Journal of the American Control Association, 13(2):171-183, 1997 Copyright O 1997 by the American Mosquito Control Association, Inc. /*%6d %?Si-q+W PESTICIDE AVOIDANCE BEHAVIOR IN ALBIMANUS, A MALARIA VECTOR IN THE AMERICAS’ THEERAPHAP CHAREONVIRIYAPHAP,? ONALD R. ROBERTS,” RICHARD G. ANDRE,-’ J. J. HAROLD HARLAN: SyLvIE/LANGUINs AND MICHAEL BANGS3

ABSTRACT The behavioral responses of 4 populations of A~iophelesalbinianus females to DDT, permethrin, and deltamethrin were characterized in excito-repellency tests. One test population (ST) from El Salvador has been maintained as a coIony for 20 years. A second population (ES) from Guatemala was colonized in 1992. Third and fourth populations consisted of field-caught specimens from Toledo District (TO) of southern Belize in 1994 and Coroza1 District (CO) of northern Belize, respectively. Females of ES, TO, and CO populations rapidly escaped from direct contact with treated surfaces for each of the 3 insecticides. Similarities in escape responses of insecticide-resistant (ES) versus insecticide-susceptible populations (TO, CO) suggest that there is no relationship between physiological and behavioral responses of Ai2. albitnanus populations to DDT, penneth- rin, and deltamethrin. Females from all but the ST colony escaped in greater numbers from chambers without direct contact with treated surfaces than from control chambers (P < 0.05). Few females from the ST colony escaped from test chambers, regardless of which insecticide was used or whether contact was allowed, indicating that the ST colony has lost its capability to respond to insecticides. Repellent responses were significant; but they were not pronounced in 30-min exposures, and they were very pronounced in 4-h exposures. We conclude that irritant and repellent responses of malaria vectors to insecticides are important components of malaria control operations.

INTRODUCTION . describe behavior that is stimulated by some com- bination of irritancy and repellency, with irritancy Anopheles albimanus Wiedemann is a primary occumng after physical contact and repellency oc- vector of malaria in many areas of Central and curring without physical contact with insecticide. South America (Breeland 1974). DDT has been Excito-repellency, like avoidance behavior, also is used extensively to control malaria transmitted by a broad classification of behavioral responses in- this vector. Today, resistance of An. albitnanus to DDT occurs in several countries (Brown 1986), but cluding both irritancy and repellency. it does not occur in others in spite of regular DDT Pyrethroids elicit behavioral responses in use (Roberts and Andre 1994). Behavioral avoid- (Threlkeld 1985). Mosquito control through the use ance of DDT has also been reported to occur in of pyrethroid-impregnated bed nets and intradomi- some An. albimanus populations (Rachou et al. ciliary spraying of pyrethroids has been initiated in 1963). In combination, findings of DDT avoidance some countries, including a few countries of Cen-. and DDT resistance in conspecific populations raise tra1 and South America (Beach et al. 1989, Curtis questions about the role of avoidance behavior in et al. 1989, World Health Organization 1989). The preventing malaria transmission and in selecting for increased use of pyrethroids should be a major insecticide resistance in malaria vectors. Avoidance stimulus for extensive tests and field studies on py- of DDT by malaria vectors has been recorded in rethroid avoidance behavior in New World. vectors the presence and absence of physiological resis- of malaria. tance (Lockwood et al. 1984), but the relationships, The complexities of excito-repellency testing, in- if any, between physiological resistance and behav- cluding methods of analyzing and interpreting test ioral avoidance are unknown. data, have resulted in no test method being ade- The term “avoidance behavior” will be used to quate or fully accepted. No test recommended by the World Health Organization will discriminate be- tween contact irritancy and noncontact repellency. This research was supported by grant R087EK from However, an experimental test system described by of the Uniformed Services University the Health Sciences. Roberts et al. (1997) addresses a number of dcfi- Bethesda, MD. The views of the authors do not purport ciencies attributed to existing behavioral tests. The to reflect the positions of the U.S. Department of Defense or the Uniformed Services University of the Health Sci- new test system was used in this series of studies ences. on relationships of avoidance behavior and physi- Kampheangsaen Campus, Kasetsart University, Fac- ological resistance in colonized and wild-caught ulty of Liberal Arts and Sciences, Nakhon Prathom 73 140, populations of An. albimanus mosquitoes from Thailand. Central America. The Arz. albimanus populations Department of Preventive Medicine and Biometrics, were characterized for isozymes, for esterases, for Uniformed Services University of the Health Sciences, insecticide susceptibilities, and for the irritancy and 4301 Jones Bridge Rd., Bethesda, MD 20814-4799. repellency effects of DDT, permethrin, and delta- 4 621 Maple Hill Lane, Crownsville, MD 21032-1062. Laboratoire de Lutte Contre Les Insectes Nuisibles methrin (Chareonviriyaphap et al., unpublished (L.I.N.), 9 11 Ave. Agropok B.P 5045, 34032 Montpel- data). Behavioral responses of four An. albimanus lier cedex, France. 1 populations were compared using three different in- .'

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172 JOURNALOF THE AMERICANMOSQUITO CONTROL ASSOCIATION VOL. 13, No. 2 I secticides and with or without physical contact with impregnated with 0.0003 and 0.0019 g AVm2 insecticides. (Char eo nviri y aph ap et al., unpublished data). 3. DDT (Dichloro-diphenyl-trichloroethane) (99% purity). This chemical was purchased from MATERIALS AND METHODS the Entomological Sciences Division, United States Army Center for Health Promotion and Preventive Anopheles albimaniis test populations: 1. Santa Medicine (USACHPPM), Aberdeen Proving Tecla colony (ST). This colony has been maintained Ground, MD, in October 1994. Based on estab- in laboratory colonies for 20 years. It was originally lished LD,, and LD,, toxicities for DDT, test papers collected from an stable in La Libertad Vil- (12 X 15 cmz) were impregnated with 0.4069 and lage along the Pacific Coast of El Salvador, Central 0.7593 g Nm2(Chareonviriyaphap et al.,' unpub- America, in 1975. It was maintained at the U.S. lished data). Additionally, papers were impregnated Department of Agriculture, Gainesville, FL, and at levels of DDT used in malaria control, i.e., 2 g later at the Walter Reed Army Institute of Research AUm2. (WRAIR), Washington, DC (Seawright, personal Insecticide-impregnated papers were received communication). The colony was obtained from the from the Entomological Sciences Division, WRAIR and was maintained at the Uniformed Ser- USACHPPM, Aberdeen Proving Ground, MD. All vices University of the Health Sciences (USUHS), papers were treated at the rate of 2.75 rnl of the Bethesda, MD, during these studies. insecticide solution per 180 cm'. 2. El Semillero colony (ES). This colony was Mosquito rearing: Anopheles albimanus colo- originally collected from a cattle corral in El Sem- nies were reared following the methods of Ford and illero near the Pacific Coast of Guatemala in Oc- Green (1972), with only minor modifications. All tober 1992. It had been maintained in the Labora- life stages were reared in an environmentally con- tory of Medical Entomology Research and Training trolled (25 -t. 5"C, 80 k 10% relative humidity) Unit (MERTU/G) in Guatemala for 1 yr before we insectary at USUHS. Adult mosquitoes were pro- obtained colony specimens in 1993. This colony vided cotton pads soaked with 10% sugar solution exhibited resistance to permethrin (C. Cordon-Ro- from the day of emergence and adults were main- sales, personal communication). tained in a 12 X 12 X 12-in. screened cage. 3. Toledo field population (TO). This population Behavioral tests: The test method consisted of was obtained from human-landing collections in a enclosing 25 female mosquitoes in a chamber lined rice cultivation area of San Felipe Village, Toledo with insecticide-treated or untreated (control) test District, southern Belize, in September 1994. The papers. Each chamber had a single portal for mos- field-caught females were susceptible to permethrin quitoes to escape to a receiving cage. The exposure and deltamethrin but demonstrated resistance to chamber accommodated a screened insert (inner DDT (Chareonviriyaphap et al., unpublished data). chamber) that, when placed in the first chamber, 4. Coroza1 field population (CO). This popula- prevented the mosquitoes from making physical tion was obtained from human-landing collections contact with test papers. Under test conditions, in a marsh area of Chan Chen Village, Corozal Dis- mosquitoes were enclosed within the chamber, and trict, northern Belize, in February 1995. The wild- the only source of light came from the exit portal. caught females were susceptible to DDT, permeth- A full test consisted of a pair of treatment chambers rin, and deltamethrin (Chareonviriyaphap et al., un- and a pair of control chambers. published data). One treatment chamber permitted tarsal contact Insecticides: Three insecticides were used in be- with insecticide-treated papers, i.e., there was no havioral tests. inner chamber. The second treatment chamber in- 1. Permethrin [3-phenoxybenzyl (IRS)-cis-trans- cluded the inner chamber, so mosquitoes could not 3-(2, 2-dichlorovinyl)-2, 2-dimethylcyclopropane- make contact with insecticide-treated papers. Treat- carboxylate] (approximately 75% trans, 25% cis ment chambers were lined with test papers that isomers) (94.7% purity). This chemical was re- were impregnated with insecticide and an oil-based ceived from AgrEvo Environmental Health (UK), carrier (risella oil). Control chambers were lined United Kingdom, in January 1994. Based on estab- with papers that were impregnated with carrier lished LD,, and LD,, doses for permethrin, test pa- alone. For brevity, tests with or without the inner X pers (12 15 cm') were impregnated with 0.0092 chambers, for either treatment or control papers, and 0.0462 g of active ingredient (AI)/m2 (Char- will be referred to as contact trials (no inner cham- eonviriyaphap et al., unpublished data). ber) or noncontact trials (with inner chamber). This 2. Deltamethrin [(S)-a-cyano-3-phenoxybenzl test system, including methods of data analysis, has (IR)-cis-trans-3-(2,2-dibromovinyl)-2, 2-dimethyl- been described by Roberts et al. (1997). All cage cyclopropane carboxylate] (99.7% purity). This components, with the exception of a rear panel, chemical was obtained from the same company were constructed of metal so they could be chem- (AgrEvo Environmental Health [UK]) in January ically cleaned and used repeatedly with different 1994. Based on established LD,, and LD,, doses chemicals and doses without risk of contamination. X for deltamethrin, test papers (12 15 cm') were A full test required 4 separate cardboard pint JUNE1997 PESTICIDEAVOIDANCE BEHAVIOR 173

(0.473 liters) cages of 25 mosquitoes (a test popu- “survivals.” The time in minutes for 50 and 90% lation) each. About 1 min was needed for 2 inves- of the test population to escape was estimated with tigators, using oral aspirators, to introduce a test the life table method and these estimates were used population into each of the 4 test chambers. After as the “escape time” summary statistics (ET,, and a 3-min holding period, the escape funnel in each ET,,). The log-rank method was used to compare chamber was opened to initiate observations. Num- patterns of escape behavior (Mantel and Haenzel bers escaping from exposure chambers into l-gal- 1959). A statistical software package, STATA@, lon (3.785 liters) cardboard receiving cages were was used for this analysis. These methods of ana- recorded in 1-min intervals; after 5 min of obser- lyzing excito-repellency data have been described vation, receiving cages were replaced with empty by Roberts et al. (1997). cages. The change of receiving cages facilitated the accurate counting of numbers of mosquitoes that RESULTS escaped. Tests pe~onned:Only An. albimaizus females Escape responses of An. albirizanus to DDT, per- were used in excito-repellency tests. Each test was methrin, and deltamethrin were tested in contact replicated at least 3 times. To fulfill the goals of and noncontact exposure chambers. Mortalities of this research, tests were performed to compare the females after a 24-h holding period, from contact 3 insecticides, concentrations of insecticides, insec- and noncontact trials, are given in Tables 1 and 2. ticide-resistant vs. insecticide-susceptible popula- Higher mortalities were observed in contact trials tions, colony vs. field-caught populations, insecti- than in noncontact trials, and higher mortalities cide contact vs. noncontact, and short-term (30 were observed in noncontact trials than in controls. min) vs. long-term (4 h) noncontact exposure. With 2.00 g/m2 of DDT in contact trials, no mor- Observations on mortality of test populations tality of escaped ES and TO females was observed, were made immediately after completion of each while 6.8% mortality was observed in ST females. test, i.e., the number of dead or knockdown speci- A high mortality (38.5%) of nonescaped ST mos- mens inside the exposure chamber was recorded. quitoes (96% remained in the chamber) was found Additionally, specimens that escaped and the re- compared to that of nonescaped ES and TO mos- maining test specimens collected from the exposure quitoes. With permethrin in contact trials (Table l), chamber were held separately for observation of 11% or less of the escaped ES and TO mosquitoes mortalities after 24-h holding periods. died, while almost 40% of the escaped ST mos- A shortage of CO specimens (Corozal, Belize) quitoes died. No mortality occurred in contact trials resulted in no test of DDT at 2.00 g/m2. Likewise, with the CO mosquitoes or in any noncontact trials a shortage of TO specimens (Toledo, Belize) re- (Table 2). In contact trials with deltamethrin (Table sulted in no tests of DDT at 0.4060 and 0,7593 g/m2 l), higher mortality was observed in ST specimens or of deltamethrin at 0.0003 g/m2. The ES colony than in the other populations tested. (Guatemala) was lost before tests of DDT at 0.4069 Times in minutes for mosquitoes to escape from and 0.7593 g/m2 could be conducted. treated chambers are given in Table 3. The escape We were concerned that the 30-min exposure pe- patterns from chambers containing insecticides riods were insufficient for measuring noncontact be- were defined as times for 50 and 90% of the pop- havioral responses (repellency). To increase the sen- ulation to leave the treated chambers (ET,, and sitivity of the test system, tests were conducted to ET,,). For DDT at 2.00 g/m’, the ES population had compare noncontact treatments with noncontact con- an ET,, of 8 min and an ET,, of 19 min. The TO trols with 4-h exposure periods. To prevent insecticide population had an ET,, of 2 min and an ET,, of 16 particles from falling from test papers above the ex- min. At the lower dose of permethrin, the ES, TO, posure chamber, clean white paper was placed on top and CO test populations had ET,, values of 2, 4, of the inner chamber for each test. Four-hour expo- and 9 min, respectively. The ET,, values for ES and sures were conducted with all 3 insecticides against CO populations were 8 and 17 min, respectively. the CO population only. Similar tests were not per- The ET,, of the TO population could not be cal- formed with the other 3 test populations since our culated. Likewise, the numbers of ST specinìens goal was just to test for increased noncontact repel- escaping from exposure chambers were low, and lency during longer exposure periods. ET,, and ET,, values could not be estimated for any Datu analyses: We used test data in a life table of the 3 insecticides tested (Table 3). survival analysis approach (Roberts et al. 1997) to Multiple comparisons among 4 test populations estimate mosquito escape rates (or rates of mos- in contact, noncontact, and control trials against quitoes staying in the chambers) and then compared 2.00 g/m2 DDT, 0.0092 g/m2 permethrin, 0.0462 differences in mosquito escape among populations, g/m2 permethrin, 0.0003 g/mz deltamethrin, and insecticides, and concentrations (doses) of insecti- 0.0019 g/m2 deltamethrin are shown in Tables 4-6. cides. With this method, the mosquito escape rate The patterns of escape behavior were tested with was estimated at 1-min intervals. Mosquitoes that the log-rank method, and significance was estab- escaped were treated as “deaths,” and those that lished at the 0.05 level of probability. Marked dif- remained in the exposure chamber were treated as ferences in escape responses were found when the I w' ' 174 JOURNALOF THE AMERICANMOSQUITO CONTROL ASSOCIATION VOL. 13, No. 2 i l Table 1. Mortalities of Anopheles nlbìmnntrs females after a 24-h holding period following exposures in contact trials of excito-repellency tests.

Number (%) 70 mortalityx Test Insecticide/ population' dose2 Tested Escaped Escaped Not escaped i ST colony, El Salvador Per- 1 1O0 23 (23) 39.1 5s (45/77) O Per-C 1O0 3 (3) O Per-2 1O0 13 (13) 7.6 12 (10/87) O Per-C 100 0 (0) O Del- 1 150 28 (19) 17.8 27 (33/122) Del-C 150 l(0.6) O O ! Del-2 75 7 (9.3) O 3 Del-C 75 l(1.3) O O i DDT- 1 125 29 (23) 6.8 39 (37/96) DDT-C 125 7 (5.6) O O ES colony, Guatemala Per- 1 1O0 97 (97) 11 O Per-C 100 o (0) O rl Per-2 150 150 (100) 8.6 O O Per-C 150 9 (6) O Del- 1 75 71 (94.5) 5.6 O O Del-C 75 3 (4) O Del-2 75 75 (100) 2.6 O O Del-C 75 . 6 (8) O DDT- 1 75 72 (96) O O DDT-C 75 4 (5.3) O O TO population, Belize Per- 1 90 90 (100) 2.2 O Per-C 75 4 (5.3) O O Per-2 76 67 (88.1) 2.9 44 (4/9) Per-C 76 8 (10.5) O O Del-1 75 70 (93.3) 1.42. 40 (2/5) Del-C 75 4 (5.3) O O DDT- 1 115 113 (98) O 50 (1/2) O DDT-C 1O0 17 (17) O CO population, Belize Per- 1 75 74 (98.7) O O O O Per-C 75 6 (8) Per-2 75 75 (100) O O O O Per-C 75 3 (4) Del- 1 75 75 (100) O O Del-C 75 2 (2.7) O O Del-2 75 75 (100) O O Del-C 75 7 (9.3) O O DDT-2 75 70 (93.3) 1.4 20 (US) DDT-C 75 8 (10.7) O O DDT-3 75 71 (94.7) O 25 (1/4) DDT-C 75 2 (2.7) O O

I ST Santa Tecla colony from EI Salvador; ES: El Semillero colony from Guatemala; TO: field population from Toledo District. Belize: CO: field population from Corozalastrict, Belize. Codes for insecticides and doses (g/m2): Per = permethrin at doses of (1) 0.0462 and (2) 0.0092; Del = deltamethrin at doses of (1) 0.0019 and (2) 0.0003; DDT = DDT at doses of (1) 2, (2)0.7593, and (3) 0.4069. Per-C, Del-C, and DDT-C are controls (without insecticides). Mortalities based on numbers dead following 24-h holding periods after specimens were removed from exposure chambers or after females escaped from exposure chambers.

ST test population was compared with the other test (P < 0.05) were observed in ES, CO, and TO pop- populations (P < 0.05) (Table 6). Significant dif- ulation escape responses between contact and non- ferences in escape probabilities were seen for all contact trials (Table 4). There were no significant compounds when the ST test population was com- differences in the ST population responses in con- pared to the ES population (P < 0.05). tact vs. noncontact exposures at lower doses of per- Contact vs. noncontact responses of An. albi- methrin and deltamethrin (P > O.OS), but significant manus to 2.0 g/m2 DDT, 0.0092 and 0.0462 g/m2 differences were found at higher doses (P < 0.05) permethrin, and 0.0003 and 0.0019 g/m2 deltameth- (Table 5). Also, significant differences in escape re- rin were compared. Escape probabilities in contact sponses were found in all four populations when trials were significantly higher than in controls for contact responses were compared to noncontact re- all populations (P < 0.05). Significant differences sponses (Tables 4 and 6). ~ ', 4' .

I JUNE 1997 PESTICIDEAVOIDANCE BEHAVIOR 175 1 Table 2. Mortalities of Aiioplzeles albimaiuLs females after a 24-h holding period following exposures in noncontact trials of excito-repellency tests. Number (%) 70 mortality3 Í Test Insecticide/ I population1 dose? Tested Escaped Escaped Not escaped I ST colony, El Salvador Per- 1 100 O O Per-C 100 O O Per-2 1O0 O O Per-C 1O0 O O Del- 1 150 O O Del-C 150 O O Del-2 75 O O Del-C 75 O O DDT- 1 125 O O DDT-C 125 O O ES colony, Guatemala Per- 1 75 O' 2 (1/67) Per-C 75 O O Per-2 125 4: 1 1 (mol) Per-C 125 O O Del-1 75 O O Del-C 75 O O DeI-2 75 O O Del-C 75 O O DDT- 1 75 O O DDT-C 75 O O TO population, Belize Per- 1 75 O O Per-C 75 O O Per-2 75 O O Per-C 75 O O Del-1 75 O O Del-C 75 O O O O DDT- 1 1O0 DDT-C 1O0 O O CO population, Belize Per- 1 75 O O Per-C 75 O O Per-2 75 O O Per-C 75 O O Del- 1 75 O O Del-C 75 O O Del-2 75 O O Del-C 75 O O DDT-2 75 O O I DDT-C 75 O O DDT-3 75 O O DDT-C 75 O O

I ST: Santa Tecla colony from EI Salvador; ES: El Semillero colony from Guatemala: TO: field population from Toledo District, Belize; CO: field population from Corozal District, Belize. Codes for insecticides and doses (g/mz): Per = permethrin at doses of (1) 0.0462 and (2) 0.0092; Del = deltamethrin at doses of (1) 0.0019 and (2) 0.0003; DDT = DDT at doses of (1) 2, (2) 0.7593, and (3) 0.4069. Per-C, Del-C, and DDT-C are controls (without insecticides). ' Mortalities based on numbers dead following 24-h holding periods after specimens were removed from exposure chambers or after females escaped from exposure chambers. b-

Statistical comparisons between 2 doses, LD,, were no significant differences between 2 doses of and LD,,, of DDT, permethrin, and deltamethrin, in the 3 chemicals in noncontact trials. contact and noncontact trials showed significantly Figures 1-4 show the proportions of mosquitoes stronger escape responses for higher doses of per- remaining in the exposure chambers under different methrin in contact trials for the ES, CO, and TO test conditions. These proportions are used to show populations (Table 5). The escape response for the patterns of escape rates. The patterns are indicative CO population was also significantly greater in con- of escape probabilities between contact and non- tact trials with the higher dose of deltamethrin. All contact trials (Figs. 1 and 3) and between noncon- other differences in escape responses between dos- tact and control trials (Figs. 2 and 4) with 4 pop- age levels were not significant (P> O.OS), and there ulations of An. albimanus. Significant differences 176 JOURNALOF THE AMERICANMOSQUITO CONTROL ASSOCIA~ON VOL. 13, No. 2

Table 3. Time in minutes for 50 (ET,,) and 90% (ET,,) of Anopheles nlbirnnizus females to escape from exposure chambers (in excito-repellency tests) treated with DDT, permethrin, or deltamethrin. DDT' Per- 1 Per-2J Del-lS Del-26 Population/ colony1 ET90 ETso ET,, ETSO ET,, ETso ET,, ET," ET," ST colony nc8 nc nc nc nc nc nc nc nc nc ES colony 8 19 2 8 1 7 7 11 2 6 TO population 2 16 4 nc 1 7 9 - 1 6 CO population - - 9 17 4 11 9 19 4 10 - ST Santa Tecla colony from El Salvador; ES: El Semillero colony from Guatemala; TO: field population from Toledo District, Belize; CO: field population from Corozal District, Belize. DDT at 2 g/mz. Permethrin at 0.0462 g/mz. Permethrin at 0.0092 g/mz. Deltamethrin at 0.0019 g/mz. Deltamethrin at 0.0003 -,elm2. ' Survival analysis was used to estimate the time in minutes for 50 and 90% of test populations to escape from exposure chambers. Very few specimens (0-2370) escaped from exposure chambers, so the ET,, and ET,, estimates could not be calculated for a 30-min -exposure:periud3orthrT@-p opul~ti~~h~-~T~c~~~dn~r~-~l~l~~-be~a~~~l~SS%~~~~~~hr30rtrrirrexpasnreperiml. Mosquitoes were not available for testing. I

in escape patterns were seen when contact trials were similar to escape responses for permethrin I were compared with control and noncontact trials (Figs. 3 and 4). Escape patterns of the ES, TO, and i (P < 0.05). The patterns of escaping females from CO test populations in noncontact trials were higher contact vs. noncontact trials and from noncontact than those from control trials. Significant noncon- vs. control trials for deltamethrin (not illustrated) tact repellency to DDT and synthetic pyrethroids I (P < 0.05) was seen in trials with the ES test spec- I imens. I Table 4. Comparison of escape responses (in excito- Tests for increased levels of escape response with repellency tests) between contact and control trials, 4-h exposures in noncontact trials were conducted contact and noncontact trials, and noncontact and control with CO populations (Fig. 5). In 4-h noncontact I trials for 4 colonies or populations of Anopheles l albiinnnrrs mosquitoes. tests, the CO test populations showed statistically i significant responses for all insecticides and doses Popu- Contact Contact Noncontact compared to the controls (P < 0.05). Four-hour la- vs. vs. vs. contact trials were not performed, since all CO test i tion/ control noncontact control specimens in contact trials escaped from the treated I colo- dose dose dose chambers within the standard (30-min) exposure ! Chemical ny' (g/m') (gimz) (g/m2) period. DDT ST 2.0000"' 2.0000* 2.0000 ES 2.0000" 2.0000" 2.0000" i CO 0.4069" 0.4049" 0.4069" DISCUSSION I CO 0.7593" 0.7593" 0.7593 Two forms of "behavioral avoidance," contact TO 2.0000" 2.0000" 2.0000" irritability and noncontact repellency, have been de- Permethrin ST 0.0092" 0.0092 0.0092 scribed (Davidson 1953, Rawlings and Davidson ST 0.0462" 0.0462" 0.0462 I 1982). Irritability occurs when an is stimu- I ES 0.0092" 0.0092" 0.0092" ES 0.0462" 0.0462" 0.0462" lated to move away from an insecticide-treated sur- face after direct physical contact with the insecti- CO 0.0092" 0.0092" 0.0092" i cide residue. In contrast, repellency occurs when CO 0.0462" 0.0462" 0.0462" !i TO 0.0092" 0.0092" 0.0092 the insect detects and avoids treated surfaces with-- l TO 0.0462" 0.0462" 0.0462 out physical contact (Roberts and Andre 1994). In this study, both contact irritability and noncontact Deltamethrin ST 0.0003'6 0.0003" 0.0003 i ST 0.0019'k 0.0019" 0.0019 repellency were documented to occur with An. al- I ES 0.0003" 0.0003" 0.0003" bimanus in the presence of DDT, permethrin, and ES 0.0019" 0.0019" 0.0019" deltamethrin. 1 CO 0.0003" 0.0003" 0.0003" Our laboratory and field results demonstrated CO 0.0019" 0.00 19:k 0.0019" that An. albimanus females from Guatemala (ES TO 0.0019" 0.0019" 0.00 I9 population) and from Belize (TO and CO popula- i * ST Santa Tecla colony from El Salvador; ES: El Semillero tions) showed dramatic escape responses from ex- 1 colony from Guatemala; TO: field population from Toledo District, posure chambers that permitted direct contact with Belize; CO: field population from Corozal District, Belize. insecticide-treated surfaces. This irritancy response 'The * identifies results of log-rank tests with statistically sig- nificant (0.05 level of probability) differences in patterns of escape was not observed in mosquitoes that had been in behavior. colony for 20 years (ST population). For all 4 test Il JUNE 1997 PESTICIDEAVOIDANCE BEHAVIOR 177

Table 5. Comparison of escape resonses for 2 doses (LD,, and LD,,) of different insecticides and populations of Anopheles albimaizus mosquitoes in contact and noncontact trials. Insecticide Type of trial Populationlcolony Compared dose level

DDT Contact CO 0.4069 vs. 0.7593 Noncontact CO 0.4069 vs. 0.7593 Permethrin Contact ST 0.0092 vs. 0.0462 Contact ES 0.0092 vs. 0.0462*? Contact CO 0.0092 vs. 0.0462* Contact TO 0.0092 vs. 0.0462" Noncontact ST 0.0092 vs. 0.0462 Noncontac t ES 0.0092 vs. 0.0462 Noncontact CO 0.0092 vs. 0.0462 Noncontact TO 0.0092 vs. 0.0462 Deltamethrin Contact ST 0.0003 vs. 0.0019 Contact ES 0.0003 vs. 0.0019 Contact CO 0.0003 vs. 0.0019* Noncontact ST 0.0003 vs. 0.0019 Noncontact ES 0.0003 vs. 0.0019 Noncontact CO 0.0003 vs. 0.0019

I ST Santa Tecla colony from El Salvador; ES: El Semillero colony from Guatemala; TO: field population from Toledo District, Belize; CO: field population from Corozal District, Belize. The 'b identifies results of log-rank tests with statistically significant (0.05 level of probability) differences in patterns of escape behavior between doses of the insecticides.

populations, there was a lower escape rate from Table 6. Comparison of escape responses between chambers that did not permit physical contact with populations of Anopheles albiinaiius females in contact treated surfaces. Regardless, the numbers escaping and noncontact trials by dose of insecticide. from noncontact test conditions were significantly Noncontact greater than the numbers escaping from control Contact trial trial chambers. This suggests that both contact irritancy Dose (population (population Insecticide (g/m2) or colony) or colony)

DDT 2.0000 ST vs. ES:bz ST vs. ES* ST vs. TO" ST vs. TO* ES vs. TO* ES vs. TO Permethrin 0.0092 ST vs. ES* ST vs. ES* ST vs. CO* ST vs. CO ST vs. TO" ST vs. TO ES vs. CO* ES vs. CO ES vs. TO* ES vs. TO CO vs. TO CO vs. TO 0.0462 ST vs. ES* ST vs. ES* ST vs. CO* ST vs. CO ST vs. TO* ST vs. TO DDT 2g/m2 .-L ES vs. CO ES vs. CO" OS 0.4- ES vs. TO'k ES vs. TO* 5- CO vs. TO" CO vs. TO 0.3- 5c Deltamethrin 0.0003 ST vs. ES* ST vs. ES* .-0- - :0.2- ST vs. CO* ST vs. CO* 0. ES vs. CO ES vs. CO P- a 0.1 - 0.0019 ST vs. ES'k ST vs. ES'k ST vs. CO* ST vs. CO* o;,,,,,,, , I,IIII , 11,,1,~,,(,,I ST vs. TO ST vs. TO* O 5 10 15 20 25 30 ES vs. CO ES vs. CO* Time (Minutes) ES vs. TO* ES vs. TO* CO vs. TO CO vs. TO Fig. 1. Proportions of Aizophefes albirnaizus females remaining in exposure chambers in contact vs. noncontact * ST Santa Tecla colony from El Salvador; ES: El Semillero colony from Guatemala; TO: field population from Toledo District, trials with 2 g/m? DDT. (ST: Santa Tecla colony; ES: EI Semillero colony; TO: field populations from Toledo Dis- Belize; CO: field population from Corozal District, Belize. ST; ST; The * identifies results of log-rank tests with statistically sig- --E-:trict, Belize; contact . . . 0 . . .: noncontact nificant (0.05 level of probability) differences in patterns of escape contact-.-: ES; . . ' -: noncontact ES; -A-: behavior between populations and trials. contact TO; . . . A . - .: noncontact TO.) 178 JOURNALOF THE AMERICANMOSQUITO CONTROL ASSOCIATION VOL. 13, No. 2

may have evolved gradually as adaptations for avoiding classes or families of toxic chemicals pro- duced by plants. In contrast to the behavioral responses of field- caught or recently colonized populations, an older colonized population (ST colony) showed little to no behavioral response to the insecticides. Large numbers of ST test specimens died in contact trials. After 2 decades of laboratory maintainance, the ST colony has lost its genetic variability (Chareonvi- riyaphap et al., unpublished data) and, as a conse- quence, its natural ability to respond behaviorally to the 3 insecticides. A similar phenomenon was seen in earlier studies with a Gorgas Panama lab- L oratory population of An. albimanus that was main- DDT 2glm2 tained as a colony for 20 years. The Panama mos- quitoes showed excitation times (minutes to first flight following DDT exposure) that were 2 times 0.71 III 11111111111Il Il O longer than those of field populations (Brown B1 5 10 15 20 25 30 1958). In combination, these results caution against Time (Minutes) the use of colony populations as “standards” for Fig. 2. Proportions of Anopheles albimanris females studying behavioral responses of mosquitoes to in- remaining in exposure chambers in noncontact vs. control secticides. trials with 2 g/m2 DDT. (ST Santa Tecla colony, ES: El In our studies, ES, TO, and CO test specimens Semillero colony, TO: field populations from Toledo Dis- quickly escaped exposure chambers without receiv- trict, Belize; -a-: noncontact ST, . . .O . . .: control ST, ing a lethal dose of DDT, demonstrating strong nat- -M-: noncontact ES; .-.O..e: control ES; -A-: noncontact TO; . . . A . . .: control TO.) ural behavioral avoidance of DDT. Additionally, the ES, TO, and CO test specimens were able to escape unharmed from permethrin-treated chambers. Pop- and noncontact repellency are involved in An. al- ulations of An. albimanus from Guatemala (ES) and bimanus escape responses. Belize (CO and TO) that escaped from deltameth- Smyth and Roys (1955) and Soliman and Cut- rin-treated surfaces were still alive 24 h later. Two komp (1963) reported that DDT had a specific ef- out of 5 nonescaped TO test specimens died within fect on antennal chemoreceptors and on sensory 24 h, but escaped and nonescaped ES mosquitoes hairs of tarsal segments. The effect of DDT on sen- showed very low mortality. Bown et al. (1987) re- sory hairs of tarsi could form the basis of irritability ported that An. albimanus departed deltamethrin- (Soliman and Cutkomp 1963). In combination, the treated huts in Mexico with low mortality. The es- function of antennal chemoreceptors and sensory cape patterns of the ES and TO test populations in hairs on tarsal segments probably results in avoid- the presence of DDT were totally different from ance behaviors that involve both contact irritability escape patterns of the ST test population, as well and noncontact repellency. as being different from all control chamber escape Physiological resistance and behavioral avoid- patterns. The rate of escape of TO specimens ex- ance are considered to be products of selective posed to DDT was quicker than that of the ES pop- pressure from insecticide use (Lockwooa et al. ulation (Fig. 2). This may be due to differences in 1984). As derived characteristics, the presence and test conditions, i.e., laboratory vs. field conditions, level of one response in a population is thought to and age of mosquitoes at the time of testing. Rob- influence selective pressure for the presence and erts et al. (1984) reported that freshly fed An. dar- level of the other. However, identical behavioral re- lingì Root females showed lower rates of escape;- sponses in both resistant and susceptible popula- than did unfed or late fed females when exposed to tions indicate that there is no relationship between DDT. Hamon and Eyraud (1961) found that older these 2 response variables in An. albimanus popu- An. gambiae and An. fiinestris Giles mosquitoes lations. This finding suggests 2 hypotheses, one be- demonstrated less irritability than young mosqui- ing that behavioral avoidance and physiological re- toes. In our studies, laboratory tests were conducted sistance are controlled by different genes. As a sec- with 3- to 5-day-old unfed female mosquitoes (ST ond hypothesis, we propose distinctly different or- and ES colonies), while the physiological age of igins for physiological vs. behavioral responses of field mosquitoes was unknown. Hecht et al. (1960) An. albimanus populations to the 3 insecticides. It reported that An. albimaniis is more active at higher is probable that physiological resistance is a recent temperatures. Tests conducted in the field were per- development due to selective pressures from agri- formed at higher ambient temperatures and humid- cultural uses of insecticides. Alternatively, the be- ities, which are conditions that might favor greater havioral responses of An. albimanus populations avoidance of DDT. .. ‘W., ., ,* JUNE1997 PESTICIDEAVOIDANCE BEHAVIOR 179

0.0092 gim2 permethrin

.-m .-K .r I (dE LI:a, 0.9 $ 0.8 .-O 5 0.7 Q 0.6

0.5 0.4 0.0462 g/m2 permethrin

0.3 0.2

0.1 O O 20 5 10 15 25 30 Time (Minutes)

Fig. 3. Proportions of Anopheles albitnanus females remaining in exposure chambers in contact and noncontact trials with 0.0092 and 0.0462 g/m2 permethrin. (ST: Santa Tecla colony; ES: El Semillero colony; TO: wild-caught ST; from Toledo District, Belize; CO: field populations from Coroza1 District, Belize; contact . . . 0.. .: non- ST; -E-: -a-: contact contact ES; a . .O. . .: noncontact ES; -A-: contact CO; . . . A . . .: noncontact CO; -+-: contact TO; . . . O . .: noncontact TO.) i .” Ø

A more gradual escape response to permethrin quitoes were bloodfed, which may have caused de- and deltamethrin was observed in CO test speci- layed escape patterns. mens compared to that in ES and TO test specimens In contact trials, more mosquitoes escaped at higher (Table 3). However, all CO test specimens eventu- concentrations of insecticides. At lower concentra- ally escaped from contact exposure chambers. The tions, synthetic pyrethroids produced poor escape re- differences in escape patterns among these popu- sponses in ST test specimens. This relationship be- lations may have been influenced by differences in tween concentration of insecticide and degree of be- physiological age or gonotrophic status of female havioral response was consistent with findings of Ree 964), mosquitoes, as described by Busvine (1 or due and Loong (1989), who reported an increased irrita- to ambient test conditions. Some of the field mos- bility response of An. nzaculutzis Theobald with in- VOL. 13, No. 2 I 180 JOURNAL OF THE AMERICAN MOSQUITOCONTROL ASSOCIATION I l l

I I

I , I i i I i iI l

I .-tl: , .-E!c i a, I cn 0.95 c i .-O I !

i i i

Time (Minutes) !

Fig. 4. Proportions of Anopheles albimanus females remaining in exposure chambers in noncontact and control trials with 0.0092 and 0.0462 g/m2 permethrin. (ST Santa Tecla colony; ES: El Semillero colony; TO: wild-caught ! ST; from ToledoST District, Belize; CO: field populations from Coroza1 District, Belize; -e--: noncontact . . . O . . .: control --a-: noncontact ES; . . . * .: control ES; -A-: noncontact CO; . . . A . .: control CO; -+-: non- contact TO; . . - O . . .: control TO.) i creasing concentrations of permethrin. Brown (1958) was seen in TO test populations since numbers es- also found that time to first flight of An. albimnus caping from the treated chambers were relatively i after exposure to DDT was short at higher concentra- low and similar to those of the controls. l tions compared to that at lower concentrations. Different insecticide concentrations appeared to Noncontact repellency of DDT may play a role have no influence on escape patterns in short-term I in reducing human-vector contact, as shown by exposures with noncontact trials. As seen in the ES, studies on An. crilicifacies Giles in India (Shalaby TO, and CO test specimens, irritancy contributed to 1966). In our tests, the ES and CO test populations a strong and immediate response, while short-term showed noncontact repellency to DDT, even though exposure in noncontact trials (repellency) produced the exposure period was only 30 min. This suggests a weak, but statistically significant, escape re- that the repellency effect of DDT is also involved sponse. Both irritancy and repellency were presum- l in An. albimunzis escape responses. In short-term ably additive properties that produced an overall I exposures, no repellency to synthetic pyrethroids stronger avoidance response. I

D *I bt , JUNE 1997 PESTICIDEAVOIDANCE BEHAVIOR 181

-...... 0...... mntrols ...... o...... 0 ...... o ...... *.,A ...... A...... A controls \ \-

xQ- I

0.5 1 I I 1 I I

" ...... -4. o*-...... 0 ...... 0 controls

' controls \\ .. -a...... :&\ ::::....& ...... A ...n controls L 0.0462 g/m2 deltamethrin

\-0.0019 g/m2 permethrin

O 4 0.2 11 2 3 5 Time (Hours) i. Fig. 5. Proportions of Anopheles albinianus females remaining in exposure chambers in noncontact vs. control trials for DDT, permethrin, and deltamethrin. Test specimens were caught in Coroza1 (CO) District, Belize.-A-: (-a-: DDT treatment; ...0 .. .: DDT control; --a-: deltamethrin treatment; ... 0 .. .: deltamethrin control; per- l methrin control; ... A.. .: permethrin treatment.)

Results from long-term exposure (4 h) to each of crim (1991), who reported a strong repellent action the 3 insecticides suggest an even more important of DDT residues in houses. A repellent exerts an role for noncontact repellency in An. albimanus area effect, and if it is sufficient to reduce indoor avoidance of insecticide residues. Overall, delta- biting then it will also reduce indoor transmission methrin was the most repellent insecticide, fol- of malaria. However, other investigators have pro- lowed by permethrin and DDT. Unlike with short- posed that the irritant properties of permethrin and term exposures in noncontact trials, greater escape deltamethrin in treated huts have an unsatisfactory activity was seen at higher doses of all 3 com- impact on malaria vectors (e.g., Rishikesh et al. I pounds with 4-h exposures. These differences seem 1978). Similar reasoning led to the termination of to indicate that a 30-min exposure is not adequate DDT use in many countries in Soviet Central Asia, for a meaningful test of noncontact repellency. Asia, and South Africa after DDT was shown to Our study showed that DDT, permethrin, and del- produce irritant effects in vectors (Bondareva et al. tamethrin irritate and repel An. albimanus females 1986, Sharp et al. 1990). Unfortunately, the use of and that most specimens escaping insecticide ex- this chemical may have been stopped because of posure will survive. Our findings are in agreement the very property that made it historically effective with the results of field studies by Roberts and Ale- in malaria control, namely, strong excito-repellency , ___-ll--.ll ..” . ,_ .., __- “Y--

” 182 JOURNALOF THE AMERICANMI 3sourro CONTROLASSOCIATION VOL. 13, No. 2 I1. action. Our tests suggest that behavioral responses Washington, DC, for providing the An. albimaniis l elicited by DDT, permethrin, or deltamethrin might colonies. We are grateful to Paul Hshieh of the Di- interrupt indoor An. albimanru-human contact. vision of Biostatistics, Department of Preventive In this study, we used survival analysis tech- Medicine and Biometrics, USUHS, for his assis- i niques for treatment of the data, as described by tance in data analysis. Finally, we extend our I Roberts et al. (1997). The power of this analysis thanks to J. E Invest of AgrEvo Environmental relates to the use of escape probabilities over time Health in the United Kingdom for providing ex- for comparing the responses of different test pop- perimental quantities of permethrin and deltameth- i ulations. The escaped mosquito was classified as rin . l “dead,” while the nonescaped mosquito was clas- sified as a “survivor.” We believe that survival REFERENCES CITED analysis minimizes the loss of valuable information and is the method of choice for a biological inter- Beach, R. E, C. Cordon-Rosales and W. G. Brogdon. pretation of excito-repellency test results. 1989. Detoxifying esterases may limit the use of py- Busvine (1964) reported that the degree of irri- rethroids for malaria control in the Americas. Parasitol. Today 5:326-327. 1 tability in mosquitoes varies with the type of insec- I ticide. Both DDT and pyrethroids generally cause Bondareva, N. I., M. M. Artem’ev and G. V. Gracheva. I 1986. Susceptibility and irritability caused by insecti- mosquitoes to leave treated surfaces before being cides to malaria mosquitoes in USSR. Part Anopheles 1. I knocked down. However, in our tests, the pyre- pulcherrimus. Med. Parazitol. Parazit. Bolezni 652-55. 1 l throids produced a more immediate irritant effect [In Russian.] I than DDT. For these comparisons, the ET,, and ET,, Bown, A. N., E. C. Frederickson, G. A. del Cananas and values proved to be powerful estimators of insec- J. E. Mendez. 1987. An evaluation of bendiocarb and 1l ticidal effects on vector escape behavior and served deltamethrin applications in the same Mexican village as useful summary statistics for comparing insec- and their impact on populations of Anopheles albiman- US. PAHO Bull. 21:121-135. ticides and doses. Earlier laboratory tests with this S. vector (Brown 1958, Rachou et al. 1963) in Panama Breeland, G. 1974. Population patterns of Anopheles albimaniis and their significance to malaria abatement. and El Salvador showed that DDT has pronounced 1 Bull. W.H.O. 50:307-315. 1 behavioral effects. In our comparisons of ET val- Brown, A. W. A. 1958. Laboratory studies on the behav- ues, DDT at 2 g/m2 (the field dose) again showed iouristic resistance of Anopheles albimanus in Panama. 1 a powerful behavioral effect with An. albitnanus Bull. W.H.O. 19:1053-1061, populations from Guatemala and Belize. The lower Brown, A. W. A. 1986. Insecticide resistance in mosqui- I ET values, i.e., shorter escape times, for An. albi- toes: a pragmatic review. J. Am. Mosq. Control Assoc. nianils when tested against the 2 pyrethroids at LD,, 2: 123- 140. I and LD,, dosage levels were remarkable. If DDT Busvine, J. R. 1964. The significance of DDT-irritability I elicits an important behavioral response from An. tests on mosquitoes. Bull. W.H.O. 3 1:645-656. i albimanus females by reducing man-vector contact Curtis, C. E, J. D. Lines, €? Carnevale, V. Robert, C. Bou- din, J. M. Halna, L. Pazart, €? Gazin, A. Richard, J. inside houses, then we can assume that this might Mouchet, J. D. Charlwood, F! M. Graves, M. I. Hossain, also be the primary action of permethrin and del- T. Kurihara, K. Ichimori, L. Zuzi, L. Baolin, G. Majori, i tamethrin when used in malaria control applica- G. Sabatinelli, M. Coluzzi, K. J. Njunwa, T. J. Wilkes, tions. Frequently, mosquitoes do not enter sprayed R. W. Snow and S. W. Lindsay. 1989. Impregnated I houses, or, if they enter, they often escape before bednets and curtains against malaria mosquitoes, pp. 5- feeding on humans. Consequently, a strong avoid- 46. In: C. E Curtis (ed.). Appropriate technology for ance behavior can reduce human-vector contact vector control. C.R.C. Press, Boca Raton, FL. I Davidson, G. 1953. Experiments on the effect of residual and disease transmission. l In conclusion, behavioral responses of malaria insecticides in houses against Anopheles gainbiae and vectors to insecticides are important components of Anopheles fiinestus. Bull. Entomol. Res. 44:23 1-254. Ford, H. R. and E. Green 1972. Laboratory rearing of the insecticide-malaria control equation. More field Anopheles albimanus. Mosq. News 32:509-5 13. I research is needed on the behavioral responses of Hamon, J. and M. Eyraud. 1961. Study of physiological vector populations from different geographic loca- factors conditioning in Anopheles irritability to DDT. I tions to various insecticides. Riv. Malariol. 40:219-640. Hecht, O., O. Mancera and A. Barrera. 1960. Relation of DDT imtation threshold to knockdown of three species ACKNOWLEDGMENTS of anopheline mosquitoes. J. Econ. Entomol. 53:634- l We thank the staff of the Epidemiology Research 640. I Center, Belize City, Belize, Central America, and Lockwood, J. A., T. C. Sparks and R. N. Story. 1984. I l Shilpa Hahe in particular, for assistance in mos- Evolution of insect resistance to insecticides: a reeval- uation of the roles of physiology and behavior. Bull. I quito collections during 1994 and 1995. We also Entomol. Soc. Am. 30:41-5 1. I thank Celia Cordon-Rosales, Medical Entomology Mantel, N. and W. Haenzel. 1959. Statistical aspects of Il Research and Training Unit (MERTU), Guatemala, the analysis of data from retrospective studies of dis- and the insectary staff, Department of Entomology, l (-IR), eases. J. Natl. Cancer Inst. 22:719-748. Walter Reed Army Institute of Research Rachou, R. G., M. Moura-Lima, J. F! Duret and J. A. Kerr. I l

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1963. Experiences with the excito-repellency test F! Hshieh. 1997. Methods for testing and analyzing ex- J. box-model OPS. Proc. 50th N. Mosq. Exterm. As- cito-repellency responses of malaria vectors to insecti- I SOC. 50i442-447. I cides. J. Am. Mosq. Control Assoc. 13 (in press). Rawlings, l? and G. Davidson. 1982. The dispersal and Shalaby, A. M. 1966. Observations on some responses survival of Anopheles cidicifacies in a Sri Lankan vil- of Anopheles culicifacies to DDT in experimental huts lage under malathion spraying. Bull. Entomol. Res. 72: in Gujarat state, India. Ann. Entomol. Soc. Am. 59: I 139-144. 938-944. i Ree, H. I. and K. P. Loong. 1989. Irritability of Anopheles Sharp, B. L., D. le Sueur and l? Bekker. 1990. Effect of farauti, Anopheles tnaciilatus, and Culex quitigriefascia- DDT on survival and blood feeding success of Anoplz-

tus to permethrin. Jpn. J. Sanit. Zool. 40:47-5 1. eles arabiensis in Northern Kwazula, Republic of South f Rishikesh, N., J. L. Clarke, H. L. Mathis, J. S. King and Africa. J. Am. Mosq. Control Assoc. 6:197-202. J. 1 Pearson. 1978. Evaluation of decamethrin and per- Smyth, T., Jr. and C. C. Roys. 1955. Chemoreceptor in methrin against Anopheles gambiae and Anopheles fci- insects and the action of DDT. Biol. Bull. 108:66-76. tiestus in a village trial in Nigeria. WHONBC/78.689. Soliman, S. A. and L. K. Cutkomp. 1963. A comparison Roberts, D. R. and R. G. Andre. 1994. Insecticide resis- of chemoreceptor and whole- responses to DDT and tance issues in vectors. Am. J. Trop. Med. Hyg. parathion. J. Econ. Entomol. 56:492-494. 50(SLlppl.):21-34. Threlkeld, S. E H. 1985. Behavioral responses in Dro- Roberts, D. R. and W. D. Alecrim. 1991. Behavioral re- sophila melanogaster associated with permethrin and sponse of Anopheles darliiigi to DDT sprayed house ectiban, pp. 29-36. In: Proceedings of the thirty-second walls in Amazonia. PAHO Bull. 25:210-217. annual meeting, Canadian Pest Management Society, Roberts, D. R., W. D. Alecrim, A. M. Tavares and K. M. Charlottetown, Prince Island, 24-26 June, 1985. Can. McNeill. 1984. Influence of physiologica1 condition on Pest Manage. Soc. the behavioral response of Anopheles darlingi to DDT. World Health Organization. 1989. The use of impregnat- Mosq. News 44:357-361. ed bednet and other materials for vector-borne disease Roberts, D. R., T. Chareonviriyaphap, H. H. Harlan and control. WHONBCB9.98 1, Geneva.

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