Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the...

Insect Molecular Biology (2002) 11(4), 329–336

© 2002 The Royal Entomological Society 329

Blackwell Science, LtdResistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae

J. G. Vontas*, M. J. Hejazi†, N. J. Hawkes*, N. Cosmidis‡, M. Loukas‡ and J. Hemingway*

*Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK; †Department of Plant Protection, College of Agriculture, University of Tabriz, Tabriz, Iran; and ‡Agricultural University of Athens, Athens, Greece

Abstract

A 2.2-kb full length cDNA containing an ORF encodinga putative acetylcholinesterase (AChE) precursorof 673 amino acid residues was obtained by a com-bined degenerate PCR and RACE strategy from anorganophosphate-susceptible Bactrocera oleae strain.A comparison of cDNA sequences of individual insectsfrom susceptible and resistant strains, coupled with anenzyme inhibition assay with omethoate, indicated anovel glycine-serine substitution (G488S), at an aminoacid residue which is highly conserved across species(G396 of Torpedo californica AChE), as a likely cause ofAChE insensitivity. This mutation was also associatedwith a 35–40% reduction in AChE catalytic efficiency.The I199V substitution, which confers low levels ofresistance in Drosophila, was also present in B. oleae(I214V) and in combination with G488S produced up toa 16-fold decrease in insecticide sensitivity. This is thefirst agricultural pest where resistance has been asso-ciated with an alteration in AChE, which arises frompoint mutations located within the active site gorge ofthe enzyme.

Keywords: Bactrocera oleae, AChE cDNA, mutations,insecticide resistance.

Introduction

Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzymein the nervous system, terminating nerve impulses bycatalysing the hydrolysis of the neurotransmitter acetylcho-line. In insects it is a glycosylated dimer which is attachedto a membrane via a glycolipid anchor (Fournier et al., 1988;Chaabihi et al., 1994). AChE is the major target for organo-phosphate and carbamate insecticides, which inhibitenzyme activity by covalently phosphorylating or car-bamylating the serine residue within the active site gorge(Corbett, 1974). Quantitative and qualitative changes inAChE confer resistance to insecticides (Fournier et al.,1992, 1993). Fournier et al. (1992) showed that insecticideresistance is correlated with the over-expression of AChEin laboratory Drosophila strains, in a similar manner to thatdescribed for scavenger or metabolizing proteins. However,point mutations usually accompanied by a modification ofthe kinetic parameters of acetylcholine hydrolysis havemore often been identified as being responsible for insect-icide resistance (Mutero et al., 1994; Zhu et al., 1996;Devonshire et al., 1998; Walsh et al., 2001; Kozaki et al.,2001). Resistance-associated mutations involve substitu-tions at key sites located within the active site gorge of theenzyme. The altered amino acids (which are often largerthan the wild-type residues) have a steric effect or alter theorientation of the active site residues. The only documentedexception to this is in the Colorado potato beetle Leptino-tarsa decemlineata, where a substitution to a smalleramino acid, remote from the catalytic region of the enzyme,has been associated with insensitivity (Zhu et al., 1996).In general the levels of organophosphate insensitivityconferred by each individual replacement are low, but incombination they produce increasingly resistant enzymes(Mutero et al., 1994; Walsh et al., 2001). Many potentialresistance-associated point mutations may result in AChEinsensitivity, without significantly reducing AChE catalyticefficiency, as confirmed by site-directed mutagenesisstudies (Vaughan et al., 1997; Villatte et al., 2000; Chenet al., 2001). However, while these studies indicate thatinsects can acquire insensitivity to insecticides through

Received 19 November 2001; accepted after revision 19 April 2002.Correspondence: Dr John Vontas, Institute of Molecular Biology andBiotechnology (IMBB-FORTH), PO Box 1527, Heraklion, Crete 71110,Greece. E-mail: [email protected]

330 J. G. Vontas et al.

© 2002 The Royal Entomological Society, Insect Molecular Biology, 11, 329–336

Insensitive acetylcholinesterase in Bactrocera oleae 331


AChE point mutations, to date such changes have onlybeen reported from Drosophila melanogaster (Muteroet al., 1994), Musca domestica (Devonshire et al., 1998;Walsh et al., 2001; Kozaki et al., 2001) andL. decemlineata (Zhu et al., 1996). The isolation andcomparison of AChE sequences from other species suchas Nephotettix cincticeps and Boophilus microplus, indicatethat an insensitive AChE phenotype does not result fromamino acid substitutions in the AChE protein itself, suggest-ing the involvement of other mechanisms (Baxter & Barker,1998; Tomita et al., 2000).

An altered acetylcholinesterase (AChE) with a 35–40%reduced catalytic efficiency for acetylthiocholine iodide(ATChI) and a 5–16-fold lower sensitivity to inhibitionby omethoate is the major organophosphate resistancemechanism in two resistant strains of the olive fruit flyBactrocera oleae (Vontas et al., 2001). Here we reportcDNA sequences which encode AChE in susceptible andorganophosphate resistant B. oleae and identify possibleamino acid substitutions associated with resistance.

Results

cDNA and deduced amino acid sequence of the B. oleae AChE

By applying a PCR strategy using degenerate primers forinsect AChE as well as utilizing RACE techniques, weobtained the full length sequence of the B. oleae precursorAChE mRNA (accession no. AF452052). Boache has anopen reading frame of 2022 bp which encodes a putative673 amino acid protein, with high identity to dipteran AChEsand highest similarity to D. melanogaster AChE (79%identity). An alignment of boache with other insect AChEsis shown in Fig. 1. The three components of the catalytictriad (Ser, Glu, His) and their surrounding amino acidsequences, the seven Cys residues which form the intra-and inter-subunit disulphide bonds, and all known aromaticresidues of the catalytic gorge are present in the olive fruitfly AChE (Fig. 1). As all these components are conserved,it is predicted that the B. oleae AChE has a similar three-dimensional structure to that of Drosophila AChE (Harelet al., 2000).

Southern analysis

Southern blots of genomic DNA digested with EcoRV, Pvu IIor EcoRI (restriction sites absent from the coding region ofboache) were probed with a 450-bp boache cDNA, derived

from a highly conserved region across insect AChEs(equivalent to exon 4 in Drosophila, Mutero et al., 1994), andeach revealed a single hybridizing fragment (Fig. 2). Evenunder lower stringency hybridization conditions, a single bandwas observed for each digestion (data not shown), indicatingthat there is a single AChE gene in the B. oleae genome.

Comparison of AChE sequences of insecticide susceptible and resistant insects

Specific activities of AChE preparations extracted from theheads of B. oleae adults with the substrate ATChI were95.3 ± 11 nmol/min/mg protein for the LS strain, 58.6 ±4.1 nmol/min/mg for the LR strain and 57.1 ± 11.1 nmol/min/mg for the Attiki insects (n = 24 insects for each strain,values are the mean ± SEM of two determinations). Thisreduced AChE catalytic efficiency of both resistant strains(which did not differ significantly from each other) wasassociated with a marked insensitivity to omethoate. Inhibi-tion of AChE activity by 0.03 mM omethoate ranged from 70to 95% for individuals of the LS strain, 50–62% for the LRstrain and 10–30% for the Attiki strain. Following AChEactivity determinations (Fig. 3A), the remaining body

Figure 2. Southern blot analysis of Bactrocera oleae LS genomic DNA. Genomic DNA was extracted from B. oleae adults, digested with EcoRI (lane 1), EcoRV (lane 2) and PVU II (lane 3) and separated by gel electrophoresis. After transfer to nylon membranes the DNA was hybridized with 32P-labelled boache cDNA. The final wash was 65 °C in 0.1 SSC and 1% SDS for 30 min.

Figure 1. Alignment of the deduced amino acid precursor sequence of boache cDNA with other insect AChE. Gaps introduced to maximize sequence identity are shown by a horizontal dash. Asterisks show the seven cysteines which form disulphide bonds, solid circles the residues of the catalytic triad, and crosses the aromatic residues. The hydrolytic peptide essential for cleavage of the precursor is indicated by arrows (Mutero & Fournier, 1992). Open triangles show the sites for amino acid substitution polymorphisms known to affect insecticide sensitivity, and a solid triangle the position where the novel mutation was found in this study. All insect sequences were retrieved from GENBANK (Aa = Aedes aegypti AS66236, Ld = Leptinotarsa decemlineata L41180, Md = Musca domestica AJ310134, Nc = Nephotettix cincticeps AF145235, Dm = Drosophila melanogaster X05893, As = Anopheles stephensi P56161, Lc = Lucilia cuprina U88631, Bo = Bactrocera oleae AF452052).



segments (thorax and abdomen) of six individuals with themost sensitive AChE from the LS strain and six individualswith the least sensitive AChE from each of the LR and Attikiresistant strains were used for molecular analysis, wheretheir cDNA sequences were compared. Several silentnucleotide substitutions were observed (data not shown).However, only the polymorphism at bases 640 and 1462resulted in amino acid substitutions (I214V and G488S,respectively) in the insecticide resistant strains (Fig. 3B).All alleles sequenced from the LS strain encoded G488,while all alleles from both resistant strains encoded aserine at this residue (Fig. 3B). This was consistent withthe AChE activity data from the same individuals (Fig. 3A),indicating that the glycine-serine substitution is a likelycause for the observed 35–40% lower AChE activity and itsreduced sensitivity to inhibition by omethoate. All individualscarrying the S488 had similar AChE activity; however, theAChE insensitivity from insects of the Attiki strain washigher than that from the LR strain (Fig. 3A). All individuals

analysed from the Attiki strain encoded V214, in contrast toI214 in the LR strain (Fig. 3B), which indicated that thismutation (equivalent to I199V, the known resistance-associated mutation in Drosophila AChE, Mutero et al., 1994),was responsible for the difference in the AChE insensitivityof the Attiki compared to LR strain (16-compared to 5-fold)(Vontas et al., 2001). The effect of I214V alone was notevaluated in B. oleae AChE (since this mutation wasalways found in combination with S488), however, it isunlikely to account for the entire 16-fold AChE insensitivity,as I199V in the highly homologous Drosophila AChE confersapproximately twofold organophosphate insensitivity(Mutero et al., 1994).

Mutation G488S

G488 (equivalent to G396 in Torpedo; Sussman et al.,1991) is a highly conserved residue across species, and isfound in all 20 available AChE sequences, includinghuman, mouse, torpedo, cat, nematodes and insects.

Figure 3. Comparison of cDNA sequences coupled with AChE biochemical determinations in individual Bactrocera oleae adults. (A) Profile of AChE remaining activity, after inhibition by 0.03 mM omethoate and AChE specific activities (in parenthesis) in enzyme preparations from heads of individual insects. (B) Summary of the amino acid substitutions found in the AChE cDNA sequences, isolated from thorax and abdomens of the same B. oleae individuals (the coding nucleotide sequence is given in parenthesis). The location in the first column indicates where the change of the amino acid occurs in B. oleae AChE and the equivalent residues in Torpedo for cross reference.



Since all major structural and functional features of AChE,including the residues facing the active site gorge, arehighly conserved in B. oleae, we analysed the G488Samino acid substitution based on the three-dimensionalstructure of Drosophila AChE (Harel et al., 2000; accessionno. ID1Q09). G488 is adjacent to the active site of theenzyme (Fig. 4A), which (in combination with its highconservation) indicates, that it may offer direct structuralsupport or is essential to preserve the shape of the activesite. Interestingly, G488 is located very close to the wellcharacterized G365A mutation in M. domestica (EMBLaccession no. AJ310134, equivalent to G328 in Torpedo),which similarly reduces the catalytic efficiency of theenzyme (30–40% decrease in specific activity for ATChI,due to slightly higher Km values in both cases) and conferscomparable levels of insecticide resistance (2.5 to 13-foldto various organophosphate oxon analogues from G365A,fivefold to omethoate from G488S, no insensitivity tocarbamates in both cases) (Vontas et al., 2001; Walshet al., 2001). The two residues (equivalent to G368 andG436 in the Drosophila AChE, accession no. 1QO9) are

alongside each other on adjacent helices in the structure ofthe native Drosophila AChE (Fig. 4A). The distancebetween alpha carbon atoms is only 3.5 Å. This means thatany mutation in this region of a side chain, no matter howsmall, will impose some steric hindrance to the close prox-imity of the two helices, resulting in their displacement, anda consequent rearrangement of the structure. Figure 4Bshows the close proximity and overlapping Van der Waalssurfaces of the two glycines, and demonstrates the sterichindrance that results, using modelling of the S488 inDrosophila AChE (S436).

Discussion

A novel glycine-serine substitution (G488S) in a highlyconserved residue (equivalent to G396 in Torpedo) wasstrongly correlated with the altered AChE-based organo-phosphate resistance mechanism that was recentlyreported in the olive fruit fly B. oleae (Vontas et al. 2001).The G488S mutation was present in all resistant insectsanalysed, but in none of the susceptible ones (from the

Figure 4. Location and modelling of the G488S Bactrocera oleae resistance mutation. (A) Stereo representation which shows the positions of residue G488 (equivalent to G396 in Torpedo and G436 in Drosophila 1QO9) and G365 (equivalent to G290 in Torpedo and G368 in Drosophila 1QO9) in relation to the active site of Drosophila AChE. (B) The Van der Waals dotted surfaces (green) of Drosophila G436 and G368 (equivalent to G488 and G365) showing their close proximity to one another in the native Drosophila AChE (accession no. 1QO9, Harel et al., 2000), with the mutation G436S (G488S in B. oleae) modelled using SYBYL, showing clearly that steric hindrance results from this mutation.



same origin), and was 100% consistent with AChE insens-itivity to omethoate and an associated decrease in AChEcatalytic efficiency. Based on the X-ray crystal structure ofthe highly homologous Drosophila AChE (Harel et al.,2000), we suggest that G488 is located in adjacent helicesand within 3.5 Å of the well-characterized G365A resist-ance associated mutation in M. domestica (Walsh et al.,2001). Modelling of S488 (S436 in Drosophila) into Dro-sophila AChE clearly showed the steric hindrance whichis likely to result from this mutation (Fig. 4B). The effect ofthese neighbouring mutations (G365A in M. domestica andG488S in B. oleae), both on the catalytic efficiency of theenzyme and to its insensitivity to insecticides, is verysimilar. The proposed role of G365A in AChE insensitivity isthrough an alteration in the configuration (orientation) of theadjacent glutamate in the catalytic triad, which results in anenhancement of the nucleophilic attack of water on thecarbonyl group of the phosphorylated serine (Walsh et al.,2001). We speculate that the G488S substitution identifiedhere has a similar effect to that of G365A in M. domestica,although functional expression and further molecularmodelling work will clarify its exact role. The I214V substitution,equivalent to the well characterized I199V resistance-associated mutation in Drosophila, which confers loworganophosphate resistance levels by itself but higherlevels in combination with other mutations (Mutero et al.,1994; Chen et al., 2001), was also present in B. oleaeAChE. In combination with G488S it resulted in the 16-foldAChE insensitivity of the Attiki strain.

All dipteran AChE precursor sequences known to date,including boache, are regarded as D. melanogaster Ace-orthologous genes, while it is difficult to find paralogousAChE genes, even in hemipteran and lepidopteran insects(Tomita et al., 2000). However, some insects have more thanone AChE gene which are possible sources of resistance(Bourguet et al., 1996; Malcolm et al., 1998). Southernanalysis showed only a single AChE locus in the B. oleaegenome, which is in line with our previous biochemicalanalysis (Vontas et al., 2001). The product of this geneshould be the main target of organophosphate insecticides.

Altered AChE-based insecticide resistance does notalways result from changes in the protein structure byamino acid substitution, suggesting that other mechanismssuch as alternate transcriptional variation and/or post-transcriptional modifications might be involved in AChEinsensitivity (Baxter & Barker, 1998; Tomita et al., 2000).The possibility that B. oleae AChE insensitivity results fromsuch modifications, although unlikely given the strongcorrelation between the biochemical properties and theposition of the novel mutation identified, cannot be dis-counted at this stage.

The high number of resistance-associated point muta-tions in AChE (each responsible for weak resistance)contrasts with the very restricted number of mutations

(responsible for strong resistance) found in other alteredtarget sites (such as GABA or kdr). This is attributed tothe commonality of the substrate-analogue insecticideand the acetylcholine binding sites. For the selection of amutation to occur, the resultant amino acid change mustreduce the binding of the insecticide without dramaticallydecreasing the rate of neurotransmitter hydrolysis. Thisresults in minor modifications of residues at severalpositions around the active site, which in combinationresults in high levels of resistance (Mutero et al., 1994). Inaccordance with these observations, the evidence for anovel insensitive AChE-associated mutation provided inthis study suggests that more ‘natural’ resistant mutationsare likely to occur in insects (Vaughan et al., 1997; Chenet al., 2001).

One of the resistance associated mutations (I214V) inB. oleae AChE is common to those reported previouslyin Drosophila, which adds to the growing list of highlyconserved target site insecticide resistance-associatedmutations, from a wide range of species (french-Constantet al., 1998). However, the identification of the novel G488Ssubstitution in B. oleae, in a very conserved residue acrossspecies, indicates that multiple further mutations of differ-ent origin may yet confer AChE insensitivity.

Experimental procedures

Chemicals

The insecticides used in this study were of analytical grade andpurchased from British Greyhound (Birkenhead, Merseyside, UK).All other reagents were purchased from Sigma (UK) except asstated.

Insects

The insecticide susceptible LS strain and the organophosphateresistant Attiki and LR B. oleae strains (obtained by selection withdimethoate; Vontas et al., 2001) were used in this study.

AChE inhibition assay

The heads were separated from thorax and abdomen of individualB. oleae adults. AChE enzyme preparations and AChE activitywere conducted as detailed previously (Vontas et al., 2001). Thesensitivity of AChE to the dimethoate oxon analogue omethoatewas determined in the absence of substrate. AChE enzymepreparations from B. oleae heads in duplicates were pre-incubated for 15 min with 0.03 mM omethoate or buffer alone.Activity was measured by placing the inhibition mixture into anexcess of ATChI and dithiobis 2-nitrobenzoic acid (DTNB).Enzyme inhibition was expressed as the mean percentage of theremaining activity.

Interaction studies of carboxylesterases with omethoate, basedon a comparison of bimolecular rate constants Ka, showed homo-geneity among the strains tested, which suggests that the differ-ences in AChE sensitivity observed were genuine and not a resultof a differential binding of the inhibitor by other esterases (Vontaset al., 2001).



Extraction of DNA, total RNA and mRNA, and cDNA synthesis

After the AChE biochemical determinations, total RNA wasisolated from the separated thorax and abdomen from selectedB. oleae individuals using TRI reagent (Sigma), as describedby Vontas et al. (2000). Poly(A)+ RNA was isolated from totalRNA (mass sampling of insects was used in the initial amplifica-tions) with the PolyATract mRNA Isolation System IV (Promega),following the manufacturer’s instructions. First strand cDNAsynthesis was carried out with an oligo-(dT) adaptor primer[5′-GACTCGAGTCGACATCGA-(dT)17-3′ ], using SuperScript II(Gibco-BRL), in the presence of 10% DMSO and at 50 °C, to preventsecondary structure formation. Genomic DNA was isolated from1 g B. oleae adults by the method of Vaughan et al. (1995).

Cloning and sequencing of B. oleae AChE

Degenerate primers were designed based on the sequence ofinsect AChE sequences available in the database and were usedto amplify a homologous region from B. oleae cDNA. The 50 µlPCR reaction contained 50 ng of first strand cDNA, 0.5 µmolof each primer, Fd1 [5′-GARATITGGAAYCCIAAYAC] R1d [5′-CCCATCCAYTCICCCCA], 0.5 mM dNTPs, 3 mM MgCl2, and 2.5Units of HotStar Taq polymerase (Qiagen) in the manufacturer’sreaction buffer. Thirty-five cycles of amplification were carried out(50 °C 30 s, 72 °C 1:30 min, 94 °C 30 s). A second round of semi-nested PCR was performed with the nested forward Fd2 [5′-GTIGGIGCNTTYGGITT] primer and the reverse Rd1 or the Fd1forward primer and the reverse Rd2 [5′-CCiGCiGAYTTCiCCRAA],with a similar reaction mixture composition as those for the firstround of PCR and cycling conditions of 48 °C 30 s, 72 °C 1:30 min,94 °C 30 s. PCR products were cloned into pGEM T-easy(Promega) and sequenced in both directions. DNA sequenceswere analysed using DNA STAR software (Lasergene). An align-ment of partial sequences obtained to the sequences of otherinsect AChEs indicated that a putative partial AChE cDNA hadbeen isolated. Anti-sense oligonucleotides specific to this partialAChE sequence were designed and used in 5′ and 3′ RACE (rapidamplification of cDNA ends) procedures. The 3′ end of the cDNAwas amplified by PCR with a specific F1 forward primer [5′-GGGCGCCAGCGAAAGCGCG] and the adaptor primer [5′-GACTCGAGTCGACATCGA] and walked with a semi-nested PCRusing the specific F2 forward primer [5′-AGTTTTCCATCT-GCGCCGACC] and the same adaptor primer. The 5′-end of thecDNA sequence for B. oleae AChE (boache) was obtained usinga modified 5′-RACE methodology described by Tomita et al.(2000). Briefly, first strand cDNA was synthesized as abovefrom polyA(+)RNA with 5′-phosphorylated R1 [5′-CTCCTCG-TAGCCCGGCATAGCG]. After RNA degradation with RNAse Hand purification of the single stranded cDNA using the Glass MaxDNA Isolation system (Gibco-BRL), the single stranded cDNA wasligated with T4 RNA ligase (Promega). First round PCR was per-formed by using the resulting re-circulated cDNA (or concatamer)as a template with the F1 specific primers and the R2 primer[5′-CGGAGACATTTGTGTTAGGATTCC]. PCR conditions were:3 mM MgCl2, 0.5 mM dNTPs, 0.4 µM of each primer, 2.5 Units ofHotStar Taq polymerase (Qiagen) in the manufacturer’s reactionbuffer; 95 °C 15 min then 35 cycles of 50 °C 30 s, 72 °C 1 min, 94 °C30 s and finally 72 °C 5 min. A second round of PCR was performedwith the nested forward F3 primer [5′-ATTTGGATTTACGGTGGT-GGC] and the reverse R3 primer [5′-CTCTTCGCCAGAGAAAC-CGG], under the same conditions. PCR products were cloned into

pGEM T-easy and sequenced in both directions. The forward primerf0 [5′-CCGGATTCATGGCTCACCCAACGTCCTTGC] and thereverse primer R0 [5′-CGGGATCCTATTGCCTGTCTGTAGCTCC]encompassing the putative start codon and termination codonof the boache cDNA, respectively, were used to amplify the fullcoding region from cDNA isolated from individual susceptible andresistant insects. A slightly internal sequence of ≈ 1.8 kb wasamplified, PCR products were purified using Microcon YM-100centrifugal filter devices (Millipore) and sequenced in both directions.

Southern hybridization

Southern hybridization was performed as detailed by Vontas et al.(2002). Briefly, separate 10 µg aliquots of genomic DNA weredigested to completion with EcoRV or Pvu II, size-fractionated byelectrophoresis on an 0.8% (w/v) agarose gel, transferred to acharged nylon membrane (Amersham Corp.) and hybridizedwith 32P-labelled boache probe (specific activity > 2 × 106 c.p.m./µg)at 65 °C for 16 h in hybridization buffer [5 × Denhardt’s solution,6 × SSC, 0.1% (w/v) SDS, 0.1% (w/v) sodium pyrophosphate, 5%(w/v) polyethylene glycol 8000, and 100 µg/ml boiled, shearedherring sperm DNA]. The final washes were at 65 °C in 0.1 × SSCand 1% (w/v) SDS for 30 min.

Analysis of AChE structure

The structure of D. melanogaster AChE was obtained from theProtein Data Bank (ID ref 1Q09) and viewed and analysed withSYBYL and SWISSPDBVIEWER <http://www.expasy.ch/spdbv>programs. Mutations were introduced using the ‘mutate monomer’function within SYBYL.

Acknowledgements

We would like to acknowledge Dr Robert Janes (Universityof London) and Prof. Robert John (Cardiff University) fortheir help with the molecular modelling aspects of this work,and Dr Hilary Ranson for a critical reading of this manu-script. J.V. was supported by a European Union MarieCurie TMR grant (MCFI-1999-00259).

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Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the...

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