Location: Tick and Biting Fly Research2010 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
Significant progress has been made towards objective completion during this first year of the project. Under sub-objective 1.A, a pyrosequencing gene database was screened to identify genes related to the stable fly's chemosensory and nervous systems. Twenty-five transcripts were selected for further characterization, several of which will be used for gene silencing experiments in the second year of the project. A horn fly EST database was screened, and ten odorant binding protein-like sequences were identified. We have made progress towards developing a stable fly linkage map. Isogenic lines were established from two laboratory fly strains, and we have designed a protocol for paired mating in order to obtain the segregating families desired for constructing the linkage map. We have made significant progress on immunohistological detection of key neurotransmitters, serotonin and octopamine, in the feeding and reproductive systems under sub-objectives 1.B. and 1.C. Serotonin is found to be present in neurons innervating the cibarial pump muscles. These neurotransmitters are found to differentially present in male and female reproductive tissues, indicating different roles in mating and egg-laying behaviors. The finding provided a foundation for the next phase of study in determining the roles of such neurotransmitter receptors in blood feeding and reproduction through behavioral and pharmacological experiments. Progress was also made toward development of an in vitro membrane feeding system for stable flies under sub-objective 1.B. Different membranes were evaluated for fly blood feeding. The amount of blood ingestion was found to be related to the age of the flies. This finding allows selection of flies of proper age group for behavioral assays to determine effect of pharmacological agents on fly blood feeding. Progress was also made toward development of behavioral assays to measure reproductive success under the sub-objective 1.C. The egg-laying pattern was determined for the normal stable flies, and will be used to compare with treated flies to determine effects of pharmacological agents. Progress was made toward introduction of dsRNA and validation of gene silencing success for stable fly AChE, ribosomal protein P1, and beta-actin, as part of objective 2. Gene cDNA sequences were identified, cloned, and used to design primer pairs for QRT-PCR and synthesis of dsRNA. We expect to extend this result to other life stages by construction of bacterial expression vectors for the RNAi and incorporation into larval medium to elicit gene silencing in various larval and pupal stages. In addition, the sequence encoding the nicotinic acetylcholine receptor subunit Da4 was identified, characterized, and PCR primers designed and synthesized to enable QRT-PCR and synthesis of dsRNA for gene silencing experiments.
Olafson, P.U., Dowd, S.E., Lohmeyer, K.H. 2010. Analysis of expressed sequence tags from a significant livestock pest, the stable fly (Stomoxys calcitrans), identifies transcripts with a putative role in chemosensation and sex-determination. Archives of Insect Biochemistry and Physiology. 74(3):179-2004.