Chemical Composition and Acaricidal Activity of the Essential Oils of Some Plant Species of Lamiaceae and Myrtaceae against the Vector of Tropical Bovine Theileriosis: Hyalomma scupense (syn. Hyalomma detritum)

The present study aimed to investigate the acaricidal properties of six essential oils. They were extracted from some plant species (Lamiaceae and Myrtaceae) using the technique of hydrodistillation with the Clevenger apparatus. The chemical compositions of the essential oils under study were determined by gas chromatography–mass spectrometer (GC-MS). An Adult Immersion Test (AIT) and a Larval Immersion Test (LIT) were used to evaluate the acaricidal activity of these essential oils against the adults and larvae of Hyalomma scupense. GC-MS analysis showed the major constituents of each essential oil: 25.49% of α-thujone (lavender); 46.82% of carvacrol (oregano); 78.78% of carvacrol (thyme); 40.27% of 1,8-cineole (blue gum); 17.45% of p-cymene (river red gum); and 26.96% of 1,8-cineole (rosemary). The biotests on the essential oils revealed that they inhibit the reproduction of H. scupense engorged females at a rate of 100 % with doses of 0.781 μl/ml of rosemary, 1.562 μl/ml of thyme, 3.125 μl/ml of lavender and oregano, and 6.250 μl/ml of blue gum and river red gum. After a treatment that lasted for 24 hours, essential oils showed a larvicidal activity with respective values of lethal concentrations (LC): LC50, LC90, and LC95 (0.058, 0.358, and 0.600 μl/ml for thyme; 0.108, 0.495, and 0.761 μl/ml for rosemary; 0.131, 0.982, and 1.740 μl/ml for oregano; 0.155, 2.387, and 5.183 μl/ml for blue gum; 0.207, 1.653, and 2.978 μl/ml for river red gum; and 0.253, 2.212, and 4.092 μl/ml for lavender). This is the first report on the acaricidal activity of these essential oils against H. scupense. The results obtained showed that the essential oils with chemotype carvacrol, 1,8-cineole, α-thujone, and p-cymene are highly acaricidal, and they can be used for ticks control. However, further studies on their toxicity in nontarget organisms are required.


Introduction
Ticks and the diseases they transmit have long been recognized as one of the major constraints of livestock development in various countries. Ticks feeding on domestic animals can result in various adverse effects including anemia, paralysis, toxicosis, decreased quality of the leather, and transmission of many diseases of diverse etiology. The causative agent of these diseases can be a virus, rickettsia, bacterium, or protozoan [1]. Therefore, the damage directly caused by ticks (and induced pathology) is a serious animal health problem that can significantly reduce overall livestock productivity.
In Algeria, cattle herds cost a heavy price if infected with piroplasmosis, a major tick transmitted disease of livestock in the country [2]. Another tick-borne disease, tropical bovine theileriosis transmitted by Hyalomma scupense (syn. Hyalomma detritum), is also present in this area. This species has been the most frequently recorded from several surveys conducted in Algeria [3,4]. Many approaches have been used for tick management such as biological control using pathogens or predators, pheromone-assisted control, herbal pour-on or dip preparations including green manufactured nanoparticles [5], and vaccination [6]. Acaricides and repellents are still regarded as the easiest method for control but applications involve several drawbacks like cost, toxicity, waiting times, and acaricide resistance [7]. In Algeria, the most widely used means to control cattle ticks are conventional acaricides. Despite of the absence of studies on the chemoresistance of marketed acaricides, Algerian veterinary practitioners have observed resistance to these compounds in recent years. Therefore, it is prudent that effective compounds be discovered and evaluated for their acaricidal potential. In this study, we investigated the effectiveness of various essential oils (EO) against H. scupense obtained from six aromatic plant species: Eucalyptus camaldulensis Dehnh (river red gum), Eucalyptus globulus Labill (blue gum), Lavandula stoechas L. (lavender), Origanum floribundum Munby (oregano), Rosmarinus officinalis L. (rosemary), and Thymus capitatus L. (thyme).

Essential Oils Extraction and Analyses.
Extraction of EO from plant parts was carried out by hydrodistillation for three hours using a Clevenger-type hydrodistillation apparatus. GC-MS analysis of the oils was performed on an HP-MS HP Model 6980 inert MSD (Agilent Technologies, USA), with HP-5MS column (30 × 0.25 mm ID × 0.25 m film thickness). Temperature of the injector was maintained at 280 ∘ C. Oven temperature was maintained at 60 ∘ C for 1 min and then increased to 280 ∘ C at 5 ∘ C/min and remained constant at this temperature for 8 min. Flow rate of the helium carrier was 1 ml/min and split mode 1/100 was used. Identification of components in the EO was accomplished by comparison of their Kovats index and GC mass fragmentation with those of Wiley Mass Spectral data (Agilent Technologies 7th edition, Inc.) and NIST 05 MS library data. Each analysis was executed in duplicate.

Ticks Collection.
Engorged females of H. scupense, with an average weight of 0.50 g, were collected from naturally infested cattle. Ticks were washed with 2% sodium hypochlorite solution and were then rinsed with distilled water before being dried with paper towels [8]. Engorged females used for Adult Immersion Test (AIT) and larvae for Larval Immersion Test (LIT) were tested on the same day of collection.

Adult Immersion Test (AIT)
. Essential oils concentrations used in Adult Immersion Tests (AIT) were prepared in series diluted in Tween 80 at 2% ranging from 12.5 to 0.097 l/ml. Engorged females were each immersed in a concentration for 5 minutes and were then removed and dried with paper towels. Ticks were individually incubated at 27 ∘ C and 90% relative humidity with a 12:12 light: dark photoperiod until the end of oviposition [9]. Eggs of each female were weighed and transferred to tubes that were covered with Tissue-Non-Tissue (TNT) fabric (Inotis: LICIAL5, spunlace nonwovens, Algeria) and fastened by elastic bands. Each bioassay was repeated three times. A similar group of ticks as untreated controls was prepared in parallel by the immersion of ticks in 2% Tween 80. The oviposition rates were assessed after the end of oviposition. Reproductive efficiency and reproduction inhibition were calculated using the following equations [10]: where OR is the oviposition rate, IFW is the initial female weight, EW is the egg weight, E is egg eclosion, RE is the reproductive efficiency, and RI is reproduction inhibition.

Larval Immersion Test (LIT).
Larval immersion bioassays utilized the syringe test technique [9] to evaluate the larvicidal activity of the EO. Approximately 200 eggs (0.01 g) were transferred to a 2.5 ml open-end syringe whose plunger was withdrawn to the line of 2 ml. The syringe was then sealed with a TNT fabric and incubated at 27 ∘ C with 90% RH in darkness. Bioassays started 14 days after eclosion. Essential oils were serially diluted in 2% Tween 80 to obtain ten concentrations ranging from 12.5 l/ml to 0.024 l/ml. Larvae were exposed to 2 ml of a concentration per syringe replicated twice. Larvae were exposed to each concentration for 5 min. Mortality in one syringe was recorded at 24 h, while larval mortality in the second syringe was recorded at 6 days. Each bioassay was repeated three times. After removing the TNT parts and emptying the syringes contents, the number of the living and dead larvae was counted in both syringes incubated at 24 h and 6 days.  Control groups were handled similarly and were exposed to 2% Tween 80 solution only.

Statistical Analysis.
The results of mean oviposition rate, egg eclosion rate, reproduction efficiency, and reproduction inhibition for adult ticks (H. scupense) were subjected to nonparametric tests using the Kruskal Wallis test [11]. Estimated LC 50 , 90 , and 95 of larvae of each EO were determined at 24 h and 6 days. Lethal concentration data were transformed according to Finney's probit analysis method [12].

Results
According to the results of GC-MS chromatographic analysis (Table 1), the major components of river red gum EO are p-cymene, (+) spathulenol, (E, E) -farnesol,pinene, cuminic aldehyde, 1-phellandrene, sabinene, carvacrol, and p-cymen-7-ol. For blue gum EO, 1,8-cineole, -pinene, viridiflorol, camphene, d-pinocarvone, and (+) -aromadendrene are the major components. The major constituents of lavender EO were -thujone, camphor, camphene, D-fenchyl alcohol, l-bornyl acetate, terminalol L, dl -limonene, -pinene, and linalool L. The main chemical components of oregano EO were carvacrol, p-cymene, andterpinene followed by -myrcene, o-cymene, thymol, transcaryophyllene, -pinene, and -terpinene. Essential oil of rosemary contained 1,8-cineole and l-camphor as major components; other recorded compounds were -pinene, borneol L, camphene, -terpineol, -pinene, trans-caryophyllene, lbornyl acetate, -myrcene, and -terpinene. In addition to the major components of thyme EO (carvacrol, p-cymene, and -terpinene), other compounds from fractionation included transcaryophyllene, m-thymol, -myrcene, andterpinene. Thyme EO completely inhibited tick oviposition at 100% at a concentration of 1.562 l/ml whereas the other EO induced this same effect at greater concentrations ranging from 3.125 to 6.25 l/ml (Table 2). At the same concentration (1.562 l/ml), thyme EO completely inhibited reproduction of H. scupense. This concentration is significantly lower than what was recorded with the other EO (3.125 -6.25 l/ml). The eclosion rates of the eggs in all treatments were significantly different as compared to controls (p < 0.05). Note that thyme and rosemary EO are more inhibitors of eclosion than other EO such as those of river red gum and blue gum. At 24 hours, thyme EO proved to be the most larvicidal, whereas the least larvicidal was blue gum EO. On the 6th day, the most and the least larvicidal EO remained the same as those obtained in 24 h (Table 3, Figures 1-6). We observed no mortality in control groups in LIT and AIT.
As far as the present study is concerned, we have noticed that each EO we evaluated for acaricidal activity was composed of a combination of 14 to 37 chemical compounds. This may indicate that associations between  constituent compounds should be studied mainly in terms of possible additive, synergistic, or even probable antagonistic properties.

Conclusion
We found excellent acaricidal activity of several EO constituents that were evaluated against H. scupense in this study.
Our results indicate that these compounds may provide real Mortality of larvae (%) Figure 6: Percentage mortality for H. scupense larvae exposed to concentrations of thyme essential oil.
alternatives to the current conventional acaricidal products, especially against resistant tick populations. However, further investigations are warranted including toxicological studies on nontarget species regarding the usefulness of these components for tick control.

Data Availability
No data were used to support this study.

Disclosure
This research did not receive any specific grant from funding from agencies in the public, commercial, or not-for-profit sectors.

Conflicts of Interest
The authors declare that there are no conflicts of interest.