Abstract
Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was >95% at 4 h. For both mosquitoes and black flies, deet alone provided <90% protection at 6 h, whereas AI3-37220 provided >95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.
Personal protective measures, including repellents, are the primary means of vector-borne disease prevention available to U.S. military troops deployed into areas where vector control is not practical (Gupta and Rutledge 1994, Copeland et al. 1995). Even when chemoprophylaxis or vaccines are available, repellents offer advantages in that they can be applied with minimal prior planning against a broad spectrum of vectors. The U.S. military continues to have an interest in developing new repellents with improved efficacy and, especially, acceptability to the user (Hooper and Wirtz 1983, Gambel et al. 1998, Strickman et al. 1999).
One promising new repellent is the piperidine compound AI3-37220. Unlike the related compound, AI3-35765, AI3-37220 does not produce a warming sensation when applied to the skin. Recent field evaluations of deet and AI3-37220 have shown that AI3-37220 is equal to or significantly better than deet in repelling Prosimulium mixtum Symes and P. fuscum Symes & Davies in Massachusetts (Robert et al. 1992), Anopheles dirus Peyton & Harrison in Thailand, and An. farauti s.s. Laveran in Australia (Frances et al. 1996, 1998), An. arabiensis Patton and An. funestus Giles in western Kenya (Walker et al. 1996), Culex pipiens L. in Saudi Arabia (Coleman et al. 1994), Leptoconops americanus Carter in Utah (Perich et al. 1995), and Amblyomma americanum (L.) in New Jersey (Solberg et al. 1995).
The purpose of our study was to determine if the combination of AI3-37220 and deet provides more effective and longer lasting protection than either deet or AI3-37220 used alone against black flies and mosquitoes in the field. Field evaluation of new repellent compounds is necessary because behavioral responses to repellents differ between feral populations and laboratory-reared mosquitoes (Frances et al. 1993, 1996). The response of arthropod vectors to deet is the standard against which the efficacies of new repellents are evaluated, but data on the response of Aedes communis sensu lato (DeGeer) and Simulium venustum sensu lato Say to deet are lacking. The current field study evaluated the response of Ae. communis and S. venustum to deet, AI3-37220, and the combination of deet + AI3-37220.
Materials and Methods
Study Site
The study was conducted in the Adirondack Mountains at Adirondack Park located on Route 3, which is 5.6 km north of Cranberry Lake, St. Lawrence County, NY, from 22 to 29 June 1994. The site consisted of scattered open areas surrounded by mixed coniferous and deciduous forest. The Grass River ran along one side of the study area.
Test Repellents
The three repellent compounds and mixture were as follows: (1) N,N-diethyl-3-methylbenzamide (deet) (Morflex, Greensboro, NC); (2) the piperidine compound 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220, synthesized by Terrance P. McGovern, Insect Chemical Ecology Laboratory, U.S. Department of Agriculture, Beltsville Agricultural Research Center, Beltsville, MD); and (3) a 1:1 ratio (volume) of deet and AI3-37220.
Insects
The black fly S. venustum and the mosquito Ae. communis were abundant, whereas the black fly Prosimulium mixtum Symes & Davies, three other species of mosquitoes [Ae. canadensis (Theobald), Ae. excrucians (Walker), Coquillettidia perturbans (Walker)], and five species of deer flies (Chrysops ater Macquart, C. carbonarius Walker, C. excitans Walker, C. mitis Osten Sacken, and C. sordidus Osten Sacken) were collected infrequently. Voucher specimens were deposited in the U.S. National Museum, Smithsonian Institution, Washington, DC. Simulium venustum and Ae. communis were the only species collected in sufficient numbers to evaluate repellency.
Field Repellent Tests
Tests were performed under a minimal risk human use protocol approved by the Walter Reed Army Institute of Research Human Use Research Review Committee (on file in our laboratory). Trials were conducted using six volunteers (two females and four males) age 21–55 yr with no known history of allergic reactions to arthropod bites. Each volunteer wore the U.S. Army Battle Dress Uniform printed with a four-color (green, loam, sand, and brown) woodland camouflage pattern and not treated with permethrin. A screen jacket (Bug Out Outdoor Wear, Wauwatosa, WI) and surgical gloves were worn to limit biting on untreated areas of the upper body, hands, and head.
Repellent solutions were applied at a rate of 0.25 mg/cm2 of surface area on the forearms of the volunteers. Four treatments were applied: (1) deet, (2) AI3-37220, (3) mixture of deet and AI3-37220 (combined application rate of 0.25 mg/cm2), or (4) ethyl alcohol (i.e., negative control). After application, the solutions were allowed to dry on the skin for 15-30 min before the first exposure at the study site.
Biting insects were collected individually in scintillation vials. Samples were taken until 30 insects were collected or 15 min elapsed, whichever came first. Any insect observed biting was collected regardless of whether it fed to repletion or whether it was standing on an untreated area while biting a treated area. Volunteers worked in pairs, with one volunteer keeping the screen jacket sleeves down and collecting biting insects from the exposed forearms of the other volunteer. At the conclusion of the test period, the volunteer who had been bitten would roll down his sleeves and the volunteer who had collected would roll up his sleeves, performing an additional 15-min test. Tests were initiated immediately after the application dried and were continued each hour for 8 h. All tests were conducted in daylight between 0800 and 2030 hours.
Trials were conducted during nine consecutive days. On the first day, treatments were randomly assigned to the six volunteers (12 arms). Thereafter, treatments were rotated so that replication was equivalent for each volunteer. By the end of the study, each volunteer had tested each of the four treatments three times.
Analysis
Using the 3-d totals for each volunteer, the nine hourly samples were grouped into three time periods: P1 = total count for hours 0–4, P2 = total count for hours 5 and 6, and P3 = total counts for hours 7 and 8.
Percent protection [100 × (control count minus repellent count)/control count] was calculated from the daily collection totals summed over volunteers for each of the nine hourly time intervals. These hourly totals were plotted at each period to show change in repellency over time after application. An analysis of variance procedure for a two-factor experiment (repellent group × test period) with repeated measurements was used to compare repellent effects over time. An arcsine transformation was used to stabilize the variance of percent protection (Little and Hills 1978, p. 158).
Results and Discussion
Aedes communis represented the majority (98%) of the 1,765 mosquitoes collected. Other species attracted to humans were Ae. excrucians (0.6% of total), Ae. canadensis (0.5%), and Coquillettidia perturbans (1.0%). The biting rates for Ae. communis ranged from 10.8 to 18.4 bites per 15 min per person (Table 1). S. venustum was the only black fly species collected in sufficient numbers (96% of 558 total black flies; 4% were P. mixtum) to determine percent protection. The range for the biting rate of S. venustum was 3.2–8.1 bites per 15 min per person (Table 1). Unfortunately, the 76 deer flies collected (61 specimens of Chrysops niger, five of C. ater, four of C. carbonarius, four of C. sordidus, one of C. excitans, and one of C. mitis) were insufficient for repellent evaluation.
Mean percent protection (95% confidence limits) against Ae. communis and Simulium venustum for three repellent treatments evaluated for duration of repellency at 4, 6, and 8 h after application
Mean percent protection (95% confidence limits) against Ae. communis and Simulium venustum for three repellent treatments evaluated for duration of repellency at 4, 6, and 8 h after application
All three repellent formulations provided average protection >95% against biting from both Ae. communis and S. venustum for the first 4 h (Table 1). AI3-37220 was the only repellent that maintained >95% protection from mosquitoes for 5 h (Fig. 1) and from black flies for 6 h (Fig. 2) after application. Protection against Ae. communis fell below 90% at 8 h after repellent application for all three treatments. By 8 h after application, at least half of the test volunteers experienced <80% protection against both insects (Fig. 3).
Percent protection provided by three different repellent treatments (concentration on skin 0.25 mg/cm2) against Ae. communis. A bold longitudinal line indicates the 90% protection level.
Percent protection provided by three different repellent treatments (concentration on skin 0.25 mg/cm2) against S. venustum.Abold longitudinal line indicates the 90% protection level.
Comparison of repellency at 8 h after application among the three treatments against Ae. communis and S. venustum (each dot represents the result from one person on one day of the study).
For the purposes of analysis, the average profiles of protection over the three time periods (i.e., 4, 6, and 8 h) were examined to test the following null hypotheses: (1) Protection declined over time at the same rate for the three repellent formulations. (2) Duration of repellency was the same. (3) Mean percent protection for each treatment was the same over time. First, the duration of repellency among treatments for mosquito and black fly species appeared to be parallel, because there was no time × repellent treatment interaction (Ae. communis,F = 1.15; df = 4, 30; P = 0.35; S. venustum,F = 1.10; df = 4, 30; P = 0.37). Second, there was no overall significant difference in repellency among the three treatments for either Ae. communis (F = 1.80; df = 2, 10; P = 0.21) or S. venustum (F = 2.63; df = 2, 10; P = 0.12). However, confidence limits of percentages were not overlapping, indicating that AI3-37220 was more repellent at 8 h against Ae. communis and at 6 h against S. venustum (Table 1). Finally, we found that although there were no overall significant differences among the three repellents against either the mosquito or the black fly, protection declined significantly over time (Ae. communis:F = 15.82; df = 2, 30; P < 0.001; S. venustum:F = 17.84; df = 2, 30; P < 0.001; Figs. 1 and 2).
Only one other study has examined the efficacy of deet + AI3-37220 in combination (Debboun et al. 1999). Testing against laboratory-reared Aedes aegypti (L.) and Anopheles stephensi Liston by using an in vitro membrane blood-feeding system, the repellency from the combination of deet + AI3-37220 was similar to that of deet, although there was some evidence (not confirmed in statistical tests) of synergistic interaction against An. stephensi. The current study showed that the overall repellency of the combination of deet + AI3-37220 was similar to an equivalent concentration of deet against Ae. communis and S. venustum.
Overall, our field study showed that the piperidine compound AI3-37220 used alone or in combination with deet provided about equal protection as deet against the mosquito Ae. communis and the black fly S. venustum. Applied as simple alcohol solutions at a dosage of 0.25 mg/cm2, these compounds would have to be reapplied every 5–6 h to maintain ≥90% protection from these two species. The repellent compound AI3-37220 could be an effective alternative to deet in the United States, as has been shown in many other parts of the world. Incorporating AI3-37220 into an appropriate formulation probably would result in a useful repellent product.
An additional repellent product would be welcome in the chemical armamentarium against biting arthropods. When vector control is not possible, repellents provide an inexpensive means of protecting individuals from insect bites (WHO 1995). Effective new repellents may encourage broader acceptability and use, thereby preventing bites that can lead to illness ranging from irritation to death (Gudgel and Grauer 1954, Pinheiro et al. 1974).
Acknowledgements
We gratefully acknowledge the support of Phillip Lawyer, Jerold Sadoff, and Alice Boarman, and the technical assistance of Ronnie Essex, Karl Korpal, and Dina Ibrahim (Walter Reed Army Institute of Research). We also thank Dan Molloy (New York State Museum, Albany, NY), Andrea Malik, and Richard Matzell (Environmental Conservation Officer, NY, State) for assistance in site selection and identifying seasonal abundance of black fly populations in the Adirondack State Park, and Henry and Dianne Ford for their support throughout the study.
References Cited
Author notes
Department of Entomology, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307–5100.
Walter Reed Biosystematics Unit, Department of Entomology, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307–5100.
U.S. Army Center for Health Promotion and Preventive Medicine, Directorate of Clinical Preventive Medicine, Aberdeen Proving Ground, MD 21010.
Division of Biometrics, Walter Reed Army Institute of Research, Washington, DC 20307–5100.
In conducting this research, the investigators adhered to the guidelines established by the National Institutes of Health for tests involving human subjects.
