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ARS Home » Plains Area » Kerrville, Texas » Knipling-Bushland U.S. Livestock Insects Research Laboratory » LAPRU » Research » Research Project #427991

Research Project: Management of Flies Associated with Livestock

Location: Livestock Arthropod Pests Research

2019 Annual Report

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.

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
The terminating project entitled, “Management of Flies Associated with Livestock," is the combination of former projects “Flies Associated with Livestock Production Systems” and “Area–Wide Screwworm Eradication” combined in March, 2017. Objectives were to reduce economic and health impacts of biting flies and screwworms, through studies of biting fly and screwworm genetics, biochemistry, physiology, and responses to various compounds evaluated for insecticidal, repellent, or physiologically disruptive capability. Notable progress was accomplished, including demonstration of significant repellent, insecticidal, or reproductive inhibition of the natural compounds, p-anisaldehyde, azadirachtin, and limonene, against multiple species of biting flies. Essential oils conditionally exempt from EPA registration as insecticides and their active chemical components were identified [e.g., Lemongrass, Cornmint, Cinnamon, Citronella, Clove, and Spearmint oils; with highly active oil components including: Trans-Cinnamaldehyde, Citronellol, Citral, Geraniol, N-propyl d-Camphor lactam, EZ-Nepetalactone, Citronellal, Diethyl (2-methylally)Phosphonate and Menthol] to exhibit significant activity as repellents and/or insecticides against horn flies, stable flies, house flies, and sand flies expanding the list of effective compounds potentially useful to reduce impact of biting flies on human and animal health. p-Anisaldehyde is particularly potent against fly eggs, providing total mortality against horn fly eggs at extremely low concentrations (<0.0001%) and was also a potent repellent against house flies. In commercial neem formulations, azadirachtin is often accompanied by other bioactive compounds that account for differences in activity and potency. Limonene, at low concentrations, was attractive to horn flies and might be useful for trapping the pest. A fly larval feeding assay was developed using fluorescent paramagnetic microparticles to identify chemicals that altered feeding and growth of larval horn flies and sand flies. Feeding stimulants may control flies by promoting toxicant ingestion while feeding inhibitors may reduce larval growth and reproductive capacity. The assay will be used in the subsequent research project to identify fly larval feeding stimulants and inhibitors. Researchers at Kerrville, Texas, identified kdr-his, a mutation in the stable fly sodium channel gene associated with resistance to permethrin. The knock-down resistant allele, kdr, which is prevalent in other insects resistant to permethrin was not identified in stable fly populations within the United States. New studies were extended to evaluate stable fly populations from additional U.S. and international locations, such as Costa Rica, France, Thailand, and Australia, where there are reports of stable fly resistance to pyrethroid insecticides. Researchers at Kerrville, Texas, scientists identified prevalence of kdr-his in Costa Rica, France, Thailand, and Australia, while kdr was identified in France and Australia. Insecticide resistance to organophosphate (OP) and carbamate pesticides often results from mutations in genes encoding acetylcholinesterase (AChE). Mutations producing high level OP-resistance in mosquitoes were engineered for expression in recombinant AChE of sand fly. One mutation, G119S, is suspected to be present in some sand fly populations, where it can arise by a single nucleotide substitution in the AChE gene. In addition, the mosquito mutations G119S, F290V and F331W were expressed individually and in combination in recombinant sand fly AChE and used to screen novel synthetic carbamates to identify those that have potential for use against OP-resistant flies and mosquitoes. All of the recombinant AChEs containing one of the OP-resistance mutations were resistant to OP-inhibition, while those containing more than one mutation lacked enzymatic activity. Several synthetic carbamates were identified as effective inhibitors of the OP-resistant AChEs, suggesting they might be effective in Africa or other areas of high sand fly or mosquito-borne disease as indoor residual sprays (IRS) or insecticide-treated bednets or screens. The novel synthetic carbamate compounds were also effective against other arthropods, including horn flies and ticks. Manual annotation of odorant receptor (Or) and odorant binding protein (Obp) gene families in the Stable fly genome was completed, revealing 74 Or and 90 Obp genes. Comparison with the Drosophila genome identified five stable fly genes that are related to Drosophila Or67d, which has a role in regulating mating behaviors. While three of these stable fly genes are expressed 15-20 times higher in mated females versus unmated/mated males, their function is unclear requiring further study. Expansion of the house fly Obp gene family was evident in comparison with Obps from Drosophila. Obp expression was detected in both sensory and nonsensory tissues, suggesting a multi-functional role for these odorant binding transport proteins. Studies at Kerrville, Texas, revealed that Salmonella enterica serovar Senftenberg could be transmitted to cattle peripheral lymph nodes upon simulation of a heavy horn fly infestation actively feeding on its bovine host for at least 11 days. Examination of the horn fly gut transcriptome identified members of the epithelial immune system, including those involved in microbe recognition (peptidoglycan recognition proteins and Gram negative binding proteins) and downstream effector molecules (antimicrobial peptides). Given that house flies act in dissemination of microorganisms, an ARS Kerrville scientist guided a Master’s student research project to identify fungal pathogens from house flies collected on south Texas cattle; identifying several species for the first time on house flies. The most frequent fungus recovered was Cladosporium cladosporodes, known as a ubiquitous airborne allergen. As part of a USDA-ARS, multi-university collaboration, the stable fly genome project was completed and the data was provided to the scientific community on bioRxiv, an open access preprint repository for biological sciences. The manuscript, submitted to a peer-reviewed journal, is under revision. Genome data enabled identification of gene families that appear to be expanded in stable flies relative to Drosophila, including those involved in chemosensation, vision, immune response, and metabolic detoxification. Genome data enabled development of targeted functional genomics studies for inclusion in another Kerrville, TX 2019-2014 project plan. Access to the stable fly and house fly genomes prompted the face fly genome sequencing project to be included as part of the USDA-ARS AgPest100 Initiative, and biological material for single insect sequencing of three muscid flies (horn fly, stable fly and face fly) was provided by researchers at Kerrville, Texas, supporting expanded comparative genomic studies within Muscidae and between other insect families of veterinary importance. Completed bioengineering construction of a transgenic male-only strain of screwworms ready for production and distribution, coordinating a critical path to development. The genetically engineered male-only strains were transferred to Methods and Development section of the Panama-U.S. Commission for the Eradication and Prevention of Cattle Screwworm, known as COPEG, for further evaluation in field trials. Use of male-only strains is expected to reduce production costs and biological waste by 50%. Demonstration of successful performance compared to non-engineered strains will enable use of the male-only strains for full production, sterilization, and release, providing considerable savings in expense and waste generation. 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. One of the single component male-only lines was used for small-scale releases in the field in Panama. Previous samples from Central and South America were subjected to genotyping by sequencing and analyses were conducted. Samples from the Dominican Republic and Trinidad and Tobago were obtained. Experiments using secondary screwworm identified four volatile ovipositional attractants. Replication for primary screwworm is expected to improve production efficiency by increasing the average number of eggs successfully produced for inoculation of larval medium used in screwworm production, important for the male-only strain to reduce the number of fertile females needed and their production cost. Replacement of the gel formulation for screwworm production with cellulose fiber resulted in significant cost savings and a more environmentally friendly waste material from screwworm production; however, the cellulose fiber medium formulation resulted in increased release of toxic ammonia gas, which was mitigated by addition of Yucca extract and potassium permanganate and is currently in use for production of the male-only strain. These improvements allow use of the less costly cellulose fiber larval medium formulation, reduce the production of toxic ammonia gas, and provide more environmentally friendly alternative waste disposal procedures. Remote sensing was utilized to identify screwworm collection sites for Caribbean Islands and was also used to identify screwworm collection sites for northern Peru and several South American countries. It is anticipated that collection of screwworm samples from these diverse locations will enable genetic analyses to identify genetic markers with location-specific variations suitable for epidemiological purposes.

1. Effects of limonene formulations on horn flies. ARS scientists at Kerrville, Texas, determined that laboratory grade limonene and a commercial formulation of limonene reduced horn fly egg viability, contact exposure to adults caused up to 100% knockdown, and laboratory grade limonene caused adult contact mortality. Limonene was attractive to horn flies at low concentrations of less than 0.1%. Limonene, a new botanically-based tactic for control of horn flies, is likely to be useful for trapping horn flies away from cattle and other livestock at low concentrations, and is a potentially useful repellent and insecticide when applied to livestock at higher concentrations.

2. Effects of p-anisaldehyde on house flies. ARS scientists at Kerrville, Texas, determined that p-anisaldehyde, a botanical, strongly repels adult houseflies at concentrations of 0.075%, is lethal by contact exposure to eggs and adults, and it curtailed larval development to the pupal stage. p-Anisaldehyde presents a new botanically-based tactic for control of house flies by inducing mortality at the egg, larval, and adult life stages.

3. New early-female-lethal and female-transformations strains. Screwworms larvae infest open wounds in humans, livestock, and other animals, consuming living tissue resulting in devastating injury or death to the host. Use of the sterile insect technique successfully eliminated screwworm infestations from North America, and a biological barrier to re-entry from South America depends on release of sterile male flies produced, sterilized and released in Panama. Production cost and efficiency to rear, treat and release the sterile insects is expected to be dramatically reduced by use of new genetically engineered lines of screwworms created by ARS scientists in Kerrville, Texas, in collaboration with scientists at North Carolina State University that prevent maturation of female screwworms, producing only male flies. ARS scientist continue research improving the genetic sexing mechanisms in bioengineered screwworm lines. Elimination of female embryos prior to hatching will have the most dramatic reduction in cost for mass rearing transgenic New World screwworm, however, for these systems to work effectively, the promoters must be embryo specific to reduce negative side effects. Two-component systems (where females are removed very early in development) have been created using three different promoters with early embryo expression and are now being tested in the laboratory. An alternative transgenic strategy which silences the female sexual development pathway, resulting in intersex or phenotypically-male genotypically-female flies, has also been used to create additional genetically engineered lines in development. Further research will compare engineered screwworm lines for functionality and competitiveness for use in sterile male releases to maintain the biological barrier at greatly reduced cost.

4. Development and validation of CRISPR-Cas9 for gene knockout in screwworms. ARS scientists at Kerrville, Texas collaborated with scientists at the University of North Carolina and the University of Campinas in Brazil to develop and validate methods for using genome editing technology CRISPR-Cas9 in primary screwworm. CRISPR-Cas9 can be used to selectively knock out target gene and insert transgenes in specific locations in the genome. This technology is a key tool in developing gene drive strains and can be used to understand gene function. The technique was verified by knocking out genes for body color, olfaction, and sex determination. This method can be used to advance the sterile control program with gene drives or female-to-male transformation systems.

Review Publications
Dupuis, J.R., Guerrero, F., Skoda, S.R., Phillips, P.L., Welch, J.B., Schlater, J.L., Azeredo-Espin, A.L., Perez De Leon, A.A., Geib, S.M. 2018. Molecular characterization of the 2016 new world screwworm fly outbreak in the Florida Keys. Journal of Medical Entomology. 55(4):938-946.
Mckay, L., Delong, K.L., Schexnayder, S., Griffith, A.P., Taylor, D.B., Olafson, P.U., Trout Fryxell, R. 2019. Cow-calf producers' willingness to pay for bulls resistant to horn flies, Haematobia irritans (L.) (Diptera: Muscidae). Journal of Economic Entomology. Vol 112(3):1476-1484.
Olafson, P.U., Kaufman, P.E., Duvallet, G., Solorzano, J., Taylor, D.B., Trout Fryxell, R. 2019. Frequency of kdr and kdr-his alleles in stable fly (Diptera: Muscidae) populations from the United States, Costa Rica, France, and Thailand. Journal of Medical Entomology. Vol 56(4):1145-1149.
Showler, A., Harlien, J.L. 2018. Effects of the botanical compound p-anisaldehyde on horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae) repellency, mortality, and reproduction. Journal of Medical Entomology. 55:183-192.
Showler, A. 2017. Botanically based repellent and insecticidal effects against horn flies and stable flies(Dipera: Muscidae). Integrated Pest Management. 8:1-11.
Paulo, D.F., Williamson, M., Arp, A.P., Li, F., Sagel, A., Skoda, S.R., Sanchez-Gallego, J., Vasquez, H., Quintero, G., Perez De Leon, A.A., Belikoff, E.J., Azeredo-Espin, A.M., Mcmillan, W.O., Concha, C., Scott, M.J. 2019. Specific gene disruption in the major livestock pests Cochliomyia hominivorax and Lucilia cuprina using CRISPR/Cas9. Genes, Genomes, Genetics. Volume 9, Issue 7.