Performing Department
(N/A)
Non Technical Summary
Genetically modified crops incorporating Bt traits targeting insect pests are widely adopted in the US, but they are now threatened by the evolution of resistance. A common method growers can use to delay resistance is to plant non-Bt refuges, but adoption is low. An easy way to ensure adoption is to blend non-Bt refuge seed with Bt seed (refuge in a bag, RIB). However, these plants can cross pollinate in the field, potentially accelerating the rate of resistance evolution for ear-feeding pests, like Helicoverpa zea. Since there are no detailed studies of the effect of Bt on field-collected H. zea mating success and behavior, our proposal seeks to 1) characterize the impact of seed-blend versus structured refuge on H. zea female mating success and fitness; 2) characterize the impact of seed-blend versus structured refuge on H. zea male ability to respond to mating pheromones before mating; 3) characterize the impact of seed-blend versus structured refuge on H. zea male spermatophore production; and 4) develop amodel of resistance in H. zea to Bt-toxins in the southern U.S. using information generated from Objectives 1-3. Our most important findings will impact the assumption of the importance of non-Bt plant refuge and the assumption of random mating in resistance models. This could impact regulations concerning the spatial placement of non-Bt host refuges or the adoption of blended refuge to replace structured refuge in southern U.S. corn.
Animal Health Component
5%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
(N/A)
Goals / Objectives
This proposal will address the research priority listed in the FY 2021 RFA of the fifth program for BRAG: Other Research Topics Designed to Further the Purposes of this Program, and specifically target section "i", research to understand the frequency and mechanisms by which pests or diseases overcome plant pest or disease resistance traits conferred by engineered genes.The overall goal of these studies is to provide the EPA scientific information to inform their creation of regulations concerning the deployment, use of refuges, and sustainability of Bt crops by focusing on H. zea, a species of regulatory concern in the southern U.S. This proposal seeks to address these concerns by describing H. zea biology in field settings of non-Bt and Bt corn as it relates to resistance and the impacts of RIB versus structured refuge on the development of resistance. Our approach will combine ecological experiments in the field, with laboratory measurements, to characterize the phenotype of individuals on non-Bt and Bt crops and to correlate these with effects on mating success. Our proposed studies, focusing on the mating success of H. zea reared in different refuge scenarios will inform: a) the fundamental understanding of the impact of blended refuge, structured refuge, and Bt crops on H. zea resistance to Bt toxins, b) gaps in the knowledge base of several steps in the insect resistance management (IRM) decision-making process for Bt crops, and c) appropriate regulations concerning non-Bt plant refuge.The specific objectives for this proposal are to:1. Characterize the impact of seed-blend versus structured refuge on H. zea female mating success and fitness2. Characterize the impact of seed-blend versus structured refuge on H. zea male ability to respond to the female sex pheromone3. Characterize the impact of seed-blend versus structured refuge on H. zea male spermatophore production4. To develop a stochastic, spatially explicit model of resistance in H. zea to Bt-toxins in the southern U.S. using information generated from Objectives 1-3From a behavioral perspective, a strength of our proposal is the inclusion of males in fitness studies and the inclusion of time-course studies. Often, researchers compare fitness-related traits from a single event per male or female (i.e., single mating, single flight in a wind tunnel, single spermatophore transfer). Including traits that relate to recovery from mating (e.g, size of spermatophore over time, ability to produce spermatophores over time), tests more realistic, field-relevant scenarios.
Project Methods
Objective 1. Characterize the impact of seed-blend versus structured refuge on H. zea female mating success and fitnessCorn hybrid, Bt traits, treatments, and experimental field information. For this objective, PDs will plant the following Bt and non-Bt corn hybrids in pure stands and in RIB. Independent field trials will be located in Florence (SC), Clayton (NC), and Plymouth (NC) during two consecutive years (2024, 2025). Treatments will be arranged as a randomized complete block design, with four blocks per trial, and 25 ft alleys between blocks.Adult collection for mating tests from hibernation boxes. We will collect 50 ears with larvae when late instar larvae are observed. We will collect 100 non-Bt and 100 Bt ears from each RIB treatment plot.Following well-established procedures, modified hibernation boxes will be placed underneath a shelter and filled with a 10-inch layer of washed sand. The 100 ears collected from each pure varietal stand plot, and the 100 non-Bt and 100 Bt ears from each RIB plot, will be inserted into the sand in an upright orientation in the bottom of separate boxes. For blended refuge treatments, additional boxes will be used to separate non-Bt and Bt ears. We will carefully sift pupae weekly from the soil of each hibernation box one to two weeks after ear placement.Pupae from SC will be shipped, and all pupae from NC will be transported to the Reisig laboratory at NC State University in Raleigh, NC. Pupae from each location will be identified to sex, stored, and kept in growth chambers at 27°C, 60% RH, and 14:10 light: dark cycle. Adults that eclose will be provisioned with a cotton ball soaked with 10% sucrose solution.Evaluation of mating success and fitness. Within the 2-5 d from adult eclosion and when calling behavior begins in H. zea females, we will briefly cold anesthetize female moths and clip the fore and hind wing of each female and place them in a separate container with a cotton ball soaked with 10% sucrose. Experiments will be conducted between 21:00 to 4:00h. Once calling behavior is observed, each calling female will be transferred to a 1 x 1 m horizontal mating table. Each calling female will be used one time, allowed to call for an hour, and, once mated, will be removed from the table along with the copulating male, and placed in a small container to complete copulation. The duration of copulation will be recorded.Females will be allowed to oviposit for three nights and then provisioned with a lab-reared male and allowed to oviposit until death. We will count the number of eggs laid on each ovipositional substrate and place them in containers with a cotton ball and a drop of water. We will count the number of neonates that hatch daily. We will measure the abdominal width of each female and her field-collected mate.Objective 2. Characterize the impact of seed-blend versus structured refuge on H. zea male Evaluation of male behavior. Adult males will be used for flight tunnel experiments at 2-5 days after eclosion. Flight bioassays will be conducted between the fifth and eighth hour of scotophase. Approximately 1 h prior to flight, individual males will be exposed to red light in wire screen cages. Moths will be flown under a mixture of red and white light (0.5 lux) at 50 cm/s wind speed and at 25°C. Quiescent males will be individually placed in an open screen cage on a stage at a height of 25 cm, 170 cm downwind from the pheromone source. If the male remains quiescent, a calling female will be introduced upwind and the male's behavior recorded. The latency from introducing the pheromone to male take off will be recorded. Males will be allowed 5 min to take off from the stage. The following male behaviors will be recorded: percent of moths that take off, percent of moths that orient to the pheromone, time to reach 50% of the way to the pheromone, time to reach the pheromone, % of males that reach the pheromone, and % of males that touch the pheromone source.To test the relative vigor of males that eclosed from seed-blend ears versus structured refuge, each male will be flown in the wind tunnel three times toward three different colony-reared females (pheromone source introduced upon initiation of calling), with five minutes in clean air between each trial. This design will enable us to estimate the average parameters of each male's response as well as the change in male response over the three trials.A calling female will be placed in a vented jar outside the wind tunnel. Humidified air will carry her pheromone through a solenoid valve. When clean air is required in the wind-tunnel, the solenoid valve will divert the pheromone to a fume hood. At the start of the experiment, the solenoid valve will direct the pheromone to a glass pipette at the upwind end of the wind-tunnel. Multiple females will be available, so that a calling female is always available for each trial.Objective 3. Characterize the impact of seed-blend versus structure refuge on H. zea male spermatophore production Evaluation of spermatophore production. One day following eclosion and two hours before the onset of scotophase, a single virgin male that emerged from our field-collected ears will be paired with a single virgin female from a laboratory susceptible colony in 250 ml plastic containers. We will record the latency to mating and mating duration for each pair. Following release from coupling, we will store the female at -4°C for dissection. We will provision the male with 10% sucrose after the female is removed. This process will be repeated every 24 hours for seven days to assess the male's ability to synthesize new spermatophores. We will dissect all females and, if a spermatophore is recovered we will measure its length and width, as well as weigh it.Data analysis. We will analyze number of spermatophores per male, percent of males that successfully transferred a spermatophore, length x width of spermatophore, and spermatophore weight using the generalized linear mixed models approach described in the first objective. We will compare males collected from both non-Bt and Bt ears in the RIB treatments.Objective 4. To develop a stochastic, spatially explicit model of resistance in H. zea to Bt-toxins in the southern U.S. using information generated from Objectives 1-3Corn hybrid, Bt traits, treatments, and experimental field information. Parameters will be developed using data from previous objectives for use in a stochastic, spatially explicit model of resistance in H. zea to Bt-toxins in the southern U.S. The model will explore the risk assessment null hypothesis that altered fecundity parameters in moths emerging in Bt-crops will not impact the rate of resistance evolution in H. zea. The model will be used to simulate alternative IRM strategies to assess the interaction of altered fecundity rates with different IRM strategies.To determine the impact of altered fecundity rates on the evolution of resistance to Bt toxins expressed in corn and cotton, the data from objective 1 will be used to update the model with current mortality and fecundity rates for various crops. While the model is capable of simulating any number of resistance loci, the time required to complete each simulation increases exponentially with the addition of each locus, so we will limit simulations to crop combinations with four or fewer Cry toxins. Fecundity and male mating success rates can vary with the genotype of individuals as well as the toxins fed on during development. These rates will vary with larval diet of plants expressing Bt-toxins (the results of objectives 1-3). The null hypothesis is that the rate of resistance evolution (measured as the time taken for all resistance allele frequencies to exceed 50% in fields planted to Bt-expressing plants) will not vary with the fitness/mating rates that vary with the larval diet in Bt-fields.