Journal of the American Control Association,12(2):172_176, 1996

FIELD EVALUATION OF REPELLENTS. DEET AND A PIPERIDINE COMPOUND. AI3-3722O. AGAINST FUNESTUS AND ANOPHELES ARABIENSISIN WESTERN KENYA'

TODD W. WALKER,' LEON L. ROBERT3 ROBERT A. COPELAND.4 ANDREW K. GITHEKO,s ROBERT A. WIRTZ,6 JOHN I. GITHURE? rNo TERRY A. KLEIN6

ABSTRACT A field evaluation of the repellents N,N-diethyl-3-methylbenzamide (deet) and 1-(3-cy- clohexen-1-yl-carbonyl)-2-methylpiperidine (AI3-3722O, a piperidine compound) was conducted againsr Anopheles funestus and An. arabiensis in Kenya. Both repellents provided significantly more protection (P < 0.001) than the ethanol control. AI3-3722O was significantly more effective (P < 0.001) than deet in repelling both species of mosquitoes. Aller t h, O.l mg/cm, of Al3-3722O provided 89.8Vo and,Tl.lVo protection against An. arabiensis and An. funestzs, respectively. Deet provided >807o protection for only 3 h, and protection rapidly decreased after this time to 6O.2Vo and 35.1Vo for An. irabienvi and Az. funestus, respectively, after t h. Anopheles funestu.r was significantly less sensitive (P < 0.001) to both repellents than An. arabiensis. The results of this study indicate that Al3-3722O is more effective than deet in repelling anophetine mosquitoes in westem Kenya.

INTRODUCTION concerning the susceptibility of these mosquito species to arthropod repellent compounds. The vectors of human parasites in The United States Army is currently evaluat- western Kenya are Anopheles gambiae Glles, ing a novel piperidine compound (AI3-3722O) An. arabiensir Patton, and An. Giles funestus that has been shown to be effective in the lab- (Githeko et al. 1993). Anopheles gambiae s.s. oratory against several anopheline species, in- and An. arabiensis are morphologically indistin- cluding laboratory-reared An. gambiae (Cole- guishable members of the An. gambiae complex man et al. 1993, Frances et al. 1993). In a recent (Gillies and de Meillon 1968, Thylor et al. field test, this compound was 1993). shown to be ef- fective against wild populations of Anopheles di- Githeko et al. (1993) reported that the intense rzs Peyton and Harrison in Thailand (S. P Fran- perennial transmission of malaria at Ahero, a ces, unpublished data) Al3-3722O is currently village in western Kenya, was primarily due to under consideration, either alone or in combi- An. funestus, not members of the An. gambiae nation with deet, as a U.S. Army repellent for- complex. Anopheles arabiensis, although pres- mulation. ent in this area, appears to play a minor role in The objective of this study was to determine malaria transmission. No field data are available if AI3-3722O provides more effective and longer lasting protection than deet against An. funestus rThe views of the authors do not purport to reflect and An. arabiensis mosquitoes in the field. Deet the position of the Department of the Army or the is the standard against which the efficacies of Department of Defense. Mention of a commercial candidate skin repellents are evaluated. Field product does not constitute an endorsement of the evaluation of new repellent compounds is nec- product by the Department of Defense. The volunteers essary because behavioral responses to repel- gave informed consent to participate in the study. lents differ between populations 'United States Army Center for Health Promotion feral arthropod and Preventive Medicine, Divisional Support Activity- and laboratory-reared populations (Frances et al. West, ATTN: MCHB-AW-ES, Aurora, CO 80045- 1993). 5001. 3 Department of Chemistry, United States Military MATERIAL AND METHODS Academy, West Point, New York 10996. 4 United States Army Medical Research Unit-Ke- Two repellents, MN-diethyl-3-methylbenza- nya, Unit 641O9, Box 401, APO AE 09831-4109. mide (deet), lOO7o (Morflex 5 Inc., Greensboro, Vector Biology and Control Research Centre, Ke- NC) and 1-(3-cyclohexen-l-yl-carbonyl)-2- nya Medical Research Institute, P O. Box 1578, Kis- methylpiperidine (AI3-3722O), 99Vo (synthe- umu, Kenya. sized by T, P McGovern, Ecol- 6 Department of Entomology, Walter Reed Army In- Chemical stitute of Research, Washington, DC 2O3O7-510O- ogy Laboratory, USDA-ARS, Beltsville, MD) 7 Biomedical Sciences Research Centre, Kenya were evaluated. The study was conducted in Ny- Medical Research Institute, P O. Box 5484O, Nairobi, anza Province, western Kenya, adjacent to the Kenya. Ahero rice irrigation scheme. Ahero is located

t72 Juue 1996 REPELLEI.i-TS PON ATTI. ARABIENSIS AND AN. FUNESTUS 173

23 km SE of Kisumu. located on the shore of 2100 h, I h prior to the start of each repellent Lake Victoria. test. At 22Wh. the volunteers entered their des- Twelve adult male residents (>21 years old) ignated houses, sat on chairs, and collected all of the area volunteered to participate in this mosquitoes biting them on their lower legs in study. Repellents were evaluated on the lower 20-mm scintillation vials for 45 min during each legs, using the area between the knee and ankle. hour of the night. A 15-min break followed each Both trouser legs were rolled up to just above collection period. The time, collector, and house the knees and a line was drawn at the knee with were recorded for each captured mosquito. This an ink marker to provide a line of demarcation collection procedure was repeated each hour un- for repellent application and mosquito collec- til approximately 0600 h the next morning so tion. Low-top canvas shoes, all of the same that 8 biting collections were made by each vol- brand, provided a demarcation line at the ankle. unteer. Ambient temperature and humidity were Volunteers wore hooded screened jackets (Bug recorded hourly throughout the test nights. This Out Outdoor Wear@Ltd., Wauwatsoa, WI) to re- 9-h repellent efficacy test was replicated for 6 strict mosquito feeding to the lower legs. test nights. Repellents were prep:red as 5Vo solutions in The collected mosquitoes were transported to absolute ethanol (50 mg/ml) and applied at a rate the Kenya Medical Research Institute (KEMRI), of 0.1 mg/cm2. The amount of repellent varied Vector Biology and Control Research Centre, at among volunteers and was based on the surface Kisian for processing and identification. In a area of application, previous study at this site, Githeko et al. (1993) reported that An. arabiensis comprised lNVo of A:[(a+b+c)t3]h, the mosquitoes captured from the An. gambiae calculated from the averaged measurements of complex. The ribosomal DNA polymerase chain leg height (ft = length between the crease of the reaction (PCR) amplification method described knee and the ankle bone) and circumference ([a by Taylor et al. (1993) was used to confirm the + b + cl/3, where a : circumference just below identification of mosquitoes identified morpho- the knee, b : circumference at mid-calf, and c logically as An. gambiae s.l. = circumference just above the ankle bone). Pri- Nightly collection totals were determined for or to treatment, the lower legs of each volunteer each of the 8 collection times. Totals for each were washed with TOqo ethanol to ensure that 45-min period were then summed and the per- the treatments were applied directly onto the cent protection at each collection time, defined skin. The ethanol was then allowed to evaporate as the number of bites received by an individual before the treatments were applied to the vol- in the treatment group relative to that of the con- * unteers' legs. The legs of the control volunteers trol, was calculated flO0(control treatment)/ were treated with absolute ethanol. contro[. Results of preliminary repellent tests The repellent tests were conducted inside lo- indicated that the data did not meet the distri- cal mud-walled houses, because An. arabiensis butional assumptions of an analysis of variance and An. funestus primarily bloodfeed indoors and that transformations would not normalize (Githeko et al. 1993). Due to the design of hous- the data. Statistical comparisons of repellent ef- es in western Kenya, mosquitoes have free ac- ficacy were made among the treatment groups cess to the interior through the open eves. Mos- using Kruskal-Wallis nonparametric analysis of quitoes can enter and exit freely even though all variance (Statistix 1987). doors and windows may be closed. Bednets were provided to the house occupants so that the RESULTS AND DISCUSSION volunteers were the only accessible -meal source. Nightly weather conditions throughout the The study was arranged in a 3-way factorial study were relatively constant. The mean tem- design (2 repellent treatments plus a control X perature was 20.6oC (range: 16.7-23.4"C) arld 8 time intervals X 6 collection nights). The 12 the mean percent relative humidity was 8l7o volunteers were selected at random for treatment (range: 63-lOOVo RH). There was little or no and house so that for each collection night there wind. were 4 participants per treatment and 3 treatment Three genera of mosquitoes (Anopheles, Ae- participants per house (deet, AI3-3722O, and des, and Culex) were collected. Ttwo species of control). Once assigned to a house, volunteer Anopheles (An. funestus and An. gambiae s.l.), treatment groups were evaluated in that house representing 72Vo of the total number, were col- throughout the study. Treatments were changed lected, providing sufficient numbers (5,029) to nightly so by the end ofthe study each volunteer evaluate repellent efficacy. Initially, 3,668 An. had received each treatment twice. funestus and 1,361 An. gambiae s./. were iden- The treatments were applied at approximately tified. Twenty percent (272) of the An. gambiae t74 JounNel-or rse Avtnrcltr Mosquno Cournol AssocnrloN Vor-.12, No. 2

ANOPHELESARABIENSIS 100 z 90 o F 80 ogJ F 70 o E o 60 zF uJ 50 o E 40 rlu z 30 ...€.-. At3-57220 -+ DEET ul 20 = 10 0

HOURSAFTER REPELLENT APPLICATION Fig. 1. Mean percent protection (+ SE) provided by A13-37220 and deet against Anopheles arabiensis. s.l. were analyzed by using ribosomal DNA; all The incidence of Plasmodiumfalciparum in- were subsequently identified as An. arabiensis. fection of the collected Anopheles in the study Based upon these results, we therefore assumed and the biting behavior of infected versesunin- that all An. gambiae s.l. were An. arabiensis. fectedAnopheles on humans using mosquito re- This agrees with earlier work by Githeko et al. pellentsis discussedby Copelandet al. (1995). (1993) who reported that An. gambiae s.s. rarely Deet and AI3-3722O were significantly more occurs in the Ahero area. effective (Kruskal-Wallis statistic [KWSI

ANOPHELESFUNESTUS 100 90 z o 80 oF uJ 70 oF tr 60 r = 50 ()UI t 40 IIJ 4 30 z ----o--- Aa3-37220 uJ 20 -O- DEET = 10 0

HOURSAFTEB REPELLENT APPLICATION

Fig.2. Mean percent protection (+ SE) providedby N3-37220 and deet aga\nst Anopheles funestus JUNE 1996 REPELLEI\TS FoR AN. ARABIENSIS AND AN. FUNESTI.JS DEET 100 90 z o 80 F (t { uJ 70 I oF E 60 o. zF 50 lu (, E 40 ul o. 30 z IIJ 20 ".o?" AN.ABABIENSIS = * AN.FUNESTUS 10 0

HOURSAFTER REPELLENT APPLICATTON

Fig. 3. Mean percentprotection (+ SE) provided by deet againstAnopheles arabiensis and An. funestus.

17.I, P < 0.001) than the ethanol control in re- nestus. There was no significant difference in the pelling An. arabiensis and,An. funestrs. Over the repellency of A13-37220 and deet during the 9-h test period, AI3-3722O was significantly first 4 h after repellent application. However, at more effective (KWS : 9.O, P < 0.fi)3) than 7 h postapplication, Al3-3722O was significantly deet in repelling both An. arabiensis and An. fu- more effective at repelling An. arabiensis (Fig. Al3-37220 100 z 90 o t- 80 o u,l F 70 o E q 60 F = IJJ 50 o tr 40 UJ o. 30 z ...-o--. AN.ARAB|ENSIS {- I! 20 AN.FUNESTUS = 10 0

HOURSAFTER REPELLENT APPLICATION

Fig. 4. Mean percent protection (* SE) provided by AI3-3722O against Anopheles arabiensis and An- fu- nestus. 176 JounNel or rnr AvgntcAN MoseulTo Cor.trpol Assoctertor.r Vol. 12,No. 2 l) and An. funestus (Fig. 2) (KWS : 8.4, P < ACKNOWLEDGMENTS 0.004) compared to deet. From 5 to t h, AI3- The voluntary participation of the village res- 3722O continued to provide the volunteer with idents in this study is gratefully acknowledged. >9OVo and >6OEa protection against biting of We would also like to specifically thank Fred An. arabiensis and An. funestus, respectively. In Onyango, Alex Masinya, Samuel Wangowe, and contrast, t h after repellent application, the per- Sammy Ligonzo for their assistance. This manu- cent protection provided by deet was only 6O.2Vo script is published with the approval of the di- and 35.17o for An. arabiensis and An. funestus, rectors, Walter Reed Army Institute of Research, respectively. and the Kenya Medical Research Institute. From 7 to t h after repellent application, both deet (Fig. 3) and Al3-3722O (Fig. 4) provided REFER"ENCES CITED : significantly better protection (KWS 11.6, P Coleman, R. E., A. L. Richards, G. J. Magnon, C. S. < 0.002) against An. arabiensis (6O.2Vo and Maxwell, M. Debboun, T, A. Klein and R. A. Wirtz. 89.8Vo at t h, respectively) compared to An. fu- 1994. Laboratory and lield trials of four repellents nestus (35.l%o and 7l.l%o at t h, respectively). with Culex pipiens (Diptera: Culicidae). J. Med. En- These data have serious implications concerning tornol. 3l:.17-22. Coleman, R. 8., L. L. Robert, L. W. Roberts, J. A. repellent efficacy relating to protection against Glass, D. C. Seeley, A. Laughinghouse, P V. Perkins parasites vectors of malaria in the Ahero area. and R. A. Mrtz. 1993. Laboratory evaluation of Anopheles funestus is the primary vector of ma- repellents against four anopheline mosquitoes (Dip- laria in this area, with An. arabiensis playing a tera: Culicidae) and two phlebotomine sand secondiuy role. This study indicates that AI3- (Diptera: Psychodidae). J. Med. Entomol- 30449- 3722O is clearly a better repellent for use in this 502. Copeland, R. S., T W. Walker, L. L. Robert, J. I. Gi- area, as compared to deet. Further studies are thure, R. A. Wirtz and T, A. Klein. 1995. Response warranted to evaluate the effectiveness of AI3- of wild Anopheles funestus to repellent-protected 3722O against other species of the An. gambiae volunteers is unaffected by malaria infection of the complex in western Kenya. vector. J. Am. Mosq. Control Assoc.4:438-440. Frances, S. P, N. Eikarat, B. Spipongsai and C. Eam- sila. 1993. Response of Anopheles dirus and Aedes CONCLUSIONS albopicas to repellents in the laboratory. J. Am. Mosq. Controf Assoc. 9:474-47 6. Gillies, M. T. and B. de Meillon. 1968. The Ano- In initial studies using laboratory-reared mos- phelinae of Africa south of the Sahara (Ethiopian quitoes, Al3-3722O was as effective as deet Zoogeographical Region). Publ. No. 54. S. Afr. Inst. against An. gambiae and other anopheline spe- Med. Res., Johannesburg. cies (Coleman et al. 1993). We also found that Githeko, A. K., M. W. Service, C. M. Mbogo, E K. Al3-3722O provided as good or better protection Atieli and E O. Juma. 1993. falcipa- than deet in duration tests. In a field study, S. P rum sporozoite and entomological inoculation rates at the Ahero rice irrigation scheme and the Miwani Frances (unpublished data) found that AI3- sugar-belt in western Kenya. Ann. Trop. Med. Par- 3722O provided significantly better protection asitol.87:379-391. than deet against wild populations of An. dirus Statistix II. 1987. An interactive statistical analysis in Thailand. Coleman et al. (1994) reported that program for microcomputers. NH Analytical Soft- Al3-3722O provided better protection than deet ware. Roseville. MN. from biting of Culex pipiens Linn. in Saudi Ara- Taylor, K. A., S. M. Paskewitz, R. S- Copeland, J. Ko- ros, R. E Beach, J. I. Githure and E H. Collins. bia. Our data and previous findings indicate that 1993. Comparison of two ribosomal DNA-based AI3-3722O provides significantly better protec- methods for differentiating members of the Anoph- tion from biting of several mosquito species eles gambiae complex (Diptera: Culicidae). J. Med. from different genera. Entomol. 3O:457-461.