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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 Monteiro Departmento de Medicina Tropical, FIOCRUZ, Rio de Janeiro, Brasil. Chagas Disease. FACTS Impact: 16-18 million cases At risk: 100 million in 21 countries

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Molecular phylogeography of Chagas disease vectors in the Amazon: vector control implications

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  1. Molecular phylogeography of Chagas disease vectors in the Amazon: vector control implications Fernando Monteiro Departmento de Medicina Tropical, FIOCRUZ, Rio de Janeiro, Brasil

  2. ChagasDisease FACTS Impact: 16-18 million cases At risk: 100 million in 21 countries Agent:Trypanosoma cruzi Vector: Triatomine bugs Distribution:The Americas Control: Three multi-national control programs (Southern Cone, Andean Pact, and Central American)

  3. Taxonomy and Theories • 1859 – R. prolixus Stal was described based on insects collected from houses in La Guaira, Venezuela • 1927 – R. robustus Larrousewas 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)

  4. Previous observations on R.prolixus and R. robustus R. prolixus 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...

  5. Model 1. prolixus=robustus prolixus/robustus prolixus/robustus

  6. Model 2.prolixusrobustus “If it comes from a house it’s prolixus, if it comes from a palm it’s robustus” prolixus x robustus

  7. T. pallidipennis 95 Combined mtlsurRNA and mtcyt b (782 bp) 99 T. dimidiata T. sanguisuga T. nitida 61 Dipetalogaster maxima 91 T. protracta Panstrongylus megistus 81 T. sordida 97 100 T. infestans R. pallescens 100 R. ecuadoriensis 100 R. brethesi 100 R. pictipes Psammolestes coreodes 64 100 R. robustus 100 R. prolixus R. neglectus Arilus cristatus Monteiro et al. Trends in Parasitology 17: 344-347 (2001) Triatomine Phylogenetic Analyses

  8. Allozymes: lack of diagnostic loci between prolixus and robustus Monteiro et al. Med Vet Entomol 16: 83-90 (2002)

  9. IQM WCY ND2 origin COI srRNA L V COII KD lrRNA ATPase8 ATPase6 L ND1 COIII ? Triatoma dimidiata mitochondrial genome 17020 bp G ND3 S A R CytB N S E F ND6 ND5 ND4L ND4 PT H Mitochondrial 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 Dotson & Beard Insect Mol Biol 10: 205-215 (2001)

  10. Mitochondrial 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 • Weaknesses • expensive • represents a limited amount of the organisms genetic makeup • introgression

  11. Materials 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

  12. Monteiro et al. Mol Ecol12: 997-1006 (2003)

  13. 0.01 100 99 100 99 100 99 74 80 Phylogenetic tree obtained R. prolixus MtCytB (662 bp) R. robustus from Orinoco basin I I II II R. robustus from Amazon basin III III IV IV mt cytb (662 bp)

  14. I 1000 km Geographic distribution of R. prolixus and R. robustus populations and percentage of sequence divergence among clades 3.4% 3.3% IV III 2.3% II 4.0%

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

  16. 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

  17. 3. Proposed model x robustus prolixus prolixus

  18. 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?

  19. 0.01 100 99 100 99 100 99 74 80 Phylogenetic tree obtained R. prolixus MtCytB (662 bp) R. robustus from Orinoco basin I I II II R. robustus from Amazon basin III III IV IV mt cytb (662 bp)

  20. Sequence alignment of the D2 variable region of 28S rDNA • 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 robustus I prolixus

  21. 0.01 100 99 100 99 100 99 74 80 Phylogenetic tree obtained R. prolixus MtCytB (662 bp) I TC R. robustus from Orinoco basin I I II II R. robustus from Amazon basin III III IV IV mt cytb (662 bp)

  22. 0.01 100 99 100 99 100 99 74 80 Phylogenetic tree obtained R. prolixus MtCytB (662 bp) “R. venezuelensis” II II R. robustus from Amazon basin III III IV IV mt cytb (662 bp)

  23. How old is R. prolixus? molecular clock calibration: 1 ma = 2.3%; Brower PNAS 91: 6491-6495 (1994)

  24. 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.

  25. Conclusions • 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

  26. But could the sequence information generated with this study be used in a practical way?

  27. 663 pb cyt b fragment used and clade-specific primers Forward Reverse 1 (R. robustus II-IV) Reverse 2 (R. prolixus) Reverse 3 (R. robustus I)

  28. 349pb 285pb 239pb R. robustus II-IV R. prolixus R. robustus I Diagnostic multiplex PCR for discriminating among R.prolixus and R. robustus clades

  29. Diagnostic multiplex PCR for discriminating among R.prolixus and R. robustus clades 349pb 285pb 239pb R. prolixus R. robustus II-IV R. robustus I P P R1 R2 R1 R2 R2 R1 P R1 R2 P P R1

  30. Comparison of Triatoma brasiliensis populations from northeast Brazil

  31. brasiliensis juazeiro melanica macromelasoma Triatoma brasiliensis Neiva, 1911 T. brasiliensis melanica Neiva & Lent, 1941 T. brasiliensis macromelasoma Galvão, 1956 T. brasiliensis brasiliensis Neiva, 1911 T. brasiliensis (Lent & Wygodzinsky 1979) T. brasiliensis chromatic forms(Costa et al. 1997, 1998)

  32. brasiliensis melanica macromelasoma juazeiro Triatoma brasiliensis chromatic forms Forms are identified based on color differences of the pronotum

  33. Diagnostic allozyme loci between the four chromatic forms 1. brasiliensis 2. melanica 3. macromelasoma 4. juazeiro Costa et al. Mem Inst Oswaldo Cruz 92: 459-464 (1997)

  34. T. brasiliensis chromatic formscollected 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)

  35. 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

  36. Triatoma brasiliensis populations

  37. Monteiro et al. Mol Phylogenet Evol32: 46-56 (2004) Deduced NJ phylogenetic tree based on 510 bp of the cyt b gene Triatoma brasiliensis populations The 35 haplotypes observed are subdivided in four main phylogenetic lineages

  38. Maximum 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

  39. Nested clade analysis of brasiliensis haplotypes

  40. brasiliensis juazeiro melanica macromelasoma Triatoma brasiliensis Neiva, 1911 T. brasiliensis melanica Neiva & Lent, 1941 T. brasiliensis macromelasoma Galvão, 1956 T. brasiliensis brasiliensis Neiva, 1911 T. brasiliensis (Lent & Wygodzinsky 1979) T. brasiliensis chromatic forms(Costa et al. 1997, 1998)

  41. Triatoma brasiliensis Neiva, 1911 T. brasiliensis melanica Neiva & Lent, 1941 T. brasiliensis macromelasoma Galvão, 1956 T. brasiliensis brasiliensis Neiva, 1911 T. brasiliensis (Lent & Wygodzinsky 1979) brasiliensis juazeiro melanica macromelasoma “T. juazeirensis” “T. melanica” T. brasiliensis

  42. Conclusions • Geographic distribution of haplotypes does not follow a genetic gradient (or cline) • Chromatic forms present very high levels of genetic differentiation, suggesting (together with other evidence), that they might represent different species • Forms can be treated as isolated targets in vector control programs

  43. Collaborators Universidad del Valle de Guatemala Celia Cordon-Rosales CDC C. Ben Beard FIOCRUZ José Jurberg Jane Costa INPA Toby Barrett LSTM Martin Donnelly ECLAT Chris Schofield

  44. Obrigado!

  45. Comparison of Triatoma infestans and T. melanosoma populations

  46. Brazil BA Bolivia CO SA Dom Sil DM T mel PR RS ARG Argentina Origin of samples sequenced for 412bp of the cytochrome b gene

  47. Phylogenetic relationship of T. infestans populations and T. melanosoma 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. Monteiro et al. Mem Inst Oswaldo Cruz 94: 229-238 (1999)

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