Repellent Effects of the Essential Oil of Lavendula Angustifolia against Adults of Hyalomma Marginatum Rufipes

INTRODUCTION Ticks and the diseases they transmit to man and animals continue to be a problem particularly in developing countries 1,6. The current tick control measures rely largely on the use of synthetic acaricides such as amitraz 11,12 and N,N-diethyl-m-toluamide (DEET) 5. However, the persistence of problems resulting from ticks and the diseases they transmit indicate that these tick control methods are at the most partially effective. In addition, the positive effects of these synthetic chemical products are marred by the problems that accompany their widespread use which include the emergence of tick strains resistant to acaricides 14 , the accumulation of toxic substances in the environment and in products destined for human consumption 2,7 and the high cost of these chemicals 15 making them inaccessible by small scale farmers. This situation raises the need for alternative tick control methods which pose lesser problems to the environment , animals and man. Recently, some plant-based products have been evaluated for tick repellent properties 10,16,18. The results obtained from these studies are quite promising and as a result present plants as possible sources of anti-tick agents. Furthermore, the recent


INTRODUCTION
Ticks and the diseases they transmit to man and animals continue to be a problem particularly in developing countries 1,6 .The current tick control measures rely largely on the use of synthetic acaricides such as amitraz 11,12 and N,N-diethyl-mtoluamide (DEET) 5 .However, the persistence of problems resulting from ticks and the diseases they transmit indicate that these tick control methods are at the most partially effective.In addition, the positive effects of these synthetic chemical products are marred by the problems that accompany their widespread use which include the emergence of tick strains resistant to acaricides 14 , the accumulation of toxic substances in the environment and in products destined for human consumption 2,7 and the high cost of these chemicals 15 making them inaccessible by small scale farmers.This situation raises the need for alternative tick control methods which pose lesser problems to the environment, animals and man.
Recently, some plant-based products have been evaluated for tick repellent properties 10,16,18 .The results obtained from these studies are quite promising and as a result present plants as possible sources of anti-tick agents.Furthermore, the recent intensification of research on anti-arthropod properties of plant-based products is motivated by the belief by many researchers 3,19 that plant-based anti-arthropod products are biodegradable and may be affordable for use even by small scale farmers.Most recently, Jaenson et al. 8 showed that the essential oil of Lavendula angustifolia diluted to 30 % in 1,2-Propanediol had 100 % repellencies against the nymphs of Ixodes ricinus.In order to gain additional insight on the repellant strengths of the essential oil of L. angustifolia against ticks, the present study compared its tick repellant properties against those of DEET using adults of Hyalomma marganitum rufipes.

Ticks, hydrodistillation and climbing repellency bioassay
Colonies of Hyalomma marganitum rufipes used in this study were bred on Himalayan rabbits at the Animal Production unit of the Department of Biology, Medunsa campus of the University of Limpopo.Off-host stages of this tick species were kept at 25 ± 1 °C and 75 ± 5 % relative humidity (RH) in glass humidity chambers under natural day and night regimen.
The extraction of the essential oil of L. angustifolia, was done by hydrodistillation.L. angustifolia plants were obtained from Plant Land Akasia Malanseuns nurs-ery in Pretoria North.Fresh leaves, soft branches and inflorescences weighing 170 g were sliced into smaller pieces and together with 700 m distilled water were introduced into the round-bottom flask and hydrodistilled for 5 hours using a Clevenger-type apparatus.The apparatus was equipped with a thermometer attached on top of the round-bottom flask.A heat-mantle was used to maintain a temperature of 90 °C.The distillate was collected with the essential oil forming a band above water.The essential oil yield was 0.56 m /170 g × 100 = 0.33 % v/w.This procedure was repeated in order to have more essential oil of L. angustifolia.Dichloromethane dilutions (concentrations) of the distillate were prepared by making 2 m of solution with 100, 200 and 400 µ quantities of the essential oil in varied amounts of dichloromethane.The following concentrations were consequently obtained: 5, 10 and 20 % v/v.To allow for comparison, similar concentrations were prepared for N,N-diethyl-mtoluamide (DEET) (DEET Technical, Volcano Agroscience, 95 %), a commercialised tick repellent.DEET is viewed as the standard ingredient of commercial repellents against which the performance of other compounds can be generally evaluated 13,20 .
The climbing repellency bioassay used in this study, was a modification of that described by Carroll 4 .However, the modification was substantial enough necessitating a full re-description.The bioassay is based on the climbing behaviour of ticks.Except for the genus Amblyomma, ticks naturally climb up vegetation to quest for a host 17 .A glass vial (height of 7.2 cm and diameter of 2.3 cm) filled with polystyrene was firmly inserted on the centre of the glass beaker.The polystyrene provided support to the vertically inserted glass rod (length 22.1 cm) and also served as a platform on which ticks were placed.Additional polystyrene was used to support the glass vial, preventing it from falling in the glass beaker (see Fig. 1).Water was poured into the beaker to completely surround the glass vial and to almost reach its height.This was done to discourage ticks from crawling away from the polystyrene platform and to stabilise humidity 4 .The top 5 cm of the glass rod was covered with Whatman number 1 filter paper (2.5 × 5 cm) on which the plant extract or DEET was released.The second filter paper of the similar kind and size was fixed just below the top one to serve as neutral filter paper.No essential oil of L. angustifolia or DEET was released on this filter paper.The neutral filter paper provided an alternative questing place comparable with the treatment filter paper.Similarly, a control was set up save that only an appropriate solvent was released on the top filter paper (Fig. 1).The solvent in both the treatment and control filter papers was allowed to evaporate for 15 minutes before the start of the experiment.

Experimental procedure
Ten unsexed H. m. rufipes ticks (3-5 weeks old) were placed on a platform of the treatment apparatus and subsequently the same was done on the control apparatus.Prior to the start of the experiment the ticks were allowed 15-minute acclimatisation period, following which their position on the glass rod was noted at 10-minute intervals for 60 minutes and thereafter at 30-minute intervals until the 120th minute.After each 10-minute interval ticks on the glass rods were moved back to the polystyrene platform.
Ticks on the treatment or control filter paper were considered not repelled while ticks not found on the treatment or control filter paper (i.e.those found on the neutral filter paper or glass rod) were considered repelled.Ticks that dropped into surrounding water were dried using Kimberly-Clark paper towel and replaced onto the platform using forceps.Five replications were done for each concentration of the essential oil and DEET.The effective concentration to repel 50 % (EC50) of the ticks was calculated using Probit analysis a free software package (US Environmen-tal Protection Agency; http://www.epa.gov/nerleerd/nerleerd/stat2.htm) 21.Percentage repellency was calculated using the formula 9 : Percentage repellency = 100 -[mean number of ticks on test]/[Mean number of ticks on control] × 100.
Mann-Whitney U-tests (two-tailed) (using Analyse-it for Microsoft Excel © 1997-2000; Analyse-it Software: http:// www.analyse-it.com/order.asp)were used to determine the significance of the differences between treatment and control.Student's t-tests were used to calculate the significance of the differences between the repellent effects of DEET and essential oil of L. angustifolia.

RESULTS
The results obtained in this study are summarised in Tables 1 and 2. Figure 2 shows that ticks avoided filter paper treated with essential oil of L. angustifolia.They settled on the lower neutral filter paper.This is unlike ticks that quested at the top of the control glass rods on the control filter paper.Similar, observations were made for DEET.
High percentage repellency (70 -100 %) against adults of H. m. rufipes was recorded in all 3 concentrations (i.e. 5, 10 and 20 % v/v) of L. angustifolia used in this study, although for 5 % v/v concentration it only persisted for 40 minutes compared to 120 minutes of the other (10 and 20 % v/v) concentrations (see Tables 1, 2).In general the repellent strength of the essential oil of L. angustifolia against adults of H. m. rufipes was similar (P > 0.05) to that of DEET against the same tick species.EC50 could not be calculated at the 10th, 20th and 30th minutes probably because data did not contain at least two concentrations for which the percenge responding is between 0 and 100 % for both DEET and L. angustifolia.From the 40th minute to the 120th minute, the EC50 generally increased with increasing time for both L. angustifolia and DEET.

DISCUSSION AND CONCLUSIONS
The present study describes the repellent properties of the essential oil of L. angustifolia diluted in dichloromethane against adults of H. m. rufipes ticks.As  indicated in our introduction, the recent study by Jaenson et al. 8 showed that the essential oil of L. angustifolia diluted to 30 % in 1,2-Propanediol had 100 % repellencies against the nymphs of Ixodes ricinus.Our results support these findings and further indicate that the repellent effects of the essential oil of L. angustifolia are not only specific to I. ricinus but are also effective against other tick species such as H. m. rufipes.Tick repellence in our study was demonstrated for each of 5 %, 10 % and 20 % concentrations established by diluting the essential oil of L. angustifolia in dichloromethane.The repellency appears to be dose-dependent since high repellence persisted for longer periods in higher concentrations than lower once.Also, data obtained from this study show that the percentage repellency recorded for L. angustifolia compared favourably with those obtained for DEET.
Previously, Lwande et al. 13 also showed that the essential oil of Gynandropsis gynadra compared favourably with DEET in repelling Rhipicephalus appendiculatus ticks.These findings clearly suggest that some plant species may contain anti-tick agents that may be equally effective against ticks as some of the commercially traded synthetic acaricides.When properly identified and characterised, such plant-based anti-tick agents may enjoy widespread use since it is generally believed that they pose less of a threat to the environment 3,19 than their synthetic counterparts.Furthermore, the use of plant-based products, particularly those from indigenous plants, may save countries the high costs of importing synthetic acaricides 10 .
However, in spite of the promising results obtained in this study and elsewhere 8 regarding the anti-tick properties of L. angustifolia, more studies are necessary in order to further clarify the anti-tick properties of this plant.Furthermore, toxicity tests on animals should be conducted to determine the side-effects of the extracts of this plant species.

Fig. 2 :
Fig. 2: Typical example showing questing position of ticks on treatment and control glass rods.(A) DEET and (B) essential oil of Lavendula angustifolia.TFP, treatment filter paper; CFP, control filter paper; NFP, neutral filter paper; GR, glass rod; T: treatment; C, control.