Genetic Variability and Geographical Diversity of the Main Chagas ...

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 ROCIÓ KLISIOWICZ,1,2 LUIZ GUSTAVO RODRIGUES OLIVEIRA,2,3 CHRISTIAN COLLINS KUEHN,2,3 ROGE´RIO LUIZ KOPP,4 VANETTE THOMAZ-SOCCOL,1 JOAÕ ARISTEU DA ROSA,5 ENNIO LUZ,1 SANTIAGO MAS-COMA,2 2,6 AND MARIÁ DOLORES BARGUES

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 complete internal transcribed spacer (ITS)-1, 5.8S, and ITS-2 sequences, of populations of the triatomine Panstrongylus megistus, the most important vector of ChagasÕ disease in Brazil since Triatoma infestans eradication. Specimens were from 26 localities of Rio Grande do Sul, Santa Catarina, Parana´, Saõ 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 was 1,513Ð1,522-bp-long (mean 1,516.9 bp), providing 26 combined haplotypes. The combination of microsatellites found in both ITSs may be of applied usefulness, to assess interpopulation specimen exchange and potential recolonizations after vector elimination by control implementation. Network results suggest that Saõ Paulo may be considered one of the spreading centers of this species. Molecular clock datation suggests that P. megistus populations are diversifying at least since 4.54 million years ago, with diversiÞcation still ongoing today by geographical isolation of populations. Evidence is provided about the relationship of genetic diversity with geographical spread that characterizes a major vector and explains its ability to colonize distant areas and different ecotopes, including human habitats, and consequently its importance in ChagasÕ disease epidemiology. KEY WORDS Panstrongylus megistus, ChagasÕ disease, rDNA intergenic region, haplotype diversity, Brazil

Among the 19 neglected tropical diseases, ChagasÕ disease is the sixth most important tropical infection in terms of global burden of disease, with high social and economic impact (WHO 2010). ChagasÕ disease, originally restricted to Latin America, is now becoming a global public health concern owing to human migrations to developed countries (Coura and Albajar 2010). The main mode of transmission of the etiologic agent, Trypanosoma cruzi, is vectorial via feces of infected hematophagous bugs (Hemiptera, Reduviidae, Triatominae). The vectors are crucial in establishing the geographical distribution of the disease, its local 1 Departamento de Patologia Ba ´sica, Setor de Cieˆ ncias Biolo´ gicas, Universidade Federal do Parana´, Centro Polite´ cnico, Curitiba, Parana, Brazil. 2 Departamento de Parasitologõá, Facultad de Farmacia, Universidad de Valencia, Burjassot - Valencia, Spain. 3 Faculdade de Cie ˆ ncias Farmaceˆ uticas de Ribeiraõ Preto, Universidade de Saõ Paulo, FCFRP-USP, Ribeiraõ Preto, Saõ Paulo, Brazil. 4 Departamento de Patologia Me ´ dica, Setor de Cieˆ ncias da Sau´ de, Universidade Federal do Parana´, Curitiba, Parana, Brazil. 5 Departamento de Cie ˆ ncias Biolo´ gicas da Faculdade de Cieˆ ncias Farmaceˆ uticas, Universidade Estadual Paulista - UNESP, Araraquara, Saõ Paulo, Brazil. 6 Corresponding author, e-mail: [email protected].

transmission patterns, and main epidemiological characteristics. Given the absence of effective drugs, the vectors become the key target for intervention activities (WHO 2012). In Brazil, about 2Ð3 million people are estimated to be infected (Dias et al. 2008), 600,000 of them with chronic heart or digestive complications, causing death in about 5,000 individuals each year (Coura and Dias 2009). The number of new ChagasÕ disease cases in Brazil has been reduced dramatically in recent years, but showing a great regional diversity in terms of morbidity and mortality (Coura and Albajar 2010, Martins-Melo et al. 2012). After Triatoma and Rhodnius, Panstrongylus is the third most speciose genus of the Triatominae subfamily. It includes 13 species with a wide geographical distribution from Mexico to Argentina (Curto de Casas et al. 1999). Twelve Panstrongylus species were found to be infected naturally by Tr. cruzi. High infection rates might be an indicator of close proximity to reservoir hosts and high susceptibility to Tr. cruzi, as seems to be the case for Panstrongylus megistus in Brazil, Panstrongylus lignarius in Peru, and Panstrongy-

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

May 2014

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

lus geniculatus in several countries (Patterson et al. 2009). P. megistus shows a wide geographical distribution, ecological valence, and great potential of colonization of artiÞcial ecotopes (Patterson et al. 2009). The Atlantic Forest seems to represent the center of distribution of P. megistus (Forattini 1980), although the species is also broadly distributed in humid areas of the Cerrado and Caatinga (Barbosa et al. 2006). The ubiquitous sylvatic populations persistently domiciliate, sporadically invading human housing and, therefore, demanding continuous surveillance (Coura and Dias 2009). The broad geographical distribution, the capacity to invade and colonize domiciles, and the high levels of Tr. cruzi infection indicate that P. megistus is the vector species of the greatest epidemiological importance in Brazil after the control of T. infestans (Gurgel-Gonç alves et al. 2012). Molecular markers generate a large amount of information about genetic diversity and phylogenetic relationships of the organisms. In Triatominae, DNA markers proved to be useful for this endeavor in general, although disadvantages and limitations of the each marker should be taken into account (Mas-Coma and Bargues 2009, Bargues et al. 2010). In the past decades, several studies have contributed to increase the genetic knowledge about Panstrongylus species from Brazil as well as other Latin American countries based on DNA markers (Garcõá and Powell 1998; Lyman et al. 1999; Bargues et al. 2000, 2002; Garcõá et al. 2001; Marcilla et al. 2002; Mas-Coma and Bargues 2009; Patterson and Gaunt 2010; Blando´ n-Naranjo et al. 2010; Zuriaga et al. 2012). Internal transcribed spacers (ITSs) of the nuclear ribosomal DNA (rDNA) have proved 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 about triatomine vectors inferred from sequences of the intergenic rDNA region, including ITS-1, 5.8S gene, and ITS-2, are scarce and only known in the infestans subcomplex species, in Triatoma rubrovaria populations, and in species of the Mepraia genus (Pacheco et al. 2003, 2007; Bargues et al. 2006; Calleros et al. 2010). The aim of the current study concerns the genetic characterization by molecular haplotyping of different populations of P. megistus from Parana´ and other neighboring states of Brazil, based on sequences of the complete rDNA intergenic region, to analyze the combined haplotype diversity and relationships. This is the most extensive molecular study of this triatomine so far and the Þrst time that the ITS-1 is analyzed in this crucial vector.

Materials and Methods Triatomine Specimens. In total, 90 P. megistus specimens were collected representing populations from 26 localities covering a geographical representation of most of the states where P. megistus is present in Brazil (Carcavallo et al. 1999). rDNA presents the peculiarity of following a concerted evolution, which homogenizes the many copies of nuclear rDNA among both homologous and nonhomologous chromosomes containing rDNA clusters within a genome. This gives rise to a uniformity inside all individuals of a population and becomes extremely useful (Mas-Coma and Bargues 2009). In total, 43 specimens were sequenced, including more than one specimen from given populations to verify certain single nucleotide polymorphisms, mainly appearing in poly-A regions and dinucleotide microsatellite repeats (Figs. 1 and 2; Table 1).

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

Sequencing of rDNA ITS-1, 5.8S, and ITS-2. For DNA 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). Total DNA was isolated by standard techniques, resuspended in 50 ␮l of TE buffer (10 mM TrisÐHCl, 1 mM EDTA, pH 7.6), and stored at ⫺20⬚C until use. The nuclear rDNA intergenic fragment corresponding to the ITS-1, 5.8S, and ITS-2 regions was polymerase chain reaction (PCR) ampliÞed with primers Eu1657 and Infer for ITS-1 and 5.8T and 28T for ITS-2, following the methods outlined before (Bargues et al. 2006) and using 4 Ð 6 ␮l of genomic DNA for each 50 ␮l reaction. AmpliÞcations were generated in a Mastercycler ep gradient (Eppendorf, Hamburg, Germany), by 30 cycles of 30 s at 94⬚C, 30 s at 50 Ð55⬚C, and 1 min at 72⬚C, preceded by 30 s at 94⬚C and followed by 7 min at 72⬚C. PCR products were puriÞed using the UltraClean PCR Clean-up DNA PuriÞcation System (MoBio, Solana Beach, CA, USA) according to the manufacturerÕs protocol and resuspended in 50 ␮l of 10 mM TE buffer (pH 7.6). Sequencing was performed on both strands by the dideoxy chain-termination method, with the Taq dye-terminator chemistry kit for ABI 3130 and ABI 3700 (Applied Biosystems, CA, USA), using the same PCR ampliÞcation primers. Given the importance of the recent discovery of a pseudogenic 5.8S⫹ITS-2 sequence, designated as “ps(5.8S⫹ITS-2),” widely distributed in triatomines of North, Central, and South America (Bargues et al. 2014), special efforts were made to ensure that no double signal was present in the chromatograms, to conÞrm that variable positions in the intergenic region were not due to an underlying paralogous sequence. The haplotype (H) terminology used for both ITSs follows the nomenclature for composite haplotyping (CH) previously proposed (Bargues et al. 2006, Mas-

Coma and Bargues 2009). Accordingly, ITS-2 haplotypes are noted by numbers and ITS-1 haplotypes are noted by capital letters. Sequences were aligned using CLUSTAL W2 (Larkin et al. 2007) and MEGA 5.2 (Tamura et al. 2011), using default settings. Minor corrections for a better Þt of nucleotide or indel correspondences were made. DnaSP v.5.1 (Librado and Rozas 2009) was used to evaluate the number of haplotypes (h), haplotype diversity (Hd), nucleotide diversity expressed as the average number of nucleotide differences between two sequences by site (␲), average number of nucleotide differences between sequences (k), and number of polymorphisms and insertions/deletions (S). Genetic distances were measured using parameters provided by PAUP v.4.0 b10 (Swofford 2002). Calculations for datation were based on both ITS-2 and ITS-1 molecular clock rates obtained for Triatomini (Bargues et al. 2000, 2006). Estimates were obtained by computing PAUP differences, and the dating ranges were obtained for the divergences that appeared in the pairwise distance matrix of nucleotide divergences for each one of the two spacers. The following sequences from GenBankÐEMBL have been used for phylogenetic analyses: ITS-1 of Panstrongylus herreri (AM949584) and Panstrongylus geniculatus (AM949585) (Mas-Coma and Bargues 2009); ITS-2 of P. herreri (AJ306550) and P. geniculatus (AJ306543) (Marcilla et al. 2002); and complete intergenic 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 phylogenetic network estimation using median-joining (MJ) network algorithm using default parameters (equal character weight ⫽ 10, transitions/transversions weight ⫽ 1:1, and connection cost as a criterion) was

Simaõ Dias Simaõ Dias (2) Saõ Felipe (2) Belo Horizonte (3) Varginha Saõ Joaõ da Boa Vista (col.) Araraquara (colonia) Araraquara, Sõ´tio Caranda´ Araraquara, Sõ´tio Caranda´ Mogi Guaç u Piraquara Bacacheri, Curitiba Campo Magro Cerro Azul Rio Branco do Sul (3) Rio Branco do Sul Rio Azul Rebouç as Castro (2) Tomazina Wenceslau Braz Santana do Itarare´ Siqueira Campos Londrina Arapongas Arapongas (2) Goioxim Palmitopolis Palmitopolis Florianopolis (2) Santa Rosa Santa Rosa (colonia) Santa Rosa Senador Salgado Filho

1 1 2 3 4 5 6 6 6 7 8 9 10 11 12 12 13 14 15 16 17 18 19 20 21 21 22 23 23 24 25 25 25 26

Sergipe Sergipe Bahia Minas Gerais Minas Gerais Saõ Paulo Saõ Paulo Saõ Paulo Saõ Paulo Saõ Paulo Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Parana´ Santa Catarina Rio Grande do Sul Rio Grande do Sul Rio Grande do Sul Rio Grande do Sul

State Sylvatic Sylvatic Domestic Peridomestic Peridomestic Sylvatic Sylvatic Peridomestic Peridomestic Domestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Peridomestic Sylvatic Sylvatic Sylvatic Sylvatic Sylvatic

Habitat ITS1-S ITS1-S ITS1-T ITS1-C ITS1-U ITS1-S ITS1-T ITS1-S ITS1-S ITS1-O ITS1-O ITS1-H ITS1-G ITS1-P ITS1-A ITS1-O ITS1-F ITS1-H ITS1-A ITS1-E ITS1-E ITS1-F ITS1-M ITS1-J ITS1-N ITS1-P ITS1-D ITS1-L ITS1-I ITS1-R ITS1-M ITS1-Q ITS1-M ITS1-K

ITS-1 haplotype 762 762 762 764 761 762 762 762 762 760 760 766 765 762 763 760 762 766 763 764 764 762 758 766 763 762 762 761 765 760 758 761 759 765

Length (bp)

ITS-1

68.8 68.8 69.1 69.0 69.0 68.8 69.1 68.8 68.8 68.7 68.7 68.9 68.6 69.0 68.7 68.7 68.6 68.9 68.7 68.8 68.8 68.6 68.6 69.1 68.8 69.0 68.9 69.2 68.8 69.1 68.6 69.0 68.5 69.0

AT (%) ITS2Ð9 ITS2Ð1 ITS2Ð2 ITS2Ð10 ITS2Ð4 ITS2Ð4 ITS2Ð2 ITS2Ð4 ITS2Ð1 ITS2Ð1 ITS2Ð1 ITS2Ð3 ITS2Ð12 ITS2Ð2 ITS2Ð1 ITS2Ð1 ITS2Ð1 ITS2Ð3 ITS2Ð3 ITS2Ð3 ITS2Ð3 ITS2Ð1 ITS2Ð1 ITS2Ð1 ITS2Ð1 ITS2Ð5 ITS2Ð8 ITS2Ð9 ITS2Ð1 ITS2Ð11 ITS2Ð7 ITS2Ð9 ITS2Ð6 ITS2Ð1

ITS-2 haplotype 598 600 598 598 600 600 598 600 600 600 600 601 600 598 600 600 600 601 601 601 601 600 600 600 600 599 600 598 600 601 600 598 600 600

Length (bp)

ITS-2

75.2 75.2 75.4 75.0 75.0 75.0 75.4 75.0 75.2 75.2 75.2 75.3 75.0 75.4 75.2 75.2 75.2 75.3 75.3 75.3 75.3 75.2 75.2 75.2 75.2 75.3 75.0 75.2 75.2 75.0 75.0 75.2 75.2 75.2

AT (%) P.meg-CH9S P.meg-CH1S P.meg-CH2T P.meg-CH10C P.meg-CH4U P.meg-CH4S P.meg-CH2T P.meg-CH4S P.meg-CH1S P.meg-CH1O P.meg-CH1O P.meg-CH3H P.meg-CH12G P.meg-CH2P P.meg-CH1A P.meg-CH1O P.meg-CH1F P.meg-CH3H P.meg-CH3A P.meg-CH3E P.meg-CH3E P.meg-CH1F P.meg-CH1M P.meg-CH1J P.meg-CH1N P.meg-CH5P P.meg-CH8D P.meg-CH9L P.meg-CH1I P.meg-CH11R P.meg-CH7M P.meg-CH9Q P.meg-CH6M P.meg-CH1K

1,515 1,517 1,515 1,521 1,516 1,517 1,515 1,517 1,517 1,515 1,515 1,522 1,521 1,515 1,518 1,515 1,517 1,522 1,519 1,520 1,520 1,517 1,513 1,521 1,518 1,516 1,517 1,514 1,520 1,514 1,513 1,514 1,513 1,520

Length (bp)

Intergenic region Combined haplotype 68.6 68.6 68.8 68.6 68.6 68.5 68.8 68.5 68.6 68.5 68.5 68.7 68.4 68.8 68.5 68.5 68.5 68.7 68.5 68.6 68.6 68.5 68.5 68.7 68.6 68.7 68.6 68.8 68.6 68.6 68.7 68.4 68.4 68.7

AT (%)

Number of the geographical location according to the maps of Figures 1 and 2. Numbers in brackets indicate the number of specimens sequenced when more than one individual.

Geographical location (no. of specimens sequenced)

No. in map

HF678472 HF678473 HF678474 HF678475 HF678477 HF678471 HF678474 HF678471 HF678473 HF678465 HF678465 HF678460 HF678461 HF678464 HF678458 HF678465 HF678459 HF678460 HF678457 HF678456 HF678456 HF678459 HF678453 HF678468 HF678462 HF678463 HF678452 HF678466 HF678467 HF678476 HF678455 HF678470 HF678454 HF678469

ITS-1, 5.8S, ITS-2 GenBank accession no.

Table 1. Geographical localities and habitats of the Panstrongylus megistus specimens analyzed, haplotypes obtained for rDNA ITS-1 and ITS-2 markers, and GenBank accession numbers of the sequences obtained

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constructed, for the ITS-1 and ITS-2 combined haplotypes, using Network 6.4.1.1 (http://www.ßuxusengineering.com/). Sites with alignment gaps or missing data were considered. Hypothetical median vectors (a hypothesized sequence that is required to connect existing sequences within the network with maximum parsimony) were added to the network for shortest connection between the data set. The relationships between P. megistus and other Triatominae species whose complete intergenic region was available were inferred by phylogenetic tree reconstruction using PAUP 4.0b10 and the maximum likelihood (ML) method based on the HasegawaÐ KishinoÐYano model. ML parameters and the evolutionary model best Þtting our data set were determined using Akaike and Bayesian information criteria, implemented in jModeltest version 0.1.1. Starting branch lengths were obtained using the least-squares method with ML distances. The sequences of Mepraia spinolai FN396516 and Triatoma eratyrusiformis FN396537 (Calleros et al. 2010) were used as outgroup. Four CHs were used to represent P. megistus; the selection was made to include representatives of each one of the groups distinguished by the network analysis (with two CHs for the most diversiÞed group and only one for each of the two other groups), and simultaneously a representative from the spreading center of the species. Statistical support for the nodes was evaluated with 1,000 bootstrap replicates, using heuristic search. Other phylogenetic trees were also reconstructed by including all species of Triatoma and Mepraia of which the complete intergenic region is known, together with all the CHs of the P. megistus populations studied to compare with network results. When reconstructing such trees, it was not forgotten that mathematical methods for phylogenetic analyses are made for the assessment of relationships between watertight compartments (i.e., noncrossing units, as it is the case of different species and higher taxa) but not for population relationships within a species. For this purpose, ML and neighbor-joining (NJ) methods were implemented in MEGA 5.2 program. A discrete gamma distribution was used to model evolutionary rate differences among sites (four categories). Evolutionary distances were computed using the Kimura 2-parameter method. To provide an assessment of the reliability of the nodes in trees, statistical support was evaluated with 1,000 bootstrap replicates. Results Sequence Analysis of rDNA ITS Haplotypes. Sequence length and AT content for each ITS with their corresponding haplotype codes and new GenBank accession numbers are listed in Table 1. The complete intergenic region revealed the existence of 26 combined haplotypes (CH) (Table 1). Their alignment generated a 1,523-bp data set that contained 31 variable positions (2.03%), of which 6 were singleton sites (0.39%), 11 parsimony informative positions (0.72%), and 14 insertions or deletions

Vol. 51, no. 3

(indels) (0.92%) (Table 2). The length of this region varied from 1,513 to 1,522 bp (mean 1,516.9 bp). The 155-bp-long 5.8S was identical in all specimens. When comparing the sequences of the 26 CHs, the pairwise ITS-1 and ITS-2 distance matrix obtained with PAUP (only parsimony informative sites considered) shows that the number of nucleotide differences is high when comparing haplotypes from Minas Gerais and Santa 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 variable positions appeared (3.00%), of which 10 were substitutions (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 was AT-biased (68.5Ð 69.2%; mean 68.9%). Thirteen of these haplotypes were present in Parana´, of which only two were also present in Rio Grande do Sul and Saõ Paulo (Table 2). The sequences of the ITS-2 provided 12 ITS-2 haplotypes (ITS2Ð1 to ITS2Ð12). In the 488-bp-long ITS-2 alignment, 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 length ranged between 598 and 601 bp (mean 599.6 bp), with an AT-biased nucleotide composition (average 75.2%). In Parana´, seven different haplotypes were detected, of which only three were shared with other localities in Rio Grande do Sul, Sergipe, Saõ Paulo, and Bahia (Table 2). A comparative analysis of the haplotype characteristics and information provided by ITS-1 and ITS-2 markers is shown in Table 4. Sequence repeats were present in the intergenic rDNA region. ITS-1 shows minisatellite repeats in all haplotypes and populations (Fig. 3), plus only one variable dinucleotide repeat (one, two, or three repeats of TG). In ITS-2, only interrupted microsatellites and dinucleotide repeats were found, with (GC) as the only one showing variations (two or three repeats) according to haplotypes (Fig. 3). Combined Haplotype Diversity. In Parana´, 15 different CHs were detected. Only one of them (1O) is also present in samples from Saõ Paulo. In the other six states, 11 different CHs were found, of which two are present in two states: CH-1S in Sergipe and Saõ Paulo; CH-2T in Saõ Paulo and Bahia (Tables 1 and 2; Fig. 4). The genetic variability among the 26 CHs showed a diversity of 0.946 (variance ⫽ 0.000047; standard deviation ⫽ 0.022), a nucleotide diversity of 0.00316 (variance ⫽ 0.0000; standard deviation ⫽ 0.00022), an average number of nucleotide differences of 4.2655, and a number of polymorphisms and insertions/deletions of 3.3995. An analysis was performed to assess a potential correlation of the CH diversity with the three geomorphological units of Parana´ state. Five CHs appeared in the Þrst plateau (planalto de Curitiba), Þve in the second (planalto de Ponta Grossa), and six in

May 2014 Table 2.


621

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 103Ð 605 and 984 Ð1517 contain variable positions in the ITS-1 and ITS-2, respectively.

the third (planalto de Guarapauva) (Table 1; Fig. 2). Neither a correlation nor a signiÞcant result was found. However, a few data may be highlighted: 1) only one haplotype is present throughout the three plateaus (ITS-2 H1); 2) only one haplotype is shared by the Þrst and second plateaus (CH-3H); 3) the third plateau is the one presenting a larger variability, despite the pronouncedly lower number of populations studied (six CHs from only four localities); and 4) only one CH of Parana´ is present in another state (CH-1O also in Saõ Paulo). Network Analysis. The CH network constructed using MJ network algorithm shows the existence of three groups: 1) a big group with a high diversity of haplotypes (13 CHs), including samples from different states (Sergipe, Saõ Paulo, Rio Grande do Sul, Santa Catarina, and Bahia) and from localities of Parana´ (Arapongas, Palmitopolis, Cerro Azul, and Piraquara); 2) a second group including haplotypes from Parana´, with the exception of only one from Rio Grande do Sul; and 3) one small group including two haplotypes from Rio Grande do Sul and one from Parana´ (Fig. 4). Between the Þrst and second groups, the intermediate position of CH 8D (from the locality of Goioxim in Parana´) should be emphasized, showing

similar mutational steps with regard to haplotypes 9L and 1I (both from Palmitopolis, Parana´), located in Þrst and second groups, respectively. Only three haplotypes (1S, 1O, and 2T) appear at least in two states simultaneously. Parana´ and Rio Grande do Sul are the only states appearing in the three groups (Fig. 4). The majority of haplotypes found in Saõ Paulo, all of them included within the Þrst group, are also present in other states. Phylogenetic Analyses. For the tree including the only four P. megistus CHs selected to represent the three different groups (distinguished in the aforementioned network), the ML model best Þtting the ITSs combined data was HKY85⫹I⫹G using the ts/tv ratio of 1.619 (kappa ⫽ 3.307; base frequencies for A, C, G, and T of 0.32369, 0.12524, 0.15439, and 0.35707, respectively; a proportion of invariable sites of 0.12; and a gamma distribution of 4.031). The resulting phylogeny (-Ln ⫽ 6413.5595) was evaluated using the leastsquares method with ML distances. In the ML tree obtained, the four P. megistus CHs cluster together with the maximum support and appear distributed in two clades, one including the representatives of groups B and C and the other including the two representatives of group A (Fig. 5).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

MG SC PR PR SP, BA RS MG PR PR, SP SE, SP SP SE PR RS RS PR PR PR PR PR PR PR PR PR RS PR

5 4 5 7 4 5 7 7 7 8 6 5 9 8 9 8 10 10 10 10 9 9 8 8 5

1

0.00368 4 4 6 3 4 6 6 6 5 5 4 8 7 8 9 11 11 11 11 10 10 9 9 6

2

0.00294 0.00294 1 3 2 3 3 3 3 4 4 3 5 4 5 9 9 9 8 8 8 7 6 6 5

3

0.00368 0.00295 0.00074 2 1 2 2 2 2 3 3 2 6 5 4 7 7 7 7 7 6 6 5 5 4

4

0.00515 0.00442 0.00221 0.00147 3 4 4 4 4 5 5 4 8 7 6 9 5 5 5 5 4 4 3 3 6

5

0.00294 0.00221 0.00147 0.00074 0.00221 3 3 3 3 4 2 1 7 6 5 6 8 8 8 8 7 7 6 6 3

6 0.00368 0.00295 0.00221 0.00147 0.00294 0.00221 4 4 4 3 5 4 8 7 6 9 9 9 9 9 8 8 7 7 6

7 0.00515 0.00442 0.00221 0.00147 0.00294 0.00221 0.00294 1 2 3 3 4 6 5 4 7 7 7 7 7 6 6 7 7 4

8 0.00516 0.00443 0.00221 0.00147 0.00295 0.00221 0.00295 0.00074 0 1 1 4 4 3 2 5 5 5 5 5 6 6 7 7 4

9 0.00515 0.00442 0.00221 0.00147 0.00294 0.00221 0.00294 0.00147 0.00000 1 1 4 4 3 2 5 5 5 5 5 6 6 7 7 4

10 0.00588 0.00368 0.00294 0.00221 0.00368 0.00294 0.00220 0.00220 0.00074 0.00073 2 5 5 4 3 6 6 6 6 6 7 7 8 8 5

11 0.00442 0.00368 0.00294 0.00221 0.00368 0.00147 0.00368 0.00221 0.00074 0.00074 0.00147 3 5 4 3 4 6 6 6 6 7 7 8 8 3

12 0.00368 0.00295 0.00221 0.00147 0.00294 0.00074 0.00294 0.00294 0.00295 0.00294 0.00368 0.00221 6 5 4 5 7 7 7 7 6 6 5 5 2

13 0.00664 0.00591 0.00369 0.00443 0.00590 0.00517 0.00590 0.00442 0.00295 0.00295 0.00368 0.00369 0.00443 1 2 5 5 5 5 5 6 6 7 7 6

14 0.00590 0.00517 0.00295 0.00369 0.00517 0.00443 0.00516 0.00368 0.00221 0.00221 0.00295 0.00295 0.00369 0.00074 1 4 4 4 4 4 5 5 6 6 5

15 0.00664 0.00591 0.00369 0.00295 0.00443 0.00369 0.00442 0.00295 0.00148 0.00147 0.00221 0.00221 0.00295 0.00147 0.00074 3 3 3 3 3 4 4 5 5 4

16 0.00587 0.00662 0.00661 0.00515 0.00662 0.00442 0.00661 0.00514 0.00368 0.00367 0.00441 0.00294 0.00368 0.00368 0.00295 0.00221 4 4 4 4 5 5 6 6 3

17 0.00736 0.00811 0.00663 0.00516 0.00368 0.00590 0.00663 0.00515 0.00368 0.00368 0.00442 0.00442 0.00516 0.00368 0.00295 0.00221 0.00293 0 0 0 1 1 2 2 5

18

19 0.00736 0.00811 0.00663 0.00516 0.00368 0.00590 0.00663 0.00515 0.00368 0.00368 0.00442 0.00442 0.00516 0.00368 0.00295 0.00221 0.00293 0.00000 0 0 1 1 2 2 5

Pairwise distances between concatenated ITS-1 and ITS-2 sequences of the P. megistus populations analyzed according to PAUP 20 0.00736 0.00812 0.00590 0.00516 0.00368 0.00590 0.00663 0.00515 0.00368 0.00368 0.00442 0.00442 0.00516 0.00368 0.00295 0.00221 0.00294 0.00000 0.00000 0 1 1 2 2 5

21 0.00736 0.00812 0.00590 0.00516 0.00368 0.00590 0.00663 0.00515 0.00368 0.00368 0.00442 0.00442 0.00516 0.00368 0.00295 0.00221 0.00294 0.00000 0.00000 0.00000 1 1 2 2 5

22 0.00661 0.00736 0.00588 0.00441 0.00294 0.00515 0.00588 0.00440 0.00441 0.00441 0.00514 0.00515 0.00442 0.00442 0.00368 0.00295 0.00366 0.00073 0.00073 0.00073 0.00073 0 1 1 4

Below diagonal ⫽ total character differences; above diagonal ⫽ mean character differences (adjusted for missing data). Composite haplotype (CH) codes listed in Table 1. PR, Parana´; SP, Saõ Paulo; RS, Rio Grande do Sul; SE, Sergipe; MG, Minas Gerais; BA, Bahia; SC, Santa Catarina.

CH10C CH11R CH5P CH2P CH2T CH9Q CH4U CH1N CH1O CH1S CH4S CH9S CH9L CH6M CH7M CH1M CH12G CH3E CH3A CH1A CH1F CH3H CH1I CH1J CH1K CH8D

Table 3.

23 0.00661 0.00736 0.00515 0.00441 0.00294 0.00515 0.00588 0.00441 0.00442 0.00441 0.00514 0.00515 0.00442 0.00442 0.00368 0.00295 0.00366 0.00073 0.00073 0.00073 0.00073 0.00000 1 1 4

24 0.00587 0.00663 0.00441 0.00368 0.00221 0.00442 0.00514 0.00514 0.00515 0.00514 0.00587 0.00588 0.00368 0.00515 0.00442 0.00368 0.00440 0.00147 0.00147 0.00147 0.00147 0.00073 0.00073 0 5

25 0.00587 0.00663 0.00441 0.00368 0.00221 0.00442 0.00514 0.00514 0.00515 0.00514 0.00587 0.00588 0.00368 0.00515 0.00442 0.00368 0.00440 0.00147 0.00147 0.00147 0.00147 0.00073 0.00073 0.00000 5

26 0.00368 0.00442 0.00368 0.00294 0.00442 0.00221 0.00441 0.00294 0.00295 0.00294 0.00367 0.00221 0.00147 0.00442 0.00368 0.00295 0.00220 0.00368 0.00368 0.00368 0.00368 0.00294 0.00294 0.00367 0.00367 -

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

623

PR-RS; PR-SP; SP-SE; SP-BA PR-RS, SE, MG, SP; PR-SP; PR-SE; MG-SP

In the phylogenetic analyses including the 26 CHs of P. megistus, both ML (HKY⫹I⫹G, matrix of pairwise distances estimated using the maximum composite likelihood approach, proportion of invariable sites ⫽ 0.407, and a discrete gamma distribution ⫽ 0.04) and NJ (Kimura 2-parameter method) furnished a similar topology distributing the CH according to the same groups distinguished by the network analysis and with the CH 8D appearing between groups A and B. Both trees show, however, low bootstrap values, as expected because of dealing with different populations of the same species (tree not shown).

PR, Parana´; SP, Saõ Paulo; RS, Rio Grande do Sul; SE, Sergipe; MG, Minas Gerais; BA, Bahia; SC, Santa Catarina.

ITS1-M, ITS1-O, ITS1-S, ITS1-T ITS2Ð1, ITS2Ð2, ITS2Ð4, ITS2Ð9 13 (1.70%) 3 (0.50%) 10 (1.30%) 5 (0.83%) 23 (3.00%) 8 (1.33%) 762.3 599.6 ITS1-A to ITS1-U ITS2Ð1 to ITS2Ð12 21 12 ITS-1 ITS-2

Haplotype codes

Average length (bp)

Nucleotide differences No. (%)

Substitutions No. (%)

Indels No. (%)

Haplotypes found in more than one state

Discussion

Total haplotypes

Table 4.

States sharing haplotypes


Comparison of the characteristics of the ITS-1 and ITS-2 sequences of the Panstrongylus megistus specimens from 26 geographical localities of seven states of Brazil

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Analysis of Sequences. The whole intergenic region comprising ITS-1, 5.8S, and ITS-2 provides a data set with an average length and AT content of 1,516.9 bp and 68.6%, respectively. This region was considerably longer than the 1,377 bp described for T. rubrovaria (Pacheco et al. 2003, 2007), the 1,375 bp of the infestans subcomplex (Bargues et al. 2006), and the 1,371 bp in the genus Mepraia (Calleros et al. 2010). The total of 26 combined haplotypes described for this complete intergenic region provides a genetic variability of 2.03% in the P. megistus populations analyzed, allowing for inter-populational differentiation. This suggests evolving divergence processes. A similar situation, with a different haplotype in almost each different locality, was also found in T. rubrovaria, although with a pronounced lower variability (1.31%) (Pacheco et al. 2003). The ITS-1 in P. megistus proved to be markedly longer than ITS-2, and its bias in AT composition was lower than in ITS-2, which agrees with previous observations 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 470 Ð 600 bp observed in different Panstrongylus species from different countries (Marcilla et al. 2002). In ITSs of several Triatominae species, sequence length varies according to the presence of repeated sequences such as microsatellites and/or minisatellites, which have been proved to furnish valuable information at population level (Bargues et al. 2006). However, such short sequence repeats have been shown to be constant among different populations within other species (Marcilla et al. 2001, Pacheco et al. 2007). In the P. megistus specimens, ITS-1 sequences showed both: 1) constant microsatellites of 2Ð 4 nucleotides tandemly repeated and scattered along the sequence, among which only one variable (TG) allowed differentiation between populations: almost all populations presented only one repeat; two repeats distinguished the locality of Goioxim in Parana´; three repeats are present in one locality of Minas Gerais (Belo Horizonte), seven localities of Parana´ (Campo Magro, Rio Azul, Reboucas, Castro, Wenceslao Braz, Londrina, and Palmitopolis), and one locality in Rio Grande do Sul (Senador Salgado Filho); and 2) minisatellites, one tandem repeat of

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Vol. 51, no. 3

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

12 nucleotides and another nontandem repeat of 17 nucleotides, which appeared to be constant in all specimens studied and consequently noninformative. These minisatellites differ from those detected

in the infestans subcomplex, within which their number of repeats clearly distinguished between sylvatic and domestic populations of T. infestans (Bargues et al. 2006).

Fig. 4. Median network analysis of Panstrongylus megistus combined haplotypes based on rDNA ITS-1 and ITS-2 sequences. The area of each haplotype is proportional to the total sample. Mutational steps between haplotypes are represented 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.)

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

With regard to ITS-2, microsatellites have usually been described in Triatominae, but never minisatellites (Mas-Coma and Bargues 2009). In the P. megistus populations analyzed, microsatellites proved to belong to two types: 1) among the several tandemly repeated dinucleotide microsatellites, only (CG) shows variations, allowing to discriminate between some populations: two repeats are present in three localities in Parana´ (Cerro Azul, Arapongas, Palmitopolis), one in Minas Gerais (Belo Horizonte), and one in Santa Catarina (Florianopolis); three repeats are present in one locality of Minas Gerais (Belo Horizonte), one in Saõ Paulo (Saõ Joao de Boa Vista), four in Parana´ (Campo Magro, Castro, Siqueira Campos, Goioxim), and one in Rio Grande do Sul (Santa Rosa); and 2) one interrupted microsatellite (AT), which appears as (AT)6TTT(AT)1AATGT(AT)5 or as (AT)4AC(AT)1TTT(AT)1AATGT(AT)5 owing to a T-C transition; haplotypes including this transition appear to show a wide geographical distribution but appear to be rare, i.e., only in seven localities among the total of 26: Simaõ Dias (Sergipe); Belo Horizonte (Minas Gerais); Campo Magro, Goioxim, and Palmitopolis (Parana´); Florianopolis (Santa Catarina); and Santa Rosa (Rio Grande do Sul). The same interrupted microsatellite in the same region of the ITS-2 is also present, but with some punctual nucleotide differences, in T. infestans haplotypes (Bargues et al. 2006). The combination of the aforementioned microsatellites in both ITSs of P. megistus may be of future applied usefulness, as for instance to assess interpopulation specimen exchange and potential recolonizations after vector elimination by control implementation. The ITS-1 sequences showed a large haplotype variability throughout the distribution range of P. megistus. IntraspeciÞc nucleotide differences appear to be higher in ITS-1 (3.00%) than in the ITS-2 (1.33%), concerning both substitutions and indels. The nucle-

625

otide divergence considering only substitutions is also higher in ITS-1 (1.30%) than in ITS-2 (0.83%). These results are in agreement with the molecular clock dating proposed for triatomines, according to which ITS-1 evolves 1.12Ð2.60 times faster than ITS-2 (Bargues et al. 2000, 2002, 2006). Thus, the number of ts and tv in the ITS-1 enables for a better population differentiation than when considering them in ITS-2. However, the intraspeciÞc variability of the ITS-2 in P. megistus is relatively high when compared with what is known in other triatomines. Despite the long ITS-2 sequence (Panstrongylus is the triatomine genus presenting the longer ITS-2; Mas-Coma and Bargues 2009), its variability enters among those more variable, although far away from the wide variability of 2.70% in T. infestans (Bargues et al. 2006), and especially that of 5.62% in Triatoma dimidiata (including all subspecies and excluding the species T. sp. aff. dimidiata) (Bargues et al. 2008). It should be highlighted, however, that T. infestans and T. dimidiata are two species that were transported by humans up to cover a geographical distribution pronouncedly wider than that of P. megistus. Diversity, Spread, and Datation. The 26 P. megistus CHs found represent a large variability, among which the populations of Florianopolis (Santa Catarina) and Belo Horizonte (Minas Gerais) appear to be the most genetically divergent ones. This is in agreement with results from other studies on P. megistus (Barbosa et al. 2003, 2006), which emphasized that Santa Catarina populations occurred in a differentiated morphoclimatic domain. When considering both markers separately, four ITS-1 (M, O, S, T) and four ITS-2 (1, 2, 4, 9) haplotypes appear simultaneously in four (Parana´, Rio Grande do Sul, Saõ Paulo, Sergipe) of the seven Brazilian states. Minas Gerais and Santa Catarina do not share haplotypes with any other state, and only the haplotype ITS1-T from Bahia is also present in Saõ Paulo. In Parana´, no haplotypeÐ geographical association was found, suggesting a spread and reshufßing of haplotype distribution. The third plateau had a strong human immigration in the past, making the northern and southwestern areas to become true pioneers, as a consequence of the acceleration of deforestation, the high rate of land modiÞcation for agriculture and open roads, and also the development of villages and towns linked together (Balhana et al. 1969). This may have facilitated passively receiving bugs from other states. Haplotype 1 of the ITS-2 appears to be the most spread one, found in all plateaus of Parana´ and in four other states. Thus, it may be considered the haplotype more close to that which initiated the spread of the species from its endemism center (Forattini 1980). The lack of geographical separation and isolation of P. megistus of Parana´ from other states was also evident in multiple isoenzyme analysis (Kopp et al. 2009). The population distribution in Brazil should be affected by analyses based on points of origin, and the events undergone by these habitats during the past 18,000 yr suggest that P. megistus populations were already formed and deÞned by this time (Forattini 1980, Pat-

626


terson et al. 2009). The ITS analyses suggest a relatively old origin of P. megistus, based on the high genetic variability and geographical diversity detected among populations studied throughout the distribution of this species in Brazil. When applying the molecular clock of 0.41Ð 0.99% variation per 1 million years (my) based on ITS-2 evolutionary rates in Triatominae (Bargues et al. 2000), the two most divergent ITS-2 haplotypes H1 and H11 furnish a divergence of 0.84 Ð2.02 my when considering all nucleotide differences and 0.33Ð 0.80 my when only considering mutations. On the contrary, the two closest ITS-2 haplotypes H1 and H3 furnish a divergence of 0.39 Ð 0.16 my when considering all nucleotide differences and nowadays (⫽0.00 my) when only considering mutations. When applying the molecular clock of 0.46 Ð2.58% variation per 1 my based on ITS-1 evolutionary rates in Triatominae (Bargues et al. 2006), the two most divergent ITS-1 haplotypes HA and HU furnish a divergence of 0.81Ð 4.54 my when considering all nucleotide differences and 0.55Ð3.11 my when only considering mutations. On the contrary, the two closest ITS-1 haplotypes HA and HE furnish a divergence of 0.05Ð 0.28 my when considering all nucleotide differences and nowadays (⫽0.00 my) when only considering mutations. Hence, these data suggest that the populations of the species P. megistus are diversifying at least since 4.54 my ago, the closest only in the recent 50,000 yr. These results Þt the aforementioned data obtained by other authors (Forattini 1980, Patterson et al. 2009). However, ITS sequence data do suggest diversiÞcation still ongoing today by geographical isolation of populations, probably caused by human transport. Such an evolution appears similar to that observed in T. infestans, a vector species whose datation of origin and spread (evolution between 5.05 my and nowadays) show an evident parallelism with that of P. megistus (Bargues et al. 2006). In P. megistus, all haplotypes found in intradomiciles (2T or 1O) were also found in both the peridomiciliary and sylvatic ecotopes, highlighting the role played by these environments as sources of invasive populations. The very low genetic variability of Tr. cruzi isolated from various hosts and vectors from Parana´ also highlights this fact, suggesting an active ChagasÕ disease sylvatic cycle of recent origin in that state (ThomazSoccol et al. 2002). The relatively old origin of P. megistus could have favored the spread and differentiation of populations throughout different areas and may explain dwelling reinvasion phenomena after insecticide control action, with intradomicile colonizing bugs originating from sylvatic or peridomestic foci. This high haplotype diversity is in agreement with results obtained in the intra- and interpopulational analyses by RAPD during a context of paleovegetation reconstruction, which revealed a high level of differentiation among P. megistus populations from several Brazilian states (Barbosa et al. 2006). Network and Phylogenetic Analyses. The topology obtained with the MJ network shows P. megistus populations distributed in three groups. The results of the network show: 1) the close relationships between the

Vol. 51, no. 3

majority of haplotypes from Parana´ (with a maximum distance shown by the western samples of Palmitopolis and Goioxim); 2) Parana´ and Rio Grande do Sul as the only states in which haplotypes of the three groups are present; 3) the close relationships between haplotypes of the neighboring states Parana´ and Saõ Paulo, but interestingly also with the northern state of Sergipe; and 4) Saõ Paulo as the state whose haplotype majority is shared with other states. The aforementioned network results suggest that Saõ Paulo may be considered one of the spreading centers of this species. This is in agreement with results from other studies that suggested that Saõ Paulo could have been the center of spread for P. megistus, together with parts of the Brazilian states of Pernambuco, Bahia, and Rio de Janeiro (Forattini et al. 1978, Forattini 1980). However, nothing a priori excludes the diversity in Saõ Paulo being the consequence of recent passive importation of specimens from abroad owing to human migratory activities. The species P. megistus apparently spread westward from this area, reaching different zones of humid forests surrounded by open habitats (Forattini 1980). The state of Minas Gerais has also been suggested to have additionally played 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 furnished the most complete tree based on intergenic region data sets (complete ITS-1, 5.8S, ITS-2 sequences) so far obtained in triatomines. In this phylogenetic tree, the genus Panstrongylus and its species P. megistus appear both supported by maximum bootstrap values. With regard to the 26 P. megistus CHs, the obtaining of a topology with both ML and NJ similar to that obtained with the MJ network should be highlighted. The P. megistus intergenic region results here obtained provide evidence about the relationship of genetic diversity with geographical spread that characterizes a major vector species and allow to explain its ability to colonize distant areas and different ecotopes, including human habitats, and consequently its great importance in the epidemiology of ChagasÕ disease. Acknowledgments Study funded by projects: ISCIII-RETIC RD06/0021/0017 and RD12/0018/0013, Red de Investigacio´ n de Centros de Enfermedades Tropicales Ð RICET, of the Program of Redes Tema´ticas de Investigacio´ n Cooperativa RETICS/FEDER, Ministry of Health and Consumption, Madrid, Spain; PROMETEO/2012/042, of the program of Ayudas para Grupos de Investigacio´ n de Excelencia, Generalitat Valenciana, Valencia, Spain; and PP/2009, of the program of Infraestrutura para Jovenes Pesquisadores, Fundadaç aõ Arauca´ria, Parana´, Brazil. F.B.C. beneÞted from a funding by CAPES (Coordenacaõ de Aperfeiç oamento de Pessoal de Nõ´vel Superior), Brazil. D.R.K. beneÞted from a Mobility Grant for Brazilian public university professors from Fundacio´ n Carolina, Madrid, Spain. C.C.K. and L.G.R.O. beneÞted from an institutional scholarship within the Sandwich Program for Foreign PhD (PDSE Nos. BEX 1225/12-0 and BEX 1271/12-1). F. Mello, from Laborato´ rio Central de Sau´ de Pu´ blica do Rio Grande do

May 2014


Sul, provided samples from Rio Grande do Sul. Technical support provided by the Servicio Central de Secuenciacio´ n para la Investigacio´ n Experimental (SCSIE) of the University of Valencia, Spain.

References Cited Balhana, A. P., B. P. Machado, and C. M. Westphalen. 1969. Histo´ ria do Parana´, vol I, 2nd ed. Gra´Þca Editora Parana´ Cultural Ltda., Curitiba, p. 184. Barbosa, S. E., C. J. Belisario, R.C.M. Souza, A. S. Paula, P. M. Linardi, A. J. Romnaha, and L. Diotaiuti. 2006. Biogeography of Brazilian populations of Panstrongylus megistus (Hemiptera, Reduviidae, Triatominae) based on molecular marker and paleo-vegetational data. Acta Trop. 99: 144 Ð154. Barbosa, S. E., J. P. Dujardin, R.P.P. Soares, H.H.R. Pires, C. Margonari, A. J. Romanha, F. Panzera, P. M. Linardi, M. Duque-de-Melo, P.F.P. Pimenta, et al. 2003. Interpopulation variability among Panstrongylus megistus (Hemiptera: Reduviidae) from Brazil. J. Med. Entomol. 40: 411Ð420. Bargues, M. D., A. Marcilla, J. M. Ramsey, J. P. Dujardin, C. J. Schofield, and S. Mas-Coma. 2000. Nuclear rDNA-based molecular clock of the evolution of Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease. Mem. Inst. Oswaldo Cruz 95: 567Ð574. Bargues, M. D., A. Marcilla, J. P. Dujardin, and S. Mas-Coma. 2002. Triatomine vectors of Trypanosoma cruzi: a molecular perspective based on nuclear ribosomal DNA markers. Trans. R. Soc. Trop. Med. Hyg. 96: 159 Ð164. Bargues, M. D., D. R. Klisiowicz, F. Panzera, F. Noireau, A. Marcilla, R. Perez, M. G. Rojas, J. E. O’Connor, F. Gonzalez-Candelas, C. Galvao, et al. 2006. Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size. Infect. Genet. Evol. 6: 46 Ð 62. Bargues, M. D., D. R. Klisiowicz, F. Gonzalez-Candelas, J. M. Ramsey, C. Monroy, C. Ponce, P. M. Salazar-Schettino, F. Panzera, F. Abad-Franch, O. E. Sousa, et al. 2008. Phylogeography and genetic variation of Triatoma dimidiata, the main Chagas disease vector in Central America, and its position within the genus Triatoma. PLoS Negl. Trop. Dis. 2: e233. Bargues, M. D., C. J. Schofield, and J. P. Dujardin. 2010. ClassiÞcation and phylogeny of Triatominae, pp. 115Ð149. In J. Telleria and M. Tibayrenc (eds.), American Trypanosomiasis: Chagas disease. One hundred years of research. Elsevier Inc., Burlington, MA. Bargues, M. D., M. A. Zuriaga, and S. Mas-Coma. 2014. Nuclear rDNA pseudogenes in Chagas disease vectors: evolutionary implications of a new 5.8S⫹ITS-2 paralogous sequence marker in triatomines of North, Central and northern South America. Infect. Genet. Evol. 21: 134 Ð156. Blando´ n-Naranjo, M., M. A. Zuriaga, G. Azofeifa, R. Zeledo´ n, and M. D. Bargues. 2010. Molecular evidence of intraspeciÞc variability in different habitat-related populations of Triatoma dimidiata (Hemiptera: Reduviidae) from Costa Rica. Parasitol. Res. 106: 895Ð905. Calleros, L., F. Panzera, M. D. Bargues, F. A. Monteiro, D. R. Klisiowicz, M. A. Zuriaga, S. Mas-Coma, and R. Perez. 2010. Systematics of Mepraia (Hempitera - Reduviidae): cytogenetic and molecular variation. Infect. Genet. Evol. 10: 221Ð228. Carcavallo, R. U., S. I. Curto de Casas, I. A. Sherlock, I. Galindez Giron, J. Jurberg, C. Galvao, C. Mena Segura, and F. Noireau. 1999. Geographical distribution and altilatitudinal dispersion, pp. 747Ð792. In R. U. Carcavallo, I. Galõńdez Giro´ n, J. Jurberg, and H. Lent (eds.), Atlas of

627

ChagasÕ disease vectors in the Americas, vol. III. Fiocruz, Rio de Janeiro, Brazil. Coura, J. R., and P. Albajar. 2010. ChagasÕ disease: a new worldwide challenge. Nature 456: s6 Ðs7. Coura, J. R., and J.C.P. Dias. 2009. Epidemiology, control and surveillance of Chagas disease: 100 years after its discovery. Mem. Inst. Oswaldo Cruz 104: 31Ð 40. Curto de Casas, S. I., R. U. Carcavallo, G. I. Galıńdez, and J. J. Burgos. 1999. Bioclimatic factors and zones of life, pp. 793Ð 838. In R. U. Carcavallo, I. Galõńdez Giro´ n, J. Jurberg, and H. Lent (eds.), Atlas of ChagasÕ disease vectors in the Americas, vol. III. Fiocruz, Rio de Janeiro, Brazil. Dias, J.C.P., A. Prata, and D. Correia. 2008. Problems and perspectives for ChagasÕ disease control: in search of a realistic analysis. Rev. Soc. Bras. Med. Trop. 41: 193Ð196. Forattini, O. P. 1980. BiogeograÞa, origem e distribuiç aõ da domiciliaç aõ de triatomõńeos no Brasil. Revista de Sau´ de Pu´ blica 14: 265Ð299. Forattini O. P., A. O. Ferreira, E. O. Rocha e Silva, and E. X. Rabello. 1978. Aspectos ecolo´ gicos da tripanossomõáse americana. XII. Variaç o˜ es mensais da tendeˆ ncia de Panstrongylus megistus a` domiciliaç aõ. Revista de Sau´ de Pu´ blica. 12: 209 Ð233. Garcıá, B. A., and J. R. Powell. 1998. Phylogeny of species of Triatoma (Hemiptera: Reduviidae) based on mitochondrial DNA sequences. J. Med. Entomol. 35: 232Ð238. Garcıá, B. A., E. N. Moriyama, and J. R. Powell. 2001. Mitochondrial DNA sequences of triatomines (Hemiptera: Reduviidae): phylogenetic relationships. J. Med. Entomol. 38: 675Ð 683. Gurgel-Gonç alves, R., C. Galvao, J. Costa, and T. Peterson. 2012. Geographic distribution of Chagas disease vectors in Brazil based on ecological niche modeling. J. Trop. Med. 2012: 1Ð15. Kopp, R. L., V. Thomaz-Soccol, D. R. Klisiowicz, N. Membrive, J. M. Soares Barata, J. Jurberg, M. Steindel, D. C. Trevisan Kopp, E. Alcaˆ ntara de Castro, and E. Luz. 2009. Phenetic analysis of Panstrongylus megistus Burmeister, 1835 (Hemiptera: Reduviidae: Triatominae) in the State of Parana´-Brazil. Braz. Arch. Biol. Technol. 52: 349 Ð357. Larkin, M., G. Blackshields, N. Brown, R. Chenna, P. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, et al. 2007. ClustalW and Clustal X version 2.0. Bioinformatics 23: 2947Ð2948. Librado, P., and J. Rozas. 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25: 1451Ð1452. Lyman, D. F., F. A. Monteiro, A. A. Escalante, C. CordonRosales, D. M. Wesson, J. P. Dujardin, and C. B. Beard. 1999. Mitochondrial DNA sequence variation among triatomine vectors of Chagas disease. Am. J. Trop. Med. Hyg. 60: 377Ð386. Marcilla, A., M. D. Bargues, J. M. Ramsey, E. MagallonGastelum, P. M. Salazar-Schettino, F. Abad-Franch, J. P. Dujardin, C. J. Schofield, and S. Mas-Coma. 2001. The ITS-2 of the nuclear rDNA as a molecular marker for populations, species and phylogenetic relationships in Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease. Mol. Phylogenet. Evol. 18: 136 Ð142. Marcilla, A., M. D. Bargues, F. Abad-Franch, F. Panzera, R. U. Carcavallo, F. Noireau, C. Galva˜ o, J. Jurberg, M. A. Miles, J. P. Dujardin, et al. 2002. Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi. Infect. Genet. Evol. 26: 1Ð11. Martins-Melo, F. R., C. H. Alencar, A. N. Ramos, Jr., and J. Heukelbach. 2012. Epidemiology of mortality related

628


to ChagasÕ disease in Brazil, 1999 Ð2007. PLoS Negl. Trop. Dis. 6: e1508. Mas-Coma, S., and M. D. Bargues. 2009. Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA. Acta Trop. 110: 112Ð136. Pacheco, R. S., C. E. Almeida, J. Costa, D. R. Klisiowicz, S. Mas-Coma, and M. D. Bargues. 2003. RAPD analyses and rDNAintergenic-spacer sequences discriminate Brazilian populations of Triatoma rubrovaria (Reduviidae: Triatominae). Ann. Trop. Med. Parasitol. 97: 757Ð768. Pacheco, R. S., C. E. Almeida, D. R. Klisiowicz, J. Costa, M. Q. Pires, F. Panzera, M. E. Bar, S. Mas-Coma, and M. D. Bargues. 2007. Genetic variability of Triatoma rubrovaria (Reduviidae: Triatominae) from Brazil, Argentina and Uruguay as revealed by two different molecular markers. Parasite 14: 231Ð237. Patterson, J. S., and M. W. Gaunt. 2010. Phylogenetic multilocus codon models and molecular clocks reveal the monophyly of haematophagous reduviid bugs and their evolution at the formation of South America. Mol. Phylogenet. Evol. 56: 608 Ð 621. Patterson, J. S., S. A. Barbosa, and D. Feliciangelli. 2009. On the genus Panstrongylus Berg 1879: evolution, ecology and epidemiological signiÞcance. Acta Trop. 110: 187Ð 199. Swofford, D. L. 2002. PAUP*: phylogenetic analysis using parsimony (*and other Methods), version 4. Computer

Vol. 51, no. 3

program distributed by the Smithsonian Institution. Sinauer, Inc, Sunderland, MA. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei, and S. Kumar. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731Ð2739. Thomaz Soccol, V., C. Bernabe, E. Castro, E. Luz, and M. Tibayrenc. 2002. Trypanosoma cruzi: isoenzyme analysis suggests the presence of an active Chagas sylvatic cycle of recent origin in Parana´ State, Brazil. Exp. Parasitol. 100: 81Ð 86. (WHO) World Health Organization. 2010. Working to overcome the global impact of neglected tropical diseases: Þrst WHO report on neglected tropical diseases. WHO, Geneva, Switzerland. (WHO) World Health Organization. 2012. Accelerating work to overcome the global impact of neglected tropical diseases: a roadmap for implementation. WHO, Geneva, Switzerland. Zuriaga, M. A., M. Blando´ n-Naranjo, I. Valerio-Campos, R. Salas, R. Zeledo´ n, and M. D. Bargues. 2012. Molecular characterization of Trypanosoma cruzi and infection rate of the vector Triatoma dimidiata in Costa Rica. Parasitol. Res. 111: 1615Ð1620. Received 9 April 2013; accepted 13 February 2014.