Location: Livestock Arthropod Pests Research
2017 Annual Report
Objectives
Objective 1: Develop new attractants, repellents, and behavior-modifying chemicals based on physiology of chemical reception in house, stable and horn flies. 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 house, stable and horn 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.
Objective 4: Complete development of a transgenic male-only strain of screwworms ready for production and distribution, coordinating a critical path to development.
Objective 5: Complete development of screwworm attractants and oviposition stimulants to be used in baits and to help synchronize rearing procedures.
Objective 6: Perform research to accomplish efficiency in the screwworm rearing process. Subobjective 6A: Develop and transfer technology for reducing ammonia emissions from screwworm larval media when cellulose fiber is used as the bulking agent. Subobjective 6B: Determine where optimum rearing environments exists within the large rooms of the screwworm mass rearing facility so to ensure maximum efficiency of the rearing process.
Objective 7: Develop ecological niche models of screwworm flies and genetic subpopulations with the practical objectives of scaling release rates to habitat and to provide projections of potential range changes in response to climate change. Subobjective 7A: Determine genetic variation of screwworms from different geographic origins across their current range. Subobjective 7B: Use remote sensing and geographical information systems to relate genetic variation of screwworms to differences in landscape across their range.
Approach
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.
Measure fitness parameters of transgenic screwworm lines and determine if transgenic males are competitive with wild type screwworm males. Confirm stability of transgenic line(s), and screen for mobilization of the transgene in bio-secure facility. Examine influence of genetic background on level of female lethality by crossing transgenic males with females collected from different locations. Record female screwworm antennal responses to chemical stimuli. Transfer transgenic line(s) with favorable fitness to COPEG. Test active chemicals for fly attraction and oviposition stimulation to improve field surveillance and enhance egg production during mass-rearing. Determine optimum dose of potassium permanganate and Yucca extract in screwworm larval media for ammonia reduction and good fly quality yields in diet bulked with cellulose fiber. Yucca schidigera (powder extract) will be added to larval diet at select intervals to determine synergistic activity in ammonia reduction. Measure temperature and humidity in separate rooms, each containing first 3 developmental stages of the screwworm life cycle. Design and develop GIS-based methodology for spatial analysis of each room. Determine general landscape patterns using satellite images at multiple locations across endemic areas from which screwworms were sampled and genotyped. Climatological data, along with general soil information, vegetation data, host composition and density, and land use patterns, will be collected and analyzed using remote sensing technologies and landscape genetics models to understand interactions between gene flow and geographic variation. This will help assess risk for screwworm cases in the barrier zone in Panama, and to prevent outbreaks in the U.S.
Progress Report
This research project on Management of Flies Associated with Livestock resulted from the establishment in March 2017 of the Livestock Arthropod Pest Research Unit (LAPRU) at the Knipling-Bushland U.S. Livestock Insects Research Laboratory. LAPRU resulted from the consolidation of the former “Tick and Biting Fly” and “Screwworm” Research Units.
Objective 1: An insect juvenile hormone analog was observed to repel horn flies on pastured cattle and will be further tested in the laboratory as a potential horn fly repellent. A manuscript was accepted for publication reporting that two neem-based compounds were shown to be repellent to adult horn flies and also exhibited strong anti-feedant properties. Each of two neem-based commercial products contained different amounts of azadiractin and other bioactive compounds, and exhibited significantly different repellency against horn flies, demonstrating that commercial neem-based products may differ substantially in composition and efficacy. Although p-anisaldehyde did not appear to exhibit repellency to horn flies in a static olfactometer, the compound completely deterred feeding from cotton pads soaked in bovine blood at concentrations of 0.6% or higher in ventilated containers. In contrast to horn flies, p-anisaldehyde was strongly repellent to house flies in laboratory studies. A number of essential oils and natural product chemicals were tested for repellency to sand flies, horn flies, stable flies and house flies. Many of the compounds that were active repellents for sand flies were also repellent to horn flies, stable flies, and house flies, but were less effective than DEET. Essentria, a natural product formulation strongly repellent with volatile insecticidal properties to sand flies, was also shown to be repellent to horn flies, stable flies, and house flies. Although Essentria knocked down horn flies, stable flies and house flies, these flies were not killed and were able to recover full mobility, in contrast to the smaller sand flies. IR3535, an insect growth regulator previously used in Europe, was not repellent to any of the flies tested, but did have strong behavior altering effects of unknown pharmacology, altering their ability to coordinate their movements or to fly. A fly larval feeding assay was developed utilizing fluorescently labeled paramagnetic microparticles with utility to identify chemicals that altered feeding and growth of larval horn flies and sand flies. Feeding stimulants may control flies by promoting ingestion of toxicants, while feeding inhibitors may reduce larval growth, inhibiting reproductive capacity. Manual annotation of odorant receptor (Or) and odorant binding protein (Obp) gene families in the Stable fly genome was completed, revealing a 74 Or and 90 Obp genes. Comparison with the Drosophila genome identified five stable fly genes that are related to Drosophila Or67d, which has a role in regulating mating behaviors. While three of these stable fly genes are expressed 15-20 times higher in mated females versus unmated/mated males, their function is unclear and requires further experimentation. Specific expansion of the Obp gene family in house flies was evident upon comparison with Obps from Drosophila. Expression of Obps was detected in both sensory and non-sensory tissues, suggesting a multi-functional role for these odorant binding transport proteins. A recombinant Obp, ScalObp47, was used in a binding assay to assess its affinity for chemicals known to produce a positive response in an electroantennogram or to attract/repel other fly species. ScalObp47 was shown to have a high affinity for dimethyl trisulfide and linalool, and low binding affinity for long chain alcohols such as nonanol and 2-decanol. We are continuing assessment of binding affinity for recombinant Obps that we have prepared.
Objective 2: Two neem-based commercial products were shown to be strongly toxic to adult horn flies as either a contact or volatile spray and exhibited strong growth regulatory effects against horn fly larvae developing in treated manure. In addition, sublethal neem exposures reduced reproductive potential of surviving flies. A plant-based compound (natural product), p-anisaldehyde, was also demonstrated to be highly toxic as either a contact or volatile spray, and bioassays demonstrated sublethal effects reducing reproductive capacity of surviving flies. Lethality of the p-anisaldehyde is particularly acute against horn fly eggs, with complete mortality at 0.00001%. Further studies testing the p-anisaldehyde against house flies and other biting or nuisance flies are underway. Additional studies tested a number of different essential oils and other compounds for mortality. Aureothin did not exhibit substantial toxicity to horn flies or stable flies.
Objective 3: Surveys were substantially completed for prevalence of enteric pathogens associated with nuisance flies collected from dairy and feedlot settings in Texas. A previous study reported that acetylcholinesterase in tick saliva was a likely modulator of host immune response to parasite or pathogen presence. We have found that mosquito and sand fly saliva also contain measurable acetylcholinesterase activity, unlike saliva from horn flies, stable flies or house flies, suggesting a strong link between salivary acetylcholinesterase and ability to vector pathogens.
Objective 4: Completed bioengineering construction of a transgenic male-only strain of screwworms ready for production and distribution, coordinating a critical path to development. The genetically engineered male-only strains were transferred to Methods and Development section of the Comision Panamá Estados Unidos para la Erradicacion y Prevencion del Gusano Barrenador del Ganado (translation: Panama-U.S. Commission for the Eradication and Prevention of Cattle Screwworm), referred to herein as COPEG, for further evaluation in field trials scheduled for the coming year. Production use of male-only strains is expected to reduce production costs and biological waste by approximately 50%. Demonstration of successful performance compared to non-engineered strains will enable use of the male-only strains for full production, sterilization, and release, providing huge savings in expense and generation of waste.
Objective 5: Experiments utilizing secondary screwworm succeeded in identification of four volatile ovipositional attractants. Replication of these results for primary screwworm is expected to improve production efficiency by increasing the average number of eggs successfully produced for inoculation of the larval medium used for screwworm production. This is particularly important for the male-only strain as it will reduce the quantity of fertile females needed and reduce the cost of their production.
Objective 6: Previous replacement of the gel formulation for screwworm production with cellulose fiber resulted in significant cost savings and resulted in a much more environmentally friendly waste material from screwworm production; however, the cellulose fiber medium formulation resulted in significantly increased release of toxic ammonia gas. Increased release of ammonia from the cellulose fiber-based larval medium was successfully mitigated by addition of Yucca extract and potassium permanganate and is currently in use for production of the male-only strain. These improvements allow utilization of the less costly cellulose fiber larval medium formulation, reduce the production of toxic ammonia gas, and provide for alternative waste disposal procedures that are more environmentally friendly.
Objective 7: Remote sensing was utilized to identify screwworm collection sites for Caribbean Islands with endemic screwworm populations. Remote sensing was also used to identify screwworm collection sites for northern Peru and several South American countries. It is anticipated that collection of screwworm samples from these diverse locations will enable genetic analyses to identify genetic markers with location-specific variations suitable for epidemiological purposes. A report of the locations and methodology was submitted to National Program Leaders.
Accomplishments
1. Construction of transgenic male-only screwworm strain. ARS researchers based in Kerrville, Texas, in collaboration with researchers at North Carolina State University and the Comision Panamá Estados Unidos para la Erradicacion y Prevencion del Gusano Barrenador del Ganado (translation: Panama-U.S. Commission for the Eradication and Prevention of Cattle Screwworm, COPEG), completed bioengineering construction of a transgenic male-only strain of screwworms ready for production and distribution, coordinating a critical path to development. The genetically engineered male-only strains were transferred to Methods and Development section of COPEG for further evaluation in field trials scheduled for the coming year. Production use of male-only strains is expected to reduce production costs and biological waste by approximately 50%. Demonstration of successful performance compared to non-engineered strains will enable use of the male-only strains for full production, sterilization, and release, providing huge savings in expense and generation of waste.
2. Effects of p-anisaldehyde on horn fly repellency, mortality, and reproduction. The horn fly, Haematobia irritans irritans (L.), is an economically important blood-feeder that mainly attacks cattle worldwide. As resistance to conventional insecticides increases, alternative control tactics are being investigated. p-Anisaldehyde occurs in many plants and it is bioactive against some arthropods. ARS researchers at Kerrville, Texas developed a series of bioassays that are effective for assessing a range of horn fly responses to chemicals. In our study, p-anisaldehyde was lethal to horn fly eggs at 0.00001% and possibly less. Mixed into cow manure, 5,000 – 20,000 ppm p-anisaldehyde reduced horn fly larvae by 85.4% – 100%. p-Anisaldehyde caused some immobilization of adult horn flies when exposed by direct contract with spray droplets and by volatiles. Adult mortality was 90% – 100% in response to 5% – 10% concentrations by 30 min, and the concentration at which 50% of the horn flies died and the concentration at which 90% of the flies died. Complete horn fly mortality was achieved by exposure to volatiles from 0.75% p-anisaldehyde by 3 h in an enclosed space; exposure to volatiles is more lethal to adult horn flies than droplets. Although horn flies were not repelled, the compound completely deterred feeding from cotton pads soaked in bovine blood. Exposure to sublethal concentrations of p-anisaldehyde did not affect horn fly egg production and hatching. These early findings about p-anisaldehyde indicate that horn fly eggs are very vulnerable to p-anisaldehyde, hence, delivery approaches might now be developed such that horn fly eggs can be exposed to the compound. Adult horn flies are also susceptible, making p-anisaldehyde a potential organic tactic for controlling horn fly infestations.
3. Identification of screwworm ovipositional attractants. ARS researchers at Lincoln, Nebraska and Kerrville, Texas completed experiments utilizing secondary screwworm that succeeded in identification of four volatile ovipositional attractants. Replication of these results for primary screwworm is expected to improve production efficiency by increasing the average number of eggs successfully produced for inoculation of the larval medium used for screwworm production. This is particularly important for the male-only strain as it will reduce the quantity of fertile females needed and reduce the cost of their production.
4. Association of salivary acetylcholinesterase with arthropod disease vector capacity. ARS researchers at Kerrville, Texas previously reported that acetylcholinesterase in tick saliva was a likely modulator of host immune response to parasite or pathogen presence. Additional studies by ARS researchers at Kerrville, Texas found that mosquito and sand fly saliva also contain measurable acetylcholinesterase activity, unlike saliva from horn flies, stable flies or house flies, suggesting a strong link between salivary acetylcholinesterase and ability to vector pathogens. This finding suggests that salivary acetylcholinesterase may be an important factor in disease transmission, presenting a new paradigm and identifying a novel target for studies to understand and prevent disease transmission by arthropod vectors.
Review Publications
Concha, C., Palavesam, A., Guerrero, F., Sagel, A., Li, F., Pardo, T., Hernandez, Y., Quintero, G., Vasquez, M., Phillips, P.L., McMillan, W., Skoda, S.R., Scott, M.J. 2016. A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique. Proceedings of the Royal Society of London B. 14:72.
Skoda, S.R., Phillips, P.L., Sagel, A., Chaudhury, M.F. 2017. Distribution and persistence of sterile screwworms (Diptera: Calliphoridae) released at the Panama-Colombia border. Journal of Economic Entomology. 110(2):783-789.
Tomberlin, J.K., Crippen, T.L., Tarone, A.M., Chaudhury, M.F., Singh, B., Cammack, J.A., Meisel, R. 2017. A review of bacterial interactions with blow flies (Diptera: Calliphoridae) of medical, veterinary, and forensic importance. Annals of the Entomological Society of America. 110(1):19-36.
Gross, A.D., Temeyer, K.B., Day, T.A., Perez De Leon, A.A., Kimber, M.J., Coats, J.R. 2017. Interaction of plant essential oil terpenoids with the southern cattle tick tyramine receptor: A potential biopesticide target. Chemico Biological Interactions. 263:1-6.
Zhu, J.J., Chaudhury, M.F., Durso, L.M., Sagel, A., Skoda, S.R., Jelvez-Serra, N.S., Santanab, E.G. 2017. Semiochemicals released from five bacteria identified from animal wounds infested by primary screwworms and their effects on fly behavioral activity. PLoS One. 12(6):e0179090. doi:10.1371/journal.pone.0179090.
Chaudhury, M.F., Zhu, J.J., Skoda, S.R. 2017. Physical and physiological factors influence behavioral responses of Cochliomyia macellaria (Diptera: Calliphoridae) to synthetic attractants. Journal of Economic Entomology. 110(4):1929-1934.
Olafson, P.U., Temeyer, K.B., Lohmeyer, K.H., Edrington, T.S., Loneragan, G. 2017. Persistence of two Salmonella enterica ser. Montevideo strains throughout horn fly (Diptera: Muscidae) larval and pupal development. Annals of the Entomological Society of America. 110(4):54-60.
