Evaluation of Essential Oils as Spatial Repellents Against Aedes aegypti in an Olfactometer

Spatial repellents are becoming an integral part of the integrated mosquito management program. Norris and Coats (2017) expressed optimism that spatial repellents are promising alternatives to the currently utilized contact repellents for the prevention of mosquito-borne diseases if properly incorporated into an integrated pest management approach. Choi et al. (2016) reported that spatial repellents have been gaining wider attention as a potential malaria and dengue transmission breaking strategy. For spatial repellents to be effective in imparting a deterrent effect on mosquitoes at a distance, the product needs to disperse volatile compounds homogeneously and continuously into the space around it (Obermayr et al. 2015).

Current vector control practices are losing their viability due to resistance development in arthropods against these practices (Norris and Coats 2017). Vongsombath et al. (2012) reported in Southeast Asia that the search for alternative methods for preventing vector-borne diseases has become more important because of the increasing resistance among dengue and malaria vector populations to commonly used insecticides. Due to the lack of economic feasibility attributed to cost of development of new active ingredients and their approval by regulatory agencies, there is a push towards search for natural products for their potential as spatial repellents. The traditional use of plant products as natural repellents and insecticides generated great interest in the search for new natural active ingredients for vector control (Boer et al. 2010). Recently, many natural products have been evaluated for their repellent properties (Trongtokit et al. 2005, Erler et al. 2006, Nerio et al. 2010, Phasomkusolsil and Soonwera 2010, Kumar et al. 2011, Manimaran et al. 2013, Kayedi et al. 2014, Soonwera 2015, Peach et al. 2019, Qualls et al. 2021).

Spatial repellents are developed to keep the disease vectors at a distance from the host and act as a barrier (Farooq et al. 2022), prevent them from entering a space (Achee et al. 2012), and become a roadblock in the disease transmission cycle. Achee et al. (2012) also described spatial repellents as vector behavior modification techniques. World Health Organization used the term to refer to insect behaviors induced by airborne chemicals, in the form of moving away from the stimulus, as attraction inhibition, and feeding response (WHO 2013). According to Ogoma et al. (2014), mosquito behaviors elicited in response to airborne compounds, including movement away from a chemical stimulus, loss of host detection, antifeeding as well as knockdown and mortality, are collectively referred to as spatial repellency. Spatial repellents do not require physical contact of the mosquito with treated surfaces such as insecticides used in indoor residual spraying and long-lasting insecticidal nets, but act in the vapor state at a distance (Ogoma et al. 2014).

Although spatial repellents have been in use for quite a while, their availability as commercial development is still limited. And their effectiveness concern has recently brought its attention to the research community (El-garja et al. 2015). Choi et al. (2016) found that Aedes aegypti (L.) exposed to transfluthrin spatial repellent were more attracted to experimental oviposition sites than were nonexposed mosquitoes. Muller et al. (2008) evaluated essential oils–based candles as repellents by means of human landing assays and found that 5% geraniol candles indicated 85% repellency against mosquitoes. Vongsombath et al. (2012) tested essential oils of Hyptis suaveolens (Poit) (Lamiaceae), Croton roxburghii Balakr. (Euphorbiaceae), and Litsea cubeba (Lours.) Pers (Lauraceae) in the field on humans for repellent activity against mosquitoes and found that all the plant oils were significantly more repellent than the negative control but less repellent than deet (N,N-diethyl-m-toluamide). Palsson and Jaenson (1999) evaluated 8 commonly utilized plants and their parts as repellents along with 2 commercial repellents and 1 negative control in a field study using human volunteers. They reported that 7 of the 8 (87%) materials repelled significantly more mosquitoes than control but not better than commercial repellents. Sharma and Ansari (1994) evaluated repellency of burning neem oil and kerosene oil mixture against Anopheles and Culex mosquitoes inside living rooms and concluded that 1% neem oil mixed with kerosene provided economical personal protection from mosquito bites.

In a continuation effort to find natural compounds as suitable spatial repellents, different volumes of 5 essential oils were evaluated for their repellency against Ae. aegypti mosquitoes in this study.

The evaluations were conducted in a True Choice olfactometer (Sigma Scientific, Micanopy, FL) at Anastasia Mosquito Control District. The olfactometer consisted of 2-choice chambers (front and rear), a mosquito release chamber, 2 odor release chambers, and flow control valves (Fig. 1). Flow control valves regulated clean and dry airflow to 2 odor release chambers and a mosquito release chamber. Airflow to odor release chambers was set at 6.8 liters/min each and to mosquito release chamber at 3.6 liters/min. Air passing through odor release chambers picked up vapors from repellent or attractant and moved through respective choice chamber. One odor release chamber fed air to rear choice chamber (referred to as fresh air side in this study) and the other to front choice chamber (referred to as repellent/attractant side in this study). The choice chambers were connected to 2 sides of the mosquito release chamber and by simple rotation of the choice chambers, they could be connected to or disconnected from the mosquito release chamber. When connected, mosquitoes in the release chamber had the option to move to either of the choice chambers.

The study comprised 3 parts. In the 1st part, Biogents (BG) lure (Biogents USA, Moorefield WV) on the attractant/repellent side and fresh air on the fresh air side were evaluated as control. Clove oil and RepelCare (containing turmeric oil and eucalyptus oil) (PerdYaThai Industry, Pharnomsarakam, Thailand) were the test repellents in this part of the study. The rates of 0.125, 0.25, and 0.5 ml of each repellent poured on a filter paper in a small plastic cup were used as repellents. The highest volume completely soaked the filter paper whereas lower rates did it partially. The BG lure on attractant/repellent side and 1 rate of one of the repellents on the fresh air side were evaluated as treatments. All tests were replicated 5 times. For each run, 15–20 female Ae. aegypti were released into the mosquito release chamber. Fifteen minutes later, the number of mosquitos in the fresh air side, attractant/repellent side, and in the mosquito release chamber were counted and recorded. The percentage of mosquitoes on the attractant/repellent side indicated the presence or absence of repellency.

In the 2nd part, clove oil was used on one choice chamber and RepelCare on the other chamber for direct comparison between the 2 repellents. All 3 rates (0.125, 0.25, and 0.5 ml) of the 2 repellents were compared with the respective rate of the other. All tests were replicated 5 times. For each run, 15–20 female Ae. aegypti were released into the mosquito release chamber. Fifteen minutes later, the number of mosquitos in the fresh air side, attractant side, and in the mosquito release chamber were counted and recorded. The side showing smaller number of mosquitoes indicated stronger repellency than the other.

In the 3rd part, 5 natural oils—clove oil, eucalyptus oil, geraniol, Immortelle oil, and lemongrass oil—were evaluated for their potential as spatial repellents. Four different quantities of each natural oil, 0.0625, 0.125, 0.25, and 0.5 ml, were poured onto filter paper in a small plastic cup. For the control test, BG lure was placed in the front odor release chamber feeding attractant vapors to attractant/repellent side choice chamber while rear odor release chamber was empty feeding fresh air to fresh air side chamber. For repellency tests, the repellent-containing cup was added to chamber having BG lure. For each run, airflow was turned on and 24-h-starved, 15–20 female Ae. aegypti were released into the mosquito release chamber. Fifteen minutes later, the number of mosquitos in the fresh air side, attractant side, and in the mosquito release chamber were counted and recorded. All control and treatment runs were replicated 5 times.

After confirming that the data followed normal distribution, analysis of variance was performed to assess the significance of difference in percent activation and percent repellency of mosquitoes between repellents and control and between different natural oils using JMP edition 14 (SAS Institute Inc., Cary, NC). The means were compared using Tukey's multiple comparison test at 95% level of significance.

The movement of mosquitoes to the attractant/repellent side was affected by the presence of different types of repellents (F = 3.64, df = 6, P = 0.0085) on the fresh air side. As shown in Table 1, there were significantly more mosquitoes on attractant/repellent side when any of the repellent was placed on the fresh air side against BG lure on attractant/repellent side compared with control when only fresh air was used against BG lure. However, 0.25-ml volume of clove oil and 0.125- and 0.25-ml volumes of RepelCare did not have significantly more mosquitoes on the attractant/repellent side. There was no difference in activation of mosquitoes between control and repellents and it ranged from 75–89% (Table 1). Both repellents significantly repelled mosquitoes to the attractant/repellent side except by 0.125- and 0.25-ml volumes of RepelCare. The BG lure showed only 39.5% repellency, whereas clove oil had 65.7–73.0% repellency and RepelCare showed 49.2–69.3% repellency. Comparing different rates of each repellent, 0.25 ml of clove oil resulted in highest repellency of 73.0% and 0.5 ml of RepelCare was 69.3%.

The movement of mosquitoes after release was affected by the presence of different repellents (F = 15.64, df = 2, P < 0.0001). As shown in Table 2, there were generally more mosquitoes on RepelCare side than on the clove oil side. However, the difference in repellency was only significant at lower rates of 0.125 and 0.25 ml (Table 2). These results indicated that clove oil was relatively stronger repellent than the RepelCare.

Overall, the essential oils significantly affected activation (F = 13.5, df = 5, P < 0.0001) and repellency (F = 7.6, df = 5, P < 0.0001). All natural oils and control, except lemongrass, activated >80% of mosquitoes (Fig. 2). Statistically, there was no difference in activation between natural oils and none was different from the control. The activation was not affected by volume of repellent used for any of the essential oils used (Fig. 3). None of the repellents provided complete repellency. On average, clove oil, geraniol, and lemongrass oil showed >60% repellency, eucalyptus oil >50%, and Immortelle oil did not show any repellency. Clove oil, eucalyptus oil, geraniol, and lemongrass oil had statistically similar repellency and all but eucalyptus oil had significantly higher repellency than control and Immortelle oil (Fig. 2). Immortelle oil had statistically similar repellency as control, which is an attractant, meaning that it did not have any repellency. It was also evaluated against fresh air alone and the presence of Immortelle oil in the attractant/repellent side did not affect mosquito behavior (Table 3).

As shown in Fig. 3, activation by clove oil was approximately 90% for all application volumes while repellency was >65% for volumes ≥0.125 ml. Activation by eucalyptus oil was approximately 85% for all application volumes while repellency was >65% for volumes ≥0.25 ml. Activation by geraniol was approximately 80% for all volumes tested, while repellency was >60% for volumes up to 0.25 ml but reduced with increase in application volume to 0.5 ml. Activation by Immortelle oil was approximately 80% for all application volumes while repellency was <40% for all volumes. Activation by lemongrass oil showed increase with increase in volume used and was approximately 70% for volumes ≥0.25 ml, while repellency reduced with increase in volume and was >65% for up to 0.125-ml volumes.

A large percentage of mosquitoes were active during this study either due to attractant or repellents. The activity ranged from 75–89% for all the tests, which indicated that mosquitoes were influenced by various stimulants tested and did not remain stationary. This activation rate is an indication of validity and appropriateness of the methodology used.

All the natural oils including a mixture of 2 oils (RepelCare), except Immortelle oil, showed reasonable repellency. All oils or mixtures tested except Immortelle and lemongrass oils showed increase in repellency with increase in application volume. However, minimum application volume to be effective was different for each oil. Lemongrass is the only oil that showed increase in repellency up to 0.125 ml and then decreased. Geraniol provided 78% repellency with application volume of 0.125 ml, which is in close confirmation with 85% repellency shown by Muller et al. (2008) from burning a candle containing 5% geraniol. Eucalyptus oil had 72% repellency with application volume of 0.25 ml, which was similar to repellency provided by burning eucalyptus leaves (Palsson and Jaenson 1999). These results are also supported by report from Batish et al. (2008), indicating spatial repellent activity of eucalyptus oil against mosquitoes and other insects. It is further supported by Omara et al. (2018), who determined the potential of eucalyptus oil to act as repellent, toxicant, and fumigant against maize weevil (Sitophilus zeamais, Motschulsky). RepelCare provided 70% spatial repellency, whereas it has shown strong contact repellency as has been shown by Tawatsin et al. (2006) against various mosquito species including Ae. aegypti. Lemongrass oil application volume of 0.0625 ml provided 80% repellency, which resembles mosquito repellency reported by Peach et al. (2019). Overall, all oils except Immortelle oil have shown potential to be used as a spatial repellent, which is in agreement with a conclusion by Norris and Coats (2017). The results of this study indicated that all essential oils tested except Immortelle oil have the potential to be developed as spatial repellents.

This is a research report only and specific mention of any commercial products does not imply endorsement by the Anastasia Mosquito Control District (AMCD). The authors acknowledge A. M. Grancaric for providing a couple of essential oils and assistance from Kai Blore of AMCD for providing all the mosquitoes for testing.