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Molecular Biology and Physiology of Biting Flies Affecting Livestock

Objective 1. Explore the genetic and physiological mechanisms of stable fly feeding and reproduction to identify novel control targets and to develop more efficient behavior modifying compounds. Sub-objective 1.A. Identify and characterize genes that have a role in the olfactory and gustatory pathways of biting flies. Sub-objective 1.B. Elucidate the mechanisms of blood-feeding in biting flies by characterizing the structure and neurophysiology of the cibarial pump, a key component of the feeding system for blood ingestion in the stable fly and other blood-feeding fly species. Sub-objective 1.C. Identify key neurotransmitters and/or receptors from biting flies and characterize their roles in mating and egg-laying behaviors.

Objective 2. Develop gene silencing tools to facilitate the functional characterization of novel control targets in biting flies, with a particular emphasis on genes that play a role in feeding and reproduction.

Objective 3. Develop genomic resources to support the initiation of a genome sequencing project for biting flies that impact livestock.

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Approach: The objectives of this project will be achieved using multidisciplinary approaches including molecular biology, immunohistochemistry, neurophysiology, and behavioral assays. Genes that play a critical role in olfaction and gestation of biting flies will be identified and characterized using pyrosequencing technology. Messenger RNA will be isolated from dissected olfactory and gustatory organs of the stable fly and used as template in the synthesis of double-stranded cDNA. Annotation of the stable fly transcriptome database representing genes expressed at different developmental stages will be accomplished by comparison to Drosophila sequences. Sequences encoding putative chemoreceptors will be isolated. The temporal and spatial expression patterns of the chemosensory gene sequences will be characterized using non-quantitative reverse transcriptase PCR and in situ hybridization techniques. The mechanisms of blood feeding in biting flies will be determined by identifying neurotransmitters in the feeding system and characterizing the cibarial pump function. Immonohistological techniques will be used to localize the specific neurotransmitters in neurons innervating the cibarial muscles. An in vitro blood feeding system will be developed and used in conjunction with the electrophysiological recording system to characterize cibarial pump function. Neurotransmitters (receptors) that are critical for blood feeding will be determined through pharmacological experiments involving agonists and antagonists. Neurotransmitters (receptors) that are critical to biting fly reproduction will be similarly identified and characterized. Immunohistological techniques will be used to identify specific neurotransmitters in neurons innervating testes in males and ovary/oviduct in females. Roles of specific neurotransmitters (receptors) in sperm transfer and egg-laying will be determined through behavioral and pharmacological experiments. Neurotransmitters that are critical for egg-laying behaviors will be further characterized by electrophysiological recordings of oviduct contraction in reduced fly preparations. Genes encoding receptors of key neurotransmitters in the sensory, feeding and reproductive systems will be identified. Gene-silencing tools will be developed to facilitate the functional characterization of novel control targets, particularly on genes that play critical roles in blood feeding and reproduction of biting flies. The double-stranded RNA (dsRNA) of a target gene will be synthesized and used for gene silencing. Microinjection techniques that are suitable for injecting dsRNA will be adopted from available insect protocols and be optimized for injecting the stable fly. The effects of gene silencing will be evaluated by measurement of transcript reduction using quantitative real-time PCR and/or by monitoring changes in key behaviors, including responses to chemical cues and mating /egg-laying success. Finally, a first generation genetic linkage map will be developed and a bacteria artificial chromosome (BAC) library will be constructed to support the initiation of a genome sequencing project for biting flies affecting livestock.
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Guerrero, Felix
Li, Andrew
Lohmeyer, Kimberly
Olafson, Pia
Perez De Leon, Adalberto
Showler, Allan
Temeyer, Kevin
Project Reports:
Published Journal Articles USDA ARS (NP 108):
Polar cuticular lipids differ in male and female sandflies (Phlebotomus papatasi)
Renthal R, Li A, Gao X, Perez De Leon A.
J Med Entomol. 2014 Nov;51(6):1237-41.
Simultaneous detection of Pyrethroid, Organophosphate and Cyclodiene target site resistance in Haematobia irritans (Diptera: Muscidae) by multiplex Polymerase chain reaction
Domingues LN, Guerrero FD, Foil LD.
J Med Entomol. 2014 Sep;51(5):964-70.
Survival and fate of Salmonella enterica serovar Montevideo in adult horn flies (Diptera: Muscidae)
Olafson PU, Lohmeyer KH, Edrington TS, Loneragan GH.
J Med Entomol. 2014 Sep;51(5):993-1001.
Horn Fly, Haematobia irritans irritans (L.), overwintering
Showler AT, Osbrink WLA, Loymeyer KH.
Int J Insect Sci. 2014 May 1;2014(6):43-7.
Discovery of the Rdl mutation in association with a cyclodiene resistant population of horn flies, Haematobia irritans (Diptera: Muscidae)
Domingues LN1, Guerrero FD, Becker ME, Alison MW, Foil LD.
Vet Parasitol. 2013 Nov 15;198(1-2):172-9.
In vitro and in vivo evaluation of cypermethrin, amitraz, and piperonyl butoxide mixtures for the control of resistant Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in the Mexican tropics
Rodriguez-Vivas RI, Li AY, Ojeda-Chi MM, Trinidad-Martinez I, Rosado-Aguilar JA, Miller RJ, Pérez de León AA.
Vet Parasitol. 2013 Oct 18;197(1):288-96.
Effects of reserpine on reproduction and serotonin immunoreactivity in the stable fly Stomoxys calcitrans (L.)
Liu SS, Li AY, Witt CM, Pérez de León AA.
J Insect Physiol. 2013 Sep;59(9):974-82.
Discovery of microRNAs of the stable fly (Diptera: Muscidae) by High-throughput sequencing
Tuckow AP, Temeyer KB, Olafson PU, Pérez de Léon AA.
J Med Entomol. 2013 Jul;50(4):925-30.
Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi
Swale DR, Tong F, Temeyer KB, Li A, Lam PC-H, Totrov MM, Carlier PR, Pérez de León AA, Bloomquist JR.
Pestic Biochem Phys. 2013 Jul;106(3):85-92.
Molecular characterization and immunolocalization of the olfactory co-recepter Orco from two blood-feeding muscid flies, the stable fly (Stomoxys calcitrans, L.) and the horn fly (Haematobia irritans irritans, L.)
Olafson PU.
Insect Mol Biol. 2013 Apr;22(2):131-42.
Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties
Temeyer KB, Brake DK, Tuckow AP, Li AY, Pérez de León AA.
Parasit Vectors. 2013 Feb 4;6:31.
Effects of pyriproxyfen and buprofezin on immature development and reproduction in the stable fly
Liu SS, Li AY, Lohmeyer KH, Pérez De León AA.
Med Vet Entomol. 2012 Dec;26(4):379-85.
Laboratory evaluation of novaluron as a development site treatment for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae)
Lohmeyer KH, Pound JM.
J Med Entomol. 2012 May;49(3):647-51.
Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): baculovirus expression, biochemical properties, and organophosphate insensitivity of the G262A mutant
Temeyer KB, Brake DK, Schlechte KG.
J Med Entomol. 2012 May;49(3):589-94.
Acetylcholinesterase of Stomoxys calcitrans (L.) (Diptera: Muscidae): cDNA sequence, baculovirus expression, and biochemical properties
Temeyer KB, Chen AC.
Vet Parasitol. 2012 Feb 28;184(1):92-5.
Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)
Liu SS, Li AY, Witt CM, Pérez de León AA.
Arch Insect Biochem Physiol. 2011 Aug;77(4):199-219.
High levels of insecticide resistance in introduced horn fly (Diptera: Muscidae) populations and implications for management
Oyarzún MP, Li AY, Figueroa CC.
J Econ Entomol. 2011 Feb;104(1):258-65.
Identification of a mutation associated with permethrin resistance in the para-type sodium channel of the stable fly (Diptera: Muscidae)
Olafson PU, Pitzer JB, Kaufman PE.
J Econ Entomol. 2011 Feb;104(2):250-7.
Evaluation of the VetCap® treatment method for horn fly control in cattle
Li AY, Ross D, Pérez de León AA.
Int J Appl Res Vet M. 2011;9(2):198-203.
Full text available in the NAL Digital Collections.
Detection of target site resistance to pyrethroids and organophosphates in the horn fly using multiplex polymerase chain reaction
Foil LD, Guerrero FD, Bendele KG.
J Med Entomol. 2010 Sep;47(5):855-61.
Laboratory evaluation of verbutin as a synergist of acaricides against larvae of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)
Li AY, Davey RB, Miller RJ.
J Econ Entomol. 2010 Aug;103(4):1360-4.
Knockdown resistance in pyrethroid-resistant horn fy (Diptera: Muscidae) populations in Brazil
Sabatini GA, Ribolla PE, Barros AT, Guerrero FD, Schumaker TT.
Rev Bras Parasitol Vet. 2009 Jul;18(3):8-14.
Non-Journal Publications:
Recombinant acetylcholinesterase 1 of the sand fly Phlebotomus papatasi (Scopoli): expression, biochemical properties, and insensitivity to organophosphate inhibition - (Abstract Only)
Are biting fly larvae biological reservoirs of Salmonella? - (Abstract Only)
Effect of storage method on manure as a substrate for filth fly development - (Abstract Only)
Expression and Biochemical Properties of a Recombinant Acetylcholinesterase 1 of the Sand Fly, Phlebotomus papatasi (Scopoli) Insensitive to Organophosphate Inhibition - (Abstract Only)
Evaluation of insecticides and repellents for the control of the sand fly Phlebotomus papatasi to protect deployed U.S. Military personnel- (Abstract Only)
Accepted Publication (20-May-13)
Bioassay improvements for assessing pyrethroid resistance in horn flies - (Abstract Only)
Accepted Publication (21-Nov-12)
Stable Fly Project in Campo Grande, Brazil - (Abstract Only)
Accepted Publication (19-Jun-12)
Discovery and functional analysis of small RNAs (miRNA/siRNA) of livestock ectoparasites. - (Abstract Only)
Accepted Publication (11-Jun-12)
North American soft ticks (Ornithodoros spp.): biology and feral swine parasitism as risks for the emergence of African swine fever in the U.S.A. - (Abstract Only)
Accepted Publication (12-Apr-12)
Innovative technologies targeting vector populations to mitigate the risk of exposure to leishmaniasis and protect deployed U.S. Military personnel in the Middle East - (Abstract Only)
Accepted Publication (10-Nov-11)
Innovative technologies targeting vector populations to mitigate the risk of exposure to leishmaniasis and protect deployed U.S. Military personnel in the Middle East - (Abstract Only)
Accepted Publication (11-Oct-11)
Challenges for the Control and Eradication of the Tropical Cattle Tick Rhipicephalus microplus and Perspectives of Anti-tick vaccines - (Abstract Only)
Accepted Publication (23-Jun-11)
Biogenic amines in the stable fly Stomoxys calcitrans L. (Diptera: Muscidae): tissue localization and roles in feeding and reproduction- (Abstract Only)
Accepted Publication (23-Jun-11)
Effect of biogenic amines on the mating and egg-laying behaviors in the stable fly - (Abstract Only)
Accepted Publication (31-May-11)
Effects of pyriproxyfen and buprofezin on immature development, female oviposition, and egg-hatching in the stable fly - (Abstract Only)
Accepted Publication (13-May-11)
Immunohistological localization of 5-HT in the CNS and feeding system of the Stable Fly - (Proceedings)
Accepted Publication (15-Sep-10)
Insight into stable fly larvae: Salivary gland-specific polypeptides and evidence for genes that may have a role in the stable fly innate immune system - (Proceedings)
Accepted Publication (02-Jul-10)
Food Safety Categories:
On-Farm Food Safety
Farm-to-Table Categories:
On-farm food production