molecular phylogeography of chagas disease vectors in the amazon: vector control implications

Molecular phylogeography of Chagas disease vectors in the Amazon: vector

control implications

Molecular phylogeography of Chagas disease vectors in the Amazon: vector

control implications

Fernando MonteiroDepartmento de Medicina Tropical,FIOCRUZ, Rio de Janeiro, Brasil

ChagasDisease

ChagasDisease

FACTSImpact: 16-18 million casesAt risk: 100 million in 21 countriesAgent: Trypanosoma cruziVector: Triatomine bugsDistribution: The AmericasControl: Three multi-national control programs (Southern Cone, Andean Pact, and Central American)

FACTSImpact: 16-18 million casesAt risk: 100 million in 21 countriesAgent: Trypanosoma cruziVector: Triatomine bugsDistribution: The AmericasControl: Three multi-national control programs (Southern Cone, Andean Pact, and Central American)

Taxonomy and TheoriesTaxonomy and Theories 1859 – R. prolixus Stal was described based on insects

collected from houses in La Guaira, Venezuela

1927 – R. robustus Larrouse was described based on two insects from the Tefé river and French Guiana

Therefore: “...if it comes from a house it’s prolixus, and if it comes from a palm it’s robustus”

“R. prolixus derived from R. robustus 500 years ago” (Schofield 1999)

1859 – R. prolixus Stal was described based on insects collected from houses in La Guaira, Venezuela

1927 – R. robustus Larrouse was described based on two insects from the Tefé river and French Guiana

Therefore: “...if it comes from a house it’s prolixus, and if it comes from a palm it’s robustus”

“R. prolixus derived from R. robustus 500 years ago” (Schofield 1999)

R. prolixusR. prolixus R. robustusR. robustusPrevious observations on R. prolixus and R. robustusPrevious observations on R. prolixus and R. robustus

“…large R. prolixus and small R. robustus can be difficult to distinguish” (Lent & Wygodzinsky 1979)

Considerable overlap in many key morphological characteristics (Hurtado-Guerrero 1992, Harry 1994)

No allozyme differences between R. prolixus and R. robustus from Venezuela (Harry et al. 1992)

Vector control implications...

“…large R. prolixus and small R. robustus can be difficult to distinguish” (Lent & Wygodzinsky 1979)

Considerable overlap in many key morphological characteristics (Hurtado-Guerrero 1992, Harry 1994)

No allozyme differences between R. prolixus and R. robustus from Venezuela (Harry et al. 1992)

Vector control implications...

Model 1. prolixus = robustusModel 1. prolixus = robustus

prolixus/robustusprolixus/robustus

prolixus/robustusprolixus/robustus

Model 2. prolixus robustusModel 2. prolixus robustus

robustus

prolixus

“If it comes from a house it’s prolixus, if it comes from a palm it’s robustus” “If it comes from a house it’s prolixus, if it comes from a palm it’s robustus”

xx

Triatomine Phylogenetic AnalysesTriatomine Phylogenetic Analyses

Arilus cristatus

T. protractaDipetalogaster maxima

Psammolestes coreodes

T. nitida

R. robustus

T. sanguisuga

Panstrongylus megistus

T. pallidipennisT. dimidiata

T. sordidaT. infestans

R. pallescensR. ecuadoriensis

R. brethesiR. pictipes

R. prolixusR. neglectus

100

91

100

64

61

100100

99

81

95

97

100

100

Combined mtlsurRNA and mtcyt b (782 bp)

Monteiro et al. Trends in Parasitology 17: 344-347 (2001)

Allozymes: lack of diagnostic loci between prolixus and robustusAllozymes: lack of diagnostic loci between prolixus and robustus

Monteiro et al. Med Vet Entomol 16: 83-90 (2002)

IQM

ND4

ND5

H

WCYND2

COI

LC

OII

KD

ATPase8

AT

Pa

se6

CO

IIIG

FESNRA

ND4LN

D3PT

ND6

CytB

S?

ND

1

L

lrR

NA

V srR

NA

origin

Triatoma dimidiata

mitochondrial genome

17020 bp

Triatoma dimidiata

mitochondrial genome

17020 bp

Mitochondrial DNAMitochondrial DNA

Single circular chromosome (approxi. 16kb)

Standardized organization 13 protein coding genes 22 tRNA genes 2 rRNA genes A+T-rich control region

contains highly variable regions, flanked by highly conserved regions, thus allowing the use of heterologous PCR primers

Single circular chromosome (approxi. 16kb)

Standardized organization 13 protein coding genes 22 tRNA genes 2 rRNA genes A+T-rich control region

contains highly variable regions, flanked by highly conserved regions, thus allowing the use of heterologous PCR primers

Dotson & Beard Insect Mol Biol 10: 205-215 (2001)

Mitochondrial DNA Sequence AnalysisMitochondrial DNA Sequence Analysis

Strengths primers readily available genome organization and

inheritance known a minimum of molecular

knowledge required for organism

theoretically not under selection

fast evolving

Strengths primers readily available genome organization and

inheritance known a minimum of molecular

knowledge required for organism

theoretically not under selection

fast evolving

Weaknesses expensive represents a limited amount

of the organisms genetic makeup

introgression

Weaknesses expensive represents a limited amount

of the organisms genetic makeup

introgression

Materials and MethodsMaterials and Methods

84 specimens were analyzed, representing 12 populations of R. prolixus and 14 populations of R. robustus, from seven Latin American countries

Genomic DNA was extracted from individual bug legs

A 663 bp fragment of the mitochondrial cytochrome b (cyt b)

gene was sequenced

Sequences were analyzed by parsimony and neighbor-joining

methods

84 specimens were analyzed, representing 12 populations of R. prolixus and 14 populations of R. robustus, from seven Latin American countries

Genomic DNA was extracted from individual bug legs

A 663 bp fragment of the mitochondrial cytochrome b (cyt b)

gene was sequenced

Sequences were analyzed by parsimony and neighbor-joining

methods

Table 1 List of samples used in the study

Species

Collection site

N

Field/ Colony

Domestic/Sylvatic

Date collected

R. prolixus Orica, Francisco Morazan, Honduras 7 F D 1999 Las Palmas, Guatemala 6 F D June 1995 Tituque, Guatemala 9 F D June 1995 Tuticopote, Guatemala 3 F D June 1995 Modesto Loaiza, Coyaima, Colombia 1 C D September 1996 Ibague, Colombia 1 C D February 1995 Pampanito, Trujillo, Venezuela 3 C D 1997 Pampan, Trujillo, Venezuela 1 C D 1987 Pampanito, Trujillo, Venezuela 2 C D 1960 San José Tiznados, Guárico, Venezuela 3 C S 1988 Ortiz, Guárico, Venezuela* 4 F S July 2001 Cojedes, Venezuela 1 C D 1995 R. robustus Pampanito, Trujillo, Venezuela 4 C S 1997 Candelaria, Trujillo, Venezuela 3 C S 1988 Napo, Ecuador 2 C S - Carauarí, Amazonas, Brazil 4 F S February 2000 Porto Velho, Rondonia, Brazil 1 C S 1985 Apuí, Amazonas, Brazil 4 C S September 1996 Itupiranga, Pará, Brazil 1 C S 1984 Purupurú, Amazonas, Brazil 4 C S December 1995 Novo Repartimento, Pará, Brazil 5 C S August 1998

Barcarena, Pará, Brazil 5 C S 1996 Cayenne, French Guiana 1 F S March 2000 Balbina, Amazonas, Brazil 1 C S November 1983

UHE Paredão, Roraima, Brazil 5 C S March 1987 Rio Mapuera, Pará, Brazil 3 C S June 1986 R. nasutus Teresina, Piauí, Brazil 2 C D -

Monteiro et al. Mol Ecol 12: 997-1006 (2003)

Table 2 Polymorphic sites observed in 20 R. prolixus and R. robustus haplotypes. Hapotypes a-f are from the Orinoco basin, haplotypes g-t are from the Amazon region 0000000000011111111111111222222222222223333333344444444444444455555555566666666666 0112456667800122334566779223344447778891245679900112223455667812334466902223333445 3388840138759119087425148252535890298942751626925140365769581302146948401271469451 Haplotype * * * ** ** * * * * * a GAG....C.A.TC.........C....A.T.....TCCACC.AT...G.T.CC..GC..CGTAT...TT.T........... b G.G....C.A.TC.........C....A.T.....TCCACC.AT...G.T.CC..GC..CGTAT...TT.T........... c G.G....C.A.TC.........C....A.T.....TCCACC.AT.A.G.T.CC..GC..CGTAT...TT.T........... d G.G.CCGCGA.T...T....G.C..A...T...G.T..ACC.ATG..G.T.CCG..C.CC.TATGG.TT.T..A....T... e G.G.CCGCGA.T...T....G.C..A...T...G.T..ACC.ATG..G.T.CCG.GC.CC.TATGG.TT.T..A....T... f G.G.CCGCGA.T...T....G.C..A...T...G.T..ACC.ATG..G.T.CCG.GCTCC.TATGG.TT.T..A....T... g ...............TC.CCG.....T.........C.............................A...TTC.......TG h ...............TCCCCG.....T.........C.............................A....TC.......TG i ...............TC..C......T.........CC............................A....TC.......TG j ...............TC..CG.....TA........CC............................A....TC.......TG k ...............TC..CGT....T.........CC..........G.G....................TC...C.T.T. l .............G.TC..CGT.C..T..............A........................A....TC.......T. m ...............TC..CGT.C..T.......................................A....TC.......T. n ...A.....AG.............T.....C.T...C.........G..T.....G.....T...........AC....T.. o ...A.....AG.............T.....C.T.............G..T.....G.....T...........AC....T.. p ........................T......CT...C.........G...G...T..................A.G..TT.. q ..............A.........T.......T...C.............G...T..................A.G.GT... r ........................T.......T.............G.......T..................A.G.GTTT. s .......................CT...A...T.T...........G.......T..............A...A.G.GTT.. t ........................T.......T.............G.......T......TA..........A.G.GTTT. Consensus AGAGTTATAGACTTGCTTTTACATCCCGGCTTCACCTTGTTGGCAGAAACATAACATCTTACGCAAGCCGCCTGTATACCCA

Phylogenetic tree obtainedPhylogenetic tree obtained

MtCytB(662 bp)

0.01

I

II

III

IV

R. prolixus

R. robustus fromOrinoco basin

R. robustus fromAmazon basin

99

100

99

100

99

74

100

80mt cytb(662 bp)

II

III

IV

I

Geographic distribution of R. prolixus and R. robustus populations and percentage of sequence divergence among clades

Geographic distribution of R. prolixus and R. robustus populations and percentage of sequence divergence among clades

III

IV

I

II

4.0%

2.3%

3.3%

3.4%

1000 km

• “Results show identical fertility (number of females that laid eggs) between intra and interspecific crosses. However, fertility (number of eggs laid per female) was greatly reduced in inter-specific crosses” (Galindez-Giron et al. 1994). • Crossings between members of the three Amazonian clades led to either reduced fertility of sterility (Barrett 1995).

• “Results show identical fertility (number of females that laid eggs) between intra and interspecific crosses. However, fertility (number of eggs laid per female) was greatly reduced in inter-specific crosses” (Galindez-Giron et al. 1994). • Crossings between members of the three Amazonian clades led to either reduced fertility of sterility (Barrett 1995).

Cross-mating experiments with R. prolixus and R. robustus from Venezuela (Orinoco basin) and the Amazon

Cross-mating experiments with R. prolixus and R. robustus from Venezuela (Orinoco basin) and the Amazon

Inferences #1:Inferences #1: R. robustus (morphologically-defined) appears to be composed of 4

distinct evolutionary lineages, from 2 major regions, the Amazon Basin and the Orinoco Basin

R. prolixus is primarily a domestic species but is occasionally found in sylvatic habitats (i. e. palm trees)

In certain regions of Venezuela there is risk of recolonization of treated houses by sylvatic R. prolixus

R. prolixus and R. robustus are separate taxa, but R. robustus is a paraphyletic assemblage

R. prolixus from around South and Central America are very homogeneous, suggesting a recent bottleneck

R. robustus (morphologically-defined) appears to be composed of 4 distinct evolutionary lineages, from 2 major regions, the Amazon Basin and the Orinoco Basin

R. prolixus is primarily a domestic species but is occasionally found in sylvatic habitats (i. e. palm trees)

In certain regions of Venezuela there is risk of recolonization of treated houses by sylvatic R. prolixus

R. prolixus and R. robustus are separate taxa, but R. robustus is a paraphyletic assemblage

R. prolixus from around South and Central America are very homogeneous, suggesting a recent bottleneck

3. Proposed model3. Proposed model

robustus

prolixus

xxprolixus

Additional Questions:Additional Questions:

Could the paraphyly of R. robustus be a result of mt DNA introgression?

Is it possible to estimate the age of R. prolixus?

Could the paraphyly of R. robustus be a result of mt DNA introgression?

Is it possible to estimate the age of R. prolixus?


MtCytB(662 bp)

0.01

I

II

III

IV

R. prolixus



99

100

99

100

99

74

100

80mt cytb(662 bp)

II

III

IV

I

181 270 naBR ........-- ....------ ----...... .......... .......... .......... .......... .......... .......... roBR4 .......... .......... .......... .......... .......... .......... .......... .......... .......... roEC .......... .......... .......... .......... .......... .......... .......... .......... .......... roBR8 .......... .......... .......... .......... .......... .......... .......... .......... .......... roVE2 .......... .......... .......... .......... .......... .......... .......... .......... .......... prVE5 .......... .......... .......... .......... .......... .......... .......... .......... .......... prCO1 .......... .......... .......... .......... .......... .......... .......... .......... .......... Consensus AAGTTATACC GTTAAGGTAT TTTCTTTAAA ACAGTTTTAG CCGTTTTATA TACTGGATAA AATTGACAGT AACGAATTAT GGTGTTGAGC 271 360 naBR .......... .......... .......... .......... .......... .......... .......... .......... .......... roBR4 ...A...... .......... .......... .......... .......... .......... .......... .......... .......... roEC ...A...... .......... .......... .......... .......... .......... .......... .......... .......... roBR8 ...A.A.... .......... .......... .......... .......... .......... .......... .......... .......... roVE2 ...A...... .......... .......... .......... .......... .......... .......... .......... .........C prVE5 ...A...... .......... .......... .......... .......... .......... .......... .......... .........C prCO1 ...A...... .......... .......... .......... .......... .......... .......... .......... .........C Consensus CACTTGAAAT TATATATATG TAAAAATATA TATAATGGAA AGTGTCCTAA AATATGGCTG TTTGCAAGTG GGTTGGTAAA AAATAGTTTT 361 450 naBR .......... .......... .......... .......... .......... .......... .......... .......... .......... roBR4 .......... .......... .......... .......... .......... .......... ........T. .......... .......... roEC .......... .......... .......... .......... .......... .......... ........T. .......... .......... roBR8 .......... .......... .......... .......... .......... .......... ........T. .......... .......... roVE2 .......... .......... .......... .......... .......... .......... ........T. .......... .......... prVE5 .......... .......... .......... .......... .......... .......... ........T. .......... .......... prCO1 .......... .......... .......... .......... .......... .......... ........T. .......... .......... Consensus AATTCGGATT TTTAACCGGT TAACTATTCC GCCTACTGTT GGTAAACTGT TCCTAGGACT GTGCTTATAA TCACCGGTCG GCAGCGATTC

Sequence alignment of the D2 variable region of 28S rDNASequence alignment of the D2 variable region of 28S rDNA

robustus I

prolixus


MtCytB(662 bp)

0.01

I

II

III

IV

R. prolixus



99

100

99

100

99

74

100

80mt cytb(662 bp)

II

III

IV

I

IT C


MtCytB(662 bp)

0.01

II

III

IV

R. prolixus


99

100

99

100

99

74

100

80mt cytb(662 bp)

II

III

IV

“R. venezuelensis”

How old is R. prolixus?How old is R. prolixus?Comparisons: Divergence (%): Divergence (My):

Within clades

R. prolixus 0.2 (0.2 – 0.3) -

R. robustus I 0.2 (0.2 – 0.3) -

R. robustus II 1.0 (0.3 – 1.5) 0.4

R. robustus III 0.2 (0.2 – 0.2) -

R. robustus IV 1.0 (0.3 – 1.4) 0.4

Between Orinoco clades 3.3 (3.0 – 3.3) 1.4

Among Amazonian clades

robustus II vs. robustus III 4.0 (3.6 – 4.4) 1.7

robustus II vs. robustus IV 3.4 (3.0 – 3.9) 1.5

robustus III vs. robustus IV 2.3 (2.0 – 2.8) 1.0

Amazon vs. Orinoco clades 7.2 (5.6 – 8.5) 3.1molecular clock calibration: 1 ma = 2.3%; Brower PNAS 91: 6491-6495 (1994)

Inferences #2:Inferences #2:

No indication of mtDNA introgression

Estimated time of separation of R. prolixus and R. robustus I from common ancestor = 1.4 mya

Little within group variation. Large between group variation.

No indication of mtDNA introgression

Estimated time of separation of R. prolixus and R. robustus I from common ancestor = 1.4 mya

Little within group variation. Large between group variation.

ConclusionsConclusions

R. prolixus and R. robustus are separate taxa

R. robustus is a paraphyletic species complex

R. prolixus and R. robustus I occur in sympatry in Trujillo, Venezuela

R. prolixus originated arround 1.4 mya

Major clades are compatible with a Pleistocene origin

R. prolixus is the species most often found colonizing houses, whereas R. robustus is found in palm trees

R. prolixus and R. robustus are separate taxa

R. robustus is a paraphyletic species complex

R. prolixus and R. robustus I occur in sympatry in Trujillo, Venezuela

R. prolixus originated arround 1.4 mya

Major clades are compatible with a Pleistocene origin

R. prolixus is the species most often found colonizing houses, whereas R. robustus is found in palm trees

But could the sequence information generated with this

study be used in a practical way?

But could the sequence information generated with this

study be used in a practical way?

663 pb cyt b fragment used and

clade-specific primers

663 pb cyt b fragment used and

clade-specific primers

Reverse 1

(R. robustus II-IV)

Reverse 1

(R. robustus II-IV)

Reverse 2

(R. prolixus)

Reverse 2

(R. prolixus)

Reverse 3

(R. robustus I)

Reverse 3

(R. robustus I)

ForwardForward

Diagnostic multiplex PCR for discriminating among R. prolixus and R. robustus clades


349pb349pb285pb285pb239pb239pb

R. prolixus R. robustus IR. robustus II-IV

R. prolixus R. robustus I R. robustus II-IV



P P R1 R2 R1 R2 R2 R1 P R1 R2 P P R1

349pb349pb285pb285pb239pb239pb

Comparison of Triatoma brasiliensis populations from northeast Brazil

Comparison of Triatoma brasiliensis populations from northeast Brazil

Triatoma brasiliensis Neiva, 1911Triatoma brasiliensis Neiva, 1911

T. brasiliensis melanica Neiva & Lent, 1941T. brasiliensis melanica Neiva & Lent, 1941 T. brasiliensis macromelasoma Galvão, 1956T. brasiliensis macromelasoma Galvão, 1956

T. brasiliensis (Lent & Wygodzinsky 1979)T. brasiliensis (Lent & Wygodzinsky 1979)

T. brasiliensis brasiliensis Neiva, 1911T. brasiliensis brasiliensis Neiva, 1911

melanicamelanicamacromelasomamacromelasoma juazeirojuazeirobrasiliensisbrasiliensis

T. brasiliensis chromatic forms (Costa et al. 1997, 1998)T. brasiliensis chromatic forms (Costa et al. 1997, 1998)

brasiliensisbrasiliensis macromelasomamacromelasoma juazeirojuazeiro melanicamelanica

Triatoma brasiliensis chromatic formsTriatoma brasiliensis chromatic forms

Forms are identified based on color differences of the pronotum

Forms are identified based on color differences of the pronotum

1. brasiliensis

2. melanica

3. macromelasoma

4. juazeiro

1. brasiliensis

2. melanica

3. macromelasoma

4. juazeiro

Diagnostic allozyme loci between the four chromatic forms

Diagnostic allozyme loci between the four chromatic forms

Costa et al. Mem Inst Oswaldo Cruz 92: 459-464 (1997)

T. brasiliensis chromatic forms collected from their type localities are genetically and ecologically different (Costa et al., 1997, 1998, 2002)T. brasiliensis chromatic forms collected from their type localities are genetically and ecologically different (Costa et al., 1997, 1998, 2002)

“Melanic forms of this species which occur in various areas have been described as two different subspecies, but intergrading forms are frequent” (Lent and

Wygodzinsky, 1979)

“Melanic forms of this species which occur in various areas have been described as two different subspecies, but intergrading forms are frequent” (Lent and

Wygodzinsky, 1979)

Could the forms represent the extremes of a morphological and

chromatic gradient ?

Could the forms represent the extremes of a morphological and

chromatic gradient ?

136 specimens of the four forms from 16 geographic populations were sequenced for a 510bp fragment of

mt cyt b gene

136 specimens of the four forms from 16 geographic populations were sequenced for a 510bp fragment of

mt cyt b gene

Triatoma brasiliensis populationsTriatoma brasiliensis populationsTable 2 Variable sites for a 510 bp fragment of the cytochrome b gene observed in 35 T. brasiliensis haplotypes. Also shown are the distribution of thehaplotypes per phenotype (br = brasiliensis, ma = macromelasoma, ju = juazeiro, and me = melanica), and the absolute number of each haplotype. The br*phenotype represents putative natural hybrids between br and ma individuals. Sequences representing the four genetic groups observed (haplotypes a, w, B, andH) have been deposited in the GeneBank (accession nos. AY336524, AY336525, AY336526 and AY336527).

Variable sites Phenotype 11111 1111111111 1111111222 2222222222 2222222223 3333333333 3333333333 3444444444 444444444Hapl. 11112234 4589900112 2233345566 7778899001 1222333444 5556788990 0122334445 6777888999 9001122233 444556889 br ma ju me br* N 3602895765 6116959470 3725840308 1470459473 9589124469 2571626477 9547082565 7238137013 6891706912 145398498 a GAGTACGGCC CAGTTCTTCC CCTCGAAAGC TCTTTCGTCC ATCGTGCACT AATATTTAAG CCTCTCGTCG TACTTTTCTA GTTATATTTC CATTTATCC * * * * 7 b .......... .......... .......... ..C....... .......... .......... .......... .......... ........C. ......... * 1 c ....G..... .......... .......... .......... .......... .......... .......... .......... .......... ......... * 1 d ..A.G..... .......... .......... .......... .......... .......... .......... .......... .......... ......... * 1 e .......... .......... .......... .......... .......... .......... .......... .....C.... .......... ......... * 1 f .......... .......... .......... .......... .......... .......... .......... .....C.... .......... ........T * 2 g .......... T......... .......... .......... .......... .......... .......... .......... .......... ......... * 1 h .......... .......... .......... C......... .......... ....C..... .......... .......... .......... ......... * 1 i .......... .......... .......... .......... .......... .......... .......... ...C...... .......... ......... * 1 j .......... .......... .......... .......... .......... .......... ..C....... .......... .......... ......... * * 9 k .......... .......... ........A. .......... .......... .......... ...T...... .......... .......... ......... * 4 l .......... .........T .......... .......... T......... .......... .......... ......C... .......... ......... * 5 m .....T.... .......... .......... .......... .......G.. .......... .......... .......... .......... ......... * 16 n .......A.. .......... ....A..... .......... .......... .......... .......... .......... .......... ......... * 1 o .......A.. .......... .......... .......... .....A.... .......... ......A... .......... .......... .....G... * 2 p .......... .......... ....A..... .......... .......... ...G...... .......... .......... .....G.... ......... * 1 q .......... .......... ....A..... .......... .........C .......... .......... .......... .......... ......... * 3 r .......... .......... .......... .......... .........C .......... .......... .......... .......... ...G..... * 1 s .......... .......... .......... .......... .........C .......... ....C..... .......... .......... ...G..... * 1 t A......... .......... .......... .......... .......... .......... .......... .......... .......... ......... * 1 u A......... ..A....... .......... .......... .......... .......... .......... .......... .......... ......... * * 3 v A......A.. .......... .......... .......... .......G.. .......... ......A... .......... .......... ......... * 1 x A.A....... .......... ....A..... .......... .......GT. .......... ......A... .......... A......... ......... * 2 y A......... .......... ....A..... .......... .......GT. .......... ........T. .......... .......... ......... * 14 w A......AA. .GA....... ......G... .......C.. .........C T......... .......... .GT....... ..C....... T........ * 5 z A......A.T ..AC...... .......... .......... .........C T......... .......... ..T...C... ..C....... T........ * 2 A A......A.T ..AC...... .......... .......... .........C T........A .......... ..T...C... ..C....... T........ * 3 B A......AA. ..AC..C..T ...TA..... .......C.T .C....T... .T..C.C.T. T..T.TACT. ..T.....CG ACC.C.C... TTC...CTT * 1 C A......AA. ..AC..C..T ...TA..... .......C.T .C....T... .T..C.C.T. T..T.TAC.. ..T.....CG ACC.C.C... TTCC..CTT * 1 D A......AA. ..AC..C..T ...TA..... .......C.T .C....T... .T..C.C.T. T..T.TACT. ..T.....CG ACC.C.C... TTCC..CTG * 1 E A.....AAA. ..AC..C..T ...TA..... ......AC.T .C....T.T. .TC.C.CGT. T..T.TACT. ..T.....CG ACC.C.C... TCCC..CTT * 5 F A......AA. ..AC..C..T ...TA..... .......C.T .C....T... .T..C.C.T. T..T.TACT. ..T.....CG ACC.C.C... TTCC..CTT * 3 G A......AA. ..AC..CC.T ...TA..... .......C.T .C....T... .T..C.C.T. T..T.TACT. ..T.....CG ACC.C.C... TTCC..CTT * 1 H AG.C...CA. ..CACTC... ATCTAG...T .TCCCT.CT. ..T.C..... T....C.... ..CT.TACT. C.TCC..T.. ACCGC.CC.T T...C.CT. * 8 I AG.C...CA. ..CACTC.T. ATCTAG.G.T .TCCCT.CT. ..TAC..... T....C.... .TCT.TACTA C.TCC..T.. ACCGC.CC.T T..CC.CT. * 26

Triatoma brasiliensis populationsTriatoma brasiliensis populations

The 35 haplotypes observed are subdivided in four main phylogenetic lineages

The 35 haplotypes observed are subdivided in four main phylogenetic lineages

Deduced NJ phylogenetic tree based on 510 bp of the cyt b gene

a 2/8(br br* ma ju)

b 8(ma)

c 1(br)

d 2(br)

e 1(br)

f 2/3(br)

g 8(ma)

h 8(br*)

i 8(ma)

j 6/7/8(br ma)

k 5(br)

l 8(br*)

m 1/2(br)

n 5(br)

o 8(br*)

p 5(br)

q 5(br)

r 8(ma)

s 8(br*)

t 8(br*)

u 8(br ma)

v 3(br)

x 3(br)

y 4(br)

w 10/11(ma)

z 9/10(ma)

A 10(ma)

B 9(ma)

C 13(ju)

D 13(ju)

E 14(ju)

F 12(ju)

G 13(ju)

H 15/16(me)

I 16(me)

T. sordida

0.02

94

100

100

100

86

juazeiro

melanica

macromelasoma

brasiliensis

Monteiro et al. Mol Phylogenet Evol 32: 46-56 (2004)

Maximum parsimony networks of T. brasiliensis haplotypesMaximum parsimony networks of T. brasiliensis haplotypes

The four chromatic forms are clearly separated in three networks

Some macromelasoma insects fall within the brasiliensis group which is suggestive of introgression

The four chromatic forms are clearly separated in three networks

Some macromelasoma insects fall within the brasiliensis group which is suggestive of introgression

Nested clade analysis of brasiliensis haplotypes

Nested clade analysis of brasiliensis haplotypes






T. brasiliensis chromatic forms (Costa et al. 1997, 1998)T. brasiliensis chromatic forms (Costa et al. 1997, 1998)






“T. melanica”“T. melanica”“T. juazeirensis”“T. juazeirensis”T. brasiliensisT. brasiliensis

ConclusionsConclusions

1. Geographic distribution of haplotypes does not follow a genetic gradient (or cline)

2. Chromatic forms present very high levels of genetic differentiation, suggesting (together with other evidence), that they might represent different species

3. Forms can be treated as isolated targets in vector control programs

1. Geographic distribution of haplotypes does not follow a genetic gradient (or cline)

2. Chromatic forms present very high levels of genetic differentiation, suggesting (together with other evidence), that they might represent different species

3. Forms can be treated as isolated targets in vector control programs

Collaborators

CDCC. Ben Beard

Universidad del Valle de Guatemala Celia Cordon-Rosales

INPAToby Barrett

LSTMMartin Donnelly

ECLATChris Schofield

FIOCRUZJosé JurbergJane Costa

Obrigado!Obrigado!

Comparison of Triatoma infestans and T. melanosoma populations

Origin of samples sequenced for 412bp of the cytochrome b gene

Argentina

Brazil

BoliviaCO SADomSil

RS

PR

BA

T mel

DM

ARG

63 183 186 243 288 297 354 384

T. infestans ARG A T T T T A T C

T. infestans BRA-BA A T T T T A T C

T. infestans BRA-RS A T T T T A T C

T. infestans BRA-PR A T T C T A T C

T. melanosoma A T T C T A T C

T. infestans BOL-Sil G T C T C A T T

T. infestans BOL-Dom G T C T C A T T

T. infestans BOL-SA G T C T C A T T

T. infestans BOL-CO G T C T C A T T

T. infestans BOL-DM A C C T C G C T

T. brasiliensis

T. infestans (BOL-DM)

T. infestans (BOL-Sil)T. infestans (BOL-Dom)T. infestans (BOL-SA)T. infestans (BOL-CO)

T. infestans (BRA-BA)T. infestans (BRA-RS)T. infestans (ARG)

T. melanosomaT. infestans (BRA- PR)

297

354

183

63

384

288

243

186

72

100

86

*

Phylogenetic relationship of T. infestans populations and T. melanosoma

Monteiro et al. Mem Inst Oswaldo Cruz 94: 229-238 (1999)

Domestic and silvatic T. infestans populations from Bolivia belong to the same evolutionary lineage.

Specimens examined can be subdivided in 2 groups: Bolivian and Brazilian/Argentinian.

T. melanosoma is genetically identical to T. infestans, suggesting that it may actually be a (melanic) population of the latter.

Taxonomical curiosities...Taxonomical curiosities...

R. robustus colection sites at the mouth of the Tefé river and French Guyana

R. robustus colection sites at the mouth of the Tefé river and French Guyana

III

IV

I

II

4.0%

2.3%

3.3%

3.4%

1000 km


MtCytB(662 bp)

0.01

I

II

III

IV

R. prolixus



99

100

99

100

99

74

100

80mt cytb(662 bp)

II

III

IV

I

molecular phylogeography of chagas disease vectors in the amazon: vector control implications

Documents

prolixus stal

robustus haplotypes

robustus larrouse

populations of

haplotypes gt

venezuela harry

variable regions

mol ecol