Location: Livestock Arthropod Pests Research
2016 Annual Report
Objectives
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.
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.
Progress Report
In support of objective 1, investigations of chemicals with fly behavior-modifying properties are continuing. In support of the hypothesis that animal based and analog compounds will repel horn flies, we have begun discussions with TNG Company proposing cooperative assessment of horn fly salivary-based antigens that confer an immune response in cattle that acts to an extent as a repellent. An insect juvenile hormone analog has been observed in a pilot experiment to repel horn flies from cattle under pasture conditions. Investigations of plant extracts and essential oils have been tested for behavior modifying properties in horn flies, stable flies and sand flies. A plant-based compound has been determined to be repellent against adult horn flies and strongly reduced blood feeding. Bioassays using the same compound have commenced on the stable fly. A methanol extract from Osage orange is being assessed and early bioassays have indicated that the extract is repellent. Local flora, chinaberry, Melia azedarach, and Texas lilac, Vitex agnus-castus, have been grown for =1 year and the foliage will be used during 2017 to obtain carbon dioxide extracts for testing as repellents against horn flies. One plant-based compound was determined to be only weakly repellent against horn flies, therefore bioassays using this compound were discontinued. Essential oils were assessed and ranked for attraction, repellency, or knock-down activity against sand flies, horn flies and stable flies. Previous studies validated repellent activity of a commercial formulation (Essentria IC3) of essential oils against sand flies. Similar studies of Essentria IC3 using horn flies, stable flies and house flies demonstrated substantial fly repellency and knock-down at higher concentrations; however, unlike sand flies, some of the knocked-down house flies were able to recover full mobility within a few hours. Investigations of two neem-based commercial products were assayed for behavior-modifying effects on horn flies. Each of the two products contained substantially different amounts of azadiractin, and three other bioactive limonoid compounds, and repellent effects against the horn fly were also substantially different, showing that commercial neem-based products can differ in their constituents and effects. The panel of mutations previously shown to be associated with organophosphate resistance in R. microplus were individually expressed and biochemically characterized in recombinant tick acetylcholinesterase 1 (BmAChE1). The rBmAChE1 constructs containing selected individual mutations generally exhibited similar inhibition by paraoxon as the wild type rBmAChE1 lacking the mutations, suggesting that the combination of mutations may be more important than individual mutations in producing insensitivity to inhibition. Recombinant sand fly (P. papatasi) acetylcholinesterases containing mutations known to produce organophosphate-resistance in mosquitoes were constructed and expressed in the baculovirus system. Biochemical characterization of the G119S, F290V, and F331W mutations demonstrated various levels of resistance to inhibition by various organophosphate and carbamate pesticides. These constructs are valuable models of mutant acetylcholinesterases expressed in mosquito vectors of malaria and other important diseases, and were further utilized to screen novel synthetic carbamates under Objective 2.
In support of Objective 1 studies of physiology of fly chemical reception, we are defining where neurons present in horn fly and stable fly antennae and maxillary palps project to the fly brain in order to understand the neurophysiology of chemical reception. Within both species, neurons from antennae project to the antennal lobe, while neurons from the maxillary palps project to the subesophageal ganglion. Further, tracking neurons from one antenna with a fluorescently-labeled neuronal tracer resulted in staining of both antennal lobes, highlighting the interconnectedness of the lobes via the antennal commissure. The antennal lobe is comprised of neuronal structures called glomeruli, and we are assembling three-dimensional reconstructions of each lobe from females and males of each fly species to compare glomerular composition within and between species.
Objective 2: (Evaluate efficacy of novel technologies for control of flies.) In support of Objective 2, two neem-based commercial products were bioassayed against horn flies and determined to be strongly toxic to adults either by contact or as a volatile agent, and were particularly toxic to fly larvae developing in manure. Sublethal dose exposure to neem was observed to reduce fly reproductive potential. In addition, a plant-based compound was bioassayed for lethal activity against horn flies. The compound was determined to be highly toxic as a contact spray or in volatile form, and bioassays for sublethal effects on reproductive capacity were completed but not yet fully analyzed. We have begun bioassays using the compound against stable flies. Two application methods of an analog juvenile hormone were bioassayed in the laboratory and in the field against stable flies, house flies, and the little house fly (Fannia canicularis) using cow, swine, and chicken manure and was found to effect complete control for at least 4 wk. One plant-based compound was bioassayed against horn flies and found to have relatively weak lethal impacts; further bioassays of this compound were discontinued for the present in order to concentrate on more promising candidate compounds. Seven commercial essential oil and plant extract products were bioassayed against bed bugs for knockdown and lethal effects. Five of the products were found to induce 80% to 100% mortality within 24 h. This study has been completed and was published in 2016. The bioassays were conducted in part to identify candidate products for use against biting flies. In vitro bioassay of mosquitoes fed on blood intoxicated with imidacloprid were completed. A number of essential oils were bioassayed and ranked for insecticidal activity against sand flies, horn flies, stable flies or house flies. In general, the essential oils were much more active against sand flies than against the larger horn flies, stable flies and house flies and did exhibit some insecticidal properties, but appeared to be much more effective as repellents. Studies to identify fly attractant compounds have been unsuccessful to date.
Recombinant sand fly acetylcholinesterases were utilized to screen and successfully identify novel synthetic carbamate compounds effective against each of the mutations present in mosquito populations vectoring malaria and viral diseases in Africa and China. Further, two of the synthetic carbamates appeared to be effective inhibitors of all three major mosquito mutations (G119S, F290V, F331W). One of the two carbamate inhibitors appeared to exhibit high inhibition of mammalian acetylcholinesterase, suggesting probable mammalian toxicity, however, the other one may be a chemical lead or candidate compound for control of disease-carrying mosquitoes and sand flies.
An active horn fly cell culture was obtained from the University of Minnesota and established in our laboratory. Studies to adapt the dual luciferase reporter previously constructed for in vitro assessment of gene silencing in tick cell culture for use in horn fly cell culture were initiated. Transfection of the dual luciferase reporter into horn fly cell culture revealed that both of the two luciferase promoters in the tick reporter construct lacked sufficient promoter activity for use to drive reporter luciferase expression in horn fly cell culture. We anticipate utilization of the reporter construct to identify and validate promoters active within the horn fly cell culture environment.
Objective 3: (Determine interactions between flies (of all stages) and microorganisms that significantly affect survival of the insects and their capability to transmit pathogens.) In support of Objective 3, we are actively defining the temporal and spatial pattern of genes expressed in the horn fly gut that have a role in the insect’s immune response to bacterial ingestion. These include genes that detect presence of bacteria (pattern recognition receptors), as well as genes that are downstream in the response pathway and have a role in clearing bacteria from the system (antimicrobial peptides). These are being compared with counterparts of these genes in the related stable fly. Work with the stable fly genome is ongoing. The genome has been annotated in silico, and it is currently being manually curated by a group of 17 research scientists with interests in chemosensory, salivary, immune response, metabolic detoxification, reproduction, sex chromosome, and sex determination pathways.
Accomplishments
1. Horn flies are mechanical vectors of Salmonella. Insects have been implicated in the dissemination of microbial pathogens within livestock production systems, and this is of increasing importance from an animal and human health perspective. Cattle peripheral lymph nodes contaminated with Salmonella can be inadvertently processed along with the beef carcass, resulting in tainted beef products. This contamination is proposed to occur via a transdermal route of entry, suggesting that the bacteria may be introduced to cattle by biting arthropods. ARS scientists identified a role for the horn fly as a mechanical vector of Salmonella and demonstrated transmission of the bacteria to cattle peripheral lymph nodes upon extended feeding by a heavy infestation of horn flies. Understanding the role that biting flies play in the spread of microbial pathogens within livestock production systems will inform producers about the importance of fly management practices.
2. Novel carbamates are potential resistance-breaking compounds for insect control. Recombinant sand fly acetylcholinesterases were utilized to screen and successfully identify novel synthetic carbamate compounds effective against each of the mutations present in mosquito populations vectoring malaria and viral diseases in Africa and China. Further, two of the synthetic carbamates appeared to be effective inhibitors of all three major mosquito mutations (G119S, F290V, F331W). One of the two carbamate inhibitors appeared to exhibit high inhibition of mammalian acetylcholinesterase, suggesting probable mammalian toxicity; however, the other one may be a chemical lead or candidate compound for control of disease-carrying mosquitoes and sand flies.
Review Publications
Borges, L.M., Li, A.Y., Olafson, P.U., Renthal, R., Bauchan, G.R., Lohmeyer, K.H., Perez De Leon, A.A. 2016. Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum. Revista Brasileira de Parasitologia Veterinaria. 25(2):217-224.
Temeyer, K.B., Tuckow, A.P. 2016. Tick salivary acetylcholinesterase: A probable immunomodulator of host-parasite interactions. Journal of Medical Entomology. 53:500-504.
