Location: Livestock Arthropod Pest Research Unit
2024 Annual Report
Objectives
Objective 1: Develop more accurate models of fly dispersal by incorporation of population genetics, remote sensing, and GIS into the surveillance of stable flies, horn flies, and screwworm flies.
Objective 2: Develop and evaluate the efficacy of novel control strategies for house, stable, horn and New World screwworm flies.
Approach
Muscid and calliphorid pests of livestock are of veterinary and medical importance worldwide, as they negatively impact both livestock production efficiency and human and animal health. The overall goal of this project is to diminish the impact of muscid and calliphorid pests by reducing host-pest interactions. Populations of stable, horn, and house flies have traditionally been managed by application of insecticides, but development of resistance to chemicals and a desire for more environmentally conscious approaches have shifted our research emphasis to identify more sustainable tactics. Chemical ecology, toxicology, molecular biology, and gene editing/genetic engineering methods will be employed to identify behavior modifying compounds and biological pathways regulating host orientation, larval survival, and insecticide resistance. This will enable development of mating disruption strategies and biologically-based management tools. One of the foci of this project, the New World screwworm (NWS), remains endemic to the Caribbean and South America, and a permanent barrier is maintained at the Panama-Colombia border to prevent re-introduction northward. Improved technologies to support population suppression and outbreak prevention would be beneficial to the bi-national commission that manages the permanent barrier. This project will blend geographic information system technologies with reduced genome sequencing approaches to characterize current and to model future pest distribution, as it relates to climate and landscape features. This will allow the scaling of sterile fly release rates and projections of NWS dispersal range, which are critical to maintaining the permanent barrier. Promising leads will be pursued to move towards development of applications that reduce negative impacts of these muscid and calliphorid pests.
Progress Report
Objective 1: New World Screwworm fly (NWS) sampling efforts in the Caribbean were conducted throughout the life of this project and, coupled with genetic data from these samples, enabled the development of a more accurate NWS distribution map in this region. Models were developed to predict the prevalence of NWS, a tool that can aid eradication program personnel to target their future surveillance efforts. The current predictive model can be further improved with broader sampling efforts in ‘unknown’ areas. Data acquired from surveillance of flies of veterinary importance are important for developing more accurate distribution models, but the traps used for surveillance tend to collect both target and non-target, closely related fly species. ARS researchers at Kerrville, Texas, are collaborating with the University of Kentucky to develop improved diagnostic tools based on unique genetic signatures of the various fly species that occupy the same landscape as the NWS. To date, ~1,300 flies have been collected, representing 19 different species.
Objective 2: Throughout this project, a broad panel of essential oils, select compounds, and novel chemistries were assayed for activity against horn flies, stable flies, and house flies. These assays were intended to identify additional tools for use within an integrated pest management (IPM) strategy for fly pests of livestock. A few essential oil compounds were as repellent as N,N-diethyl-meta-toluamide (DEET) towards horn flies (cinnamon bark oil and citronellol) and stable flies (citronellol, clove oil, and trans-cinnamaldehyde), while geraniol and eugenol had twice as high repellent activity as DEET against stable flies. The repellent activity observed against horn flies and stable flies was short-lived, exhibiting repellency for 24 hours or less, even at the highest concentrations tested. None of the essential oils exhibited significant repellent activity against house flies. The compound p-anisaldehyde had repellent activity towards adults and lethality in embryos of horn flies and house flies. Further, it was a feeding deterrent to adult horn flies and house flies. Fluralaner, a novel isoxazoline compound, exhibited insecticidal activity toward horn flies, stable flies, and house flies. In support of developing tools to enhance IPM strategies, a larval bioassay was developed to detect substances that stimulate larval feeding. The assay is based on labeled paramagnetic microparticles (1-2 microns in diameter). Including yeast extract or ethanol into larval food resulted in a higher recovery of these microparticles relative to a control. This indicated that these substances stimulated ingestion (phagostimulatory) and supported the use of this assay to identify additional substances that can be added to toxicant baits for enhanced larval mortality.
ARS researchers at Pacora, Panama, continued efforts to develop and maintain transgenic, male-only lines of NWS in support of Objective 2. Difficulties were encountered when rearing strains with elements that drive the expression of lethal genes in the embryonic stage of NWS development. The antibiotic doxycycline was substituted for tetracycline in colony maintenance protocols, drastically increasing these transgenic lines' performance and stability. However, observed lethality occurred in the pupal rather than the predicted embryonic stage upon shifting the antibiotic. This was resolved by removing doxycycline from the diet during the later stages of larval development, rescuing the embryonic lethality, and improving fitness in all strains. Evaluation of the gut microbial community (microbiome) and the genes expressed (transcriptome) by larvae reared on tetracycline revealed that being fed a diet with doxycycline results in observable changes in both microbiome and transcriptome, though there are few observable effects on larval fitness. There were few differences between flies reared with or without tetracycline, indicating there are no long-term effects caused by the rearing system required for transgenic male-only strains of screwworm.
Throughout this project, ARS researchers at Kerrville, Texas, improved genomic resource availability for pests associated with livestock settings, including whole genome sequences for the face fly, the black dump fly, and the lesser house fly. In collaboration with researchers at Texas A&M University, advances were made in protocols for consistently injecting stable fly embryos to conduct gene editing studies for characterizing the function of genes of interest. In a separate collaboration with Texas A&M University, protocols were optimized to identify the host bloodmeal source of stable flies collected from field settings, improving our understanding of the interaction between host availability and fly preference.
Objective 3: ARS researchers at Kerrville, Texas, established and continued improvement of an NWS population genetic reference database, which was undertaken over the life of this project. The database was designed to enable source identification of NWS outbreaks encountered within and outside the eradication buffer zone. This effort included optimizing DNA extraction protocols and data analysis pipelines; coordinating field collections originating from a wide range of localities, including now eradicated countries; identifying genetic markers unique to 12 populations across the geographic range; and developing an assay that can distinguish NWS flies originating from the Outer Caribbean, Inner Caribbean, or South America. The assay has been critical to providing same-day results in a standard laboratory without the need for sequencing, and it is being employed to address questions about the geographic origin of the surge in NWS outbreak cases that have occurred since 2022. Importantly, collaborations were established in 2024 to receive wild-caught NWS samples from Brazil, Colombia, and Cuba to continue to expand the database in future project cycles.
In further support of this objective, ARS researchers at Kerrville, Texas, improved the formulation of a lure used to attract NWS, reducing cost and improving transportability. Specialty equipment was designed and fabricated to study NWS behaviors, including an olfactometer to test attraction/repellency and an apparatus that catalogs the daily activity patterns of NWS. Equipment for macro- and microanalyses of NWS olfaction was obtained, comprising rigs for acquiring the output of the NWS antenna to its brain in response to an odor (electroantennogram) and the output from a single sensory neuron on the antenna in response to an odor. ARS scientists in Kerrville, Texas, identified eight odorant receptors that are highly female-biased in NWS antennae. Five of these have been cloned into a model fly antenna system (Drosophila) for screening with a panel of chemical compounds. These tools support planned studies to design an improved, NWS female-specific lure that can be deployed in traps for population reduction.
Accomplishments
1. Doxycycline is a viable alternative to tetracycline for rearing lines of male-only, transgenic screwworm. The production of sterile screwworm flies used in the sterile insect technique (SIT) release program relies on irradiating both female and male adults. The sterile males that are released mate with wild females to produce sterile progeny, reducing the size of the fly population. The released females, although sterile, may still cause damage. Transgenic male-only insect lines offer vast improvements to the SIT release programs by increasing efficacy and lowering costs. This genetic system is designed to turn ‘on’ a female-lethality gene, eliminating females from production. The fly diet includes the antibiotic tetracycline to turn ‘off’ the female-lethality gene to produce generations of flies for a colony. The transgenic flies would need to be produced in massive quantities for a release program, requiring large amounts of tetracycline. ARS researchers at Pacora, Panama, evaluated the use of doxycycline, an alternative antibiotic. They demonstrated that doxycycline is effective at substantially lower dosages and is more stable than tetracycline, making it a promising alternative in a mass-rearing setting with significant cost savings for the program.
2. Genomic assay confirms that screwworm outbreak in Panama originated from South America, most likely Colombia. For 16 years, a biological “barrier zone” for screwworm flies has been maintained at the Panama—Colombia border in the Darién Gap. This zone is a buffer between South America, where screwworm flies are widespread, and Central America/North America, which is free of them. In the “barrier zone,” there are usually only about 60 outbreak cases per year. Of significant concern, cases started to be detected outside this area in 2022, and over 8,000 outbreak cases have now been recorded throughout Panama and into Costa Rica and Nicaragua. These outbreak flies were either transported to the area by boats arriving in Panama from the Caribbean, introducing flies from outside the “barrier zone,” or the flies had come from across the Colombian border, overcoming the “barrier zone.” To better understand the origin of these outbreak screwworm populations, fly outbreak samples were collected from seven regions of Panama for analysis using tools developed by an ARS researcher in Kerrville, Texas. Fly DNA was extracted from all samples, analyzed using a screwworm diagnostic genomic assay, and compared to a screwworm genetic reference database based on geographic collections. The results indicated that outbreak flies indeed came from South America, most likely Colombia, suggesting that resources be invested in more stringent surveillance of the “barrier zone” region.
3. An alternative chicken-based diet for mass-rearing screwworm flies. The control of screwworm flies is achieved using the sterile insect technique (SIT), which involves the mass-rearing and releasing of sterile screwworm flies along the Panama-Colombia border to prevent reinfestation of Central and North America. The diet used for mass-rearing greatly impacts the quality and quantity of sterile flies produced, both of which are necessary to successfully implement SIT. The diet currently used to mass-rear screwworm flies in Panama contains dried bovine red blood cells, dried bovine plasma, egg powder, milk replacement powder, cellulose (thickening agent), formaldehyde (antimicrobial), and water. ARS researchers at Kerrville, Texas, and Pacora, Panama, developed an alternative diet containing two chicken by-products that can be used as a substitute for the egg and milk components of the current diet. The alternative diet performs similarly to the current diet; the chicken by-products cost less and are locally available, which would provide significant cost savings to the program.
Review Publications
Temeyer, K.B., Schlechte, K.G., Coats, J.R., Cantrell, C.L., Rosario-Cruz, R., Lohmeyer, K.H., Perez De Leon, A.A., Li, A.Y. 2024. In vitro evaluation of essential oils and saturated fatty acids for repellency against the old-world sandfly, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Insects. https://doi.org/10.3390/insects15030155.
Arp, A.P., Williamson, M.E., Vasquez, M., Quintero, G., Vargas Lowman, A., Sagal, A., Scott, M.J. 2024. Doxycycline is a viable alternative to tetracycline for use in insect Tet-Off transgenic sexing systems, as assessed in the blowflies Cochliomyia hominivorax(Dipteria:Calliphoridae) and Lucilia Cuprina(Diptera:Calliphoridae). Journal of Economic Entomology. https://doi.org/10.1093/jee/toae023.
Hickner, P.V., Sagel, A., Quintero, G., Vasquez, M., Tietjen, M., Lohmeyer, K.H., Arp, A.P. 2023. An alternative chicken-based diet for mass-rearing screwworm flies, Cochliomyia hominivorax (Diptera: Calliphoridae). Journal of Economic Entomology. https://doi.org/10.1093/jee/toad219.
Showler, A., Harlien, J.L. 2024. Effects of three desiccant dusts and two dusts augmented with botanicals on house fly (Diptera: Muscidae) eggs, larvae, pupae, and adults. Journal of Economic Entomology. http://dx.doi.org/10.59467/BI.2024.20.73.