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 Livestock Arthropod Pest Research Unit: Kerrville, TX
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ARS Home Plains Area Kerrville, Texas Knipling-Bushland U.S. Livestock Insects Research Laboratory Livestock Arthropod Pest Research Unit Research





- Olafson, Pia
- Showler, Allan
- Arp, Alex
- Tietjen, Mackenzie
- Hickner, Paul
- Temeyer, Kevin
- 2023
- 2022
- 2021
- 2020
- Publications
- Animal Health (103)
- Veterinary, Medical and Urban Entomology (104)
- Food Safety, (Animal and Plant Products) (108)

Research Project: Integrated Pest Management of Flies of Veterinary Importance **Location:
                            Livestock Arthropod Pest Research Unit** 2020 Annual Report **Objectives**
                                

Objective 1: Develop more accurate models of fly dispersal by incorporation of genetics, remote sensing, and GIS into the surveillance of screwworm flies. 
Objective 2: Identify, develop and evaluate the efficacy of novel surveillance and control strategies, including genomic-based strategies for house, stable, horn and New World screwworm flies. 
•	Subobjective 2A: Assess compounds for behavior modifying and insecticidal properties to control muscid flies. 
•	Subobjective 2B: Develop and expand tools for functional genomic investigations of muscid flies for identification and validation of control targets. 
•	Subobjective 2C: Develop germ-line transformation strains for muscid and calliphorid flies to evaluate potential for suppressing fly populations. 
•	Subobjective 2D: Elucidate mechanisms of muscid fly insecticide resistance utilizing available genome sequence databases and develop molecular surveillance assays to monitor gene fixation and flow in natural populations.
•	Subobjective 2E: Develop muscid and calliphorid larval feeding bioassays to identify and characterize phagostimulant and phagoinhibitory substances.
•	Subobjective 2F: Assess the effect of tetracycline on the gut transcriptome and microbiome of NWS. 
•	Subobjective 2G: Assess fly proteins selected from genomic data as potential immunogens for control of muscid flies.
Objective 3: Characterize population genetics and population ecology of New World screwworms and develop approaches to mitigate range expansion and accidental introduction into new locations. 
•	Subobjective 3A: Develop a population genetics database of screwworms from the Caribbean region. 
•	Subobjective 3B: Isolate and identify attractants optimized for male NWS. **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**
                                

In support of Objective 1, to develop more accurate models of screwworm fly dispersal, New World screwworm (NWS) samples were collected in Trinidad and Tobago and the Dominican Republic. Geospatial data from these samples will be used to make predictive models for NWS presence in the Caribbean. Additionally, samples will be used in genotyping by sequencing analyses to identify potential genetic markers associated with host preference, population, or migration routes. These genetic markers will provide population-specific signatures for the different NWS populations and support source identification efforts should future NWS incursions occur as well as to aid in identification of loci potentially associated with insecticide resistance. 
Research addressing Objective 2, to develop novel surveillance and fly control strategies resulted in the identification of natural and synthetic products that are effective as insecticides or repellents of filth flies affecting livestock. Molecular databases and tools are being developed to identify and functionally characterize genes critical to stable fly and face fly biology, and continued advancements were made in developing germ-line transformation strains for use in suppression of NWS populations, including NWS strains that result in all-male offspring. 
Multiple essential oils and their components were tested for repellent efficacy to horn flies, in support of Subobjective 2A to investigate their insecticidal and behavior modifying properties. Citronellol was as effective a repellent as N,N-Diethyl-meta-toluamide (DEET) to both sexes of horn flies, while cinnamon oil and spearmint oil were each as repellent as DEET to male horn flies but were much less repellent to females. Filth flies are difficult to manage in cattle production systems. ARS scientists in Kerrville, Texas and Gainesville, Florida in collaboration with partners at Northern Illinois University, Pennsylvania State University, and Cornell University, determined that fluralaner is effective for filth fly control because of its performance against horn flies and multiple strains of house flies, and because its mode of action differs from any filth fly insecticides currently on the market.  In addition, the botanical compounds p-Anisaldehyde and limonene were each found to exhibit repellency and insecticidal effects to various life stages of house flies and horn flies, suggesting the potential use as an aid in controlling insecticide resistant house and horn fly populations. p-Anisaldehyde was active as a larvicide to house flies at final concentrations in bovine manure as low as 0.15%. Taken together, natural compounds and fluralaner present new opportunities for development of effective fly control strategies to potentially mitigate increasing pesticide resistance in house and horn fly populations.  
In support of Subobjective 2B to expand functional genomics of flies, a database of genes expressed by the face fly, Musca autumnalis, was assembled. The face fly is a non-biting, muscid livestock pest that is characterized by a larval stage that develops in dung, which is similar to the larval stage of the blood-feeding, biting horn fly. This interesting biology provides an opportunity for comparative gene expression analyses. The face fly database represents genes expressed by newly emerged and unfed adults, fed and mated adults, and the male reproductive tract. This data will form the basis for identifying genes that are unique to these conditions and those that exhibit differences in levels of expression between these conditions. Further, molecular components were developed to target and disrupt stable fly genes that control eye color and body pigment, in preparation for proof-of-concept gene disruption studies. 
Strains of NWS containing the DR7 driver construct were created and bred to homozygosity to address Subobjective 2C, developing germ-line transformation strains to evaluate potential use in suppression of NWS populations. Strains with DR7 were crossed with strains homozygous for a female lethal effector construct and bred to homozygosity. Preliminary testing with these strains proved that they do produce only males in the absence of the repressor tetracycline. Further fitness testing is required to evaluate the effectiveness of these strains for mass rearing and to ensure that death occurs in the embryo as predicted. Further, strains of NWS that result in all-male offspring were developed by suppressing the female sex determination pathway. The strains contain an effector construct that produces inverted repeats to essentially silence transformer, a gene that promotes female development. Two different constructs have been bred to homozygosity and crossed with DR7 driver strains. Preliminary testing has resulted in flies with female heads and male abdomens. 
In support of Subobjective 2E to develop fly larval feeding bioassays for identification of substances that act as feeding stimulants (phagostimulants) or feeding deterrents (phagoinhibitors), fluorescent-labeled paramagnetic particles were utilized to measure larval ingestion of the bacteria-sized particles.  Aqueous extract of bovine feces, yeast extract, and ethanol were all found to be feeding stimulants to 2nd instar horn fly larvae. 
Research was completed to address Subobjective 2F, assessing the gut transcriptome and microbiome of NWS. Samples of field-collected NWS larvae, adults, and myiasis swabs were acquired from sites in Panama, while samples from transgenic and production NWS strains and their associated diet were collected from the COPEG (Panama – United States Commission for the Eradication and Prevention of Screwworm) mass rearing facility. These samples will form the basis of comparisons between the microbial community of field-collected versus laboratory-reared NWS populations. All samples were dissected, with genomic DNA from gut and body extracted independently, and these samples will be sequenced in Year 2 of the current project.  
Finally, in support of Subobjective 2G, a select number of horn fly antigens were formulated for cattle immunization and in vitro assays were completed to assess potential fly immunogens for development of anti-fly vaccines for cattle.  
Research related to Objective 3 was conducted to assess population genetics and ecology of NWS in the Caribbean region and to isolate and identify NWS attractants. Samples from the Dominican Republic, Trinidad and Tobago were collected. All specimens were labeled with associated location and host metadata. Data analysis protocols and software were prepared for use in downstream analyses once sample collection is completed and sequencing data are prepared. Odor chemical signatures from the cuticles of mated and unmated male and female NWS adults were collected for testing to identify components that exhibit attractant properties to NWS. **Accomplishments**
                                

1. Fluralaner effective in controlling filth flies impacting cattle production systems. Filth flies continue to be a challenge to manage in cattle production systems. ARS scientists in Kerrville, Texas and Gainesville, Florida in collaboration with partners at Northern Illinois University, Pennsylvania State University, and Cornell University, determined that fluralaner is effective for control of horn flies and multiple strains of house flies. The mode of action for fluralaner differs from any filth fly insecticides currently on the market, indicating it can be used to control insecticide-resistant populations of horn flies and house flies. 2. Natural compounds as insecticides and fly repellents. Fly resistance to chemical pesticides is an increasing problem highlighting the need to develop alternatives to existing chemical pesticides for fly control. ARS scientists at Kerrville, Texas demonstrated the effectiveness of a number of natural compounds, including essential oils and other botanical compounds as repellents or insecticides when utilized in laboratory bioassays against various life stages of horn flies and house flies. Cinnamon oil, spearmint oil, citronellol, and limonene each exhibited repellent activity comparable or greater than N,N-Diethyl-meta-toluamide (DEET) to horn flies. Unlike DEET, these natural plant compounds also exhibited insecticidal activity against horn flies. These compounds can be incorporated into integrated pest management strategies to suppress populations of horn flies associated with livestock production, reducing losses to livestock producers and potential annoyance to nearby human populations. **Review Publications**
                            *Dupuis, J., Ruiz, R., Barr, N., Thomas, D.B., Geib, S.M. 2019. Range-wide population genomics of the Mexican fruit fly: Toward development of pathway analysis tools. Evolutionary Applications. 12(8):1641-1660. https://doi.org/10.1111/eva.12824.*
                                *Domingues, L.N., Solis, G.D., Bendele, K.G., Perez De Leon, A.A., Guerrero, F. 2020. Sequence and transcript expression of the super-kdr locus of the horn fly, Haematobia irritans. Medical and Veterinary Entomology. https://doi.org/10.1111/mve.12442.*
                                *Temeyer, K.B., Schlechte, K.G., Dandeneau, L. 2020. Sand fly colony crash tentatively attributed to nematode infestation. Journal of Medical Microbiology. 57(4):1301-1304. https://doi.org/10.1093/jme/tjz256.*
                                *Temeyer, K.B., Schlechte, K.G., McDonough, W.P. 2019. Baculoviral expression of presumptive OP-resistance mutations in BmAChE1 of Rhipicephalus (Boophilus) microplus (Ixodida:Ixodidae) and biochemical resistance to OP inhibition. Journal of Medical Entomology. 56(5):1318-1323. https://doi.org/10.1093/jme/tjz062.*
                                *Burgess, E.R., Geden, C.J., Lohmeyer, K.H., King, B., Machtinger, E.T., Gaillard, E.R., Scott, J.G. 2020. The companion animal insecticide fluralaner outperforms industry-leading agricultural insecticides in resistant and susceptible strains of filth flies. Scientific Reports. 10(1):1-8. https://doi.org/10.1038/s41598-020-68121-z.*
                                *Showler, A., Harlien, J.L., Perez De Leon, A.A. 2019. Effects of laboratory grade limonene and a commercial limonene-based insecticide on Haematobia irritans irritans (L.) (Muscidae: Diptera): deterrence, mortality, and reproduction. Journal of Medical Entomology. 56(4):1064-1070. https://doi.org/10.1093/jme/tjz020.*
                                *Romero-Salas, D., Cruz-Romero, A., Aguilar-Dominguez, M., Ibarra-Priego, N., Barradas-Pina, F.T., Nogueira-Domingues, L., Castro-Arellano, I., Lohmeyer, K.H., Perez De Leon, A.A. 2018. Seroepidemiology of bovine herpes virus-1 infection in water buffaloes from Mexico. Tropical Biomedicine. 35(2):541-552.*
                                *Showler, A., Harlien, J.L. 2018. Lethal and repellent effects of the botanical p-anisaldehyde on Musca domestica L. (Diptera: Muscidae). Journal of Economic Entomology. 112:485-493. https://doi.org/10.1093/jee/toy351.*
                                *Domingues, L.N., Guerrero, F., Foil, L.D. 2019. Impacts of long-term insecticide treatment regimes on skdr and kdr pyrethroid resistance alleles in horn fly field populations. Parasitology Research. 118:2485–2497. https://doi.org/10.1007/s00436-019-06386-8.*
                                *Domingues, L.N., Guerrero, F., Cameron, C., Farmer, A., Bendele, K.G., Foil, L.D. 2018. The assembled transcriptome of the adult horn fly, Haematobia irritans. Data in Brief. 19:1933-1940.*
                                *Meisel, R.P., Olafson, P.U., Guerrero, F., Konganti, K., Benoit, J.B. 2020. High rate of sex chromosome turnover in muscid flies. G3, Genes/Genomes/Genetics. 10(4):1341-1352. https://doi.org/10.1534/g3.119.400923.*
                                *Temeyer, K.B., Schlechte, K.G., Olafson, P.U., Drolet, B.S., Tidwell, J.P., Osbrink, W.L., Showler, A., Gross, A., Perez De Leon, A.A. 2020. Association of salivary acetylcholinesterase with arthropod vectors of disease. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjaa096.*
                                *Li, Z., Guerrero, F., Perez De Leon, A.A., Foil, L.D., Swale, D.R. 2020. Small-molecule inhibitors of inward rectifier potassium (Kir) channels reduce bloodmeal feeding and have insecticidal activity against the horn fly (Diptera: Muscidae). Journal of Medical Entomology. 57(4):1131-1140. https://doi.org/10.1093/jme/tjaa015.*
                                *Showler, A., Garcia, A.R., Caesar, R.M. 2020. Lethal effects of a silica gel + pyrethrins (Drione) on Amblyomma americanum (L.) (Ixodida: Ixodidae) larvae and nymphs. Veterinary Parasitology. https://doi.org/10.1093/jme/tjaa119.*
                                *Barros, A.T., Rodrigues, V.D., Cancado, P.H., Nogueira Domingues, L. 2019. Resistance of the stable fly, Stomoxys calcitrans (Diptera: Muscidae), to cypermethrin in outbreak areas in Midwestern Brazil. Brazilian Journal of Veterinary Parasitology. 28(4):802-806. https://doi.org/10.1590/s1984-29612019089.*


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