ASSESSMENT OF THE LARVAL TARSAL TEST AND SEARCH FOR SINGLE NUCLEOTIDE MUTATIONS IN PYRETHROID-RESISTANT RHIPICEPHALUS MICROPLUS
Tick and Biting Fly Research
2012 Annual Report
1a.Objectives (from AD-416):
Evaluate the resistance profile of cattle tick strains, and identify the presence of point mutations (PM) in cattle tick larvae.
1b.Approach (from AD-416):
Perform in vitro bioassays to determine susceptibility of four laboratory strains to ten acaricidal compounds with the Larval Tarsal Test (LTT); susceptibility to ivermectin of an ivermectin-resistant strain and susceptibility to fipronil of a fipronil-resistant strain using the Larval Packet Test (LPT); and susceptibility to ivermectin of an ivermectin-resistant strain using the Klafke Larvel Immersion Test (LIT). LC50s will be calculated from which resistance ratios will be estimated based on the comparison with a susceptible reference strain (Munoz). Molecular bioassays will be either run individually or investigated through a multiplex PCR newly developed in the CFTRL. Thirty larvae per population will be analyzed individually. The presence and frequency of the point mutation in these field strains will be compared with in vitro results on their resistance status previously assessed with the LTT. For some of the strains, if the presence of a point mutation is identified, there will be the possibility to investigate the frequency of the mutation among larvae having survived or died at a specific dose of flumethrin and cypermethrin.
A new bioassay technique, the Larval Tarsal Test, was evaluated at the Cattle Fever Tick Research Laboratory against pesticide-resistant and -susceptible ticks. The bioassay performed very well, allowing for increased efficiencies in labor and pesticide use. The test was able to detect resistance to all compounds used with a precision equal to or greater than standard bioassay techniques currently available in the laboratory. Plans are in place to use the larval tarsal technique to test all outbreak samples sent to the laboratory for pesticide resistance testing. Additionally, a multiplex PCR procedure was developed to detect three distinct mutations on the sodium channel. These three mutations independently confer resistance to pyrethroid pesticides in ticks. Currently, there are individual tests for each mutation. The results of this research will allow the Cattle Fever Tick Research Laboratory to perform bioassays on ticks collected within the Cattle Fever Tick Eradication Zone more efficiently to gain much more detailed information regarding the magnitude of the resistance than before with our standard testing methods. Additionally, with one PCR procedure we can now test for three separate mutations that confer resistance to pyrethoid pesticides instead of running three separate tests.