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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

2009 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.


3.Progress Report
Research conducted over the past five years of this project culminated in significant discoveries of genetic mutations in the southern cattle tick sodium channel and acetylcholinesterase genes that confer or are associated with acaricide resistance. These results enabled us to design molecular diagnostic assays for these mutations, and these assays were used to provide convincing evidence of widespread resistance to pyrethroids in Mexico. This technology was transferred to national and international collaborators to enable rapid assessment of acaricide resistance. This project also furthered what is known about the function of tick acetylcholinesterase, the target of organophosphate acaricides used by the USDA's Cattle Fever Tick Eradication Program. Of significance was the demonstration that suppressing the expression of one form of the enzyme did not affect tick viability, but that suppression of all three forms of the enzyme resulted in tick mortality. Results from this research provided critically needed information to the USDA's Cattle Fever Tick Eradication Program and lays the foundation for future work aimed at developing novel acaricides for resistance management to ensure the continued success of the eradication program.


4.Accomplishments
1. Validation of a molecular assay for detecting pyrethroid resistance in ticks from Mexico: There is a growing body of evidence suggesting that populations of the southern cattle tick in Mexico are developing widespread resistance to currently available acaricides. Traditional bioassays for resistance detection require specific tick stages and are labor-intensive and time-consuming. We developed a rapid and cost effective molecular diagnostic assay to detect a mutation in the tick sodium channel gene that is believed to result in resistance to pyrethroids. To validate its effectiveness, we conducted a study with Mexican collaborators using both the traditional bioassay and the molecular assay to assess acaricide resistance in tick samples from Yucatan, Mexico. Data generated from this study validated the use of the assay, confirmed that the sodium channel mutation was the major mechanism of resistance to pyrethroids, and revealed that the gene mutation frequency was related to the level of tick resistance to pyrethroids. These results are significant because they support the value of the molecular diagnostic assay as a tool for rapid detection of pyrethroid resistance in the southern cattle tick.

2. Silencing tick acetylcholinesterase genes leads to tick mortality: The cattle fever tick has developed resistance to all currently used acaricides, necessitating the development of novel acaricides that are both effective against ticks and safe to humans and the environment. Gene silencing is a powerful tool that can aid in new pesticide development by identifying novel targets for chemical control. Acetylcholinesterase (AChE) is the target for the organophosphate (OP) coumaphos, which is the only acaricide approved for use in the U.S. as part of the Cattle Fever Tick Eradication Program. Understanding the function of tick AChE will allow us to design new molecular pesticides that can be more effective against OP-resistant ticks. There are at least three genes that produce the AChE enzyme in the tick. Using gene silencing, we have demonstrated that having multiple genes may enable the tick to compensate for mutations present in one of the genes. This finding is of significance because this compensation may allow for increasing levels of OP resistance, a threat to the Cattle Fever Tick Eradication Program.


6.Technology Transfer

Number of the New/Active MTAs (providing only)1

Review Publications
Rosario-Cruz, R., Almazan-Garcia, C., Miller, R., Dominquez-Garcia, D., Hernandez-Ortiz, R., De La Fuente, J. 2009. Genetic basis and impact of tick acaricide resistance. Frontiers in Bioscience. 14:2657-2665

Chen, A.C., He, H., Temeyer, K.B., Jones, S., Green, P., Barker, S.C. 2009. A survey of Rhipicephalus microplus populations for mutations associated by pyrethroid resistance. Journal of Economic Entomology. 120(1):373-380.

Last Modified: 8/22/2014
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