VECTOR CONTROL,PEST MANAGEMENT,RESISTANCE,REPELLENTS Laboratory Evaluation of the Response of and Uninfected and Infected With Dengue Virus to Deet

1 2 STEPHEN P. FRANCES, RATANA SITHIPRASASNA, AND KENNETH J. LINTHICUM

Department of Entomology, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok, 10400,

J. Med. Entomol. 48(2): 334Ð336 (2011); DOI: 10.1603/ME10120 ABSTRACT Laboratory studies were conducted to compare the response of Aedes aegypti (L.) and Aedes albopictus (Skuse) adults, uninfected and infected with four serotypes of dengue virus to a repellent containing 5% N, N-diethyl-3-methylbenzamide (deet). The results showed that mosquitoes infected with the four serotypes of dengue responded similarly to uninfected mosquitoes.

KEY WORDS N, N-diethyl-3-methylbenzamide; Aedes aegypti; Aedes albopictus; dengue; Thailand

Mosquitoes infected with some arboviruses exhibit Vincke and Lips did not affect their response to deet behavior different from that observed for uninfected and the synthetic pyrethroid permethrin in the labo- mosquitoes (Mims et al. 1966, Grimstad et al. 1980, ratory. Another study demonstrated that infection of Lambrechts and Scott 2009). Arboviral infections in Edhazardia aedis (Kudo) (Microsporidia: Culico- mosquitoes may affect survival, fecundity, and feeding sporidae) in Ae. aegypti resulted in reduction in blood behavior (Turell et al. 1985, Faran et al. 1987). Studies feeding and fecundity, but increased deet repellency using ßuorescent antibody techniques indicate that (Barnard et al. 2007). Dengue (DEN)-2 virus-infected Aedes albopictus In southeast Asia, Ae. aegypti is the principal vector (Skuse) and splendens (Wiedemann) of dengue viruses in urban areas, and Ae. albopictus is develop signiÞcant infection of the nervous system a vector in rural areas, in the absence of Ae. aegypti. In (Kuberski 1979, Yamamoto et al. 1987). DEN-3 virus this short communication, we report laboratory stud- infections in Aedes aegypti (L.) have been shown to ies that investigated the impact of dengue virus infec- have an effect on the time required for locating and tions in Ae. aegypti and Ae. albopictus on the response taking a blood meal (Platt et al. 1997). Infection of the to the repellent deet. nervous system may alter the mosquitoesÕ response to repellents or insecticides. Materials and Methods The chemical N, N-diethyl-3-methylbenzamide

(deet) was Þrst marketed commercially as an Ae. aegypti were the F2ÐF4 progeny from eggs ovi- repellent in 1954 (McCabe et al. 1954) and is now the posited by adults collected as larvae in Bangkok, Thai- most widely used repellent compound applied to hu- land. Ae. albopictus were the F1ÐF3 progeny from eggs man skin for protection against biting and nuisance oviposited by adults collected in Krabi Province, Thai- mosquitoes (Curtis et al. 1990, Fradin 1998, Fradin and land. Mosquitoes were reared at 30ЊC and at 75Ð85% Day 2002). Despite its widespread use, there have only RH. Viral titers were calculated by determining the been a few evaluations of the effectiveness of repel- infective dose 50/ml of the virus assayed in lents containing deet conducted against mosquitoes adult Tx. splendens mosquitoes by the methods de- infected with human pathogens. A study by Robert et scribed by Rosen and Gubler (1974). The four dengue al. (1991) showed that infection of stephensi virus strains used were all isolated from human sera Liston with the parasite berghei and are designated as follows: DEN-1, D82-041 strain (SM-1, C6/36-4), Bangkok, viral titer ϭ 109.2; DEN-2, Mention of a commercial product does not constitute its endorse- New Guinea strain (SM-35, C6/36-7), New Guinea, ment by the Australian Defense Force, the United States Department viral titer ϭ 107.2; DEN-3, strain CH53489 (LLC- of Agriculture, or the United States Department of Defense. The MK2Ð1, C6/36-2), Bangkok, viral titer ϭ 108.2; DEN-4, opinions expressed in this study are those of the authors and do not necessarily reßect those of the Australian Defense Force or any extant strain 816689 (C6/36-3), Dominican Republic, viral policy. titer ϭ 109.0. 1 Corresponding author: Australian Army Malaria Institute, Galli- Groups of 5- to 7-d-old adult female mosquitoes poli Barracks, Enoggera Queensland 4051, Australia (e-mail: were placed into two groups of equal numbers (100Ð [email protected]). 2 USDAÐARS Center for Medical Agricultural & Veterinary Ento- 200 specimens) and intrathoracically inoculated with mology, 1600/1700 SW 23rd Drive, Gainesville, FL 32608. 0.28 ␮l of either undiluted stock dengue virus seeds or March 2011 FRANCES ET AL.: DEET AGAINST Aedes SP.INFECTED WITH DENGUE 335 phosphate-buffered saline. Mosquitoes were then Table 1. Number of Aedes aegypti and Aedes albopictus adult placed in 500-ml paper cups with screen tops and sugar females attracted to a forearm treated with 5% deet pads, and held for 14 d at 30ЊC. Before testing, mos- No. mosquitoes attracted/no. tested (%) quitoes were held without a sugar source for 12Ð24 h. Dengue Aedes aegypti Aedes albopictus For the feeding test mosquitoes were transferred to a virus wire mesh cage, measuring 30 - 30 - 30 cm, on which Infected Uninfected Infected Uninfected two parallel strips of tape (spaced 5 cm apart) were 1 5/40 (12.5) 6/51 (11.8) 1/36 (2.8) 1/92 (1.1) placed diagonally on two sides. 2 33/248 (13.3) 43/312 (13.8) 2/160 (1.3) 2/82 (2.4) 3 26/168 (15.5) 11/64 (17.2) 5/315 (1.6) 0/130 (0.0) Because mosquitoes were viremic, a method was 4 84/400 (21.0) 57/288 (19.8) 12/496 (2.4) 15/492 (3.0) used to observe the behavior of mosquitoes exposed to deet-treated forearms. A human volunteer applied 5% deet (Colbar, Melbourne, Australia) in ethanol (1 ml) to one arm, whereas the other was left untreated. Each species were attracted to untreated forearms. Varia- trial consisted of exposing untreated and deet-treated tion in the response of different mosquitoes to repel- forearms to a cage containing virus-infected and virus- lent active ingredients is well known (Frances 2007), uninfected mosquitoes. To determine the level of and studies conducted with Ae. albopictus against a feeding behavior (avidity) exhibited by the mosqui- range of deet and dimethylphthlate formulations toes in each cage for each of the trials, the untreated showed that deet provided extended protection arm was placed 1 cm away from the screen mesh of the against uninfected Ae. albopictus compared with cage, directly over the diagonal tape strips on one side Anopheles dirus Peyton and Harrison (Frances et al. on the cage for 60 s. After 60 s, the number of mos- 1993). quitoes that landed on the screen mesh between the A majority of laboratory tests conducted with for- tape strips and remained there was recorded. Imme- mulations of deet and other active ingredients use diately after exposure of the mosquitoes to the un- uninfected Ae. aegypti adults as a test mosquito (Fran- treated arm, the treated arm was placed similarly over ces 2007). The results of the current study showed that the tape strips on the other side of the cage for a period mosquitoes infected with the four serotypes of dengue of 120 s, and then the number of mosquitoes between responded similarly to uninfected mosquitoes. This the tape strips was counted. For each trial, the pro- suggests that deet may be used in the Þeld with equal cedure was repeated for both cages three more times success against dengue-infected and uninfected Ae. at 1-h intervals. aegypti and Ae. albopictus mosquitoes. However, tests After completion of the trial, all mosquitoes were with other active ingredients, such as picaridin (2-(2- frozen at Ϫ70ЊC, awaiting individual specimen assay in hydroxyethyl)-piperidine carboxylic acid 1-methyl es- a ßavivirus antigen capture enzyme-linked immu- ter), IR3535 (ethyl butylacetylaminopropionate), and nosorbent assay (ELISA) following the methods de- para-methane-3,8-diol, are warranted, as these active scribed by Sithiprasasna et al. (1994). This ELISA uses ingredients, along with deet, are recommended for use a monoclonal antibody that recognizes all ßaviviruses. in the United States by the Centers for Disease Con- Specimens were considered positive when optical trol and Prevention. It is warranted to evaluate the density was equal to the mean of the negative controls response of mosquitoes infected with other important plus 3 SDs. arboviruses, such as West Nile virus in the United States, where repellent chemicals are recommended to reduce contact between humans and mosquitoes. Results and Discussion All virus-inoculated mosquitoes were found to be Acknowledgments infected with the dengue viruses after individual assay in the ELISA, and each had a disseminated infection We thank Larp Panthursiri for technical assistance, and R. D. Cooper for comments on the manuscript. because of the intrathoracic route of infection. Mos- quitoes inoculated with phosphate-buffered saline were not found to be infected with dengue after assay References Cited in ELISA. The response of uninfected and dengue- Barnard, D. R., R.-D. Xue, M. A. Rotstein, and J. J. Becnel. infected Ae. aegypti and Ae. albopictus to a forearm 2007. Microsporidiosis (Microsporidia: Culicosporidae) treated with 5% deet is shown in Table 1. Ae. aegypti alters blood-feeding responses and DEET repellency in were attracted to the human forearm treated with 5% Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 44: deet, with 12.5Ð21.0% of infected mosquitoes and 1040Ð1046. 11.8Ð19.8% of uninfected mosquitoes attracted. In Curtis, C. F., J. D. Lines, L. Baolin, and A. Renz. 1990. contrast, fewer Ae. albopictus were attracted, with Natural and synthetic repellents, pp. 75Ð92. In C. F. Curtis 1.3Ð2.8% of infected and 0Ð3.0% uninfected mosqui- (ed.), Appropriate Technology in Vector Control. CRC, toes attracted to a deet-treated forearm. There were Boca Raton, FL. Faran, M. E., M. J. Turell, W. S. Romoser, R. G. Routier, P. H. no statistical differences in the proportion of infected Gibbs, T. L. Cannon, and C. L. Bailey. 1987. Reduced and uninfected Ae. aegypti (␹2 ϭ 12.8, df ϭ 7, P ϭ 0.08) 2 survival of adult Culex pipiens infected with Rift Valley and Ae. albopictus (␹ ϭ 6.7, df ϭ 7, P ϭ 0.46) attracted fever virus. Am. J. Trop Med. Hyg. 37: 403Ð409. to a forearm treated with 5% deet. Between 40 and 60% Fradin, M. S. 1998. Mosquitoes and mosquito repellents: a of both uninfected and infected mosquitoes from both clinicianÕs guide. Ann. Intern. Med. 128: 931Ð940. 336 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 48, no. 2

Fradin, M. S., and J. F. Day. 2002. Comparative efÞcacy of Platt, K. B., K. J. Linthicum, K.S.A. Myint, B. L. Innis, K. insect repellents against mosquito bites. N. Engl. J. Med. Lerdthusnee, and D. W. Vaughn. 1997. Impact of den- 347: 13Ð18. gue virus infection on feeding behavior of Aedes aegypti. Frances, S. P. 2007. EfÞcacy and safety of repellents con- Am. J. Trop. Med. Hyg. 57: 119Ð125. taining deet, 1st ed., pp. 311Ð325. In M. Debboun, S. P. Robert, L. L., I. Schneider, and R. A. Wirtz. 1991. Deet and Frances, and D. Strickman (eds.), Insect Repellents: Prin- permethrin as protectants against malaria-infected and ciples, Methods & Uses. CRC, Boca Raton, FL. uninfected mosquitoes. J. Am. Mosq. Frances, S. P., N. Eikarat, B. Sripongsai, and C. Eamsila. 1993. Control Assoc. 7: 304Ð306. Response of Anopheles dirus and Aedes albopictus to re- Rosen, L., and D. Gubler. 1974. The use of mosquitoes to pellents in the laboratory. J. Am. Mosq. Control Assoc. 9: detect and propagate dengue viruses. Am. J. Trop. Med. 474Ð476. Hyg. 23: 1153Ð1160. Grimstad, P. R., Q. E. Ross, and G. B. Craig, Jr. 1980. Aedes Sithiprasasna, R., D. Strickman, B. L. Innis, and K. J. Lin- triseriatus (Diptera: Culicidae) and La Crosse virus. II. thicum. 1994. Enzyme linked immunosorbent assay for ModiÞcation of mosquito feeding behaviour by virus in- detecting dengue and Japanese encephalitis viral an- fection. J. Med. Entomol. 17: 1Ð7. tigen in mosquitoes. Ann. Parasitol. Trop. Med. 88: Kuberski, T. 1979. Fluorescent antibody studies on the de- 397Ð404. velopment of dengue-2 virus in Aedes albopictus (Diptera: Turell, M. J., T. P. Gargan, and C. L. Bailey. 1985. Culex Culicidae). J. Med. Entomol. 16: 343Ð349. pipiens (Diptera: Culicidae) morbidity and mortality as- Lambrechts, L., and T. W. Scott. 2009. Mode of transmission sociated with Rift Valley fever infection. J. Med. Entomol. and the evolution of arbovirus virulence in mosquito 22: 332Ð337. vectors. Proc. R. Soc. B 276: 1369Ð1378. Yamamoto, N., T. Kimura, and A. Ohyama. 1987. Multipli- McCabe, E. T., W. F. Barthel, S. I. Gertler, and S. A. Hall. cation and distribution of type 2 dengue and Japanese 1954. Insect repellents. III. N, N-diethylamides. J. Org. encephalitis viruses in after in- Chem. 19: 493Ð498. trathoracic inoculation. Arch. Virol. 97: 37Ð47. Mims, C. A., M. F. Day, and I. D. Marshall. 1966. Cytopathic effects of Semiliki forest virus in the mosquito Aedes aegypti. Am. J. Trop. Med. Hyg. 15: 775Ð784. Received 6 May 2010; accepted 7 October 2010.