Non Technical Summary
Alfalfa weevil, the key economic pest of alfalfa throughout the continental US, has developed resistance to the pyrethroid group of insecticides (Warrior ®, Mustang® Maxx and others) used to control them, making its economic damage even more severe. Our progress during the last 1.5 years has quantified and mapped pyrethroid resistant alfalfa weevil populations in several western US states.In two affected areas some producers have abandoned alfalfa as a crop due to the lack of effective controls for insecticide-resistant weevils. This threatens not only the economics of the $20 billion alfalfa forage industry, but also the $67 billion and $38 billion beef and milk commodities that depend upon on-farm alfalfa forage production. A research and Extension effort is needed to mitigate economic loses by prolonging the useful life of the pyrethroid insecticides through resistance management. Research will continue to map the geographic spread of insecticide resistance using dose response experiments, extend testing to central and eastern US states and determine mechanisms of resistance. Science-based results will be incorporated into Extension media that provides resistance management recommendations to stakeholders. A multi-state team of research scientists and Extension specialists will develop and deliver resistance management recommendations in the Western US. This research and Extension proposal addresses ASAFS program area 1, "Increase alfalfa forage and seed yields and forage quality through improved management practices ... to reduce biotic and abiotic stresses and costs of production" (100%) and is broadly supported by stakeholders.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	1640	1130	100%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1130 - Entomology and acarology;

Goals / Objectives
Overall goal: Complete the implementation of an insecticide resistance management planin the western region of the US and extend the results nationally. Determine the mechanisms of resistance to further customize region-specific management recommendations. Evaluate the use of higher throughput molecular methods to diagnose resistant weevil populations.Develop a plan, based on sound science and delivered by Extension, to manage insecticide resistance and its economic damage, thus prolonging the efficacy of insecticides for alfalfa weevil control.Specific objectives:1) Monitor pyrethroid resistance in alfalfa weevil populations across the western US to map its geographic spread using a vial assay mail-out kit.2) Extend research nationally by confirming reports of pyrethroid resistance in the Midwestern and Eastern US regions.3) Determine mechanisms of resistance that impact resistance management recommendations.4) Deliver resistance management recommendations through multi-state Extension.

Project Methods
Monitor pyrethroid resistance in alfalfa weevil populations across the western US to map its geographic spread using a vial assay.Field reports of resistance will be solicited in the western US by through the Resistant Alfalfa Weevil webpage (https://www.montana.edu/resistantalfalfaweevil/), trade magazines and at trade shows, through state Extension system alerts and mailing lists, the alfalfa weevil working group network and other media.Dose-Response Experiments:Technical-grade active ingredients (lambda-cyhalothrin and zeta-cypermethrin) were provided by manufacturers and purchased from Sigma-Aldrich.Active ingredients aredissolved in 95% acetone to make working stocks that are serially diluted seven times to produce concentrations for testing. One mL of each concentration is pipetted into sterile vials (Discount Vials, Madison, WI) and dried on a hot dog roller, delivering concentrations of active ingredient ranging from 3.3 µg/cm2to 0.0033µg /cm2 of vial surface area.Larvae are collected by sweep netting and transported (or shipped from a collaborator) to the laboratory for testing.Prior to bioassay testing,larvae are placed in five-gallon buckets (covered with no-see-um number 20 mesh, Quest Outfitters, Sarasota, FL) with fresh alfalfa (grown in Montana State University's greenhouse) for 24-48 hours at 21°C.Each bioassay consisted of sevenconcentrationsand a 95% acetone control for each of the active ingredients (n = 5 vials for each concentration). Ten 3rdto 4thinstar larvae are placed in each treated vial. Vials are then stored in the dark for24-hours at 21°C. After 24 hours the bioassays are evaluated for larval mortality.Probit analysis(Finney 1952)is used to quantify the lethalconcentrationthat generates 50% mortality (LC50).Extend research nationally by confirming reports of pyrethroid resistance in the Midwestern and Eastern US regions.Contacts willing to ship insects will be solicited from areas where failure of control using pyrethroids has been observed.Methods outlined for the vial bioassay will be used.Determine mechanisms of resistance that impact resistance management recommendations.Both susceptible and resistant (to pyrethroid active ingredients)alfalfa weevil populations fromseveral different states have now been characterized. Alfalfa weevils from these populationswill be used in bioassays to determine mehcanisms of resistnace.Metabolic resistance:The standard method is to bioassay resistance and susceptible populations in the presence and absence of inhibitors of the detoxification enzymes (i.e. susceptible population in the presence and absence of inhibitors vs resistant population in the presence and absence of inhibitors). If detoxification enzymes are involved in resistance, mortality of resistant alfalfa weevils will increase in the presence of inhibitors.The vial bioassay methods will be used. Alfalfa weevil larvae will be exposed to inhibitors coated on the surface of glass vials for one hour prior to being transferred to pyrethroid treated vials. Inhibitors will include piperonyl butoxide, triphenyl phosphate and diethyl maleate. Susceptible and resistant alfalfa weevils from Montana and California will be collected and assayed using concentration-response assays. LC50 values will be calculated using probit analysis. Concentration-response curves will be tested for statistically significant differences (i.e. with and without inhibitor) using Polo PC software.Target site insensitivity:Mutations to the sodium ion channel are detected by PCR and sequencing of the sodium ion channel gene. The steps include isolating total RNA and genomic DNA from susceptible and resistant populations, converting RNA to first strand cDNA, PCR amplifying and sequencing the sodium ion channel gene. Standard bioinformatics software (ClustalX; MEGA X) is used to align the nucleotide sequences to detect differences between susceptible and resistant insects and to identify know resistance mutations. The identification of mutations to the sodium ion channel confirms the presence of target site insensitivity as a mechanism. Individual weevils from different western region states can then be analyzed by PCR to determine the frequency of resistance mutations in different populations.Deliver resistance management recommendations through multi-state Extension.This objective is a continuance of progress demonstrated during the last 1.5 years, as outlined in section 1.4.3.Research results will be integrated into Extension programing to provide multi-state recommendations to manage insecticide resistance in forage and seed alfalfa production systems. Changing management practices to delay the spread and severity of pyrethroid resistance requires a compelling argument. Science-based data and case examples from this project will be incorporated into Extension programing. Extension efforts and resource materials will be coordinated using a central webpage, www.montana.edu/resistantalfalfaweevil/. This approach will develop compelling and consistent recommendations and supporting Extension media that will be delivered to stakeholders in the western US.Science-based Recommendations for Western US Forage and Seed Alfalfa ProducersRecommendations will be developed consistent with multi-state criteria in collaboration with the established network of Extension specialists. This will provide an opportunity for individual states to customize recommendations to their specific production systems. Recommendations will need to be comprehensive and incorporate the need to control all insect pests, includingaphids and caterpillars in forage alfalfa and lygus bugs and seed infesting pests in seed alfalfa crops.Up-to-date research results will be incorporated into a variety of extension media designed to deliver multi-state recommendations to stakeholder groups. The extension media will be made available for download from the central website www.montana.edu/resistantalfalfaweevil/. Extension media will include:• A color alfalfa weevil management brochure that includes a section on resistance management and state-specific sections.• A guide for insecticide use in forage and seed alfalfa with a resistance management focus.• A one-page leaflet summarizing resistance management recommendations.• A PowerPoint slide show on alfalfa weevil biology and resistance management.• A turning point presentation that include change of knowledge survey questions.• News letters.