In vitro metabolism of pyriproxyfen by microsomes from susceptible and resistant housefly larvae

Archives of Insect Biochemistry and Physiology 37:215–224 (1998)

© 1998 Wiley-Liss, Inc.

In Vitro Metabolism of Pyriproxyfen by Microsomes FromSusceptible and Resistant Housefly Larvae

Li Zhang, Shinji Kasai, and Toshio Shono*

Laboratory of Applied Zoology, Institute of Agriculture and Forestry, University of Tsukuba,Tsukuba, Ibaraki, Japan

Levels of cytochrome P450 and b5 were investigated in mi-crosomal enzymes of houseflies from the gut and fat body ofthe third instar larvae of a pyriproxyfen-resistant strain(YPPF) and two pyriproxyfen-susceptible strains (YS and SRS).In comparison to the YS and SRS strains, YPPF microsomeshad higher levels of total cytochrome P450s in both the gutand fat body. Furthermore, microsomes from the gut and fatbody of YPPF larvae were found to have a much greater abil-ity to hydroxylate aniline than YS larvae. In vitro metabo-lism studies of pyriproxyfen indicated that the metabolic rateswere much higher in both the gut and fat body of YPPF lar-vae than of YS and SRS larvae. The major metabolites ofpyriproxyfen in houseflies were identified to be 4´-OH-pyriproxyfen and 5´´-OH-pyriproxyfen. Cytochrome P450inhibitors, piperonyl butoxide (PB) and 2-propynyl 2,3,6-trichlorophenyl ether (PTPE), decreased the metabolic ratessignificantly in all three strains. This study confirmed thatmicrosomal cytochrome P450 monooxygenases play an impor-tant role in the pyriproxyfen resistance of the housefly. Fur-thermore, it suggests that the fat body must be as importantas the gut for the metabolism of pyriproxyfen in resistanthousefly larvae. Arch. Insect Biochem. Physiol. 37:215–224,1998. © 1998 Wiley-Liss, Inc.

Key words: pyriproxyfen; resistance; housefly; metabolism; cytochrome P450monooxygenase

Abbreviations used: DTT = dithiothreitol; EDTA = ethyl-enediaminetetraacetic acid; HDA = hexane:diethyl ether:aceticacid (50:50:0.1); JH = juvenile hormone; JHA = juvenile hor-mone analog; LSC = liquid scintillation counting; p-APMSF =(p-amidinophenyl) methanesulfonyl fluoride hydrochloride; PB= piperonyl butoxide; POP = 4-phenoxyphenol; POPA = 4-phenoxyphenyl (RS)-2-hydroxypropyl ether; PTPE = 2-propynyl2,3,6-trichlorophenyl ether; PTU = 1-phenyl-2-thiourea; TEA =toluene:ethyl acetate:acetic acid (75:25:1); TLC = thin-layer chro-matography; 4´-OH-POP = 4,4´-oxydiphenol; 4´-OH-POPA = 4-(4-hydroxyphenoxy) phenyl (RS)-2-hydroxypropyl ether;4´-OH-pyr = 4-(4-hydroxyphenoxy) phenyl (RS)-2-(2-pyri-

dyloxy) propyl ether; 5´´-OH-pyr = (RS)-5-hydroxy-2-[1-methyl-2-(4-phenoxyphenoxy)] ethoxy pyridine.

Contract grant sponsor: Ministry of Education, Science andCulture of Japan; Contract grant numbers: 07406003 and07556014; Contract grant sponsor: University of TsukubaResearch Projects.

*Correspondence to: Toshio Shono, Laboratory of Applied Zo-ology, Institute of Agriculture and Forestry, University ofTsukuba, Tsukuba, Ibaraki 305, Japan.

Received 20 May 1997; accepted 21 August 1997

216 Zhang et al.

INTRODUCTION

Pyriproxyfen, 4-phenoxyphenyl (RS)-2-(2-pyridyloxy) propyl ether, is a juvenile hormoneanalog (JHA) which has been confirmed to havejuvenile hormone (JH) activity (Hatakoshi et al.,1986) and shares common mechanisms of actionwith another JHA, methoprene (Riddiford andAshburner, 1991). This chemical has insecticidalactivities against various pest insects, such ashouseflies (Kawada et al., 1987), mosquitoes(Schaefer et al., 1988), and scales (Peleg, 1988).It has also shown potential for practical use inthe control of cockroaches (Koehler and Pat-terson, 1991), tsetse flies (Hargrove and Lan-gley, 1993), whiteflies (Ishaaya and Horowitz,1995), and chironomids (Ali et al., 1993).

Resistance to pesticides is one of the mostsevere problems in the management of agricul-turally and medically important pests (Brattstenet al., 1986; Mullin and Scott, 1992; Feyereisen,1995). Previous studies have shown that manyspecies of insects have already developed resis-tance to JHAs (Sparks and Hammock, 1983;Hammock, 1985). Development of resistance topyriproxyfen will be an obstacle to further useof this chemical as a pesticide. A recent studyshowed that a housefly strain (the Rmp strain)which was highly resistant to methyl parathionwas about 32-fold more insensitive to pyri-proxyfen than a susceptible strain (Bull andMeola, 1994). As part of a study of strategiesfor resistance management, we established apyriproxyfen-resistant strain of housefly (theYPPF strain) by laboratory selection of an or-ganophosphate-resistant housefly colony withthis chemical to investigate the resistancemechanism of the insect. Third instar larvae ofYPPF strain developed 4,900-fold resistance asevaluated by the medium mixing method. Theseflies developed 1,400- and 400-fold resistance inthe third instar larvae and white pupae, respec-tively, when assayed by topical application(Zhang et al., 1997; Zhang and Shono, 1997).Genetic analysis and synergism studies of re-sistance led to the indication that cytochromeP450 monooxygenase plays an important rolein resistance of the housefly to pyriproxyfen(Zhang et al., 1997).

Cytochrome P450s are important in themetabolism of many endogenous and foreignsubstances and are well known as a major fac-tor in detoxification of DDT, synthetic pyre-throids, organophosphates, carbamates, and

JHAs (Agosin, 1985; Feyereisen, 1993). Bull andMeola (1994) reported that a P450 inhibitor, pip-eronyl butoxide (PB), strongly reduced the ex-tent of the in vivo metabolism of pyriproxyfenby female adult houseflies and thus indicatedthat cytochrome P450 monooxygenase plays animportant role in in vivo degradation of pyri-proxyfen in houseflies.

Studies on cytochrome P450 monooxy-genase–mediated resistance of insects showedthat cytochrome P450s are usually expressed athigh levels in adults, although resistance to in-secticides is expressed in many stages of the in-sect life (Agosin, 1985). Yu and Terriere (1978)showed that the activity of P450 monooxygenasein metabolizing JH I was much higher in adultsthan in larval stages of diazinon-resistant Rut-gers houseflies. They also showed that the thirdinstar larvae possessed the highest activityamong all the larval stages. JHAs are especiallyeffective on the stages before metamorphosis,and the selection of the YPPF housefly led tohigh resistance in the third instar larvae (Zhanget al., 1997). Therefore, the third instar larvaeof houseflies were used to provide useful infor-mation on resistance mechanisms in the devel-opmental stages of insects. Because cytochromeP450s are detectable in a wide range of insecttissues and their activities may differ accordingto tissues (Scott, 1993), we dissected the gut andfat body from housefly larvae and discuss therole of cytochrome P450 monooxygenases in themetabolism of pyriproxyfen.

MATERIALS AND METHODS

Chemicals

[Phenoxyphenyl-14C] pyriproxyfen (specificactivity 109 mCi/mmol) was kindly provided bySumitomo Chemical Co., Ltd. (Osaka, Japan). Thischemical showed more than 98% radiochemicalpurity by thin-layer chromatography (TLC).

The following authentic standards (Table 1)were also kindly supplied by Sumitomo ChemicalCo., Ltd.: 4-phenoxyphenol (POP), 4,4´-oxydiphenol (4´-OH-POP), 4-phenoxyphenyl(RS)-2-hydroxypropyl ether (POPA), 4-(4-hy-droxyphenoxy) phenyl (RS)-2-hydroxypropylether (4´-OH-POPA), 4-(4-hydroxyphenoxy)phenyl (RS)-2-(2-pyridyloxy) propyl ether (4´-OH-pyr), and (RS)-5-hydroxy-2-[1-methyl-2-(4-phenoxyphenoxy)] ethoxy pyridine (5´´-OH-pyr).Cytochrome P450 inhibitors, PB (purchased from

Pyriproxyfen Metabolism by Housefly Microsomes 217

Wako Pure Chemical Industries, Ltd., Osaka,Japan) and 2-propynyl 2,3,6-trichlorophenyl ether(PTPE) (provided by Nihon Bayer Agrochem K.K.,Yuki, Japan), were also used in the in vitrometabolism. All other chemicals were obtainedcommercially.

Insects

Three strains of houseflies (Musca domes-tica L.) were used in this study: 1) SRS, a WHOstandard reference susceptible strain, 2) YS, anorganophosphate-resistant colony collected fromthe third Yumenoshima dumping island in To-kyo, Japan (this strain was susceptible topyriproxyfen) and 3) YPPF, a pyriproxyfen-re-sistant strain established from the YS strain af-ter successive selections with pyriproxyfen for17 generations which showed a 4,900-fold resis-tance to pyriproxyfen by the medium mixingmethod in larvae (Zhang et al., 1997).

Isolation of Gut and Fat Body

Third instar larvae of houseflies at thewandering stage were used for preparing mi-crosomes from the gut and fat body by the fol-lowing procedure. Larvae, removed from thebreeding container and washed with water, werekept under wet conditions without food for 1 dayto completely clear the guts. The larvae werethen chilled by transferring them to a plasticdish on ice. After the anterior tip of each larvawas cut off with a small scalpel, the gut and fatbody were extruded through the anterior endby squeezing them out with a forceps while hold-ing the posterior end with another forceps. Thegut and fat body tissues were immediately putinto a dish on ice containing several millilitersof modified homogenization buffer (Lee andScott, 1989): 0.1 M sodium phosphate buffer (pH7.5) containing 10% (v/v) glycerol, 1 mM ethyl-enediaminetetraacetic acid (EDTA), 0.1 mM

dithiothreitol (DTT), 1 mM (p-amidinophenyl)methanesulfonyl fluoride hydrochloride (p-APMSF), and 1 mM 1-phenyl-2-thiourea (PTU)(dissolved in diethylene glycol). Guts and fat bod-ies were separated and placed in centrifuge tubeswith homogenization buffer (20 tissues/millili-ter of buffer).

Preparation of Microsomes From Gutand Fat Body

The separated guts and fat bodies were ho-mogenized with an IKA Ultra-Turrax T25(Janke & Kunkel GmbH & Co. KG, Staufen,Germany) homogenizer at 9,000 rpm for 10 s and5 s, respectively. Homogenates were centrifugedat 10,000g for 15 min in a Tomy RS-205 centri-fuge (Tomy Seiko Co., Ltd., Tokyo, Japan) equip-ped with a 4N-II rotor. The supernatant wasfiltered through glass wool and centrifuged at105,000g for 1 h in a Beckman TL-100 tabletopultracentrifuge (Beckman Instruments, Palo Alto,CA) equipped with a TLA-100.4 fixed angle rotor.The microsomal pellets were resuspended byhomogenizing in resuspension buffer made ac-cording to Lee and Scott (1989): 0.1 M sodiumphosphate buffer (pH 7.5) containing 20% (v/v)glycerol, 1 mM EDTA, 0.1 mM DTT, and 1 mMp-APMSF. The above procedures were done at0–4°C. Microsomes were freshly prepared beforeeach experiment.

Enzyme and Protein Assays

Cytochrome P450 and b5 were quantitativelyanalyzed by the method of Omura and Sato (1964)and Ronis et al. (1988) with a Shimadzu UV-160Aspectrophotometer (Shimadzu Co., Kyoto, Japan)using extinction coefficients of 91 mM cm–1 between450 and 490 nm for P450 and 185 mM cm–1 be-tween 423 and 409 nm for b5. Proteins were deter-mined by the method of Bradford (1976). Bovineserum albumin was used as a standard. Data wereobtained from at least three replicates.

Bioassay of Aniline Hydroxylase Activity

Assay of aniline hydroxylation by micro-somes was modified from that of Imai et al.(1966). The 1 ml reaction mixture contained 0.7ml of 0.1 M sodium phosphate buffer (pH 7.5, con-tained 1 mM EDTA), 0.1 ml of 10 mM anilinehydrochloride, and 0.1 ml of microsome (about10 mg protein/milliliter). The reaction was initi-ated by the addition of 0.1 ml of 10 mM β-NADPH. The reaction was carried out for 30 minat 30°C in a shaking water bath. Reactions were

TABLE 1. TLC Rf Values of Authentic Standards*

Solvent systemCompound TEA HDA

Pyriproxyfen 0.69 0.60POP 0.47 0.384´-OH-pyr 0.43 0.255´´-OH-pyr 0.40 0.25POPA 0.36 0.234´-OH-POP 0.26 0.174´-OH-POPA 0.18 0.10

*Solvent systems: TEA, toluene:ethyl acetate: acetic acid(75:25:1); HDA, hexane:diethyl ether:acetic acid (50:50:0.1).

218 Zhang et al.

terminated by adding 1 ml of 0.1 M trichloroaceticacid. After centrifugation at 2,500 rpm for 10 min,a 1.5 ml aliquot of the supernatant was taken outand 0.5 ml of 10% Na2CO3 was added, followed byaddition of 1 ml of 2% phenol in 0.2N NaOH. Aftershaking for another 30 min, the absorbance of themixture was measured at 630 nm. Distilled waterinstead of aniline hydrochloride was used as a con-trol, and p-aminophenol was used as a standard.Each experiment was replicated at least three timeswith two sets of reaction mixtures per replication.

In Vitro Metabolism by Microsomes

Each incubation mixture in a 10 ml tube(PYREX®; Iwaki Glass, Chiba, Japan) contained0.9 ml of 0.1 M sodium phosphate buffer (pH 7.5,with 1 mM EDTA), two guts or fat bodies in 0.1ml volume (enzyme), and 110,000–150,000 dpm(0.15–0.2 µg) of [phenoxyphenyl-14C]pyriproxy-fen in 10 µl ethanol. For the inhibition study, 5µl of 2 mM PB or PTPE in ethanol was added tothe reaction mixture (a final concentration of0.01 mM in the mixture). The reaction mixturewas then preincubated at 30°C for 10 min in ashaking water bath. Reactions were started byadding 0.2 ml of 10 mM β-NADPH. After 30 minof incubation, metabolism was stopped by addi-tion of 0.2 ml 1 N HCl and 0.5 g (NH4)2SO4.Organosoluble metabolites were extracted threetimes with 3 ml diethyl ether and the extractspooled. After drying with Na2SO4, the extractswere concentrated to 500 µl under a N2 stream,and 10 µl was assayed by liquid scintillationcounting (LSC). The extracts were concentratedagain and adjusted to 20,000 dpm/10 µl; then10 µl extract was spotted on TLC plates anddeveloped in a toluene:ethyl acetate:acetic acid(75:25:1) (TEA) solvent. Radioactive spots wereidentified by autoradiography for 12 h using aBAS-III Fuji Imaging Plate (Fuji Photo FilmCo., Ltd., Kanagawa, Japan), and areas of ra-

dioactivity were located and quantified using aBAS 2000 Bio Image analyzer (Fuji). Metaboliteswere identified by cochromatography with authen-tic compounds.

Thin-Layer Chromatography

TLC was carried out on Merck (Darmstadt,Germany) plates of silica gel 60 F254 (20 × 20cm; layer thickness, 0.25 mm). Radioactive me-tabolites were identified by two-dimensionalcochromatography with unlabelled authentic com-pounds using TEA and hexane:diethyl ether:acetic acid (50:50:0.1) (HDA) solvent systems.Unlabelled authentic compounds were detectedby viewing with ultraviolet light (254 nm), andthe Rf of each compound is shown in Table 1.

RESULTSCytochrome P450 and b 5 Contents of Gutand Fat Body Tissues

Levels of cytochrome P450 and cytochromeb5 in guts and fat bodies from the three strainsof houseflies are shown in Table 2. The proteincontent of fat bodies was 2.5–3-fold higher thanthat of guts. The cytochrome P450 level of YPPFgut was 1.7-fold higher than that of YS and SRSgut. The cytochrome P450 content in fat bodieswas also different in the three strains: in YPPFand YS strains they were 5.2- and 3.0-fold higher,respectively, than in the SRS strain. Interestingly,cytochrome P450 in YPPF fat bodies was alsoabout 1.7-fold of that in YS fat bodies. Unlike cy-tochrome P450, levels of cytochrome b5 in guts ofthe three strains were almost the same. Cyto-chrome b5 levels in fat bodies of YPPF and YSwere also the same, although they were 1.6-foldhigher than that of SRS. The data also showedthat the content of cytochrome P450 or b5 fromguts of each strain was much higher than thatfrom fat bodies. The YPPF strain was a pyri-

TABLE 2. Levels of Cytochrome P450 and b5 in the Gut and Fat Body of Houseflies of Pyriproxyfen-ResistantYPPF and -Susceptible YS and SRS Strains*

Protein P450 b5

Strain (µg/larva) nmol/mg protein Ratioa nmol/mg protein Ratioa

Gut YPPF 11.5 0.429 ± 0.055 1.7 0.237 ± 0.037 0.8YS 11.6 0.252 ± 0.043 1.0 0.248 ± 0.013 0.8SRS 10.6 0.248 ± 0.025 1.0 0.297 ± 0.012 1.0

Fat body YPPF 33.2 0.265 ± 0.026 5.2 0.134 ± 0.012 1.6YS 28.8 0.155 ± 0.027 3.0 0.134 ± 0.018 1.6SRS 35.1 0.051 ± 0.004 1.0 0.082 ± 0.013 1.0

*All values are mean ± SE of at least three replicates.aRatio is based on a value of 1.0 for the SRS strain.


proxyfen-resistant strain selected with this chemi-cal from the third instar larvae of the pyri-proxyfen-susceptible YS strain (Zhang et al.,1997). The data indicate that pyriproxyfen selec-tion led to an increase in the amount of cyto-chrome P450 in both guts and fat bodies of thethird instar larvae.

Activities of Aniline Hydroxylation

Microsomal monooxygenase activities ofaniline hydroxylation from guts and fat bodies ofthe three strains are summarized in Table 3. Theactivity was undetectable in microsomes from theSRS strain. The YPPF strain possessed muchgreater ability to hydroxylate aniline than the YSstrain. It is obvious that, after selection withpyriproxyfen, housefly larvae exhibited 2.7- and1.8-fold higher hydroxylation activity of anilinein gut and fat body, respectively. The results sug-gest that increased aniline hydroxylation activityin the YPPF strain may be associated with highlevels of pyriproxyfen resistance which wasthought to be related to cytochrome P450 mono-oxygenase (Zhang et al., 1997). Thus, it appearsthat the high level of pyriproxyfen resistance inthe YPPF strain is best correlated to the anilinehydroxylation activity of cytochrome P450 mono-oxygenase.

In Vitro Metabolism of Pyriproxyfen

Pyriproxyfen was unmetabolized whenNADPH was not added to the reaction mixture(data not shown). When NADPH was added,pyriproxyfen was metabolized at a very highrate by microsomal enzymes from guts and fatbodies of the third instar larvae of resistantYPPF houseflies (Table 4). The Rf values for un-known A, B, and C metabolites were 0.49, 0.25,and 0.22, respectively (developed with the TEAsolvent system). Recoveries of radioactivities

were 85% and 81% for the gut and fat body, re-spectively. More than 30% of pyriproxyfen wasmetabolized within 30 min. The major metabo-lites were 4´-OH-pyr, 5´´-OH-pyr, and an un-known metabolite at the TLC origin. Anothermajor metabolite was unknown C. Two-dimen-sional cochromatography using two solvent sys-tems (toluene:diethyl ether [3:2] and chloroform:methanol [95:5]) showed that unknown C con-tained two metabolites, one of which had a Rfvalue near authentic 5´´,4´-OH-pyr described byMatsunaga et al. (1995). Addition of cytochromeP450 inhibitors PB or PTPE to the enzyme mix-ture before reaction inhibited the oxidase ac-tivities of microsomal enzymes, and thus themetabolized pyriproxyfen decreased to less than8% in both the gut and fat body. Accordingly,the hydroxylation ability (represented as thepercentages of 4´-OH-pyr and 5´´-OH-pyr) of in-hibited enzymes decreased to much lower lev-els than that of enzymes without inhibitors.Furthermore, both PB and PTPE also caused adecrease or disappearance in all unknown me-tabolites and POPA and 4´-OH-POPA.

Compared to resistant YPPF houseflies, sus-ceptible YS and SRS houseflies showed muchweaker ability to degrade pyriproxyfen (Tables5, 6). Recoveries of radioactivities were morethan 90% for both the gut and fat body. Lessthan 10% of pyriproxyfen was metabolized bymicrosomal enzymes from both guts and fat bod-ies of the two housefly strains. The main me-tabolites were still 4´-OH-pyr and 5´´-OH-pyr,as in the YPPF strain. It is also clear that themetabolite at the TLC origin was less than 1.0%,and unknown metabolites B and C and 4´-OH-POPA did not appear in both strain. In addi-tion, PB or PTPE almost completely inhibitedthe metabolic reaction in the two strains. Thus,the results of in vitro metabolism as well as thedata of cytochrome P450 levels and aniline hy-droxylation activity in all three strains indicatedand supported the hypothesis of our previousstudy that increased cytochrome P450 mono-oxygenase plays an important role in the metabo-lism of pyriproxyfen in resistant YPPF houseflies(Zhang et al., 1997).

DISCUSSION

Pyriproxyfen bears little apparent struc-tural resemblance to the natural JHs and otherJHAs such as methoprene and ethoprene (R-20458), it possesses neither ester bond nor ep-

TABLE 3. Microsomal Monooxygenase Activity ofAniline Hydroxylation in the Third Instar Larvaeof Houseflies

pmole/mg protein/mina

Strain Gut Ratiob Fat body Ratiob

YPPF 657 ± 26.7 2.7 278 ± 27.3 1.8YS 244 ± 25.7 1.0 156 ± 25.0 1.0SRS UDc UDaAll values represent the mean ± SE of three or more experi-ments, each replicated twice.bRatio is based on a value of 1.0 of the YS strain.cUD, undetectable.

220 Zhang et al.

oxide moiety, and thus insect JH esterase andJH epoxide hydrolase, which are the two majorenzymes responsible for JH degradation(Hammock, 1985), are not involved in the me-tabolism of pyriproxyfen. On the other hand,cytochrome P450 monooxygenase plays an im-portant role in pyriproxyfen metabolism of thehousefly, as this study showed. The major me-tabolites are 4´-OH-pyriproxyfen and 5´´-OH-pyriproxyfen. The proposed metabolic pathwaysof pyriproxyfen in the housefly are shown in Fig-ure 1. The preferred sites of metabolic reaction,based on the percentage of identified metabo-lites in this study, are 1) hydroxylation of theterminal phenyl ring at position 4, 2) hydroxy-lation of the pyridyl ring at position 5, and 3)cleavage of the propyl pyridyl ether bond (seemetabolites POPA and 4´-OH-POPA in Fig. 1).Studies on the in vivo and in vitro metabolismof pyriproxyfen in rats and mice (Matsunaga etal., 1995; Yoshino et al., 1996) indicated thatmetabolic reactions also included hydroxylation

of the terminal phenyl ring at position 2,desphenylation, and cleavage of the propyl phe-nyl ether. Cleavage of the propyl phenyl ether(metabolites as POP or 4´-OH-POP) was notfound in this study; thus, the metabolic path-ways of pyriproxyfen are slightly different ininsects and mammals.

Compared to the susceptible YS and SRSstrains, microsomal enzymes from YPPF larvaeproduced more polar metabolites of pyriproxyfenwhich remains at the TLC origin. Consideringthe fact that pyriproxyfen was unmetabolizedwithout NADPH and that the production of po-lar metabolites at the origin was almost inhib-ited by cytochrome P450 inhibitors, we concludedthat the polar metabolite was produced as a sec-ondary metabolite of pyriproxyfen by NADPH-dependent cytochrome P450 enzymes.

Bull and Meola (1994) observed that therewas no obvious difference in the in vivo metabo-lism of pyriproxyfen by female adult housefliesof the Rmp strain (an organophosphate-resistant

TABLE 5. Metabolites of Pyriproxyfen in Microsomal Enzymes From the Gut and Fat Body of the Third InstarLarvae of Susceptible YS Houseflies*

Percentage of total recovered radioactivityGut Fat body

Compound No inhibitor +PB +PTPE No inhibitor +PB +PTPE

Unmetabolized 94.68 100 100 95.33 100 100Unknown A 0.63 0.544´-OH-pyr 2.58 2.075´´-OH-pyr 1.25 1.03POPA 0.33 0.34Unknown BUnknown C4´-OH-POPAOrigin 0.52 0.69

*All values represent the mean of three replicates.

TABLE 4. Metabolites of Pyriproxyfen in Microsomal Enzymes From the Gut and Fat Body of the Third InstarLarvae of Resistant YPPF Houseflies*



Unmetabolized 63.75 97.57 95.13 69.85 94.65 92.46Unknown A 1.93 1.114´-OH-pyr 12.95 1.73 3.23 8.08 2.72 5.235´´-OH-pyr 5.48 0.70 0.62 2.35 1.05 0.62POPA 2.59 0.47 1.58 0.62Unknown B 1.25 0.93Unknown C 5.16 3.824´-OH-POPA 1.10 1.25Origin 5.79 0.55 11.04 1.59 1.06



strain) and the Rpyr strain which was selectedwith pyriproxyfen from Rmp houseflies and de-veloped a fifteenfold higher resistance than Rmphouseflies. The YPPF strain was establishedfrom the YS strain with pyriproxyfen selectionfor 17 generations and reached high resistanceto this chemical (Zhang et al., 1997). Comparedwith YS larvae, YPPF larvae increased their cy-tochrome P450 level in both guts and fat bodiesand also increased their hydroxylation activityand ability to metabolize pyriproxyfen. There-fore, selection of houseflies with this chemicalmay lead to different results according to thehousefly strains and growth stages used.

Increased levels of total cytochrome P450and/or P450 monooxygenase activities are oftenassociated with monooxygenase-mediated resis-tance in insects (Scott, 1993). Our results show

that, although YPPF housefly larvae may haveonly 1.7-fold of total cytochrome P450s in boththe gut and fat body of YS larvae and may el-evate only 2.7- and 1.8-fold of total hydroxyla-tion activities, the metabolic activity in YPPFlarvae may be much higher because more than30% of total radioactive pyriproxyfen was de-graded by YPPF larvae while only about 5% wasmetabolized by YS larvae. The above discrepancybetween the increased amount of cytochromeP450s and the elevated metabolic activities mayindicate the possibility that pyriproxyfen resistancein YPPF larvae is related not only to the increasein cytochrome P450 level but also to the alterationof cytochrome P450 monooxygenases which pre-fer pyriproxyfen as a substrate.

This study also showed that 0.01 mM PB orPTPE inhibited the enzyme activities of resistant

TABLE 6. Metabolites of Pyriproxyfen in Microsomal Enzymes From the Gut and Fat Body of the Third InstarLarvae of Susceptible SRS Houseflies*



Unmetabolized 90.95 97.94 97.40 98.23 100 100Unknown A 0.504´-OH-pyr 6.73 2.06 2.19 1.065´´-OH-pyr 0.95 0.41 0.38POPA 0.33 0.33Unknown BUnknown C4´-OH-POPAOrigin 0.54


Fig. 1. Proposed major metabolic pathways of pyriproxyfen in microsomes of housefly larvae.

222 Zhang et al.

YPPF larvae to very low levels, almost the samelevel as the activities of susceptible flies withoutinhibitors. Thus, it was shown that cytochromeP450 inhibitors completely inhibited the elevatedcytochrome P450 activities. PB or PTPE also com-pletely inhibited the activities of YS microsomesand of microsomes from SRS fat bodies. Theseresults also confirm that resistance to pyri-proxyfen in YPPF houseflies is associated withelevated activity of cytochrome P450s. Our pre-vious study (Zhang et al., 1997) showed thatthe resistance to pyriproxyfen in YPPF larvaewas depressed by PTPE but not PB; thus, theresults of the in vivo toxicity bioassay of insec-ticides and in vitro metabolism may sometimesbe incongruous, and further studies on in vivometabolism of pyriproxyfen in the housefly areneeded to resolve this discrepancy.

It is obvious that selection of YS larvae withpyriproxyfen led to an increased level of cyto-chrome P450, but not b5, in both the gut andfat body of YPPF larvae (Table 2). Zhang andScott (1996) showed that cytochrome b5 is in-volved in certain cytochrome P450 monooxy-genase–mediated resistance of the houseflies.Valles and Yu (1996) also suggested that theelevated cytochrome b5 level in a multiple-resis-tant strain of German cockroach plays an impor-tant role in the detoxification of insecticides.Although cytochrome b5 of microsomal enzymesfrom the YPPF fat body is 1.6-fold of that fromthe SRS fat body, we do not think cytochrome b5is important in resistance to pyriproxyfen inYPPF flies because there were no changes in thelevels of cytochrome b5 in gut and fat body whencompared to YS larvae.

The distribution of cytochrome P450 mono-oxygenases and their activities to different sub-strates vary among different tissues of insects,and the fat bodies of insects are one of the maintissues in which many substrates can be me-tabolized by cytochrome P450 monooxygenases(Feyereisen, 1983; Lee and Scott, 1992; Rose etal., 1995). Our study showed that fat bodies aswell as the gut of housefly larvae play an im-portant role in degradation of pyriproxyfen. Infact, the fat body of insects is not only an im-portant tissue for the metabolism of JHA butalso a site of JH receptors and of JH bindingproteins (Shemshedini and Wilson, 1990, 1993;Braun et al., 1995). Shemshedini and Wilson(1990) reported that resistance to JH III andmethoprene in Drosophila is associated with aJH binding protein in the fat body. Therefore,

the fat body must be a very important materialfor studies on the detoxification and the modeof action of JHs and JHAs.

Insecticide resistance is a dynamic and mul-tidimensional phenomenon. The most importantresistance mechanisms in insects are the en-hancement of the capacity to metabolicallydetoxify insecticides and alterations in targetsites that prevent insecticides from binding tothem (Brattsten et al., 1986). Accordingly, re-sistance mechanisms to pyriproxyfen in YPPFhousefly larvae likely involve an altered JH re-ceptor or binding protein. Our genetic and syn-ergist studies indicated the existence of multipleresistance mechanisms in YPPF larvae (Zhanget al., 1997); thus, it is undeniable that alteredJH receptor or binding protein is associated withthe resistance to pyriproxyfen.

ACKNOWLEDGMENTS

We are grateful to Dr. D. Taylor, College ofAgrobiological Resources, University of Tsu-kuba, Japan, for his reading of the manuscriptand to Dr. H. Honda of our laboratory for hishelpful comments on the work. We also thankMr. S. Sone of Nihon Bayer Agrochem K.K., Yuki,Japan, for providing the synergist and SumitomoChemical Co., Ltd. (Osaka, Japan) for generouslysupplying the [phenoxyphenyl-14C] pyriproxyfenand authentic chemicals. This work was partlysupported by a grants in aid for DevelopmentalScientific Research from the Ministry of Educa-tion, Science, and Culture of Japan (07406003 and07556014) and by the University of TsukubaResearch Projects.

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In vitro metabolism of pyriproxyfen by microsomes from susceptible and resistant housefly larvae

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