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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

2007 Annual Report


1a.Objectives (from AD-416)
Widespread acaricide-resistance in the southern cattle tick, Boophilus microplus, in Mexico and worldwide has made the management of ticks increasingly difficult and poses a serious threat to the U.S. cattle industry due to the possibility of re-infestation of resistant ticks carried by imported cattle from Mexico. The overall goal of this research is to identify mechanisms involved in resistance to chemical acaricides and to develop rapid, accurate, and sensitive molecular diagnostic probes for the early detection and assessment of resistance status in tick populations so that an effective pest management strategy can be designed. Specifically, objectives for the proposed research are: 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.

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.


1b.Approach (from AD-416)
Use modern molecular biological techniques to clone genes from the southern cattle tick, Boophilus microplus that are possible targets of chemical acaricides. Mutations in target genes could render the targets refractory to acaricide treatments. After these genes are cloned from the reference (susceptible) strain of ticks, the same genes from various acaricide resistant strains will be compared to the reference to detect any mutations. If mutations are found, both the wild-type and mutant genes will be expressed in vitro and biochemical/physiological functions of the expressed proteins will be examined to determine if the mutations could account for resistance. Additional genes encoding detoxifying enzymes will also be cloned. We will determine if detoxifying enzyme genes are amplified or the rate of transcription of these genes is increased in acaricide-resistant ticks treated with acaricide, both of which could accelerate the detoxification of acaricides, leading to resistance. Molecular probes will be developed for genes found responsible for resistance and evaluated first in the laboratory strains and then in field-collected populations. After these probes are validated, they will be used to study the fitness cost of acquiring resistance, and in conjunction with classical Mendelian genetic manipulations, to determine the mode of inheritance of resistance traits.


4.Accomplishments
Expression of cattle tick acetylcholinesterase (AChE) genes: Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have shown the presence of three biochemically active acetylcholinesterase (AChE) genes, first time in any arthropod. Acetylcholinesterase is critical in the normal function of the nervous system and is the major target for organophosphate (OP) pesticides. Multiple AChEs expressed in the southern cattle tick suggest multiple potential target sites for action of OP pesticides in this organism. Further, we have found a mutation in one of the AChE genes that generates an AChE enzyme resistant to OP inactivation, but insufficient to account for the OP-resistance. These results suggest that OP-resistance in the cattle tick is complex, and potentially involves multiple genes. (NP104; Component 2, Detection and Surveillance Technology; Goal 2.1).

Developed rapid assay to monitor mutations in octopamine receptor gene: Octopamine receptor is the major site of action of a class of pesticides known as formamidines. Mutation in this receptor is suspected to be the primary mechanism of resistance to these pesticides. Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have developed a rapid assay based on molecular biology to monitor the octopamine receptor gene. This rapid assay provides a necessary tool for the study of the mode of formamidine pesticide resistance in the southern cattle tick. It also can be used to monitor the prevalence of the octopamine receptor mutation in field populations of the cattle tick which is a reflection of the extent of pesticide resistance. (NP104; Component 2, Detection and Surveillance Technology; Goal 2.1).

Discovery of the synergistic effect of permethrin and amitraz mixtures: Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have found synergism between permethrin and amitraz against the southern cattle tick, and this is the first report of such synergism in ticks. Field populations of the cattle tick have developed very strong resistance to permethrin and moderate to strong resistance to amitraz. These findings provide a new way to control resistant ticks with existing pesticides that would have otherwise been abandoned. Pesticide mixtures had not been explored to control the southern cattle tick. This work not only demonstrates the enhanced toxicity of these mixtures to tick larvae in laboratory bioassays, but also the enhanced efficacy against pyrethroid-resistant ticks. (NP104; Component 2, Detection and Surveillance Technology; Goal 2.1).

Invalidation of bioassay: Recently, adult immersion tests have been modified to greatly speed the detection of resistance. A study was designed to independently evaluate the reliability of these modifications in Australia and the United States. Scientists at the Cattle Fever Tick Research Laboratory found that the modified techniques were not a reliable predictor of resistance and should not be used. Unfortunately, the modified test is currently used extensively in Mexico by the pharmaceutical industry, and it is impossible to make informed tick management decisions based on faulty data. This leads to improper treatment of cattle for tick control and quickens the pace of resistance development in Mexico. A reversion to the proper techniques should lead to better management decisions in Mexico and in turn slow the development of resistance. This will reduce the development of resistance in Mexican cattle ticks and will reduce the threat that resistant ticks pose to the Cattle Fever Tick Eradication Program in the United States. (NP104; Component 2, Detection and Surveillance Technology; Goal 2.1).

Selection of bioassay for amitraz resistance: Recently, three bioassay techniques were developed for the determination of amitraz susceptibility in the southern cattle tick. Scientists at the Cattle Fever Tick Research Laboratory led a study to compare these techniques and determined which were adequate for the various important studies needed to further the study of amitraz resistance in the cattle tick. Additionally, the scientists demonstrated various techniques that will greatly enhance the accuracy of the lethal concentration estimates generated by statistical analysis. (NP104; Component 2, Detection and Surveillance Technology; Goal 2.1).


6.Technology Transfer

Number of U.S. patents granted1

Review Publications
Klafke, G.M., Sabatini, G.A., de Albuquerque, T.A., Martins, J.R., Kemp, D.H., Miller, R.J., Schumaker, T.T.S. 2006. Larval immersion tests with ivermectin in populations of the cattle tick Rhipicephalus (Boophilus microplus) (Acari: Ixodidae) from State of Sao Paulo, Brazil. Veterinary Parasitology. 142(3-4):386-390.

Jonsson, N.N., Miller, R.J., Robertson, J.L. 2007. Critical evaluation of the modified-adult immersion test with discriminating dose bioassy for Boophilus microplus using American and Australian isolates. Veterinary Parasitology. 146:307-315.

Li, A.Y., Guerrero, F.D., Pruett, J.H. 2007. Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae). Journal of Pesticide Biochemistry and Physiology. 87:147-155

Miller, R. J., Davey, R.B., George, J.E. 2007. First report of pemethrin-resistant Boophilus microplus (Acari: Ixodidae) collected within the United States. Journal of Medical Entomology. 44(2):308-315.

Miller, R.J., Davey, R.B., White, W.H., George, J.E. 2007. A comparison of three bioassay techniques to determine amitraz susceptibility in Boophilus microplus (Acari: Ixodidae). Journal of Medical Entomology. 44(2):283-294.

Temeyer, K.B., Chen, A.C. 2007. Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae). DNA Sequence. 18(2):85-91.

Guerrero, F., Barros, T. 2006. Role of kdr and esterase-mediated metabolism in pyrethroid resistant populations of Haematobia irritans irritans (Diptera: Muscidae) in Brazil. Journal of Medical Entomology. 43(5):896-901.

Oremus, G., Guerrero, F.D., Alison, Jr, M.W., Kimball, M.M., Kim, J.H., Foil, L.D. 2006. Effects of mid-season avermectin treatments on pyrethroid resistance in horn fly (Diptera: Muscidae) populations at three locations in Louisiana. Veterinary Parasitology. 141(1-2):156-164.

Li, A.Y., Chen, A.C., Miller, R., Davey, R.B., George, J.E. 2007. Acaricide resistance and synergism between permethrin and amitraz against susceptible and resistant strains of Boophilus microplus (Acari: Ixodidae). Pest Management Science. 63(9):882-889.

Chen, A.C., He, H., Davey, R.B. 2007. Mutations in a putative octopamine receptor gene in amitraz-resistant cattle ticks. Veterinary Parasitology. 148:379-383.

Last Modified: 4/18/2014
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