Pesticide Biochemistry and Physiology 87 (2007) 62–72 www.elsevier.com/locate/ypest

Comparative study on glutathione S- activity, cDNA, and gene expression between malathion susceptible and resistant strains of the tarnished plant bug, Lygus lineolaris

Yu Cheng Zhu a,¤, Gordon L. Snodgrass a, Ming Shun Chen b

a Jamie Whitten Delta States Research Center, ARS-USDA, Stoneville, MS 38776, USA b ARS-USDA-GMPRC, Manhattan, KS 66502, USA

Received 4 April 2006; accepted 5 June 2006 Available online 9 June 2006

Abstract

Control of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), in cotton in the mid-South relies heavily on pesticides, mainly organophosphates. Continuous and dominant use of chemical sprays has facilitated resistance development in the tarnished plant bug. A natural population in Mississippi with resistance to malathion was studied to examine whether and how glutathione S- (GST) played an important role in the resistance. Bioassays were Wrst conducted to examine synergism of two GST inhibitors. Both ethac- rynic acid (EA) and diethyl maleate (DM) eVectively abolished resistance and increased malathion toxicity against two resistant strains by more than 2- and 3-fold, whereas incorporation of GST inhibitors did not signiWcantly increase malathion toxicity against a susceptible strain. GST activities were compared in vitro between malathion susceptible and resistant strains by using 1-chloro-2,4-dinitrobenzene as a GST substrate. The resistant strain had signiWcantly higher (1.5-fold) GST activity than the susceptible strain. Up to 99%, 75%, and 85% of the GST activities were inhibited by EA, sulfobromophthalein (SBT), and DM, respectively. The GST activities tended to increase from May to October by 1.76-fold. All three inhibitors signiWcantly suppressed the GST activity to a constant low level over the season. Further examination of GST cDNA indicated that in the coding region only one nucleotide variation was revealed between the suscepti- ble and resistant strain. This variation did not cause a protein sequence change, and an identical amino acid sequence was predicted for both strains. Multiple sequence alignment and phylogenetic analysis showed that the 216-residue GST from the tarnished plant bug was highly similar (up to 68% of amino acid sequence identity) to the GSTs from other insects, which conferred organophosphate resistance. GST gene expression levels were examined using real-time PCR, and the results indicated that GST gene transcripts were elevated in the resistant strain by 1.3-fold. Published by Elsevier Inc.

Keywords: Malathion; Resistance; cDNA; mRNA; Expression; Glutathione S-transferase; GST; Inhibitor; Tarnished plant bug; Lygus lineolaris

1. Introduction on cotton, and this has resulted in widespread resistance to pyrethroid and increased resistance to organophosphate Although Bt cotton that produce Cry toxins eVectively insecticides [1–3]. Zhu et al. [3] showed that more than 11- control many important lepidopteran species, many suck- fold malathion resistance was present in a natural popula- ing insects, especially the tarnished plant bug Lygus lineo- tion of the tarnished plant bug collected in Mississippi, laris (Palisot de Beauvois), have emerged in recent years as which was also resistant (4.5-fold) to the pyrethroid insecti- economically important pests. Chemical spray with insecti- cide permethrin. cides has been the dominant measure for plant bug control Development of resistance to pyrethroid and tolerance or resistance to organophosphate insecticides in the tar- * Corresponding author. Fax: +1 662 686 5421. nished plant bug is major factor for its increasing pest sta- E-mail address: [email protected] (Y.C. Zhu). tus on cotton. Understanding resistance mechanisms is

0048-3575/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.pestbp.2006.06.002 Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 63 essential for developing strategies to manage the resistance. grown near Crossett and plant bugs from this location were A previous study [3] indicated that elevated esterase gene found to be very susceptible to most insecticides used on expression and esterase activity was associated with mala- cotton [22]. The resistant strain was originally collected thion resistance in a resistant strain. By mutation and/or from weeds in or near cotton Weld near Mound Bayou elevated gene expression, esterases play an important role (Bolivar County), MS. The Weld collected adults from in organophosphate resistance development in many resis- Mound Bayou were selected with malathion in the labora- tance cases [4–8]. Moreover, esterases can also be involved tory to produce a test population for activity assays in pyrethroid resistance. Valles [9] found a signiWcant corre- and molecular analyses made up of individuals more lation between general esterase activity and the pyrethroid homozygous for malathion resistance. They were selected resistance level in Blatella germanica. In the mid-South by exposing adults for 3 h in 20-ml glass vials that had been area, pyrethroid insecticides are no longer recommended treated with 100 g of malathion per vial. Preliminary tests for control of plant bugs because of resistance. Organo- showed that 100 g of malathion would kill about 50% of phosphate insecticides, including malathion, are the major the plant bugs in 3 h of exposure. The LC50 of the selected chemicals used for plant bug control on cotton. The natural adults was not determined. Two adults were placed in each population of tarnished plant bugs we studied had devel- vial and survivors were held for 24 h to eliminate any adults oped resistance to both pyrethroid and organophosphate that subsequently died. Another malathion resistant popu- insecticides. However, resistance levels to malathion were lation was collected in Bruce (Calhoun County), MS. This more prominent than the resistance to permethrin [3]. We colony was used for the synergist bioassay and was unse- assumed that factors other than elevated esterase activity lected. Susceptible and resistant plant bugs were tested in might contribute to malathion resistance development in the bioassays within one week after they collected in the this population. It was also thought that increased esterase Weld. gene expression, rather than a mutation, was the underlying mechanism for the resistance to malathion. 2.2. Bioassay and synergist test Glutathione S-transferases (GSTs) are detoxifying found in vertebrates, plants, insects, yeasts, and A glass-vial bioassay [22] was used to determine resis- aerobic bacteria [10,11]. The primary function of GSTs is tance levels and synergism of two GST inhibitors. Twenty- generally considered to be the detoxiWcation of both endog- ml glass scintillation vials were Wrst treated with malathion enous and xenobiotic compounds either directly or by cata- (798% technical grade, Chem Service, West Chester, PA) lyzing the secondary metabolism of a vast array of or GST inhibitors, ethacrynic acid (EA) (Sigma, St. Louis, compounds oxidized by the cytochrome P450 family [12]. MO) and diethyl maleate (DM) (Aldrich, Milwaukee, WI). GSTs catalyze the conjugation of glutathione (GSH) to Acetone was used as the solvent, and was included as a con- hydrophobic substrates, such as herbicides and insecticides trol. Malathion and GST inhibitors were applied by pipet- [13], which facilitates the metabolism of target compounds ting 0.5 ml of the insecticide or an inhibitor diluted in [10,14–16]. Several studies indicated that GSTs have an acetone into each vial. Each vial was rolled on its side until important role in the acquisition of resistance to insecti- an even layer of insecticide or inhibitor dried on its inner cides. Some resistant insects have been found to metabolize surface. Malathion and GST inhibitors were applied to the insecticides more eYciently via a GSH-dependent route vials on the same day the test was performed. A small piece [17]. High levels of GST activity have been detected in some of green bean pod, Phaseolus vulgaris L. (cut transversely) resistant insect strains [18–20], and the resistance develop- about 3 mM thick was added to each vial as food for the ment had been correlated with enhanced GST activity and adults, and a cotton ball was used to seal each vial. Adult GST-dependent insecticide metabolism [21]. Because GSTs plant bugs were placed into treated vials (2 per vial). Syner- are capable of catalyzing the secondary metabolism follow- gists were tested in the glass vials by exposing adults to ing the oxidation by the cytochrome P450s [12], it is impor- 400 g/vial DM or 400 g/vial EA for 24 h. Adults were tant to understand whether and how the GSTs play a role then transferred to vials (2 per vial) treated with malathion. in resistance development in the tarnished plant bug, which Vials were held during a test in an upright position at labo- can be resistant to both pyrethroid and organophosphate ratory conditions of 24–26 °C, and humidity was not insecticides. In this study, we assessed synergism of GST controlled. Mortality was determined after 24 h, and adults inhibitors and compared GST cDNA sequences, enzyme were considered dead if they were unable to stand normally activities, and GST gene expression levels between suscepti- or walk, or there was no movement when they were prod- ble and resistant strains. ded. All bioassays had three replications for each treatment, and each replication contained 10 vials with two adults in 2. Materials and methods each vial. In the Wrst experiment, malathion was used at 25 g/vial, and resistant bugs were collected from Mound 2.1. Insect Bayou, MS. In the second experiment, malathion at 5 g/ vial was used for susceptible bugs collected from Crossett, The malathion-susceptible strain was collected from AR and malathion at 25 g/vial was used for resistant bug weeds near Crossett (Ashley County), AR. Cotton is not collected from Bruce, MS. The concentrations were 64 Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 determined based on LC50 values obtained from pretrial CNTAYYT and GST2F: CARMGNYTNTAYTTYGAY experiments. ATGGGNAC, were designed. These two primers were expected to anneal to corresponding sequences coding for 2.3. Enzyme preparation two conserved regions (ESRAIMTYL, QRLYFDMGT) of a GST from the brown planthopper, Nilaparvata lugens Individual tarnished plant bugs were homogenized in [27]. These two regions are highly conserved in other insect 340l, 0.1M sodium phosphate buVer, pH 8.0. Homogenates GSTs, including those from Drosophila simulans (Gen- were then centrifuged at 10,000g for 5min at 4°C, and the Bank: AAK66764), Drosophila melanogaster (AAM52032), supernatant was used for enzyme analysis. Protein concentra- Drosophila mauritiana (P30105), Musca domestica tions were determined using the Bradford method [23]. (P28338), Lucilia cuprina (P42860), Bactrocera papayae Bovine serum albumin was used to obtain the standard curve. (AAO19738), Anopheles gambiae (Q93113), Anopheles dirus (AAB41104), Culicoides variipennis (AAB94639), and Gal- 2.4. Glutathione S-transferase (GST) assays leria mellonella (AAK64362). RT-cDNA was used as the template for the Wrst PCR ampliWcation with GST1F and To determine glutathione transferase activity, microtiter an oligo(dT) primer (no band was obtained). The second plate assays were conducted using 1-chloro-2,4-dinitroben- semi-nested PCR was conducted to amplify the Wrst PCR zene (CDNB, Aldrich, Milwaukee, WI). The pre-reaction product (GST1F + dT) with GST2F + dT primers. A dis- mixture (100l) consisted of the following: 8mM glutathione tinct fragment, approximately 600 bp, was revealed from (Sigma, St. Louis, MO), 10l enzyme homogenate, and 0.1 M the second PCR ampliWcation. The cDNA fragment band phosphate buVer pH 8.0. Reaction was started with the addi- was sliced from a low-melting point gel and extracted using tion of 200l CDNB in 0.1M phosphate buVer pH 8.0 con- the Promega (Madison, WI) Wizard PCR Preps DNA puri- taining 15% glycerol with a Wnal concentration of 0.5 mM. A Wcation system. PuriWed DNA was cloned into a pGEM-T Bio-Tek plate reader ELx808IU equipped with a 340nm Wlter vector (Promega) and then it was sequenced. The fragment was used to monitor the activity at 25°C, for 10min with mea- was conWrmed as a part of the GST cDNA by similarity surements taken every 15s [24]. Inhibitors for glutathione searching [28] the GenBank database (BlastX, NR) of the transferase activity were EA (Wnal concentration 0.19mM), National Center for Biotechnology Information (NCBI). sulfobromophthalein sodium hydrate (Aldrich, Milwaukee, To obtain the 5Ј-end of the GST cDNA, three reverse WI) (Wnal concentration 0.01mM), and DM (Wnal concentra- primers, LLGSTR1, LLGSTR2, and LLGSTR3 (Fig. 4), tion of 0.712mM). Inhibitor concentrations were referred were synthesized. Reverse transcription was performed from those of Prapanthadara et al. [25]. Inhibitors were added using the reverse primer LLGSTR1, and the 5Ј-end of to enzyme homogenate and allowed to incubate at 37°C for GST cDNA was isolated and C-tailed using the 5Ј-RACE 10min before the addition of substrate [26]. (rapid ampliWcation of cDNA end) system (Invitrogen). Subsequent semi-nested ampliWcations were performed 2.5. Seasonal survey of GST activities using a forward abridged anchor primer (5Ј-RACE kit) and the speciWc reverse primers LLGSTR2 and Through the months of May to October of 2003, tar- LLGSTR3. The DNA fragment resolved from the 5Ј- nished plant bugs were collected from weeds in or near a RACE ampliWcation was cloned into a pGEM-T vector cotton Weld in SunXower County, Mississippi. Ten individ- and sequenced from both directions. GST cDNA was ual adults for each month were tested for levels of GST assembled, and it was conWrmed that it contained the full activity as previously described in duplicate. coding sequence for the GST-like protein. To obtain full GST-like cDNA from the susceptible and malathion- 2.6. RNA extraction and cDNA synthesis selected strains of the tarnished plant bug, total RNA was extracted from these strains and used for RT-cDNA syn- Five tarnished plant bug adults (approximately 30mg) thesis. A forward primer LLGSTF0 and a reverse primer were homogenized in 500l Trizol solution (Invitrogen, Carls- LLGSTR1 were used in PCR to Xank the 5Ј and 3Ј non- bad, CA). Total RNA was precipitated with isopropanol and coding regions, respectively (Fig. 4). To eliminate potential  resuspended in double distilled-H2O. Five g of RNA was error created by Taq DNA polymerase, a thermal-stable used as a template for cDNA synthesis using an oligo(dT) proof-reading Pfu DNA polymerase (Promega, Madison, primer in the reverse transcription (RT) reaction (SuperScript WI) was used to reamplify a full length GST coding First Strand cDNA Synthesis System, Invitrogen). The proce- region cDNA from the RT-cDNAs of the susceptible and dures were performed using insects from the susceptible strain resistant strains. The resolved cDNA fragments (700 bp) and repeated using insects from the malathion-selected strain. were puriWed from low-melting point agarose gel, and then A-tailed for 30 min at 72 °C using the nucleotide ade- 2.7. Cloning glutathione S-transferase cDNA nine and Taq DNA polymerase. They were then cloned into a pGEM-T vector (Promega), and the resolved clones To clone GST cDNA fragments, two degenerate for- were sequenced from both directions on an ABI 3700 ward primers GST1F: GARWSNMGNGCNATHATGA sequencer. Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 65

The BlastX program was used to search the sequence bug (F D 25.55, df D 2, P < 0.05). Addition of both DM and database of the National Center for Biotechnology Infor- EA increased 24-h mortality by more than 3-fold. In the mation Internet server for proteins with amino acid second experiment (Fig. 2), susceptible bugs showed sequence similarity to the GSTs [28]. The ExPASy Proteo- 41.7 § 14.2% mortality after 24 h treatment with malathion mics tools of the ExPASy Molecular Biology Server of at 5 g/vial (Fig. 2A). Malathion with DM and EA Swiss Institute of Bioinformatics (http://www.expasy.ch/ increased mortality to 50 § 4.9% and 46.7 § 10.0%. But, the tools/) were used to process data of the deduced protein mortality increase was not signiWcant (F D 0.16, df D 2, sequences. DNAStar software (version 6; ClustalW P > 0.05). In contrast to the susceptible strain, resistant bugs method; [29], parameters were set as default: gap showed 25 § 4.9% mortality after treatment with malathion penalty D 10, gap length penalty D 0.2, delay divergent at 25 g/vial (Fig. 2B). Similarly, treatment with either GST seqs(%) D 30, and Gonnet series was used for protein inhibitor signiWcantly increased eYcacy against the resis- weight matrix.) or an Internet Server (http://pbil.ibcp.fr) tant tarnished plant bug (F D 13.63, df D 2, P < 0.05). Addi- was used to conduct multiple sequence alignment. tion of both DM and EA increased 24-h mortality by more than 2-fold. 2.8. Quantitative reverse transcription (QRT) PCR analysis of GST mRNA expression 3.2. Comparison of GST activities between S and R strains

The expression levels of GST mRNAs were determined Glutathione S-transferase activities were examined quantitatively in the susceptible and resistant strains using in vitro using 1-chloro-2,4-dinitrobenzene as the GST sub- a real-time PCR system (ABI 7500, Applied Biosystems, strate (Fig. 3). Protein was also treated with three GST Foster City, CA) and iScript One-Step RT-PCR Kit with inhibitors, EA, sulfobromophthalein (SBT), and DM, to SYBR Green (Bio-Rad, Hercules, CA). Two primers, examine suppression of GST activities. The baseline of LLGSTF3 and LLGSTR6 (Fig. 4), were designed to GST activity in the susceptible strain was 90.05 § 8.8. The amplify a 93 bp GST cDNA fragment. A clear single band resistant strain had 134.4 § 8.27 baseline activity, which was was obtained from a pre-trial gradient PCR ampliWcation signiWcantly (1.5-fold) higher than that of the susceptible W of GST cDNA within Tm ranging from 55 to 68 °C. RNA strain. Pooled data also showed signi cant greater activity was individually extracted from adults of the susceptible in the resistant strain (57.65) than that in the susceptible and resistant strains of the tarnished plant bug. The RNA strain (31.69) (F D 39.27, df D 1, P < 0.0001). Inhibitor treat- was reverse transcribed using a SuperScript First Strand ments also signiWcantly suppressed GST activities in both cDNA Synthesis System (Invitrogen). The RT cDNA was strains (F D 128.61, df D 3, P < 0.0001). EA treatment signiW- spectrophotometrically quantiWed and equal amount of the cantly suppressed GST activity by 99% and 96% in the sus- cDNA was added to each real-time PCR. An absolute ceptible and resistant strains, respectively. SBT inhibited quantiWcation protocol was adopted and a series of concen- 75% GST activity in the susceptible strain and 65% GST trations of the cloned GST cDNA were used as standards. activity in the resistant strains. Similarly, DM suppressed Two step PCR (95 °C for 10 s and 60 °C for 1 min) was con- 85% GST activity in the susceptible strain and 67% GST ducted for 40 cycles and data were collected at 60 °C. activity in the resistant strains.

2.9. Data analysis 3.3. Seasonal change of the GST activity

Means for each treatment were compared using ANOVA The tarnished plant bug adults were collected once a and separated using Fisher’s protected least signiWcant diVer- month from May to October in 2003. GST activities were ence procedure at the D0.05 level (PROC GLM, SAS ver- measured using 1-chloro-2,4-dinitrobenzene as the sion 9.1, SAS Institute Inc., Cary, NC). Regression analysis substrate in the presence or absence of inhibitors. Results was conducted with the SAS Proc Reg procedure. demonstrated that without an inhibitor the GST activity increased as the season progressed (Fig. 4). The lowest GST 3. Results activity (31.11 § 4.58) was obtained in May, the earliest month that samples were collected from the Weld. The high- 3.1. Synergism of GST inhibitors est GST activity (54.85 § 3.93) was found in the insects col- lected in October, the last samples for the year. The GST In the Wrst experiment, no plant bug died 24 h after treat- activities increased 1.76-fold over the season. The GST ment with either DM alone (400 g/vial) or EA alone activities obtained in September and October were signiW- (400 g/vial) (Fig. 1). Malathion alone (25 g/vial) killed cantly higher than those obtained in May and June approximate 20% of the plant bugs. The same concentra- (F D 3.52, df D 5, P < 0.05). Regression analysis indicated tion of the malathion with DM killed 61.7 § 1.7% of treated that the linear relationship between GST activity and plant bugs, and the malathion with EA killed 68.3 § 4.4% of season was signiWcant (F D 16.95, P < 0.01). Unlike the con- treated plant bugs. Treatment with either GST inhibitor trol, the GST treated with any of three inhibitors showed signiWcantly increased toxicity against the tarnished plant signiWcantly lower activity (F D 235.49, df D 3, P < 0.0001). 66 Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72

80 obtained, a full GST coding region (700 bp) was ampliWed a from both susceptible and resistant RT-cDNAs using for- a ward primers LLGSTF0 and LLGSTR1 and proof-reading 60 Pfu DNA polymerase. Combined full length cDNA from the susceptible strain contained 889 nucleotides (LLSGST, Fig. 5). BlastX search of the GenBank indicated that the 40 LLSGST was matched to insect GST and its open reading

b frame (76–723 bp, Fig. 5) encoded a full GST protein Mortality (%)±SE sequence (216 amino acid residues). A GST fragment was 20 also sequenced from the resistant strain, and sequence alignment indicated that the cDNA fragment ampliWed from the resistant strain had one nucleotide diVerence from 0 Acetone DM EA Malathion Malathion+DM Malathion+EA the cDNA of the susceptible strain at position 477 (Fig. 5). Treatments A cytosine was obtained from the susceptible strain and a Fig. 1. Response of the tarnished plant bug adults (resistant strain from thymine from the resistant strain. However, the cDNAs Bayou, MS) to malathion only (25 g/vial) or malathion in combination from both strains coded for an identical protein sequence. with the glutathione S-transferase inhibitors diethyl maleate (DM) and ethacrynic acid (EA). DiVerent letters on the top of bars indicate signiW- 3.5. GST genomic DNA sequence cant diVerence (P < 0.05) between treatments. A 700-bp fragment was ampliWed from genomic DNA of EA almost completely suppressed GST activity over whole the susceptible strain using primers LLGSTF0 + season. The GST treated with SBT showed irregular Xuctu- LLGSTR1. Pair-wise alignment with corresponding cDNA ations over the season, whereas the GST treated with DM of the same strain showed exactly identical sequences, indi- showed a decreasing trend over the season, but no signiW- cating no intron exists in the corresponding region of the cant linear relationship was detected (F D 1.68, P >0.05). GST gene (Fig. 5).

3.4. GST cDNA sequence 3.6. Deduced GST

A GST cDNA fragment was successfully ampliWed by Predicted GST-like protein consisted of 26 positively using two degenerate primers with oligo(dT) reverse charged residues (Arg + Lys) and 26 negatively charged res- primer. Five clones were sequenced and they contained idues (Asp + Glu). Deduced GST-like protein had a molec- identical sequences. After the 5Ј-end of the GST cDNA was ular mass of 23.95 kDa and a theoretical pI of 6.72. It had

A B 70 Susceptible strain Resistant strain

60 a a a a a 50

40

b 30 Mortality (%)±SE

20

10

0

Malathion Malathion Malathion+DM Malathion+EA Malathion+DM Malathion+EA Treatments

Fig. 2. (A) Response of the tarnished plant bug adults (susceptible strain from Crossett, AR) to malathion only (5 g/vial) and the combination of mala- thion with the glutathione S-transferase inhibitors, diethyl maleate (DM) and ethacrynic acid (EA). DiVerent letters on the top of bars indicate signiWcant diVerence (P < 0.05) between treatments. (B) Response of the tarnished plant bug adults (resistant strain from Bruce, MS) to malathion only (25 g/vial) and the combination of malathion with glutathione S-transferase inhibitors. DiVerent letters on the top of bars indicate signiWcant diVerence (P <0.05) between treatments. Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 67

160

a 140 Susceptible Resistant 120

100 b

80

60 c c

40 d GST Kinetics (Mean Velocity±SE) 20 de e e 0 No Inhibitor Ethacrynic acid Sulfobromophthalein Diethyl maleate 1-Chloro-2,4-Dinitrobenzene

Fig. 3. Comparison of glutathione S-transferase activities between malathion susceptible and resistant strains of the tarnished plant bug, and suppression of the activities by three glutathione S-transferase inhibitors (ethacrynic acid, sulfobromophthalein, and diethyl maleate). The activities are shown as kinetic velocity calculated using Bio-Tek KC4 software. DiVerent letters on the top of bars indicate signiWcant diVerence (P < 0.05) between treatments.

70 domestic silkworm Bombyx mori [35] (GenBank: BAD60789). Multiple sequence alignment of these ten 60 GST with no inhibitor GST with EA GSTs with the GST from the tarnished plant bug using the GST with SBT 50 GST with DM ClustalW method (GapOpen D 10, GapLength D 0.2) exhibited over 34% sequence identity (Fig. 6). Seventy-four 40 residues, including many residues for protein folding and GSH binding, were conserved among all 11 insect GSTs. 30 Pair-wise sequence comparison showed that the GST from

20 the tarnished plant bug shared 57.4% to 68.1% identity with these ten insect GSTs (Fig. 7A). The phylogenetic tree gen- 10

GST Kinetics (Mean Velocity±SE) erated by using DNAStar software (Fig. 7B) showed that the GSTs from Hemiptera and Diptera were clearly sepa- 0 rated from the GSTs from Lepidoptera. The GST from the May Jun Jul Aug Sep Oct tarnished plant bug was closely related to the GST from the Months planthopper, both of which were clearly separated from X Fig. 4. Seasonal variation of the glutathione S-transferase activities and GSTs of ies and mosquitoes (Fig. 7B). corresponding response (kinetic velocity) to three glutathione S-transfer- ase inhibitors [ethacrynic acid (EA), sulfobromophthalein (SBT), and 3.7. GST mRNA expression levels diethyl maleate (DM)] over the season. GST mRNA expression levels were compared using no signal peptide when it was examined using SignalP 3.0 real-time PCR. Results showed that the malathion suscepti- program [30]. Homology search of GenBank using BlaxtP ble strain had 2.83 § 0.28, and the resistant strain had revealed that the deduced 216-residue protein was similar 3.60 § 1.02 pg per 25 l reaction. The resistant strain had to several insect GSTs which were believed to be responsi- 1.3-fold higher GST expression levels than the susceptible ble for detoxiWcation and insecticide resistance develop- strain. ment. Ten GST sequences were downloaded from the GenBank, including the GSTs from the rice brown plant 4. Discussion hopper N. lugens [27] (GenBank: AAM21563), from the African malaria mosquito A. gambiae [31] (GenBank: Glutathione transferases are enzymes involved in CAB03593; GenBank: EAA44713), from the greater wax detoxiWcation mechanisms of many endogenous and moth G. mellonella [32] (GenBank: AAK64362), from the xenobiotic compounds [36]. They act by catalyzing the marmalade hoverXy Episyrphus balteatus (Vanhaelen, conjugation of glutathione with numerous potentially unpublished; GenBank: CAH58743), from A. dirus [33] toxic compounds to form a S-substituted glutathione. (GenBank: AAG38507), from D. simulans (Le GoV et al., DetoxiWcation of insecticides occurs via a dealkylation in unpublished; GenBank: AAK66764), from the houseXy M. which glutathione is conjugated with the alkyl portion of domestica (Syvanen, unpublished; GenBank: CAA43599), the insecticide [37], or via a dearylation reaction which is from A. dirus [34] (GenBank: AAB41104), and from the the reaction of glutathione with the leaving group [38]. 68 Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72

Fig. 5. cDNA and amino acid sequences of glutathione S-transferase isolated from a susceptible (LLSGST) and a resistant strain (LLRGST) of the tar- nished plant bug. Dot “.” was used to show identical nucleotide between LLSGST and LLRGST. The start codon ATG and termination codon TGA are shown in bold. The polyadenylation signal AATAAA is shown in gray background. Solid arrows indicate PCR ampliWcation range of cDNAs of both sus- ceptible ad resistant strains using Pfu DNA polymerase and PCR ampliWcation of the genomic DNA of the susceptible strain using Taq DNA polymerase. Dash-line arrows indicate real-time PCR ampliWcation range. Underlined amino acid sequences were used for designing degenerate primers. Forward primer sequences used for cloning and real-time PCR are underlined and labeled on the top. Complementary sequences of the reverse primers are indi- cated by italic and underlined letters. The boxed sequences with black background at 5Ј- and 3Ј-ends are cloned only from the susceptible strain. The boxed letters with black background at position 477 are nucleotide variation between the susceptible and resistant strains. The sequences have been depos- ited in GenBank with Accession No. DQ315381 for the susceptible strain LLSGST and DQ315382 for the resistant strain LLRGST.

This catalytic action can be suppressed by many agents. ment with malathion only. All these results indicated that GST inhibitors, such as ethacrynic acid and diethyl male- GSTs in the tarnished plant bug are involved in malathion ate, covalently bind and modify GSTs at the N-terminal detoxiWcation and resistance development. domain which results in inactivation of the GSTs [39,40]. Although GST in vitro activities were signiWcantly sup- This phenomenon was conWrmed in our bioassay experi- pressed in both susceptible and resistant strains by inhibitor ments. First, we found that EA and DM signiWcantly treatments, it is not clear why treatment of the susceptible increased susceptibility to malathion. Further examina- strain with GST inhibitors did not signiWcantly increase mor- tion indicated that malathion resistant strain had signiW- tality as was found in the resistant strains. It is likely that inhi- cantly higher activity than the susceptible strain, and the bition was concentration-dependent on both the inhibitor activity was suppressible. It is possible that the treatment reaching the target and the abundance of the target. A live of live insects with GST inhibitors substantially sup- insect bioassay and an in vitro enzyme activity assay are two pressed GSTs. Reduced GST activity in the bugs sup- diVerent systems. The enzyme activity assay only measured pressed or slowed down the detoxiWcation of malathion, GST activity with a speciWc substrate. If susceptible and resis- which resulted in higher mortality as compared to treat- tant bugs have identical GST proteins, the inhibitors would Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 69

Fig. 6. Predicted amino acid sequence LLSGST (DQ315381) of glutathione S-transferase from L. lineolaris and alignment with 10 other insect glutathione S-transferase sequences, including NL_AAM21563 from N. lugens, AG_CAB03593 and AG_EAA44713 from A. gambiae, GM_AAK64362 from G. mellonella, EB_CAH58743 from E. balteatus, AD_AAG38507 and AD_AAB41104 from A. dirus, DS_AAK66764 from D. simulans, MD_CAA43599 from M. domestica, and BM_BAD60789 from B. mori. Conserved residues are indicated with the background. The residues for binding are marked with ᭜ at the top of the sequences. The residues for forming the hydrophobic pocket are indicated with ᭡ at the bottom of the sequences. The residues for folding are indicated with a ᮀ at the bottom of the sequences. The catalytic active residue is marked with ᭹ at the bottom of the sequences. Hyphens represent sequence alignment gaps. non-preferentially suppress the GSTs in both susceptible and should have had a larger impact on the resistant strain than resistant strains. Because cloned GST cDNAs encode identi- on the susceptible strain because relatively greater amounts cal GST proteins in susceptible and resistant strains (Fig. 5), of GSTs were suppressed in the resistant strain, and subse- increased enzyme activity in the resistant strain might result quently more of the resistant insects died after treatment from a higher concentration of the GST proteins. A higher with a GST inhibitor and malathion. In the susceptible concentration of GST proteins could have been generated by insects, relatively lower amounts of GSTs were suppressed elevated GST gene expression, increased GST mRNA stabil- by GST inhibitors, and only a marginal increase in mortal- ity conferred from nucleotide substitution (Fig. 5), or modiW- ity was obtained in our bioassay (Fig. 2). In addition to the cation of the upstream regulatory region. These hypotheses role of GSTs, esterases may play another key role in the must be tested in future studies. malathion detoxiWcation and resistance development [3]. In the bioassay system, the situation was more compli- From the result in Fig. 2, we can see that treatments of sus- cated as compared to an enzyme activity assay using a spe- ceptible bugs with GST inhibitors did not increase mortal- ciWc substrate. Many enzymes may be involved in the ity substantially. It is possible that in the susceptible insects detoxiWcation of malathion, including GSTs, esterases, and esterases and other detoxiWcation enzymes play a key role other potential proteins. Although the resistant strain had and GSTs play a minor role in malathion detoxiWcation, higher GST activity, presumably conferred from elevated since only a marginal increase of mortality was obtained GST gene expression or mRNA stability, GST inhibitors after the GSTs were suppressed. However, in the resistant could have depressed the GSTs to a level which was no insects, both GSTs and esterases may play an important longer important in detoxifying malathion. This inhibition role, because (1) inhibition of GSTs signiWcantly increased 70 Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72

A

B

Fig. 7. (A) Amino acid sequence identity (up-right corner) and divergence (bottom-left corner) among eleven insect glutathione S-transferases. (B) Phylo- genetic tree, which was constructed using DNAStar software, showing the relationship among eleven insect glutathione S-transferases. toxicity of malathion and (2) resistant insects tolerated a 5- to an increase in the amount of one or more GST enzymes, fold higher dose of malathion (25 vs. 5 g/vial) and still either as a result of gene ampliWcation or more commonly maintained survival rates similar to susceptible insects after through increases in the transcriptional rate, rather than by GST inhibitor/malathion treatment. qualitative changes in the individual enzymes [41,42]. They The malathion resistant strain tended to have higher further concluded that elevated GST activity was impli- GST gene expression levels than the susceptible strain. By cated in resistance to at least four classes of insecticides. using real-time PCR, we detected approximately 1.3-fold Other evidence also suggested that the level of expression of higher GST gene expression levels in the resistant strain. GST was a crucial factor in determining the sensitivity of The resistant strain also had a relative large amount of var- cells to a broad spectrum of toxic chemicals [15]. iation in its GST expression levels than the susceptible In summary, this study provides information on resistance strain. This might have been caused by large variation gene regulation in malathion-resistant strains of the tar- among resistant individuals. Strain impurity and heterozy- nished plant bug. GST inhibitors eVectively abolished the gous genotypes might also have contributed to the large resistance and signiWcantly increased susceptibility in resis- variation among resistant individuals. The ratio would be tant strains. In addition, we found that the GST activity higher than 1.3-fold if a homogeneous resistant strain could increased over the season. This phenomenon was well syn- be obtained (though it is diYcult) and used for real-time chronized with the movement of the bugs into cotton, where PCR analysis. Another possibility is that several GSTs may they were exposed to organophosphate insecticides. The con- exist in the tarnished plant bug. Elevated GST gene expres- sistency of reduced sensitivity to malathion and increased sion needs to be further studied by cloning and character- GST activity, may have resulted from elevated gene expres- ization of other GST members in the tarnished plant bug. sion, and demonstrated that GSTs were involved in mala- Increased transcription in the resistant strain may be an thion detoxiWcation and resistance development. underlying cause of GST-mediated insecticide resistance in the tarnished plant bug. The consistency between bioassay Acknowledgments data and enzyme activity assay data supports this hypothe- sis. This phenomenon was also observed by other research- The authors are grateful to Dr. Xuming Liu of Kansas ers who found that higher enzyme activity was usually due State University and Dr. Omaththage Perera of USDA-ARS Y.C. Zhu et al. / Pesticide Biochemistry and Physiology 87 (2007) 62–72 71

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