Joumal of the American Mosquito Control Association, 12(3):483-486, 1996 EFFECTS OF SUBLETHAL DOSAGES OF METHOPRENE ON ANOPHELES DIRUS SPECIES A AND 81 RATANA SITHIPRASASNA, EKAWAN LUEPROMCHAI ENO KENNETH J. LINTHICIIM Department of Entomology, U.S. Army Medical Component, Armed Forces Research Institute of Medical Sciences, 31516 Rajvithi Road, Bangkok lO4O0 (from USA use USAMC-AFRIMS, APO AP 96546) ABSTRACT, Anopheles dlrzs species A and B individuals were exposed as 4th-stage larvae to sub- lethal concenhations ofmethoprene. Median lethal concentrations were 0.21 ppb for species A and 0.17 ppb for species B. When exposed to 0.10 ppb methoprene the sex ratio of species A changed from fewer males to more males; no effect was observed in the sex ratio of species B. Exposure to methoprene had no effect on wing length and survival of either species. Results indicate that exposure to methoprene significantly affected fecundity of both species of An. dirus. INTRODUCTION 1989, Sawby et al. 1992, Beehler and Mulla 1993). Anopheles dirus Peyton and Harrison is a In the present study, specimens of An. dirus mosquito species that is widely distributed species A and B were exposed in the laboratory throughout Southeast Asia and is one of the as 4th-stage larvae to sublethal concentrations of most important vectors of falciparum and vivax methoprene. Possible effects on adult sex ratio, malaria (Rosenberg et al. 1990). Control of An. wing length, survival, feeding success, oviposi- dirus adults is extremely difficult. The species is tion, and egg hatching were measured. largely unaffected by indoor spraying of houses with DDT and fenitrothion because it is primar- ily exophilic (Prasittisuk 1985). Possible control MATERIALS AND METHODS of immature stages has not been seriously con- Mosquitoes: sidered because larvae inhabit small, temporary Anopheles dlrus species A were from a colony ground pools in forested areas, often in inacces- established from Chonburi, Thai- land, in 1968, that was maintained sible regions in hills along international borders using forced- mating techniques. This colony (Rattanarithikul and Panthusiri 1994). Conse- has been peri- odically outbred with field-collected specimens. quently, larval habitats are very difficult to lo- The most recent outcross cate and treat with larvicides. occurred I month be- fore the start of this study. Anopheles dirus spe- Characteristics of pellet and granule formu- cies B were from a self-mating colony estab- lations of methoprene, such as its ability to be lished from Sabah, Malaysia, in 1978. Both col- used as a sustained release larvicide (Linthicum -+ onies were reared at 27 2oC,75-8OVo RH. in et al. 1989, Kramer and Beesley l99l), its abil- natural light for multiple generations. ity to retain activity for several months during Methoprene: Altosid@ (4Vo methoprene) sus- pretreatment in dry areas (Logan et al. l99O), tained-release pellets used in this study were and its potential ability to penetrate canopy cov- provided by Zoecon Corporation, Dallas, TX. ers, may make it suitable for aerial application Pellets were dissolved in acetone before serial as a larvicide for An. dirus in areas where ma- dilutions were made in water (Robert and Olson laria is endemic. In addition to the lethal effects 1989). For each dilution, approximately 100 ear- of methoprene, sublethal effects on adult mos- ly 4th-stage larvae were placed in 6 plastic pans quito populations exposed to low doses as larvae (25.4 x 30.5 X 5.1 cm) containing 1.5 liters of also have been observed (Robert and Olson water and either 0, 0.01, or 0.10 ppm concentra- tions of methoprene. An abundant amount of a rThe crushed locally purchased fish food was added views of the authors do not purport to reflect every afternoon to each pan and the excess in- the positions of the Department of the Army or the fusion removed each morning. Department of Defense. Research was conducted in Each species was compliance with the Animal Welfare Act and other exposed to each methoprene dose, and each ex- Federal statutes and regulations relating to animals and posure was repeated 7 times. experiments involving animals and adheres to the Experimental procedures: After exposure of Guide for Use of Laboratory Animals, NIH publication 4th-stage larvae to methoprene for 36-48 h, pu- 86-23. 1985 ed. pae were removed from plastic pans and placed Jounxlt- op tnp AurnrceN Moseuno CoNrnor-Assocrar.rox Vor-. 12. No. 3 Thble l' Sex ratio inAnopheles dirus adtits and wing lengths in ovipositing females after exposure of 4th-stage larvae to sublethal doses of methoprene. + Methoprene Mean sex ratio SE (male:i female)rvursrv/ ryrc4rr -r concentra_ \4..erv Mean wingwrttE lengthrEtrBur \tIIIr,(mm) I SEJE tion (ppb) SpeciesA SpeciesB Species A Species B + 0 O.75 O.2lal 0.37 + 0.ol 3.O3+ O.t2 3.1I -i-0.05 + 0.01 0.76 0.10a o.24+ O.tl 2.99+ O.ll 3.13+ O.O4 -f -r 0.10 1.50 0.10b 0.35 0.20 2.95 -l- 0. l0 3.12+ 0.06 I Means within a column followed by different letters are significmtly different (ANovA, p < 0.05) directly in a paper cup in a 0.3-m3 wire mesh lex quinquefosciatus Say (Robert and Olson cage. Percent mortality of pupae was determined 1989). Exposure of larvae to concentrations of by counting the number of adults successfully methoprene of 0.01 and 0. l0 ppb was equivalent emerging and the number of dead pupae and to an LC3r and LCoo in species A and an LC.,, adults. The sex of all emerging adults was re- and LC., in species B, respectively. There was corded. In each trial adult survival was mea- significantly more mortality in specimens ex- sured by placing l0 of the male and l0 of the posed to methoprene than in those of the control female adults that emerged in each of 2 O.25- group (chi-square, P I 0.0001). Exposure of liter plastic cups covered with nylon mesh and species A to 0.10 ppb of methoprene signifi- counting the number dead each day until all in- cantly changed the sex ratio from fewer males dividuals had died. To measure survival under to more males (Table 1, chi-square, P < 0.01), stressful and normal conditions cups were pro- but had no effect on species B. Conversely, Rob- vided with either a water or a sucrose source and ert and Olson (1989) observed a significant in- placed at room temperature (26"C). The remain- crease in the proportion of females of Cx. quin- der of the emerging adult females were exposed quefasciatus exposed to methoprene. Wing 7 days after emergence to an anesthetized adult length of ovipositing females was not affected golden Syrian hamster in a 0.3-m3 cage. The by exposure to methoprene in either species (Th- number feeding, based upon the appearance of ble 1). Methoprene has been documented to any amount of blood in the abdomen, was re- cause both longer and shorter wing length in Cz- corded. All An. dirus species A (after force-mat- lex pipiens Linn. and Cx. quinquefasciatus (Ke- ing) and species B individuals that had fully en- lada et al. 1981, Robert and Olson 1989) at con- gorged were placed individually in 25-ml glass centrations near the LC.o. No change in survival bottles containing 5-cm strips of filter paper and in groups given water or sucrose of either spe- 5 ml of water and allowed to oviposit. The num- cies was observed following exposure to meth- ber of eggs oviposited and the number hatching oprene (Table 2). At slightly higher concentra- was recorded. The wing length of all mosquitoes tions of methoprene reduced survival has been that oviposited was measured. reported for Cx. quinquefasciat s (Robert and Statistical analysis: The median lethal con- Olson 1989) and Culex tarsalis coq. (Arias and centration (LC.o) of methoprene for each species Mulla 1975). was determined by probit analysis (SPSS Inc. Although methoprene did not have any effect 1993). Estimates of mean survival time in days on the number of An. dirus species A feeding were determined by Kaplan-Meier survival (Table 3), feeding was significantly reduced in analysis (SPSS Inc. 1993). Comparisons of species B by lOVo and 25Vo after exposure to mean sex ratio, wing length, and number of eggs 0.01 and O.lO ppb of methoprene, respectively oviposited were made using ANOVA. Compar- (Table 3, chi-square, P < O.Ol). Similarly, ex- isons of pupal mortality, feeding success, ovi- posure to methoprene did not affect the number position, and egg hatching were made using of fully engorged species A females that ovi- multiple contingency table tests for indepen- posited; however, the number of species B fe- dence (Centers for Disease Control 1994). males that oviposited was reduced by approxi- mately l9Vo and 22Vo after exposure to O.Ol and 0.10 ppb of methoprene, respectively (Table 3, RESULTS AND DISCUSSION chi-square, P < 0.001). There was no effect on The LC.o of methoprene for An. dirus was the mean number of eggs from ovipositing fe- 0.21 ppb for species A and 0.17 ppb for species males of either species. Methoprene has been B. The LCro was similar to that reported for Ae- found to reduce the number of Cx. quinquefas- des aegypti (Linn.) (Sawby et al. 1992) and Cu- ciatus eggs produced (Robert and Olson 1989). Sep,reirlnrn 1996 SusLrrHaL Dosecps or MprnopnrNe 485 Table 2. Survival of Anopheles dirzs adults after exposure of 4th-stage larvae to sublethal doses of methoprene.