Location: Tick and Biting Fly Research2012 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Progress was achieved during this third year of the project. Under sub-objective 1.A, the odorant co-receptor (Orco) was isolated and characterized from the stable fly and the horn fly, representing the first description for blood-feeding muscid flies. The finding provides a foundation to further study the functional role of ligand-specific odorant receptors in order to understand how repellents/bait attractants alter the stable fly olfactory response. In support of a stable fly whole genome sequencing project (objective 3), RNA has been generated and provided to a Yale University researcher for transcriptome analysis of stable fly adult females, males, and third instar larvae. An inbred strain is also being developed for sequencing by a collaborator at Washington University. In addition, the fate/survivability of ingested GFP-expressing E. coli in the stable fly midgut was determined in a collaborative project with other ARS scientists in Manhattan, KS. The immunohistological analysis of biogenic amines in the central nervous system and reproductive systems of adult stable flies under sub-objectives 1.B. and 1.C. was completed. A manuscript reporting the results of pharmacological experiments that demonstrate the roles of serotonin and octopamine in sperm transfer and mating success was generated (sub-objective 1C1). Problems initially encountered with the GABA immunehistology protocol were solved to allow completion of GABA experiments in the coming months. A physiological recording technique was developed to record both ingestion and salivation processes during stable fly blood feeding. The same recording system was also used to test the effects of various neuroactive molecules on stable fly feeding (sub-objective 1B). Significant technical difficulties were encountered in gene silencing in flies (Objective 2); therefore, research activities were redirected to develop a system to enable the determination of gene silencing efficiency in vitro using target genes cloned into a dual luciferase reporter plasmid. Development of this system was deemed necessary because unexpectedly high mortality (50-60%) prevented production of usable data for evaluating silencing efficiency prior to functional genomics or target validation. Development of the new dual luciferase reporter system will enable the precise determination of silencing efficiency in cells for comparison and selection of silencing constructs for use in functional genomics or target validation. Excellent progress has been made in developing a novel dual luciferase reporter plasmid, which entailed selection and optimization of monitoring conditions for two different luciferase genes expressed in arthropod cell culture. The new dual luciferase reporter is expected to enable us to select efficient gene silencing constructs in a reliable and timely manner, greatly facilitating subsequent improvements of in vivo gene silencing experimental procedures.
Lohmeyer, K.H., Pound, J.M. 2012. Laboratory evaluation of novaluron for controlling larval horn flies, house flies, and stable flies (Diptera: Muscidae). Journal of Economic Entomology. 49(3):647-651.