Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

PESTICIDE Biochemistry & Physiology

Pesticide Biochemistry and Physiology 89 (2007) 65–72 www.elsevier.com/locate/ypest

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae) Lan Zhang, Xiwu Gao *, Pei Liang Department of Entomology, China Agricultural University, Beijing 100094, PR China Received 24 December 2006; accepted 6 March 2007 Available online 12 March 2007

Abstract A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.  2007 Elsevier Inc. All rights reserved. Keywords: Musca domestica; Beta-cypermethrin; Cross-resistance; Carboxylesterase; Monooxygenases; Glutathione S-transferase; Synergism

1. Introduction Pyrethroids, a group of insecticides derived from natural compounds (pyrethrins) isolated from the Chrysanthemum genus of plants [1], have been used in agriculture and home formulations for over 40 years and account for about 25% of the worldwide insecticide market [2]. Two distinct classes of pyrethroids have been identified based on different behavioral, neuropsychological and biochemical profiles [3]. Type I pyrethroids (e.g., permethrin and tetramethrin) mainly cause hyper-excitation and fine tremors, while Type II pyrethroids (e.g., cypermethrin and deltamethrin) possess an a-cyano group and produce more complex syndromes, including clonic seizures [4]. Pyrethroids were initially highly effective on the housefly control [5]. Unfortunately, very high levels of resistance to

*

Corresponding author. Fax: +86 10 62732974. E-mail address: [email protected] (X. Gao).

0048-3575/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.03.001

this class of insecticides have developed worldwide due to intensive use over the last 30 years [6–9]. Cytochrome P450-mediated detoxification and target site insensitivity are two major mechanisms accounting for the pyrethroids resistance in houseflies [10–18]. The increased level of detoxification by monooxygenase was found in many housefly strains and well-documented in the LPR strain [13–15]. Liu and Scott [13] found that, CYP6D1, a gene of cytochrome P450, was responsible for pyrethroid resistance in the LPR strain, and the molecular basis was due to increased mRNA transcription level of CYP6D1. The second mechanism, the reduced sensitivity of the insect nervous system to pyrethroids, was first recognized in the housefly [10], which was resulted from point mutations (kdr and super-kdr) within the voltage-gated sodium channel, and has been identified to be one of the major mechanisms in several resistant housefly strains to pyrethroids [11,16–18]. Currently, most of the works about the mechanisms for the pyrethroid resistance developed by houseflies are

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carried out by using type I pyrethroids. However, little insight is given to type II pyrethroids, which have been used extensively for the housefly control worldwide and houseflies also have developed resistance to these pyrethroids [19–21]. The esterase-mediated pyrethroid resistance has been reported in many pest insects [22–25]. Interestingly, esterase-mediated resistance played no or only a little role for pyrethroid resistance in houseflies according to these works. In China, the exploitation of pyrethroids started from the beginning of the 1970s and the application of this class of insecticides has flourished in recent years. The area treated with pyrethroids occupies more than one-third of the total insecticide-treated area [26], however, the rapid development of resistance to pyrethroids in many insect pests led to a significant and increasing threat to their effective use in China. Beta-cypermethrin, an insecticide of type II pyrethroids, is a synthetic pyrethroid containing 4 of the 8 isomers that constitute technical cypermethrin, and has been produced and applied originally to agricultural pests control in China since 1988 [27], which occupies more than 50% of the total production of pyrethroids market [28]. In recent years, it has been used extensively for the control of health pests as the only product of cypermethrin in China [29]. Unfortunately, different levels of resistance to beta-cypermethrin has been observed in field strains of the housefly from 5 districts of Tianjin (from 10- to 49-fold) and 11 cities of Zhejiang Province (from 7.60- to 55.25fold) [30,31]. Therefore, it is critical to study the resistance development and mechanisms to beta-cypermethrin in houseflies to delay or control beta-cypermethrin resistance in China. In the present study, we established a resistant strain of the housefly and determined the pattern of resistance development and cross-resistance. The synergism of PBO and DEF to beta-cypermethrin was determined and the biochemical assays were carried out, including carboxylesterases, monooxygenases and glutathione S-transferases. 2. Materials and methods 2.1. Housefly strains Three strains were used in this study: CSS, a susceptible strain reared in the laboratory for many years without exposure to insecticides; CRR, a beta-cypermethrin resistant strain established by selection with beta-cypermethrin for 25 generations in the laboratory from a field housefly strain (CFD) which was collected from a dump in Beijing, China in 1998 and maintained in the laboratory without exposure to insecticides until selected by the topical application in October 2003. 2.2. Chemicals Beta-cypermethrin (95.2%), dichlovos (DDVP, 98.7%) and phoxim (99.0%) were obtained from Tianjin Longdeng

Chemical Co., Ltd. Alpha-cypermethrin (94.4%) and zetacypermethrin (94.2%) were obtained from Suzhou Fumeishi Chemical Co., Ltd. Tetramethrin (95.0%), cyhalothrin (96.0%), deltamethrin (99%) and bifenthrin (95%) were provided by Jiangsu Yangnong Chemical Group Co., Ltd. Propoxur (99%) was supplied by Bayer. Abamectin (97.1%) was supplied by North China Pharmaceutical Group Aino Co., Ltd. Piperonyl butoxide (PBO, 98%) and S,S,S-tributylphosphorotrithioate (DEF, 98%) were purchased from Chem Service (West Chester, PA). a-Naphthyl acetate (a-NA), b-naphthyl acetate (b-NA), a-naphthyl butyrate (a-NB), a-naphthyl caproate (a-NC), a-naphthyl propionate (a-NP), eserine, a-naphthol, b-naphthol, 1chloro-2,4-dinitrobezene (CDNB), reduced glutathione (GSH), phenylmethylsulfonyl (PMSF), dithiothreitol (DTT), phenylthiourea (PTU), fast blue B salt, sodium dodecyl sulfate (SDS) and bovine serum albumin (BSA) were products of Sigma Chemical Co. (St. Louis, MO) at the highest purity available. The other chemicals were of analytical quality and purchased from commercial suppliers. 2.3. Selection For selection, uncoupled houseflies less than 24 h old were treated with beta-cypermethrin solution (in acetone) to the thoracic notum by topical application [32]. The dose for the selection resulted in 60–80% mortality in each generation. The survived houseflies after 24 h treatment with the selected dose were released into the rearing cage as parents of the next generation and kept at 25±1 C, 60–80% RH, and 16 h: 8 h LD photoperiod and supplied with water, sugar and milk powder [17]. 2.4. Bioassays Bioassays were conducted using the topical application to assess the resistance of 4-day-old female houseflies according to the method of Scott and Georghiou [11]. Insecticides were dissolved to 5–7 concentrations in acetone that gave >0% and <100% mortality with 30 houseflies used at each dose. Control houseflies were treated with acetone. The same topical application method was used in the cross-resistance and synergists studies. Synergists, PBO and DEF, were applied at the maximum sublethal dose (1 lg per fly) 1 h before the insecticide treatment [16]. All tests were repeated three times and performed at 25 ± 1 C. Mortality was assessed after 24 h and bioassay data were pooled. 2.5. Biochemical assays 2.5.1. Carboxylesterase assays Carboxylesterase (CarE) activities were determined with five naphthyl-substituted substrates as described by van Asperen [33] with modifications. Four-day-old houseflies (heads were removed) with similar size from each strain

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were homogenized in ice-cold buffer (0.04 M phosphate buffer, pH 7.0). The homogenate was centrifuged at 4 C, 10,000g for 15 min and the supernatant was filtered to remove the lipid layer. For each reaction, 1.8 ml phosphate buffer (0.04 M, pH 7.0) containing 3 · 10 4 M substrate and 3 · 10 4 eserine, and 0.5 ml crude enzyme diluted from the enzyme source with homogenization buffer were incubated at 30 C for 15 min. The reaction was stopped by the addition of 0.9 ml of fast blue B salt solution (2 parts of 1% fast blue B salt and 5 parts of 5% SDS). The optical density (OD) was measured at 600 nm for the hydrolysis production, a-naphthol, of a-NA, a-NB, a-NC and a-NP and at 550 nm for b-naphthol of b-NA. Absorbance values were converted to nmol naphthol/min/mg protein using naphthol standard curves and protein contents. Michaelis constants (Km) and maximal velocities (Vmax) were measured for each substrate which was diluted serially to 5–7 concentrations in homogenization buffer. Values of Km and Vmax were calculated using Enzfit software (Elservier, Cambridge, United Kingdom).

The homogenate was centrifuged at 10,000g for 30 min at 4 C. The filtered supernatant was served as enzyme source. Reaction mixtures contained GSH (final concentration 1.0 mM), enzyme homogenate buffer (0.1 M phosphate buffer, pH 6.5), and CDNB (final concentration 1.0 mM) in a total volume of 0.9 ml. The reaction was started by adding CDNB. Subsequently, the rate of change in optical density (OD) at 340 nm during the initial 2 min was measured using a UV/VIS Spectrometer Lambda Bio-40 (Perkin-Elmer, USA) and converted to activity using the extinction coefficient of 9.6 mM 1 cm 1 for the reaction and the estimated protein content of the enzyme homogenate. The kinetic constants were determined as described above with the GSH concentration, which was kept constant at 1.0 mM, and the CDNB was diluted to 5–7 concentrations.

2.5.2. Cytochrome P450 assays Microsomes from houseflies of the CRR and CSS strains were prepared by the method of Lee and Scott [34]. Only abdomens of 4-day-old houseflies from each strain were homogenized in ice-cold homogenization buffer (0.1 M sodium phosphate buffer, pH 7.5, containing 10% glycerol, 1.0 mM EDTA, 0.1 mM DTT, 1.0 mM PTU and 1.0 mM PMSF) and centrifuged at 4 C, 10,000g for 20 min using a 5417R centrifuge (Eppendorf, Germany). The supernatant was filtrated and centrifuged again at 4 C, 100,000g for 1 h using a HimacCP 80b super-centrifuge (Hitachi Koki, Japan). The microsomal pellets were re-suspended with re-suspension buffer (0.1 M sodium phosphate buffer, pH 7.5, containing 20% glycerol, 1.0 mM EDTA, 0.1 mM DTT and 1.0 mM PMSF) and then diluted to a final protein concentration of 1 mg/ml. The cytochrome P450 was quantitatively analyzed by the method of Omura and Sato with modifications [35]. The microsomal re-suspension was poured into the reference and test cuvettes (1 ml/cuvette) and reduced for 2–3 min by a few milligrams of solid sodium dithionite. Both cuvettes were set in UV/VIS Spectrometer Lambda Bio-40 (Perkin-Elmer, USA) and the baseline was recorded between 400 and 500 nm. Then CO was passed through the test cuvette for 1 min and the difference spectrum between 400 and 500 nm was recorded. The content of cytochrome P450 was calculated with a molecular extinction coefficient of 91 mM 1 cm 1.

2.6. Statistical analysis

2.5.3. Glutathione S-transferase assays Activity of glutathione S-transferase (GSTs) toward CDNB was measured by the method of Habig et al. [36] with modifications. Abdomens from each strain were homogenized in ice-cold buffer (0.1 M phosphate buffer, pH 6.5, containing 1.0 mM EDTA and 1.0 mM DTT).

2.5.4. Protein assays Protein concentration was determined by the method of Bradford [37], using bovine serum albumin as the standard.

To estimate parameters of the dose-mortality regression line for each bioassay, the probit analysis was conducted with POLO software (LeOra Software Inc., Cary, NC) which automatically corrected for control mortality [38]. The resistance ratio was calculated for each insecticide by dividing the LD50 of the CRR strain by the LD50 of the CSS strain or the original CFD strain. All biochemical assays were run in at least triplicate. Student’s t-tests were performed with significance reported for P < 0.05. 3. Results 3.1. Selection As the effect of continuous selection with beta-cypermethrin for 25 generations, the value of LD50 was increased from 4.72 ng/fly to 8131.08 ng/fly and the slope of the log dose-probit line increased from 1.90 to 2.30 in the CRR strain (Table 1). This result demonstrated that the selection with beta-cypermethrin caused considerable change in resistance level, and the increase of slope suggested that the CRR strain became more genetically homogeneous. As shown in Table 1, three characteristic phases could be discerned in the process of resistance development. At the first stage, the fluctuation of LD50 value for beta-cypermethrin was slight and the resistance to beta-cypermethrin was less than 10-fold before the eleven generation of selection (Table 1). After the first stage, the LD50 value increased sharply from 12.36 ng/ fly for F11 to 2355.86 ng/fly for F19 generation, and then finally reached a plateau phase at the third stage. This resistance development pattern indicated that the initial frequency of resistance gene was low in the CRR strain [39].

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Table 1 History of selections with beta-cypermethrin of the resistant house fly strain Generation

LD50 (95% FL) (ng/fly)

Slope (SE)

v2 (df a)

Ratiob

SS F0 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21c F22 F23 F24 F25

1.84 (1.43–2.23) 4.72 (3.61–5.87) 1.52 (1.10–1.94) 4.48 (3.19–5.60) 3.92 (3.12–4.75) 5.13 (3.55–6.70) 7.60 (4.80–10.12) 7.66 (5.17–10.07) 13.95 (10.55–17.35) 6.11 (4.54–8.76) 9.01 (7.03–11.22) 12.36 (9.84–14.98) 18.33 (14.96–21.79) 18.55 (13.00–23.95) 39.66 (25.27–54.09) 138.80 (71.84–223.36) 141.36 (106.81–179.67) 188.44 (155.96–227.51) 446.68 (311.32–649.12) 949.52 (707.04–1.18436) 2355.86 (1670.48–3436.86) 2831.17 (1926.05–3906.86) — 2726.59 (1825.29–3797.99) 6258.35 (4199.16–8222.18) 8328.73 (6156.00–10325.56) 8131.08 (6581.03–10269.13)

2.79 1.90 1.87 2.68 2.40 1.95 2.18 2.54 2.20 2.03 2.43 2.29 3.24 2.76 1.98 1.19 2.49 1.89 1.49 1.93 1.33 2.05 — 2.04 2.40 2.91 2.30

0.75 1.74 0.88 3.89 0.95 1.00 2.99 2.99 0.93 0.99 0.43 1.35 1.09 0.59 3.87 3.66 3.39 0.66 2.92 3.22 2.95 0.62 — 1.06 2.36 2.07 2.36

1.00 2.57 0.82 2.44 2.13 2.78 4.11 4.16 7.59 3.59 4.90 6.71 9.99 10.08 21.55 75.47 76.74 102.41 242.76 516.04 1280.35 1538.67 — 1481.84 3401.23 4526.45 4419.07

a b c

(0.40) (0.19) (0.22) (0.42) (0.22) (0.23) (0.31) (0.33) (0.28) (0.26) (0.23) (0.25) (0.03) (0.35) (0.25) (0.20) (0.25) (0.17) (0.20) (0.29) (0.14) (0.21) (0.23) (0.32) (0.36) (0.20)

(4) (4) (4) (4) (4) (4) (3) (3) (3) (3) (3) (4) (3) (3) (4) (4) (5) (4) (3) (4) (3) (4) (4) (3) (4) (4)

Degree of freedom. Resistance ratio = LD50 of the CRR strain/LD50 of the CSS strain. Not determined.

3.2. Cross-resistance pattern The cross-resistance pattern for different groups of insecticides, pyrethroids, organophosphates, carbamates and abamectin was determined with F25 generation houseflies (Table 2). In the group of pyrethriod insecticides, the highest level of resistance was developed to deltamethrin (14,403.81fold), which is very similar to cypermethrin in structure (i.e., only the chlorine atom at the vinyl of cypermethrin is substituted by the bromine in deltamethrin). The resistance to alpha-cypermethrin and zeta-cypermethrin was evolved to 532.23- and 1,587.17-fold, respectively. However, the resistance was only 54.16- and 10.70-fold compared with the CFD strain for tetramethrin and bifenthrin. The resistance to antiacetylcholinesterase agents was detected in the CRR strain, and the resistance ratio was 20.74 for DDVP, 14.12 for phoxim and more than 35.00 for propoxur, respectively. The CRR strain also developed a low level of cross-resistance to abamectin (6.40-fold), a member of macrocyclic lactone anthelminitics used as miticide and insecticide which interacted with GABA receptor and glutamate-gated chloride channels [40]. 3.3. Synergism The effect of PBO and DEF on beta-cypermethrin toxicity to the CRR and CSS strains was shown in Table 3. The

synergistic potential of PBO to beta-cypermethrin in the CRR strain was very similar to that in the CSS strain, and the synergistic ratio was 21.88 for the CRR strain and 15.33 for the CSS strain, respectively. The resistance of the CRR strain was decreased to 3309.75-fold. However, the synergistic pattern of DEF to beta-cypermethrin was very different from that of PBO in both CRR and CSS strain. DEF caused 364.29-fold synergism in the CRR strain, however, only 2.35-fold in the CSS strain. The resistance to beta-cypermethrin was deduced dramatically from 4419.07- to 28.61-fold in the CRR strain. These results suggested that the esterase played much more important role than cytochrome P450 in the CRR strain to beta-cypermethrin resistance. 3.4. Biochemical assays Activities and maximal velocities (Vmax) of carboxylesterases were significantly greater in the CRR strain than that in the CSS strain (P < 0.05) when five naphthyl-substituted chemicals were used as substrates. The ratio of the carboxylesterase activity was 4.25 for a-NA, 6.91 for bNA, 5.20 for a-NC, 5.25 for a-NP and 5.75 for a-NC compared with the CSS strain, respectively, and the ratio of maximal velocities ranged from 3.21 for a-NA to 7.16 for a-NC (Table 4). However, there were no significant differences in the Michaelis constants (Km) for all tested

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Table 2 Responses of the CRR strain to different insecticides Strain

Insecticide

LD50 (95% FL) (ng/fly)

Slope (SE)

v2 (df a)

RRb

CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR CFD CRR

Beta-cypermethrin Beta-cypermethrin Alpha-cypermethrin Alpha-cypermethrin Zeta-cypermethrin Zeta-cypermethrin Deltamethrin Deltamethrin Tetramethrin Tetramethrin Bifenthrin Bifenthrin DDVP DDVP Phoxim Phoxim Propoxur Propoxur Abamectin Abamectin

4.72 (3.61–5.87) 6258.36(4199.16–8222.18) 9.03 (5.47–15.78) 4806.06 (3909.54–5990.44) 2.41 (1.69–3.12) 3825.09 (3102.86–1550.44) 0.75 (0.35–1.12) 10802.86(8839.01–17600.91) 83.96 (54.87–125.73) 4547.41 (3123.99–6325.75) 6.60 (3.33–10.05) 70.64 (61.07–82.28) 28.15 (18.98–38.01) 584.11 (480.53–703.36) 104.36 (70.28–136.40) 1472.83 (1071.48–2017.92) 160.24 (100.64–226.68) >5600.00 7.08 (5.30–9.98) 45.32 (24.08–66.06)

1.90 2.40 1.17 1.88 1.97 2.60 1.43 3.31 1.39 1.59 1.78 2.96 2.00 3.73 2.69 2.23 1.44 — 1.82 1.55

1.74 2.36 0.44 1.34 2.29 3.60 2.78 0.40 1.75 2.52 3.56 0.53 0.78 0.36 2.56 2.32 2.21 — 2.35 0.65

1.00 1325.92 1.00 532.23 1.00 1587.17 1.00 14403.81 1.00 54.16 1.00 10.70 1.00 20.74 1.00 14.12 1.00 >35.00 1.00 6.40

a b

(0.19) (0.32) (0.21) (0.26) (0.20) (0.27) (0.22) (0.83) (0.24) (0.33) (0.24) (0.25) (0.37) (0.35) (0.53) (0.27) (0.20) (0.18) (0.24)

(4) (4) (4) (4) (4) (5) (5) (3) (4) (5) (5) (4) (4) (3) (4) (4) (4) (5) (4)

Degree of freedom. Resistance ratio = LD50 of the CRR strain/LD50 of the CFD stain.

Table 3 Synergism of PBO and DEF on beta-cypermethrin in the CSS and CRR strain Compound

Beta-cypermethrin +PBO +DEF a b

CSS

CRR 2

a

b

LD50(95% FL) (ng/fly)

Slope (SE)

v (df )

SR

LD50 (95% FL) (ng/fly)

Slope (SE)

v2(df a)

SRb

1.84 (1.43–2.23) 0.12 (0.08–0.16) 0.78 (0.55–0.98)

2.79 (0.40) 1.83 (0.24) 1.96 (0.26)

0.75 (5) 2.30 (4) 1.59 (4)

1.00 15.33 2.35

8131.08 (6581.02–10269.13) 371.49 (173.56–589.11) 22.32 (13.77–31.37)

2.40 (0.32) 1.05 (0.19) 1.54 (0.20)

2.36 (4) 4.55 (6) 1.15 (4)

1.00 21.88 364.29

Degree of freedom. Synergism ratio = LD50 of beta-cypermethrin/LD50 of PBO or DEF+ beta-cypermethrin.

Table 4 Carboxylesterase activities and kinetic parameters to five substrates in the CSS and CRR strain Substrate

CSS

CRR a

CarE activity a-NA b-NA a-NB a-NP a-NC

34.56 ± 12.83 13.41 ± 1.96 81.45 ± 26.58 69.05 ± 1.16 25.96 ± 4.24

Kmb 25.07 ± 5.49 34.48 ± 1.50 43.28 ± 4.45 45.24 ± 1.28 29.48 ± 4.47

Vmaxc 46.06 ± 3.02 17.73 ± 2.47 105.64 ± 7.68 82.89 ± 7.69 30.09 ± 1.38

CarE activitya *

146.54 ± 15.98 92.84 ± 28.51* 422.79 ± 97.75* 362.03 ± 97.47* 149.24 ± 5.27*

Kmb

Vmaxc

23.81 ± 5.05 31.33 ± 4.04 34.65 ± 3.74 18.55 ± 2.58* 37.29 ± 6.73

148.24 ± 8.97* 121.78 ± 4.81* 615.76 ± 20.96* 463.20 ± 17.29* 215.46 ± 12.40*

Values are shown as means ± SD of 3 determinations. a CarE activity = lmol product/min/mg protein. b Km (Michaelis constant) = nM. c Vmax (Maxinum velocity) = lmol product/min/mg protein. * Indicates the value is significantly different form that of the CSS strain (Student’s t test, P < 0.05).

substrates except for a-NP (P < 0.05) between the CRR and CSS strains. The CRR strain exhibited higher GSTs activity and Vmax (1.27- and 1.36-fold, respectively) as compared to the CSS strain (Table 5). However, statistical analysis (Student’s t test) indicated that there was significant difference in the Vmax values of GSTs but not the specific activities of GSTs between the CRR and CSS strains, indicating that

GSTs played a very limited role in beta-cypermethrin resistance in the CRR strain. Interestingly, the content of cytochrome P450 was 53.08 ± 1.33 nmol/mg protein in the CRR strain, which was a little lower than that of the CSS train, 66.81 ± 8.93 nmol/mg protein, and the student’s t test indicated there was no significant difference in the level of total cytochrome P450 between the CRR and CSS strains.

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Table 5 Activity, kinetic parameters of glutathione S-transferase and the level of total cytochrome P450 in the CRR and CSS strain Strain

GSTs

Cytochrome P450 a

CSS strain CRR strain

Activity

Kmb

Vmaxc

Contentd

65.97 ± 12.70 84.16 ± 3.77

0.12 ± 0.05 0.10 ± 0.03

61.34 ± 2.86 83.69 ± 11.58*

66.81 ± 8.93 53.08 ± 1.33

Values are shown as means ± SD of 3 determinations. a Activity = nmol/min/mg protein. b Km (Michaelis constant) = mM, detected with the different concentrations of CDNB. c Vmax (Maxinum velocity) = nmol/min/mg protein. d Content of the total cytochrome P450 = nmol/mg protein. * Indicates the value is significantly different form that of the CSS strain (Student’s t test, P < 0.05).

The above results of both the synergisms and the biochemical assays suggested that the enhanced esterase activity played an important role in the beta-cypermethrin resistance compared with GSTs and cytochrome P450 in the CRR strain. 4. Discussion Pap and Toth [41] used the susceptible reference strain (WHO/SRS) to design a beta-cypermethrin resistant housefly strain and studied the selection procedures, the resistance development and the cross-resistance pattern as well as synergism of PBO. The resistance ratios to betacypermethrin in the females and males were only 164.9 and 190.2, respectively, when compared to the susceptible strain after continuous selection for 25 generations. The authors also deduced that esterase mediated metabolism and kdr- and/or super-kdr mechanisms presented likely in the resistant strain from results of cross-resistance. However, our results here showed that the continuous selection with beta-cypermethrin resulted in very high resistance (RR = 4419.07), and we studied further the mechanisms responsible for the resistance to beta-cypermethrin by bioassays of synergists and biochemical assays which suggested that carboxylesterases play an important role for the pyrethroid resistance in the CRR strain. The selection with beta-cypermethrin resulted in crossresistance to an array of insecticides, some of which act on different target sites. In pyrethroid insecticides, which alter the function of voltage-sensitive sodium channels, the CRR strain developed various degrees of resistance to pyrethroids tested. The relative order of the resistance developed in the CRR strain was deltamethrin > alphacypermethrin > zeta-cypermethrin > tetramethrin > bifenthrin. This result was supported by the previous report of Huang and Ottea [42] that resistance to pyrethroids with 3-phenoxybenzyl alcohols (i.e., permethrin, cypermethrin, deltamehrin) was greater than that of pyrethroids lacking this group (i.e., tefluthrin, bifenthrin, teramethrin), indicating the penoxybenzyl group contained targets for metabolism in pyrethroids resistant insects. Interestingly, our results showed that the CRR strain selected by beta-cypermethrin had very high level of cross-resistance to deltamethrin (14,403.81-fold) as compared to the CSS strain.

Ru et al. [43] reported the resistance ratio was 30.6 and 337.0 to cyhalothrin and fenvalerate, respectively, in a strain of Helicoverpa armigera that was selected by cyhalothrin, and the difference in kdr to fenvalerate and cyhalothrin mainly accounted for the difference in the resistance level to these two pyrethroids. In the CRR strain, there might also be some differences in the major resistance mechanism to deltamethrin and beta-cypermethrin. The resistance to different isomers of cypermethrin, (beta-cypermethrin, alpha-cypermethrin and zeta-cypermethrin) was significantly different. Similar result was observed in other strains selected with pyrethroid insecticides [44], suggesting the importance of constitutional and stereo-chemical structure of pyrethroids for the development of resistance or cross-resistance in insects. The CRR strain also exhibited resistance to two organophosphates (DDVP and phoxim) and one carbamate (propoxur), which are inhibitors of acetylcholinesterase in cholinergic synapses of insect nerve systems. Sawicki [45] considered that this pattern of cross-resistance to organophosphates and carbamates induced by pyrethroids selection suggested that esterases and/or monooxygenase-mediated detoxification played an important role in resistance of insects to these three types of insecticides. At the same time, CRR developed 6.40-fold resistance to abamectin. Scott [46] studies indicated that LPR, a strain selected with permethrin, developed higher level (25-fold) cross-resistance to abamectin that was mainly mediated by monooxygenase. This difference in cross-resistance to abamectin suggested monooxygenase-mediated detoxification play a limited role in beta-cypermethrin resistance in CRR. Therefore, we conclude that esterase is the key factor for the resistance and/ or cross-resistance to organophosphates, carbamates and pyrethroids in the CRR strain. Synergist bioassays are commonly used to test for the involvement of metabolic mechanisms in insecticides resistance. In this study, PBO, a synergist inhibiting the monooxygenase in oxidative metabolic system, increased beta-cypermethrin toxicity by 21.88-fold in the CRR stain in contrasted to 15.33-fold in the CSS strain and the synergism resistance ratio (SRR = SR of resistance strain/SR of susceptible strain) was only 1.43. DEF, an esterase inhibitor, increased the beta-cypermethrin toxicity by 364.29-fold and significantly reduced the resistance ratio from 4419.07

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to 28.62, indicating that esterases-mediated resistance was an important mechanism for beta-cypermethrin in the CRR strain as compared to monooxygenases mediatedresistance and the residual resistance measured might be due to the presence of additional mechanisms (e.g., the enhanced monooxygenase activity and the reduced target site sensitivity). These findings were consistent with those from the biochemical assays about activities of carboxylesterases. The specific activities of carboxylesterases for all tested substrates were significantly higher in the CRR strain than in the CSS strain. However, there was no significant difference in the activity of GSTs and the level of total cytochrome P450 between the CRR and CSS strains. The esterase-based resistance mechanisms characterized to date predominantly include the elevation of activity through gene amplification and point mutations within the esterase structural genes which change their substrate specificity [47]. In this paper, carboxylesterases activities and Vmax toward five substrates were much higher in the CRR strain than in the CSS strain, but there were no significant differences in the Km for all tested substrates except for a-NP (P < 0.05) between the CRR and CSS strain, suggesting that carboxylesterases in these two strains were quantitatively different but qualitatively similar [48]. It suggested that the CRR strain developed resistance through the gene amplification of the esterase. Over-production of non-specific carboxylesterases as an evolutionary response to the selection pressure of organophosphate and carbamate insecticides has been documented in numerouse arthropod species [49]. There are also some reports of enhanced esterase activities responsible for pyrethroids resistance in insects. However, no clear pattern was further revealed [50–52]. In our studies, although results from bioassay of synergists and biochemical assays indicate that the esterase-meditated resistance due to esterase gene(s) amplification is a major mechanism for high level resistance to beta-cypermethrin, our conclusions were necessarily tenuous and further studies were required since the information about molecule biology properties of esterase-mediated pyrethroids resistance in houseflies were not studied. Therefore, studies and results in this paper provided a foundation for subsequent efforts for studying the potential role of esterase involved in the resistance to pyrethroids in insects. Acknowledgments This research was supported by National Basic Research Program of China (Contract No. 2006CB102003) and National Natural Science Foundation of China (Contract No. 30530530, 30571232, 30471153, and 30170621). References [1] J.E. Casida, Pyrethrum flowers and pyrethroid insecticides, Environ. Health Perspect. 34 (1980) 189–202.

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