Location: Livestock Arthropod Pests Research
2018 Annual Report
Objectives
Objective 1: Develop new attractants, repellents, and behavior-modifying chemicals based on physiology of chemical reception in house, stable and horn flies. Subobjective 1A: Assess compounds for potential behavior-modifying properties. Subobjective 1B: Elucidate biting fly chemosensory protein function.
Objective 2: Evaluate efficacy of novel technologies for control of house, stable and horn flies. Subobjective 2A: Evaluate the efficacy of various compounds as insecticides to control biting flies. Subobjective 2B: Identify and evaluate novel approaches for existing molecular targets and tools for assessment of new targets for biting fly control.
Objective 3: Determine interactions between flies (of all stages) and microorganisms that significantly affect survival of the insects and their capability to transmit pathogens. Subobjective 3A: Characterize the horn fly gut innate immune response to microbial infection. Subobjective 3B: Define the reservoir and vectorial capacity of biting flies for microorganisms that are pathogenic to livestock and humans.
Objective 4: Complete development of a transgenic male-only strain of screwworms ready for production and distribution, coordinating a critical path to development.
Objective 5: Complete development of screwworm attractants and oviposition stimulants to be used in baits and to help synchronize rearing procedures.
Objective 6: Perform research to accomplish efficiency in the screwworm rearing process. Subobjective 6A: Develop and transfer technology for reducing ammonia emissions from screwworm larval media when cellulose fiber is used as the bulking agent. Subobjective 6B: Determine where optimum rearing environments exists within the large rooms of the screwworm mass rearing facility so to ensure maximum efficiency of the rearing process.
Objective 7: Develop ecological niche models of screwworm flies and genetic subpopulations with the practical objectives of scaling release rates to habitat and to provide projections of potential range changes in response to climate change. Subobjective 7A: Determine genetic variation of screwworms from different geographic origins across their current range. Subobjective 7B: Use remote sensing and geographical information systems to relate genetic variation of screwworms to differences in landscape across their range.
Approach
Identify new attractants, repellents, and behavior-modifying chemicals based on assessment of natural and synthetic compounds for behavior-modifying properties.
Identify and elucidate structure activity relationships of biting fly chemosensory proteins and behavior-modifying chemicals. Identify lead compounds for further development based on behavior-modifying properties and structure activity relationships. Identify physiological pathways for development of novel control technologies by targeting key components. Evaluate the efficacy of natural and synthetic compounds as insecticides for control of biting flies.
Modify structure of lead compounds and assess effects on compound efficacy to identify structural attributes contributing to and enhancing biological activity. Evaluate efficacy of gene silencing based on key physiological targets for biting fly control. Evaluate efficacy of vaccines based on key physiological targets for biting fly control. Elucidate interactions between flies (of all stages) and microorganisms that significantly affect survival of the insects and their capability to transmit pathogens, including the innate immune response of biting flies to microorganisms in the fly gut. Elucidate the reservoir and vector competence of biting flies for microorganisms that are pathogenic to livestock and humans.
Measure fitness parameters of transgenic screwworm lines and determine if transgenic males are competitive with wild type screwworm males. Confirm stability of transgenic line(s), and screen for mobilization of the transgene in bio-secure facility. Examine influence of genetic background on level of female lethality by crossing transgenic males with females collected from different locations. Record female screwworm antennal responses to chemical stimuli. Transfer transgenic line(s) with favorable fitness to COPEG. Test active chemicals for fly attraction and oviposition stimulation to improve field surveillance and enhance egg production during mass-rearing. Determine optimum dose of potassium permanganate and Yucca extract in screwworm larval media for ammonia reduction and good fly quality yields in diet bulked with cellulose fiber. Yucca schidigera (powder extract) will be added to larval diet at select intervals to determine synergistic activity in ammonia reduction. Measure temperature and humidity in separate rooms, each containing first 3 developmental stages of the screwworm life cycle. Design and develop GIS-based methodology for spatial analysis of each room. Determine general landscape patterns using satellite images at multiple locations across endemic areas from which screwworms were sampled and genotyped. Climatological data, along with general soil information, vegetation data, host composition and density, and land use patterns, will be collected and analyzed using remote sensing technologies and landscape genetics models to understand interactions between gene flow and geographic variation. This will help assess risk for screwworm cases in the barrier zone in Panama, and to prevent outbreaks in the U.S.
Progress Report
Objective 1: Previous studies indicated that p-anisaldehyde exhibited significant insecticidal activity to horn flies, and although it was not an effective repellent, its presence in bovine feces was strongly inhibitory to fly oviposition. This year, further studies expanded available data on p-anisaldehyde insecticidal and avoidance efficacy to developmental stages of horn flies and extended studies to house flies and sand flies where p-anisaldehyde exhibited strong repellent activity to both fly species. Various formulations of neem exhibited negligible repellency to house flies. Several essential oils conditionally exempt from EPA registration as minimum risk pesticide products under 40 CFR 152.25 and their major components were found to exhibit very strong repellency to the sand fly, Phlebotomus papatasi, a major vector of cutaneous leishmaniasis that significantly reduced readiness of deployed military personnel in Iraq and Afghanistan. The conditionally exempt status from EPA registration could potentially allow immediate military use of these oils as area, special, or personal repellents that exhibited experimental repellent efficacy at concentrations substantially lower than DEET. Ten additional recombinant odorant binding proteins were expressed from stable flies and their identity will be confirmed by protein sequencing prior to use in competitive binding studies to characterize their biochemical properties.
Objective 2: Completed studies of synthetic carbamates for inhibition of recombinant fly acetylcholinesterases (rAChEs) containing known organophosphate (OP)-resistance mutations from sand flies and mosquitoes. Identified novel synthetic carbamates exhibiting substantial inhibition of OP-insensitive AChEs that are effective inhibitors at low concentrations, effectively establishing their potential utility for control of OP-resistant flies and mosquitoes, including OP-resistant mosquitoes that vector malaria in Africa. The low-level OP-resistant G262A mutation in horn fly AChE is scheduled for assessment of inhibition by synthetic carbamates. Studies to identify novel toxicants are underway including: i) an IGR (insect growth regulator) study against horn fly, house fly, and stable fly; ii) assessment of p-anisaldehyde to house flies, horn flies and stable flies, including fly eggs – the compound is highly toxic against horn fly eggs. Gene silencing studies using RNA interference for identification and validation of novel targets in various fly species have presented significant difficulty due to high mortality associated with handling stress, including microinjection. Initial studies to adapt the dual luciferase reporter plasmid originally developed for use in tick cell culture for use in fly cell culture revealed that existing promoters utilized in the tick reporter construct were inactive in horn fly cell culture, indicating the need for substantial work to identify and characterize promoters active in horn fly cell culture.
Objective 3: Studies to define the reservoir and vectorial capacity of biting flies for microorganisms that are pathogenic to livestock and humans were delayed due to difficulty in obtaining fluorescently-tagged bacterial strains from the cooperator. These studies are anticipated to be completed in the coming year.
Objective 4: The male-only transgenic screwworms were tested for mating against their closest taxonomic relative, Secondary Screwworm, Cochliomyia macellaria (Fabricus); no mating occurred, demonstrating normal species-selective mating behavior of the transgenic flies. Small-scale field releases of one male-only line were completed; analyses are ongoing.
Objective 5: Tests of attractiveness of bacterial volatile compounds for gravid females for development of baits and to aid in rearing of sterile flies was completed with secondary screwworm but not replicated with primary screwworm due to retirement of M. Chaudhury. (Completion with primary screwworm was planned for the coming year.) No progress or changes. No research goals for this FY. The scientist leading this work has left ARS.
Objective 6: Replacement of formaldehyde by potassium permanganate in screwworm diet acts as an antimicrobial and provides control of ammonia. Potassium permanganate is successfully being used by the customer at a pilot scale to rear the transgenic male-only screwworm line. The customer is proceeding to transition to replacement of formaldehyde by potassium permanganate for factory level production of screwworms for sterile male releases. This will result in substantial improvement of the working environment within the plant due to reduced emission of ammonia gas.
Objective 7: One of the single component male-only lines was used for small-scale releases in the field in Panama. Data are still being analyzed but the males did distribute similarly to the wild type flies. A 2-component system (where females are removed very early in development) is now being tested in the laboratory.
Accomplishments
1. Essential oil repellents for sand flies. Several essential oils conditionally exempt from EPA registration as minimum risk pesticide products under 40 CFR 152.25 and their major components were tested at ARS in Kerrville, Texas and found to exhibit very strong repellency to the sand fly, Phlebotomus papatasi, a major vector of cutaneous leishmaniasis that significantly reduced readiness of deployed military personnel in Iraq and Afghanistan. The conditionally exempt status from EPA registration could potentially allow immediate military use of these oils as area, spacial, or personal repellents that exhibited experimental repellent efficacy at concentrations substantially lower than N,N-Diethyl-meta-toluamide (DEET).
2. Novel carbamates may remediate organophosphate resistance mutations. Completed studies at ARS in Kerrville, Texas of synthetic carbamates for inhibition of recombinant fly acetylcholinesterases (AChEs) containing known organophosphate resistance mutations from sand flies and mosquitoes. Identified novel synthetic carbamates exhibiting substantial inhibition of organophosphate (OP)-insensitive AChEs that are effective inhibitors at low concentrations, effectively establishing their potential utility for control of OP-resistant flies and mosquitoes, including OP-resistant mosquitoes that vector malaria in Africa.
3. Natural compound potentially useful for fly control. Para-anisaldehyde is a natural compound used commercially as a flavoring ingredient and is found in American cranberry, anise oil, fennel and vanilla. ARS research at Kerrville, Texas found that para-anisaldehyde exhibited significant insecticidal activity against several fly species, including horn fly, stable fly, housefly and at least one species of sand fly, Phlebotomus papatasi. The compound also exhibited ovipositional inhibitory activity when incorporated into fly larval medium, suggesting possible use as an ovipositional deterrent for treatment of round hay bale cattle feeding stations, which are believed to be a prime reproductive environment for stable flies.
4. Increased efficiency and reduced cost of screwworm production. Improved rearing procedures were developed by ARS in Kerrville, Texas and successfully demonstrated at a pilot scale and are being incorporated at the production facility for large-scale rearing of screwworm flies for sterile male release, resulting in decreased release of toxic ammonia gas within the production plant. In addition, testing of the transgenic male-only screwworm strain has progressed to small field-scale releases to test its capacity to successfully compete for mating with wild screwworm flies. If fully successful, replacement of rearing fertile screwworm lines with the transgenic male-only strain is expected to reduce fly production costs by up to 50% and potentially eliminate requirement for irradiation to sterilize the flies, further improving fly vigor and competitiveness in the sterile male release program.
Review Publications
Prado-Rebolledo, O.F., Lezama-Gutierrez, R., Molina-Ochoa, J., Garcia-Marquez, L.J., Minchaca-Llerenas, Y.B., Morales-Barrera, E., Tellez, G., Hargis, B., Skoda, S.R., Foster, J.E. 2017. Effect of Metarhizium anisopliae (Ascomycete), Cypermethrin, and D-limonene, alone and combined, on larval mortality of Rhipicephalus sanguineus (Acari: Ixodidae). Journal of Medical Entomology. 54(5):1323-1327.
Molina-Ochoa, J., Galindo-Velasco, E., Rosales-Gutierrez, A., Gonzalez-Ramirez, M., Lezama-Gutierrez, R., Chan-Cupul, W., Skoda, S.R., Ullah, M.I., Foster, J.E. 2018. Susceptibility of adult engorged ticks, Rhipicephalus microplus (Acari: Ixodidae) to a native heterorhabditid isolate (Nematoda: Heterorhabditidae) in Colima, Mexico. Pakistan Journal of Zoology. 50:767-770. https://doi.org/10.17582/journal.pjz/2018.50.2.sc2.
Tiroesele, B., Skoda, S.R., Hunt, T.E., Lee, D.J., Molina-Ochoa, J., Foster, J.E. 2018. Morphological and genetic analysis of four color morphs of bean leaf beetle, Cerotoma trifurcata (Coleoptera: Chrysomelidae). Journal of Insect Science. 18:2.
Gundersen, D.E., Adrianos, S.L., Allen, M.L., Becnel, J.J., Chen, Y., Choi, M.Y., Estep, A., Evans, J.D., Garczynski, S.F., Geib, S.M., Ghosh, S.B., Handler, A.M., Hasegawa, D.K., Heerman, M.C., Hull, J.J., Hunter, W.B., Kaur, N., Li, J., Li, W., Ling, K., Nayduch, D., Oppert, B.S., Perera, O.P., Perkin, L.C., Sanscrainte, N.D., Sim, S.B., Sparks, M., Temeyer, K.B., Vander Meer, R.K., Wintermantel, W.M., James, R.R., Hackett, K.J., Coates, B.S. 2017. Arthropod genomics research in the United States Department of Agriculture-Agricultural Research Service: Applications of RNA interference and CRISPR gene editing technologies in pest control. Trends in Entomology. 13:109-137.
Skoda, S.R., Welch, J., Phillips, P.L. 2018. Screwworms (Diptera: Calliphoridae) in the U.S.: response to and elimination of the screwworm outbreak on the Florida Keys. Journal of Medical Entomology. 55(4):777-786.
Temeyer, K.B. 2018. Molecular biology of tick acetylcholinesterases. Frontiers in Bioscience. 23:1320-1337. https://doi.org/10.2741/4646.
Tiroesele, B., Skoda, S.R., Hunt, T.E., Lee, D.J., Molina-Ochoa, J., Foster, J.E. 2014. Population structure, genetic variability, and gene flow of the bean leaf beetle, Cerotoma trifurcata, in the Midwestern United States. Journal of Insect Science. 14:62.
