In vitro acaricidal activity of different ectoparasiticide classes against Amblyomma sculptum larvae.

Zoonoses are major causes of morbidity and mortality worldwide. Among them, Brazilian Spotted Fever (BSF) is an important one that occurs in some regions of South America and can be transmitted by the "star tick" Amblyomma sculptum. Application of acaricides against the larval stage is important as strategy of population control. However, there is still a deficiency of studies on chemical control of A. sculptum and the present work aims to evaluate the in vitro acaricidal activity of cypermethrin, flumethrin, deltamethrin, fipronil, coumaphos and chlorpyrifos against A. sculptum larvae. Bioassays were performed using the larval immersion test method. A discriminatory analysis between the antiparasitic classes most used for tick control was carried out, which made it possible to determine the classes with higher potential for controlling A. sculptum larvae. Our results showed that A. sculptum larvae present highest sensitivity to the synthetic pyrethroid group, followed by the phenylpyrazole, organophosphate and macrocyclic lactone groups. These findings may support studies on improvement of tick control as in animals as in the environment.


Introduction
Ticks are widely distributed in Brazil with 70 species, being the Amblyomma genera (32 spp.) the most representative (Dantas-Torres et al., 2019). They need to feed on the blood of vertebrates (mainly mammals) and are responsible for causing cutaneous lesions, anemia, inoculation of toxins and transmission of pathogens. These occurrences can lead to host death (Prata, 2005;Rodrigues et al., 2015;Moraes-Filho, 2017). Amblyomma sculptum Berlese, during a long time cited as Amblyomma cajennense (Fabricius), also called by "star tick", the most important tick species, given that it has the capacity to parasitize several animal species, including humans. Infected ticks can transmit the bacterium Rickettsia rickettsii, etiological agent of Brazilian Spotted Fever (BSF), an important zoonosis and the most lethal rickettsiosis in the world (Bechah et al., 2008;Labruna et al., 2002;Labruna, 2009).
Although A. sculptum has low parasitic specificity, especially in the immature phases, capybaras and horses are the preferred hosts for all stages of this species (Labruna et al., 2001). For effective control over BSF, strategic tick control could be useful, in order to reduce the number of ectoparasites both among animals and in the environment. The immature stages of ticks are more sensitive to acaricides than is the adult stage. Therefore, reduction of the immature tick population consequently provides a reduction in the number of adults (Rodrigues et al., 2015). The main classes of acaricides currently used in Brazil, via the topical route, are macrocyclic lactones, phenylpyrazoles, pyrethroids and organophosphates. However, there is still a deficiency of studies on chemical control over A. sculptum.
Given the need for more information about A. sculptum control and to enable and ensure the efficacy and safety of the use of active ingredients for controlling ticks, it is first necessary to determine the effective concentrations. Evaluation of in vitro activity and estimation of LC 50 and LC 90 values are important tools at this stage. Therefore, the aim of the present study was to evaluate the in vitro acaricidal activity of cypermethrin, flumethrin, deltamethrin, fipronil, coumaphos and chlorpyrifos against A. sculptum larvae.

Preparation of test solutions
The emulsifiable concentrates of each technical-grade active ingredient were prepared as described in Table 1. The surfactant Triton-x was added to all the emulsifiable concentrates at 2%. Different solvents (ethanol, N-methylpyrrolidone, xylene and acetone) were used to ensure complete solubilization of the active ingredients. Stock solutions were prepared through dilutions (1:100) of the emulsifiable concentrates in pure water. Working concentrations (n = 10) were prepared using the diluents at the concentration range described in Table 1. The test solutions were prepared in accordance with the recommendations of the FAO Guidelines for Resistance Management and Integrated Parasite Control in Ruminants (FAO, 2004).

In vitro larvicidal assay
The experiments followed the standards established by the Ethics Committee for Animal Use (CEUA) of the Veterinary Institute, Federal Rural University of Rio de Janeiro (UFRRJ). The larvae of A. sculptum (CEUA/IV no. 7699190418) that were used in the experiment were obtained from colonies maintained in rabbits in the Laboratory for Experimental Chemotherapy in Veterinary Parasitology of UFRRJ.
The colony of A. sculptum was created from fed females collected from horses in the UFRRJ herd that did not receive any carrapaticidal treatment in the last 6 months. For the in vitro assay, 12th generation larvae were used.
The bioassays were performed using the larval immersion test (LIT) method (Shaw, 1966), as adapted by Leite (1988) and Chagas et al. (2002). For each concentration, approximately 100 non-fed 35-day-old larvae of A. sculptum were deposited on a 2 cm x 2 cm filter paper sandwich, which was impregnated with 0.5 mL of the test solution. The filter paper sandwich was wrapped in a filter paper envelope (6 cm x 6 cm) that was then properly sealed with binder clips. The envelopes were kept in a climatized chamber at 27 ± 1 °C and relative humidity of 80 ± 10%. The mortality assessment was performed after 24 hours for organophosphates, phenylpyrazole and synthetic pyrethroids and after 48 hours for macrocyclic lactones (FAO, 2004). The evaluation criterion used was motility, i.e. any larva that presented minimal movement was considered alive. The mean number of live larvae per concentration was evaluated with the aid of a stereomicroscope. The tests were performed in duplicate for each concentration. Mortality was calculated in accordance with the following formula proposed by Abbott (1925): Mortality (%) = dead larvae x 100 / total larvae.

Statistical analysis
The Probit analysis method was used to assess LC 50 and LC 90 lethal concentration values together with their 95% confidence interval (95% CI) (μg.mL -1 ) and the slope ± SE of the concentration curve, with the χ 2 test to determine the accuracy of data fitting. The goodness-of-fit test showed that the values did not present any significant heterogeneity at the level of p ≥ 0.05. The Probit analysis estimates were calculated using the IBM SPSS statistical software, version 23.

Mortality
The bioassay results showed that all the active ingredients tested exhibited acaricidal activity against A. sculptum larvae ( Table 2). The best efficacy results were found for the synthetic pyrethroid group, which achieved 100% efficacy at the concentrations of 0.625 μg.mL -1 , 5 μg.mL -1 and 20 μg.mL -1 , for flumethrin, deltamethrin and cypermethrin respectively. Organophosphates and phenylpyrazole also presented good results with 100% efficacy at 500 μg.mL -1 , 75 μg.mL -1 and 60 μg.mL -1 , for chlorpyrifos, coumaphos and fipronil respectively. Ivermectin achieved 100% efficacy at the highest concentration (5500 μg.mL -1 ). The LC 50 and LC 90 values for the active ingredients evaluated, against A. sculptum larvae, are described in Figure 1. The LC 50 ranged from 0.06 μg.mL -1 to 187.275 μg.mL -1 , while the LC 90 ranged from 0.216 μg.mL -1 to 711.084 μg.mL -1 . The linear regression coefficient (r) was higher than 0.85 and the p-value of the goodness-of-fit test was higher than 0.05 for all the active ingredients evaluated. This demonstrated through the Probit analysis that the data fitting was accurate, without any significant heterogeneity ( Table 3).

Discussion
The scarcity of studies for the control of A. sculptum is perhaps one of the reasons that in the last 30 years few products have been launched with instructions for use in horses, with the use of off-label products in these animals being common. Labruna et al. (2004) reported that only a few pyrethroid formulations would be available for the control of A. cajennense in horses.
Ticks used in both studies (ours and Bittencourt et al., 1989) obtained their colonies from specimens collected from horses in the UFRRJ herd. After 30 years, the LC 50 levels for flumethrin and deltamethrin do not increase, indicating that the susceptibility of this population over time has not undergone significant changes in sensitivity. A possible explanation for this fact in Brazil would be the use of fipronil off-label, since its commercial presentation is intended for use in cattle.
Alonso-Díaz et al. (2013), carried out several in vitro studies in Mexico to determine the levels of susceptibility of populations of A. cajennense to different synthetic acaricides, observing high frequencies of resistance to organophosphates and amitraz. The authors highlight the need for more in vitro studies to be carried out to monitor susceptibility levels.
Despite the relevance that this parasite has for horses and human's health, there is still a lack of in vitro and in vivo studies to evaluate synthetic acaricides that can be used in the control of horse ticks. The determination of the acaricidal action of the active ingredients used in the present study against A. sculptum is relevant, as it indicates that all of them presented efficacy levels, however it is emphasized that the results found do not allow us to state that they can be used to control this tick.
The study was evaluated with non-fed larvae and as stated by Bittencourt et al. (1989), this stage is the one that presents the greatest susceptibility to pyrethroids. Other studies should be conducted with the other stages (not fed and fed) in order to be able to affirm that an active ingredient has high potential to be used in the control of A. sculptum. The bioassays with A. sculptum larvae can serve as initial tests for the selection of promising active ingredients, as well as for the monitoring of resistant populations.

Conclusion
The synthetic pyrethroids deltamethrin, cypermethrin and flumethrin and the phenylpyrazole fipronil demonstrated high larvicidal efficacy in in vitro assays against the larvae of Amblyomma sculptum.