Potential for Resistance to Pyriproxyfen: a Promising

SEPTEMBER 199I RnsrsreNcn PornNtrer, To PyRrpRoxyFEN

POTENTIAL FOR RESISTANCETO PYRIPROXYFEN:A PROMISING NEW MOSQUITO LARVICIDE

C. H. SCHAEFER eNo F. S. MULLIGAN III1

Mosquito Control Researchlnboratory, IJniuersity of California, 9240 S. Riuerbend,Auenue, Parli.er, CA 93648

ABSTRACT. An organophosphorus-resistantstrain of Culcx quin4u.efasciaturwas pressured with pyriproxyfen for 1? genbrations.Egg viability began declining in the F? generation and becamelower as the ielection procesi continued; by the F17generation egg viabilif was too low to proceed further. Susceptibility tests on larvae of the Fs, Fro, Frr, and Frz generationsshowed no indication of increased tolerance to pyriproxyfen.

INTRODUCTION of chlorpyrifos and related compounds due to insecticide resistance(Stewart 1975). Pyriproxyfen, 2-[1-methyl-2-(4-phenoxy- Another consideration is the potential for phenoxy)ethoxyl pyridine, is also known as S- cross-resistanceof strains which have already 31183and by the trademark names Nylar@and been selectedfor insecticide resistance.For ex- [email protected] compound acts as a juvenile ample, when a new benzamide larvicide which hormone mimic; it doesnot producedirect larval had a high degreeof efficacy against mosquitoes toxicity but disrupts the normal processof insect was used to pressure an organophosphorus-re- development, which results in pupal mortality sistant (OP-R) strain of Cx. quirquefasciatu.s, or in the production of abnormal adults. Pyri- cross-resistancebecame apparent after only 4 proxyfen is highly active against a variety of generationsof selection (Schaeferet al. 1981). insects of public health importance including Information on how quickly insecticide-resist- cockroaches (Chow and Yang 1990), fleas ant strains might develop tolerance to pyri- (Palma and Meola 1990),the tsetsefly (Langley proxyfen was sought.Insecticide pressure exper- et al. 1990) and mosquitoes(Estrada and Mulla iments using pyriproxyfen were initiated in 1987 1986,Mulla et al. 1986,Schaefer et al. 1988). and were continued through 1990 and are sum- Mulligan and Schaefer (1990) showed that marized in this report. pyriproxyfen could be usedto treat animal waste lagoons for Culex quinEtefasciatlrzs Say larvae and that single treatments provided up to 2 MATERIALS AND METHODS months' control. The active ingredient readily Insecticide:A,technical standard of pyriproxy- leaves the water and adsorbs onto organic mat- fen (99.6%) was provided by the McGlaughlin, ter, where it then decaysat an exponential rate Gormley, King Company, Minneapolis, MN. while also providing biological activity (Schaefer Mosquitocolnnies: An OP-R stuainof Cx. quin' et al. 1991). Such highly polluted habitats are a qucfoscintw was obtained from undergxound well known sourcefor the breeding of Cx. quin- pipelines in Fresno, CA, in 1985 and colonized; quefasciatw and closely related Culer speciesall they were occasionallypressured with fenthion. over the world. The very large populations fre- A (OP-S) strain of the samespecies, quently encountered in such habitats often re- susceptible been maintained at this location for quire regular insecticide applications. Large which has 20 years, was used for initial comparisonsof populations and high selection pressure over ca. relatively long time periods lead to insecticide susceptibility. tesfs.'For larval sus- resistance, especially when only one agent, or Insectici.desusceptibility tests to pyriproxyfen, 25 4th-instar closely related chemical control agents, are uti- ceptibility placed in 250 ml tap water in Pyrex@ Iized. For example, Schaeferand Dupras (1970) larvae were (80 x 100 mm). A series of 10 showed that when sewagelagoons were treated storage dishes (range 0.001-0.000002ppm) was with chlorpyriphos, the active ingredient ad- concentrations 10 pl from acetone solu- sorbed onto organic debris and the biological obtained by applying tions to the water. Each concentration was made activity against Cx. quinErcfasciatu.s lawae for a test. Three separatetests were lasted for 4-8 weeks. However, repeated treat- in duplicate and the lot ments ofthese sourcesled to loss ofeffectiveness conducted,each on a different date, were combined in a single probit analysis. Test containers were held at 27"C at a photoperiod of 14:10(L:D). Mortality counts were made after I Current address: Consolidated Mosquito Abate- adult emergence was complete; the data were ment District. P.O. Box 278. Selma. CA 93662. adjustedfor control mortality (Abbott 1925)and 410 JounNer,oF THEAnpnrclx Mosqurro Coxrnor,Assocreuor.r VoL. 7, No. 3

analyzed by probit analysis (SAS Institute Table2. A summaryof larvalnumbers and mortality 1985). duringpressuring" with pyriproxyfen. prior pressuring Just to initiation of of the No. No. Mortality OP-R strain with pyriproxyfen, the susceptibil- Generation exposed survivors (%) ity to malathion, parathion, fenthion and chlor- Parent 1,762 pyrifos was determined, as previously described 619 47 (Wilder Fr 1,996 824 59 and Schaefer 1972').For comparison, F2 2,0t3 862 57 the same compounds were also run against a F3 953 2t2 78 susceptiblestrain. F4 t 1e, 530 81 Pressure techni4uz: Pressuring of the OP-R I'- r,226 495 60 strain of Cx. quinqtrcfascintuswas initiated in Fe 1,950 622 68 September 1987. Mid- to late 4th-instar larvae Fz 2,714 693 74 were placed in a glass aquarium containing 4 Fs 2,198 523 ,o liters of water treated with 0.00001ppm pyri- Fs 492 55 88 Fto 85C proxyfen. Pupae were removedas agecohorts at 193 77 Frt 889 252 72 various time intervals and reared as separate Fu 384 72 81 groups.For those groups in which the combined Frt 600 156 74 final mortality was 507oor greater,the surviving Ftn 483 t62 66 adults were added to the next generation adult Fts 750 84 cage. Fru 183 80 56 Becauseof reducedegg production and viabil- uPressure dose: parent through F10generations, ity (see Discussion), the method was changed 0.0000fppmi Frr throughFre generations, 0.00002 to beginning in the F1 generation.Thereafter, Iots 0.00007ppm. of 50 late, 4th-instar larvae were placed in 250 ml water in Pyrex storage dishes and treated with pyriproxyfen at 0.00002, 0.00004 and Results pressurirry multiplp gencratinns: 0.00007ppm. Adults successfullyemerging from from Table 2 summarizesthe numbers of larvae ex- treatments where mortality exceeded50Vo wete posed addedto the cageof the next generation. and the resulting mortality for each generation. Beginning in the F7 generation, egg production and viability became reduced and RESULTS AND DISCUSSION adults from unpressured larvae were returned back to the cage of the same generation to Susceptibility of laboratory strains o/ Culex quinquefasciatusto pyriproxyfen.'A comparison maintain the colony. As sufficient numbers of of the susceptibility of OP-S and OP-R labora- viable eggswere obtained, the pressuresequence tory strains to pyriproxyfen and 4 organophos- was continued. The problem of obtaining viable phorus larvicides is summarizedin Table 1. The eggs became worse, and by the F11generation OP-R strain was most tolerant to fenthion as the method had to be changed (as noted under would be expectedsince this compoundhad been Methods) as too few larvae could be obtained used to maintain pressureto this colony. There for pressuring at a given time. However, in the was no apparent difference in susceptibility to F17generation, egg viability becametoo low to pyriproxyfen based on the LCso",but there was continue. Enough larvae were obtained to run a a possibility of a small increase in tolerance of single susceptibility test, but attempts to obtain the OP-R strain basedon the LCes". sufficient larvae for additional tests failed.

Table 1. Susceptibility of larvae of Cubx quirqwfasciatus laboratory strains prior to selectionwith pyriproxyfen (in ppm). Strain Resistance ratio OP-S OP-R

Insecticide LCro LCru LCoo LCru LCuo' LCruo Chlorpyrifos 0.0019 0.oo27 0.0070 0.030 3.7 11.1 Fenthion 0.0033 0.005? 0.021 0.097 6.4 17.0 Malathion 0.052 0.089 0.14 0.43 2.6 4.8 Parathion 0.0035 0.0058 0.0087 0.030 2.5 5.2 Pyriproxyfen 0.000018 0.00016 0.000022 0.00042 r.2 2.6 'LCso OP-R/LC5oOP-S. oLCes OP-R/LCg5 OP-S. SEPTEMBER1991 RssrsreNcn Porenrlu ro PyRtpRoxyt'EN

As pressuring with pyriproxyfen continued, Estrada,J. G. and M. S. Mulla. 1986.Evaluation of the survivors becameincreasingly homogeneous, two new insect growth regulators against mosqui- as is apparent from the slope values (Table 3). toes. J. Am. Mosq. Control Assoc.2:57-60. This processresulted in the selectionfor reduced Langley,P. A., T. Felton, K. Stafford and H. Oouchi. 1990. pyriproxyfen, juvenile hor- eggviability, as described Formulation of a above,but there were mone mimic, for tsetsecontrol. Med. Vet. Entomol. no indications of increased tolerance to pyri- 4:127-I33. proxyfen. Mulla, M. S., H. A. Darwazeh,B. Kennedyand D. M. Interpretation of results:This study is encour- Dawson. 1986. Evaluation of new insect growth aging with respectto the responseof the genetic regulatorsagainst mosquitoeswith notes on nontar- pool of one OP-R strain. However, there is no get organisms.J. Am. Mosq. Control Assoc.2:314- question that mosquitoeshave the potential for 320. development of resistance to pyriproxyfen, or Mulligan, F. S. III and C. H. Schaefer.1990. Efficacy juvenile any other new control agent, if large numbers of a hormone mimic, pyriproxyfen (S- for wastewater are exposed to high selection pressure 31183), mosquito control in dairy over lagoons.J. Am. Mosq. Control Assoc.6:89-92. enough time. The only practical method for Palma,K. G. and R. W. Meola.1990. Field evaluation prolonging the life expectancy of a new, prom- of Nylar for control of cat fleas in home yards. J. ising chemical agent is for judicious use. In Med. Entomol. 27:1045-1049. habitats having continual breeding of large SAS Institute. 1985.SAS user's guide: statistics, ver- numbers of mosquitoes,we feel that it is prudent sion 5. SAS Institute, Cary, NC. that pyriproxyfen not be used as a sole means Schaefer,C. H. and E. F. Dupras, Jr. 1970.Factors of control but rather that its use be alternated affecting the stability of Dursban in polluted water. with applications of control agent(s) having a J. Econ.Entomol. 63:701-705. Schaefer.C. H.. T. Miura and W. H. Wilder. 1981. different mode(s) of action. Biological activity of two new substituted benzam- ides against mosquitoes and nontarget organisms. ACKNOWLEDGMENTS J. Econ.Entomol. 74:658-661. Schaefer,C. H., T. Miura, E. F. Dupras, Jr., F. S. The cooperation of the Consolidated Mos- Mulligan III and W. H. Wilder. 1988. Effrcacy, quito Abatement and the Delta Vector Control nontarget effects, and chemical persistence of S- Districts is gratefully acknowledged.This study 31183:a promisingmosquito control agent.J. Econ. Entomol.81:1648-1655. was supported in part from USDA Cooperative Schaefer,C. H., E. F. Dupras, Jr. and F. S. Mulligan AgreementNo. 82-CRSR-2-1010. III. 1991.Studies on the environmentalpersistence of S-31183(pyriproxyfen): adsorption onto organic REFERENCES CITED matter and potential for leachingthrough soil. Eco- tox. Environ. Safety 1991.2l.,207-274. Abbott, W. S. 1925.A method for computingthe Stewart, J. P. 1975. Culex pipiens quinquefasciatus: effectiveness of an ingecticide. J. Econ. Entomol. resistanceto chorpyrifos and other O.P. compounds. 78:265-267. Proc. Pap. Calif. Mosq.Control Assoc.43:37-40. Chow, Y. S. and H. T. Yang. 1990. The application of Wilder, W. H. and C. H. Schaefer. 1972. Factors sex pheromones and juvenile hormone analogs on affecting the insecticide susceptibility of Aedes ni- cockroaches (In Chinese). Kaohsiung J. Med. Sci. gromaculis larvae in laboratorv tests. Mosq. News 6:389-401. 32:390-398.

Table 3. Susceptibility of the OP-R sttain Culex quinquefasciatus pressuring .of before and after with pynproxyten (rn ppm).

Generation LCuo LCnu Slope Date tested Parent 0.000022 0.00042 r.29 Jun. 1987 F5 0.000015 0.000099 1.98 Mar. 1988 Fru 0.000032 0.00031 1.68 Apr. 1989 Frt 0.000015 0.000049 3.18 Mar. 1990 Frr" 0.000018 0.000055 O.DD Sep. 1990 'Single test; all othersin triplicate.