Acute and Sublethal Effects of (S)-Methoprene on Some Australian

Home , Culex annulirostris

1997 Journal of the American Mosquito Control Association, 13(2):153-155' Copyrighi @ lgg7 by the American Mosquito Control Association, Inc'

ACUTE AND SUBLETHAL EFFECTS OF (^'-METHOPRENE ON SOME AUSTRALIAN MOSQUITOES

SCOTT A. RITCHIE,T'' MICHAEL ASNICAR3 rNo BRIAN H' KAY'

7 species ABSTRACT. Laboratory bioassays were used to determine the efficacy of (S)-methoprene against to 6'54 of Australian mosquitoes. The gOVo lethal concentration (LC*) ranged from 0.17 ppb for Aedes vigilax ppb for Culex sitiins. The survival of adults exposed as larvae to 2 sublethal dosages of (S)-methoprene was co-purea to a control group. Little effect was noted for Cx. sitiens and Culex annulirostris. However, survival of male and female Ae. vigilax was significantly reduced and appeared to be dose related. Bloodfeeding success of female Ae. vigilctx was also significantly reduced.

INTRODUCTION iprasasna et al. 1996). Methoprene is also docu- mented to affect body size (wing length) and sex The insect growth regulator methoprene has been ratio (Robert and Olson 1989). We investigated the successfully used to control many species of mos- effects of sublethal amounts of (S)-methoprene on (for quitoes, with minimal nontarget toxicity a re- adult longevity in Ae. vigilax, Cx. annulirostis, and view see Ross et al. 1994a, 1994b). Methoprene Cx. sitiens, and on bloodfeeding success in Ae. vi- occurs in 2 forms, the (R) and (S) isomers, with gilax, the primary target for mosquito control pro- (.f)-methoprene having the greatest insecticidal ac- grams in coastal Australia. tivity. (,S)-methoprene is available in many formulations. Liquid formulations (ALTOSID@ Liquid MATERIALS AND METHODS Larvicide tA.L.L.l) provide control over a limited period, whereas sustained-release formulations Laboratory bioassays: Laboratory bioassays such as ALTOSID@ Pellets and XR Briquets can were used to determine the effectiveness of (S)- control mosquitoes for several months (Kramer et methoprene (A.L.L., 5Vo active ingredient) against al. 1993, Ross et al.1994a). 7 common Australian mosquitoes. Aedes aegypti, laboratory bioassays to determine We conducted Ae. notoscriptus, Ae. vigilax, An. farauti, Cx. an- the efficacy of A.L.L. against 7 common Australian nulirostris, and Cx. sitiens were from colonies mosquitoes. Aedes aegypti (L.) is a vector of den- maintained at the Queensland Institute of Medical gue in northern (Cleland et al. 1918); Queensland Research, whereas Ae. funereus eggs were obtained (Theobald), Aedes notoscriptus Aedes funereus from wild mosquitoes collected in Cairns, Queens- (Skuse), Aedes vigilax (Skuse), Culex annulirostris land. Twenty 3rd-instar larvae were exposed to dif- Skuse, and Culex sitiens Wiedemann are all known ferent concentrations of A.L.L. in 200 ml of deion- or potential vectors of Ross River virus (Russell ized water within a capped, 250 ml Schott glass 1994; Ritchie et al., in press), with Cx. annulirostris (Ae. funereus and Cx. sitiens, and Ae. vigilax larvae a vector of Murray Valley encephalitis (Mclean were reared in 16 and 33Eo seawater, respectively). 1953) and probably Japanese encephalitis (Ritchie Preliminary trials indicated that survival of Ae. fu- et al. 1997). Anopheles (Laveran) s./. is con- farauti nereus and An. farauti was improved using 100 ml sidered a major vector of malaria throughout Aus- of water; thus 100 ml of water was used in bioas- tralasia, including Queensland north of the 19th says with these species. A control using IOO ppb parallel (Lee et al. 1989). of the A.L.L. emulsion without (^f)-methoprene was Poor application of methoprene, coupled with included in each trial. All trials featured 3-4 dose loss of efficacy of sustained-release formulations, replicates. Larvae were fed a slurry offish food and can lead to adult emergence. Nonetheless, adults liver powder (An. farauti were fed on a powder diet from larvae exposed to sublethal concentrations of that spread across the water surface) and reared at methoprene can be adversely affected. Sawby et al. 28"C with a 14:10 light:dark cycle. (1992) found that sublethal doses of (S)-metho- The 7o emergence inhibition (VoEI) was calculat- prene decreased survival of adult Ae. aegypti. How- ed as: ever, adult survival was not significantly influenced in Anopheles dirus Peyton and Harrison, although VoEI: 7OO - bloodfeeding and oviposition were depressed (Sith- x [ (number of emerging adults/20)]. Because the Schott jar was capped, emerged adults included eclosed adults that were incapable of tQueensland Institute Research, of Medical 300 Her- flight. For each trial, VoEI was corrected for control ston Road, Brisbane 4006 Australia. 2 mortality using Abbott's formula (Abbott 1925). Present address: Tropical Public Health Unit, Queens- Probit analysis (Finney l97l) was used to deter- land Health, P O. Box 1103, Cairns, Queensland 4870 Australia. mine the respective median lethal concentration 3 Redcliffe City Council, P O. Box 66, Redcliffe, (LC.o) and 9OVo letl;;al concentration (LCro) of (,S)- Queensland 4020 Australia. methoprene using Probit 5 for Windows@ (P Gil-

153 t54 Jounrsar- oF THE AMERTcaN Mosqurro CoNTRoL AssoclA.rroN Vor-. 13, No. 2

Table I ' Lethal concentration of (s)-methoprener, in. parts billion (ppb), _per to kill 50 (LC.n) and 90zo (LC*) of the larvae of 7 Australian mosquitoes.r No. Dose range Minimum Mosquitospecies tested2 (ppb AI) LCro (95Vo Cl) LC* (95Vo Cl) lethal dose3 Aedes aegypti 420 0.1s6-10.0 0.397(0.318-0.496) 1.308(0.963-1.779) 2.50 Aedes funereus 430 0.016-2.5 0.072(o.051-o.103) 0.68s(0.s36-1.076) 2.5 Aedes notoscriptus 360 0.031-4.0 o.3s9 (0.2884.447) l .117 (0.824-1.513) 4.O Aedes vigilax 560 0.004-0.50 o.o22(o.or7-o.o28) 0.167(0. t0s-o.265) 0.50 Anopheles farauti 720 0.016-4.0 0.057 (0.o40--0.080) 0.986(0.587-1.657) 4.O Culex annulirostris 720 0.003-1.25 0.089 (0.07H.105) o.34r (0.2624.443) o.62s Culex sitiens 780 0.039-40.0 t.124 (0.924-1.367\ 6.544(4.944-8.664) 40.0 'As determined by probit analysis (Finney 1971). ' Excluding controls. r The dose (in ppb) inclusive and beyond which all tesr lilvae died.

Iespe, P M. B. 10, Rydalmere, New South Wales ppb. Vector control programs have had great success 2116 Australia). controlling this mosquito with (S)-methoprene in The effect of sublethal dosages of (S)-metho- southeast Queensland, with treatments of ALTO- prene: The impact of sublethal dosages of (,S)- SAND@ (Biorational Resources, Scarborough, methoprene on the survival of adult Ae. vigilax, Cx. Queensland, Australia) (O.4Vo AI) at 2-3 kglha pro- annulirostris, and Cx. sitiens was determined by viding control of Ae. vigilax for 7-10 days (S. A. exposing 300-2,000 3rd-instar larvae to A.L.L. at Ritchie, D. McGinn, and N. Woods, unpublished concentrations of 0 (control) and the respective data). However, Cx. sitiens is not as susceptible to LCrn and LC* determined from the laboratory bio- (S)-methoprene, with an LC* 39 times greater than assays. Pupae were placed in 60-ml beakers placed thatfor Ae. vigilax. Field studies with ALIOSANDo in a 5-liter cardboard bucket (Kentucky Fried and ALTOSID@ Pellets confirm this (S. A. Ritchie, Chicken Family Size@) covered with screen and a unpublished data) and suggest that treatment failures moist paper towel to maintain high humidity. The are likely to occur from March to May when Cx. effective VoEI was determined as described earlier, sitiens is most abundant in the salt marsh. but because the emergence jar was open within the The effect of sublethal doses of (S)-methoprene: bucket, dead adults on the water surface were not Sublethal doses of (S)-methoprene significantly (P included in the emerged adults total. The beakers < 0.01) affected the survival of adult Ae. vigilax were removed from the bucket and emerged adults (Table 2). Mortality of Ae. vigilax males and fe- were maintained at 28'C with a l4:lo light: dark males was directly related to dosage. Generally, cycle and given access to lOVo sucrose and sliced sublethal doses of (S)-methoprene did not signifi- apple. Dead adults were removed and tallied daily. cantly affect the survival of the Culex tested except For each sex of all species tested, survival functions for male Cx. annulirosrrls (Table 2). Interestingly, for the 3 treatments were compared by the log-rank sublethal effects seemed to be related to suscepti- test (Armitage and Berry 1987) using PROC LIFE- bility in the bioassay. Larval Ae. vigilax were af- TEST in SAS (SAS Institute Australia Pty. Ltd., fected at relatively lower doses, with surviving Private Bag No. 52, Lanecove, New South Wales adults suffering reduced survival, whereas larval 2066 Australia; Release 6.13 licensed to the Trop- Cx. sitiens had relatively low susceptibility with no ical Public Health Unit;. noticeable effects on adult survival. For the bloodfeeding experiment, 15 adult fe- In Ae. vigilax, (S)-methoprene significantly af- male Ae. vigilax from larvae exposed to 0.12 ppb fected bloodfeeding success. Adult females surviv- (S)-methoprene (ca. LCru) or a control group were ing O.l2 ppb of (,9)-methoprene, which prevented placed in a 400-ml glass beaker (covered with cloth eclosion of 89.2Vo of larvae, bloodfed at a signifi- gauze) ca. 4 days after emergence. Mosquitoes cantly lower (t : 5.34, df : 16, P < 0.001) rate were starved for 12 h, then, at dusk. offered a hu- (mean + SD : 23 + l1%o) than the control group man blood meal for 6 min. The experiment was 65 + l6Vo). replicated 9 times. The percentage (arcsine trans- These data, particularly at the higher doses, sug- formed) of bloodfed mosquitoes for each group was gest that Ae. vigilax exposed to nearly lethal doses compared by a Student's t-test. of (.)-methoprene may be a minimal pest and disease threat. Bloodfeeding success may be reduced, and the survival of females may be insufficient to RESULTS AND DISCUSSION develop viremia (ca. 4 days at 28'C, for Ross River Bioassays of (S)-methoprene: Most of the mos- virus [Kay 1982]). However, careful field studies of quitoes were very susceptible to (S)-methoprene, this phenomenon :re needed before the findings of with LC*s generally < 2 ppb (Table l). Aedes vi- the study reported here are applicable to operational gilax was especially susceptible, with an LCeo < O.2 situations. 155 Junr 1997 Iupecr or MBtuopnnrvroN AUSTRALIANMosQunoss

of (S)-methoprene Table 2. Mean (+ SE) survival in days of adult mosquitoes exposed to different doses (ALTOSID@ Liquid Larvicide).'

Species Dose (ppb AI) Aedes vigilax o.l0 o.12 VoEl 5.O7o 4O.2Vo 89.2Vo + Male 74, 12.3+ O.5a,23 480,6.1+ O.lb,20 71,2.3 O.2c,7 + Female 82, l7.3 + O.75a,31 687,9.O+ 0.13b,2l 71,5.0 0.2c,l0 Dose (ppb AI)

Culex annulirostris (r.) 1.2 '7l.2%o VoEl 13.4Vo 43.7Vo + Male 71,2O.4+ 1.4a,47 92,26.8+ 1.2b,51 50,17.6 1.1c,28 + Female 163,28.6+ 0.8a,49 53,28.O + 1.8a,54 37,27.'7 1.7a,42 Dose (ppb AI)

Culex sitiens lo VaEI 1.67o 46.O7o 85.LVa Male 314,223 + 0.5a,5O 115.21.3+ O.7a,49 17,19.5 + 2.4a,41 Female 351.27.7 + O.6a,62 l2O. 25.5 + I .0a.55 3l, 23.4 + 2.5a,45

I VoEl = 7o emergence inhibition. Data for male and female are given as n, mean a SE days survived, maximum days survived. Mems followed by a different letter are significantly different (P < 0.05) by the log-rank test (Armitage and Bery 1987).

ACKNOWLEDGMENTS prene pellets in a saltwater marsh. J. Am. Mosq. Con- trol Assoc. 9:127-130. We thank Kay Marshall and Tonya Watson of the Lee D. J., M. M. Hicks. M. L. Debenham, M. Griffiths, Queensland Institute of Medical Research and An- E. N. Marks, J. H. Bryan and R. C. Russell. 1989. The drew van den Hurk of the Tropical Public Health Culicidae of the Australasian Region. Entomology Unit for providing some of the mosquitoes, and Monograph No. 2, Volume 5. Australian Government Chris Stafford of Biorational Resources Pty. Ltd. Press Service. Canberra. Australia. for supplying (S)-methoprene. We are especially Mclean, D. M. 1953. Transmission of Murray Valley en- grateful to Paul Hughes for his support and sug- cephalitis virus by mosquitoes. Aust. J. Exp. Biol. Med. gestions and to Wei Luo of the Tropical Public Sci.3l:481-49O. Health Unit and Prof. Geoffrey Berry of The Uni- Ritchie, S. A., D. Phillips, A. Broom, J. Mackenzie, M. A. van den Hurk. 1997. Isolation of versity of Sydney for statistical advice. Finally, we Poidinger, and Japanese encephalitis from Culex annulirostris in Aus- thank two anonymous reviewers for their insightful tralia. Am. J. Trop. Med. Hyg. 56:80-84. comments. This work was funded by grants from Robert, L. L. and J. K. Olson. 1989. Effects of sublethal Queensland Arbovirus Advisory Committee, the dosages of insecticides on Culex quinquefasciatus. l. Local Authorities Research Committee. and Sandoz Am. Mosq. Control Assoc. 5:239-246. Australia Pty. Ltd. Ross, D. H., D. Judy, B. Jacobson and R. Howell. 1994a. Methoprene concentrations in freshwater microcosms REFERENCES CITED treated with sustained-release altosid@ formulations. J. Am. Mosq. Control Assoc. 10:202-21O. Abbott, W. S. 1925. A method of computing the effec- Ross, D. H., P Cohle, P. R. Blase, J. B. Bussard and K. tiveness of an insecticide. J. Econ. Entomol. 18:265- Neufield. 1994b. Effects of the insect growth regulator 267. (S)-methoprene on the early life stages of the fathead Armitage, P and G. Berry. 1987. Statisticalmethods in minnow Pimephales promelas in a flow-through labo- medical research,2nd ed. Blackwell Scientific Publi- ratory setting. J. Am. Mosq. Control Assoc. 10:211- cations, Kingdom. Oxford, United 22r. Cleland,J. B., B. Bradleyand W. McDonald. 1918.Den- R. 1994. Ross River virus: disease trends and gue fever in Australia. Its history and clinical course, Russell, C. vector ecology in Australia. Bull. Soc. Vector Ecol. 19: its experimental transmission by Stegomyiafasciata, and the results of inoculation and other experiments.J. 73-8l Hyg.Camb. l6:317-418. Sawby, R., M. J. Klowden and R. D. Sjogren. 1992. Sub- Finney, D. J. 1971. Probit analysis, 3rd ed. Cambridge lethal effects of larval methoprene exposure on adult University Press,Cambridge, United Kingdom. mosquito longevity. J. Am. Mosq. Control Assoc. 8: Kay, B. H. 1982. Three modesof transmissionof Ross 290-292. River virus by Aedesvigilax (Skuse).Aust. J. Exp. Biol. Sithiprasasna, R., E. Luepromchai and K. J. Linthicum. Med. Sci. 6O:339-344. 1996. Effects of sublethal dosages of methoprene on Krame! V. L., E. R. Carper and C. Beesley. 1993. Con- Anopheles d/ras species A and B. J. Am. Mosq. Control trol of Aedes dorsalis with sustained release metho- Assoc. l2:483-486.