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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPING AND USING MOLECULAR AND BIOCHEMICAL METHODS FOR THE DIAGNOSIS OF ACARICIDE RESISTANCE IN BOOPHILUS MICROPLUS

Location: Tick and Biting Fly Research

2005 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter?
The U.S. livestock industry generates about 50% of the annual agricultural income of the United States. Together, horn flies and ticks account for an estimated annual potential loss to the U.S. cattle industry of several billion dollars. This potential loss is presently being averted, in part, by control of these ectoparasites with good animal husbandry practices and chemical pesticides. In addition to the control of ectoparasites, chemical pesticides are critical in maintaining an import barrier to Boophilus microplus, a vector of cattle fever (babesiosis). Boophilus ticks were eradicated from the U.S. following a 50-year program of dipping cattle and vacating tick-infested pastures. Boophilus ticks and babesiosis are still widespread in Mexico, and a quarantine zone along the U.S. border with Mexico is maintained to prevent their reintroduction into the U.S. Loss of the import barrier and reintroduction of Boophilus ticks and their associated diseases would cause great losses to the U.S. cattle industry. In Mexico, populations of B. microplus that are resistant to organophosphate (OP) and pyrethroid acaricides are widespread, and the emergence of resistance to the formamidine pesticide amitraz has been observed within the last few years.

Pesticide (acaricide) resistant populations of B. microplus, the southern cattle tick, in Mexico will compromise the Cattle Fever Tick Eradication Program of USDA, APHIS, Veterinary Services, if technology is not available to diagnose resistance in outbreaks of ticks from Mexico and then enable the choice of an acaricide to eradicate them. Infestations of southern cattle ticks from Mexico occur in south Texas along the Rio Grande River as a result of tick-infested livestock or wildlife that cross the river into the U.S. and disseminate ticks. Rapid, accurate methods are needed to diagnose acaricide resistance in a population of ticks. These methods could provide the basis for a rapid response with an acaricide to which the ticks are vulnerable.

This research project seeks to elucidate the mechanisms of pesticide resistance to pyrethroid OP and amitraz resistance in the southern cattle tick, B. microplus. Rapid and sensitive molecular tests will be developed to allow identification and monitoring of resistance to these pesticides after the elucidation of the resistance mechanisms. Mechanisms of emerging resistance to newer pesticides, such as ivermectin, will also be pursued.

The project has four specific goals: .
1)Identify and characterize mechanisms involved in tick resistance to different acaricides;.
2)Develop rapid, accurate and sensitive diagnostic protocols employing molecular probes or best available technology based upon information on resistance mechanisms to detect resistant genotypes and determine resistance status in tick populations;.
3) Determine the reliability, sensitivity, and utility of molecular and biochemical methods for the diagnosis of acaricide resistance in populations of B. microplus to multiple chemical groups of acaricides; and.
4)Use molecular and biochemical assays for resistance diagnosis in assessments of the fitness and mode of inheritance in organophosphate (OP), pyrethroid (P), and formamidine (F) resistance in B. microplus. The research falls under NP 104, Action Plan Goal 2.1 Detection and Diagnostics. The research addresses Goal 3, Objective 3.2 of the ARS Strategic Plan, Develop and Deliver Science-Based Information and Technologies to Reduce the Number and Severity of Agricultural Pest, Insect, Weed, and Disease Outbreaks.

The research provides assays for resistance diagnosis that will facilitate the selection and use of efficacious pesticides that will be used to protect dairy and beef producers from losses caused by pesticide-resistant blood-feeding flies and ticks, and tick-borne disease agents.


2.List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY 2005)

Analysis of adenylyl cyclase involved in the regulation of cellular function that might play a role in resistance to acaricides.

Cloning of octopamine receptor that is suspected to be responsible for acaricide resistance.

Development of probes for mutations in octopamine receptor associated with acaricide resistance.

Year 2 (FY 2006)

Electrophysiological studies on nervous and muscles.

Cloning of acetylcholinesterase, an enzyme critical for proper nerve functions and suspected to be responsible for resistance to certain pesticides.

Cloning of esterases, enzymes that break down pesticides, suspected to have increased activity resulting in resistance.

Identification of genes responsible for pesticide resistance.

Correlation of molecular diagnosis of resistance with bioassay.

Selection and study of genetics of pesticide resistant strains.

Year 3 (FY 2007)

Further electrophysiological studies.

Cloning of two families of enzymes (cytochrome P450s and glutathione-S-transferases) responsible of the breakdown of pesticides.

Further cloning of esterases.

Continued identification of genes associated with pesticide resistance.

Analysis of function of octopamine receptor.

Development of molecular probes for enzymes that break down pesticides.

Investigation on the inheritance of pesticide resistance.

Year 4 (FY 2008)

Cloning of ligand-gated ion channels.

Further identification of genes responsible for pesticide resistance.

Further development of molecular probes for enzymes that break down pesticides.

Evaluation of resistance of field-collected ticks and correlate with results obtained by molecular assays.

Study on whether mutations that confer pesticide resistance on the tick would make them less fit under natural selection.

Investigation on inheritance of pesticide resistance.

Year 5 (FY 2009)

Further cloning of ligand-gated ion channels.

Development of diagnostic probes for mutations in ligand-gated ion channels that lead to pesticide resistance.

Further identification of genes responsible for pesticide resistance.

Further development of molecular probes for enzymes that break down pesticides.

Continued analysis of functions of octopamine receptor.

Adaptation of molecular diagnostic tools developed in the laboratory for field application.

Further investigation on whether mutations that confer pesticide resistance on the tick would make them less fit in natural selection.

Further investigation on inheritance of pesticide resistance.


4a.What was the single most significant accomplishment this past year?
Discovery of Coumaphos-Resistant Boophilus microplus in South Texas ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory found, for the first time ever, coumaphos-resistant southern cattle ticks submitted by the USDA, APHIS-VS, Cattle Fever Tick Eradication Program (CFTEP) in south Texas. Coumaphos is currently the only pesticide that is registered for the control of the southern cattle tick in the U.S. Discovery of coumaphos-resistant ticks has a huge impact on the CFTEP because an alternate acaricide will have to be used if coumaphos resistance is established in Texas, but no other pesticide is currently registered for the control of this tick. These findings were reported to APHIS-VS for appropriate action to eradicate these outbreak populations. Consequences of establishment of coumaphos-resistant tick populations in the U.S. would be extremely serious to the cattle producers and consumers.


4b.List other significant accomplishments, if any.
Identification of Novel Pesticide Resistance-Associated Genes. A set of novel genes encoding proteins, which are involved in the development of acaricide resistance, were identified in Boophilus microplus. Acaricide-resistant Boophilus microplus ticks in Mexico are a threat to the APHIS-VS CFTEP that maintains the Boophilus-free status of the U.S. ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory sequenced Boophilus microplus expressed genes and computational analysis of the DNA sequences identified a set of genes that are involved in the development of pesticide resistance in other organisms and are likely involved in B. microplus resistance. An understanding of the genes responsible for acaricide resistance will assist the development of effective eradication strategies when outbreaks of organophosphate-resistant ticks occur in the U.S.

Development of Electrophysiological System for Recording Tick Feeding Activity. It is critical to understand the physiological processes that control tick behaviors, such as feeding, and how chemical agents (acaricides) interrupt particular behaviors of ticks. It is exceedingly difficult to study elements of tick feeding, such as suction of blood and salivation, in details due to the small size of ticks. Consequently, very little is known about the neurophysiological mechanisms that control tick feeding. ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory developed an in vitro system that led to the identification of particular pharyngeal nerve/muscle electrophysiological bursting patterns that are associated with bloodsucking phase of feeding. Such a system provides a novel approach for pharmacological and toxicological studies of the action of various acaricides on ticks, which can provide the basis for the development of new acaricides.

Identification of Mutation in Acetylcholinesterase (AChE) in Organophosphorus (OP) Resistant Horn Fly and Development of PCR Assay for the Mutation. A specific mutation causing OP-insensitivity in AChE of field-collected, diazinon-resistant horn flies from Louisiana was identified. ARS scientists at the Knipling-Bushland U.S. Livestock Insects Research Laboratory developed a PCR assay to diagnose presence of the mutant allele in individual flies. Assay will enable quantitation of resistance gene frequency in populations. Use of the assay will a) enable choice of alternative treatment strategies for effective IPM; b) allow assessment of field-collected OP-resistant flies to enable identification of OP-resistant strains utilizing a different mutation or mechanism; and c) enable assessment of alternative treatment strategies for effect on resistance gene frequency in development of resistance mitigation strategies.


4c.List any significant activities that support special target populations.
None.


5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
Reporting of the first case of coumaphos-resistant ticks found inside the USA USDA, APHIS-VS, Cattle Fever Tick Eradication Program (CFTEP) uses the results of the research to develop effective eradication strategies for the southern cattle tick. Of the 29 outbreak populations in Texas submitted by the APHIS-VS-CFTEP, one strain was determined to be resistant to organophosphate acaricides and another strain to pyrethroid acaricides. Coumaphos is currently the only pesticide that is registered for the control of southern cattle tick in the U.S. In addition, these results were important to personnel of the CFTEP because they showed that acaricide-resistant ticks were likely to occur within the U.S. at any time. Discovery of coumaphos-resistant ticks in south Texas should help APHIS-VS in the development of eradication strategies when outbreaks of organophosphate resistant ticks occur in the U.S. This accomplishment addresses Milestone 4.1 in the Project Plan and the ARS Action Plan Component 2, Detection and Surveillance Technology, Goal 2.1, Detection and Diagnostics. The accomplishment contributes to Performance Measure 3.2.1 in the ARS Strategic Plan.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
None.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
Temeyer, K.B., Lohmeyer, K.H., Chen, A.C., Foil, L.D. 2005. Recent progress in understanding organophosphate resistance in horn flies and cattle fever ticks. 49th Annual Livestock Insect Workers Conference, June 19-22, 2005, Bozeman, Montana.


Review Publications
Temeyer, K.B., Pound, J.M., Miller, J.A., Chen, A.C., Pruett Jr, J.H., Guerrero, F., Davey, R.B., Untalan, P.M., Lohmeyer, K.H., Li, A.Y., Miller, R., George, J.E. 2004. Organophosphate resistance in Mexican strains of Boophilus microplus: A major threat to the U.S. cattle industry. Southern Association of Agricultural Scientists Bulletin of Biochemistry and Biotechnology. 17:43-51.

Li, A.Y. 2005. Use of synergists for elucidation of resistance mechanisms and management of resistant ticks and horn flys. In: Proceedings of 30th Anos al Servicio de la Granderia Nacional, 1975-2005, June 8-9, 2005, Jiutepec, Morelos, Mexico. 2005 CDROM.

Li, A.Y., Davey, R.B., George, J.E. 2005. Carbaryl resistance in Mexican strains of the southern cattle tick Boophilus microplus (Acari: Ixodidae). Journal of Economic Entomology. 98(2):552-556.

Foil, L.D., Coleman, P., Eisler, M., Fragoso-Sanchez, H., Garcia-Vasquez, Z., Guerrero, F., Jonsson, N.N., Li, A.Y., Miller, R., Pruett Jr, J.H., S. Toor. 2004. Factors that influence the prevalence of acaricide resistance and tick-borne diseases. Veterinary Parasitology. 125:163-181.

Li, Andrew Y., Pruett, John H., Davey, Ronald B., George, John E. 2005. Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari:Ixodidae). Pesticide Biochemistry and Physiology. 81:145-153.

Tan, J., Liu, Z., Wang, R., Huang, Z., Chen, A.C., Gurevitz, M., Dong, K. 2005. Identification of amino acid residues in the insect sodium channel critical for pyrethroid binding. Molecular Pharmacology. 67(2):513-522.

Sammataro, D., Untalan, P., Guerro, F., Finley, J. The resistance of Varroa mites (Acari: Varroidae) to acaricides and the presence of esterase. 2005. Internat. J. Acarol. Vol. 31, No. 1. 67-74.

Last Modified: 9/1/2014
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