Estudo populacional de Anopheles darlingi utilizando análise de RAPD e seqüenciamento de genes mitocondriais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determinação da infecção em Anopheles por diagnóstico molecular

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estrutura populacional de Anopheles darlingi em diferentes localidades de Rondônia ao longo do Rio Madeira através da genotipagem de microssatélites

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identificação molecular de espécies do complexo Anopheles albitarsis (Diptera, Culicidae, Anophelinae), coletadas em dois municípios da Amazônia brasileira, com análise de suscetibilidade natural a plasmódios humanos

Anofelinos membros de complexos de espécies crípticas podem exibir diferenças comportamentais, de susceptibilidade a infecção malárica, e resistência a inseticidas. Assim, a identificação de espécies vetoras tem relevância epidemiológica, o que nem sempre é possível por critérios morfológicos. Métodos alternativos têm sido empregados para tal, como os que analisam regiões altamente conservadas do DNA ribossômico, variável entre as espécies, conhecidas como espaçadoras internas transcritas (ITS). Considera-se atualmente que o complexo Anopheles c seja composto por seis espécies: An. albitarsis s.s., An. oryzalimnetes, An. albitarsis F, An. marajoara, An. deaneorum, e An. janconnae. Destas, pelo menos as três últimas são incriminadas como vetores de malária na Amazônia brasileira. O objetivo deste estudo foi realizar identificação molecular de espécies do complexo An. albitarsis, por análise da seqüências do ITS2 do rDNA, com vistas a analisar sua importância na transmissão de malária nos municípios de Macapá, Amapá e Peixe-Boi, Pará, inclusive investigando pela primeira vez a ocorrência do An. albitarsis F nestas duas áreas epidemiologicamente distintas: a primeira com histórico de alto risco de transmissão de malária e a segunda não. O estudo foi realizado entre janeiro de 2009 e abril de 2010, e consistiu de capturas de anofelinos de 12 horas de duração (ecostofase) no peridomicílio. Todas as fêmeas coletadas foram morfologicamente identificadas e apenas os An. albitarsis s.l. tiveram cabeça e tórax separadas para análise da infecção natural por ELISA; ovários para análise de paridade e patas, asas e carcaça para identificação molecular. Em Macapá foram realizadas seis coletas, obtendo-se um total de 584 anofelinos, sendo 366 An. albitarsis s.l. (62,7%), 167 An. darlingi (28,6%), 33 An. triannulatus s.l (5,6%), 15 An. braziliensis (2,6%) e 3 An. nuneztovari (0,5%). Pela PCR foi possível visualizar a banda específica de An. marajoara em 320 espécimes dos An. albitarsis s.l testados. Do restante, 33 foram negativos e 13 amplificaram um fragmento de ~490 pb nos iniciadores empregados, não permitindo chegar ao diagnóstico específico. O An. marajoara apresentou características biológicas e comportamentais que ratificam sua importância epidemiológica na transmissão de malária em Macapá, tais como: ser a espécie mais prevalente, com maior proporção de fêmeas paridas (73,0%), e portanto com maiores chances de se infectarem com o plasmódio, ocorrer tanto na estação menos quanto na mais chuvosa, e apresentar atividade hematofágica durante toda a ecostofase, alem disso, foi encontrado naturalmente infectado por P. vivax e P. falciparum (taxa de infecção natural de 3,1%). Em Peixe-Boi, foram capturados 43 anofelinos: An. triannulatus s.l (20 espécimes, 46,5 %), An. albitarsis s.l. (13: 30,2 %), An. darlingi (8: 18,6%), e An. nuneztovari (2: 4,7%). Todos os An. albitarsis s.l. coletados foram identificados pela ITS2 como An. oryzalimnetes. Nenhum deles foi encontrado infectado pelos plasmódios testados, e a maioria das fêmeas era parida (84,6%). São necessários levantamentos entomológicos sistemáticos que analisem a importância deste anofelino na transmissão de malária na cidade. O An. albitarsis F não foi encontrado nas duas áreas estudadas. Nossos resultados contribuem para o entendimento da epidemiologia da malária na região Amazônica brasileira.

A importância do Anopheles darlingi Root, 1926 e Anopheles marajoara Galvão e Damasceno, 1942 na transmissão de malária no município de Macapá/AP - Brasil

A malária no município de Macapá é principalmente peri urbana, áreas estas caracterizadas por ressaca, pela presença de fragmentos de floresta e assentamentos desordenados (invasões). O objetivo deste estudo foi verificar a importância dos Anopheles darlingi e Anopheles marajoara na transmissão de malária em Macapá. O estudo foi realizado de outubro de 2007 a setembro de 2008, na comunidade de Lagoa dos Índios, Macapá. Foram coletados 4.601 mosquitos, dos quais 3.029 foram Anopheles marajoara (65,8%), 917 Anopheles darlingi (19,9%), 429 Anopheles braziliensis (9,3%), 208 Anopheles triannulatus (4,5%), 18 Anopheles peryassui (0,4%) e cinco Anopheles nuneztovari (0,1%). Apenas 32,8% dos espécimes foram coletadas no intradomicilio (1.511) e 67,2% no peridomicilio (3.090). O Indice de Picada Homem Hora do An. darlingi no intradomicilio variou entre 0 a 6,5 e no peridomicilio de 0 a 22 picadas homem hora. Já para o An. marajoara a variaçao foi de 0 a 22 no intradomicilio e de 0 a 175,5 no peridomicilio. A analise das exúvias e da dissecção de genitália resultaram na confirmaçao das duas espécies estudadas, An. darlingi e An. marajoara, e que o An. marajoara é a única espécie do complexo albitarsis circulante na área. A abundância dos vetores flutuou associada com o padrão sazonal das chuvas. An. darlingi é mais abundante no final e inicio das chuvas, enquanto o An. marajoara esteve presente em alta densidade durante todo o período de chuvas. Dos 4.601 mosquitos testados, 100 foram positivos para plasmódios humanos pelo método de ELISA, resultando em uma taxa de infecção de 2,17%. Dos 3.029 An. marajoara testados 71 (2,34%) foram positivos e dos 917 An. darlingi, 28 (3,05%). Este estudo demonstrou que as duas espécies estudadas mantêm a transmissão de malária durante todo o ano, ratificando assim a importância das mesmas.

Susceptibilidade experimental do Anopheles (Nyssorhynchus) nuneztovari Galbadon, 1940 ao Plasmodium vivax Grassi&Feletti, 1890 e Plasmodium falciparum Welch, 1897

A importância do An. nuneztovari como vetor primário de malária já foi comprovado em países da América do Sul como Venezuela, Colômbia e Peru. Na Amazônia brasileira, embora tenha sido encontrado naturalmente infectado com Plasmodium vivax e P. falciparum e em alta densidade, é ainda considerado vetor secundário desta doença. O objetivo deste presente trabalho foi avaliar a susceptibilidade do An. nuneztovari à infecção por plasmódios humanos. Para isso exemplares da geração F1, obtida em laboratório, de An. nuneztovari e An. darlingi (espécie controle) foram alimentados, em alimentador artificial, com sangue de pacientes com diagnóstico inicial de malária causada por P. falciparum, cuja revisão resultou no diagnóstico de infecção mista. Todas as amostras sangüíneas dos pacientes infectaram espécimes das duas espécies, não mostrando diferença significativa entre elas quanto à susceptibilidade. Para detecção de infecção malárica nos mosquitos foi usado o teste ELISA (Enzime – Linked Imunosorbent Assay) cujos resultados foram discordantes do diagnóstico laboratorial, já que o teste detectou infecções pelo P. falciparum, P. vivax VK210 ou P. vivax VK247entre os mosquitos positivos sugerindo que os pacientes apresentavam infecção mista. Também foi observado o curto período de desenvolvimento de oocistos e esporozoítos, de quatro a cinco dias, o que pode ser explicado pela alta temperatura (>30°C) que os mosquitos foram expostos. Assim nossos resultados sugerem possível envolvimento do An nuneztovari na transmissão de malária humana na área estudada e alertam para o papel deste, como possível vetor principal de malária humana na região amazônica brasileira.

Development of the BG-Malaria trap as an alternative to human-landing catches for the capture of Anopheles darlingi

Although the human-landing catch (HLC) method is the most effective for collecting anthropophilic anophelines, it has been increasingly abandoned, primarily for ethical considerations. The objective of the present study was to develop a new trap for the collection of Anopheles darlingi . The initial trials were conducted using the BG-Sentinel trap as a standard for further trap development based on colour, airflow direction and illumination. The performance of the trap was then compared with those of the CDC, Fay-Prince, counterflow geometry trap (CFG) and HLC. All trials were conducted outdoors between 06:00 pm-08:00 pm. Female specimens of An. darlingi were dissected to determine their parity. A total of 8,334 anophelines were captured, of which 4,945 were identified as An. darlingi . The best trap configuration was an all-white version, with an upward airflow and no required light source. This configuration was subsequently named BG-Malaria (BGM). The BGM captured significantly more anophelines than any of the other traps tested and was similar to HLC with respect to the number and parity of anophelines. The BGM trap can be used as an alternative to HLC for collecting anophelines.

Susceptibility of Anopheles aquasalis and An. darlingi to Plasmodium vivax VK210 and VK247

The susceptibility of Anopheles aquasalis (F₃ generation) and An. darlingi (F₁ generation) to Plasmodium vivax circumsporozoite protein phenotypes from a limited number of blood samples of malaria patients in Belém, state of Pará, Brazil, was examined. A polymerase chain reaction was used to determine the P. vivax phenotypes in blood samples and the blood-fed infected mosquitoes were dissected and tested by ELISA. In all patient infections, more infected An. aquasalis and An. darlingi were positive for VK210 compared with VK247.

Seasonal genetic partitioning in the neotropical malaria vector, Anopheles darlingi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

NEW RECORDS OF ANOPHELES HOMUNCULUS IN CENTRAL AND SERRA DO MAR BIODIVERSITY CORRIDORS OF THE ATLANTIC FOREST, BRAZIL

Two new records of Anopheles homunculus in the eastern part of the Atlantic Forest are reported. This species was found for the first time in Barra do Ouro district, Maquine municipality, Rio Grande do Sul state, located in the southern limit of the Atlantic Forest. The 2nd new record was in the Serra Bonita Reserve, Camacan municipality, southeast Bahia state. These records extend the geographical distribution of An. homunculus, suggesting that the species may be widely distributed in coastal areas of the Atlantic Forest. It is hypothesized that the disjunct distribution of the species may be caused by inadequate sampling, and also difficulties in species identification based only on female external characteristics. Species identification was based on morphological characters of the male, larva, and pupa, and corroborated by DNA sequence analyses, employing data from both 2nd internal transcribed spacer of nuclear ribosomal DNA and of mitochondrial cytochrome c oxidase subunit I.

Wing geometry of Anopheles darlingi Root (Diptera: Culicidae) in five major Brazilian ecoregions

We undertook geometric morphometric analysis of wing venation to assess this character's ability to distinguish Anopheles darlingi Root populations and to test the hypothesis that populations from coastal areas of the Brazilian Atlantic Forest differ from those of the interior Atlantic Forest, Cerrado, and the regions South and North of the Amazon River. Results suggest that populations from the coastal and interior Atlantic Forest are more similar to each other than to any of the other regional populations. Notably, the Cerrado population was more similar to that from north of the Amazon River than to that collected of south of the River. thus showing no correlation with geographical distances. We hypothesize that environmental and ecological factors may affect wing evolution in An. darlingi. Although it is premature to associate environmental and ecological determinants with wing features and evolution of the species, investigations on this field are promising. (C) 2012 Elsevier B.V. All rights reserved.

BRAZILIAN MOSQUITO (DIPTERA: CULICIDAE) FAUNA. I. Anopheles SPECIES FROM PORTO VELHO, RONDONIA STATE, WESTERN AMAZON, BRAZIL

This study contributes to knowledge of Anopheles species, including vectors of Plasmodium from the western Brazilian Amazon in Porto Velho, Rondonia State. The sampling area has undergone substantial environmental changes as a consequence of agricultural and hydroelectric projects, which have caused intensive deforestation and favored habitats for some mosquito species. The purpose of this study was to diagnose the occurrence of anopheline species from collections in three locations along an electric-power transmission line. Each locality was sampled three times from 2010 to 2011. The principal adult mosquitoes captured in Shannon trap were Anopheles darlingi, An. triannulatus, An. nuneztovari l.s., An. gilesi and An. costai. In addition, larvae were collected in ground breeding sites for Anopheles braziliensis, An. triannulatus, An. darlingi, An. deaneorum, An. marajoara, An. peryassui, An. nuneztovari l.s. and An. oswaldoi-konderi. Anopheles darlingi was the most common mosquito in the region. We discuss Culicidae systematics, fauna distribution, and aspects of malaria in altered habitats of the western Amazon.

Morphometrical diagnosis of the malaria vectors Anopheles cruzii, An. homunculus and An. bellator

Background: Anopheles (Kerteszia) cruzii is a primary vector of Plasmodium parasites in Brazil's Atlantic Forest. Adult females of An. cruzii and An. homunculus, which is a secondary malaria vector, are morphologically similar and difficult to distinguish when using external morphological characteristics only. These two species may occur syntopically with An. bellator, which is also a potential vector of Plasmodium species and is morphologically similar to An. cruzii and An. homunculus. Identification of these species based on female specimens is often jeopardised by polymorphisms, overlapping morphological characteristics and damage caused to specimens during collection. Wing geometric morphometrics has been used to distinguish several insect species; however, this economical and powerful tool has not been applied to Kerteszia species. Our objective was to assess wing geometry to distinguish An. cruzii, An. homunculus and An. bellator. Methods: Specimens were collected in an area in the Serra do Mar hotspot biodiversity corridor of the Atlantic Forest biome (Cananeia municipality, State of Sao Paulo, Brazil). The right wings of females of An. cruzii (n= 40), An. homunculus (n= 50) and An. bellator (n= 27) were photographed. For each individual, 18 wing landmarks were subjected to standard geometric morphometrics. Discriminant analysis of Procrustean coordinates was performed to quantify wing shape variation. Results: Individuals clustered into three distinct groups according to species with a slight overlap between representatives of An. cruzii and An. homunculus. The Mahalanobis distance between An. cruzii and An. homunculus was consistently lower (3.50) than that between An. cruzii and An. bellator (4.58) or An. homunculus and An. bellator (4.32). Pairwise cross-validated reclassification showed that geometric morphometrics is an effective analytical method to distinguish between An. bellator, An. cruzii and An. homunculus with a reliability rate varying between 78-88%. Shape analysis revealed that the wings of An. homunculus are narrower than those of An. cruzii and that An. bellator is different from both of the congeneric species. Conclusion: It is possible to distinguish among the vectors An. cruzii, An. homunculus and An. bellator based on female wing characteristics.

Kerteszia subgenus of Anopheles associated with the Brazilian Atlantic rainforest:current knowledge and future challenges

Abstract Background The Atlantic rainforest ecosystem, where bromeliads are abundant, provides an excellent environment for Kerteszia species, because these anophelines use the axils of those plants as larval habitat. Anopheles (K.) cruzii and Anopheles (K.) bellator are considered the primary vectors of malaria in the Atlantic forest. Although the incidence of malaria has declined in some areas of the Atlantic forest, autochthonous cases are still registered every year, with Anopheles cruzii being considered to be a primary vector of both human and simian Plasmodium. Methods Recent publications that addressed ecological aspects that are important for understanding the involvement of Kerteszia species in the epidemiology of malaria in the Atlantic rainforest in the Neotropical Region were analysed. Conclusion The current state of knowledge about Kerteszia species in relation to the Atlantic rainforest ecosystem was discussed. Emphasis was placed on ecological characteristics related to epidemiological aspects of this group of mosquitoes. The main objective was to investigate biological aspects of the species that should be given priority in future studies.

A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

Abstract Background The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup. Methods Larvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches. Results Bayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach. Conclusions Our study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.