Introduction

The Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) cattle tick is a blood-sucking ectoparasite that occurs in tropical and subtropical regions (^{PEREIRA et al., 2008}). This species has caused economic losses to the world livestock, inducing intense irritation of the animals, leather depreciation, decreased weight gain, decreased production of meat and milk, and transmitting Babesia spp. and Anaplasma spp. (^{GOMES 1998}; ^{FURLONG, 2005}; ^{RECK et al., 2014}). In Brazil, these losses exceed 3 billion dollars annually (^{GRISI et al., 2014}).

The most widely used control method is the use of synthetic chemical agents. However, chemical control can lead to selected populations of resistant ticks (^{GUERRERO et al., 2012}) and are costly and contaminate the environment with residues harmful to the hosts and also humans (^{FREITAS et al., 2005}). Therefore, new approaches are needed, and natural products are potential candidates for acaricidal drugs. These compounds generally have low environmental impact and can lead to slower tick resistance (^{BORGES et al., 2003}; ^{HU & COATS, 2008}).

Lippia sidoides is a Verbenaceae plant known in Brazil as “alecrim-pimenta”. Despite the occurrence in northeastern of Brazil, it is now cultivated in several Brazilian states due to its herbal characteristics (^{MATOS & OLIVEIRA, 1998}). L. sidoides leaves are approximately constituted with 4% essential oil with carvacrol and thymol as major constituents, depending on the evaluated germplasm (^{LORENZI & MATOS, 2008}; ^{SANTOS et al., 2015}). The essential oils of L. sidoides have bactericidal, fungicidal and molluscicidal properties which are generally attributed to the major components (^{MATOS, 2000}, ²⁰⁰²; ^{CARVALHO et al., 2003}; ^{BOTELHO et al., 2007}).

In addition to the activities mentioned above, the acaricide activity of the essential oil of L. sidoides with high concentration of thymol and carvacrol has been used against Tetranychus urticae (^{CAVALCANTI et al., 2010}), R. (B.) microplus, R. sanguineus, Amblyomma cajennense and Dermacentor nitens (^{GOMES et al., 2012}, ²⁰¹⁴). However, different genotypes of plants show differences in their chemical composition and thus different bioactivities (^{CRUZ et al., 2013}; ^{COSTA-JÚNIOR et al., 2016}). Therefore, the aim of this study was to evaluate the activity of the essential oil of different germplasms of L. sidoides plants against larvae and females of R. (B.) microplus.

Materials and Methods

Results

Twenty six components of the L. sidoides essential oil were identified (Table 2). The LSID006, LSID102, LSID103, and the LSID104 genotypes presented, respectively, 21, 14, 19 and 16 compounds. The most abundant chemical compound in LSID006, LSID102, and LSID103 was thymol (54.4%; 38.7%; 64.8%, respectively), and the most abundant compound in LSID104 was the thymol isomer, carvacrol (43.7%).

Lippia sidoides oils showed efficacy against larvae and engorged female ticks (Table 3). The LSID104 genotype, which uniquely presented carvacrol as its major constituent, had one of the worst larvicide effects. LSID006 had the highest larvicide effect. LSID103 had the highest amounts of thymol and presented a lower larvicide activity than LSID006. The LSID103 and LSID006 are in different clusters based on acaricidal activity (Figure 1).

Figure 1 Clustering of Lippia sidoides genotypes based on acaricidal activity with the Euclidean distances. 

LSID102 presented higher efficacy against engorged females (LC50 = 2.81 mg/mL) (Table 3). Similar to the results with larvae, no direct relationship between clustering analysis was found based on the chemical constituents and the acaricidal effect because LSID006 was the least effective compound against engorged females and is chemically similar to the LSID102 genotype (Figure 1). Thymol acetate is present only in LSID102 and LSID103 (Table 2), the two genotypes with the largest acaricidal efficacy against engorged females (Table 3), which could indicate the possibility of a synergistic effect of this compound.

Clustering analysis of L. sidoides genotypes based on acaricidal activity showed that LSID102 and LSID103 are closely related (Figure 1). These genotypes were the most effective against engorged females (Table 3). Both genotypes are more closely related to thymol than carvacrol, although all of them represent the same cluster (Figure 1).

Discussion

The difficulties in preparing proper formulations, differences in the chemical composition of plants of the same species due to extrinsic and intrinsic factors and differences on the activity of the formulations from the same vegetal species are hindrances that need to be addressed in order to enable progress to transposing the efficacy obtained from the laboratory to the field (^{BORGES et al., 2011}). This study selected L. sidoides genotypes with highest efficacies on R. (B.) microplus advancing knowledge for the standardization of a compound.

The susceptibility to acaricidal compounds is related to the life stages of the tick, as well as the physiological process involving blood feeding. In general, the immature stages of ticks seem to be more susceptible to synthetic acaricide effects than others stages (^{PINHEIRO, 1987}). The essential oil of L. sidoides was most efficient against larvae than nymphs of R. sanguineus and A. cajennense (^{GOMES et al., 2014}). However the animals are always infested by ticks at different life stages and the best compound should be effective against all of them. In the present study a cluster analysis was performed to select L. sidoides genotypes with the highest efficacies against R. (B.) microplus larvae and engorged female.

Several phytochemical studies demonstrated the presence of thymol as the major compound of L. sidoides (^{CAVALCANTI et al., 2010}; ^{VERAS et al., 2012}; ^{GOMES et al., 2014}). The exception observed in LSID104 (^{CAVALCANTI et al., 2010}) could be a result of the chemical distance explained by the clustering analysis of L. sidoides genotypes, where LSID104 was grouped alone and the other genotypes showed considerable chemical similarity (^{SANTOS et al., 2015}).

Carvacrol was the most efficient compound against larvae mainly organophosphate resistant strain larvae (^{CRUZ et al., 2013}; COSTA-JUNIOR et al., 2016), the essential oil that presented as its major constituent, had one of the worst larvicide effects. This result suggests that despite carvacrol alone having elevated larvicide action, it is not the main bioactive compound, and the different oil constitutions play an important role in the acaricidal action. We hypothesize that the high levels of thymol (54.4%) in LSID006 are responsible for this action, but LSID103 has higher amounts of thymol and presented a lower larvicide activity because LSID103 and LSID006 are in the same cluster based on chemical constituents (^{SANTOS et al., 2015}) but are in different clusters based on acaricidal activity (Figure 1). β-bisabolene is present only in LSID103 and could have antagonistic activity. Additionally, some compounds exist only in LSID006 (β-pinene; 1,8 cineole; ipsdienol; aromadrendene; β-selinene) and could contribute to the elevated larvicide action of this genotype. The synergistic effects of β-pinene, 1,8 cineole and aromadendrene have recently been reported (^{MULYANINGSIH et al., 2010}; ^{RODENAK et al., 2014}; ^{ZHANG et al., 2015}), but to the best of our knowledge, the minor compounds of L. sidoides essential oil have not been studied to evaluate their synergistic capabilities in R. (B.) microplus.

The synergy studies conducted in Lippia spp. corroborate our results. The activity of the essential oil of L. sidoides and L. gracilis and chemical components against the fungus Thielaviopsis paradoxa recently have been reported. The compounds p-cymene, 1.8 cineole, α-terpinene and β-caryophyllene had no efficiency when tested alone, and the authors proposed that they act in synergy with other compounds because the thymol concentration required to control the fungus was higher than the concentration of the L. sidoides essential oil (^{CARVALHO et al., 2013}).

Different plant genotypes can present distinct essential oil profiles (^{GIL et al., 2002}; ^{DRAGLAND et al., 2005}; ^{PEIXOTO et al., 2015}). In this context, it is important to study the relation between these composition variations and the interference in the bioactivity. For example, LSID102 and LSID104 presented similar leishmanicidal activity (Concentration that inhibits 50% - IC₅₀ = 74.1 and 54.8 μg/mL, respectively) (^{FARIAS-JUNIOR et al., 2012}), although both genotypes are in different clusters based on their chemical constitution (^{SANTOS et al., 2015}). In addition, no significant differences were observed in the acaricidal activity of the essential oils of different L. sidoides genotypes against T. urticae, and after acaricidal analysis with selected compounds it was suggested that the evaluated components act synergistically to achieve the acaricidal effect (^{CAVALCANTI et al., 2010}).

The bioactivity of thymol has been reported against ticks and insects (^{NOVELINO et al., 2007}; ^{WALIWITIYA et al., 2010}; ^{CRUZ et al., 2013}), and the larvicide activity of L. sidoides essential oil against Aedes aegypti was attributed to this monoterpene (^{CARVALHO et al., 2003}). The LSID102 and LSID103 genotypes were the most effective against engorged females (Table 3). Both genotypes are more closely related to thymol than carvacrol, although all of them represent the same cluster (Figure 1). The presence of thymol in these genotypes is associated with the activity against engorged females.

Although the genotypes with higher efficiency against larvae (LSID006) and engorged females (LSID102) have thymol as their major constituent, which could be an indicative that this monoterpene is involved in the acaricidal effect, the general chemical balance among the essential oil compounds leads to different acaricidal effects. Both genotypes are in different clusters based on acaricidal activity (Figure 1), which suggests that there could be different action modes of these essential oils during different life stages of the R. (B.) microplus. Although the activity of L. sidoides essential oil has been described on R. (B.) microplus, to our knowledge, this is the first evaluation of the relationship among the activity of different genotypes of L. sidoides and the acaricide effect.

Conclusions

The results indicated that the chemical differences in the L. sidoides genotypes influence the acaricidal activity against R. (B.) microplus. In addition, the clustering analysis of L. sidoides genotypes based on acaricidal activity suggests that the essential oils have different modes of action in larvae and in engorged females. We conclude that the different constitutions of the essential oils, as well as the relationships among the compounds, play important roles in the acaricidal action. However, further studies are needed to verify the global acaricidal effects of the minor compounds of L. sidoides essential oil. The findings of this work facilitate the understanding and the development of innovative strategies aimed to control the cattle tick R. (B.) microplus.