Acaricide activity of Piper macedoi Yunck essential oil against Rhipicephalus sanguineus Atividade acaricida do óleo essencial de Piper macedoi Yunck contra Rhipicephalus sanguineus Actividad acaricida del aceite esencial de Piper macedoi Yunck contra Rhipicephalus sanguineus

This study evaluated the acaricide efficacy of Piper macedoi essential oil on larvae of ticks of the species Rhipicephalus sanguineus. The essential oil was extracted by hydrodistillation in a Clevenger-type apparatus. The test consisted of six treatments: from the group I to IV, samples corresponded to different concentrations of essential oil (500 μg.mL; 250 μg.mL; 100 μg.mL and 50 μg.mL) diluted in Tween 80 at 2%. Groups V and VI corresponded to the negative controls (with distilled water and Tween 80 to 2%) and the positive control (with acaricide Amitraz at 12.5%), respectively. The essential oil was rich in apiole (39.81%) and dillapiole (26.47%). The essential oil of P. macedoi presented an activity against the larvae of R. sanguineus, with a better efficiency observed for concentrating 500 μg.mL, mortality of 80.67%, indicating a dose-dependent response.


Introduction
Ticks have called the interest of public health and the scientific community due to their impact on the transmission of diseases. These parasites are part of the group of important vectors of infectious agents for animals and the second in importance for humans, only behind the group of mosquitoes (Araújo et al., 2015a). Species such as Ambyoma cajennense and Rhipicephalus sanguineus are important vectors of Rickettsia rickettsii, the etiological agent of spotted fever in Brazil and other Latin American countries (Moraes-Filho, 2009;Vélez et al., 2012).
The canine babesiosis, zoonosis transmitted by tick bites whose agents are intraerythrocytic hemoprotozoa of the genus Babesia, can also be transmitted to humans (Dantas-Torres & Figueredo, 2006;Araújo et al., 2015b). Another tickrelated zoonosis is the Lyme-like infection syndrome (Baggio-Yoshinari disease) caused by bacteria of the genus Borrelia, which, although uncommon in Brazil, has confirmed cases in some regions, such as in the States of Tocantins and Espírito Santo Mantovani et al., 2007;Yoshinari et al., 2010). The occurrence of Rhipicephalus sanguineus in humans is common, especially among people who work in veterinary clinics and kennels (Louly et al., 2006) because dogs from urban or rural areas, raised in kennels, with limited space, are the natural hosts of R. sanguineus. An engorged female of this species can make oviposition within human dwellings, leading to the development of adults (Massard & Fonseca, 2004) and, despite the great preference for the dog, R. sanguineus can also sting people (Mentz et al., 2016).
Tick control is usually performed with the application of organo-synthetic acaricides indiscriminately, which often results in poisoning of animals and applicators; in acaricidal residues in animal products; development of resistance of ticks to chemical acaricides; as well as environmental pollution (Campos et al., 2012).
An alternative that is increasing in appeal, late, is the search for bioacaricidals of natural source, derived from substances of special plant metabolism that produces a great diversity of compounds with acaricidal action. These resources can be explored using essential oils or compounds as a model for the synthesis of synthetic pesticides (Nwanade et al., 2020). Research, Society andDevelopment, v. 11, n. 1, e18911124610, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.24610 3 The use of natural and phyto-therapeutic products show lower toxicity to mammals, slower development of parasitic resistance, and are biodegradable, thus providing low environmental impact and a reduced number of residues in products of animal origin (Chagas, 2004;Veríssimo & Katiki, 2015).
Piperaceae is a plant family possessing a rich diversity in essential oils, that is composed by monoterpenes, sesquiterpenes and arylpropanoids responsible for interesting biological properties (Santos et al., 2001;Oliveira et al., 2013;Salehi et al., 2019). Piper macedoi Yunck is a native species of the Brazilian Atlantic Forest, without reports in the literature on pharmacological studies or biological tests. In this context, this evaluate the acaricide efficacy of P. macedoi essential oil on R. sanguineus larvae. This study is a pioneer for biological assays of P. macedoi essential oil. For extracting the essential oil of P. macedoi, used in the acaricide assay, fresh leaves (100 g) were submitted to hydrodistillation in a Clevenger-type apparatus for 2 h, in the Interdisciplinary Laboratory of the Federal University of Southern Bahia-UFSB, with a yield of 0.4%. Chemical analysis of the essential oil (solution in dichloromethane at 1mg.mL -1 ) was performed by gas chromatography coupled to the flame ionization detector (GC-FID) and by GC coupled to mass spectrometry (GC-MS) at the Analytical Platform of Farmaguinhos, FIOCRUZ, Rio de Janeiro. The CG-FID analysis conditions were HP-5MS column (30-m x 0.32 mm i.d. x 0.25 μm film thickness), temperature programming from 60 °C to 240 °C, with an increment of 3 °C.min -1 , using hydrogen and synthetic air as carrier gases, with a flow rate at 1.0 mL.min -1 .

Samples of Piper macedoi
The retention indexes (IR) were determined from the retention time of a homologous series of hydrocarbons (C8-C28, Sigma-Aldrich Brazil), obtained by GC-FID, under the same conditions of analysis of the essential oils. The column and temperature conditions by GC-MS analysis were the same for GC-FID. Helium (> 99.99%) was used as the carrier gas with a flow rate at 1.0 mL.min -1 .
The substances present in the essential oil were identified by comparing their mass spectra with database registration (WILEY7n) and by comparing the Retention Indexes (RI) calculated with those in the literature (Adams, 2017). The quantification of substances in the essential oil was estimated by the chromatogram area obtained using GC-FID.

Piper macedoi essential oil acaricide activity
For the acaricide assay, engorged females of Rhipicephalus sanguineus were collected from dogs in veterinary clinics, NGOs and free-living animals from different neighborhoods of the municipality of Teixeira de Freitas.
The females were sanitized with distilled water and dried with sterilized filter paper, then placed in a Petri dish and incubated in an incubator at 25 °C and relative humidity of 85% for oviposition (Labruna et al., 2002). After 14 days, the eggs were collected and packed in a plastic syringe adapted to observe the beginning of hatching and, after that, another 14 days were waited before the acaricide test with the larvae.
The larvae were transferred to a Petri dish containing distilled water and with the aid of a brush. A total of 100 larvae were placed between two pieces of filter paper (2 x 2 cm) impregnated with the treatment solutions (0.4 mL for each repetition). This "sandwich" was placed in a filter paper envelope (6 x 6 cm), sealed by a double tape and stapled (Lambert et Research, Society and Development, v. 11, n. 1, e18911124610, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i1.24610 4 al., 2021). The envelopes were placed in an air-conditioned incubator (± 27 °C and RH > 80%) and the recording of live and dead larvae was performed after 24 h.
The test consisted of six treatments, from the group I to IV corresponding to the different concentration of P. macedoi essential oil (500; 250; 100 and 50 μg.mL -1 ) diluted in Tween 80 at 2%. Groups V and VI corresponded to the negative control (with distilled water and Tween 80 at 2%) and the positive control (Amitraz at 12.5%), respectively. The experiment was conducted in triplicate.
To calculate mortality, the following formula was used: Mortality (%) = Dead larvae x 100/Total larvae. The mean mortality was defined as the mean mortality (%) = repetition mortality 1 + repetition mortality 2 + repetition mortality 3.
The chemical study of P. macedoi collected in an Atlantic Forest area is a pioneer work, and there are no reports in the literature of biological assays performed neither with extracts nor with essential oils of this species. However, chemical analyses of the essential oil of P. macedoi leaves were performed using specimens collected in a bush savanna (Cerrado) area in the State of Minas Gerais (Oliveira et al., 2016). This study showed that monoterpenes were identified as the major compounds, both for the wild plant sample (46.2%) as for a cultivated sample (46.7%), being (E)-β-ocimene the substance present in the highest concentration (wild plant 28.5%; cultivated plant 24.9%). The class of arylpropanoids also appeared in great percentual content (wild plant 25.3%; cultivated plant 23.5%), being partisan the majority compound (wild plant 14.1%; cultivated plant 17.8%). Dilapiolle was identified in this sample at low concentration (wild plant 1.1%; cultivated plant 1.8%) and apiole was not registered in the essential oil of P. macedoi from Cerrado site. The sesquiterpene fraction (wild plant 20.9%; cultivated plant 22.6%) was also found in reasonable content, with germacrene D as the major compound for this group (wild plant 3.7% and cultivated plant 3.2%) (Oliveira et al., 2016). The differences between the major compounds found in P.

Piper macedoi Yunch essential oil acaricidal activity
The essential oil from leaves of P. macedoi was shown to be effective, presenting an acaricidal activity against the larvae of R. sanguineus. After 24 h of larvae exposure to different essential oil concentrations, a mortality of 80.67% was observed at a concentration of 500 μg.mL -1 , presenting a result similar to the one observed for amitraz acaricide, which produced 82.33% of mortality. Concentrations of 250, 100, and 50 μg.mL -1 showed mortality rates of 55.00%; 52.67% and 58.00%, respectively (Table 2). Table 2 -Percentage of mortality of R. sanguineus larvae exposed to different concentrations of P. macedoi essential oil.
*Averages followed by the same letters do not differ from each other by the Tukey test at 1 % probability (p<0.001).
There was an increase in the efficacy of the mortality rate of R. sanguineus larvae proportional to the increase in the concentration of essential oil, indicating a dose-dependent response (Figure 1). The LC50 and LC90 for the essential oil of P.
macedoi were calculated at 221.946 μg.mL -1 and 557.084 μg.mL -1 , respectively ( Figure 1). Thus, the data suggest a higher dose of essential oil to present a mortality rate of more than 90% and to present results higher than Amitraz. The drug of choice for the environmental treatment, where 95% of the tick population can be found, has long been based on pyrethroids because this class of acaricide has greater residual power (Labruna & Machado, 2006). However, recently, due to the increase in resistance to pyrethroids, these were replaced by formamidines (Amitraz), to which, in the same way, some tick populations are creating resistance (Farias et al., 2008;Santos et al., 2009 between the essential oil of P. macedoi (500 μg.mL -1 ) and the drug Amitraz suggests the possibility of an alternative product to be used in environments and facilities where tick larvae are usually found, and even in the dog, after appropriate toxicological tests.
As observed in this study, the chemical composition of many essential oils, showing monoterpenes and sesquiterpenes, is responsible for repellency and biocide activities against the tick R. (B.) microplus (Facey et al., 2005;Gazim et al., 2011). These components, with arylpropanoids, especially represented by apiole, dillapiole, myristicin, safrole and sarisan, are found in species of the family Piperaceae, also show interesting biological properties (Santos et al., 2001, Salehi et al., 2019. Indeed, species of Piperaceae rich in arylpropanoids, such as Piper aduncum L., are also bioactive, demonstrating the activity against Schistosoma sp. As well as insecticide, larvicide and fungicide activities (Almeida et al., 2009). According to the literature, the major compounds of the essential oil of P. aduncum from Amazon region are the arylpropanoid dillapiole, responsible for most of the biological activities of the species (Almeida et al., 2009). Another study also conducted with P.
aduncum showed excellent activity against R. (B.) microplus. The essential oil recovered from the hexane extract of this plant presented, in its chemical constitution, the arylpropanoid dillapiole as the main substance (94.84%) and an activity capable of causing the mortality of 100% of R. microplus larvae at low concentrations (0.1 mg.mL -1 ; 0.5 mg.mL -1 ; 1 mg.ml -1 ) ).
Comparing the data presented in the literature and considering that in this study the essential oil of P. macedoi showed 66.28% of the chemical constitution of its essential oil represented by arylpropanoids (apiole -39.81% and dillapiole -26.47%), it is possible to associate the larvicidal activity with the presence of these compounds. A synergistic effect between then may be considered. In fact, the synergy between compounds from plant extracts and essential oils is one of the main advantages in the use of natural products in relation to synthetic drugs, as they can have more than one active ingredient acting synergistically, potentiating the biological activity in question, also hindering the formation of resistance of ticks, unlike synthetic products (Mgbojikwe & Okoye, 2004).

Conclusion
It was possible to conclude that the essential oil from fresh leaves of P. macedoi showed an activity against the larvae of R. sanguineus, with a better efficiency observed for concentrating 500 μg.mL -1 , also indicating a dose-dependent response.
Also, it is possible to suggest that the larvicidal activity against R. sanguineus presented here may be associated with apiole and dillapiole, not ruling out synergy between all compounds.