Infection, Genetics and Evolution 27 (2014) 105–113

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Infection, Genetics and Evolution

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Evolutionary and dispersal history of , main vector of , by chromosomal markers ⇑ Francisco Panzera a, , María J. Ferreiro a, Sebastián Pita a, Lucía Calleros a, Ruben Pérez a, Yester Basmadjián b, Yenny Guevara c, Simone Frédérique Brenière d, Yanina Panzera a a Sección Genética Evolutiva, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay b Departamento de Parasitología y Medicina, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay c Laboratorio de Citogenética Alberto Tellería Cáceres, Universidad Nacional Mayor de San Marcos, Lima, Peru d INTERTRYP (Interactions hôtes-vecteurs-parasites dans les infections par trypanosomatidae), Institut de Recherche pour le Développement (IRD), Montpellier, article info abstract

Article history: Chagas disease, one of the most important vector-borne diseases in the Americas, is caused by Received 30 May 2014 Trypanosoma cruzi and transmitted to humans by insects of the subfamily Triatominae. An effective Received in revised form 1 July 2014 control of this disease depends on elimination of vectors through spraying with insecticides. Genetic Accepted 5 July 2014 research can help insect control programs by identifying and characterizing vector populations. In Available online 11 July 2014 southern Latin America, Triatoma infestans is the main vector and presents two distinct lineages, known as Andean and non-Andean chromosomal groups, that are highly differentiated by the amount of Keywords: heterochromatin and genome size. Analyses with nuclear and mitochondrial sequences are not Triatoma infestans conclusive about resolving the origin and spread of T. infestans. rDNA variability C-heterochromatic polymorphism The present paper includes the analyses of karyotypes, heterochromatin distribution and chromosomal Pyrethroid resistance mapping of the major ribosomal cluster (45S rDNA) to specimens throughout the distribution range of Hybrid zone this species, including pyrethroid-resistant populations. A total of 417 specimens from seven different Hybridization countries were analyzed. We show an unusual wide rDNA variability related to number and chromosomal position of the ribo- somal genes, never before reported in species with holocentric chromosomes. Considering the chromo- somal groups previously described, the ribosomal patterns are associated with a particular geographic distribution. Our results reveal that the differentiation process between both T. infestans chromosomal groups has involved significant genomic reorganization of essential coding sequences, besides the changes in heterochromatin and genomic size previously reported. The chromosomal markers also allowed us to detect the existence of a hybrid zone occupied by individuals derived from crosses between both chromosomal groups. Our genetic studies support the hypothesis of an Andean origin for T. infestans, and suggest that pyrethroid-resistant populations from the Argentinean-Bolivian border are most likely the result of recent secondary contact between both lineages. We suggest that vector control programs should make a greater effort in the entomological surveillance of those regions with both chromosomal groups to avoid rapid emergence of resistant individuals. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction extending over more than 6 million km2, including parts of seven South American countries, and was responsible for well over half Triatoma infestans (Hemiptera, Reduviidae, Triatominae) is an of the 18 million people affected by Chagas disease (WHO, 1991). important vector of the flagellate protozoan Trypanosoma cruzi, Since then, control interventions in the framework of the ‘‘South- causative agent of Chagas disease or American trypanosomiasis. ern Cone Initiative’’ have substantially reduced the distribution of In the early 1990s, T. infestans had a wide geographical distribution T. infestans to less than 1 million km2 and 9.8 million people infected (Schofield et al., 2006). At present, domestic T. infestans mainly persists in the Andean valleys of Bolivia, southern Peru, ⇑ Corresponding author. Address: Sección Genética Evolutiva, Facultad de Cien- and parts of the Gran Chaco region of , Bolivia and cias, Universidad de la República, Calle Iguá 4225, 11400 Montevideo, Uruguay. Tel.: +598 25258619; fax: +598 25258617. Paraguay. One of the challenges facing the control of T. infestans E-mail addresses: [email protected], [email protected] (F. Panzera). in these regions is the recent detection of pyrethroid resistance http://dx.doi.org/10.1016/j.meegid.2014.07.006 1567-1348/Ó 2014 Elsevier B.V. All rights reserved. 106 F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113

(Depickère et al., 2012; Lardeux et al., 2010; Picollo et al., 2005). T. de Plagas e Insecticidas CITEFA-CONICET, Argentina), within the infestans is known to comprise two main evolutionary lineages framework of the project SSA/ATU. Pyrethroid resistance in recognized by molecular markers (nuclear and mitochondrial individuals here analyzed from Salvador Mazza has also been sequences) (Bargues et al., 2006; Giordano et al., 2005; Monteiro experimentally confirmed by Cardozo et al. (2010). Furthermore et al., 1999; Piccinali et al., 2009, 2011; Quisberth et al., 2011; several reports using triatomine samples collected in this area Torres-Pérez et al., 2011) and phenetic characteristics (Calderón- since 2002 showed high resistance levels, with resistance ratios Fernández et al., 2012; Hernández et al., 2008). These lineages, (RRs) ranging from 50.5 to 183 (Lardeux et al., 2010; Picollo known as the Andean and non-Andean groups, are defined by et al., 2005; Toloza et al., 2008). substantial differences (from 30% to 50%) in the amount of nuclear DNA due to different quantities of highly repeated DNA revealed as 2.2. Chromosome preparations and banding procedures heterochromatin by C-banding (Panzera et al., 2004). Based on the existence of sylvatic populations, two main hypotheses have been Gonads from adult insects were removed and fixed in ethanol– proposed to clarify the origin and spread of T. infestans throughout acetic acid (3:1). Chromosome squashes were prepared in 45% ace- . The ‘‘Andean’’ hypothesis suppose that the high- tic acid. C-banding was used to establish the diploid chromosome altitude valleys of Bolivia is the center of origin and dispersal number (2n) and the number of C-heterochromatic chromosomes (Schofield, 1988; Usinger et al., 1966), while the ‘‘Chaco’’ hypothe- (Panzera et al., 2004). We applied the fluorescence in situ hybrid- sis assumes that the origin is the subtropical lowlands regions of ization (FISH) to determine the chromosome location of the 45S southern Bolivia, north-western Paraguay and north of Argentina ribosomal clusters (Pita et al., 2013). (Carcavallo, 1998). Several reports using different nuclear and For each specimen, at least 20 cells were analyzed to determine mitochondrial sequences are not conclusive about resolving the chromosome traits. Slides were examined under a Nikon Eclipse origin and spread of T. infestans (for review sees Torres-Pérez 80i microscope and the images were obtained with a DS-5Mc-U2 et al., 2011). To better understand their chromosomal organization digital camera. In males, mitotic (prometaphase) and meiotic and population differentiation, we analyzed the karyotypes, het- (metaphase I or II) plates were observed. In total, 425 T. infestans erochromatin distribution and chromosomal location of the major specimens were examined by C-banding and 105 by FISH ribosomal cluster (45S rDNA) in specimens from all countries of its techniques (Table 2, Figs. 2 and 3). Of the latter, 70% of them were original distribution, with emphasis on sylvatic and pyrethroid- analyzed by both techniques (data not shown). resistant populations not previously studied. In Triatominae, these chromosomal markers are the main source of karyological variation in population differentiation and speciation processes (Panzera 3. Results et al., 2010, 2012; Pita et al., 2013). We try to respond to the following questions: (i) what are the main karyological changes 3.1. Chromosome number that occurred during the dispersal of T. infestans? and (ii) do the pyrethroid-resistant individuals detected along the Argentina- All specimens showed the same normal diploid number Bolivia border represent ancient or recent populations? Our (2n = 22) constituted by 20 autosomes and two sex chromosomes findings provide further information about the origin and spread (XY in males, XX in females) (Fig. 2). However, in some individuals of T. infestans, including the origin of the pyrethroid resistance we observed the occurrence of chromosomal fragments or extra- populations in non-Andean regions. chromosomes (Fig. 2E). These are the smallest of the chromosome complement, both euchromatic and heterochromatic, and their frequency varied from 1 to 3 amongst individuals. These chromo- 2. Methods somal fragments appear in gonial mitotic prometaphases, both in males and females, but we did not detect any alteration in the 2.1. Material analyzed meiotic segregation of carrier individuals. The frequencies of individuals with chromosomal fragments were very variable, but All specimens of T. infestans came from natural populations and the populations from Salvador Mazza showed the highest most of them were collected within the framework of the project frequency (more than 80% of individuals) (Table 2). SSA/ATU INCOCT 2004 515942 (Catalá et al., 2007). These speci- mens were also analyzed by cuticular hydrocarbon and antennal 3.2. Distribution of C-heterochromatin phenotypes (Calderón-Fernández et al., 2012; Hernández et al., 2008, respectively). The geographic origin of each population, year Each specimen exhibited a specific C-banding pattern. All of collection and habitat (domiciliary, peridomiciliary and sylvatic) populations were polymorphic, with variations in the number of are given in Table 1 and Fig. 1. It is noteworthy that in several col- chromosomes with C-heterochromatin and in the chromosome lection sites here analyzed (Uruguay, , Chile and some from position of C-blocks (one or both chromosomal ends). All Argentina and Paraguay), currently there are no specimens of this individuals show a C-heterochromatic Y chromosome while the X species, being eliminated by vector control activities. Andean syl- chromosome is euchromatic or heterochromatic. The number of vatic specimens were mainly collected amongst rock piles, while autosomes with C-heterochromatin differentiates three distinct the ‘‘dark morphs’’ from the Bolivian Chaco (non-Andean region) groups, two of them previously described (Panzera et al., 2004) were caught in hollow trees (Fig. 1, map reference 29) (Noireau and a third group here named as ‘‘Intermediate group’’ (Table 2 et al., 2000). and Fig. 2): Since 2002, control services for Chagas disease from Argentina and Bolivia reported low efficiency of deltamethrin and other pyre- throids for the treatment of rural sites close to Salvador Mazza 3.2.1. Andean group (Salta Province, Argentina) and Yacuiba cities (Tarija Department, Includes 102 insects from Bolivia and Peru (Fig. 1, map refer- Bolivia) (Fig. 1, map references 17–28). Pyrethroid resistance of ences 1–16). The number of heterochromatic autosomes with insects collected from Yacuiba (Tarija, Bolivia) and Salvador Mazza C-blocks varies from 14 to 20, with a mean of 15.64 and a standard (Salta, Argentina) were determined with topical application bioas- deviation (SD) of 1.35 (Table 2, Fig. 2A and B). All individuals have says (WHO, 1994), by María Inés Picollo (Centro de Investigaciones X chromosome with C-heterochromatin. F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113 107

Table 1 Geographical location of Triatoma infestans individuals analyzed in this study. P, peridomiciliary; D, domiciliary; S, sylvatic.

Map reference Country Department or province Locality, habitat Altitude (masl) Latitude South Longitude West Year of collection 1 Peru Arequipa Arequipa valleys, P 2336 16° 310 5900 71° 280 2700 1997/2009 2 Peru Ica Nazca city, P 588 14° 500 2100 74° 560 25 2005/09 3 Bolivia La Paz Sapini, D-S 1880 16° 480 5500 67° 420 2100 2010 4 Bolivia La Paz Chivisini, S 2892 16° 570 5300 67° 520 5300 2008 5 Bolivia Cochabamba Cotapachi, D-P-S 2750 17° 250 2900 66° 150 5300 2004 6 Bolivia Cochabamba 20 de Octubre, S 2596 17° 290 0300 66° 060 4400 2008 7 Bolivia Cochabamba Tabacal, S 2182 17° 560 0100 65° 230 0600 2008 8 Bolivia Cochabamba Mataral, D-P-S 1700 18° 360 1100 65° 080 6000 2005/08 9 Bolivia Cochabamba Jamach´ Uma, D 2707 17° 300 5200 66° 050 1100 1997 10 Bolivia Chuquisaca Carapari, P 1892 18° 370 1800 65° 100 3000 2005 11 Bolivia Chuquisaca Uyuni, D-S 2542 19° 260 0000 64° 500 0700 1997/2008 12 Bolivia Potosí Thago Thago, P 2000 18° 000 4400 65° 480 3100 2010 13 Bolivia Potosí La Deseada, S 2921 21° 310 1900 65° 410 3400 2005 14 Bolivia Potosí Viscachani, S 3041 21° 360 4100 65° 480 2200 2010 15 Bolivia Potosí Palquiza, S 2908 21° 310 4100 65° 450 0400 2010 16 Bolivia Potosí Urulica, S 3129 21° 350 4000 65° 490 5600 2010 17 Bolivia Tarija Palmar Chico, D-P 610 21° 530 5000 63° 360 4500 2004 18 Bolivia Tarija El Barrial, D 387 22° 020 2200 63° 400 5400 2004 19 Bolivia Tarija Yaguacua, D 650 21° 420 0000 63° 360 0000 2005 20 Bolivia Tarija San Francisco del Inti, D-P 610 21° 490 4100 63° 360 3500 2005 21 Bolivia Tarija Estación Sunchal, D-P 558 21° 410 1700 63° 220 0100 2005 22 Bolivia Tarija Quebrada Busuy, D-P 387 21° 160 7500 63° 270 8700 2005 23 Bolivia Tarija San Antonio, D 429 21° 150 3900 63° 280 4700 2005 24 Bolivia Tarija Suarurito, D-P 853 21° 160 5600 63° 560 3300 2005 25 Bolivia Tarija Tacuarandy, P 958 21° 230 0300 63° 590 5700 2005 26 Argentina Salta Salvador Mazza, La Toma, D 834 22° 010 5600 63° 420 3100 2005 27 Argentina Salta S. Mazza, El Chorro, D-P 820 22° 010 3700 63° 410 1400 2005 28 Argentina Salta S. Mazza, El Obraje, D-P 840 22° 040 4100 63° 410 4700 2005 29 Bolivia Santa Cruz Tita, S 300 18° 340 3100 62° 400 0500 1997/2008 30 Bolivia Santa Cruz Tita, P 300 18° 800 0000 63° 120 6700 1997/2008 31 Argentina Salta La Unión, D 169 23° 450 38000 63° 080 1500 2005 32 Argentina Salta San Carlos, P 2088 25° 480 3300 65° 570 5300 2005 33 Argentina Chaco Pampa Ávila, D-P 107 26° 590 5000 61° 300 4400 2005 34 Argentina Chaco Tres Estacas, P 122 26° 540 3000 61° 400 2300 2005 35 Argentina Santiago del Estero Huachana, P 165 28° 130 2500 64° 070 1300 2005 36 Argentina Santiago del Estero Silípica, P 138 28° 060 1200 64° 070 0800 2005 37 Argentina La Rioja 4 Esquinas, P 320 31° 480 0300 65° 860 6600 2002 38 Argentina La Rioja San Antonio, P 326 29° 280 3300 66° 150 0000 2002 39 Argentina La Rioja Anillaco, P 1419 28° 800 0000 66° 950 0000 1997 40 Argentina Catamarca Palo Blanco, P 1828 27° 200 2600 67° 450 3500 2005 41 Argentina Formosa Villa Escobar, P 66 26° 610 6600 58° 660 6600 2001 42 Argentina Córdoba Los Leones, P 250 30° 430 1600 64° 480 3500 2000 43 Paraguay Boquerón Jericó, D 115 22° 350 4600 59° 480 4100 2005 44 Paraguay Boquerón Tiberia, D 115 22° 370 1700 59° 550 5000 2005 45 Paraguay Presidente Hayes Niya Toyish, D 126 22° 570 2700 59° 520 3200 2005 46 Paraguay Pres. Hayes Estancia Salazar, D 126 23° 040 4100 59° 160 5300 2005 47 Paraguay San Pedro San Pedro, D 213 24° 100 0000 57° 080 330 2005 48 Paraguay Cordillera San Bernardino, D 334 25° 180 2600 57° 170 4300 2005 49 Uruguay Rivera Several localities, D-P 0–250 30° 530 5500 55° 330 0100 1988–95 50 Uruguay Soriano Several localities, P 0–250 30° 420 1500 57° 900 2000 1988–95 51 Chile IV Region: Coquimbo Pulpica, P 740 30° 510 3000 70° 460 200 2002 52 Brazil Paraiba Monteiro, D 602 07° 530 2900 37° 070 0000 1997

3.2.2. Non-Andean group Considering the number of C-heterochromatic autosomes, sig- Includes 238 specimens from Argentina, Bolivia (non-Andean nificant statistical differences by Student t test were determined region), Brazil, Chile, Paraguay, and Uruguay (Fig. 1, map references between Andean and non-Andean groups (p < 0.001). 29–52). The number of heterochromatic autosomes varies from 4 to 6 (5.86 ± 0.52) (Table 2, Fig. 2C and D), but most individuals 3.3. Chromosome location of 45S rDNA cluster (86.2%) have six C-heterochromatic autosomes. In all individuals, the X chromosomes are euchromatic. T. infestans showed marked variability between populations and between individuals, both in the number of rDNA loci and in the 3.2.3. Intermediate group type of chromosomes (autosomes and sex chromosomes) that car- Restricted to Argentina-Bolivia border, including 85 individuals ried the rDNA genes (Table 2 and Fig. 3). The number of rDNA loci from localities from Salvador Mazza (Argentina) and Tarija per male haploid genome ranged from 1 to 4 sites. The 45S rDNA (Bolivia) (Fig. 1, map references 17–28). The number of heterochro- cluster is located on one sex chromosome (X chromosome) and matic autosomes varies from 7 to 11 (8.20 ± 1.08) (Table 2, Fig. 2E even on up to three autosomal pairs. Due to the similar size of and F). In 72 insects (84.7%), the X chromosomes are euchromatic the autosomes is very difficult to determine exactly which chromo- (similar to non-Andean group), while in the remaining 13 are some pair presents the ribosomal cluster. However, one ribosomal heterochromatic (similar to Andean group). signal is always located in at least one of the four largest autosomal 108 F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113

Fig. 1. Location of the collection sites of Triatoma infestans analyzed in this study. See number identification in Table 1. D Collection sites of individuals from Andean Group, Intermediate Group, non-Andean Group.

Table 2 Chromosomal results in Triatoma infestans obtained with C-banding (number of autosomes with C-bands and % of chromosomal fragments) and by fluorescent in situ hybridization (chromosome location of the ribosomal genes). Pattern 1A means rDNA on one autosomal pair, 1A + X means on one autosomal pair plus X chromosome, 2A means on two autosomal pairs, 2A + X means on two autosomal pairs plus X, 1A + 1/2 A + X means on two autosomal pairs (with one heterozygote) plus X, 1/2 A + X means on one heterozygote autosomal pair plus X, pattern 1/2 A + 1/2 A + X means on two autosomal pairs (both heterozygotes) plus X, 1/2 A + 1/2 A + 1/2 A + X means on three heterozygote autosomal pairs plus X chromosome and X pattern means rDNA only on the X chromosome. SD, Standard Deviation. Between parentheses we included the number of individuals studied by C-banding and FISH.

Map reference No. of C-autosomes Chromosomal fragments rDNA pattern locationb mean ± SD (%) 1 15.93 ± 1.16 (15)a 0.0 1A (2); 2A (1) 2 15.55 ± 1.21 (11) 0.0 1A + X (1) 3–4 14.90 ± 0.88 (10) 0.0 1A + X (6) 5–9 15.62 ± 1.37 (34) 0.0 1A (16); 1A + X (11); 2A + X (2); 1A + 1/2 A + X(1) 10–11 16.29 ± 1.65 (17)a 0.0 1A (3)b; 2A (2); 1A + X (1); 1A + 1/2 A + X (1) 12–16 15.20 ± 1.21 (15)a 0.0 1A (5)b; 1A + X (3) Andean Group 15.64 ± 1.35 (102) 0.0 55 individuals: 1A (26); 1A + X (22); 2A (3); 2A + X (2); 1A + 1/2 A + X (2) 17–25 8.16 ± 1.19 (32) 18.8 X (1); 1/2 A + X (6); 1/2 A + 1/2 A + X (1); 1/2 A + 1/2 A + 1/2 A + X (1) 26–28 8.23 ± 1.01 (53) 83.0 X (9); 1/2 A + X (4) Intermediate 8.20 ± 1.08 (85) 58.8 22 individuals: X (10); 1/2 A + X (10); 1/2 A + 1/2 A + X (1); 1/2 A + 1/2 A + 1/2 A + X Group (1) 29–30 6.15 ± 0.38 (13)a 0.0 X (5) 31–32 6.17 ± 0.41 (6) 16.7 X (2) 33–34 5.58 ± 0.56 (33) 24.2 X (3) 35–36 5.27 ± 0.77 (22) 0.0 X (3) 37–39 6.20 ± 0.56 (15)a 0.0 X (4) 40 6.05 ± 0.23 (19) 15.8 X (2)b 41 6.00 ± 0.00 (5) 0.0 ND 42 5.17 ± 0.58 (12)a 0.0 X (2) 43–46 6.04 ± 0.52 (27) 0.0 X (3) 47–48 6.00 ± 0.00 (12) 0.0 X (1) 49–50 5.99 ± 0.12 (70)a 0.0 X (2)b 51 6.00 ± 0.00 (2) 0.0 ND 52 6.00 ± 0.00 (2) 0.0 X (1)b Non-Andean 5.86 ± 0.52 (238) 4.4 % 28 individuals: all X pattern Group

a Partial C-banding data from Panzera et al. (2004). b Partial FISH data from Panzera et al. (2012). pairs. In total, we observed nine ribosomal location patterns: on pairs plus X chromosome (1/2 A + 1/2 A + 1/2 A + X, Fig. 3H) and one autosomal pair (pattern 1A, Fig. 3A); on one autosomal pair only on the X chromosome (pattern X, Fig. 3I). In all cases, the plus X chromosome (1A + X, Fig. 3B); on two autosomal pairs hybridization signals were located at a terminal or subterminal (2A, Fig. 3C); on two autosomal pairs plus X (2A + X, Fig. 3D); on chromosome position. two autosomal pairs (with one heterozygote) plus X (1A + 1/2 Considering the three chromosomal groups previously A+X, Fig. 3E); on one heterozygote autosomal pair plus X (1/2 described, the ribosomal patterns are associated with a particular A+X,Fig. 3F); on two autosomal pairs (both of them heterozygotes) geographic distribution (Table 2). The Andean group is the most plus X (1/2 A + 1/2 A + X, Fig. 3G); on three heterozygote autosomal variable, with five patterns exclusively detected in this group F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113 109

Fig. 2. Representative C-banding patterns observed in the three chromosomal groups of Triatoma infestans: 2n = 22 chromosomes (20 autosomes plus XY in males/XX in females). (A), (C), (E) Mitotic prometaphases. (B), (D), (F) Fist meiotic metaphase. (A, B): Andean Group: Most of the 20 autosomes, and both sex chromosomes shown C- heterochromatic blocks. (C, D): non-Andean Group: In (C), oogonial prometaphase with only 6 autosomes with C-blocks. The two X chromosomes are euchromatic and indistinguishable for the euchromatic autosomes. In (D), three autosomal pairs are C-heterochromatic, while the Y chromosome appears C-positive and the X chromosome euchromatic. (E, F): Intermediate Group: Nine autosomes are C-heterochromatic, while that both sex chromosomes are also C-positive. Arrowhead pointed out a small euchromatic chromosomal fragment. Scale bar = 10 lm.

(Fig. 3A–E). Two of them (patterns 1A and 1A + X) involved 90% of (Hughes-Schrader and Schrader, 1961). Unlike what is observed the individuals analyzed (48/55). Most of the collection sites are in monocentric chromosomes, chromosomal fragments in holocen- polymorphic, being the populations from Cochabamba and Chu- tric systems attach to spindle fibres and migrate normally during quisaca the most variable (Table 2) and the populations from La cell divisions, becoming what are known as B chromosomes Paz the most homogeneous. (Mola and Papeschi, 2006). In true bugs however, B chromosomes The non-Andean group showed only one ribosomal pattern: seem very uncommon and have been detected in only 12 of the rDNA loci located on the X chromosome (Fig. 3I and Table 2), 465 species so far studied (Kuznetsova et al., 2011). In Triatominae, involving 28 individuals from six countries, including sylvatic chromosomal fragments usually occur as byproducts of a translo- and peridomestic specimens from the Bolivian Chaco region cation among non-homologous autosomes, as has been observed (Fig. 1, map references 29–30). in Mepraia gajardoi and T. infestans (Pérez et al., 2004; Poggio The Intermediate group shows four ribosomal patterns in the 22 et al., 2013). In these mutant individuals, an autosomal trivalent individuals analyzed (Table 2). One of them is similar to that and chromosomal fragments were observed both in mitosis and observed in the non-Andean group: on the X chromosome (10 indi- meiosis. However, the individuals of T. infestans with chromosomal viduals)(Fig. 3I). The other three patterns are exclusively found in fragments here analyzed are quite different, as they show normal this group: 1/2 A + X pattern in 10 individuals (Fig. 3F), and two meiotic segregation without autosomal associations. These chro- patterns showing 2 and 3 heterozygote autosomal pairs plus X mosomal fragments may reflect the occurrence of small fissions chromosome found in one individual for each one (Fig. 3G and H). or the presence of unstable chromosome regions (fragile sites) that are prone to breakage in mitotic metaphases. The frequency of 4. Discussion individuals with chromosomal fragments varies greatly according to the populations studied (Table 2), and its appearance probably 4.1. Chromosome number is associated with populations resistant to pyrethroids (see below Intermediate group and pyrethroid resistance). All individuals have 20 autosomes plus two sex chromosomes (2n = 22). However, some individuals have chromosomal frag- 4.2. Chromosome location of 45S rDNA genes ments or extra-chromosomes (Table 2 and Fig. 2E). One distinctive cytogenetic feature of heteropteran insects, including Triatominae, The striking intraspecific rDNA variability detected in T. infestans is the holocentric or holokinetic nature of their chromosomes is unusual in insects, and exceptional in holocentric chromosomes. 110 F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113

Fig. 3. Nine ribosomal patterns showing the intraspecific variability in the chromosome location of the 45S rDNA observed in first meiotic metaphases of Triatoma infestans discriminated by chromosomal groups. Andean patterns: (A) rDNA on one autosomal pair = pattern 1A; (B) on one autosomal pair plus X chromosome = pattern 1A + X, (C) on two autosomal pairs = pattern 2A, (D) on two autosomal pairs plus X = pattern 2A + X, (E) on two autosomal pairs (with one heterozygote) plus X = pattern 1A + 1/2 A + X. Intermediate patterns: (F) on one heterozygote autosomal pair plus X = pattern 1/2 A + X, (G) on two autosomal pairs (both heterozygotes) plus X = pattern 1/2 A + 1/2 A + X, (H) on three heterozygote autosomal pairs plus X chromosome = pattern 1/2 A + 1/2 A + 1/2 A + X. Intermediate and non-Andean pattern: (I) only on the X chromosome = X pattern. The autosomal pairs with heterozygotes signals are pointed out by arrows. Scale bar = 10 lm. A = autosome.

Considering almost 100 heteropteran species analyzed to date, the 1400 masl) in various countries including Bolivia (Chaco region) chromosome position of the ribosomal genes appears to be a spe- but also at higher altitudes in some localities of Argentina such cies-specific character. The number of rDNA loci described in T. infe- as Palo Blanco (1800 masl) or San Carlos (2088 masl) (Fig. 1). These stans (from 1 to 4 sites) is very high in comparison with the 1 or 2 data confirm that Andean insects are able to colonize lowlands, and loci found in most heteropteran species analyzed to date (Bardella that non-Andean insects would be able to colonize highlands, as et al., 2013; Panzera et al., 2012). suggested previously (Panzera et al., 2004). Thus, the occurrence The existence of individuals with ribosomal genes located on of a chromosomal group in a particular region may be associated four different chromosomes (X chromosome and up to three auto- with selection mechanisms linked to altitude as well as with the somal pairs) allows clear recognition of hybrid individuals revealed historical dispersal routes of each chromosomal group. Our chro- by the occurrence of heterozygous patterns for a particular locus mosomal data from Peru and Chile are consistent with the pres- (Fig. 3E–H). The high rDNA variation in T. infestans also suggests ence of the two lineages in Chile, as suggested by mitochondrial that some structural characteristic of their chromosomes promotes sequences (Torres-Pérez et al., 2011). The colonization of northern changes in the number and position of ribosomal genes. The heter- Chile by the Andean group has probably also occurred from the ologous chromosome associations between several autosomes and coastal regions of Peru, and not only through the Andes. By con- sex chromosomes during cell divisions in T. infestans could favor trast, we have not detected both lineages in Argentina, as has been the high mobility of rDNA loci through of transposition and/or suggested by microsatellite markers (Pérez de Rosas et al., 2011). ectopic recombination, which are the principal mechanisms suggested for rDNA changes in other organisms (Schubert and 4.4. Origin and dispersal of T. infestans in the light of chromosomal Wobus, 1985). markers

4.3. Geographic distribution of Andean and non-Andean groups Two main hypotheses have been proposed to explain the origin and spread of T. infestans throughout South America. The ‘‘Andean’’ The Andean group of T. infestans is distributed in high-altitude hypothesis assumed that the Andean valleys of Bolivia represent regions (above 1700 masl) of the Andean valleys of Bolivia and the geographic area of the most ancient populations (Schofield, Peru, and also in lower altitudes such as the city of Nazca (588 1988; Usinger et al., 1966), where sylvatic populations are found masl). The non-Andean group is found in lowland regions (0 to amongst rock piles associated with wild rodents (Dujardin et al., F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113 111

1987; Waleckx et al., 2011). This hypothesis also suggests that the sylvatic T. infestans associated with different habitats and hosts is domiciliation of T. infestans began in this region, associated with in an agreement of differentiation of sensilla patterns observed the domestication of wild guinea-pigs by pre-Columbian Andean between both chromosomal groups (Hernández et al., 2008). cultures and was then spread in association with human migra- tions along two main routes: a north-western one towards Peru 4.5. The Intermediate chromosomal group and then to north of Chile, and a southern route to Argentina, Chile, Paraguay, Brazil, and Uruguay (Schofield, 1988). The second theory, One of the most significant results of this study was the detec- called ‘‘Chaco’’ hypothesis, assumed that T. infestans originated in tion of a third chromosome group (Intermediate), restricted to a the dry subtropical woodlands of southern Bolivia, Paraguay, and small lowland region about 70 km wide along the Argentina-Boli- north Argentina (Carcavallo, 1998). Several studies have confirmed via border (Fig. 1, map references 17–28). This Intermediate chro- the presence of sylvatic populations in this region, usually occupy- mosomal group has three chromosome characteristics. First, the ing hollow trees, probably associated with birds, and presenting number of heterochromatic autosomes (7–11) is lower than low or no infection with T. cruzi (Ceballos et al., 2009; Noireau Andean group (14–20) but higher than non-Andean group (4–6). et al., 2000; Rolón et al., 2011; Waleckx et al., 2011). Analyses with Second, the Intermediate group includes individuals both with nuclear and mitochondrial sequences show that the sylvatic and and without heterochromatic X chromosome, as observed in domestic populations from Andean-Bolivia and Chaco regions Andean and non-Andean individuals. Third, about 50% of individu- show similar values of haplotype diversity (Bargues et al., 2006; als have an exclusive rDNA pattern (1/2 A + X). Considering these García et al., 2013; Piccinali et al., 2009, 2011; Quisberth et al., three chromosomal characteristics and Mendelian inheritance of 2011; Waleckx et al., 2011; 2012). Furthermore, molecular phylog- C-heterochromatin as both of the ribosomal genes, two opposing eographic including coalescent analyses are not conclusive about hypotheses can be suggested for the origin of the Intermediate resolving the Andean or Chaco origin of T. infestans (Torres-Pérez group. et al., 2011). However in this paper, the high rDNA diversity The first hypothesis, that we called ‘‘ancestral origin’’, assumes observed in the Andean group compared to the monomorphic that the Intermediate group represents a middle step of gradual non-Andean group strongly suggests that the Andean group is heterochromatin variance that occurred during the early diver- the ancestral in T. infestans, with the non-Andean group being a gence between Andean and non-Andean groups. The three chro- derivative. Considering the two most common patterns of the mosomal groups of T. infestans might reflect the ancestral Andean group (1A and 1A + X patterns in 90% of the individuals processes of chromosomal differentiation, forming a north–south analyzed), a single transposition event (from an autosome to X cline or gradient of heterochromatin. The current geographic dis- chromosome) or autosomal loss of rDNA loci (in 1A + X pattern) tribution of the Intermediate group along the Argentina-Bolivia could explain the X pattern in the non-Andean group. An ancestral border would thus represent the original area where the Andean origin from non-Andean group (Chaco hypothesis) would have to and non-Andean groups diverged. imply several events of transposition and duplication of ribosomal The alternative hypothesis, called ‘‘secondary contact’’, assumes genes occurring simultaneously, which is highly unlikely. that this group originated recently from crosses between Andean In light of our chromosomal data, we support the hypothesis and non-Andean individuals, which were previously isolated. Fol- that T. infestans originated in the Andean valleys of Bolivia, as a syl- lowing divergence between the Andean and non-Andean groups, vatic species with polymorphic populations in terms of high the two forms would have continued to differentiate in isolation amounts of heterochromatic autosomes (14–20) and variable ribo- – for example through adaptation to different environments. How- somal cluster positions. The most variable populations were those ever, when the two groups were subsequently brought back of Cochabamba and Chuquisaca, suggesting these as the original together, for example through anthropogenic activities such as regions. Interestingly, the populations from La Paz are the most human migration and domestic vector control, interbreeding homogeneous in their ribosomal patterns (Table 2), similar as between them would have given rise to the Intermediate group. observed with cytochrome b sequences, suggesting a possible This second hypothesis assumes that the Intermediate group is ancient isolation and bottleneck in this area (Waleckx et al., very recent formation and not a relic of the ancient process of 2011). The emergence of the non-Andean group involved a deep divergence between Andean and non-Andean groups. genomic rearrangement reflected in a reduction of heterochroma- The C-heterochromatin traits observed in the Intermediate tin to 3 autosomal pairs (and consequently in the overall genome group, namely the number of heterochromatic autosomes and size), lost the C-heterochromatin in the X chromosome and a ribo- the X chromosomes with and without C-bands, are consistent with somal cluster situated only on the X chromosome. Molecular data either hypothesis. The same statement was achieved by analysis based on nuclear and mitochondrial sequences suggests that this with antennal phenotypes (Hernández et al., 2008). However, the divergence occurred between 59,000 and 588,000 years ago high frequency of the heterozygous rDNA pattern (1/2 A + X, (Bargues et al., 2006; Torres-Pérez et al., 2011) although this would Fig. 3F) exclusively detected in the Intermediate group can only be long before human presence in these regions. Perhaps each be explained assuming the hypothesis of a secondary contact. This chromosome group would have independently undergone a pro- pattern would be easily derived by crosses between non-Andean cess of diversification as sylvatic populations associated with dif- individuals (X pattern) with Andean individuals having the ferent habitats and hosts – the Andean group with rock piles and 1A + X and 1A patterns (the two most frequently observed in rodents, and the non-Andean group associated with trees and Andean individuals). All F1 progeny would have the heterozygous birds. Subsequent human influence could have led to multiple pattern, and crossing between them would give offspring showing and independent domestication processes in both groups, as sug- heterozygous and parental patterns. By contrast, it is very unlikely gested by molecular markers (Waleckx et al., 2011). A ribosomal that a population that includes a high frequency of heterozygotes divergence based on ecological preference can be tested because (about 50%) could be maintained for long time, as the ancestral it predicts that sylvatic populations in non-Andean regions would hypothesis presupposes. This high frequency strongly indicates have ribosomal genes only on the X chromosome, as observed in these individuals are of recent origin (hypothesis of secondary con- sylvatic specimens from Bolivian Chaco (Table 2). FISH analyses tact) and suggests that Argentina-Bolivia border represents a very of sylvatic specimens from Argentinean Chaco with putative ances- recent hybrid zone formed by crosses of two parental populations tral mitochondrial haplotypes (Piccinali et al., 2011) may help to (Andean and non-Andean groups) generating intermediate or clarify this issue. A hypothesis of ancestral diversification of ‘‘mixed’’ genotypes (with chromosomal characteristics of the two 112 F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113 main chromosome groups). Experimental crosses between these heterochromatin (Andean and non-Andean groups) may modify groups showed that the F1 progeny have intermediate amounts the gene expression of euchromatic regions adjacent to the hetero- of heterochromatin (Panzera et al., 2004). Epidemiological data chromatin (position-effect variegation). from vector control activities also support a recent origin of the The emergence of resistant populations along the Argentina- Intermediate group (see below). In conclusion, our FISH data Bolivia border supports a close relationship between the Interme- strongly suggests that the Intermediate chromosomal group diate genomes and pyrethroid resistance. According to this idea, detected along the Argentina-Bolivia border is due to a recent sec- we can predict that nearby regions occupied by Andean and non- ondary contact between Andean and non-Andean groups, rather Andean groups, or non-Andean regions experiencing human than an ancient variation resulting from north–south cline of migration from Andean regions (such as Gran Chaco region), would heterochromatin. have a greatest likelihood of developing pyrethroid resistance. Recently, several domestic populations of T. infestans from the 4.6. Intermediate group and pyrethroid resistance Bolivian Chaco (Izozog, Santa Cruz Department) appear to be the result of a mixture of Andean and non-Andean groups probably Considering that the Intermediate group is situated at relatively generated by the passive transport of triatomines from the Andean low altitudes (387–958 masl), it seems most likely to have been to the Gran Chaco region (Quisberth et al., 2011). Although T. infe- occupied by non-Andean individuals prior to secondary contact stans populations from Santa Cruz Department are currently con- with Andean populations. This assumption is supported by the sidered amongst the most susceptible to deltamethrin (Depickère high frequency of individuals with ribosomal X pattern (similar et al., 2012), these ones could have a high probability of developing to non-Andean specimens). It thus seems likely that insects from pyrethroid resistant in the near future. Andean regions moved to southern Bolivia, reaching the Argen- tina-Bolivia border, perhaps by passive human transport. When 5. Conclusions these Andean individuals cross with non-Andean, producing Inter- mediate individuals resistant to pyrethroids, which was not previ- This paper reveals that the differentiation process between the ously present in this region. Epidemiological information supports two T. infestans chromosomal groups has involved significant geno- this scenario. Data provided by the Vector Control Program of Salta mic reorganization of heterochromatin and essential coding Province point out that, in the localities of Salvador Mazza, the sequences. Furthermore, the detection of pyrethroid-resistant spraying of houses with deltamethrin between 1995 and 1998 insects in a hybrid zone between both chromosomal groups sug- led to very low house infestation rates (ranging between 0.5 and gests a correlation between genomic variability and insecticide- 0.8%). This indicates that this region was then occupied by popula- resistant populations. Vector control programs should make a tions susceptible to deltamethrin. Since 1998 however, and despite greater effort in hybrid-zones surveillance to avoid rapid emer- continued vector control activities, there has been a gradual gence of resistant individuals. As an effective control of Chagas dis- increase of insects in houses, reaching house infestation levels of ease depends on vector elimination, our study underscores the 50 to 80% in 2004. Similar control failures were reported at the importance of chromosomal and genomic studies to provide a bet- same time in the Bolivian city of Yacuiba, close to Salvador Mazza, ter understanding of the dynamics of domestic insect population. across the Argentina-Bolivian border (Cardozo et al., 2010). The elimination of T. infestans by pyrethroid insecticides in Bra- zil, Uruguay, Chile, and its drastic reduction in large parts of Para- Authors’ contributions guay and Argentina, clearly indicates that pyrethroid resistance was very uncommon in non-Andean regions. By contrast, in the F.P., R.P., Y.P.: Design, coordination and wrote the different ver- Bolivian Andes, pyrethroid resistant populations appear to be sions of the manuscript. F.P., M.J.F., S.P., I.F., L.C., Y.B., Y.G., S.F.B., much more frequent, being detected in several localities from the Y.P.: performed data and their analyses, helped to draft the departments of Cochabamba, Chuquisaca, Sucre, and Potosí manuscript. F.P., Y.B., Y.G., S.F.B.: contributed to the triatomine (Depickère et al., 2012; Lardeux et al., 2010; Roca Acevedo et al., collection. All authors read and approved the final version of the 2011). This suggests that Andean populations have particular manuscript. genetic backgrounds that enable the development of resistance to pyrethroid insecticides more rapidly than non-Andean popula- Acknowledgements tions. Although, there are probably different mechanisms responsi- ble for resistance to pyrethroids in T. infestans (Germano et al., Partial financial supports were received from CSIC (Udelar) pro- 2010), genetic analyses of experimental crosses between resistant jects, PEDECIBA and ANII from Uruguay and European Community and susceptible individuals showed that the inheritance of delta- project SSA/ATU INCO-CT 2004 515942. This study benefited from methrin resistance is autosomal and with incomplete dominance, international collaboration of the European Community Latin involving at least three genes (Cardozo et al., 2010). This inheri- American Network for Research on the Biology and Control of tance fashion indicates that, under pyrethroid selective pressure, Triatominae (ECLAT). We are particularly grateful for epidemiolog- the resistant character may spread easily to susceptible insects. ical data provided by Alberto Gentile (Director General de A striking observation is the high frequency of chromosome Coordinación Epidemiológica, Ministerio de Salud Pública, Salta, fragments in the pyrethroid resistant populations from Salvador Argentina), the coordination of European project by Antonieta Mazza (Table 2). In other insects, chromosome rearrangements Rojas de Arias (Paraguay) and Silvia Catalá (CRILAR, Argentina) have been suggested to be involved in insecticide resistance. In and the kindly cooperation to elaborate the map by Gabriela Ferná- Myzus persicae, an aphid with holocentric chromosomes, an auto- ndez (Faculty of Sciences, Uruguay). To Nidia Acosta, Pilar Alderete, somal translocation is responsible for organophosphate and carba- Rubén Cardozo, Alberto Gentile, Blanca Herrera, Abraham Jemio, mate resistance by the amplification of esterase gene E4 due to a François Noireau, Elsa López, Ximena Porcasi, David Rivera, Mirko position effect caused by the repositioning of heterochromatin Rojas and Raul Stariolo are thanked for their contributions to the (Blackman et al., 1978). A similar phenomenon may have occurred collection of specimens. We dedicate this paper to the memory in T. infestans, where changes in position and amount of of our friend François Noireau who contributed substantially to heterochromatin are very frequent. It is likely that homologous the understanding of the genetic and ecological variation of recombination between individuals with different amounts of T. infestans. F. Panzera et al. / Infection, Genetics and Evolution 27 (2014) 105–113 113

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