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United States Department of Agriculture

Agricultural Research Service


Location: Tick and Biting Fly Research

2008 Annual Report

1a.Objectives (from AD-416)
The overall objective of this research is to develop effective and environmentally safe tools and sustainable strategies for the integrated management of livestock pests. Toward that goal our specific objectives for the next 5 years are to: .
1)Develop and improve methods of chemical control of horn flies and stable flies;.
2)Develop alternatives to classical chemical control of horn flies and stable flies; and.
3)Develop and evaluate control strategies to provide sustainable pest management practices.

1b.Approach (from AD-416)
Use in-vitro and in-vivo bioassays to evaluate new classes of chemical control agents against horn flies and stable flies. Develop and improve both conventional and controlled release chemical delivery systems to increase the effectiveness, improve efficiency, reduce the quantity of pesticide needed, and improve the safety of chemical control. Develop biological and physical control technologies for blood-feeding flies on cattle. Create and use simulation models to serve as a framework to guide both research and implementation of sustainable control programs. Develop and test sustainable horn fly and stable fly management strategies for pastured cattle. Develop computer assisted decision-aid programs for use by extension personnel and producers. Develop protocols for the selective breeding of cattle resistant to horn flies.

3.Progress Report
In 2008 we made several important contributions to our milestones. New control measures that have been evaluated for controlling stable and horn flies include a novel insecticide, Novaluron (an IGR) and a promising new formulation of the fungi Metharhizium anisopliae. In addition, significant strides have been made toward determining the biochemical characteristics of different thrombostasin variants in horn flies. We have also produced a bacterially expressed, recombinant protein for thrombostasin variant, TS-8. Due to the loss of our primary grazing pasture for cattle, we have had to redirect affected milestones. Without grazing lands, it is impossible to phenotype cattle as high or low horn fly carriers. As a contingency, we have begun working on sequencing the genome of the stable fly. To date we have used flow cytometry to estimate the genome sizes of the stable fly and have constructed a stable fly library of normalized cDNAs that represent genes expressed at the embryonic, larval, and pupal stages as well as in adult female and male heads. In addition, work has begun to identify sequences that have a role in stable fly olfaction as well as to construct a stable fly library of cDNAs that represent genes expressed in the antennae and maxillary palps of adult males and females. A senior scientist retired in January of 2008. Thus, some of the milestones related to the retired scientist's area of responsibility have not met completion. (NP104; Component 3, Goal 3.1.1; Component 4, Goal 4.1.1; Component 4, Goal 4.2.1)

1. Frequency distribution of horn fly thrombostasin variants: A study to evaluate the frequency distribution of variants of thrombostasin unique to a Texas field collection was conducted. The frequency for all variants in horn flies collected from cattle hosts specifically phenotyped as high- or low-carriers of horn flies was evaluated, demonstrating a significant difference between thrombostasin variants of flies collected from the high- versus low-carrier hosts. These results suggest that high- and low-carrier hosts attract flies with common variants of the anticoagulant thrombostasin. (NP104; Component 3; Goal 3.1.1)

2. Successful cloning and bacterial expression of TS-8, a thrombostasin variant: TS-8 was selected for expression because it was present at a significantly higher frequency within the Texas field collection relative to reports for horn flies from Alabama and our colony strain. The recombinant protein has been purified, enabling us to evaluate its biochemical characteristics, i.e., anti-thrombin activity, as part of FY2009. In parallel, we will be producing recombinant proteins of thrombostasin variants that were present at a lower frequency to evaluate differences in anti-thrombin activity, if any. Cloning and expression of thrombostasin variant proteins allows us to investigate differences in anticoagulation function and provides potential candidate proteins for development of vaccines. (NP104; Component 3; Goal 3.1.1)

3. Experiments designed to estimate the genome size for the stable fly and horn fly: In collaboration with Texas A&M University, Dept. of Entomology, we used flow cytometry to estimate the genome sizes of the stable fly and the horn fly. Individual heads from females and males of the stable fly and horn fly were analyzed separately, and the results were calculated using an internal standard (known genome size) that was co-prepared with each sample, in this case D. virilis. This data was collected in support of our laboratory's interest in sequencing the genome of the stable fly, a significant livestock pest. (NP104; Component 3; Goal 3.1.1)

4. Construction of a stable fly library of normalized cDNAs from various life stages: We elected to normalize the sample to reduce the representation of superprevalent expressed genes, facilitating the discovery of rare genes. In collaboration with Research and Testing Laboratories at Medical Biofilm Research Institute, Lubbock, TX, we have initiated sequencing of the stable fly normalized library using the 454 Life Sciences™ pyrosequencing platform. The Institute maintains a Roche Genome Sequencer FLX instrument that is capable of producing 350,000 reads, averaging 200-250 bases per read, providing at least 70 million bases of sequence in a single day. This sequence information will be included in a gene index as a genomic resource for researchers in the field of stable fly genomics. (NP104; Component 3; Goal 3.1.1)

5. Investigation of the molecular pathways associated with sensory-mediated behaviors of the stable fly: Understanding molecular pathways associated with sensory-mediated behaviors of the stable fly can provide targets for the development of novel/alternative control technologies. In an effort to identify sequences that have a role in stable fly olfaction, we have constructed a stable fly library of cDNAs that represent genes expressed in the antennae and maxillary palps of adult males and females. A small-scale sequencing project, conducted at our lab, of 186 randomly selected clones from this library resulted in the identification of several genes expressed exclusively in stable fly antennae. These include an odorant receptor, two odorant binding proteins (OBPs), and an odorant degrading enzyme (cytochrome P450). We have also identified an additional seven genes that are known to mediate olfaction in other dipterans. Understanding these pathways can provide targets for the development of novel/alternative control technologies. This data was the basis for an NRI grant proposal, entitled "Molecular biology of olfaction in the stable fly, Stomoxys calcitrans". (NP104; Component 3; Goal 3.1.1)

6. Evaluation of entomopathogenic fungi for the control of horn flies: Due to insecticide resistance problems and growing concerns over the environmental impact of synthetic insecticides, a need for more natural, alternative types of control for biting flies on cattle has arisen. Entomopathogenic fungi are naturally occurring fungi that infect a specific, limited group of host insects without negatively affecting the environment. As previously reported, three strains of entomopathogenic fungi, Beauveria bassiana (strain GHA), Metarhizium anisopliae (strain ESCI), and Paecilomyces fumosoroseus, were tested in the laboratory for control efficacy against horn flies. Preliminary evaluations of all three strains on cattle in the field showed that B. bassiana may be an effective horn fly control treatment when used in a dust or spray-on application. In addition, a new formulation of M. anisopliae was recently evaluated in the laboratory against horn flies and was found to be as effective as B. bassiana. Both of these promising strains need to be formulated for field application and tested on cattle in the pasture. If these fungi can be formulated to prevent ultra-violet light degredation their half-life under field conditions can be extended, offering an alternative to insecticide control. (NP104; Component 4; Goal 4.2.1)

7. Evaluation of Novaluron for the control of immature stable and horn flies: Experiments were conducted to determine the efficacy of Novaluron against the immature stages of stable flies and horn flies. The lethal concentration of Novaluron that kills 50% (LC-50) and 90% (LC-90) of test subjects was determined for both species by bioassay techniques. Interestingly, immature horn flies were less sensitive to Novaluron than immature stable flies. Additionally, a subsequent field experiment was conducted whereby hay and manure mix was treated with Novaluron granules and cattle walking on the mixture was simulated. In these bioassays, it was determined that ca. 5000 ppb was necessary to eliminate emergence of pupae harvested from the samples. These results demonstrate the efficacy of Novaluron against horn flies and stable flies, suggesting that treatment with Novaluron should have potential for control of stable flies in the area around round bales. (NP104; Component 4; Goal 4.1.1)

6.Technology Transfer

Number of the New MTAs (providing only)4
Number of Non-Peer Reviewed Presentations and Proceedings2

Last Modified: 9/23/2014
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