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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** 2022 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, researchers at Kerrville, Texas, and Pacora, Panama, created ecological niche models for New World Screwworm (NWS) in the Caribbean using the algorithms, Maximum Entropy, Generalized Linear Model, Generalized Additive Model, and an Ensemble model. Model evaluation will improve targeting of NWS control efforts and identify areas at risk for NWS, thus increasing control efficacy at reduced cost.  A genetic distance matrix incorporating genetic variation will be included in the best fit model after evaluation completion. By incorporating genetics in the model, connection corridors can be identified to reveal how and where flies are dispersing to allow management units to know if populations can be eradicated at separate times or if dispersal between areas requires simultaneous eradication. Understanding areas at risk for screwworm and connections between fly populations will improve eradication efficiency.
ARS researchers at Kerrville, Texas, expanded assessment of compounds exhibiting repellent or insecticidal activity toward muscid flies, as part of Sub-objective 2A. Repellent activity of p-anisaldehyde, previously found to be repellent to house flies, remained stable for prolonged storage at ambient temperature, and for at least a week exposed to direct sunlight, suggesting that p-anisaldehyde would retain repellent activity in field trials. In addition, spraying food sources attractive to house flies substantially reduced fly landing on the treated food source compared to an untreated food source. P-anisaldehyde was shown to be lethal to eggs of horn flies, stable flies, and house flies, suggesting that p-anisaldehyde, a compound found in vanilla, fennel, and cranberries, may be useful for filth fly control on or in oviposition substrates. Ongoing research is examining use of inert desiccant dusts to control house fly adults and larvae. ARS researchers tested components of essential oils that previously exhibited greater repellency to sand flies than diethyltoluamide (DEET), for horn fly, stable fly and house fly repellency. Oils and components tested included catnip oil, cinnamon oil, citral, citronella oil, citronellol, clove oil, cornmint oil, DEET, Essentria-IC3, eugenol, geraniol, IR3535, lemongrass oil, menthol, p-anisaldehyde, sesame oil, spearmint oil, thyme oil and trans-cinnamaldehyde. Horn flies were repelled by citronellol or DEET at 0.06125%, while cinnamon oil and spearmint oil were equally effective only on male flies. Stable flies exhibited greater repellent sensitivity to eugenol, geraniol, trans-cinnamaldehyde and citronellol at 0.5% than to 1% DEET. An inverse relationship between fly size and repellent efficacy of essential oils and components was apparent. Lastly, laboratory bioassays revealed that topical and oral formulations of fluralaner applied to cattle were highly effective against horn flies, house flies, and stable flies. Laboratory bioassays also evaluated efficacy of topical cedarwood oil extract against stable flies. 
In support of Sub-objective 2B to expand functional genomics of flies, researchers at Kerrville, Texas, designed and assembled constructs to replace a gene cluster encoding defensin proteins with antimicrobial properties. It is anticipated that microinjections of the replacement cluster will be completed in the first quarter of Year 4 (FY23) to evaluate if removal of the defensin genes affect stable fly ability to survive and thrive in microbe-rich substrates. In addition, Kerrville researchers collaborated with a University of Texas San Antonio researcher to characterize a stable fly odorant binding protein that exhibited an affinity for metal ions (i.e., copper, nickel, and zinc), suggesting that it may have a role to protect the fly nervous and immune systems from negative effects of circulating metal ions.
Studies continued in support of Sub-objective 2C to develop germ-line transformation strains for calliphorid flies. Tetracycline is used in the NWS conditional gene expression systems to control lethality of female flies in colony propagation and maintenance; however, ARS researchers at Kerrville, Texas, and Pacora, Panama, found that tetracycline is unstable in larval diet, resulting in variable fly yields and frequent strain death in active female-lethal strains. Doxycycline successfully replaced tetracycline for colony maintenance & production of the conditional expression, transgenic strains. Importantly, doxycycline has increased bioavailability, molecule stability, and product availability in Panama. Doxycycline dosages for colony maintenance and production of male-only stains were effective at less than one quarter of tetracycline dosage, producing increased strain performance and stability. As part of this sub-objective, NWS strains with DR3 “all-in-one” constructs were created, intended to produce constant, low expression of double-stranded RNA (dsRNA) targeting transformer, resulting in XX male flies. Two variations of DR3 “all-in-one” were developed, but none produced any phenotypic effect. Using a different strategy, double component strains were created by crossing a line driving expression of an effector in early embryos with lines comprising two variations of the effector (transformer dsRNA). The resulting DR7 “all-in-one” strains were unreliable, producing double homozygous lethality, no effect, or partial function. These data highlight the need for more research on transformer expression and suppression to design novel driver strains that reliably silence transformer under controlled rearing conditions. As this will require significant time and resources, continued efforts to pursue this sub-objective have been paused.  
Transgenic NWS strains are reared in diet containing antibiotics to suppress the transgene, but effects of antibiotic on gut homeostasis is unexplored. In support of Sub-objective 2F, ARS researchers at Kerrville, Texas, and Pecora, Panama, collected gut tissue samples from adult and larval NWS reared with or without tetracycline in the diet. Gut samples were also collected from a simulated “male-only” condition where the previous generation was reared on tetracycline, but the test generation was reared without tetracycline. Total RNA isolated from these gut samples will be used in RNAseq differential gene expression analyses to determine if transgenic NWS strain fitness is impacted by rearing with antibiotics. In parallel, total DNA from samples of the same conditions were submitted for 16s metagenome sequencing in support of Sub-objective 2F. These results will reveal bacterial communities present, allowing comparison between adult male NWS of the current production strain and the transgenic male-only strain and differences that occur due to antibiotic use.
Molecular tools were unavailable to identify population origins for NWS outbreaks, critical to stop fly dispersion pathways that could expand fly range. As part of Sub-objective 3A, researchers at Kerrville, Texas, and Pacora, Panama, analyzed single nucleotide polymorphisms (SNPs) from NWS using population genetic tools to identify markers specific to 12 populations across the geographic range. Results indicated that NWS in the 2016 Florida outbreak likely originated from Trinidad and Tobago or the Dominican Republic (Publication #387497). Sampled populations included Jamaica, Brazil, Colombia, Peru, Argentina (two populations), Mexico, Costa Rica, Panama, Production Strain, Trinidad and Tobago, and the Dominican Republic. The current database consists of 114 samples and 9,877 SNPs and will be continualy expanded with flies from additional geographic locations, enabling source identification in future outbreaks. In addition, a diagnostic laboratory assay was developed to identify the regional origin of a NWS fly. The assay is based on differential detection of 6 SNPs by qPCR to determine if a fly is from the Outer Caribbean, Inner Caribbean, or South America, providing same day results in a standard laboratory without need for sequencing.  
In efforts to isolate and identify attractants for male NWS (Sub-objective 3B), cuticular hydrocarbons (CHCs) from mated or unmated males and females were extracted by hexane wash of adults (3 days post-eclosion). CHC extracts were evaluated by mass-spectrometry, yielding four distinct hydrocarbon profiles, with mated females having a more masculine profile. These results suggest males transferred CHCs to females during mating, which may signal subsequent males that the female has been mated. CHCs differing in abundance between males and females were identified and confirmed using synthetics. An olfactometer was designed and built to test sex biased CHCs for short range attraction/repellency. In addition, CHCs will be tested as a contact pheromone by “perfuming” adults and evaluating mating success. The bioassays could identify male-specific attractants as they will be tested on males and females. Behavioral assays were also developed but were not conducted due to Covid-related travel restrictions. These studies may identify factors influencing mating success in NWS, important to control program implementation based on the sterile insect technique.  
In additional NWS-related research, the genome of the secondary screwworm was sequenced to advance NWS genomic studies. Electroantennography on the secondary screwworm fly was conducted to test physiological response to novel compounds. Among novel compounds eliciting a physiological response were two diamines (putrescine and cadaverine) and thiophenol. A substitute for dimethyl disulfide, a component of Swormlure with significant shipping restrictions, was identified and field tested. Swormlure components were evaluated in paraffin wax (hydrophobic chemicals) and agar gel (hydrophilic chemicals) to produce more consistent and longer lasting bait. Electrophysiological tests suggest the “solid” baits are feasible and will be field tested within the next year. **Accomplishments**
                                

1. Microbiome of wild and domestic screwworm fly sequenced. The New World screwworm fly (NWS) is an ectoparasite of warm-blooded animals, primarily livestock, that was eradicated from the U.S., Mexico, and Central America, resulting in an estimated annual savings of $1 billion. Eradicated areas are protected from incursions with a barrier zone along the Panama-Colombia where millions of sterile flies are released weekly. ARS researchers in Pacora, Panama, and Kerrville, Texas collaborated with USDA-APHIS and Panamanian scientists and technicians from the NWS eradication program to obtain NWS samples. The team characterized significant differences in the microbiology of wild and domestic NWS used for sterile releases as well as life stages. These results provide a basis for future microbiological studies in NWS that could aid in improved domestic rearing techniques and provided insight into the ecology of wild NWS flies. Two potential bacteria of veterinary importance were found on wild flies, suggesting NWS may be a mechanical vector of animal pathogens. 2. Demonstration of an inexpensive in vitro system for maintenance of the Asian tiger mosquito, Aedes albopictus. ARS researchers in Kerrville, Texas, developed and demonstrated an inexpensive system was developed and demonstrated to successfully feed and maintain wild-caught Asian tiger mosquitoes.  The system is ideal for experimental assessment of wild mosquito populations, including determination of pathogens, repellent and insecticide susceptibility, growth regulators, effects of sublethal treatments, or other studies on the F1 generation. (Publication log# 378403.) **Review Publications**
                            *Rochon, K., Hogsette, Jr, J.A., Kaufman, P.E., Olafson, P.U., Swiger, S.L., Taylor, D.B. 2021. Stable Fly (Stomoxys calcitrans [Diptera: Muscidae]) – biology, management, and research needs. Journal of Integrated Pest Management. 12(1). Article 38. https://doi.org/10.1093/jipm/pmab029.*
                                *Arp, A.P., Quintero, G., Sagel, A., Gonzales, R., Phillips, P.L., Hickner, P.V. 2022. Insights into the microbial ecology of the New World screwworm, Cochliomyia hominivorax, from wild collections and under mass-rearing for sterile insect releases. Scientific Reports. 12. Article 1042. https://doi.org/10.1038/s41598-022-04828-5.*
                                *Shah, J., Buckmeier, B.G., Griffith, W., Olafson, P.U., Perez De Leon, A.A., Renthal, R. 2021. Fly odorant-binding protein with high-histidine N-terminal extension binds to transition metals. Insect Biochemistry and Molecular Biology. 141. Article 103707. https://doi.org/10.1016/j.ibmb.2021.103707.*
                                *Tietjen, M., Perez De Leon, A.A., Sagel, A., Skoda, S.R., Phillips, P.L., Mitchell III, R.D., Duran, U., Tortosa, S., Arp, A.P. 2022. Geographic population genetic structure of the New World screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae), using SNPs. Journal of Medical Entomology. 59(3):874-882. https://doi.org/10.1093/jme/tjac024.*
                                *Bendele, K.G., Bodine, D.L., Xu, Q., Foil, L.D., Cameron, C., Perez De Leon, A.A., Farmer, A., Retzel, E., Moore, V., Lohmeyer, K.H., Guerrero, F. 2022. The adult horn fly transcriptome and the complement of transcripts encoding cytochrome P450s, glutathione S-transferases, and esterases. Veterinary Parasitology. https://doi.org/10.1016/j.vetpar.2022.109699.*
                                *Osbrink, W.L. 2021. An inexpensive In Vitro blood-feeding system for wild Aedes albopictus (Diptera: Culicidae). Journal of Entomological Science. https://doi.org/10.18474/JES21-35.*
                                *Showler, A., Harlien, J.L. 2021. Lethal effects of commercial Kaolin Dust and Silica Aerogel Dust with and without botanical compounds on Horn Fly eggs, larvae, pupae, and adults in the laboratory. Journal of Medical Entomology. https://doi.org/10.1093/jme/tjab140.*


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