Mosqu it o R e pellen c y o f E s sen t ia l O i ls Deriv e d f r o m L a o P l a nts Ka r i n A n dersson Degr e e p r oje c t i n b i o lo g y , B a chel o r o f s c i enc e , 2 0 10 Examensarbe t e i b i o lo g i 1 5 h p t i l l k a ndidatexame n , 2 0 10 Biolo g y E d ucati o n C e nt r e a n d D e partme n t o f S y stema t i c B io lo g y , U p ps a l a U n iversity Superviso r s : P r ofess o r T h om a s Ja e ns o n a n d C h an d a V o ngsomba t h , M . S c . 1. Abstract Essential oils or extracts obtained from four plant species growing in Laos were tested for anti-mosquito activity in the field. Solutions of alcohol or acetone containing different concentrations of essential oil; 5, 10 or 19 v/v %, were prepared and tested. Mosquitoes were collected by human baits wearing oil impregnated mosquito nets around their shanks. The number of attracted mosquitoes was compared to the number attracted to positive and negative controls, i.e., human baits wearing nets impregnated with 19 % N,N-diethyl-meta- toluamide (DEET, NN-diethyl-methyl-benzamide) or untreated nets, respectively. A one way ANOVA analysis was conducted on the log-transformed total number of mosquitoes collected using each treatment. The statistical analysis showed that significantly fewer mosquitoes were attracted to the treatment harbouring 19 % oil of Scutellaria angustifolia than to the negative control. No significant decrease in attracted mosquitoes could be detected for the treatments containing oil from the plant Litsea cubeba. However, a tendency of repellency was indicated. Therefore, an investigation running over a longer period of time is desired for Litsea cubeba. No conclusions could be drawn for the remaining two species; Tagetes patula and Citrus sinensis. 1 2. Introduction Mosquitoes are vectors of many infectious agents causing potentially severe diseases, including malaria, dengue fever, filariasis, yellow fever and different types of encephalitis. Out of these diseases, malaria is the one with the largest impact on global health, being endemic in 109 countries and leaving half of the world’s population at risk of contracting the infection. Malaria is transmitted by Anopheles mosquitoes and is caused by protozoan parasites of the genus Plasmodium. The disease is annually causing around one million deaths globally, striking hardest against young children and pregnant women in tropical developing countries (Roll Back Malaria Partnership 2009). Laos is one of the countries in which malaria is endemic where nearly 100 % of the cases are due to Plasmodium falciparum. This is the most virulent species of the malaria parasites, being responsible for the majority of the fatal cases around the world. It was estimated that approximately 60 % of the Lao population was in high risk of getting malaria in 2008, even though the confirmed cases of the infection decreased by over 50 % between 2000 and 2008 (WHO 2009). Therefore, there is still an urgent need of good preventive measures in order to control or eradicate the disease from this area. Since there is no effective vaccine available against malaria, and because of an increasing problem with resistance against conventional anti-malaria drugs, vector control plays a very important role in the fight against this, and other, mosquito-borne infections. Prevention of mosquito bites by natural insect repellents has been used for hundreds of years, and commercial repellents have been on the market since the early 20th century. The first product containing N,N-diethyl-meta-toluamide (DEET) was released in 1956 and still today, around 50 years later, DEET-containing products are the most commonly used commercial mosquito repellents globally (Peterson and Coats 2001). Though DEET is considered to be rather safe and has proved to function excellent as a mosquito repellent, there are still a few drawbacks with the substance. There have for example been reports of toxic effects, including impacts on the central nervous system, following usage (for example Katz et al. 2008, Imperial College 2002 and Briassoulis et al. 2001). Furthermore, it is not recommended to use DEET- containing products during longer periods of time, which makes DEET a repellent more suitable for usage by tourists than local inhabitants living in malaria endemic areas. Many plants have evolved aromas that repel insects and other herbivores (Langerheim 1994 and Pålsson and Jaenson 1999); making essential oils extracted from plants potential sources of anti-mosquito material. Eucalyptus and citronella oils are two well documented natural substances which have proved to be efficient mosquito repellents (for example Batish et al. 2008, Trongtokit et al. 2005). These oils have thus become ingredients in a number of commercial mosquito repellents (Thavara et al. 2002). Other species found to have resident anti-mosquito activities are for example Syzigum aromaticum, Cymbopogon nardus, Pogostemon cablin and Zanthoxylum limonella (Trongtokit et al. 2005). Since both the flora and the mosquito reservoir differ among countries, evaluations of the anti-mosquito activity of numerous plant species are needed in order to find efficient, useful repellents for the people living in areas with high prevalence of mosquito-borne diseases. In this study, the mosquito repellency of essential oils or extracts from four plant species commonly found in Laos is evaluated through collection of mosquitoes using human bait.       2 3. Material and Methods 3.1.1 Selection of plant species Before the beginning of these experiments, people from 63 villages in Laos were interviewed about their traditional usage of botanicals products as mosquito repellents (C. Vongsombath and H. de Boer, unpublished data). Based on the results from these interviews, the following plants were selected for oil extraction: Croton caudatus, Litsea cubeba, Scutellaria angustifolia, Citrus sinensis and Tagetes patula. 3.1.2 Collection of plant material L. cubeba, and a small fraction of the material of C. caudatus, was collected in the Xaysomboun district, Vientiane province, Laos, in June 2009. This material was kept in the freezer until steam distilled in December 2009. The remaining amount of C. caudatus was brought from outside Vang Vieng, Laos, on 11th of December 2009. T. patula was bought at a market in Vientiane Capital, Laos, on 22nd of December 2009 and S. angustifolia was picked outside Fuang, Laos, on 25th of December 2009. Also C. sinensis was brought from outside Vang Vieng, Laos, but in November 2009. The material was refrigerated until steam distilled in December. 3.2 Extraction and preparation of the essential oils As demonstrated in Table 1, the essential oils were obtained from the bark of C. caudatus, the fruit of L. cubeba, all parts of S. angustifolia and the peel of C. sinensis. Attempts were also done to extract oil from the flowers of T. patula. An amount of 0.3-1.1 kg at a time of the chosen plant parts was cut into small pieces and placed in a distillation flask. Water was added until approximately 80 % of the material was covered, whereupon the flask was placed in a distillation chamber and heated until boiling. Steam distillation was carried out for 2 hours, before the oily phase of the distillate was collected in a glass tube. The number of distillation sessions for the different species depended on the yield of essential oils. The extracted oils were stored refrigerated (in about +4 oC) until their mosquito repellent capacities were tested in the field. Table 1: Yield of essential oils obtained from steam distillation of plant material from five different Lao plant species. Species Parts used Fresh weight (kg) Volume of oil (ml) Yield (ml oil/kg material) L. cubeba Fruit 2.4 45 19 S. angustifolia All parts 1.3 6 4.6 C. sinensis Peel 2.0 4 2.0 C. caudatus Bark 2.1 2 0.95 T. patula Flowers 1.0 0 0.00 Since the yield of oil from C. caudatus was so low (Table 1), the species was removed from the experiment and its potential as a mosquito repellent therefore remains to be tested. Steam distillation of T. patula did not generate any oil (Table 1); therefore extractions of the flowers were prepared in three solvents possessing different polarity characteristics. 3 A total of 100 g of the plant material was put into either 100 ml of water, hexane or 70 % ethanol. The mixtures were left standing at room temperature for one week. The plant material was removed from the solvents through two filtration sessions; one using a mosquito net and the other using filter paper. The oils to be tested in the field were diluted in 99.5 % ethanol (L. cubeba) or in pure acetone (S. angustifolia, C. sinensis) until each one had a final volume of 12 ml and final oil concentrations of 5, 10 and 19 v/v %. To avoid rapid evaporation when applied to cloths, an additional volume of 20 µl coconut oil was added to each of the nine solutions. 3.3.1 Night-time experiment The mosquito repellency of the essential oils extracted from L. cubeba and S. angustifolia was evaluated in the surroundings of the village Ban Nalang (Fuang district, Vientiane province, Laos) between 25th and 29th of December 2009. During three hours each night (6 p.m to 9 p.m.), two groups consisting of five volunteers each served as human bait and collected mosquitoes that landed below their knees. The other parts of the bodies, except hands and head, were covered by clothes. Test tubes and torches emitting red light were used during the collection. The mosquitoes caught were transferred to plastic boxes in which a wad of cotton soaked in ethyl acetate was put, resulting in death of the insects. All members in both groups had before the beginning of the collection washed their legs with water and soap and tied a mosquito net (approximately 30 · 50 cm) to their shanks. Three persons in each group had both of their nets impregnated with 0.5 ml of the solutions containing 5, 10 or 19 v/v % of the essential oil in question. Besides, one person in every group served as a negative control and used untreated nets, while the remaining ones were positive controls and wore nets impregnated with Myggspray US 622 (Swedish commercial mosquito repellent) containing 19 % DEET. The volunteers switched treatment every night, so everyone in the groups tested all the concentrations of the oils and was both negative and positive controls before the experiment was completed. Scattered trees and houses were present in the area in which the experiment took place. The volunteers were not sitting closer than 5 metres from each other. During the five days of the experiment, the temperature ranged from 24.0 to 27.3◦C at the starting time and from 17.0 to 19.0◦C at the time for finish. The humidity increased towards the ending time for the experiment every day and was never below 36 %, nor above 68 %. There was no wind or rain at the time for the experiment. 3.3.2 Day-time experiment The same methods and procedures as for the night-time experiment were applied when testing the repellency of extracts of T. patula (diluted in hexane, ethanol or water) and the essential oil extracted through steam distillation of C. sinensis. However, these tests were performed during day-time in the Huay Yang reservation forest (15 km outside Vientiane Capital, Laos) between the 3rd and 5th of January 2010. During the initial two days, two collections of three hours each were carried out per day; one between 9 and 12 a.m. and one between 2 p.m. and 5 p.m. During the period before noon, the temperature ranged from 23.9 to 28.0◦C and the 4 humidity from 42 to 61 % whereas on the afternoons, it ranged from 27.1 to 31.4◦C and from 32 to 53 %, respectively. There was no rain or wind at the time for the experiment. 3.4 Identification of mosquitoes After the completion of the field testing, time was spent on identification of the collected mosquitoes. For this purpose, the computer program KeyMosq98 (Illustrated Key of Mosquitoes in Thailand, developed by the Department of Medical Science, Ministry of Public Health, Thailand, 1998) was used. Mosquitoes that proved difficult to identify were brought to the Malaria Control Centre, Vientiane, for comparison with specimens in their collection. 3.5 Calculations and statistical analyses The mosquito repellency was calculated for all the different concentrations and solutions of the tested plant products through usage of the following formula: % Repellency = [(No. control – No. test product) / No. control]* 100 (Pålsson and Jaenson 1999) The efficacies of the oils as mosquito repellents were evaluated through a one-way ANOVA analysis of the log-transformed total number of mosquitoes caught for each treatment. If significant differences between the treatments were found, they were tested by Tukey’s test. The same methods were used to assess if there were any significant differences between the days on which the experiments took place and in the number of mosquitoes attracted to the individuals functioning as human bait, respectively. Dissimilar data generated by one volunteer evaluating S. angustifolia was removed before performing the analyses, meaning that 56 collected mosquitoes were excluded. On the data from the night-time experiment, separate ANOVA-analyses were also run on the log-transformed total number of mosquitoes belonging to the genera Culex, Anopheles and Mansonia. 3.6 Theoretical experiment Based on the data collected in the field, a theoretical experiment was conducted in order to simulate what efficacy of the oils, obtained from S. angustifolia and L. cubeba, that would have been observed if the field work had proceeded for twice as many days. It was assumed that the same number of mosquitoes as recorded originally would have been attracted to the different treatments in a prolonged study; i.e. ANOVA analyses were performed in the same manner as before but on duplicates of the datasets. 5 3.7 Chemical analyses of the essential oils Gas chromatography was performed to analyse the chemical compositions of the essential oils of L. cubeba and S. angustifolia. The analyses were run in a Varian 3400 gas chromatograph (Varian, Palo, Alto, CA) containing a DB-Wax capillary column of the length 30 metres, an inner diameter of 0.25 mm and a film thickness of 0.25 µm (produced by J&W Scientific, USA). The chromatograph was linked to a Finnigan SSQ 700, releasing electronic ionization. The analyses were conducted using the ion source at 150◦C. The gas used was helium, having an inlet pressure of 67 kPa. During the first minute of the analysis, the initial temperature of 40◦C was unaltered. It was later increased by 5◦C per minute until reaching 220◦C, where status quo was allowed for 12 minutes. An injector temperature of 215◦C was used. The retention times of the essential oils’ volatiles were the basis for their separation. Identification was enabled using the reference databases Nist Library and Massfinder. 4. Results 4.1 Night-time experiment 4.1.1 Abundance and distribution of mosquito genera A total number of 122 mosquitoes was collected (3 escaped) by the group evaluating the mosquito repellency of S. angustifolia. Out of the collected ones, 109 were identified to genus or species. As visualized in Table 2 the majority belonged to the genera Mansonia, Anopheles and Culex. The group testing L. cubeba collected 148 mosquitoes (24 escaped). Out of these, 132 were identified to genus or species. The majority belonged to the same genera as those collected by the other group (Table 2). A one-way ANOVA confirmed that there was no significant difference in catching efficiency among the collectors. Evaluation of the distribution of mosquito genera through a one-way ANOVA demonstrated that there was no significant difference in the number of Anopheles and Mansonia attracted to any of the different S. angustifolia treatments. However, a significant difference could be detected for the number of Culex (F=7.95, df=4, 19, P=0.001). Tukey’s test revealed that the negative control in this case differed significantly from all the other treatments, being greatest when compared to the solution with the highest concentration of oil. However, no significant difference in either the number of attracted Anopheles, Mansonia or Culex could be demonstrated for any of the different L. cubeba treatments. 6 Table 2: Proportions and numbers of mosquitoes in different genera, collected by all the human baits using cloths impregnated with essential oils of S. angustifolia and L. cubeba. Genus Fraction (%) No. of mosquitoes S. angustifolia L. cubeba S. angustifolia L. cubeba Armigeres 0.00 2.70 0 4 Aedes 0.00 0.00 0 0 Anopheles 15.6 25.7 19 38 Culex 30.3 24.3 37 36 Mansonia 43.4 36.5 53 54 Unidentified 10.7 10.8 13 16 4.1.2 Efficacy of L. cubeba and S. angustifolia as mosquito repellents The total number of mosquitoes attracted to the differently treated mosquito nets, including both the collected and the missed ones, is along with the plants’ calculated mosquito repellency shown in Table 3. Notable is that the negative control in the group testing L. cubeba on average attracted at least twice as many mosquitoes as the others. In the case of S. angustifolia, the difference between the negative control and the other treatments is even greater; in comparison to the 19 % oil solution, the negative control attracted nearly twelve times as many mosquitoes. Table 3: Total number of mosquitoes attracted to human baits. The baits were using differently impregnated shank cloths, according to the demonstrated scheme. The calculated mosquito repellency is shown for each treatment. Treatment No. of mosquitoes Repellency (%) Total Per occasion Non-adjusted Compared to 19% DEET L. cubeba 5 % 24 4.8 64.7 91.6 L. cubeba 10% 26 5.2 61.8 87.5 L. cubeba 19 % 34 6.8 50.0 70.8 DEET, 19% 20 4.0 70.6 100 Negative control 68 14 --- --- S. angustifolia 5 % 25 6.3 64.8 73.0 S. angustifolia 10 % 15 3.8 78.9 88.9 S.angustifolia 19% 6 1.5 91.5 103 DEET, 19% 8 2.0 88.7 100 Negative control 71 18 --- --- The differences between the number of mosquitoes attracted to the treatments in the group testing L. cubeba, were through a one-way ANOVA shown to be non-significant (F=1.42, df=4, 24, P=0.265). In contrast, it could be confirmed that there indeed was a significant difference between the treatments in the group testing S. angustifolia (F=4.70, df= 4, 19, P = 0.012). Tukey’s test further revealed that the positive control and the solution containing the highest concentration of oil were clearly separated from the negative control, as shown in Fig. 1. 7 Fig. 1: The log-transformed total number of mosquitoes subjected to Tukey’s statistical test. The vertical bars show the 95 % confidence interval. The mosquitoes were caught when trying to take a blood meal from the legs of human baits covering their shanks with mosquito nets either impregnated with different concentrations of essential oil of S. angustifolia, or with 19 % DEET (pos). The negative control group (neg), using untreated nets, is also shown. 4.1.3 Simulation of the anti-mosquito activity of S. angustifolia and L. cubeba during a longer period of field work In the theoretical experiment aiming to investigate how it possibly would look like if more data had been available, new one-way ANOVAs and additional Tukey’s analyses were performed on duplicates of the data sets. With this amount of data, the number of mosquitoes attracted to the treatments containing both 5 %, 10 % and 19 % of S. angustifolia was significantly less than the number attracted to the negative controls (F=10.96, df= 4, 39, P<0.0005). The same scenario could be demonstrated for the Culex mosquitoes alone (F=18.54 df=4, 39, P<0.0005). For Mansonia, a significant reduction of attraction was observed for the 19 % oil treatment (F=9.00, df=4, 39, P<0.0005). It could further be demonstrated that there were, in comparison to the negative control, significantly fewer Anopheles mosquitoes attracted to all of the different S. angustifolia treatments (F=6.81, df= 4, 39, P<0.0005). The results from all the Tukey’s test here described is shown in Fig. 2. For L. cubeba, analysis of the duplicated data set showed that there was a clear difference in mosquitoes number between the negative control and the treatment constituting of 5 % essential oil (F=3.19, df=4, 49, P=0.022). 8 Fig. 2: Simulation of the differences in mosquito attraction to human baits covering their legs with mosquito nets impregnated with different concentrations of essential oil of S. angustifolia. The positive and negative control groups are shown. Data from five days of mosquito collection have here been duplicated, log-transformed and subjected to one way ANOVA analyses followed by Tukey’s statistical test. As seen in the graphs, the tests show that solutions of S. angustifolia both repel mosquitoes in general and those belonging to the genera Culex, Mansonia and Anopheles. The horizontal bars show the 95 % confidence intervals. 9