ORIGINAL PAPER

Activity of increased specific and non-specific esterasesand glutathione transferases associated with resistanceto permethrin in pediculus humanus capitis(phthiraptera: pediculidae) from Argentina

Silvia Barrios & Eduardo Zerba & Maria I. Picollo &

Paola Gonzalez Audino

Received: 6 October 2009 /Accepted: 28 October 2009 /Published online: 17 November 2009# Springer-Verlag 2009

Abstract Enhanced metabolism by oxidative enzymes is amajor cause of pyrethroid resistance in insects. In this work,we evaluated the role of specific and non-specific esterasesin head louse populations from Buenos Aires with differentlevels of resistance to permethrin. As esterase activity issubstrate-dependent, four different esters were used asunspecific substrates in order to obtain a better characteriza-tion of the possible role of these enzymes in the resistancephenomenon. The unspecific substrates were phenylthioace-tate, 1- and 2-naphtyl-acetate, and p-nitrophenyl acetate. A 7-coumaryl permethrate was synthesized and used as a specificsubstrate to measure pyrethroid esterases by a very sensitivemicrofluorometric method. The results on pyrethroid esteraseactivity obtained with this substrate showed that theseenzymes contribute to the detoxifying activity in resistantpopulations, although no correlation was found betweenpyrethroid esterase activity and resistance ratios. In thisstudy, we established that the activity of esterase againstspecific and non-specific substrates is increased in pyrethroid-resistant populations of head lice from Buenos Aires. Also,dichlorodiphenyltrichloroethane (DDT) resistance valuesdemonstrated that there is a DDT cross-resistance phenom-

enon in pyrethroid-resistant head louse populations andsuggested that an alteration in the receptor of the nervoussystem (kdr gen) is a key factor of the resistance phenomenain these head louse populations.

Introduction

In Argentina, field populations of the human head lousePediculus humanus capitis De Geer (Phthiraptera: Pediculidae)have developed resistance to permethrin and other pyrethroidsafter the extensive use of these insecticides since 1990 (Picolloet al. 1998). Recently, an extensive resistance survey in BuenosAires showed significant levels of resistance in lice on childrenfrom schools (Toloza et al. 2009) and seasonal fluctuations inhead lice infestations are also common (Bauer et al. 2009). Thepermethrin resistance ratios (RR) obtained by the filter paperexposure method in these highly resistant populations rangedfrom 5.4 to >88.7 compared with a previously unexposedreference population (Vassena et al. 2003).

Recent studies on permethrin-resistant head lice focusedon the possible mechanisms of resistance (Bartels et al.2001). Metabolic enzyme systems known to be involved inresistance include monooxygenases, esterases and glutathi-one transferases. Hemingway et al. (1999) reported highglutathione S-transferase and monooxygenase activities inhead lice from Israel with resistance to dichlorodiphenyltri-chloroethane (DDT) and permethrin. Lee et al. (2000) andTomita et al. (2003) reported a molecular analysis of kdr-like resistance in permethrin-resistant head louse popula-tions from the USA and UK. By molecular cloning andsequencing, these authors identified two point mutationsassociated with permethrin resistance in the head lice. In a

S. Barrios : E. Zerba :M. I. Picollo : P. G. Audino (*)Centro de Investigaciones de Plagas e Insecticidas(CITEFA-CONICET),Juan Bautista de La Salle 4397-(B1603ALO),Villa Martelli, Provincia de Buenos Aires, Argentinae-mail: pgonzalezaudino@citefa.gov.ar

E. Zerba : P. G. AudinoUniversidad Nacional de General San Martín.Escuela de Postgrado,Avenida 53 3563, (1650),San Martín, Provincia de Buenos Aires, Argentina

Parasitol Res (2010) 106:415–421DOI 10.1007/s00436-009-1677-5

previous study, we demonstrated the importance of enhancedmetabolism by monooxygenases in head louse populationsfrom Buenos Aires, Argentina, that exhibited different levelsof resistance to permethrin. A positive correlation wasestablished between enzyme activity and permethrin lethaldose 50 (LD50) in the resistant populations (GonzalezAudino et al. 2005).

In the present study, we evaluated the role of specificand non-specific esterases in head louse populations ofBuenos Aires with different levels of resistance to per-methrin. Taking into account that esterase activity issubstrate-dependent, four different esters were used asunspecific substrates in order to obtain a better characteriza-tion of the possible role of these enzymes in the resistancephenomenon. These unspecific substrates were phenylthioa-cetate, 1- and 2-naphtyl acetate, and p-nitrophenyl acetate.However, unspecific substrates are possibly not a goodenough model of real pyrethroid esterase activity. Thus, inorder to obtain a specific substrate for pyrethroid esterases,we synthesized 7-coumaryl permethrate (7-CP), an esterstructurally similar to permethrin and with the same acidmoiety. The hydrolysis of this ester yields fluorescent 7-hydroxycoumarin (7-OHC) that allows using microfluoro-metric methods to quantify esterase activity (Santo Orihuelaet al. 2006).

Materials and methods

Biological material Head lice were collected from infestedchildren in randomly selected schools from each citydistrict in and around Buenos Aires, Argentina, wherepermethrin-based pediculicides have been used intensivelysince 1990. Live head lice were obtained from 6,250children aged from 6 to 12 years old, using a fine-toothedanti-louse comb (Nopucid, Laboratorio ELEA, BuenosAires, Argentina) according to a protocol approved by thead hoc Committee of CIPEIN and archived at our laboratory.Lice were grouped and named in separate populationsaccording to the school from which they were collected basedon our previous work (Picollo et al. 1998; Picollo et al. 2000;Vassena et al. 2003). Five permethrin-resistant populationswith different levels of resistance were collected, namelyBandera Argentina (BA), Hogar Loyola (HL), República deTurquía (RT), Hogar Mitre (HM), Guardia de Honor (GH)and Ricardo Guiraldes (RG). Adults and third instar wereselected at the laboratory for the bioassays (Mumcuogluet al. 1995; Picollo et al. 2000; Vassena et al. 2003; Gallardoet al. 2009). Once collected, the lice were kept in anenvironmental chamber (Lab-Line Instruments, MelrosePark, IL) at 18±0.5°C and 70–80% RH, in the dark andwithout food for a maximum of 2 h before the toxicologicalbioassays and 15 h before biochemical assays.

Resistant populations were collected from children whohad been previously exposed to insecticide treatments.These populations were named according to the districtfrom which they were collected: BA, GH, RT, RG, HL andHM. The resistance ratio of these populations has beenpreviously characterized (González Audino et al. 2005),but as the application of insecticides in schools is highlyvariable from year to year, we decided to determine thecurrent levels of resistance.

As pyrethroid susceptible strains of head lice were notavailable for reference, a susceptible strain of body lice(S-BL) was used instead based on the similarity betweenpyrethroid susceptibility in body lice and head lice with nohistory of exposure (Downs et al. 2002; Mougabure Cuetoet al. 2006). Our body louse colony, originated from acolony reared in the Department of Parasitology at theUniversity of Queensland, Australia, was maintained at29+/−1°C and 50–60% RH. Bioassays were performed onboth adults and third instars because the two stages haveshown to be equally susceptible (Mumcuoglu et al. 1995).

Chemicals Technical-grade permethrin (42.5% cis and54.2% trans) was donated by Chemotecnica (Buenos Aires,Argentina). Technical grade DDT was purchased fromPolyscience Corp. Thiophenol acetate (PTA) and 5,5′dithiobis-2-nitrobenzoic acid (Ellman's reagent) were purchased fromAldrich. Eserine hemisulfate, 1- and 2-napthyl acetate (1-NAand 2-NA), Diazo blue, p-nitro phenyl acetate (p-NPA),p-chloro-2, 4-dinitrobencene, glutathione and the Bradfordprotein measurement kit were purchased from Sigma.All solvents and general reagents were analytical grade.The synthesis of 7-coumaryl (1R,S)-cis-trans-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate (7-coumarylpermethrate) was carried out according to Santo Orihuela etal. (2006). (1R,S) cis-trans- (2,2-dichloro vinyl)-2,2- dimethylcyclopropane carboxylate acid chloride (cis-, trans- and cis-trans- permethrinic acid chloride) was from Chemotecnica,Buenos Aires, Argentina, 7-OHC was from Sigma, USA,toluene was from Merck and triethylamine from Aldrich,USA. Identity was confirmed by GC-MS (Shimadzu MS-QP5050A) in electron impact mode. The 1H-NMR and13C-NMR analyses were performed in a Bruker advanceDPX 400.

Bioassays Serial dilutions of permethrin and DDT (1, 1, 1-trichloro-2, 2-bis (p-chloro phenyl) ethane) were preparedin acetone and applied with a 5 µl Hamilton syringe with arepeating dispenser. Each head louse was treated on thedorsal abdomen with 0.1 µl of the solution according toVassena et al. (2003). The final dose ranged from 0.03 to3,000 ng/insect for permethrin and 0.01 to 5 µg/insect forDDT. Each concentration was replicated three times with atleast ten insects per replicate. Control insects were treated

416 Parasitol Res (2010) 106:415–421

with acetone alone. Treated insects were placed into a Petridish over a 9 cm Whatman No. 1 filter disc moistened with0.5 ml of water and maintained in an environmentalchamber (Lab-line Instruments, Melrose Park, IL) in thedark at 18±0.5°C and 70–80% RH. Mortality was recordedafter 18 h of treatment (Picollo et al. 1998). The criterionfor mortality was the inability of lice to walk from thecenter to the edge of a 7 cm filter paper disc.

Enzyme assays Adult lice and third instar homogenateswere subjected to esterase, glutathione S-transferase (GST)and protein assays. At least 300 adults from each populationwere tested. Insects were homogenized in groups of fivein 500 µl of ice-cold phosphate buffer 0.2 M pH 7.2. Thesupernatant was used immediately after a brief centrifugation(100 g for 2 min) to sediment any suspended materials(Yu and Nguyen 1982). To obtain specific values of enzymeactivity and protein concentration, the individual homoge-nates were determined by BIO-RAD protein determinationkit (BIO-RAD, UK) using bovine serum albumin as thestandard protein. General esterase activity was determinedusing standard colorimetric methods. A Shimadzu UV-160spectrophotometer was used in all UV-Visible determinations.

PTA activity was measured with Ellman's (1961)colorimetric method. Briefly, eserine inhibition studieswere performed by incubating the homogenate with 50 μlof eserine 10−4 M during 15 min. The activity of 1- and2- naphtyl acetate carboxylesterases was measured asdescribed by van Asperen (1962), p-nitro phenyl acetatewas determined according to Riddles et al. (1983) and GSTwas measured by the spectrophotometric method reportedby Habig et al. (1974). The activity of pyrethroid esterases,7-CP hydrolysis fluorescence was determined using a micro-plate fluorescence reader (Packard Fluorocount) (SantoOrihuela et al. 2006). Results were analyzed with Fluorocount®and Excel 2000 software.

Hydrolysis of synthesized substrate by head louse esteraseswas performed on individual homogenates. For this, theinsects were cooled and then each one was homogenized in220 μl phosphate buffer (pH 7.2, 0.05 M). Assays werestarted by adding 10 μl 7-CP (3.5 mM, 2-methoxy ethanol) to190 μl of the homogenate in black microtiter plates (Packard,Meriden, USA) at 25°C. The production of 7-OHC was

monitored with the excitation wavelength at 400 nm andemission band at 440 nm. Fluorescence was measured after30 min and the total amount of fluorescent product wascalculated using a calibration curve. All activities werecorrected for background hydrolysis and expressed as nano-grams of 7-OHC per minute.

Statistical analysis Mortality data were collected usingAbbot’s formula (Abbott 1925). Dose-mortality data fromeach head louse population was subjected to probit analysis(Litchfield and Wilcoxon 1949). After probit analysis,lethal dose ratios and 95% confidential limits (95% CL)were calculated as described by Robertson and Preisler(1992). Enzymatic activities were analyzed using an F-testand a t test for two samples to compare the activities of asusceptible population with each of the resistant ones. ABonferoni test was carried out to analyze the differencesbetween resistant populations.

Frequency profiles for 7-CP activity from individualinsects were analyzed for pyrethroid esterases.

Results

The results of the permethrin toxicity tests in field populationsof head lice from Buenos Aires are summarized in Table 1.LD50 values are expressed as nanograms of permethrin perinsect. The values of permethrin RR of the five resistantpopulations compared with the reference population (S-BL)ranged from 71.8 to 556.

General esterases and GST activity PTA esterase activitywas significantly higher in each of the resistant populationscompared with the activity in the susceptible population(p<0.05, F-test for variances of two samples, t test for twosamples considering unequal or equal variances as revealedby the F-test). However, no differences were observedbetween the resistant populations (p>0.05, Bonferroni test).The results of eserine inhibition showed there is a similarcontribution of cholinesterase activity in all the populations.

Esterase activity towards 1-NA and pNPA was signifi-cantly higher in resistant populations compared with thesusceptible population (p<0.05, F-test and t test). However,

Population Number Slope ± SE LD50 (ng/insecto; (95% CL) RRa (95% CL)

S-BL 150 2.18±0.18 0.7 (0.6–0.8) –

BA 140 1.13±0.1 51 (15–121) 71.8 (51.9–99.5)

HL 100 1.19±0.13 81 (58–105) 113.7 (80.7–160.2)

RG 100 0.74±0.11 120 (74–179) 166.5(104.7–264.9)

RT 150 1.12±0.11 393 (183–1645) 556 (386-799)

Table 1 Resistance ratios (RRs)to permethrin in resistant headlice and susceptible body lice

a RR calculated for LD50 valuesfor S-BL strain, calculatedaccording to Robertson andPreisler (1992).

Parasitol Res (2010) 106:415–421 417

no differences were observed between the resistant popula-tions (p>0.05, Bonferroni test). The activity towards 2-NAwas also significantly higher in the resistant populations(p<0.05, F-test and t test), although in this case the activitybetween the different resistant populations was significantlydifferent (RT vs. BA, RT vs. HL and RG vs. BA; p<0.05).Furthermore, a positive correlation was found between theactivity of 2-NA and permethrin resistance (r=0.92, p=0.01;Fig. 1).

GST activities towards CDNB were significantly higherin the resistant populations compared with the susceptiblepopulation (p<0.005, F-test and t test).

Permethrate esterase activity Pyrethroid esterase activitytowards 7-CP was significantly higher in resistant popula-tions compared with the susceptible one (p<0.05; Table 2).Differences in the frequency profiles for 7-CP activity fromindividual insects were measured between strains. Theactivities of the S-BL population were distributed uniformlywith more than 80% of the individuals expressing less than15 ng 7-OHC/insect (Fig. 2). In contrast, the distributionsof activity measured in samples of RT, RG, HL and BAinsects were broad, with values of over 15 ng 7-OHC/insectin more than 13%, 18%, 8% and 6% of the insects,respectively.

DDT resistance Table 3 shows the DDT LD50 valuesobtained for each permethrin resistant head louse popula-tion. LD50 for susceptible P. humanus humanus was0.0354 μg/insect. However, it was not possible to deter-mine a definite value of LD50 for the resistant populations.The values obtained demonstrated that there is a DDT-resistance phenomenon in the pyrethroid resistant popula-tions of head lice. Similarly, the intervals of confidencecould not be determined for the BA, HL and RT populations.Taking S-BA as reference, the resistant ratios were >141.2 forall the resistant populations.

0

0,1

0,2

0,3

0,4

0,5

0,6

-3,5 -3 -2,5 -2 -1,5 -1 -0,5 0

Log LD50

Lo

g a

ctiv

ity

Fig. 1 Correlation between permethrin LD50 and the activity of2-naphtyl acetate esterases (y ¼ 0:1448xþ 0:4749, r=0.92, p<0.05) T

able

2Non

-specificandspecific

esterase

andglutathion

etransferaseactiv

ities

ofhu

man

andbo

dyhead

lice

Pop

Non

-specificesterases

Specificesterases

Glutathione

transferases

PTA

(N;µmol/m

inmgp

)PTA

+Ese

(N;µmol/m

inmgp

)1-NA

(N;µmoles/m

inmgp

)2-NA

(N;µmoles/m

inmgp

)pN

PA(N;µmoles/m

inmgp

)7-CP

(N;ng

min

ins)

CDNB

(N;µmoles/m

inmgp

)

S-BL

2.28

±0.02

(40)

a1.78

±0.02

(40)

a1.21

±0.04

(80)

a1.10

±0.02

(100

)a0.97

3±0.00

2(48

)a0.27

4±0.02

3(57)

a0.16

±0.01

(90)

a

BA

3.24

±0.05

(45)

b2.72

±0.04

(45)

b2.12

±0.06

(50)

b1.45

±0.03

(55)

b1.45

±0.05

(87)

b0.93

6±0.07

8(36

)b0.66

±0.01

(72)

b

HL

2.91

±0.04

(30)

b2.31

±0.03

(30)

b2.16

±0.07

(75)

b1.87

±0.03

(55)

bd

1.30

±0.07

(42)

b0.97

8±0.05

1(36

)b0.70

±0.02

(144

)b

RT

3.12

±0.02

(55)

b2.55

±0.03

(55)

b1.98

±0.04

(125

)b3.03

±0.04

(50)

cd1.81

±0.08

(51)

b0.90

0±0.04

5(60

)b0.84

±0.02

(48)

b

RG

3.02

±0.02

(55)

b2.41

±0.03

(55)

b1.69

±0.05

(80)

b2.53

±0.06

(50)

ce1.32

±0.04

(48)

b0.56

3±0.05

9(24

)b0.73

±0.03

(60)

b

Ntotalnumberof

insectsused,PTA

phenyl

tioacetate,1-NA,1-naphtylacetate,2-NA2-naphtylacetate,pN

PAp-nitrophenylacetate,7-CP7-coum

aryl

perm

ethrateCDNB,1-chloro-2

4-dinitrobencene

Valuesin

thesamecolumnfollo

wed

bydifferentletters

aresign

ificantly

different(P

<0.05

).

418 Parasitol Res (2010) 106:415–421

Discussion

Increased esterase activity has been correlated with anincrease in insecticide resistance in many species ofinsects. Increased esterase activity has been reported forpyrethroid resistant Blatella germanica: using 1-NA assubstrate, activity was 1.7 fold higher in the resistant

population and with PNPA the increase was 2.1 fold(Anspaugh et al. 1994). Rose et al. (1995) found a Tobaccobudworm (Heliothis virescens) population resistant to cyper-methrin (RR91), with higher esterase activities in the resistantpopulation compared with a susceptible one using both1-NA and P-NPA as substrates. Also, Delorme et al. (1988)demonstrated the role of increased detoxification by esterasesin a deltamethrin-resistant strain of Spodoptera exigua using1- and 2-NA as substrates. Furthermore, activity towards2-NA was increased in resistant populations of Helicoverpaarmighera compared with susceptible populations (Bues etal. 2005) and a resistant strain of Aedes aegypti, generated bydeltamethrin showed significant elevation in the activity ofalpha- and beta-esterases and glutathione-S-transferase(Jagadeshwaran and Vijayan 2009).

In a previous study by our laboratory, Picollo et al.(2000) showed that treating permethrin-resistant head licewith a carboxyesterase inhibitor produced a partial rever-sion of the permethrin resistance, suggesting that esterases

Table 3 Resistance to DDT in P. h. humanus and P. h. capitispopulations from Buenos Aires

Population Slope ± SE LD50 (ųg/insect; 95% CL) RR

S-BL 2.01±0.17 0.0354 –

BA – >5 >141.2

HL – >5 >141.2

RT _ >5 >141.2

RG _ >5 >141.2

0

5

10

1520

25

30

7-EC activity (ng/ins)

RT

02468

101214

7-EC activity (ng/ins)

RG

02468

10121416

Fre

qu

ency

(%

)

7-EC activity (ng/ins)

HL

02468

10121416

0,0-

5,0

10,0

-15,

020

,0-2

5,0

30,0

-35,

040

,0-4

5,0

50,0

-55,

060

,0-6

5,0

0,0-

5,0

10,0

-15,

020

,0-2

5,0

30,0

-35,

040

,0-4

5,0

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-55,

060

,0-6

5,0

0,0-

5,0

10,0

-15,

020

,0-2

5,0

30,0

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040

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5,0

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,0-6

5,0

0,0-

5,0

10,0

-15,

020

,0-2

5,0

30,0

-35,

040

,0-4

5,0

50,0

-55,

060

,0-6

5,0

7-EC activity (ng/ins)

BA

05

10152025303540

7-EC activity (ng/ins)

S-BL

Fre

qu

ency

(%

)F

req

uen

cy (

%)

Fre

qu

ency

(%

)

Fre

qu

ency

(%

)

0,0-

5,0

10,0

-15,

020

,0-2

5,0

30,0

-35,

040

,0-4

5,0

50,0

-55,

060

,0-6

5,0

Fig. 2 Frequency histogramsfor pyrethroid esterases. A7-CP activities measured as ng7-OHC/insect in individualP. capitis from different popula-tions (S-BL, BA, HL, RG andRT). Frequency is expressedrelative to the total numberof insects tested

Parasitol Res (2010) 106:415–421 419

contribute to the detoxification of pyrethroids in theseresistant head lice from Buenos Aires.

Considering that GST has no critical role in pyrethroiddetoxification, the increase in GST activity suggests it couldbe associated, although not directly involved, with theresistance phenomenon of the studied populations. Theincrease in GST activity could be related to the increase inoxidase and esterase activity, probably owing to theregulation by the same gene. Plapp (1984) reported that inMusca domestica, several genes that code for detoxifyingenzymes (P450s, GST) are regulated by a main genelocalized on chromosome 2.

Changes in the frequency distribution of activity towardssubstrate 7-CP has previously been associated with pyre-throid resistance and can be used as a biochemical markerof this resistance in T. infestans when the percentage ofindividuals with high esterase levels is analysed in thedifferent populations (Santo Orihuela et al. 2008). However,the high levels of resistance we observed cannot beexplained only in terms of detoxifying enzymes, evenconsidering esterase and oxidase activities. A positivecorrelation was observed between the levels of monooxyge-nase and resistance to pyrethroids (Gonzalez Audino et al.2005), but the present results suggest there is anothermechanism of permethrin resistance in these populations.This leads us to consider the presence of a modified voltage-dependant sodium channel (a kdr-type mechanism). Scottand Matsmura (1981, 1983) discovered that nerve insensi-tivity measured as kdr was the only apparent mechanism ofpyrethroid cross-resistance in a DDT-resistant strain of theGerman cockroach. As DDT and pyrethroids share the sametarget, the development of a cross-resistance between them ispossible. The strategy of detecting cross-resistance providesan indication of the possible mechanism involved in theevent.

Hemingway et al. (1999) found increased GST activityin P. humanus capitis populations from Israel resistantto DDT, permethrin and fenothrin, although this activitywas only correlated with DDT-resistance. DDT-resistancesuggests that an alteration in the receptor of the nervoussystem (kdr mechanism) is the main factor of the resistancephenomenon in head louse populations from Buenos Aires.The best way to demonstrate this is by electrophysiologicalstudies in order to detect differences in the transmission ofimpulses between resistant and susceptible populations(Hemingway et al. 1999). Another option is to work withmolecular biology methods in order to detect pointmutations in the voltage-dependant sodium channel. Leeet al. (2000) reported a kdr-type mechanism and in secondplace an increased activity of the P450 complex as the mainresistance mechanisms in resistant populations of P. capitisfrom USA and UK. The authors identified two pointmutations on the sodium channel in the resistant popula-

tions. Yoon et al. (2004) detected permethrin resistance inhead lice from Florida and California and established DDTcross-resistance. Susceptible populations from Panamá andEcuador do not posses mutations T9291and L932F whileall the resistant populations have both, indicating that inthis case permethrin resistance is based on a kdr-typemechanism.

The whole resistant head louse populations studied inthis work showed a significantly higher detoxifying esteraseactivity compared with the susceptible populations for allthe substrates used for the determination. Therefore,esterase activity seems to be a mechanism contributing topyrethroid detoxification in resistant head lice.

Acknowledgements We are especially grateful to Mercedes Mantesi,Guillermo Valenzuela, Laura Galicio, Rosa Flores, Isabel Cañete andthe authorities of the elementary schools where the head lice werecollected. MIP, EZ and PGA are members of the National ResearchCouncil (CONICET).This investigation received financial support fromLaboratorio ELEA S.A. (Argentina) and the Agencia Nacional dePromoción Científica ANPCyT (Argentina).

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