The Effectiveness of Botanical Extracts as Repellents Against Aedes aegypti Mosquitoes

Introduction

I live in southwestern Louisiana, where because of the abundance of water and the warm climate, mosquitoes are active year-round. All over the world, people are at risk from mosquito-borne diseases such as malaria, dengue, yellow fever, West Nile virus, and several forms of encephalitis (Gubler 1989, Monath 1989). Personal protection from mosquito bites is currently the most important way to prevent transmission of these diseases (Fradin 1998).

Throughout the world, there are about 3,500 species of mosquitoes. The female mosquito bites people and animals because they need the protein found in blood to help develop their eggs. Mosquitoes are attracted to people by skin odors and the carbon dioxide from breath (Bowen 1991). The use of repellents makes a person unattractive for feeding and therefore repels the mosquito (Maibach et al. 1966).

The most common mosquito-repellent formulations available on the market contain a synthetic chemical called N, N-diethyl-3-methylbenzamide (DEET). It was developed and patented by the U.S. Army in 1946 for use by military personnel in insect-infested areas. DEET was recognized as one of the few products effective against mosquitoes and biting flies. It was registered for use by the general public in the U.S. in 1957 (EPA 1980). The efficacy of DEET in providing long-lasting protection against a wide variety of mosquito species has been documented in several studies that have shown excellent repellency against mosquitoes. (Schreck and McGovern 1989, Fradin and Day, 2002, Roberts and Reigart 2004).

Table 1. Biological Activities in Tested Plants. (Dr. Duke’s Phytochemical and Ethnobotanical database library was used for identification of the plants’ biological activities.)

Although DEET is an effective repellent against mosquitoes, there are concerns associated with its use. It is irritating to mucous membranes, and concentrated formulations dissolve plastic. Some human toxicity effects have been reported after applications of DEET, varying from mild to severe (Briassoulis et al. 2001; Bell and Veltri 2002). Because of these undesirable side effects, research on repellents derived from plant extracts is needed to find alternatives that would be safer but still effective.

The repellent properties of plants to mosquitoes and other pest insects were well known before the use of synthetic chemicals. Traditionally, people used natural compounds to protect themselves against insect bites. Some plant species contain insecticidal and/or insect-repellent substances. A review by Sukumar (1991) highlighted the potential of plants for use in mosquito control, either as repellents, larvicides, or insecticides. Extracts of several plants—neem (Azadirachta indica), basil (Ocimum basilicum), (Mentha piperata), and lemon eucalyptus (Corymbia citriodora)—have been studied as possible mosquito repellents and have demonstrated good efficacy against some mosquito species (Sharma et al. 1993; Ansari et al. 2000; Trigg and Hill 1996).

The research is promising, but the number of plants that has been extensively studied is relatively small. Plants contain a wide range of chemical compounds. When extracted from the plant material, these compounds show useful biological activities such as repelling insects or altering insect feeding behavior, killing larvae, or disrupting growth (Duke 2000). My research investigated the possible potential of various botanicals: Mexican marigold (Tagetes minuta lucida), lemongrass (Cybnopogon citratus), rosemary (Rosmarinus officinalis), and citrosa (Pelargonium citrosum) as natural repellents against Aedes aegyptimosquitoes.

Table 2. Chemical compounds present in the plants. (Dr. Duke’s Phytochemical and Ethnobotanical database library was used for all plant compound indentifications.)

Mexican marigold, or Tagetes minuta lucida in the family Asteraceae, is an annual herb native to Central and South America, but it also grows widely in Africa, India, Spain, and the United States. The plant grows in any well-cultivated site, even in poor dry soil. This plant produces clusters of fragrant orange-yellow flowers, and the leaves have an aroma similar to fennel. The leaves are used locally in Africa and India to repel blowflies and safari ants (Scholtz and Holm 1986). The active compounds in the plant which act as antifeedants are alpha-pinene, limonene, and borneol. Borneol is also a component of many essential oils and is a natural insect repellent. The compound alpha-terpineol is also responsible for the insecticidal and pesticidal properties of the plant (Duke 2000).

Lemongrass, or Cybnopogon citrates in the family Poaceae, is a perennial herb widely cultivated in the tropics and subtropics. Native to Southeast Asia, lemongrass can also be found growing in India, South America, Australia, Africa, and the United States. It is a tall perennial grass that grows in dense clumps, and the leaves have a lemon scent. Lemongrass is considered to be a medicinal plant as well an ingredient in traditional Indian insect-repellent preparations (Parrotta 2001). Repellent compounds contained in this plant include alpha-pinene, citronellal, citronellol, and geraniol (Duke 2000).

Rosemary, or Rosmarinus officinalis in the family Lamiaceae, is an evergreen shrub that in the southern European countries around the Mediterranean region. It is widely cultivated all over the world. It is strongly aromatic and toxic to insects due to the many compounds extracted from its leaves and flowers (Palsson and Jaenson 1999).

Here mosquitoes are removed from a rearing cage with an aspirator.

The compounds with insect-repellent activities in this plant are borneol and camphor (Duke 2000).

Citrosa, or Pelargonium citrosum in the family Geraniaceae, is a genetically engineered geranium hybrid. It is an evergreen found in southern Africa, Australia, and New Zealand. In North America and Europe, the citrosa plant is being marketed as a mosquito-repelling plant. The compounds limonene, citrosa, and geraniol, found in the citrosa plant, are responsible for the strong odor when the leaves are rubbed (Duke 2000). These compounds have also demonstrated insect repelling capabilities (Jeyabalan, Arul, and Thangamathi, 2003).

Materials And Methods

Mosquitoes

The mosquito type used in this study was female Aedes aegypti , as they are easy to raise and are avid biters (Schreck 1977). The eggs were obtained from a research biologist at the Calcasieu Parish Mosquito Control laboratory. Larvae were reared and maintained on liver powder. Adults were maintained in screened cages and provided a sucrose solution (10% water). Larvae and adults were continuously available for the experiments.

Commercial Repellents

Table 3. Name, ingredients, and formulations of two natural-based commercial repellents. (Concentration of ingredients as stated on product label.)

Two commercial natural-product-based repellents, Repel and Natrapel, made primarily with plant extracts and/or essential oils, were tested as a comparison to the plant-extract repellents.

Plant Extracts

Test sample extracts were made from the four plants: Mexican marigold, lemongrass, rosemary, and citrosa. Leaves of each plant were used to develop three different concentrations of extract: 3.4%, 10.1%, and 17%. For this procedure, the leaves

Lemongrass leaves are cut and weighed into one, three, and five grams.

were cut and weighed into one, three, and five grams. The leaves were then washed, diced up thoroughly, and 30 milliliters of bottled water was added to each sample. The samples were filtered into separate containers, covered, labeled, and allowed to sit for 24 hours then filtered again to remove any remaining plant material. Afterwards, each sample was placed into labeled spray bottles.

Testing Procedures

For the testing procedures, the duration of protection provided by each plant extract sample and repellent was tested by means of arm-in-cage studies (EPA 1999), in which a treated arm is inserted into a cage with a fixed number of unfed mosquitoes, and the elapsed time to the first bite is recorded. For each test, ten A. aegypti female mosquitoes that were between seven and 14 days old were removed from their rearing cage with an aspirator and placed into a cage measuring 28 cm by 28 cm by 28 cm. Tests were conducted with a low density of mosquitoes per cage rather than a high density to more accurately reflect the typical biting environment encountered during most outdoor activities.

Prior to each test, a control arm, washed from elbow to fingertips and air-dried, was inserted into the cage in

Prior to the test, the arm is sprayed from the elbow to fingertips with plant extract.

order to provide a standard for comparing mosquito-biting activity during the experiment. Once bit, the arm was
removed from the cage, and the plant extract sample or repellent being tested was sprayed evenly from the elbow to the fingertips.

The testing procedures shown in Figure 1 were followed. For the first test, the treated arm was inserted into the cage for one minute every five minutes for up to 15 minutes. If no bites occurred, then the arm was reinserted for one minute every 15 minutes until the first bite occurred.

For Tests 2–20, if the plant extract or repellent had initially worked for less then 15 minutes, the treated arm was inserted into the cage for one minute every five minutes until the first bite occurred. If the plant extract or repellent had initially worked for 15 minutes to four hours, the treated arm was inserted into the cage for one minute every 15 minutes until the first bite occurred. If the plant extract or repellent had initially worked for more than four hours, the treated arm was inserted into the cage for one minute every hour for four hours, then one minute every 15 minutes thereafter, until the first bite occurred.

Figure 1. Testing procedures.

Twenty trials were run for each plant sample extract and repellent. For each trial, test mosquitoes were used that had not been exposed to either the plant sample extract or the repellent. Testing was done with insertions at limited intervals because continuous exposure might have induced prolonged blockage of their antennal chemoreceptors, which would prevent further biting.

Results

Table 4. Mean time of protectiveness of repellents against A. aegypti. (At the 0.05 significance level, the means of any two groups with the same letter are not significantly different.)

After all testing was complete, an analysis of variance (ANOVA) was performed using the data to compare the mean protection times. A P-value of less than 0.05 was considered to indicate statistical significance. The analysis showed that all 12 plant extract samples and the two commercial repellents tested resulted in a P-value of 0.001, showing significant differences. A Tukey’s multiple comparison test was then run to determine specific pair wise differences. The results showed no significant differences in protection time between lemongrass and citrosa 3.4% concentrations; lemongrass and citrosa 10.1% concentrations; and rosemary 10.1% concentration, lemongrass 17% concentration, Repel, and Natrapel.

There were overall differences in protection time (PT) among the extracts, shown in Figure 2. The higher concentrations, 17%, in each plant exhibited longer protection times when compared to the 3.4% and 10.1% concentrations. When comparing the four plants, the rosemary extract provided the longest protection time in each of the concentrations: 3.4% (1:50), 10.1% (3:57), and 17% (5:57). When comparing the protection times of the commercial natural-based repellents, Repel (4:03) and Natrapel (4:05) had a lower repellency then the 17% concentrations of Mexican marigold (5:37), rosemary (5:57), and citrosa (4:37), which provided 34 minutes to one hour, or 54 minutes longer protection. However, the 3.4% and 10.1% plant concentrations had lower repellency compared to Repel

Figure 2. Comparison of repellency protection times. (Mean time of protection was converted from decimal time into hours and minutes.)

and Natrapel.

Discussion And Conclusion

The purpose of this research was to evaluate the potential of plant extracts as natural repellents. All 12 concentrations of plant extracts from Mexican marigold, lemongrass, rosemary, and citrosa confirmed that their broad spectrum of chemicals were effective as repellents against A. aegypti mosquitoes.

Mosquitoes are responsible for a variety of diseases. Despite the advances in techniques and products used for their control, the mosquito continues to pose serious public health problems. An insect repellent of plant origin should be harmless to humans as well as providing a means of personal protection. Therefore, use of these botanical derivatives in mosquito protection instead of chemicals could reduce the costs and environmental effects. The obvious benefits of these plant extracts would provide public health protection and an environmentally safer alternative. The significant impact could be worldwide because of existence of these types of plants in countries where production could possibly be done simply and economically to provide a viable form of personal protection from disease vectors.

The world has a variety of plant species, and more studies should be carried out. Identification of other compounds found in different plant species could be discovered and used in pest control as a safer alternative to synthetic chemicals and pave the way for further use of natural repellents against insects. However, plants developed for repellents need to be sustainable. Ideally, they would be fast-growing, naturally abundant, and easy to cultivate. The source of the repellent should be obtained preferably from replaceable parts of the plant, such as the leaves rather than parts that, when removed, kill or damage the plant, such as the roots or shoots. Abundance, and survival after parts have been harvested, is important for sustainability, because useful plants may become scare due to over-harvesting. The plant parts utilized also need to be available when needed, or be easy to harvest and store.

My hope is that one day these plants may serve as a source for managing the control of mosquitoes. However, no one should think that success is at hand and that botanical insecticides will replace all synthetic products. They are only alternatives to be used in integrated pest-management programs, and they should be used together with other available control measures. The general public, however, must become more vigilant in the prevention methods to eliminate the growth process of mosquitoes and “fight the bite.”

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