In vitro evaluation of the acaricidal activity of Syzygium aromaticum (L.) essential oil and eugenol against non-fed larvae of Rhipicephalus sanguineus Avaliação in vitro da atividade acaricida do óleo essencial de Syzygium aromaticum (L.) e eugenol contra larvas de Rhipicephalus sanguineus não alimentadas

Copyright Lambert et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution and reproduction in any medium provided the original work is properly cited. In vitro evaluation of the acaricidal activity of Syzygium aromaticum (L.) essential oil and eugenol against non-fed larvae of Rhipicephalus sanguineus

These ticks are difficult to control, due to their heterogeneous life cycle, since only about 5% of the total population of R. sanguineus is on animals, while the remainder is in the environment. Controlling these ticks is usually done using conventional acaricides based on chemicals, which are applied to the environment and/or to dogs (Dantas-Torres, 2008). Over the years, many ticks have managed to survive treatments and there have been reports that some ectoparasiticides, such as pyrethroids, amitraz and organophosphates, are no longer as effective for controlling R. sanguineus as they used to be (Eiden et al., 2015;Rodriguez-Vivas et al., 2017).
Plant-based products with ectoparasiticidal activity have shown significant results in vitro over recent years (Ellse & Wall, 2014). The diversity of these plants, combined with their properties of biodegradability, low cost of production, low toxicity and low environmental waste, confirms the potential for use of these parasite control products (Barreto et al., 2006;Chagas et al., 2012).
Syzygium aromaticum (L.) Merr. & L.M. Perry (Myrtaceae), is popularly known as clove. Eugenol (EG) is the major compound in its essential oil and can reach a proportion of up to 95% (Mbaveng & Kuete, 2017). Both the essential oil (EO) of Syzygium aromaticum (EOSA) and EG have already been evaluated regarding their insecticidal activity (Bagavan et al., 2011;Jairoce et al., 2016) and also regarding their pulicidal activity (Lambert et al., 2020). There is a lack of reports about the use of EOSA against R. sanguineus in the literature. However, the acaricidal activity of EOSA and EG and of other EOs containing EG as the major component has already been described (Araújo et al., 2016;Ferreira et al., 2019).
Therefore, the aim of this study was to evaluate the in vitro activity of EOSA and EG against R. sanguineus larvae.

Material and methods
Essential oils from the flower buds of S. aromaticum (Kitano®, São Paulo, Brazil) were obtained by means of hydrodistillation in a Clevenger apparatus for 3 h and they were dried over anhydrous Na 2 SO 4 . And chemical characterization was performed through gas chromatography (GC-FID and GC-MS) analysis, either proceedings described by Lambert et al. (2020). Technical-grade eugenol (99%) was purchased from Sigma-Aldrich®, code E51791 (St. Louis, USA).
The experiments followed the standards established by the Ethics Committee for Animal Use (CEUA) of the Veterinary Institute of the Federal Rural University of Rio de Janeiro (UFFRJ), under the approval number CEUA/IV 090/14. The larvae of R. sanguineus used in the experiment were obtained from colonies that were maintained in rabbits in the Laboratory for Experimental Chemotherapy in Veterinary Parasitology of UFRRJ.
To carry out bioassays, stock solutions of EG and EOSA were prepared at a concentration of 40 mg.mL -1 using as diluent a solution containing 20% of acetone (Synth) and 3% of Tween-80 (Vetec) in distilled water. Serial dilutions (1:2) were performed from stock solutions, using the same diluent mentioned above, which gave rise to ten solutions over a concentration range from 0.0781 to 40 mg.mL -1 . The diluent was also used as negative control. To ensure viability of the colony, fipronil at 0.400 mg.mL -1 was used as a positive control.
The bioassays were performed using the larval immersion test (LIT) method adapted for Chagas et al. (2002). Approximately 100 non-fed 21 days old larvae of R. sanguineus were deposited on a 2 cm × 2 cm filter paper sandwich, which was impregnated with 0.5 mL of the solution under test. The filter paper sandwich was wrapped in a filter paper envelope (6 cm × 6 cm) properly sealed with binder clips. The envelopes were kept in a climatized chamber at 27 ± 1 °C and relative humidity of 80 ± 10% for 24 h for subsequent mortality assessment. The evaluation criterion used was motility, any larva that presented minimal movement was considered alive. The mean number of live larvae per concentration was evaluated in periods of mainly 24 hours with the help of a stereoscopic microscope. The tests were performed in duplicates for each concentration. Mortality was calculated according to the following formula: Data obtained for both EG and EOSA were submitted to Probit analysis to estimate Lethal Concentrations (LC 50 and LC 90 ) using the IBM SPSS Statistics program, version 26.0 April, 2019. The evaluation of the relative potency of EOSA and eugenol activity on R. sanguineus was also calculated out in the same statistics program. All analyzes were calculated considering the 95% confidence interval (p ≤ 0.05).
The mortality results from non-fed larvae of R. sanguineus that were observed for EOSA and EG are shown in Table 1. EOSA showed increasing acaricide activity starting from the concentration of 1.25 mg.mL -1 , and reached approximately 90% mortality at 5 mg.mL -1 and 100% at 40 mg.mL -1 . The mortality results for EG did not shown linearity in the concentration range between 0.15 and 2.5 mg.mL -1 , with rates ranging from 11.49% to 32.54%. There was a substantial increase in mortality from 2.5 to 5.0 mg.mL -1 , to reach 86.78% at the latter concentration. Linear increases were observed thereafter, until reaching mortality of 99.42% at the highest concentration tested (40 mg.mL -1 ). In both tests, no mortality was observed in the negative controls (0%), while in the positive control mortality was higher than 98%, thus demonstrating that the method was applied correctly. Table 1. Percentage mortality of non-fed larvae of Rhipicephalus sanguineus exposed to the essential oil of Syzygium aromaticum and eugenol. The Probit analysis on the acaricidal activity for EOSA and EG is described in Table 2. The LC 50 and LC 90 estimates found were 3.29 and 6.74 mg.mL -1 and 1.42 and 6.39 mg.mL -1 for EOSA and EG, respectively. Pearson correlation coefficient (r) values were calculated for both assays (EOSA and EG) and were close to 1 (r > 0.9). This indicated that there was a strong linear correlation over the concentration range tested. EOSA showed higher slope values (4.127) than EG (1.965), thus showing greater sensitivity. However, the analysis on relative potency for LC 90 (1.05) showed that there was no significant difference between EG and EOSA, regarding activity against these non-fed larvae of R. sanguineus.

Discussion
Eugenol is the chemotype of S. aromaticum and it shows several properties such as pulicidal and insecticidal activity (Lambert et al., 2020). However, the activity of EOSA against R. sanguineus has not yet been reported. The activity of EOSA and EG against R. microplus larvae was described by Ferreira et al. (2018), who showed that they caused 100% mortality at concentrations of 2.5 mg.mL -1 and 5 mg.mL -1 for EG and EOSA, respectively. Our results demonstrated that higher concentrations (10, 20 and 40 mg.mL -1 ) were necessary to achieve R. sanguineus larvae mortality above 95% for EOSA and EG.
The difference in mortality rates obtained can be explained in terms of differences in susceptibility between tick species. Greater susceptibility of R. microplus than of R. sanguineus has already been shown by Monteiro et al. (2012) and Araújo et al. (2016) to EG, and Ferreira et al. (2019) to Ocimum gratissimum EO, whose major compound is EG.
The LC 50 values ranged from 1.7 mg.mL -1 (Valente et al., 2014) to 2.77 mg.mL -1  and 4.76 mg.mL -1 (Araújo et al., 2016). Mortality above 99% was reached at higher concentrations for R. sanguineus than for R. microplus (Araújo et al., 2016;Coelho et al., 2020;Senra et al., 2013). We found mortality above 99% at a higher concentration (40 mg.mL -1 ) than what was reported by Senra et al. (2013) (10 mg.mL -1 ) and Coelho et al. (2020) (20 mg.mL -1 ). On the other hand, our LC 50 values were lower (1.42 mg.mL -1 ) than those reported by Araújo et al. (2016) (5.19 mg.mL -1 ). Although EG is the major compound of EOSA, we did not find any significant superiority of EG activity against non-fed larvae of R. sanguineus, compared with EOSA, thus corroborating the findings of Ferreira et al. (2018) in relation to R. microplus. The activity of essential oils on arthropods is generally related to the major compound, however there is evidence that oil compounds may have synergistic activities when together, this fact is described by Ellse and Wall (2014).

Conclusion
The essential oil of S. aromaticum and eugenol showed larvicidal efficacy on R. sanguineus larvae in vitro. Even though in vivo efficacy and safety assessments are still needed, these findings are promising for development of products to control these ticks. Table 2. Probit analysis on Rhipicephalus sanguineus larvae exposed to different concentrations of the essential oil of Syzygium aromaticum and eugenol after 24 hours: LC 50 (mg.mL -1 ), LC 90 (mg.mL -1 ), R 2 and slope. Probit analyses were performed for all data using IBM SPSS Statistics software (version 26.0 April, 2019); LC 50 (mg. mL-1) (95% CI): 50% lethal concentration values followed with their 95% confidence interval; LC 90 (mg.mL -1 ) (95% CI): 90% lethal concentration values followed with their 95% confidence interval; R: Pearson correlation coefficient; Slope (SE): slope of the concentration curve and Standard Error.