SUPLEMENTARY MATERIAL Efficacy of carvacrol on Rhipicephalus (Boophilus) microplus engorged female ticks (Canestrini, 1887) (Acari: Ixodidae): effects on mortality and reproduction.

The activity of carvacrol was evaluated in R. microplus female ticks by estimating the mean lethal concentration (LC 50 ) and the acaricidal efficacy. Ticks were subjected to the Adult Immersion Test (AIT) to calculate LC 50 . From this value, the AIT was performed again at the concentrations of 20%, 40%, 60%, 80% and 100% of the LC 50 . Two control groups were established: one treated with distilled water and one with the solvent (ethanol 50%). The following parameters were evaluated: female weight before oviposition, egg mass weight, pre-oviposition and incubation period, hatching percentage, egg production index, fecundity rate, estimated reproduction, reduction in oviposition and hatching, and product efficacy. The obtained LC 50 was 20.11 mg/mL. Egg mass weight, egg production index, pre-oviposition and fecundity rates of groups treated with 80% and 100% of LC 50 were statistically different when compared to control groups. The product efficacy in groups treated with 100% of LC 50 was 61.10%.


Experimental
This experiment was conducted at the Laboratory of Parasite Biology (BIOPAR), located in the Federal University of Lavras (UFLA), Lavras/MG, Brazil.
Adult R. microplus female ticks (Acari: Ixodidae) were taken from naturally infected cattle of the municipality of Lavras/MG, Brazil and region. These animals did not receive any acaricidal treatment in the last three months before the experiments.
Females were carefully removed according to standardized procedures (Gammons and Salam 2002) and selected according to their integrity and ability to move.
The Adult Immersion Test (AIT) followed the procedures proposed by Drummond et al. (1973). Each group of ticks was immersed in their respective solutions for 5 minutes. The parasites were then dried on absorbent paper and placed in identified Petri dishes in a BOD incubator under controlled conditions (28 ± 1°C, 80% humidity, 12-hour photoperiod). The animals were observed for a total period of seven days, as proposed by Oliveira et al. (2008).
After the detection of the mortality interval, carvacrol was diluted again and tested in a new series of dilutions. Ticks with homogeneous body weight (p<0.05) were divided in 12 experimental groups with 10 individuals each: 2 Control Groups (C I e C II ), in which females were exposed to distilled water (C I ) and solvent alone (C II ), and 10 Treatment Groups (T I-T X ), in which ticks were exposed to the concentrations of 12, 53,16,33,19,15,21,96,23,82,26,61,29,37,31,21,34,85,37,57 mg/mL of carvacrol diluted in ethanol 50%, based in the mortality interval obtained previously.
The AIT was performed again and the ticks were then kept in a BOD incubator under controlled conditions (28 ± 1°C, 80% humidity, 12-hour photoperiod), also during seven days. Experimental groups were tested in duplicates. The obtained results were submitted to Probit Analysis (Statistica v.7.0), in order to calculate LC 50 .

Evaluation of the reproductive parameters of R. microplus
Adult females were washed in a sieve under running water and then dried on soft absorbent paper. The experimental protocol was based on the AIT proposed by Drummond, Ernst, Trevino, Gladney and Graham (1973). A total of 84 ticks, with homogeneous weights (p<0.05), were divided into 7 experimental groups: 2 Control Groups (C I , C II ), in which ticks were exposed to distilled water and solvent (ethanol at 50% v/v in distilled water), respectively, and 5 treatment groups (T I , T II , T III , T IV e T V ), in which ticks were exposed to concentrations of 20, 40, 60, 80 and 100% of the mean lethal concentration (LC 50 ) of carvacrol diluted in ethanol 50%. After immersion, female ticks were dried on absorbent paper and kept individually in identified Petri dishes in a BOD incubator, under controlled conditions (28 ± 1°C, 80% humidity, 12hour photoperiod).
The following parameters were evaluated: a) female weight (FW) after detachment; b) total egg mass weight of each female (EW); c) pre-oviposition period (POP), the number of days counted from the moment they detach from host to the beginning of oviposition; d) incubation period (IP), the time period from oviposition to the beginning of hatching; e) egg hatchability rate (HR), determined visually by estimating the proportion of larvae in relation to unhatched eggs.
From these values, the egg production index (EPI) was determined, according to Statistical analysis was performed using GraphPad Prism 5 (v.5.03). All obtained values were statistically analyzed by Analysis of Variance (ANOVA), and the comparison of means was performed using Tukey's test (p<0.05).