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

Authors: Somia Djebir; Samir Ksouri; Mohamed Trigui; Slim Tounsi; Awatif Boumaaza; Youssef Hadef; Ahmed Benakhla
Journal: BioMed Research International
 (2019)

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## Abstract

The present study aimed to investigate the acaricidal properties of six essential oils. They were extracted from some plant species (Lamiaceae andMyrtaceae) 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 ofHyalomma 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 ofH. 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 againstH. 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.


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## Body

## 1. 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 byHyalomma 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) againstH. 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), andThymus capitatus L. (thyme).


## 2. Materials and Methods

### 2.1. Plant Materials

During April through July 2015, leaves and flowering tops were collected from their natural habitats from Wilaya of Guelma: lavender, Ain Safra region of Djebel Maouna, latitude: 36.403237, longitude: 7.387801; oregano, Djebel Haouara region, latitude: 36.544436, longitude: 7.523108; thyme, Ouled Chiha region of Hammam Ouled Ali, latitude: 36.588422, longitude: 7.467377; and blue gum and river red gum, Djebel Beni Salah area, latitude: 36.476148, longitude: 7.854270. Leaves and flowering tops of rosemary were harvested on April 2015 from the Wilaya of Tebessa in Ouenza region (Gora Range) latitude: 35.917372, longitude: 8.127908.


### 2.2. 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 × 0.25μm 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.


### 2.3. Ticks Collection

Engorged females ofH. 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.


### 2.4. Bioassays

#### 2.4.1. 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 0.097μl/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: LICIAL®, 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]:(1)OR=EWIFWOR%=ORcontrol-ORtreatedORcontrol×100RI%=REcontrol-REtreatedREcontrol×100RE=EWIFW×%Ewhere 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.


#### 2.4.2. 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 12.5μl/ml to 0.024 μl/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.


### 2.5. 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 LC50, 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]. The Tukey test was used to identify differences between mean values of the LC50, LC90, and LC95 which were obtained at 24 h and after 6 days for each EO. All these statistical analyses were performed using the Social Science Statistics Software (SPSS) for Windows, version 20.0. [13] (IBM Corp. released 2011 IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.)


## 2.1. Plant Materials

During April through July 2015, leaves and flowering tops were collected from their natural habitats from Wilaya of Guelma: lavender, Ain Safra region of Djebel Maouna, latitude: 36.403237, longitude: 7.387801; oregano, Djebel Haouara region, latitude: 36.544436, longitude: 7.523108; thyme, Ouled Chiha region of Hammam Ouled Ali, latitude: 36.588422, longitude: 7.467377; and blue gum and river red gum, Djebel Beni Salah area, latitude: 36.476148, longitude: 7.854270. Leaves and flowering tops of rosemary were harvested on April 2015 from the Wilaya of Tebessa in Ouenza region (Gora Range) latitude: 35.917372, longitude: 8.127908.


## 2.2. 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 × 0.25μm 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.


## 2.3. Ticks Collection

Engorged females ofH. 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.


## 2.4. Bioassays

### 2.4.1. 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 0.097μl/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: LICIAL®, 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]:(1)OR=EWIFWOR%=ORcontrol-ORtreatedORcontrol×100RI%=REcontrol-REtreatedREcontrol×100RE=EWIFW×%Ewhere 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.


### 2.4.2. 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 12.5μl/ml to 0.024 μl/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.


## 2.4.1. 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 0.097μl/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: LICIAL®, 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]:(1)OR=EWIFWOR%=ORcontrol-ORtreatedORcontrol×100RI%=REcontrol-REtreatedREcontrol×100RE=EWIFW×%Ewhere 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.


## 2.4.2. 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 12.5μl/ml to 0.024 μl/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.


## 2.5. 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 LC50, 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]. The Tukey test was used to identify differences between mean values of the LC50, LC90, and LC95 which were obtained at 24 h and after 6 days for each EO. All these statistical analyses were performed using the Social Science Statistics Software (SPSS) for Windows, version 20.0. [13] (IBM Corp. released 2011 IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.)


## 3. Results

According to the results of GC-MS chromatographic analysis (Table1), the major components of river red gum EO are p-cymene, (+) spathulenol, (E, E) –farnesol, α-pinene, cuminic aldehyde, 1-phellandrene, sabinene, carvacrol, and p-cymen-7-ol. For blue gum EO, 1,8-cineole, α-pinene, viridiflorol, camphene, d-pinocarvone, and (+) - aromadendrene are the major components. The major constituents of lavender EO were α-thujone, camphor, camphene, D-fenchyl alcohol, l-bornyl acetate, terminalol L, dl -limonene, α-pinene, and linalool L. The main chemical components of oregano EO were carvacrol, p-cymene, and γ-terpinene followed by β-myrcene, o-cymene, thymol, trans-caryophyllene, α-pinene, and α-terpinene. Essential oil of rosemary contained 1,8-cineole and l-camphor as major components; other recorded compounds were α-pinene, borneol L, camphene, α-terpineol, β-pinene, trans-caryophyllene, l-bornyl acetate, β-myrcene, and γ-terpinene. In addition to the major components of thyme EO (carvacrol, p-cymene, and γ-terpinene), other compounds from fractionation included transcaryophyllene, m-thymol, β-myrcene, and α-terpinene.Table 1
Chemical composition of the essential oils tested.
No. Compoundsa KIb Area (%) EC c EG d LS e OF f RO g TC h 1 Tricyclene 921 - - 0.66 - 0.15 - 2 α-thujene 928 - - - 0.51 - 0.33 3 α-pinene 941 4.41 14.23 1.75 1,93 12.06 0.71 4 camphene 953 - 4.60 7.31 0.39 6.39 - 5 sabinene 977 2.43 - - - - - 5 β-pinene 978 - 0.35 0.08 0.22 3.61 - 6 β-myrcene 992 0.76 - - 3.57 2.23 1.24 7 α-phellandrene 998 4.04 - - - - 0.22 8 Δ,3-carene 1004 - - - - - 1.01 9 α-terpinene 1015 - - 0.10 1.37 - 1.15 10 p-cymene 1018 17.45 - - 18.35 - 6.62 11 o-cymene 1026 - - - 3.53 - - 12 dl-limonene 1030 - - 2.13 - - - 13 β-ocimene 1040 0.14 - - 0.07 - - 14 d-pinocarvone 1042 - 4.31 - - - - 15 1,8-cineole 1046 - 40.27 - - 26.90 - 16 (E)-ocimene 1054 0.08 - - - - - 17 α-terpinolene 1063 0.76 0.46 - - 0.57 - 18 γ-terpinene 1065 0.84 0.25 0.10 11.32 1.37 3.96 19 trans-sabinene hydrate 1070 - - - - 0.03 - 20 α-terpinolene 1088 - - - 0.32 - - 21 α-thujone 1099 - - 25.49 - - - 22 linalool L 1102 0.64 - 1.43 0.50 0.41 - 23 d-fenchyl alcohol 1139 - - 6.85 - - - 24 l-camphor 1152 - - 20.06 - 19.00 - 25 pinocarvone 1165 - - 0.18 - - - 26 borneol L 1168 - - 3.99 - 11.76 - 27 4-terpinenol 1178 - 0.39 0.83 - - 0.54 28 p-cymene-8-ol 1182 - - 0.94 - - - 29 α-terpineol 1190 - - - - 5.77 - 30 myrtenal 1193 - - 0.68 - - - 31 myrtenol 1195 - 0.28 0.56 - - - 32 verbenone 1205 - - 0.88 - - - 33 fenchyl acetate 1210 - - 0.97 - - - 34 trans-(+)-carveol 1212 0.16 0.51 - - - - 35 β-citronellol 1217 - - 0,36 - 0.09 - 36 isobornyl formate 1233 - - 0,13 - - - 37 pulegone 1237 - - - - 0.26 - 38 cuminic aldehyde 1240 4.29 - - - - - 39 l-carvone 1241 - - 0.51 - - - 40 carvacrol methyl ether 1244 - 0.30 0.96 - 0.09 - 41 citrol 1255 - 0.11 - - - - 42 piperitone 1258 0.57 - - - - - 43 l-bornyl acetate 1285 - 0.08 5.51 - 3.00 - 44 thymol 1286 - - - 2.04 0.10 2.14 45 p-cymen-7-ol 1291 1.56 - - - - - 46 carvacrol 1299 1.59 - - 46.82 0.36 78.78 47 piperitenone 1339 - - - 1.18 0.16 - 48 γ-pyronene 1345 0.92 - - - - - 49 α-cubebene 1351 - - 0.09 - - - 50 eugenol 1359 - - - - 0.05 - 51 (+)-cyclosativene 1362 - - 0.25 - - - 52 copaene 1375 0.05 0.05 0.15 - 0.14 - 53 carvacryl acetate 1391 - - - - - 0.80 54 methyl eugenol 1398 - - - - 0.14 - 55 calarene 1409 - 0.19 - - - - 56 α-humulene 1413 - 0.17 - - 0.41 - 57 transcaryophyllene 1418 - - 0.11 2.00 2.30 2.27 58 (+)-aromadendrene 1440 0.21 2.53 - - 0.02 - 59 β-patchoulene 1441 - - - - 0.04 - 60 β-elemene 1445 0.17 - - - - - 61 caryophyllene 1454 - 0.16 - 0.21 - 0.09 62 aromadendrene 1467 1.86 - - - - - 63 α-gurjunene 1475 0.06 0.31 - - - - 64 trans-β-farnesene 1479 0.69 - - - - - 65 γ-gurjunene 1481 - 0.31 - - - - 66 α-curcumen 1482 - - - 0.15 - - 67 germacrene-D 1485 0.07 - 0.11 - - - 68 ledene 1489 - 0.95 - 0.15 - - 69 α-guaiene 1491 - 0.04 - - - - 70 β-selinene 1493 0.24 0.04 - - 0.02 - 71 eremophilene 1502 - 0.41 - - - - 72 α-muurolene 1504 - - - - 0.03 - 73 δ-guaiene 1505 - 1.37 - - - - 74 bicyclogermacrene 1505 1.28 - - - - - 75 β-bisabolene 1506 - - 0.03 0.41 0.03 - 76 α-amorphene 1511 0.05 0.04 - 0.06 0.19 - 77 γ-cadinene 1513 - 0.16 - - - - 78 Δ-cadinene 1524 0.20 0.09 0.53 - 0.16 - 79 β-sesquiphellandrene 1525 - - - 1.34 - - 80 zingiberene 1526 - - - - - - 81 cadina-1,4-diene 1532 - - 0.13 - - - 82 ledol 1560 0.66 - - - - - 83 (-)-allospathulenol 1576 0.89 - - - - - 84 spathulenol 1581 13.45 - - - - - 85 caryophyllene oxide 1582 - - 0.48 0.54 0.49 - 86 viridiflorol 1587 - 8.14 1.89 - - - 87 caryophyllenol-I 1641 - - 0.34 - 0.36 - 88 t-muurolol 1641 1.03 - - - - - 89 torreyol 1643 - - 0.22 - - - 90 isospathulenol 1644 1.57 - - - - - 91 cadalin 1652 - - 0.15 - - - 92 (Z,Z)-farnesal 1720 0.72 - - - - - 93 trans-farnesol 1722 5.37 - - - - - 94 farnesal 1738 1.08 - - - - - 95 farnesyl acetate 3 1825 0.43 - - - - - a Identification of components based on GC-MS Wiley 7.0 version library and National Institute and Technology 05 MS (NIST) library data. b KI: Kovats Indices on HP-5MS capillary column. c River red gum. d Blue gum. e Lavender. f Oregano. g Rosemary. h Thyme.Thyme EO completely inhibited tick oviposition at 100% at a concentration of 1.562μl/ml whereas the other EO induced this same effect at greater concentrations ranging from 3.125 to 6.25 μl/ml (Table 2). At the same concentration (1.562 μl/ml), thyme EO completely inhibited reproduction ofH. scupense. This concentration is significantly lower than what was recorded with the other EO (3.125 - 6.25 μl/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.Table 2
Oviposition rates (%OR), hatching of eggs (%E), percentage of reproductive efficiency (%RE), and percent reproduction inhibition (%RI) of femaleH.scupense ticks with different concentrations of essential oils tested.
Essential oil (μl/ml) OR (%) E (%) RE (%) RI (%) EC 0.097 31.37 ± 7.12 g 65.99 ± 0.03 fe 28.41 ± 3.79 hg 54.71 ± 3.84 g 0.195 38.54 ± 0.56 hg 62.86 ± 5.74 e 24.24 ± 6.28 gfe 61,37 ± 7.09 hg 0.390 42.84 ± 3.89 i 61.86 ± 1.18 e 22.19 ± 2.18 f 64.64 ± 5.26 ih 0.781 52.02 ± 4.42 j 59.42 ± 3.87 dc 17.89 ± 2.19 e 71.49 ± 4.52 jih 1.562 64.56 ± 9.11 k 54.69 ± 7.88 c 12.16 ± 1.82 d 80.62 ± 1.86 k 3.125 81.22 ± 0.59 l 8.52 ± 2.69 b 1.00 ± 0.10 b 98.40 ± 0.36 m 6.25 94.49 ± 1.19 m 0.00 a 0.00 a 100.00 n 12.50 100.00 n 0.00 a 0.00 a 100.00 n  EG 0.097 2.69 ± 0.47 b 99.53 ± 0.14 j 64.71 ± 6.17 nml 3.15 ± 1.48 a 0.195 4.85 ± 0.28 c 99.38 ± 0.04 j 63.19 ± 6.92 ml 5.43 ± 2.63 ba 0.390 5.93 ± 1.58 c 98.64 ± 0.17 ih 62.00 ± 4.69 l 7.21 ± 1.92 cb 0.781 19.20 ± 3.61 d 98.57 ± 0.18 ih 53.22 ± 2.55 kj 20.35 ± 6.77 d 1.562 21.76 ± 5.91 ed 97.84 ± 1.88 h 51.15 ± 6.09 ji 23.45 ± 8.02 ed 3.125 36.86 ± 0.53 hg 8.04 ± 2.69 b 3.39 ± 1.18 c 94.92 ± 2.09 l 6.25 43.02 ± 3.06 i 0.00 a 0.00 a 100.00 n 12.50 100.00 n 0.00 a 0.00 a 100.00 n  LS 0.097 1.53 ± 0.09 a 99.44 ± 0.09 e 65.43 ± 0.08 l 2.08 ± 0.12 a 0.195 3.09 ± 1.59 a 99.11 ± 0.54 e 64.18 ± 0.49 k 3.95 ± 0.74 b 0.390 3.23 ± 0.91 a 95.51 ± 1.48 d 61.76 ± 1.35 j 7.57 ± 2.02 c 0.781 33.36 ± 6.21 ecd 94.21 ± 2.83 d 41.95 ± 1.78 f 37.21 ± 2.66 gi 1.562 60.01 ± 6.53 g 93.40 ± 3.32 d 24.96 ± 1.25 d 62.65 ± 1.87 igfh 3.125 99.88 ± 0.01 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 6.250 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 12.50 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l  OF 0.097 10.90 ± 2.46 bc 91.80 ± 2.48 d 54.66 ± 2.09 i 18.21 ± 3.13 d 0.195 21.15 ± 9.18 cd 90.58 ± 4.58 d 47.73 ± 3.41 g 28.58 ± 5.11 fi 0.390 41.53 ± 0.57 f 89.68 ± 1.13 d 35.04 ± 0.62 e 47.57 ± 0.93 hi 0.781 45.48 ± 3.66 f 70.37 ± 7.33 c 25.64 ± 3.78 d 61.64 ± 5.65 i 1.562 47.98 ± 4.57 f 4.31 ± 0.99 b 1.50 ± 0.49 b 97.76 ± 0.73 k 3.125 100 ± 0.00 i 0.98 ± 0.01 a 0.00 ± 0.00 a 100 ± 0.00 l 6.250 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 12.50 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l  RO 0.097 0.36 ± 0.08 a 97.33 ± 1.18 h 64.80 ± 6.45 nml 3.02 ± 0.17 a 0.195 21.25 ± 2.64 ed 96.90 ± 0.59 h 50.99 ± 2.09 ji 23.70 ± 2.94 ed 0.390 23.75 ± 2.00 fe 93.13 ± 2.91 g 47.45 ± 3.56 i 28.99 ± 1.06 fe 0.781 31.79 ± 3.48 g 95.55 ± 1.01 g 43.55 ± 1.63 i 34,82 ± 1.59 g 1.562 95.72 ± 1.91 m 0.00 a 0.00 a 100.00 n 3.125 100.00 n 0.00 a 0.00 a 100.00 n 6.25 100.00 n 0.00 a 0.00 a 100.00 n 12.50 100.00 n 0.00 a 0.00 a 100.00 n  TC 0.097 9.04 ± 2.33 b 99.77 ± 0.02 e 60.65 ± 0.02 j 9.24 ± 0.03 c 0.195 21.42 ± 1.09 c 99.57 ± 0.11 e 52.28 ± 0.08 hi 21.76 ± 0.12 e 0.390 27.45 ± 5.07 de 98.47 ± 0.87 e 47.74 ± 0.60 g 28.55 ± 0.89 fi 0.781 67.79 ± 0.82 h 98.23 ± 0.66 e 21.14 ± 0.20 c 68.36 ± 0.30 j 1.562 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 3.125 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 6.25 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l 12.50 100 ± 0.00 i 0.00 ± 0.00 a 0.00 ± 0.00 a 100 ± 0.00 l Control - 100 ± 0.00 f 66.82 ± 0.00 m - Means within a column followed by the same letter are not significantly different by the Kruskal Wallis test (p ≥ 0.05).Table 3
Lethal concentrations of 50%, 90%, and 95% ofH. scupense larvae with different doses of essential oils tested.
Essential oil LC50 (μl/ml) LC90 (μl/ml) LC95 (μl/ml) (μl/ml) 24 h 6j 24 h 6j 24 h 6j EC 0.207 a 0.003 a 1.653 b 0.066 a 2.978 b 0.151 a EG 0.155 a 0.081 c 2.387 b 0.705 a 5.183 c 1.301 b LS 0.253c 0.039b 2.212c 0.324c 4.092c 0.587c OF 0.131b 0.030b 0.982b 0.176b 1.740 b 0.290b RO 0.108 a 0.017 b 0.495 a 0.073 a 0.761 a 0.110 a TC 0.058a 0.016a 0.358a 0.058a 0.600a 0.083a Means within a column followed by the same letter are not significantly different by the Tukey test (p ≥ 0.05).Figure 1
Percentage mortality forH.scupense larvae exposed to concentrations of river red gum essential oil.Figure 2
Percentage mortality forH.scupense larvae exposed to concentrations of blue gum essential oil.Figure 3
Percentage mortality forH.scupense larvae exposed to concentrations of lavender essential oil.Figure 4
Percentage mortality forH.scupense larvae exposed to concentrations of oregano essential oil.Figure 5
Percentage mortality forH.scupense larvae exposed to concentrations of rosemary essential oil.Figure 6
Percentage mortality forH.scupense larvae exposed to concentrations of thyme essential oil.


## 4. Discussion

In general, most of the major components of the EO tested in our study (carvacrol, 1,8-cineole,α-thujone, borneol L, α-pinene, p-cymene, and α-terpinene) have been identified as major acaricidal agents around the world [14–20]. Acaricide and insecticidal properties of other Thymus species have been noted by several authors [21, 22]. Many other studies on the acaricidal properties of EO extracted from other Origanum species have been carried out by Ramzi et al. [23], Koc et al. [24], Cetin et al. [15], and Coskun et al. [16]. Moreover, the larvicidal activity of α-pinene (from river red gum) has also been documented againstAedes aegypti,Aedes albopictus [25], andAnopheles stephensi [26]. In our study, 1,8-cineole from rosemary and blue gum EO exhibited excellent acaricidal activity againstH. scupense. Indeed, according to Pirali-Kheirabadi et al. [27], this EO showed an ovicidal, larvicidal, and adulticidal activity againstRhipicephalus annulatus. Furthermore, Martinez-Velazquez et al. [28] asserted that the EO of rosemary plant is lethal to another tick species (Rhipicephalus microplus). We found significant acaricidal activity of α-thujone in lavender EO on the reproductivity of adults as well as larvicidal action againstH. scupense.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.


## 5. Conclusion

We found excellent acaricidal activity of several EO constituents that were evaluated againstH. scupense in this study. Our results indicate that these compounds may provide real 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.



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*Source: 7805467-2019-02-07.xml*