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Genetic Variability and Geographical Diversity of the MainChagas' Disease Vector Panstrongylus megistus (Hemiptera:Triatominae) in Brazil Based on Ribosomal DNA IntergenicSequencesAuthor(s): Francelisse Bridi Cavassin, Debora Do Rocío Klisiowicz, Luiz GustavoRodrigues Oliveira, Christian Collins Kuehn, Rogério Luiz Kopp, Vanette Thomaz-Soccol, João Aristeu Da Rosa, Ennio Luz, Santiago Mas-Coma, and María DoloresBarguesSource: Journal of Medical Entomology, 51(3):616-628. 2014.Published By: Entomological Society of AmericaURL: http://www.bioone.org/doi/full/10.1603/ME13073

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MOLECULAR BIOLOGY/GENOMICS

Genetic Variability and Geographical Diversity of the Main Chagas’Disease Vector Panstrongylus megistus (Hemiptera: Triatominae) in

Brazil Based on Ribosomal DNA Intergenic Sequences

FRANCELISSE BRIDI CAVASSIN,1 DEBORA DO ROCIO KLISIOWICZ,1,2

LUIZ GUSTAVO RODRIGUES OLIVEIRA,2,3 CHRISTIAN COLLINS KUEHN,2,3 ROGERIO LUIZ KOPP,4

VANETTE THOMAZ-SOCCOL,1 JOAO ARISTEU DA ROSA,5 ENNIO LUZ,1 SANTIAGO MAS-COMA,2

AND MARIA DOLORES BARGUES2,6

J. Med. Entomol. 51(3): 616Ð628 (2014); DOI: http://dx.doi.org/10.1603/ME13073

ABSTRACT Studies were made on the ribosomal DNA intergenic region, comprising completeinternal transcribed spacer (ITS)-1, 5.8S, and ITS-2 sequences, of populations of the triatominePanstrongylus megistus, the most important vector of ChagasÕ disease in Brazil since Triatoma infestanseradication. Specimens were from 26 localities of Rio Grande do Sul, Santa Catarina, Parana, Sao Paulo,Minas Gerais, Bahia, and Sergipe states. In total, 21 ITS-1 and 12 ITS-2 haplotypes were found.Nucleotide differences were higher in ITS-1 (3.00%) than in ITS-2 (1.33%). The intergenic region was1,513Ð1,522-bp-long (mean 1,516.9 bp), providing 26 combined haplotypes. The combination ofmicrosatellites found in both ITSs may be of applied usefulness, to assess interpopulation specimenexchange and potential recolonizations after vector elimination by control implementation. Networkresults suggest that Sao Paulo may be considered one of the spreading centers of this species. Molecularclock datation suggests that P. megistus populations are diversifying at least since 4.54 million yearsago, with diversiÞcation still ongoing today by geographical isolation of populations. Evidence isprovided about the relationship of genetic diversity with geographical spread that characterizes amajor vector and explains its ability to colonize distant areas and different ecotopes, including humanhabitats, and consequently its importance in ChagasÕ disease epidemiology.

KEYWORDS Panstrongylusmegistus,ChagasÕ disease, rDNA intergenic region, haplotype diversity,Brazil

Among the 19 neglected tropical diseases, ChagasÕdisease is the sixth most important tropical infection interms of global burden of disease, with high social andeconomic impact (WHO 2010). ChagasÕ disease, orig-inally restricted to Latin America, is now becoming aglobal public health concern owing to human migra-tions to developed countries (Coura and Albajar2010). The main mode of transmission of the etiologicagent, Trypanosoma cruzi, is vectorial via feces of in-fected hematophagous bugs (Hemiptera, Reduviidae,Triatominae). The vectors are crucial in establishingthe geographical distribution of the disease, its local

transmission patterns, and main epidemiological char-acteristics. Given the absence of effective drugs, thevectors become the key target for intervention activ-ities (WHO 2012).

In Brazil, about 2Ð3 million people are estimated tobe infected (Dias et al. 2008), 600,000 of them withchronic heart or digestive complications, causingdeath in about 5,000 individuals each year (Coura andDias 2009). The number of new ChagasÕ disease casesin Brazil has been reduced dramatically in recentyears, but showing a great regional diversity in termsof morbidity and mortality (Coura and Albajar 2010,Martins-Melo et al. 2012).

After Triatoma and Rhodnius, Panstrongylus is thethird most speciose genus of the Triatominae subfam-ily. It includes 13 species with a wide geographicaldistribution from Mexico to Argentina (Curto de Ca-sas et al. 1999). Twelve Panstrongylus species werefound to be infected naturally by Tr. cruzi. High in-fection rates might be an indicator of close proximityto reservoir hosts and high susceptibility to Tr. cruzi,as seems to be the case for Panstrongylus megistus inBrazil,Panstrongylus lignarius in Peru, andPanstrongy-

1 Departamento de Patologia Basica, Setor de Ciencias Biologicas,Universidade Federal do Parana, Centro Politecnico, Curitiba,Parana, Brazil.

2 Departamento de Parasitologõa, Facultad de Farmacia, Universi-dad de Valencia, Burjassot - Valencia, Spain.

3 Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Univer-sidade de Sao Paulo, FCFRP-USP, Ribeirao Preto, Sao Paulo, Brazil.

4 Departamento de Patologia Medica, Setor de Ciencias da Saude,Universidade Federal do Parana, Curitiba, Parana, Brazil.

5 Departamento de Ciencias Biologicas da Faculdade de CienciasFarmaceuticas, Universidade Estadual Paulista - UNESP, Araraquara,Sao Paulo, Brazil.

6 Corresponding author, e-mail: M.D.Bargues@uv.es.

0022-2585/14/0616Ð0628$04.00/0 � 2014 Entomological Society of America

lus geniculatus in several countries (Patterson et al.2009).P. megistus shows a wide geographical distribution,

ecological valence, and great potential of colonizationof artiÞcial ecotopes (Patterson et al. 2009). The At-lantic Forest seems to represent the center of distri-bution of P. megistus (Forattini 1980), although thespecies is also broadly distributed in humid areas of theCerrado and Caatinga (Barbosa et al. 2006). The ubiq-uitous sylvatic populations persistently domiciliate,sporadically invading human housing and, therefore,demanding continuous surveillance (Coura and Dias2009). The broad geographical distribution, the ca-pacity to invade and colonize domiciles, and the highlevels of Tr. cruzi infection indicate that P. megistus isthe vector species of the greatest epidemiological im-portance in Brazil after the control of T. infestans(Gurgel-Goncalves et al. 2012).

Molecular markers generate a large amount of in-formation about genetic diversity and phylogeneticrelationships of the organisms. In Triatominae, DNAmarkers proved to be useful for this endeavor in gen-eral, although disadvantages and limitations of theeach marker should be taken into account (Mas-Comaand Bargues 2009, Bargues et al. 2010). In the pastdecades, several studies have contributed to increasethe genetic knowledge about Panstrongylus speciesfrom Brazil as well as other Latin American countriesbased on DNA markers (Garcõa and Powell 1998; Ly-man et al. 1999; Bargues et al. 2000, 2002; Garcõa et al.2001; Marcilla et al. 2002; Mas-Coma and Bargues 2009;Patterson and Gaunt 2010; Blandon-Naranjo et al.2010; Zuriaga et al. 2012). Internal transcribed spacers(ITSs) of the nuclear ribosomal DNA (rDNA) haveproved to be useful for the classiÞcation of species,subspecies, hybrids, and populations of Triatominae,and for inferring their phylogenetic relationships

(Bargues et al. 2008, Mas-Coma and Bargues 2009).Nowadays, studies that provide complete data abouttriatomine vectors inferred from sequences of theintergenic rDNA region, including ITS-1, 5.8S gene,and ITS-2, are scarce and only known in the infestanssubcomplex species, in Triatoma rubrovaria popula-tions, and in species of theMepraia genus (Pacheco etal. 2003, 2007; Bargues et al. 2006; Calleros et al. 2010).

The aim of the current study concerns the geneticcharacterization by molecular haplotyping of differ-ent populations of P. megistus from Parana and otherneighboring states of Brazil, based on sequences of thecomplete rDNA intergenic region, to analyze the com-bined haplotype diversity and relationships. This is themost extensive molecular study of this triatomine sofar and the Þrst time that the ITS-1 is analyzed in thiscrucial vector.

Materials and Methods

Triatomine Specimens. In total, 90 P. megistus spec-imens were collected representing populations from26 localities covering a geographical representation ofmost of the states where P.megistus is present in Brazil(Carcavallo et al. 1999). rDNA presents the peculiar-ity of following a concerted evolution, which homog-enizes the many copies of nuclear rDNA among bothhomologous and nonhomologous chromosomes con-taining rDNA clusters within a genome. This gives riseto a uniformity inside all individuals of a populationand becomes extremely useful (Mas-Coma and Bar-gues 2009). In total, 43 specimens were sequenced,including more than one specimen from given popu-lations to verify certain single nucleotide polymor-phisms, mainly appearing in poly-A regions and dinu-cleotide microsatellite repeats (Figs. 1 and 2; Table 1).

Fig. 1. Maps of Panstrongylus megistus: (A) geographical distribution of this vector species in Brazil; (B) distribution oflocalities of Brazilian states from where specimens were obtained (for Parana see Fig. 2). (Online Þgure in color.)

May 2014 CAVASSIN ET AL.: GENETIC STUDIES OF P. megistus IN BRAZIL 617

Sequencing of rDNA ITS-1, 5.8S, and ITS-2. ForDNA extraction, a leg of each specimen Þxed in 70%ethanol was processed using methods outlined before(Bargues et al. 2000, 2002; Marcilla et al. 2001). TotalDNA was isolated by standard techniques, resus-pended in 50 �l of TE buffer (10 mM TrisÐHCl, 1 mMEDTA, pH 7.6), and stored at �20�C until use. Thenuclear rDNA intergenic fragment corresponding tothe ITS-1, 5.8S, and ITS-2 regions was polymerasechain reaction (PCR) ampliÞed with primers Eu1657and Infer for ITS-1 and 5.8T and 28T for ITS-2, fol-lowing the methods outlined before (Bargues et al.2006) and using 4Ð6 �l of genomic DNA for each 50�l reaction. AmpliÞcations were generated in a Mas-tercycler ep gradient (Eppendorf, Hamburg, Ger-many), by 30 cycles of 30 s at 94�C, 30 s at 50Ð55�C, and1 min at 72�C, preceded by 30 s at 94�C and followedby 7 min at 72�C.

PCR products were puriÞed using the UltraCleanPCR Clean-up DNA PuriÞcation System (MoBio, So-lana Beach, CA, USA) according to the manufacturerÕsprotocol and resuspended in 50 �l of 10 mM TE buffer(pH 7.6). Sequencing was performed on both strandsby the dideoxy chain-termination method, with theTaqdye-terminatorchemistrykit forABI3130andABI3700 (Applied Biosystems, CA, USA), using the samePCR ampliÞcation primers. Given the importance ofthe recent discovery of a pseudogenic 5.8S�ITS-2sequence, designated as “ps(5.8S�ITS-2),” widely dis-tributed in triatomines of North, Central, and SouthAmerica (Bargues et al. 2014), special efforts weremade to ensure that no double signal was present inthe chromatograms, to conÞrm that variable positionsin the intergenic region were not due to an underlyingparalogous sequence.

The haplotype (H) terminology used for both ITSsfollows the nomenclature for composite haplotyping(CH) previously proposed (Bargues et al. 2006, Mas-

Coma and Bargues 2009). Accordingly, ITS-2 haplo-types are noted by numbers and ITS-1 haplotypes arenoted by capital letters.

Sequences were aligned using CLUSTAL W2 (Lar-kin et al. 2007) and MEGA 5.2 (Tamura et al. 2011),using default settings. Minor corrections for a better Þtof nucleotide or indel correspondences were made.DnaSP v.5.1 (Librado and Rozas 2009) was used toevaluate the number of haplotypes (h), haplotypediversity (Hd), nucleotide diversity expressed as theaverage number of nucleotide differences betweentwo sequences by site (�), average number of nucle-otide differences between sequences (k), and numberof polymorphisms and insertions/deletions (S). Ge-netic distances were measured using parameters pro-vided by PAUP v.4.0 b10 (Swofford 2002). Calcula-tions for datation were based on both ITS-2 and ITS-1molecular clock rates obtained for Triatomini (Bar-gues et al. 2000, 2006). Estimates were obtained bycomputing PAUP differences, and the dating rangeswere obtained for the divergences that appeared inthe pairwise distance matrix of nucleotide divergencesfor each one of the two spacers.

The following sequences from GenBankÐEMBLhave been used for phylogenetic analyses: ITS-1 ofPanstrongylus herreri (AM949584) and Panstrongylusgeniculatus (AM949585) (Mas-Coma and Bargues2009); ITS-2 ofP. herreri (AJ306550) andP. geniculatus(AJ306543) (Marcilla et al. 2002); and complete in-tergenic region of Triatoma sordida (AJ576063), T.infestans (AJ576051), Triatoma platensis (AJ576061),Triatoma delpontei (AJ576057) (Bargues et al. 2006),and T. rubrovaria (AJ557258) (Pacheco et al. 2007).Network and Phylogenetic Analyses. A phyloge-

netic network estimation using median-joining (MJ)network algorithm using default parameters (equalcharacter weight � 10, transitions/transversionsweight � 1:1, and connection cost as a criterion) was

Fig. 2. Geographical localities of the state of Parana, Brazil, from where Panstrongylus megistus specimens were capturedand analyzed. Note differentiation of state areas according to the geomorphological units of the plateau. (Online Þgure incolor.)

618 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3

Tab

le1

.G

eogr

aphi

call

ocal

itie

san

dha

bita

tsof

the

Pan

stro

ngyl

usm

egis

tus

spec

imen

san

alyz

ed,

hapl

otyp

esob

tain

edfo

rrD

NA

ITS-

1an

dIT

S-2

mar

kers

,an

dG

enB

ank

acce

ssio

nnu

mbe

rsof

the

sequ

ence

sob

tain

ed

No.in

map

Geogra

ph

ical

loca

tion

(no.of

speci

men

sse

quen

ced)

Sta

teH

abit

at

ITS-1

ITS-2

Inte

rgen

icre

gio

nIT

S-1

,5.

8S,

ITS-2

Gen

Ban

kac

cess

ion

no.

ITS-1

hap

loty

pe

Len

gth

(bp)

AT

(%)

ITS-2

hap

loty

pe

Len

gth

(bp)

AT

(%)

Com

bin

ed

hap

loty

pe

Len

gth

(bp)

AT

(%)

1Sim

aoD

ias

Serg

ipe

Sylv

atic

ITS1-

S76

268

.8IT

S2Ð

959

875

.2P

.meg-C

H9S

1,51

568

.6H

F67

8472

1Sim

aoD

ias

(2)

Serg

ipe

Sylv

atic

ITS1-

S76

268

.8IT

S2Ð

160

075

.2P

.meg-C

H1S

1,51

768

.6H

F67

8473

2Sao

Felipe

(2)

Bah

iaD

om

est

icIT

S1-

T76

269

.1IT

S2Ð

259

875

.4P

.meg-C

H2T

1,51

568

.8H

F67

8474

3B

elo

Hori

zon

te(3

)M

inas

Gera

isP

eri

dom

est

icIT

S1-

C76

469

.0IT

S2Ð

1059

875

.0P

.meg-C

H10

C1,

521

68.6

HF

6784

754

Var

gin

ha

Min

asG

era

isP

eri

dom

est

icIT

S1-

U76

169

.0IT

S2Ð

460

075

.0P

.meg-C

H4U

1,51

668

.6H

F67

8477

5Sao

Joao

da

Boa

Vis

ta(c

ol.)

Sao

Pau

loSylv

atic

ITS1-

S76

268

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460

075

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1,51

768

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F67

8471

6A

rara

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olo

nia

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Pau

loSylv

atic

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T76

269

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259

875

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1,51

568

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F67

8474

6A

rara

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Pau

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eri

dom

est

icIT

S1-

S76

268

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S2Ð

460

075

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H4S

1,51

768

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F67

8471

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da

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Pau

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eri

dom

est

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S76

268

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160

075

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1,51

768

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7M

ogi

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est

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O76

068

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160

075

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H1O

1,51

568

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F67

8465

8P

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568

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acac

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Peri

dom

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668

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360

175

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H3H

1,52

268

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8460

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ampo

Mag

roP

aran

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eri

dom

est

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G76

568

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1260

075

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H12

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521

68.4

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6784

6111

Cerr

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ana

Peri

dom

est

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S1-

P76

269

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259

875

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H2P

1,51

568

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F67

8464

12R

ioB

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)P

aran

aP

eri

dom

est

icIT

S1-

A76

368

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160

075

.2P

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H1A

1,51

868

.5H

F67

8458

12R

ioB

ran

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Par

ana

Peri

dom

est

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S1-

O76

068

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160

075

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H1O

1,51

568

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F67

8465

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ana

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268

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160

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768

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8459

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360

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368

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968

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468

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1,52

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tan

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Par

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dom

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1,51

368

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20L

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dri

na

Par

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Peri

dom

est

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160

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168

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dom

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368

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160

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975

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668

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8463

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Peri

dom

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268

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075

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768

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8452

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alm

itopolis

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ana

Peri

dom

est

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875

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468

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alm

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Par

ana

Peri

dom

est

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568

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S2Ð

160

075

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1,52

068

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8467

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lori

anopolis

(2)

San

taC

atar

ina

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atic

ITS1-

R76

069

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1160

175

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H11

R1,

514

68.6

HF

6784

7625

San

taR

osa

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Gra

nde

do

Sul

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atic

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M75

868

.6IT

S2Ð

760

075

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1,51

368

.7H

F67

8455

25San

taR

osa

(colo

nia

)R

ioG

ran

de

do

Sul

Sylv

atic

ITS1-

Q76

169

.0IT

S2Ð

959

875

.2P

.meg-C

H9Q

1,51

468

.4H

F67

8470

25San

taR

osa

Rio

Gra

nde

do

Sul

Sylv

atic

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M75

968

.5IT

S2Ð

660

075

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.meg-C

H6M

1,51

368

.4H

F67

8454

26Sen

ador

Sal

gad

oF

ilh

oR

ioG

ran

de

do

Sul

Sylv

atic

ITS1-

K76

569

.0IT

S2Ð

160

075

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H1K

1,52

068

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F67

8469

Num

ber

of

the

geogra

ph

ical

loca

tion

acco

rdin

gto

the

map

sof

Fig

ure

s1

and

2.N

um

bers

inbra

ckets

indic

ate

the

num

ber

of

speci

men

sse

quen

ced

wh

en

more

than

on

ein

div

idual

.

May 2014 CAVASSIN ET AL.: GENETIC STUDIES OF P. megistus IN BRAZIL 619

constructed, for the ITS-1 and ITS-2 combined hap-lotypes, using Network 6.4.1.1 (http://www.ßuxus-engineering.com/). Sites with alignment gaps or miss-ing data were considered. Hypothetical medianvectors (a hypothesized sequence that is required toconnect existing sequences within the network withmaximum parsimony) were added to the network forshortest connection between the data set.

The relationships between P. megistus and otherTriatominae species whose complete intergenic re-gion was available were inferred by phylogenetic treereconstruction using PAUP 4.0b10 and the maximumlikelihood (ML) method based on the HasegawaÐKishinoÐYano model. ML parameters and the evolu-tionary model best Þtting our data set were deter-mined using Akaike and Bayesian information criteria,implemented in jModeltest version 0.1.1. Startingbranch lengths were obtained using the least-squaresmethod with ML distances. The sequences ofMepraiaspinolai FN396516 and Triatoma eratyrusiformisFN396537 (Calleros et al. 2010) were used as out-group. Four CHs were used to represent P. megistus;the selection was made to include representatives ofeach one of the groups distinguished by the networkanalysis (with two CHs for the most diversiÞed groupand only one for each of the two other groups), andsimultaneously a representative from the spreadingcenter of the species. Statistical support for the nodeswas evaluated with 1,000 bootstrap replicates, usingheuristic search.

Other phylogenetic trees were also reconstructedby including all species of Triatoma and Mepraia ofwhich the complete intergenic region is known, to-gether with all the CHs of the P. megistus populationsstudied to compare with network results. When re-constructing such trees, it was not forgotten that math-ematical methods for phylogenetic analyses are madefor the assessment of relationships between watertightcompartments (i.e., noncrossing units, as it is the caseof different species and higher taxa) but not for pop-ulation relationships within a species. For this pur-pose, ML and neighbor-joining (NJ) methods wereimplemented in MEGA 5.2 program. A discretegamma distribution was used to model evolutionaryrate differences among sites (four categories). Evo-lutionary distances were computed using the Kimura2-parameter method. To provide an assessment of thereliability of the nodes in trees, statistical support wasevaluated with 1,000 bootstrap replicates.

Results

Sequence Analysis of rDNA ITS Haplotypes. Se-quence length and AT content for each ITS with theircorresponding haplotype codes and new GenBankaccession numbers are listed in Table 1.

The complete intergenic region revealed the exis-tence of 26 combined haplotypes (CH) (Table 1).Their alignment generated a 1,523-bp data set thatcontained 31 variable positions (2.03%), of which 6were singleton sites (0.39%), 11 parsimony informa-tive positions (0.72%), and 14 insertions or deletions

(indels) (0.92%) (Table 2). The length of this regionvaried from 1,513 to 1,522 bp (mean 1,516.9 bp). The155-bp-long 5.8S was identical in all specimens. Whencomparing the sequences of the 26 CHs, the pairwiseITS-1 and ITS-2 distance matrix obtained with PAUP(only parsimony informative sites considered) showsthat the number of nucleotide differences is highwhen comparing haplotypes from Minas Gerais andSanta Catarina with haplotypes present in other states(Table 3).

Twenty-one ITS-1 haplotypes (ITS1-A to ITS1-U)were found. In their 766-bp-long alignment, 23 vari-able positions appeared (3.00%), of which 10 weresubstitutions (1.30%), including six transitions (ts)and four transversions (tv), and 13 were indels (1.7%).The ITS-1 length ranged between 758 and 766 bp(mean 762.3 bp), and the nucleotide composition wasAT-biased (68.5Ð69.2%; mean 68.9%). Thirteen ofthese haplotypes were present in Parana, of whichonly two were also present in Rio Grande do Sul andSao Paulo (Table 2).

The sequences of the ITS-2 provided 12 ITS-2 hap-lotypes (ITS2Ð1 to ITS2Ð12). In the 488-bp-long ITS-2alignment, only eight variable positions appeared(1.33%), of which Þve were substitutions (0.83%),including two transitions (ts) and three transversions(tv), and three were indels (0.50%). ITS-2 lengthranged between 598 and 601 bp (mean 599.6 bp), withan AT-biased nucleotide composition (average75.2%). In Parana, seven different haplotypes weredetected, of which only three were shared with otherlocalities in Rio Grande do Sul, Sergipe, Sao Paulo, andBahia (Table 2).

A comparative analysis of the haplotype character-istics and information provided by ITS-1 and ITS-2markers is shown in Table 4.

Sequence repeats were present in the intergenicrDNA region. ITS-1 shows minisatellite repeats in allhaplotypes and populations (Fig. 3), plus only onevariable dinucleotide repeat (one, two, or three re-peats of TG). In ITS-2, only interrupted microsatellitesand dinucleotide repeats were found, with (GC) asthe only one showing variations (two or three repeats)according to haplotypes (Fig. 3).Combined Haplotype Diversity. In Parana, 15 dif-

ferent CHs were detected. Only one of them (1O) isalso present in samples from Sao Paulo. In the other sixstates, 11 different CHs were found, of which two arepresent in two states: CH-1S in Sergipe and Sao Paulo;CH-2T in Sao Paulo and Bahia (Tables 1 and 2; Fig. 4).

The genetic variability among the 26 CHs showed adiversity of 0.946 (variance � 0.000047; standard de-viation � 0.022), a nucleotide diversity of 0.00316(variance � 0.0000; standard deviation � 0.00022), anaverage number of nucleotide differences of 4.2655,and a number of polymorphisms and insertions/dele-tions of 3.3995.

An analysis was performed to assess a potentialcorrelation of the CH diversity with the three geo-morphological units of Parana state. Five CHs ap-peared in the Þrst plateau (planalto de Curitiba), Þvein the second (planalto de Ponta Grossa), and six in

620 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3

the third (planalto de Guarapauva) (Table 1; Fig. 2).Neither a correlation nor a signiÞcant result wasfound. However, a few data may be highlighted: 1)only one haplotype is present throughout the threeplateaus (ITS-2 H1); 2) only one haplotype is sharedby the Þrst and second plateaus (CH-3H); 3) the thirdplateau is the one presenting a larger variability, de-spite the pronouncedly lower number of populationsstudied (six CHs from only four localities); and 4) onlyone CH of Parana is present in another state (CH-1Oalso in Sao Paulo).Network Analysis. The CH network constructed

using MJ network algorithm shows the existence ofthree groups: 1) a big group with a high diversity ofhaplotypes (13 CHs), including samples from differ-ent states (Sergipe, Sao Paulo, Rio Grande do Sul,Santa Catarina, and Bahia) and from localities ofParana (Arapongas, Palmitopolis, Cerro Azul, and Pi-raquara); 2)a secondgroup includinghaplotypes fromParana, with the exception of only one from RioGrande do Sul; and 3) one small group including twohaplotypes from Rio Grande do Sul and one fromParana (Fig. 4). Between the Þrst and second groups,the intermediate position of CH 8D (from the localityof Goioxim in Parana) should be emphasized, showing

similar mutational steps with regard to haplotypes 9Land 1I (both from Palmitopolis, Parana), located inÞrst and second groups, respectively.

Only three haplotypes (1S, 1O, and 2T) appear atleast in two states simultaneously. Parana and RioGrande do Sul are the only states appearing in thethree groups (Fig. 4). The majority of haplotypesfound in Sao Paulo, all of them included within the Þrstgroup, are also present in other states.Phylogenetic Analyses. For the tree including the

only four P. megistus CHs selected to represent thethree different groups (distinguished in the aforemen-tioned network), the ML model best Þtting the ITSscombined data was HKY85�I�G using the ts/tv ratioof 1.619 (kappa � 3.307; base frequencies for A, C, G,and T of 0.32369, 0.12524, 0.15439, and 0.35707, re-spectively; a proportion of invariable sites of 0.12; anda gamma distribution of 4.031). The resulting phylog-eny (-Ln � 6413.5595) was evaluated using the least-squares method with ML distances. In the ML treeobtained, the four P. megistus CHs cluster togetherwith the maximum support and appear distributed intwo clades, one including the representatives ofgroups B and C and the other including the two rep-resentatives of group A (Fig. 5).

Table 2. Nucleotide differences found in the complete intergenic rDNA sequence of Panstrongylus megistus specimens studied

Position � numbers (to be read in vertical) refer to variable positions obtained in the alignment made with MEGA 5.2.Identical � .; indel � -.Positions 103Ð605 and 984Ð1517 contain variable positions in the ITS-1 and ITS-2, respectively.

May 2014 CAVASSIN ET AL.: GENETIC STUDIES OF P. megistus IN BRAZIL 621

Tab

le3

.P

airw

ise

dist

ance

sbe

twee

nco

ncat

enat

edIT

S-1

and

ITS-

2se

quen

ces

ofth

eP

.m

egis

tus

popu

lati

ons

anal

yzed

acco

rdin

gto

PA

UP

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

1C

H10

CM

G-

0.00

368

0.00

294

0.00

368

0.00

515

0.00

294

0.00

368

0.00

515

0.00

516

0.00

515

0.00

588

0.00

442

0.00

368

0.00

664

0.00

590

0.00

664

0.00

587

0.00

736

0.00

736

0.00

736

0.00

736

0.00

661

0.00

661

0.00

587

0.00

587

0.00

368

2C

H11

RSC

5-

0.00

294

0.00

295

0.00

442

0.00

221

0.00

295

0.00

442

0.00

443

0.00

442

0.00

368

0.00

368

0.00

295

0.00

591

0.00

517

0.00

591

0.00

662

0.00

811

0.00

811

0.00

812

0.00

812

0.00

736

0.00

736

0.00

663

0.00

663

0.00

442

3C

H5P

PR

44

-0.

0007

40.

0022

10.

0014

70.

0022

10.

0022

10.

0022

10.

0022

10.

0029

40.

0029

40.

0022

10.

0036

90.

0029

50.

0036

90.

0066

10.

0066

30.

0066

30.

00590

0.00

590

0.00

588

0.00

515

0.00

441

0.00

441

0.00

368

4C

H2P

PR

54

1-

0.00

147

0.00

074

0.00

147

0.00

147

0.00

147

0.00

147

0.00

221

0.00

221

0.00

147

0.00

443

0.00

369

0.00

295

0.00

515

0.00

516

0.00

516

0.00

516

0.00

516

0.00

441

0.00

441

0.00

368

0.00

368

0.00

294

5C

H2T

SP

,B

A7

63

2-

0.00

221

0.00

294

0.00

294

0.00

295

0.00

294

0.00

368

0.00

368

0.00

294

0.00

590

0.00

517

0.00

443

0.00

662

0.00

368

0.00

368

0.00

368

0.00

368

0.00

294

0.00

294

0.00

221

0.00

221

0.00

442

6C

H9Q

RS

43

21

3-

0.00

221

0.00

221

0.00

221

0.00

221

0.00

294

0.00

147

0.00

074

0.00

517

0.00

443

0.00

369

0.00

442

0.00

590

0.00

590

0.00

590

0.00

590

0.00

515

0.00

515

0.00

442

0.004

420.

0022

1

7C

H4U

MG

54

32

43

-0.

0029

40.

0029

50.

0029

40.

0022

00.

0036

80.

0029

40.

0059

00.

0051

60.

0044

20.

0066

10.

0066

30.

0066

30.

0066

30.

0066

30.

0058

80.

0058

80.

0051

40.

0051

40.0

0441

8C

H1N

PR

76

32

43

4-

0.00

074

0.00

147

0.00

220

0.00

221

0.00

294

0.00

442

0.00

368

0.00

295

0.00

514

0.00

515

0.00

515

0.00

515

0.00

515

0.00

440

0.00

441

0.00

514

0.00

514

0.00

294

9C

H1O

PR

,SP

76

32

43

41

-0.

0000

00.

0007

40.

0007

40.

0029

50.

0029

50.

0022

10.

0014

80.

0036

80.

0036

80.

0036

80.

0036

80.

0036

80.

0044

10.

0044

20.

0051

50.

0051

50.

0029

5

10C

H1S

SE

,SP

76

32

43

42

0-

0.00

073

0.00

074

0.00

294

0.00

295

0.00

221

0.00

147

0.00

367

0.00

368

0.00

368

0.00

368

0.00

368

0.00

441

0.00

441

0.00

514

0.00

514

0.00

294

11C

H4S

SP

85

43

54

33

11

-0.

0014

70.

0036

80.

0036

80.

0029

50.

0022

10.

0044

10.

0044

20.

0044

20.

0044

20.

0044

20.

0051

40.

0051

40.

0058

70.

0058

70.

0036

7

12C

H9S

SE

65

43

52

53

11

2-

0.00

221

0.00

369

0.00

295

0.00

221

0.00

294

0.00

442

0.00

442

0.00

442

0.00

442

0.00

515

0.00

515

0.00

588

0.00

588

0.00

221

13C

H9L

PR

54

32

41

44

44

53

-0.

0044

30.

0036

90.

0029

50.

0036

80.

0051

60.

0051

60.

0051

60.

0051

60.

0044

20.

0044

20.

0036

80.

0036

80.

0014

7

14C

H6M

RS

98

56

87

86

44

55

6-

0.00

074

0.00

147

0.00

368

0.00

368

0.00

368

0.00

368

0.00

368

0.00

442

0.00

442

0.00

515

0.00

515

0.00

442

15C

H7M

RS

87

45

76

75

33

44

51

-0.

0007

40.

0029

50.

0029

50.

0029

50.

0029

50.

0029

50.

0036

80.

0036

80.

0044

20.

0044

20.

0036

8

16C

H1M

PR

98

54

65

64

22

33

42

1-

0.00

221

0.00

221

0.00

221

0.00

221

0.00

221

0.00

295

0.00

295

0.00

368

0.00

368

0.00

295

17C

H12

GP

R8

99

79

69

75

56

45

54

3-

0.00

293

0.00

293

0.00

294

0.00

294

0.00

366

0.00

366

0.00

440

0.00

440

0.00

220

18C

H3E

PR

1011

97

58

97

55

66

75

43

4-

0.00

000

0.00

000

0.00

000

0.00

073

0.00

073

0.00

147

0.00

147

0.00

368

19C

H3A

PR

1011

97

58

97

55

66

75

43

40

-0.

0000

00.

0000

00.

0007

30.

0007

30.

0014

70.

0014

70.

0036

8

20C

H1A

PR

1011

87

58

97

55

66

75

43

40

0-

0.00

000

0.00

073

0.00

073

0.00

147

0.00

147

0.00

368

21C

H1F

PR

1011

87

58

97

55

66

75

43

40

00

-0.

0007

30.

0007

30.

0014

70.

0014

70.

0036

8

22C

H3H

PR

910

86

47

86

66

77

66

54

51

11

1-

0.00

000

0.00

073

0.00

073

0.00

294

23C

H1I

PR

910

76

47

86

66

77

66

54

51

11

10

-0.

0007

30.

0007

30.

0029

4

24C

H1J

PR

89

65

36

77

77

88

57

65

62

22

21

1-

0.00

000

0.00

367

25C

H1K

RS

89

65

36

77

77

88

57

65

62

22

21

10

-0.

0036

7

26C

H8D

PR

56

54

63

64

44

53

26

54

35

55

54

45

5-

Belo

wdia

gon

al�

tota

lch

arac

ter

dif

fere

nce

s;ab

ove

dia

gon

al�

mean

char

acte

rdif

fere

nce

s(a

dju

sted

for

mis

sin

gdat

a).C

om

posi

teh

aplo

type

(CH

)co

des

list

ed

inT

able

1.P

R,P

aran

a;SP

,Sao

Pau

lo;R

S,R

ioG

ran

de

do

Sul;

SE

,Serg

ipe;M

G,M

inas

Gera

is;B

A,B

ahia

;SC

,San

taC

atar

ina.

622 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3

In the phylogenetic analyses including the 26 CHsof P. megistus, both ML (HKY�I�G, matrix of pair-wise distances estimated using the maximum compos-ite likelihood approach, proportion of invariablesites � 0.407, and a discrete gamma distribution �0.04) and NJ (Kimura 2-parameter method) furnisheda similar topology distributing the CH according to thesame groups distinguished by the network analysis andwith the CH 8D appearing between groups A and B.Both trees show, however, low bootstrap values, asexpected because of dealing with different popula-tions of the same species (tree not shown).

Discussion

Analysis of Sequences.The whole intergenic regioncomprising ITS-1, 5.8S, and ITS-2 provides a data setwith an average length and AT content of 1,516.9 bpand 68.6%, respectively. This region was considerablylonger than the 1,377 bp described for T. rubrovaria(Pacheco et al. 2003, 2007), the 1,375 bp of the infes-tans subcomplex (Bargues et al. 2006), and the 1,371bp in the genus Mepraia (Calleros et al. 2010). Thetotal of 26 combined haplotypes described for thiscomplete intergenic region provides a genetic vari-ability of 2.03% in the P. megistus populations ana-lyzed, allowing for inter-populational differentiation.This suggests evolving divergence processes. A similarsituation, with a different haplotype in almost eachdifferent locality, was also found in T. rubrovaria, al-though with a pronounced lower variability (1.31%)(Pacheco et al. 2003).

The ITS-1 in P. megistus proved to be markedlylonger than ITS-2, and its bias in AT composition waslower than in ITS-2, which agrees with previous ob-servations in other Triatominae (Bargues et al. 2006,Pacheco et al. 2007, Mas-Coma and Bargues 2009,Calleros et al. 2010). Regarding ITS-2, average length(599.6 bp) agreed with the range of 470Ð600 bp ob-served in different Panstrongylus species from differ-ent countries (Marcilla et al. 2002). In ITSs of severalTriatominae species, sequence lengthvaries accordingto the presence of repeated sequences such as mic-rosatellites and/or minisatellites, which have beenproved to furnish valuable information at populationlevel (Bargues et al. 2006). However, such short se-quence repeats have been shown to be constantamong different populations within other species(Marcilla et al. 2001, Pacheco et al. 2007).

In the P. megistus specimens, ITS-1 sequencesshowed both: 1) constant microsatellites of 2Ð4 nu-cleotides tandemly repeated and scattered along thesequence, among which only one variable (TG) al-lowed differentiation between populations: almost allpopulations presented only one repeat; two repeatsdistinguished the locality of Goioxim in Parana; threerepeats are present in one locality of Minas Gerais(Belo Horizonte), seven localities of Parana(Campo Magro, Rio Azul, Reboucas, Castro, Wenc-eslao Braz, Londrina, and Palmitopolis), and onelocality in Rio Grande do Sul (Senador SalgadoFilho); and 2) minisatellites, one tandem repeat of

Tab

le4

.C

ompa

riso

nof

the

char

acte

rist

ics

ofth

eIT

S-1

and

ITS-

2se

quen

ces

ofth

eP

anst

rong

ylus

meg

istu

ssp

ecim

ens

from

26

geog

raph

ical

loca

litie

sof

seve

nst

ates

ofB

razi

l

Tota

lh

aplo

types

Hap

loty

pe

codes

Avera

ge

len

gth

(bp)

Nucl

eoti

de

dif

fere

nce

sN

o.(%

)

Subst

ituti

on

sN

o.(%

)In

dels

No.(%

)H

aplo

types

foun

din

more

than

on

est

ate

Sta

tes

shar

ing

hap

loty

pes

ITS-1

21IT

S1-

Ato

ITS1-

U76

2.3

23(3

.00%

)10

(1.3

0%)

13(1

.70%

)IT

S1-

M,I

TS1-

O,I

TS1-

S,I

TS1-

TP

R-R

S;P

R-S

P;SP

-SE

;SP

-BA

ITS-2

12IT

S2Ð

1to

ITS2Ð

1259

9.6

8(1

.33%

)5

(0.8

3%)

3(0

.50%

)IT

S2Ð

1,IT

S2Ð

2,IT

S2Ð

4,IT

S2Ð

9P

R-R

S,SE

,M

G,SP

;P

R-S

P;P

R-S

E;M

G-S

P

PR

,P

aran

a;SP

,Sao

Pau

lo;R

S,R

ioG

ran

de

do

Sul;

SE

,Serg

ipe;M

G,M

inas

Gera

is;B

A,B

ahia

;SC

,San

taC

atar

ina.

May 2014 CAVASSIN ET AL.: GENETIC STUDIES OF P. megistus IN BRAZIL 623

12 nucleotides and another nontandem repeat of 17nucleotides, which appeared to be constant in allspecimens studied and consequently noninforma-tive. These minisatellites differ from those detected

in the infestans subcomplex, within which theirnumber of repeats clearly distinguished betweensylvatic and domestic populations of T. infestans(Bargues et al. 2006).

Fig. 3. Sequence repeats detected in ITS-1 and ITS-2 haplotypes of Panstrongylus megistus from Brazil: (A) distributionof minisatellites and of the variable microsatellite in the ITS-1; (B) distribution of two types of variable microsatellites in theITS-2. Thick lines represent sequence of ITS-1 and ITS-2 in the 5�-3� sense. Numbers refer to alignment positions of the ITS-1and ITS-2, including all haplotypes found. Nucleotides in boxes correspond to mini- and microsatellite repeats.

Fig. 4. Median network analysis of Panstrongylus megistus combined haplotypes based on rDNA ITS-1 and ITS-2sequences. The area of each haplotype is proportional to the total sample. Mutational steps between haplotypes arerepresented by line length. Small red-Þlled circles represent suggested intermediate haplotypes not present in the sample.Combined haplotype codes and corresponding geographical localities listed in Table 1. (Online Þgure in color.)

624 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 3

With regard to ITS-2, microsatellites have usuallybeen described in Triatominae, but never minisatel-lites (Mas-Coma and Bargues 2009). In the P. megistuspopulations analyzed, microsatellites proved to be-long to two types: 1) among the several tandemlyrepeated dinucleotide microsatellites, only (CG)shows variations, allowing to discriminate betweensome populations: two repeats are present in threelocalities in Parana (Cerro Azul, Arapongas, Palmito-polis), one in Minas Gerais (Belo Horizonte), and onein Santa Catarina (Florianopolis); three repeats arepresent in one locality of Minas Gerais (Belo Hori-zonte), one in Sao Paulo (Sao Joao de Boa Vista), fourin Parana (Campo Magro, Castro, Siqueira Campos,Goioxim), and one in Rio Grande do Sul (Santa Rosa);and 2) one interrupted microsatellite (AT), whichappears as (AT)6TTT(AT)1AATGT(AT)5 or as(AT)4AC(AT)1TTT(AT)1AATGT(AT)5 owing to aT-C transition; haplotypes including this transitionappear to show a wide geographical distribution butappear to be rare, i.e., only in seven localities amongthe total of 26: Simao Dias (Sergipe); Belo Horizonte(Minas Gerais); Campo Magro, Goioxim, and Palmi-topolis (Parana); Florianopolis (Santa Catarina); andSanta Rosa (Rio Grande do Sul). The same interruptedmicrosatellite in the same region of the ITS-2 is alsopresent, but with some punctual nucleotide differ-ences, in T. infestans haplotypes (Bargues et al. 2006).The combination of the aforementioned microsatel-lites in both ITSs of P. megistus may be of futureapplied usefulness, as for instance to assess interpop-ulation specimen exchange and potential recoloniza-tions after vector elimination by control implementa-tion.

The ITS-1 sequences showed a large haplotype vari-ability throughout the distribution range of P. megis-tus. IntraspeciÞc nucleotide differences appear to behigher in ITS-1 (3.00%) than in the ITS-2 (1.33%),concerning both substitutions and indels. The nucle-

otide divergence considering only substitutions is alsohigher in ITS-1 (1.30%) than in ITS-2 (0.83%). Theseresults are in agreement with the molecular clockdating proposed for triatomines, according to whichITS-1 evolves 1.12Ð2.60 times faster than ITS-2 (Bar-gues et al. 2000, 2002, 2006). Thus, the number of ts andtv in the ITS-1 enables for a better population differ-entiation than when considering them in ITS-2.

However, the intraspeciÞc variability of the ITS-2 inP. megistus is relatively high when compared withwhat is known in other triatomines. Despite the longITS-2 sequence (Panstrongylus is the triatomine genuspresenting the longer ITS-2; Mas-Coma and Bargues2009), its variability enters among those more variable,although far away from the wide variability of 2.70% inT. infestans (Bargues et al. 2006), and especially that of5.62% in Triatoma dimidiata (including all subspeciesand excluding the species T. sp. aff. dimidiata) (Bar-gues et al. 2008). It should be highlighted, however,that T. infestans and T. dimidiata are two species thatwere transported by humans up to cover a geograph-ical distribution pronouncedly wider than that of P.megistus.Diversity, Spread, and Datation. The 26 P. megistus

CHs found represent a large variability, among whichthe populations of Florianopolis (Santa Catarina) andBelo Horizonte (Minas Gerais) appear to be the mostgenetically divergent ones. This is in agreement withresults from other studies onP.megistus (Barbosa et al.2003, 2006), which emphasized that Santa Catarinapopulations occurred in a differentiated morphocli-matic domain.

When considering both markers separately, fourITS-1 (M, O, S, T) and four ITS-2 (1, 2, 4, 9) haplotypesappear simultaneously in four (Parana, Rio Grande doSul, Sao Paulo, Sergipe) of the seven Brazilian states.Minas Gerais and Santa Catarina do not share haplo-types with any other state, and only the haplotypeITS1-T from Bahia is also present in Sao Paulo.

In Parana, no haplotypeÐgeographical associationwas found, suggesting a spread and reshufßing of hap-lotype distribution. The third plateau had a stronghuman immigration in the past, making the northernand southwestern areas to become true pioneers, as aconsequence of the acceleration of deforestation, thehigh rate of land modiÞcation for agriculture and openroads, and also the development of villages and townslinked together (Balhana et al. 1969). This may havefacilitated passively receiving bugs from other states.

Haplotype 1 of the ITS-2 appears to be the mostspread one, found in all plateaus of Parana and in fourother states. Thus, it may be considered the haplotypemore close to that which initiated the spread of thespecies from its endemism center (Forattini 1980).The lack of geographical separation and isolation of P.megistus of Parana from other states was also evidentin multiple isoenzyme analysis (Kopp et al. 2009). Thepopulation distribution in Brazil should be affected byanalyses based on points of origin, and the eventsundergone by these habitats during the past 18,000 yrsuggest that P. megistus populations were alreadyformed and deÞned by this time (Forattini 1980, Pat-

Fig. 5. Phylogenetic ML tree of Panstrongylus megistuscombined ITS-1 and ITS-2 haplotypes with two other speciesof the genus Panstrongylus and Þve South American speciesof genus Triatoma. Mepraia species used as outgroup. Sup-ports for nodes indicated by bootstrap values obtained with1,000 replicates using heuristic search in PAUP. Groups A, B,and C correspond to groupings obtained with the networkanalysis (Fig. 4).

May 2014 CAVASSIN ET AL.: GENETIC STUDIES OF P. megistus IN BRAZIL 625

terson et al. 2009). The ITS analyses suggest a rela-tively old origin of P. megistus, based on the highgenetic variability and geographical diversity detectedamong populations studied throughout the distribu-tion of this species in Brazil. When applying the mo-lecular clock of 0.41Ð0.99% variation per 1 millionyears (my) based on ITS-2 evolutionary rates in Tri-atominae (Bargues et al. 2000), the two most divergentITS-2 haplotypes H1 and H11 furnish a divergence of0.84Ð2.02 my when considering all nucleotide differ-ences and 0.33Ð0.80 my when only considering mu-tations. On the contrary, the two closest ITS-2 haplo-types H1 and H3 furnish a divergence of 0.39Ð0.16 mywhen considering all nucleotide differences and now-adays (�0.00 my) when only considering mutations.When applying the molecular clock of 0.46Ð2.58%variation per 1 my based on ITS-1 evolutionary ratesin Triatominae (Bargues et al. 2006), the two mostdivergent ITS-1 haplotypes HA and HU furnish a di-vergence of 0.81Ð4.54 my when considering all nucle-otide differences and 0.55Ð3.11 my when only consid-eringmutations.Onthecontrary, the twoclosest ITS-1haplotypes HA and HE furnish a divergence of 0.05Ð0.28 my when considering all nucleotide differencesand nowadays (�0.00 my) when only consideringmutations. Hence, these data suggest that the popu-lations of the species P. megistus are diversifying atleast since 4.54 my ago, the closest only in the recent50,000 yr. These results Þt the aforementioned dataobtainedbyotherauthors(Forattini 1980,Pattersonetal. 2009). However, ITS sequence data do suggestdiversiÞcation still ongoing today by geographical iso-lation of populations, probably caused by humantransport. Such an evolution appears similar to thatobserved in T. infestans, a vector species whose data-tion of origin and spread (evolution between 5.05 myand nowadays) show an evident parallelism with thatof P. megistus (Bargues et al. 2006).

InP.megistus, all haplotypes found in intradomiciles(2T or 1O) were also found in both the peridomiciliaryand sylvatic ecotopes, highlighting the role played bythese environments as sources of invasive populations.The very low genetic variability of Tr. cruzi isolatedfrom various hosts and vectors from Parana also high-lights this fact, suggesting an active ChagasÕ diseasesylvatic cycle of recent origin in that state (Thomaz-Soccol et al. 2002). The relatively old origin of P.megistus could have favored the spread and differen-tiation of populations throughout different areas andmay explain dwelling reinvasion phenomena after in-secticide control action, with intradomicile colonizingbugs originating from sylvatic or peridomestic foci.This high haplotype diversity is in agreement withresults obtained in the intra- and interpopulationalanalyses by RAPD during a context of paleovegetationreconstruction, which revealed a high level of differ-entiation among P. megistus populations from severalBrazilian states (Barbosa et al. 2006).Network and Phylogenetic Analyses. The topology

obtained with the MJ network shows P. megistus pop-ulations distributed in three groups. The results of thenetwork show: 1) the close relationships between the

majority of haplotypes from Parana (with a maximumdistance shown by the western samples of Palmito-polis and Goioxim); 2) Parana and Rio Grande do Sulas the only states in which haplotypes of the threegroups are present; 3) the close relationships betweenhaplotypes of the neighboring states Parana and SaoPaulo, but interestingly also with the northern state ofSergipe; and 4) Sao Paulo as the state whose haplotypemajority is shared with other states.

The aforementioned network results suggest thatSao Paulo may be considered one of the spreadingcenters of this species. This is in agreement with re-sults from other studies that suggested that Sao Paulocould have been the center of spread for P. megistus,together with parts of the Brazilian states of Pernam-buco, Bahia, and Rio de Janeiro (Forattini et al. 1978,Forattini 1980). However, nothing a priori excludesthe diversity in Sao Paulo being the consequence ofrecent passive importation of specimens from abroadowing to human migratory activities. The species P.megistus apparently spread westward from this area,reaching different zones of humid forests surroundedby open habitats (Forattini 1980). The state of MinasGerais has also been suggested to have additionallyplayed a role in the original spread of this species,based on the large genetic variability found in three P.megistus populations (Barbosa et al. 2003).

The phylogenetic analysis performed has furnishedthe most complete tree based on intergenic regiondata sets (complete ITS-1, 5.8S, ITS-2 sequences) sofar obtained in triatomines. In this phylogenetic tree,the genus Panstrongylus and its species P. megistusappear both supported by maximum bootstrap values.With regard to the 26 P. megistus CHs, the obtainingof a topology with both ML and NJ similar to thatobtained with the MJ network should be highlighted.

The P. megistus intergenic region results here ob-tained provide evidence about the relationship of ge-netic diversity with geographical spread that charac-terizes a major vector species and allow to explain itsability to colonize distant areas and different ecotopes,including human habitats, and consequently its greatimportance in the epidemiology of ChagasÕ disease.

Acknowledgments

Study funded by projects: ISCIII-RETIC RD06/0021/0017and RD12/0018/0013, Red de Investigacion de Centros deEnfermedades Tropicales Ð RICET, of the Program of RedesTematicas de Investigacion Cooperativa RETICS/FEDER,Ministry of Health and Consumption, Madrid, Spain; PRO-METEO/2012/042, of the program of Ayudas para Grupos deInvestigacion de Excelencia, Generalitat Valenciana, Valen-cia, Spain; and PP/2009, of the program of Infraestrutura paraJovenes Pesquisadores, Fundadacao Araucaria, Parana, Bra-zil. F.B.C. beneÞted from a funding by CAPES (Coordenacaode Aperfeicoamento de Pessoal de Nõvel Superior), Brazil.D.R.K. beneÞted from a Mobility Grant for Brazilian publicuniversity professors from Fundacion Carolina, Madrid,Spain. C.C.K. and L.G.R.O. beneÞted from an institutionalscholarship within the Sandwich Program for Foreign PhD(PDSE Nos. BEX 1225/12-0 and BEX 1271/12-1). F. Mello,from Laboratorio Central de Saude Publica do Rio Grande do

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Sul, provided samples from Rio Grande do Sul. Technicalsupport provided by the Servicio Central de Secuenciacionpara la Investigacion Experimental (SCSIE) of the Universityof Valencia, Spain.

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Received 9 April 2013; accepted 13 February 2014.

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