J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 372 http://jad.tums.ac.ir Published Online: January 06, 2016 Original Article Behavioral Response of Aedes aegypti Mosquito towards Essential Oils Using Olfactometer *Ashish Uniyal 1, Sachin N Tikar 1, Murlidhar J Mendki 1, Ram Singh 1, Shakti V Shukla 2, Om P Agrawal 3, Vijay Veer 1, Devanathan Sukumaran 1 1Vector Management Division, Defence R and D Establishment Jhansi Road, Gwalior, India 2Fragrance and Flavour Development Center, G. T. Road, Makrand Nagar, Kannuaj, India 3School of Studies in Zoology, Jiwaji University, Gwalior, India (Received 23 Aug 2014; accepted 6 Dec 2014) Abstract Background: Aedes aegypti mosquito is responsible for transmitting human diseases like dengue and chikungunya. Personal or space protection with insect repellents is a practical approach to reducing human mosquito contact, thereby minimizing disease transmission. Essential oils are natural volatile substances from plants used as protective measure against blood-sucking mosquitoes. Methods: Twenty-three essential oils were evaluated for their repellent effect against Ae. aegypti female mosquito in laboratory conditions using Y-tube olfactometer. Results: The essential oils exhibited varying degree of repellency. Litsea oil showed 50.31%, 60.2 %, and 77.26% effective mean repellency at 1 ppm, 10 ppm and 100 ppm respectively, while DEET exhibited 59.63%, 68.63%, 85.48% and DEPA showed 57.97%, 65.43%, and 80.62% repellency at respective above concentrations. Statistical analysis revealed that among the tested essential oils, litsea oil had effective repellency in comparison with DEET and DEPA against Ae. aegypti mosquito at all concentration. Essential oils, DEET and DEPA showed significant repellence against Ae. aegypti (P< 0.05) at all 3 concentration tested. Conclusion: Litsea oil exhibited effective percentage repellency similar to DEET and DEPA. The essential oils are natural plant products that may be useful for developing safer and newer herbal based effective mosquito repellents. Keywords: Essential oils, Aedes aegypti, Repellent, Y-maze olfactometer, Flight orientation Introduction Mosquito borne diseases such as malaria, Japanese encephalitis and dengue are a ma- jor concern in both developed and develop- ing countries. Dengue and chikungunya are important diseases transmitted by Aedes ae- gypti mosquito which lives in urban habitats and breeds mostly in man-made and natural containers (Julian 2009, WHO 2012). Its peak host seeking activity is early in the morning and in the evening before dusk (WHO 2012). These diseases have many economical and sociological adverse effects on human, do- mestic animal populations (WHO 2013) and Dengue fever is one of the main causes of child mortality in Asia and Africa (Gupta et al. 2012). Since there is no effective vaccine available for the control of these deadly dis- eases (Norashiqin et al. 2008, Gu et al. 2009), mosquito control programs are essential to prevent spread of these diseases. Insect re- pellents are used as a personal protection that can provide a practical and economical means of preventing mosquito-borne diseases. The most common mosquito repellent DEET (N,N Diethyl-m-toluamide), a gold-standard of synthetic repellent, which is currently available in the market has shown repellency for 6 to 8 hours against mosquito (Yap 1986, Kweka et al. 2012) and other blood sucking insects (Browne et al. 1997). Even *Corresponding authors: Mr Ashish Uniyal, E-mail: ashish_uniyal5@gmail.com J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 373 http://jad.tums.ac.ir Published Online: January 06, 2016 though DEET is mostly used as a mosquito repellent, but it has the side effects on hu- man being such as skin irritation, affects the central nervous system, mucous membranes and mild toxic (Maibach and Johnson 1975, Reuveni and Yagupsky 1982, Phas- omkusolsil et al. 2010, Miot et al. 2011) and strong solvent for plastic and other synthetic products (Kazembe et al. 2012). Every year more than 29 billion mosquito coils are sold for personal protection which contain pyre- thrum daisy a plant product obtained from the flower of Asteraceae family basically used for repellent and insecticidal purpose against blood sucking insects (WHO 1998, Lawrance et al. 2004). Due to these adverse effects, attempts are made to find safe and ecofriendly repellents derived from plant extracts/ materials. Several characteristics such as insecticidal, repellent and growth reducing properties are found in plant essential oils. The major fami- lies, which contain essential oils as insecticidal and repellent properties, include Lamiceae, Asteraceae, Myrtaceae and Lauraceae. Plant essential oil from Myrtaceae family has ef- fective repellent activity against mosquitoes (Tapondjou et al. 2003, Maia et al. 2011). However, Labiatae, Apiaceae, Lamiaceae contain insect repellent, antifeedent activity and insecticidal properties (Regnault-Roger et al. 1994, Vasilakoglou et al. 2007). Plant family Poaceae, Rutaceae also showed ef- fective repellent activity against mosquitoes (Regnault-Roger 1997, Maia et al. 2011). Essential oil from plant family such as Ge- raniaceae, Oleaceae, Piperaceae, Cupressaceae and Burseraceae also exhibited repellent ac- tivity against blood sucking mosquitoes (Amer and Mehlhorn 2006). According to Moore et al. (2006) plant families such as Lamiaceae, Myrtaceae and Poaceae are best known species as insect repellent. However, p- menthone-3,8- diol (PMD) from Euca- lyptus maculate citrodon a lemon eucalyptus and citronella oil from Cymbopogon nardus are effective natural repellent preferred by various users (Curtis et al. 1987, Trigg et al. 1996, Trigg 1996, Trongtokit et al. 2005, Hsu et al. 2013) and no adverse effect re- ported since 1984 (US EPA 1999) Essential oils are the alternative source against synthetic repellent because they are non-toxic for human and other organisms (Das et al. 2003, Tarek et al. 2012). Essential oils from several plants have shown effec- tive repelling against mosquitoes (Barnard 1999). Natural plant based repellents have also demonstrated good efficacy against some mosquito species in tests examining as adul- ticidal activity (Yang et al. 2005, Manimaran et al. 2012), larvicidal activity (Ansari et al. 2000, Adebajo et al. 2012), repellent activity (Amer and Mehlhorn 2006, Gleiser et al. 2011) and adulticidal, repellent, larvicidal, oviposition deterrent activity (Prajapati et al. 2005). The effectiveness of any plant essen- tial oil as repellent depends on several fac- tors such as quality, type of repellent, mode of action, temperature, humidity, biting re- sponse of mosquito, volatility, methods of extractions (Tawatsin et al. 2001). Factors affecting the quality of essential oils include plant species, cultivating conditions, and mat- uration of harvested plants, plant storage, plant preparation and methods of extraction (Tawatsin et al. 2001, Norashiqin et al. 2010). Behavioral studies on essential oils are very important research to identify effective plant oils and their constituent responsible for ex- hibiting repellent effects against mosquitoes and there is a need to develop eco-friendly, safe, cost effective repellants for blood suck- ing insects. In the present study, an attempt has been made to study repellency of 23 various es- sential oils obtained from Fragrance and Flavour Development Center, Kannuj, U P India, against Ae. aegypti mosquitoes in la - boratory condition. J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 374 http://jad.tums.ac.ir Published Online: January 06, 2016 Material and Methods Test Insect The test mosquitoes Ae. aegypti were reared in the laboratory conditions in wooden cages (750 X600 X600 mm) for feeding and egg laying on a filter paper strip in a plastic con- tainer/bowl containing 250 ml of water. Cot- ton with 10% sugar solution was provided for nourishment and the female mosquitoes were fed on rabbits for blood meal initially for 2 days and then at every alternative days. The eggs were collected and transferred to a bowl containing two liters of water, for rear- ing of hatched larvae up to adult stage. Brewer’s yeast powder was provided as food for larvae and water was changed on alter- nate days. The pupae were collected and kept in small cages (550 X450 X 50 mm) covered with cotton cloth for emerging into the adult. Aedes aegypti (five to six days old) adults were drawn from the stock colony maintained at 27±2 ˚C and 70±5% RH for all the evaluations. Essential Oils and Synthetic repellents Twenty-three essential oils as mentioned in Table 1 were obtained from the Fragrance and Flavour Development Center (FFDC), Kannuj, Uttar Pradesh, India. The synthetic repellent N, N- diethyl-m-toluamide (DEET) 98.5% pure was purchased from Sigma Al- drich chemicals and N, N-diethyl phenyl ac- etamide (DEPA) 99% pure was synthesized by chemists from Synthetic Chemistry Divi- sion of DRDE Gwalior. Flight orientation The repellent behavior response of Ae. aegypti exposed to 23 essential oils was studied using Y- maze olfactometer is shown in Fig. 1 described by Erler et al. (2006) slightly modified. It is made up of glass tube having internal diameter of 2.5 cm and 45 cm length from main arm containing one arm for testing repellent and the other arm for control. The olfactometer was kept on table, pressurized air was flow continuously into the olfactometer at the rate of 1.5 L/min. Fil- ter paper strips (1cmx 5cm) were loaded with 100 µ l of different concentration viz, 1 ppm, 10 ppm, 100 ppm of individual essen- tial oils, DEET, and DEPA dissolved in iso- propanol. DEET and DEPA were used as positive controls and the paper strips treated with solvent isopropanol alone was used as control. After the test oil was applied on treated paper, it was fixed in one arm and solvent treated paper in another arm of Y- maze olfactometer as control. Twenty Ae. aegypti females (5 to 6 days old) were used for each test. The mosquitoes were released into the main arm located at the other end of the olfactometer has a circular tube (1 inch) having circular (1cm diameter) access hole for introduction of mosquitoes and cotton was used the keep the access hole closed. Numbers of mosquitoes present in test and control arm of Y -maze were counted after three minutes of exposure and all mosqui- toes were removed soon after completion of experiment. After completion of one experi- ment the treated and control were inter- changed, for each assay at different concen- trations six replication were taken for test mosquito. The experiment was carried out during day time from 1000 hrs to 1600 hrs at room temperature 27±2 ˚C, Relative Hu- midity 70±5% with light intensity of 120– 125 lux (bioassay room was illuminated by three 36 W fluorescent lamps). Six replicates of tests were conducted for each treatment and for each replicate a new set of twenty female mosquitoes were used. After every treatment, Y-maze and set up connections were washed with acetone and dried soon to avoid any interference of other essential oils scents. J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 375 http://jad.tums.ac.ir Published Online: January 06, 2016 Data Analysis The percent repellency of essential oils was calculated as per Erler et al. (2006). Where PR= (C-T)/(C+T) X 100 where C= number of insects in control arm and T= number of insects in treated arm, insects that remained in the main arm were not taken into account. The data were subjected to sta- tistical analysis for comparison analyzed by using statistical software (Sigma Stat V2.03). The values for repellency and mean were analyzed using one way ANOVA for deter- mination of variance ratio and least sig- nificant difference (LSD). Results The results obtained from behavioral re- sponse of Ae. aegypti to essential oils using Y-maze olfactometer are present in Table 2. At 1 ppm concentration, litsea, geranium and rosewood exhibited significantly higher re- pellency (50.31%, 48.74% and 46.00%) in comparison with the other oils and similar repellency was observed for DEET (59.63%) and DEPA (57.97%) (F= 16.948, df= 149, P< 0.001). At higher concentration of 10 ppm, litsea (60.26%), rosewood (59.22%) and ge- ranium (56.84%) also showed significant repellency over other oils but similar to that of DEET (68.63%) and DEPA (65.432%) (F= 18.238, df= 149, P< 0.001) followed by lemon scented (45.12%). A further increase in the concentration to 100ppm also showed similar trend, where litsea (77.26%), rose- wood (66.90%) and geranium (66.11%) ex- hibited significantly higher repellence’s of mosquitoes over other oils but lesser effects than DEET (85.48%) and DEPA (80.62%), (F= 12.677, df= 149, P< 0.001) followed by lemongrass (57.49%). Based on the repellency of essential oils against Ae. aegypti mosquitoes, the order of effective percentage repellency can be ar- ranged as follows, litsea > rosewood > gera- nium > lemongrass > lemon scented > cam- phor > citronella > galbanum > dill > cinna- mon > basil > frankincense > lavender > black pepper > thyme > rosemary > jasmine > catnip > peppermint > chamomile > juni- per > amyris > tagetes. Fig. 1. ‘Y’ maze olfactometer showing behavioural bioassay assembly air flow meter and regulating unit J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 376 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 1. List of essential oils obtained from different plant sources used for the repellent study against Aedes aegypti mosquitoes using “Y” maze olfactometer (Source of oils: Fragrance and Flavour Development Center, Kannuj, U P, India) Name of Material Plant Name Family Origin of plant Distillation Part Used Amyris Amyris balsamifera Rutaceae Haitai, Jamaica Steam wood Basil Ocimum basilicum Lamiaceae India Steam Fresh Plant Black pepper Piper nigrum Piperaceae India Steam Seed Camphor Cinnamomum cam- phora Lamiaceae China steam Leaves Catnip Nepeta cataria Lamiaceae France, Canada steam Leaves Chamomile Anthemis nobilis Asteraceae France, Italy Steam Leaves Cinnamon Cinnamamomus zeylanicum Lauraceae Sri Lanka Steam Bark Citronella Cymbopogon win- terianus Poaceae Indonesia, Central Nepal Steam Leaves Dill Anethum graveo- lens Apiaceae Hungary Steam Seed Frankincense Boswellia carteri Burseraceae Somalia Steam Tree resin Galbanum Ferula galbaniflua Apiaceae Turkey Steam Tree resin Geranium Pelargonium grav- eolens Geraniaceae South Africa, Egypt Steam Leaves, Stalk Jasmine Jasminum grandi- florum Oleaceae India, South Asia Hydro Flower Juniper Juniperus com- munis Cupressaceae India Steam Fruit Lavender Lavendula an- gustifolia Lamiaceae France Steam Flower Lemon grass Cymbopogon cit- rates Poaceae Southeast Asia. Hydro, Steam Leaves Lemon scented Eucalyptus citri- odora Myrtaceae Australia Steam Leaves Litsea Litsea cubeba Lauraceae China Steam Fruit Peppermint Mentha piperita Lamiaceae India Steam Leaves, Flower Rosemary Rosmarinus offici- nalis Lamiaceae Spian, Tunisia Steam Shrub Rosewood Aniba rosaeodora Lauraceae Brazil Steam Wood Tagetes Tagetes minuta Asteraceae South America Steam Flower Thyme Thymus serpyllum Labiatae Europe and North Africa Steam Leaves J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 377 http://jad.tums.ac.ir Published Online: January 06, 2016 Table 2. Repellency of 23 essential oils against Aedes aegypti mosquitoes using “Y” maze olfactometer in comparison with synthetic insect repellents N, N-diethyl phenyl acetamide (DEPA) and N, N- diethyl-m-toluamide (DEET) S. No Compound Mean% repellency ± SE 1 ppm 10 ppm 100 ppm 1 Litsea 50.31 ± 5.03a 60.26 ± 2.91a 77.26 ± 2.94ab 2 Rosewood 46.00 ± 4.85ab 59.22 ± 2.56a 66.90 ± 4.23b 3 Geranium 48.74 ± 4.55a 56.84 ± 2.97ab 66.11 ± 2.16bbc 4 Lemongrass 21.45 ± 3.83cd 43.60 ± 3.15c 57.49 ± 1.83c 5 Lemon scented 33.99 ± 2.49bc 45.12 ± 2.91bc 55.39 ± 3.8c 6 Camphor 23.12 ± 3.35c 41.5 ± 2.98c 54.21 ± 1.73c 7 Citronella 24.56 ± 3.18c 34.22 ± 3.03c 52.95 ± 4.75c 8 Galbanum 13.54 ± 2.95d 30.77 ± 3.37d 51.38 ± 3.04c 9 Dill 8.10 ± 2.56d 33.29 ± 2.38cd 51.04 ± 2.98c 10 Cinnamon 13.75 ± 4.23d 27.67 ± 3.62d 50.82 ± 2.3c 11 Basil 20.29 ± 3.66d 39.88 ± 3.5c 50.56 ± 5.14c 12 Frankincense 23.70 ± 4.67c 40.68 ± 3.73c 50.46 ± 2.29c 13 Lavender 19.62 ± 3.17d 28.74 ± 2.42d 49.92 ± 4.06c 14 Black pepper 22.22 ± 4.14c 33.90 ± 2.67c 48.96 ± 4.29c 15 Thyme 20.11 ± 3.11d 36.14 ± 4.16c 48.61 ± 2.6c 16 Rosemary 18.78 ± 5.22d 31.07 ± 3.29d 46.01 ± 2.49c 17 Jasmine 15.70 ± 3.35d 24.55 ± 3.32de 46.08 ± 3.85c 18 Catnip 20.34 ± 5.53d 34.16 ± 5.21c 44.30 ± 3.63c 19 Peppermint 27.64 ± 6.02c 34.72 ± 2.8c 42.15 ± 3.55c 20 Chamomile 11.65 ± 4.39d 30.31 ± 2.7d 40.73 ± 3.86c 21 Juniper 11.04 ± 4.38d 29.98 ± 4.46d 39.92 ± 3.11cd 22 Amyris 12.28 ± 3.28d 20.85 ± 3.43e 31.37 ± 3.75d 23 Tagetes 13.94 ± 3.67d 16.65 ± 3.16e 30.52 ± 5.29d 24 DEPA 57.97 ± 2.8a 65.43 ± 2.84a 80.62 ± 2.48a 25 DEET 59.63 ± 2.28a 68.63 ± 2.53a 85.48 ± 2.3a Mean percentage repellency ±SE, Data followed by the different letters are significantly different (P< 0.05, by one-way ANOVA and Least Significance Difference). The values showed by the same letter are statistically non-significant (P> 0.05) Discussion Essential oils are composition of volatile components having minor constituents con- tain pleasant fragrance which are responsible for mosquito repellency and inhibit the ori- entation of blood sucking insects (Campbell et al. 2010). Host-seeking insects, orient to a host by using chemical stimuli precede vis- ual and thermal stimuli emanating from the host. Different mosquito species develop dif- ferent host preferences, and it is generally as- sumed that host selection and discrimination is mainly based on olfactory cues (Takken 1991). Results obtained in the present study using olfactometer bioassay showed that vol- atile essential oils exhibited concentration de- pendent spatial repellency against Ae. aegypti. Litsea, rosewood and geranium oil showed effective repellency against Ae. aegypti mos- quito but the effectiveness of essential oils was not superior over synthetic insect repel- lents DEET and DEPA. The result of litsea oil repellent efficacy was supported by Vong- J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 378 http://jad.tums.ac.ir Published Online: January 06, 2016 sombath et al. (2012) against Armigeres, Cu- lex and Aedes, likewise, Amer and Mehlhorn (2006) against Ae. aegypti, An. stephensi and Cx. quinquefasciatus. They have also sup- ported the repellent efficacy of geranium and rosewood. In this study, DEET and DEPA were used as positive controls and the effec- tive repellent activities of DEET against mosquitoes as compared with other essential oils are supported by Kazembe et al. (2012). The effective repellent activity of DEET and DEPA against Ae. aegypti and An. stephensi mosquito was reported by Debboun and Wagman (2004). Hu et al. (2011) reported that Z- citral and limonene as the predominant component of litsea oil extracted from the plant Litsea cu- beba an evergreen tree found in Japan, southern China, and some parts of Southeast Asia. However, Rosewood oils from the plant Aniba rosaeodora contains linalool and 1,8-Cineole as the main chemical constituent in rosewood oil (Guilherme et al. 2007). More- over, Geranium oil from Pelargonium grav- eolenes reported to contains β-Citronellol as main component (Campbell et al. 2010). Es- sential oils contain more than 20 to 80 minor and major highly volatile chemical constitu- ents of which the major components showed effective repellent against Ae. aegypti mos- quito (Campbell et al. 2010). Plant oil which contains limonene, linalool, citronellol showed effective repellent activity against different mosquito species (Barnard 1999, Tawatsin et al. 2001, Kline et al. 2003, Kang et al. 2009, Hsu et al. 2013). Due to high volatile proper- ty, essential oil exhibited effective but short duration of protection against mosquitoes. Synthetic repellent has low rate of vapor- ization and more effective than essential oils but they cause adverse effect on human health (Maibach and Johnson 1975, Choochote et al. 2007). Plant based repellents are safe, non- toxic and ecofriendly. Therefore most of the repellent manufacturers use different sub- stances, chemicals or natural products as fix- ative compounds such as vanillin, salicylic acid, coconut oil, mustard oil with essential oils for reducing the rate of evaporation of volatile components and for improvement of long lasting repellency against mosquitoes (Tawatsin et al. 2001, Das et al. 2003, Kongkaew et al. 2011, Sritabutra et al. 2013). However, Amer and Mehlhorn (2006) found that several essential oils namely litsea, niaouli and catnip prepared in vanillin demonstrated good repellent efficacy against different mosquito species as compared to synthetic chemical DEET. Moreover, Adeni- ran and Fabiyi (2012) using formulated cream base lemongrass oil against Ae. ae- gypti mosquito. Whereas, several essential oils with synthetic chemicals as formulated cream, spray, lotion showed effective repel- lency against Ae. albopictus, Cx. nigripalpus and Ochlerotatus triseriatus were reported by Barnard and Xue (2004) and formulated neem cream exhibited effective repellency against Aedes, Culex and Anopheles mosqui- toes (Dua et al. 1995). Hence, essential oils are alternative source as a mosquito repellent as compared to synthetic mosquito repellent. Present olfactometer bioassay elicited sensory response of Aedes mosquito in the absence of any skin emanations. However, mosquito response to essential oils applied on skin may give a different result. Behavior of mosquito towards oils in the presence of skin emanations and other unidentified hu- man odor components were found different (Bernier et al. 2005, Hao et al. 2012). Alt- hough many essential oils have been found to be potently repellent, because of the high volatility of major constituents of most oils, very few have found potential in personal protection. On the other hand, they have shown great potential in space protection. Using an olfactometer test is quick and ef- fective way to evaluate the behavioural re- sponses of mosquitoes towards volatile J Arthropod-Borne Dis, September 2016, 10(3): 372–382 A Uniyal et al.: Behavioral Response of … 379 http://jad.tums.ac.ir Published Online: January 06, 2016 stimuli of essential oil. These effective es- sential oils can be used as plant based prod- uct for provided a protection against various mosquito- borne diseases. There is a need for promoting the use of herbal products be- cause of their safety to individual and com- munities. Conclusion Based on the above studies on repellent study of essential oils, litsea, rosewood and geranium showed nearly effective repellency like synthetic repellent DEET and DEPA against Ae. aegypti female mosquitoes. Our research is continued in this line of work for searching effective essential oils with higher repellent activity against blood sucking mos- quitoes to develop a newer anti mosquito re- pellent from herbal-based product as an alter- native repellent to synthetic insect repellents. Acknowledgements The authors are grateful to Prof (Dr) M P Kaushik, Director, Defence Research and Development Establishment (DRDE), Gwa- lior and all the colleagues in the Department of Vector Management Division for their help in maintenance of the Ae. aegypti mosquito colony and their support while carrying out the above study on essential oils. The authors declare that there is no conflict of interests. 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