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.
1. Characterizing an odorant co-receptor from the stable fly and the horn fly critical to insect olfaction. In insects, odors are perceived and converted from a blend of chemicals into an electrical signal to the brain center by way of a complex consisting of a ligand-selective odorant receptor (Or) and a conserved odorant co-receptor (Orco). Many studies in insects have demonstrated that 'knocking out' Orco directly affects odor-based behavior, emphasizing how critical Orco is to insect olfaction. An ARS-Kerrville scientist identified and localized Orco on the antennae of both the stable fly and the horn fly, both of which are major pests of livestock in the United States. The finding of Orco lays the foundation to study the functional role of Or-Orco complexes, an approach that may enhance our understanding of how repellents/bait attractants alter the stable fly and horn fly olfactory response.
2. Determining roles of biogenic amines in stable fly reproductive behaviors. It is believed that the biogenic amines serotonin and octopamine play a role in reproductive behaviors in stable flies. If so, manipulating them might offer a way to reduce these pests. We applied reserpine, a substance that depletes amines, to both male and female stable flies. Treated female flies laid fewer eggs, and eggs hatching was similarly reduced, based on the amount of reserpine applied. In males, sperm transfer was blocked. This study confirms that biogenic amines do play a role in stable fly reproduction and that manipulating their function could be the basis for an effective control strategy.
3. A critical enzyme identified in a sand fly species. Sand flies transmit disease agents that can be deadly to humans, such as leishmaniasis, a painful and debilitating skin condition. Chemical insecticides play a critical role in sand fly control. Acetylcholinesterase (AChE) is an enzyme in the nervous system of insects that is targeted by chemical insecticides technically known as organophosphates. This important enzyme has been identified, cloned, and sequenced from a sand fly species transmitting the organisms causing leishmaniasis. The active recombinant sand fly AChE was created successfully and used to screen novel synthetic compounds as part of insecticide discovery research efforts. Collaborative research with University of Florida investigators resulted in the identification of new compounds showing significantly improved activity against the sand fly AChE. These findings offer the opportunity to develop new insecticides for effective sand fly control.
4. MicroRNA characterized in the stable fly. The stable fly, Stomoxys calcitrans, is a blood-feeding fly pest affecting cattle. It is difficult to control this economically important pest with conventional insecticides. ARS-Kerrville scientists searched for new molecules in the stable fly that can potentially be used to develop novel fly control technology. They have identified a number of unique microRNAs in the stable fly. Understanding the roles of microRNA in the stable fly may lead to discovery of novel control target.
5. Development of a rapid diagnostic assay to detect endosulphan resistance in horn fly. Insecticide resistance is a major problem to cattle producers attempting to control horn flies, and novel methods of control are needed. Recently developed horn fly treatments incorporate the endosulphan class of insecticides; however, it is likely that an endosulphan resistance-associated gene mutation, termed rdl, is pre-existing in horn fly populations, which means that the effectiveness of these treatments will be short-lived. ARS scientists at Kerrville, Texas, in collaboration with scientists at Louisiana State University, have developed a rapid DNA-based assay to detect the presence of the rdl mutation in individual flies. Testing results of field-collected horn fly samples confirmed that this resistance gene mutation was responsible for control failure associated with use of this insecticide. This assay will provide a valuable tool to detect endosulphan resistance in other populations of the horn fly, so that more effective insecticides can be used in a fly control program.
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.