Introduction

Rhipicephalus sanguineus (s.l.) is the most widespread tick in human dwellings and of great importance as a biological vector of several pathogens that cause severe infections in canines as well in humans (Otranto et al. 2009). The control of the tick is quite difficult due to its ethology: R. sanguineus has an endophilic behavior; a fully engorged adult female may oviposit up to 4000 eggs which are deposited in hidden places and almost inside the ground (Dantas-Torres 2010); due to the cryptic behavior and the small size of the larvae and nymphs, low-level indoor infestations can be difficult to detect, allowing populations to increase rapidly (Eiden et al. 2015).

Industrial, synthetic acaricides have long been used to effectively control the parasitic stages of the tick. However, these molecules have several disadvantages, including development of tick resistance and permanence of residues in the animals and in the environment. Hence, the effect of plant derivatives on several cellular and animal models has been intensively studied in the last years (Bakkali et al. 2008; Castelblanco et al. 2013; Pavela et al. 2016). Compounds from plants are an alternative to industrial, synthetic products; tick resistance usually develops slowly because they are a mixture of several active agents with different mechanisms of action (Borges et al. 2011). Plants accumulate organic substances in significant quantities and concentrations and are a renewable source of these substances; hence, they can be exploited economically and sustainably (Da Silva et al. 2016). The use of plant derivatives in the control of veterinary ectoparasites is an area which holds considerable potential for the future and research into their use is still at an early stage (Ellse and Wall 2014).

Essential oils (EOs) are natural compounds with an important component of volatile molecules with strong odor and generally soluble in organic solvents. EOs are composed primarily of terpenes, terpenoids and other aromatic compounds; two or three major components are at fairly high concentrations (20–70%) compared to others components present in trace amounts. Antimicrobial, insecticide and acaricide effects have been exhaustively demonstrated (Andreotti et al. 2013; Kačániová et al. 2014; Martins et al. 2014; Wanzala et al. 2014).

Schinus molle L. (Anacardiaceae) is known as Peru tree, bolilla, pirú, preconcuahuitl, copalquahuitl, yag lachi ntaka (popoloca), molle, Californian pepper and pink pepper. The molle was the sacred tree of the Incas; they used to plant it on the periphery of their palaces and public buildings (De Mendonça et al. 2012). The tree grows from sea level to 3500 m a.s.l. from Southern Mexico to Northern Chile and Central Argentina in America and in many other tropical and subtropical regions. The red fruits are aromatic and have a peppery, pungent and sharp flavor; fruits contain an essential oil rich in hydrocarbon monoterpenes which contribute to the citrus aroma and sesquiterpenes that provide the pungent woody odor (Bernhard et al. 1983). The “molle” has been used in ethnobotanical medicine to treat bad air and a myriad of disorders, from toothache to labor pain because of its antibacterial, antiviral, antiseptic, topical, antifungal, antioxidant, anti-inflammatory, antitumor and analgesic activities (Tene et al. 2007). The antimicrobial activity of S. molle essential oil (EOSm) has been demonstrated in Gram +, Gram − and in some fungi (Martins et al. 2014), as well as on Trypanosoma cruzi (Molina-Garza et al. 2014). There are reports on the activity of molle as an insecticide on mosquito species such as Aedes aegypti or the housefly Musca domestica (Wimalaratne et al. 1996; Chantraine et al. 1998).

In order to exploit an alternative mechanism of control of dog ticks using plant derivatives, this work aimed at evaluating the acaricidal effect of the EO obtained from fruits of S. molle L. on the larval stages and engorged adult females of the brown tick of the dog, R. sanguineus.

Materials and methods

Isolation and characterization of the essential oil of Schinus molle (EOSm)

Fruits of S. molle in maturity state were collected in three locations from Catamayo (3°58′S, 79°21′O), Province of Loja; Cuenca (2°54′S, 79°02′O), Province of Azuay and Salcedo (1°02′S, 78°35′O), Province of Tungurahua, Ecuador. The EOSm  was isolated from 300 g of mesocarp from fresh fruits by hydrodistillation using a Clevenger-type apparatus for three hours and dried over anhydrous sodium sulfate. EOSm was stored in sealed vials protected from the light at 4 °C until further analysis. Characterization of EOSm was accomplished by (a) quantitative analyses (gas chromatography with a flame ionization detector), (b) qualitative analyses (gas chromatography and mass spectrometry) and (c) determination of physical properties (density and refraction index) as is completely described in Rey-Valeirón et al. (2017).

Obtention of engorged females of Rhipicephalus sanguineus

One-hundred adult engorged females of R. sanguineus were collected manually from the ears, interdigital spaces, neck, groin and base of the tail of two cross-bred, male dogs with no acaricide treatment for at least 45 days. Specimens were kept in plastic flasks with small holes until assays, approximately 1 h after. R. sanguineus females were identified by standard keys and used for the assays.

Larval package test (LPT)

To obtain larvae, eight adult engorged females were stuck to the lid of a glass Petri dish with double-sided sticky tape and maintained in an incubator at 29 ± 1 °C and relative humidity (RH) 80% for 20 days. Eggs were then collected and transferred to a glass tube with a cotton lid to allow air and moisture exchange and incubated to allow hatching in the same conditions as above. Larvae of 21 days old were used for LPT.

The LPT was carried out as by Stone and Haydock (1962). One-hundred larvae were placed in the center of a sheet of filter paper measuring 6 × 6 cm with pencil drawn grids. Five concentrations of EOSm were used (0.125, 0.25, 0.50, 1 and 2%) diluted in anionic detergent Tween 80 (v/v). Tween 80 diluent alone had been previously evaluated for not causing mortality of larvae. Each sheet with the 100 larvae inside was folded and moistened with 1 mL of each concentration to be tested. The sheet was then placed into an envelope of same filter paper. Additionally, cypermethrin 1:1000 in water was tested as synthetic acaricide. EOSm control group were treated with 1 mL of 2% Tween 80 and cypermethrin control groups with 1 mL of deionized water. Each treatment was repeated five times. To prevent effects due to volatility of some compounds from EOSm, the control groups were kept apart in the same conditions (27 ± 1 °C and RH > 80 ± 10%). After 24 h, the packets were opened and the number of living and dead larvae was counted manually. Only larvae capable of locomotion were considered alive. The percentage of mortality was calculated as Abbott (1925). Mortality in control groups was calculated as number of dead larvae/total number of larvae × 100%.

Adult immersion test (AIT)

AIT was carried out as by Drummond et al. (1973) with minor modifications. Groups of eight engorged adult females were weighed and immersed for 5 min in 10 mL of each EOSm dilution (0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 20% v/v in 2% Tween 80) into a 25 mL beaker which was gently agitated at room temperature. The detergent solution was used as the negative control of EOSm. Cypermethrin at dilution 1:1000 (as recommended by manufacturer) was used as synthetic, commercial acaricide and deionized water as negative control of cypermethrin. The weight of the groups was homogeneous. After the immersion, the females were placed on paper sheets for 15 min to allow the solvents and excess solution to evaporate. Each female was stuck to the lid of glass Petri dishes with double-sided sticky tape. The groups were maintained in a climate-controlled chamber (27 ± 1 °C and RH > 80 ± 10%) and the egg masses were collected until the death of the females, around day fourteen. To obtain larvae, eggs were treated as described above in  "Results" section. Larvae and unhatched eggs of each group were counted in a 24-wells culture plates under a stereo microscope to estimate the percentage of hatching.

To establish the acaricidal efficacy of the EOSm or cypermethrin, several biological parameters were calculated using the following formulas (FAO 2004):

  1. (a)

    Surviving period: number of ticks which remained alive after treatment

  2. (b)

    Percentage of egg hatching (EH) = (number of larvae)/(total number of unhatched eggs and larvae) × 100

  3. (c)

    Reproductive index (RI) = egg mass weight/engorged adult female weight before oviposition

  4. (d)

    Inhibition of oviposition (IOv%) = (RI control group − RI treated group/RI control group) × 100.

  5. (e)

    Reproductive efficiency (RE) = egg mass weight/engorged adult female weight before oviposition × egg hatching

Statistical analysis

Statistical differences between biological parameters and concentrations were calculated by analysis of variance (α =  0.05). Pearson correlation test was used to calculate the correlations between the EOSm concentration and mortality of larvae or reproductive parameters of adult females. Shared variance (r2) was also estimated to account the effect of EOSm concentrations on reproductive parameters. Calculation of LC90 and LC50 (concentration necessary for 90% and 50% lethality) was carried out. In all the cases, Prism v6.4 for Windows was used (GraphPad Software, USA).

Results

Characterization of the EOSm

Twenty-one components comprising more than 97% of the EOSm were identified. The major components were p-cymene (40.0%) followed by limonene (19.5%), myrcene (7.7%), and camphene (5.6%). The yield (wt/wt) was 3.50 ± 0.30% (Table 1).

Table 1 Composition of Schinus molle essential oil used as acaricide against unengorged larvae and engorged adult females of Rhipicephalus sanguineus
Full size table

LPT

The results of the larval package test are shown in Table 2. Mortality rate was dose-dependent (p < 0.05); the highest value of mortality (> 99%) was achieved with 2% EOSm, but no statistical differences were found between 1 and 2%. LC50 and LC90 were calculated as 0.21 and 0.80%, respectively. Correlation between concentrations and larval mortality was strong and positive (r = 0.849). Cypermethrin in dilution 1:1000 caused 23.1% larval mortality.

Table 2 Mortality of Rhipicephalus sanguineus larvae treated with different concentrations of Schinus molle essential oil or cypermethrin
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Effect of EOSm on biological and reproductive parameters of Rhipicephalus sanguineus

Surviving period

At all the concentrations tested, immersion in EOSm was not lethal to engorged females: the specimens remained alive until the end of oviposition. Identical results were observed in ticks treated with cypermethrin and in control groups immersed in detergent or in deionized water.

Inhibition of oviposition (IOv), egg hatching (EH) and reproductive efficiency (RE)

The highest value of IOv (29.6%) was achieved by 20% EOSm (p < 0.05). Concentrations below 16% resulted in negligible values of IOv (data not shown). A moderate coefficient of correlation (0.681) was found between EOSm concentrations and IOv values and the shared variance was over 46%. IOv in the group treated with cypermethrin was 13.7% (Table 3).

Table 3 Effect of essential oil of Schinus molle on reproductive parameters of Rhipicephalus sanguineus
Full size table

The efficacy of the EOSm was also evaluated on the egg hatchability of R. sanguineus. EH ranged between 53 and 98.3% with concentrations of 16 and 20% EOSm, resulting in the lowest egg hatching values (53 and 59.4%, respectively). A strong negative correlation (r = − 0.948) was found between EOSm concentrations and EH; shared variance was over 89%. In the group treated with cypermethrin, EH was only 73% (Table 3).

RE decrease was dose-dependent starting at a concentration of 2% (Table 3). Lowest RE value (22.6%) was achieved with concentration of 20%. Concentrations below 2% resulted in negligible values of RE (data not shown). A negative and strong correlation (r = − 0.985) was found between EOSm concentrations and RE; shared variance was over 97%. In the group treated with cypermethrin, RE was 61.6% (Table 3).

Discussion

The major components in Ecuadorian EOSm were characterized by monoterpenes p-cymene, limonene, myrcene and camphene, partially consistent with the findings of other studies. Bernhard et al. (1983) reported myrcene, α-phellandrene, δ-cadinene, limonene, α-cadinol and β-phellandrene as major components in the EOSm from California. Zahed et al. (2011) identified limonene and β-phellandrene, α-phellandrene, myrcene and α-pinene in EOSm collected from four locations in Tunisia. Fruit EOSm from Portugal was characterized mainly by β-myrcene, limonene, α-phellandrene and β-phellandrene (Martins et al. 2014). Limonene and myrcene are the common components in all those essential oils. However, Batista et al. (2016) did not detect the presence of myrcene, phellandrene and limonene in samples from Rio de Janeiro, Brazil. Differences may be due to genotype, plantation origin, age, physiology or ecological conditions as seasonality, water availability and soil nutrients (dos Santos et al. 2015, Bhattacharya 2015).

The results presented in this paper proved the efficacy of the phytotherapy upon R. sanguineus in vitro. At a concentration of 2%, EOSm was lethal to R. sanguineus larvae. The results were not surprising because the EOSm obtained from fruits showed 99.9% of mortality of R. (B.) microplus larvae at concentration of 2.5% (Rey-Valeirón et al. 2017). A fraction of the EOSm obtained from the whole plant demonstrated acaricidal effect in vitro on R. (B.) microplus (Cidade-Torres et al. 2012). A recently report showed insecticidal effect of EOSm from leaves and fruits on adult stages of Ctenocephalides felis felis in vitro (Batista et al. 2016).

Although comparisons are difficult due the units used to record the concentrations and it is extremely difficult to compare the efficacy of oils tested in one study with that of oils tested in another (Ellse and Wall 2014), several reports focused on acaricidal effect of EOs on ungorged larvae. Ribeiro et al. (2008) observed that the oil of bitter cinnamon, Drimys brasiliensis Miers (Winteraceae) at 6.25 µL/ml was lethal, killing 100% of the larvae of R. sanguineus. Lippia sidoides (Verbenaceae) EO caused 96% of mortality at 14.10 mg/mL (Gomes et al. 2014). However, a concentration of 20% of Tagetes minuta (Asteraceae) EO was required to cause 100% of mortality in larvae (da Silva et al. 2016). The hydrophobic nature of the oils may exert mechanical effects on the parasite—particularly larvae—by disrupting the cuticular waxes and blocking the spiracles, which leads to death by water stress or suffocation (Ellse and Wall 2014).

This is the first report about the effect of EOSm on adult stages of R. sanguineus. Although the susceptibility of R. sanguineus engorged females was lower than that of the larvae and EOSm was not lethal, the oil had a remarkable effect over biological parameters as shown by decreases in oviposition, EH and RE if compared with untreated group. In this work, the best results were obtained with 20% EOSm: 29.6% of IOv, 59.4% of EH and 22.6% of RE. The EO of Tagetes patula caused no deaths in engorged adult females of R. sanguineus subjected to treatment but reduced the oviposition (Politi et al. 2012). The effect on oil over tick reproductive system was showed by Sampieri et al. (2012). They reported ultrastructural changes in the somatic and germ cells of ovaries, in females engorged on rabbits with castor oil diets. Vendramini et al. (2012) demonstrated that andiroba (Carapa guianensis) seed oil was able to cause severe changes in the oocytes and the reproductive system of R. sanguineus.

The results were obtained from female ticks manually collected before they had completely finished blood feeding; nonetheless, the control group was able to lay viable eggs with a hatching rate of 95%. Compared with the untreated group, the EH value of 59.43% with EOSm concentration of 20% was certainly due to the essential oil.

The low impact of cypermethrin on larval mortality and the regular efficacy on reproductive parameters of R. sanguineus obtained in the present study requires further revision in upcoming research, because cypermethrin is one of the most used in tick control on dogs from Venezuela and Ecuador (data unpublished). Rodriguez-Vivas et al. (2017) reported high levels of cypermethrin resistance in R. sanguineus populations in dogs in Yucatán, Mexico.

The results obtained in this work with the EOSm suggest that effects on mortality and reproductive parameters observed in the assays are related to the presence of hydrogenated monoterpenoids (MH), since major components of EOSm were MH (limonene, p-cymene, α-phellandrene, myrcene, and camphene). Monoterpenoids were the first inhibitors from plants which were considered to have anticholinesterasic properties; advantageously, these compounds penetrate quickly the membranes due to volatile and lipophilic characteristics. Limonene has also been reported as broad spectrum arthropod and insect repellent due to a potent acetylcholinesterase (AChE) inhibitory activity whereas other compounds as geraniol or R-carvone have strong insecticidal activity but are weak inhibitors of AChE (López and Pascual-Villalobos 2010). As the AChE from insects differs from the mammalian for a single residue, known as the insect-specific cysteine residue, AChE can be an insect-selective target (Pang et al. 2012) for one or several major components of EOs. Prado-Rebolledo et al. (2017) reported up to 80.9% of mortality in larvae treated with d-limonene. p-Cymene, the major constituent of EOSm, was found to be toxic against two stored pests (Sitophilus granarius and Tribolium confusum) (Kordali et al. 2008).

In arthropods, the group of biogenic amine messengers consists of dopamine, tyramine, octopamine, serotonin and histamine (Blenau et al. 2012); the tyraminergic/octopaminergic pathway is the main regulatory system for oviposition control in insects, arachnids, crustaceans and mollusks (Li et al. 2015). The identification of the orthologous octβ 2R octopamine receptor in R. microplus as well as the inhibition of oviposition by adrenergic ligands strongly suggest a role in ticks (Cossió-Bayúgar et al. 2015). Importantly, EOs can be considered as agonists of all types of octopamine and tyramine receptors (Jankowska et al. 2018). Hence, the study of the effects of EOSm on insect-specific octopamin receptor should be considered in the quest of candidates for the control of R. sanguineus. However, there are many potential targets in the tick nervous system that propose EO components as candidates for acaricides.

In addition to the toxicity of a plant against arthropods, the product should not be toxic to mammals. Plant derivatives are not always harmless; in fact, it has been shown that some compounds have good antioxidants or antimutagenic activities at low concentrations but at higher concentrations induce cellular DNA damage or can have deleterious effects on the behavior and health of the host (Bakkali et al. 2008; George et al. 2008); some essential oils or some of their constituents may be considered as secondary carcinogens after metabolic activation (Guba 2001). Most studies on acaricides report the bioactivity of EOs and their compounds in vitro, but there is a minority of reports about the use in vivo of natural acaricides (Manzoor et al. 2013). In the case of S. molle, Bras et al. (2011) studied the acute dermal exposure to ethanolic and hexanic extracts in rats. A slight and reversible skin irritation was observed, which indicated that the topical use of these extracts was safe, either as a therapeutic agent or as an insect repellent in that animal model.

This study presents an evaluation of the acaricidal effect of EOSm in vitro and the results showed an important effect on larvae and female reproductive parameters of R. sanguineus. However, before their use on dogs, several issues concerning acaricide potential of EOSm have to be evaluated. A toxicology profiling including excipient development, research into residual activities and the effects on the behavior of dogs are required. Graham et al. (2005) showed that dog’s behavior changed significantly with the length of exposure to odours of lavender (Lavandula angustifolia), chamomile (Anthennis nobilis), rosemary (Cymbopogon citrates) and peppermint (Mentha piperata) essential oils.

EOs have a limited half-life because their volatile components with repellent effect tend to being short-lived in their effectiveness (Nerio et al. 2010). However, 2.5% turmeric essential oil sprayed on dogs demonstrated repellency comparable to 20% DEET and was more effective than 5% PMD (para-Menthane-3,8-diol, a monotorpene product of Corymbia citriodora ssp. citriodora) (Goode et al. 2018). The use of EOs is less difficult to manage in pet habitat than in livestock, because pets are usually kept singly and owners are often willing to reapply products (Ellse and Walls 2014).

A drawback in the development of EOs as acaricides is the high cost of extraction and low yields. Nonethless, it is possible to develop synthetic analogs of the major components with acaricide or repellent effects. TT-4302, a plant-based repellent with 5% geraniol, showed 100% of efficacy against R. sanguineus in vitro (Bissinger et al. 2014).

Finally, the results obtained in the present study contribute to a further research in acaricidal/repellent effects of synthetic components based on EOSm, which would be better controlled in terms of preparation, reproducibility and a higher economical viability.