Effect of essential oils against acaricide‐susceptible and acaricide-resistant Rhipicephalus ticks

The indiscriminate use of acaricides is a problem worldwide and has increased the selection of acaricide-resistant tick populations. The goal of this study was to evaluate the acaricide effects of two essential oils (from Schinus molle and Bulnesia sarmientoi) using the larval immersion test on three Rhipicephalus tick species. Rhipicephalus evertsi, Rhipicephalus appendiculatus and Rhipicephalus pulchelus ticks collected in Kenya, without history of acaricide exposure, were tested, as well as individuals from two populations of Rhipicephalus microplus (with or without history of acaricide exposure), for comparison. The sample most resistant to the treatments was a population of R. microplus with previous acaricide exposure, whereas the least tolerant sample was a strain of the same species that never had contact with acaricides (Porto Alegre strain). Interestingly, the field tick samples without previous acaricide exposure responded to essential oils with a mortality profile resembling that observed in the acaricide-resistant R. microplus field population, and not the susceptible Porto Alegre strain. The essential oil of B. sarmientoi and its two components tested (guaiol and bulnesol) caused the highest mortality rates in the tested species and are potential molecules for future studies on control methods against these species.


Introduction
Ticks are important disease vectors, affecting both human and animal populations, and transmitting a wide range of pathogens (Silva and Silva 1999;Corson et al. 2004;Sonenshine and Roe 2006). These arthropods cause considerable losses to the cattle industry worldwide (Grisi et al. 2014). Infestation generates several deleterious effects on the host including blood loss, reduced weight gain and milk production, and skin damage at the site of attachment (Fular et al. 2018;Souza Conceição et al. 2017;Souza et al. 2012).
In addition to the direct effects caused by blood feeding, ticks also transmit pathogenic organisms of great economic importance. The bovine tick Rhipicephalus microplus is the main vector of Babesia protozoans (Babesia bovis and Babesia bigemina) and the bacterium Anaplasma marginale (Barbosa et al. 2013;Souza Conceição et al. 2017). Rhipicephalus appendiculatus is the main vector of the protozoan Theileria parva, the agent of East Coast fever in cattle, a clinical syndrome of great economic importance in 12 sub-Saharan countries (Nene et al. 2016). Rhipicephalus evertsi is the vector of Rickettsia africae (Hedimbi et al. 2011;Walker et al. 2014), whereas Rhipicephalus pulchellus transmits benign bovine theileriosis caused by Theileria taurotragi, in addition to sheep's milk virus. To humans, these ticks can transmit Rickettsia conorii, a bacterium that causes tick typhus, as well as the Crimean-Congo hemorrhagic fever virus (Walker et al. 2014).
The most common method for tick control is the use of synthetic acaricides, however, the misuse and overuse use of these products has accelerated the selection of resistant tick populations (Daher 2011;Souza et al. 2012;Biegelmeyer et al. 2014). An important strategy explores the use of bioactive compounds from plants to develop new acaricides (Roh et al. 2003;Santos et al. 2012a, b;Štefanidesová et al. 2017;Rodriguez-Vivas et al. 2018;Vinturelle et al. 2017). Research on plant bioactive compounds can lead to the identification of new molecules presenting different modes of action, biological targets and synergy in comparison to current commercial products (Benelli and Pavela 2018;Pavela 2015;Rattan 2010;Agwunobi et al. 2020).
Bulnesia sarmientoi and Schinus molle are plants native to South America belonging to the order Sapindales. The essential oils of the two species present high antimicrobial activity (Abd-allah et al. 2015;Pegard 2015), fungicidal activity (Castillo et al. 2012;Pawlowski et al. 2012) and repellent and insecticidal actions against several pests (De Batista et al. 2016;Ferrero et al. 2006;Rizwan-ul-Haq and Aljabr 2015;Rodilla et al. 2011;Torres et al. 2012). These properties, associated with low toxicity to mammals , may contribute to the development of new products to control infestations by different arthropods of economic importance.
The present work investigated the effect of essential oils from B. sarmientoi and S. molle on larvae from four Rhipicephalus species of economic importance. These oils and the two main components of both (α-phellandrene and sabinene of S. mole, bulnesol and guaiol of B. sarmientoi) may have potential for future studies on control methods against these species.

Schinus molle
Schinus molle is a tree native to the Peruvian Andes and its resin is exuded from the trunk to obtain a liquid rubber that was used for embalming (Adorno and Boserup 2016). The leaves of this species are composed and have serrated margins, the yellow flowers are gathered in inflorescences and its fruits show reddish color of the drupe type (IBF 2021). Fruits and leaves were processed by steam distillation in the Clevenger apparatus according to the European Pharmacopeia. The resulting sample was analyzed and components were identified by GC and GC-MS. Polar products not detectable by GC-MS were isolated by CC and identified by 1 H-NMR, 13 C-NMR and high-resolution mass spectrometer.
Two of the essential oil components, the monoterpenoids α-phellandrene and sabinene, were selected based on previous functions described ( Espinosa-García and Lanhenheim 1991;Zhang et al. 2017) and purchased from Sigma-Aldrich and kept as a 20% (w/v) stock solution for further tests. The essential oil and its components were solubilized in methanol 100%.

Bulnesia sarmientoi
Bulnesia sarmientoi commonly known as sacred wood (palo santo in Spanish) is a tree that grows in woods in the region of Chaco, belonging to a portion of the territory of Argentina and Paraguay. The leaves of this plant are short, light green and oval. Its small flowers are white and the fruits yellow-orange. The trunk is protected by a thick gray bark, traditionally used as incense due to its aromatic property. B. sarminetoi essential oil is extracted from wood and sawdust by steam distillation. The obtained oil has a yellowish-green coloration, containing a high concentration of two sesquiterpene alcohols: bulnesol and guaiol (Rodilla et al. 2011).
Essential oil from B. sarmientoi was extracted using vapor distillation of wood by the Cooperative Colonizing Society Chortitzer Komitee (Loma Plata, Paraguay). The essence of palo santo was dissolved in n-hexane with mild heating. This solution was allowed to cool and placed in a refrigerator to form guaiol crystals, which were then harvested by filtering. From the remaining solution, mother liquor, the solvent was evaporated to obtain the essential oil with low guaiol content. The product was chromatographed on flash chromatography with hexane and hexane-ethyl acetate mixtures of increasing polarity. This system allowed us to obtain different fractions with mixtures of minor products, and fractions containing pure guaiol and bulnesol that were used for the tests in this work (Rodilla et al. 2011).

Ticks
Rhipicephalus microplus from acaricide susceptible strain named Porto Alegre was reared in bovines, which were brought from a tick-free area and maintained in insulated individual boxes at Faculdade de Veterinária of Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Calves between 5 and 10 months old were infested with 15-day-old tick larvae. Twenty-two days after infestation, female ticks were collected from calves (Reck et al. 2009). Animal care was conducted in accordance with institutional guidelines (institutional approval number 27559).
For R. microplus field populations samples, fully engorged females were collected from cattle in a farm in Campos dos Goytacazes, Rio de Janeiro, Brazil (21° 45′ 21″ S, 41° 19′ 57″ W). In this farm, the acaricides cypermethrin, chlorpyrifos, fenthion and fipronil have been extensively used during the last 3 years for tick control.
The African ticks R. evertsi, R. appendiculatus and R. pulchellus were obtained from the tick colony of the Tick Vector Laboratory of International Livestock Research Institute (ILRI) in Nairobi, Kenya. These ticks were originally collected from field animals without history of acaricide treatment, and posteriorly maintained under standard laboratory conditions in the absence of acaricide exposure for multiple generations.

Larval immersion test
Collected females were maintained in climatic chambers at 28 ºC and 80% relative humidity for oviposition. Eggs were collected every day in microtubes closed with a thin screen (10 mg per tube; i.e., approx. 200 eggs/tube). Hatching occurred approximately 21 days after the onset of oviposition. Bioassays were performed in 7-to 10-day-old larvae.
The larval immersion test (LIT) was chosen due to its higher sensitivity in comparison to other methods (Castro-Janer et al. 2010, 2011. Compounds were tested at the following concentrations: 0.01, 0.1, 1 and 10% (v/v). Ten days after egg hatching, about 1.5 mL of the test solutions were added to the tube until larvae were totally immersed. After 5 min, the liquid was removed, larvae were transferred to filter paper to remove excess solution, then placed in Petri dishes for 24 h. Mortality was evaluated by counting live larvae after this period. Methanol was used as solvent along with essential oils in the LIT.

Statistical analysis
The results represent means based on experiments performed in triplicate (independent samples), according to Shaw (1966). Effect of treatments on larval mortality was analyzed by one-way ANOVAs using the SAS statistical software (SAS Institute, Cary, NC, USA). Tukey's test was used for post-hoc analysis (α = 0.05).

Mortality of Rhipicephalus microplus larvae from acaricide-exposed or non-exposed populations
Both oils and the isolated compounds caused almost 100% mortality in larvae of R. microplus from the susceptible Porto Alegre strain at concentrations above 0.1% (Fig. 1). At 0.01%, sabinene, bulnesol and guaiol induced 40, 15 and 50% larval mortality, respectively, whereas all other compounds reached 100% at this concentration. The R. microplus field population has been exposed at least for 3 years to acaricides like cypermethrin, chlorpyrifos, fenthion and fipronil. Larvae from this population could better tolerate all treatments compared with the Porto Alegre strain (Fig. 2). Immersion of larvae in S. molle essential oil, α-phellandrene, sabinene, and guaiol at concentrations below 10% did not show statistically significant changes in mortality compared to control (Fig. 2a-c, e). Bulnesia sarmientoi essential oil and bulnesol induced mortality at 1% concentration (Fig. 2d, e). The mortality induced by guaiol was markedly different between acaricide-susceptible ticks and the field population, being effective at 0.01% against larvae from Porto Alegre strain but only at 10% concentration against field ticks (Figs. 1f and 2f).
Mortality after the various treatments is summarized as a heat map (Fig. 6). It shows that the essential oils and their components are similarly active against African ticks (R. evertsi, R. appendiculatus and R. pulchellus) and the R. microplus field population (Fig. 6b-e). Rhipicephalus microplus from the Porto Alegre strain presented a greater sensitivity to essential oils and their components when compared to African ticks, despite being equally free of previous acaricide exposure (Fig. 6a). Heat map of essential oil effects on Rhipicephalus spp. Data are presented as percentage mortality (0-100%) after treatment with essential oils or isolated compounds at concentrations from 0.01 to 10% in larval immersion tests of a laboratory maintained R. microplus strain; b R. microplus field population; c R. evertsi wild population; d R. appendiculatus wild population; and e R. pulchellus wild population. The map shows the drastic difference in xenobiotic resistance between samples with or without history of acaricide exposure, and the resistance in African ticks. Sm, Schinus molle oil; Ap, α-phellandrene; Sn, sabinene; Bs, Bulnesia sarminetoi oil; Bl, bulnesol; Gl, guaiol 1 3

Discussion
Cases of resistant tick populations started to be reported in 1937 for arsenical acaricides, and nowadays there is resistance to all known classes of acaricide (Angus 1996;George et al. 2008;Rodriguez-Vivas et al. 2018). In the present study, it was observed that R. microplus ticks previously exposed to acaricides (Fig. 2) were less susceptible to treatment with essential oils and their selected compounds, whereas ticks that have never been exposed to acaricides suffered high larval mortality upon these treatments (Fig. 1). However, African ticks without contact with acaricides displayed a mortality profile closer to the acaricide-resistant field R. microplus strain, as opposed to the susceptible Porto Alegre strain.
In the present study we show that the R. microplus field population has lower susceptibility to essential oils and its components when compared to a strain that did not suffer the same selective pressure (Porto Alegre strain) (Figs. 1, 2; 6a, b). The immersion test with S. molle and B. sarmientoi essential oil revealed an unexpected tolerance in Rhipicephalus ticks from East Africa (without acaricide exposure) almost higher than in the Brazilian strain with a history of exposure to acaricide (Figs. 2a,d;3a,d;4a,d;5a,d;6).
The toxicity of the essential oils used in this study against various arthropod species was also documented by other researchers. The S. molle essential oil presented repellent and insecticidal activity against Trogoderma granarium and Tribolium castaneum with mortality rates of 90 and 76.7%, respectively, at a concentration of 10% (Abdel-sattar et al. 2010). The biological activity observed in the essential oil of S. molle can be attributed to the concentrations of α-phellandrene and sabinene (Matias et al. 2016;Zhang et al. 2017). Hexane extracts from leaves and fruits of S. molle also had a repellent effect on Triatoma infestans and Cydia pomonella larvae (Ferrero et al. 2006). The repellent activity found for Oriental cockroach (Blatta orientalis) was attributed to the major components (germacrene D and β-caryophyllene) (Deveci et al. 2010). Schinus molle essential oil presented a high level of toxicity against Artemia salina, with LC 50 values of 47 and 67 mg/ml for leaf and fruit, respectively ). Other parasites of economic importance are also affected by these essential oils. For instance, the flea Ctenocephalides felis, the most important ectoparasite of dogs and cats, as well as the cattle tick R. microplus showed high mortality after treatments with fractions of this essential oil, in line with the present results (De Batista et al. 2016;Torres et al. 2012). Rhipicephalus microplus without previous acaricide exposure were tested using S. molle essential oil in a larval immersion test, and the mortality rate was similar to the one observed for the field strain in the present study (Torres et al. 2012).
In African species, substances isolated from B. sarmientoi essential oil-guaiol and bulnesol-were not as active as was the original essential oil, suggesting a synergistic effect between these and other substances from the plant. In the present study, this essential oil induced 100% larval mortality for all tested tick species at 10% concentration, demonstrating that, besides being able to inhibit tick feeding, B. sarmientoi also presents acaricide action (D panels in Figs. 1, 2, 3, 4, 5, and 6). Further studies on the effect of B. sarmientoi on other arthropods and mammals are needed to assess their specificity and the possibility for the development of new products for tick control.
Author contributions Conceived and designed the experiments: CL, IVJ, NG, JR, SP. Performed the experiments: DL. Analyzed the data: DL, RM, CL. Contributed reagents/materials/analysis tools: CL, NG. Wrote the paper: RM, CL.
Funding This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brazil (CAPES)-Finance Code 001. We acknowledge the support by grants from the CNPq-Instituto Nacional de Ciência e Tecnologia de Entomologia Molecular, FAPERJ, and FAPERGS (Brazil).

Compliance with ethical standards
Conflicts of interest There is no conflicts of interest/competing interests.
Ethical approval All experiments were performed in accordance with the standards and ethics of animal experimentation, established by Brazilian federal Law No. 11794/08 after approval of the Ethics Committee of Animal Use (CEUA) of NUPEM -UFRJ. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
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