THE REPELLENT AND DETERRENT EFFECTS OF DEET, BAYREPEL, SS220, LEMON EUCALYPTUS OIL AND CATNIP OIL AGAINST AEDES AEGYPTI (DIPTERA: CULICIDAE)

MEREDITH G. MORROW

INTRODUCTION The threat of mosquito-borne disease transmission continues to be a serious public health problem worldwide, particularly in tropical and subtropical climates (WHO, 2007). Although progress has been made in recent years in the development of effective mosquito control technologies, personal protective measures remain a critical element in preventing mosquito bites in humans (Fradin and Day, 2002). In addition, multiple chemical, botanical, and “alternative” repellent products (such as Catnip and Lemon Eucalyptus oil) are currently being marketed to consumers as being effective repellents (Bernier et al., 2005). Using Aedes aegypti as a model, in this study I determined the deterrent and repellent effects of Deet, Bayrepel, SS220, Lemon Eucalyptus oil and Catnip oil and have shown which provides the most significant protection from biting mosquitoes.

MATERIALS AND METHODS

Houston strain Aedes aegypti eggs were obtained from the USUHS insectary and were vacuum hatched for approximately 15 minutes. Fifty larvae were pipetted into each of the 77 cups, so as to minimize the effect of crowding and were reared to adult. Mosquitoes were kept in constant darkness (DD) and in a temperature ranging from (22º26ºC). Humidity was not measured during the rearing process. Cups were fed daily with a

mixture of ground fish food, and water was changed as needed to maintain cleanliness and to prevent accumulation of bacteria. Adult females were allowed to age approximately 10 days and were starved 24 hours before experimentation. Repellency tests were carried out through the use of the K&D module following methods described by Klun and Debboun (2000). The bioassay involved application of each of the five substances on human skin underneath the corresponding K&D module (with one control per test using acetone). Using washable-ink marker, volunteers were instructed to use a skin-marking template which represented the 3cm by 4cm floor openings of the six cells of the K&D module. Each template was outlined on the outer top and inner thigh positions of the right left. Each cell was randomly treated with 110 µl of solution containing 25 nM of acetone (for controls), Deet, Bayrepel, SS220, Lemon Eucalyptus oil or Catnip oil.

Each of the six modules housed five female mosquitoes which were exposed to human skin for two minutes and was replicated four times per volunteer every four hours (Fig. 1). The number of feeding mosquitoes was observed as a way of determining the substances’ role in mosquito contact chemoreception. In order to determine how long one application to the skin will continue to repel mosquitoes, odds ratios based on each of the three time points and the associated number of blood-feeding mosquitoes were also calculated. All tests were performed in ambient fluorescent lighting from 0600 to 1800 over 1d. The insects were aspirated in a bleach solution after being used once per test.

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Fig. 1. Photo Credit: USDA - Chemicals Affecting Insect Behavior Lab (2007)

RESULTS Analysis was carried out through binary logistic regression where odds ratios were obtained for each time period and repellent utilizing SPSS 12.0.1 for Windows (SPSS, 2003).

This study determined that a mosquito exposed to Deet has .070 times the odds of taking a blood meal compared to the Control. That is, a mosquito is 93% less likely to take a blood meal from a person who is wearing Deet. When it came to our second repellent, we found that a mosquito exposed to Bayrepel has .020 times the odds of taking a blood meal compared to the Control. In other words, a mosquito is 98% less likely to take a blood meal from a person who is wearing Bayrepel, showing that Bayrepel is somewhat more protective than Deet. A mosquito exposed to SS220 has .011 times the odds of taking a blood meal compared to the Control. That is, a mosquito is

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99% less likely to take a blood meal from a person who is wearing SS220, again showing a higher repellency effect compared to both Deet and Bayrepel.

The two final repellents (Lemon Eucalyptus oil and Catnip oil) were studied to determine if natural, “alternative” repellents were as effective as commercialized, chemical repellents. Our study shows that a mosquito exposed to Lemon Eucalyptus oil has .056 times the odds of taking a blood meal compared to the Control. Therefore, a mosquito is 94% less likely to take a blood meal from a person who is wearing Lemon Eucalyptus oil. While this shows a lower protection odds ratio compared to SS220 and Bayrepel, Lemon Eucalyptus oil showed slightly more repellency than Deet in this experiment (Table 1). Lastly, a mosquito exposed to Catnip oil has .363 times the odds of taking a blood meal compared to the Control. Consequently, a mosquito is 64% less likely to take a blood meal from a person wearing Catnip oil, thus showing that out of the five repellents tested, Catnip oil is the least effective repellent. However, all compounds differed significantly from the control.

Four hours after initial application of the repellents, a mosquito was 64% more likely to take a blood meal. Eight hours after initial application of the repellents, a mosquito had 22 times the odds of taking a blood meal compared to Time 0 (initial application of repellent). Twelve hours after initial application of the repellents, a mosquito had 24 times the odds of taking a blood meal compared to the Time 0. The statistical analysis of each of the three time periods compared to the initial application showed a dramatic drop in efficiency from Time 0 to Time 1 (4 hours after application),

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with a gradual decline in repellency continuing toward Time 2 (8 hours after application) and to Time 3 (12 hours after application).

Table 1. Repellent Odds Ratios and 95% Confidence Intervals Repellent Odds Ratio 95% C.I. Deet 0.070 (0.024, 0.197) Bayrepel 0.020 (0.007, 0.061) SS220 0.011 (0.004, 0.036) Lemon Eucalyptus oil 0.056 (0.019, 0.160) Cat nip oil 0.363 (0.132, 0.998)

DISCUSSION The fact that each of the repellent odds ratios were less than one and had a 95% Confidence interval not including 1, show that there is a negative association between exposure (repellent) and outcome (blood feeding) at the 5% significance level. However, one must call into account the fact that the upper bound for Catnip oil was 0.998, much closer to 1 compared to the other repellents used in this study showing that the odds ratio produces larger errors as the outcome rate rises above 1%. This follows with the observation that Catnip oil seemed to be the least effective repellent due to the high number of feeding mosquitoes we observed feeding on each of the four volunteers. Overall, SS220 repelled and deterred blood feeding behavior the most followed by Bayrepel, Lemon Eucalyptus oil, Deet and lastly Catnip oil. Based on these preliminary results, I agree with Klun et al. (2003) and surmise that SS220 is a candidate to serve as a new and effective alternate repellent for protection against vectors of disease.

Observations from this experiment reflect the results of a study carried out by Chauhan et al. in 2005 which states that Catnip oil is significantly less effective when

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compared to Deet or SS220 in deterring the biting of Aedes aegypti (Chauhan et al., 2005). In addition, another study mirrors this result by stating that Deet is a more effective contact repellent than Catnip oil when used with Aedes aegypti, Anopheles albimanus, and Anopheles quadrimaculatus (Bernier et al., 2005).

Deet was found to be the second weakest repellent in our study which follows Dogan and Rossignol’s (1999) observation that Deet does not act as a true repellent, but instead acts as a mild inhibitor. However, while SS220 was observed to be the strongest repellent in this experiment, another study by Klun et al. (2002 & 2006) stated that SS220 proved to be as effective as Deet against Aedes aegypti, but was more effective than Deet against Anopheles stephensi (Klun et al., 2002 & 2006). This is clearly in contrast with our results which show SS220 significantly more effective as a repellent compared to Deet against Aedes aegypti.

There were a number of additional variables that were not accounted for in this experiment since they were both outside the scope of this study and also proved too complex for SPSS statistical analysis. Multiple factors play a part in determining how effective any repellent will be. These factors include the species of the biting organisms and the density of organisms in the immediate surroundings (Maibach et al., 1966). Host factors that affect the number of questing females include: the age, sex, level of activity, and biochemical attractiveness to biting arthropods (Golenda et al., 1999; Maibach et al., 1966; Muirhead-Thomson, 1951). Further, environmental factors that also play a role in attraction and repellency include: the ambient temperature, humidity, and wind speed

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(Maibach et al., 1966). As a result, a given repellent will not protect all users equally from all mosquitoes equally, given the added environmental factors that play a role in appetitive searching and host attractiveness. Therefore, protection against questing arthropods is best achieved by avoiding infested habitats, wearing protective clothing, in addition to applying insect repellent (Fradin and Day, 2002).

Furthermore, while volunteers were instructed to touch their experimental leg as little as possible, contact with clothing and/or itching may have served to smear the repellents off, or spread repellents outside of their corresponding K&D module cell. If this study were to be replicated in the future, analyses based on different species of mosquitoes would be of interest (Klun et al., 2004). In addition, if enough volunteers could be utilized (compared to our 2 male and 2 female volunteers), repellent effectiveness studies dependent on gender would also be of interest. Additional information could also be gained by replicating the experiment in varying environmental conditions (i.e. dark versus light) or looking at the possible differences between young and old mosquitoes. Lastly, looking at and altering host attractiveness by changing specific food intake, menstruating versus non-menstruating females would also be of significance. While host attractiveness still lacks a standardized description or quantification, a study similar to the aforementioned may demonstrate important changes in mosquito behavior that would be of benefit to a future student in Physiological Parameters of Vector Competence.

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ACKNOWLEDGMENTS

I would like to thank Nicole L. Achee, John P. Grieco, and David M. Claborn for volunteering to be bitten in the repellency tests; Nicole L. Achee for supplying both the Houston strain mosquitoes and the repellents; and Cara Olsen for technical assistance and statistical analysis in this project.

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LITERATURE CITED

Bernier et al.. (2005) Comparison of Contact and Spatial Repellency of Catnip Oil and N,N-Diethyl-3-methylbenzamide (Deet) Against Mosquitoes. Journal of Medical Entomology. 42(3): 306-311. Chauhan et al.. (2005) Feeding Deterrent Effects of Catnip Oil Components Compared with Two Synthetic Amides Against Aedes aegypti. Journal of Medical Entomology. 42(4): 643-646. Dogan EB, and Rossignol PA. (1999) An Olfactometer for Discriminating Between Attraction, Inhibition, and Repellency in Mosquitoes (Diptera: Culicidae). Journal of Medical Entomology. 36(6): 788-793. Fradin MS, and Day JF. (2002) Comparative Efficacy of Insect Repellents against Mosquito Bites. The New England Journal of Medicine. 347(1): 13-18. Golenda et al. (1999) Gender-related efficacy difference to an extended duration formulation of topical N,N-diethyl-m-toluamide (DEET). American Journal of Tropical Medicine and Hygiene. 60(4): 654-657. Klun et al. (2006) Biting and Deterrent Activity of a Deet Analog, Two DEPA Analogs, and SS220 Applied Topically to Human Volunteers Compared with Deet Against Three Species of Blood-Feeding Flies. Journal of Medical Entomology. 43(6): 1248-1251. Klun et al. (2004) Comparative Resistance of Anopheles albimanus and Aedes aegypti to N,N-Diethyl-3-methylbenzamide (Deet) and 2-Methylpiperidinyl-3-cyclohexen1-carboxamide (AI3-37220) in Laboratory Human-Volunteer Repellent Assays. Journal of Medical Entomology. 41(3): 418-422.

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Klun et al. (2003) Synthesis and Repellent Efficacy of a New Chiral Piperidine Analog: Comparison with Deet and Bayrepel Activity in Human-Volunteer Laboratory Assays Against Aedes aegypti and Anopheles stephensi. Journal of Medical Entomology. 40(3): 293-299. Klun et al. (2002) Effectiveness of Optically Active Insect Repellent (1S,2S')-2methylpiperidinyl-3-cyclohexene-1-carboxamide (SS-220) compared to Deet and Bayrepel®. The 2002 Annual Meeting and Exhibition at the Entomological Society of America Conference: Section D Medical and Veterinary Entomology. Klun, JA and Debboun M. (2000) A New Module for Quantitative Evaluation of Repellent Efficacy Using Human Subjects. Journal of Medical Entomology. 37(1): 177-181. Maibach et al. (1966) Factors that attract and repel mosquitoes in human skin. The Journal of the American Medical Association. 196(3): 263-266. Muirhead-Thomson RC. (1951) The distribution of anopheline mosquito bites among different age groups: a new factor in malaria epidemiology. British Medical Journal. 1(4715): 1114-1117. SPSS. (2003) SPSS 12.0.1 for Windows, SPSS Inc. USDA. (2007) Chemicals Affecting Insect Behavior Laboratory- K&D module modeling photograph. Avail from: http://www.ars.usda.gov/main/site_main.htm?modecode=1275-17-00. Accessed: 1/17/08.

World Health Organization. (2007) WHO: Global Malaria Programme. Avail from: http://www.who.int/malaria. Accessed: 1/17/08.

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