Location: Livestock Arthropod Pest Research Unit
2021 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 New World screwworm range and spatial ecology, a database of screwworm presence data was created using data collected by sampling efforts, published literature, and historical sources. To build a predictive model, mapping layers possibly contributing to screwworm prevalence such as temperature, altitude, land use, and livestock, were collected and integrated into mapping software. Preliminary models were generated using this data. Further sampling in more areas to fill in unknown areas and validation of the predictions are needed to complete the niche model.
In support of Subobjective 2A, to assess compounds for behavior modifying and insecticidal properties to control muscid flies, one compound, p-anisaldehyde was demonstrated to be repellent to house fly for more than 24 hours, but less than 48 hours. After exposure to sunlight for 2 hours, p-anisaldehyde was no longer repellent. p-Anisaldehyde was sufficiently repellent to keep house flies from landing on three different food sources (milk, orange juice, chicken broth) when applied as a mist to the food sources and also when applied in close proximity to, but not in contact with the food sources. In addition, components of essential oils that had been previously shown by bioassay to be “more repellent than DEET” to Phlebotomus papatasi were bioassayed against horn flies and stable flies. Results appeared to be dependent on fly size, with stable flies being generally less repelled by all of the tested components compared to horn flies. The same dependency on fly size was also exhibited between sexes, with males being less repelled than (larger) female flies. The sole exception was the response of stable flies to clove oil, in which females were repelled at 0.5%, compared to males at 1%. Stable fly males were repelled at 1% DEET, while females were not repelled at 1%. Stable flies were slightly repelled by citronellol and trans-cinnamaldehyde. Horn flies were equally repelled by citronellol and DEET, but were more sensitive to DEET than to all other essential oil components tested.
In support of Subobjective 2B, to expand functional genomics of flies, a database was developed representing genes expressed in the sensory organs of horn flies, i.e., antennae, maxillary palps, and mouthparts. This database was compared with genes expressed in horn fly bodies devoid of these organs, enabling us to highlight gene families with upregulated expression in tissues with a role in horn fly chemosensation. Members of the odorant, gustatory, and ionotropic receptor gene families were identified, along with representative odorant binding protein genes. These data were used to evaluate phylogenetic relationships of these gene families between houseflies, stable flies, and horn flies, providing a framework for focusing on genes that are expanded in this fly lineage.
In support of Subobjective 2C, developing germ-line transformation strains to evaluate their potential use in suppression of new world screwworm (NWS) populations, genetically-modified strains were produced containing an all-in-one construct consisting of the DR3 driver and one of two transformer inverse repeat effectors. Strains were bred to homozygosity and tested for function. None of the strains with either effector produced mutations, lethality, or other effect in females in the absence of the repressor tetracycline. Additionally, DR6 driver was crossed with both effector strains. In testing combinations DR7 and DR6 strains with one of the transformer inverse repeat effectors, mutations have been overserved in all combinations but not all strains. Some combinations have no mutations, some resulted in complete mortality of males and females, while in others roughly half of the females express some degree of reproductive masculinization while the other half are normal.
Research was completed to address Subobjective 2F, assessing the gut transcriptome and microbiome of NWS. Microbiome samples from the mass-rearing facility, transgenic, and wild captured flies were previously collected, and DNA was isolated. Samples were submitted for total microbiome sequencing. Sequence libraries obtained were high quality and analysis has begun using a microbiome analysis software suite. Major differences between mass-reared and wild flies are significant, with a reduction in diversity under colony conditions. Samples of screwworm larvae and adults reared with or without tetracycline, or on generation separated from tetracycline to mimic a male-only transgenic release, have been collected for 3 subsequent generations. Guts will be extracted from these samples and submitted for RNA sequencing to assess the impact tetracycline has on transgenic NWS fitness.
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. In support of Objective 3A, DNA was isolated from 140 NWS samples from field collections, and inbred colonies from a diverse range of locations, including now eradicated countries. The inclusion of these samples will improve the resolution of population marker selection. New DNA extraction protocols were developed to reduce contamination from host tissues in larval guts, and to improve DNA yields and quality. Analysis of the chemical ecology of mated and unmated male and female NWS was completed in support of Objective 3B. Cuticular hydrocarbon profiles from male and female flies are significantly different and hydrocarbon profiles in mated females are more masculine. Chemical differences identified here will be tested for attractant or repellant properties to develop sex-specific lures.
Accomplishments
1. Genome of the stable fly sequenced. The stable fly is a blood-feeding pest of economic significance to U.S. cattle producers, reducing cattle productivity by an estimated $2 billion annually. Management of this pest is challenging, and novel methods of targeting stable flies are desirable to enhance a producer’s ability to suppress fly populations. ARS researchers in Kerrville, Texas, and Manhattan, Kansas, collaborated with scientists from 12 U.S. and three foreign universities to sequence and describe the genome of the stable fly. The team identified gene families involved in stable fly olfaction and vision, blood-feeding, reproduction, and metabolism of pesticide compounds. This important resource can be used to identify and target pathways that are critical to stable fly biology with the ultimate goal of developing unique strategies to reduce the burden of these flies on livestock production settings.
Review Publications
Altuna, M., Hickner, P.V., Castro, G., Mirazo, S., Perez De Leon, A.A., Arp, A.P. 2021. New World screwworm (Cochliomyia hominivorax) myiasis in feral swine of Uruguay: One Health and transboundary disease implications. Parasites & Vectors. 14:26. https://doi.org/10.1186/s13071-020-04499-z.
Olafson, P.U., Askoy, S., Attardo, G.M., Buckmeier, B.G., Chen, X., Coates, C.J., Davis, M.C., Dykema, J., Emrich, S., Friedrich, M., Holmes, C.J., Ioannidis, P., Jansen, E.N., Jennings, E.M., Lawson, D., Martinson, E.O., Maslen, G.L., Meisel, R.P., Murphy, T.D., Nayduch, D., Nelson, D.R., Oyen, K.J., Raszick, T., Ribeiro, J.M., Robertson, H.M., Rosendale, A.J., Sackton, T.B., Saelao, P., Swiger, S.L., Sze, S., Tarone, A., Taylor, D.B., Warren, W.C., Waterhouse, R.M., Weirauch, M.T., Werren, J.H., Wilson, R.K., Zdobnov, E.M., Benoit, J.B. 2021. The genome of the stable fly, Stomoxys calcitrans, reveals potential mechanisms underlying reproduction, host interactions, and novel targets for pest control. BMC Biology. 19:41. https://doi.org/10.1186/s12915-021-00975-9.
Olafson, P.U., Saski, C.A. 2021. Chemosensory-related gene family members of the horn fly, Haematobia irritans irritans, identified by transcriptome analysis. Insects. 13:252-260. https://doi.org/10.1016/j.ijppaw.2020.11.002.
Showler, A., Harlien, J.L. 2021. Repellency of p-anisaldehyde against Musca domestica L. (Diptera: Muscidae) in the laboratory. Journal of Medical Entomology. 1-7. https://doi.org/10.1093/jme/tjab097.
