2012 Annual Report 1a.Objectives (from AD-416): Objective 1: Determine the ability of screwworm flies to cross major geographic barriers. Objective 2: Develop a strain of screwworm fly that produces only males. Sub-objective 2a: Develop a genetic sexing system in the horn fly, a species closely related to screwworm, as a model system to be applied to screwworms. Sub-objective 2b: Using information and techniques developed with horn flies, develop a genetic sexing, males-only strain of screwworms. Objective 3: Identify chemical oviposition attractants that can be used in attract-and-kill and for improved rearing. Sub-objective 3a: Identify the natural volatiles from bacteria-inoculated and incubated bovine blood that are responsible for attracting gravid screwworm flies to oviposit. Sub-objective 3b: Identify synthetic compounds with equivalent attraction to gravid screwworm flies for depositing eggs as natural volatiles from bacteria-inoculated and incubated bovine blood. Objective 4: Develop new surveillance methods based on oviposition attractants. 1b.Approach (from AD-416): For our first objective we will identify four islands, in the Republic of Panama, where sterile screwworm flies marked with fluorescent powder will be released. Traps, placed on the mainland, will be used to detect if the screwworms crossed the water 'barrier'. The second objective, development of a male-only strain of screwworm, will be approached by an ARS collaborator first developing transgenic techniques in a related species, the horn fly. Once genetic transformation is accomplished in horn flies the techniques will be transferred to the screwworm. This two-step approach is necessary because. 1)the screwworm is a quarantined pest and adequate facilities for its study are not present in the U.S., and. 2)the collaborator has extensive genetic information/experience with horn flies that will directly transfer to screwworms. Volatiles from bacteria-infested wounds that previously were shown to attract female screwworms will be collected and identified with standard gas chromatograph and mass spectrometry to approach our third objective. Existing 'synthetic' chemicals that are equivalent to the 'natural' volatiles identified as most attractive will then be evaluated for attractiveness to female screwworms using standard bio-assay techniques. Our approach to the fourth objective will be a combination of evaluating the attractiveness of trap design (color, shape, and size) along with the use of volatiles, natural or synthetic as determined from objective 3, that are attractive to female screwworms under field conditions. 3.Progress Report: A greater than expected workload involved in Objectives 2 and 3 and a lack of personnel have led to curtailment of work in Objective 1. Furthermore, after meetings that included representatives from our main customer, Panama – US Commission for Eradication of Screwworms (COPEG), the NP-104 Program Leader, and the Unit, it was determined that higher priority be given to work relative to our contingency of "Determining genetic variation of screwworms." To that end, contacts were developed with potential collaborators in locations with extant screwworm populations to provide samples for genetic analyses. Substantial progress, accelerated from the originally projected milestones, has been achieved in Objective 2. Microinjection and electroporation were successfully applied for injecting foreign DNA into screwworm eggs. Five different genetic 'constructs' have been inserted into thousands of screwworm embryos; all resulting putative lines are 'marked' with genes for fluorescent proteins. Two of the genetic 'constructs' were designed using information from the screwworm genetic 'library' constructed during this project. We continue crossing and screening putative transgenic lines of screwworm for genetic sexing traits. Progress continues on schedule in Objective 3. Assays were done using individual, synthesized chemicals and chemical blends. Volatiles were also identified from decomposing animal liver, an attractant to wild females in previous field trials, and successfully assayed with the secondary screwworm as the model insect. A trap designed for use with other blow flies was modified and assayed with screwworm using blends of the synthetic chemicals attractive to female screwworms. Results were promising; further trap designs will be evaluated with and without blends of synthetic chemical attractants. Review Publications Belay, D.K., Clark, P.L., Skoda, S.R., Isenhour, D.J., Molina-Ochoa, J., Gianni, C., Foster, J.E. 2012. Spatial genetic variation among Spodoptera frugiperda (Lepidoptera: Noctuidae) sampled from the United States, Puerto Rico, Panama, and Argentina. Annals of the Entomological Society of America. 105(2): 359-367. Lezama-Gutiérrez, R., Molina-Ochoa, J., Chávez-Flores, O., Ángel-Sahagún, C.A., Skoda, S.R., Reyes-Martínez, G., Barba-Reynoso, M., Rebolledo-Dominguez, O., Ruiz-Aguilar, G.M., Foster, J.E. 2012. Use of the entomopathogenic fungi Metarhizium anisopliae, Cordyceps bassiana and Isaria fumosorosea to control Diaphorina citri (Hemiptera: Psylidae) in Persian lime under field conditions. International Journal of Tropical Insect Science. 32(1):39-44. Chaudhury, M.F., Sagel, A., Skoda, S.R. 2012. Evaluation of waste artificial larval rearing media as oviposition attractant for New World screwworm (Diptera: Calliphoridae). Journal of Medical Entomology. 49(2):293-298.