classes of insecticides. Further research is needed to determine heritability and resistance mechanism(s).

Previous studies have indicated that the expression of insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) in cotton can have a significant influence on the behavior of bollworm larvae (Helicoverpa zea, Lepidoptera:Noctuidae). This suggests that the particular Bt protein produced by a cotton variety may need to be considered when determining the most ideal scouting methods to utilize for bollworm. NonBt, WideStrike (producing Cry1Ac + Cry1F Bt insecticidal proteins), and Bollgard II (Cry1Ac + Cry2Ab) cotton varieties were planted and either treated with an insecticide or left untreated. The presence of H. zea larvae and their feeding injury were recorded according to their location in the canopy and type of floral structure where they were found. Results from comparison of larval and injury distributions indicated no significant differences between the different cotton varieties tested, and that insecticide treatment had minimal impact on this distribution. Larval size was generally associated with location in the canopy, suggesting that larvae tend to move towards the middle of the canopy as they age. The effect of different Bt cotton technologies appears to associate with how quickly larvae move to preferred feeding sites rather than their preference for particular feeding sites. These results suggest that scouting methods could be standardized independently of the presence of a Bt cotton trait or previous insecticide application. Focusing scouting efforts on the middle portion of the canopy (i.e., nodes 6-9) should increase the detection of small larvae and ‘fresh’ injury and be less influenced by previous insecticide applications.

The wide occurrence of resistance to Cry1A and Cry2A insecticidal toxins from Bacillus thuringiensis (Bt) in the corn earworm/bollworm Helicoverpa zea (Boddie) leaves the Vip3A toxin produced during the vegetative stage of Bt as the only fully active toxin expressed in transgenic crops to control H. zea in the U.S.A. During 2021, the first unexpected survival of H. zea and injury (UXI) on a maize hybrid expressing Cry1A.105, Cry2Ab2, and Vip3Aa in Louisiana, U.S.A. were observed in two sentinel plots used for resistance monitoring. A follow-up intensive investigation was conducted with two H. zea populations established from larvae collected from the two UXI plots. The main goal of this study was to reveal if the unexpected damage was due to resistance development in the insect to the Bt toxins expressed in the maize hybrid. Diet-overlay bioassays showed that the two populations were highly resistant to Cry1A.105, moderately resistant to Cry2Ab2, but still highly susceptible to Vip3Aa when compared to a reference susceptible strain. In 10 d assays with detached ears, the larvae of the two UXI populations exhibited survival on ears expressing only Cry toxins but presented near 100% mortality on maize hybrids containing both cry and vip3A transgenes. Multiple field trials over three years demonstrated that natural H. zea populations in Louisiana were highly resistant to maize expressing only Cry toxins but remained susceptible to all tested hybrids containing cry and vip3A genes. Altogether, the results of this study suggest that the observed UXIs in Louisiana were associated with a resistance to Cry toxins but were not due to a resistance to Vip3A. The possible causes of the UXIs are discussed. The results generated and procedures adopted in this study help in determining thresholds for defining UXIs, assessing resistance risks, and documenting field resistance.