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Funded Project

Funding Program: Regional IPM Grants (S-RIPM)

Project Title: Novel Feeding Disruption Assay for Monitoring Insecticide Resistance in Adult Lepidopteras

Project Director (PD): Michael Roe [1]

Lead State: NC Lead Organization: NC State University

Research Funding: $126,300

Start Date: May-16-2008 End Date: May-15-2011

Pests Involved: lepidoptera

Summary: This is a research project. Resistance to chemical insecticides has been a common and continuous problem facing agriculture since the first reports of resistance to DDT. Because of the significant cost of new insecticide development, the potential of resistance and cross resistance eliminating new and old insecticide chemistries, the impact of resistance on increased use of pesticides in the environment, and the fact that resistance monitoring technologies is an under studied area of Ag research, it appears prudent that we invest in better methods for resistance monitoring of the major pest species. The National Road Map for IPM listed the following need relevant to this proposal: Develop new diagnostic tools& for detection of pesticide resistance in pest populations. The 2007 SERA003-IPM Priorities included Resistance management for pesticides, Development of decision aids for management of pests and Research on evaluation techniques for IPM. This proposal exactly addresses these needs by providing easier to use and more cost effective monitoring tools for insect resistance; the preservation of insecticidal control technologies; reductions in insecticide failures and the cost of new insecticide development; and reductions of insecticides in the environment because of reduced pesticide efficacy. This research proposal is a multi-state effort, is biology-based (not sub-organismal), and has been acknowledged in the previous review as regionally important. A response to the previous review of this proposal is provided in the project narrative. The assay method developed in this proposal will be validated in North Carolina, Georgia, Mississippi and Texas and should be applicable to most crop systems and chemical insecticides in the SE. In this grant we will develop a novel adult feeding disruption test (FDT) for the diagnosis of insecticide resistance in Lepidoptera, which can be expanded to other insects including sucking pests. The assay will be based on a diagnostic dose of insecticide in a novel hydrateable (nectar-based) meal pad where the disruption of nectar consumption and reduction of fecal production will be monitored with a blue indicator dye. Assay development will include the design of a novel assay delivery system, determining feasibility to different insecticides and Lepidoptera, field evaluation of the technology in four area of the SE US and the development of a finished kit with instructions for beta testing with different end users. This laboratory previously conducted research on a larval FDT kit with funding from the SRIPM program. This work produced two patents and several publications and was part of the Ph.D. work of three Entomology graduate students. These kits are now being used as a standard method by industry and government researchers in the US, Mexico, South American, Australia, and Japan and represent the first high throughput, standardized assay method for resistance monitoring. We will apply our experience with larval FDT to research on an adult FDT assay, which will require the development of a novel hydrateable nectar source, novel assay architecture and other new technologies to accommodate moth feeding. The advantages of an adult FDT assay are the following: assay is applied to the adult stage, which is easy to collect from the field in light and pheromone traps; assay will monitor area-wide levels of insect resistance to pesticides; assay will work for insecticides that have reduced or no larval or adult contact activity; assay will have an easy to read end point of feeding disruption (appearance of blue color) and can also be used with a mortality end point; assay should be rapid (as little as 4 h) and monitor resistance in individual or groups of insects; assay method includes providing adult insects a nectar source, which will support normal physiological functions during the course of the assay; assay apparatus will facilitate easy kit formulation for different insect species and insecticides using a single format and no specialized equipment; the assay system provides a standard format for insect resistance monitoring and which is designed for high throughput kit production at a minimal cost per assay; and the assay will be ready to use of the shelf (just add a drop of water). Objectives: Objective 1. Laboratory validate the adult feeding disruption test (FDT) against the tobacco budworm and diamondback moth and with multiple insecticides (Year 1). Objective 2. Develop a field diagnostic dose using different populations of the tobacco budworms established in the laboratory from the SE US; and validate the assay using adults collected directly from the field (Years 2-3). Objective 3. Develop novel adult FDT assay architecture including optimum adult assay chamber volume and geometry, a hydrateable nectar/insecticidal meal and holder, and assay instructions; and beta test the kit (Years 2-3).

Final Report:

Results From report submitted by the PI to USDA CRIS report system PROGRESS: 2009/06 TO 2010/05 OUTPUTS: OUTPUTS: (1) Developed a prototype device to measure the disruption of nectar feeding in moths to evaluate the use of this approach for monitoring insecticide resistance; (2) Evaluated the use of dyes as a marker for measuring nectar feeding in moths and that could be used to determine feeding disruption; (3) Evaluated the effect of nectar feeding followed by starvation on additional nectar feeding; (4) Examined the dose response of insecticides in nectar on moth feeding and mortality; and (5) Evaluated the use of feeding disruption as determined by the production of blue feces for the diagnosis of insecticide susceptibility. The work conducted is being presented at scientific meetings, published in scientific journals, used to support intellectual property disclosures and support advanced degrees in Entomology at NC State University. PARTICIPANTS: The research is the Ph.D. work in Entomology of Mr. Jaap B. van Kretschmar. This has provided training in bioassay development, insect resistance to pesticides, field and laboratory research related to the project, insect rearing, and applications of the work into other fields like transcriptomics and sucking pests. The expansion work has led to new intellectual property and the submission of a US patent and associated training. The PI, R. M. Roe, has been responsible for the idea of the project and its implementation including publications completed and in process, intellectual property disclosures, patents submitted, chair of Mr. van Kretschmar's graduate committee, expansion of the work into other fields as described in this report and presentations at scientific meetings. Many other graduate students, postdocs and other professors listed on the publications in this reporting also contributed to this project. The work also has been in cooperation with Cotton Inc. of Cary, NC (USA). TARGET AUDIENCES: The target audiences for the work include Ph.D. scientists, extension agents, students training for advanced degrees in Entomology and related fields, and private industry, especially companies developing insecticides and small companies interested in technology development. The work is being presented at local, national and international scientific meetings, presented in courses taught at NC State University, published in the scientific literature, submitted as NC State University intellectual property, submitted for patenting in the US and abroad, and marketed by NC State University for commercial development. PROJECT MODIFICATIONS: No major changes in the project were made. As a result of the research, new project areas have been developed as described in the progress report. IMPACT: 2009/06 TO 2010/05 (1) We were successful in developing a prototype device that can be used in proof of concept research and from which a final device for resistance assay can be modeled; (2) We developed a blue marker dye that can be added to nectar and that will not affect nectar feeding by moths and which is not absorbed from the nectar by the moth after feeding; (3) For partial proof of concept, it was necessary to show that if moths are collected from the field that have already fed on a nectar source, that starvation in the lab will initiate nectar feeding again. This was demonstrated successfully in the lab, i.e., that nectar fed moths when starved will feed again; (4) We showed that a dose response was possible for moths feeding on nectar containing in separate experiments, two different insecticide chemistries with different modes of action and with the end point being reductions in nectar consumption and mortality; and (5) We showed that feeding reductions or feeding disruption due to intoxication with insecticide in the nectar could be determined by reductions in the production of blue feces deposited on the assay device. In summary, we now have a prototype architecture for a feeding disruption assay for detecting insecticide resistance in moths and proof of concept that the assay method can detect insecticide susceptibility on laboratory reared insects. We also have traveled for training to rear a new insecticide resistance strain of the tobacco budworm for the final proof of concept for the technology under development and have developed assay architecture for production of a self running, field assay kit. In addition, the technology has been extended to proof of concept for larval and adult plant bugs and stink bugs and this work is being expanded. Furthermore, the assay has been extended to adult mosquitoes for the management of resistance to treated bed nets and area wide control using insecticides both abroad and in the US; this has resulted in the submission of a proposal to the military (pending)and the successful funding of a graduate student position in the lab by the US Navy to work on this application. Furthermore, we have additional proof of concept that the assay method will diagnose resistance using a laboratory pyrethroid resistant strain of the tobacco budworm. The work is part of a Ph.D. thesis with an anticipated completion of the thesis for graduation in December 2010.

Impacts From report submitted by the PI to USDA CRIS report system (1) We were successful in developing a prototype device that can be used in proof of concept research and from which a final device for resistance assay can be modeled; (2) We developed a blue marker dye that can be added to nectar and that will not affect nectar feeding by moths and which is not absorbed from the nectar by the moth after feeding; (3) For partial proof of concept, it was necessary to show that if moths are collected from the field that have already fed on a nectar source, that starvation in the lab will initiate nectar feeding again. This was demonstrated successfully in the lab, i.e., that nectar fed moths when starved will feed again; (4) We showed that a dose response was possible for moths feeding on nectar containing in separate experiments, two different insecticide chemistries with different modes of action and with the end point being reductions in nectar consumption and mortality; and (5) We showed that feeding reductions or feeding disruption due to intoxication with insecticide in the nectar could be determined by reductions in the production of blue feces deposited on the assay device. In summary, we now have a prototype architecture for a feeding disruption assay for detecting insecticide resistance in moths and proof of concept that the assay method can detect insecticide susceptibility on laboratory reared insects. We also have traveled for training to rear a new insecticide resistance strain of the tobacco budworm for the final proof of concept for the technology under development and have developed assay architecture for production of a self running, field assay kit. In addition, the technology has been extended to proof of concept for larval and adult plant bugs and stink bugs and this work is being expanded. Furthermore, the assay has been extended to adult mosquitoes for the management of resistance to treated bed nets and area wide control using insecticides both abroad and in the US; this has resulted in the submission of a proposal to the military (pending)and the successful funding of a graduate student position in the lab by the US Navy to work on this application. Furthermore, we have additional proof of concept that the assay method will diagnose resistance using a laboratory pyrethroid resistant strain of the tobacco budworm. The work is part of a Ph.D. thesis with an anticipated completion of the thesis for graduation in December 2010.

Outcomes N/A

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