Material and methods
Ticks: Engorged R. sanguineus s. l. and A. sculptum females were collected from naturally infested dogs and equines, respectively. At the laboratory they Apoptosis Compound Library
were placed in an acclimatized-chamber (27±1°C, RH >80%) to obtain eggs. Egg mass obtained was kept within the chamber until the larval hatching was completed, and then used for infestation. Naïve rabbits (Oryctolagus cuniculus) were used to feed tick larvae and nymphs following a protocol approved by the Committee on Ethical Animal Use of the Federal University of Goiás (CEUA/UFG, protocol number 024/2014). Unfed adult ticks (seven to 21days after ecdysis) were used in the bioassays.
The choice test and head-to-head bioassays employing a Petri dish were adapted from previously described methodology by Bissinger et al. (2009). Tests were conducted in an acclimatized room (27°C, RH=70%) and under complete darkness. A circular filter paper (Whatman qualitative, cat n° 1001 − 90mm) was placed in a Petri dish (total area=63.6cm2). One half of the filter paper (area=31.8cm2) was treated with 87μl (2.75μl.cm−2) of test material at a specific concentration, and the other half was treated with the same volume with solvent or another test material and concentration for pairwise mean comparisons. The treated filter papers were dried in a fume hood (Permution, E. J. Krieger e Cia Ltda) for 10min before they were used for testing. Ten replicates consisting of six ticks (three males and three females) were done. The ticks were placed in the center of each Petri dish and their position was evaluated at 5, 10, and 30min after their release on the arena.
For the choice test, four concentrations of DEET (C12H17NO, 97% pure, batch MKBH0428V) and β-citronellol (ReagentPlus, C10H20O, 95% pure, batch MKBF5458V) were evaluated using ethanol 99.5% alone as the negative control.
Stock solutions (7.2%; 0.200mg in 2.75μl) were prepared in 99.
5% ethanol and were used to prepare additional test concentrations in 2.75μl: 3.6% (0.100mg), 1.8% (0.050mg) and 0.9% (0.025mg). The reference concentration of 7.2% for the stock solutions was chosen because it mimics what is used in some commercial formulations of DEET. Solvent vs. blank were also assayed with both tick species. A head-to-head bioassay was performed using varying concentrations of β-citronellol (7.2, 3.6, 1.8, and 0.9%) against DEET at 7.2% which allowed repellency comparisons of DEET: β-citronellol ratios: 1:1, 2:1, 4:1 and 8:1.
Statistical analysis: Proportional (binary) data on the repellency response from the two tick species exposed to DEET and β-citronellol were fitted to generalized linear (logistic) models with binomial distribution for errors and logit link-function according to analysis of deviance (Warton and Hui, 2011). This model sought to test whether the concentration-repellency response was different between the compounds for each tick species. Another model was produced to identify the most susceptible tick species to each compound
by comparing concentration-repellency response trends (i.e., based on slope contrasts) using the same statistical approach mentioned above. Comparison between means within each concentration was performed using Chi-square test and considered to be statistically different at <0.05.</div>