Field evaluation of Deet and Pip

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         Field Evaluation of Deet and a Piperidine Repellent Against
       Ae&s communis (Diptera: Culicidae) and Simdium venustum
      (Diptera: Simuliidae) in the Adirondack Mountains of New York

             MUSTAPHA DEBBOUN,’               DANIEL      STRICKMAN,’           VICTORIA     B. SOLBERG,’
RJ~~ARl-j C. WILKERSON,”          KENNETH                      .
                                                  R. MCPHERSON,‘           3 CLAUDIA                ,
                                                                                            GOLENDA,‘       * LISA   KEEP,’
                  ROBERT               ~
                              A. WIRTZ,‘      ’ ROBERT       BURGE,’      AND   TERRY      A. KLEINI,   ’

                 Department of Entomology, Division of Communicable Diseases and Immunology,
                       Walter Reed Army Institute of Research, Washington, DC 20307-5100

                                           J. Med. Entomol. 37(6): 919-923 (2000)
          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:l ratio of deet + AI3-37220 were
          evaluated topically (0.25 mglcm’ applied in ethanol solution) on human volunteers against the
          mosquito Aed+z.s  communis   (DeGeer) and the black fly Simulium. venusturn Say. The average repel-
          lency 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.

          KEY WORDS                     Simuliumuerwtum, repellents, deet, piperidine, AI3-37220
                          Aedes communis,

hso~h~    PROTE~       MEASURES,   including I’ dentS,
                                              q                      minimal prior planning against a broad spectrum of
are the primary means of vector-borne       disease pre-             vectors. The U.S. military continues to have an interest
vention available to U.S. military troops deployed into              in developing new repellents with improved efficacy
areaswhere vector control is not practical (Gupta and                and, especially, acceptability to the user (Hooper and
Rutledge 1994, Copeland et al. 1995). Even when che-                 Wirtz 1983, Gambel et al. 1998, Strickman et al. 1999).
moprophylaxis or vaccines are available, repellents                     One promising new repellent is the piperidine com-
of’er advantages in that they can be applied with                    pound AI3-37220.       Unlike the related compound,
                                                                     AI3-35765, AI3-37220 does not produce a warming
  Inconducting research,the investigators adhered to the guide-
                                                                     sensation when applied to the skin. Recent field eval-
IIws  established the National Institutes of Health for testsinvolv-
                 by                                                  uations of deet and AI3-37220 have shown that AI3-
9 b_yan subjects.                                                    37220 is equal to or significantly better than deet in
    _=wtment of Entomoioav. Dwwon of Commumcabie Diseases            repeiiing Yrosimuiium mirtum Symes and Y. &scum
ad Immunology,     Walter Rezd Atiy Institute of Research, Wash-
&on, DC 20307-5100.                                                  Symes & Davies in Massachusetts (Robert et al. 1992))
  2W&er ReedBiosystematicsUnit, Department of Entomology, Anoph-eles             dir-w Peyton & Harrison in Thailand, and
DivisionCommunicable        Diseases and Immunology, Walter Reed     An. faruuti S.S. Laveran in Australia (Frances et al.
hY Instituteof Research,Washington, DC 20307-5100.                   1996, 1998), An. arubiensis Patton and An. fine&us
  3Cmt%t address:U.S. Army Medical Department Center and
schoola  Medical Zoology Branch, Fort Sam Houston, TX 78234-6142.    Giles in western Kenya (Walker et al. 1996)) Culer
  C went address:
   ‘                 JointVaccine Acquisition Program, Project Man-  pipiens L. in Saudi Arabia (Coleman et al. 1994)) Lep-
%Trnmt    Office,Fort Detrick MD 217024041.                          tocunops um-er-icunus   Carter in Utah (Perich et al.
     u.s.h~   Center for Heal;h Promotion and Preventive Medicine,   1995), and Amblyomma umericanum (L.) in New Jer-
     atorate of Clinical Preventive Medicine, Aberdeen Proving
 ??4 MD 21010.                                                       sey (Solberg et al. 1995).
   “Current  address: Entomology Branch, Division of Parasitic Dis-     The purpose of our study was to determine if the
 w centers for Disease Control and Prevention, Atlanta, CA           combination     of AI3-37220 and deet provides more
   ’ b*ii    of Biometrics Walter Reed Army Institute of Research,
                                                                     effective and longer lasting protection than either
 \Vuhi%on.DC 20307-5i00.                                             deet or AI3-37220 used alone against black flies and
   EC  went address:  HHC, 18th MedicalCommand, Unit 15281,APO       mosquitoes in the field. Field evaluation of new re-
                                                                     pellent compounds is necessary because behavioral
920                                    JOURNAL    OF MEDICAL       ENTOMOLOGY                                                            Vol. 37, no.6

responses to repellents differ between feral popula-                solutions were allowed to dry on the skin for 15_ati.
tions and laboratory-reared  mosquitoes (Frances et al.             before the first exposure at the study site.
1993,1996). The response of arthropod vectors to deet                   Biting insects were collected individually in sch.
is the standard against which the efficacies of new                 tillation vials. Samples were taken until 30 insects                             w,r,

repellents are evaluated, but data on the response of               collected or 15 min elapsed, whichever came first.Any
Aedes communis sensu lato (DeGeer)       and Simulium               insect observed biting was collected regardless of
zxnustum sensu lato Say to deet are lacking. The cur-               whether it fed to repletion or whether it was stacl:
rent field study evaluated the response of Ae. commu-               on an untreated area while biting a treated area yif
nis and S. tienustum to deet, AI3-37220, and the com-               unteers worked in pairs, with one volunteer keeping
bination of deet + AI3-37220.                                       the screen jacket sleeves down and collecting bih,
                                                                    insects from the exposed forearms of the other vol_
                                                                     unteer. At the conclusion of the test period, the vol_
                  Materials   and Methods
                                                                     unteer who had been bitten would roll down his
   Study Site. The study was conducted in the Adiron-                sleeves and the volunteer who had collected would
dack Mountains at Adirondack Park located on Route                  roll up his sleeves, performing an additional lsi_mb
3, which is 5.6 km north of Cranberry Lake, St. Law-                test. Tests were initiated immediately after the appb_
rence County, NY, from 22 to 29 June 1994. The site                  cation dried and were continued each hour for 8 h.All
consisted of scattered open areas surrounded                  by     tests were conducted in daylight between 0800 and
mixed coniferous and deciduous forest. The Grass                     2030 hours.
River ran along one side of the study area.                             Trials were conducted during nine consecutive
   Test Repellents. The three repellent             compounds        days. On the first day, treatments were randomly as-
and mixture were as follows: (1) N,N-diethyl-3-meth-                 signed to the six volunteers      (12 ms).    Thereafter,
ylbenzamide       (deet) (Morflex, Greensboro, NC) ; (2)             treatments were rotated so that replication was equiv-
the piperidine       compound      l- [3-cyclohexen-l-ylcar-         alent for each volunteer. By the end of the study, each
bony1 ] -2-methylpiperidine        (AI3-37220,      synthesized      volunteer had tested each of the four treatments three
by Terrance P. McGovern, Insect Chemical Ecology                     times.
Laboratory, U.S. Department of Agriculture, Beltsville                  Analysis. Using the 3-d totals for each volunteer, the
Agricultural     Research Center, Beltsville, MD); and               nine hourly samples were grouped into three time
 (3) a 1:l ratio (volume) of deet and AI3-37220.                     periods: Pl = total count for hours O-4, P2 = total
    Insects. The black fly S. oenustumand the mosquito               count for hours 5 and 6, and P3 = total counts for hours
Ae. communis were abundant, whereas the black fly                   7 and 8.
Prosimulium mixtum Symes & Davies, three other spe-                    Percent protection    [ 100 X (control count minus
cies of mosquitoes [Ae. canadensis (Theobald),               Ae.    repellent count) /control count] was calculated from
excrucians (Walker), Coquillettidia per-turbans(Walk-               the daily collection totals summed over volunteers                                      for
er) 1,and five species of deer flies (Chrysops ater Mac-            each of the nine hourly time intervals. These hourly
quart, C. carbonarks Walker, C. excitans Walker, C.                 totals were plotted at each period to show changein
mitis Osten Sacken, and C. sordidus Osten Sacken)                   repellency over time after application. An analysisOf
were collected infrequently. Voucher specimens were
deposited in the U.S. National Museum, Smithsonian
Institution, Washington, DC. Simulium wnustum and
                                                                       Table     1.     Mean     percent   protection      (95%         contidence    Km@
Ae. communis were the only species collected in suf-                against    Ae.    communis     and Simulium        senustum      for three   reden’
ficient numbers to evaluate repellency.                             treatments evaluated          for duration       of repellency       at 4, 6, aad 8 b
    Field Repellent Tests. Tests were performed under               after   application
a minimal            ilman
              risk h_______ law pmtncQ!
                             ___             qprQ”ed     by &e
                                                                                                                         Time     periods             -
Walter Reed Army Institute of Research Human Use                     Repellent treatment
                                                                                                           4h                   6h                   8h
Research Review Committee             (on file in our labora-
tory). Trials were conducted using six volunteers (two                                                Aeaks communis
females and four males) age 21-55 yr with no known                  Control
history of allergic reactions to arthropod bites. Each              (bites/l5 minlperson)                  18.7                 10.8
                                                                    AK-37220                               98%
volunteer wore the U.S. Army Battle Dress Uniform                                                                                              (76.$.5!
                                                                                                       (94.3-99.5)         (9O.?z3.9)
printed with a four-color         (green, loam, sand, and           Deet + AD-37220                        98%                  95%
brown)     woodland      camouflage pattern and not treated                                                                (88.7-98.4)
with pemlethrin.        A screen jacket (Bug Out Outdoor            Deet                                   98%                  74%
                                                                                                       (94.3-99.5)         (64.3-82.2)
Wear, Wauwatosa, WI) and surgical gloves were worn
 to limit biting on untreated areas of the upper body,                                              Simulium      venusturn

 hands, and head.                                                   Control
     Repellent solutions were applied at a rate of 0.25             (bites/15 minIperson)                   3.2                   6.0
 mg/cm’ of surface area on the forearms of the volun-                                                                      (86~~9.5)
 teers. Four treatments were applied: (1) deet, (2)                 Deet + AI3-37220                                          84%
 AI3-37220, (3) mixture of deet and AI3-37220 (com-                                                    (88E9.2)            (70.9-92.8)
 bined application rate of 0.25 mg/cm2), or (4) ethyl               Deet                                  98%                 68%
                                                                                                       (82.3-99.9)         (51.2-78.8)
 alcohol (i.e., negative control). After application, the
                                                     ~EB~OUNETAL.:F&ELL.ENTSANDA~.                                                     wrnmunis                          AND                 S. venusturn

     loo--              -- ---
       90    -4
.z    a0 -
al     70-
1      60-                  b    AKI-37220
E      50-                  0    Deet + Al337220
                                                                                                                                                               0    AK&37220
3      40-                  *    Deet
$      30-
                                                                                                                                                                    Deet+ Af3-37220
       20 -
                                                                                                                                                               .    Deet

       10 -
                        I             I       I          I          I          I          I           I                                                        I                 I                   I            I          I   I   I   I
                  0     1            2       3           4         5           6          7           8                                      0                 1                 2                   3            4          5   6   7   a
                            Hours After Repellent Application                                                                                                      Hours After Repellent Application

  Fig. I.  Percent protection provided by three different                                                        Fig. 2. Percent protection provided by three different
repllent treatments (concentration on skin 0.25 mg/cm*)                                                       repellent treatments (concentration on skin 0.25 mg/cm”)
againstAe. communis. A bold longitudinal line indicates the                                                   againstS. uenustum.A bold longitudinal line indicates the 90%
90%protection level.                                                                                          protection level.

variance procedure for a two-factor experiment (re-          All three repellent formulations provided average
pellent group X test period) with repeated measure-       protection >95% against biting from both Ae. commu-
mentswas used to compare repellent effects over           nis and S. venusturn for the first 4 h (Table 1). AI3-
time. An arcsine transformation was used to stabilize     37220 was the only repellent that maintained >95%
the variance of percent protection   (Little and Hills    protection from mosquitoes for 5 h (Fig. 1) and from
1978, p. 158).                                            black flies for 6 h (Fig. 2) after application. Protection
                                                          against Ae. communis fell below 90% at 8 h after re-
                                                          pellent application for all three treatments. By 8 h after
                 Results and Discussion                   application, at least half of the test volunteers expe-
   Aedes communis represented the majority (98%) of       rienced <80% protection against both insects (Fig. 3).
the 1,765 mosquitoes collected. Other species at-            For the purposes of analysis, the average profiles of
tracted to humans were Ae. ezruciulzs (0.6% of total),    protection over the three time periods (i.e., 4,6, and
Ae. canadensis (0.5%)) and Coquilkttidia per-turbans 8 h) were examined to test the following null hypoth-
 (1.0%).The biting rates for Ae. communisranged from      eses: (1) Protection declined over time at the same
10.8 to 18.4 bites per 15 min per person (Table 1). S.    rate for the three repellent formulations. (2) Duration
venusturn   was the only black fly species collected in   of repellency was the same. (3) Mean percent pro-
sufficient numbers (96% of 558 total black flies; 4%      tection for each treatment was the same over time.
were P. mirtum) to determine percent protection. The      First, the duration of repellency among treatments for
range for the biting rate of S. venusturnwas 3.2-8.1      mosquito and black fly species appeared to be parallel,
bites per 15 min per person (Table 1) . Unfortunately,    because there was no time X repellent treatment
the 76 deer flies collected (61 specimens of Chrysops interaction (Ae. wmmunis, F = 1.15; df = 4, 30; P =
niger, five of C. ater, four of C. cur-bon&us, four of C. 0.35; S. venusturn, F = 1.10; df = 4, 30; P = 0.37).
sordidus,  one of C. excitans,and one of C. mitis) were   Second, there was no overall significant difference in
insufficient for repellent evaluation.                    repellency among the three treatments for either Ae.

                                 zl                     .
                                              _...~___._~.._..__.....~...~.....~...~......_. .
                                          oel?t                                       _____.__._.__._______~._...____________
                                                                                                                                                                   -1                    l

                                          Deet .+ .AU-37220. . . . . .._.....‘ ....................       .                               .                               .
                                 F              . ........
                                                                                                                            . . . . . . . . . . . . . . . . . . . . . . . . . . . ..‘ .........’
                                          A13-37220                                .                                               . .                                                                            .
                                               _.~--.__~_..--.__.....-...-...-.-.---.----.-.-                                                           ..
                                                                                                                            ..-...-.....................‘ ‘ .........
                                                                                                                1---.                                                                                               -
                                           Deet     .                                 .         l                                                                                    .                       ..
                                 ii                  I                                                          I                                                                                                     I
                                           Deet + AK%-37220                                                    .                                       . .                   .               .           .
                                 E                   I                                                             I                                                                                                  I
                                   .      AB-37220                                                                      .                                  .        .I                           .                :
                                                I                                                               I                                                        I                                         I
                                 2             0.0                                                             50                                                   80               -                            100
                                                                                                    Percent Protection

     Fig. 3.      Comparison ofrepellency                     at 8      h after application among the three treatments againstAe. cornmurk and S. uenustum
 (each represents the result from one person on one day of the study).

922                                 JOURNAL OF MEDICAL ENTOMOLOGY                                                Vol. 37, &i, 6

communis (F = 1.80; df = 2,lO; P = 0.21) or S. venusturn            ratory and field trials of four repellents with &h%
(F = 2.63; df = 2,lO; P = 0.12). However, confidence                (Diptera: Culicidae). J. Med. Entomol. 31: 17-22.
limits of percentages were not overlapping, indicating          C~p&nd, R. S., T. W. Walker, L. L. Robert, J. 1. Gi&
that AI3-37220 was more repellent at 8 h against Ae.                R. A. Wirtz, and T. A. Klein. 1995. Response of ,$
                                                                    An.opFRh jhestus to repellent-protected voluriters is
wmmunis and at 6 h against S. venusturn (Table 1).
                                                                    unaffected by malaria infection of the vector, J. Am
Finally, we found that although there were no overall
                                                                    Mosq. Control Assoc. 11: 438-440.
significant differences among the three repeiients
                                                                Debboun, M., D. Str-ickman, T. A. Klein, J. A. G&s,E.wylie
against either the mosquito or the black fly, protection            A. Laughinghouse, R. A. Wirtz, and R. K. Gupta 1%:
declined significantly over time (Ae. communis: F =                 Laboratory evaluation of AI3-37220, AI3-35765, c~c-a,
15.82; df = 2,30; P < 0.001; S. venusturn: = 17.84; df =            and Deet repellents against three species of mosquitoes.
2, 30; P < 0.001; Figs. 1 and 2).                                   J. Am. Mosq. Control Assoc. 15: 342-347.
    Only one other study has examined the efficacy of           Frances, S.P., N. Eikarat, B. Sripongsai, and C. Earn& 1%.
deet + AI3-3’ 220 in combination         (Debboun     et al.        Response of Arwpheks dir-us       and Aedes albopim tore_
1999). Testing against laboratory-reared     Aedes uegypti          pellents in the laboratory. J. Am. Mosq. Control Assoc.    9:
                                                                    474 - 476.
 (L.) and Anopheles stephensi   Liston by using an in vitro
                                                                Frances, S. P., T. A. Klein, D. W. Hildebrandt, R. Burg%c
membrane blood-feeding system, the repellency from
                                                                    Noigamol, N. Eikarat, B. Sripongsai, and R. A. W&
the combination of deet + AI3-37220 was similar to
                                                                     1996. Laboratory and field evaluation of deet, GIG4, and
that of deet, although there was some evidence (not                 AI337220 against Anophh dim (Diptera: C&i&)                in
confirmed in statistical tests) of synergistic interaction          Thailand. J. Med. Entomol. 33: 511-515.
against An. stephensi.The current study showed that             Frances, S. P., R. D. Cooper, and k W. Sweeney. 13”d.
the overall repellency of the combination of deet +                 Laboratory and field evaluation of the repellents Deet,
AI3-37220 was similar to an equivalent concentration                CIC4, and AI337220 against Anopheksfarauti (Diptera:
of deet against Ae. communis and S. venustum.                       Culicidae) in Australia. J. Med. Entomol. 35: 690-693.
    Overall, our field study showed that the piperidine         Gambel, J. M., J. F. Brundage, R. F. Burge, R. F. DeFraites,
                                                                    B. L. Smoak, and R. A. Wirtz. 1998. Survey of U.S.Army
compound AI3-37220 used alone or in combination
                                                                    soldiers’ knowledge, attitudes, and practices regarding
with deet provided about equal protection as deet
                                                                    personal protection measures to prevent arthropod-re-
against the mosquito Ae. communisand the black fly S.
                                                                    lated diseasesand nuisance bites. Mil. Med. 163:695-701.
venusturn. Applied as simple alcohol solutions at a             Gudgel, E. D., and F. H. Grauer. 1954. Acute and chronic
dosage of 0.25 mg/cm’  ,   these compounds would have               reactions to black fly bites (Simulium fly). Ann. Med.
 to be reapplied every 5-6 h to maintain 190% pro-                   Assoc. Arch. Dermastol. Syphiol. 70: 609-615.
 tection from these two species. The repellent com-             Gupta, R. K., and L. C. Rutledge. 1994. Role of repellentsia
pound AI3-37220 could be an effective ahernative to                 vector control and disease prevention. Am. J. Trop. Med.
 deet in the United States, as has been shown in many                Hyg. 50 (suppl.) : 82- 86.
 other parts of the world. Incorporating AI3-37220 into         Hooper, R. L., and R. A. Wirtz. 1983. Insect repellent used
 an appropriate formulation probably would result in a               by troops in the field: results of a questionnaire. Mil. Med.
                                                                     148: 34 -38.
 useful repellent product.
                                                                Little, T, A., and F. J. Hills. 1978. Agricultural expeiimea-
    An additional repellent product would be welcome
                                                                     tation. Wiley, New York.
 in the chemical armamentarium against biting arthro-           Martin, S., J. Gambel, J. Jackson, N. Aronson, R. Gupta, B
 pods. When vector control is not possible, repellents               Rowton, M. Perich, P. McEvoy, J. Berman, A. Magill, and
 provide an inexpensive means of protecting individ-                 C. Hoke. 1999. Leishmaniasis in the United States&-
 uals from insect bites (WHO        1995). Effective new             tary. Mil. Med. 163: 801-807.
 repellents may encourage broader acceptability and             Perich, M. J., D. Strickman, R. A. Wirtz, S. A. Stockwell, J.1.
 use, thereby preventing bites that can lead to illnev               Click, R. Burge. G. Hunt, and P. G. Lawyer. 1995. Field
 ranging from irritation to death (Gudgel and Grauer                 evaluation of four repellents against Zkptoconws avE+
  1954, Pinheiro et al. 1974).                                       canus (Diptera: Ceratopogonidae) biting midges. J. bled.
                                                                     Entomol. 32: 306-309.
                                                                Pinheiro, F. P., G. Bensabath, D. Costa, Jr., 0. M. hIan-&
                   Acknowledgments                                   Z. C. Lins, and A.H.P. Andrade. 1974. Haemorrh@
                                                                     syndrome of Altamira. J_ancet859: 639-642.
    We gratefully acknowledge the support of Phillip Lawyer,    Robert, L. L., R. E. Coleman, D. A. Lapointe, P.J.S.~arti”,
Jerold Sadoff, and Alice Boarman, and the technical assis-           R Kelly, and J. D. Edman. 1992. Laboratory and field
tance of Ronnie Essex, Karl Korpal, and Dina Ibrahim                 evaluation of five repellents against the black flies Pm-
(Walter Reed Army Institute of Research). We also thank              imulium mixturn and P. &.scztm (Diptera: Simuliidae),
Dan Molloy (New York State Museum, Albany, NY), Andrea               J. Med. Entomol. 29: 267-272.
Malik, and Richard Matzell (Environmental Conservation          Solberg, V. B., T. A. Klein, K. R. McPherson, B. A. Bradford
Officer, NY, State) for assistancein site selection and iden-        J. R. Burge, and R. A. Wirtz. 1995. Field evaluation of
tifying seasonal abundance of black fly populations in the          deet and a piperidine repellent (AI337220)       againstA*-
Adirondack State Park, and Henry and Dianne Ford for their                ametinum ( Atari: Ixodidae)J. Med. Entoniol,
support throughout the study.                                       32: 870 - 875.
                                                                Strickman, D., M. E. Miller, L. L. Kelsey, W. J. Lee, He #”
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~ov,der ~OOO                   DEFWXJN ET AL.: I~EI.LE~       AND Ae. wmmunis     AND S.   wnustum                923

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