� Corresponding author. Tel./fax: +

E-mail address: wyd@njau.edu.cn

0965-1748/$ - see front matter # 200

doi:10.1016/j.ibmb.2004.04.001

86-25-84396062.

(Y. Wu).

4 Elsevier Ltd. All rights reserved.

Insect Biochemistry and Molecular Biology 34 (2004) 763–773

www.elsevier.com/locate/ibmb

The involvement of microsomal oxidases in pyrethroid resistancein Helicoverpa armigera from Asia

Y. Yang a, Y. Wu a,�, S. Chen a, G.J. Devine b, I. Denholm b, P. Jewess c, G.D. Moores c

a Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Weigang No.1, Nanjing 210095, PR Chinab Plant and Invertebrate Ecology Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

c Biological Chemistry Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

Received 11 February 2004; accepted 15 April 2004

Abstract

Five contemporary strains of the bollworm Helicoverpa armigera Hubner from China, Pakistan and India, all with high resist-ance to pyrethroids, were compared with a standard susceptible strain that originated from the Cote D’Ivoire in the 1970s(‘SCD’). Two of the Chinese strains (‘YGF’ and ‘YGFP’) were derived by laboratory selection from a third, field collected strain(‘YG’). The strain ‘YG’ exhibited 7-, 14- and 21-fold resistance to fenvalerate, cypermethrin and deltamethrin, respectively. Afterselection with fenvalerate for 14 generations (‘YGF’), this increased to 1690-, 540- and 73-fold. Selection with a mixture of fenva-lerate and piperonyl butoxide (PBO) for 14 generations (‘YGFP’) resulted in resistance ratios of 2510, 2920 and 286. The syner-gistic ratios to fenvalerate that resulted from pre-treatment of PBO were 5-, 462- and 12-fold in YG, YGF and YGFP strains,respectively. Resistance ratios for a Pakistani strain (PAK) were 2320-, 4100- and 223-fold to fenvalerate, cypermethrin and delta-methrin, respectively. The synergistic ratio of PBO to these pyrethroids was 450-, 950- and 11-fold. The strong synergism of pyre-throids by PBO implied that an oxidative metabolism could be involved in pyrethroid resistance in these resistant strains. Theactivities of cytochrome P450 monooxygenases from midguts of final instar larvae to p-nitroanisole (PNOD), ethoxycoumarin(ECOD), methoxyresorufin (MROD) significantly increased in all the resistant strains when compared with the susceptible strain.This further implies that cytochrome P450 monooxygenases are involved in pyrethroid resistance in Asian H. armigera. Compara-tive in vitro studies of the metabolism of 14C-deltamethrin by midgut microsomes of the resistant PAK and susceptible SCDstrains showed that the resistant strain had a much greater capacity than the susceptible strain for the metabolic degradation ofdeltamethrin. This enhanced metabolic degradation occurred in the presence of NADPH which suggested an oxidative detoxifica-tion. In the resistant strains, minor increases in glutathione S-transferase activity (to the substrates CDNB and DCNB), andesterase activity (to the substrate a-naphthyl acetate) further suggested that, of the putative metabolic mechanisms, oxidases arethe most important. This study provides the first evidence that cytochrome P450 monooxygenases are a major metabolic mech-anism responsible for pyrethroid resistance in H. armigera from Asia.# 2004 Elsevier Ltd. All rights reserved.

Keywords: Insecticide resistance; Pyrethroids; Monooxygenases; Glutathione S-transferases; Esterases; Helicoverpa armigera

1. Introduction

The cotton bollworm, Helicoverpa armigera (Hubner)(Lepidoptera: Noctuidae), is one of the most seriousagricultural pests in Asia, Africa, southern Europe andAustralia. Due to heavy selection pressure over the past40 years, it has developed resistance to organochlorine,carbamate, organophosphate and pyrethroid insecti-

cides in many countries (McCaffery, 1998) and hasbegun to show some tolerance to newer chemistriessuch as fipronil, chlorfenapyr, spinosad and indoxacarb(Ahmad et al., 2003). The failure of insecticides to con-trol resistant H. armigera has been a strong incentivefor the development and adoption of Bt cotton geneti-cally engineered to withstand bollworm attack (Huanget al., 2002; Fakrudin et al., 2003).All the major mechanisms of resistance such as

delayed penetration, nerve insensitivity and metabolicdetoxification have been implicated as the cause of

764 Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773

pyrethroid resistance in H. armigera (Ahmad et al.,1989; Gunning et al., 1991; Forrester et al., 1993; Wuet al., 1995, 1997; McCaffery, 1998; Ahmad andMcCaffery, 1999; Tan and McCaffery, 1999; Martinet al., 2002). Studies on the metabolic mechanismsresponsible for pyrethroid resistance in H. armigerahave been the subject of controversy regarding the rela-tive roles of oxidation by microsomal cytochromeP450 monooxygenases and hydrolysis by esterases(McCaffery, 1998; Scott, 1999). Oxidative metabolismby cytochrome P450 monooxygenases in H. armigerahas previously been implicated as a major metabolicmechanism to pyrethroid resistance in Asia (Ahmadand McCaffery, 1991; Wu et al., 1995, 1997; Kranthiet al., 1997, 2001). However, esterases have also beenfound to be involved (Kranthi et al., 1997; Chen et al.,2002). Elsewhere in the world, an increase in mono-oxygenases titres was thought to be the main resistancemechanism in a deltamethrin resistant strain of H.armigera from the Cote D’Ivoire (Martin et al., 2002).In Australia, oxidative metabolism of monooxygenaseswas thought to be the major metabolic mechanism offield resistance in the 1980s (Gunning et al., 1991; For-rester et al., 1993). However, further studies haveproved that current pyrethroid-resistant H. armigera inAustralia have overproduced esterase isoenzymes thatcan both sequester and hydrolyse pyrethroids. Itappears, therefore, that detoxification by hydrolysis,together with sequestration is the major metabolicmechanism of pyrethroid resistance in Australia (Gun-ning et al., 1996, 1999). Interestingly most publishedreports on metabolic mechanisms of pyrethroid resist-ance in the same strain of H. armigera have implicatedonly one major mechanism, oxidases or esterases.In the present study, we provide further evidence for

an oxidative mechanism of pyrethroid resistance inAsian H. armigera populations from China, India andPakistan by determining, in vitro, the differences inmetabolic enzyme activities between pyrethroid resist-ant and susceptible strains and by metabolic studiesusing a radiolabelled pyrethroid.

2. Materials and methods

2.1. Insects

The susceptible strain (SCD) that originated fromthe Cote D’Ivoire in the 1970s was supplied by theAgrochemicals Division, Bayer CropScience, Germany.The Chinese field strain (YG) was collected fromYanggu County, Shandong Province, China in 2001.This was maintained in the laboratory withoutexposure to insecticides. The strain ‘YGF’ was selectedfrom a subpopulation of ‘YG’ by the topical appli-cation of fenvalerate for 14 generations. A further

subpopulation of YG was used to derive the strain‘YGFP’ by pre-treatment with the synergist piperonylbutoxide (PBO) (8 lg/larva) 1 h prior to treatmentwith fenvalerate for 14 generations. The YGF andYGFP strains were tested within two generations afterselection. The PAK strain was collected in July 2003from okra (Hibiscus esculenta) in Lahore, Pakistan. Itwas supplied by the Central Cotton Research Institute,Multan, Pakistan. The IND strain was supplied by theCentral Institute for Cotton Research, Nagpur, Indiaand was collected from the local area around Nagpur,India in 2003. Both PAK and IND strains were testedwithin two generations reared in the laboratory with-out selection.Larvae of H. armigera were reared on an artificial

diet at 27� 1vC with a 16:8 (L:D) photoperiod.

Adults were held under the same temperature and lightconditions at an RH of 60% and supplied with a 10%sugar solution.

2.2. Chemicals

The insecticides used were of technical grade: fenva-lerate (93%, Changzhou Pesticide plant, China), delta-methrin (99%, Bayer CropScience, Germany),cypermethrin (98%, Rothamsted Research, UK),phoxim (80.5%, Nantong Chemical Plant, China),monocrotophos (80%, Qingdao Pesticide Plant, China),piperonyl butoxide (PBO) (95%, Endura, Italy), S,S,S-tributylphosphorothioate (DEF) (98%, Sigma, UK).14C-deltamethrin (56.8 mCi/mmol, labelled in the ben-zyl carbon) was supplied as a stock solution in acetonefrom Rothamsted Research, UK. Radiochemical purityof 14C-deltamethrin was >99%.Aldrin was received from Dr. Ehrenstorfer (Augs-

burg, Germany) and dieldrin was from the Institute ofIndustrial Organic Chemistry (Warsaw, Poland). Othersubstrates used to measure enzyme activity werepurchased from original manufactures, i.e. methoxyr-esorufin from Molecular Probes Inc (Eugene, OR), p-nitroanisole, 7-ethoxycoumarin, a-naphthyl acetate andNADPH from Sigma (UK), 1-chloro-2,4-dini-trobenzene (CDNB) from Aldrich (UK), and 3,4-dichloronitrobenzene (DCNB) from BDH (UK).

2.3. Bioassay

Technical grade insecticide was dissolved in acetoneand a series of concentrations was prepared. For eachconcentration, at least 30 third instar larvae (10–15 mg)were treated topically on the thorax with 0.25-ll insec-ticide by Hamilton syringe. When appropriate, PBO(an inhibitor of monooxygenases) or DEF (an inhibitorof esterases) was applied at 8 lg/larva 1 h before appli-cation of insecticide. After 48 h, the mortality was

Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773 765

assessed. Bioassay data were analysed using Poloplus1

software (LeOra Software, California, 2003).

2.4. Enzyme preparation

Two-day-old final instar larvae (300� 50 mg) wereplaced on ice. That portion of the body anterior to thefirst pair of thoracic legs and posterior to the fourthpairs of abdominal legs was discarded. The remainingpart of the body was split open along the dorsum. Themidgut was removed and its contents were washed andrinsed in ice-cold 0.1 M sodium phosphate buffer (pH7.6). Midguts were homogenized, on ice, in homo-genization buffer (0.1 M phosphate buffer, pH 7.6, con-taining 1 mM EDTA, 1 mM DTT, 1 mM PTU, 1 mMPMSF and 20% glycerol). Five midguts were homo-genized in 400 ll buffer and the homogenizer rinsedwith a further 800 ll of buffer. Both rinses were com-bined (1200 ll). The homogenate was centrifuged at

4vC, 10,000g for 20 min. The supernatant was

decanted into a clean eppendorf tube, placed on iceand used immediately for monooxygenase, glutathioneS-transferase (GST) and esterase assays. For the prep-aration of microsomes from midguts, 50 midguts werehomogenized in the homogenization buffer. After cen-trifugation at 10,000g for 20 min, the supernatant wasfiltered through glass wool and centrifuged at 100,000gfor 75 min. The resulting pellet of microsomes wasre-suspended in the homogenization buffer and stored

at �80vC.

2.5. Enzyme assays

Assays examining the O-demethoxylation of p-nitroanisole (PNOD) and methoxyresorufin (MROD),and the O-deethoxylation of 7-ethoxycoumarin (ECOD)by cytochrome P450 monooxygenases were conductedusing the procedures developed by Rose et al. (1995);Mayer et al. (1977) and Ullrich and Weber (1972),respectively.For the PNOD assay, 100 ll of 2 mM p-nitroanisole

solution and 90 ll enzyme were added to each well. Themicroplate was incubated for 3 min at 30

vC and the

reaction initiated by the addition of 10 ll of 9.6 mMNADPH. The absorbance was read in a Thermomax

microplate reader (Molecular Devices) at 405 nm and30

vC for 15 min. The activity of PNOD was presented

as nmole p-nitrophenol min�1 mg protein�1.For the MROD assay, methoxyresorufin was dis-

solved in DMSO to make a 1 mM stock solution andthen in 0.1 M pH 7.8 sodium phosphate buffer to acti-vate the solution. Fifty microliter of the enzyme and100 ll of 0.01 mM methoxyresorufin added to eachwell. The microplate was incubated for 3 min at 30

vC

and the reaction initiated by the addition of 10 ll of9.6 mM NADPH. The plate was placed in a lumi-

nescence spectrometer (Perkin Elmer LS50B). Enzymeactivity was measured at excitation wavelength of 530nm (slit 5 nm), emission wavelength 585 nm (slit 5 nm)in 15 cycles and 2 sec for each well. The activity of theenzyme was presented as pmole resorufin min�1 mgprotein�1.For the ECOD assay, ethoxycoumarin was dissolved

in ethanol to make a 20 mM stock solution and acti-vated by the addition of 0.1 M pH 7.8 sodium phos-phate buffer. Fifty microliter of the enzyme and 80 llof 0.5 mM ethoxycoumarin were added to each well.The microplate was incubated for 3 min at 30

vC and

the reaction initiated by the addition of 10 ll of 9.6mM NADPH. The plate was placed in a luminescencespectrometer (Perkin Elmer LS50B). Enzyme activitywas measured at an excitation wavelength 380 nm (slit5 nm), and an emission wavelength 460 nm (slit 2.5nm). The activity of the enzyme was presented aspmole 7-hydroxycoumarin min�1 mg protein�1.Monooxygenase epoxidation activity to aldrin (AE)

was measured as follows. The 1-ml reaction mixturecontaining 0.7 ml 0.1 M pH 7.8 sodium phosphate buf-fer, 0.2 ml enzyme and 20 ll 1 mg/ml aldrin (in etha-nol) was incubated at 30

vC in a shaker bath for 3 min

before adding 0.1 ml 8 mg/ml NADPH. The reactionmixture was incubated for another 15 min at 30

vC

before terminating by the addition of 0.8 ml acetone.The product, dieldrin, was extracted twice by 2 ml ofredistilled hexane and then the supernatants were pooledand dried using anhydrous sodium sulphate. Theextractions were made up to 5 ml using redistilled hex-ane. The sample was analysed under constant pressure(25 psi) using a HP 6890 gas chromatograph with ECDand a HP-5MS capillary column (J&W Scientific, UK).The injector, detector and column temperatures were250, 300 and 240

vC, respectively. The activity of AE

was presented as nmole dieldrin min�1 mg protein�1.For assays quantifying glutathione S-transferase

(GST) activity, the substrates 1-chloro-2,4-dini-trobenzene (CDNB) and 3,4-dichloronitrobenzene(DCNB) were used. For CDNB, 10 ll of the enzymesolution (diluted 20-fold in 0.1 M pH 7.6 sodium phos-phate buffer) was mixed with 100 ll 1.2 mM CDNBand 100 ll 6 mM glutathione (GSH). For DCNB, 25 llenzyme solution was mixed with 25 ll 0.1 M sodiumphosphate buffer and 100 ll 1.2 mM DCNB and 6 mMGSH. Enzyme activity was measured in a Thermomax

microplate reader at 340 nm and 30vC using the kin-

etic mode for 10 min. The activity of GST was determ-ined using the extinction coefficient of 9.6 mM�1 cm�1

for CDNB and 8.5 mM�1 cm�1 for DCNB.Non-specific esterase activity was measured using

a-naphthyl acetate (a-NA) as a substrate as follows.Ten microliter of enzyme solution (diluted 10-fold in0.1 M pH 7.6 sodium phosphate buffer) was mixedwith 200 ll of substrate solution (5 ml 0.2 M pH 6.0

766 Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773

phosphate buffer, 10 mg Fast Blue RR salt and 0.1 ml

100 mM a-NA). Enzyme activity was measured at 450

nm and 25vC using a Thermomax microplate reader in

the kinetic mode for 10 min. The activity of the ester-

ase is presented as nmole naphthol min�1 mg

protein�1.Total protein content was determined by the Bradford

method (Bradford, 1976) using bovine albumin (Sigma)

as a standard and a Bradford dye reagent (Sigma).

2.6. In vitro metabolism of deltamethrin

The in vitro metabolism of deltamethrin was mon-

itored using the method of Zhang and Scott (1996).

For a 1 ml reaction mixture, 0.7 ml 0.1 M pH 7.8

phosphate buffer, a 0.2 ml microsome suspension

(about 1 mg protein) and 4 ll 14C-deltamethrin (6600

dpm/ll) were added to a glass vial. In the control incu-

bation a 0.2 ml pH 7.8 phosphate buffer was added

instead of the microsome suspension. The reaction was

initiated by the addition of 0.1 ml NADPH (8 mg/ml)

and the contents were incubated at 30vC and shaken

at 100 oscillations/min for 4 h. The reaction was stop-

ped by the addition of 200 ll 2 N HCl. Two milliliter

hexane:ethyl acetate(1:1) was triply added to extract

the remaining deltamethrin and its metabolites. The

organic fraction of three extracts were combined

together and evaporated to dryness under nitrogen.

Fifty microliter chloroform was added to redissolve the

extracts which were stored on ice. Each sample was

spotted onto a TLC plate (0.25 mm thick, Whatman

LK6DF) using deltamethrin as a standard. The plate

was chromatographed using toluene (saturated with

formic acid):ether (5:1) (Bigley and Plapp, 1978). After

chromatography, the plate was scanned by the TLC-

Linear Analyzer (Berthold) or exposed to a Kodak sto-

rage phospher screen for 72 h. The phospher screen

was scanned using the Personal Molecular Imager2

FX System (Bio-Rad). The radioactive areas of delta-

methrin and metabolites were located by the scan

graph and quantified by gently scraping relevant areas

of the TLC plates and suspending the fine powder in

1 ml distilled water and then 5 ml emulsifier scintillator

cocktail. These suspensions were then counted by LSC

(Kontron).

2.7. Statistical analyses

Laboratory data on the evaluation of different rates

of enzyme assays were subjected to analysis of variance

(ANOVA) followed by Student’s t-test (a ¼ 0:05).

3. Results

3.1. Bioassays

The toxicities by topical application of differentinsecticides to the susceptible and four of the resistantstrains are presented in Table 1. YGF, YGFP andPAK resistant strains had extremely high resistance tothe pyrethroids fenvalerate, cypermethrin and deltame-thrin. However, resistance to two organophosphorusinsecticides (phoxim and monocrotophos) in theseresistant strains was low to moderate. Under differentselection pressures, H. armigera could develop differentpatterns of resistance to insecticides. Selection with acombination of fenvalerate and PBO made the YGFPstrain significantly more resistant to cypermethrin anddeltamethrin compared with the YGF strain that wasselected with fenvalerate alone.When the oxidase inhibitor, PBO was used as a

synergist in the YGF, YGFP and PAK resistantstrains, the synergistic ratios were 462-, 12- and 450-fold for fenvalerate, and 54-, 46- and 950-fold forcypermethrin, respectively. The strong synergism ofpyrethroids by PBO implied that oxidative metabolismcould be involved in pyrethroid resistance in theseresistant strains. The esterase inhibitor, DEF only gave2- to 11-fold synergism ratios for fenvalerate andcypermethrin in the YGF and YGFP strains. This sug-gested that esterases might be unimportant or lessimportant in conferring pyrethroid resistance in theseresistant strains.

3.2. Enzyme assays

In order to make more specific assertions regardingthe role of monooxygenases in pyrethroid resistance inAsian H. armigera, the interaction of enzyme prepara-tions from final instar larval midguts with severalmodel substrates was examined. The substrates chosenwere two O-demethylation substrates, p-nitroanisoleand methoxyresorufin, one O-deethylation substrate,ethoxycoumarin and one epoxidation substrate, aldrin.Against the former three substrates, activity levelsincreased along with increasing pyrethroid resistance inthe five resistant strains from the three countries. How-ever, there was no positive correlation between theepoxidation activity and pyrethroid resistance (Table 2).Among the resistant strains, the YG strain, with thelowest phenotypic pyrethroid resistance in bioassays,showed the lowest activity and the strain IND, withextremely high level of cypermethrin resistance (Dr.Keshav Kranthi, personal communication), exhibitedthe highest activity against all the substrates exceptaldrin. Among the three Chinese strains, YGF strainhad the highest activity in the PNOD assay whereasYGFP strain showed the highest activity in ECOD and

Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773 767

Table 1

Topical toxicity of different insecticides to susceptible and resistant Helicoverpa armigeraa

Strain T

est LD50 (lg/larva) (95%FL) S lope R Rb S Rc

SCD F

envalerate 0.012 (0.010–0.015) 2 .924 1

Fenvalerateþ PBO

0.016 (0.013–0.020) 3 .148 1.3 0.8

Cypermethrin

0.0058 (0.0047–0.0071) 3 .229 1

Deltamethrin

0.0015 (0.0012–0.0019) 2 .587 1

Phoxim

0.023 (0.019–0.028) 4 .171 1

Monocrotophos

0.254 (0.194–0.327) 2 .148 1

YGF F

envalerate 20.313 (14.997–31.283) 1 .567 1 690

Fenvalerateþ PBO

0.044 (0.032–0.059) 1 .769 4 4 62

FenvalerateþDEF

4.807 (3.148–7.341) 1 .258 400 4

Cypermethrin

3.129 (2.394–4.668) 1 .696 540

Cypermethrinþ PBO

0.058 (0.045–0.079) 2 .177 10 54

CypermethrinþDEF

1.105 (0.821–1.471) 1 .855 190 3

Deltamethrin

0.110 (0.078–0.146) 1 .793 73

Deltamethrinþ PBO

0.027 (0.021–0.035) 2 .440 18 4

Phoxim

0.445 (0.367–0.551) 2 .178 19

Monocrotophos

11.257 (8.789–14.047) 1 .974 44

YGFP F

envalerate 30.118 (17.678–41.795) 1 .039 2 510

Fenvalerateþ PBO

2.537 (1.457–3.773) 1 .358 211 12

FenvalerateþDEF

2.722 (1.938–3.572) 1 .972 226 11

Cypermethrin

16.951 (13.199–22.966) 2 .199 2 920

Cypermethrinþ PBO

0.366 (0.295–0.450) 3 .069 63 46

CypermethrinþDEF

7.712 (4.603–11.851) 1 .163 1 330 2

Deltamethrin

0.429 (0.305–0.575) 1 .764 286

Deltamethrinþ PBO

0.027 (0.009–0.050) 1 .448 18 16

Phoxim

0.857 (0.660–1.115) 2 .334 37

Monocrotophos

3.495 (1.062–5.950) 0 .822 14

YG F

envalerate 0.088 (0.060–0.118) 1 .765 7

Fenvalerateþ PBO

0.018 (0.014–0.022) 3 .242 1.5 5

Cypermethrin

0.079 (0.061–0.099) 2 .640 14

Deltamethrin

0.031 (0.023–0.040) 2 .682 21

Phoxim

0.306 (0.219–0.430) 1 .570 13

Monocrotophos

2.628 (1.938–3.613) 1 .725 10

PAK F

envalerate 27.875 (19.690–48.379) 1 .587 2 320

Fenvalerateþ PBO

0.062 (0.033–0.088) 1 .796 5 4 50

Cypermethrin

23.752 (15.464–48.627) 1 .592 4 100

Cypermethrinþ PBO

0.025 (0.015–0.035) 1 .989 4 9 50

Deltamethrin

0.335 (0.236–0.508) 1 .432 223

Deltamethrinþ PBO

0.031 (0.021–0.047) 1 .903 21 11

Phoxim

0.431 (0.241–0.597) 1 .831 19

a n ¼ 150 210, 30 larvae for each of five to seven doses.b RR ðresistance ratioÞ ¼ LD50 of resistant strain=LD50 of SCD strain.c SR ðsynergism ratioÞ ¼ LD50 of insecticide alone=LD50 of insecticideþ PBO.

Table 2

Cytochrome P450 activities of midgut toward different substrates in susceptible and resistant Helicoverpa armigeraa

Strain P

NODb (nmole min�1

mg protein�1)

R/S E

CODb (pmole

min�1 mg protein�1)

R

/S M RODb (pmole

min�1 mg protein�1)

R

/S A Ec (nmole

min�1 mg protein�1)

R

/S

YGF (n ¼ 25)

0:4� 0:09 ab 11.7 8 :65� 1:67 c 2.9 0 :19� 0:04 b 3 .2 0 :34� 0:03 a 4 .3

YGFP (n ¼ 18) 0

:22� 0:07 b 6.5 1 5:7� 2:58 bc 5.2 0 :37� 0:08 ab 6 .2 0:1� 0:01 b 1 .3

YG (n ¼ 10) 0:0

72� 0:04 c 2.1 3 :28� 0:89 d 1.1 0 :22� 0:05 b 3 .7 0 :09� 0:02 b 1 .1

IND (n ¼ 5) 0

:52� 0:14 a 15.3 7 9:9� 14:73 a 2 6.6 0 :46� 0:03 a 7 .7 0 :24� 0:02 a 3 .0

PAK (n ¼ 10) 0

:46� 0:11 ab 13.5 24 :43� 6:7 b 8.1 0 :23� 0:06 b 3 .8 0 :12� 0:02 b 1 .5

SCD (n ¼ 15) 0:0

34� 0:02 c 3� 0:58 d 0 :06� 0:04 c 0 :08� 0:01 b

a Results are shown as mean� SE. Means in the column followed by the same letter are not significantly different (a ¼ 0:05, ANOVA)

(PNOD: F ¼ 5:798; df ¼ 5; 77; P ¼ 0:0001. ECOD: F ¼ 20:503; df ¼ 5; 77; P < 0:0001. MROD: F ¼ 5:429; df ¼ 5; 77; P ¼ 0:0002. AE:

F ¼ 21:813; df ¼ 5; 20; P < 0:0001).b PNOD, p-nitroanisole O-demethylation; ECOD, ethoxycoumarin O-deethylation; MROD, methoxyresorufin O-demethylation.c AE: aldrin epoxidation (n ¼ 3 7).

768 Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773

MROD assays. It was interesting that the YG andYGF strains demonstrated similar activity in theMROD assay in comparison with the YGFP strainwhich exhibited double their activity.In the DCNB assay, the GST activity in the midguts

of H. armigera was significantly higher in all the resist-ant strains tested as compared with the susceptibleSCD strain (Table 3). In the CDNB assay, the GSTactivity in the YGF, YGFP and PAK strains washigher than in the susceptible strain. This suggestssome involvement of GSTs in pyrethroid resistance inH. armigera. Vontas et al. (2001) demonstrated thatGSTs could act as antioxidant defence agents, notbinding protein, to confer pyrethroid resistance in thebrown planthopper (BPH), Nilaparvata lugens Stal. Itis possible that elevated GSTs in pyrethroid-resistantstrains of H. armigera play a similar role to that inBPH. In the a-NA assay for esterases, the activity wasslightly higher in the YGFP strain but the other strainswere not significantly different to the SCD strain. Thissuggests that midgut esterases (or at least ones detect-able using a-NA) play little role in resistance to pyre-throids in Asian populations of H. armigera.

3.3. In vitro metabolism of 14C-deltamethrinby microsomes

Microsomes from the final instar larval midguts ofthe PAK and SCD strains were extracted for the invitro metabolism work. The microsomal activities ofmonooxygenases, GSTs and esterases in both strainsare shown in Table 4. Activities of monooxygenases(ECOD and MROD) and GSTs (CDNB) were signifi-cantly higher in the PAK strain compared with theSCD strain. However, there was no difference in theesterase activity (to a-NA) between both strains. This isconsistent with our results from the midgut prepara-tions.Data for in vitro metabolism of 14C-deltamethrin by

microsomes of midguts in the PAK and SCD strainsare shown in Table 5. The average recovery of radioac-tive material was about 76% in both strains. WithNADPH (the co-factor of monooxygenase), six meta-

bolites (A, B, C, D, E and F) were shown to be presentin the organosoluble fraction when the extracts wererun on TLC plates (shown in Fig. 1). In the absence ofNADPH, only two metabolites A and F were detected(shown in Table 5). The metabolism of fenvalerate inthe house fly Musca domestica L. similarly showed adecreased number of metabolites without NADPH(Bull and Pryor, 1990). It is suggested that at least fourmetabolites B, C, D, E were produced via oxidativeroutes. This clearly shows that oxidation by mono-oxygenases was involved in the metabolism of deltame-thrin.Based on the proportion of unmetabolised deltame-

thrin, combined oxidase and esterase metabolism inmicrosomal preparations fortified with NADPH is ca.2.6 times more active in the PAK strain (15.7%) thanthe SCD strain (41.3%); the putative esterase metab-olism in microsomal preparations lacking NADPH wassimilar between the PAK (48.6%) and SCD strain(54.5%). This suggests that the difference in thecapacity for the metabolic degradation of deltamethrinin microsomal fractions between the PAK and SCDstrains is mainly caused by their different ability in oxi-dation.

4. Discussion

Cytochrome P450 monooxygenases are a complexgroup with numerous isoenzymes that can have eitherbroad or extremely narrow substrate specificity. Forexample, P450 activities in resistant and susceptiblestrains of the lepidopteran pests Spodoptera frugiperdaSmith and Plutella xylostella L. have been studiedusing several substrates (Yu, 1991,1992; Yu andNguyen, 1992). The results of enzyme assays for P450activity differed between species. In S. frugiperda, O-demethylation towards p-nitroanisole (PNOD) washigher in three resistant strains, but O-dealkylations tomethoxyresorufin and ethoxyresorufin (MROD andEROD) were not consistently higher in these resistantstrains. In P. xylostella, the P450 activity againstPNOD, MROD and EROD was higher in the resistant

Table 3

Glutathione S-transferase and esterase activities of midgut in susceptible and resistant H. armigeraa

Strain G

ST–CDNB (nmole

min�1 mg protein�1)

R

/S G ST–DCNB (nmole

min�1 mg protein�1)

R

/S E sterase-aNA (nmole

min�1 mg protein�1)

R/S

YGF (n ¼ 3) 6

72:2� 126:3 b 1 .7 1 6:8� 0:47 b 2 .9 1 76:3� 17:1 bc 1.0

YGFP (n ¼ 3) 13

46:6� 92:5 a 3 .5 1 8:8� 1:07 ab 3 .2 2 82:1� 15:6 a 1.6

YG (n ¼ 5) 3

56:8� 32:1 c 0 .9 1 3:5� 0:47 c 2 .3 2 01:9� 15:2 bc 1.1

IND (n ¼ 3) 4

14:3� 48:8 c 1 .0 9:6� 0:29 d 1 .6 1 66:9� 6:5 c 0.9

PAK (n ¼ 3) 12

59:8� 83:1 a 3 .3 2 0:6� 1:1 a 3 .5 2 12:6� 7:7 b 1.2

SCD (n ¼ 3) 3

86:7� 35:8 c 5:8� 0:3 e 1 80:4� 4:9 bc

a Results are shown as mean� SE. Means in the column followed by the same letter are not significantly different (a ¼ 0:05, ANOVA) (GST–

CDNB: F ¼ 25:652; df ¼ 5; 14; P < 0:0001: GST–DCNB: F ¼ 114:441; df ¼ 5; 14; P < 0:0001. Esterase-aNA: F ¼ 7:266; df ¼ 5; 14; P ¼ 0:0015).

Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773 769

strain than the susceptible strain. MROD showed thegreatest increase in P450 activity in the resistant strain.Similar differences have been reported for the house flyM. domestica and the tobacco budworm Heliothis vir-escens Fab. (Scott et al., 1990; Rose et al., 1995).Ethoxycoumarin O-deethylation (ECOD) of P450s hasbeen used as a measure of general P450 activity inmany insect and mite species, for example, codlingmoth Cydia pomonella L. (Bouvier et al., 2002), themite Tetranychus urticae Koch (Stumpf and Nauen,2001) and Drosophila melanogaster Meigen. The elev-ated activity of ECOD in D. melanogaster has beenassociated with resistance to DDT, chloropyrifos anddeltamethrin (Sousa et al., 1995). However, accordingto Frank et al. (1997), the ECOD assay in Drosophilacould not be used as an indicator of the activity ofmonooxygenases to the natural toxin compound, car-negine. In an anti-P450lpr inhibitory experiment fromthe LPR strain of M. domestica, ECOD was only par-tially inhibited by a specific antibody developed againsta purified P450 protein. However, the same antibodyinhibited 98% of MROD activity. Methoxyresorufinwas therefore demonstrated as a characteristic sub-strate of P450lpr protein. This protein proved to begenerated by the Cyp6d1 gene and therefore MRODhas been identified as a CYP6D1 specific assay andECOD for one or more other P450s (Wheelock andScott, 1992a,b). In a pyrethroid resistant German cock-roach strain, a purified P450MA protein had a strongconnection with cytochrome P450-mediatedN-demethylation which metabolised 4-chloro-N-methy-laniline (Scharf et al., 1998). Most experiments ininsects have proved the diversity of specificities of cyto-chrome P450 monooxygenases to different substrates.It is therefore necessary to use multiple model sub-strates to detect P450 activity and to evaluate P450-mediated metabolism in insecticide resistance. Inaddition to the use of model substrates, studies ofinsecticide metabolism in vivo and in vitro should beused whenever possible to confirm monooxygenase-mediated resistance in insects (Scott, 1999).There have been numerous studies on the detection

and quantification of P450s in many insects. Apartfrom aldrin epoxidation few studies have been carried

out on P450 assays against different specific substratesin H. armigera. Tang et al. (2000) showed elevatedO-demethylation to p-nitroanisole and the aldrin epox-idation of monooxygenases in a phoxim-resistant strainof H. armigera were associated with phoxim resistance.O-demethylation to p-nitroanisole and aldrin epoxida-tion by monooxygenase in H. armigera have beenfound to be higher in the midgut of H. armigera andthe monooxygenases could be induced by penta-methylbenzene and naphathalene in both the midgutand fatbody (Qiu et al., 2003). There are reports ofmonooxygenase involvement in resistance to pyre-throids in many insects based on ECOD and MRODassays but no such studies exist with reference to H.armigera.Most evidence that implicates the role of P450s in

pyrethroid resistance in H. armigera, is circumstantialand is based on the synergism of pyrethroids by PBOin vivo (Ahmad and McCaffery, 1991; Gunning et al.,1991; Barden et al., 1992; Forrester et al., 1993; Wuet al., 1995; Kranthi et al., 1997, 2001; McCaffery,1998; Martin et al., 2002). The present studies clearlyindicated that pyrethroid resistant strains from China,India and Pakistan had a more active monooxygenasesystem than the susceptible strain and moreover that(for the MROD and ECOD) these showed a positivecorrelation with increasing pyrethroid resistance. WhenPBO was used as a synergist to pre-treat larvae, thesynergism ratios varied from 4-fold to 950-fold in theresistant strains. In both YGF and PAK strains, resist-ance to fenvalerate dropped dramatically from 1690-and 2320-fold to 4- and 5-fold respectively, implyingthat the resistance was almost completely suppressed.In the present study, such data are given further cre-dence by the fact that in vitro enzyme assays indicatean enhanced P450 activity in all the resistant strainsusing three substrates (p-nitroanisole, methoxyresorufinand ethoxycoumarin), especially PNOD and ECOD ofhomogenates of midguts were highly elevated from 2.9-fold to 26.6-fold. In vitro metabolism of 14C-deltame-thrin by microsomes derived from those midgutsdemonstrated that, without NADPH, there wasless metabolism of deltamethrin. With NADPH, theresistant strain could metabolize more deltamethrin

Table 4

Metabolic enzymes activities of microsomes of midguts in Helicoverpa armigeraa

Strain M

FO–ECOD (pmole

min�1 mg protein�1)

R

/S M FO–MROD

(pmole min�1

mg protein�1)

R/S E

sterase-aNA

(nmole min�1

mg protein�1)

R

/S G ST–CDNB (nmole

min�1 mg protein�1)

R/S

PAK (n ¼ 3) 1

14:4� 20:5 a 1 1.2 3 :2� 0:59 a 2.5 1 94:5� 12:5 a 1 .2 1 35:0� 14:3 a 7.5

SCD (n ¼ 2)

10:2� 3:4 b 1 :3� 0:09 b 1 68:8� 10:1 a 18:0� 1:6 b

a Results are shown as mean� SE. Means in the column followed by the same letter are not significantly different (a ¼ 0:05, ANOVA) (MFO–

ECOD: F ¼ 45:494; df ¼ 1; 5; P ¼ 0:0011. MFO–MROD: F ¼ 22:453; df ¼ 1; 3; P ¼ 0:017. Esterase-aNA: F ¼ 2:276; df ¼ 1; 3; P ¼ 0:228. GST–

CDNB: F ¼ 154:391; df ¼ 1; 3; P ¼ 0:0011).

Table5

Invitro

metabolism

of14C-deltamethrinbymicrosomes

ofmidgutsin

Helicoverpaarm

igera

Strain

NADPH

aDistributionofradioactivematerial,%

ofinitialradiolabelleddeltamethrin(�

SE)

Totalrecovery

(%)

Vial

H2O

fraction

Organosolublefraction

AB

CD

EF

Deltamethrinb

PAK

+12:0�2:3

16:5�1:2

12:6�2:6

5:6�2:4

2:5�0:8

2:6�0:6

2:3�0:1

5:8�0:3

15:7�2:2

d75:5�2:0

�7:6�0:3

12:4�1:1

7:4�0:8

ND

cND

ND

ND

0:2�0:04

48:6�3:5

bc

76:2�3:5

SCD

+8:1�0:1

10:4�1:5

7:7�0:4

2:2�0:6

3:0�0:5

2:3�0:7

0:2�0:1

0:7�0:1

41:3�3:0

c76:0�3:3

�7:3�0:7

5:0�0:9

9:7�1:3

ND

ND

ND

ND

0:1�0:02

54:5�5:6

ab

76:6�4:1

CK

+11:0�1:1

0:8�0:1

1:6�0:7

ND

ND

ND

ND

ND

63:7�0:5

a77:1�1:1

aForPakandSCD,withNADPH,n¼

3;withoutNADPH,n¼

2.ForCK

(controlincubation),n¼

2.

bResultsare

shownasmean�SE.Meansin

thecolumnfollowed

bythesameletter

are

notsignificantlydifferent(a

¼0:05,ANOVA)(F

¼37:043;df¼

4;7;P¼

0:0001).

cND

meansnotdetectable(thelimitofdetectionis20dpm).

770 Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773

Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773 771

than the susceptible strain. In the absence of NADPH,the putative main metabolism is hydrolysis by estera-ses; in the presence of NADPH, oxidation is triggered.This suggests that the increased metabolism of pyre-throid in the resistant strain was monooxygenase-mediated. The results from synergism experiments,monooxygenase activity and in vitro metabolism studyconsistently suggested that elevated cytochrome P450monooxygenases (at least at midgut microsomes) are amajor metabolic mechanism responsible for pyrethroidresistance in H. armigera from Asia.It is clear from the present study that different selec-

tion pressures select different resistance patterns. Thefenvalerateþ PBO selected strain (YGFP) showedhigher resistance to pyrethroids than the fenvalerateselected strain (YGF). These two strains had differentresponses to PBO synergism. Fenvalerate was highlysynergisable in the strain selected with fenvaleratealone. A similar result also was found in P. xylostella(Chen and Sun, 1986). Our original purpose of usingfenvalerateþ PBO selection regime was to select fornerve insensitivity resistance mechanism. It is surpris-ing that this regime resulted in higher monooxygenaseactivity in the YGFP strain than in the YGF strain.The multiplicity of monooxygenases makes it likelythat certain monooxygenase isoenzymes are associatedwith specific resistance characteristics and that theapplication of specific insecticides might select for oneor more contributing isoenzymes (Lee and Scott, 1989;Yu, 1992; Hodgson et al., 1993). Comparison of theresults of the monooxygenase enzyme assays on theYG, YGF and YGFP strains, shows that PNOD and

AE were highest in the YGF strain, and that ECODand MROD were highest in the YGFP strain. Selectionwith fenvalerate alone appeared to enhance PNOD andAE. Selection by fenvalerate plus PBO increasedECOD and MROD. These strain specific increases sug-gested that the YGF and YGFP strains possess differ-ent patterns of elevated monooxygenase activity thathave resulted from different selection regimes. We havecloned two P450 genes, Cyp9a12 and Cyp9a14 from theYGF strain (GenBank Accession No. AY371318 andAY487948) and found overexpression of both genes inthe YGF strain compared with the YG and SCDstrains using semi-quantitative PCR (unpublisheddata). Further studies will be directed to express thesetwo genes in vitro and determine their monooxygenaseactivities to different model substrates and pyrethroidinsecticides.Neurophysiologically detectable nerve insensitivity

resistance to pyrethroids has been documented in fieldpopulations from Thailand, Australia and India(Ahmad et al., 1989; Gunning et al., 1991; Kranthiet al., 2001). Strong synergism of PBO to pyrethroidssuggested nerve insensitivity might not be important inthe resistant strains employed in present study. A num-ber of recent studies have demonstrated association ofmutations in para-homologous sodium channel geneswith nerve insensitivity resistance in many pest insectsand the most common mutation is the leucine (L) tophenylalanine (F) substitution in domain II segment 6(IIS6) (Soderlund and Knipple, 2003). Head et al.(1998) made sequence comparisons between resistantand susceptible strains of both Heliothis virescens and

-deltamethrin metabolism by microsomes from the midgut of final instar larva in H

Fig. 1. Phospher image of 14C elicoverpa armigera. Lanes 1–2:

SCD susceptible strain with NADPH; Lanes 3–4: PAK resistant strain with NADPH.

772 Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773

H. armigera and showed consistent aspartic acid to

valine (D1549V) and glutamic acid to glycine (E1553G)

substitutions in the cytoplasmic linker region between

domains III and IV of the para-homologous sodium

channel sequence of resistant insects. However, we

have compared sequences of both IIS4–S6 and III–IV

linker of para-homologous sodium channel gene of

pyrethroid-resistant strains of H. armigera from China,

India and Pakistan (including the YG, YGF and

YGFP strains) and no mutation has been found

(unpublished data).

Acknowledgements

The authors thank Dr. Naghmy Khan for her tech-

nical support in rearing insects, Dr. Mushtaq Ahmad

(CCRI, Pakistan) and Dr. Keshav Kranthi (CCIR,

Inida) for providing the PAK and IND strains, respect-

ively. Dr. Mushtaq Ahmad and Dr. Derek Russell

(NRI) are thanked for revising the manuscript. The

work was funded by the Common Fund for Commodi-

ties project CFC/ICAC-014 and partially supported by

the Teaching and Research Award Program for Out-

standing Young Teachers in Higher Education Institu-

tions of MOE, PR China (TRAPOYT). Y. Yang also

received support from China Scholarship Council.

Rothamsted Research receives grant-aided support

from the Biotechnology and Biological Sciences

Research Council of the United Kingdom (BBSRC).

References

Ahmad, M., McCaffery, A.R., 1991. Elucidation of detoxication

mechanisms involved in resistance to insecticides in the third

instar larvae of a field-selected strain of Helicoverpa armigera with

the use of synergists. Pestic. Biochem. Physiol. 41, 41–52.

Ahmad, M., McCaffery, A.R., 1999. Penetration and metabolism of

trans-cypermethrin in a susceptible and a pyrethroid-resistant

strain of Helicoverpa armigera. Pestic. Biochem. Physiol. 65,

6–14.

Ahmad, M., Gladwell, R.T., McCaffery, A.R., 1989. Decreased nerve

insensitivity is a mechanism of resistance in a pyrethroid-resistant

strain of Helicoverpa armigera from Thailand. Pestic. Biochem.

Physiol. 35, 165–171.

Ahmad, M., Iqbal Arif, M., Ahmad, Z., 2003. Susceptibility of Heli-

coverpa armigera (Lepidoptera: Noctuidae) to new chemistries in

Pakistan. Crop Prot. 22 (3), 539–544.

Barden, G., Rose, H., Gunning, R., 1992. Cytochrome P-450 content

and aldrin epoxidase activity in larvae of a susceptible and a pyre-

throid-resistant strain of Helicoverpa armigera (Hubner)

(Lepidoptera: noctuidae). J. Aust. Entomol. Soc. 31, 350.

Bigley, W.S., Plapp Jr., ., F.W., 1978. Metabolism of cis- and trans-

[14C]permethrin by the tobacco budworm and the bollworm. J.

Agric. Food Chem. 26 (5), 1128–1134.

Bouvier, J., Boivin, T., Beslay, D., Sauphanor, B., 2002. Age-depen-

dent response to insecticides and enzymatic variation in suscep-

tible and resistance codling moth larvae. Arch. Insect Biochem.

Physiol. 51, 55–66.

Bradford, W.W., 1976. A rapid and sensitive method for the quanti-

tation of microgram quantities of protein utilizing the principle of

protein-dye binding. Anal. Biochem. 72, 248–254.

Bull, D., Pryor, N., 1990. In vivo and in vitro fate of fenvalerate in

house-flies. Pestic. Biochem. Physiol. 38, 140–152.

Chen, J.S., Sun, C.N., 1986. Resistance of diamond back moth (Lepi-

doptera: Yponomeutidae) to a combination of fenvalerate and

piperonyl butoxide. J. Econ. Entomol. 79, 22–30.

Chen, S., Yang, Y., Wu, Y., 2002. Frequency distributions of esterase

activity and insecticide resistance in third-instar larvae in different

strains of Helicoverpa armigera Hubner. Acta Entomol. Sin. 45,

733–738.

Fakrudin, B., Vijaykumar, Krishnareddy, K.B., Badariprasad, Patil,

B.V., Kuruvinashetti, M.S., 2003. Insecticide resistance in cotton

bollworm, Helicoverpa armigera (Hubner): a mini review. Resist-

ant Pest Manage. Newsl. 13, 1.

Frank, M.R., Danielson, P., Fogleman, J., 1997. Comparison of

Drosophila cytochrome P450 metabolism of natural and model

substrates. J. Insect Physiol. 43, 953–957.

Forrester, N., Cahill, M., Bird, L., Layland, J., 1993. Management of

pyrethroid and endosulfan resistance in Helicoverpa armigera

(Lepidoptera: Noctuidae) in Australia. Bull. Entomol. Res. Suppl.

Ser. 1, 1–132.

Gunning, R., Easton, C., Balfe, M., Ferris, I., 1991. Pyrethroid resist-

ance mechanisms in Australian Helicoverpa armigera. Pestic. Sci.

33, 473–490.

Gunning, R., Moores, G., Devonshire, A., 1996. Esterases and esfen-

valerate resistance in Australian Helicoverpa armigera (Hubner)

Lepidoptera: Noctuidae. Pestic. Biochem. Physiol. 54, 12–23.

Gunning, R., Moores, G., Devonshire, A., 1999. Esterase inhibitors

synergise the toxicity of pyrethroids in Australian Helicoverpa

armigera (Hubner) (Lepidoptera: Noctuidae). Pestic. Biochem.

Physiol. 63, 50–62.

Head, D., McCaffery, A., Callaghan, A., 1998. Novel mutations in

the para-homologous sodium channel gene associated with pheno-

typic expression of nerve insensitivity resistance to pyrethroids in

Heliothine lepidoptera. Insect Molecular Biology 7, 191–196.

Hodgson, E., Rose, R.L., Goh, D.K.S., Rock, G.C., Roe, R.M.,

1993. Insect cytochrome P450-: metabolism and resistance to

insecticides. Biochem. Soc. Trans. 21, 1060–1065.

Huang, J., Rozelle, S., Pray, C., Wang, Q., 2002. Plant biotechnology

in China. Science 25, 674–677.

Kranthi, K., Armes, N., Rao, N., Raj, S., Sundaramurthy, V., 1997.

Seasonal dynamics of metabolic mechanisms mediating pyrethroid

resistance in Helicoverpa armigera in Central India. Pestic. Sci. 50,

91–98.

Kranthi, K., Jadhav, D., Wanjari, R., Kranthi, S., Russel, D., 2001.

Pyrethroid resistance and mechanisms of resistance in field strains

of Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ. Ento-

mol. 94, 253–263.

Lee, S., Scott, J., 1989. Microsomal cytochrome P450 mono-

oxygenases in the house fly (Musca domestica L.): biochemical

changes associated with pyrethroid resistance and phenobarbital

induction. Pestic. Biochem. Physiol. 35, 1–10.

Martin, T., Chandre, F., Ochou, O., Vaissayre, M., Founrnier, D.,

2002. Pyrethroid resistance mechanisms in the cotton bollworm

Helicoverpa armigera (Lepidoptera: Noctuidae) from West Africa.

Pestic. Biochem. Physiol. 74, 17–26.

Mayer, R., Jermyn, J., Burke, M., Prough, R., 1977. Methoxyresor-

ufin as a substrate for the fluorometric assay of insect microsomal

O-dealkylases. Pestic. Biochem. Physiol. 7, 349–354.

McCaffery, A.R., 1998. Resistance to insecticides in heliothine Lepi-

doptera: a global view. Philos. Trans. R. Soc. Lond. B 353, 1735–

1750.

Qiu, X., Li, W., Tian, Y., Leng, X., 2003. Cytochrome P450 mono-

oxygenases in the cotton bollworm (Lepidoptera: Noctuidae):

tissue difference and induction. J. Econ. Entomol. 96, 1283–1289.

Y. Yang et al. / Insect Biochemistry and Molecular Biology 34 (2004) 763–773 773

Rose, R., Barbhaiya, L., Roe, R., Rock, G., Hodgson, E., 1995.

Cytochrome P-450-associated insecticide resistance and the devel-

opment of biochemical diagnostic assays in Heliothis virescens.

Pestic. Biochem. Physiol. 51, 178–191.

Scharf, M., Neal, J., Marcus, C., Bennett, G., 1998. Cytochrome P450

purification and immunological detection in an insecticide resistant

strain of German cockroach. Insect Biochem. Mol. Biol. 28, 1–9.

Scott, J., 1999. Cytochromes P450 and insecticide resistance. Insect

Biochem. Mol. Biol. 29, 757–777.

Scott, J., Lee, S., Shono, T., 1990. Biochemical changes in the cyto-

chrome P450 monooxygenases of seven insecticide-resistance

house fly (Musca domestica L.) strains. Pestic. Biochem. Physiol.

36, 127–134.

Soderlund, D., Knipple, D., 2003. The molecular biology of knock-

down resistance to pyrethroid insecticides. Insect Biochem. Mol.

Biol. 33, 563–577.

Sousa, G., Cuany, A., Brun, A., Amichot, M., Rahmari, R., Berge,

J., 1995. A microfluorometric method for measuring ethox-

ycoumarin-O-deethylase activity on individual Drosophila melano-

gaster abdomens: interest for screening resistance in insect

populations. Anal. Biochem. 229, 86–91.

Stumpf, N., Nauen, R., 2001. Cross-resistance, inheritance, and bio-

chemistry of mitochondrial electron transport inhibitor-acaricide

resistance in Tetranychus urticae (Acari: Tetranychidae). J. Econ.

Entomol. 94, 1577–1583.

Tan, J., McCaffery, A.R., 1999. Expression and inheritance of nerve

insensitivity resistance in larvae of Helicoverpa armigera (Lepi-

doptera: Noctuidae) from China. Pestic. Sci. 55, 617–625.

Tang, F., Yue, Y., Hua, R., 2000. The relationships among MFO,

glutathione S-transferases, and phoxim resistance in Helicoverpa

armigera. Pestic. Biochem. Physiol. 68, 96–101.

Ullrich, V., Weber, P., 1972. The O-dealkylation of 7-ethox-

ycoumarin by liver microsomes, a direct fluorometric test. Hoppe-

Seyler’s Z. Physiol. Chem. Bd. 353, S.1171–1177.

Vontas, J., Small, G., Hemingway, J., 2001. Glutathione S-trans-

ferases as antioxidant defence agents confer pyrethroid resistance

in Nilaparvata lugens. Biochem. J. 357, 65–72.

Wheelock, G., Scott, J., 1992a. Anti-P450lpr antiserum inhibits spe-

cific monooxygenase activities in LPR house fly microsomes. J.

Exp. Zool. 264, 153–158.

Wheelock, G., Scott, J., 1992b. The role of cytochrome P450lpr

in deltamethrin metabolism by pyrethroid resistant and sus-

ceptible strains of house flies. Pestic. Biochem. Physiol. 43,

67–77.

Wu, Y., Shen, J., Tan, F., You, Z., 1995. Mechanism of fenvalerate

resistance in Helicoverpa armigera Hubner. J. Nanjing Agric.

Univ. 18, 63–68.

Wu, Y., Shen, J., Chen, J., Zhou, W., Li, A., Shen, W., 1997. Deter-

mination and tissue distribution of microsomal cytochrome P450

and cytochrome b5 in sixth-instar larva of Helicoverpa armigera

Hubner. J. Agric. Biotechnol. 5, 297–301.

Yu, S., 1991. Insecticide resistance in the fall armyworm, Spodoptera

frugiperda (J.E. Smith). Pestic. Biochem. Physiol. 39, 84–91.

Yu, S., 1992. Detection and biochemical characterization of insecti-

cide resistance in fall armyworm (Lepidoptera: Noctuidae).

J. Econ. Entomol. 85, 675–682.

Yu, S., Nguyen, S., 1992. Detection and Biochemical characterization

of insecticide resistance in the diamondback moth. Pestic. Bio-

chem. Physiol. 44, 74–81.

Zhang, M., Scott, J.G., 1996. Cytochrome b5 is essential for cyto-

chrome P450 CYP6D1-mediated cypermethrin resistance in LPR

house flies. Pestic. Biochem. Physiol. 55, 150–156.