Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T14:01:20Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_IdentificacaoMolecularEspecies.pdf: 3577435 bytes, checksum: 1ec06b9895715025d7c02ddfc1768072 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-12T13:49:08Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_IdentificacaoMolecularEspecies.pdf: 3577435 bytes, checksum: 1ec06b9895715025d7c02ddfc1768072 (MD5) / Made available in DSpace on 2014-02-12T13:49:08Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Tese_IdentificacaoMolecularEspecies.pdf: 3577435 bytes, checksum: 1ec06b9895715025d7c02ddfc1768072 (MD5) Previous issue date: 2010 / 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. / Members of Anopheles cryptic species complexes may exhibit behavioral differences among them, the susceptibility to malaria infection, and resistance to insecticides, thus, the specific identification has epidemiological relevance, which is not always possible by morphological criteria. Alternative methods have been employed such as those that analyze highly conserved regions of ribosomal DNA, which varies among species, known as internal transcribed spacer (ITS). Anopheles albitarsis complex is currently composed of six species: An. albitarsis s.s., An. oryzalimnetes, An. albitarsis F, An. marajoara, An. deaneorum and An. janconnae. At least the last three are incriminated as malaria parasite vectors in the Brazilian Amazon. The aim of this study was to perform molecular identification of An. albitarsis complex species, for analysis of ITS2 rDNA sequences, in order to assess their importance in malaria transmission in the cities of Macapá, Amapa and Peixe-Boi, Para, including investigating the first time the occurrence of An. Albitarsis F in two epidemiologically distinct areas: the first with a history of high risk of malaria transmission and the second not (Peixe-Boi). All-night human landing catches of mosquitoes were carried out in the peridomestic environment, from January 2009 and April 2010. The females collected were morphologically identified and only An. albitarsis s.l. was dissected: head and thorax were separated for analysis of natural infection by ELISA; ovaries for parity analysis and the carcass, legs and wings for molecular identification. In Macapá were collected 584 mosquitoes: 366 An. albitarsis s.l. (62.7%), 167 An. darlingi (28.6%), 33 An. triannulatus s.l. (5.6%), 15 An. braziliensis (2.6%) and 3 An. nuneztovari (0.5%). It was possible to visualize the specific band An. marajoara in 320 specimens of An. albitarsis s.l. tested. Of the remainder, 33 were negative and 13 amplified a fragment of ~ 490pb, not allowing to reach the specific diagnosis. The An. marajoara specimens showed biological and behavioral characteristics that confirm their epidemiological importance in malaria transmission in Macapa, such as being the most prevalent species, with a higher proportion of parous females (73.0%), and therefore with the greatest chances of infected with Plasmodium, occur in both seasons (dry and wet), presenting biting activity in all hours worked, moreover, was found infected by P.vivax and P. falciparum (natural infection rate of 3.1%). In Peixe-Boi, were captured 43 anophelines: An. triannulatus s.l. (20 specimens, 46.5%), An. albitarsis s.l. (13, 30.2%) An. darlingi (8, 18.6%), and An. nuneztovari (2, 4.7%). All An. albitarsis s.l. collected were identified by ITS2 as An. oryzalimnetes. None was found infected by Plasmodium, and the vast majority was parous females (84.6%). Systematic entomological surveys are needed to analyse the importance of An. oryzalimnetes in malaria transmission in the city. The An. albitarsis F was not found in the two areas studied. Our results contribute to our understanding of the epidemiology of malaria in the Brazilian Amazon region.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-13T12:58:00Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao_SusceptibilidadeExperimentalAnopheles.pdf: 428229 bytes, checksum: abd7aec5037cb5ccb35ee10d8bd4e6ee (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-22T17:03:57Z (GMT) No. of bitstreams: 2 Dissertacao_SusceptibilidadeExperimentalAnopheles.pdf: 428229 bytes, checksum: abd7aec5037cb5ccb35ee10d8bd4e6ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-04-22T17:03:58Z (GMT). No. of bitstreams: 2 Dissertacao_SusceptibilidadeExperimentalAnopheles.pdf: 428229 bytes, checksum: abd7aec5037cb5ccb35ee10d8bd4e6ee (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2006 / 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. / The importance of the An. nuneztovari as malaria primary vector had already been confirmed in different South America countries such as Venezuela, Colombia and Peru. In the Brazilian Amazonian, although this specie had been found naturally infected by Plasmodium vivax and P. falciparum and in high densities, it is considered a secondary vector of this disease. The aim of this study was to evaluate the susceptibility of the An. nuneztovari to the infection by human Plasmodium species. For that, mosquitoes specimens of F1 progeny, obtained under laboratory conditions, of the An. nuneztovari and An. darlingi (control) were fed on an artificial feeding apparatus, using human blood that had been found carrying the malaria parasite, P. falciparum, whose revision had resulted as mixed infection. All blood samples from patients had infected specimens of both mosquitoes species tested, and no significant statistical difference was found related to the susceptibility. For the detection malaria infection in mosquitoes specimens it was conducted the ELISA (Enzime – Linked Imunosorbent Assay) test, whose results were divergent from the diagnostic one, since it has identified mosquitoes specimens infected by P. falciparum, P. vivax VK210 and/or P. vivax VK247, which suggests that the donors patients had mixed infection. It was also observed the short period of development of the oocysts and sporozoites, from four to five days, fact that can be explained by the high temperature (>300C) that the mosquitoes were exposed. Thus, our results suggest the probable enrollment of the An. nuneztovari in the human malaria transmission in the study area and point out the role of this mosquito specie as human malaria primary vector in the Brazilian Amazon region.

Resultados anteriores sugerem que existem diferenças biológicas entre espécimens de Anopheles oswaldoi capturados no Estado do Acre e os do Estado de Rondônia, Brasil. Esta espécie tem sido apontada como um importante vetor de malária em localidades do Peru e Acre. Entretanto, em Rondônia, somente um número pequeno de A. oswaldoi alimentados em pacientes com malária desenvolveram infecção nas glândulas salivares. Além disso, há suspeita de que espécimens identificados como A. oswaldoi, capturados em áreas abertas em Costa Marques, Rondônia, são na verdade A. konderi, e que A. oswaldoi sensu stricto estaria restrito a áreas de florestas. Estes dados, juntamente com as dificuldades encontradas na identificação de anofelinos do grupo Oswaldoi, baseadas em critérios morfológicos, sugerem que espécimens de A. oswaldoi são membros de um complexo de espécies crípticas. A distinção de espécies crípticas de insetos vetores é de grande importância, já que diferentes membros em um complexo podem exibir diferenças na ecologia, capacidade vetorial e resposta a medidas de controle. Análise de sequências de DNA, particularmente da região do ITS2 do cistron do DNA ribossômico, tem sido usada como um caracter diagnóstico em alguns grupos de espécies crípticas, tornando-se um instrumento para estudos taxonômicos e filogenéticos. A primeira parte deste estudo teve o objetivo de determinar as diferenças encontradas nas sequências de ITS2 de espécimens de A. oswaldoi capturados em várias localidades da América do Sul. As regiões dos ITS2 destes anofelinos foram amplificadas usando oligonucleotídeos iniciadores conservados das regiões 5.8S e 28S e os produtos de PCR foram clonados e sequenciados. Os ITS2 de todos os mosquitos capturados tiveram tamanho aproximado de 350 nucleotídeos, com aproximadamente 53% de conteúdo de GC. Análise do alinhamento destas sequências, mostrou similaridade variando entre 87% e 100%, e a análise de uma árvore de similaridade, neighbor-joining, produzida com p-distance usando as diferenças nas sequências de ITS2, separou estes espécimens em quatro grupos. Um deles está provavelmente relacionado ao A. oswaldoi sensu stricto, e um outro pode estar relacionado à espécie A. konderi. Os outros dois grupos podem corresponder a espécies cuja identificação morfológica permanece para ser esclarecida no complexo A. oswaldoi. Estes dados são evidências de que espécimens de A. oswaldoi estão incluídos em um complexo de espécies crípticas e que identificação por DNA pode resolver estas questões taxonômicas. A. konderi tem sido considerado uma sinonímia de A. oswaldoi. Embora estágios adultos e imaturos destas espécies de anofelinos apresentem características morfológicas idênticas, aspectos encontrados na genitália masculina podem distinguir estes taxa. A segunda parte deste estudo foi conduzida com o objetivo de comparar a susceptibilidade de A. oswaldoi s.s. e de A. konderi à infecção com Plasmodium vivax. A susceptibilidade foi baseada na proporção de mosquitos com oocistos e esporozoítos. Os anofelinos foram capturados no Acre e em Rondônia para obtenção de progênies F1. Após a emergência dos adultos, as genitálias masculinas dos mosquitos de cada progênie foram dissecadas e examinadas. Todas progênies originadas dos mosquitos capturados em Rondônia corresponderam a A. konderi, enquanto que cerca de 85,0% das progênies do Acre eram A. oswaldoi s.s.. Estas progênies F1 de A. oswaldoi s.s., A. konderi e A. darlingi foram alimentadas simultaneamente com sangue infectado com P. vivax. Estes mosquitos foram dissecados 10-12 dias após infecção e examinados para verificação da presença de oocistos e esporozoítos. Tanto A. oswaldoi s.s. como A. konderi apresentaram oocistos nos tratos digestivos, entretanto, a porcentagem de tratos digestivos positivos para oocistos foi maior em A. oswaldoi s.s. (13,8%) do que em A. konderi (3,3%). Esporozoítos foram encontrados somente nas glândulas salivares de A. oswaldoi s.s., com 6,9% de positividade. As taxas de infecção nos controles A. darlingi foram de 22,5% a 30,0%, para ambos oocistos e esporozoítos. Estes resultados indicam que A. oswaldoi s.s. pode transmitir P. vivax e sugerem que esta espécie é mais susceptível que A. konderi. Embora A. oswaldoi s.s. seja uma espécie exofílica e zoofílica, este anofelino pode estar envolvido na transmissão da malária humana como parece estar ocorrendo no Estado do Acre. / Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

Malaria occurs mostly in the tropical regions of the world. Sub-Saharan Africa is the area most affected. The occurrence of a very efficient mosquito vectors Anopheles gambiae complex and Anopheles funestus group sustain high transmission of the Plasmodium falciparum, the most predominant and deadly malaria parasite species. Local weather conditions are appropriate and often transmission occurs throughout the year. Limited resources and socio-economic instability constitute the major factors impeding efficient malaria control activities. The worldwide malaria eradication programme carried out during the 1950's focused mainly on insecticide residual spraying with DDT, anti-malarial drug treatment, and surveillance.Regional eradication of the disease was achieved, nevertheless, in many endemic regions of sub-Saharan Africa excluded from the eradication campaign, the disease is still afflicting their inhabitants. The malaria disease burden estimation in tropical Africa relies on mortality and morbidity data collected by the health system information. Conservative estimates of the burden of disease claim for more than 300 million clinical episodes and 1 - 3 million deaths every year and young children harbour the largest and most important portion of this bulk.Currently, the situation is deteriorating, increasing malaria-related morbidity and mortality have been reported. The rapid development and widespread of parasites strains resistant to almost all anti-malarial drugs, and vector resistance are the major contributing factors.In addition, global climate change is affecting the health of human populations, including changes in the transmission and seasonality of vector-borne diseases. The range of factors affecting transmission and distribution of vector-borne diseases, particularly malaria, include those related to temperature, humidity and precipitation. In Mozambique, malaria is endemic throughout the country, due to a multitude of factors such as climatic/environmental (favourable temperatures and rain patterns, abundant breeding sites) and socio-economical (poverty related improper housing/shelter, unaffordable preventive means). Transmission is perennial, with peaks during and after rainy seasons. The intensities of transmission may vary depending on the amount of rain and air temperature. However, at present there is a lack of good quality and updated information on the endemicity levels in the country.The country-wide malaria survey carried out between 2002 and 2003 aimed to determine the prevalence and intensity of Plasmodium infections, the prevalence and the severity of anaemia in children under 10 years of age and in pregnant women across different ecological settings, in order to characterize the malaria transmission intensities and to estimate the disease burden in Mozambique. The last comprehensive malaria survey in the country was carried out in 1952. For that reason, this survey was an unique opportunity to perform a sound methodological assessment of the current epidemiological malaria situation in the country. Based on altitude and on geographical region differences samples were collected from stratified areas distinguished as coastal, plateau and highland strata, in the northern, centre-northern, central and southern regions. For sampling at community level, in each of those stratified areas, a modified cluster sampling method with 30 clusters, used by WHO for evaluation of coverage of the Expanded Programme of Immunization was adopted. The study consisted of house-to-house survey, in 24 districts randomly selected. A total of 12,002 subjects including children less than ten years of age and pregnant women were enrolled. The malariometric survey consisted of finger pricking and blood collection to prepare thick and thin film for malaria parasite species identification, and respective density and determination of haemoglobin concentration. Measurement of axillary temperature and in those with fever a rapid enzyme test for malaria diagnosis was performed. The entomologic survey consisted of pyrethrum spray knock down mosquito collections. In total 6,557 female anopheline mosquitoes caught in 1,440 dwellings, were analysed for sporozoite infection using PCR techniques and the entomological inoculation rates were determined for each strata across regions. / La malaria se encuentra preferentemente en las regiones tropicales del mundo, siendo África sub-sahariana el área con más afectación. La gran eficiencia de los mosquitos vectores Anopheles gambiae complex y Anopheles funestus favorece una gran transmisión del Plasmodium falciparum, el parásito más predominante y más maligno de las especies causante de malaria. Las condiciones ambientales locales son apropiadas y a menudo la transmisión se da a lo largo todo el año.Los recursos limitados y la inestabilidad socio-económica constituyen los principales factores que impiden la total eficacia de las estrategias de control de la malaria. El programa de erradicación mundial de la malaria se se llevó a cabo durante los años 50 focalizándose básicamente en la fumigación con DDT, el tratamiento con fármacos anti-maláricos y la vigilancia.La erradicación regional de la enfermedad se conseguió en muchas regiones endémicas del África sub-sahariana, no obstante en zonas excluidas de la campaña de erradicación, la enfermedad continua afectando a sus habitantes.La estimación de la carga de enfermedad por malaria en el África tropical se obtiene de los datos de mortalidad y morbilidad recogidos por el sistema de informació de la salud.Los datos conservadores estiman que la carga de esta enfermedad causa más de 300 millones de episodios clínicos y entre 1 - 3 millones de muertes cada año, siendo los niños los más afectados.En la actualidad, la situación se esta deteriorando, observándose un incremento de la morbilidad y mortalidad por malaria. El rápido desarrollo, la propagación de cepas del parásito resistentes ante todos los fármacos anti-maláricos y la presencia de vectores resistentes son los factores que más han contribuido a este incremento.A demás, el cambio climático global está afectando la salud de las poblaciones, incluyendo cambios en la transmision y estacionalidad de las enfermedades mediadas por vectores. Los factores que afectan la transmisión y la distribución de este tipo de enfermedades, particularmente la malaria, incluyen los relacionados con la temperatura, la humedad y las precipitaciones. En Mozambique, la malaria es una enfermedad endémica en todo el país, debido a la multitud de factores, como los climáticos/medioambientales (temperaturas favorables y patrones de precipitación, abundantes espacios para la reproducción) y socio-económicos (pobreza relacionada con vivienda inadecuada, medios preventivos inasequibles). La transmisión es perenne, con picos durante y después de la estación de lluvias. Las intensidades de transmisión pueden variar dependiento de la cantidad de precipitación y la temperatura en el ambiente. Sin embargo, actualmente, hay una falta de información actualizada y de calidad sobre los niveles de endemicidad del país. El estudio llevado acabo por todo el país entre los años 2002 y 2003 tenía como objetivo determinar la prevalencia y la intensidad de las infecciones por Plasmodium, la prevalencia y la severidad de la malaria en niños menores de 10 años de edad y en mujeres embarazadas a través de diferentes nichos ecológicos, para poder caracterizar la intensidad de transmisión por malaria y estimar la carga de esta enfermedad en Mozambique. El último estudio exahustivo de estas características en el país fue realizado en 1952. Por esta razón, este estudio era una oportunidad única para realizar un asesoramiento metodológico profundo de la situación epidemiológica actual de la malaria el país. Las muestras fueron recogidas basándose en la altitud y diferencias geográfica de cada región. Las areas estratificadas se clasificaron en: estrato costero, plateau y de montaña, y según la región en: norte, centro-norte, central y sur. Para el muestreo a nivel de comunidad, en cada una de esas áreas estratificadas, se utilizó un método por muestreo modificado por grupos con 30 grupos, ya usado por la OMS para la evalución de la cobertura del Programa Ampliado de Vacunación.El estudio consisitió en visitar casa por casa en 24 distritos seleccionados aleatóriamente. Se incluyeron un total de 12,002 individuos, tanto niños menores de 10 años de edad como mujeres embarazadas. El estudio malariométrico consistió en recoger sangre por punción en el dedo para preparar una lámina delgada y otra gruesa para la identificación de la especie del parásito de la malaria y una estimación de la densidad, y para la determinación de la concentración de hemoglobina. Se tomó la tempreatura axilar y en presencia de fiebre se realizaba un test enzimático rápido para el diagnóstico de malaria.El estudio entomológico consistió en la recogida de mosquitos rociados con piretrum. En total 6,557 mosquitos anófeles hembra de 1,440 viviendas fueron analizados para infeción de esporozoito usando técnicas de PCR, y la tasa de inoculación entomológica fue determinada para cada estrato a través de las diferentes regiones.

Resultados anteriores sugerem que existem diferenças biológicas entre espécimens de Anopheles oswaldoi capturados no Estado do Acre e os do Estado de Rondônia, Brasil. Esta espécie tem sido apontada como um importante vetor de malária em localidades do Peru e Acre. Entretanto, em Rondônia, somente um número pequeno de A. oswaldoi alimentados em pacientes com malária desenvolveram infecção nas glândulas salivares. Além disso, há suspeita de que espécimens identificados como A. oswaldoi, capturados em áreas abertas em Costa Marques, Rondônia, são na verdade A. konderi, e que A. oswaldoi sensu stricto estaria restrito a áreas de florestas. Estes dados, juntamente com as dificuldades encontradas na identificação de anofelinos do grupo Oswaldoi, baseadas em critérios morfológicos, sugerem que espécimens de A. oswaldoi são membros de um complexo de espécies crípticas. A distinção de espécies crípticas de insetos vetores é de grande importância, já que diferentes membros em um complexo podem exibir diferenças na ecologia, capacidade vetorial e resposta a medidas de controle. Análise de sequências de DNA, particularmente da região do ITS2 do cistron do DNA ribossômico, tem sido usada como um caracter diagnóstico em alguns grupos de espécies crípticas, tornando-se um instrumento para estudos taxonômicos e filogenéticos. A primeira parte deste estudo teve o objetivo de determinar as diferenças encontradas nas sequências de ITS2 de espécimens de A. oswaldoi capturados em várias localidades da América do Sul. As regiões dos ITS2 destes anofelinos foram amplificadas usando oligonucleotídeos iniciadores conservados das regiões 5.8S e 28S e os produtos de PCR foram clonados e sequenciados. Os ITS2 de todos os mosquitos capturados tiveram tamanho aproximado de 350 nucleotídeos, com aproximadamente 53% de conteúdo de GC. Análise do alinhamento destas sequências, mostrou similaridade variando entre 87% e 100%, e a análise de uma árvore de similaridade, neighbor-joining, produzida com p-distance usando as diferenças nas sequências de ITS2, separou estes espécimens em quatro grupos. Um deles está provavelmente relacionado ao A. oswaldoi sensu stricto, e um outro pode estar relacionado à espécie A. konderi. Os outros dois grupos podem corresponder a espécies cuja identificação morfológica permanece para ser esclarecida no complexo A. oswaldoi. Estes dados são evidências de que espécimens de A. oswaldoi estão incluídos em um complexo de espécies crípticas e que identificação por DNA pode resolver estas questões taxonômicas. A. konderi tem sido considerado uma sinonímia de A. oswaldoi. Embora estágios adultos e imaturos destas espécies de anofelinos apresentem características morfológicas idênticas, aspectos encontrados na genitália masculina podem distinguir estes taxa. A segunda parte deste estudo foi conduzida com o objetivo de comparar a susceptibilidade de A. oswaldoi s.s. e de A. konderi à infecção com Plasmodium vivax. A susceptibilidade foi baseada na proporção de mosquitos com oocistos e esporozoítos. Os anofelinos foram capturados no Acre e em Rondônia para obtenção de progênies F1. Após a emergência dos adultos, as genitálias masculinas dos mosquitos de cada progênie foram dissecadas e examinadas. Todas progênies originadas dos mosquitos capturados em Rondônia corresponderam a A. konderi, enquanto que cerca de 85,0% das progênies do Acre eram A. oswaldoi s.s.. Estas progênies F1 de A. oswaldoi s.s., A. konderi e A. darlingi foram alimentadas simultaneamente com sangue infectado com P. vivax. Estes mosquitos foram dissecados 10-12 dias após infecção e examinados para verificação da presença de oocistos e esporozoítos. Tanto A. oswaldoi s.s. como A. konderi apresentaram oocistos nos tratos digestivos, entretanto, a porcentagem de tratos digestivos positivos para oocistos foi maior em A. oswaldoi s.s. (13,8%) do que em A. konderi (3,3%). Esporozoítos foram encontrados somente nas glândulas salivares de A. oswaldoi s.s., com 6,9% de positividade. As taxas de infecção nos controles A. darlingi foram de 22,5% a 30,0%, para ambos oocistos e esporozoítos. Estes resultados indicam que A. oswaldoi s.s. pode transmitir P. vivax e sugerem que esta espécie é mais susceptível que A. konderi. Embora A. oswaldoi s.s. seja uma espécie exofílica e zoofílica, este anofelino pode estar envolvido na transmissão da malária humana como parece estar ocorrendo no Estado do Acre. / Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

Les moustiques anophèles sont les vecteurs du parasite Plasmodium l’agent du paludisme. Le parasite subit des pertes massives pendant son cycle de développement chez l’anophèle, ce qui suggère que les moustiques sont capables de développer une réaction immunitaire efficace contre le parasite. La connaissance de l’immunité et de la résistance des moustiques au genre Plasmodium provient principalement de systèmes de laboratoire qui utilisent des espèces de parasites de rongeurs ou d’oiseaux comme modèles du paludisme humain. Les observations présentées dans cette thèse suggèrent que certains gènes comme Tep1 et LRIM1 sont des médiateurs de réponses antiparsitiques contre différents Plasmodiums dans différents vecteurs. Cependant, le degré d'efficacité avec laquelle un moustique est capable de réduire le nombre de parasites peut être variable surtout entre combinaison de souche de moustique et de souche de parasite différentes, selon que la paire soit hautement compatible ou non.<p><p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

>Magister Scientiae - MSc / Tsetse flies (Glossina spp) are vectors of African trypanosome (Trypanosoma spp) parasites, causative agents of Human African trypanosomiasis (sleeping sickness) and Nagana in livestock. Research suggests that tsetse fly immunity factors are key determinants in the success and failure of infection and the maturation process of parasites. An analysis of tsetse fly immunity factors is limited by the paucity of genomic data for Glossina spp. Nevertheless, completely sequenced and assembled genomes of Drosophila melanogaster, Anopheles gambiae and Aedes aegypti provide an opportunity to characterize protein families in species such as Glossina by using a comparative genomics approach. In this study we characterize thioester-containing proteins (TEPs), a sub-family of immunity-related proteins, in Glossina by leveraging the EST data for G.morsitans and the genomic resources of D. melanogaster, A. gambiae as well as A.aegypti.A total of 17 TEPs corresponding to Drosophila (four TEPs), Anopheles (eleven TEPs) and Aedes aegypti (two TEPs) were collected from published data supplemented with Genbank searches. In the absence of genome data for G. morsitans, 124 000 G.morsitans ESTs were clustered and assembled into 18 413 transcripts (contigs and singletons). Five Glossina contigs (Gmcn1115, Gmcn1116, Gmcn2398, Gmcn2281 and Gmcn4297) were identified as putative TEPs by BLAST searches. Phylogenetic analyses were conducted to determine the relationship of collected TEP proteins.Gmcn1115 clustered with DmtepI and DmtepII while Gmcn2398 is placed in a separate branch, suggesting that it is specific to G. morsitans.The TEPs are highly conserved within D. melanogaster as reflected in the conservation of the thioester domain, while only two and one TEPs in A. gambiae and A. aegypti thioester domain show conservation of the thioester domain suggesting that these proteins are subjected to high levels of selection. Despite the absence of a sequenced genome for G. morsitans, at least two putative TEPs where identified from EST data.

Le contrôle du paludisme ne semble aujourd'hui envisageable que par stratégies combinées ciblant différents stades du parasite. Chez le vecteur, certains mécanismes de la réponse immunitaire pourraient être manipulés pour bloquer le développement sporogonique du parasite. Cette thèse examine les effets de la diversité du vecteur et du parasite dans le couple le plus important en termes d'épidémiologie, A. gambiae - P. falciparum. Des polymorphismes de gènes de l'immunité du moustique contrôlant le niveau d'infection ont été identifiés par étude d'association. Certains d'entre eux ont un effet spécifique selon les isolats de parasites, suggérant de potentielles interactions génotype X génotype. Nous avons déterminé un déséquilibre de liaison très bas dans les populations naturelles de vecteurs, validant notre approche par gènes candidats. Les caractéristiques et les forces évolutives faisant d'A. gambiae un vecteur du paludisme majeur sont discutées. Les diverses populations de vecteurs et parasites peuvent interagir de manière spécifique. Pour tester cela, des infections par des isolats de parasites sympatriques et allopatriques ont été comparées, montrant des intensités plus faibles dans les couples sympatriques. Les profils d'expression des gènes montrent cependant peu de régulations spécifiques aux populations, mais plutôt des différences extrêmes selon les isolats de parasites. Ces résultats suggèrent des effets importants de la diversité entre populations et individus. En conclusion, cette thèse souligne l'importance de la prise en compte de la diversité naturelle des vecteurs et parasites dans les recherches futures sur leurs interactions. / To achieve malaria control a variety of approaches must be combined targeting different stages of the parasites life cycle. With better understanding of mosquito immunity, it is hoped that aspects of natural resistance can be manipulated to prevent parasite development. This thesis investigates the effect of both mosquito and parasite diversity on the mosquitoes response to malaria using the most important human malaria system; Anopheles gambiae-Plasmodium falciparum in natural/semi-natural conditions. Mosquito loci are identified that significantly control infection phenotype, some of which act in a parasite isolate specific manner, highlighting their potential involvement in genotype by genotype interactions. Such research is moving towards genomewide studies; however, on finding very low linkage disequilibrium in wild mosquitoes, it favors candidate gene association studies. A. gambiae characteristics that make it such a good malaria vector are discussed and the evolutionary forces driving these traits. Selection behind vector-parasite interactions can differ spatially and temporally causing specificities in sympatric couples. Sympatric and allopatric mosquito infections with malaria are compared, showing that sympatric infections develop lower infection intensities suggesting local adaptation. Mosquito gene expression profiles highlight a small number of genes differentially regulated between sympatric and allopatric infections, however extreme differences in gene regulation are observed within populations, probably driven by the variable nature of malaria parasites. This thesis highlights the importance of taking into account natural diversity in future research.

Sindbis virus (SINV) is an arthropod-borne Alphavirus in the family Togaviridae. Sindbis virus has a broad host range that includes avian, mammalian, and human hosts; therefore, its receptor(s) is/are highly conserved. When the mosquito imbibes a viremic blood meal, the virus infects the midgut cells, disseminates into the hemolymph, and eventually infects the salivary glands. The midgut is an organ of transmission and the virus must overcome the midgut epithelia infection- and escape-barriers. Sindbis virus infection is determined by the chance collision of the glycoproteins with a compatible receptor. Research has supported the involvement of high-affinity laminin receptor and heparan sulfate in SINV binding to host cells. However, it has been suggested that not all strains of SINV are dependent on heparan sulfate for attachment/entry and that SINV could be utilizing multiple receptors. A study using Drosophila demonstrated that, of the nine genes that encode for proteins that enhance SINV infection, only natural resistance-associated macrophage protein (NRAMP) was conserved. A symporter of divalent metals and hydrogen ions, NRAMP is ubiquitously expressed. Overexpression of NRAMP led to an increase in SINV infection of human cells while deletion of NRAMP in mouse and Drosophila decreased SINV infection. Sindbis virus could be utilizing this protein to overcome the infection barriers of mosquito midgut epithelia. In this study, NRAMP was localized to Aedes aegypti and Anopheles quadrimaculatus tissues via immunofluorescence assay and TR339-TaV-eGFP was detected in the midgut epithelia and visceral muscles. We suspect that NRAMP was detected on midguts and/or Malpighian tubules of Aedes aegypti and Anopheles quadrimaculatus. The similarities between the pattern of NRAMP labeling and TR339-TaV-eGFP infection of the midgut suggest that SINV infection is influenced by NRAMP in the midgut epithelia. Because NRAMP is ubiquitously expressed, this research provides insight into the attachment and entry phase of the arbovirus lifecycle.

Memory of chirality (MOC) is an emerging strategy for asymmetric synthesis which relies upon the intermediacy of transiently non-racemic reactive species. In these reactions the configuration of the sole stereogenic center of the enantiopure starting material is "memorized" by a chiral non-racemic conformation in the intermediate; trapping then captures the stereochemical information, and generates a new stereogenic center with high fidelity. We experimentally and computationally studied the highly retentive deprotonation/alkylations of 1,4-benzodiazepin-2-ones (BZDs) that rely upon this strategy. We captured a transiently non-racemic BZD enolate intermediate in enantiopure form, then released the enolate and observed its subsequent reaction. This approach allowed the first ever step-wise observation of the stereochemical course of such a MOC process. Approximately 2 million deaths are caused by malaria every year in the world. In total roughly 3.2 billion people are living under the risk of malaria transmission. Current use of anticholinesterase insecticides has been limited by their toxicity to human beings. A major African malaria insect vector, Anopheles gambiae (Ag), was targeted. Based on sequence alignment and homology models of AgAChE, a strategy of dual-site binding was adopted that targets Trp84 in the active site and Cys286 at the peripheral site. Selective AChE inhibitors have been designed and synthesized. / Ph. D.