Laboratory and Field Evaluation of Five Repellents Against the Black Flies Prosimulium mixtum and P. fuscum (Diptera: Simuliidae) LEON L. ROBERT, RUSSELL E. COLEMAN, DENNIS A. LAPOINTE, 1 PAULA J. S. MARTIN,1 ROSE KELLY,1 AND JOHN D. EDMAN 1 Department of Entomology, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100
KEY WORDS Insecta, Prosimulium, repellents, deet
BLACK FLY BITES can produce allergic responses
ranging from transitory local irritation to severe systemic illness that may require hospitalization (Gudgel & Grauer 1954) or cause death (Pinheiro et al. 1974). Because of the lack of control programs and the severity and length of duration of allergic responses to black fly bites, it is important that repellents significantly reduce biting. An early report (Fedder et al. 1962) indicated that deet, when applied to the skin, was ineffective against Simulium galeratum Edwards. However, a number of subsequent studies have shown deet to be effective against black flies in laboratory (Mokry 1980, Bernardo & Cupp 1986) and field (Schreck et al. 1979) tests. Both in-vivo and in-vitro studies have shown that deetimpregnated mesh jackets provide 3-6 h of protection from black fly biting, depending on the concentration of deet used (Frommer et al. 1975, Renz & Enyong 1983). Bernardo & Cupp (1986) found a statistically significant, inverse correlation between biting activity and percentage deet. Unfortunately, a number of biting Diptera are tolerant to deet (Buescher et al. 1987, Rutledge et al. 1983). In addition, there is increasing concern about the safety of deet-based repellents
In conducting the research described in this report, the investigators adhered to the guidelines established by the National Institutes of Health for experiments involving human subjects. Human subjects in this study gave free and informed voluntary consent. 1 Department of Entomology, University of Massachusetts, Amherst, Mass. 01003.
(Anonymous 1988, Moody 1989, Oransky et al. 1989). For these reasons, new compounds continually are screened for repellent activity. The ultimate goal of this process is to identify compounds that are safer and more effective against a broader range of arthropods than deet. Schiefer et al. (1976) tested twelve experimental repellents impregnated into mesh polyester-cotton jackets against black flies in Costa Rica and New York. In Costa Rica, deet provided statistically better protection from black fly landings than did the eight experimental compounds. In New York, the compound AI3-70087 (2-hydroxyethyl cyclohexane carboxylate) provided better protection than deet, whereas the compound AI3-8118 (tetrahydrofurfuryl octanoate) provided protection similar to that of deet. Schreck et al. (1979) reported that five repellent compounds tested against black flies were as effective as deet. The objective of our study was to compare the sensitivity of the black flies Prosimulium mixtum Symes & Davies and P. fuscum Symes & Davies to deet and three experimental (CIC-4, AI3-37220, AI3-35765) repellents in laboratory and field tests. In addition, a new U.S. military standard issue deet formulation was tested. The three experimental repellents are in the advanced stages of testing and are effective against a variety of mosquitoes and phlebotomine sand flies (R.E.C., L.L.R., unpublished data). These compounds may prove useful as a replacement for deet or may be used in conjunction with deet in a composite repellent.
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J. Med. Entomol. 29(2): 267-272 (1992) ABSTRACT Deet (N,IV-diethyl-3-methyIbenzamide), the lactone CIC-4 ([2-hydroxymethyl-cyclohexyl] acetic acid lactone), the USDA Proprietary Chemicals AI3-37220 (1[3-cyclohexen-l-ylcarbonyl]-2-methylpiperidine) and AI3-35765 (l-[3-cyclohexen-l-ylcarbonyl] piperidine), and the U.S. military extended duration repellent formulation (EDRF) of deet were evaluated for repellency in the laboratory and field against the black flies Prosimulium mixtum and P. fuscum. CIC-4, AI3-37220, and AI3-35765 were as effective as deet at repelling P. mixtum and P. fuscum in laboratory and field experiments. Only the EDRF provided significantly longer protection than the deet standard against these black flies in the field.
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flies biting on each test site were recorded each min for 5 min. Three tests were conducted on each repellent-treated area, so that all tests were completed within 20 min of repellent application on the skin. In subsequent trials, the range of dosages applied was adjusted to bracket the median effective dosage of the test repellent. The test was then replicated at that range of dosages until a valid estimate of the ED 5 0 and ED 9 5 was obtained. All tests were conducted inside a walk-in incubator (28°C and 75% RH) using three human volunteers. Adult female P. mixtum and P. fuscum used in the laboratory tests were nettea near the field study site as they swarmed around the collector's head. Flies were aspirated into 4-liter (1-gal) cardboard containers with a moist paper towel inside to maintain humidity and transported to the laboratory in styrofoam coolers. Flies were allowed to feed ad libitum on 3% sucrose solution for =4 h, then were held overnight (—10 h) with water only at ~20°C in a dark room to increase the percentage of flies that would blood feed (Bernardo & Cupp 1986). Flies were transferred without anesthetization from holding cages to the repellent test cages 1 h before the start of each repellent test. Field Tests. Procedures used were a modification of those described by Schreck et al. (1979). All repellents (except the EDRF) were applied as 1-ml aliquots of a 25% ethanol solution and spread evenly over the forearm of the subject from wrist to elbow. Ethanol solutions were formulated on a weight-volume basis; therefore, 250 mg of repellent was applied in each test. The other forearm of each subject was treated with 1 ml of ethanol only (control). Label instructions on the EDRF indicate that 2.5 ml are enough to cover both forearms; therefore, 1.25 ml were used (416 mg deet) to treat one forearm. Control arms were untreated. Because of the amount of the EDRF applied, results obtained using this compound cannot be directly compared with those obtained using the other four test repellents. For each day of testing, each individual was randomly assigned a repellent application. During the first 3 d of testing, the subjects were treated in the field; however, black fly biting activity was limited to the warmest hours of the day (1100—1700 hours) and repellent failure (defined as the time to the 10th bite on the treated arm) was not always achieved before the flies stopped biting. Therefore, on subsequent days the repellent was applied early in the morning (0800 hours) in the laboratory, 4 h before the start of the field tests. Treated and untreated (control) arms were exposed continuously to natural populations of flies. Subjects sat in folding chairs with arms raised. Subjects wore U.S. military battle dress uniforms, headnets, and latex gloves to prevent attack on exposed, untreated parts of the body.
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Black Fly Species and Location of Field Site. Prosimulium mixtum, P. fuscum, and Simulium venustum Say (Rothfels & Freeman 1977, Simmons 1985) are the most abundant and widely distributed black fly species in western Massachusetts (Holbrook 1967). The P. mixtum group consists of seven sibling species that cannot be distinguished from one another morphologically (Rothfels & Freeman 1977). P. fuscum is a distinct species; however, adults cannot be distinguished morphologically from members of the P. mixtum group (Kim & Merritt 1987). P. mixtum and P. fuscum were used in all of the laboratory repellent tests and were the predominant black flies during field studies conducted between 1 and 10 May 1990. The study site was a 1-ha field covered by short grass. It was surrounded by mature mixed coniferous-deciduous forest and located 200 m from the outlet of Lake Wyola, Franklin County, Mass. The outlet was characterized by shallow, fast-moving water flowing over flat bedrock and gravel, and is a breeding site for P. fuscum, P. mixtum, and S. venustum (Simmons 1985). Test Repellents. The following technical grade chemicals were tested in laboratory and field studies: N,JV-diethyl-3-methyl-benzamide (deet) (Virginia Chemical Company, Portsmouth, Va.); USDA piperidine compounds AI3-37220 (1- [3 - cyclohexen-1-ylcarbonyl] - 2 - methylpiperidine) and AI3-35765 (l-[3-cyclohexen-l-ylcarbonyl]piperidine), synthesized by Terrence P. McGovern (Insect Chemical Ecology Laboratory, USDA, Beltsville Agricultural Research Center, Beltsville, Md.); and (2-hydroxymethylcyclohexyl)acetic acid lactone (CIC-4, Angus Chemical Company, Northbrook, 111). Also, the U.S. military standard issue Insect-Arthropod Repellent Lotion containing 33.3% deet and 66.7% inert ingredients, commonly referred to as "extended duration repellent formulation" (EDRF) (3M Consumer Specialties Division, St. Paul, Minn.), was tested in the field studies. Laboratory Tests. The test procedure used to determine the effective dosages of the repellents was a modification of the American Society for Testing and Materials Standard ED951-83 (1983). The specific test methods were based on the variable dose-fixed time, "free choice" method described by Buescher et al. (1982). Five circular test areas (29 mm diameter) were outlined on the volunteer's forearm using a plastic template and a felt-tipped pen. These test areas were treated randomly with 25 /A of the diluent (control) and four serial dilutions of the test repellent in absolute alcohol. After the diluent was allowed to dry for 5 min, a plastic cage (4 by 5 by 18 cm) with matching cutouts on its floor and containing 20 female black flies was secured to the area with rubber bands. The number of black
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Table 1. Laboratory siensitivity of the repellents AI3-37220, AI3-35765, CIC-4, and Deet against the black flies P. mixtum and P. fuscum Repellent
n
Slope
ED 50 (95% CL)a
AI3-37220 AI3-35765 CIC-4 DEET
30 30 30 30
-2.38 -2.90 -1.88 -2.14
6.2a (5.1-7.8) 12.5b (9.9-16.2) 8.8ab (7.7-9.9) 10. lab (6.1-35.6)
ED 95 (95% CL)a 30.7a 46.3a 65.8a 59.1a
(20.5-59.5) (31.5-89.7) (51.1-91.9) (21.9-3,651.7)
Initial repellent sensitivity determined using a dose-response testing procedure with field-collected P. mixtum and P. fuscum. " fig repellent/cm2 skin. Mean dosages (95% confidence limits in parentheses) followed by the same letter are not significantly different (P < 0.05) from each other by comparing 95% CL.
to separate mean protection time values (Snedecor & Cochran 1967).
Results Laboratory Tests. Results of 120 tests conducted to estimate the ED 50 s and ED 95 s of AI337220, AI3-35765, CIC-4, and deet against P. mixtum and P. fuscum are presented in Table 1. The 95% confidence limits are included to indicate statistical differences between the effective dosage values. The ED 5 0 of AI3-37220 (6.2 /xg/cm2) was significantly lower (P < 0.05) than the ED 5 0 of AI335765 (12.5 /ug/cm2). The ED 50 s of deet (10.1 jag/cm2) and CIC-4 (8.8 /xg/cm2) were not statistically different from either AI3-37220 or AI335765. There were no significant differences in repellency among compounds at the ED 9 5 level. Field Tests. Of the 1,141 black flies collected while biting human subjects during the field repellent tests, 94.9% (n = 1,083) were P. mixtum and P. fuscum and 5.1% (n = 58) were S. venustum. Although the number of S. venustum collected was small, the percentage of the daily total collected gradually increased during the study from 0.0% on days 1 and 2 to 15.6% on day 6. In total, 7,669 black flies landed on the test subjects over the course of the study, with 2,191 of these landings resulting in bites (Table 2). Significantly fewer (P < 0.05) black flies landed on the treated arms than on the control arms. More landings on control arms (31.6%) than treated arms (12.6%) resulted in biting (Table 2). The mean protection time provided by the EDRF (7.18 h) was statistically longer (P < 0.01) than that provided by the four other repellents tested (Table 3). However, 416 mg of deet was applied to each forearm when using EDRF compared with only 250 mg of each of the other four compounds. No statistically significant differences in protection time were found between the three experimental repellents and deet (when tested at a concentration of 25% in ethanol). Although AI3-37220 was not statistically more effective than the deet standard, the mean protection time for AI3-37220 was greater than that for deet by =1.2 h.
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The effectiveness of each repellent was determined by the protection time. Schreck et al. (1979) defined protection time as time to first bite followed by another bite within 30 min (protection time as defined is therefore distinct from repellent failure, which was considered as the time to 10th bite). In this study, the number of flies landing and biting each forearm were determined and recorded every 15 min. Black flies that landed were aspirated from the arm as soon as they began probing and were stored in ethanol for subsequent identification. The subjects changed places at the end of each 15-min period. Each individual test was not terminated until 10 bites were recorded on the treated forearm (repellent failure) or until the test was terminated at the end of the day. Because test subjects differ in attractiveness to flies (Travis 1950), the best measure of effectiveness of a repellent is the ratio of protection time and percent protection compared with a standard repellent (deet). Six human subjects (four male, two female) were utilized during 6 d of testing conducted during a 2-wk period. Statistical Analysis. Data collected during laboratory testing were analyzed by the method of probit analysis using a computer program developed at Letterman Army Institute of Research, Presidio of San Francisco, Calif. Calculation of the confidence limits was based on the method of Goldstein (1964). Analysis for each test yielded median effective dose (ED 50 ), ED 9 5 , 95% confidence intervals (calculated by Fieller's Theorem) and slope value. Significance differences were determined by comparing the 95% confidence intervals among effective doses. Data from field tests were analyzed using the Statistix statistical analysis program (Statistix 1987). The Wilk-Shapiro-Rankit Plots test (Shapiro & Francia 1972) was used to determine whether data obtained from the control arms conformed to a normal distribution. Differences in the protection times then were compared statistically using an analysis of variance (Sokal & Rohlf 1981). This statistical technique was used because biting activity was normally distributed over the course of the test period and, therefore, met the distributional assumptions of analysis of variance. Student-Newman-Keuls Test was used
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Table 2. Landing and biting of black flies during field repellent studies conducted between 1 and 10 May 1990 at Lake Wyola, Mass.
1 2 3 4 5 6 Totals
Landing
Control arm Biting
% Biting
Landing
Treated arm Biting
% Biting
952 1,694 1,440 1,030 906 434 6,456
290 455 393 433 292 175 2,038
30.5 26.9 27.3 42.0 32.2 40.3 31.6
279 221 283 151 251 28 1,213
36 29 30 13 43 2 153
12.9 13.1 10.6 8.6 17.1 7.1 12.6
Black flies were 94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum.
Discussion In a series of tests conducted in Maine against S. venustum and P. mixtum, Schreck et al. (1979) compared five candidate repellents with deet. Mean protection time in tests using 25% (wt/vol) Table 3. Protection time of 4 repellent compounds and the military Extended Duration Repellent Formation (EDRF) of deet against black fly bites received between 1
Repellent
Protection time, h° Meanb Range
DEET AI3-35765 CIC-4 AI3-37220 EDRF
2.82a 2.75a 3.10a 4.07a 7.18b
1.25-4.00 1.25-4.50 1.50-5.25 2.25-6.75 5.25-9.00
Ratio to deet 1.00 0.98 1.10 1.44 2.55
94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum. " Protection time is the time from repellent application to the first confirmed bite (followed by a second bite within 30 min). b Mean protection times followed by a different letter are significantly different (P < 0.05) using Student-NewmanKeuls Test.
repellent ranged from 314 min for AI3-30180 to 505 min for AI3-35765, with deet providing 426 min protection time. None of the candidate repellents provided significantly longer protection time than that provided by deet. In our study, the only repellent that provided significantly longer protection time than deet was EDRF of deet. This may have resulted from the higher concentration used in EDRF (416 mg per arm compared with 250 mg per arm with the other compounds) or from the slow-release polymer formulation used in the EDRF. The protection time provided by deet and AI3-35765 against S. venustum and P. mixtum and P. fuscum in our study was much shorter (7 h) than that found by Schreck et al. (1979) with the same compounds against the same species of black flies. Why the protection time varied so markedly cannot be ascertained; however, S. venustum and P. mixtum were found in a 2:1 ratio in Maine compared with a 1:20 ratio recorded in this study. Specific differences in the sensitivity of these two black flies to repellents were not determined and may have affected the results of these tests. Das et al. (1985) found that deet (20% wt/vol) provided protection time of =7 h and N-benzoyl piperidine and N-toluyl piperidine provided >8 h protection time against Simulium himalayense (Puri). The efficacy of the U.S. military EDRF against black flies clearly was demonstrated in this Table 4. Time to 1st, 3rd, 5th, and 10th bite as a means of evaluating the efficacy of 5 repellents against black flies Repellent DEET AI3-35765 CIC-4 AI3-37220 EDRF
1st Bite 2.57a 2.25a 1.63a 4.07a 6.50b
Mean protection time. h° 3rd Bite 5th Bite 10th Bite 3.25a 3.82a 3.95a 4.93ab 7.78b
3.93 4.18 5.07 >5.32 >7.82
4.70 5.00 >5.63 >5.68 >7.82
turn. " Mean protection times followed by a different letter are significantly different (P < 0.05) from each other using Student-Newman-Keuls test. Times to 5th and 10th bite only represent lower limits in protection time detected during the study; therefore, statistical analyses were not carried out for these samples.
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Table 4 represents a summary of protection time to 1st, 3rd, 5th, and 10th bites. The ranking of repellents from best to worst based upon the different lengths of protection time was generally the same. For each measure of protection time, EDRF provided statistically longer protection (P < 0.01) compared with the other repellents. Of the four repellents tested in a 25% (wt/vol) formulation in ethanol, AI3-37220 consistently provided the longest protection time whereas deet (except in time to first bite) provided the shortest protection time (Table 4). Percent landing protection among the five repellents during each test period varied widely (Table 5). Although significantly fewer (P < 0.05) flies landed on the treated arms than on the control arms (Table 2), there was no apparent degradation in landing protection over time (Table 5). EDRF provided exceptional (percent) biting protection for the entire 9-h test period. Of the other compounds tested, AI3-37220 provided protection for the longest period (Table 5).
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Table 5. Percent protection of 5 repellents against landing and biting of black flies during field repellent studies conducted between 1 and 10 May 1990 at Lake Wyola, Mass. Time after treatment, h 1 2 3
4 5 6 7 8 9 1
2 3 4
CIC-4
82.4 87.1 72.7 71.1 77.6 83.0
92.0 96.1 90.7 87.7 63.2 52.3
—
—
100.0 95.4 88.9 88.8 90.9 76.5
91.3 95.6 89.7 93.2 88.9 85.3
—
—
AI3-35765
AI3-37220
Percent landing protection 86.0 75.4 84.3 77.9 67.8 74.3 —
—
Percent biting protection 100.0 78.4 93.7 91.2 96.7 88.6 —
84.6 86.4 80.9 80.8 77.9 68.7 82.9
100.0 100.0 94.4 91.7 94.4 76.0 62.9 —
EDRF 63.6 84.2 93.7 77.8 90.2 90.5 90.6 86.0 91.9 100.0 100.0 100.0 100.0 100.0 99.0 97.6 100.0 97.9
94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum.
study. Although data obtained using this repellent formulation cannot be directly compared with those obtained with the other four repellents, the protection provided by EDRF when label instructions were followed was impressive. The protection time provided by EDRF was 2.55 times that provided by the 25% wt/vol deet formulation. If a level of
KEY WORDS Insecta, Prosimulium, repellents, deet
BLACK FLY BITES can produce allergic responses
ranging from transitory local irritation to severe systemic illness that may require hospitalization (Gudgel & Grauer 1954) or cause death (Pinheiro et al. 1974). Because of the lack of control programs and the severity and length of duration of allergic responses to black fly bites, it is important that repellents significantly reduce biting. An early report (Fedder et al. 1962) indicated that deet, when applied to the skin, was ineffective against Simulium galeratum Edwards. However, a number of subsequent studies have shown deet to be effective against black flies in laboratory (Mokry 1980, Bernardo & Cupp 1986) and field (Schreck et al. 1979) tests. Both in-vivo and in-vitro studies have shown that deetimpregnated mesh jackets provide 3-6 h of protection from black fly biting, depending on the concentration of deet used (Frommer et al. 1975, Renz & Enyong 1983). Bernardo & Cupp (1986) found a statistically significant, inverse correlation between biting activity and percentage deet. Unfortunately, a number of biting Diptera are tolerant to deet (Buescher et al. 1987, Rutledge et al. 1983). In addition, there is increasing concern about the safety of deet-based repellents
In conducting the research described in this report, the investigators adhered to the guidelines established by the National Institutes of Health for experiments involving human subjects. Human subjects in this study gave free and informed voluntary consent. 1 Department of Entomology, University of Massachusetts, Amherst, Mass. 01003.
(Anonymous 1988, Moody 1989, Oransky et al. 1989). For these reasons, new compounds continually are screened for repellent activity. The ultimate goal of this process is to identify compounds that are safer and more effective against a broader range of arthropods than deet. Schiefer et al. (1976) tested twelve experimental repellents impregnated into mesh polyester-cotton jackets against black flies in Costa Rica and New York. In Costa Rica, deet provided statistically better protection from black fly landings than did the eight experimental compounds. In New York, the compound AI3-70087 (2-hydroxyethyl cyclohexane carboxylate) provided better protection than deet, whereas the compound AI3-8118 (tetrahydrofurfuryl octanoate) provided protection similar to that of deet. Schreck et al. (1979) reported that five repellent compounds tested against black flies were as effective as deet. The objective of our study was to compare the sensitivity of the black flies Prosimulium mixtum Symes & Davies and P. fuscum Symes & Davies to deet and three experimental (CIC-4, AI3-37220, AI3-35765) repellents in laboratory and field tests. In addition, a new U.S. military standard issue deet formulation was tested. The three experimental repellents are in the advanced stages of testing and are effective against a variety of mosquitoes and phlebotomine sand flies (R.E.C., L.L.R., unpublished data). These compounds may prove useful as a replacement for deet or may be used in conjunction with deet in a composite repellent.
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J. Med. Entomol. 29(2): 267-272 (1992) ABSTRACT Deet (N,IV-diethyl-3-methyIbenzamide), the lactone CIC-4 ([2-hydroxymethyl-cyclohexyl] acetic acid lactone), the USDA Proprietary Chemicals AI3-37220 (1[3-cyclohexen-l-ylcarbonyl]-2-methylpiperidine) and AI3-35765 (l-[3-cyclohexen-l-ylcarbonyl] piperidine), and the U.S. military extended duration repellent formulation (EDRF) of deet were evaluated for repellency in the laboratory and field against the black flies Prosimulium mixtum and P. fuscum. CIC-4, AI3-37220, and AI3-35765 were as effective as deet at repelling P. mixtum and P. fuscum in laboratory and field experiments. Only the EDRF provided significantly longer protection than the deet standard against these black flies in the field.
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flies biting on each test site were recorded each min for 5 min. Three tests were conducted on each repellent-treated area, so that all tests were completed within 20 min of repellent application on the skin. In subsequent trials, the range of dosages applied was adjusted to bracket the median effective dosage of the test repellent. The test was then replicated at that range of dosages until a valid estimate of the ED 5 0 and ED 9 5 was obtained. All tests were conducted inside a walk-in incubator (28°C and 75% RH) using three human volunteers. Adult female P. mixtum and P. fuscum used in the laboratory tests were nettea near the field study site as they swarmed around the collector's head. Flies were aspirated into 4-liter (1-gal) cardboard containers with a moist paper towel inside to maintain humidity and transported to the laboratory in styrofoam coolers. Flies were allowed to feed ad libitum on 3% sucrose solution for =4 h, then were held overnight (—10 h) with water only at ~20°C in a dark room to increase the percentage of flies that would blood feed (Bernardo & Cupp 1986). Flies were transferred without anesthetization from holding cages to the repellent test cages 1 h before the start of each repellent test. Field Tests. Procedures used were a modification of those described by Schreck et al. (1979). All repellents (except the EDRF) were applied as 1-ml aliquots of a 25% ethanol solution and spread evenly over the forearm of the subject from wrist to elbow. Ethanol solutions were formulated on a weight-volume basis; therefore, 250 mg of repellent was applied in each test. The other forearm of each subject was treated with 1 ml of ethanol only (control). Label instructions on the EDRF indicate that 2.5 ml are enough to cover both forearms; therefore, 1.25 ml were used (416 mg deet) to treat one forearm. Control arms were untreated. Because of the amount of the EDRF applied, results obtained using this compound cannot be directly compared with those obtained using the other four test repellents. For each day of testing, each individual was randomly assigned a repellent application. During the first 3 d of testing, the subjects were treated in the field; however, black fly biting activity was limited to the warmest hours of the day (1100—1700 hours) and repellent failure (defined as the time to the 10th bite on the treated arm) was not always achieved before the flies stopped biting. Therefore, on subsequent days the repellent was applied early in the morning (0800 hours) in the laboratory, 4 h before the start of the field tests. Treated and untreated (control) arms were exposed continuously to natural populations of flies. Subjects sat in folding chairs with arms raised. Subjects wore U.S. military battle dress uniforms, headnets, and latex gloves to prevent attack on exposed, untreated parts of the body.
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Black Fly Species and Location of Field Site. Prosimulium mixtum, P. fuscum, and Simulium venustum Say (Rothfels & Freeman 1977, Simmons 1985) are the most abundant and widely distributed black fly species in western Massachusetts (Holbrook 1967). The P. mixtum group consists of seven sibling species that cannot be distinguished from one another morphologically (Rothfels & Freeman 1977). P. fuscum is a distinct species; however, adults cannot be distinguished morphologically from members of the P. mixtum group (Kim & Merritt 1987). P. mixtum and P. fuscum were used in all of the laboratory repellent tests and were the predominant black flies during field studies conducted between 1 and 10 May 1990. The study site was a 1-ha field covered by short grass. It was surrounded by mature mixed coniferous-deciduous forest and located 200 m from the outlet of Lake Wyola, Franklin County, Mass. The outlet was characterized by shallow, fast-moving water flowing over flat bedrock and gravel, and is a breeding site for P. fuscum, P. mixtum, and S. venustum (Simmons 1985). Test Repellents. The following technical grade chemicals were tested in laboratory and field studies: N,JV-diethyl-3-methyl-benzamide (deet) (Virginia Chemical Company, Portsmouth, Va.); USDA piperidine compounds AI3-37220 (1- [3 - cyclohexen-1-ylcarbonyl] - 2 - methylpiperidine) and AI3-35765 (l-[3-cyclohexen-l-ylcarbonyl]piperidine), synthesized by Terrence P. McGovern (Insect Chemical Ecology Laboratory, USDA, Beltsville Agricultural Research Center, Beltsville, Md.); and (2-hydroxymethylcyclohexyl)acetic acid lactone (CIC-4, Angus Chemical Company, Northbrook, 111). Also, the U.S. military standard issue Insect-Arthropod Repellent Lotion containing 33.3% deet and 66.7% inert ingredients, commonly referred to as "extended duration repellent formulation" (EDRF) (3M Consumer Specialties Division, St. Paul, Minn.), was tested in the field studies. Laboratory Tests. The test procedure used to determine the effective dosages of the repellents was a modification of the American Society for Testing and Materials Standard ED951-83 (1983). The specific test methods were based on the variable dose-fixed time, "free choice" method described by Buescher et al. (1982). Five circular test areas (29 mm diameter) were outlined on the volunteer's forearm using a plastic template and a felt-tipped pen. These test areas were treated randomly with 25 /A of the diluent (control) and four serial dilutions of the test repellent in absolute alcohol. After the diluent was allowed to dry for 5 min, a plastic cage (4 by 5 by 18 cm) with matching cutouts on its floor and containing 20 female black flies was secured to the area with rubber bands. The number of black
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Table 1. Laboratory siensitivity of the repellents AI3-37220, AI3-35765, CIC-4, and Deet against the black flies P. mixtum and P. fuscum Repellent
n
Slope
ED 50 (95% CL)a
AI3-37220 AI3-35765 CIC-4 DEET
30 30 30 30
-2.38 -2.90 -1.88 -2.14
6.2a (5.1-7.8) 12.5b (9.9-16.2) 8.8ab (7.7-9.9) 10. lab (6.1-35.6)
ED 95 (95% CL)a 30.7a 46.3a 65.8a 59.1a
(20.5-59.5) (31.5-89.7) (51.1-91.9) (21.9-3,651.7)
Initial repellent sensitivity determined using a dose-response testing procedure with field-collected P. mixtum and P. fuscum. " fig repellent/cm2 skin. Mean dosages (95% confidence limits in parentheses) followed by the same letter are not significantly different (P < 0.05) from each other by comparing 95% CL.
to separate mean protection time values (Snedecor & Cochran 1967).
Results Laboratory Tests. Results of 120 tests conducted to estimate the ED 50 s and ED 95 s of AI337220, AI3-35765, CIC-4, and deet against P. mixtum and P. fuscum are presented in Table 1. The 95% confidence limits are included to indicate statistical differences between the effective dosage values. The ED 5 0 of AI3-37220 (6.2 /xg/cm2) was significantly lower (P < 0.05) than the ED 5 0 of AI335765 (12.5 /ug/cm2). The ED 50 s of deet (10.1 jag/cm2) and CIC-4 (8.8 /xg/cm2) were not statistically different from either AI3-37220 or AI335765. There were no significant differences in repellency among compounds at the ED 9 5 level. Field Tests. Of the 1,141 black flies collected while biting human subjects during the field repellent tests, 94.9% (n = 1,083) were P. mixtum and P. fuscum and 5.1% (n = 58) were S. venustum. Although the number of S. venustum collected was small, the percentage of the daily total collected gradually increased during the study from 0.0% on days 1 and 2 to 15.6% on day 6. In total, 7,669 black flies landed on the test subjects over the course of the study, with 2,191 of these landings resulting in bites (Table 2). Significantly fewer (P < 0.05) black flies landed on the treated arms than on the control arms. More landings on control arms (31.6%) than treated arms (12.6%) resulted in biting (Table 2). The mean protection time provided by the EDRF (7.18 h) was statistically longer (P < 0.01) than that provided by the four other repellents tested (Table 3). However, 416 mg of deet was applied to each forearm when using EDRF compared with only 250 mg of each of the other four compounds. No statistically significant differences in protection time were found between the three experimental repellents and deet (when tested at a concentration of 25% in ethanol). Although AI3-37220 was not statistically more effective than the deet standard, the mean protection time for AI3-37220 was greater than that for deet by =1.2 h.
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The effectiveness of each repellent was determined by the protection time. Schreck et al. (1979) defined protection time as time to first bite followed by another bite within 30 min (protection time as defined is therefore distinct from repellent failure, which was considered as the time to 10th bite). In this study, the number of flies landing and biting each forearm were determined and recorded every 15 min. Black flies that landed were aspirated from the arm as soon as they began probing and were stored in ethanol for subsequent identification. The subjects changed places at the end of each 15-min period. Each individual test was not terminated until 10 bites were recorded on the treated forearm (repellent failure) or until the test was terminated at the end of the day. Because test subjects differ in attractiveness to flies (Travis 1950), the best measure of effectiveness of a repellent is the ratio of protection time and percent protection compared with a standard repellent (deet). Six human subjects (four male, two female) were utilized during 6 d of testing conducted during a 2-wk period. Statistical Analysis. Data collected during laboratory testing were analyzed by the method of probit analysis using a computer program developed at Letterman Army Institute of Research, Presidio of San Francisco, Calif. Calculation of the confidence limits was based on the method of Goldstein (1964). Analysis for each test yielded median effective dose (ED 50 ), ED 9 5 , 95% confidence intervals (calculated by Fieller's Theorem) and slope value. Significance differences were determined by comparing the 95% confidence intervals among effective doses. Data from field tests were analyzed using the Statistix statistical analysis program (Statistix 1987). The Wilk-Shapiro-Rankit Plots test (Shapiro & Francia 1972) was used to determine whether data obtained from the control arms conformed to a normal distribution. Differences in the protection times then were compared statistically using an analysis of variance (Sokal & Rohlf 1981). This statistical technique was used because biting activity was normally distributed over the course of the test period and, therefore, met the distributional assumptions of analysis of variance. Student-Newman-Keuls Test was used
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Table 2. Landing and biting of black flies during field repellent studies conducted between 1 and 10 May 1990 at Lake Wyola, Mass.
1 2 3 4 5 6 Totals
Landing
Control arm Biting
% Biting
Landing
Treated arm Biting
% Biting
952 1,694 1,440 1,030 906 434 6,456
290 455 393 433 292 175 2,038
30.5 26.9 27.3 42.0 32.2 40.3 31.6
279 221 283 151 251 28 1,213
36 29 30 13 43 2 153
12.9 13.1 10.6 8.6 17.1 7.1 12.6
Black flies were 94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum.
Discussion In a series of tests conducted in Maine against S. venustum and P. mixtum, Schreck et al. (1979) compared five candidate repellents with deet. Mean protection time in tests using 25% (wt/vol) Table 3. Protection time of 4 repellent compounds and the military Extended Duration Repellent Formation (EDRF) of deet against black fly bites received between 1
Repellent
Protection time, h° Meanb Range
DEET AI3-35765 CIC-4 AI3-37220 EDRF
2.82a 2.75a 3.10a 4.07a 7.18b
1.25-4.00 1.25-4.50 1.50-5.25 2.25-6.75 5.25-9.00
Ratio to deet 1.00 0.98 1.10 1.44 2.55
94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum. " Protection time is the time from repellent application to the first confirmed bite (followed by a second bite within 30 min). b Mean protection times followed by a different letter are significantly different (P < 0.05) using Student-NewmanKeuls Test.
repellent ranged from 314 min for AI3-30180 to 505 min for AI3-35765, with deet providing 426 min protection time. None of the candidate repellents provided significantly longer protection time than that provided by deet. In our study, the only repellent that provided significantly longer protection time than deet was EDRF of deet. This may have resulted from the higher concentration used in EDRF (416 mg per arm compared with 250 mg per arm with the other compounds) or from the slow-release polymer formulation used in the EDRF. The protection time provided by deet and AI3-35765 against S. venustum and P. mixtum and P. fuscum in our study was much shorter (7 h) than that found by Schreck et al. (1979) with the same compounds against the same species of black flies. Why the protection time varied so markedly cannot be ascertained; however, S. venustum and P. mixtum were found in a 2:1 ratio in Maine compared with a 1:20 ratio recorded in this study. Specific differences in the sensitivity of these two black flies to repellents were not determined and may have affected the results of these tests. Das et al. (1985) found that deet (20% wt/vol) provided protection time of =7 h and N-benzoyl piperidine and N-toluyl piperidine provided >8 h protection time against Simulium himalayense (Puri). The efficacy of the U.S. military EDRF against black flies clearly was demonstrated in this Table 4. Time to 1st, 3rd, 5th, and 10th bite as a means of evaluating the efficacy of 5 repellents against black flies Repellent DEET AI3-35765 CIC-4 AI3-37220 EDRF
1st Bite 2.57a 2.25a 1.63a 4.07a 6.50b
Mean protection time. h° 3rd Bite 5th Bite 10th Bite 3.25a 3.82a 3.95a 4.93ab 7.78b
3.93 4.18 5.07 >5.32 >7.82
4.70 5.00 >5.63 >5.68 >7.82
turn. " Mean protection times followed by a different letter are significantly different (P < 0.05) from each other using Student-Newman-Keuls test. Times to 5th and 10th bite only represent lower limits in protection time detected during the study; therefore, statistical analyses were not carried out for these samples.
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Table 4 represents a summary of protection time to 1st, 3rd, 5th, and 10th bites. The ranking of repellents from best to worst based upon the different lengths of protection time was generally the same. For each measure of protection time, EDRF provided statistically longer protection (P < 0.01) compared with the other repellents. Of the four repellents tested in a 25% (wt/vol) formulation in ethanol, AI3-37220 consistently provided the longest protection time whereas deet (except in time to first bite) provided the shortest protection time (Table 4). Percent landing protection among the five repellents during each test period varied widely (Table 5). Although significantly fewer (P < 0.05) flies landed on the treated arms than on the control arms (Table 2), there was no apparent degradation in landing protection over time (Table 5). EDRF provided exceptional (percent) biting protection for the entire 9-h test period. Of the other compounds tested, AI3-37220 provided protection for the longest period (Table 5).
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ROBERT ET AL.: REPELLENCY OF COMPOUNDS AGAINST BLACK FLIES
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Table 5. Percent protection of 5 repellents against landing and biting of black flies during field repellent studies conducted between 1 and 10 May 1990 at Lake Wyola, Mass. Time after treatment, h 1 2 3
4 5 6 7 8 9 1
2 3 4
CIC-4
82.4 87.1 72.7 71.1 77.6 83.0
92.0 96.1 90.7 87.7 63.2 52.3
—
—
100.0 95.4 88.9 88.8 90.9 76.5
91.3 95.6 89.7 93.2 88.9 85.3
—
—
AI3-35765
AI3-37220
Percent landing protection 86.0 75.4 84.3 77.9 67.8 74.3 —
—
Percent biting protection 100.0 78.4 93.7 91.2 96.7 88.6 —
84.6 86.4 80.9 80.8 77.9 68.7 82.9
100.0 100.0 94.4 91.7 94.4 76.0 62.9 —
EDRF 63.6 84.2 93.7 77.8 90.2 90.5 90.6 86.0 91.9 100.0 100.0 100.0 100.0 100.0 99.0 97.6 100.0 97.9
94.9% P.fuscum and P. mixtum and 5.1% Simulium venustum.
study. Although data obtained using this repellent formulation cannot be directly compared with those obtained with the other four repellents, the protection provided by EDRF when label instructions were followed was impressive. The protection time provided by EDRF was 2.55 times that provided by the 25% wt/vol deet formulation. If a level of
