INFLUENCE OF SEX RATIO AND DENSITY OF MOSQUITOES ON THE EFFICACY OF IR3535® AND DEET 205 The infl uence of mosquito sex ratio and density on the effi ca cy of test chemicals was ex- pressed as CPT for Ae. aegypti, Cx. quinquefasciatus, and An. aquasalis. Consistent with the baseline comparison, and regardless of the sex ratio and density, Culex was repelled the longest, followed by Aedes and Anopheles. Within each species, the numbers and propor- tions of males and females present had a marked infl uence on CPT. For both Aedes and Anopheles, CPT was longest against 100 fem ales:100 males, often be- ing close to double than those observed for other treatments. In marked contrast, Culex appeared to bite more avidly in the presence of males. Across species, higher densities also appeared to reduce CPTs. Mosquito behavior likely interacts with mosquito density and repellent action to deter- mine protection time under the different demographic conditions (2,3,15). Mating activ- ity by males and perhaps male-avoidance behaviors of females may reduce biting rates in Aedes and Anopheles. By contrast, in Culex, the presence of males or mating may serve as a cue to seek a blood meal, increasing female motivation and rendering the repellents less effective. The generally lower repellent performance when more females were present, regardless of the sex ratio, might also represent a change in female avidity due to density, or simply an increase in the absolute number of females with inherently lower thresholds for feeding in the presence of the repellent. Differences in treatment effects observed within and among species are informative for cage testing practices and in relation to natural variation in fi eld mosquito demography. First, cage test results can vary strikingly in response to population density, male:female ratio, and species. Second, because mosquito demography varies in space and time in nature, it is important to consider its infl uence in the conduct and interpretation of fi eld test results. Although it is diffi cult to infer the extent to which these factors may underlie performance variation in past effi cacy evaluations reported in the literature, predictions of the infl uence of density and sex ratio based on species-specifi c knowledge of mosquito feeding and mating behavior may deserve additional attention in fi eld studies (19). In addition, knowledge of these factors may be useful in designing laboratory assays that take greater account of population variables that infl uence repellent performance in con- sumer and other public health contexts. In addition, a long-standing focus of non-pesticidal mosquito population control is the release of large numbers of males, often at high densities. For control of disease-vectoring populations of Ae. aegypti, fi eld studies (7,20) estimated the optimal density of release points for large-scale sterile male release programs to be at 50 meter intervals. Such approaches, when successful, may be self-limiting due to population declines, requiring frequent releases into the future to maintain population suppression. However, even more self-sustaining approaches, such as mass release of mosquitoes carrying multi-locus deleterious transgenes Figure 5. Structures of the repellents. (A) DEET (CAS no. 134-62-3) and (B) IR3535® (CAS no. 52304-36-6).

JOURNAL OF COSMETIC SCIENCE 206 (21), or ultimately microbes or transgenes with active drive mechanisms (21), may still strongly impact local and regional mosquito demography in ways that merit greater at- tention in repellent development. CONCLUSION Further consideration of mosquito sex ratio and density may be important for optimal development and modeling of mosquito repellent effi cacy in both laboratory and fi eld conditions. We tested the infl uence of mosquito density and female:male ratio in the laboratory with the topical repellent IR3535®. Notably, the direction of male infl uence in Culex species tested was opposite to that in Aedes and Anopheles species tested. These study results are important because they extend the study of density factors beyond DEET, and further point to striking variations among species in the outcomes of sex ratio manipulations. If confi rmed, these results suggest that sex ratio and density merit greater attention in fi eld studies, and the question of whether female-only guidelines for cage testing best model fi eld conditions for all mosquito species should be revisited. REFERENCES (1) S. P. Onyanga and S. J. Moore, “Evaluation of Repellent Effi cacy in Reducing Disease Incidence,” in Repellents: Principles, Methods and Uses, 2nd Ed. M. Debboun, S. P. Frances, and D. Strickman. Eds. (CRC Press, Boca Raton, 2015) p117. (2) D. R. Barnard, Biological assay methods for mosquito repellents. J. Am. Mosq. Control Assoc., 21(sp1), 12–16 (2005). (3) S. P. Carroll, “Evaluation of Topical Insect Repellents and Factors that Affect Their Performance,” in Insect Repellents, Principles, Methods, and Uses, M. Debboun, S P. Frances, and D. Strickman. Eds. (CRC Press, Boca Raton, 2006), pp. 245–259. (4) United States Environmental Protection Agency, Product Performance Test Guidelines OPPTS 810.3700: Insect Repellents to be Applied to Human Skin. Technical Document 712-C-10-001, accessed July 7, 2010, https://www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0150-001. (5) European Commission Directorate, Technical Notes for Guidance. Insecticides, Acaricides and Products to Control Other Arthropods (PT 18) and Repellents and Attractants (only Concerning Arthropods) (PT19), accessed July 7, 2010, https://EuropeanCommission,Directorate-GeneralEnvironment,CA- Sept10-Doc.6.2b https://circabc.europa.eu/sd/a/d7363efd-d8fb-43e6-8036-5bcc5e87bf22/CA-Sept13- Doc%205.1.e%20(Rev1)%20-%20treated%20articles%20guidance.doc (6) World Health Organization, Guidelines for Effi cacy Testing of Mosquito Repellents for Human Skin, WHO/HTM/WHOPES/2009.4, accessed July 7, 2010, https://www.eho.ont/iris/handle/10665/70072. (7) A. A. Khan, H. I. Maibach, and D. L. Skidrnore, Insect repellents: effect of mosquito and repellent re- lated factors on protection time. J. Econ. Entomol., 68, 43–45 (1975). (8) D. R. Barnard, Mediation of DEET repellency in mosquitoes (Diptera: Culicidae) by species, age, and parity. J. Med. Entomol., 35(3), 340 (1998). (9) R. D. Xue and D R. Barnard, Effects of partial blood engorgement and pretest carbohydrate availability on the repellency of DEET to Aedes albopictus. J. Vector Ecol., 24, 111–114 (1999). (10) D. R. Barnard, K. H. Posey, D. Smith, and C. E. Schreck, Mosquito density, biting rate and cage size effects on repellent tests. Med. Vet. Entomol., 12(1), 39–45 (1998). (11) G. Puccetti, “IR3535® (Ethyl Butylacetylaminoproprionate),” in Insect Repellents, Principles, Meth- ods and Uses, M. Debboun, S. P. Frances and D. Strickman. Eds. (CRC, Boca Raton, 2006), pp. 53–60. ( 12) United States Environmental Protection Agency, Pesticides: regulating Pesticides.3-[N-Butyl-N- acetyl]-aminopropionic acid, ethyl ester (113509). Technical Document, United States Environmental Protection Agency, Washington, D.C., accessed July 13, 2011. http://.epa.gov/opp00001/biopesticides/ ingredients. ( 13) S. P. Carroll, Prolonged effi cacy of IR3535 repellents against mosquitoes and blacklegged ticks in North America. J. Med. Entomol. 45(4), 706–714 (2008).