DEVELOPING AND USING MOLECULAR AND BIOCHEMICAL METHODS FOR THE DIAGNOSIS OF ACARICIDE RESISTANCE IN BOOPHILUS MICROPLUS
Tick and Biting Fly Research
Project Number: 6205-32000-028-00
Start Date: Oct 01, 2004
End Date: Sep 30, 2009
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.
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.