Baccharis dracunculifolia DC (Asteraceae) leaf and flower essential oils to control Rhipicephalus microplus Canestrini (Arachnida: Ixodidae) in the free-living stage

Baccharis dracunculifolia, native to Brazil and the main source of “green propolis”, has been reported with several biological activities, and may be a source of bovine tick control substituting synthetic acaricides. Objective: to evaluate the in vitro and ex situ acaricidal activity of B. dracunculifolia leaf and flower essential oils against Rhipicephalus microplus. Methodology: the essential oils were extracted by hydrodistillation and analyzed by a gas chromatography coupled to mass spectrometry; the acaricidal activity of the essential oil was evaluated in vitro against adult females and against the egg hatchability; moreover, the Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788 3 acaricidal activity against tick larvae was evaluated in vitro and ex situ. Results: the major class of the essential oils was oxygenated sesquiterpene (55.1% leaves 50.4% flowers) and the main compounds were (21.5% leaves; 20.6% flowers) and spathulenol (21.8% leaves; 20.3% flowers). The essential oil at 500 mg/mL was effective to control egg hatchability with a reduction of egg laying capacity and decrease of number of adult ticks and larvae. The larvicidal activity of the essential oil had LC99.9 from 35 to 37 mg/mL by probit analysis, and the essential oil from 11 to 14 mg/mL presented 85 to 95% of treatment efficiency in the ex situ test. Conclusion: B. dracunculifolia leaf and flower essential oils are stable and have application potential to control bovine ticks.

(Meliaceae), among others (Callejon et al., 2016). Other secondary metabolites from plants such as extracts, essential oils and isolated compounds have shown promising biological activities (Ribeiro et al., 2010;Bispo, Almeida, & Nunes, 2020) such as the sesquiterpene nerolidol, a major compound found in B. dracunculifolia essential oil to control bovine tick .
Considering that natural compounds from plants are still of interest to control bovine tick and to reduce tick resistance to synthetic acaricides, and that there have been no studies on the acaricidal activity of B. dracunculifolia essential oil in ex situ conditions, this study aimed to evaluate the chemical composition and in vitro and ex situ acaricidal activity of B. dracunculifolia leaf and flower essential oil against R. microplus. Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.

Essential oil extraction and chemical composition
Baccharis dracunculifolia leaf or flower essential oil was extracted separately by hydrodistillation in a Clevenger apparatus for 2 h (Miranda, Cardoso, Batista, Rodrigues, & Figueiredo, 2016). At the end of distillation, the essential oil was removed from the apparatus, transferred to amber vials, and stored at -20 °C (Pereira, Costa, Liporoni, Rego, & Jorge, 2016).
The essential oil chemical identification was carried out by a gas chromatographer (Agilent 7890B) coupled to a mass spectrometer (Agilent 5977A MSD) and a HP5-MS UI column (Agilent fused silica capillary of 30 m × 250 µm × 0.25 µm; Agilent Technologies), with initial oven temperature from 40 °C (2 min) to 230 ºC (3 ºC/min), and kept at this temperature for 20 min. Helium was utilized as the carrier gas at the linear speed of 1 mL/min up to 300 °C, and pressure release of 56 kPa. The injector temperature was 250 ºC; the injection volume was 1 µL; the injection occurred in split mode (20:1). Temperatures of the transfer line, ion source, and quadrupole were 280, 230, and 150 ºC, respectively. The mass spectrometry detection system was utilized in "scan" mode at the mass/charge rate/load (m/z) of 40-600 with "solvent delay" of 3 min. The compounds were identified by comparing them to mass spectra found in Wiley 275 libraries and by comparing the retention indices (RI) obtained by a homologous series of n-alkane standard (C7-C28) (Adams, 2017). Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788 7

Adult immersion test
The adult immersion test (AIT) was performed according to Drummond, Ernst, Trevino, Gladney and Graham (1973). Engorged female adult ticks (900) from dairy cattle of Northeastern region of Paraná state, Brazil, which had not been exposed to acaricides for 60 days, were utilized. The ticks were washed with ultrapure water and selected according to their healthy appearance, body integrity, and full engorgement (Leite, Labruna, Oliveira, Monteiro & Caetano Junior, 1995).
Groups of 30 engorged female ticks had body mass measured and immersed for 5 min, at 28 ºC, in 10 mL essential oil suspension, synthetic solution, or 2% polysorbate-80 emulsion, and then transferred to Petri dishes (10 ticks per plate) in a chamber at 28 °C with 80% relative humidity for 14 days until oviposition. After 14 days, the egg mass of each female tick was recorded, placed in assay tubes, and kept at 28 °C in a chamber with 80% relative humidity for 21 days until hatching. After 21 days, the larvae were killed by immersion in sulfuric ether and counted in order to obtain the hatching rate. All the tests were performed in triplicate. The estimated reproduction (ER) and the product efficacy (PE) were calculated by the tick mass of engorged adult females, eggs, and egg hatching rate, according to Equations 1 and 2 (Drummond, Ernst, Trevino, Gladney, & Graham, 1973

Larval packet test
Engorged adult female ticks without previous treatment with acaricides were kept in a controlled environment to produce larvae. The obtained larvae were placed in a closed paper Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788 8 filter envelope (2 × 2 cm) impregnated with essential oil, positive or negative control solutions according to the larval packet test (LPT) (Fernandes, Bessa, & Freitas, 2008;Chagas et al., 2012). The essential oil was applied at final concentrations of 50.00, 25.00, 12.50, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09, 0.04, and 0.02 mg/mL. The positive and negative controls were the same ones utilized in AIT. The filter paper containing larvae was kept in a Petri dish in a chamber at 28 °C and after 24 h the living larvae were separated from the dead ones (Leite, Labruna, Oliveira, Monteiro & Caetano Junior, 1995). The treatments were carried out in triplicate and the larval mortality was determined according to Equation 3. Mortality (%) = dead larvae / total larvae × 100 (Equation 3) All the tests were done in triplicate. The essential oil with lethal concentration to kill 99.9% (LC99.9) of larvae was utilized for the ex situ test of tick control in vases in a protected environment.

Ex situ test (free-living stage)
Plastic vases (n = 9), 25 cm height and 25 cm diameter, were filled up with 2.2 kg soil, After 24 h, the larval migration to the apex of the grass leaves was observed (Araújo et al., 2015). Each treatment consisted of a group of three vases. The obtained LC99.9 in LPT (in vitro test) for the essential oils and positive control were used for the treated and control group.
A 1.25 mL/L Colosso® commercial solution (150.00 mg/mL cypermethrin, 250.00 mg/mL chlorpyrifos, and 10.00 mg/mL citronellal) was utilized as positive control and 2% polysorbate-80 (mass: volume) emulsion was utilized as negative control in the same LPT and AIT concentrations utilized before. For each treatment, 4 mL of each solution per vase was sprayed starting from the leaf apex until the soil in order to simulate l commercial applications of acaricide in pastures for the herd. After 24 h, the grass leaves were trimmed with the help of an entomological lens and larvae without movement after touching were considered dead. Next, the number of living larvae in the negative group, and living larvae in Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788 9 the treated group with the essential oils, or with positive control Colosso® were determined.
From these data, the efficacy of treatments of essential oils were calculated by the Equation 4 and according to Bittencourt, Bahiense, Fernandes and Souza (2003).
Efficacy of treatment of essential oil (%) = (A -B) / A × 100 (Equation 4) where A = number of living larvae in the negative control group and B = number of living larvae in the test or control group.

Statistical analysis
The experiment, a quantitative study (Pereira, Shitsuka, Parreira, & Shitsuka, 2018), had a completely random design. The data were submitted to analysis of variance (ANOVA) and the differences among arithmetic averages with standard deviations were determined by Scott-Knott test at 5% significance. The lethal concentrations that killed 50% (LC50) and 99.9% (LC99.9) of adult and larvae ticks with the respective confidence interval (CI; = 0.05) were calculated by probit analysis (ED 50 Plus version 1.0). All the tests were carried out in triplicate.

Results
Forty-eight compounds were identified in B. dracunculifolia leaf and flower essential oil such as oxygenated sesquiterpenes with 55.1% (leaf) and 50.4% (flower), hydrocarbon sesquiterpenes with 28.6% (leaf) and 29.9% (flower), and hydrocarbon monoterpenes with 10.9% (leaf) and 14.9% (flower) ( Table 1). The major compounds of the essential oil were nerolidol with 21.5% (leaf) and 20.6% (flower), and spathulenol with 21.8% (leaf) and 20.3% (flower) with low variation between the amount of each compound for the leaf and the flower essential oil (Table 1 and Figure 1 and 2).
In addition, there is a reduction of the tick oviposition from 38 to 80% for leaf essential oil from 400 to 500 mg/mL and reduction from 27 to 74% for flower essential oil from 400 to 500 mg/mL ( Table 2). The leaf essential oil from 300 to 400 mg/mL has ER values of 73 and 81% ( Figure 3); for the flower essential oil the ER was of 38 and 63%, respectively ( Figure 3). It suggests that the leaf essential oil is more efficient at a lower concentration than the flower essential oil. The leaf essential oil compared to the flower essential oil had slightly higher amounts of each chemical compound such as nerolidol, spathulenol, and δ-cadinene that might be responsible for a more efficient activity. The leaf essential oil at 400 mg/mL had PE greater than 80%, but for the flower essential oil the PE was 64% against adult female ticks (Figure 4). The adult female tick mortality was more effective at lower concentrations of leaf essential oil compared to flower essential oil. Thus, B. dracunculifolia essential oil should be used at the concentration of 500 mg/mL to be considered an acaricidal product with efficiency over 95% according to the Brazilian Ministry of Agriculture (Brasil, 1997). Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788  The larvicidal activity of 95% calculated for B. dracunculifolia essential oils (probit analysis) were effective at 13.36 mg/mL of leaf essential oil and 31.06 mg/mL of flower essential oil ( Figure 5). The acaricidal activity of essential oils was more effective against larvae and at a lower concentration than against adults. Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788  For tick larvae, it was determined by probit analysis that the essential oils had LC50 ranging from 10.5 to 13.5 mg/mL and LC99.9 from 34.6 to 36.6 mg/mL (Table 3). The greatest efficiency at the lowest concentration with larvicidal activity was found for B. dracunculifolia leaf essential oil with LC50 of 10.5 mg/mL and LC99.9 of 34.6 mg/mL (Table 3). Baccharis dracunculifolia leaf and flower essential oils had treatment efficacy from 85 to 95% for the ex situ test in a protected environment against tick larvae (Table 4). The leaf essential oil was 34.6 mg/mL and the flower essential oil was at 36.6 mg/mL according to probit analysis (Table 3). In the ex situ test, the flower essential oil was more effective than the leaf essential oil probably because of a better stability of this essential oil in adverse conditions.

Discussion
The major compounds of B. dracunculifolia leaf and flower essential oils in our study were mainly nerolidol and spathulenol, corroborating the literature on this plant (Massignani et al., 2009;Queiroga et al., 2014). In addition, Lage et al. (2015) reported that the essential oil from B. dracunculifolia fresh aerial parts (leaves) from Viçosa, Brazil, had nerolidol tick mortality rate was dosage-dependent. The acaricidal activity by AIT showed 7.8% hatchability and reduction in the amount and quality of produced eggs at 60 mg/mL essential oil. The modified acaricidal activity by LPT showed 99.6% larval mortality of the essential oil at 15 mg/mL. These results are in accordance to the ones found in our study because the highest activity of the leaf and flower essential oils occurred against bovine tick larvae. The susceptibility of adult bovine ticks seems to be substantially lower compared to larval bovine ticks submitted to essential oils (Castro et al., 2018). Bovine larvae are more vulnerable than adult females because their cuticle is thinner, allowing the active compounds to penetrate in the tick (El Amri et al., 2014). Moreover, the cuticle of engorged female ticks can increase from 32 to 43% in the feeding phase (Flynn and Kaufman, 2011); thus, the thinner cuticle in larvae could explain the higher acaricidal activity against ticks in the larval phase than in the adult phase (Chagas, Leite, Furlong, Prates, & Passos, 2003).
Baccharis dracunculifolia leaf essential oil in our study was more active to inhibit egg hatchability of ticks probably due to the difference in the chemical composition of the essential oils. The oxygenated monoterpenes and sesquiterpenes were found at greater concentrations in the leaf essential oil (4.6% and 55.1%, respectively) than in the flower essential oil (3.9% and 50.4%, respectively). Oxygenated sesquiterpenes have shown greater acaricidal potential than hydrocarbon sesquiterpenes because the presence of oxygenated functional groups in the molecule and the capacity to form hydrogen bindings can potentialize the biological activity of these compounds (Eldoksch, Ayad, & El-Sebae, 2009;Amaral et al., 2017). Gross, Temeyer, Day and Léon (2017) reported that oxygenated terpenes acted as agonists (pulegone) or modulators (piperonyl alcohol), (1,4-cineole, carvacrol and isoeugenol) of R. microplus tyramine receptors, causing anatomical alterations in the digestive tract and in tick mortality.
The emergence of resistant mite populations to acaricides has been increasing in all regions where the parasite finds favorable conditions to its development (Klafke et al., 2017;Reginato, Cadore, Menezes, Sangioni, & Vogel, 2017). In the field, after a maturation period on pasture, larvae move to leaf extremities to increase the chances to reach the livestock and complete its development cycle (Gonzales, 1974). This phase of the tick reproductive cycle corresponds to the free-living stage of larvae when 95% of the larvae are distribute in pasture and only 5% are parasites of the animal (Powell & Reid, 1982). In our study, the spraying of B. dracunculifolia leaf and flower essential oils killed 85.3% and 95.1% of R. microplus larvae on B. eminii leaves, simulating the conditions of free-living stage of the parasite in the larval phase. Moreover, when exposed to open environment conditions, the essential oils can Research, Society and Development, v. 9, n. 10, e5049108788, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i10.8788 20 volatize or oxidize, reducing the efficiency reported in in vitro tests to control ticks (Borges, Souza, & Barbosa, 2011). Therefore, the results in our study suggest that the flower essential oil instead of the leaf one is more stable under adverse conditions of the ex situ test and, thus, has greater utilization potential.

Final Considerations
Baccharis dracunculifolia leaf and flower essential oils have potential to control R. microplus in two important life cycles of ticks, reducing their egg hatchability and killing larvae. The stability of the essential oils from this plant makes them an alternative to synthetic chemical products against bovine ticks, and also for further studies on the effect on non-target organisms and residual effect on the environment.