Experimental Parasitology 126 (2010) 184–186
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Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control Khodadad Pirali-Kheirabadi a,*, Jaime A. Teixeira da Silva b a b
Department of Pathobiology, Faculty of Veterinary Medicine and Research Institute of Zoonotic Diseases, University of Shahrekord, Shahrekord 115, Iran Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken 761-0795, Japan
a r t i c l e
i n f o
Article history: Received 19 August 2009 Received in revised form 26 January 2010 Accepted 20 April 2010 Available online 28 April 2010 Keywords: Lavandula angustifolia Rhipicephalus (Boophilus) annulatus Bioacaricide
a b s t r a c t Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC50, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatus in vitro. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Ticks are blood-sucking external parasites of mammals, birds and reptiles throughout the world and can transmit a wide variety of pathogens (Samish and Rehacek, 1999), including bacteria, rickettsiae, protozoa and viruses to humans and animals. They are becoming more resistant to acaricides (Onofre et al., 2001; Ducornez et al., 2005). In addition, chemical substances that are widely used for pest control have a considerable geno- and cytotoxic effect on human target cells (Ündeg˘er and Basßaran, 2005). Since the degradation of synthetic acaricides takes considerable time, they remain as residues in agricultural environments and induce resistant strains of ticks (Drummond, 1977; Onofre et al., 2001). Thus, there is increasing interest in alternative methods for tick control and a growing demand for non-chemical products in agriculture that are safer to animals and the environment (Georghiou and Lagunes-Tejada, 1991; Samish and Rehacek, 1999). The use of herbal medicine as acaricides is one option. Lavandula angustifolia (Lamiaceae), native to the western Mediterranean region, is a strongly aromatic shrub growing 1–2 m in height and is a commonly used household herb (Chevallier, 1996). The essential oil * Corresponding author. Fax: +98 3814424427. E-mail addresses:
[email protected],
[email protected] Kheirabadi).
(K.
0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.04.012
Pirali-
(EO) obtained from lavender flowers has several medicinal properties and biological activities: antihalitosis, powerfully antiseptic, antispasmodic, aromatic, carminative, cholagogue, diuretic, nervine, sedative, stimulant, stomachic and tonic (Chiej, 1984; Grieve, 1984; Bown, 1995). Its powerful antiseptic properties are able to kill many common microorganisms such as typhoid, diphtheria, Streptococcus and Pneumococcus; it also has antiviral properties and is a powerful antidote to some snake venoms (Phillips and Foy, 1990). It is also widely used in the fragrance, cosmetics and food industries (Porto et al., 2009). Very few field experiments to kill ticks using any EOs have been performed. In order to prove their value as commercial tick-control agents (i.e. as a biocontrol agent), we aimed, for the first time ever, in this study to evaluate the effect of L. angustifolia EO on adult stages of Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) in in vitro trials. 2. Material and methods 2.1. Tick rearing Adult R. (B.) annulatus were collected from at least five naturally infested Holstein calves in the forests of Mazandaran province, the northern district of Iran near the Caspian Sea. Engorged female ticks were selected and maintained in the laboratory at 25 °C and 80% relative humidity (RH) in cotton-capped test tubes (5 ticks/
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10 ml tube) for laying eggs. Males were discarded since they die spontaneously after copulation with female ticks. After the eggs hatched, 10-day old larvae were placed on healthy 2–3 monthsold Holstein calves to feed. Engorged female ticks were collected after 15–22 days from the infected calves and transferred to the laboratory within 3–4 h, washed in 70% ethanol and dried (1 min at room temperature) to perform subsequent experiments. 2.2. Essential oil EO prepared from the aerial parts of L. angustifolia was purchased from Barij Essence Co., Kashan, Iran. These oils were prepared according to the hydrodistillation method of Jalali-heravi et al. (2006). 2.3. Bioassay on ticks Serial EO concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v using 60% ethanol as solvent) was prepared and the acaricidal effect of each concentration were tested on five engorged R. (B.) annulatus female ticks. This experiment was repeated in triplicate. The ticks were immersed in each concentration for 1 min then transferred to Petri dishes containing moist filter paper and incubated for 24 h at 25 °C and 80% RH in a germinator (Pars Novin Asa, Iran). For all concentrations, a control group of five ticks was also treated in the same manner, except that they were immersed in 60% ethanol instead of lavender EO solution. Ticks were observed at 6, 15 and 24 h post inoculation (hpi) with a stereo microscope (Olympus CX41, Japan) and the mortality rate and mass of eggs produced in each group was recorded. Failure in egg laying and EO lethal concentrations (LC/EC50 and LC/EC99) were also calculated using a Probit test Ver. 1.5. The dead ticks were diagnosed based on three specific signs and features: increasing cuticle darkness, stopped malpighian tube movement and hemorrhagic skin lesions. Failure in egg laying was then calculated and was based on direct observation. To quantify egg-laying ability, egg masses were weighed using an analytical scale at the end of the experiment after having removed tick cadavers. 2.4. Statistical analysis Data were expressed as the mean ± standard error of the mean (SEM). Groups were compared using one-way ANOVA and means were compared by post hoc tests using SPSS Ver. 14. Values of P < 0.05 were considered significant.
reached 100%. L. angustifolia EO-induced mortality was significant for concentrations more than 4.0% compared with the non-treated control (P < 0.05). There was egg-laying failure (ELF) and incapacity to mass-produce eggs (MPE) by adult female ticks exposed to lavender EO compared with non-treated controls (Tables 2 and 3). MPE decreased in a dose-dependent manner in ticks exposed to the EO but was only significant at 4.0%. Egg-laying failure was qualitatively noticed via direct observation. All groups, including treatments and controls, were checked every 8 h and data was recorded accordingly. Any changes in ticks, such as laying of eggs, were added to previous data. The highest ELF (100%) was observed for groups treated with 6.0 and 8.0% EO while ticks in non-treated control could lay eggs (Table 3). At EO concentrations greater than 2.0%, highest mortality was observed early, i.e., at 6 hpi, and subsequently decreased. In contrast, the mortality rate was 73.26% at 24 hpi in ticks treated with 4.0% EO but there was no significant mortality at [EO] less than 4.0% (Table 1). The LC/EC50 was 2.76% while the LC/EC99 was 8.84%. 4. Discussion The value of medicinal plants as commercial tick-control agents has yet to be proved and their high degree of safety and effectiveness make them promising candidates for use as commercial tick biocontrol agents, although additional formulations, chemical and photostability and studies on their application routes are needed to support this statement. Different microorganisms have now been introduced as pathogens or predators of ticks, e.g. bacteria (Hassanain et al., 1997; Zhioua et al., 1999), entomopathogenic fungi (Onofre et al., 2001; Pirali-Kheirabadi et al., 2007a,b), surprisingly Some studies have documented the biocontrol of ticks by medicinal plants and have used herbal medicine such as EOs of aromatic plants e.g., Cunila spp. (Lamiaceae), Pelargonium roseum and Eucalyptus globulus against adult stage of R. (B.) annulatus and R. (B.) micropolus in vitro (Apel et al., 2009; Pirali-Kheirabadi et al., 2009). Also limonene, limonene oxide, b-amino alcohol derivatives and thymol were used on the cattle tick R. (B.) micropolus and engorged nymphs and females of Rhipicephalus
Table 2 Mass of produced eggs (mean ± SEM; n = 15) in engorge female of R. (B.) annulatus treated by L. angustifolia 24 h post inoculation.
3. Results L. angustifolia EO showed strong acaricidal effects against R. (B.) annulatus and the mortality rate of ticks was dose-dependent (Table 1), showing significant acaricidal activity even at 6 hpi. After 1 min exposure of adult female ticks to 8.0% EO, the mortality rate
Table 1 Mortality (mean ± SEM; n = 15) in engorge female of R. (B.) annulatus treated by L. angustifolia oil 24 h post inoculation.
*
*
*
Essential oil concentration (%)
Produced egg (g)
Control 0.5 1.0 2.0 4.0 6.0 8.0 ND = not detected
0.1220 ± 5.033E 03 0.1233 ± 3.528E 03 0.1040 ± 7.211E 03 0.1028 ± 4.338E 03 2.777E 02 ± 2.295E 02* ND (zero) * ND (zero) *
Indicates significant difference between treatments and control (P = 0.05).
Table 3 Failure in egg laying (mean ± SEM; n = 15) in engorge female of R. (B.) annulatus treated by L. angustifolia oil 24 h post inoculation.
Essential oil concentration (%)
Mortality (%)
Essential oil concentration (%)
Egg-laying failure (%)
Control 0.5 1.0 2.0 4.0 6.0 8.0
0±0 0±0 6.6 ± 6.6 33.3 ± 6.6 73.3 ± 17.6* 100 ± 0* 100 ± 0*
Control 0.5 1.0 2.0 4.0 6.0 8.0
0±0 0±0 6.6 ± 6.6 33.3 ± 6.6 80 ± 11.7* 100 ± 0* 100 ± 0*
Indicates significant difference between treatments and control (P = 0.05).
*
Indicates significant difference between treatments and control (P = 0.05).
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sanguineus, respectively (Ferrarini et al., 2008; de Oliveira Monteiro et al., 2009). Results from this study indicate clear and irrefutable evidence that L. angustifolia EO had a good acaricidal effect against R. (B.) annulatus females. Over the past decades, plant EOs were widely used against phytophagous pests and mosquitoes (Çalmasßur et al., 2006), ticks (Iori et al., 2005) and mites (Kim et al., 2004). Despite the anti-microbial and antiviral activities of L. angustifolia (Kim and Lee, 2002), the acaricidal activity of this medicinal plant against cattle fever tick, R. (B.) annulatus, had never been tested or previously reported. The primary components of lavender EO are linalool (51%) and linalyl acetate (35%) (Prashar et al., 2004). In a study by Perrucci et al. (1998) the acaricidal activities of L. angustifolia EO and linalool (i.e. its main component), were investigated against Psoroptes cuniculi, a mite. Both the EO and linalool exhibited acaricidal properties and linalool was detected in the ether extract of treated mites. The acaricidal activity of L. angustifolia against R. (B.) annulatus observed in our study might thus be attributed to linalool and linalyl acetate as the major plant constituents. The mode of action of lavender EO resembled that of geranium and peppermint oils, which also have acaricidal activity (Lis-Balchin and Hart, 1999; Pirali-Kheirabadi and Razzaghi-Abyaneh, 2007). Chemical analysis of Tanacetum vulgare extracts indicated that B-thujone is the major compound of the EO (87.6%) and probably contributes significantly to the acaricidal activity of this EO (Chiasson et al., 2001). The acaricidal efficacy of the herbal ectoparasiticide aerosol spray, ECTOZEE was evaluated by spraying dogs heavily infested with ticks (Boophilus microplus). A single spray resulted in 100% mortality of adult males, nymphs and larvae and no apparent toxicity was developed (Das and Bhatia, 1993). In a previous study, we showed a low to moderate acaricidal effect of chamomile (Matricaria chamomile) flower extract against the adult stage (females) of R. (B.) annulatus (Pirali-Kheirabadi and Razzaghi-Abyaneh, 2007). This finding encouraged us to search other medicinal plants as potential biocontrol agents of ticks. In conclusion, this study shows the potent effect of L. angustifolia EO as a powerful acaricidal agent against R. (B.) annulatus. We are now testing whether this EO is a candidate agent in the control of cattle fever ticks in the field.
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