Location: Southern Insect Management Research
2024 Annual Report
Objectives
1. Determine ecological characteristics and insect-plant interactions, such as susceptibility and fitness, to help identify components that can be manipulated to minimize the evolution of resistance to Bt toxins in Helicoverpa zea.
1.A. Determine the status of Bt resistance in H. zea and develop methods to measure the subsequent fitness costs associated with survival on Bt crops relative to other wild and cultivated hosts.
1.B. Evaluate the impact of Bt corn as a primary host on subsequent H. zea damage and fitness on Bt cotton.
2. Characterize genomic, transcriptomic, and population genetic components of H. zea relative to their contribution to evolution of resistance to Bt toxins and develop computational methods for identifying interactions between gene co-regulatory networks that modulate resistance loci.
2.A. Develop computational methods to identify any gene regulatory networks that interact in responding to intoxication with Bt toxins in H. zea.
2.B. Characterize genomic, transcriptomic, and population genetic components of H. zea relative to their contribution to evolution of resistance to Bt toxins.
3. Develop and optimize early detection methods for invasive polyphagous pests of cotton.
Approach
Recent failures of transgenic crops producing Bacillus thuringiensis (Bt) insecticidal toxins to control noctuid pests and reports of field-evolved resistance to Bt toxins indicate an increase in tolerance to certain Bt toxins in pest populations. To gain a better understanding of factors contributing to the evolution of resistance and to identify components that could be manipulated to minimize the development of resistance to Bt toxins, ecological characteristics of bollworm (BW), Helicoverpa zea, and its interactions with host plants will be investigated. The long-term objective of this project is to identify ecologically sustainable approaches and develop new strategies for the efficient management of BW resistance to Bt toxins. A large-scale Bt resistance survey will be conducted by collecting BW populations from a range of wild and crop hosts across the southern U.S. Progeny from these populations will be assayed using discriminating doses of Bt toxins that compare susceptibility of field insects with control insects from laboratory colonies. This survey will serve as a basis for quantifying the incidence of Bt resistant in BW in areas where Bt corn and cotton production coexists. The fitness parameters will examine the impacts of Bt crops on tolerant insects and fitness of their offspring. This information will assist in determining the status of susceptibility of BW to Bt crops in the southern US. Impacts of Bt corn as a primary host of BW on subsequent damage and fitness on Bt cotton will be assessed using correlations between Bt toxin levels in kernels and larval survival on Bt field corn. Toxin levels in kernels and larval survival on different Bt corn hybrids will facilitate the inference of selection pressure on BW by Bt corn, which will be vital for developing insect resistance management (IRM) strategies in Bt cotton. Contribution of genetic components to Bt toxin resistance evolution will be studied using empirical and computational methods. Genetic loci linked Bt resistance will be evaluated using computational methods such as weighted gene co-regulatory network analysis to predict interactions between gene co-regulatory networks that modulate resistance. Genetic loci predicted to have a high probability of participation in modulating mode of action of Bt toxins will be used in quantitative, comparative, and population genetic studies to evaluate their roles in to Bt toxin resistance. This approach is expected to identify novel genetic loci involved in the toxin mode of action and those contributing to resistance to Bt toxins. This project will also develop novel technologies or improve upon those currently available to facilitate rapid detection of invasive pests. Species-specific antibody-based lateral flow immune assays (LFIA) will be used for rapid identification of species. When LFIA is not possible due to lack of species-specific antigens (targets) in proteins to develop antibodies, isothermal recombinase polymerase amplification (RPA) technology that can amplify species-specific DNA tagged with artificial antigens will be used to detect invasive species.
Progress Report
Substantial progress on laboratory and field research components was made during the past year by building upon the research accomplishments of the past few years. On research related to Objective 1: Determine ecological characteristics and insect-plant interactions, such as susceptibility and fitness, to help identify components that can be manipulated to minimize the evolution of resistance to Bacillus thuringiensis (Bt) toxins in Helicoverpa zea, scientists in Stoneville, MS, evaluated the relative expression of two crystalline Bt proteins present in a popular, transgenic cotton variety. Plant reproductive parts across development were screened, including blooms, squares (young fruits), and bolls.
As expected, both proteins were positively correlated with bollworm larval mortality and negatively correlated with penetration of all three tissues by larvae. Protein optical density, a standard measure of protein concentration, accurately predicted bollworm larval mortality. Techniques developed in this study will also be applied to Vip3A, a third-generation Bt protein. Future work will help us understand bollworm larval mortality in transgenic cotton varieties carrying the Vip3A gene recently released in the United States.
Sampling of wild host plants for corn earworm and tobacco budworm was continued. Non-cultivated plants can host cotton pests thus allowing the pests to increase in numbers without being exposed to Bt cultivars. This arrangement helps reduce the spread of Bt resistance in the environment. These reservoirs increase the frequency of susceptible genes, reducing the long-term risk of Bt resistance. Crimson clover continues to be a key host for early-season populations of corn earworm, but it had exceptionally low populations of tobacco budworm. Hophornbeam copperleaf harbored mixed populations of each species from late June through the fall harvest season. We demonstrated that non-crop host plants are a valuable safeguard in resistance management.
We also sampled non-cotton crop hosts. Various corn hybrid lines (non-Bt and Bt) are planted annually in April and May in cooperation with collaborators. Fields are monitored for insect survival and damage to kernels to assess insect fitness. This data will be used to understand the impact of Bt corn variety choice on bollworm survival in cotton.
Bollworms were also assayed for survival on various fruiting structures of Bt cottons expressing contrasting Bt protein combinations. This research quantifies the economic benefit of stacked Bt varieties for growers. Even non-lethal effects of Bt toxins on bollworm growth and development can provide economic benefit. We are also exploring this research area. Newer, vegetatively produced toxins, such as Vip3A, might impact insect fitness differently than toxins expressed in the reproductive parts. Our work on third-generation toxins will guide the development of future insect resistance management strategies.
Some bollworm larvae can survive exposure to Bt toxins, particularly those with decreased susceptibility. How does survival after exposure to Bt alter interactions of pests with other insect species? We studied how non-deadly (sublethal) exposure to Bt impacts interactions between bollworms and lady bugs (the convergent lady beetle), a predatory natural enemy. Predatory natural enemies can reduce pest densities in Bt and non-Bt cotton fields. However, we did not know how Bt toxins affected this predator-prey relationship. The sources of Bt included a commercially available sprayable product and transgenic cotton flowers. We chose Bt cotton flowers because they have long been expected to be a weak point for plant protection. For instance, previous reports have documented bollworm larvae survival in blooms.
Our research indicated that sublethal levels of exposure to Bt can increase predation from the convergent lady beetle. The increased predation is potentially due to the bollworm larvae’s reduced size and development following sublethal exposure to Bt. Next, we will focus on understanding how various sources of nutrition impact sublethal effects of Bt and insect interactions in Bt cotton.
Genetic analysis of bollworm populations across the United States was continued under the Objective 2: Characterize genomic, transcriptomic, and population genetic components of H. zea relative to their contribution to the evolution of resistance to Bt toxins and develop computational methods for identifying interactions between gene co-regulatory networks that modulate resistance loci. ARS researchers at Stoneville, Mississippi, collaborated with university partners at the University of Texas at Tyler, Texas, to develop an interface to facilitate gene network analysis. Gene network analysis uses advanced statistical techniques to understand how genes relate to one another in living organisms. This technique is an ideal approach for understanding pest resistance mechanisms and how to defeat them.
ARS researchers at Stoneville, Mississippi, conducted population genetic studies of native bollworm populations from six states: Colorado, Florida, New York, Pennsylvania, Texas, and Florida. Screening of more than 2000 insects identified cross-species transfer (introgression) of genes from invasive old-world bollworm, Helicoverpa armigera into native bollworm H. zea.
Alarmingly high rates of an old-world bollworm gene that gives insects resistance to pyrethroid insecticides (CYP337B3) were detected in native bollworm populations. We concluded that hybridization (inter-mating) of these two Helicoverpa species is rapidly increasing. A variant of the CYP337B3 gene that was previously reported exclusively in Africa was also identified about 38% of the native bollworm carrying the CYP337B3 gene. Therefore, at least two populations of old-world bollworm have transferred the CYP337B3 gene to native bollworm. Continued genetic monitoring of old-world bollworm and native bollworm is crucial for resistance management in United States cotton.
Accomplishments
1. Documented bollworm larval mortality on various fruiting structures of transgenic cotton. The relative expression of two crystalline proteins present in squares, blooms, and bolls of a Bollgard II cotton variety was determined over time, and the effects of these proteins on bollworm survival were evaluated. Both crystalline proteins were positively correlated with bollworm larval mortality and negatively correlated with larval penetration across all fruiting structures tested. ARS researchers at Stoneville, Mississippi, successfully utilized optical density measurements to quantify the effects of protein expression on bollworm larval mortality in various cotton fruiting structures. Techniques developed during this study will next be used to understand the contribution of Vip3A to bollworm larval mortality in newer, third-generation transgenic cottons recently released in the United States.
2. Documented an important natural refuge of corn earworm and tobacco budworm in the Southern United States. In the Southern United States, tobacco budworm and corn earworm are the two primary caterpillar pests controlled by crop plants expressing insecticidal toxins and synthetic insecticide applications. Both insect species have developed resistance to synthetic insecticides, and the survival of corn earworms on some transgenic crop varieties is reportedly higher than when these crops were introduced. One of the key elements to combat the development of resistance for these insects is to ensure that a portion of the insect population is unexposed to the insecticidal toxins in a “refuge.” This makes susceptible insects available to mate with potentially resistant moths that survive insecticide exposure as nymphs. A seven-year study examining the suitability of wild host plants for both corn earworm and tobacco budworm was completed by ARS researchers at Stoneville, Mississippi. Hophornbeam copperleaf was found to harbor populations of both insects in high densities during the summer and through the fall. This is a key period of insecticide exposure, and hophornbeam copperleaf appears to be an essential wild host that contributes to the delay in resistance. The plant inhabits ditch banks and other non-cropland, and the preservation and utilization of this wild host can aid in delaying resistance development in both insect pests.
3. Demonstrated sublethal effects of Bt on the native bollworm, including impacts on predation from a natural enemy. A set of experiments was completed by ARS researchers at Stoneville, Mississippi, to a) evaluate the sublethal effects of Bt on bollworm larvae and b) determine how this impacted predation from the Convergent lady beetle, a common natural predator in cotton fields of the southern United States. Results showed significant sublethal effects caused by Bt proteins incorporated into an artificial diet and from Bt cotton flowers. Sublethal effects of Bt were found to significantly increase the duration in which a bollworm larva was susceptible to predation from the convergent lady beetle. These findings demonstrate how the sublethal effects Bt can alter pest suppression from biological control agents in Bt cotton.
4. Documented transfer of genes responsible for insecticide resistance from invasive old-world bollworm to native bollworm. The invasive old-world bollworm (Helicoverpa armigera) was discovered in South America in 2013 and is known to carry genes conferring resistance to insecticides. Bollworm control failure in Olathe, Colorado prompted an investigation to screen for genes specific to old-world bollworm in native bollworms (Helicoverpa zea). Analysis of DNA by ARS researchers at Stoneville, Mississippi, from native bollworm populations from Colorado, Florida, New York, Pennsylvania, Texas, and Virginia identified the presence of old-world bollworm genes responsible for pyrethroid resistance in the native bollworm at frequencies as high as 28%. The findings were communicated to stakeholders, growers, and state and federal regulatory agencies.
5. Characterized the impacts of fungal endophytes on a cotton pest. The effect of fungal endophytes (microbes that live inside of plants) on cotton was examined. ARS researchers at Stoneville, Mississippi, measured cotton yield and plant growth parameters at distinct plant growth stages. The ability of these fungi to harm insects was studied in diet cup bioassays using Lygus lineolaris (tarnished plant bug) adults. The behavior of tarnished plant bug adult males and females toward endophytic cotton squares was analyzed using olfactometer assays. Field experiments studied the effect of fungal endophytes on feeding and infestation rates of L. lineolaris and Helicoverpa zea under field conditions and how it impacts cotton yield. Experiments showed that the fungal endophytes colonized multiples structures in cotton plants. Plants with the endophytic fungus had an increase in the number of cotton squares, plant height, and weight compared to control plants. Beauveria bassiana strains/isolates such as GHA, NI-8, and JG-1 showed significant mortality in Lygus adults compared to controls. Also, male and female Lygus adults exhibited repellence behavior towards endophytic cotton squares containing JG-1 isolate of B. bassiana and to other B. bassiana strains such as NI-8, GHA, and SPE-120. Our results support the further study of fungal endophytes as a biological control for insect pests on cotton.
Review Publications
Sandhi, R.K., Briar, S.S., Reddy, G.V. 2024. Recent advancements in use of entomopathogens and nematophagous mites for management of plant parasitic nematodes. Sustainability in Plant and Crop Production. 02:151-182. https://doi.org/10.1007/978-3-031-52557-5_6.
Sharma, A., Mendu, V., Reddy, G.V. 2024. Host plant resistance to insect pests in wheat. Book Chapter. 1:123-140. https://doi.org/10.1007/978-981-99-7520-4_5.
Allen, K.C., Elkins, B.H., Little, N. 2023. calypha ostryifolia: a natural refuge for Chloridea virescens and Helicoverpa zea (Lepidoptera: Noctuidae) in the Southern United States. Annals of the Entomological Society of America. https://doi.org/10.1093/aesa/saad034.
Elkins, B.H., Portilla, M., Allen, K.C., Little, N., Mullen, R.M., Paulk, R.T., Read, Q.D. 2024. Sublethal effects of a commercial Bt product and Bt cotton flowers on the bollworm (Helicoverpa zea) with impacts to predation from a lady beetle (Hippodamia convergens). PLOS ONE. 19(5):e0302941. https://doi.org/10.1371/journal.pone.0302941.
Little, N., Paulk, R.T., Elkins, B.H., Allen, K.C. 2024. Relative Expression of Crystalline Proteins among the Fruiting Structures of Transgenic Cotton Over Time and Its Impact on Bollworm Mortality. Southwestern Entomologist. 49(2):1-14. https://doi.org/10.3958/059.049.0215.