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
Work during FY 2008 resulted in significant progress in the following areas:.
1)determining the fitness cost of amitraz resistance;.
2)the correlation of bioassay and biochemical assays for the documentation of resistance to organophosphate (OP) pesticides;.
3)the characterization of resistant ticks collected close to the cattle fever tick quarantine zone; and.
4)the documentation and initial characterization of a strain of ticks resistant to four different classes of acaricide. In addition, many scientists have speculated that a fitness cost is associated with amitraz resistance, but our work with temperature stress shows no evidence to prove this conclusion. Additionally, traditional OP bioassay results that take weeks to obtain, correlated well with a rapid biochemical (AChE inhibition) assay, when evaluated with a coumaphos and diazinon-resistant strain of R. microplus. These results demonstrate that the more rapid biochemical assay technique can successfully discriminate between the magnitudes of resistance to different OP acaricides. A highly pyrethroid-resistant strain of R. microplus was characterized with bioassay, synergists, and PCR. This stain was collected in Mexico, 50 km south of the US Cattle Fever Tick Eradication Program, and is a significant threat to the program's success if these ticks were to migrate north. Lastly, a strain of ticks was collected in northern Mexico that is resistant to 4 classes of acaricides. This is the first time a multi-resistant tick strain has been shown to be resistant to this many acaricide classes, and is the first demonstration of fipronil resistance in Mexico. This work will provide critical information for federal and state tick inspectors regarding threats to the Eradication Program, and lead to better choices of acaricides for use in control strategies needed to efficiently eradicate tick outbreaks within the US. (NP104; Component 4; Goal 4.1.1; Component 3, Goal 3.2.1; Component 2, Goal 2.1.1)

4.Accomplishments
1. Experiments designed to validate an acaricide mixture as an effective tool to control resistant Boophilus microplus: Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have demonstrated through on-animal trials in New Caledonia that deltamethrin-amitraz mixtures had significantly improved control efficacy against resistant ticks. The results validated the observations of synergistic effect of pyrethroid and amitraz mixtures in a previous laboratory study. The acaricide mixtures have the potential to be used as an effective, economic and practical way to control pyrethroid-resistant tick populations in Mexico and elsewhere. (NP104; Component 4; Goal 4.1.1)

2. Octopamine and octopamine receptors were discovered in the brain and reproductive tissues of ticks: Octopamine receptors have been postulated as the target of amitraz, a member of the formamidine class of pesticides. Little is known about the presence or function of octopamine, one of the key neuromodulators, and its receptors in the nervous system or other tissues of ticks. Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have used immunohistological techniques to detect octopamine and octopamine receptor in the nervous system (synganglion) and key tissues of Boophilus microplus and two other ixodid tick species. Octopaminergic neurons were detected mostly in the synganglion, while the octopamine receptors were found primarily in the reproductive tissues of female ticks, including ovary, oviduct and Gene's organ. These are the first evidence of octopamine and octopamine receptors in ticks, and may help elucidate the function of octopamine and its receptors in tick physiology, particularly reproduction, therefore helping in development of novel acaricides that attack such systems to achieve control of ticks. (NP104; Component 3; Goal 3.2.1)

3. Physiological fitness cost to Boophilus microplus as a result of amitraz resistance: Development of resitance to a toxicant by an organism generally results in that individual being less competitive with others in the population. The resistant individual is therefore considered less biologically fit. The acquisition of resistance often comes with a "fitness cost". For many years scientists have speculated that there was a fitness cost to amitraz resistance. Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have completed two experiments to examine this problem. The first was a free-running cage experiment where an amitraz-resistant strain was split into two groups. One group was selected each generation with amitraz while the other was not exposed to amitraz. After each generation, each group was tested for susceptibility to amitraz using bioassay. After two years (8 generations) of treatment no difference was observed between the two groups relative to amitraz-resistance, that is, in the absence of amitraz exposure amitraz-resistance was maintained. A second study examined the influence of low temperatures on the survival of amitraz-resistant and susceptible ticks. No difference could be detected. To date, there does not seem to be a measurable fitness cost to amitraz resistance. This should be taken into account when amitraz is heavily used in field situations. (NP104; Component 2; Goal 2.1.1)

4. An experiment designed to compare traditional bioassay with a biochemical AChE inhibition assay for the diagnosis of organophosphate (OP) resistance: In order to determine if ticks are resistant to organophosphates, bioassay has traditionally been used. Unfortunately, these techniques require six weeks to produce results. Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have developed a rapid assay to detect OP-resistance, and recently this assay was used to detect resistance to diazinon and coumaphos in a field-collected strain of cattle tick. This work demonstrated that the results accurately detect resistance and provide results in only 2 days. This has many beneficial applications especially to support the rapid eradiation of ticks in the cattle fever tick eradication program. (NP104; Component 2; Goal 2.1.1)

5. Rapid characterization of pyrethroid resistance in a strain of ticks collected from Coahuila, Mexico: Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory have used PCR and bioassay with synergists to rapidly describe the magnitude of pyrethroid resistance in a strain of Rhipicephalus (Boophilus) microplus ticks from Coahuila, Mexico. The State of Coahuila has been previously reported to be "tick-free"; however, during this reporting period cattle fever ticks were found and collected from this state. These ticks were also found to be resistant to pyrethroids. The collection site was in close proximity to the United States. This study showed that the risk of resistant ticks migrating into the United States is real, and eradication efforts by the US cattle fever tick eradication program should plan for this eventuality. (NP104; Component 2; Goal 2.1.1)

6. Involvement of mutations in Boophilus microplus acetylcholinesterase 1 (BmAChE1) or BmAChE3 in organophosphate (OP) resistance: Scientists at the Knipling-Bushland US Livestock Insects Research Laboratory found multiple transcripts for each of the BmAChEs expressed in synganglion of individual ticks, suggesting that there are more than 2 copies for each of the BmAChE genes or that there are mechanisms to generate transcript diversity such as alternative splicing (known to account for structural and functional diversity of AChE in vertebrates). In addition, quantitative-PCR (Q-PCR) was utilized to assess gene copy number, which demonstrated a copy number of about 4 for each of the three BmAChEs in Boophilus microplus. This finding substantiates that gene duplication contributes to the complexity of BmAChE expression in vivo. Further, mutations in BmAChE1 present in OP-resistant ticks were found to reduce OP-sensitivity of recombinant BmAChE1 by approximately 40-fold. Additional mutations from OP-resistant ticks were found to increase substrate specificity. These and previous results indicate potential involvement of BmAChE1 or BmAChE3 mutations in resistance development. Acetylcholinesterase is the major target for OP acaricides, these results strongly suggest that the mechanism for resistance to OPs is complex. (NP104; Component 2; Goal 2.1.1)

6.Technology Transfer

Number of Non-Peer Reviewed Presentations and Proceedings

3

Review Publications
Miller, R., Esparaza Rentaria, J.A., Quiroz Martinez, H., George, J.E. 2007. Characterization of permethrin-resistant Boophilus microplus (Acari: Ixodidae) collected from the State of Coahuila, Mexico. Journal of Medical Entomology. 44(5):895-897.