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Anais do IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais


Recife, 13 a 17 de setembro 2010

Anais do IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais


Recife, 13 a 17 de setembro 2010

Paulo Ernani Gadelha


Presidente da FIOCRUZ

Claude Pirmez
Vice-Presidente de Pesquisa e Laboratrios de Referencia

Eduardo Maia Freese de Carvalho


Diretor do CPqAM

CPqAM:
Centro de Pesquisas Aggeu Magalhes CPqAM/Fiocruz Av. Professor Moraes Rego, s/n Cidade Universitria - Recife - PE - Brasil CEP: 50.670-420 Caixa Postal: 7472 www.cpqam.fiocruz.br

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

APRESENTAO E AGRADECIMENTOS
O IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais (Entomol4), realizado no Auditrio Frederico Simes Barbosa, do Centro de Pesquisas Aggeu Magalhes/FIOCRUZ, Recife, no perodo de 13 a 17 de setembro de 2010, teve a participao de 53 palestrantes de instituies do Brasil e do exterior, com 180 participantes inscritos, entre estudantes, pesquisadores e profissionais vinculados a servios de vigilncia entomolgica e epidemiolgica. Esta edio contar tambm com o II simpsio satlite sobre Status taxonmico de Lutzomyia longipalpis e o III Curso de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais, que antecede o workshop, realizado entre 08 e 13 de setembro no Departamento de Gentica da UFPE. O objetivo central do Entomol promover a discusso e o debate de temas contemporneos da biologia e do controle de insetos vetores de doenas tropicais, visando o fortalecimento e integrao dos grupos de pesquisa atuantes nesta rea do conhecimento, com a participao dos mais importantes pesquisadores do Brasil e alguns convidados do exterior. Por outro lado, o evento tem a participao expressiva de estudantes de doutorado e mestrado que integram programas de psgraduao no pas em reas de concentrao direta ou indiretamente relacionadas ao tema, com especial nfase em entomologia molecular. Agradecemos os apoios recebidos da CAPES, CNPq, FACEPE, Fundao Oswaldo Cruz, Universidade Federal de Pernambuco e da Sociedade Brasileira de Medicina Tropical (Regional Pernambuco) e Sociedade Brasileira de Parasitologia, sem os quais no seria possvel o sucesso alcanado pelo evento, que vem constituindo-se em um dos mais tradicionais do pas na rea de vetores de doenas infecciosas. Os textos dos resumos esto de acordo com a ordem de apresentao do programa.

Sinval Pinto Brando Filho (CPqAM/FIOCRUZ, Recife), coordenador geral Maria Helena Lobo Neves (CPqAM/FIOCRUZ, Recife) Valdir de Queiroz Balbino (UFPE, Recife) Paulo Filemon Pimenta (CPqRR/FIOCRUZ, Belo Horizonte) Reginaldo Peanha Brazil (IOC/FIOCRUZ, Rio de Janeiro) Marcelo Ramalho Ortigo (Kansas State University, USA)

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

PROGRAMAO
Dia 13/09 (segunda-feira)
Horrio: 18:00 - 20:00 Solenidade de abertura Dr. Sinval Pinto Brando Filho (Coordenador Geral do ENTOMOL 4) Dr. Eduardo Freese (Diretor do Centro de Pesquisas Aggeu Magalhes/FIOCRUZ) Dr. Ansio Brasileiro (Pr-Reitor de Pesquisas UFPE) Carlos Henrique Nery Costa (VicePresidente da SBMT) Ana Nilce Maia Elkhoury (Diretoria de Doenas Transmitidas por Vetores da SVS/MS)) Palestra de abertura Dr. Gregory Lanzaro The Society for Vector Ecology (SOVE): Explanatory conference about the Brazilian Chapter

Dia 14/09 (tera-feira)


08:30 - 12:30 Sesso Temtica I Coordenador: Sinval Pinto Brando Filho Carlos Brisola Marcondes Ovos de mosquitos: negligenciados, mas importantes (e bonitos) Ftima Ximenes Parmetros da transmisso de Leishmania chagasi em rea periurbana Eunice Galati Capacidade vetorial de vetores permissivos de Leishmania Carlos Henrique Nery Costa O papel de Lutzomyia longipalpis na urbanizao do calazar no Brasil Edelberto Santos Dias Eco-epidemiologia da leishmaniose visceral em Minas Gerais Elizabeth Rangel Lutzomyia whitmani e a eco-epidemiologia da leishmaniose tegumentar no Brasil Sinval Pinto Brando Filho Infecciosidade de roedores silvestres experimentalmente infectados com Leishmania braziliensis para o flebotomneo 14:00 - 18:00 Sesso Temtica II Coordenador: Andr Furtado Leda Rgis Dados recentes sobre o controle mecnico do Aedes aegypti Walter Leal Oviposition attractans for Culex quinquefasciatus: their receptors, reception, and use in vector management Joo Pinto Como pode a gentica populacional ajudar no controle de vetores?

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

James Gordon Hamilton Synthetic Lutzomyia longipalpis pheromone: where do we go from here? Orin Courtenay A long-lasting topical deltamethrin treatment of dogs reduces the vectorial capacity of Lutzomyia longipalpis 20:00 h - Sesso Temtica Especial - Mar Hotel, Boa Viagem, Recife Coordenador: Reginaldo Peanha Brazil Elyes Zhioua Eco-epidemiology of zoonotic cutaneous leishmaniasis in North Africa Reginaldo Peanha Brazil Impacto das mudanas climticas e degradao de habitats sobre a distribuio dos flebotomneos e disperso das leishmanioses no Brasil Jeffrey Jon Shaw Mudanas climticas e seus impactos na ecoepidemiologia de doenas tropicais Alexandre Peixoto Cronobiologia molecular de insetos vetores

Dia 15/09 (quarta-feira)


08:30 - 12:30 Sesso Temtica III Coordenador: Maria Helena Lobo Neves Marcos Horcio Processo hematofgico nos insetos vetores de doenas Felipe Arley Pessoa Mansonelose, uma filariose neglicenciada na Amaznia Srgio Luz Sensibilidade da armadilha Adultrap e ovitrampas para Aedes aegypti em Manaus/Amazonas Cecilia de Oliveira Cudischevitch Hematophagy and cell signaling in mosquitoes: the role of protein tyrosine phosphatases as modulators of Aedes aegypti immunity Maria Helena Lobo Neves Caracterizao da resistncia de mosquitos a larvicidas biolgicos Alessandra Gutierrez Estudo da disperso de Lutzomyia longipalpis em Campo Grande, Mato Grosso do Sul Constncia Junqueira Ayres Bases moleculares da resistncia ao temephos em Aedes aegypti 14:00 - 18:00 Sesso Temtica IV Coordenador: Lda Narcisa Rgis Marcos Sorgine Interao Aedes aegypti dengue vrus

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

Ngila Costa Secundino Proteofosphoglicano (PPG) conferindo Leishmania major resistncia a enzimas digestivas induzidas pela alimentao sanguinea em flebotomneos vetores Jesus Valenzuela Molecular interactions at the Host-Vector-Parasite interface Nicolau Maus Serra Freire Aspectos da reproduo de Ixodida e a transmisso vertical de riqutsias patognicas Pedro Marcos Linardi Epidemiologia das pulgas 20:00 h (Mar Hotel) - II Simpsio Satlite Sobre o Status Taxonmico de Lutzomyia longipalpis Coordenadores: Eunice Galati e Jeffrey Shaw Paul Ready Sandfly taxonomy is an imperfect predictor of vectorial traits Alexandre Peixoto Complexo Lutzomyia longipalpis James Gordon Hamilton Sex pheromone types in Lutzomyia longipalpis complex Gregory Lanzaro Genetic variation in the salivary protein maxadilan among population of Lutzomyia longipalpis Valdir Balbino Complexo Lutzomyia longipalpis: em busca de novas solues para um antigo problema

Dia 16/09 (quinta-feira)


08:30 - 12:30 Sesso Temtica V Coordenador: Filipe Dantas Torres Beatriz Ferreira Identificao de constituintes no-proteicos na saliva do carrapato Rhipicephalus sanguineus com propriedades imunomoduladoras Filipe Dantas Torres Ticks as vectors of Leishmania: facts and speculations Tereza Magalhes RNAi em mosquitos: defesa natural antiviral e ferramenta tecnolgica Carlos Logullo Molecular strategies to ectoparasite control Rodrigo Soares Aspectos moleculares da interao de lipofosfoglicanos de Leishmania com o intestino mdio de flebotomneos

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

Andre Nbrega Pitaluga Busca de alvos moleculares em Lutzomyia longipalpis com potencial de interveno vetor-patgeno Clarissa Teixeira Understanding the initial inflammatory response to sand fly bites 14:00 - 18:00 Sesso Temtica VI Coordenador: Marcelo Ramalho Ortigo Paul Ready Is there an arms race involving salivary peptides of the vectors of zoonotic visceral leishmaniasis? Aldina Barral Evolution of Lutzomyia recombinant proteins as epidemiological markers of sandfly exposure David W. Severson A. aegypti research in the post-genomics era Paulo Filemon Pimenta O processo de interao do Lutzomyia (Lutzomyia) longipalpis com a Leishmania (Leishmania) chagasi Marcelo Ramalho Ortigo RNA interference in sand flies

Dia 17/09 (sexta-feira)


08:30 - 12:30 Sesso Temtica VII Coordenador: Valdir de Queiroz Balbino Daniel Bray Synthetic pheromone: a potential tool for L. longipalpis Fernando Arajo Monteiro Sistemtica molecular de triatomneos: novas hipteses e descobertas Valdir de Queiroz Balbino Sandfly Database: integrando dados biolgicos e moleculares de flebotomneos Wanderli Pedro Tadei Malria: perspectiva do controle com novas tecnologias e o cenrio genmico Jos Carlos Miranda Criadouros naturais de flebotomineos Jos Dilermando Andrade Filho Flebotomineos: mitos ou verdades

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

14:00 - 18:00 Sesso Temtica VIII Coordenador: Paulo Filemon Pimenta Gregory Lanzaro Challenges in defining the complex genetic structure of Anopheles gambiae Margareth Capurro Controle da dengue por mosquitos transgnicos: paper ou realidade? Mary Ann McDowell Sandfly genome project Iliano Vieira Coutinho Abreu Gomes Ecological genetics of sand fly salivary gland genes Pedro Lagerblad de Oliveira Analysis of transcriptome of Rhodnius prolixus midgut

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

PALESTRANTES
1. Aldina Barral (CPqGM/Fiocruz/BA) 2. Alessandra Gutierrez Oliveira (UFMS) 3. Alexandre Peixoto (IOC/Fiocruz/RJ) 4. Andre Nbrega Pitaluga (IOC/Fiocruz/RJ) 5. Beatriz Gomes Brazil (UFMS) 6. Beatriz Rossetti Ferreira (USP) 7. Carlos Brisola Marcondes (UFSC) 8. Carlos Henrique Nery Costa (UFPI) 9. Carlos Logullo (UENF) 10. Cecilia de Oliveira Cudischevitch (UFRJ); 11. Clarissa Teixeira (NIAID/NIH, USA) 12. Constncia Junqueira Ayres (CPqAM/Fiocruz/PE) 13. Daniel Bray (Keele University, UK) 14. David W. Severson (University of Notre Dame, USA) 15. Edelberto Santos Dias (CPqRR/Fiocruz/MG) 16. Elizabeth Rangel (IOC/Fiocruz/RJ) 17. Elyes Zhioua (Pasteur Institute of Tunis, Tunsia) 18. Eunice Galati (USP) 19. Felipe Arley Pessoa (CPqLMD/Fiocruz/AM) 20. Fernando Arajo Monteiro (IOC/Fiocruz/RJ) 21. Filipe Dantas Torres (Universit degli Studi di Bari, Itlia) 22. Gregory Lanzaro (University of California Davis, USA) 23. Iliano Vieira Coutinho Abreu Gomes (Kansas State University, EUA) 24. James Gordon C. Hamilton (Keele University, UK) 25. Jeffrey Jon Shaw (USP) 26. Jesus Valenzuela (NIAID/NIH, USA)

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

27. Joo Pinto (Instituto de Higiene e Medicina Tropical, Lisboa, Portugal) 28. Jos Carlos Miranda (CPqGM/Fiocruz/BA) 29. Jos Dilermando Andrade Filho (CPqRR/Fiocruz/MG) 30. Lda Narcisa Rgis (CPqAM/Fiocruz/PE) 31. Marcelo Ramalho Ortigo (Kansas State University, USA) 32. Marcos Horcio (UFMG) 33. Marcos Sorgine (UFRJ) 34. Margareth Capurro (USP) 35. Maria de Fatima Freire Ximenes (UFRN) 36. Maria Helena Lobo Neves (CPqAM/Fiocruz/PE) 37. Mary Ann McDowell (University of Notre Dame, USA) 38. Ngila Costa Secundino (CPqRR/Fiocruz/MG) 39. Nicolau Maus Serra Freire (IOC/Fiocruz/RJ) 40. Orin Courtenay (University of Warwick, UK) 41. Paul Ready (National History Museum, UK) 42. Paulo Filemon Pimenta (CPqRR/Fiocruz/MG) 43. Pedro Lagerblad de Oliveira (UFRJ) 44. Pedro Marcos Linardi (UFMG) 45. Reginaldo Peanha Brazil (IOC/Fiocruz/RJ) 46. Rodrigo Soares (CPqRR/Fiocruz/MG) 47. Sergio Luz (CPqLMD/Fiocruz/AM) 48. Sinval Pinto Brando Filho (CPqAM/Fiocruz/PE) 49. Tereza Magalhes (CPqAM/Fiocruz/PE) 50. Valdir de Queiroz Balbino (UFPE) 51. Vladimir Costa (UFPI) 52. Walter Leal (University of California Davis, USA) 53. Wanderli Pedro Tadei (INPA)

NDICE
Carlos Brisola Marcondes Ovos de mosquitos: negligenciados, mas importantes (e bonitos).............................................15 Eunice Galati Capacidade vetorial de vetores permissivos de Leishmania......................................................19 Carlos Henrique Nery Costa The role of Lutzomyia longipalpis in the urbanization of kala-azar in brasil...............................................................................................20 Edelberto Santos Dias Eco-epidemiologia da leishmaniose visceral em Minas Gerais..................................................23 Elizabeth Rangel Lutzomyia (N.) whitmani and the eco-epidemiology of american cutaneous leishmaniasis in brazil ...........................................................................25 Sinval Pinto Brando Filho The infectiousness of experimental infections of Leishmania (Viannia) braziliensis in wild and synanthropic rodents to the sand fly Lutzomyia longipalpis........................................................29 Leda Rgis Dados recentes sobre impacto da remoo mecnica de ovos e adultos sobre densidades populacionais de Aedes aegypti em dois municpios de Pernambuco ........................................................................................................31 Walter Leal Oviposition attractans for Culex quinquefasciatus: their receptors, reception, and use in vector management.........................................................35 Joo Pinto Genetica populacional e controlo de vectores............................................................................40 Reginaldo Peanha Brazil Impact of climatic changes and habitat degradation on phlebotominae (Diptera: Psychodidae) distribution and leishmaniases dispersion in brazil........................................................................................44 Jeffrey Jon Shaw Climatic changes and its impact on the eco-epidemiology of tropical diseases........................................................................................46 Alexandre Peixoto Molecular chronobiology of insect vectors..................................................................................52 Felipe Arley Pessoa Mansonella spp e vetores no Amazonas....................................................................................55 Srgio Luz Sensitivity of ovitraps and adultraps for detecting household infestation by the dengue vectors Aedes aegypti and Aedes albopictus..........................................................................................59 Cecilia de Oliveira Cudischevitch Hematophagy and cell signaling in mosquitoes: he role of protein tyrosine phosphatases as modulators of Aedes aegypti immunity.......................................................................................62

Alessandra Gutierrez Estudo da disperso de Lutzomyia longipalpis em Campo Grande, Mato Grosso do Sul....................................................................................65 Constncia Junqueira Ayres Bases moleculares da resistncia ao temephos em Aedes aegypti ......................................................................................................................69 Ngila Costa Secundino Proteofosphoglicano (PPG) conferindo Leishmania major resistncia a enzimas digestivas induzidas pela alimentao sanguinea em flebotomneos vetores........................................................................................76 Nicolau Maus Serra Freire Aspectos da reproduo de Ixodida e a transmisso vertical de riqutsias patognicas...........................................................................78 Pedro Marcos Linardi Epidemiologia das pulgas...........................................................................................................82 Paul Ready Sandfly taxonomy is an imperfect predictor of vectorial traits.....................................................90 Alexandre Peixoto Molecular chronobiology of insect vectors..................................................................................91 Gregory Lanzaro Genetic variation in the salivary protein maxadilan among population of Lutzomyia longipalpis................................................................................94 Beatriz Ferreira Tick saliva contains novel non-proteic molecules with Immunomodulatory properties....................................................................................................95 Tereza Magalhes RNAi em mosquitos: defesa natural antiviral e ferramenta tecnolgica...........................................................................................................97 Carlos Logullo Molecular strategies to ectoparasite control.............................................................................101 Rodrigo Soares Aspectos moleculares da interao de lipofosfoglicanos de Leishmania com o intestino mdio de flebotomneos..........................................................................................103 Clarissa Teixeira Comparing the initial immune response in the skin of animals exposed to uninfected and Leishmania infected sand flies..................................................................................................107 Paul Ready Is there an arms race involving salivary peptides of the vectors of zoonotic visceral leishmaniasis?....................................................................109 Paulo Filemon Pimenta O processo de interao do Lutzomyia (Lutzomyia) longipalpis com a Leishmania (Leishmania) chagasi................................................................112 Marcelo Ramalho Ortigo Reverse genetics in sand flies: targeting midguts molecules affects Leishmania development.............................................................................115

Fernando Arajo Monteiro Triatomine molecular systematics: new findings and new hypotheses...................................................................................................117 Valdir de Queiroz Balbino Sandfly database: development of an integrated platform of biological and molecular data of sandflies (Diptera: Psychodidae) of medical and veterinary importance ................................................120 Wanderli Pedro Tadei Anlise epidemiolgica da malria em manaus/am considerando a estratificao por distrito sanitrio..................................................................122 Jos Carlos Miranda Criadouro de Lutzomyia longipalpis em rea endmica para leishmaniose visceral no estado da Bahia.......................................................124 Jos Dilermando Andrade Filho Flebotomineos: mitos ou verdades ..........................................................................................126 Gregory Lanzaro Challenges in defining the complex genetic structure of Anopheles gambiae...............................................................................................129 Margareth Capurro Mosquitos transgnicos, do paper a realidade.......................................................................130 Mary Ann McDowell Sandfly genome project............................................................................................................132 Iliano Vieira Coutinho Abreu Gomes Ecological genetics of sand fly salivary gland genes................................................................135 Pedro Lagerblad de Oliveira Analysis of transcriptome of Rhodnius prolixus midgut.............................................................137 Filipe Dantas Torres Ticks as vectors of Leishmania Parasites: facts and speculations.........................................................................................140 Maria Helena Lobo Neves Silva Filha Caracterizao de mecanismos de resistncia de mosquitos a larvicidas biolgicos.........................................................................................145

IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

RESUMOS
MOSQUITO EGGS: NEGLECTED BUT IMPORTANT (AND BEAUTIFUL) Carlos Brisola Marcondes
Departamento de Microbiologia, Centro de Cincias Biolgicas, UniversidadeFederal de Santa Catarina, Florianpolis, SC, Brasil

Mosquito eggs have been studied, initially utilizing light microscopy (LM), and since 1970's also by Scanning Electron Microscopy (SEM). These studies, many of them developed by J. R. Linley, revealed the exquisite structural detail of mosquito eggs, and his work generated 27 publications describing the ultrastructure of eggs of 56 mosquito species, of nine genera, before his premature decease (Lounibos 1995), besides several others published after his demise (e.g., Lounibos et al. 1997). In addition to the importance for studies of biology and systematics, the sheer aesthetic beauty of his photographs (Lounibos 1995) deserves appreciation. The study of mosquito eggs is useful for species differentiation in breeding sites and for systematics, but because a small number has been studied, compared to the total number of known mosquito species (almost 3,400), this information has rarely been utilized. For example, from 218 species of Sabethini in the American continent (Marcondes & Zillikens 2006), only two (Trichoprosopon digitatum (Rondani) and Tr. compressum Lutz) had their eggs studied by SEM (Linley 1990; Linley et al. 1991), and only about 12% of eggs of 1,275 species of Aedini have been described, most of them only by LM (Reinert 2005), making the utilization of egg morphology for systematics difficult (e.g., Reinert et al. 2004). Even in Anopheles, the most extensively studied genus, a small proportion of its 472 species have their eggs characterized. Egg external structure contributed, for example, to differentiate some species of Anopheles albimanus complex (Lounibos et al. 1997) and An. antunesi Galvo & Amaral from An. pristinus n. sp. (Nagaki et al. 2010).

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IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

Eggs of six of 28 species of Haemagogus were described, and they can be differentiated by the shape of their chorionic cells and distribution of tubercles. In Hg. capricornii Lutz, these cells have a porous appearance, symmetrically elongated in the periphery, with great rounded tubercles (Alencar et al. 2005a). Hg. tropicalis Cerqueira & Antunes has always a central greater tubercle surrounded by smaller ones, contrasting to other species of Haemagogus (Alencar et al. 2008). The description of Ochlerotatus terrens (Walker) eggs (Alencar et al. 2005b), which also occur in tree holes, is useful to differentiate them from Haemagogus eggs. Oc. albifasciatus (Macquart) and Oc. scapularis (Rondani) eggs, which develop in ground pools, can be easily differentiated by the shape and distribution of tubercles (Santos-Mallet et al. 2009, 2010). Contrastingly, eggs of An. cruzii (Dyar & Knab) from An. bellator (Dyar & Knab) could not be differentiated (Forattini & Marucci 1993). The shape of the micropyle and its collar and of tubercles, besides the grouping of tubercles in chorionic cells, is very important to discriminate species, and multivariate analysis has also been utilized for this purpose. Eggs of An. aquasalis Curry from two Venezuelan localities (Maldonado et al. 1997) and species of An. quadrimaculatus complex (Linley et al., 1993) were differentiated, utilizing discriminant analysis of egg structures. Multivariate analysis of Culex quinquefasciatus Say eggs from four ecologically different localities in the West of India indicated a strong positive correlation (0.95) between the cluster distance of egg attributes and geographical distance (Suman et al. 2009). Besides usual analysis of exochorium (=chorium), the endochorium (=vitelline membrane) of Psorophora confinnis complex mosquitoes from 12 localities in seven states of United States revealed at least 13 distinct variations in endochorionic pattern (Bosworth et al. 1983). Studies on eggs of similar species and/or living in similar breeding places, like Oc. scapularis and Oc. rhyachophilus (Costa Lima) and the 22 known Phoniomyia

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IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

species, should be prioritized. Assessment of changes in egg morphology may be used for toxicity studies. Garlic damaged the structure of eggs of Aedes aegypti (Jarial 2001), and this effect should be checked on tests or substances potentially useful to the control of mosquitoes. Eggs can be obtained from wild-collected or laboratory bred females, maintained in vials with plaster of Paris or water. Females should be untouched in the first 24 hours after feeding, due to formation of peritrophic matrix, and observed for 3-4 days. Egg-laying may be stimulated by pulling one (WRBU 2010) or both (A. Fernandes- pers. commun.) wings from gravid females that were previously anesthetized by exposition to low temperatures (~4oC) for some minutes. In our laboratory, eggs of Ocherotatus spp. and An. cruzii have been very easily obtained by the maintenance of females in a small plastic tube, with plaster of Paris, but the same technique has had low productivity for Sabethini species, even wetting the plaster with water from bamboo internodes and bromeliads. Mondet (1997) proposed a method to obtain eggs from Haemagogus mosquitoes, not yet tested for other species; he modified the usual technique of maintenance of female in a simple vial with water, providing a second one, in which it can hide in the dark.

REFERENCES Alencar J, Degallier N, Guimares AE, Costa JM, Marques WA, Silva VC, Santos-Mallet JR 2008. Scanning electron microscopy of the egg of Haemagogus tropicalis. J Am Mosq Control Assoc 24: 16-20. Alencar J, Guimares AE, Mello RP, Lopes CM, Degallier N, Santos-Mallet JR 2005a. Scanning electron microscopy study of the egg of Haemagogus (Haemagogus) capricornii Lutz, 1904 (Diptera: Culicidae). J Med Entomol 42: 1-6. Alencar J, Guimares AE, Gil-Santana HR, Santos-Mallet JR 2005b. Scanning electron microscopy of eggs of Ochlerotatus (Protomacleaya) terrens Walker. J Am Mosq Control Assoc 21: 355-359. Bosworth AB, Meola SM, Thompson M, Olson JK 1998. Chorionic morphology of eggs of the Psorophora confinnis complex in the United States. II. Pre- and post deposition studies of Psorophora columbiae (Dyar and Knab) eggs. J Am Mosq Control Assoc 14: 46-57. Forattini OP & Marucci D 1993. Scanning electron microscopy of the eggs of two species of Anopheles (Kerteszia) (Diptera:Culicidae). Mem Inst Oswaldo Cruz 88: 349-352.

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Jarial MS 2001. Toxic effect of garlic extracts on the eggs of Aedes aegypti (Diptera: Culicidae): a scanning electron microscopic study. J Med Entomol 38: 446-450. Linley JR 1991. The egg of Trichoprosopon compressum (Diptera: Culicidae). Mosq System 23: 161-169. Linley JR, Lounibos LP, Linley PA 1990. Fine structure of the egg of Trichoprosopon digitatum (Diptera: Culicidae) and its relationship to egg raft formation. J Med Entomol 27: 578-585. Linley JR, Kaiser PE, Cockburn AF 1993. A description and morphometric study of the eggs of species of the Anopheles quadrimaculatus complex (Diptera: Culicidae). Mosq System 25: 124147. Lounibos LP 1995. Obituary: John R. Linley. J Am Mosquito Control Assoc 11: 244-245. Lounibos LP, Duzak D, Linley JR 1997. Comparative egg morphology of six species of the Albimanus section of Anopheles (Nyssorhynchus) (Diptera: Culicidae). J Med Entomol 34: 136155. Lounibos LP, Duzak D, Linley JR 1997. Comparative egg morphology of six species of the Albimanus section of Anopheles (Nyssorhynchus) (Diptera: Culicidae). J Med Entomol 34: 136155. Maldonado V, Finol HJ, Navarro JC 1997. Anopheles aquasalis eggs from two Venezuelan localities compared by scanning electron microscopy. Mem Inst Oswaldo Cruz 92: 487-491. Marcondes CB, Zillikens A 2006. Mosquitos-borboletas. Cincia Hoje 38: 66-67. Mondet B 1997. Condies de sobrevivncia em laboratrio de Haemagogus janthinomys Dyar, 1921 (Diptera: Culicidae). Rev Soc Bras Med Trop 30: 11-14. Nagaki SS, Motta MA, Sallum MA 2010. Redescription of Anopheles (Nyssorhynchus) antunesi Galvo & Amaral and description of a new species of the Myzorhynchella Section (Diptera: Culicidae) from Serra da Mantiqueira, Brazil. Mem Inst Oswaldo Cruz 105: 27885. Reinert JF 2005. List of species described in the egg stage of tribe Aedini (Diptera: Culicidae) with their literature citations. J Am Mosq Control Assoc 21: 252-262. Reinert JF, Harbach R, Kitching IJ 2004. Phylogeny and classification of Aedini (Diptera: Culicidae), based on morphological characters of all life stages. Zool J Linn Soc 142: 289-368. Santos-Mallet JR, Gleiser RM, Alencar J, Marques WA, Sarmento JS, Marcondes CB 2009. Scanning electron microscopy of the egg of Ochlerotatus albifasciatus (Diptera: Culicidae). J Med Entomol 46: 980-985. Santos-Mallet JR, Mller GA, Gleiser RM, Alencar J, Marques WA, Sarmento JS, Marcondes CB 2010. Scanning electron microscopy of the eggs of Aedes scapularis from southern South America. J Am Mosq Control Assoc 26: 205-209. Suman DS, Shrivastava AR, Parashar BD, Pant SC, Agrawal OP, Prakash S 2008. Scanning electron microscopic studies on egg surface morphology and morphometrics of Culex tritaeniorhynchus and Culex quinquefasciatus (Diptera: Culicidae). Parasitol Res 104: 173-176. WRBU 2010. http://www.wrbu.org/techniques.html (September 30, 2010)

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IV Workshop de Gentica e Biologia Molecular de Insetos Vetores de Doenas Tropicais

CAPACIDADE VETORIAL DE VETORES PERMISSIVOS DE LEISHMANIA Eunice A B Galati


Departamento de Epidemiologia, Faculdade de Sade Pblica, Universidade de So Paulo. Av. Dr. Arnaldo, 715, 01246904, So Paulo. E-mail: egalati@usp.br

A leishmaniose visceral americana (LVA) vem acometendo populaes caninas e humanas de vrios centros urbanos e encontra-se em expanso. Em algumas reas tem sido identificada a infeco pela Leishmania (L.) infantum chagasi na populao canina e felina, sem que a comprovada espcie vetora, Lutzomyia longipalpis, tenha sido encontrada. Em contrapartida, esto presentes outras espcies de flebotomneos em densidade elevada e estreita relao com reservatrios desse parasita que talvez Situao

possam estar atuando como vetoras permissveis da L. (L.) i. chagasi.

semelhante pode ser encontradas em relao aos agentes da leishmaniose tegumentar americana (LTA). Portanto, necessria a investigao da capacidade vetorial dessas espcies de flebotomneos na transmisso da Leishmania (L.) infantum chagasi e dos agentes da LTA, Leishmania spp, comparando-as com a de espcies que so reconhecidamente vetoras desses agentes, para se dimensionar o risco de transmisso dessas parasitoses em uma dada rea. Para a investigao da capacidade vetorial, os seguintes parmetros necessitam ser identificados: densidade das espcies de flebotomneos em relao aos hospedeiros e infeco experimental pelos parasitas a partir da alimentao dos flebotomneos em fontes de infeco das Leishmania spp existentes na localidade, identificao da probabilidade diria de sobrevivncia dos flebotomneos, durao do ciclo gonotrfico, perodo de incubao extrnseca dos parasitas nos flebotomneos, proporo de flebotomneos infectantes e transmisso experimental dos parasitas para novos hospedeiros. A comparao entre a capacidade vetorial das espcies (V) dar-se- pelo valor obtido na seguinte frmula: V = ma2pn b / loge p, onde: m = densidade do vetor em relao ao hospedeiro; a = AI/GC, onde AI a proporo de fmeas se alimentando no hospedeiro e GC a durao do ciclo gonotrfico; b = proporo de vetores portando um agente infeccioso que efetivamente infectivo; p = probabilidade de sobrevivncia diria do vetor; n = durao do perodo de incubao extrnseca, em dias e, 1/- loge p = expectativa de vida.

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THE ROLE OF LUTZOMYIA LONGIPALPIS IN THE URBANIZATION OF KALA-AZAR IN BRASIL Carlos H. N. Costa
Federal University Of Piaui

American visceral leishmaniasis was described in the past in South America as a rural disease, mostly from the semi-arid Brazilian Northeast. Although smaller towns were described with urban transmission earlier, only in the beginning of the 80s the urban transmission spread to larger cities. Starting in Teresina and So Lus, the capitals of the Mid-North, the disease appeared in very far apart regions in epidemic waves spreading to other larger cities in the Northeast, the North, the Center-West, the Southeast and finally the South, leaving few states free of the disease. Therefore, the understanding of the causes that led to the phenomenon of urbanization of kala-azar in Brazil is of foremost priority to discover critical strategies to control the disease. At least two paths of explanation are reasonable: those related to the agent Leishmania infantum and those associated with the vector Lutzomyia longipalpis. Both can be genetically or environmentally determined. The parasite parameters that can affect transmission are those that determine the ability to reach the vector, and those factors that influence the capability to infect the host. The vector parameters that might be involved with the urbanization are those related to the allowance of parasite attachment in the midgut, the midgut environment for parasite development, the biting rate, and mortality. However, the history of the urbanization suggests that parasite factors are not in the game. Indeed, mapping the involvement of larger cities with the urbanization shows that the emergence of this process was not from a place to closer places as the transmission of a new strain from host to host would be, but rather it emerged as a multicentric process hitting far apart cities from the Northeast (Teresina and So Lus, in 1981 and 1982), North (Santarm in 1984),

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Southeast (Montes Claros in 1984) and Center-West (Corumb, in 1984). Additionally, some cities close to older endemic foci were never hit, like Belm, Recife and Salvador. Sand flies linked factors look more logic to explain the urbanization of kalaazar. Due to the same reasons as for parasites, vector genetic factors by themselves may not be able to justify the process. However, if coupled to environmental changes, they may have been helpful for the urbanization. While Leishmania infantum is new in the Americas, with little genetic variability, Lu. longipalpis evolved in the New World and its genetic diversity is broad. If an extensive environmental change happened, one of such a population could be selected, finding a new and unoccupied niche in the cities. Two categories of environmental changes have been proposed. One is linked to social events and suggests that migrations of people from endemic rural areas into cities, where surrounding woodland has been devastated and living conditions are very poor, might have created the conditions for sand fly proliferation. However, cities typically with these characteristics and close to endemic areas were never hit by kala-azar, like Recife and Salvador, and cities with recent kala-azar transmission have not been subjected to such a wave of migration and poverty like Campo Grande, Araatuba and Bauru. Therefore, another type of environmental change has to be proposed, which may be easily established and that may be common to many geographically and economically apart cities. One such a change is urban landscaping. Non-endemic species of trees can be rapid introduced, bring with them a particular community of living organisms. Thus, landscaping may have altered vectors lives by several ways. Since sand flies depend on vegetation to feed and for protection, recently introduced trees may have provided some sort of special advantage to sand flies by the absence of associated predators or by offering advantageous nutrients; these factors might increase the biting rate by expanding the sand fly population or by

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bringing them to the proximity of humans and dogs. Also, they can change the most critical parameter for the transmission of vector borne diseases, which is the probability to survive the extrinsic incubation period. This can be achieved by either getting plant-associated sugars that could fasten the development of L. infantum to metacyclic promastigotes, or by reducing the daily sand fly mortality. The main candidates to be the factor of urbanization of kala-azar in Brazil are the acacias. Acacia seyal was described as associated to urban epidemics in Sudan and Acacia podalyriifolia (Mt. Morgan wattle or Queensland silver wattle) is widespread in Brazilian cities and is recommended by landscapers for urban treeplanting. However, to prove it one has to show the association of urban Lu. longipalpis to particular species of plants and to demonstrate the genetic diversity of this vector from urban epidemic areas relative to those from rural non-epidemic settings.

References Costa CH 2008. Characterization and speculations on the urbanization of visceral leishmaniasis in Brazil. Cad Saude Publica. 24: 2959-2963. de Queiroz Balbino V, Coutinho-Abreu IV, Sonoda IV, Melo MA, de Andrade PP, de Castro JA, Reblo JM, Carvalho SM, Ramalho-Ortigo M 2006. Genetic structure of natural populations of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) from the Brazilian northeastern region. Acta Trop. 98: 15-24. Costa CH, Pereira HF, Arajo MV 1990. Epidemia de leishmaniose visceral no Estado do Piau, Brasil, 1980-1986. Rev Saude Publica. 24: 361-72. Rogers DJ 1988. The dynamics of vector-transmitted diseases in human communities. Philos Trans R Soc Lond B Biol Sci. 321: 513-39, 1988. Elnaiem DEA, Mukhawi AM, Hassan MM, Osman ME, Osman OF, Abdeen MS, et al. Factors affecting variations in exposure to infections by Leishmania donovani in eastern Sudan 2003. East Mediterr Health J 9: 827-36. Yamamoto MA, Schimidt ROL, Couto HTZ, Silva Filho DF 2004. rvores urbanas. Piracicaba: Prefeitura Municipal de Piracicaba, 22pp.

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ECO-EPIDEMIOLOGIA DA LEISHMANIOSE VISCERAL EM REAS ENDMICAS DO ESTADO DE MINAS GERAIS, BRASIL Edelberto Santos Dias A Leishmaniose Visceral Americana (LVA) uma zoonose em processo de expanso tanto em nmero de casos, quanto em sua distribuio geogrfica, apresentando elevada capacidade no de novos focos de transmisses em regies rurais e urbanas. Alm de se manter endmica em reas de transmisso antigas. Nas Amricas, cerca de 90% dos casos humanos tm sido registrados no Brasil. A leishmaniose visceral americana (LVA), est notificada em 23 dos 27 estados brasileiros, distribudos em todas as regies do pas. As alteraes no ambiente natural e os constantes movimentos migratrios das populaes carentes do campo para a periferia das cidades tm facilitado este processo. Em levantamento sobre os mtodos de controle para as leishmanioses cutnea e visceral pelo Ministrio da Sade, foi destacada a dificuldade para avaliar em que a extenso os mtodos de controle empregados tem reduzido o nmero de casos em uma ou outra forma da doena. Segundo o levantamento, existe a necessidade urgente de aproximar os profissionais envolvidos na rea de pesquisa e sade para, juntos, reverem as atuais estratgias de controle e redefinirem procedimentos e medidas capazes de assegurar um impacto real sobre o controle desta doena. As medidas preconizadas pelo programa brasileiro de controle da LVA, iniciado h mais de 40 anos, composto de trs medidas bsicas de sade pblica: a distribuio gratuita do medicamento para tratamento especfico dos casos humanos notificados; o controle de reservatrios domsticos e o uso de inseticidas visando o controle vetorial. Entretanto, muitos pontos da epidemiologia e profilaxia da LVA no esto, ainda, devidamente esclarecidos, o que dificulta a escolha das melhores estratgias de controle. Para tal, estamos desenvolvendo projetos de Pesquisa Aplicada em colaborao multinstitucional e multidisciplinar envolvendo pesquisadores e tcnicos das seguintes instituies: Instituto de Cincias Biolgicas da UFMG; Escola de Farmcia da UFOP; Centro de Pesquisas Ren Rachou - FIOCRUZ; Faculdade de Medicina do Tringulo Mineiro UFTM; e com o apoio logstico da Fundao Nacional de Sade; Secretaria Estadual de Sade de Minas Gerais, tendo como coordenadores o Dr. Aluizio Prata na rea humana, Dr. Joo Carlos Frana da Silva na rea canina, Dr. Edelberto Santos Dias na rea entomolgica, Dr. George Luiz Lins Machado-Coelho nas anlises epidemiolgicas.

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A proposta geral de nossa apresentao divulgar alguns resultados obtidos de uma maneira sincronizada das trs variveis envolvidas na transmisso da doena (casos humanos, caninos e a fora de infeco vetorial) visando apontar s principais reas de risco e direcionar de uma forma contundente as aes de controle da LV em municpios endmicos do estado de Minas Gerais.

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Lutzomyia (N.) whitmani AND THE ECO-EPIDEMIOLOGY OF AMERICAN CUTANEOUS LEISHMANIASIS IN BRAZIL

Elizabeth F. Rangel
Laboratrio de Transmissores de Leishmanioses Instituto Oswaldo Cruz - Fiocruz

Colaboradores: Simone M. Costa & Daniel Motta-Silva, Lab. de Transmissores de Leishmanioses, IOC; Christovam Bracellos, Instituto de Comunicao e Informao Cientfica e Tecnolgica em Sade/ FIOCRUZ; Michela Cechinel, Secretaria de Vigilncia em Sade, MS

American Cutaneous Leishmaniasis (ACL) has been registered in all Brazilian States, it is in process of territorial expansion, and the disease incidence is increasing in various regions of the country at the same time it shows changes in its epidemiological profile. Moreover even the dynamics of transmission is associated to a variety of parasites and vectors, in a close relationship in restricted ecological niches. Currently, we can admit Lutzomyia (N.) whitmani as the most important vector of ACL in Brazil, where it has been recorded in all geographic regions in association with different vegetation types, which explains its presence in biomes including Amazonian Forest, Savanna (campos cerrados), Northeastern Savanna (Caatinga) and Atlantic Forest. It is clearly adapted to different climatic conditions and to preserved and impacted areas, where presents different habits in a diversity of habitats, ranging from the wild environment to the interior of the residences (Rangel & Lainson 2009). L. (N.) whitmani is incriminated as vector of two leishmania related to ACL: Leishmania (V.) shawi only in the Amazon, and Le. (V.) braziliensis in all Brazilian geographic regions. Still, on the vector competence, recent studies report that the adhesion of parasites (Le. (V.) braziliensis) in the midgut of L. (N.) whitmani was more evident when compared to L. (N.) intermedia (Soares et al. 2010). The literature also shows eco-epidemiological studies involving this vector, describing different biological aspects in several regions of Brazil. The studies on feeding habits of this sand fly reveals that this species is an opportunist feeder, including the anthropophily, and shows the usual

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feeding pattern for sand flies, starting at dusk and may extend until dawn (Rangel & Lainson 2009); but in Southern Brazil studies have shown that L. (N.) whitmani has diurnal activity in an urban area in Paran State (Santos et al. 2009). In investigations conducted in the Northeast, Southeast, South and Central Brazil this sand fly species often is close related to domestic environment, being able to be collected inside the house, and feeding in domestic animals (Rangel & Lainson 2009) In Amazon region, in several areas of Par (PA) State, studies recorded different habits for L. (N.) whitmani from those observed in other regions: it was found to be essentially silvatic and could be collected at the base of the trunk, and especially in the canopy of trees, associated with wild animals, besides showing little tendency to bite man. The impact of environmental natural changes as well those determined by man actions, has caused changes and degradation in ecosystem, enabling the emergence of epidemics and maintenance of ACL transmission cycles. Different aspects of ecology, in association with climatic changes, probably play an important role in the spread of leishmaniases in Brazil in recent years and in this context, L. (N.) whitmani seems to be the sand fly species that best fits the new environments, in association with humans and domestic animals in both, rural and peridomiciliary habitats (Costa et al. 2007, Shaw 2007, 2008) Facing with the magnitude of the health problem represented by ACL, the Brazilian National Programme of Leishmaniases, of the Brazilian Ministry of Health, has been analyzing the spatialization in the perspective of epidemiological circuits of disease, represented by a concentration of human cases during a determinate period; the circuits are composed by areas with different levels in density of cases, which may show a tendency to either expand or contract. Initial evidence has shown that L. (N.) whitmani occurs in most ACL epidemiological circuits (Costa et al. 2007). The definition of determinant factors in the expansion of ACL passes through the dispersion analysis of L. (N.) whitmani and geoprocessing technology can be useful in obtaining data. Recent studies to better assess the existence of spatial aggregation of L. (N.) whitmani and ACL through

Geographic Information System (GIS) have highlighted the existence of spatial aggregation
of this sand fly vector and human cases. The Brazilian regions North (Acre, Amazonas, Rondnia, Par, Tocantins and Amap), Central West (Mato Grosso) and Northeast (Cear and Pernambuco) represent the highest number of ACL cases concomitant with the presence of L. (N.) whitmani.

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Considering the hypothesis proposed by Lainson (1988) that L. (N.) whitmani could be a complex of cryptic species of two or more taxa, based on evidence from such diverse habitats and habits, studies have been discussed about the taxonomic identity of this sand fly vector (Rangel et al. 1996, Ready et al. 1997, 1998, Margonari et al. 2004). In the matter involving L. (N.) whitmani with the transmission of two different species of Leishmania, from the viewpoint of specificity between parasite and vector it would be reasonable to assume that this sand fly would actually be a species complex, based on all evidences of behavior and vector competence. In this context, to give support to the hypothesis that L. (N.) whitmani s.l. from the Amazon region (Dom Elizeu-Paragominas/PA and Buriticupu/MA), could represent a lineage isolated from others (Northeast and Southeast) corroborating the existence of "whitmani complex, some suggested evidences in the literature should be considered: silvatic habits and habitats, transmission of L. (V). shawi, besides the differences presented in morphometric and molecular studies.

Phylogenetic analyses based on molecular markers strongly suggest a

correlation of populations / clades with climatic and geographic determinants.


However, this aspect is not yet sufficiently clear and deserves further study.
Supported by CNPq.Edital Universal and Fiocruz

References Costa SM, Cechinel M, Bandeira V, Zannuncio JC, Lainson R, Rangel EF 2007. Lutzomyia (Nyssomyia) whitmani s.l. (Antunes & Coutinho, 1939) (Diptera: Psychodidae: Phlebotominae) and the Epidemiology of American Cutaneous Leishmaniasis in Brazil. Mem Inst Oswaldo Cruz 102(2): 149-153. Lainson R 1988. Ecological interactions in the transmission of the leishmaniasis. Philosophical Transactions of the Royal Society London Serie B 321: 389-404. Margonari CS, Dias-Forte CL, Dias ES 2004. Genetic variability in geographical populations of Lutzomyia whitmani elucidated by RAPD-PCR. J Med Entomol 41: 187-192. Rangel EF, Lainson R 2009. Proven and putative vectors of American cutaneous leishmaniasis in Brazil: aspects of their biology and vectorial competence. Mem Inst Oswaldo Cruz 104(7): 937-954. Rangel EF, Lainson R, Souza AA, Ready P, Azevedo ACR 1996. Variation

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between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera: Psychodidae: Phlebotominae) in Brazil. Mem Inst Oswaldo Cruz 91: 43-50. Ready PD, Day JC, Souza AA, Rangel EF, Davies CR 1997. Mitochondrial DNA characterization of populations of Lutzomyia whitmani (Diptera: Psychodiade) incriminated in the peridomestic and silvatic transmission of Leishmania species in Brazil. Bull Ent Res 87: 187-195. Ready PD, Souza AA, Reblo JMM, Day JC, Silveira FT, Campbell-Lendum D, Davies CR, Costa JML 1998. Phylogenetic species and domesticity of Lutzomyia whitmani at the south-east boundary of Amazonian Brazil. Bull Entomol Res 87: 187-195. Santos DR, Santos AR, Santos ES, Oliveira O, Poiani LP, Silva AM 2009. Observaes sobre a atividade diurnal de Nyssomyia whitmani (Diptera: Psychodidae), na rea urbana de Maring, Paran, Brasil. Epidemiol Serv Sade, Braslia 18(3): 223-232. Shaw 2007. The leishmaniasis survival and expansion in a changing world. A mini-review. Mem Inst Oswaldo Cruz 102: 541-547. Shaw 2008. How climatic and environmental variations affect the ecoepidemioly of the leishmaniases and their control. III Workshop de Gentica e Biologia Molecular de Insetos Vetores, 13pp. Soares RP, Margonari C, Secundino NC, Macedo ME, da Costa SM, Rangel EF, Pimenta PF, Turco SJ 2010. Differential midgut attachment of Leishmania (Viannia) braziliensis in the sand flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia. J Biomed Biotechnol 827-851.

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THE INFECTIOUSNESS OF EXPERIMENTAL INFECTIONS OF Leishmania (Viannia) braziliensis IN WILD AND SYNANTHROPIC RODENTS TO THE SAND FLY Lutzomyia longipalpis Maria S. Andrade1, Maria E. F. Brito1, Francisco G. Carvalho1 , Hlio F. Valena1 , Ericka L. Almeida1, Edeneide Maria Xavier1, Fbia C. Soares1, Jos C. Miranda2 , Jeffrey J. Shaw3, Sinval P. Brando-Filho 1
1 Centro de Pesquisas Aggeu Magalhes, Fundao Oswaldo Cruz, Recife, PE, Brazil 2 Centro de Pesquisas Gonalo Moniz, Fundao Oswaldo Cruz, Salvador, BA, Brazil 3 Departamento de Parasitologia, Universidade de So Paulo, So Paulo, SP, Brazil

Determining the infectiousness of mammals incriminated as reservoirs is a crucial step in understanding their relative importance in the enzootic cycle. Colonies were established of 3 rodents (Rattus rattus, Necromys lasiurius , Nectomys squamipes) that had previously been incriminated as potential reservoirs by parasitological and molecular methods (Brando-Filho et al 2003). The animals were bred and held after infection in the Aggeu Magalhes Research Centre level 3 biosecurity animal facility. In the first experiment 10 animals of each species were used, each animal was inoculated with a total of 5.5 x 106 log phase promastigotes of L.(V.) braziliensis (strain MNEC/BR/2000/CPQAM95 zymodeme IOC-Z74). The inoculum was divided into three and inoculated subcutaneously into the ear, the hind foot pad and intraperitoneally. The same experiment was repeated in 10 more animals of each species with an inoculum of 2.8 x 105 log phase promastigotes per animal. Six months after being infected laboratory reared Lu. longipalpis were fed on each animal by restraining it in a cage containing male and female sandflies. A total of 1,248 females were successfully fed and of these 805 were dissected. In the first experiment, in which each animal received a total of 5.5 x 10 6 promastigotes, flagellates were detected in at least one sand fly fed on 7 N. lasiurus, 4 N. squamipes and 1 R. rattus. In the second experiment, in which each animal received 2.8 x 10 5 , all the animals proved to be infectious to sand flies. The difference between the results of the first and second

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experiment could be due to differences in the distribution and number of parasites. This possibility is presently be investigated by quantifying the number of parasites in the different tissues using RealTime PCR technology.

References (1) Brando-Filho SP, Brito ME. Carvalho FG, Ishikawa, EA, Cupolillo E, Floeter-Winter L, Shaw JJ, 2003. Trans R Soc Trop Med Hyg v.97, 291-296.

Financial support: CNPq, FACEPE.

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DADOS RECENTES SOBRE IMPACTO DA REMOO MECNICA DE OVOS E ADULTOS SOBRE DENSIDADES POPULACIONAIS DE AEDES AEGYPTI EM DOIS MUNICPIOS DE PERNAMBUCO
LEDA REGIS1, RIDELANE VEIGA ACIOLI2, CANDIDA MARIA N. RIBEIRO3, JULIANA SERAFIM4, KARINA BRITTO4, JOS CONSTANTINO SILVEIRA JR1, WAYNER VIEIRA DE SOUZA1 , PAULO JUSTINIANO RIBEIRO JR5, MARIA ALICE V. MELO-SANTOS1, ANTONIO MIGUEL V. MONTEIRO6
1 Centro de Pesquisas Aggeu Magalhes, FIOCRUZ, Recife-PE. e-mail: leda@cpqam.fiocruz.br. Secretaria Estadual de Sade de Pernambuco. 3Secretaria Municipal de Sade de Sta Cruz do Capibaribe-PE. Secretaria Municipal de Sade de Ipojuca. UFPR, Curitiba-PR. 6INPE, So Jos dos Campos-SP.
2 4 5

Aedes aegypti, principal vetor dos virus DENV, uma espcie bem adaptada a ambientes urbanos. Esta adaptao evolutiva ao sinantropismo inclui a hematofagia muito freqente (quase diria) e preferencialmente em humanos, bem como adaptaes ao desenvolvimento ps-embrionrio em habitats aquticos muito instveis, que incluem, notadamente: a disperso de ovos, protegidos por crion extremamente resistente, em variadssimos tipos de recipientes; baixa seletividade quanto s propriedades fsico-qumicas da gua; comportamento (larvas e pupas) de fuga gil e capacidade respiratria para permanncia prolongada em submerso. Estes aspectos da reproduo e do desenvolvimento das formas aquticas nesta espcie tornam muito rduas as tarefas de localizar os muito numerosos stios de criao, e de visualizar as formas imaturas, o que parece ser a principal causa da baixa eficincia dos programas que se baseiam na pesquisa larvria e uso de larvicidas para monitoramento e controle deste mosquito. Diferentemente do que ocorre com Culex quinquefasciatus, culicideo que desenvolveu outras formas de adaptao ao ambiente urbano, ha evidencias cada vez mais claras de que o desenvolvimento larval no a fase do ciclo biolgico mais apropriada como alvo das aes em programas de controle do vetor da dengue. Um sistema de monitoramento e controle populacional do Ae. aegypti (SMCP-Aedes), que se fundamenta nas especificidades biolgicas deste

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mosquito, foi desenvolvido por pesquisadores e tcnicos da Fiocruz, INPE, UFPR, UFPE, e Secretarias de Sade de Pernambuco e de trs municpios pernambucanos (Monteiro et al 2005, Regis et al 2008, Bonat et al 2010). Neste sistema, o monitoramento populacional tem como base a deteco de fmeas reprodutivamente ativas, pela identificao e quantificao de ovos depositados em armadilhas georefenciadas, distribudas no espao urbano. O sistema se apia em tecnologias da informao espacial com uso intensivo da web e de software livres para analisar e gerar automaticamente relatrios sobre a distribuio espao-temporal da densidade populacional do Aedes, com base em amostras continuamente coletadas com ovitrampas redesenhadas, contendo larvicida microbiano (Bti). Ele inclui um conjunto de intervenes integradas para controle populacional do vetor, planejadas com base na vigilncia entomolgica, e pactuadas com os habitantes (Regis et al 2009). As aes de controle se baseiam na remoo e incinerao em massa de ovos nos momentos e locais priorizados segundo os dados de vigilncia, e so reforadas pela remoo mecnica de mosquitos adultos com aspiradores portveis. As ovitrampas modificadas para esta funo contm Bti como larvicida e como suporte de oviposio utilizado tecido em algodo, com base em estudos anteriores (Lenhart et al 2005). Alm de proporcionar maior espao para deposio dos ovos, os tecidos so mais prticos para transporte e incinerao do que as palhetas rgidas utilizadas nas ovitrampas-sentinela. A adio de um estimulante de oviposio (Barbosa et al 2010) poder, no futuro, ampliar a capacidade desta armadilha para remoo em massa de ovos do ambiente. Avaliado em escala real em dois municpios (Santa Cruz do Capibaribe e Ipojuca-PE), o SMCP-Aedes teve confirmada sua alta sensibilidade para detectar e quantificar o vetor, e identificar locais com maior concentrao populacional, o que permite orientar as aes de controle. Foram detectados nveis altssimos de infestao dos espaos urbanos por Ae. aegypti, em ambos os municpios, com percentuais de positividade >90%, semelhantes aos anteriormente revelados em

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sete bairros do Recife. O sistema permitiu mensurar o impacto da remoo mecnica de ovos e adultos sobre a populao do mosquito, indicando redues significativas das densidades populacionais do vetor nos dois municpios, como resultado da eliminao de cerca de 5 milhes de ovos removidos do ambiente durante 6 meses, com > 8400 ovitrampas-controle, e da eliminao, com aspiradores, de 12,7 mil mosquitos adultos (aproximadamente 75% Culex e 25% Aedes). Observou-se que os instrumentos utilizados para remoo mecnica de ovos e de adultos ensejam a aproximao com as ferramentas e estratgias de controle, favorecendo a compreenso da biologia do vetor pelos agentes e habitantes. Palavras-chave: controle do vetor, dengue, vigilncia entomolgica, ovitrampas, aspiradores. Referencias BARBOSA, R. M. ; FURTADO, A. F. ; REGIS, L. ; Leal WS 2010.. Evaluation of an oviposition-stimulating kairomone for the yellow fever mosquito, Aedes aegypti, in Recife, Brazil. Journal of Vector Ecology 35: 204-207.
BONAT W.H. ; DALLAZUANNA H.S. ; RIBEIRO JR, P. J. ; MONTEIRO, A. M. ; REGIS, L. N. ; SILVEIRA JR, J. C. ; ACIOLI R.V. ; SOUZA W.V. 2009. Investigando fatores associados a contagens

de ovos de Aedes aegypti coletados em ovitrampas em Recife. Revista Brasileira de Biometria. 27: 519-537. 2005. Building a better ovitrap for detecting Aedes aegypti oviposition. Acta Trop 96: 56-59.
LENHART AE, WALLE M, CEDILLO H, KROEGER A MONTEIRO AMV, CARVALHO MS, ASSUNO R, SOUZA WV, RIBEIRO JR PJ, DAVIS JR C, REGIS L, the SAUDAVEL Project Team 2005. SAUDAVEL: Bridging the Gap between

Research and Services in Public Health Operational Programs by MultiInstitutional Networking Development and Use of Spatial Information Tech nology I n n o v a t i v e To o l s . [ c i t e d 2 0 0 8 J a n 1 0 ] . A v a i l a b l e f r o m : http://www.dpi.inpe.br/saudavel/publicacoes.html REGIS, L. ; SOUZA W.V. ; Furtado A.F. ; FONSECA, C.D. ; SILVEIRA JR, J. C. ; RIBEIRO JR, P. J. ; MELO-SANTOS M.A.V. ; CARVALHO, M. S. ; MONTEIRO, A. M. 2009. An Entomological surveillance system based on open spatial Information for participative Dengue control. Anais da Academia Brasileira de Cincias 81: 655-662. REGIS, L. ; MONTEIRO, A. M. ; SANTOS, M. A. V. M. ; SILVEIRA JR, J. C. ; FURTADO, A. F. ; ACIOLI, R. V. ; SANTOS, G. M. ; NAKAZAWA, M. ; CARVALHO, M. S. ; RIBEIRO JR, P. J. ; SOUZA, W. 2008. Developing new approaches for

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detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system. Memrias do Instituto Oswaldo Cruz, 103: 50-59.
Apoio: Fiocruz-PDTSP/Rede Dengue, CNPq, MS-DECIT, FACEPE, SES-PE

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OVIPOSITION ATTRACTANTS FOR Culex quinquefasciatus: THEIR RECEPTORS, RECEPTION, AND USE IN VECTOR MANAGEMENT STRATEGIES Walter S. Leal
Department of Entomology, University of California Davis, California, USA, Email: wsleal@ucdavis.edu

Culex mosquitoes are vectors of pathogens including the human filarial nematode, Wuchereria bancrofti, and encephalitis-causing viruses, such as St. Louis, Japanese, Venezuela equine, Western equine encephalitis, and West Nile virus . Given the resistance of Culex populations to modern insecticides, alternative methods of controls are sorely needed. Larval development is a particularly vulnerable phase in the life cycle of Culex mosquitoes, as eggs are laid in rafts from which hundreds of larvae emerge in confined areas -- thus facilitating management. Gravid females rely on environmental oviposition attractants to locate oviposition sites. In general, insects detect chemical signals (referred to as semiochemicals in chemical ecology and odorants in olfaction) with specialized sensory structures, the olfactory sensilla, present on different chemosensory tissues such as antennae, maxillary palp and proboscis. Odorant receptors (ORs) are expressed in the dendritic membrane of olfactory receptor neurons (ORNs) housed in these sensilla. Hydrophobic odorant molecules have to pass through an aqueous medium, the sensillar lymph, surrounding the dendrites and separating the port of entry on the sensilla (the pore tubules) and receptors neurons. There is growing evidence indicating that odorant-binding proteins (OBPs) are involved in the uptake, transport, and delivery of odorants to ORs , whereas odorantdegrading enzymes (ODEs) inactivate chemical signals . The first mosquito OBP (CquiOBP1) was isolated from antennae of female Cx. quinquefasciatus by native gel electrophoresis and further cloned from cDNA to obtain a full-length sequence .

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This protein was shown to bind the mosquito oviposition pheromone MOP in a pHdependent manner and to be expressed in a subset of sensilla including one type responding to this pheromone . Taken together, these experiments suggest that this protein is involved in the detection of semiochemicals involved in mosquito oviposition behavior. Indeed, gene silencing of CquiOBP1 by RNA interference led to a decrease in electrophysiological responses to MOP and other oviposition attractants . We take advantage of our understanding of the molecular basis of mosquito olfaction to screen compounds on the basis of their affinity to OBPs, an approach dubbed reverse chemical ecology . As a proof-of-concept, the first mosquito OBP, CquiOBP1 was employed as a molecular target for screening potential oviposition attractants. These OBP-based screenings led to the identification of nonanal, which albeit missed in the conventional approach, was demonstrated in the field to be an oviposition attractant . To augment this reverse chemical ecology approach we have also characterized odorant receptors (ORs) from this mosquito species. Whereas CquiOR2 is highly sensitive to indole and moderately sensitive to skatole , CquiOR10 is narrowly tuned to skatole . In female antennae of the Southern House mosquito, skatole is detected by a small-spikeamplitude ORN housed in A1 sensilla , which is also sensitive to a lower degree to geranylacetone and ethyl hexanoate, but does not respond to indole or 2methylphenol; see Figs. S5,S6, supporting information in ). In the Xenopus oocyte system CquiOR10 was unresponsive to geranylacetone . Although expression of ORs in heterologous systems, such as the Xenopus oocyte system, is an invaluable tool for de-orphanizing and characterizing receptors, it does not completely mimic the insect olfactory system. Typically, these systems are devoid of OBPs, odorant-degrading enzymes, sensory neuron membrane proteins, and other olfactory proteins that may play a part in the selectivity and sensitivity of the olfactory system. Thus, it is conceivable that heterologously expressed CquiOR10 and the receptor in its native environment differ in the detection of geranylacetone

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because the former is devoid of OBPs. However, one cannot rule out the possibility that a separate ORN responding to geranylacetone has the same spike amplitudes of the skatole-detecting ORN thus rendering them indistinguishable by single unit recordings. Alternatively, the same small-spike neuron sensitive to skatole may express another OR along with CquiOR10. In marked contrast to mammalian olfactory system, co-expression of ORs has been documented in Drosophila melanogaster. Co-expression of CquiOR10 and another OR would not be entirely surprising given the number of putative odorant receptors in the Southern House mosquito genome and the number of ORNs in their sensory system . In summary, identification of olfactory proteins (OBPs and ORs) involved in the reception of these semiochemicals may open the door for development of better oviposition attractants. Recently, we have identified an oviposition attractant mixture of trimethylamine and nonanal, which performed equivalent to the cumbersome infusion ; a synthetic formulation of this oviposition attractant is now commercially available:

http://www.chemtica.com/ChemtIngles/PDF%20files/PDF%20Traps/gravid%20 mosquito.pdf. Previously, skatole - a natural product found in animal excreta and also a product of fermentation of organic material - has been previously identified as an oviposition attractant for the Southern House mosquito, Culex quinquefasciatus . Field studies demonstrated that traps baited with optimal doses of skatole collected significantly more eggs and gravid females than control traps. Recently, we have demonstrated that Cx. quinquefasciatus laid significantly more eggs in traps loaded with Bacillus thuringensis var. israelensis and baited either with a synthetic oviposition attractant (skatole) or a natural attractant (infusion) than in control water traps loaded only with Bti . Given that this agent is toxic to the offspring , deploying oviposition traps loaded with Bti should in theory lead to reduction of Culex populations. Although attract-and-kill is by no means a panacea, this environmentally friendly strategy is an untapped insect vector

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management resource in part because better oviposition attractants are indispensable.

Acknowledgments This work was supported in part by the National Institutes of Health, the National Science Foundation and gifts from Bedoukian Research Inc. and Fuji Flavor Co.

References BARBOSA, R. M. R., REGIS L., VASCONCELOS R., LEAL W. S. 2010. Culex mosquitoes (Diptera: Culicidae) egg laying in traps loaded with Bacillus thuringensis variety israelensis and baited with skatole. J. Med. Entomol. 47:345-348. BIESSMANN, H., ANDRONOPOULOU E., BIESSMANN M. R., DOURIS V., DIMITRATOS S. D., ELIOPOULOS E., GUERIN P. M., IATROU K., JUSTICE R. W., KROBER T. and others. 2010. The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes. PLoS ONE 5:e9471. HUGHES, D. T., PELLETIER J., LUETJE C. W., LEAL W. S. 2010. Odorant Receptor from the Southern House Mosquito Narrowly Tuned to the Oviposition Attractant Skatole. J Chem Ecol. ISHIDA, Y., CORNEL A. J., LEAL W. S. 2001. Development of a system for high throughput screening of attractants and repellents for Culex mosquitoes. Mosquito Control Research, Annual Report. p 26-28. ISHIDA, Y., CORNEL A. J., LEAL W. S. 2002. Identification and cloning of a female antenna-specific odorant-binding protein in the mosquito Culex quinquefasciatus. J Chem Ecol 28:867-71. ISHIDA, Y., LEAL W. S. 2005. Rapid inactivation of a moth pheromone. Proc Natl Acad Sci U S A 102:14075-9. LAURENCE, B. R., PICKETT J. A. 1982. erythro-6-Acetoxy-5-hexadecanolide, the major compound of a mosquito attractant pheromone. J. Chem. Soc., Chem. Commun.:59-60. LEAL, W. S. 2005. Pheromone reception. Top. Curr. Chem. 240:1-36. LEAL, W. S. 2007. Molecular-based chemical prospecting of mosquito attractants and repellents. In: Debboun M, Frances SP, Strickman D, editors. Insect Repellents: Principles, Methods, and Uses. New York: CRC Press. p 229-242. LEAL, W. S., BARBOSA R. M., XU W., ISHIDA Y., SYED Z., LATTE N., CHEN A. M., MORGAN T. I., CORNEL A. J., FURTADO A. 2008. Reverse and conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes. PLoS ONE 3:e3045.

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MBOERA, L. E., TAKKEN W., MDIRA K. Y., PICKETT J. A. 2000. Sampling gravid Culex quinquefasciatus (Diptera: Culicidae) in Tanzania with traps baited with synthetic oviposition pheromone and grass infusions. J Med Entomol 37:172-6. MILLAR, J. G., CHANEY J. D., MULLA M. S. 1992. Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions. J Am Mosq Control Assoc 8:11-7

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GENETICA POPULACIONAL E CONTROLO DE VECTORES Joo Pinto


Unidade de Entomologia Mdica, Centro de Malria e outras Doenas Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa. Rua da Junqueira 100, 1349-008 Lisboa, Portugal. (jpinto@ihmt.unl.pt)

Nos ltimos anos, tm vindo a proliferar estudos sobre a estrutura gentica de populaes de vectores de malria. Estes estudos tm como principal objectivo caracterizar a variabilidade e diferenciao gentica de populaes naturais, de modo a compreender aspectos biolgicos to diversos como o estatuto taxonmico de espcies ou grupos intra-especficos, a disperso de genes de interesse, ou o impacte de medidas de controlo na reduo das populaes de vectores. Neste contexto, o complexo Anopheles gambiae tem sido o mais estudado. Este complexo inclui os principais vectores de malria (Anopheles gambiae sensu stricto e Anopheles arabiensis) da frica Subsaariana. Este continente concentra perto de 90% dos ca. 300-500 milhes de casos de malria que ocorrem anualmente no mundo. A elevada importncia epidemiolgica deste complexo levou a que a espcie nominal, A. gambiae s.s., tenha sido o primeiro mosquito vector a ter o genoma sequenciado (Holt et al. 2002). Este avano cientfico trouxe uma nova era aos estudos de gentica populacional, com a implementao de scannings genmicos, que implicam a genotipagem de milhares de marcadores genticos espalhados pelo genoma, e abordagens de genmica populacional.

Estrutura populacional e disperso de genes

Na frica ocidental, A. gambiae s.s. encontra-se subdividido em duas formas moleculares designadas por M e S. As formas foram identificadas por polimorfismos nas regies IGS e ITS do rDNA e so consideradas as unidades de um processo de especiao incipiente em curso em A. gambiae s.s (della Torre et

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al. 2002). Com excepo do extremo ocidental da sua distribuio (Oliveira et al. 2008), a frequncia de hbridos entre as duas formas bastante reduzida (<1%), apesar das extensas reas de simpatria. Scannings genmicos demonstraram que o maior grau de diferenciao gentica entre as formas ocorre em apenas trs regies genmicas relativamente pequenas (Turner et al. 2005). Este padro de diferenciao genmica em mosaico sugere a aco de divergncia selectiva no processo de especiao simptrica. A nvel do controlo de vectores, a importncia da subdiviso populacional em A. gambiae s.s. tem a sua maior expresso na distribuio de genes associados resistncia a insecticidas. A frequncia de mutaes associadas resistncia knockdown (kdr) bastante elevada em populaes da forma S na frica ocidental. No entanto, estas mutaes so menos frequentes em populaes da forma M, mesmo quando as duas formas se encontram em simpatria (Santolamazza et al. 2008). Estudos recentes demonstraram ainda que as duas mutaes kdr descritas em A. gambiae s.s. tero tido mltiplas origens em cada forma molecular e que fenmenos de introgresso gentica, embora raros, tero contribudo para a transferncia dos alelos associados resistncia entre formas (Pinto et al. 2007; Etang et al. 2009).

Monitoramento gentico do controlo de vectores Um dos objectivos do controlo de vectores passa pela reduo das populaes de mosquitos. Uma reduo populacional drstica vai perturbar a estabilidade gentica da populao, perturbao que pode ser detectada atravs da anlise de marcadores genticos neutros (i.e. no sujeitos a seleco). Assim, o impacte de medidas de controlo poder ser avaliado atravs do uso de ferramentas analticas que permitam avaliar contraces/expanses populacionais. Estas incluem estimativas do tamanho efectivo populacional e testes de equilbrio mutao-deriva (MDE). Esta abordagem de monitoramento gentico foi j utilizada, com resultados promissores, na avaliao de

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intervenes piloto com redes mosquiteiras impregnadas com insecticidas (Wondji et al. 2005). Contudo, existem ainda muitas dificuldades inerentes aplicabilidade desta abordagem (Pinto et al. 2003). Estas incluem aspectos relacionados com natureza dos marcadores genticos (e.g. taxa de mutao), a biologia do vector (e.g. elevada fecundidade e geraes sobrepostas) e o tipo de mtodos analticos disponveis para estimar o tamanho efectivo populacional e detectar desvios ao MDE (e.g. janelas temporais de sensibilidade). Estes mtodos foram muitas vezes desenvolvidos no mbito da biologia da conservao, para detectar flutuaes demogrficas em espcies ameaadas. Este um conceito diametralmente oposto ao que se pretende com o monitoramento gentico de estratgias de controlo. O desenvolvimento de novos mtodos de genotipagem high-throughput, com a anlise de um elevado nmero de marcadores genticos (e.g. SNPs), bem como de novas ferramentas analticas para a deteco de alteraes genticas associadas a perturbaes demogrficas, poder trazer uma nova esperana para a aplicao destes mtodos na avaliao do impacte de programas de controlo de vectores. Referncias Della Torre A, Costantini C, Besansky NJ, Caccone A, Petrarca V, Powell JR, Coluzzi M 2002. Speciation within Anopheles gambiae--the glass is half full. Science 298: 115-117. Etang J, Vicente JL, Nwane P, Chouaibou M, Morlais I, Do Rosario VE, Simard F, Awono-Ambene P, Toto JC, Pinto J 2009. Polymorphism of intron-1 in the voltage-gated sodium channel gene of Anopheles gambiae s.s. populations from Cameroon with emphasis on insecticide knockdown resistance mutations. Mol Ecol 18: 3076-3086. Holt RA, Subramanian GM, Halpern A, Sutton GG, et al. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298: 129-149. Oliveira E, Salgueiro P, Palsson K, Vicente JL, Arez AP, Jaenson TG, Caccone A, Pinto J 2008. High levels of hybridization between molecular forms of Anopheles gambiae from Guinea Bissau. J Med Entomol 45: 1057-1063. Pinto J, Donnelly MJ, Sousa CA, Malta-Vacas J, Gil V, Ferreira C, Petrarca V,

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do Rosrio VE, Charlwood JD 2003. An island within an island: genetic differentiation of Anopheles gambiae in So Tom, West Africa, and its relevance to malaria vector control. Heredity 91: 407-414. Pinto J, Lynd A, Vicente JL, Santolamazza F, Randle NP, Gentile G, Moreno M, Simard F, Charlwood JD, do Rosrio VE, Caccone A, Della Torre A, Donnelly MJ 2007. Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae. PLoS One 2: e1243. Santolamazza F, Calzetta M, Etang J, Barrese E, Dia I, Caccone A, Donnelly MJ, Petrarca V, Simard F, Pinto J, della Torre A 2008. Distribution of knockdown resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa. Malar J 7: 74. Turner TL, Hahn MW, Nuzhdin SV 2005. Genomic islands of speciation in Anopheles gambiae. PLoS Biol 3: e285. Wondji C, Simard F, Lehmann T, Fondjo E, Sam-Ekobo A, Fontenille D 2005. Impact of insecticide-treated bed nets implementation on the genetic structure of Anopheles arabiensis in an area of irrigated rice fields in the Sahelian region of Cameroon. Mol Ecol 14: 3683-3693.

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IMPACT OF CLIMATIC CHANGES AND HABITAT DEGRADATION ON PHLEBOTOMINAE (DIPTERA: PSYCHODIDAE) DISTRIBUTION AND LEISHMANIASES DISPERSION IN BRAZIL Reginaldo P. Brazil1, Ursula Brazil-Rocha2 & Beatriz G.Brazil3
1 - Instituto Oswaldo Cruz- Fiocruz, RJ, Brazil (rpbrazil@ioc.fiocruz.br); 2 - Universidade Federal Rural do Rio de Janeiro, RJ, Brazil; 3 - Universidade Federal de Mato Grosso do Sul, MS, Brazil

We examined changes in the phlebotomine fauna and the spread of Cutaneous and Visceral Leishmaniasis resulting from human intervention and climatic changes in the biomes of 5 regions of Brazil. Areas of natural undisturbed or highly degraded forest are compared with the overall abundance and richness of sand fly species. Overall abundances were higher in undisturbed forests (Amazonian and Atlantic Forest) with species richness greater than in degraded habitat. This seems to maintain a natural balance for Leishmania infection in sylvatic animals. Moreover, several studies point to important changes such as drought in the Amazon and extreme rainfall in the south of Brazil thereby contributing to dispersion of Leishmaniasis endemicity. In disturbed areas a few species, mainly Leishmania vectors, are dominant and highly adaptable to this environment. In areas of Visceral Leishmaniasis, the sand fly fauna were dominated by Lutzomyia longipalpis (Lutz & Neiva), vector of Leishmania infantum chagasi (Cunha & Chagas), independent of its natural biome. The same is observed for Nyssomyia intermedia / N. neivai vectors of Leishmania braziliensis (Vianna), both with tendency to dominate degraded environment in south and southeast Brazil. Species such as N. whitmani, another common vector of Cutaneous Leishmaniasis, in less degraded areas are now moving to transmitting the disease at degraded environment in several regions of Brazil. The zoophilic species Bichromomyia flaviscutellata (Mangabeira), a natural forest vector of Leishmania amazonensis, may be spreading to new biomes and may represent a new threat to man.

Keywords:Phlebotominae;Leishmaniasis;Climatic Change; Forest Degradation; vector dispersion.

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References: Dias-Lima A, Guedes MLS, Sherlock IA. 2003 Horizontal Stratification of the Sand Fly Fauna (Diptera: Psychodidae) in a Transitional Vegetation between Caatinga and Tropical Rain Forest, State of Bahia, Brazil. Mem. Inst. Oswaldo Cruz 98: 733-737 Dorval MEC, Alves TP, Oliveira AG, Brazil RP, Galati EAB, Cunha RV 2007. Modification of Disney trap for capture of sand flies (Diptera: Psychodidae: Phlebotominae). Mem Inst Oswaldo Cruz 102: 877-878 Oliveira AG, Andrade-Filho JD, Falco AL, Brazil RP 2003. Estudo de flebotomneos (Diptera, Psychodidae, Phlebotominae) na zona urbana da cidade de Campo Grande, Mato Grosso do Sul, Brasil, 1999-2000. Cad Saude Publica 19: 933-944. Peterson AT; Shaw J 2003.Lutzomyia vectors for cutaneous leishmaniasis in Southern Brazil: ecological niche models, predicted geographic distributions, and climate change effects. International Journal for Parasitology 33: 919931 Ready PD. 2008 Leishmaniasis emergence and climate change. Rev Sci Tech. 27:399-412. Rogers DJ, Randolph SE. 2006.Climate change and vector-borne diseases. Adv Parasitol ; 62:345-81. Sutherst RW. 2004. Global change and human vulnerability to vector-borne diseases. Clin Microbiol Rev, 17: 136173. Silva DF, Freitas RA, Franco AMR. 2007. Diversidade e abundncia de flebotomneos do gnero Lutzomyia (Diptera: Psychodidae) em reas de mata do nordeste de Manacapuru, AM. Neotrop Entomol 36: 138-144

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CLIMATIC CHANGES AND ITS IMPACT ON THE ECO-EPIDEMIOLOGY OF TROPICAL DISEASES Jeffrey Shaw
Parasitology Department, So Paulo University, So Paulo, Brazil

Climatic changes can be linked to natural or human activity but these all result in modifications of the microclimate of ecological niches in which vectors breed. Understanding how these niches are modified by climatic changes is the key to predicting changes in distributions and successful vector control. During recent years an increasing amount of scientific data indicates that temperatures throughout the world are increasing. This phenomenon generally, known as global warming, has led to extremes in weather systems, such as changes in precipitation patterns that result in droughts or flooding. It has been suggested that mans' activities are responsible for global warming but this opinion is not universally accepted and some consider that it could be part of a natural cycle. Besides this the rate of destruction of natural habitats and the modification of ecological niches has also increased alarmingly during the last century and is clearly related to the global increase in the human population. This coupled with global warming has lead to unprecedented changes in the distribution of the insect vectors of many viral and parasitic diseases. Table 1 lists the potential effects of global warming on the principal vector borne diseases. In all cases the major consideration is that microclimatic changes caused by global warming will expand suitable vector breeding sites. However, it must be remembered that mans own activities, such as deforestation and urbanization, also result climatic changes at various levels which are responsible increase the number of ecological niches that are favourable for vector reproduction. Different species of mosquitoes thrive in ecological niches created by man. One of the best examples of this is the adaption of Aedes aegypti to the

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peridomestic habitats

and the corresponding epidemics of Dengue, in such

countries as Brazil. Control attempts have been marginally successful but in most cases they fail due to the lack of collaboration of local populations. In these situations man is responsible for creating microclimatic conditions in and around unprotected water vessels which are perfect breeding sites for A.aegypti. Other examples of how man's activities change climatic conditions are the creation of artificial lakes for hydroelectric and irrigation schemes. In Rondnia State, Brazil there has been an increase in the incidence of malaria due to the proliferation of breeding sites that are suitable for the local vector, Anopheles darlingi . Similar situations are linked to adaption of other mosquito species, such as A. scapularis, to syanthropic environments which are potential vectors of viral and parasitic pathogen. Leishmaniasis is one of the most complex vector borne diseases. In most regions it is zoonotic and more rarely anthroponotic. One of the greatest concerns is a migration of zoonotic cycles to peridomestic environments which potentially can lead to man to man transmission. The difficulty in these situations is determining if the climatic changes that are responsible for this are because of mans immediate activities or more general climate changes such as El Nio. In Costa Rica there was an increase cutaneous leishmaniasis (CL), in spite of continued deforestation between 1991 and 2001. During this period there was a 3 year periodicity that was coincidental to the southern oscillations in El Nio . In other regions, such as Bahia, Brazil, El Nio has been associated with a fall in the transmission rate of visceral leishmaniasis (VL) . The reason for this is that thought to be due to an extended dry season which has a negative effect on the vector (Lutzomyia longipalpis) population. This associated with other factors such as the waning of herd immunity related to lower transmission rates leads to an increase in VL cases when the sand fly population increases during the rainy season that follows El Nio. Contrary to this El Nio has been associated with an increase in malaria transmission in Venezuela and the Indian subcontinent . (Bouma and Dye, 1997; Bouma and van der Kaay, 1996).

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Table 1. Potential effect of Global Warming on the major vector borne ropical diseases* Disease Chances of Increase with Global Warming extremely likely very likley likley likley very likley very likely very likely

Malaria Leishmaniasis Chagas' disease Lymphatic filariasis Onchoceriasis Schistosomiasis Dengue


Based on (Patz et al., 1996)

It is postulated that El Nio episodes will increase with global warming. Such changes in frequency could affect vectors differently causing either an increase or decrease in the frequency of the diseases that they transmit. The literature contains many references to fluctuations of sand fly populations that are associated with rainfall. However, the exact reasons for these fluctuations are unknown but they are possibly linked to variations in the microclimate of larval habitats. In the Serra das Carajs of Par State, Brazil there is a complete absence the principal vector, Psychodopygus wellcomei, during the dry season. . In this case pupal diapause is stimulated by the lack of precipitation which is clearly related to the level of humidity in the soil. In the another area of the same State it was found that the vector of Leishmania (Leishamnia) amazonensis occurred in greater numbers in forests in which the ground water levels where highest . However, in this region the very high a monthly rainfall above 200 to 250 mm reduced the L. flaviscutellata population, therefore, declines during the rainy season and steadily builds up during the dry season. In British Honduras L. olmeca disappeared during the dry season and reappeared during the wet season . At first sight, it would appear that L. olmeca and L. flaviscutellata differ in their seasonal fluctuations. In British Honduras, however, the rainfall did not exceed 160 mm. per

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month during the peak population period while in Belm during the peak period the average rainfall was below 250 mm. From this comparison it would seem then that both are, in fact, affected similarly by rainfall. In a forest habitat in Rondnia State, Brazil L.longipalpis was only detectable during a few months of the year. These months coincided with the spring period in which the rainfall was increasing after a cooler period. However, as the rains increased the populations fell and disappeared completely during the rainy season. In an endemic VL area of Maraj Island the L.longipalpis population was higher in both silvatic and peridomestic environments during the wet season . However, at the end of the wet season the population in the forest fell drastically but remained high within the domestic environment. In the urban environment of the capital of Mato Groso do Sul, Campo Grande, this same species was present in greatest numbers during the months of highest rainfall . In the same urban environment in Belo Horizonte, Brazil the L.longipalpis populations peaked just after the maximum period of rains, which was December to February , which was the same as Campo Grande. One of the biggest challenges in Brazil is to understand why the vector of VL is spreading and adapting to new areas throughout the country, particularly within cities of varying sizes. For many years the vector did not occur within urban areas of Brazils capital, Brasilia. The natural environment of this region is cerrado which experiences long dry periods. Urban development has changed this and increased the soil humidity throughout the year as well as increasing enormously potential sources food for both larvae and adults. This could be one reason why VLs vector has colonized the capital. Prediction models can be used to investigate the effect of global warming on the distribution of any plant or animals species. Using such models projections have been made on how increases in global temperatures would affect the

distribution of CL vectors in Brazil and its neighbours. Projections of potential geographic distributions across scenarios of global climate change suggested that

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only L. whitmani is likely to experience dramatic improvements in conditions in south-eastern Brazil, where cutaneous leishmaniasis appears to be re-emerging. However L. intermedia and L. migonei may be seeing more subtle improvements. The overall conclusion is that variations in the distribution and abundance of vector populations are intimately linked to the ecological niches in which the larval stages develop. A change in the suitability of these niches depends on micro and macro climatic variations caused by both global weather variations and modifications of local environments by man himself. The nature of the alterations caused by man are infinite and range from simply building a house or a small lake to destroying vast areas of forest or building enormous damns for hydroelectric schemes. The resultant combinations of these factors are similarly so great that each situation needs to be careful assessed by detailed studies on each vector species.

References

Bouma, M.J., Dye, C., 1997, Cycles of malaria associated with El Nio in Venezuela. J Amer Med Assoc 278, 17721774. Bouma, M.J., van der Kaay, H.J., 1996, . El Nio Southern Oscillation and the historic malaria epidemics on the Indian subcontinent and Sri Lanka: an early warning system for future epidemics?:. Trop Med Int Health 1, 8696. Chaves, L.F., Pascual, M., 2006, Climate cycles and forecasts of cutaneous leishmaniasis, a nonstationary vector-borne disease. PLoS Med 3, e295. Disney, R.H.L., 1968, Observations on a zoonosis: leishmaniasis in British Honduras. J Annim Ecol 5, 1-59. Forattini, O.P., Kakitani, I., Massad, E., Marucci, D., 1993, Studies on mosquitoes (Diptera: Culicidae) and anthropic environment. 2. Immature stages research at a rice irrigation system location in south-eastern Brazil. Rev Saude Publica 27, 227-236. Franke, C.R., Ziller, M., Staubach, C., Latif, M., 2002, Impact of the El Nino/Southern Oscillation on visceral leishmaniasis, Brazil. Emerg. Infect. Dis. 8, 914-917.

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Gil, L.H., Tada, M.S., Katsuragawa, T.H., Ribolla, P.E., da Silva, L.H., 2007, Urban and suburban malaria in Rondonia (Brazilian Western Amazon) II. Perennial transmissions with high anopheline densities are associated with human environmental changes. Mem Inst Oswaldo Cruz 102, 271-276. Lainson, R., Dye, C., Shaw, J.J., MacDonald, D.W., Courtenay, O., Souza, A.A.A., Silveira, F.T., 1990, Amazonian visceral leishmaniasis - Distribution of the vector Lutzomyia longipalpis (Lutz & Neiva) in relation to the fox Cerdocyon thous (Linn.) and the efficiency of this reservoir host as a source of infection. Mem. Inst. Oswaldo. Cruz 85, 135-137. Medronho, R.A., Macrini, L., Novellino, D.M., Lagrotta, M.T., Camara, V.M., Pedreira, C.E., 2009, Aedes aegypti immature forms distribution according to type of breeding site. Am J Trop Med Hyg 80, 401-404. Oliveira, A.G., Galati, E.A., Fernandes, C.E., Dorval, M.E., Brazil, R.P., 2008, Seasonal variation of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in endemic area of visceral leishmaniasis, Campo Grande, state of Mato Grosso do Sul, Brazil. Acta Trop. 105, 55-61. Patz, J.A., Epstein, P.R., Burke, T.A., Balbus, J.M., 1996, Global climate change and emerging infectious diseases. Jama 275, 217-223. Peterson, A.T., Shaw, J., 2003, Lutzomyia vectors for cutaneous leishmaniasis in Southern Brazil: ecological niche models, predicted geographic distributions, and climate change effects. Int. J. Parasitol. 33, 919-931. Ready, P.D., Lainson, R., Shaw, J.J., 1984, Habitat and seasonality of Psychodopygus wellcomei help incriminate it as a vector of Leishmania braziliensis in Amazonia and northeast Brazil. Trans R Soc Trop Med Hyg 78, 543-544. Resende, M.C., Camargo, M.C., Vieira, J.R., Nobi, R.C., Porto, M.N., Oliveira, C.D., Pessanha, J.E., Cunha Mda, C., Brando, S.T., 2006, Seasonal variation of Lutzomyia longipalpis in Belo Horizonte, State of Minas Gerais. Rev Soc Bras Med Trop 39, 51-55. Shaw, J.J., Lainson, R., 1972, Leishmaniasis in Brazil. VI. Observations on the seasonal variations of Lutzomyia flaviscutellata in different types of forest and its relationship to enzootic rodent leishmaniasis (Leishmania mexicana amazonensis). Trans R Soc Trop Med Hyg 66, 709-717.

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MOLECULAR CHRONOBIOLOGY OF INSECT VECTORS Alexandre A. Peixoto


Laboratory of Insect Molecular Biology Instituto Oswaldo Cruz, FIOCRUZ Rio de Janeiro, Brazil

The behaviour of insect vectors plays a vital role in the dynamics of disease transmission (Klowden and Zwiebel 2004). One aspect of vector behaviour particularly relevant in this respect is the control of daily patterns of activity and blood-feeding. These rhythms are controlled by an endogenous biological clock (Saunders 2002), which is in turn under genetic control. However, we still know little about the molecular chronobiology of blood-sucking insects. The molecular pacemaker of the insect model species Drosophila melanogaster involves a number of genes that take part in interlocked negative transcriptional feedback loops that control the circadian rhythms in behaviour and physiology (reviewed in Hardin 2005). In the two main interacting feedback loops, the genes Clock (Clk) and cycle (cyc) encode transcription factors that form a heterodimer that binds to upstream E-box sequences (CACGTG) and activates the transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). The heterodimer Per/Tim (or Per alone) interacts with Clk/Cyc inhibiting its function. Meanwhile, Vri and PDP1 regulate Clk transcription by competing for the same site in its promoter. These two interlocked loops drive the circadian gene expression of its components, except in the case of cyc, which is constitutively expressed. Kinases have also an important role controlling the cyclic stability and nuclear entry of clock proteins. The cycles in the expression and abundance of negative and positive elements of the clock are important for the generation of the circadian rhythms in hundreds of outputs genes controlling different aspects of physiology and behaviour. Finally, a light-resetting mechanism involving the protein encoded by the cryptochrome (cry) gene allows the synchronization of endogenous rhythms to the environmental light-dark cycles.

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We have been studying the molecular chronobiology of sandflies and mosquitoes for the last few years. Initially, we carried out an analysis of the activity rhythms and circadian expression of per, tim, Clk and cyc in the sandfly Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Latin America. The analysis of the activity rhythms in L. longipalpis, a crepuscular/nocturnal insect in the wild, revealed that, compared to D. melanogaster, this sandfly shows more nocturnal activity under controlled laboratory conditions. Circadian expression of the four clock genes also revealed interesting differences between fruitflies and sandflies. Even though L. longipalpis per and tim show peaks of mRNA abundance in the early evening, as observed in D. melanogaster, the expression of sandfly Clk and cyc differ strikingly (Meirelles-filho et al 2006a; b). L. longipalpis Clk mRNA levels peak around the light-dark transition, while in the fruitfly the peak occurs in the late night / early morning. In addition, cyc shows a rhythm in mRNA abundance in Lutzomyia, even though it is constitutively expressed in Drosophila. Another interesting difference between fruitflies and sandflies is the fact that L. longipalpis Cyc protein has a C-terminal activation domain that is missing in Drosophila (Meirelles-filho et al 2006a;b). We also examined the effect of blood-feeding in the expression levels of these four clock genes in L. longipalpis. While Clk and cyc sustain their levels in blood-fed female sandflies, we observed down-regulation of per and tim expression, which is correlated with a reduction in locomotor activity (Meirellesfilho et al 2006a;b). These results suggest that the endogenous circadian clock and its control over the activity rhythms in this vector are modulated by blood intake. We are currently concentrating our efforts on the behavioural and molecular analysis of mosquito activity rhythms and we are investigating the differences between the diurnal Aedes aegypti, a vector of dengue and yellow fever viruses, and the nocturnal Culex quinquefasciatus, a vector of filariasis and West-Nile fever virus. We analysed the circadian expression patterns of

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clock genes in both species. The results revealed conserved expression profiles in most genes except cryptochrome2 (cry2) (Gentile et al 2009). Cry2 is a negative transcriptional regulator related to mammalian cryptochromes, that is found in a number of insects but is absent in Drosophila. We observed that cry2 mRNA abundance shows a single peak in Cx. quinquefasciatus but a bimodal pattern in Ae. aegypti, suggesting differences in the way the gene is regulated in the two species (Gentile et al 2009).
References:

Hardin PE 2005. The Circadian Timekeeping System of Drosophila. Curr Biol 15: R714-R722 Gentile C, Rivas GBS, Meireles-Filho ACA, Lima JBP, Peixoto AA 2009. Circadian expression of clock genes in two mosquito disease vectors: cry2 is different. J Biol Rhythms 24: 444-51. Klowden MJ, Zwiebel L 2004. Vector Olfaction and Behavior. In Biology of Disease Vectors 2nd Edition. ed. Marquardt WC et al. Elsevier Academic Press. Amsterdam. Meireles-Filho AC, da S Rivas GB, Gesto JS, Machado RC, Britto C, de Souza NA, Peixoto AA. 2006a. The biological clock of an hematophagous insect: locomotor activity rhythms, circadian expression and downregulation after a blood meal. FEBS Lett. 580: 2-8. Meireles-Filho AC, Amoretty PR, Souza NA, Kyriacou CP, Peixoto AA 2006b. Rhythmic expression of the cycle gene in a hematophagous insect vector. BMC Mol Biol. 7: 38. Saunders DS 2002. Insect Clocks. 3rd edition Elsevier Science. Amsterdam.

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Mansonella spp E VETORES NO AMAZONAS

Felipe Arley Costa Pessoa1, Marilaine Martins2 e Jansen Fernandes Medeiros3


1Centro de Pesquisa Lenidas e Maria Deane-Fiocruz, Manaus, AM, Brasil Rua Terezina, 476. Adrianpolis. Manaus -AM, 69.057-070. 2. Fundao de Medicina Tropical do Amazonas, Universidade do Estado do Amazonas, 3. Instituto Nacional de Pesquisas da Amaznia/Universidade do Estado Amazonas.

A regio amaznica caracterizada pela sua imensido e seu isolamento. Apesar de ser pouco povoada em relao a outras regies do Brasil, as comunidades ribeirinhas esto espalhadas em todos os grandes rios. Geralmente em reas distantes entre si e de seus respectivos municpios e muito pouco assistidos quanto a sade. Entre as diversas doenas endmicas encontradas nessa regio, existe a mansonelose que uma filariose, causada pelo acmulo de microfilrias de Mansonella spp (Nematoda: Onchocercidae) nos vasos sanguneos e perifrico do homem. Provavelmente a mais negligenciada, apesar de existir estudos epidemiolgicos feitos por Deane (1949) e outros pesquisadores desde o final da dcada de 40. A M. ozzardi possui distribuio limitada ao continente americano e no Brasil j foi encontrada nos estados do Amazonas, Roraima e Mato Grosso. No estado do Amazonas, acomete em grande escala as populaes ribeirinhas e indgenas (Medeiros & Py-Daniel 2009). Em 2009, Damasceno & Py-Daniel assinalaram a primeira ocorrncia de M. perstans para o alto rio Negro, municpio de So Gabriel da Cachoeira, Amazonas e encontraram Culicoides paraensis (Diptera: Ceratopogonidae) infectado.. Essa filariose possui uma sintomatologia muito discutida, embora vrios pesquisadores que conviveram em reas endmicas, argumentem com convico que a mansonelose apresenta sintomas bem evidentes nos indivduos com elevada quantidade de microfilrias no sangue. Os principais sintomas observados nos indivduos com a doena so: febre moderada sem causa aparente, frieza nas pernas, dores nas articulaes e cefalia (dor de cabea).

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Branco et al (1998) e Cohen et al (2008) observaram problemas oculares em pacientes que estava infectados com Mansonella spp. e levantaram a possibilidade da associao entre leses oculares e esse filardeo. Martins et al (2010) encontraram diferenas significativas aos sintomas de dores articulares, frieza nas pernas, febre em pacientes com mansonelose, comparando com pessoas que no tinham a filariose na mesma rea. Muitas vezes a sintomatologia confundida com outra doena da regio, a malria, pela presena de febre e calafrios (Medeiros et al 2008), diminuindo a qualidade de vida da pessoa com a doena.. A transmisso da M. ozzardi realizada por duas famlias de Diptera dentro de Culicomorpha: Ceratopogonidae (maruim ou mosquitos-plvora) e Simuliidae (pium ou borrachudo). No Brasil at o momento, somente os simuldeos esto apontados como transmissores de M. ozzardi, sendo quatro espcies do gnero Cerqueirellum envolvidas na transmisso: C. amazonicum, C. argentiscutum, C. oyapockense e o recentemente descrito C. pydanieli (Pessoa el al 2008a) como provvel transmissor. Esses simuldeos so extremamente parecidos quanto a suas formas adultas e muitas vezes necessrio que se faa um estudo morfolgico associando-se larvas e pupas. Seus criadouros so em grandes rios brasileiros, particularmente na regio amaznica. So diurnos, e atacam em grande nmero, chegando por vezes a 2.500 picadas hora (Py-Daniel, comunicao pessoal). Medeiros & Py-Daniel (2004) calcularam uma taxa de picada anual de aproximadamente 87 mil por habitante no municpio de Manacapuru, Amazonas, Ros-Velsquez (comunicao pessoal) calculou taxa de picada anual de 80 mil em uma localidade em Roraima. O estudo da taxonomia e sistemtica dos simuldeos no Brasil no tem ainda uma uniformidade quanto a nomenclatura, sendo que uma escola mais tradicionalista prefere manter as espcies em um mega gnero Simulium (Shelley et al 2009); Em 1994, Py-Daniel & Moreira-sampaio, propuseram, baseado em estudo filogentico, que o gnero Simulium parafiltico e elevaram os subgneros existentes a gnero. E diversos

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grupos de espcies em novos gneros. Em 2008b, Pessoa et al fizeram um estudo filogentico baseado em 42 caracteres de larvas, pupas e adultos e confirmaram a monofilia do gnero Cerqueirellum. De diversos trabalhos epidemiolgicos j feitos para reas endmicas de mansonelose, encontrou-se prevalncias que variavam entre 12 a mais de 60% (e.g. Lawrence et al 1979, Medeiros et al 2008, Medeiros et al 2009a, Medeiros et al. 2009b, Martins et al 2010). Levando-se em conta que somente o estado do Amazonas foi relativamente bem amostrado, com cerca de quase um milho de habitantes que habitam em reas endmicas e que casos tambm esto ocorrendo em reas urbanas (Martins et al 2010) e que ainda no existem bons levantamentos epidemiolgicos nos estados vizinhos, pode-se apenas dimensionar que existem uma grande quantidade de pessoas infectadas por essa filariose. Existem apenas trs grupos formais cadastrados no Diretrio de pesquisa do CNPq e ainda o Ministrio da Sade no reconhece como doena e apesar de ser de fcil tratamento, a dificuldade de acesso ao remdio grande, principalmente para populaes isoladas. A sensibilizao dos tomadores de decises e das autoridades de sade para tratamento torna-se urgente para a regio amaznica. Referncias Branco, B.C.; Chamon, W.; Belfort Neto, R.; Belfort, Jr.; Costa, A.J.A. Achados oculares entre habitantes do municpio de Pauini e possvel associao entre leses corneanas e mansonelose na Amaznia. Arquivo Brasileiro de oftalmologia, 61(6):674-642, 1998. Cohen JM, Ribeiro JAS, Martins M 2008. Acometimento ocular em pacientes com mansonelose. Arq Bras Oftalmol 71: 167-171. Deane MP 1949. Sobre a incidncia de filrias humanas em Manaus, estado do Amazonas. Rev Serv Esp Saude Publ 2: 849-858. Damasceno CP 2009. Estudos sobre a transmisso de Mansonella spp. (Nematoda: Onchocercidae) por Culicoides spp. (Diptera: Ceratopogonidae) na comunidade de Assuno, Rio Iana, Amazonas.. 2009. Dissertao- Instituto Nacional de Pesquisas da Amaznia. Lawrence ND, Erdtmann B, Peet JW, Nunes de Mello JA, Hearl GR, James V,

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Neel MD, Salsano FM 1979. Estudos epidemiolgicos entre populaes indgenas da Amaznia. II. Prevalncia da microfilaremia de M. ozzardi: Comparao de dois mtodos de diagnstico. Acta Amazonica, 10: 763-769. Martins M, Pessoa FAC, Medeiros, MB, Andrade EV, Medeiros JF 2010 . Mansonella ozzardi in Amazonas, Brazil: prevalence and distribution in the municipality of Coari, in the middle Solimes River. Mem Inst Oswaldo Cruz 105: 246-253. Medeiros, JF, Py-Daniel V. Seasonality, parity and transmission indices of Mansonella ozzardi (Manson) (Nematoda: Onchocercidae) by Cerqueirellum argentiscutum (Shelley & Luna Dias) (Diptera: Simuliidae) in a lower Solimes River community, Amazonas, Brazil. Acta Amazonica, 34: 201-207, 2004. Medeiros JF, Py-Daniel 2009. Mansonelose. In: Carlos Brisola Marcondes. (Org.). Doenas transmitidas e causadas por artrpodes. 1 ed. So Paulo: Atheneu, p. 274-280. Medeiros JF, Py-Daniel V, Barbosa UC, Ogawa GM 2008. Current profile of Mansonella ozzardi (Nematoda: Onchocercidae) in communities along the Ituxi river, Lbrea municipality, Amazonas, Brazil. Mem Inst Oswaldo Cruz, 103 409411. Medeiros JF, Py-Daniel V, Barbosa UC, Izzo TJ 2009a. Mansonella ozzardi in Brazil: prevalence of infection in riverine communities in the Purus region, in the state of Amazonas. Mem Inst Oswaldo Cruz, 104: 74-80.
Medeiros JF, Py-Daniel V, Barbosa UC, Ogawa GM 2009b. Ocorrncia da Mansonella ozzardi (Nematoda, Onchocercidae) em comunidades ribeirinhas do rio Purus, municpio de Boca do Acre, Amazonas, Brasil. Cadernos de Sade Pblica, v. 25, n. 6, p. 1421-1426.

Pessoa FAC, Barbosa UC, Medeiros JF 2008a. A new species of Cerqueirellum Py-Daniel, 1983 (Diptera: Simuliidae) and proven new vector of mansonelliasis from the Ituxi River, Amazon basin, Brazil. Acta Amazonica, 38: 569-582. Pessoa FAC, Py-Daniel V, Ros Velsquez CM 2008. Cladistic analysis of the Neotropical genera Cerqueirellum Py-Daniel, 1983, Coscaroniellum Py-Daniel, 1983 and Shelleyellum PyDaniel & Pessoa, 2005 (Diptera: Simuliidae). Acta Amazonica 38: 551-568. Py-Daniel V, Sampaio RTM 1994. Jalacimgomyia gen. N. (Culicomorpha); a ressurreio de Gymnopaidinae; a eliminao do nvel tribal; apresentao de novos caracteres e a redescrio dos estdios larval e pupal de Simulium colombaschense (Fabricius, 1787) (Diptera: Simuliidae). Mem CAICET 4: 101148. Shelley AJ, Hernndez LM, Maia-Herzog M, Luna Dias APA, Garritano PR 2010 The Blackflies (Dipetra: Simuliidae) of Brazil Vol.6, Pensoft, Sofia, 821pp Tavares, A.M.; Fraiha Neto, H. Mansonelose. IN. Doenas infecciosas e parasitrias. Enfoque Amaznico. 737p, 1997.

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SENSITIVITY OF OVITRAPS AND ADULTRAPS FOR DETECTING HOUSEHOLD INFESTATION BY THE DENGUE VECTORS Aedes aegypti and Aedes albopictus Srgio LB Luz1, Fernando Abad-Franch1, Gonalo Ferraz2 Research Program on Infectious Disease Ecology in the Amazon (RP-IDEA), Instituto Lenidas e Maria Deane Fiocruz Amaznia, Manaus, Brazil 2 Biological Dynamics of Forest Fragments Project (BDFFP), Smithsonian Tropical Research Institute/Instituto Nacional de Pesquisas da Amaznia, Manaus, Brazil
1

Background Vector surveillance is at the root of Dengue control and prevention, with many methods available for detecting the presence of the primary mosquito vectors, Aedes aegypti and Ae. albopictus, in or around human residences. The ideal detection device should be highly sensitive, cheap, easily operated, and acceptable to householders. Oviposition traps (Ovitraps) are considered to meet these requirements reasonably, but a new adult mosquito trap (Adultrap) was recently proposed as an alternative or supplementary method for detecting infestation. In the absence of a 'gold standard' capable of indentifying infested households without error under field conditions, measuring the sensitivity of mosquito detection devices remains a high-priority challenge. Here, we offer a comparative assessment of the performance of Ovitraps and Adultraps, along with a statistical estimate of time-varying infestation rates for both vector species.

Methods Building upon analytical methods developed by wildlife biologists to study animal occurrence under imperfect detection, we investigated the sensitivity of Ovitraps and Adultraps in 50 households of Manaus, the largest Central Amazon city. Households were randomly assigned to one of three treatments: 2 Ovitraps one week plus 2 Ovitraps the following week (N=25 households); 2 Ovitraps one week plus 2 Adultraps the following week (N=12); and, finally, only 2 Adultraps only in the

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second week (N=13). This 50-site, two-week sampling strategy was applied monthly from September 2008 to September 2009. Individual trap results (positive/negative) were recorded separately, yielding a monthly 'detection history' for each household. Detection histories were then used to obtain estimates of the probabilities of detecting Ae. aegypti and Ae. albopictus in households that were actually infested during each two-week sampling period. Estimation was based on a hierarchical logistic model implemented in a Bayesian framework.

Results Ovitraps were overall much more sensitive (~68%, 95% Confidence Bounds [CB95%] 64-72) than Adultraps (~17%, CB95% 12-23) in detecting A. aegypti when it was actually present in a household. Ovitrap sensitivity was even higher for Ae. albopictus (~81%, CB95% 77-84), whereas Adultraps performed similarly poorly (~12%, CB95% 8-16). When comparing Ovitrap performance between weeks within each month, first-week traps were slightly more sensitive than second-week ones for Ae. aegypti (68.8%, CB95% 65.2-72.1 vs. 67.6%, CB95% 63.3-71.8); the reverse was true for Ae. albopictus, with second-week Ovitraps being more sensitive (78.2%, CB95% 75.1-80.9 vs. 83%, CB95% 79.7-86.3). Adultrap performance was somewhat worse in households where Ovitraps had been operated in the previous week (mean sensitivity: Ae. aegypti 13.4%, CB95% 9.3-18.1, Ae. albopictus 9%, CB95% 5.7-12.7) than at sites where only Adultraps were deployed (Ae. aegypti 21%, CB95% 15.5-28, Ae. albopictus 14.8%, CB95% 10.4-20.1). Monthly infestation rate estimates varied from 97.2% (CB95% 90.2-99.9, September 2008) to 69.3% (CB95% 53.7-84, September 2009) for Ae. aegypti; this is over one order of magnitude higher than the official Dengue surveillance figures (2-8.2%) for our study area and period. Ae. albopictus infestation estimates were always above 80%, with a lower CB95% limit of 68.6% in March 2009.

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Conclusions Even though individual Ovitraps failed to detect infestation in just over 30% of occasions, they proved reasonably sensitive for A. aegypti detection under field conditions, even when operated for a single week. When the goal is to obtain a binary measure of infestation, Ovitraps overwhelmingly outperform both the regular surveillance scheme based on household inspections by trained personnel and the newer Adultraps. These results were even clearer for Ae. albopictus, with Ovitrap sensitivity in excess of 80% in the second week of operation. In contrast, the more costly and cumbersome Adultraps performed very poorly for both species with detection failures documented in over 80% of sites where the vectors were actually present. Adultrap use in Dengue vector surveillance is therefore questionable, particularly when detecting household infestation by A. aegypti is the operational goal. Our results suggest that infestation indices currently used for both risk stratification and operational decision-making are extremely inaccurate. The use of Ovitraps for ascertaining the presence or absence of the target Aedes species in households, combined with a sampling and analytical approach that takes detection failures into account, could critically improve the efficiency of Dengue vector control and surveillance.

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HEMATOPHAGY AND CELL SIGNALING IN MOSQUITOES: THE ROLE OF PROTEIN TYROSINE PHOSPHATASES AS MODULATORS OF AEDES AEGYPTI IMMUNITY. Ceclia de O. Cudischevitch1, Raquel S. Telhado1, Rodrigo D. Nunes1, Alan B. Silveira1, Felipe Gazos-Lopes1, Gergia C. Atella1, Marcos H. Sorgine1, Lalima L. Madan2, Rafael Mesquita3 and Mrio Alberto C. Silva-Neto1
1-Programa de Biologia Molecular e Biotecnologia, Instituto de Bioqumica Mdica, Universidade Federal do Rio de Janeiro, UFRJ. 2-Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India. 3-Instituto Federal de Educao, Cincia e Tecnologia do Rio de Janeiro, RJ.

Tyrosine phosphorylation is a key modulator of signal transduction in living cells. In fact due to their extreme role on cell biology intracellular phosphotyrosine levels are quite low and are strictly regulated by the balance between tyrosine kinases and tyrosine phosphatases (PTPs). This later enzymes play a key role in the fine tunning of immune system in mammalian cells but their role in mosquito defense mechanisms against pathogens remains largely unknown. Mosquito immunity is continuously challenged both by its continuously oscillating microbiota as well by invading pathogens such as malaria parasite. Thus it turns out to understand how intracellular mechanisms eventually based on tyrosine phosphorylation may regulate such complex panel. We have identified a Protein Tyrosine Phosphatase (PTP) activity in Aedes aegypti midgut. Total enzyme activity against an artificial substrate using sugar-fed mosquitoes is strongly inhibited by classical PTP inhibitors and also by a specific substrate peptide of the mammalian classical PTPs subtype as PTP 1B. Gel filtration chromatography of midgut extracts showed a major peak of PTP activity with apparent molecular mass of 112 kDa whose activity is also supressed by the overall PTP inhibitor sodium vanadate. Once upon blood feeding total midgut PTP activity is partially supressed and such decrease is followed by an increase in total phosphotyrosine phosphorylation profile in this tissue. However, when mosquitoes were fed with

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blood infected with the malaria parasite Plasmodium gallinaceum total PTP activity against PTP 1B substrate is only partially supressed and remains close to the levels determined in sugar-fed mosquitoes. In order to determine the specific profile of different PTP expression under different meals we have identified 43 genes coding for mosquito PTPs in Aedes aegypti genome. 16 genes code for type I classical PTPs which are enzymes totally devoted to the regulation of phosphotyrosine levels in living cells. Such 16 genes code for 18 transcripts and thus those transcripts were submitted to a structural analysis in order to identify the ones which are truly catalytically active PTPs. 10 PTPs coded by only 08 genes fall into this category. Profiling the expression of these 10 PTP genes under different meals showed that the gene AAEL003108 codes for a PTP whose RNA is the most abundant in all tested conditions. Also transcription of such gene is enhanced by blood feeding and maximally enhanced after a malaria-enriched blood feeding. Once midgut microbiota is largely affected in blood and malaria-fed mosquitoes we have hypothesized whether the expression of such PTP is modulated by bacterial populations. Treatment of an Aedes aegypti cell culture, Aag2 with different molecules that either mimics viruses or bacteria lead to a huge increase on the synthesis of AAEL003108 mRNA. Also expression of such gene follows the same immune profile of the antimicrobial peptide Defensin. We have next established whether there is a molecular link between the expressions of such genes. Antibiotic treatment of mosquito midgut suppressed both the expression of AAEL003108 and defensin. Silencing 3108 gene through RNAi assays in live mosquitoes also lead to suppression of defensin mRNA synthesis. In conclusion, these data show that a mosquito PTP is able to control the expression of an antimicrobial peptide. Altogether, these data show for the first time that bloodfeeding and malaria parasite infection manipulate diferential sets of mosquito PTPs probably involved in mosquito immunity and enroll these enzymes as intracellular signalling sensors of hematophagy.

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References: Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T 2004. Protein tyrosine phosphatases in the human genome. Cell 117: 699-711. Cross J L, Johnson P 2009. Tyrosine phosphorilation in immune cells: direct and indirect effects on Toll-like receptor-induced proinflamatory cytokine production. Critical Reviews in Immunology. Lemaitre B, Hoffman J 2007. The host defense of Drosophila Melanogaster. Annual Review of Immunology 25: 697-743. Olsen J V, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P Mann M. 2006. Global, in vivo, and site-specific phosporylation dynamics in signaling networks. Cell. 127: 635-648. Tonks N K 2006. Protein tyrosine phosphatases: from genes, to function, to disease. Nature Reviews Molecular Cell Biology. 7: 833-846.

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ESTUDO DA DISPERSO DE Lutzomyia longipalpis EM CAMPO GRANDE, MATO GROSSO DO SUL

Everton Falco de Oliveira1, Elaine Arajo e Silva2, Antonio Conceio Paranhos Filho3,Carlos Eurico Fernandes4, Alison Andr Ribeiro3, Roberto Macedo Gamarra3, Reginaldo Peanha Brazi5, Alessandra Gutierrez de Oliveira1,4
1. Universidade Federal de Mato Grosso do Sul - Programa de Ps-graduao em Doenas Infecciosas e Parasitrias, Campo Grande, MS. 2. Centro de Controle de Zoonoses, Secretaria de Sade do Municpio de Campo Grande, MS. 3. Universidade Federal de Mato Grosso do Sul - Laboratrio de Geoprocessamento para Aplicaes Ambientais/DHT/CCET, Campo Grande, MS. 4. Universidade Federal de Mato Grosso do Sul - Laboratrio de Parasitologia/DPA/CCBS, Campo Grande, MS. 5. Instituto Oswaldo Cruz - LBII, Rio de Janeiro, RJ.

Aspecto importante para a disseminao da doena e ainda pouco conhecido, diz respeito disperso de seus vetores. No entanto, estudos dessa natureza em ambientes florestais e principalmente urbanos, so escassos no Brasil (BRAZIL; BRAZIL, 2003). Poucos estudos de disperso realizados com flebotomneos do gnero Phlebotomus mostraram deslocamento superior a 1500m (DOHA et al., 1991; KILLICK-KENDRICK et al., 1984), no entanto, para espcies da Amrica, no ultrapassaram 200m, com exceo de Lu. longipalpis, que alcanou at 960m na Colmbia e 260m na regio Amaznica (ALEXANDER, 1987; ALEXANDER; YOUNG 1992; CASANOVA et al., 2005; DYE; DAVIES; LAINSON, 1991; KELLY, DYE, 1997; MORRISON et al. 1993). A partir de 2005 iniciaram-se os estudos de disperso na rea urbana de Campo Grande, na ocasio foram marcados 1.034 espcimes de Lu. longipalpis, sendo que 99,4% foram recapturados no prprio local de soltura e 0,6% nos 50 e 100m de distncia juntos aps 24h (OLIVEIRA, 2006). Em dois experimentos realizados, um na Colmbia e outro no Brasil, foi observado que esta espcie capaz de grandes deslocamentos em curto espao de tempo. Morrison et al. (1993) recapturaram um exemplar a 960m em 48 horas, e no Brasil, Dye, Davies e Lainson (1991) recuperaram em poucos dias espcimes de Lu. longipalpis a 20, 85, 200 e 700m de distncia.

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A elevada taxa de recaptura no ponto de soltura, constatada por Oliveira (2006), sugere que os flebotomneos, em reas urbanas, apresentam pouca atividade de disperso, permanecendo no prprio local, devido presena abundante de alimento e abrigo. Fundamenta esta afirmativa, a anlise do sangue ingerido pelas fmeas de Lu. longipalpis capturadas no ectopo deste experimento, que detectou 86,4% de sangue de ave no trato digestivo dos espcimes. Assemelham-se a este achado, aqueles de Dye, Davies e Lainson (1991) que obtiveram uma taxa de recaptura de 83% no prprio local de soltura. Em 2008, outro estudo de disperso de Lu. longipalpis foi iniciado com auxlio das geotecnologias. Na ocasio foram georreferenciadas 20 residncias numa rea perifrica da cidade. Os resultados demonstraram deslocamento de 156, 169 e 223m de distncia (dados no publicados). Com base nos dados apresentados acima, objetivou-se analisar a distribuio espacial de Lu. longipalpis, em seis experimentos de captura, marcao e soltura ao longo de um ano, relacionando-os aos parmetros de cobertura do solo, ndice de vegetao normalizado (NDVI) e variaes climticas (temperatura, umidade relativa do ar, precipitao pluviomtrica, direo e velocidade do vento). Estes dados foram gerados por meio de um densimetro, de imagens IKONOS-2 de 05 de maro de 2006 e de uma estao meteorolgica Vantage Duo instalada no prprio local, respectivamente. Os resultados preliminares de trs destes experimentos mostraram deslocamento mximo de 55m, sendo que a maioria dos insetos foram recapturados no mesmo local de soltura. Atravs da anlise de varincia linear simples foi evidenciado que houve o predomnio de machos recapturados. A anlise de correlao revelou nveis de baixa intensidade entre a cobertura vegetal e o total de machos marcados (r= 0,20), machos no marcados (r= 0,25), fmeas marcadas (r= 0,12) e fmeas no marcadas (r= 0,22). Para o NDVI as correlaes foram r= 0,11 para machos marcados; r=0,20 para machos no marcados; e r= 0,17 para fmeas no marcadas. Todas as correlaes foram

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significativas (p< 0,05, modelo de Pearson). Embora os resultados sejam preliminares, possvel estimar que o deslocamento dos insetos em rea urbana apresenta um padro focal com efeito importante da cobertura vegetal e ndice de vegetao normalizado. Os dados climticos esto sendo colhidos e processados para futuras anlises. REFERNCIAS ALEXANDER, J. B.; YOUNG, D. G. Dispersal of phlebotomine sand flies (Diptera: Psychodidae) in a Colombian focus of Leishmania (Viannia) braziliensis. Memrias do Instituto Oswaldo Cruz, Rio de Janeiro, v. 87, n. 3, p. 397-403, July/Sept. 1992. ALEXANDER, J. B. Dispersal of phlebotomine sand flies (Diptera: Psychodidae) in a Colombia coffe plantation. Journal of Medical Entomology, Lanham, v. 24, n. 5, p. 552-558, Sept. 1987. BRAZIL, R. P.; BRAZIL, B. G. Biologia de flebotomneos neotropicais. In: RANGEL, E. F.; LAINSON, R. Flebotomneos do Brasil. Rio de Janeiro: Fiocruz, 2003. cap. 4, p. 257-274. CASANOVA, C.; COSTA, A. I. P.; NATAL, D. Dispersal pattern of tha sand fly Lutzomyia neivai (Diptera: Psychodidae) in a cutaneous leishmaniasis endemic rural area in Southeastern Brazil. Memrias Instituto Oswaldo Cruz, Rio de Janeiro, v. 100, n. 7, p. 719-724, Nov. 2005. DOHA, S.; SHEHATA, M. G.; EL SAID, S.; EL SAWAF, B. Dispersal of Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu in El Hammam, Matrouh Governorate, Egypt. Annales de Parasitologie Humaine et Compare, Paris, v. 66, n. 2, p. 69-76, Mar. 1991. DYE, C.; DAVIES, C. R.; LAINSON, R. Communication among phlebotomine sandflies: a field study of domesticated Lutzomyia longipalpis populations in Amazonian Brazil. Animal Behaviour, St. Louis, v. 42, n. 2, p. 183-192, Aug. 1991. KELLY, D. W.; DYE, C. Pheromones, kairomones and the aggregation dynamics of the sandfly Lutzomyia longipalpis. Animal Behaviour, St. Louis, v. 53, n. 4, p. 721-731, Apr. 1997. KILLICK-KENDRICK, R.; RIOUX, J. A.; BAILLY, M.; GUY, M. W.; WILKES, T. J.; GUY, F. M.; DAVIDSON, I.; KUCHILI, R.; WARD, R. D.; GUILVARD, E.; PERIERES, J.; DUBOIS, H. Ecology of leishmaniasis in the South of France. 20. Dispersal of Phlebotomus ariasi Tonnoir, 1921 as a factot in the spread of the visceral leishmaniasis in the Cvennes. Annales de Parasitologie Humaine et Compare, Paris, v.59, n. 6, p. 555-572, Nov./Dec.1984.

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MORRISON, A. C.; FERRO, C.; MORALES, A.; TESH, R.; WILSON, M. L. Dispersal of the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) at an endemic focus of visceral leishmaniasis in Colombia. Journal of Medical Entomology, Lanham, v. 30, n. 2, p. 427-35. Mar. 1993. OLIVEIRA, A. G. Estudos ecolgicos de Phlebotominae (Dptera: Psychodidae) na rea urbana do municpio de Campo Grande, Mato Grosso do Sul. 2006. 133 f. Tese (Doutorado em Biologia Parasitria) Instituto Oswaldo Cruz, Fundao Oswaldo Cruz, Rio de Janeiro, 2006.

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BASES MOLECULARES DA RESISTNCIA AO TEMEPHOS EM Aedes aegypti Constncia Ayres


Departamento de Entomologia, Centro de Pesquisas Aggeu Magalhes/ FIOCRUZ

Introduo A dengue continua como um grave problema de sade pblica no Brasil. Para exemplificar, somente no estado de Pernambuco at o momento (Julho 2010) houve um aumento de 459,96% no nmero de casos da doena notificados em relao ao mesmo perodo do ano anterior. No existem vacinas disponveis para o combate da doena e por isso, o controle do vetor, o mosquito Aedes aegypti, ainda a nica forma de prevenir a disseminao da dengue. O controle do A. aegypti feito, segundo recomendaes do Programa Nacional de Controle do Dengue, atravs do uso de inseticidas e eliminao de criadouros no ambiente. Durante muito tempo o larvicida organofosforado, temephos, foi o inseticida de escolha para eliminao das larvas do mosquito. Entretanto, a resistncia a este composto foi registrada em diversas populaes do pas e o mesmo foi substitudo pelo inseticida biolgico, Bti, larvicida base da bactria entomopatognica Bacillus thuringiensis israelensis. A idia seria substituir pelo Bti, para que o status de susceptibilidade ao temephos fosse restaurado e este pudesse ser novamente empregado. Entretanto, foi verificada em campo que a susceptibilidade a este composto revertia de forma lenta, e mesmo aps 4 anos de interrupo do uso deste composto, a resistncia ainda estava presente nas populaes (Ministrio da Sade, 2009). Desenvolvemos, portanto, uma linhagem de A. aegypti

resistente ao temephos, que apresentou no final uma Razo de Resistncia (RR) em torno de 180 vezes (Melo-Santos et al., 2010), para que fosse possvel estudar o processo de evoluo e reverso da resistncia ao temephos de forma mais aprofundada, e conhecer as bases moleculares que levam ao aparecimento da

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resistncia a este composto. Esta linhagem foi denominada de RecR e no momento encontra-se em sua 23 gerao. A resistncia aos inseticidas qumicos provocada basicamente por dois principais mecanismos no excludentes, a insensibilidade do stio-alvo do inseticida (alterao do stio alvo) ou por alterao na estrutura ou nvel de expresso das enzimas que metabolizam os inseticidas (resistncia metablica). No primeiro caso, um nmero pequeno de mutaes j foi descrito na literatura, que reporta alteraes no canal de sdio, alvo dos piretrides e DDT, no receptor do cido gama amino butrico (GABA), stio de ao dos ciclodienos e na acetilcolinesterase (Ace), alvo dos organofosforados e carbamatos. Estas

mutaes j so bem descritas e mtodos de diagnstico molecular foram especificamente desenvolvidos para a realizao de screening em populaes de campo. Vale ressaltar que no caso da Ace, nunca foram descritas mutaes neste gene em populaes naturais de A. aegypti (Weill et al., 2004) que estivessem envolvidas com a resistncia a organofosforados, como j descrito em outros insetos. Acredita-se que como estas molculas alvo desempenham funes essenciais no organismo, as mesmas no podem suportar um grande nmero de mutaes, pois estas poderiam ser letais. Por outro lado, a resistncia metablica envolve um grande nmero de genes pertencentes a superfamlias gnicas, que evoluem em sua maioria por duplicao gnica, gerando genes redundantes em funo e passveis de acumular variaes que poderiam levar inclusive a novas funes gnicas. Em A. aegypti, por exemplo, foram identificados 235 membros, classificados nas trs principais famlias de enzimas detoxificadoras: Citocromo P450, Glutationa stransferases (GSTs) e esterases (Strode et al., 2008). Com este amplo arsenal de genes metablicos que poderiam estar envolvidos com os diversos mecanismos de resistncia, identificar mutaes pontuais responsveis por estes mecanismos e desenvolver mtodos de diagnostic-los torna-se uma tarefa quase impossvel. Com o intuito de facilitar a identificao do papel das enzimas de

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detoxificao nos diferentes mecanismos de resistncia metablica em A. aegypti, Strode et al (2008) desenvolveram o Detox Chip, o qual j havia sido desenvolvido previamente tambm para o principal vetor da malria, o Anopheles gambiae (David et al., 2005). Este chip permite avaliar atravs da tcnica de microarranjos (microarrays) a expresso de mais de 200 genes de detoxificao simultaneamente. Isto facilitou enormemente a comparao dos padres de expresso destes genes em linhagens resistentes e susceptveis a inseticidas qumicos, alm de identificar os genes individuais envolvidos em cada mecanismo. Este chip j foi empregado em alguns trabalhos, onde se avaliou populaes de A. aegypti resistentes a piretrides e DDT. Entretanto, a base molecular da resistncia ao temephos ainda permanece desconhecida. O objetivo do presente estudo foi, portanto, caracterizar a base gentica da resistncia na linhagem de A. aegypti RecR.

Materiais e Mtodos A linhagem RecR, cerca de 180 vezes resistente ao temephos, foi usada nos experimentos de microarranjos para identificao dos possveis genes envolvidos na resistncia. Esta anlise foi feita de forma pareada com a colnia Rec-Lab, considerada padro de susceptibilidade. Os detalhes do processo de seleo da Rec-R, assim como da reverso da resistncia nesta linhagem est descrito em Melo-Santos et al. (2010). A tcnica de microarranjo foi executada na Liverpool School of Tropical Medicine, seguindo a metodologia descrita em Strode et al. (2008). A validao dos resultados obtidos por microarranjos foi feita atravs da tcnica de PCR quantitativa seguindo a metodologia descrita em Magalhes et al. (2008), utilizando amostras (somente pools de larvas) da linhagem RecR, da RecLab (susceptvel) e tambm da RecReversa (RecRev), a qual representa uma sublinhagem da RecR, cuja resistncia foi parcialmente revertida por supresso do uso do inseticida (interrupo do processo de seleo). Bioensaios e testes bioqumicos foram feitos para acompanhar o status de susceptibilidade das

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linhagens e avaliar a atividade das enzimas metablicas, respectivamente. Estes testes foram feitos de acordo com Montella et al. (2007). Alm disso, uma populao natural de Araripina, situada a 690 Km de Recife e que deu origem a RecR tambm foi avaliada pelos testes bioqumicos e bioensaios.

Resultados: Os testes bioqumicos demonstraram atividades elevadas das GSTs, esterases (alfa e beta) e em menor grau as oxidases de funo mista (MFO) na RecR, sendo as alteraes das GSTs as de maior importncia, tanto em larvas como em adultos. Os resultados do microarranjo com o Detox Chip revelaram a existncia de genes superexpressos na RecR, tanto em larvas quanto em mosquitos adultos. No estgio larval foram identificados 6 genes (1 CYP, 3 GSTs, 1 esterase e 1 peroxinectina) enquanto que nos adultos 13 genes mostraram-se super expressos na RecR (dos quais sete foram CYPs). A superexpresso nestes genes variou de 2.10 a 7.03 vezes nas larvas e de 2.12 a 5.8 nos adultos quando comparados RecLab. Um nico gene CYP450 foi super expresso nos dois estgios (larvas e adultos). Os resultados da PCRq confirmaram os dados do microarranjo e mostraram que a quantidade de transcritos tambm cai em torno de 8 vezes na linhagem RecRev para o gene com o maior nvel de expresso. Quando comparadas amostras da RecRev exposta e no exposta ao temephos, os nveis de expresso gnica foram similares. Considerando a populao de Araripina, apenas a -esterase apresentou um padro modificado pelo ensaio bioqumico.

Discusso O temephos o inseticida de escolha para controle de mosquitos vetores e apesar da resistncia a este composto j ter sido registrada em diversas populaes de A. aegypti do Brasil (Carvalho et al., 2004, Luna et al., 2004, Lima et al., 2006, Macoris et al., 2003, 2007) e do mundo (Lazcano et al., 2009, Llins et

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al., 2010), o mesmo continua ainda a ser utilizado em vrias regies. A seleo da resistncia ao temephos em laboratrio tambm j foi feita em outras populaes do mundo (Georghiou et al., 1987; Wirth e Georghiou, 1999; Rodriguez et al., 2002; Hamdan et al., 2005; Tikar et al., 2009), porm raramente se atingiu nveis to altos de resistncia, como o observado em RecR (180 vezes, obtida aps 17 geraes). Wirth e Georghiou (1999) observaram nveis similares em uma linhagem do Caribe, porm neste caso, os autores iniciaram a seleo desta linhagem a partir de uma RR com valor j bastante elevado (RR=46.8) e aps 13 geraes a RR alcanou o valor de 180 vezes, representando um aumento de apenas 4 vezes. Nos outros trabalhos, o mximo observado foi de 51 vezes aps 32 geraes em uma populao de A. aegypti da Malsia (Handam et al., 2005). O alvo dos organofosforados, como o caso do temephos, a acetilcolinesterase, enzima que cataliza a hidrlise do neurotransmissor acetilcolina, presente nas sinapses nervosas. Mutaes no gene que codifica esta enzima (Ace) levam a resistncia aos inseticidas da classe dos organofosforados. Uma das mutaes neste gene que leva resistncia, j bem descrita em outros insetos, improvvel de ocorrer em A. aegypti, pois seria necessria a troca em dois nucleotdeos no mesmo cdon (Weill et al., 2004) e, portanto, sua deteco em campo nesta espcie nunca foi registrada. Em RecR, um estudo preliminar tambm descartou o envolvimento deste gene no mecanismo de resistncia selecionado (Melo-Santos et al., 2010). Isto sugere que o mecanismo metablico o principal responsvel pela resistncia ao temephos nesta linhagem. Os trabalhos relatados acima que acompanharam as atividades das enzimas de detoxificao nas populaes resistentes mostram que, na maioria das vezes, as esterases so as enzimas que apresentam maior alterao de atividade enzimtica. A anlise por microarranjos na RecR revelou somente uma esterase alterada nas larvas e nenhuma em adultos. O nosso trabalho mostrou que o gene CYP que super expresso tanto nas larvas como nos adultos, reduziu sua expresso em torno de 8 vezes na linhagem

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com a resistncia revertida (RecRev). Isto sugere que este gene est possivelmente envolvido com o mecanismo de resistncia que foi selecionado em laboratrio, em um grau muito maior que os outros genes superexpressos. Os outros genes diminuram sua expresso em torno de 1.6 a 4.1 e esto tambm provavelmente associados resistncia, porm numa escala menor. Estes resultados mostram que durante o processo de seleo vrios genes podem ser selecionados, principalmente quando a linhagem foi previamente exposta a vrios inseticidas. Alm disso, a comparao com a RecRev exposta mostrou um dado importante: a superexpresso destes genes no ocorre de forma imediata em resposta ao contato com o inseticida e sim que ela foi selecionada ao longo do processo de seleo e herdada. Este trabalho representa o primeiro estudo a avaliar a base molecular da resistncia ao temephos e sua reverso em A. aegypti e abre novos caminhos para a compreenso dos mecanismos de resistncia mltipla e o desenvolvimento de novas ferramentas de diagnstico e monitoramento da resistncia em campo, facilitando o seu manejo, o que pode de certa forma, aumentar a eficincia dos programas de controle de vetores.

Bibliografia

Carvalho Mdo S, Caldas ED, Degallier N, Vilarinhos Pde T, Souza LC, Yoshizawa MA, Knox MB, Oliveira C. 2004. Susceptibility of Aedes aegypti larvae to the insecticide temephos in the Federal District, Brazil. Rev Saude Publica, 38:623-9. Hamdan H, Sofian-Azirun M, Nazni WA, Lee HL. 2005. Insecticide resistance development in Culex quinquefasciatus (Say), Aedes aegypti (L.) and Aedes albopictus (Skuse) larvae against malathion, permethrin and temephos. Trop Biomed, 22:45-52. Lazcano JA, Rodrguez MM, San Martn JL, Romero JE, Montoya R. 2009. Assessing the insecticide resistance of an Aedes aegypti strain in El Salvador. Rev Panam Salud Publica, 26:229-34.

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Lima EP, de Oliveira Filho AM, de Oliveira Lima JW, Ramos Jnior AN, de Ges Cavalcanti LP, Pontes RJ. 2006. Aedes aegypti resistance to temefos in counties of Cear State. Rev Soc Bras Med Trop. 39(3):259-63. Llins GA, Seccacini E, Gardenal CN, Licastro S. 2010. Current resistance status to temephos in Aedes aegypti from different regions of Argentina. Mem Inst Oswaldo Cruz,105:113-6. Luna JE, Martins MF, Anjos AF, Kuwabara EF, Navarro-Silva MA. 2004. Susceptibility of Aedes aegypti to temephos and cypermethrin insecticides, Brazil. Rev Saude Publica, 38:842-3. Macoris Mde L, Andrighetti MT, Takaku L, Glasser CM, Garbeloto VC, Bracco JE. 2003. Resistance of Aedes aegypti from the state of So Paulo, Brazil, to organophosphates insecticides. Mem Inst Oswaldo Cruz, 98:703-8. Macoris, M.L., Andrighetti, M.T., Otrera, V.C., Carvalho, L.R., Caldas Junior, A.L., Brogdon, W.G., 2007. Association of insecticide use and alteration on Aedes aegypti susceptibility status. Mem Inst Oswaldo Cruz. 102, 895-900. Magalhaes, T., Oliveira, I. F., Melo-Santos, M. A., Oliveira, C. M., Lima, C. A., Ayres, C. F. J. 2008. Expression of defensin, cecropin, and transferrin in Aedes aegypti (Diptera: Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae), and the abnormal development of nematodes in the mosquito. Exp Parasitol, 120: 364-371. Melo-Santos M. A. V., Melo J. J. M. V., Arajo A. P, Paiva M. H. S., Gomes, T. C. S., Regis L. N, Furtado A. F., Magalhaes T, Macoris M. L. G., Andrighetti M. T. M., Ayres CFJ. 2010. Resistance to the organophosphate temephos: mechanisms, evolution and reversion in an Aedes aegypti laboratory strain. Acta Tropica, 113: 180-189. Ministrio da Sade, 2009. Reunio de avaliao do monitoramento da resistncia das populaes de Aedes aegypti do Brasil. Ministrio da Sade, Braslia. Montella, I.R., Martins, A.J., Viana-Medeiros, P.F., Lima, J.B., Braga, I.A., Valle, D. 2007. Insecticide resistance mechanisms of Brazilian Aedes aegypti population from 2001 to 2004. Am. J. Trop. Hyg, 77: 467-477. Strode, C., et al. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. 2008. Insect Biochem. Mol. Biol,.38: 113-23. Weill, M., Berthomieu, A., Berticat, C., Lutfalla, G., Ngre, V., Pasteur, N., Philips, A., Leonetti, JP., Fort, P., Raymond, M. 2004. Insecticide resistance: a silent base prediction. Current Biology, 14: R552-R553.

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PROTEOFOSPHOGLICANO (PPG) CONFERINDO LEISHMANIA MAJOR RESISTNCIA A ENZIMAS DIGESTIVAS INDUZIDAS PELA ALIMENTAO SANGNEA EM FLEBOTOMNEOS VETORES Ngila Francinete Costa Secundino
Laboratrio de Entomologia Medica, Centro de pesquisas Ren Rachou/ Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, Brazil.nagila@cpqrr.fiocruz.br

As leishmanioses se encontram distribudas em 88 pases atravs de 4 continentes colocando em risco cerca de 350 milhes de pessoas. Mais de 90% dos casos ocorrem nos pases da frica, sia e Amrica Latina. Dependendo da espcie de Leishmania transmitida, as infeces humanas podem acarretar manifestaes clnicas que podem ser resumidas em duas formas, a leishmaniose cutnea e a visceral, denominada de acordo com o local preferencial de desenvolvimento do parasito. A leishmaniose cutnea pode causar leses desfigurantes e a leishmaniose visceral pode ser fatal, podendo causar milhares de mortes. A competncia vetorial dos flebotomneos Phlebotomus duboscqi e Lutzomyia longipalpis foi testada utilizando L. major cepa silvestre e cepas mutantes, ou seja, deficiente em fosfoglicanos (LPG2 ou LPG5A-/5B-) ou LPG sozinho (lpg1) juntamente com seus respectivos controles gene adicionar novamente. Nossos resultados confirmam que a LPG, a molcula mais abundante na superfcie dos promastigotas de Leishmania e conhecida por mediar ligao ao intestino do vetor so necessria para evitar a perda de infeco durante o processo de excreo do sangue no vetor natural P. duboscqi, mas no em um vetor natural L. longipalpis. As enzimas digestivas produzidas e induzidas pela alimentao sangnea representam uma potencial barreira para a sobrevivncia da Leishmania. Nossos resultados demonstram que 36-72 h aps a alimentao infecciosa, todos os parasitas desenvolveram bem, exceto o lpg2 e lpg5A-/5B- mutantes, que mostraram uma reduo significativa de sobrevivncia e crescimento. O uso de inibidores de protease promoveu a sobrevida e

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crescimento nos tempos iniciais aps o repasto infectante com a promastigota deficiente Lpg2. O proteofosfoglicano (PPG) mostrou ser a molcula chave conferindo resistncia a enzimas digestivas do intestino mdio, uma vez que impediu a morte de promastigotas de lpg2- quando expostos a lisados do intestino mdio, os quais foram preparados a partir de uma alimentao com sangue no infectado e os intestinos foram dissecados 36 horas aps a alimentao. A proteo no foi associada com a inibio das atividades enzimticas, mas com a aquisio da molcula (PPG) na superfcie celular do parasito. Este fato parece semelhante funo das mucinas em mamferos. O PPG promastigotas contra danos proteolticas. protege a superfcie das

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ASPECTOS DA REPRODUO DE IXODIDA E A TRANSMISSO VERTICAL DE RIQUTSIAS PATOGNICAS Nicolau Maus Serra-Freire

A reproduo nos carrapatos sexuada interna, diica com participao de dois sexos, mas em algumas espcies acontece de forma unissexuada. Fmeas podem se originar unissexuadamente por partenognese telitoca, mas os machos praticamente s acontecem aps conjuno dos dois sexos. No desenvolvimento do ciclo vital passam pelas fases de ovo, larva, ninfa e adultos, que se alimentam, realizam mudas, e fazem ecdises o que d origem a diferentes estdios conhecidos por: neolarva, metalarva, neoninfa, metaninfa, neandro, gonandro, negina, partengina, telegina e quengina. Esta a dinmica da reproduo dos carrapatos duros, famlia Ixodidae, que difere muito da dinmica reprodutiva dos carrapatos moles, famlia Argasidae e que ser abordado mais adiante. A famlia Ixodidae dividida em dois grandes grupos cuja principal caracterstica morfolgica que permite seus reconhecimentos a posio do sulco anal. Nos Prostriata o sulco anal antecede a abertura anal, neste grupo esto os carrapatos da subfamlia Ixodinae, enquanto nos Metastriata o sulco anal fica depois da abertura anal, e a esto todas as outras subfamlia que acontecem no Brasil. Do ponto de vista reprodutivo, nos Metastriata, o encontro entre machos e fmeas para a transferncia de gametas masculinos do macho para a fmea acontece, obrigatoriamente sobre o hospedeiro, enquanto nos Prostriata esse encontro pode acontecer antes dos espcimes estarem no hospedeiro; pode-se ser entendido que os Prostriata amadurecem as gnadas antes mesmo da alimentao de sangue, e os Metastriata precisam ingerir um pouco de sangue para que as gnadas iniciem a produo de gametas. O acasalamento no um encontro casual entre macho e fmea, mas

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induzido por trocas de estmulos por feromnios, principalmente eliminados pela fmea que sinaliza j estar em condies de reproduo, para indicar onde est localizada, e para indicar ao macho onde est a abertura genital (vulva) para que o macho saiba onde introduzir o pacote de gametas (espermatforo). A comunicao semioqumica espcie especfica, mas para efeito de descrio aqui, estaremos generalizando que acontece a atuao do hormnio 20hidroxiecdisona (20E) e de fatores adicionais, presentes na hemolinfa da fmea telegina, na captao de vitelogenina (Vg) pelos ocitos. Isto porque o desenvolvimento do embrio depende da disponibilidade do material do vitelo estocado nos ocitos. Enzimas participam ativamente do processo reprodutivo como a cistena endopeptidase envolvida na degradao de vitelina (Vt) nos ovos, denominada Vitellin Degrading Cysteine endopeptidase (VTDCE). Estas enzimas da classe das cistena endopeptidases esto amplamente distribudas nas teleginas do carrapato. Estudos com VTDCE mostraram que enzima de origem materna, com provvel sntese extra-ovariana e transportada pela hemolinfa, at ser internalizada pelos ocitos. A VTDCE est presente durante todo o desenvolvimento embrionrio, assim como em larvas. Interessantemente, esta cistena endopeptidase apresenta a propriedade de se associar a Vt por um outro stio, alm do stio ativo. A VTDCE a enzima mais eficiente na degradao de vitelina (Vt) com grande importncia fisiolgica, inclusive nas tentativas de controle imunolgico por vacinao. A efetivao dos ciclos vitais consecutivos, que constitui o convencionado como ciclo biolgico da espcie, para os Ixodidae pode acontecer com todas as mudas e ecdises acontecendo sobre o hospedeiro (ciclo homoxeno), ou acontecer de que cada estdio ingurgitado (metalarva, e metaninfa) abandone o hospedeiro para realizar a muda e ecdise fora do mesmo (ciclo heteroxeno); tambm acontece de determinadas espcies realizar o ciclo vital praticamente sempre na mesma espcie de hospedeiro (espcie monoxvica), ou no mostrar

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tal seletividade podendo se desenvolver em diferentes hospedeiros eficientemente (espcie polixvica). A composio do sangue do hospedeiro fator importante para a eficincia da reproduo do carrapato, da a maior ou menor seletividade da espcie. Aps a transferncia do espermatforo, que o macho introduziu pela vulva da fmea at o posicionamento do receptculo seminal, a fmea produz o fator ativador dos espermiforos (gameta masculino). Ativados os espermiforos, sequencialmente da superfcie para o interior vo se desprendendo do espermatforo e se movimentando em direo a vagina cervical; esta movimentao e a modificao qumica do espermatforo liberam o fator ativador da ovulao, e os ocitos V passam do estroma ovariano para a luz do oviduto (ovulao), e na luz deste so empurrados por movimentos peristlticos em direo a vagina. Na vagina o ocito V recebe o pro ncleo do espermiforo, e com os dois pro ncleos (feminino e masculino) passa pela vulva, recebido pelos tentculos do rgo de gene, untados com a secreo da glndula de gene, e posicionados fora do corpo da fmea. A postura continuada, e os ovos ficam aderidos uns aos outros pela secreo que os untou. A conjuno dos dois pro ncleos (fecundao) acontece em dado momento, em perodo de um a trs dias, o que explica um perodo de postura de oito a 15 dias (em mdia), e perodo de ecloso de um a trs dias, caracterizando que o desenvolvimento embrionrio no se inicia logo aps a postura. O nmero de ovos postos por cada telegina depende do carter gentico, da quantidade e qualidade de sangue ingerido, das condies ambiente (temperatura, umidade). Que estimamos atravs do IEN = ndice de eficincia nutricional, e IER = ndice de eficincia reprodutiva, PMO = peso da massa de ovos, IEO = ndice de eclodibilidade dos ovos. As riqutsias, especialmente do grupo Febre Maculosa, ingeridas pela partengina ao se alimentar em hospedeiro com riqutsemia, usam o processo digestivo intracelular na mucosa do intestino do carrapato para infectarem os

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mesmos, e valendo-se do processo de mobilizao dos fatores adicionais, presentes na hemolinfa da fmea telegina, para a captao de vitelogenina pelos ocitos, chegam e infectam estes; como o desenvolvimento do embrio depende da disponibilidade do material do vitelo estocado nos ocitos as riqutsias sobrevivem e estaro presentes no corpo das larvas formadas. As neolarvas no infectantes precisam de um tempo para endurecer o exoesqueleto, transformando-se em neolarvas infectantes, e neste perodo que as riqutsias chegam suas glndulas salivares. Nesta abordagem estamos caracterizando apenas a transmisso vertical das riqutsias (de uma gerao para outra), mas fato que tambm existe a transmisso horizontal (entre indivduos da mesma gerao), de um estdio para o outro (metalarva para neoninfa; metaninfa para negina e/ou neandro). Os Argasidae desenvolvem o ciclo vital com estdios de ovo, larva, protoninfa, deutoninfa, em algumas espcies tambm acontecem outros estdios ninfais (tritoninfa, tetraninfa e pentaninfa), e adultos. Com exceo das larvas, cada um dos outros estdio se alimenta mais de uma vez antes de acontecer a muda e ecdise, portanto inexistem os estdios de neo e metaninfa, ou os neo dos adultos; a fmea faz vrias posturas de pequeno nmero de ovos, sempre intercalada por um repasto sanguneo. A atividade vetorial para riqutsias pelos argasdeos ainda precisa ser mais bem investigada.

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EPIDEMIOLOGIA DAS PULGAS Pedro Marcos Linardi


Departamento de Parasitologia do ICB/UFMG

As pulgas esto includas na ordem Siphonaptera e seus hospedeiros so animais endotrmicos, essencialmente mamferos. A ordem Rodentia a mais importante porque contm o maior nmero de espcies parasitadas, alm de epidemiologicamente algumas destas funcionarem como reservatrios de infeces e, ecologicamente, ocuparem diversos nichos em diferentes ectopos. Atualmente so conhecidas quase 3.000 espcies e/ou subespcies (Lewis 1998). No Brasil, a despeito da riqueza de nossa mastofauna e dos diversos biomas existentes, apenas 62 espcies foram, at o presente, assinaladas (Linardi & Guimares 2000). Na fase adulta, a hematofagia realizada pelos dois sexos, com alimentao diretamente nos capilares (solenfagas). O repasto se prolonga aps a repleo para que o sangue extravasado sirva de alimento s larvas. Cada repasto dura cerca de 10 minutos, com duas a trs refeies ao dia. O ciclo de ovo a adulto completado em aproximadamente 25-30 dias, dependendo das condies de temperatura, umidade e alimentao obtida pelas larvas. A ecloso ocorre 24-36 horas aps o primeiro repasto sanguneo. Cada um dos trs instares larvrios passa por mudas a cada trs dias; uma exceo Tunga penetrans, com apenas dois instares larvrios. Os ovos so ovides ou elipsoidais, esbranquiados, geralmente depositados nos ninhos dos hospedeiros. A ecloso dos ovos tambm varivel por espcie. As larvas so eucfalas, vermiformes, podas e esbranquiadas, com aparelho bucal mastigador. Vivem livremente nas tocas e ninhos de seus hospedeiros, alimentando-se do excremento de pulgas adultas incorporados a detritos orgnicos e dejetos dos hospedeiros. A emergncia dos adultos a partir

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das pupas estimulada por presso mecnica, como p. ex. o deslocamento dos hospedeiros nas proximidades ou seu pisoteio sobre os casulos. A abertura de portas ou janelas em ambientes fechados tambm favorece a emergncia. O tempo que os adultos despendem na ao parasitria varivel, indo desde pulgas que vivem sobre o corpo dos hospedeiros, com as fmeas penetrando sob a pele dos mesmos (Tunga spp.) at as que no vivem sobre o corpo daqueles (Pulex irritans e as pulgas de aves); entretanto, a maioria das espcies alimenta-se intermitentemente, na pele e pelagem de seus hospedeiros (Ctenocephalides spp, Xenopsylla spp, Polygenis spp.). Em relao preferncia alimentar, as pulgas podem ser especficas ou eclticas. Epidemiologicamente, o ecletismo de certas espcies parmetro mais importante em razo do intercmbio de pulgas entre hospedeiros. As pulgas participam de diferentes elos na cadeia epidemiolgica: parasitos propriamente ditos, vetores biolgicos e hospedeiros invertebrados. 1. Pulgas como agentes infestantes:

Independentemente de transmisso de molstias aos respectivos hospedeiros, as pulgas exercem sobre eles diversas aes: ? Ao irritativa: pelo efeito da picada e inoculao de saliva, elas podem provocar reaes alrgicas de intensidade variada aos respectivos hospedeiros. No homem, a hipersensibilidade provocada conhecida como prurigo de Hebra (De Almeida & Croce 1990). Em ces e gatos, estima-se que mais de 50% dos casos dermatolgicos atendidos em clnicas veterinrias estejam relacionados com a irritao conhecida como dermatite alrgica (Bevier-Tournay 1989). Os animais parasitados tornam-se irriquietos e, na tentativa de se livrarem das pulgas, mordem e arranham a pele, arrancando plos e escarificando os tecidos cutneos. ? Ao espoliadora: vrias espcies de pulgas continuam a exercer a hematofagia, mesmo aps repletas, assim, altas infestaes em animais de pequeno porte podem conduzir anemia. As pulgas picam os hospedeiros

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vrias vezes ao dia. ? Ao inflamatria: em pulgas que so penetrantes ou semi-penetrantes, os orifcios deixados no corpo dos hospedeiros tornam-se passveis de infeco por agentes oportunistas do ttano, gangrena gasosa e blastomicoses. infestao por T. penetrans, seguida ou no por infeces secundrias d-se o nome de tungase. O homem e mamferos domsticos (sunos, ces, gatos e bovinos) so os principais hospedeiros, ainda que livremente, machos e fmeas podem ser encontrados em montes de esterco, areias de praia, pocilgas e interior de certas habitaes. No homem, a infestao principalmente observada na sola plantar, calcanhar, cantos dos dedos (ps e mos), bordas das unhas e espaos interdigitais e, eventualmente, tem sido noticiada no escroto, nus e plpebras. Quando as leses cutneas so numerosas e prximas entre si, localizadas ao longo do calcanhar, recebem a denominao de favo de mel. A tungase pode provocar dificuldades de postura e de locomoo dos hospedeiros, necrose ssea e tendinosa e at perda dos dedos dos ps. Casos severos, com infestaes macias por todo o corpo, tambm tm sido observados. Em bovinos, se a infestao for localizada nas patas, os reprodutores podero ficar impedidos de fecundarem as vacas, em virtude da falta de apoio para a cobertura. Alguns detalhes relativos infeco foram apresentados por Heukelbach (2005), Feldmeier et al. (2007) e Kehr et al (2007).

2.

Pulgas como transmissoras de molstias:

2.1. Mixomatose - agente etiolgico: Mixomae mollitor; reservatrios: coelhos; vetor: pulga Spilopsyllus cuniculi. A transmisso realizada mecanicamente e o vrus localiza-se no tubo digestivo. No h qualquer registro dessa virose no Brasil. 2.2. Riquetsioses tifo murino: agente etiolgico: Rickettsia typhi;

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reservatrios: roedores sinantrpicos. A transmisso do patgeno se realiza pelas fezes de Xenopsylla cheopis, especialmente quando esmagadas pelos dedos. A riqutsia infecta e se reproduz no tubo digestivo da pulga, no exercendo ao patognica sobre o sifonptero. Alguns casos humanos j foram assinalados em Minas Gerais e So Paulo. A Bartonella henselae, agente da doena da esfoladura em gatos, tambm transmitida pelas fezes de C. felis felis. Recentemente, usando tcnicas moleculares, Rickettsia felis foi diagnosticada em pulgas Ctenocephalides spp em Minas Gerais (Oliveira et al. 2000). Horta et al. (2007) diagnosticaram R. felis em C. felis felis capturadas de ces e gatos e em Polygenis atopus coletadas de gambs, no estado de So Paulo. Tambm, por meio de tcnicas moleculares, Fischer et al. (2002) evidenciaram Wolbachia sp. em T. penetrans. 2.3. Salmoneloses: as bactrias Salmonella enteritidis e S. typhimurium, ainda que se localizando e reproduzindo no tubo digestivo das pulgas X. cheopis e Nosopsyllus fasciatus, no causam prejuzos a elas. Os roedores e o homem so tidos como reservatrios e o mtodo de transmisso se faz pela picada, inoculao ou fezes do sifonptero. 2.4. Tularemia: a Francisella tularensis localiza-se no tubo digestivo de diversas espcies de pulgas, mas no se reproduz nem tampouco prejudicial a elas. Os reservatrios so roedores e lagomorfos; no foi, at o presente, aqui assinalada. 2.5. Peste: esta , sem dvida, a principal molstia transmitida pelas pulgas, dada a sua morbidade, letalidade e registro histrico. O agente etiolgico a Yersinia pestis e a transmisso se processa pela picada de pulgas infectantes aps as bactrias reproduzirem-se e bloquearem o proventrculo do inseto, impedindo-o assim de se alimentar e com isso aumentando, significativamente, o nmero de picadas. Os reservatrios so, essencialmente, roedores domiciliares e silvestres. Do ponto de vista epidemiolgico h cinco tipos bsicos de ciclos de peste, assim caracterizados: a) ratos sinantrpicos pulgas de ratos (Xenopsylla)

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homem; b) roedores silvestres homem; c) roedores silvestres

pulgas de roedores silvestres (Polygenis) pulgas de roedores silvestres (Polygenis) pulgas de roedores sinantrpicos

roedores campestres ou sinantrpicos

(Xenopsylla) homem; d) homem pulgas humanas (Pulex irritans) homem; e) homem homem (peste pulmonar). Pulgas infectadas vivem, em mdia, trs dias. Uma panormica atual da peste, encontra-se em Stenseth et al. (2008). 2.6. Tripanosomatdeos: segundo Smit (1977), 27 espcies, j foram assinaladas em pulgas, com predominncia para o gnero Trypanosoma (18 espcies). A espcie mais comum Trypanosoma lewisi, que se multiplica no interior de X. cheopis e N. fasciatus, sem contudo apresentar ao patognica sobre os hospedeiros vertebrados (roedores). Outros tripanosomatdeos heteroxnicos foram encontrados em Polygenis tripus infestantes de roedores silvestres em Belo Horizonte, MG (Botelho & Linardi 1992). Entre os

tripanosomatdeos monoxnicos, o gnero Leptomonas inclui seis espcies que parasitam pulgas (Mc Ghee & Cosgrove 1980, Beard et al. 1989). A tcnica da PCR tem tambm permitido reconhecer DNA de Leishmania chagasi em C.felis felis retiradas de ces naturalmente infectados, abrindo assim a possibilidade da transmisso mecnica do calazar canino por meio de pulgas (Coutinho & Linardi 2007). Recentemente, Ferreira et al. (2009), usando a mesma tcnica, em pulgas de ces de Araatuba/SP, chegaram s mesmas concluses de Coutinho & Linardi (2007). Conforme observado por Pacheco et al. (1998), tripanosomatdeos monoxnicos de pulgas podem causar infeces oportunistas em indivduos imunodeficientes. Relativamente Leptomonas, Avelar et al. (2007) assinalaram uma prevalncia de infeco de 1,7% em 1.500 exemplares de C. felis felis dissecados. 2.7. Dilepidiose: Dipylidium caninum cestdeo habitual em carnvoros domsticos e que eventualmente parasita o homem. A infeco das pulgas (Ctenocephalides spp., Pulex irritans) ocorre em suas fases larvrias e quando ingeridas, as pulgas liberam no tubo digestivo dos vertebrados, as larvas de

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cestdeos. A infeco patognica para as pulgas. Avelar et al. (2007) noticiaram uma prevalncia de 0,4% deste cestdeo, ao dissecarem pulgas (C. felis felis) de ces de Belo Horizonte, MG. 2.8. Himenolepases: X. cheopis, N. fasciatus e Leptopsylla segnis atuam como hospedeiros intermedirios de Hymenolepis diminuta e H. nana. Os cisticercides alojam-se no tubo digestivo e cavidade geral das pulgas, sendo prejudiciais a elas. A transmisso se processa pela ingesto ou esmagamento das pulgas infectantes. Himenolepdeos em P. tripus foram noticiados por Botelho & Linardi (1992). 2.9. Filariose de ces: o verme filarial Dipetalonema reconditum um parasito patognico e exclusivo de ces, desenvolvendo-se na cavidade geral de pulgas Ctenocephalides spp. A prevalncia de infeco entre 1.613 C. felis felis capturadas em Belo Horizonte foi de 0,8% (Linardi 1992). 2.10. Infeces por tilenqudeos: vermes da famlia Allantonematidae (Nematoda: Tylenchida) tm sido observados na cavidade geral de pulgas. Na regio Neotropical, tais registros foram assinalados pela primeira vez por Linardi et al. (1981), em P. tripus, em uma prevalncia de 11,2% das pulgas examinadas, com os autores levantando possibilidade de sua utilizao para controle biolgico e, consequentemente, profilaxia da peste. 2.11. Infeco e/ou infestao por artrpodos: alguns parasitides, como larvas de Chalcidoidea (Hymenoptera) tm sido observadas em certas espcies de pulgas. Algumas espcies de caros tambm tm sido encontradas em associao com certas espcies de pulgas, quer em foresia, quer no interior da cavidade geral. Infestaes naturais de P. tripus por Rhizoglyphus echinopus foram constatadas por Cerqueira & Linardi (1976) em Belo Horizonte, MG. 2.12. Outros parasitos de pulgas: a literatura ainda cita registros como Bacillus thuringensis, Beauveria bassiana, Escherichia coli, Hepatozoon erhardovae, Nosema sp. e gregarinas. Beard et al. (1990) listam vrios organismos como endosimbiontes de pulgas, pouco se conhecendo sobre sua

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prevalncia, biologia e grau de patogenicidade para as pulgas. Recentemente, Avelar et al. (2007) e Avelar & Linardi (2008) assinalaram Nolleria pulicis (Chitridiopsidae) e Steinina sp. (uma gregarina da famlia Actinocephalidae) em C. felis felis coletadas de ces de Belo Horizonte, MG. Referncias Avelar DM, Bussoloti AS, Ramos MCA, Linardi PM 2007. Endosymbionts of Ctenocephalides felis felis (Siphonaptera: Pulicidae) obtained from dogs captured in Belo Horizonte, Minas Gerais, Brazil. J Invert Pathol 94: 149-152. Avelar DM, Linardi PM 2008. Seasonality and prevalence rates of Steinina sp. (Eugregarinorida: Actinocephalidae) in Ctenocephalides felis felis (Siphonaptera: Pulicidae) from dogs captured in Belo Horizonte, Minas Gerais, Brazil. J Med Entomol 45: 1139-1142. Beard CB, Butler JF, Orshar EC 1989. In vitro grouth characterization and host-parasite relationship of Leptomonas pulexsimulantis n. sp., a trypanosomatid flagellate of the flea Pulex simulans. J Parasitol 75: 658-668. Beard CB, Butler JF, Hall DW 1990. Prevalence and biology of endosymbionts of fleas (Siphonaptera: Pulicidae) from dogs and cats in Alachua County, Florida. J Med Entomol 27: 1050-1061. Bevier-Tournay DE 1989. Fleas and flea control. Curr Vet Ther 10: 586-592. Botelho JR, Linardi PM 1992. Endoparasites of Polygenis tripus (Siphonaptera: Rhopalopsyllidae) of wild rodents from Belo Horizonte, Minas Gerais, Brazil. Mem Inst Oswaldo Cruz 87: 453-455. Cerqueira EJL, Linardi PM 1976. Infeco natural de Polygenis tripus (Jordan, 1933)(Siphonaptera, Rhopalopsyllidae pelo Rhizoglyphus echinopus (Fumouze & Robin, 1868)(Acarina, Sarcoptiformes). Natura UFBa 76: 153-157. Coutinho MTZ, Linardi PM 2007. Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals? Vet Parasitol 147: 320-325. De Almeida FA, Croce J 1990. Hypersensitivity in patients with Hebra's prurigo caused by flea bite. Med Cutan Ibero Lat Am 18: 132-7. Feldmeier H, Witt LH, Schwalfenberg S, Linardi PM, Ribeiro RA, Capaz R, Van Marci E, Meckes O, Mehlhorn H, Mencke N, Heukelback J 2007. Investigations on the biology, epidemiology, pathology and control of Tunga penetrans in Brazil. VI. Natural history of the infestation in laboratory-raised Wistar rats. Parasitol Res 102: 1-13.

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Ferreira MGPA, Fattori KR, Souza F, Lima VMF 2009. Potential role for dog fleas in the cycle of Leishmania spp. Vet. Parasitol 165: 150-154. Fischer P, Schmetz C, Banci C, Bonow I, Mand S, Fischer K, Bttner DW 2002. Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobactera from filarial parasites. Exp Parasitol102: 201-211. Heukelbach J 2005. Tungiasis. Rev Inst Med Trop S Paulo 47: 307-313. Horta MC, Labruna MG, Pinter A, Linardi PM, Schumaker TTS 2007. Rickettsia infection in five areas of the state of So Paulo, Brazil. Mem Inst Oswaldo Cruz 102: 793-801. Kehr JD, Heukelbach J, Mehlhorn H, Feldmeier H 2007. Morbidity assessment in sand flea disease (tungiasis). Parasitol Res 100: 413-421. Lewis, RE 1998. Resum of Siphonaptera (Insecta) of the world. J Med Entomol 35: 377-389. Linardi PM 2002. Endoparasitos de Ctenocephalides felis felis (Siphonaptera: Pulicidae) em Belo Horizonte, MG. O Biolgico 64: 65. Linardi PM, Cerqueira, EJL, Williams P 1981. Polygenis tripus (Siphonaptera: Rhopalopsyllidae) naturally infected by Allantonematidae (Nematoda: Tylenchida). J Med Entomol 18: 41-43. Linardi PM, Guimares LR 2000. Sifonpteros do Brasil. Editora Museu de Zoologia USP/FAPESP, So Paulo, 291 pp. Mc Ghee RB, Cosgrove WB 1980. Biology and physiology of the lower trypanosomatidae. Microbiol Rev 44: 140-173. Oliveira RP, Galvo MAM, Mafra CL, Chamone CB, Calic SB, Silva SU, Walker DH, 2002. Rickettsia felis in Ctenocephalides spp. fleas, Brazil. Emerg Inf Dis 8: 317-319. Pacheco RS, Marzochi MCA, Pires MQ, Brito CMM, Madeira MF, Barbosa-Santos EGO 1998. Parasite genotypically retated to a monoxenous tripanosomatid of dog's flea causing opportunistic infection in an HIV positive patient. Mem Inst Oswaldo Cruz 93: 531-537. Smit FGAM 1977. A concise list of organisms found in or on Siphonaptera. Flea News 11: 13-14. Stenseth NS, Atshabar B, Begon M, Belmain SR, Bertherat E, Carniel E, Cage KL, Leirs W, Rahalison L 2008. Plague: past, present and future. PLoS Medicine 5 (1), e3: 009-0013.

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SANDFLY TAXONOMY IS AN IMPERFECT PREDICTOR OF VECTORIAL TRAITS Paul D. Ready


Department of Entomology, Cromwell Road, Natural History Museum, London SW7 5BD, U.K. E-mail: P.Ready@nhm.ac.uk

No classification of phlebotomine sandflies is likely to suit both medical parasitologists and sandfly specialists. Choices have to be made, either maintaining a practical classification containing few vectorial genera (mostly Phlebotomus for the Old World and Lutzomyia for the Neotropics) or changing the generic names of vectors so that the classification represents an evolutionary hypothesis. However, biomedical criteria might not be sufficient for recognizing only a few genera, because sandflies transmit not only Leishmania but also arboviruses, and humans are bitten by members of non-vectorial genera (e.g. Sergentomyia in Africa and Asia). For leishmaniasis, vectorial roles are often determined by co-evolution with the parasite at the species level, with selection being maintained by ecological contacts. There is only imperfect co-cladogenesis of generic groups of sandflies and subgeneric complexes of Leishmania species, and so taxonomy is an imperfect predictor of vectorial roles. Natural hybridization between sandfly species has been recorded for several species complexes in the Mediterranean region and Latin America, highlighting the need to focus on gene flow and the distribution of phenotypes of biomedical importance, not on taxonomy. From a biomedical point of view, it is unnecessary to make frequent changes to sandfly taxonomy, either at the generic or species level. Taxonomic stability makes it easier for non-specialists to use a sandfly classification for the retrieval of information. Recommendations are expected from the next International Symposium on Phlebotomine Sandflies (ISOPS 7), to be held in Antalya, Turkey, in April 2011. Reference Ready, P.D. (2010) Should sandfly taxonomy predict vectorial and ecological traits? Journal of Vector Ecology, in press. 90

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MOLECULAR CHRONOBIOLOGY OF INSECT VECTORS Alexandre A. Peixoto


Laboratory of Insect Molecular Biology Instituto Oswaldo Cruz, FIOCRUZ Rio de Janeiro, Brazil

The behaviour of insect vectors plays a vital role in the dynamics of disease transmission (Klowden and Zwiebel 2004). One aspect of vector behaviour particularly relevant in this respect is the control of daily patterns of activity and blood-feeding. These rhythms are controlled by an endogenous biological clock (Saunders 2002), which is in turn under genetic control. However, we still know little about the molecular chronobiology of blood-sucking insects. The molecular pacemaker of the insect model species Drosophila melanogaster involves a number of genes that take part in interlocked negative transcriptional feedback loops that control the circadian rhythms in behaviour and physiology (reviewed in Hardin 2005). In the two main interacting feedback loops, the genes Clock (Clk) and cycle (cyc) encode transcription factors that form a heterodimer that binds to upstream E-box sequences (CACGTG) and activates the transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). The heterodimer Per/Tim (or Per alone) interacts with Clk/Cyc inhibiting its function. Meanwhile, Vri and PDP1 regulate Clk transcription by competing for the same site in its promoter. These two interlocked loops drive the circadian gene expression of its components, except in the case of cyc, which is constitutively expressed. Kinases have also an important role controlling the cyclic stability and nuclear entry of clock proteins. The cycles in the expression and abundance of negative and positive elements of the clock are important for the generation of the circadian rhythms in hundreds of outputs genes controlling different aspects of physiology and behaviour. Finally, a light-resetting mechanism involving the protein encoded by the cryptochrome (cry) gene allows the synchronization of endogenous rhythms to the environmental light-dark cycles.

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We have been studying the molecular chronobiology of sandflies and mosquitoes for the last few years. Initially, we carried out an analysis of the activity rhythms and circadian expression of per, tim, Clk and cyc in the sandfly Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Latin America. The analysis of the activity rhythms in L. longipalpis, a crepuscular/nocturnal insect in the wild, revealed that, compared to D. melanogaster, this sandfly shows more nocturnal activity under controlled laboratory conditions. Circadian expression of the four clock genes also revealed interesting differences between fruitflies and sandflies. Even though L. longipalpis per and tim show peaks of mRNA abundance in the early evening, as observed in D. melanogaster, the expression of sandfly Clk and cyc differ strikingly (Meirelles-filho et al 2006a; b). L. longipalpis Clk mRNA levels peak around the light-dark transition, while in the fruitfly the peak occurs in the late night / early morning. In addition, cyc shows a rhythm in mRNA abundance in Lutzomyia, even though it is constitutively expressed in Drosophila. Another interesting difference between fruitflies and sandflies is the fact that L. longipalpis Cyc protein has a C-terminal activation domain that is missing in Drosophila (Meirelles-filho et al 2006a;b). We also examined the effect of blood-feeding in the expression levels of these four clock genes in L. longipalpis. While Clk and cyc sustain their levels in blood-fed female sandflies, we observed down-regulation of per and tim expression, which is correlated with a reduction in locomotor activity (Meirellesfilho et al 2006a;b). These results suggest that the endogenous circadian clock and its control over the activity rhythms in this vector are modulated by blood intake. We are currently concentrating our efforts on the behavioural and molecular analysis of mosquito activity rhythms and we are investigating the differences between the diurnal Aedes aegypti, a vector of dengue and yellow fever viruses, and the nocturnal Culex quinquefasciatus, a vector of filariasis and West-Nile fever virus. We analysed the circadian expression patterns of

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clock genes in both species. The results revealed conserved expression profiles in most genes except cryptochrome2 (cry2) (Gentile et al 2009). Cry2 is a negative transcriptional regulator related to mammalian cryptochromes, that is found in a number of insects but is absent in Drosophila. We observed that cry2 mRNA abundance shows a single peak in Cx. quinquefasciatus but a bimodal pattern in Ae. aegypti, suggesting differences in the way the gene is regulated in the two species (Gentile et al 2009).
References:

Hardin PE 2005. The Circadian Timekeeping System of Drosophila. Curr Biol 15: R714-R722 Gentile C, Rivas GBS, Meireles-Filho ACA, Lima JBP, Peixoto AA 2009. Circadian expression of clock genes in two mosquito disease vectors: cry2 is different. J Biol Rhythms 24: 444-51. Klowden MJ, Zwiebel L 2004. Vector Olfaction and Behavior. In Biology of Disease Vectors 2nd Edition. ed. Marquardt WC et al. Elsevier Academic Press. Amsterdam. Meireles-Filho AC, da S Rivas GB, Gesto JS, Machado RC, Britto C, de Souza NA, Peixoto AA. 2006a. The biological clock of an hematophagous insect: locomotor activity rhythms, circadian expression and downregulation after a blood meal. FEBS Lett. 580: 2-8. Meireles-Filho AC, Amoretty PR, Souza NA, Kyriacou CP, Peixoto AA 2006b. Rhythmic expression of the cycle gene in a hematophagous insect vector. BMC Mol Biol. 7: 38. Saunders DS 2002. Insect Clocks. 3rd edition Elsevier Science. Amsterdam.

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GENETIC VARIATION IN THE SALIVARY PROTEIN MAXADILAN AMONG POPULATION OF Lutzomyia longipalpis Gregory C. Lanzaro
Professor, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA

Our major research effort focuses on understanding the complex interactions between insect vectors, pathogens and vertebrate hosts. We are focusing on how these interactions may be understood at the molecular level by studying salivary proteins and the genes that encode them. We work with one small protein, known as maxadilan (MAX) that occurs in the saliva of, Lu. longipalpis. MAX is a potent vasodilator. In addition, MAX inhibits macrophage function. It has been shown that MAX is important in the pathogenesis of L. chagasi and that animals immunized with MAX are protected from L. chagasi infection. We have conducted a series of experiments in which we have shown that specific antiMAX antibodies are produced by animals and humans exposed to Lu. longipalpis bites and that these antibodies neutralize the vasodilatory activity of MAX. Consequently flies feeding on animals with MAX antibodies take smaller bloodmeals and lay fewer eggs than those feeding on naive animals. We reasoned that flies in nature have likely evolved some means of avoiding the host immune system. We have demonstrated that MAX is hypervariable in natural fly populations and that this variation represents antigenic variation that has evolved in response to the challenge imposed by host antibodies. Currently we are on working variation among populations with respect to the level of MAX expression and have found that populations differ with respect to the amount of MAX in their saliva. We have found that there appears to be a relationship between the amount of MAX present in saliva and visceralization of Le. chagasi.

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TICK SALIVA CONTAINS NOVEL NON-PROTEIC MOLECULES WITH IMMUNOMODULATORY PROPERTIES Oliveira, C.J.F.; 2S-Nunes, A.; 3Francischetti, I.M.; 1Carregaro, V.; Anatriello, E.; 1Silva, J.S.; 1Santos, I.K.F.M.; 3Ribeiro, J.M.C.; 4Ferreira, B.R.
1

1
1

Department of Biochemistry and Immunology, School of Medicine of Ribeiro Preto, University of So Paulo (USP), Brazil; Department of Immunology, Instituto de Cincias Biomdicas, USP, Brazil; 3 Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD, USA; 4 Department of Maternal-Child Nursing and Public Health of Ribeiro Preto, School of Nursing of Ribeiro Preto, USP, Brazil; email: brferrei@usp.br
2

Dendritic cells (DCs) are central coordinators of the innate and acquired immune responses against pathogens, and depending how these cells are activated they can induce a susceptible state or stimulate development of protective immunity. Ticks are blood-sucking ectoparasites arthropods that can suppress host immunity to promote their feeding process and survival. In the last decades, studies have identified the presence of pharmacologically active compounds in tick saliva directed against immune cells and their effectors pathways. In the present work, molecules present in Rhipicephalus sanguineus tick saliva with modulatory effects on DC differentiation, cytokine production and co- and stimulatory molecules expression were identified. Firstly, we observed that tick saliva inhibited IL-12p40 (~60% of reduction) and TNF-alpha (~62% of reduction), while doubled IL-10 cytokine production by C57BL/6 mice bone marrow-derived DCs stimulated by TLR-2 (PGN), TLR-4 (LPS) and TLR-9 (CpG-ODN 1826) ligands. Tick saliva also decreased the percentage of CD11c/CD40+ and CD11c/CD86+ cells in activated DCs. To examine the molecules responsible for this effect, we fractionated the saliva using Microcon filtration (with a cut off of 5 kDa) and reverse-phase chromatography, and tested each fraction on DC maturation induced by LPS. Fractions with proved effects were then analyzed by tandem mass spectrometry or competition ELISA assays to identify the molecules involved. With this approach we demonstrated, for the first time in tick saliva, the purine nucleoside adenosine (~110 pmoles/L) as an antiinflammatory salivary inhibitor of DCs cytokine production. We also found in tick saliva the presence of prostaglandin-E2 (PGE2 ~100 nM) with similar effect as adenosine in modulating cytokine production by DCs. Both, adenosine as well as PGE2 was able to modulate the expression of CD40, but not CD86 expression. In addition, adenosine from tick saliva was able to stimulate the synthesis of PGE2 by LPS-stimulated mice DCs. Flow cytometry analysis revealed that the addition of saliva or PGE2, but not adenosine, inhibits the differentiation of DCs from

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bone-marrow precursor mice cells (reduction of 58%). Our results also demonstrate that adenosine and PGE2 from tick saliva have a complementary immunosuppressive property, since they induced and amplified a common intracellular signaling pathway via cAMP-protein kinase A (PKA). Taken together, the presence of the non-proteic molecules adenosine and PGE2 in saliva reveals an important evolutionary mechanism by which ticks subvert the homeostatic and immune system of their hosts to facilitate their feeding, moreover, this adaptation possibly can improve their activity as vectors for pathogens, as it induces an impaired immune response.
Financial Support: FAPESP and CNPq.

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RNAI EM MOSQUITOS: DEFESA NATURAL ANTIVIRAL E FERRAMENTA TECNOLGICA Tereza Magalhes


Centro de Pesquisas Aggeu Magalhes, Fundao Oswaldo Cruz, Recife, PE, Brazil

O RNA de interferncia (RNAi), descoberto em 1998 no organismo C. elegans, um mecanismo endgeno de regulao gnica dependente de molculas de RNA, que est presente em muitos organismos eucariotos. Sua descoberta logo desencadeou o desenvolvimento de tcnicas de silenciamento gnico in vitro e in vivo. Em insetos, o sistema de RNAi funcional e j foi parcialmente caracterizado, tendo Drosophila melanogaster como principal organismo modelo. Em 2002, um artigo foi publicado demonstrando que a simples inoculao de um fragmento de RNA fita dupla (dsRNA) em indivduos adultos do principal vetor da malria na frica, o Anopheles gambiae, causava o silenciamento in vivo do mRNA homlogo ao dsRNA, no caso o peptdeo antimicrobiano defensina. Desde ento, a tcnica de RNAi tem sido amplamente utilizada para estudos de genmica funcional em mosquitos. Estes estudos esclarecem o envolvimento de molculas individuais em diversos processos fisiolgicos como digesto, reproduo e imunidade a determinados parasitas, sendo de grande relevncia cientfica. Posteriormente aplicao da tcnica de RNAi em mosquitos para elucidao do papel de molculas em processos fisiolgicos, soube-se, utilizando modelos de laboratrio, que o RNAi age como mecanismo natural de defesa em mosquitos infectados com alguns vrus. O primeiro artigo sobre o papel do RNAi na defesa antiviral foi com A. gambiae e o vrus O'nyong-nyong, um alphavirus. Nesse estudo, uma molcula da maquinaria celular do RNAi foi silenciada atravs da prpria tcnica de RNAi para demonstrao do papel desse mecanismo na proteo do A. gambiae ao O'nyong-nyong. Aps este trabalho, vrios outros

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surgiram na mesma linha. Em nosso laboratrio, estamos interessados nos mecanismos moleculares envolvidos na interao vetor-patgeno, entre eles a resposta imune de mosquitos a diversos microorganismos. Os seguintes modelos so utilizados em nossos projetos: Aedes aegypti-Wuchereria bancrofti; A. aegypti-vrus dengue; e A. aegypti-bactria.

RNAi como ferramenta molecular: Em algumas linhas de pesquisa, utilizamos o RNAi como tcnica de silenciamento gnico para o estudo funcional de molculas especficas de mosquitos. Segue, abaixo, dois estudos baseados nessa estratgia. Aedes aegypti-Wuchereria bancrofti No Recife (PE, Brasil), o Culex quinquefasciatus transmite a Wuchereria bancrofti (verme filarial) entre a populao humana, fazendo com que o local seja endmico para a filariose linftica. Ao contrrio de C. quinquefasciatus, o mosquito A. aegypti refratrio W. bancrofti, no entanto os mecanismos moleculares responsveis por essa refratariedade so desconhecidos. Recentemente, demonstramos que trs molculas (defensina, cecropina e transferrina) de A. aegypti so mais expressas nesse inseto aps infeco com W. bancrofti. O prximo passo consiste em silenciar essas molculas atravs de RNAi para verificar sua influncia no desenvolvimento do verme dentro do inseto. Aedes aegypti-bactria H um nvel de conservao entre as principais vias de regulao do sistema imune (Toll, Imd) de D. melanogaster e mosquitos. No entanto, diferenas importantes no modo com que microorganismos ativam essas vias comearam a ser observadas. De modo a contribuir na elucidao do papel dos componentes dessas vias na proteo do mosquito a diferentes patgenos, silenciamos por RNAi um fator de transcrio (REL1) da via Toll em A. aegypti e demonstramos que essa via crucial na proteo contra Bacillus subtilis, uma bactria Gram positiva, e contra Escherichia coli, uma bactria Gram negativa,

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diferindo de estudos com D. melanogaster. No mesmo estudo, silenciamos a defensina, um peptdeo antimicrobiano cuja expresso controlada em parte por REL1, e mostramos que essa molcula no tem papel fundamental na proteo contra essas duas espcies de bactria em A. aegypti, levantando a hiptese que outra molcula controlada por REL1 exerce funo protetora no mosquito aps infeco com B. subtilis e E. coli.

RNAi como mecanismo natural antiviral: Outro estudo em andamento em nosso laboratrio sobre a atuao de mecanismos antivirais (entre eles o RNAi) na modulao da competncia vetorial de A. aegypti ao vrus dengue (DENV). Segue, abaixo, a descrio desse estudo. Aedes aegypti-DENV Estudos sobre os determinantes da transmisso da dengue so importantes para o desenvolvimento de novas estratgias de controle dessa doena. Um desses determinantes a competncia vetorial (ou nvel inato de susceptibilidade) de A. aegypti ao DENV, que varia entre populaes do mosquito. Recentemente, foi demonstrado o envolvimento de duas vias regulatrias do sistema imune do mosquito (Toll e JAK-STAT) e do RNAi na defesa anti-DENV em A. aegypti. No entanto, no se sabe ainda o real papel desses mecanismos na modulao da competncia vetorial em populaes de campo de mosquitos ao DENV. Dessa forma, um de nossos objetivos demonstrar se h correlao entre esses mecanismos e o nvel de susceptibilidade de A. aegypti provenientes de diferentes municpios de Pernambuco a diferentes sorotipos do DENV. Para isso, estamos trabalhando com quatro populaes de campo de A. aegypti e utilizando dois sorotipos de DENV (DENV-1 e DENV-2) para experimentos de infeco artificial. Para caracterizao da competncia vetorial dessas populaes ao DENV, ser feita quantificao viral em vrios tecidos do mosquito em cinco momentos aps a infeco. Para os estudos sobre a ao dos mecanismos antivirais, a expresso de uma molcula-chave de cada um dos trs mecanismos anti-DENV identificados em A. aegypti (Toll, JAK-STAT e RNAi) ser

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mensurada nas mesmas amostras utilizadas para quantificao viral. A relevncia cientfica desse estudo consiste na obteno de dados inditos sobre a ao de mecanismos antivirais nos diferentes rgos dos mosquitos (at hoje no se sabe, por exemplo, onde e quando esses mecanismos agem), bem como a relao desses mecanismos na susceptibilidade do mosquito ao DENV, isto , se mosquitos de campo mais resistentes ao DENV empregam com maior eficincia algum dos mecanismos antivirais descritos em A. aegypti. Alm disso, a diversidade gentica dessas molculas tambm ser analisada para se verificar o tipo de seleo atuando sobre elas e o nvel de diversidade inter- e intraespecfica. Por fim, se houver evidncia da relao entre as molculas e o nvel de susceptibilidade dos mosquitos ao DENV, as mesmas sero silenciadas por RNAi e o padro de infeco viral ser novamente analisado. Espera-se, com esses estudos, trazer contribuies significativas para a pesquisa sobre a interao vetor-patgeno nos modelos acima citados. Essas informaes podero ser relevantes para o desenvolvimento de novas estratgias de controle de insetos vetores.

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MOLECULAR STRATEGIES TO ECTOPARASITE CONTROL

Jorge Moraes, Evenilton P. Costa, Aoi Masuda, Itabajara da Silva Vaz Jr., and Carlos Logullo Most drugs have been discovered in random screens or by exploiting information about macromolecular receptors and structure-based approach to design couples this information with specialized computer programs to propose novel enzyme inhibitors and other therapeutic molecules. The combination of molecular structure determination and computation is emerging as an important tool for drug development. This approach will be applied to developing strategies to discovery new more efficient drugs to ectoparasite control. Ticks are the major ectoparasites in cattle raising and cause vast economical losses worldwide. These parasites transmit some pathogens to humans and animals. Our research group has searched for strategies to obtain new targets for tick control and the vaccine potential of three proteins of Rhipicephalus (Boophilus) microplus has been evaluated. We used the native and recombinant forms of the BYC protein, native VTDCE protein and recombinant Haemaphysalis longicornis GST, and all vaccinations interfered with the development of the embryo. Two of these proteins are proteinases involved in yolk processing during embryogenesis. We have also investigated the physiological bases of tick embryo formation with the aim of developing new approaches for tick control, and found that there is a relationship between the main energy source and the morphogenetic changes that occur during R. microplus embryogenesis. Embryogenesis occurs within 21 days under laboratory conditions, whereas the fundamental morphology and metabolic modifications take place on the 6thday, corresponding to the cellularization of the embryo. More significant metabolic changes take place after cellularization of the embryo, especially in the glycolytic and gluconeogenic pathways, suggesting that there are two distinct metabolic phases.

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Triosephosphate isomerase (TIM) and pyrophosphatase enzymes are important target for drug development and vaccines directed against ectoparasites. These enzymes participate in glycolysis and gluconeogenesis and involved in the central control of metabolism. Considering TIM and pyrophosphatase could using as targets for drug development against ectoparasites based mainly on the identification and structural characterization of non-conserved amino acids that are essential in catalysis or stability of the enzymes from the parasite we are proposing this study. In the present work we identified the presence of a novel TIM and pyrophosphatase in R. microplus ovaries and embryos. The sequence and molecular modeling analyses suggest, for the first time that both enzymes are sensitivity to sulfhydryl reagents. These non-conserved cysteine residues could be targets for drug development against tick infestation. With the advancements of research on the genomes of the tick and other arthropods, the strategy of speciesspecific enzyme inhibition of homologous enzymes may become an alternative for the control of disease vectors in arthropods.

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ASPECTOS MOLECULARES DA INTERAO DE LIPOFOSFOGLICANOS DE Leishmania COM O INTESTINO MDIO DE FLEBOTOMNEOS


Rodrigo Soares
Laboratory of Medical Entomology, Centro de Pesquisas Ren Rachou, Fundao Oswaldo Cruz. Av. Augusto de Lima, 1715, 30190-002. Belo Horizonte, MG, Brazil, email: rsoares@cpqrr.fiocruz.br

O Lipofosfoglicano (LPG) de Leishmania um fator de virulncia multifuncional tendo importncia na interao tanto com o hospedeiro vertebrado quanto invertebrado. Suas principais funes incluem: adeso e especificidade ao epitlio do intestino mdio do inseto vetor, proteo contra enzimas digestivas, proteo contra o sistema de complemento do hospedeiro vertebrado, opsonizao dos macrfagos, facilitao da fagocitose, inibio da fuso do lisossomo com o vacolo parasitforo, inibio da protena quinase C a agonista do receptor do tipo toll 2 (TLR2) (Descoteaux & Turco, 1999; de Veer et al., 2003). A molcula de LPG possui quatro domnios: 1) uma ncora lipdica de 1-Oalquil-2-liso-fosfatidilinositol; 2) uma poro central formada por um heptassacardeo Gal(? 1,6)Gal(? 1,3)Galf(? 1,3)[Glc(? 1)-PO4]Man(? 1,3)Man(? 1,4)-GlcN(? 1), 3) uma regio de repeties de dissacardeos fosforilados Gal(? 1,4)Man(? 1)-PO4 e 4) um pequeno oligossacardeo terminal (cap). Os polimorfismos na poro 3) e 4) so determinantes na especificidade das espcies de Leishmania por seus vetores e j foram bioquimicamente caracterizadas em espcies do Novo e Velho Mundo. Os estudos demonstrando a interao do LPG com o intestino mdio em flebotomneos comearam na dcada de 90, envolvendo o vetor Phlebotomus papatasi e Leishmania major (McConville et al., 1992). Foi observado que o LPG das formas procclicas desta espcie apresenta resduos terminais de galactose que aps a metaciclognese so substitudos por arabinose. Esta alterao resulta no desprendimento das formas metacclicas do epitlio do intestino mdio e as mesmas

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migram em direo s pores anteriores do inseto. Mutantes incapazes de sintetizar LPG no eram capazes de manter a infeco dentro do vetor (Butcher et al., 1996). Foi descrito para este flebotomneo a existncia de um receptor (PpGal-P) que reconhece estas galactoses (Kamhawi et al., 2004), o qual estaria presente nas microvilosidades das clulas epiteliais. No caso de Leishmania donovani (Sudo), um interessante mecanismo ocorre. Esta espcie no possui cadeias laterais no LPG e supe-se que a adeso ocorra atravs dos resduos de galactose presentes no regio terminal do cap (Sacks et al., 1995). Aps a metaciclognese, o LPG dobra de tamanho e devido a um encurvamento da molcula o cap se tornario crptico e ento as formas metacclicas se soltariam do epitlio. Em L. donovani (ndia), ocorre um mecanismo diferente, esta espcie possui resduos de glicoses no LPG das formas procclicas. Aps a metaciclognese estes resduos desaparecem e as formas metacclicas se soltam do intestino mdio de Phlebotomus argentipes (Mahoney et al. 1999). De modo similar, o mesmo ocorre com Leishmania infantum (cepa PP75), em Lutzomyia longipalpis (Soares et al., 2002). Esta variabilidade interespecfica no LPG das diferentes espcies resulta em especificidade na interao como demostrado por Pimenta et al. (1992; 1994). Entretanto, em Leishmania braziliensis, uma espcie peripilria, um mecanismo completamente diferente ocorre. Os LPGs das formas procclicas no possuem cadeias laterias, enquanto que o das metacclicas coloca um ou dois resdos de glicoses (Soares et al., 2005). Estes acares promovem adeso ao epitlio do intestino mdio e so capazes de inibir a adeso de formas procclicas (Soares et al., 2010). No sabemos por qu ocorre a adeso das metacclicas em L. braziliensis e provavelmente este fenmeno deve-se caracterstica peripilria desta espcie. Outra possibilidade seria a existncia de um segungo estgio metacclico, cujo desprendimento posterior deve ser confirmado por experimentos in vivo no vetor. Como parte de um grande projeto no estudo dos LPGs de espcies do Novo Mundo, nosso trabalho teve como objetivo avaliar o polimorfismo intra-especfico nas unidades repetitivas do LPG de L. infantum (syn. L. chagasi), e avaliar a sua interao com o vetor L. longipalpis (Lapinha). Foi determinado o grau de variabilidade das unidades repetitivas do

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LPG de 17 cepas de L. infantum (13 brasileiras, 2 europias e 2 africanas), avaliando o potencial de uma ferramenta bioqumica neste estudo. Os LPGs foram purificados e o tipo de carboidrato da cadeia lateral determinado por cromatografia lquida de alta performance (HPLC), eletroforese de carboidratos assistida por fluorforos (FACE) e eletroforese capilar (CE). Foram observados trs diferentes perfis para os LPGs analisados: (1) LPGs sem cadeias laterais (90% das cepas); (2) LPG com uma cadeia lateral (5%, cepa PP75) e (3) LPG com mais de uma cadeia lateral (5%, cepa BH46). Nossos resultados mostraram que o nvel de polimorfismo para o LPG de L. infantum baixo j que a maioria das cepas brasileiras e todas as cepas europias e africanas no apresentaram cadeias laterais. A anlise por HPLC revelou a presena de glicosilao proeminente para as cepas PP75 e BH46, caracterstica que parece ser constante apenas no LPG de cepas do Novo Mundo. Para se avaliar o papel destes polimorfismos na interao com o vetor, infectamos atravs de alimentao artificial fmeas de L. longipalpis com as cepas contendo LPG do tipo I (Ba262, EMO, IPT1), Tipo II (PP75) e Tipo III (BH46). Os insetos foram dissecados nos dias 2 e 6 e o nmero de parasitos contado em cmara de neubauer. No foi observada diferena na taxa de infeco do intestino mdio destes flebotomneos. O LPG crucial para a adeso e sobrevivncia dentro do vetor, porm polimorfismos intraespecficos nesta molcula em L. infantum no interferiram na sua interao com L. longipalpis. Referncias Descoteaux A, Turco, SJ 1999. Glycoconjugates in Leishmania infectivity. Biochim. Biophys. Acta 1455:341-52. de Veer MJ, Curtis JM, Baldwin TM, Didonato JA, Sexton A, McConville MJ, Handman E, Schofield L 2003. MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling. Eur. J. Immunol. 33:2822-31. Mahoney AB, Sacks DL, Saraiva E, Modi G, Turco SJ 1999. Intra-species and stagespecific polymorphisms in LPG control Leishmania donovani-sand fly interactions. Biochemistry 38:9813-23. McConville MJ, Turco SJ, Ferguson MAJ, Sacks DL 1992. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage. EMBO J. 11:3593-600.

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Pimenta PFP, Turco SJ, McConville MJ, Lawyer PG, Perkins P, Sacks DL 1992. Stage-specific adhesion of Leishmania promastigotes to the sandfly midgut. Science 234:212-14. Pimenta PFP, Saraiva EM, Rowton E, Modi GB, Garraway LA, Beverley SM, Turco SJ, Sacks DL 1994. Evidence that vectorial competence of phlebotomine sandflies for different species is controlled by structural polymorphisms in the surface of lipophosphoglycan. Proc. Nat. Acad. Sci. USA 91:9155-59. Sacks DL, Pimenta PFP, McConville MJ, Schneider P, Turco SJ 1995. Stagespecific binding of Leishmania donovani to the sand fly vector midgut is regulated by conformational changes in the abundant surface lipophosphoglycan. J. Exp. Med. 181:685-97. Soares RP, Macedo ME, Ropert C, Gontijo NF, Almeida IC, Gazzinelli RT, Pimenta PF, Turco SJ 2002. Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis. Mol. Biochem. Parasitol. 121:213-24. Soares RP, Cardoso TL, Barron T, Arajo MS, Pimenta PF, Turco SJ 2005. Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis. Int. J. Parasitol. 35:245-53. Soares RP, Margonari C, Secundino NF, Macedo ME, da Costa SM, Rangel EF, Pimenta PF, Turco SJ (2010). Differential midgut attachment of Leishmania (Viannia) braziliensis in the sand flies Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia. J. Biomed. Biotechn. 2010: Article ID 439174, 7 pages.

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COMPARING THE INITIAL IMMUNE RESPONSE IN THE SKIN OF ANIMALS EXPOSED TO UNINFECTED AND LEISHMANIA INFECTED SAND FLIES. Clarissa Teixeira, Regis Gomes, Luiz Fabiano Oliveira, Dia-Eldin Elnaiem, Claudio Meneses, Shaden Kamhawi and Jesus G. Valenzuela
Laboratory of Malaria and Vector Research, Vector Molecular Biology Unit National Institutes of Allergy and Infectious Diseases, National Institutes of Health Rockville, MD - USA, e-mail: teixeirac@niaid.nih.gov

During blood feeding sand flies inject Leishmania parasites alongside saliva into the host's skin. The saliva of sand flies contain a wide variety of molecules that modulate their host's haemostatic, inflammatory and immune response and have been implicated in facilitating the establishment of Leishmania in the host. Additionally, immunity to sand fly saliva or sand fly bites was shown to protect mice from further infection. Although protection to Leishmania infection is strongly correlated with a delayed type hypersensitivity (DTH) response in the presence of IFN-gamma little is known about the immune response responsible for the protection resulting from pre-exposure to saliva. To further understand the saliva-induced immune responses in the skin, we used a macroarray (Oligo GEArray) approach to compare the expression profile of cytokines induced following Phlebotomus duboscqi bites in nave and pre-exposed C57BL/6 mice challenged with L. majorinfected P. duboscqi flies. The relative expression of cytokines was investigated 248h after challenge. The results obtained show a rapid and strong early expression of inflammatory genes in pre-exposed compared to naive mice following challenge. To further characterize this early immune reponse and to verify if the expression of chemokines and cytokines reflected the cells found in the skin, we characterized the leukocyte populations recruited to the skin by flow cytometry 648h post-infection. Interestingly, pre-exposed mice bitten by infected sand flies

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showed a significantly increased recruitment of different leukocyte populations as compared to nave mice bitten by infected sand flies. Pre-exposed mice also presented a higher frequency of IFN- producing cells 24-48h post-infection compared to nave mice. These results strongly suggest that the host's initial immune response is dramatically modified following infected bites. Furthermore, the importance of testing Leishmania vaccine candidates with infected sand fly bites has been highlighted. Therefore, it is imperative to validate if a single sand fly salivary protein can confer protection following the powerful challenge of infected bites when considering salivary-based vaccines. To respond this important question we selected LJM11, a protective salivary molecule when tested with needle challenge with Leishmania and saliva, to test against Leishmania infected sand fly bites. Immunization with recombinant LJM11 (rLJM11) conferred protection following challenge with infected bites with significant lower parasite loads in comparison to the control group. Immunization with rLJM11 also induced IFN- gamma production in vitro from spleen cells of mice pre-exposed to bites. Importantly, immunization with rLJM11 induced a long lasting protection five months after the last immunization following challenge with infected bites. The results obtained from the work described here suggest that the immunity to specific sand fly salivary proteins can confer a powerful and lasting protection to the powerful challenge of infected sand fly bites and strongly suggest the inclusion of sand fly salivary-components when considering a Leishmania vaccine.

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IS THERE AN ARMS RACE INVOLVING SALIVARY PEPTIDES OF THE VECTORS OF ZOONOTIC VISCERAL LEISHMANIASIS? Paul D. Ready
Department of Entomology, Cromwell Road, Natural History Museum, London SW7 5BD, U.K. E-mail: P.Ready@nhm.ac.uk

Several authors have suggested that arms races occur between parasitic Leishmania (Protozoa, Trypanosomatidae) and their mammalian hosts or natural vectors, haematophagous adult females of phlebotomine sandflies (Diptera, Psychodidae). Certainly, arms races or balancing selection should be considered when developing vaccines for controlling the transmission of the species of Leishmania causing a spectrum of cutaneous and visceral leishmaniasis in Latin America, North Africa, southwest Asia and the Indian subcontinent. Female sandflies counteract host responses to capillary damage by pumping saliva into their haemorrhagic feeding pools, and some of the diverse salivary peptides help to control Leishmania infections (Th1-type cell mediated immunity) or exacerbate them (Th2-type CMI). Therefore, specific salivary peptides have been used for the experimental vaccination of mice, to protect against Old World Leishmania major by stimulating either a humoral response that neutralized the exacerbation of the infection (e.g. maxadilan from the neotropical vector Lutzomyia longipalpis) or a cellular response that killed parasites at the site of the bite, (e.g. PpSP15 from the natural vector Phlebotomus papatasi). The choice of salivary peptide for vaccination will also depend on its natural polymorphism among the geographical populations of a sandfly species and its rate of change in response to selection pressures from hosts and parasites. My team has investigated the role of selection on sandfly salivary peptides, using for a first model the natural variation of apyrase (E.C.3.6.1.5) in Phlebotomus ariasi, which is one of the European vectors of Leishmania infantum. This parasite causes zoonotic visceral leishmaniasis in the Mediterranean region as well as in Brazil and other parts of Latin America. Sandfly apyrase is an anti-platelet

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haemostatic factor, which we targeted because it is a vaccine candidate and its structure-function is known. Also, it is a low copy-number gene, and so we were able to design primers for scoring its genotypes using the PCR Amplification of Specific Alleles (PASA technique) followed by cycle sequencing. The detection of positive or balancing selection requires the use of a variety of tests, many of which do not distinguish between recent selection and neutral demographic variation (e.g. Fu & Li's D, Tajima's D). In general, recent population expansions and selective sweeps mimic signals of directional selection, and population declines and sub-divisions mimic balancing selection. Therefore, we targeted P. ariasi, because demographic variation in apyrase could be identified by reference to neutral markers. We characterized several natural populations of this sandfly, because selection may not always be detectable throughout the range of a species. No test provided support for positive directional selection, balancing selection or selective sweeps among a range of populations of P. ariasi associated with both geographical and environmental variation in Europe. The geographical pattern of genetic variation was consistent with neutral demographic processes, mainly isolation-by-distance and regional isolation resulting from multiple genetic divergences and population expansions during late Pleistocene glacial cycles. We also applied to apyrase tests (PAML branch models, and the McDonaldKreitman test) that are appropriate for detecting older selection. These tests were based on a well-supported phylogeny of all but one of the Mediterranean vectors of L. infantum in the subgenus Phlebotomus (Larroussius). Positive directional selection was identified only once, associated with an ancient gene duplication event, following which positive selection is frequent but not inevitable. It can be concluded that the apyrase of P. ariasi is not currently in an arms race with this sandfly's vertebrate hosts or parasitic L. infantum. Therefore, strong positive or diversifying selection on apyrase should not hinder its use in developing a vaccine against leishmaniasis. The control of leishmaniasis by a Th1-type CMI

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response stimulated by salivary peptides has been experimentally demonstrated for only two natural vector-parasite associations, namely Old World P. papatasi transmitting L. major to inbred mice and neotropical L. longipalpis transmitting L. infantum to its reservoir host, the domestic dog. Such studies help the rational design of vaccines but, in parallel, an empirical approach to screening for vaccine candidates might be necessary because of the complexity of the interactions among the three types of organism in each transmission cycle. For zoonotic visceral leishmaniasis, the appropriate model involves the domestic dog. The vaccination of this primary reservoir host is most likely to control not only a major veterinary problem but also human leishmaniasis, both in Latin America and in Europe.

Acknowledgements: This research was funded by EU grant GOCE-2003-010284 EDEN. It does not necessarily reflect the views of the European Commission. I thank my colleagues Dr Shazia Mahamdallie and Prof. Bernard Pesson for their invaluable contributions.

References: Mahamdallie, S.S., Pesson, B. & Ready, P.D. (2010) Multiple genetic divergences and population expansions of a Mediterranean sandfly, Phlebotomus ariasi, in Europe during the Pleistocene glacial cycles. Heredity doi: 10.1038/hdy.2010.111. Mahamdallie, S.S. & Ready, P.D. (2010) No contemporary arms race involving the sandfly salivary peptide apyrase: implications for vaccination against zoonotic visceral leishmaniasis. Proceedings of the Royal Society London series B, submitted. Ready, P.D. (2010) Leishmaniasis emergence in Europe. Eurosurveillance 15: pii=19505.

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O PROCESSO DE INTERAO DO Lutzomyia (Lutzomyia) longipalpis COM A Leishmania (Leishmania) chagasi.

Paulo F. P. Pimenta, Vanessa C. Freitas, Ana Paula M. Duarte, Rodrigo P. P. Soares, Mrcia D. Laurenti e Ngila F. C. Secundino.

Laboratrio de Entomologia Mdica, Centro de Pesquisas Ren Rachou FIOCRUZ-MG, Av. Augusto de Lima, 1715 CEP 30190-002, Belo Horizonte, M.G. Brazil.

Estudos da interao Leishmania-vetor so importantes para o entendimento dos processos de desenvolvimento e transmisso do parasito. Significativas informaes tm sido relatadas principalmente para espcies de Leishmania do Velho Mundo. No Novo Mundo, existem poucas informaes detalhadas sobre o desenvolvimento de Leishmania chagasi em seu vetor natural Lutzomyia longipalpis. O desenvolvimento desse parasito foi investigado em infeces experimentais do vetor com promastigotas de cultura e amastigotas axnicas obtidas por transformao in vitro. Ambas as formas do parasito geraram alta porcentagem de flebotomneos infectados. Foi observado um decrscimo no nmero de parasitos por intestino no terceiro dia aps o repasto. Entretanto, os parasitos sobreviventes nos dois grupos foram capazes de se multiplicar e desenvolver no intestino, apresentando densidade mxima entre seis e sete dias. Todos os morfotipos de promastigotas foram observados. Infeces iniciadas com promastigotas ou amastigotas axnicas mostraram perfis semelhantes de desenvolvimento e produziram formas infectivas. Assim, promastigotas podem ser preferencialmente utilizadas em experimentos de infeco por ser este um processo menos laborioso. Para muitas espcies de Leishmania tem sido sugerido que o lipofosfoglicano (LPG) promove a adeso do parasito ao epitlio intestinal do flebotomneo. Anlises estruturais do LPG de diferentes espcies de Leishmania tm revelado que o polimorfismo nesses glicoconjugados se deve a variao dos acares das cadeias laterais e "cap". A anlise das cadeias laterais

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em L. chagasi da cepa BH46 revelou pela primeira vez um LPG poliglicosilado (1, 2 e 3 cadeias laterais de glicose). A importncia do LPG na sobrevivncia dessa espcie em L. longipalpis foi investigada usando parasito mutante na biossntese do LPG. Foram observadas redues na sobrevivncia ou no crescimento desses mutantes no perodo que antecede a defecao e, em 60 horas, a infeco foi completamente perdida. Em todos os pares naturais Leishmania/vetor analisados at o momento o LPG requerido para a adeso do parasito ao intestino para evitar sua expulso com o bolo fecal. Diferentemente, ns observamos que a sntese de LPG foi essencial para a sobrevivncia inicial de L. chagasi no intestino de L. longipalpis, provavelmente protegendo o parasito do ataque enzimtico. Aspectos morfolgicos dessa interao foram investigados por microscopia de luz, eletrnica de varredura e transmisso. Diferente da adeso altamente especializada previamente proposta para L. chagasi em L. longipalpis, poucos parasitos foram observados com corpo e flagelo em contato com o epitlio intestinal e, esporadicamente, com o flagelo superficialmente inserido entre as microvilosidades. A saliva do vetor tem um importante papel na transmisso de Leishmania. Tem sido considerado que a saliva aumenta a infectividade. A nossa preocupao foi comparar o efeito da saliva de flebotomneos capturados no campo (SGL-W) com os de colnias (SGL-C) e entender o processo de infeco. Verificamos que as leses foram significativamente maiores em camundongos infectados com ambas as salivas quando comparado com os infectados somente com parasitas, e mais ainda, as leses por parasite+SGL-C foram maiores do que as de parasite+SGL-W. Estudos morfomtricos histopatolgicos mostraram que na fase aguda da infeco baixos nmeros de polimorfonucleares e maior nmero de mononucleares e parasitas nos camundongos infectados com SGL-C quando comparado com SGL-W. Na fase crnica, o numero de mononucleares foi mais baixo e o numero de parasitas foi maiores nos camundongos infectacdos com SGL-C do que com SGL-W. Gel de SDS-PAGE para SGL-C e SGL-W mostrou

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diferenas na composio e quantidade de bandas proticas determinadas pela densitometria. Estes resultados chamam a ateno para modelos experimentais de saliva, os quais mostram exacerbao da infeco causada pela saliva. Ns tambm investigamos os efeitos da saliva considerando a evoluo a imunomodulao da infeco. Comparando os grupos injectados com saliva, as leses dos grupos SGH-W determinou uma baixa produo de IL-4 e IL-10, mas altos nveis de IL-12 quando comparado com a saliva de SGH-C. Os nossos achados indicam um provvel bias, isto , um erro sistemtico devido ao mtodo utilizado, o uso de saliva de flebotomneos colonizados em laboratrio ao invs de saliva de vetores capturados no campo, os quais so os reais transmissores da doena.

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REVERSE GENETICS IN SAND FLIES: TARGETING MIDGUTS MOLECULES AFFECTS LEISHMANIA DEVELOPMENT Marcelo Ramalho Ortigo
Biology of Disease Vectors Laboratory, Department of Entomology, Kansas State University, Kansas, USA.

For a successful development within the midgut of the sand fly vector, Leishmania must overcome several barriers which are imposed by the vector that include an early proteolytic attack, the need to escape the peritrophic matrix (PM), and attachment to the midgut epithelia to prevent excretion with the remnants of the blood meal. The ability to overcome these barriers has been associated with species specificity, and interference with the sand fly vector-parasite balance can change the outcome of the infection in the the vector. Leishmania lipophosphoglycan (LPG) was shown to be a critical molecule in the midgut attachment process for some sand fly-Leishmania pairs. Further, blockage of a midgut LPG receptor, PpGalec, severely impaired L. major development and survival in the midgut of Phlebotomus papatasi. Such an effect supported the use of transmission blocking strategies against sand fly-transmitted leishmaniasis (TBV). Following overall analyses of the midgut transcriptome of the sand fly P. papatasi, several midgut molecules were selected as potential TBV candidates based on their response top infection with Leishmania major. Our lab is currently investigating some of these targets, which include a midgut-specific, blood induced chitinase (Ppchit1) previously characterized, several peritrophins some of which likely associated with the PM scaffolding formation, and other molecules involved in various aspects of sand fly physiology, from blood feed to interaction with pathogens. Analyses of expression profiles via real time or semi-quantitative PCR revealed tissues specificity for several of the molecules being investigated. With RNA interference (RNAi) we expect to uncover the actual physiological roles of such molecules, and more importantly, assess whether their targeting on TBV approaches is feasible.
Funding: National Institutes of Health- NIAID

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TRIATOMINE MOLECULAR SYSTEMATICS: NEW FINDINGS AND NEW HYPOTHESES


Fernando Monteiro
Instituto Oswaldo Cruz FIOCRUZ, Rio de Janeiro, Brazil

This talk presents the results of the projects of two Parasitology M.Sc. students (now Ph.D. students) I had the pleasure of mentoring at the Instituto Oswaldo Cruz, in Rio de Janeiro. Both projects focused on the molecular systematics, biogeography, and ecology, of three epidemiologically important Rhodnius species that occur in the Amazon region and in Bolivia. They are described below.

1. Phylogeography of Rhodnius pictipes (Triatominae): lessons for vector surveillance in the Amazon region

Human Chagas disease is hypoendemic in the Amazon. Foci of relatively intense transmission, related to large-scale harvesting or consumption of forest products, punctuate a widespread background pattern of low-intensity, continuous vector-borne transmission. Most transmission events, including localized outbreaks of food-borne acute cases, involve contact between Trypanosoma cruzi-infected sylvatic triatomines and susceptible humans or their foodstuffs. Because the reinvasion of insecticide-treated houses by adventitious sylvatic bugs seems inevitable, traditional control strategies are unlikely to be effective in the Amazon. An accurate knowledge of the systematics, geographic boundaries, patterns of genetic diversity, and ecological trends of Amazonian triatomines is therefore critical for the development of rational control-surveillance strategies. Phylogeographic and population genetics approaches based on mitochondrial DNA markers are very informative in such situations. Rhodnius pictipes is the most widespread sylvatic vector of Chagas disease in the Amazon. Its range largely overlaps that of the five cryptic species of the R. robustus complex. Moreover, R. pictipes and R. robustus are congeneric and share the same sylvatic ecotopes. This peculiar setting contains all the elements required for testing a basic principle of vicariance biogeography: that important vicariant events will affect

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equally all organisms with comparable dispersal capabilities that occur in a particular area. To test whether the vicariant events that isolated populations of the ancestral R. robustus stock, leading to the formation of the cryptic species complex, have had the same effect on R. pictipes. A total of 110 R. pictipes from 20 collection sites across Amazonia (Brazil, Bolivia, Colombia, and French Guiana) were sequenced for a 682bp fragment of the mitochondrial cytochrome b gene (cytb). R. stali, R. brethesi, R. amazonicus, R. pallescens, R. ecuadoriensis, and R. colombiensis cytb sequences were also analyzed. Twenty-two selected specimens, representing all collection sites were also sequenced for the second ribosomal internal transcribed spacer (ITS-2, 700bp), to provide information from a nuclear marker, and thus strengthen the study. We combined phylogenetic analyses, haplotype genealogies/networks, and palaeoecological data to investigate (i) the molecular alpha-systematics of R. pictipes, and (ii) the phylogeny, phylogeography and historical biogeography of the pictipes group. We finally compare the evolutionary patterns of the two main Amazonian Rhodnius lineages within the pictipes and robustus groups. The interpretation of the tree topology obtained for the former group (Neighbor-joining method with a K2-p nucleotide substitution model) based on a recent molecular clock calibration (Pfeiler et al. 2006) was used to test the predictions of two often evoked biogeographical hypotheses that would have generated the patterns observed: the marine incursions of the Miocene/Pliocene, and the Pleistocenic refuges. Similar to R. robustus s.l., R. pictipes is a paraphyletic assemblage of four sibling or near-sibling species, three of which are new to science. However, markedly different levels of within-complex inter-specific divergence suggest that the phylogeographic patterns of these two groups were not shaped by the same vicariant events. The ancestral pictipes group population went through at least eleven speciation events during the Miocene, Pliocene, and Pleistocene. The three cryptic species discovered in this study diverged from R. pictipes s.s. during the Mid/Late Miocene (12.6-6.8 Mya), possibly as a consequence of the Amazon-Orinoco Miocene marine transgressions. The wide

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geographic distribution and shallow phylogeographic structuring of R. pictipes s.s. suggest a sudden post-speciation, Mid-Pleistocene range expansion during an interglacial about 0.295-0.180 Mya. This work represents a key contribution to the development of a new approach for the surveillance and control of sylvatic triatomines in the Amazon. The identification of undescribed cryptic taxa within R. pictipes s.l. will allow assessing their vectorial capacity and their relevance in terms of disease transmission. Furthermore, our data reveal how the intricate historical biogeography of the Amazon differentially shaped the patterns of genetic diversity within two closely-related vector groups, providing an objective measure of the complexity inherent to defining regional surveillance strategies.

2. Molecular systematics and epidemiological relevance of Rhodnius spp. (Hemiptera: Reduviidae) in Bolivia Chagas disease, caused by the flagellate protozoan Trypanosoma cruzi, currently affects an estimated 9 million people in Latin America with 70 to 90 million living under the risk of infection. In Bolivia 60 to 80% of the territory is considered to be endemic and 28% of the population is infected. Vectorial transmission, mediated by Triatoma infestans, is the most important mode of disease transmission to humans. Consequently, other vector species have been neglected and little is known about the epidemiological role they play in Bolivia. In this study, Rhodnius populations were investigated aiming at (1) the determination of the taxonomic status of collected specimens via molecular taxonomy (DNA sequencing of a mitocondrial cytochrome b (cyt b) gene fragment and of the ribosomal second internal transcribed spacer (ITS-2); (2) the definition of the preferred natural ecotope for the detected species; (3) the assessment of the genetic structure of the populations studied; and (4) the evaluation of the epidemiological relevance of each species, through the analysis of T. cruzi infection rates. A total of 102 specimens were obtained from 10 localities distributed in the departments of La Paz, Cochabamba, Santa Cruz, and Beni. It was determined that the Rhodnius specimens collected belong to two different species: R. stali and what is believed to represent a new member of the R. robustus cryptic species

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complex (provisionally called R. robustus boliviano). Polymorphism analyses of the cyt b gene fragment revealed low nucleotide diversities for R. stali ( = 0,00067 and 0,00326, in Caranavi and Chapare, respectively). In the Alto Beni region, where the species colonizes human dwellings, it was possible to determine its main natural ecotope as Attalea phalerata palm trees; in the Alto Beni region, only one of the 12 collected specimens was infected with T. cruzi. A single infected specimen was obtained in Santa Cruz. The 84 R. robustus boliviano individuals collected belong to two highly structured populations, but with low nucleotide diversities ( = 0,00055 and 0,00112 in Santa Cruz and Chapar, respectively). Cyt b and ITS-2 data revealed an important introgression event of R. robustus II mitochondria into R. robustus boliviano. In Santa Cruz, of the 68 R. robustus boliviano obtained, infection rates per locality ranged from 40 to 100%. Interestingly, none of 17 sylvatic specimens was infected.
These studies were funded by FIOCRUZ, CNPq, and Faperj.

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SANDFLY DATABASE: DEVELOPMENT OF AN INTEGRATED PLATFORM OF BIOLOGICAL AND MOLECULAR DATA OF SANDFLIES (DIPTERA: PSYCHODIDAE) OF MEDICAL AND VETERINARY IMPORTANCE

G. Z. G. Silva1, S. M. O. Medeiros1, R. C. A. Gomes1, M. A. O. Santos1, M. V. A. Batista1, W. Cunha1, L. Gomes1, T. L. D. Lima1, T. A. E. Ferreira2, V. Q. Balbino1

1. Laboratrio de Bioinformtica e Biologia Evolutiva (LABBE), Departamento de Gentica, UFPE, Recife, Pernambuco, Brazil; 2. Departamento de Estatstica e Informtica, UFRPE, Recife, Pernambuco, Brazil.

In recent years it has been observed a notable expansion in the number of high throughput molecular studies in several species of insects vectors of tropical diseases, particularly those involved in the transmission of malaria (Anopheles gambiae), dengue and yellow fever (Aedes aegypti). The completion of these studies has resulted in the identification of several genes involved in a series of key aspects of these organisms' evolutionary success, contributing to the establishment of control strategies increasingly more specific and effective, which should minimize the impact of vector-borne diseases in the world's public health. In the case of sandflies (Diptera: Psychodidae) involved in the transmission of causative agents of leishmaniasis (Leishmania spp.), these studies remain rather incipient and consist mainly of the analysis of transcriptomes of some species. In this context, more representative species in public biological databases are Lutzomyia longipalpis (vector of Le. infantum chagasi, etiological agent of Visceral Leishmaniasis in the New World) and Phlebotomous papatasi (vector of Leishmania major, etiological agent of Cutaneous Leishmaniasis in the Old World). Owing to the need to obtain information on the biology of the sandflies' interactions with their hosts, the cDNA libraries evaluated in these studies were built using organs such as salivary glands and digestive systems. These analyses

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have enabled the identification of important molecules, opening the perspective of their future utilization in the development of more effective methods of leishmaniasis control. The information produced in these investigations demands the development of computational methods that enable the automation of functional annotation and interpretation of the resulting data. In an effort to contribute in this regard, we present the SandFly Database (SandFlyDB) an integrated platform of biological and molecular data on the most intensively studied sandfly species. The main focus of the proposed tool consisted of the computational analysis of the totality of expressed sequence tags (ESTs) of these organisms available in GenBank (42,778 ESTs of Ph. papatasi, 33,123 of Lu. longipalpis and 7,727 of other sandfly species), with a view to establishing a protocol for automatic identification and annotation of protein coding genes. Sequences were retrieved from GenBank (http://www.ncbi.nlm.nih.gov), as well as the following information: designation of cDNA libraries; organs and/or physiological stages of the organisms used in the construction of libraries. This information was stored in a MySQL database, enabling the classification of ESTs according to used tissue type (salivary glands or digestive systems) and the physiological stage (sandflies fed with blood or sugar; infected with Leishmania sp. or otherwise). The files were clustered using CAP3 software (http://seq.cs.iastate.edu/download.html), and the contigs and singlets were compared against a local copy of the NR database from Genbank using the BLASTX software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The next step consisted of the recovery of information from secondary databases in order to obtain additional information on the functions of the identified proteins. Searches were carried out against local copies of the following databases: KEGG (http://www.genome.jp/kegg); Gene Ontology (http://www.geneontology.org); and COG (http://www.ncbi.nlm.nih.gov/COG). This information will be freely available online on our web site (http://www.bioinfo.ufpe.br), for those interested in structural, functional and comparative genomics of sandflies.

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Supported by CNPq, UFPE, CAPES and FACEPE ANLISE EPIDEMIOLGICA DA MALRIA EM MANAUS/AM CONSIDERANDO A ESTRATIFICAO POR DISTRITO SANITRIO. Rosemary C Pinto, Wagner CM Terrazas, Wanderli P Tadei, Antnio EM Oliveira,

FVS-AM / INPA

Introduo:

Na Amaznia, em funo das caractersticas ambientais, comum o registro de malria urbana em decorrncia da prximidade da floresta circundante, o que facilita o contato homem-vetor. Manaus um exemplo clssico desta situao. Neste trabalho apresenta-se uma anlise epidemiolgica da malria na cidade, considerando-se uma estratificao por Distrito Sanitrio.

Objetivo:

Avaliar o perfil epidemiolgico dos trs Distritos Sanitrios de ocorrncia de malria, relacionandoos a parmetros ambientais.

Material e Mtodos: As informaes epidemiolgicas foram obtidas no Banco de Dados do Sivep_Malria_Web para os anos de 2004 a 2008, com foco principal entre 2007 e 2008. A estratificao foi por aglomerados e consolidados para os trs Distritos Sanitrios de Manaus das zonas Leste, Norte e Oeste. Levou-se em conta o risco instalado e as propores entre as reas, obtidos a partir da positividade das espcies parasitrias, segundo local de infeco.

Resultados: A malria em Manaus aumentou cerca de 15% entre 2004 e 2005 e, entre 2005-2008, os nmeros vm sucessivamente se reduzindo, apresentando uma queda total de 69%. Os trs Distritos apresentaram maior concentrao de casos em 2007 e 2008, sendo que os dois primeiros

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concentram cerca de 86% dos casos em 2008. A reduo entre 2007 e 2008 foi maior para os Distritos das Zonas Norte e Oeste, respectivamente 62% e 57%. Considerando o risco de malria, o IPA apresentou quedas de 34,70%, 62,11% e 53,30% nos trs Distritos. Comparando as zonas urbana rural h um contraste muito grande nos valores de IPA nos trs Distritos. Nas reas rurais os valores chegam a ser at 98 vezes maiores que nas reas urbanas. Todos os Distritos registraram aumento no IFA, especialmente na zona rural.

Concluso: Os resultados mostram que a malria em Manaus, nos Distritos estudados concentra-se na rea rural (cerca de 80%), o que dificulta a implementao das aes de controle em funo da alta exposio ao vetor. Em contrapartida, os Distritos Leste e Oeste diferem quanto ao nvel de risco da malria IPA: 467,36 e 276,41 respectivamente. Outra concluso importante trata-se dos valores de IFA, que reduziram aps a introduo do Coartem, associado a Primaquina. Entretanto, a partir do momento em que a Primaquina deixou de ser ministrada, detectou-se aumento nas propores. Estes resultados necessitam ser analisados em maior profundidade, em funo dos desdobramentos que podem estar envolvidos nos processos de transmisso e de ateno ao homem doente.

FINANCIAMENTO: FVS-AM/INPA/FAPEAM/CNPq/MS

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CRIADOURO DE Lutzomyia longipalpis EM REA ENDMICA PARA LEISHMANIOSE VISCERAL NO ESTADO DA BAHIA

Jos Carlos Miranda


Centro de Pesquisa Gonalo Muniz / FIOCRUZ Rua Valdemar Falco, 121 Candeal Salvador BA jmiranda@bahia.fiocruz.br

Com a finalidade de localizar os criadouros naturais de Lutzomyia longipalpis, principal vetor da Leishmaniose Visceral Americana (LVA), nas diferentes estaes climticas do ano, assim como, a densidade de insetos adultos presentes no dia e local de coleta das amostras de solos, foram coletadas 1536 amostras na rea endmica para LVA, durante o perodo de Agosto de 2008 a Julho de 2010. As amostras foram coletadas em locais com caractersticas adequadas ao desenvolvimento das formas imaturas como: pouca umidade, presena de matria orgnica em decomposio e pouca luminosidade. As coletas foram realizadas no Distrito de Cavunge, localizado no municpio de Ipecaet, Bahia. Foram coletadas mensalmente 64 amostras de solos em 5 residncias (no intra e no peri-domiclio), durante 24 meses. Para a analise das amostras foram aplicadas as tcnicas de exame sob estereomicroscpio durante 70 dias, seguido da flutuao. Das 1536 amostras coletadas, at o momento 53 foram positivas para formas imaturas de Lutzomyia longipalpis, distribudas da seguinte forma: sob fezes de galinha; em base de caixa d'gua; oco de toco; oco de razes de umbuzeiro que serve como abrigo para galinhas; sobre pedras sob umbuzeiro, terra frouxa sobre pedras; sob pote de gua potvel; buracos em residncias com paredes de barro; entre pedras e razes de umbuzeiro que serve como abrigo para galinhas. O encontro dos criadouros naturais de Lutzomyia longipalpis poder

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nortear medidas de controle para a LVA, como o direcionamento do local e a melhor estao climtica do ano para a aplicao de inseticidas ou o controle biolgico a partir da utilizao de microorganismos entomopatognicos no peridomiclio das reas endmicas.

Suporte Financeiro : CNPq ( FIOCRUZ / FAPESB )

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FLEBOTOMNEOS: MITOS OU VERDADES Jos Dilermando Andrade Filho


Coleo de Flebotomneos, Centro de Referncia Nacional e Internacional para Flebotomneos, Centro de Pesquisas Ren Rachou/Fiocruz. Av. Augusto de Lima, 1715, Barro Preto, 30.190-002, Belo Horizonte, Minas Gerais. jandrade@cpqrr.fiocruz.br

Os flebotomneos so um grupo de insetos pertencentes famlia Psychodidae. Dentro dos psicdideos, esse grupo se destaca por apresentar o hbito hematfago e, em decorrncia desse comportamento, algumas espcies tornaram-se vetores de algumas doenas, com destaque para as leishmanioses. De fato, no ano de 2012 ir se comemora um sculo de estudos com flebotomneos no Brasil. Diversos avanos foram observados nesse perodo, entretanto, vrias observaes feitas nas dcadas passadas permanecem como verdades absolutas, sem que seja contestada pelos pesquisadores atuais. Esse trabalho visa questionar alguns fatos (mitos?) que acompanham h dcadas esse fantstico grupo de insetos. fcil se ouvir dos flebotomlogos, que os flebotomneos so muito delicados, frgeis, que morrem toa. Acreditava-se que com o desmatamento, os flebotomneos seriam extintos de determinadas reas, pois no sobreviveriam as mudanas ambientais. O que se v hoje muito diferente. Em diversas capitais, eles so encontrados em casas e apartamentos, rodeados por concreto. Como um bicho frgil sobrevive a isso? Aqui no se leva em considerao a poluio a que eles so expostos nos grandes centros, mas apenas se indaga: como pode um flebotomneo sobreviver em um ambiente to inspito? Talvez ele no tenha sido avisado que frgil, delicado e que morre toa interessante notar que, quando se sacrifica um flebotomneo por congelamento, aps se iniciar a triagem ou o armazenamento dos insetos em tubos contendo lcool 70, dentre todos os insetos, antes de serem estocados, eles so os primeiros e retomar suas atividades, sendo que alguns conseguem voar e pousar em paredes prximas, enquanto os demais dpteros, colepteros, lepidpteros, etc. permanecem de pernas pro ar. Como podemos imaginar um inseto frgil ser

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encontrado no stimo andar de um apartamento? Nesse momento chegamos a outro ponto, o vo dos flebotomneos. Como os flebotomneos, que voam pouco, saltando, chegaram at ali? Prdios cercados por prdios, sem reas verdes ao redor. E como Lutzomyia longipalpis, voando to pouco consegue chegar ao quinto, sexto, stimo andar de vrios prdios em Belo Horizonte? Uma das primeiras coisas que se aprende sobre flebotomneos a capacidade limitada de vo desse grupo. O Brasil cercado de barreiras geogrficas, tais como serras e rios. Um flebotomneo conseguiria atravessar um rio com 100 metros de largura de uma margem outra? Se ele tem essa capacidade de vo to limitada, no! Ento como explicar que uma determinada espcie apresente distribuio geogrfica to ampla? E so vrias as espcies de flebotomneos que tm uma vasta distribuio geogrfica pela Amrica do Sul. Pode-se alegar a disperso passiva, principalmente pelo vento, esse fator no pode ser descartado, porm h registro de flebotomneo voando, aps se ingurgitar de sangue, por 20 metros. Como explicar que se coletou flebotomneos em um barco, no meio do Rio das Velhas, a 80 metros de cada margem? No havia como o flebotomneo chegar at ali, a no ser por vontade prpria, voando, dessa forma, creio que os flebotomneos voam sim saltado, provavelmente o gasto de energia seja menor, entretanto eles podem voar a longas distncias, atingindo a margem contrria de rios, dispersando-se por novas reas. Nesse ponto passamos aos aspectos ambientais, mais precisamente aos altos ndices pluviomtricos, que, segundo nos ensinam, influenciam de forma crucial no encontro de adultos, pois as fortes chuvas lavam, carregam as larvas, principalmente, as pupas. As fortes enchentes que assolam o Brasil, certamente devem ter um peso no encontro de formas adultas de flebotomneos, porm, se eles fossem afetados por essas catstrofes naturais, tais como enchentes, muitas populaes teriam se extinguido e hoje as leishmanioses no seriam o que so! As fortes precipitaes que atingem determinadas regies, onde se forma grandes enxurradas naturais podem lavar as larvas se elas l estiverem, mas certamente, o solo por onde

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passam essas grandes quantidades de gua j empobrecido, e os estgio imaturos dos flebotomneos no estariam ali, estando bem protegidos dessas tormentas. Em uma inundao, muitas vezes grandes quantidades de terra ficam submersas, porm, quando as chuvas param ou diminuem, os flebotomneos continuam a ser coletados, sem alterao significativa nos meses subseqentes s inundaes. As larvas certamente tm seus mecanismos de fuga para escapar dessas adversidades, porm, infelizmente, o estudo do comportamento de flebotomneos escasso, pouco se sabe sobre esse aspecto. Outro aspecto interessante relativo no s aos flebotomneos, mas diz respeito a qualquer animal ou vegetal: o nome vulgar. A nomenclatura vulgar utilizada para se referir a determinado ser vivo. Esses nomes vulgares esto relacionados a uma questo cultural, enraizada em certas regies, podendo variar para cada uma delas. Por esse motivo os taxonomistas do nomes cientficos aos animais e plantas, a fim de padronizar e se evitar as confuses oriundas dos nomes vulgares. Obviamente, por ser popular, vulgar, no pode haver regras e por incrvel que parece os especialistas criaram regras para isso! Interessante que os flebotomlogos no aceitam que um flebotomneos seja chamado de mosquito, sendo aceitos birigui, cangalha, tatuquira e mosquito-palha! Mosquito no pode, mosquito-palha pode! um nome vulgar, que vem da cultura de um povo, de uma populao, que vem passando de geraes a geraes, e deve ser respeitado pelos estudiosos. Espera-se que nos prximos 100 anos novos achados e avanos venham a desmitificar os erros que certamente acontecero no decorrer dos estudos dos flebotomneos.

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CHALLENGES IN DEFINING THE COMPLEX GENETIC STRUCTURE OF Anopheles gambiae Gregory C. Lanzaro,
Professor, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA

The African malaria vector, Anopheles gambiae, may serve as a model system for studying vector population biology. The population genetics of this species has been intensively studied. The picture that has emerged is a species consisting of populations that are highly structured genetically and that remain difficult to describe. Starting in the 1960's cytogenetic studies of polytene chromosomes revealed that An. gambiae is a complex of species that differ in ways critically important to understanding malaria epidemiology. These studies also revealed that West African populations of An. gambiae sensu stirctu are highly complex and may be in early stages of species evolution. Contemporary molecular techniques were applied to the problem beginning in the 1990's. Study of microsatellite DNA polymorphism revealed that levels of gene flow between chromosomal forms varies over the genome and that, as expected, divergence is higher in regions of the genome contained within chromosome inversions. Attempts to identify molecular markers that would replace classical cytogenetics for defining discrete populations culminated in the description of two population groups in West and Central Africa that appear to be reproductively isolated. These groups are known as the M and S molecular forms of An. gambiae and have been widely described as incipient species, that is, populations in early stages of speciation. Recent work has revealed that the M and S concepts fail to adequately describe populations, with reproductive isolation between the two complete in some localities, but nonexistent in others. A population genomics approach involving analysis of the entire genome using microarray technology has revealed islands of speciation that are hypothesized to contain genes that mediate reproductive isolation and have revealed information that contradicts earlier work regarding the significance of chromosome inversions. 129

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MOSQUITOS TRANSGNICOS, DO PAPER A REALIDADE. Soraia de Lima Oliveira, Danilo Oliveira Carvalho e Margareth Lara Capurro

Departamento de Parasitologia, Instituto de Cincias Biomdicas, Universidade de So Paulo. Instituto Nacional de Cincia e Tecnologia em Entomologia Molecular (INCT-EM), Brazil. mcapurro@icb.usp.br

Linhagens de Aedes aegypti supressoras de populaes esto prontas para testes de campo. Estudos preliminares em laboratrios foram realizados em vrios Pases do Primeiro Mundo (EUA, Reino Unido, Frana), mostrando que as fmeas vindas do Continente Americano assim como da sia, no fazem distino entre os machos selvagens (no transgnicos) e os machos transgnicos. Atualmente, estes estudos esto sendo realizados em vrios Pases Endmicos (Brasil, Mxico, Malsia, Ilhas Caimam, Cuba, Vietnam) onde alm dos experimentos em laboratrio, so realizados os estudos de Liberao Controlada (caixas externas), estudos de disperso dos machos e a prpria supresso de populaes. Para que os estudos fossem realizados no Brasil, o Comit Interno de

Biossegurana do Instituto de Cincias Biomdicas avaliou o projeto de Estudo de disperso de machos transgnicos de linhagens supressoras de populao e encaminhou o processo a Comisso Tcnica Nacional de Biossegurana (CTNBio) para a regularizao de acordo com as normas de segurana em vigor no pas. A regularizao avalia a biossegurana dos Organismos Geneticamente Modificados (OGM) e seus derivados, dos estudos realizados por terceiros, referentes proteo da sade humana, dos organismos vivos e do meio ambiente, em atividades que envolvam a construo, experimentao, manipulao, transporte, comercializao, armazenamento, liberao e descarte

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de OGM e derivados. A nossa proposta apresentar a comunidade cientfica a proposta dos projetos que esto sendo executados nos vrios Pases, inclusive no Brasil, para discusso profunda dos riscos e benefcios que estas linhagens de Aedes aegypti podem oferecer.

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SAND FLY GENOME PROJECT

Mary Ann McDowell, Eck Institute for Global Health, Department of Biological Sciences, University of Notre Dame, USA. E-mail: mcdowell.11@nd.edu. Sand flies serve as vectors for several established, emerging and re-emerging infectious agents. As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting protozoan, bacterial, and viral pathogens. Completed genome sequences of these medically important vectors will foster development of novel technologies to control these devastating diseases. Phlebotomine sand fly research has served as a key model for studies concerning vector/parasite/host interactions by revealing novel mechanisms defining vector competence, propelling the field of vaccine research into promising areas, and identifying novel therapeutics for human use. The sand fly genome project will accelerate progress in these areas, as well as complement and enhance ongoing comparative genomics efforts. Phlebotomine sand flies are members of the family Psychodidae, which includes a diverse group of vectors that vary widely in geographic distribution, ecology and the pathogens they transmit. The goal of the Sand Fly Genome Sequencing Project is to sequence the genomes of two different phlebotomine sand flies, Phlebotomus papatasi and Lutzomyia longipalpis, that exhibit distinct distributions, behavior, and pathogen specificity. A comparative approach between these two species will provide substantial added-value, both technical and scientific, and will accelerate the discovery of regulatory and biochemical pathways within this family as potential biopharmaceuticals, vaccine candidates, and targets for insecticide development. Moreover, comparative genome sequence analyses between these and other available genomes will elucidate the pathways that lead to arthropod blood-feeding and immunity, inform arthropod phylogenetic relationships and enhance our comprehension of the evolutionary mechanisms that define genome organization. Funded by United States National Human Genome Research Institute the Sand Fly Genome Project was initiated by a proposal submitted by Drs. Mary Ann McDowell, Frank Collins, Marcelo Ramalho-Ortigao, Jesus Valenzuela, Shaden Kamhawi, Rod Dillon, Paul Bates, and Michael Lehane. The L. longipalpis portion of the project is headed by Dr. Rod Dillon, Liverpool school of Tropical

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Medicine, UK (LSTM) and Dr. Stephen Richards, Baylor College of Medicine Sequencing Center, USA. The P. papatasi portion of the project is headed by Dr. Mary Ann McDowell, University of Notre Dame, USA (UND) and Dr. Sandy Clifton, Washington University Sequencing Center, USA. The specific aims of the project are to 1) generate a normalized cDNA library of P. papatasi and sequence 40,000 Expression Sequence Tags (EST); 2) generate BAC libraries for both P. papatasi and L. longipalpis and end sequence both libraries at 10x coverage; 3) sequence 5 full length randomly chosen BAC sequences for each species, and 4) complete whole genome sequences at 15x coverage for L. longipalpis and P. papatasi using 454 Titanium sequencing along with 30x coverage of 3 and 8kb insert libraries. The Jacobina strain, Bahia, Brazil of L. longipalpis and the Israeli strain of P. papatasi are being used for this sequencing effort. The L. longipalpis Jacobina colony at the Liverpool School of Tropical Medicine was originally established by Richard Ward in 1988 by flies caught in Jacobina, Bahia State, Brazil and has been maintained continuously since establishment. The Israeli strain of P. papatasi was transferred to the University of Notre Dame in 2005 from Walter Reed Army Institute of Research (WRAIR). The colony was originally established in 1970 at Hebrew University, Jerusalem and transferred to WRAIR in 1983. The colonies were chosen because the majority of genetic information currently available was generated from these strains and the relatively reduced genetic heterogeneity present in long-term colonies. A normalized cDNA library was constructed from P. papatasi flies housed at WRAIR. Total RNA was collected from the four larval stages, pupa, male and female at 1, 3, and 10 days post emergence. Additional RNA was included from females 6, 12, 24, 36, 48, 72, 94 and 120 hours post feeding on uninfected and Leishmania major (strain V1) infected mouse blood. Library construction was completed by Expressed Genomics and EST sequencing was performed by the Genome Center at Washington University, St. Louis. The EST sequences are currently available on the NCBI EST data base. Comparative analysis between P. papatasi ESTs and the previous L. longipalpis EST data set (Dillon et al, Genomics 88:831, 2006) is completed. Genomic DNA from males and females from L. longipalpis (Jacobina Strain, housed at LSTM) was used to generate a BAC library. The library construction was conducted by Clemson University Genomics Institute. Similarly, a BAC library from P. papatasi (Israeli Strain, housed at UND) genomic DNA was generated by Amplicon Express. BAC clones and Filters are available for purchase from the respective institutions and sequences can be found in the

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NCBI trace archive database. Whole genome sequencing is ongoing, but nearing completion. Sequence fragment data will be available in NCBI's Sequence Read Archive. The VectorBase Bioinformatics Resource Center will assume responsibility for display and future curation of the genomes on completion of the project.

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ECOLOGICAL GENETICS OF SAND FLY SALIVARY GLAND GENE EXPRESSION Iliano V. Coutinho-Abreu1,2, Rami Mukbel1, Mariah Wadsworth1, Gwen Stayback1, Marcelo Ramalho-Ortigo2 and Mary Ann McDowell1 The Eck Family Center for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA. 2 Department of Entomology, Kansas State University, 123 W. Waters Hall, Kansas State University, Manhattan, KS 66506, USA. Environmental factors can modulate gene expression in many organisms. In Phlebotomus papatasi, the availability of sugar-sources in natural habitats plays an important role in the physiology of this sand fly as well as modulates the activity of proteins that participate in digestion and vectorial capacity. As salivary proteins are important mediators of Leishmania infection, they also contribute to the vectorial capacity of sand flies. Thereby, we compare de expression profiles of ten P. papatasi salivary gland genes (SP12, SP14, SP15, SP28, SP29, SP30, SP32, SP36, SP42, and SP44) of specimens inhabiting different ecological habitats. Additionally, the expression profiles of these genes were also assessed between specimens collected through the sand fly season in each habitat. Influences of senescence or diet on P. papatasi salivary gland gene expression also were assessed in colonized specimens. Sand fly collections were carried out in two different sites in Egypt, Aswan (Baharif) and North Sinai (Om Shikhan). In the Middle East, sand flies were caught in Swaymeh. In regards to ecological differences among collection sites, Swaymeh and North Sinai are dry habitats whereas Aswan is an irrigated area. The comparisons between field samples collected at the same time point in the season between the three populations analyzed (geographic analyses) showed that the gene expression levels vary through the season between these populations, ranging from 2- to 25-fold. Of significance, expression differences were revealed between Swaymeh and Aswan specimens collected late in the season. However, none of populations presented higher levels of expression throughout the whole season, suggesting that genetic factors play, if any, a minor role in the expression differences between these populations. Furthermore, when the expression
1

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levels of sand flies collected in the same population at different time points through the season (seasonal analyses), the levels of expression of six out of ten genes (SP12, SP15, SP29, SP36, SP42, and SP44) were up-expressed in specimens from Swaymeh in the end of the season, when the availability of sugar sources is reduced due to water deprivation. On the other hand, the up-expression of these genes in the end of the season was less evident in specimens collected in Aswan, which is an irrigated area less susceptible to drought effects. These results indicate that expression profiles of P. papatasi salivary gland genes can be more influenced by environmental factors than by the genetic divergences between populations. Nonetheless, phenology studies point to the presence of a higher number of engorged and gravid females towards the end of the season in dry habitats, when specimens also are thought to be older. If such specimens presented higher salivary gland gene expression than sugar fed younger ones, this could explain the greater expression displayed by specimens collected late in the season in Swaymeh. Nevertheless, the expression patterns of gravid or engorged and older laboratory female specimens displayed similar or lower expression levels than sugar-fed younger ones, ruling out such biotic factor as responsible for modulation the expression profiles of field collected specimens. Many questions remain unanswered regarding the ecological and molecular bases for the P. papatasi salivary gland gene expression differences, yet greater expression of at least six salivary genes coincide with an increase in the number of human cases late in the sand fly season. Among the possibilities, a higher dose of saliva inoculated into the host skin may be modulating Leishmania development, as observed in the laboratory. Additionally, as sand fly salivary components are potential vaccine candidates, seasonal variation in saliva dose inoculated into host skin may interfere with vaccine efficacy because dose is an important component of immune responses. Therefore, the genetic plasticity of genes involved with vectorial capacity in disease vectors seems to play an important epidemiological role in the establishment of diseases in natural habitats.

Financial Support: Department of Defense (DoD) Defense Advanced Research Projects Agency (DARPA) 136

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TRANSCRIPTOME OF THE DIGESTIVE TRACT OF THE BLOOD SUCKING BUG, Rhodnius prolixus

Jos M.C. Ribeiro1, Glria RC Braz3,14, Walter R. Terra2, 14, Cllia Ferreira2,14, Raquel L L Oliveira3,14, Marcos HF Sorgine4,14, Gabriela O Paiva-Silva4,14, Marcelo Medeiros5,14, Leonardo Koerich6,14, Antonio B. Carvalho6,14, Fernando A. Genta7,14, Carla Polycarpo4,14, David Majerowicz4,15, Michelle Alves-Bezerra4,14, Katia C Gondim4,14, Rafael D. Mesquita8,14, Felipe Dias4,14, Renata Gonalves4,14, Jos H M Oliveira4,14, Ednildo Alcntara Machado5,14; Alice 3,14, Patrcia Fampa4,14, Mrio A.C. Silva-Neto4,14, Gergia C. Atella4,14, Helena Arajo9,14, Rodrigo F Fonseca9,14, Ricardo Nascimento Arajo10,14, Marcos H. Pereira10,14, Cristiano Lazoski6,14, Rolando R Pomar11, Luis Diambra11, Guenter Schaub12, Carsten Balczun12, Aparecida Tanaka13,14, Pedro L Oliveira4,14
(1) Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, 12735 Twinbrook Parkway room 2E32, Rockville MD 20852; (2) Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, So Paulo, SP; (3) Departamento de Bioqumica, Instituto de Qumica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ; (4) , Instituto de Bioqumica Mdica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ; (5) Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ; (6) Departamento de Gentica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ; (7) Instituto Oswaldo cruz, FIOCRUZ, Rio de Janeiro, RJ; (8) Instituto Federal de Educao do Rio de Janeiro, Rio de Janeiro; RJ (9) Instituto de cincias Biomdicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ; (10) Departamento de Parasitologia, Instituto de Cincias Biomdicas Universidade Federal de Minas Gerais, Belo Horizonte, MG; (11) Centro Regional de Estudios Genomicos, Universidad Nacional de La Plata, Argentina; (12) Department of Animal Ecology, Evolution and Biodiversity, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Bochum, Germany; (13) Departamento de Bioqumica, Universidade Federal do Estado de So Paulo; (14) Instituto Nacional de Cincia e Tecnologia em Entomologia Molecular, Brasil.

Most arthropod-borne infectious diseases are transmitted by insect vectors that belong to a single genera and frequently a single species has a prevalent role on spreading the pathogen. In contrast, triatomine bugs comprise several different genera. Several species are vectors of Chagas' disease in the Americas, infecting 1516 million people in Latin America today. Rhodnius prolixus is a relevant vector in Central and South America, and became a model for insect physiology and biochemistry thanks to its use in the classical work of Dr. Vincent Wigglesworth. Rhodnius prolixus has the smallest genome among all triatomine and therefore its genome was chosen for sequencing. Included in this effort was the sequencing of several organ specific cDNA libraries, using pyrosequencing technology that will be discussed here. All instars of Rhodnius prolixus feed exclusively on blood and are highly adapted to hematophagy: a 30 mg Vth instar nymph can take 10 times its own weight

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in blood in a 15 min meal, which is stored in the anterior midgut. Being an hemiptera, Rhodnius digestive system follows the basic organization plan of this order, which has lost both the chintin-based peritrophic membrane and all digestive serine proteinases during the course of evolution. Instead, the midgut epithelium is covered by layers of phospholipid membranes the so-called perimicrovillar membranes. Digestion of the large amount of protein found in its diet occurs only after the food is transferred to the posterior midgut and is accomplished by acidic proteinases similar to lysosomal proteolytic enzymes. The rectum possesses transitional epithelium that can stretch to accommodate the faeces and urine and is supposed to be the site where Trypanosoma cruzi the agent of Chagas' disease, differentiates into the infective metacyclic form. Along with the faeces, this protozoa is released on the skin of the mammalian host during rectal discharges that take place after a blood meal. Therefore, most annotation effort on Rhodnius cDNAs has focused on transcripts from the digestive apparatus. Among the libraries produced as part of the R. prolixus genome sequencing effort, there were a whole body library (WB 862,980 reads), produced from RNA deriving from insects from various instars and states post blood meal, as well as an anterior midgut (AM 156,780 reads), posterior midgut (PM 145,986 reads) and a rectum (RE 170,565 reads) library. These reads were assembled together into 65,946 contigs which had a length above 250 nt allowing. The number of reads in each contig was used for the identification of transcripts that were significantly over expressed in particular tissues (10 fold more expressed in gut tissues in comparison to the whole body library), thus allowing identification of digestive organs specific transcripts in R. prolixus The assembly had 27,751 contigs larger than 499 nt, 8,324 contigs with lengths above 999 nt and 972 above 1999 nt. Additionally, over 2,900 coding sequences (CDS) were obtained, most of which (~2,300) full length (Met to stop codon), which will help train the gene finder programs for genomic annotation of this organism. Among the contigs possibly associated with digestive function, they were classified (Table 1), in their order of abundance of digestive tract derived reads, into

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digestive enzymes, immunity-related, protease inhibitors, transporters and storage, lipocalins, mucins, other secreted products, odorant binding proteins and peritrophins. Aspartyl and cysteinyl protease were the most frequent over expressed transcripts in the gut tissues. It seems that a considerable expansion of both protein families has occurred and 17 coding sequences from aspartyl proteases, most of them full length, were identified, together with 9 cysteine proteinases. Interestingly, despite no blood digestion was detected on the AM, several of those proteinases are highly transcribed in the AM, as well as in the RE, in addition of the PM. Surprisingly 26 contigs of peritrophins, most of them full length, were found significantly over expressed in at least one segment of the gut, in spite of the lack of peritrophic membrane. Lipocalins and odorant binding proteins, two protein families capable to bind hydrophobic compounds are highly expressed in the midgut, none of them having a known physiological ligand. Ferritins are also overexpressed in the gut, showing that heme oxygenase pathway is active in the gut and that avoiding Fenton reaction is also a priority for this tissue. In addition to the expected presence of digestive lipases, analysis of lipid metabolism has shown significant expression of genes involved on beta-oxidation, suggesting that lipid degradation is a major source of energy for midgut cells. Also, genes involved in phospholipid synthesis are overexpressed, probably to cope with the secretion of perimicrovillar membranes. Genes related to immunity have been found highly expressed as lectins, lysozymes, serpins and a homlogue of the Toll ligand Spaetzle, which are potentially involved in control of intestinal microbiota and interaction with pathogens. Taken together, analysis of Rhodnius transcriptome reveal several unanticipated aspects of the physiology of the midgut of triatomine bugs, thus creating a novel landscape and opening new avenues for future research.

Supported by CNPq, FAPERJ, NIH and HHMI.

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TICKS AS VECTORS OF Leishmania PARASITES: FACTS AND SPECULATIONS

Filipe Dantas-Torres
Dipartimento di Sanit Pubblica e Zootecnia, Facolt di Medicina Veterinaria, Universit degli Studi di Bari, Str. Prov. per Casamassima km 3, 70010 Valenzano (BA) Italia. E-mail: f.dantastorres@veterinaria.uniba.it.

Introduction Leishmania protozoa are the aetiological agents of the leishmaniases, a group of parasitic diseases that is still causing a huge burden on public health worldwide. Phlebotomine sand flies are the only biological vectors of Leishmania spp. proven so far. Secondary modes of transmission have been extensively discussed in the literature, including congenital transmission, venereal transmission, via blood transfusion and by arthropods other than phlebotomine sand flies (Dantas-Torres 2009). In particular, there has been much debate about the role of fleas and ticks as vectors of Leishmania infantum (syn. L. chagasi) in recent years (Otranto & Dantas-Torres 2010). In this context, this article discusses the facts and speculations concerning the participation of ticks in the transmission of L. infantum.

Transmission of Leishmania infantum by tick bites The only evidence of transmission of L. infantum by tick bites arises from a PhD study conducted in the Oklahoma State University by the mid-1980s (McKenzie 1984). In this study, nymphs of R. sanguineus were fed on two naturally infected dogs, allowed to moult and fed as adults on two uninfected dogs. Both dogs became seropositive and experienced elevated body temperatures. Later, these dogs were splenectomised and treated with corticosteroids. After the treatment had been administered for 4 weeks, the dogs were infested with uninfected nymphs and adults of R. sanguineus. After detachment, cultures of tick gut were

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made and positive results were obtained from ticks fed on one of the dogs. Later on, lymph node samples from this dog were shown to be culture-positive for Leishmania protozoa. Based on these results, McKenzie (1984) concluded that R. sanguineus was able to transmit L. infantum to susceptible during normal feeding. More recently, a study reported the detection of L. infantum DNA in a pool of salivary glands of R. sanguineus collected from an infected dog in southern Italy (Dantas-Torres et al. 2010a).

Transmission of L. infantum by ingestion of infected ticks A recent study investigated the possibility of L. infantum transmission via ingestion of infected ticks (Coutinho et al. 2005). In this study, golden hamsters (Mesocricetus auratus) were fed with macerates of ticks collected from L. infantum-infected dogs. Remarkably, two out of 17 hamsters experimentally infected via this route were Leishmania-positive upon examination of liver and spleen stained smears. Furthermore, some hamsters became seropositive and/or PCR-positive. This study suggested that ingestion of infected ticks could represent a possible (yet to be proven) secondary way of L. infantum transmission between dogs.

Transstadial and transovarial passage of L. infantum in the tick The transstadial passage of L. infantum in the tick has been claimed by early researchers (Blanc & Caminopetros 1930, Mckenzie 1984). Remarkably, a recent study demonstrated the persistence of L. infantum DNA in unfed nymphs and adults that had been fed (as larvae and nymphs, respectively) on infected dogs (Paz et al. 2010). However, the presence of the parasites could not be confirmed either by microscopic examination or by parasite culture of tick macerates (Paz et al. 2010). Indeed, this put in doubt the validity of early studies and suggested that the transstadial passage of L. infantum in the tick needs to be confirmed.

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Early studies failed to demonstrate the occurrence of transovarial passage of L. infantum in R. sanguineus (Blanc & Caminopetros 1930, Mckenzie 1984). A recent experimental study using a highly sensible real-time PCR protocol demonstrated the presence of L. infantum DNA in larvae of R. sanguineus, near four months after the experimental infection of females (Dantas-Torres et al. 2010b).

Prevalence of Leishmania spp. infection in ticks in endemic areas The natural infection by Leishmania-like flagellates has long been reported in ticks (Machattie & Chadwick 1930, Sherlock 1964, Silva et al. 2007), but the morphological identification of trypanosomatids found in ticks is difficult and the true identity of the parasite remains doubtful. So far, no study has properly assessed the prevalence of Leishmania spp. infection in ticks in endemic areas. Indeed, published studies refer to ticks collected from a limited number of naturally infected dogs (Coutinho et al. 2005, Dantas-Torres et al. 2010a), which greatly increases the chance of finding a positive tick. As a consequence, the reported infection rates are usually higher than 10% (Coutinho et al. 2005, Dantas-Torres et al. 2010a), which is quite high if compared with infection rates in phlebotomine sand fly vectors. It is crucial to assess the actual prevalence of Leishmania infection in ticks in order to establish whether they could actually represent an important source of Leishmania infection for dogs.

Future research needs The hypothesis of ticks as vectors of Leishmania needs further research. Although there is no irrefutable proof that R. sanguineus transmit L. infantum between dogs, the available data suggest that this theory should not be neglected. A well-designed experimental study with uninfected R. sanguineus ticks and infected and naive dogs should be enough to answer definitely the question whether R. sanguineus is a vector of L. infantum. For obvious ethical and safety

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reasons, it is not an easy task to maintain a group of dogs infected by L. infantum, infest them with naive ticks, maintain fed ticks in the laboratory and, after their moulting, feed them on naive dogs. In Brazil, for example, animal ethics committees are trying to reduce the use of animals in research and to encourage the adoption of alternative methods (e.g., in vitro models). Indeed, the question whether R. sanguineus is a vector of L. infantum cannot be definitively answered unless a well-designed experimental study, with ticks and dogs, is carried out.

References Blanc G, Caminopetros J 1930. La transmission du Kala-Azar mditerranen par une tique: Rhipicephalus sanguineus. C R Acad Sci 191: 1162-1164. Coutinho MT, Bueno LL, Sterzik A, Fujiwara RT, Botelho JR, De Maria M, Genaro O, Linardi PM 2005. Participation of Rhipicephalus sanguineus (Acari: Ixodidae) in the epidemiology of canine visceral leishmaniasis. Vet Parasitol 128: 149-155. Dantas-Torres F 2009. Canine leishmaniosis in South America. Parasit Vectors 2 Suppl 1: S1. Dantas-Torres F, Lorusso V, Testini G, de Paiva-Cavalcanti M, Figueredo LA, Stanneck D, Mencke N, Brando-Filho SP, Alves LC, Otranto D 2010a. Detection of Leishmania infantum in Rhipicephalus sanguineus ticks from Brazil and Italy. Parasitol Res 106: 857-860. Dantas-Torres F, Martins TF, de Paiva-Cavalcanti M, Figueredo LA, Lima BS, Brando-Filho SP 2010b. Transovarial passage of Leishmania infantum kDNA in artificially infected Rhipicephalus sanguineus. Exp Parasitol 125: 184-185. Machattie C, Chadwick CR 1930. Notes on a trypanosome infection of the dog tick Rhipicephalus sanguineus in Iraq. Trans R Soc Trop Med Hyg 23: 417-420. McKenzie KK 1984. A study of the transmission of canine leishmaniasis by the tick, Rhipicephalus sanguineus, and an ultrastructural comparison of the promastigote. PhD Dissertation, Oklahoma State University. Otranto D, Dantas-Torres F 2010. Fleas and ticks as vectors of Leishmania spp. to dogs: caution is needed. Vet Parasitol 168: 173-174. Paz GF, Ribeiro MF, Michalsky EM, da Rocha Lima AC, Frana-Silva JC, Barata RA, Fortes-Dias CL, Dias ES 2010. Evaluation of the vectorial capacity of Rhipicephalus sanguineus (Acari: Ixodidae) in the transmission of canine visceral leishmaniasis. Parasitol Res 106: 523-528. Sherlock I 1964. Notas sbre a transmisso da leishmaniose visceral no Brasil. Rev Bras Malariol D Trop 16: 19-26.

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Silva OA, Silva PB, Silva OV, Braga GM, Albuquerque Jnior A, Queiros Neto V, Rocha ME, Silva EF 2007. Canine visceral leishmaniasis in northeast Brazil: epidemiological aspects. Bull Soc Pathol Exot 100: 49-50.

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CARACTERIZAO DE MECANISMOS DE RESISTNCIA DE MOSQUITOS A LARVICIDAS BIOLGICOS Silva Filha, M.H.N.L. Depto. de Entomologia, CPqAM-FIOCRUZ

Biolarvicidas a base das bactrias entomopatgenas Bacillus thuringiensis sorovar. israelensis (Bti) e Bacillus sphaericus tm sido produzidos e utilizados em larga escala em programas de controle de vetores (Lacey, 2007). Estes biolarvicidas possuem ao efetiva e seletiva para culicdeos e simuldeos, sendo incuos para a fauna no-alvo (Merritt et al., 2005; Lacey, 2007). A atividade inseticida destes agentes devido produo de um cristal protico, durante a esporulao bacteriana, que contm protoxinas com ao larvicida mediante a ingesto. O modo de ao dos cristais composto pelas seguintes etapas: ingesto pelas larvas, solubilizao do cristal em pH intestinal alcalino, liberao das protoxinas e ativao para a forma de toxinas pela ao de serina proteases, e finalmente ligao das toxinas a receptores especficos localizados no microvilli do epitlio intestinal. Aps a ligao aos receptores, as toxinas desencadeiam efeitos citopatlogicos que culminam com a morte das larvas (Lacey, 2007). O Bti apresenta um espectro larvicida para alguns gneros de dpteros tais como Simulium, Aedes, Culex e Anopheles e tem sido usado desde a dcada de 80, sobretudo para o controle de larvas de Simulium e Aedes (Regis et al., 2001). O cristal do Bti composto pelas toxinas Cry4Aa (125 kDa), Cry4Ba (135 kDa), Cry11Aa (68 kDa) e Cyt1Aa (28 kDa) codificadas por genes localizados em um megaplasmdio (Berry et al., 2002). As toxinas do Bti agem em sinergia e nenhuma delas, individualmente ou em combinaes, apresenta uma toxicidade to elevada quanto quela do cristal nativo (Georghiou and Wirth, 1997). As toxinas Cry so constitudas por trs domnios e os domnios II e III so responsveis pela ligao com os receptores especficos, enquanto que a toxina Cyt tem ao citoltica e capacidade de inserir-se diretamente na membrana das clulas, sem necessitar interagir com receptores (Bravo et al., 2007). Algumas molculas que servem como receptores de toxinas de Bti tm sido identificadas. A toxina Cry11Aa pode ligar-se no epitlio intestinal de larvas de Ae. aegypti a diferentes receptores j descritos, como uma fosfatase alcalina (65 kDa), uma

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aminopeptidase (140 kDa) e a uma caderina (250 kDa), enquanto que liga-se a uma ? amilase (70 kDa) em An.albimanus (Fernandez et al., 2006; Chen et al., 2009a; Chen et al., 2009b; Luna et al., 2010). A toxina a Cry4B apresenta ligao a uma caderina (200 kDa) em An.gambiae e a uma ? -amilase de (70 kDa) em An. albimanus e (Hua et al., 2008; FernandezLuna et al., 2010). Foi demonstrado que a toxina Cyt1A pode funcionar como receptor das toxinas Cry, devido a sua capacidade de ligar-se a elas e de se inserir nas membranas das clulas e, este achado elucidou como ocorre a sinergia entre as toxinas do Bti (Perez et al., 2005). A presena de quatro toxinas no cristal do Bti aliada complexidade do seu modo de ao desfavorece a seleo de resistncia pelo menos, em relao a mecanismos que envolvem a alterao dos receptores. O Bti vem sendo utilizado h mais de 25 anos e no h relatos consistentes de resistncia a este agente (Regis et al., 2001). Apesar destas caractersticas, a seleo de resistncia ao Bti deve continuar a ser investigada visto que outros mecanismos tais como alteraes proteolticas, que inviabilizam o processamento das protoxinas em toxinas, j foram reportados em insetos praga resistentes s toxinas Cry1 do Bt (Oppert et al., 1997). Embora pouco investigado a resistncia atravs de enzimas detoxificadoras tambm reconhecida como um importante mecanismo de resistncia a inseticidas de qumicos de sntese e foi recentemente reportada para toxinas do B. thuringiensis (Gunning et al., 2005; Boyer et al., 2007). O B. sphaericus possui trs classes de protenas inseticidas e a mais importante delas, que o princpio ativo dos biolarvicidas comerciais, a protoxina binria (Bin). Esta protoxina, produzida sob a forma de cristal aps a esporulao, um heterodmero, formado pelas sub-unidades BinA (42 kDa) e BinB (51 kDa), cujos genes esto localizados no cromossomo bacteriano (Charles et al., 1996). O seu espectro de ao mais limitado do que o Bti e os principais alvos so larvas dos gneros Culex e Anopheles. Aps a ingesto pelas larvas e solubilizao, as subunidades da protoxina Bin so clivadas no lmen intestinal por serina-proteases em fragmentos ativos, os que agem em sinergia sendo BinB responsvel pelo reconhecimento dos receptores e BinA pela toxicidade para as clulas (Nicolas et al., 1990; Nicolas et al., 1993). Os receptores da toxina Bin em espcies alvo como C. pipiens, C.

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quinquefasciatus e An. gambie, so ? -glicosidases (66 kDa) de membrana e foram denominados respectivamente Cpm1, Cqm1 e Agm3 (Silva-Filha et al., 1999; Darboux et al., 2001; Romo et al., 2006; Opota et al., 2008). O modo de ao do B. sphaericus baseado na ao de uma toxina que interage como um nico receptor favorece a seleo da resistncia mediante a exposio exclusiva e prolongada a este agente. A resistncia j foi registrada sob condies de laboratrio e de campo e o principal mecanismo a ausncia de receptores no epitlio intestinal que acarreta a falha de ligao da toxina Bin (Nielsen-LeRoux et al., 2002; Oliveira et al., 2004). O estudo molecular da resistncia em algumas destas colnias resultou na caracterizao de alelos com mutaes que codificam ? -glicosidases desprovidas de ncora GPI (cpm1GEO, cqm1REC, cpm1BP) ou de regies de aminocidos (cpm1BP_DEL), que as impedem de funcionar como receptor da toxina Bin (Darboux et al., 2002; Romo et al., 2006; Darboux et al., 2007). Todos os casos de resistncia estudados at o presente, mostram que este carter herdado de forma recessiva, portanto, o desenvolvimento de mtodos moleculares para a sua deteco de alelos de resistncia fundamental visto que a avaliao da suscetibilidade das populaes atravs de bioensaios no capaz de detectar indivduos heterozigotos (NielsenLeRoux et al., 2002; Amorim et al., 2007; Amorim et al., 2010). O desenvolvimento de um mtodo de PCR especfico para o alelo de resistncia cqm1REC demonstrou a sua presena em populaes naturais de C. quinquefasciatus da cidade do Recife, e a sua freqncia foi mais elevada em uma populao tratada com o B. sphaericus (Chalegre et al., 2009). A presena do cqm1REC em uma freqncia detectvel em populaes sem histrico de exposio ao biolarvicida, demonstra que este alelo no raro e que estratgias de diagnstico e de manejo de resistncia so necessrias para evitar a sua seleo. A avaliao do desempenho biolgico de duas colnias resistentes associada ao alelo cqm1REC mostrou que custo biolgico discreto e no trouxe conseqncias para a manuteno das colnias sob condies de laboratrio por perodos superiores a 50 geraes (Oliveira et al., 2003; Amorim et al., 2010). A estabilidade da resistncia, avaliada em uma prognie resultante do cruzamento de indivduos sensveis e resistentes, mostrou que a freqncia de indivduos resistentes e do alelo cqm1REC manteve-se estvel por 11 geraes,

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na ausncia da presso de seleo (Amorim et al., 2010). O alelo cqm1REC difere de alelos de resistncia que so raros e que esto frequentemente associados ao um elevado custo biolgico. As caractersticas do cqm1REC so compatveis com quelas de mutaes que so consideradas polimorfismos balanceados, visto que a seleo contra e a favor so similares alm de no estarem associados a custo biolgico, fatores que possibilitam sua manuteno na populao na ausncia de presso seletiva (Ffrench-Constant, 2007). Os dados relativos estabilidade da resistncia conferida pelo alelo cqm1REC apontam a importncia do seu diagnstico, pois, eles podem ser mantidos na populao, mesmo na interrupo de presso de seleo, se estratgias para a reduo de sua freqncia no forem introduzidas. O Bti tem uma grande importncia neste contexto pois ele txico para larvas de Culex resistentes toxina Bin, no h resistncia cruzada com as toxinas Cry/Cyt, e por esta razo recomendado para eliminar indivduos que portam alelos de resistncia ao B. sphaericus (Pei et al., 2002; Wirth et al., 2010) A seleo de resistncia um fenmeno inevitvel em vista da grande capacidade adaptativa dos insetos, portanto, essencial focalizar estudos na caracterizao de alelos que possam subsidiar a aplicao de estratgias de manejo de resistncia para garantir o uso sustentvel destas molculas inseticidas. A utilizao de biolarvicidas, em particular de bactrias entomopatgenas, representa um avano tecnolgico considervel e alternativa eficaz e segura para ser aplicada em campanhas de controle de vetores. Referncias
Amorim, L.B., de Barros, R.A., Chalegre, K.D., de Oliveira, C.M., Regis, L.N., Silva-Filha, M.H., 2010. Stability of Culex quinquefasciatus resistance to Bacillus sphaericus evaluated by molecular tools. Insect Biochem Mol Biol 40, 311-316. Amorim, L.B., Oliveira, C.M.F., Rios, E.M., Regis, L., Silva-Filha, M.H.N.L., 2007. Development of Culex quinquefasciatus resistance to Bacillus sphaericus strain IAB59 needs long term selection pressure. Biological Control 42, 155-160. Berry, C., et al., 2002. Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68, 5082-5095. Boyer, S., Tilquin, M., Ravanel, P., 2007. Differential sensitivity to Bacillus thuringiensis var. israelensis and temephos in field mosquito populations of Ochlerotatus cataphylla (Diptera: Culicidae): toward resistance? Environ Toxicol Chem 26, 157-162. Bravo, A., Gill, S.S., Soberon, M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49, 423-435. Chalegre, K.D., Romao, T.P., Amorim, L.B., Anastacio, D.B., de Barros, R.A., de Oliveira, C.M., Regis, L., de-Melo-Neto, O.P., Silva-Filha, M.H., 2009. Detection of an allele conferring resistance to Bacillus sphaericus binary toxin in Culex quinquefasciatus populations by molecular screening. Appl. Environ. Microbiol. 75, 1044-1049.

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