Location: Tick and Biting Fly Research2011 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
Continued progress has been made towards objective completion during this second year of the project. Under sub-objective 1.A, a stable fly, antennal-specific transcriptome was generated using direct RNA sequencing. The data will be annotated to identify additional chemosensory-related transcripts, a supplement to those previously identified by screening a pyrosequencing database (FY 2010). Identification of stable fly transcripts encoding the last transmembrane domain of several biogenic amine receptors was completed in support of studies proposed in subobjective 1.B and 1.C. We have made progress towards developing a stable fly linkage map, objective 3 of this project. We are conducting paired matings to obtain the segregating families desired for constructing the linkage map. We have made significant progress on immunohistological detection of serotonin and octopamine in the feeding and reproductive systems under sub-objectives 1.B. and 1.C. Serotoninergic neurons were found to innervate the feeding related systems. The findings have been published in a peer-reviewed journal. We also found that serotonin and octopamine are differentially present in male and female reproductive tissues, suggesting different roles in mating and egg-laying behaviors. Following progress we made toward development of a stable fly feeding method under sub-objective 1.B in year one, we have initiated the bioassay and pharmacological experiments to determine the effect of different pharmacological agents on stable fly feeding. We expect to complete the stable fly feeding experiments by the end of the calendar year (2011). Also under sub-objectives 1.C, we conducted a series of pharmacological experiments to further determine the roles of serotonin and octopamine. We used reserpine as an amine depleting agent to determine how the mating and egg-laying behaviors are affected by depleting biogenic amines, including serotonin and octopamine, from stable flies. Results indicate treatment of female stable flies significantly reduced the number of eggs laid. The proportion of eggs hatching was also significantly reduced in a dose-dependent manner. Similarly, treatment of male flies with reserpine led to reduced mating success or total block of mating. Progress was made toward introduction of dsRNA and validation of gene silencing success for stable fly acetylcholinesterase, 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. Quantitative RT-PCR parameters were optimized and dsRNA was synthesized for each of the 3 candidate genes. Gene silencing was attempted in adult flies but did not yield expected mortality for silencing of vital genes. We extended the studies to late instar larva and succeeded in achieving high mortality in experimental groups prior to eclosion of adults. 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.
Liu, S.S., Li, A.Y., Witt, C.M., Perez De Leon, A.A. 2011. Immunohistological localization of serotonin in the CNS and feeding system of the stable fly stomoxys calcitrans L. (Diptera: muscidae). Archives of Insect Biochemistry and Physiology. 77(4):199-219.