These dishes were kept in an incubator at 28 ± 1.5 °C and approximately 85% relative

humidity and 14 days later, eggs were collected and transferred to glass tubes sealed with hydrophobic cotton to allow larval hatching. Egg masses from many female ticks from the same farm were mixed before hatching so that larvae used in these experiments were not all siblings. Technical grade cypermethrin (93.59% purity) (Allvet®, Londrina, Brazil) was serially diluted in a mixture of trichloroethylene (Synth, Diadema, Akt inhibitor Brazil) and olive oil (Sigma–Aldrich, São Paulo, Brazil) (2:1, v/v), resulting in different concentrations (in % of active ingredient): 5, 4, 2.4, 2.04, 1.632, 0.979, 0.588, 0.353, 0.212, 0.127 for field populations and 0.1, 0.06, 0.022, 0.013, 0.008, 0.005, 0.003, 0.002 for the R. microplus ‘Porto Alegre’ strain. This strain has been maintained at the Instituto Biológico de São Paulo without Selleck MDV3100 contact to acaricides and is considered susceptible. Filter papers (Whatman n° 1) measuring 8.5 cm × 7.5 cm were impregnated with 0.67 ml of each cypermethrin concentration, including the negative control (only the mixture of trichloroethylene and olive oil). Two papers were used per concentration. Approximately 100 larvae, aged between 14 and 21 days, were added to each of these papers which were folded and sealed with bulldog clips on the sides and top. Papers were stored in

the incubator under the conditions described above and larvae mortality was assessed after 24 h of exposure. Larvae unable to move were considered dead. The same dilution and larvae exposure procedures were performed with chlorpyriphos (97.43% purity) (Ourofino, Cravinhos, Brazil). In this case the concentrations used were (in % of active ingredient): 0.128, 0.064, 0.032,

0.016, 0.008, 0.004, 0.002, 0.001, 0.0005, and 0.00025 for both field populations and ‘Porto Alegre’ strain. Mortality data were analyzed by POLO-PC (Leora Software, 1987) in order to obtain the lethal concentration for 50% of the population (LC50) with a 95% confidence interval Chlormezanone (CI 95%). The resistance ratio (RR) was calculated by dividing the LC50 obtained from the field populations by the LC50 obtained from the ‘Porto Alegre’ susceptible reference strain. Differences in LC50 were considered significant when their 95% fiducial limits did not overlap. Tests showing mortality rates between 5% and 10% in the control group were submitted to Abbott’s formula (Abbott, 1925). Larvae that were not used in LPT were stored in 99% ethanol and kept at −20 °C for later molecular analysis. DNA was purified from individual larvae with the Qiagen DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions for animal tissue. Larvae were incubated overnight at 56 °C with proteinase K to allow thorough dissolving of the tissue.