Susceptibility to Organophosphate

JouRuer,oF THEAnrpnrclli Moseurro CoNrnor,Assocreuolr VoL.6,No.2

SUSCEPTIBILITYTO ORGANOPHOSPHATEINSECTICIDES IN LARVAL AEDES ALBOPICTUS

DAWN M. WESSON

Department of Biological Sciences, Uniuersity of Notre Dame, Notre Dame, IN 46556

ABSTRACT. Larvae of 26 strains of Aedes albopictus were tested for susceptibility to 5 organophosphate insecticides. The recently collected geographic strains included 13 from the USA, 5 from Brazil, 3 from Southeast Asia and 5 from Japan. The greatest amount of variability was seen among U.S. populations, ranging from quite susceptible to potentially resistant. Brazilian samples were generally quite susceptible to all insecticides tested. Three Japanese strains showed tolerance or low level resistance to fenitrothion (2x, 3x and 8x), as did 2 U.S. strains (3x and 5x). Two U.S. strains showed tolerance to malathion (3x and 4x), while one strain from Chicago, Illinois, showed low level resistance (22X). Selection for resistance to malathion in a laboratory strain composed of multiple U.S. geographic strains resulted in a resistance ratio of 21x after 6 senerations of selection.

INTRODUCTION on the organophosphatesusceptibility of Brazil- ian populations. Continued expansion geographic ofthe range This study examined the susceptibility of a of AedesaLbopictus (Skuse) in the United States variety of geographicstrains of Ae. albopictusto (Moore al. et 1988)and recentfindings concern- 5 commonly used organophosphateinsecticides potential (Grim- ing the vector of the mosquito to provide baseline insecticide susceptibility in- stadet al. 1989,Mitchell et al. 1987)have led to formation, and to allow for comparison among public an increasenot only in awarenessof the various populations. In addition, laboratory se- mosquito, but also in attempts to control it. In lection of Ae. albopictus lawae for insecticide southern regions of the country, this mosquito resistanceto malathion was conducted. is currently a significant pest speciestying up relatively large amounts of money and laborl (Peacocket al. 1988).In the Midwest, popula- MATERIALS AND METHODS tions are generally smaller and control efforts vary greatly (Wessonet al. 1989),depending on Mosquitostrains: Mosquito strains had been Iocal vector control agencies and resources. field-collectedand colonizedwithin the past 2 Some areas have attempted no control while years before testing (Table 1). Exceptions were othershave achieved local eradication.2 2 long-establishedlaboratory strains, OAHU of Thosepopulations of Ae. albopictuswhich are Ae. aLbopictu.sand ROCK of Ae. aegypti (Linn). being exposed to control efforts are prone to The latter strains servedas standard susceptible developinsecticide resistance. Organophosphate strains for comparison. After initial tests, the insecticidesare commonly used in mosquitocon- SABAH strain of Ae. albopictus became the trol, either alone or in combinationwith other standard becauseit was more susceptiblethan types of insecticides;in the U.S. malathion, te- OAHU to all insecticidestested. mephos and chlorpyrifos are most often used, Larual bioassay:Log dosage-probitmortality either as adulticides or larvicides. tests were performed with chlorpyrifos, fenitro- Information on Ae. aLbopicfi.mlarval suscepti- thion, fenthion, malathion and temephos.AIco- bility to organophosphateinsecticides is lacking holic dilutions of analltical or technical grade for most areasin the U.S. where the mosquito insecticides were used. Test protocol followed occurs, although exceptions exist3 (Khoo et al. that outlined by the World Health Organization 1988,Robert and Olson 1989,Cilek et al. 1989). (1981).Special attention was given to produc- In addition, there is little information on organ- tion of uniform, uncrowdedlarvae (fed on a Iiver ophosphate susceptibility from Japanese and powder suspensionof 4 e/liter). Lots of 20-25 Northern Asian populations of Ae. albopictus fourth instar larvae were tested for 24 hours in (Hawley 1988).Similarly, there are no reports 250 ml of water with 1 ml of the appropriate insecticideconcentration. Tests were conducted in 16-oz (a00-ml) polyethylene-coatedpaper 1 cups (Nestyle);they consistedof at least 2 rep- Monthly report of the New Orleans Mosquito Con- licates per concentration and 4 concentrations trol Board, December, 1987, cover story. per insecticide 2 Berry, R. L. 1989. Reported at the 24th Annual series,excluding untreated con- Meeting of the Ohio Mosquito Control Association- trols. Most tests were replicated at least 4 times. Mansfield, OH. Resulting survival data were subjectedto probit 3 Annual report of the New Orleans Mosquito Con- analysis(Finney 1971),using a BASIC micro- trol Board, 1986, p. 20-24. computerprogram (Raymond1985). JuNn 1990 ORcANopHospuerp SuscrprrBrlrry IN AE. ALBozICTUS 259

Table 1. Geographicstrains ofAedes albopictustested for organophosphatesusceptibility. Strain-Origin (yearcollected) Strain-Origin (yearcollected)

USA stratns Brazilian stratre 1. ALSACE-New Alsace,IN, USA (1987) 1. ANCH-Anchieta, ESS,Brazil (1988) 2. DoM-Chicago, IL, USA (1987) 2. ANCH 2-Anchieta, ESS, Brazil (1988) 3. DUNN-Evansville, IN, USA (1987) 3. CARl-Cariaciaca, ESS, Brazil (1986) 4. ESL-East St. Louis, IL, USA (1988) 4. PAULO-Sao Paulo,SPS, Brazil (1988) 5. FAUST-Findlay, OH, USA (1987) 5. TEREZA 2-St. Tereza,ESS (1988) 6. HCMCD-Harris Co.,TX, USA (1986) Southeust A sian strairx 7. HOUSTON-Houston, TX, USA (1986) 1. SERIAN-Sarawak, E. Malaysia(1987) 8. INDY-Indianapolis, IN, USA (1986) 2. SABAH-Sabah, E. Malaysia(1986) 9. JAX-2-Jacksonville, FL, USA (1987) 10. LEX-Lexington, KY, USA (1987) (Hauaiian) 11. NO86-New Orleans,LA, USA (1986) 3. OAHU-Honolulu, HI, USA (1971) 12. Hill, OH, USA (1987) OAK-Oak Japanese sl rairc 13. OAK-2-Oak Hill, OH, USA (1988) 1. KABE-Kabeshima, Japan (1988) 2. KUBO-Kuboizumi, Japan (1988) (1988) ROCK-Rockefeller Institute (prior to 1959) 3. NAGA-Nagasaki, Japan (1988) Aedesaegypti 4. OKIN-Okinawa, Japan 5. SEBURI-Seburi, Japan (1988)

Baseline susceptibility: Using the SABAH Each geographiccomposite was then tested and strain (2-SE ASIA, Figs. 1-5), the following selectedwith all 5 insecticides. concentrationswere established as baseline sus- ceptibilities for Ae. albopictus:0.01 mglliter- RESULTS chlorpyrifos,0.02 mg/liter-fenthion, 0.20 mg/ liter-malathion, 0.02 mg/liter-temephos, and Insecticidesusceptibility: Figures 1-5 compare 0.02 mg/liter-fenitrothion. Doubling the base- LCe5 values for all geographic strains. The line concentration for each insecticide gives a ROCK strain of Ae. aegypti was highly suscep- concentration diagnostic for potential resist- tible to all 5 insecticides. ance, i.e., individuals surviving this insecticide Temephos:(Fig. 1.) Three U.S. strains (nos. dose may be considered resistant. The World 2, 5 and 13) and a Japanesestrain (no. 1) had Health Organization (1980) has proposedsimi- LCe5values at or above the diagnostic concen- lar diagnostic standards for Ae. aegypti.aThe tration of 0.04 mg/liter. The resistanceratio of greater of these 2 diagnostic values, either for the Oak Hill strain (no. 13)was 3x. All Brazilian Ae. ahopi.ctu.sor for Ae. acgypti, is listed as the and Southeast Asian strains had LCgs values diagnosticconcentration. This givesa more con- below the diagnostic concentration. servativeestimate of resistancepotential in Ae. Chlorpyrifos: (Fig. 2.) The LCgs values ob- albopictus. tained with chlorpyrifosfor 3 U.S. strains (nos. Resistanceselection: Georghiou et al. (1987) 2, 4 and 13), were equal to or just above the were successfulin selectingfor resistancein Ae. diagnosticconcentration of 0.02mg/liter. A Bra- aegpti after first combining a number of geo- zilian strain (no. 2) and a SoutheastAsian strain graphic strains to form a "synthetic" laboratory (no. 1) also displayed tolerance to this insecti- colony. That technique has also been used in cide. this study. Fenitrothion: (Fig. 3.) While many of the Geographicstrains of Ae. albopictuswere com- strains were quite susceptibleto fenitrothion, 3 bined from the 4 major regions shown in Table had LCgsvaluesin excessof the diagnostic con- 1. Fifty males and 50 females from each strain centration of 0.06 mg/liter. The 1988 Oak Hill were usedas stock material: 18 U.S., 4 Brazilian, strain from the U.S. (no. 13) had a resistance 15 SoutheastAsian and 9 Japanesestrains were ratio of 5X, while the Japanese strains from used to make a composite strain from the U.S., Kabeshima(no. 1) and Kuboizumi (no. 2) had Brazil, SoutheastAsia and Japan, respectively. resistanceratios of 3x and 8x, respectively. Malathion: (Fig. a.) The majority of strains tested were susceptibleto malathion. Three ex- aDiagnostic concentrations for Ae. aegypti:0.0I mg/ ceptionswere the U.S. strainsfrom Chicago(no. liter-chlorpyrifos, 0.05 mglliter-fenthion, 1.00 mgl 2) and Houston (nos. 6 and 7). The 2 strains liter-malathion, 0.02 mg/liter-temephos, and 0.06 from the Houston area had LCss values just mg/iter-fenitrothion. abovethe diagnostic dose (1.00 mg/liter), while Jounua.r,oF THEAunnrcnN Mosqurro Coxrnol AssocrlrroN Vor,.6, No. 2

LC 95 (95% CONFTDENCELIMIT) 0.1

c PI = o.os (, " 0.06 z o k c 0,04 F 2 ,rl (J 2 ^^^ 6 u.uz o

6 7 I 9 t0ll12l3 234 t23 l2345ROCK

USA BRAZIL SE ASIA JAPAN Fig. 1. Temephossusceptibility in 26 strains of Aedesalbopictus larvae. Bar height denotesLCgs value; point denotes95% confidencelimit. Horizontal line is at the diagnosticconcentration. Strain numbers correspondto numbersin Table 1. All strains were tested in 4 replicatetrials exceptSE ASIA nos. 2 and 3, which were replicated6 and 8 times,respectively.

LC 95 (95% CONFTDENCELIMrT) 0.1 lrl tr j 0.0I o

z 0.06

gt H z 0.04 H z 0.02 o

0 1234 5678910lll2 l3 1234 5 123 l23,t5ROCK

USA BRAZIL SE ASIA JAPAN Fig. 2. Chlorpyrifos susceptibility in 26 strains of Aedes albopictus larvae. Bar height denotes LCss value; point denotes 95% confidence limit. Horizontal line is at the diagnostic concentration. Strain numbers correspond to numbers in Table 1. All strains were tested in 4 replicate trials except USA no. 13 and SE ASIA nos. 2 and 3; these were replicated 5, 6 and 8 times, respectively. the Chicagostrain more than tripled that value in the first generation and increased to 50Vo, with a resistanceratio of 22x at the LCgs,level. 70%,70%,75% and 80% after 6 generations. While not obvious from the graph, the Chicago Selection in the U.S. composite strain resulted population was highly heterogeneous,resulting in a resistanceratio of 21x after 6 generations in a very shallowLd-pm Iine slope(1.32). of selection(Fig. 6). As comparedto the SABAH Fenthion: (Fig. 5.) With the exceptionof one strain, the U.S. compositeresistance ratio was U.S. strain (no. 13),all strains were susceptible 27x at the LCgslevel. to fenthion. The LCgsvalue for the 1988 Oak Hill strain equaledthe diagnostic concentration (0.05mg/liter) with a resistanceratio of 4x. DISCUSSION Selectionexperiment: Malathion selection of the U.S. compositeproved most successful.In- Certain trends are evident from the Ae. albo- secticideselection pressure was initiated at 40% picfus organophosphatesusceptibility results: 1) Jur.ro1990 ORceNopuospsetn SuscrprIBILITy IN AE. ALBoPIcrus

LC 95 (95% CONFIDENCELIMIT) 0.3 u BI F ,l 0.25 o o.2 z Dlqgnostlc Concentrqllon = 0.06 0.r5

F z !r 0.1 o z o o 0.05

0 12345678 910llt2 13 12345 I23 l2 345ROCr USA BRAZIL SE ASIA JAPAN

Fig. 3. Fenitrothion susceptibility in 26 strains of Aedes albopicttrc larvae. Bar height denotes LCss value; -cotrespondpoint denotes 95Vo confidence limit. Horizontal line is at the diagnostic concentration. Strain numbers to numbers in Table 1. All strains were tested in 4 replicate trials except SE ASIA nos. 2 and 3 which were replicated 6 and 8 times, respectively.

LC 95 (95% CONFIDENCE LIMIT) ^6 E l{ F JC o

2 o

gIs F tri 2 o z 8r

r234567E9lOlll2r3r234 5 123 l2345ROCr US.A BRAZIL SE ASIA JAPA,N Fig. 4. Malathion susceptibility in 26 strains of Aedes albopictus larvae. Bar height denotes LCss value; point denotes 95% confidence limit. Horizontal Iine is at the diagnostic concentration. Strain numbers correspond to numbers in the Table L. All strains were tested in 4 replicate trials except USA no. 12 and SE ASIA nos. 2 and 3; these were replicated 3, 6 and 8 times, respectively. when compared to the ROCK strain, Ae. albo- this may be one factor in explaining why Ae. plctus strains were less susceptiblethan Ae. ae- aegypti populations seem to be disappearing gypti to all 5 insecticides; 2) Brazilian strains from areaswhere the 2 coexist(Ho et al. 1989), were, in general,highly susceptibleto all ofthe although other hypothesesto explain this phe- insecticides,most notably to malathion, fenitro- nomenonhave been proposed (Nasci et al. 1989). thion and fenthion; and 3) U.S. strains showed However, in Ae. aegypfi, as in Ae. albopictus, a wide range of variability in responseto the considerable variation exists between geo- insecticides, from quite susceptible to poten- graphic strains in responseto insecticide appli- tially resistant. cation. Samples of Ae. aegypti from the Carib- The comparison of Ae. albopictussusceptibil- bean have exhibited resistance to malathion ity to that of Ae. aegypti is useful since these (Fox 1980) and temephos (Georghiou et al. mosquitoesoccur in the same habitats in many 1987).Hemingway et al. (1989),however, found areas in the southern U.S. If Ae. albopictttsis no resistanceto malathion in pyrethroid-resist- lesssusceptible to organophosphateinsecticides, ant Ae. aegyptifrom Puerto Rico. 262 Jounrel oF THE AurRrceN Mosqurro CoNrnol Assocll'rton VoL.6,No.2

LC 95 (95%CONFIDENCE LIMIT) 0.2 g Irl t-

F: o 0.15 Disgnostlc Concentrotlon = 0.05 z

0.t

F z !I o 2 0,05 u

0 2 3 4 5 6 7 6 9 r0 lll2 l3 | 2 0 4 5 | 2 3 | 2 3 4 5 ROCr USA BRAZIL SE ASIA JAPAN Fig. 5. Fenthion susceptibility in 26 strains of Aedesalbopictus larvae. Bar height denotesLCe5 value; point denotes95% confidencelimit. Horizontal line is at the diagnosticconcentration. Strain numbers correspondto numbersin the Table 1. AII strainswere tested in 4 replicatetrials exceptUSA no. 13 and SE ASIA nos. 2 and 3; thesewere replicated 5, 6 and 7 times,respectively.

F z

CONCENTRATION (MC,/LITER) Fig. 6. Responseto 6 generationsof malathion selectionin the U.S. compositestrain of Aedesulbopictus. Sn refers to each consecutivegeneration of selection.In 6/7 Ld-pm lines, all but one or fewer data points fit the line accordingto chi-squareanalysis (Finney 1971).

Comparedto U.S. and Asian populationsof number of introductions from the same source Ae. ahopictu.s,Brazilian strains show less ge- or from a single introduction. netic variation, accordingto isozymeand DNA The variation in insecticide susceptibility ev- analysis(S. Kambhampati,personal communi- ident in U.S. strains of Ae. albopictushas 2 cation).Brazilian strainsare also highly uniform possibleexplanations: 1) the populationsin the with regard to absenceof photoperiodically in- U.S. stemmedfrom a number of different Asian duced diapause(G. Craig, personal communi- sourceswith different insecticide exposurehis- cation) and laboratory transmission of La tories,or 2) insecticideselection has taken place Crossevirus (T. Streit, personal communica- sinceAe. albopictusarrived in the U. S., giving tion). Similarly, Brazilian strains were uni- rise to populations with different insecticidesus- formly insecticide-susceptiblewith the excep- ceptibilities. Perhaps a combination of these 2 tion of ANCH-2 to chlorpyrifos.This lack of factors accountsfor the observedvariability. variability supports the conceptthat the Brazil- As stated earlier, there is limited information ian populations originated either from a Iow regardingsusceptibility of Asian strains of Ae. JUNE 1990 ORGANopHospHere SuscsprIBILITY IN AE. ALBoPIC'lus 263 albopictus to organophosphate insecticides. population on an annual basis; the results of Brown (1986) reported populations from Mad- 1989susceptibility tests are not yet available. agascar,Malaysia, Singaporeand Vietnam as Preliminary attempts have been made to de- resistant,but no resistanceratios were given' tect nonspecificesterase amplification as a re- Japanesepopulations from Tokyo and Okinawa sistancemechanism in both the DOM (Chicago) were tested for susceptibility to the organophos- and malathion-selectedU. S. compositestrains. phates, fenitrothion, fenthion and temephos Amplification of detoxifying esteraseenzymes is (Takahashi et al. 1985). The Tokyo strain often the basis for organophosphateresistance showeda resistanceratio of 5x to fenthion, but in Culexmosquitoes (Mouches et al. 1986,Ray- was susceptibleto the other insecticidestested; mond et al. 1987) and has been described in the Okinawa strain was susceptibleto all 3 in- Anophel.esalbimanus Wied. (Brogdon et al. secticides.Additional information is lacking re- 1988). In Ae. albopictus,initial studies have garding mosquito susceptibility in Japan, where shown no difference in intensity of esterase U.S. populationsof Ae. albopictusprobably orig- banding between the malathion-resistant and inated (Hawley et al. 1987). However, organo- control (SABAH) strains, when using polyacryl- phosphate insecticides are commonly used in amide gel electrophoresis.Selection for insensi- Japan, not only for mosquito control, but also tive acetylcholinesterasemight, alternatively, for control of agricultural pests affecting rice account for resistance development in these and barley, among other crops (M. Mogi, per- strains. Methods for detection of this resistance sonal communication). Since the present study mechanism have recently been updated shows lowered susceptibility or resistance of (ffrench-Constantand Bonning 1989). Japanesestrains to someinsecticides, some se- Given the potential public health importance lection for resistancein Ae. albopicfusmay have of Ae. albopicifus,continued insecticide suscep- occurred in northern Asia, prior to U.S. intro- tibility surveillance is essential. Current infor- duction. mation about the effectivenessof many com- Some selection for insecticide tolerance has monly used insecticidesagainst Ae. albopictusis probably taken place since the anival of Ae. spotty and often anecdotal.Given the wide range albopictusin the U.S., especially to malathion. of responseto insecticidesseen in these popu- Khoo et al. (1988) found that 50% of adult lations, there is a particular need for testing on mosquitoesof the HCMCD strain from Harris a localbasis. County, Texas, were resistant to malathion applied topically. Robert and Olson (1989)found ACKNOWLEDGMENTS adult female Ae. albopictus from Houston and following people Liberty County, Texas, resistant to malathion. I would like to thank the mosquito Similarly, the present findings regarding the and/or organizations for supplying R. L. HCMCD, HOUSTON and especiallythe DOM strains or helping with field collections: E. (Chicago)strain point to the recentdevelopment Berry, J. E. Freier, W. A. Hawley, L. Mun- of resistance in those populations. This seems stermann,D. Sprenger,and workers from the (Fort Iikely since none of the Japanesestrains tested Centersfor DiseaseControl Collins, CO), Vector-Borne showedunusually high tolerance to malathion. the Ohio Department of Health Board of Health, the The selectionof the U.S. compositestrain re- DiseaseUnit, the Indiana the Illi- sulting in a resistance ratio of 2lx in only 6 Illinois Department of Public Health, Pan Amer- generations demonstratesthe potential for re- nois Natural History Survey and the I also thank G. B. sistancedevelopment in this species. ican Health Organization. T. Streit for Less easily explained is the apparent decrease Craig, Jr., S. Kambhampati and research,M. in the susceptibilityof the Oak Hill, Ohio, pop- allowing referenceto unpublished providing in- ulation over a one-yearperiod (1987-88).One Mogi for information on types of explanation is that heavy insecticide pressure secticidesused on agricultural crops in Japan probit com- on that population has resulted in decreased and M. Raymond for supplying the B. Craig,Jr., susceptibility to all 5 insecticides. However, puter program.The support of G. since the 1988 colony originated from 3 field- made this work possible.This study was funded collected inseminated females, genetic drift by NIH Grant AI-02753and NIH Training Pro- could have producedthat result. Continued un- gram2 T32 AI-07030. treated tire introductions to the tire yard in Oak Hill may be importing Iess susceptibleindivid- REFERENCES CITED uals from other localities-as evidencedby the Brogdon,W. G.,J. H. Hobbs,Y. St.Jean, J. R.Jacques finding of Ae. aegypti at Oak Hill in 1989where and L. B. 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