www.elsevier.com/locate/meegid

Infection, Genetics and Evolution 5 (2005) 123–129

Molecular characterisation of Trypanosoma rangeli strains isolated

from Rhodnius ecuadoriensis in Peru, R. colombiensis in

Colombia and R. pallescens in Panama, supports a

co-evolutionary association between parasites and vectors

D.A. Urreaa, J.C. Carranzaa, C.A. Cuba Cubab, R. Gurgel-Goncalvesb,F. Guhlc, C.J. Schofieldd, O. Trianae, G.A. Vallejoa,*

aLaboratorio de Investigaciones en Parasitologıa Tropical, Facultad de Ciencias, Universidad del Tolima, A.A. No. 546, Ibague, ColombiabUnidade de Parasitologia Medica e Biologıa de Vetores, Fac. Medicina, Universidade de Brasilia, Brasilia, DF, CEP 70910-900, Brasil

cCentro de Investigaciones en Microbiologıa y Parasitologıa Tropical (CIMPAT), Universidad de los Andes, Bogota, ColombiadECLAT, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1 E7HT, UK

eInstituto de Biologıa, Facultad de Ciencias Exactas y Naturales, Universidad de Antioquia, Medellın, Colombia

Received 8 May 2004; received in revised form 10 July 2004; accepted 17 July 2004

Available online 1 October 2004

Abstract

We present data on the molecular characterisation of strains of Trypanosoma rangeli isolated from naturally infected Rhodnius

ecuadoriensis in Peru, from Rhodnius colombiensis, Rhodnius pallescens and Rhodnius prolixus in Colombia, and from Rhodnius pallescens

in Panama. Strain characterisation involved a duplex PCR with S35/S36/KP1L primers. Mini-exon gene analysis was also carried out using

TrINT-1/TrINT-2 oligonucleotides. kDNA and mini-exon amplification indicated dimorphism within both DNA sequences: (i) KP1, KP2 and

KP3 or (ii) KP2 and KP3 products for kDNA, and 380 bp or 340 bp products for the mini-exon. All T. rangeli strains isolated from R. prolixus

presented KP1, KP2 and KP3 products with the 340 bp mini-exon product. By contrast, all T. rangeli strains isolated from R. ecuadoriensis, R.

pallescens and R. colombiensis, presented profiles with KP2 and KP3 kDNA products and the 380 bp mini-exon product. Combined with

other studies, these results provide evidence of co-evolution of T. rangeli strains associated with different Rhodnius species groups east and

west of the Andean mountains.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Trypanosoma rangeli; kDNA; Mini-exon; Molecular characterization; Rhodnius colombiensis; Rhodnius ecuadoriensis; Rhodnius pallescens;

Rhodnius prolixus; Co-evolution

1. Introduction

Trypanosoma rangeli (Tejera) is a protozoan flagellate

frequently found infecting wild and domestic vertebrates,

including humans, in several Latin American countries. It

seems non-pathogenic for the vertebrate host, in contrast to

the related Trypanosoma cruzi—the causative agent of

Abbreviations: kDNA, kinetoplast DNA; PCR, polymerase chain reac-

tion; RAPD, random amplified polymorphic DNA

* Corresponding author. Fax: +57 8 2669176.

E-mail address: gvallejo@ibague.cetcol.net.co (G.A. Vallejo).

1567-1348/$ – see front matter # 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.meegid.2004.07.005

Chagas disease. Both are transmitted by blood-sucking

Triatominae (Hemiptera, Reduviidae) and in some areas of

Latin America the two trypanosome species can produce

mixed infections in the insect vectors or in the vertebrate

hosts (Vallejo et al., 1988). Serological cross-reactions

between the two species can complicate specific diagnosis of

Chagasic infection (Guhl and Marinkelle, 1982; Guhl et al.,

1985, 1987), so that the biological and epidemiological

characteristics of T. rangeli are generally studied within the

context of the biology and epidemiology of T. cruzi. Most

species of Triatominae seem able to transmit T. cruzi. By

contrast, T. rangeli is mainly transmitted by species of

D.A. Urrea et al. / Infection, Genetics and Evolution 5 (2005) 123–129124

Rhodnius, although infections have occasionally been

reported in Triatoma dimidiata and Panstrongylus megistus

(Marinkelle, 1968a; Steindel et al., 1994).

Several methods for the biological, biochemical and

molecular characterisation and differentiation of T. cruzi and

T. rangeli have been developed (D’Alessandro, 1976;

Macedo et al., 1993; Steindel et al., 1994; Grisard et al.,

1999a; Vargas et al., 2000; Guhl et al., 2002; Morales et al.,

2002; Amaya et al., 2003; Chiurillo et al., 2003). A duplex

PCR reaction using three primers (S35/S36/KP1L) has also

been designed allowing amplification from mini-circles

having one conserved region (denoted as KP1), two

conserved regions (denoted as KP2) and four conserved

regions (denoted as KP3) (Vallejo et al., 1999, 2002, 2003).

Strains isolated from Rhodnius prolixus presented KP1, KP2

and KP3 mini-circle amplification products and were

denoted as T. rangeli KP1(+). On the other hand, strains

isolated from Rhodnius colombiensis, Rhodnius pallescens

or P. megistus presented amplification products derived from

KP2 and KP3 mini-circles (but not from KP1) and were

denoted as T. rangeli KP1(�).

The molecular characterisation of 18 T. rangeli strains

isolated from the salivary glands of naturally infected

R. colombiensis, R. pallescens and R. prolixus has been

recently reported, using two independent sets of molecular

markers. kDNA and mini-exon amplification indicated

dimorphism within both DNA sequences: KP1, KP2 and

KP3, or KP2 and KP3 products for kDNA mini-circles and

380 bp or 340 bp products for the mini-exon. One of two

associations was observed within individual strains: KP1,

KP2 and KP3 kDNA products with the 340 bp mini-exon

product, or the KP2 and KP3 kDNA products with the

380 bp mini-exon product. These independent mitochon-

drial and nuclear molecular markers have shown T. rangeli

clearly divided into two major phylogenetic groups

associated with specific vectors in Colombia and in other

Latin-American countries (Vallejo et al., 2003). These

results support either clonal evolution or speciation in

T. rangeli populations, possibly derived as a secondary

adaptation to their parasitic condition in triatomine vectors.

The phylogenetic groupings of T. rangeli strains may

relate to particular adaptations or historical associations with

particular species of Rhodnius. Available evidence suggests

that Rhodnius species have radiated from ancestral forms

similar to R. pictipes in the Amazon region, leading to three

main adaptive lines: (1) forest forms (pictipes, stali, brethesi,

paraensis, dallesandroi), (2) the ‘prolixus’ group, mainly in

savanna-like and woodland areas east of the Andean

mountains (prolixus, robustus, neglectus, nasutus, domes-

ticus), and (3) the ‘pallescens’ group forming a north–south

cline west of the Andes (pallescens, colombiensis, ecuador-

iensis) (Chavez et al., 1999; Dujardin et al., 1999; Schofield

and Dujardin, 1999). To test this idea of association between

parasite strain and vector groupings, we report here the

characterisation of T. rangeli strains isolated from naturally

infected R. ecuadoriensis from Peru, and compare these

with strains isolated in Colombia from R. colombiensis,

R. pallescens, and R. prolixus, and with an additional strain

isolated from R. pallescens in Panama.

2. Materials and methods

2.1. Parasites and DNA extraction

Eleven T. rangeli strains isolated from the salivary glands

of naturally infected R. ecuadoriensis, R. colombiensis,

R. pallescens and R. prolixus captured in Peru, Colombia

and Panama were used in this study (Fig. 1). Metacyclic

trypomastigotes from the salivary glands were intraperito-

neally inoculated into ICR mice. Haemoculture was done

from positive mice 5 days post-inoculation. All strains were

maintained at 28 8C in NNN medium with LIT supple-

mented with 10% heat-inactivated foetal bovine serum

through weekly passages. Genomic DNA was extracted

from cultured parasites by the phenol chloroform method

and precipitation with ethanol and sodium acetate. DNA

concentration was determined by electrophoresis for

comparison with quantification standards.

2.2. kDNA PCR amplification

A duplex PCR was used, according to Vallejo et al.

(2002). Amplification was performed at 10 ml final volume,

containing 1 ml 10� Taq polymerase reaction buffer

(GIBCO-BRL), 200 mM dNTPs mixture, 3.5 mM MgCl2,

25 mM KCl, 25 mM of primers S35: (50 AAA TAA TGT

ACG GGT GGA GAT GCA TGA 30); S36: (50 GGG TTC

GAT TGG GGT TGG TGT 30); KP1L: (50 ATA CAA CAC

TCT CTA TAT CAG G 30) and 0.5 units of Taq DNA

polymerase (GIBCO-BRL). The PCR were run in a DNA

Thermal Minicycler (M.J. Research PTC-150-16) and

subjected to 35 amplification cycles. The temperature

profiles for denaturing, primer annealing and primer

extension were 95 8C for 1 min (with a longer initial time

of 5 min), 60 8C for 1 min and 72 8C for 1 min, respectively,

and a final extension for 5 min. The reaction products were

electrophoresed in 6% polyacrylamide gels and stained with

silver nitrate (Sanguinetti et al., 1994).

2.3. Mini-exon gene intergenic region PCR amplification

T. rangeli mini-exon gene PCR was performed using

TrINT-1 and TrINT-2 oligonucleotides, according to Grisard

et al., 1999b. Amplifications were performed in a 10 ml final

volume, containing the following reagents: PCR buffer

(10 mM Tris–HCl, pH 8.0), 50 mM KCl, 1.5 mM MgCl2,

200 mM dNTPs; 10 pmol of each oligonucleotide TrINT-1:

(50 CGC CCA TTC GTT TGT CC 30); TrINT-2: (50 TCC

AGC GCC ATC ACT GAT C 30) and 1 U Taq DNA

polymerase. The following temperature profile was used:

95 8C/5 min, 60 8C/1 min, 72 8C 1 min, 29 cycles of 95 8C/

D.A. Urrea et al. / Infection, Genetics and Evolution 5 (2005) 123–129 125

Fig. 1. Proposed evolutionary lines of Rhodnius species from the Amazon forest region radiating out to the north into the llanos of Venezuela, to the northwest

into northern Colombia and to the Magdalena valley of Central Colombia and eastern Ecuador and Peru (adapted from Schofield and Dujardin, 1999). The map

shows the geographical origins of Rhodnius species where T. rangeli strains were isolated and characterised in this study: (*) R. ecuadoriensis (La Libertad

Department, Peru); (~) R. colombiensis (Tolima Department, Colombia); ( ) R. pallescens (Panama); ( ) R. pallescens (Antioquia and Sucre Departments,

Colombia); ( ) R. prolixus (Boyaca Department, Colombia).

1 min and 60 8C/1 min and a final 72 8C incubation for

10 min. The reaction products were electrophoresed in 2%

agarose gel and stained with ethidium bromide.

3. Results

The two kDNA mini-circle amplification profiles showed

that one group of T. rangeli strains, denoted as T. rangeli

KP1(+), showed a 760 bp fragment obtained with S35 and

S36 primers from mini-circles having two conserved regions

(KP2), a complex of bands, varying between 300 and

450 bp, obtained with S35 and S36 primers from mini-

circles having four conserved regions (KP3) and a 165 bp

fragment obtained with KP1L and S36 primers from the

mini-circle having one conserved region (KP1). A second

group of T. rangeli strains, denoted as T. rangeli KP1(�),

showed a similar kDNA amplification profile but without the

165 bp fragment. For both groups, the 760 bp product

showed variable intensity—very weak in some strains but

clearly strong in others—presumably due to a variable

proportion of these mini-circles in the kDNA network of

different strains.

T. rangeli strains isolated from R. prolixus showed only

the KP1(+) profile, whereas all strains isolated from R.

ecuadoriensis, R. colombiensis and R. pallescens showed the

KP1(�) profile (Fig. 2). PCR with TrINT-1/TrINT-2 primers

revealed similar dimorphism within those strains analysed.

A 340 bp mini-exon product was obtained in strains isolated

from R. prolixus, whereas a 380 bp mini-exon product

was obtained in strains isolated from R. ecuadoriensis,

R. colombiensis and R. pallescens (Fig. 3).

In summary, only one of two molecular associations was

observed for each T. rangeli strain: KP1, KP2 and KP3

kDNA products together with the 340 bp mini-exon product,

or KP2 and KP3 kDNA products together with the 380 bp

mini-exon product. All strains isolated from R. ecuador-

iensis, R. colombiensis and R. pallescens presented the

characteristic T. rangeli KP1(�) molecular profile, while

strains isolated from R. prolixus presented the T. rangeli

KP1(+) molecular profile. In addition, a T. rangeli strain

isolated from a human living in an area infested by

R. prolixus, also showed the KP1(+) profile. (Table 1).

4. Discussion

There is now strong evidence of dimorphism within

T. rangeli strains from different geographical origins, based

on DNA fingerprinting (Macedo et al., 1993), isoenzyme and

random amplified polymorphic DNA (RAPD) assays

(Steindel et al., 1994), molecular karyotypes (Toaldo

et al., 2001) and, as in the present work, mini-exon gene

characterisation (Grisard et al., 1999b) and kDNA ampli-

fication (Vallejo et al., 2002, 2003). These two forms of T.

rangeli have been denoted as KP1(+) and KP1(�) based on

D.A. Urrea et al. / Infection, Genetics and Evolution 5 (2005) 123–129126

Fig. 2. Silver stained 6% polyacrylamide gel containing polymerase chain reaction products with S36/S35/KP1L primers. Lane M corresponds to 100 bp ladder

(GIBCO-BRL). Lanes 1–3 correspond to T. rangeli 1545, 444 and 3123 strains isolated from R. prolixus (Colombia). Lanes 4–6 correspond to TrS, Rcol, P53

strains isolated from R. colombiensis (Colombia). Lanes 7–9 correspond to Peru 1, Peru 2 and Peru 1A strains isolated from R. ecuadoriensis (Peru) and lane 10

corresponds to Panama strain isolated from R. pallescens.

the clarity and consistency of differentiation by kDNA

amplification.

Including the present study, we have now analysed 20

strains of T. rangeli isolated from R. prolixus, together with

one strain isolated from a human living in an area infested

with R. prolixus. All consistently showed the KP1(+) profile.

Similarly, strains of T. rangeli recently isolated from

R. neglectus (and its associated vertebrate host, Didelphis

albiventris) captured in the state of Minas Gerais and in the

Federal District of Brazil have also all been characterised as

KP1(+) (Marquez DS, Lages-Silva F, Ramırez LE, 2002.

Annual Meeting on Basic Research in Chagas Disease. Rev

Fig. 3. Ethidium bromide staining of agarose gel containing the 340 and 380 bp PC

444, 2123 and Duran strains isolated from R. prolixus (Colombia); lanes 5–7 corres

lanes 8–10 correspond to Peru 1, Peru 2 and Peru 1A strains isolated from R. ec

pallescens. Lane M corresponds to 1 Kb ladder plus (GIBCO-BRL).

do Inst Med Trop Sao Paulo 44 (Suppl. 12), 55 and Gurgel-

Goncalves R, 2003. Distribuicao espacial de populacoes

silvestres de Triatominae (Hemiptera, Reduviidae) e

circulacao enzootica de Trypanosoma cruzi e T. rangeli

no Distrito Federal, Brasil. Dissertacao de Mestrado.

Universidade de Brasılia, Brasılia, Distrito Federal. 141p.).

In comparison, we have now analysed a total of 12 strains

of T. rangeli isolated from R. pallescens in Panama and

northern Colombia (Sucre and Antioquia), together with 25

strains isolated from R. colombiensis in central Colombia

(Tolima), and three strains isolated from R. ecuadoriensis in

northern Peru (La Libertad). All presented the KP1(�)

R products amplified from T. rangeli isolates. Lanes 1–4 correspond to 1545,

pond to TrS, Rcol and P53 strains isolated from R. colombiensis (Colombia);

uadoriensis (Peru); lane 11 corresponds to Panama strain isolated from R.

D.A. Urrea et al. / Infection, Genetics and Evolution 5 (2005) 123–129 127

Table 1

T. rangeli isolates used for molecular characterisation according to kDNA mini-circle organisation and mini-exon gene polymorphism

Strains Host Geography/ecotype Mini-circle organisation Mini-exon product (bp

Peru 1 R. ecuadoriensis Peru, La Libertad Dpt/domestic KP2, KP3 380

Peru 1A R. ecuadoriensis Peru, La Libertad Dpt/domestic KP2, KP3 380

Peru 2A R. ecuadoriensis Peru, La Libertad Dpt/domestic KP2, KP3 380

P53 R. colombiensis Colombia, Tolima Dpt/sylvatic KP2, KP3 380

Trs R. colombiensis Colombia, Tolima Dpt/sylvatic KP2, KP3 380

Rcol R. colombiensis Colombia, Tolima Dpt/sylvatic KP2, KP3 380

Panamaa R. pallescens Panama/peridomestic KP2, KP3 380

444 R. prolixus Colombia, Boyaca Dpt/domestic KP1, KP2, KP3 340

1545 R. prolixus Colombia, Boyaca Dpt/domestic KP1, KP2, KP3 340

3123 R. prolixus Colombia, Boyaca Dpt/domestic KP1, KP2, KP3 340

Duran Human Colombia, Boyaca Dpt/domestic KP1, KP2, KP3 340

a From the Cryobank of London School of Tropical Medicine and Hygiene.

profile (Vallejo et al., 2002, 2003, and present work). There

thus appears to be a consistent association of T. rangeli

KP1(+) with Rhodnius species of the prolixus group

(represented in these studies by prolixus and neglectus),

and a similarly consistent association of T. rangeli KP1(�)

with Rhodnius species of the pallescens group (pallescens,

colombiensis and ecuadoriensis).

Under current interpretations (Schofield and Dujardin,

1999) the 15 described species of Rhodnius are believed to

be monophyletic, originating from a form today represented

by R. pictipes in the Amazon-Orinoco forest. R. pictipes is a

widespread generalist species occupying a range of forest

habitats, and shares morphological characteristics with other

Triatominae that are not found in other species of Rhodnius

(except in the closely-related R. stali). There appear to have

been three main evolutionary lines from this putative

ancestral form, to give more specialised forest forms (such

as paraensis and brethesi), and to give two main groups that

are largely found in regions outside the Amazon forest itself.

One of these is through the northern and southern forms of

R. robustus (type I, and types II, III, and IV, respectively, in

the genotyping of Monteiro et al., 2003), with the northern

form giving R. prolixus as its domestic derivative, and the

southern forms giving R. neglectus and R. nasutus as

savanna derivatives, and R. domesticus in the Atlantic forest,

so that these five species (robustus, prolixus, neglectus,

nasutus and domesticus) are commonly referred to as the

prolixus group. All these species occur in regions east of the

Andes, except for some domestic R. prolixus which are

believed to have been transported by accidental human

intervention.

The third main evolutionary line is believed to have been

through the biogeographical bottleneck of northern Colom-

bia formed by the Sierra Nevada and the northern tip of the

Colombian Andes. This appears to have given rise to R.

pallescens in northwest Colombia, extending north-west-

wards into Panama, Costa Rica and southern Nicaragua, and

southwards into the central Magdalena valley of Colombia

where a closely related form, R. colombiensis, is now

recognised. R. pallescens and R. colombiensis are mainly

associated with Attalea palm trees, and it appears that as this

putative cline extended southwards into Ecuador a

differentiation occurred in association with Phytolephas

palms to give the forms now recognised as R. ecuadoriensis

(Abad-Franch et al., 2001, 2002). Further south, in northern

Peru, R. ecuadoriensis is primarily domestic as there are few

palms present (Cuba-Cuba et al., 2002). These three species

(pallescens, colombiensis, ecuadoriensis) are commonly

referred to as the pallescens group, and all occur west of the

Andean Cordillera (Jaramillo et al., 2000).

Our results with T. rangeli are consistent with these

interpretations, with the KP1(+) form associated with the

prolixus group, and the KP1(�) form associated with the

pallescens group—suggesting a degree of co-evolution

between vector and parasite. Moreover, since the suggested

derivation of the prolixus group would have involved a

generalised outward spread from the Amazon region,

whereas that of the pallescens group appears to have

involved a biogeographical bottleneck, we would deduce

that the KP1(�) form would represent a derivative of the

KP1(+) form (rather than vice versa). It is possible that T.

rangeli may also have played a role in the selective pressure

leading to differentiation of R. pallescens, since we have

never seen evidence of pathogenicity of the KP1(�) form

either to R. prolixus or to most of the species of the

pallescens group, whereas the KP1(+) form is widely

reported to be pathogenic to R. prolixus (e.g. Marinkelle,

1968b; D’Alessandro, 1976).

If the ancestral form of T. rangeli was originally

associated with the Amazonian pictipes-like ancestor, we

deduce that it would have been similar to T. rangeli KP1(+)

containing KP1, KP2 and KP3 mini-circles in its kDNA

network. Dispersal of this ancestral T. rangeli KP1(+)

population probably involved genetic simplification by

selective pressures in new vector species where T. rangeli

KP1(�) originated by KP1 mini-circle deletion or by a

transkinetoplastidy phenomenon in which bulk DNA

kinetoplast alterations involving dominance of different

mini-circle subclass copy numbers occurred. This phenom-

enon has been described in Leishmania submitted to

selective drug resistance (Sheng-Yih et al., 1992). In short,

T. rangeli KP1(+) would have given rise to T. rangeli

D.A. Urrea et al. / Infection, Genetics and Evolution 5 (2005) 123–129128

KP1(�) under selective pressure and KP2 and KP3 became

dominant mini-circles in the kDNA network. However,

although we suggest that T. rangeli strains isolated today

from R. pictipes would show the KP1(+) profile, we do not

yet have evidence concerning the molecular characterisation

of T. rangeli subpopulations circulating in R. pictipes or in

closely related species such as R. brethesi, R. stali, R.

dalessandroi and R. paraensis.

Our results demonstrate that isolates of T. rangeli can be

ordered into two primary lineages based on amplification of

kDNA and mini-exon gene, and that each lineage is

associated with different species-groups of Rhodnius.

However, recent RAPD studies of 16 T. rangeli isolates

from the Brazilian Amazon revealed that most (12) of these

isolates differed from isolates from Colombia, Venezuela,

Panama, El Salvador and from Southern Brazil, thus

apparently constituting a further group of T. rangeli (Maia

Da Silva et al., 2004). Further analysis within each of the

T. rangeli groups could reveal the phylogenetic diversity

within them, while further analysis of associations between

T. rangeli isolates and other Rhodnius species could indicate

if vector-restriction is a determinant factor in the segregation

of T. rangeli into distinct phylogenetic groups.

Acknowledgements

This work was supported by grants from the Instituto

Colombiano, Francisco Jose de Caldas (COLCIENCIAS)

(Grants 1105-04-018-99 and 1105-04-13-012) and the

University of Tolima Research Fund and partially supported

by CNPq and CAPES, Brazil, and has benefited from

international collaboration through the ECLAT network and

Chagas Disease Intervention Activities—European Com-

munity network (CDIA-EC). We are grateful to the Boyaca

Department Secretariat of Health (Colombia) for helping in

collecting domestic R. prolixus during the National

Programme for the Prevention and Control of Chagas

Disease and Children’s Cardiopathy. We would especially

like to thank Roso Loaiza Tique for assistance in collecting

R. colombiensis specimens.

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