Recipient Organization
LEPIDEXT L.L.C
1122 OAK HILL DR
LEXINGTON,KY 40505
Performing Department
(N/A)
Non Technical Summary
Insects are major pests of food and fiber crops with polyphagous larval Lepidoptera species such as Helicoverpa zea and Helicoverpa armigera among the most ubiquitous and damaging pests globally. These pests are invasive and rapidly develop resistance to conventional and transgenic pesticides, presenting additional threats due to their ability to form fertile hybrids that enable exchange of genetic resistance traits. Lepidext's first product, InsterusHz is a sexually transmitted virus that sterilizes adult moths, suppressing corn earworm (CEW) infestations and reducing size of pest populations. Preliminary data establish that InsterusHz can suppress H. zea populations. However, treatment of large acreage requires far more efficient methods than those used for the lab-scale production, which are labor, time, and space intensive. The overarching goal of this Phase II proposal is to increase the production of InsterusHz moths for release. The objectives are designed around aspects of production that inhibit mass production; Objective 1 optimizes the Phase I solution for manual injection, Objective 2 implements in-house experimentation designed to mass produce cannibalistic insects, and Objective 3 focuses on how to ship large quantities of adult moths. Anticipated outcomes of the proposal will result in the development of a commercially viable production system that allows for the efficient production and shipment of InsterusHz moths for the treatment of H. zea populations across the U.S. The commercial entry point is the seed corn production and development industry, currently experiencing significant CEW pressure both in Hawaii and Iowa, with a total addressable market of $12B.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
0%
Developmental
100%
Goals / Objectives
The overarching goal of this Phase II proposal is to increase the production of InsterusHzmoths for release and accompanying virus production. The proposed work builds upon success in Phase I and production studies supported by a Kentucky State Matching Phase I award. The objectives are designed around aspects of production that inhibit mass production; Objective 1 optimizes the Phase I solution for manual injection, Objective 2 implements in-house experimentation designed to mass produce cannibalistic insects, and Objective 3 focuses on how to ship large quantities of adult moths.Objective 1: Optimize oral feeding and infection by breeding for binary InsterusHz moth production.Task 1.1 Optimization of Virus Concentration and Oral Delivery Method.Sterility rates from oral delivery can vary from cohort to cohort with current methodologies and further refinement is needed to increase consistency among resulting F1 cohorts within a production environment. Task 1.1 will examine three parameters (virus concentration, exposure period, and oviposition time relative to oral exposure) that have been identified as critical for successful oral infection in adult moths. Success criteria: Standardize transmission methods for 89% or greater efficiency.Task 1.2 Optimized Mating Transmission.Task 1.2 will expand upon prior testing to further refine the infection process by examining the exposure times required for mating transmission of InsterusHz in a production setting. Success criteria: Mating consistently (75% CI) occurs with separated males and females.Task 1.3 Integration of Oral Virus Delivery and Mating Transmission.Optimized oral delivery and mating transmission procedures will be integrated into a single procedure to ensure consistent, high levels of virus transmission to uninfected adult moths (F0) that result in sterile, infected progeny (F1) for field releases (i.e. pesticidal applications) and subsequent infection of uninfected adults (F0) for future production cycles. Success criteria: Viral transmission >90%.Objective 2: Optimize production.Task 2.1 Optimization of grid method for growth.This task will test expansion of the rearing grid size to 0.5X, 2X, and 3X the grid current volumes, with accompanying changes in food volume. These sizes will be tested for the impact on growth and emergence of pupae, with the goal of increasing the number of grid squares that lead to 50% efficiency. This task will additionally be used to examine potential impacts on the growth cycle of the maintenance colony in addition to the InsterusHz colony. Success criteria: Adult yield ≥ 50%.Task 2.2 Optimization of air flow, humidity, and temperature to housing units. A grid base rearing system requires stacking grids to maximize space, and moisture trapping and mold has proven an issue in the preliminary grid system. Testing conducted with this task will be used to balance the potential growth of mold with the necessity of increasing production for high moth volume output. Success criteria: Adult yield from grid method with air flow ≥ 60%.Task 2.3 Implementation of egg removal and retrieval. Significant preliminary testing has been done to determine materials compatible with egg laying and removal. Testing will be conducted to refine the collection of the eggs, followed by treatment and washing. Success criteria: Build and test egg retrieval system.Task 2.4 Optimization of egg distribution. This task is to optimize the method of distribution in conjunction with achieving appropriate egg density within the grid to enhance survival to adulthood. There are numerous dispersion technologies that can be adopted and adjusted to our case-specific use. The optimal method of distribution will be chosen and integrated into the manufacturing process. Success criteria: Choice of system, implementation, and testing of an egg dispersion system that achieves 3 egg/grid +/- 10% error.?Objective 3: Optimization of shippingTask 3.1 Development of a high-density shipping unit. Existing low-density shipping units will be adapted with 3D printed inserts to increase interior surface area available for resting adult moths. Success criteria: Successful high-density prototypes will be based on resulting adult survivorship that does not significantly differ from control cohorts. Other considerations, such as the ability to reuse the shipping unit in subsequent shipments, will be evaluated.Task 3.2 Development of a cold-compacted shipping unit. Based on methodologies developed for the Pink Bollworm Eradication Program (PBEP), a cold-compacted shipping unit will be evaluated for InsterusHz moths. Success criteria: Evaluation of cold-compaction procedure will be based on ability to compact and immobilize adult moths for a period of 24 hours and resulting adult survivorship that does not significantly differ from control cohorts.Task 3.3 Shipment and field cage virus transmission assessments. Successful high-density (3.1) and cold-compacted (3.2) prototype units will be further evaluated under real world conditions. Success criteria: Successful shipping prototypes will demonstrate high adult moth survivorship (not significantly different from non-shipped moths) and ability to transmit the InsterusHz virus to wild type moths at rates consistent with infected moths not ongoing the shipment process.
Project Methods
Optimize oral feeding and infection by breeding for binary InsterusHzmoth production.Task 1.1 Optimization of Virus Concentration and Oral Delivery Method. Oral delivery of the InsterusHz virus has been previously demonstrated and with recent advancements of oral infection up to 89%. Task 1.1 will examine parameters (virus concentration, exposure period, and oviposition time relative to oral exposure) that are critical for oral infection. Specifically, we will investigate if higher viral concentrations and longer exposure periods can increase consistency. Egg collection time relative to oral exposure has also been found to dictate sterility rates and further testing will investigate egg collection periods that result in consistently greater sterility patterns relative to the other parameters to be examined. Engineering efforts for this task will focus on the development of a feeding apparatus that allows for large numbers of adult moths consume virus containing sucrose at one time. Success criteria: Standardize transmission methods for 89% or greater efficiency.Task 1.2 Optimized Mating Transmission. In the field and lab, transmission of wild type HZNV2 and the InsterusHz virus strain has been found to be spread through mating. Task 1.2 further refine the infection process by examining the exposure times required for mating transmission in a production setting. Specifically, sterile adult moths are utilized to initially transmit the InsterusHz virus to young, uninfected adults. Following exposure, newly infected mating pairs are established (F0) to produce fully sterile (F1) progeny for field releases. Exposure times and densities of uninfected mating pairs relative to sterile adult moths will be examined to maximize transmission rates and the production of F1 sterile progeny. Further examinations of oviposition time relative to exposure will also be assessed. Success criteria: Mating consistently (75% CI) occurs with separated males and females.Task 1.3 Integration of Oral Virus Delivery and Mating Transmission. Optimized oral delivery and mating transmission procedures will be integrated into a single procedure to ensure consistent, high levels of virus transmission to uninfected adult moths (F0) that result in sterile, infected progeny (F1) for field releases (i.e. pesticidal applications) and subsequent infection of uninfected adults (F0) for future production cycles. Success criteria: Viral transmission >90%.Optimize production.Task 2.1 Optimization of grid method for growth. Preliminary grid tests resulted in ~40% of the grid squares reaching adulthood. In normal rearing, the percentage is about 70%. The current grid system has not been optimized to reduce mold and the grid sizing and food requirements have not been optimized for growth. In this task we will test expansion of the grid size to 0.5X, 2X, and 3X the grid current volumes, with accompanying changes in food volume. These sizes will be tested for the impact on growth and emergence of healthy pupae, followed by further optimization steps in subsequent tasks to increase this percentage. This task will additionally be used to examine potential impacts on the growth cycle of the maintenance colony in addition to the InsterusHz colony. Success criteria: Adult yield from grid method ≥ 50%.Task 2.2 Optimization of air flow, humidity, and temperature to housing units. A grid system requires stacking grids to maximize space, and moisture trapping, and mold has proven an issue in the preliminary grid system. The first part of this task will investigate the space requirements between the grids and the air flow requirements to ensure that sufficient oxygen reaches the entirety of the grid. In this second part of this task, having established the ability to create a uniform environment in the first part, we will examine the growth rate from egg to pupation under varying humidity conditions. The results will be used to balance the potential growth of mold with the necessity of increasing production for high moth volume output. Success criteria: Adult yield from grid method with air flow ≥ 60%.Task 2.3 Implementation of egg removal and retrieval. Significant preliminary testing has been done to determine materials compatible with egg laying and removal. Preliminary testing revealed solutions to each of these issues, and this task will build, test, and troubleshoot a whole system for egg removal and retrieval. Once eggs are collected, they will be distributed across the optimized grid (Task 2.4). Success criteria: Build and test egg retrieval system.Task 2.4 Optimization of egg distribution. This task is to optimize the method of distribution in conjunction with achieving appropriate egg density within the grid. Preliminary work to determine the survival rates for single and multiple eggs per grid square indicated that survival is highest at 3 eggs per grid. There are numerous dispersion technologies that can be adopted and adjusted to our case-specific use. In this task we will explore the different options in terms of their speed, error rate, labor requirements, capital investment, and maintenance. Success criteria: Choice of system, implementation, and testing of an egg dispersion system that achieves 3 egg/grid +/- 10% error.Optimization of shippingTask 3.1 Development of a high-density shipping unit. Existing low-density shipping units will be adapted with 3D printed inserts to increase interior surface area available for resting adult moths. Shipping units will be based on commercially available packaging materials (e.g. cardboard tubes) that can be customized (e.g. length). A maximum density of 100 adult moths will be targeted per unit (proposed label maximum release rate per acre). Experimental prototypes will be packed with newly eclosed adult moths (n = 100), placed into an insulated shipping container and held for 24 hours to simulate shipping conditions. 100 moths originating from the same cohort as those used to pack the shipping prototype units will be maintained in a standard adult rearing cage and will serve as a control. After 24 hours, adult moths will be released into adult rearing cages and longevity tracked for a period of ten days. Success criteria: Successful high-density prototypes will be based on resulting adult survivorship that does not significantly differ from control cohorts.Task 3.2 Development of a cold-compacted shipping unit. Based on methodologies developed for the Pink Bollworm Eradication Program (PBEP), a cold-compacted shipping unit will be evaluated for InsterusHz moths. Cohorts of 100 adult moths will be chilled to the point of immobilization and packed into a container with minimal space remaining. Adult moths will be held in a chilled/immobilized state for a period of 24 hours, released into an adult rearing cage and survivorship tracked for a ten-day period. A control cohort, not undergoing chilling or compaction, will be used as a control group. Success criteria: Evaluation of cold-compaction procedure will be based on ability to compact and immobilize adult moths for a period of 24 hours and resulting adult survivorship that does not significantly differ from control cohorts.Task 3.3 Shipment and field cage virus transmission assessments. Successful prototype units will be further evaluated. Collaborators will be shipped prototype units to assess adult survivorship. Post shipment, adult moths will be released into adult rearing cages and survivorship monitored for ten days. Successful prototypes will be further evaluated in field cages to investigate virus transmission under near field conditions. Virus infection rates will be compared to control results conducted at Lepidext's facility using moths not undergoing shipment. Success criteria: Successful shipping prototypes will demonstrate high adult moth survivorship and ability to transmit the virus to wild type moths at rates consistent with control unshipped moths.