evolutionary and dispersal history of triatoma infestans, main vector of chagas disease, by...

1

3

4

5

6

7 Q1

8

9101112

1314

1 6

1718192021

2223242526272829

3 0

58

59

60

61

62

63

Infection, Genetics and Evolution xxx (2014) xxx–xxx

MEEGID 2014 No. of Pages 9, Model 5G

15 July 2014

Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

Evolutionary and dispersal history of Triatoma infestans, main vector ofChagas disease, by chromosomal markers

http://dx.doi.org/10.1016/j.meegid.2014.07.0061567-1348/� 2014 Published by Elsevier B.V.

⇑ Corresponding author. Address: Sección Genética Evolutiva, Facultad de Cien-cias, Universidad de la República, Calle Iguá 4225, 11400 Montevideo, Uruguay. Tel.:+598 25258619; fax: +598 25258617.

E-mail addresses: [email protected], [email protected] (F. Panzera).

Please cite this article in press as: Panzera, F., et al. Evolutionary and dispersal history of Triatoma infestans, main vector of Chagas disease, by chrommarkers. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.07.006

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, Uruguayb Departamento de Parasitología y Medicina, Facultad de Medicina, Universidad de la República, Montevideo, Uruguayc Laboratorio de Citogenética Alberto Tellería Cáceres, Universidad Nacional Mayor de San Marcos, Lima, Perud INTERTRYP (Interactions hôtes-vecteurs-parasites dans les infections par trypanosomatidae), Institut de Recherche pour le Développement (IRD), Montpellier, France

a r t i c l e i n f o a b s t r a c t

31323334353637383940414243

Article history:Received 30 May 2014Received in revised form 1 July 2014Accepted 5 July 2014Available online xxxx

Keywords:Triatoma infestansrDNA variabilityC-heterochromatic polymorphismPyrethroid resistanceHybrid zoneHybridization

44454647484950515253545556

Chagas disease, one of the most important vector-borne diseases in the Americas, is caused by Trypano-soma cruzi and transmitted to humans by insects of the subfamily Triatominae. An effective control of thisdisease depends on elimination of vectors through spraying with insecticides. Genetic research can helpinsect control programs by identifying and characterizing vector populations. In southern Latin America,Triatoma infestans is the main vector and presents two distinct lineages, known as Andean andnon-Andean chromosomal groups, that are highly differentiated by the amount of heterochromatinand genome size. Analyses with nuclear and mitochondrial sequences are not conclusive about resolvingthe origin and spread of T. infestans.

The present paper includes the analyses of karyotypes, heterochromatin distribution and chromosomalmapping of the major ribosomal cluster (45S rDNA) to specimens throughout the distribution range ofthis species, including pyrethroid-resistant populations. A total of 417 specimens from seven differentcountries 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 geographicdistribution. Our results reveal that the differentiation process between both T. infestans chromosomalgroups has involved significant genomic reorganization of essential coding sequences, besides thechanges in heterochromatin and genomic size previously reported. The chromosomal markers alsoallowed us to detect the existence of a hybrid zone occupied by individuals derived from crosses betweenboth 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 likelythe result of recent secondary contact between both lineages. We suggest that vector control programsshould make a greater effort in the entomological surveillance of those regions with both chromosomalgroups to avoid rapid emergence of resistant individuals.

� 2014 Published by Elsevier B.V.

57

64

65

66

67

68

69

1. Introduction

Triatoma infestans (Hemiptera, Reduviidae, Triatominae) is animportant vector of the flagellate protozoan Trypanosoma cruzi,causative agent of Chagas disease or American trypanosomiasis.In the early 1990s, T. infestans had a wide geographical distribution

70

71

72

73

74

extending over more than 6 million km2, including parts of sevenSouth American countries, and was responsible for well over halfof the 18 million people affected by Chagas disease (WHO, 1991).Since then, control interventions in the framework of the‘‘Southern Cone Initiative’’ have substantially reduced the distribu-tion of T. infestans to less than 1 million km2 and 9.8 million peopleinfected (Schofield et al., 2006). At present, domestic T. infestansmainly persists in the Andean valleys of Bolivia, southern Peru,and parts of the Gran Chaco region of Argentina, Bolivia andParaguay. One of the challenges facing the control of T. infestansin these regions is the recent detection of pyrethroid resistance

osomal

http://dx.doi.org/10.1016/j.meegid.2014.07.006

mailto:[email protected]

mailto:[email protected]


http://www.sciencedirect.com/science/journal/15671348

http://www.elsevier.com/locate/meegid


75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90 Q2

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

2 F. Panzera et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx


15 July 2014

(Depickère et al., 2012; Lardeux et al., 2010; Picollo et al., 2005). T.infestans is known to comprise two main evolutionary lineages rec-ognized by molecular markers (nuclear and mitochondrialsequences) (Bargues et al., 2006; Giordano et al., 2005; Monteiroet al., 1999; Piccinali et al., 2009, 2011; Quisberth et al., 2011;Torres-Pérez et al., 2011) and phenetic characteristics (Calderón-Fernández et al., 2012; Hernández et al., 2008). These lineages,known as the Andean and non-Andean groups, are defined by sub-stantial differences (from 30% to 50%) in the amount of nuclearDNA due to different quantities of highly repeated DNA revealedas heterochromatin by C-banding (Panzera et al., 2004). Based onthe existence of sylvatic populations, two main hypotheses havebeen proposed to clarify the origin and spread of T. infestansthroughout South America. The ‘‘Andean’’ hypothesis suppose thatthe high-altitude valleys of Bolivia is the center of origin and dis-persal (Schofield et al., 1988; Usinger et al., 1966), while the‘‘Chaco’’ hypothesis assumes that the origin is the subtropical low-lands regions of southern Bolivia, north-western Paraguay andnorth of Argentina (Carcavallo et al., 1998). Several reports usingdifferent nuclear and mitochondrial sequences are not conclusiveabout resolving the origin and spread of T. infestans (for review seesTorres-Pérez et al., 2011). To better understand their chromosomalorganization and population differentiation, we analyzed the kary-otypes, heterochromatin distribution and chromosomal location ofthe major ribosomal cluster (45S rDNA) in specimens from allcountries of its original distribution, with emphasis on sylvaticand pyrethroid-resistant populations not previously studied. InTriatominae, these chromosomal markers are the main source ofkaryological variation in population differentiation and speciationprocesses (Panzera et al., 2010, 2012; Pita et al., 2013). We try torespond to the following questions: (i) what are the main karyolog-ical changes that occurred during the dispersal of T. infestans? and(ii) do the pyrethroid-resistant individuals detected along theArgentina-Bolivia border represent ancient or recent populations?Our findings provide further information about the origin andspread of T. infestans, including the origin of the pyrethroid resis-tance populations in non-Andean regions.

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

2. Methods

2.1. Material analyzed

All specimens of T. infestans came from natural populations andmost of them were collected within the framework of the projectSSA/ATU INCOCT 2004 515942 (Catalá et al., 2007). These speci-mens were also analyzed by cuticular hydrocarbon and antennalphenotypes (Calderón-Fernández et al., 2012; Hernández et al.,2008, respectively). The geographic origin of each population, yearof collection and habitat (domiciliary, peridomiciliary and sylvatic)are given in Table 1 and Fig. 1. It is noteworthy that in severalcollection sites here analyzed (Uruguay, Brazil, Chile and somefrom Argentina and Paraguay), currently there are no specimensof this species, being eliminated by vector control activities.Andean sylvatic specimens were mainly collected amongst rockpiles, while the ‘‘dark morphs’’ from the Bolivian Chaco (non-Andean region) were caught in hollow trees (Fig. 1, map reference29) (Noireau et al., 2000).

Since 2002, control services for Chagas disease from Argentinaand Bolivia reported low efficiency of deltamethrin and other pyre-throids for the treatment of rural sites close to Salvador Mazza(Salta Province, Argentina) and Yacuiba cities (Tarija Department,Bolivia) (Fig. 1, map references 17–28). Pyrethroid resistance ofinsects collected from Yacuiba (Tarija, Bolivia) and Salvador Mazza(Salta, Argentina) were determined with topical application bioas-says (WHO, 1994), by María Inés Picollo (Centro de Investigaciones

Please cite this article in press as: Panzera, F., et al. Evolutionary and dispersal hmarkers. Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.0

de Plagas e Insecticidas CITEFA-CONICET, Argentina), within theframework of the project SSA/ATU. Pyrethroid resistance in indi-viduals here analyzed from Salvador Mazza has also been experi-mentally confirmed by Cardozo et al. (2010). Furthermore severalreports using triatomine samples collected in this area since2002 showed high resistance levels, with resistance ratios (RRs)ranging from 50.5 to 183 (Lardeux et al., 2010; Picollo et al.,2005; Toloza et al., 2008).

2.2. Chromosome preparations and banding procedures

Gonads from adult insects were removed and fixed in ethanol–acetic acid (3:1). Chromosome squashes were prepared in 45% ace-tic acid. C-banding was used to establish the diploid chromosomenumber (2n) and the number of C-heterochromatic chromosomes(Panzera et al., 2004). We applied the fluorescence in situ hybrid-ization (FISH) to determine the chromosome location of the 45Sribosomal clusters (Pita et al., 2013).

For each specimen, at least 20 cells were analyzed to determinechromosome traits. Slides were examined under a Nikon Eclipse80i microscope and the images were obtained with a DS-5Mc-U2digital camera. In males, mitotic (prometaphase) and meiotic(metaphase I or II) plates were observed. In total, 425 T. infestansspecimens were examined by C-banding and 105 by FISHtechniques (Table 2, Figs. 2 and 3). Of the latter, 70% of them wereanalyzed by both techniques (data not shown).

3. Results

3.1. Chromosome number

All specimens showed the same normal diploid number(2n = 22) constituted by 20 autosomes and two sex chromosomes(XY in males, XX in females) (Fig. 2). However, in some individualswe observed the occurrence of chromosomal fragments or extra-chromosomes (Fig. 2E). These are the smallest of the chromosomecomplement, both euchromatic and heterochromatic, and theirfrequency varied from 1 to 3 amongst individuals. These chromo-somal fragments appear in gonial mitotic prometaphases, both inmales and females, but we did not detect any alteration in themeiotic segregation of carrier individuals. The frequencies of indi-viduals with chromosomal fragments were very variable, but thepopulations from Salvador Mazza showed the highest frequency(more than 80% of individuals) (Table 2).

3.2. Distribution of C-heterochromatin

Each specimen exhibited a specific C-banding pattern. Allpopulations were polymorphic, with variations in the number ofchromosomes with C-heterochromatin and in the chromosomeposition of C-blocks (one or both chromosomal ends). Allindividuals show a C-heterochromatic Y chromosome while the Xchromosome is euchromatic or heterochromatic. The number ofautosomes with C-heterochromatin differentiates three distinctgroups, two of them previously described (Panzera et al., 2004)and a third group here named as ‘‘Intermediate group’’ (Table 2and Fig. 2):

3.2.1. Andean groupIncludes 102 insects from Bolivia and Peru (Fig. 1, map refer-

ences 1–16). The number of heterochromatic autosomes withC-blocks varies from 14 to 20, with a mean of 15.64 and a standarddeviation (SD) of 1.35 (Table 2, Fig. 2A and B). All individuals haveX chromosome with C-heterochromatin.

istory of Triatoma infestans, main vector of Chagas disease, by chromosomal7.006


193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

Table 1Geographical 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 1997/20092 Peru Ica Nazca city, P 588 14� 500 2100 74� 560 25 2005/093 Bolivia La Paz Sapini, D-S 1880 16� 480 5500 67� 420 2100 20104 Bolivia La Paz Chivisini, S 2892 16� 570 5300 67� 520 5300 20085 Bolivia Cochabamba Cotapachi, D-P-S 2750 17� 250 2900 66� 150 5300 20046 Bolivia Cochabamba 20 de Octubre, S 2596 17� 290 0300 66� 060 4400 20087 Bolivia Cochabamba Tabacal, S 2182 17� 560 0100 65� 230 0600 20088 Bolivia Cochabamba Mataral, D-P-S 1700 18� 360 1100 65� 080 6000 2005/089 Bolivia Cochabamba JamachUma, D 2707 17� 300 5200 66� 050 1100 199710 Bolivia Chuquisaca Carapari, P 1892 18� 370 1800 65� 100 3000 200511 Bolivia Chuquisaca Uyuni, D-S 2542 19� 260 0000 64� 500 0700 1997/200812 Bolivia Potosí Thago Thago, P 2000 18� 000 4400 65� 480 3100 201013 Bolivia Potosí La Deseada, S 2921 21� 310 1900 65� 410 3400 200514 Bolivia Potosí Viscachani, S 3041 21� 360 4100 65� 480 2200 201015 Bolivia Potosí Palquiza, S 2908 21� 310 4100 65� 450 0400 201016 Bolivia Potosí Urulica, S 3129 21� 350 4000 65� 490 5600 201017 Bolivia Tarija Palmar Chico, D-P 610 21� 530 5000 63� 360 4500 200418 Bolivia Tarija El Barrial, D 387 22� 020 2200 63� 400 5400 200419 Bolivia Tarija Yaguacua, D 650 21� 420 0000 63� 360 0000 200520 Bolivia Tarija San Francisco del Inti, D-P 610 21� 490 4100 63� 360 3500 200521 Bolivia Tarija Estación Sunchal, D-P 558 21� 410 1700 63� 220 0100 200522 Bolivia Tarija Quebrada Busuy, D-P 387 21� 160 7500 63� 270 8700 200523 Bolivia Tarija San Antonio, D 429 21� 150 3900 63� 280 4700 200524 Bolivia Tarija Suarurito, D-P 853 21� 160 5600 63� 560 3300 200525 Bolivia Tarija Tacuarandy, P 958 21� 230 0300 63� 590 5700 200526 Argentina Salta Salvador Mazza, La Toma, D 834 22� 010 5600 63� 420 3100 200527 Argentina Salta S. Mazza, El Chorro, D-P 820 22� 010 3700 63� 410 1400 200528 Argentina Salta S. Mazza, El Obraje, D-P 840 22� 040 4100 63� 410 4700 200529 Bolivia Santa Cruz Tita, S 300 18� 340 3100 62� 400 0500 1997/200830 Bolivia Santa Cruz Tita, P 300 18� 800 0000 63� 120 6700 1997/200831 Argentina Salta La Unión, D 169 23� 450 3800 0 63� 080 1500 200532 Argentina Salta San Carlos, P 2088 25� 480 3300 65� 570 5300 200533 Argentina Chaco Pampa Ávila, D-P 107 26� 590 5000 61� 300 4400 200534 Argentina Chaco Tres Estacas, P 122 26� 540 3000 61� 400 2300 200535 Argentina Santiago del Estero Huachana, P 165 28� 130 2500 64� 070 1300 200536 Argentina Santiago del Estero Silípica, P 138 28� 060 1200 64� 070 0800 200537 Argentina La Rioja 4 Esquinas, P 320 31� 480 0300 65� 860 6600 200238 Argentina La Rioja San Antonio, P 326 29� 280 3300 66� 150 0000 200239 Argentina La Rioja Anillaco, P 1419 28� 800 0000 66� 950 0000 199740 Argentina Catamarca Palo Blanco, P 1828 27� 200 2600 67� 450 3500 200541 Argentina Formosa Villa Escobar, P 66 26� 610 6600 58� 660 6600 200142 Argentina Córdoba Los Leones, P 250 30� 430 1600 64� 480 3500 200043 Paraguay Boquerón Jericó, D 115 22� 350 4600 59� 480 4100 200544 Paraguay Boquerón Tiberia, D 115 22� 370 1700 59� 550 5000 200545 Paraguay Presidente Hayes Niya Toyish, D 126 22� 570 2700 59� 520 3200 200546 Paraguay Pres. Hayes Estancia Salazar, D 126 23� 040 4100 59� 160 5300 200547 Paraguay San Pedro San Pedro, D 213 24� 100 0000 57� 080 330 200548 Paraguay Cordillera San Bernardino, D 334 25� 180 2600 57� 170 4300 200549 Uruguay Rivera Several localities, D-P 0–250 30� 530 5500 55� 330 0100 1988–9550 Uruguay Soriano Several localities, P 0–250 30� 420 1500 57� 900 2000 1988–9551 Chile IV Region: Coquimbo Pulpica, P 740 30� 510 3000 70� 460 200 200252 Brazil Paraiba Monteiro, D 602 07� 530 2900 37� 070 0000 1997

F. Panzera et al. / Infection, Genetics and Evolution xxx (2014) xxx–xxx 3


15 July 2014

3.2.2. Non-Andean groupIncludes 238 specimens from Argentina, Bolivia (non-Andean

region), Brazil, Chile, Paraguay, and Uruguay (Fig. 1, map references29–52). The number of heterochromatic autosomes varies from 4to 6 (5.86 ± 0.52) (Table 2, Fig. 2C and D), but most individuals(86.2%) have six C-heterochromatic autosomes. In all individuals,the X chromosomes are euchromatic.

212

213

214

215

216

217

218

219

220

221

3.2.3. Intermediate groupRestricted to Argentina-Bolivia border, including 85 individuals

from localities from Salvador Mazza (Argentina) and Tarija (Boli-via) (Fig. 1, map references 17–28). The number of heterochro-matic autosomes varies from 7 to 11 (8.20 ± 1.08) (Table 2,Fig. 2E and F). In 72 insects (84.7%), the X chromosomes areeuchromatic (similar to non-Andean group), while in the remain-ing 13 are heterochromatic (similar to Andean group).


Considering the number of C-heterochromatic autosomes, sig-nificant statistical differences by Student t test were determinedbetween Andean and non-Andean groups (p < 0.001).

3.3. Chromosome location of 45S rDNA cluster

T. infestans showed marked variability between populations andbetween individuals, both in the number of rDNA loci and in thetype of chromosomes (autosomes and sex chromosomes) that car-ried the rDNA genes (Table 2 and Fig. 3). The number of rDNA lociper male haploid genome ranged from 1 to 4 sites. The 45S rDNAcluster is located on one sex chromosome (X chromosome) andeven on up to three autosomal pairs. Due to the similar size ofthe autosomes is very difficult to determine exactly which chromo-some pair presents the ribosomal cluster. However, one ribosomalsignal is always located in at least one of the four largest autosomal



222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

Table 2Chromosomal results in Triatoma infestans obtained with C-banding (number of autosomes with C-bands and % of chromosomal fragments) and by fluorescent in situhybridization (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 meanson 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 oneheterozygote 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 heterozygoteautosomal pairs plus X chromosome and X pattern means rDNA only on the X chromosome. SD, Standard Deviation. Between parentheses we included the number of individualsstudied by C-banding and FISH.

Map reference No. of C-autosomesmean ± SD

Chromosomal fragments(%)

rDNA pattern locationb

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

Group8.20 ± 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

(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 ND42 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 ND52 6.00 ± 0.00 (2) 0.0 X (1)b

Non-AndeanGroup

5.86 ± 0.52 (238) 4.4 % 28 individuals: all X pattern

a Partial C-banding data from Panzera et al. (2004).b Partial FISH data from Panzera et al. (2012).

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.



15 July 2014

pairs. In total, we observed nine ribosomal location patterns: onone autosomal pair (pattern 1A, Fig. 3A); on one autosomal pairplus X chromosome (1A + X, Fig. 3B); on two autosomal pairs(2A, Fig. 3C); on two autosomal pairs plus X (2A + X, Fig. 3D); ontwo autosomal pairs (with one heterozygote) plus X (1A + 1/2A + X, Fig. 3E); on one heterozygote autosomal pair plus X (1/2A + X, Fig. 3F); on two autosomal pairs (both of them heterozy-gotes) plus X (1/2 A + 1/2 A + X, Fig. 3G); on three heterozygote


autosomal pairs plus X chromosome (1/2 A + 1/2 A + 1/2 A + X,Fig. 3H) and only on the X chromosome (pattern X, Fig. 3I). In allcases, the hybridization signals were located at a terminal orsubterminal chromosome position.

Considering the three chromosomal groups previouslydescribed, the ribosomal patterns are associated with a particulargeographic distribution (Table 2). The Andean group is the mostvariable, with five patterns exclusively detected in this group



238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

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 infemales). (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 andindistinguishable for the euchromatic autosomes. In (D), three autosomal pairs are C-heterochromatic, while the Y chromosome appears C-positive and the X chromosomeeuchromatic. (E, F): Intermediate Group: Nine autosomes are C-heterochromatic, while that both sex chromosomes are also C-positive. Arrowhead pointed out a smalleuchromatic chromosomal fragment. Scale bar = 10 lm.



15 July 2014

(Fig. 3A–E). Two of them (patterns 1A and 1A + X) involved 90% ofthe individuals analyzed (48/55). Most of the collection sites arepolymorphic, being the populations from Cochabamba andChuquisaca the most variable (Table 2) and the populations fromLa Paz the most homogeneous.

The non-Andean group showed only one ribosomal pattern:rDNA loci located on the X chromosome (Fig. 3I and Table 2),involving 28 individuals from six countries, including sylvaticand peridomestic specimens from the Bolivian Chaco region(Fig. 1, map references 29–30).

The Intermediate group shows four ribosomal patterns in the 22individuals analyzed (Table 2). One of them is similar to thatobserved in the non-Andean group: on the X chromosome (10 indi-viduals)(Fig. 3I). The other three patterns are exclusively found inthis group: 1/2 A + X pattern in 10 individuals (Fig. 3F), and twopatterns showing 2 and 3 heterozygote autosomal pairs plus Xchromosome found in one individual for each one (Fig. 3G and H).

279

280

281

282

283

284

285

286

4. Discussion

4.1. Chromosome number

All individuals have 20 autosomes plus two sex chromosomes(2n = 22). However, some individuals have chromosomal frag-ments or extra-chromosomes (Table 2 and Fig. 2E). One distinctivecytogenetic feature of heteropteran insects, including Triatominae,is the holocentric or holokinetic nature of their chromosomes


(Hughes-Schrader and Schrader, 1961). Unlike what is observedin monocentric chromosomes, chromosomal fragments in holocen-tric systems attach to spindle fibres and migrate normally duringcell divisions, becoming what are known as B chromosomes(Mola and Papeschi, 2006). In true bugs however, B chromosomesseem very uncommon and have been detected in only 12 of the465 species so far studied (Kuznetsova et al., 2011). In Triatominae,chromosomal fragments usually occur as byproducts of a translo-cation among non-homologous autosomes, as has been observedin Mepraia gajardoi and T. infestans (Pérez et al., 2004; Poggioet al., 2013). In these mutant individuals, an autosomal trivalentand chromosomal fragments were observed both in mitosis andmeiosis. However, the individuals of T. infestans with chromosomalfragments here analyzed are quite different, as they show normalmeiotic segregation without autosomal associations. These chro-mosomal fragments may reflect the occurrence of small fissionsor the presence of unstable chromosome regions (fragile sites) thatare prone to breakage in mitotic metaphases. The frequency ofindividuals with chromosomal fragments varies greatly accordingto the populations studied (Table 2), and its appearance probablyis associated with populations resistant to pyrethroids (see belowIntermediate group and pyrethroid resistance).

4.2. Chromosome location of 45S rDNA genes

The striking intraspecific rDNA variability detected in T. infestansis unusual in insects, and exceptional in holocentric chromosomes.



287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

Fig. 3. Nine ribosomal patterns showing the intraspecific variability in the chromosome location of the 45S rDNA observed in first meiotic metaphases of Triatoma infestansdiscriminated 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 + 1/2A + 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 + 1/2A + 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 Xchromosome = X pattern. The autosomal pairs with heterozygotes signals are pointed out by arrows. Scale bar = 10 lm. A = autosome.



15 July 2014

Considering almost 100 heteropteran species analyzed to date, thechromosome position of the ribosomal genes appears to be aspecies-specific character. The number of rDNA loci described inT. infestans (from 1 to 4 sites) is very high in comparison with the1 or 2 loci found in most heteropteran species analyzed to date(Bardella et al., 2013; Panzera et al., 2012).

The existence of individuals with ribosomal genes located onfour different chromosomes (X chromosome and up to three auto-somal pairs) allows clear recognition of hybrid individuals revealedby the occurrence of heterozygous patterns for a particular locus(Fig. 3E–H). The high rDNA variation in T. infestans also suggeststhat some structural characteristic of their chromosomes promoteschanges in the number and position of ribosomal genes. The heter-ologous chromosome associations between several autosomes andsex chromosomes during cell divisions in T. infestans could favorthe high mobility of rDNA loci through of transposition and/orectopic recombination, which are the principal mechanisms sug-gested for rDNA changes in other organisms (Schubert andWobus, 1985).

4.3. Geographic distribution of Andean and non-Andean groups

The Andean group of T. infestans is distributed in high-altituderegions (above 1700 masl) of the Andean valleys of Bolivia andPeru, and also in lower altitudes such as the city of Nazca (588masl). The non-Andean group is found in lowland regions (0 to


1400 masl) in various countries including Bolivia (Chaco region)but also at higher altitudes in some localities of Argentina suchas Palo Blanco (1800 masl) or San Carlos (2088 masl) (Fig. 1). Thesedata confirm that Andean insects are able to colonize lowlands, andthat non-Andean insects would be able to colonize highlands, assuggested previously (Panzera et al., 2004). Thus, the occurrenceof a chromosomal group in a particular region may be associatedwith selection mechanisms linked to altitude as well as with thehistorical dispersal routes of each chromosomal group. Our chro-mosomal data from Peru and Chile are consistent with the pres-ence of the two lineages in Chile, as suggested by mitochondrialsequences (Torres-Pérez et al., 2011). The colonization of northernChile by the Andean group has probably also occurred from thecoastal regions of Peru, and not only through the Andes. By con-trast, we have not detected both lineages in Argentina, as has beensuggested by microsatellite markers (Pérez de Rosas et al., 2011).

4.4. Origin and dispersal of T. infestans in the light of chromosomalmarkers

Two main hypotheses have been proposed to explain the originand spread of T. infestans throughout South America. The ‘‘Andean’’hypothesis assumed that the Andean valleys of Bolivia representthe geographic area of the most ancient populations (Schofield,1988; Usinger et al., 1966), where sylvatic populations are foundamongst rock piles associated with wild rodents (Dujardin et al.,



335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464



15 July 2014

1987; Waleckx et al., 2011). This hypothesis also suggests that thedomiciliation of T. infestans began in this region, associated withthe domestication of wild guinea-pigs by pre-Columbian Andeancultures and was then spread in association with human migra-tions along two main routes: a north-western one towards Peruand then to north of Chile, and a southern route to Argentina, Chile,Paraguay, Brazil, and Uruguay (Schofield, 1988). The second theory,called ‘‘Chaco’’ hypothesis, assumed that T. infestans originated inthe dry subtropical woodlands of southern Bolivia, Paraguay, andnorth Argentina (Carcavallo, 1998). Several studies have confirmedthe presence of sylvatic populations in this region, usually occupy-ing hollow trees, probably associated with birds, and presentinglow or no infection with T. cruzi (Ceballos et al., 2009; Noireauet al., 2000; Rolón et al., 2011; Waleckx et al., 2011). Analyses withnuclear and mitochondrial sequences show that the sylvatic anddomestic populations from Andean-Bolivia and Chaco regionsshow similar values of haplotype diversity (Bargues et al., 2006;García et al., 2013; Piccinali et al., 2009, 2011; Quisberth et al.,2011; Waleckx et al., 2011). Furthermore, molecular phylogeo-graphic including coalescent analyses are not conclusive aboutresolving the Andean or Chaco origin of T. infestans (Torres-Pérezet al., 2011). However in this paper, the high rDNA diversityobserved in the Andean group compared to the monomorphicnon-Andean group strongly suggests that the Andean group isthe ancestral in T. infestans, with the non-Andean group being aderivative. Considering the two most common patterns of theAndean group (1A and 1A + X patterns in 90% of the individualsanalyzed), a single transposition event (from an autosome to Xchromosome) or autosomal loss of rDNA loci (in 1A + X pattern)could explain the X pattern in the non-Andean group. An ancestralorigin from non-Andean group (Chaco hypothesis) would have toimply several events of transposition and duplication of ribosomalgenes occurring simultaneously, which is highly unlikely.

In light of our chromosomal data, we support the hypothesisthat T. infestans originated in the Andean valleys of Bolivia, as a syl-vatic species with polymorphic populations in terms of highamounts of heterochromatic autosomes (14–20) and variable ribo-somal cluster positions. The most variable populations were thoseof Cochabamba and Chuquisaca, suggesting these as the originalregions. Interestingly, the populations from La Paz are the mosthomogeneous in their ribosomal patterns (Table 2), similar asobserved with cytochrome b sequences, suggesting a possibleancient isolation and bottleneck in this area (Waleckx et al.,2011). The emergence of the non-Andean group involved a deepgenomic rearrangement reflected in a reduction of heterochroma-tin to 3 autosomal pairs (and consequently in the overall genomesize), lost the C-heterochromatin in the X chromosome and a ribo-somal cluster situated only on the X chromosome. Molecular databased on nuclear and mitochondrial sequences suggests that thisdivergence occurred between 59,000 and 588,000 years ago(Bargues et al., 2006; Torres-Pérez et al., 2011) although this wouldbe long before human presence in these regions. Perhaps eachchromosome group would have independently undergone a pro-cess of diversification as sylvatic populations associated with dif-ferent habitats and hosts – the Andean group with rock piles androdents, and the non-Andean group associated with trees andbirds. Subsequent human influence could have led to multipleand independent domestication processes in both groups, as sug-gested by molecular markers (Waleckx et al., 2011). A ribosomaldivergence based on ecological preference can be tested becauseit predicts that sylvatic populations in non-Andean regions wouldhave ribosomal genes only on the X chromosome, as observed insylvatic specimens from Bolivian Chaco (Table 2). FISH analysesof sylvatic specimens from Argentinean Chaco with putative ances-tral mitochondrial haplotypes (Piccinali et al., 2011) may help toclarify this issue. A hypothesis of ancestral diversification of


sylvatic T. infestans associated with different habitats and hosts isin an agreement of differentiation of sensilla patterns observedbetween both chromosomal groups (Hernández et al., 2008).

4.5. The Intermediate chromosomal group

One of the most significant results of this study was the detec-tion of a third chromosome group (Intermediate), restricted to asmall lowland region about 70 km wide along the Argentina-Bolivia border (Fig. 1, map references 17–28). This Intermediatechromosomal group has three chromosome characteristics. First,the number of heterochromatic autosomes (7–11) is lower thanAndean group (14–20) but higher than non-Andean group (4–6).Second, the Intermediate group includes individuals both withand without heterochromatic X chromosome, as observed inAndean and non-Andean individuals. Third, about 50% of individu-als have an exclusive rDNA pattern (1/2 A + X). Considering thesethree chromosomal characteristics and Mendelian inheritance ofC-heterochromatin as both of the ribosomal genes, two opposinghypotheses can be suggested for the origin of the Intermediategroup.

The first hypothesis, that we called ‘‘ancestral origin’’, assumesthat the Intermediate group represents a middle step of gradualheterochromatin variance that occurred during the early diver-gence between Andean and non-Andean groups. The three chro-mosomal groups of T. infestans might reflect the ancestralprocesses of chromosomal differentiation, forming a north–southcline or gradient of heterochromatin. The current geographic dis-tribution of the Intermediate group along the Argentina-Boliviaborder would thus represent the original area where the Andeanand non-Andean groups diverged.

The alternative hypothesis, called ‘‘secondary contact’’, assumesthat this group originated recently from crosses between Andeanand non-Andean individuals, which were previously isolated. Fol-lowing divergence between the Andean and non-Andean groups,the two forms would have continued to differentiate in isolation– for example through adaptation to different environments. How-ever, when the two groups were subsequently brought backtogether, for example through anthropogenic activities such ashuman migration and domestic vector control, interbreedingbetween them would have given rise to the Intermediate group.This second hypothesis assumes that the Intermediate group isvery recent formation and not a relic of the ancient process ofdivergence between Andean and non-Andean groups.

The C-heterochromatin traits observed in the Intermediategroup, namely the number of heterochromatic autosomes andthe X chromosomes with and without C-bands, are consistent witheither hypothesis. The same statement was achieved by analysiswith antennal phenotypes (Hernández et al., 2008). However, thehigh frequency of the heterozygous rDNA pattern (1/2 A + X,Fig. 3F) exclusively detected in the Intermediate group can onlybe explained assuming the hypothesis of a secondary contact. Thispattern would be easily derived by crosses between non-Andeanindividuals (X pattern) with Andean individuals having the1A + X and 1A patterns (the two most frequently observed inAndean individuals). All F1 progeny would have the heterozygouspattern, and crossing between them would give offspring showingheterozygous and parental patterns. By contrast, it is very unlikelythat a population that includes a high frequency of heterozygotes(about 50%) could be maintained for long time, as the ancestralhypothesis presupposes. This high frequency strongly indicatesthese individuals are of recent origin (hypothesis of secondary con-tact) and suggests that Argentina-Bolivia border represents a veryrecent hybrid zone formed by crosses of two parental populations(Andean and non-Andean groups) generating intermediate or‘‘mixed’’ genotypes (with chromosomal characteristics of the two



465

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

558

559

560

561

562

563

564

565

566

567

568

569Q3

570

571

572

573

574

575

576

577

578

579

580

581

582

583

584

585



15 July 2014

main chromosome groups). Experimental crosses between thesegroups showed that the F1 progeny have intermediate amountsof heterochromatin (Panzera et al., 2004). Epidemiological datafrom vector control activities also support a recent origin of theIntermediate group (see below). In conclusion, our FISH datastrongly suggests that the Intermediate chromosomal groupdetected along the Argentina-Bolivia border is due to a recent sec-ondary contact between Andean and non-Andean groups, ratherthan an ancient variation resulting from north–south cline ofheterochromatin.

4.6. Intermediate group and pyrethroid resistance

Considering that the Intermediate group is situated at relativelylow altitudes (387–958 masl), it seems most likely to have beenoccupied by non-Andean individuals prior to secondary contactwith Andean populations. This assumption is supported by thehigh frequency of individuals with ribosomal X pattern (similarto non-Andean specimens). It thus seems likely that insects fromAndean regions moved to southern Bolivia, reaching the Argen-tina-Bolivia border, perhaps by passive human transport. Whenthese Andean individuals cross with non-Andean, producing Inter-mediate individuals resistant to pyrethroids, which was not previ-ously present in this region. Epidemiological information supportsthis scenario. Data provided by the Vector Control Program of SaltaProvince point out that, in the localities of Salvador Mazza, thespraying of houses with deltamethrin between 1995 and 1998led to very low house infestation rates (ranging between 0.5 and0.8%). This indicates that this region was then occupied by popula-tions susceptible to deltamethrin. Since 1998 however, and despitecontinued vector control activities, there has been a gradualincrease of insects in houses, reaching house infestation levels of50 to 80% in 2004. Similar control failures were reported at thesame time in the Bolivian city of Yacuiba, close to Salvador Mazza,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-guay and Argentina, clearly indicates that pyrethroid resistancewas very uncommon in non-Andean regions. By contrast, in theBolivian Andes, pyrethroid resistant populations appear to bemuch more frequent, being detected in several localities from thedepartments of Cochabamba, Chuquisaca, Sucre, and Potosí(Depickère et al., 2012; Lardeux et al., 2010; Roca Acevedo et al.,2011). This suggests that Andean populations have particulargenetic backgrounds that enable the development of resistanceto pyrethroid insecticides more rapidly than non-Andean popula-tions. Although, there are probably different mechanisms responsi-ble for resistance to pyrethroids in T. infestans (Germano et al.,2010), genetic analyses of experimental crosses between resistantand susceptible individuals showed that the inheritance of delta-methrin resistance is autosomal and with incomplete dominance,involving at least three genes (Cardozo et al., 2010). This inheri-tance fashion indicates that, under pyrethroid selective pressure,the resistant character may spread easily to susceptible insects.

A striking observation is the high frequency of chromosomefragments in the pyrethroid resistant populations from SalvadorMazza (Table 2). In other insects, chromosome rearrangementshave been suggested to be involved in insecticide resistance. InMyzus persicae, an aphid with holocentric chromosomes, an auto-somal translocation is responsible for organophosphate and carba-mate resistance by the amplification of esterase gene E4 due to aposition effect caused by the repositioning of heterochromatin(Blackman et al., 1978). A similar phenomenon may have occurredin T. infestans, where changes in position and amount ofheterochromatin are very frequent. It is likely that homologousrecombination between individuals with different amounts of


heterochromatin (Andean and non-Andean groups) may modifythe gene expression of euchromatic regions adjacent to the hetero-chromatin (position-effect variegation).

The emergence of resistant populations along the Argentina-Bolivia border supports a close relationship between the Interme-diate genomes and pyrethroid resistance. According to this idea,we can predict that nearby regions occupied by Andean and non-Andean groups, or non-Andean regions experiencing humanmigration from Andean regions (such as Gran Chaco region), wouldhave a greatest likelihood of developing pyrethroid resistance.Recently, several domestic populations of T. infestans from theBolivian Chaco (Izozog, Santa Cruz Department) appear to be theresult of a mixture of Andean and non-Andean groups probablygenerated by the passive transport of triatomines from the Andeanto the Gran Chaco region (Quisberth et al., 2011). AlthoughT. infestans populations from Santa Cruz Department are currentlyconsidered amongst the most susceptible to deltamethrin(Depickère et al., 2011), these ones could have a high probabilityof developing pyrethroid resistant in the near future.

5. Conclusions

This paper reveals that the differentiation process between thetwo T. infestans chromosomal groups has involved significant geno-mic reorganization of heterochromatin and essential codingsequences. Furthermore, the detection of pyrethroid-resistantinsects in a hybrid zone between both chromosomal groupssuggests a correlation between genomic variability and insecti-cide-resistant populations. Vector control programs should makea greater effort in hybrid-zones surveillance to avoid rapid emer-gence of resistant individuals. As an effective control of Chagas dis-ease depends on vector elimination, our study underscores theimportance of chromosomal and genomic studies to provide a bet-ter understanding of the dynamics of domestic insect population.

Authors’ contributions

F.P., R.P., Y.P.: Design, coordination and wrote the differentversions of the manuscript. F.P., M.J.F., S.P., I.F., L.C., Y.B., Y.G.,S.F.B., Y.P.: performed data and their analyses, helped to draftthe manuscript. F.P., Y.B., Y.G., S.F.B.: contributed to the triatominecollection. All authors read and approved the final version of themanuscript.

Uncited reference

Waleckx et al. (2012).

Acknowledgements

Partial financial supports were received from CSIC (Udelar) pro-jects, PEDECIBA and ANII from Uruguay and European Communityproject SSA/ATU INCO-CT 2004 515942. This study benefited frominternational collaboration of the European Community LatinAmerican Network for Research on the Biology and Control of Tri-atominae (ECLAT). We are particularly grateful for epidemiologicaldata provided by Alberto Gentile (Director General de Coordina-ción Epidemiológica, Ministerio de Salud Pública, Salta, Argentina),the coordination of European project by Antonieta Rojas de Arias(Paraguay) and Silvia Catalá (CRILAR, Argentina) and the kindlycooperation to elaborate the map by Gabriela Fernández (Facultyof Sciences, Uruguay). To Nidia Acosta, Pilar Alderete, Rubén Card-ozo, Alberto Gentile, Blanca Herrera, Abraham Jemio, François Noi-reau, Elsa López, Ximena Porcasi, David Rivera, Mirko Rojas andRaul Stariolo are thanked for their contributions to the collection



586

587

588

589

590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631632633634635636637638639640641642643644645646647648649650651652653654

655656657658



15 July 2014

of specimens. We dedicate this paper to the memory of our friendFrançois Noireau who contributed substantially to the understand-ing of the genetic and ecological variation of T. infestans.

659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728

References

Bardella, V.B., Fernandes, T., Vanzela, A.L.L., 2013. The conservation of number andlocation of 18S sites indicates the relative stability of rDNA in species ofPentatomomorpha (Heteroptera). Genome 56, 425–429.

Bargues, M.D., Klisiowicz, D.R., Panzera, F., Noireau, F., Marcilla, A., Pérez, R., Rojas,M.G., O’Connor, J.E., González-Candelas, F., Galvão, C., Jurberg, J., Carcavallo,R.U., Dujardin, J.P., Mas-Coma, S., 2006. Origin and phylogeography of theChagas disease main vector Triatoma infestans based on nuclear rDNAsequences and genome size. Infect. Genet. Evol. 6, 46–62.

Blackman, R.L., Takada, H., Kawakami, K., 1978. Chromosomal rearrangementinvolved in insecticide resistance of Myzus persicae. Nature 271, 450–452.

Calderón-Fernández, G.M., Girotti, J.R., Juárez, M.P., 2012. Cuticular hydrocarbonpattern as a chemotaxonomy marker to assess intraspecific variability inTriatoma infestans, a major vector of Chagas’ disease. Med. Vet. Entomol. 26,201–219.

Carcavallo, R.U., 1998. Phylogeny of triatominae. The Triatoma infestans complex.Mem. Inst. Oswaldo Cruz 93, 68–70.

Cardozo, R.M., Panzera, F., Gentile, A.G., Segura, M.A., Pérez, R., Díaz, R.A., Basombrío,M.A., 2010. Inheritance of resistance to pyrethroids in Triatoma infestans, themain Chagas disease vector in South America. Infect. Genet. Evol. 10, 1174–1178.

Catalá, S., Gorla, D., Panzera, F., Juárez, P., Picollo, M.I., Noireau, F., Dujardin, J.P.,et al., 2007. Analytical appraisal, Southern Cone – biological and environmentalcauses of the spatial structuration in Triatoma infestans and the implications forvector control programmes. In: Proceedings SSA/EC American TrypanosomiasisUpdate Workshop, Asunción, Paraguay, pp. 13-18.

Ceballos, L.A., Piccinali, R.V., Berkunsky, I., Kitron, U., Gürtler, R.E., 2009. First findingof melanic sylvatic Triatoma infestans (Hemiptera: Reduviidae) colonies in theArgentine Chaco. J. Med. Entomol. 46, 1195–1202.

Depickère, S., Buitrago, R., Siñani, E., Baune, M., Monje, M., Lopez, R., Waleckx, E.,Chavez, T., Brenière, S.F., 2012. Susceptibility and resistance to deltamethrin ofwild and domestic populations of Triatoma infestans (Reduviidae: Triatominae)in Bolivia: new discoveries. Mem. Inst. Oswaldo Cruz 107, 1042–1047.

Dujardin, J.P., Tibayrenc, M., Venegas, E., Maldonado, L., Desjeux, P., Ayala, F.J., 1987.Isozyme evidence of lack of speciation between wild and domestic Triatomainfestans (Heteroptera, Reduviidae) in Bolivia. J. Med. Entomol. 24, 40–45.

García, B.A., Pérez de Rosas, A.R., Blariza, M.J., Grosso, C.G., Fernández, C.J., Stroppa,M.M., 2013. Molecular population genetics and evolution of the Chagas’ diseasevector Triatoma infestans (Hemiptera: Reduviidae). Curr. Genomics 14, 316–323.

Germano, M.D., Roca Acevedo, G., Mougabure Cueto, G.A., Toloza, A.C., Vassena, C.V.,Picollo, M.I., 2010. New findings of insecticide resistance in Triatoma infestans(Heteroptera: Reduviidae) from the Gran Chaco. J. Med. Entomol. 47, 1077–1081.

Giordano, R., Cortez, J.C.P., Paulk, S., Stevens, L., 2005. Genetic diversity of Triatomainfestans (Hemiptera: Reduviidae) in Chuquisaca, Bolivia based on themitochondrial cytochrome b gene. Mem. Inst. Oswaldo Cruz 100, 753–760.

Hernández, L., Abrahan, L., Moreno, M., Gorla, D., Catalá, S., 2008. Phenotypicvariability associated to genomic changes in the main vector of Chagas diseasein the southern cone of South America. Acta Trop. 106, 60–67.

Hughes-Schrader, S., Schrader, F., 1961. The kinetochore of the Hemiptera.Chromosoma 12, 327–350.

Kuznetsova, V.G., Grozeva, S.M., Nokkala, S., Nokkala, C., 2011. Cytogenetics of thetrue bug infraorder Cimicomorpha (Hemiptera, Heteroptera): a review. ZooKeys154, 31–70.

Lardeux, F., Depickère, S., Duchon, S., Chavez, T., 2010. Insecticide resistance ofTriatoma infestans (Hemiptera, Reduviidae) vector of Chagas disease in Bolivia.Trop. Med. Int. Health 15, 1037–1048.

Mola, L.M., Papeschi, A.G., 2006. Holokinetic chromosomes at a glance. BAG 17, 17–33.

Monteiro, F.A., Pérez, R., Panzera, F., Dujardin, J.P., Galvão, C., Dayse Rocha, D.,Noireau, F., Schofield, C., Beard, C.B., 1999. Mitochondrial DNA variation ofTriatoma infestans populations and its implication on the specific status of T.melanosoma. Mem. Inst. Oswaldo Cruz 94, 229–238.

Noireau, F., Flores, R., Gutierrez, T., Abad-Franch, F., Flores, E., Vargas, F., 2000.Natural ecotopes of Triatoma infestans dark morph and other sylvatictriatomines in the Bolivian Chaco. Trans. R. Soc. Trop. Med. Hyg. 94, 23–27.

729


Panzera, F., Dujardin, J.P., Nicolini, P., Caraccio, M.N., Rose, V., Tellez, T., Bermúdez,H., Bargues, M.D., Mas-Coma, S., O’Connor, J.E., Pérez, R., 2004. Genomic changesof Chagas disease vector, South America. Emerg. Infect. Dis. 10, 438–446.

Panzera, F., Pérez, R., Panzera, Y., Ferrandis, I., Ferreiro, M.J., Calleros, L., 2010.Cytogenetics and genome evolution in the subfamily Triatominae (Hemiptera,Reduviidae). Cytogenet. Genome Res. 128, 77–87.

Panzera, Y., Pita, S., Ferreiro, M.J., Ferrandis, I., Lages, C., Pérez, R., Silva, A.E., Guerra,M., Panzera, F., 2012. High dynamics of rDNA cluster location in kissing bugholocentric chromosomes (Triatominae, Heteroptera). Cytogenet. Genome Res.138, 56–67.

Pérez, R., Calleros, L., Rose, V., Lorca, M., Panzera, F., 2004. Cytogenetic studies inMepraia gajardoi (Hemiptera, Reduviidae). Chromosome behavior in aspontaneous translocation mutant. Eur. J. Entomol. 101, 211–218.

Pérez de Rosas, A.R., Segura, E.L., García, B.A., 2011. Molecular phylogeography ofthe Chagas’ disease vector Triatoma infestans in Argentina. Heredity 107, 71–79.

Piccinali, R.V., Marcet, P.L., Noireau, F., Kitron, U., Gürtler, R.E., Dotson, E.M., 2009.Molecular population genetics and phylogeography of the Chagas diseasevector Triatoma infestans in South America. J. Med. Entomol. 46, 796–809.

Piccinali, R.V., Marcet, P.L., Ceballos, L.A., Kitron, U., Gürtler, R.E., Dotson, E.M., 2011.Genetic variability, phylogenetic relationships and gene flow in Triatomainfestans dark morphs from the Argentinean Chaco. Infect. Genet. Evol. 11,895–903.

Picollo, M.I., Vassena, C., Santo Orihuela, P., Barrios, S., Zaidemberg, M., Zerba, E.,2005. High resistance to pyrethroid insecticides associated with ineffective fieldtreatments in Triatoma infestans (Hemiptera: Reduviidae) from NorthernArgentina. J. Med. Entomol. 42, 637–642.

Pita, S., Panzera, F., Ferrandis, I., Galvão, C., Gómez-Palacio, A., Panzera, Y., 2013.Chromosomal divergence and evolutionary inferences in Rhodniini based onthe chromosomal location of ribosomal genes. Mem. Inst. Oswaldo Cruz 108,376–382.

Poggio, M.G., Gaspe, M.S., Papeschi, A.G., Bressa, M.J., 2013. Cytogenetic study in amutant of Triatoma infestans (Hemiptera: Reduviidae) carrying a spontaneousautosomal fusion and an extra chromosome. 2013. Cytogenet. Genome Res. 139,44–51.

Quisberth, S., Waleckx, E., Monje, M., Chang, B., Noireau, F., Brenière, S.F., 2011.‘‘Andean’’ and ‘‘non-Andean’’ ITS-2 and mtCytB haplotypes of Triatoma infestansare observed in the Gran Chaco (Bolivia): population genetics and the origin ofreinfestation. Infect. Genet. Evol. 11, 1006–1014.

Roca Acevedo, G., Cueto, G.M., Germano, M., Santo Orihuela, P., Rojas Cortez, M.,Noireau, F., Picollo, M.I., Vassena, C., 2011. Susceptibility of sylvatic Triatomainfestans from Andean valleys of Bolivia to deltamethrin and fipronil. J. Med.Entomol. 48, 828–835.

Rolón, M., Vega, M.C., Román, F., Gómez, A., Rojas de Arias, A., 2011. First report ofcolonies of sylvatic Triatoma infestans (Hemiptera: Reduviidae) in theParaguayan Chaco, using a trained dog. PLoS Negl. Trop. Dis. 5 (5), e1026.

Schofield, C.J., 1988. Biosystematics of the Triatominae. In: Service, M.W. (Ed.),Biosystematic of Haematophagous Insects, vol. 37. Clarendon Press, Oxford, pp.284–312.

Schofield, C.J., Jannin, J., Salvatella, R., 2006. The future of Chagas disease control.Trends Parasitol. 22, 583–588.

Schubert, I., Wobus, U., 1985. In situ hybridization confirms jumping nucleolusorganizing regions in Allium. Chromosoma 92, 143–148.

Toloza, A.C., Germano, M., Cueto, G.M., Vassena, C., Zerba, E., Picollo, M.I., 2008.Differential patterns of insecticide resistance in eggs and first instars ofTriatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia. J. Med.Entomol. 45, 421–426.

Torres-Pérez, F., Acuña-Retamar, M., Cook, J.A., Bacigalupo, A., García, A., Cattan, P.E.,2011. Statistical phylogeography of Chagas disease vector Triatoma infestans:testing biogeographic hypotheses of dispersal. Infect. Genet. Evol. 11, 167–174.

Usinger, R.L., Wygodzinsky, P., Ryckman, R.E., 1966. The biosystematics ofTriatominae. Annu. Rev. Entomol. 11, 309–330.

Waleckx, E., Salas, R., Huamán, N., Buitrago, R., Bosseno, M.F., Aliaga, C., Barnabé, C.,Rodriguez, R., Zoveda, F., Monje, M., Baune, M., Quisberth, S., Villena, E., Kengne,P., Noireau, F., Brenière, S.F., 2011. New insights on the Chagas disease mainvector Triatoma infestans (Reduviidae, Triatominae) brought by the geneticanalysis of Bolivian sylvatic populations. Infect. Genet. Evol. 11, 1045–1057.

Waleckx, E., Depickère, S., Salas, R., Aliaga, C., Monje, M., Calle, H., Buitrago, R.,Noireau, F., Brenière, S.F., 2012. New discoveries of sylvatic Triatoma infestans(Hemiptera: Reduviidae) throughout the Bolivian Chaco. Am. J. Trop. Med. Hyg.86, 455–458.

WHO (World Health Organization), 1991. Control of Chagas disease. TechnicalReport. Series No. 811, Geneva. Switzerland.

WHO (World Health Organization), 1994. Protocolo de evaluación de efectoinsecticida sobre triatominos. Acta Toxicol. Argent. 2, 29–32.



evolutionary and dispersal history of triatoma infestans, main vector of chagas disease, by...

Documents