Journal of the AmericanMosquito Control Association,l8(3):196-201' 2002 Copyright @ 2002 by the American Mosquito Control Association' Inc'

EFFECTIVENESS AND RESIDUAL ACTIVITY COMPARISON OF GRANULAR FORMULATIONS OF INSECT GROWTH REGULATORS PYRIPROXYFEN AND S-METHOPRENE AGAINST FLORIDA MOSQUITOES IN LABORATORY AND OUTDOOR CONDITIONS

JAI K. NAYAR,' ARSHAD ALI2 rNo MORTEZA ZAIM3

ABSTRACT. Effectiveness and residual activity tests of granular formulations of 2 insect growth regulators (IGRs), s-methoprene and pyriproxyfen, against laboratory-reared larvae of 5 colonized mosquitoes, Aedes ae- gypti, Aedes albipictus, Aides taeniorhynchus, Anopheles quadrimaculatus, and Culex nigripalpas, were con- i""t"O i" the laboratory and outdoors in plastic tubs. Culex quinquefasciatas was exposed to these two IGRs in the laboratory only. Each IGR formulation was applied at 0.02 and 0.05 ppm active ingredient (AI) against 5 of the 6 mosquito species both in the laboratory and the outdoor evaluations, whereas Cx. quinquefasciatus was exposed to 0.2 and 0.4 ppm AI of s-methoprene, and 0.1 and 0.2 ppm AI of pyriproxyfen in the laboratory. s- Methoprene at 0.02 and 0.05 ppm AI resulted in variable levels (<39-1007o) of inhibition of adult emergence in the 5 species monitored for 6 weeks after treatment under both test conditions. Aedes taeniorhynchus was the most susceptible to s-methoprene in terms of initial and residual activity. Culex quinquefasciatus and Ae- al- bopictus wire the most tolerant to .r-methopene, with maximum emergence inhibitions amounting to 84V" in Cx- qiinquefasciatus at O.4 ppm and 44.3V" in Ae. albopictus at 0.05 ppm during the lst week in the laboratory' iyriproiyfen at comparable treatment rates to .r-methoprene caused very high levels (>80-1007o in most cases) oi itritlut--d residuai emergence inhibitions of the tested species in the laboratory as well as outdoors' In several species, pyriproxyfen induced complete inhibition of adult emergence for several weeks after treatment, even at the lowei rate of 0.02 ppm. The World Health Organization has recently recommended the use of pyriproxyfen for the control of some mosquito species at specified rates in certain habitats.

KEY WORDS Mosquitoes, Aedes spp., Anopheles quadrimaculatus, Culex spp., bioassay, pyriproxyfen, s- methoprene, granular formulations

INTRODUCTION for their immediate effectiveness and long-term re- sidual activity. The insect growth regulator (IGR) pyriproxyfen is a juvenile hormone mimic that is highly active against a wide variety of insects of public health MATERIALS AND METHODS importance, including fleas, tsetse flies, houseflies, cockroaches, imported fire ants, chironomid midg- Mosquito species: Laboratory-reared late 3rd- (Lin- es, and mosquitoes (Hirano et al. 1998). Because and early 4th-stage larvae of Aedes aegypti (colonized of the reported excellent activity of the earlier for- naeus) in 1982, Florida Medical Ento- mulations of pyriproxyfen against mosquitoes in mology Laboratory [FMEL], Vero Beach, FL), numerous laboratory and field studies worldwide Aedes alboplcras (Skuse) (colonized in 1992, (Hirano et al. 1998), a granular formulation of this Gainesville, FL), Aedes taeniorhynchas (Wiede- (colonized IGR containing 0.57o active ingredient (AI) was mann) in 1965, FMEL, Vero Beach, (Say) (colonized submitted by Sumitomo Chemical Company, Japan, FL), Anopheles quadrimaculatus to the World Health Organization Pesticide Evalu- in 1950, Malaria Research Center, Tallahassee, FL), ation Scheme (WHOPES) for testing and evalua- Culex nigripalpus Theobald (colonized in 1997, tion against mosquitoes. This paper contains the re- FMEL, Vero Beach, FL), and Culex quinquefascia- sults of this evaluation conducted in Florida and tus Say (colonized in 1997, FMEL, Vero Beach, simultaneously compares the results with the activ- FL) were used. ity of a granular formulation of the IGR ^t-metho- Test IGRs: Pyriproxyfen (Sumilarv O.5VoG,lot prene containing l.5Vo AI. The IGRs were tested at 5O99X92, Sumitomo Chemical Co., Ltd., Osaka, 2 equivalent concentrations against laboratory- Japan), supplied through WHOPES, and s-metho- reared larvae of colonized mosquito species in the prene (Altosid@ XR-G, 1.57o Al, supplied by Well- laboratory as well as in plastic tubs placed outdoors mark International, Dallas, TX) were tested. Laboratory evaluations : These evaluations were conducted in white polyethylene trays (46 x 38 X I University of Florida, IFAS, Florida Medical Ento- 8 cm), lined with 2-mil-thick polyethylene sheets, mology Laboratory, 200 9th Street SE, Vero Beach, FL containing 6 liters of well water and maintained at 32962. 25 1- loc in a rearing room (Nayar et al. 1998). 'University of Florida, IFAS, Mid-Florida Research One hundred late 3rd- and early 4th-stage larvae of and Education Centet,2725 Binion Road, Apopka, FL mosquito species were introduced into each tray 32703. a food (6:1 yeast and 3WHO (World Health Organization) PesticideEvalua- along with 200 mg of larval tion Scheme,CH-1211, Geneva 27, Switzerland. beef liver powder). After 2-3 h of larval acclima-

196 SpprpNaepn2002 lvsecr Gnowru Rrcuurons Acxusr Flonroe Mosqunoes r97

tion, the trays were randomly treated. Each IGR ing the span of these evaluations conducted be- formulation was applied at O.O2 and 0.05 ppm AI tween Septembet 1999 and June 2000. against all mosquito species except for Cx. quin- The efficacy of a formulation against a mosquito quefasciatus, which was exposed to O.2 and 0.4 species in the trays and tubs was assessed as per- ppm AI of s-methoprene, and 0.1 and 0.2 ppm AI cent inhibition of adult emergence of the species in of pyriproxyfen. Because of the percent AI differ- treatments, and adjusted for any larval or pupal ence in the 2 test formulations, different amounts mortalities in corresponding controls with the for- (by weight) of the 2 formulations were needed to mula of Mulla et al. (1974): achieve the required ppm AI concentrations of each : - IGR. Three replicates of each treatment and 3 un- Vo inhibition of emergence l0O 100(T/C), treated trays to serve as controls were maintained where T is percent emergence in treated containers for each species. Al1 trays were examined daily to and C is percent emergence in control containers. score posttreatment larval and pupal mortality or Mean percent reductions of adult emergence in survivorship, and adult emergence. A large pipette each batch of mosquito species caused by the 2 was used to collect live pupae and dead larvae and formulations in trays and tubs were analyzed by l- pupae from each tray on a daily basis. The live way analysis of variance with Tukey-Kramer mul- pupae were maintained in cups placed in mosquito tiple comparison posttests by using the computer cages to check for emergence. Survivorship in the software Instat V. 3.00 for Windows (Graphpad treated and control trays was determined by count- Software, San Diego, CA). ing the number of pupal skins remaining in the cor- responding cups. After I wk, when all larvae and pupae had either died or survived as adults in the RESULTS AND DISCUSSION control trays, a fresh batch of 100 laboratory-reared late 3rd- and early 4th-stage larvae of the same Effectiveness and residual activity of the 2 IGRs mosquito species was introduced to each treated against the test mosquitoes in the laboratory trays and control trays (if necessary, dead and live larvae and in tubs outdoors are shown in Figs. I and 2, and pupae were collected from the treated trays and respectively. Pyriproxyfen at O.O2 and 0.05 ppm discarded before the introduction of a 2nd batch of rates monitored for 6 wk after treatment induced larvae) and routine daily posttreatment mortality almost lUOVo emergence inhibition of Ae. aegypti and survivorship observations were continued. In in the laboratory as well as in the tubs, whereas s- this manner, 6 batches (weekly) of mosquito larvae methoprene was less effective, reducing emergence of a species were introduced to the trays to deter- of this species 22.3-93.7Vo in the laboratory and mine residual activity of each formulation. Two lO.3-lOOVo in tubs, even at the higher rate of 0.05 hundred milligrams of mosquito larval food was ppm (Figs. la, 1b). The activity profile of s-meth- added to each tray at the time of introduction of oprene for the lst 2 weeks after treatment in the each new batch of mosquito larvae and on alternate laboratory and I wk after treatment in the tubs at days thereafter. the high rate of 0.05 ppm was similar to that of Outdoor evaluations: These evaluations were pyriproxyfen, but thereafter, pyriproxyfen showed conducted in 1-m-diameter polyethylene tubs, lined much higher levels of sustained residual activity with 2-mil-thick polyethylene sheets, containing against Ae. aegypti in both test systems. Itoh (1993) 100 liters of well water (Ali et al. 1994). The tubs reported that a synthetic slow-release formulation were maintained outdoors under a canopy (Sun of pyriproxyfen (0.O57o AI) exhibited prolonged canopy, Sunshield II, Powell & Powell Supply activity against larvae of Ae. aegypti even when the Company, Fuquay Varina, NC) to protect from rain treatments were diluted by using and replenishing and direct sunlight. The test procedures, treatment water in the treated jars. rates, and daily observations for the 2 IGR formu- The high rate of 0.05 ppm of s-methoprene pro- lations against each mosquito species in the tubs duced a maximum of 44.3Vo (in laboratory) and were the sarne as described above for the laboratory 32Vo (in tubs) emergence inhibition in Ae. albop- trays, except that I g of the larval food was added ictus at I wk after treatment (Figs. lb and 2b). In to the tubs at the introduction of each batch of 100 comparison, pyriproxyfen induced 52.7-lOOVo (at mosquito larvae and on alternate days thereafter. 0.02 ppm) and 93-l0OVo (at 0.05 ppm) emergence Each tub was kept covered with fine-mesh plastic inhibition in the laboratory, and sustained l007o screen to protect from air-borne debris, wild in- emergence inhibition in the tubs for 6 wk after sects, and any oviposition by wild mosquitoes. treatment at both treatment rates. Pyriproxyfen Water temperature in the tubs was recorded against Ae. albopictus was distinctly superior over throughout an evaluation. Four temperature-record- s-methoprene in terms of magnitude and duration ing Onset@computers (32 K Waterproof Stowaway of activity at equal concentrations of active ingre- TidBit, Onset Computer Corporation, Pocasset, dients of the 2 IGRs. These observations concur MA) were randomly submerged in 4 separate tubs. with those of the laboratory bioassay study of Ali Minimum and maximum water temperatures in the et al. (1995), which study showed 21.5 times higher tubs recorded daily ranged from 13.4 to 30.2.C dur- toxicity of pyriproxyfen against Ae. albopictus than JounNar-or ruB AlaBrrcANMoseurro CoNrnor-AssocrnrroN VoL. 18,No.3

s - Methoprene Pyriproxyfen 0.a2ppnffi 0.05ppmffi 0.02ppm[] 0.05ppm!

(a) Aedes aegypti 100

0 z o 100 E d) = z 0 ul (c) o z trl 100 (, E. LU 0 l.tJ Culexnigripalpus F fr roo ff tIl (L 0 o Anopheles quadri maculatus U) (e) lt 100 z 1ll =O Culex quinquef asci atus * 100

0 4 WEEKS POST.TREATMENI Fig. 1. Posttreatment mean 1 SD percent inhibition of adult emergence of laboratory-reared larvae exposed to s- meth;prene and pyriproxyfen in trays. The larvae of the lst 5 mosquito species were exposed at 0.02 and 0.05 ppm active ingredient (AI) of each insect gfowth regulator (IGR). *Larvae of Culer quinquefasciat s were exposed to 0.2 and 0.4 ppm AI of each IGR. Means of percent adult emergence inhibition followed by the same letter are not statistically significant (P > 0.05). of s-methoprene, when using the technical grade of xyfen was superior over s-methoprene, producing each IGR. higher levels of emergence inhibitions of Ae. tae- Residual activity of s-methoprene against Ae. niorhynchus at O.O2 ppm as well as at 0.05 ppm in taeniorhynchrr was generally of higher magnitude the laboratory and outdoors, specifically during the and duration in outdoor tubs than in the laboratory 3rd to 6th weeks after treatment. No significant dif- trays (Figs. lc and 2c). The higher rates of s-meth- ference (P > 0.05) was found in emergence inhi- oprene did not consistently produce significantly bition of Ae. taeniorhynchus between the low and higher levels of emergence inhibitions in the week- the high rates of pryriproxyfen in both test systems. ly observations, particularly in the tubs. Pyripro- The previous laboratory study of Schaefer et al. SEPTEMBER2002 INspcr Gnowrs REcularons ActNsr Flonroa MoseurroEs r99

s - Methoprene Pyriproxyfen [email protected] 0.02ppmf, 0.0sppml

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z o (b) Aedes albopictus F.l 6' = z UJ o z UJ (c) Aedes taeniorhvnchus (9 (f,r lll I

L! zF IJJ O (E (d) LU (L 6.1 U) l!. z l! Anoph el es quad ri m acul atu s

WEEKSPOST.TREATMENI Fig. 2. Posttreatment mean :t SD percent inhibition of adult emergence of laboratory-reared larvae of 5 mosquito species exposed to s-methoprene and pyriproxyfen in tubs outdoors at 0.02 and 0.05 ppm active ingredient (AI) of each insect growth regulator. Means of percent adult emergence inhibition of a mosquito species followed by the same letter are not statistically significant (P > 0.05).

(1988), which showed pyriproxyfen median lethal gripalpus from the tubs during the 6 wk of obser- concentration and 9O7o lethal concentration values vation. of 0.01 and 0.052 ppb, respectively, against larvae Against An. quadrimaculqtus, r-methoprene at of Ae. taeniorhynchus, is indicative of the excellent 0.02 and 0.05 ppm in the laboratory as well as in activity of this IGR against this mosquito species. the outdoor tubs caused notably lower levels of The activity of s-methoprene against Cx. nigri- emergence inhibition than did pyriproxyfen used at palpus lasted for 2-3 wk in the laboratory and for same concentrations, although these differences 3-4 wk in the tubs at both rates of treatment (Figs. were not statistically significant for the lst week ld and 2d). In compaf,ison, pyriproxyfen gave con- after treatment in the laboratory and up to the 3rd sistently superior results; even the lower rate of this week after treatment in the tubs (Figs. le and 2e). IGR completely suppressed emergence of Cx. ni- The former IGR in the laboratory resulted in 14- 200 JoURNAL or tgn Avr,ntclN Mosquno CoNrnor- AssocIATIoN Vor. 18,No.3

79.1Vo (at 0.02 ppm) and l9-78.87o (at 0.05 ppm) vestigations employing pyriproxyfen (O.5Vo G) emergence inhibition of An. quadrimaculatus dlur' against mosquitoes in the genera Aedes, Anopheles, ing the l-6 wk after treatment, whereas pyripro- and Culex at rates ranging from 0.01 to I ppm AI xyfen at O.02 ppm caused 91.3-lOOVo emergence in a variety of field situations (Kerdpibule 1989, suppression (lOUVo for the lst 5 wk), and complete Kamimura and Arakawa 1991, Okazawa et al. emergence suppression for 6 wk after treatment at 1991, Thongrungiat and Kanda 1991, Adames and the higher rate of 0.05 ppm. In the outdoor tubs, s- Rovira 1993, Kawada et al. 1994). Based on the methoprene was highty effective against An. quad' present results as well as those of other WHOPES- rimaculatus for only 2 wk after treatment, whereas sponsored pyriproxyfen studies and a thorough lit- pyriproxyfen at either rate of treatment induced a erature review of laboratory and fleld activity of complete inhibition of emergence of this mosquito pyriproxyfen against mosquitoes and aquatic non- for at least 5 wk after treatment. The superior ac- target organisms (Hirano et al. 1998, WHO 2O0l), tivity of S-31 183 (pyriproxyfen) over s-methoprene WHOPES recently recommended the use of pyri- against An. quadrimaculatus was reported previ- proxyfen for the control of some mosquito species ously by Estrada and Mulla (1986). at specified rates in certain habitats (WHO 2001). Culex quinquefasciatus was relatively more tol- Also recently, the Joint FAO/WHO Meeting on erant to ,r-methoprene compared to all other mos- Pesticide Residues has considered that, because of quito species tested. Even the high rate of 0.4 ppm extremely low toxicity of pyriproxyfen to mam- of this IGR resulted in 84Vo emergence inhibition mals, pyriproxyfen GR can be safely added to of Cx. quinquefasciatus at I wk after treatment, drinking water at a rate of 0.01 mg Al/liter, for with activity rapidly declining in subsequent weeks mosquito control (FAO 2O0l). of observation (Fig. 1). Pyriproxyfen at 0.1 ppm and 0.2 ppm (rates lower than s-methoprene) com- ACKNOWLEDGMENTS pletely inhibited adult emergence of Cx. quinque- fasciatus in the laboratory for at least 4-5 wk. Such Gratitude is expressed to WHOPES for provision activity of pyriproxyfen is compatible with reports of a research grant for the reported investigation. of Adames and Rovira (1993) and Chavasse et al. The mention of specific company names or prod- (1995) showing good field control of Cx. quinque- ucts does not in any way imply that they are rec- fasciatus with O.5Vogranular and lOVo emulsifiable ommended or endorsed by WHO in preference over concentrate formulations of pyriproxyfen used at others that are not mentioned. This is Florida Ag- rates of 0.025-0.1 pm AI of the lGR. ricultural Experiment Stations Journal Series R- s-Methoprene and pyriproxyfen also were tested 08388. for effectiveness and residual activity against lab- oratory-reared larvae of Ae. albopictus and Cx. ni- REFERENCES CITED gripalpus of the fleld strains in the laboratory trays only (data are not included here). The results Adames E, Rovira J. 1993. Evaluation of the juvenile (S-31183) showed a trend very similar to that observed for growth regulatorpyriproxyfen againstthree speciesof mosquitoesin Panama.J Am Mosq Control the larvae from the laboratory colonies of these Assoc 9'.452-453. species at the same treatment rates. Because meth- Ali A, Nayar JK, Xue RD. 1995.Comparative toxicity of oprene ((rs)-methoprene and s-methoprene) has selected larvicides and insect growth regulators to a been used for mosquito control in Florida for the Florida laboratory population of Aedesalbopictus. J Am past 2 decades, the similarities of susceptibility of Mosq Control Assoc | 1:72-76. laboratory colonies to the field strains indicated a Ali A, Xue RD, Lobinske R, CarandangN. 1994.Eval- lack of any tolerance to .r-methoprene developed by uation of granularcorncob formulations of Bacillus thu- field populations of Ae. albopictus and Cx. nigri- ringiensis serovar.israelensis against mosquito larvae Am Mosq Control palpus in the Vero Beach area of Florida. using a semi-field bioassaymethod. J Assoc lO:492-495. This study clearly demonstrated the superior ac- ChavasseDC, Lines JD, Ichimori K, Majara AR, Minjas tivity of pyriproxyfen over s-methoprene, on basis JN, Marijani J. 1995.Mosquito control in Dar es Sa- of equal concentrations of the active ingredient, laam. lI. Impact of expanded polystyrene beads and against a wide variety of mosquitoes in the labo- pyriproxyfen treatmentofbreeding sites on Culex qutn- ratory and in experimental tubs placed outdoors. In quefasciatusdensities. Med Vet Entomol9:147-154. a majority of cases, pyriproxyfen at the lower rate EstradaJG. Mulla MS. 1986.Evaluation of two new in- of 0.02 ppm induced complete inhibition of adult sect growth regulators against mosquitoesin the labo- emergence of the tested species for several weeks ratory.J Am Mosq Control Assoc2:57-60. FAO and Agricultural Organization of the United after treatment. These results suggested that com- [Food Nationsl. 2OOl.Pesticide residue infood-2OOI Repott plete inhibition of adult emergence of these mos- of the Joint Meeting of the FAO Panel of Experts on quitoes may be achieved with rates even lower than Pesticide Residues in Food and Environment and the the 0.02 ppm AI of pyriproxyfen. The results of WHO Core AssessmentGroup on Pesticide Residues. this study concerning the initial and residual activ- FAO Plant Production and Protection Paper. p. 167. ity of pyriproxyfen against mosquitoes are in com- Rome,Italy. plete agreement with those of several previous in- Hirano M, Hatakoshi M, Kawada H, Takimoto Y. 1998. SEPTEMBER2002 INsgcr GnowrH Rsculnrons AcntNsr FLonroe Moseurroes 201

Pyriproxyfen and other juvenile hormone analogues. tors that may influence larvicidal activity of Bacillus Rev Toxicol 2:357-394. thuringiensis serovar. israelensis against two Florida Itoh T. 1993. Control of DF/DFH vector, Aedes mosquito, mosquito species. J Am Mosq Control Assoc 15:32-42. with insecticides. Trop Med 35:259-267. Okazawa Tl Bakote'e B. Suzuki H. Kawada H. Kere N. Kamimura K, Arakawa R. 1991. Field evaluation of an 1991. Field evaluation of an insect growth regulator, insect growth regulator, pyriproxyfen, agalnst Culex pi- pyriproxyfen, against Anopheles punctulatus on North piens pallens and Culex tritaeniorhynchus. Jpn J Sanit Guadalcanal, Solomon Islands. J Am Mosq Control As- ZooI 42:249-254. soc 7:6O4-6O7. Schaefer CH, Miura Dupras EF Jr, Mulligan FS III, Kawada H, Kohama I Abe Y. 1994. Larvicidal activity I Wilder WH. 1988. Efficacy, nontarget effects, and of a water soluble granular formulation of the insect chemical persistence of S-31183, a promising mosquito growth regulator, pyriproxyfen, against Culex mosqui- (Diptera: Culicidae) control agent. J Econ Entomol 8l'. toes. Jpn J Environ Entomol Zool 6:68-77. 1648-1655. Kerdpibule V. 1989. A field test of 2-!-methyl-2(4-phe- Thongrungiat S, Kanda T, 1991. Efficacy ofpyriproxyfen noxy-phenoxy)ethoxyl pyridine against principal vec- for the control of Culex tritaeniorhynchrr^r at an open tors of malaria in a foot-hill area in Thailand. Jpn J rice field in Bang Len, Nakhon Pathom Province, Thai- Trop Med Hyg 17:-175-183. land. Trop Biomed 8:l l3-1 16. Mulla MS, Darwazeh HA, Norland RL. 1974. Insect WHO [World Health Organization]. 2001. Review of in- growth regulators: evaluation procedures and activity sect growth regulator Pyriproxyfen GR. In: Report of against mosquitoes. J Econ Entomol 67:329-332. the fourth WHOPES working group meeting 20O0 De- Nayar JK, Knight JW Ali A, Carlson DB, O'Bryan PD. cember 4-5: Geneva. Switzerland. Geneva: WHO/ 1998. Laboratory evaluation of biotic and abiotic fac- CDS, WHOPES42OOI.2. p 5O-67.