Molecular phylogeography of Chagas disease vectors in the Amazon: vector control implications
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Transcript of 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?
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
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
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
IT C
Phylogenetic tree obtainedPhylogenetic tree obtained
MtCytB(662 bp)
0.01
II
III
IV
R. prolixus
R. robustus fromAmazon basin
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
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
Diagnostic multiplex PCR for discriminating among R. prolixus and R. robustus clades
Diagnostic multiplex PCR for discriminating among R. prolixus and R. robustus clades
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
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)
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. 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
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