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ARS Home » Plains Area » Kerrville, Texas » Knipling-Bushland U.S. Livestock Insects Research Laboratory » LAPRU » Research » Research Project #427991

Research Project: Management of Flies Associated with Livestock

Location: Livestock Arthropod Pests Research

2015 Annual Report

Objective 1: Identify new attractants, repellents, and behavior-modifying chemicals based on physiology of chemical reception. Subobjective 1A: Assess compounds for potential behavior-modifying properties. Subobjective 1B: Elucidate biting fly chemosensory protein function. Objective 2: Evaluate efficacy of novel technologies for control of flies. Subobjective 2A: Evaluate the efficacy of various compounds as insecticides to control biting flies. Subobjective 2B: Identify and evaluate novel approaches for existing molecular targets and tools for assessment of new targets for biting fly control. Objective 3: Determine interactions between flies (of all stages) and microorganisms that significantly affect survival of the insects and their capability to transmit pathogens. Subobjective 3A: Characterize the horn fly gut innate immune response to microbial infection. Subobjective 3B: Define the reservoir and vectorial capacity of biting flies for microorganisms that are pathogenic to livestock and humans.

Identify new attractants, repellents, and behavior-modifying chemicals based on assessment of natural and synthetic compounds for behavior-modifying properties. Identify and elucidate structure activity relationships of biting fly chemosensory proteins and behavior-modifying chemicals. Identify lead compounds for further development based on behavior-modifying properties and structure activity relationships. Identify physiological pathways for development of novel control technologies by targeting key components. Evaluate the efficacy of natural and synthetic compounds as insecticides for control of biting flies. Modify structure of lead compounds and assess effects on compound efficacy to identify structural attributes contributing to and enhancing biological activity. Evaluate efficacy of gene silencing based on key physiological targets for biting fly control. Evaluate efficacy of vaccines based on key physiological targets for biting fly control. Elucidate interactions between flies (of all stages) and microorganisms that significantly affect survival of the insects and their capability to transmit pathogens, including the innate immune response of biting flies to microorganisms in the fly gut. Elucidate the reservoir and vector competence of biting flies for microorganisms that are pathogenic to livestock and humans.

Progress Report
Objective 1 and 2: In support of our overall effort to reduce the impact of biting flies associated with animal production systems, our group tested a number of natural compounds for attraction or repellent activity (Objective 1) and for insecticidal activity (Objective 2) against horn flies, stable flies and sand flies. These compounds included commercially available natural product formulations, a number of plant essential oils, and derivatives that were selected for evaluation based on reported activity against other insects. Studies included static air olfactometry, two-choice avoidance assays, and insecticidal assays. Results of these studies suggested that components of some of the essential oils exhibited fly behavior-altering properties (attraction/repellency) and insecticidal activity towards horn, stable flies, and sand flies (Phlebotomus papatasi). These will be the subjects of studies to identify the bioactive components of these essential oils and, if warranted, elucidate their mode of action. One of the natural product commercial formulations exhibited substantial repellency as well as vapor and contact insecticidal activity against the sand fly, P. papatasi, and it has been targeted for field studies. Potential collaborators (Africa/Southern Europe) have been identified for these field studies, and negotiations to establish collaborative research and development agreements have begun. Interestingly, this commercial formulation also exhibited substantial knockdown activity against horn flies, stable flies, and house flies, but unlike sand flies, these larger fly species were able to recover mobility after a prolonged (4 h) knockdown period. We previously identified a novel synthetic carbamate (PRC408) as an efficacious inhibitor of biting fly and tick acetylcholinesterases with an improved mammalian safety profile. Bioassays against horn flies, stable flies, sand flies (P. papatasi) and the southern cattle tick demonstrated high insecticidal and acaricidal efficacy equivalent to carbaryl, a commercially available carbamate insecticide. Recombinant P. papatasi acetylcholinesterase (PpAChE) mutants were constructed that contained mutations known to be responsible for very high level resistance to organophosphate and carbamate insecticides in mosquitoes. Novel synthetic carbamates were identified that were efficacious inhibitors of the recombinant PpAChE mutants that were demonstrated to be highly resistant to available insecticides, strongly suggesting that these compounds could be used for control or remediation of resistant mosquito and sand fly populations. This would aid in preventing establishment of these resistance mutations in susceptible pest populations. Our laboratory also evaluated aqueous spray or granular formulations of cyromazine, an insect growth regulator, and found it to be efficacious for up to 4 weeks in the control of stable flies, house flies, and lesser house flies. In our continued efforts to understand the effects of systemic parasiticides on Aedes albopictus populations, an inexpensive in vitro blood feeding system for mosquitoes was developed, validated, and is being used to conduct insecticide bioassays with spiked citrated cattle blood. This study will characterize various chemistries fed in vitro to Ae. albopictus and assess their effect on mosquito mortality and fecundity upon exposure to various concentrations. Objective 3: The stable fly genome sequencing project has progressed substantially since it began in 2009. An assembled genome was uploaded to GenBank in May 2015 and National Center for Biotechnology Information gene annotation pipelines are currently being used to identify protein-coding genes, as well as define gene locations and gene structures. Our lab’s interest in insect-microbe interactions led us to complete a differential gene expression analysis of the horn fly gut upon ingestion of Gram-negative (Salmonella enterica) versus Gram-positive (Enterococcus faecalis) bacteria. Definition of horn fly gut innate immune response genes that have a role in the bacterial response is forthcoming. We embarked on identifying bacterial populations that are associated with adult stable fly communities on dairy production systems in north-central Texas. To date, 31 different genera of culturable bacteria have been identified. The dairies sampled consist of both free stall and drylot facilities, and we will evaluate whether certain bacterial communities are associated with a specific facility type. Ongoing studies to understand the importance of the horn fly-Salmonella relationship to infection of cattle revealed that Salmonella enterica serovar Senftenberg could be transmitted to cattle peripheral lymph nodes upon simulation of a heavy horn fly infestation actively feeding on its bovine host for at least 11 days.

1. Identification of novel synthetic carbamates that inhibit insecticide-resistant variants of sand fly acetylcholinesterase. New insecticidal chemistries are desirable for the control of blood-feeding flies, especially given their rapid development of resistance to numerous classes of insecticide. ARS scientists at Kerrville, TX, in research supporting the Deployed War Fighter Protection Research Program, produced mutant variants of a critical sand fly enzyme, acetylcholinesterase (PpAChE), which is the target of organophosphorous and carbamate insecticides. These variants contained mutations known to be responsible for very high levels of resistance in mosquitoes. An in vitro assay was utilized to screen novel synthetic carbamates in collaboration with researchers at the University of Florida and Virginia Tech. Several novel carbamates were identified that were effective at inhibiting these PpAChE mutants that were otherwise highly resistant to available insecticides. This strongly suggests that these compounds could be used to prevent establishment of these resistant alleles in susceptible populations and for control or remediation of resistant populations of mosquitoes and sand flies.

Review Publications
Gross, A.D., Temeyer, K.B., Day, T.A., Perez De Leon, A.A., Kimber, M.J., Coats, J.R. 2015. Pharmacological characterization of a tyramine receptor from the southern cattle tick, Rhipicephalus (Boophilus) microplus. Insect Biochemistry and Molecular Biology. 63:47-53.
Showler, A.T., Osbrink, W.L.A. 2015. Stable fly, Stomoxys calcitrans (L.), dispersal and governing factors. International Journal of Insect Science. 7:19-25.
Temeyer, K.B., Tong, F., Totrov, M.M., Tuckow, A.P., Chen, Q., Carlier, P.R., Perez De Leon, A.A., Bloomquist, J.R. 2014. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): construction, expression and biochemical properties of the G119S orthologous mutant. Parasites & Vectors. 7:577.
Renthal, R., Li, A.Y., Gao, X., Perez De Leon, A.A. 2014. Polar cuticular lipids differ in male and female sandflies (Phlebotomus papatasi). Comparative Biochemistry and Physiology. 51(6):1237-1241.
Rochon, K., Baker, R.B., Almond, G.2., Gimeno, I.M., Perez De Leon, A.A., Watson, D.W. 2015. Persistence and retention of porcine reproductive and respiratory syndrome virus in stable flies (Diptera: Muscidae). Journal of Medical Entomology. 52(5):1117-1123.