Assessment of different Lippia sidoides genotypes regarding their acaricidal activity against Rhipicephalus ( Boophilus ) microplus

The aim of this study was to select different genotypes of Lippia sidoides with the highest activity against larvae and engorged females of the tick Rhipicephalus ( Boophilus ) microplus . The germplasms studied were LSID006, LSID102, LSID103 and LSID104. The LSID104 genotype, that presented carvacrol as a major constituent, was the germplasm with the worst larvicide effect (LC 50 2.99 mg/mL). The LSID006 genotype was the least effective against engorged females (LC 50 12.46 mg/mL)

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(MATOS, , 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(GOMES et al., , 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.

Essential oil
Leaves of L. sidoides were collected from the active Germplasm Bank of Medicinal Plants that was established with L. sidoides plants from different geographical locations (Table 1) at the research farm of the Federal University of Sergipe, Brazil (SANTOS et al., 2015).The harvests of all genotype were performed at the same time.After manual defoliation, the leaves were dried in a forced air circulation oven for five days at 40 °C.
The essential oils were extracted by hydrodistillation in a Clevenger apparatus for 140 minutes.Each sample consisted of 75 g of dried leaves from four plants.The essential oils extraction, as well the determination and analysis of their chemical composition, were conducted according Santos et al. (2015).
Briefly, the chemical composition of the essential oils was determined using a gas chromatograph coupled to a mass spectrometer equipped with an AOC-20i auto injector and a fused-silica capillary column.Quantitative analyses were performed by flame ionization gas chromatography (FID).The essential oil components were identified by comparing their mass spectra with the available spectra in the equipment database (NIST05 and WILEY8).Finally, the measured retention indices were compared with those in the literature (ADAMS, 2007), and the retention times (RT) were determined using the Van Den Dool & Kratz (1963) equation and a homologous series of n-alkanes (C 8 -C 18 ).

Obtaining the larvae and the engorged females
The larvae and engorged females of R. (B.) microplus ticks used in this work were obtained from colonies maintained at the Biological and Health Science Center of the Federal University of Maranhão (UFMA), Brazil.This study was approved by the Ethics Committee on Animal Use of UFMA under protocol 23115018061/2011-01.Larvae between 14 and 21 days after hatching were used in the Larval Packet Test.Adult engorged female ticks (≥ 4.5 mm in length) were collected from the bodies of artificially infested cattle.

Larval packet test
The larval packet test was performed according to Stone & Haydock (1962) and modified by FAO (1984) and Leite (1988), as described below.Two sheets of filter paper (4 cm 2 ) (Whatman 80 g) were treated with 400 μL of solution containing 3% dimethyl sulfoxide (DMSO) and essential oil.Twelve concentrations, ranging from 0.0612 to 15.00 mg/mL of essential oil isolated from each of the four L. sidoides genotypes, were used for the test.Ten concentrations ranging from 0.0612 to 25.00 mg/mL of thymol (Merck) and carvacrol (Sigma-Aldrich) were performed, tested as published before (CRUZ et al., 2013).
Approximately 100 tick larvae were placed in filter papers folded to form a packet and sealed with a plastic clothespin.The packet was placed in an incubator (27 °C and relative humidity ≥ 80%) for 24 hours.After this time, alive and dead larvae were counted.Ticks that did not move were considered dead.The experiment was performed with four replicates for each treatment.Furthermore, a solution of 3% DMSO was used as the negative control.

Adult immersion test
The adult immersion test was performed as described by Drummond et al. (1973).The adult immersion test shows the activity on mortality as well as the interference in reproduction, by evaluation of oviposition and eggs hatching.Engorged female cattle ticks were collected from artificially infested calves.Groups of ten engorged female ticks were weighed to obtain groups with weights ranging from 2.24 to 2.32 g.
Each tick group was dipped in one of twelve concentrations ranging from 0.0612 to 25.00 mg/mL of essential oil isolated from one of the four L. sidoides genotypes, using 3% DMSO as solvent for five minutes.Ten concentrations ranging from 1.00 to 25.00 mg/mL of thymol (Merck) and carvacrol (Sigma-Aldrich) were performed, tested and published before (CRUZ et al., 2013).DMSO (3%) was used for the negative control group.The engorged females were subsequently dried on a paper towel, placed in Petri dishes and maintained in a biochemical oxygen demand (BOD) incubator at 27 ± 1 °C and relative humidity ≥ 80% for 35 days to allow oviposition and hatching of the larvae.The eggs mass were weighed and the hatching was evaluated.The efficacy was calculated according Drummond et al. (1973).

Statistical analysis
Lethal concentrations were calculated using GraphPad Prism 6.0.Significant differences between the average efficiency of each pair of essential oil and/or monoterpene were considered when there was no overlap between the 95% confidence limits of the LC 50 values (RODITAKIS et al., 2005).The data of the acaricidal activity (on larvae and engorged female) of the essential oils from each genotype were submitted to cluster analysis using DataLab 3.5 software.The dissimilarity matrix was simplified with dendrograms using Ward's clustering method.The dendrograms were drawn using the PHY FY website (FREDSLUND, 2006).
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).
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).

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

Table 1 .
Identification and geographical origin of the Lippia sidoides genotypes used in the present study.

Table 2 .
Essential oil composition (%) from Lippia sidoides genotypes characterized by gas chromatography associated with a mass spectrophotometer.
Cruz et al. (2013)up and published inCruz et al. (2013).Different letters represent significant differences among the essential oils or monoterpenes (p ˃ 0.05).