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                    U.S. DEPARTMENT OF AGRICULTURE
 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** 2023 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**
                                

In support of Objective 1 to 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, ARS researchers in Kerrville, Texas, and Pacora, Panama, collected New World screwworm flies (NWS), Cochliomyia hominivorax, from sites within the Caribbean Region, documented by GPS (Global Positioning System) location. GIS (Global Information System) environmental features of the fly collection sites were examined to determine fly habitat characteristics and distribution. ARS scientists together with other researchers examined the presence of NWS and the associated genetic landscape to gain insights into the characteristics and distribution of fly habitats. Collected flies were genetically characterized for each location by genetic sequencing and analysis to elucidate the genetic divergence of flies collected from each of the collection sites, revealing that flies in Trinidad and Tobago exhibit greater genetic diversity than flies in the Dominican Republic, suggesting greater isolation of fly populations in the Dominican Republic, perhaps due to proximity of Trinidad to Venezuela and dispersal of flies between Trinidad and Venezuela. This new information may be critical to the potential success of control programs within the region.
In support of Objective 2 to develop and evaluate the efficacy of novel control strategies for house, stable, horn, and New World screwworm flies (Subobjective 2A), previous research by ARS scientists in Kerrville, Texas, was expanded to demonstrate the insecticidal and repellent activity of p-anisaldehyde and desiccant dusts, some augmented with botanical compounds. Research is continuing to evaluate and extend the residual efficacy of p-anisaldehyde as a repellent to house flies. Essential oils and effective components previously identified as repellent to the sand fly, Phlebotomus papatasi strain Israeli were evaluated by bioassay against horn flies, stable flies, and house flies. Results comparing over 20 different essential oils and components exhibited an apparent inverse relationship between average fly size (by species) and repellent efficacy, that is, repellent efficacy of essential oil components generally decreased from horn flies to stable flies to house flies. None of the compounds tested were more repellent than N,N-diethyl-meta-toluamide (DEET) against horn flies, and only eugenol and geraniol were more repellent to both sexes of stable flies than DEET, exhibiting equivalent efficacy as 1% DEET at half the dose. In addition, IR3535, a synthetic insect repellent used extensively in Europe was also less repellent to horn flies, stable flies, and house flies than DEET. Laboratory bioassay data to evaluate topical and oral fluralaner demonstrated that this novel isoxazolin insecticide was highly effective against horn flies, house flies, and stable flies. Laboratory bioassays were also conducted to evaluate the efficacy of topical cedarwood oil extract against stable flies. Bioassay data were analyzed and a manuscript detailing the control efficacy of fluralaner has been published. A manuscript on the efficacy of cedarwood oil is anticipated to be submitted for peer-reviewed publication before the end of FY23. (Subobjective 2D) ARS scientists at Kerrville, Texas, continued to collaborate with Texas A&M University to establish a stable fly line originating from Texas flies; however, difficulties remain for reliably propagating flies for more than a generation, complicating efforts to pressure a population and select for pyrethroid resistance. Further collaborations between scientists from ARS and Texas A&M University are evaluating host usage by stable flies collected from a wildlife safari in Texas. A molecular assay was utilized to identify bloodmeals ingested by individual flies, and detected bloodmeals of various exotic hosts, including yak, water buffalo, elk, fallow deer, and eland. These results emphasize the cosmopolitan feeding behavior of stable flies given the availability of nearby cattle hosts. (Subobjective 2F) Transgenic New World Screwworm (NWS) strains are reared in diet containing antibiotics to suppress the transgene, but the effects of antibiotic on gut homeostasis was not previously explored. In support of Sub-objective 2F, ARS researchers at Kerrville, Texas, and Pacora, Panama, completed sequencing and analysis of bacterial communities and total RNA from the guts of larval and adult NWS reared with or without tetracycline in the diet to assess the influence of antibiotics on midgut gene expression in C. hominivorax. This work is important because tetracycline/doxycycline is required to propagate the female-lethal strains but may affect the competitiveness of the released sterile flies. Gut samples were collected from a simulated “male-only” condition where the previous generation was reared on tetracycline, but the test generation was reared without tetracycline. RNAseq data revealed only 93 genes are differentially expressed between production strain and male-only strain compared to 1516 genes differentially expressed when reared on tetracycline. Significant changes were observed in gut microbiomes of NWS larvae and adults reared with and without tetracycline, with tetracycline reducing the bacterial diversity. The “male-only” generations were more like NWS reared without tetracycline than their parental generation reared with tetracycline, suggesting a rapid shift back to a normal gut microbiome in the absence of tetracycline. We continue to conduct nutritional analyses of the components comprising the larval diets for studying and mass rearing C. hominivorax at the plant in Pacora, Panama (COPEG). These components include spray-dried bovine red blood cells, spray-dried bovine plasma, milk replacement powder, egg powder, and soy powder. Our recent studies have shown that chicken by-products are a suitable replacement for the egg and milk replacement powders, potentially reducing rearing costs. Therefore, we have recently added the chicken by-products to the nutritional analysis expected to be completed by the middle of August 2023 to conclude current studies on nutritional composition of the larval diet. 
In support of Objective 3 to characterize population genetics and population ecology of New World screwworms and develop approaches to mitigate range expansion and accidental introduction into new locations ARS scientists in Kerrville, Texas, utilized secondary screwworm, Cochliomyia macellaria, as a model for NWS. There have been recent expansions of NWS from Columbia across the permanent quarantine barrier into Panama suggesting that the release of sterile male NWS may be losing effectiveness due to reduced competitiveness with wild flies. Competition with wild flies may involve many factors. To understand what genetic and behavioral factors may be involved, more needs to be known about the biology of these flies. Because a colony of NWS cannot be maintained in the United States, ARS scientists established a colony of the secondary screwworm fly, Cochliomyia macellaria, in Kerrville, Texas, as a proxy for C. hominivorax. Comparative analysis with NWS will provide valuable insights into the evolution of parasitism in the Cochliomyia, as well as to advance genetic and behavioral studies of NWS. The genome of C. macellaria was recently sequenced using a “trio binning” approach and a chromosome level assembly was produced. RNA sequencing for gene annotations and comparative analysis with NWS are under way. **Accomplishments**
                                

1. A genetic landscape map of the New World screwworm in the Caribbean. Screwworm has been eradicated from the United States, but outbreaks can and do occur as it is still endemic in much of the Caribbean and South America. Screwworms were collected in the Caribbean by ARS researchers in collaboration with USDA-Animal and Plant Health Inspection Service (APHIS), Commission for the Eradication and Prevention of Screwworm (COPEG), and local Agricultural Ministries. The GPS coordinates of individual flies were correlated with their genetic data to produce a genetic landscape. Evidence of dispersal from mainland Venezuela to the island of Trinidad was observed while the smaller island of Tobago supports a less diverse population pointing to genetic bottleneck. The Dominican Republic was also less diverse indicating its distance from other populations being far enough to limit dispersal. The environmental correlates as they relate to this fly’s presence were also examined and show that this fly is most often found where canopy cover is 15-40% and average precipitation is 225 mm. These results aid in locating the best sites for future surveillance and understanding dispersal in this fly. 2. Blacklegged ticks and deer keds from the same deer host do not predictably share disease-causing pathogens. Ticks and tick-borne diseases are an increasing threat to human and animal health. Blacklegged ticks transmit the agent of Lyme disease in the United States, and adult ticks complete their life cycle on white-tailed deer (WTD). WTD are not a source for the Lyme disease pathogen, but they are a host to other ticks and biting flies. European deer keds are a blood-feeding fly that feeds primarily on WTD, and this introduced species is found in the northern United States and Canada and it has an expanding range. Interestingly, blacklegged ticks and European deer keds have been found feeding on the same deer host, creating a scenario where these pests might share disease-causing pathogens as they are feeding. ARS researchers in Kerrville, Texas, and Pullman, Washington, collaborated with University of Pennsylvania researchers to examine this understudied relationship. Using molecular assays to detect bacterial pathogens, the analysis of paired tick-deer ked specimens collected from individual WTD hosts indicated that there were limited incidences of shared pathogens despite pathogenic organisms being detected from each species. These results provide a framework for continued studies to define the role of deer-tick-ked interactions, as they relate to the transmission of tick-borne pathogens. 3. A new formulation of screwworm fly attractant. The New World screwworm fly (NWS) is an obligate parasite of warm-blooded animals that was eradicated from North America during the mid- to late-20th century using the sterile insect technique. Lures are an important component of the screwworm eradication program, where they are used for surveillance, sample collection, and strain evaluation in the field. The first chemical lure, swormlure, was developed based on the attractiveness of NWS to volatile organic compounds produced from decomposing animal tissues. The formulation has changed little over the years and presently contains 10 chemicals, one of which is dimethyl disulfide (DMDS). Restrictions on the transport of DMDS have recently impeded its use in swormlure-4 (SL-4). However, dimethyl trisulfide (DMTS) is not as highly restricted and can be shipped via air transportation. Both chemicals are produced by microbial decomposition of animal tissues. Field trials using sterile NWS showed that swormlure containing DMTS (SL-5) was as effective as swormlure containing DMDS (SL-4). **Review Publications**
                            *Olafson, P.U., Poh, K.C., Evans, J.R., Skvarla, M.J., Matchinger, E.T. 2022. Limited detection of shared zoonotic pathogens in deer keds and blacklegged ticks co-parasitizing white-tailed deer in the eastern United States. Medical and Veterinary Entomology. 37(2):179-188. https://doi.org/10.1111/mve.12620.*
                                *Tietjen, M., Arp, A.P., Lohmeyer, K.H. 2023. Development of a diagnostic SNP panel for identifying geographic origins of Cochliomyia hominivorax, the New World screwworm. Veterinary Parasitology. https://doi.org/10.1016/j.vetpar.2023.109884.*
                                *Tietjen, M., Pfeiffer, V., Poh, K.C. 2022. Insights into the genetic landscape and presence of Cochliomyia hominivorax in the Caribbean. Parasitology Research. 122:547-556. https://doi.org/10.1007/s00436-022-07757-4.*
                                *Hickner, P.V., Pacheco, L., Duke, S.E., Sanchez Ortiz, C., Welch, J.B., Phillips, P.L., Arp, A.P. 2023. A new formulation of screwworm attractant with reduced hazardous chemicals and transport restrictions. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjad043.*
                                *Labbe, F., Abdeladhim, M., Abrudan, J., Hickner, P.V., et al. (2023). Genomic analysis of two phlebotomine sand fly vectors of Leishmania from the New and Old World. PLOS Neglected Tropical Diseases. https://doi.org/10.1371/journal.pntd.0010862.*
                                *Bendele, K.G., Guerrero, F., Lohmeyer, K.H., Foil, L., Metz, R., Johnson, C. 2023. Horn fly transcriptomes from 10 populations from the southern United States. Data in Brief. https://doi.org/10.1016/j.vetpar.2022.109699.*


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