Student Number : 0318930A - PhD thesis - School of Animal, Plant and Environmental Studies - Faculty of Science / The widespread use of pyrethroid insecticides has led to the emergence of significant insecticide resistance in various parts of the world. An unprecedented increase in the number of annual malaria cases reported in Kwazulu Natal, South Africa in 1999 to 2000 was attributed to the re-emergence of pyrethroid-resistant Anopheles funestus Giles. Resistance was metabolic-based with increased monooxygenase (P450) metabolising the pyrethroid insecticides. This emphased the need to understand the molecular mechanisms conferring pyrethroid resistance in An. funestus. The present study aimed to firstly isolate P450 genes in An. funestus and secondly, to identify P450 gene over-expressed in a resistant (FUMOZ-R) strain compared to a susceptible (FANG) strain. A third aim was to construct an An. funestus cDNA library to lay the foundation for future studies on P450 monooxygenses. Degenerate primers based on conserved regions of three An. gambiae P450 families were used to amplify cDNAs from An. funestus. Eleven CYP4, four CYP6 and five CYP9 partial genes were isolated and sequenced. BLAST results revealed that An. funestus P450s have a high sequence similarity to An. gambiae with above 75% identity at the amino acid level. The exception was CYP9J14. The An. gambiae P450 with the closest similarity to CYP9J14 exhibited only 55% identity suggesting a recent duplication event in CYP9J14. Molecular phylogenetic analysis also supported this hypothesis. Intron positions were highly conserved between the two species. Expression studies using blot analysis implicated CYP6P9, an ortholog of CYP6P3 in An. gambiae, as the over-expressed P450. Dot blot analysis revealed a 500-fold expression higher in FUMOZ-R strain compared with FANG strain. Semiquantitative PCR revealed that CYP6P9 was developmentally regulated. Expression was not detected in eggs and was higher in larvae compared to pupae. Quantitative real time PCR showed that CYP6P9 expression was 4.5-fold higher in 3-day old FUMOZ-R males than females and 3.5-fold higher in the 14-day old males than 14- day old females. Statistically, this difference was not significant suggesting that CYP6P9 expression is not sex specific. The An. funestus cDNA library construction in λTriplEx2 vector was successful with a titre of 4.9 x108 pfu/ml and a transformation efficiency of 98%.

To meet the rising food demands of communities in Accra,Ghana, urban agriculture has been popularized as a way to increase food security and improve nutrition (Donovan et al., 2012). Urban agriculture is defined as "the cultivation of crops at both the subsistence and commercial levels including the keeping of livestock in open spaces in urban areas (Adjaye, n.d.). In Accra, urban agriculture covers 1,091 hectares, employs over one thousand people, and supplies residents with 90% of its vegetables ("Accra Metropolitan", n.d.). Further, 60% of households in Accra participate in backyard farming ("Accra Metropolitan", n.d.). Although urban agriculture provides many benefits for communities in Accra, it has been linked to the creation of suitable habitats for Anopheles gambiae complex larvae. In Accra, a spatio-temporal distribution of An. gambiae complex larvae and larvae habitats has not been established. A larval study in two urban agriculture and two non-urban agriculture sites was conducted in the months of May, July, August, and September 2014. When combined together, 3,807 An. gambiae complex larvae were collected from the urban agriculture sites of Korle Bu and Opeibea over the period of the study. When combining the urban non-agriculture sites of Madina and Ashaiman, 2,484 An. gambiae complex larvae were collected over the same period. The results of this study in Accra show that Korle Bu, an urban agriculture site, was the most productive site, with 2,604 An. gambiae complex larvae collected for the months of May, July, August, and September. July was the most productive month for Korle Bu, with 1,653 An. gambiae complex larvae collected. Further investigations of An. gambiae complex larval habitats are necessary to better understand malaria transmission attributes unique to Accra, Ghana.

Mediante trabajos en campo y laboratorio, se investigó la respuesta de Anopheles pseudopunctipennis (Theobald, 1901) a la exposición de diferentes concentraciónes de insecticidas tipo piretroides 0.1%, 0.075%, 0.050%, 0.025%, 0.0125%. Las muestras biológicas fueron colectadas dentro y fuera de las viviendas, el método utilizado para la captura fue el método del cebo humano y trampa luz, el trabajo de campo se realizó en el Anexo Pampa Baja y San Isidro. La investigación realizada con los insecticidas piretroides según la metodología de la Organización Mundial de la salud (OPS) y en el Perú por el Instituto Nacional de Salud (INS) de concentración 0,1% por 24 horas se determinó una mortalidad de 81% para insecticida Deltametrina categorizado como especie en vigilancia por la Organización Mundial; para insecticidas Lamdaciaholotrina, Ciflutrina Permetrina, Cipermetrinala mortalidad fue superior al 98 % catalogados como especie sensible a este grupo de insecticidas. En el estudio en campo de Anopheles pseudopunctipennis, se ha evaluado el índice de Picadura por Hombre/Noche (IPHN) y el Índice de Picadura Hombre/hora (IPHH), en donde se ha observado el nivel máximo en el mes de enero llegando a 23 mosquitos por hora y 272 mosquitos por noche. Los resultados observados han permitido dar la información de la situación en que se encuentra la especie en estudio de nuestra región que es un vector principal de la malaria y si no se toma medidas de un control integrado, incrementará el riesgo de transmisión por los factores ambientales favorables y las inadecuadas prácticas para su control.

Low level and variable transgene expression plague efforts to produce and characterize transgenic lines in many species. When transformation efficiency is high, productive transgenic lines can be generated with reasonable effort. However, most efforts to date in mosquitoes have resulted in suboptimal levels of transformation. This, coupled with the large space and intensive labor requirements of mosquito colony maintenance makes the optimization of transformation in mosquitoes a research priority. This study proposes two strategies for improving transgene expression and transformation efficiency. The first is to explore exogenous promoter/enhancer combinations to direct expression of either the transgene itself, or the transposase required for insertion of the transgene into the genome. An extension of this strategy is to investigate the use of a powerful viral transactivating protein and its cognate enhancer to further increase expression of these targets. The second strategy involves the identification of an endogenous boundary element for use in insulating transgenes and their associated regulatory elements. This would mitigate the inappropriate expression or silencing of many transgenes inserted into “unfavorable” genomic environments as a consequence of an inability to specifically target the integration of transposons currently used in mosquito transgenesis. The IE1 transactivating protein and its cognate enhancer from a baculovirus were shown to significantly increase expression of a reporter gene from three different promoters in cultured mosquito cells. Other heterologous enhancer/promoter combinations resulted in minimal increases or insignificant changes in expression. Orthologues of the vertebrate insulator-binding factor, CTCF, were cloned and characterized in two mosquito species, Aedes aegypti and Anopheles gambiae. The expression profile of mosquito CTCF is consistent with its role as a putative insulatorbinding protein. Preliminary binding site studies reveal a C/G-rich binding site consistent with that known in vertebrates and indicate that CTCF may bind widespread sites within mosquito genomes.

No description available.

The study assessed the impact of rice paddy water management practices and related environmental conditions that prevail in Niono, Mali, on the size of larval populations of Anopheles gambiae s.l., a major malaria vector. The longer the period of uninterrupted flooding, the greater was the larval population size. As the density of aquatic weeds increased, the size of the mosquito larval population declined, whereas the presence of rice plants enhanced the size of mosquito larval populations. Numbers of mosquito larvae initially increased as rice plant density rose, but then decreased marginally as densities reached their peak. These results confirm that water management plays a major role in the production of mosquito larvae. Nevertheless, comparison with studies conducted in other areas suggest that the impact of water management on mosquitoes varies regionally. In the Niono region, controlled irrigation and drainage should favour the reduction of Anopheles mosquito larval populations.

Thesis (Ph. D.)--Oregon State University, 1994. / Typescript (photocopy). Includes bibliographical references. Also available online.

Mestre -- Escola Nacional de Saude Publica, Rio de Janeiro, 2002.

Made available in DSpace on 2012-09-06T01:11:45Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 481.pdf: 1732518 bytes, checksum: 5331df652b55c7dbbb5a4fa1087fe16f (MD5) Previous issue date: 2002 / Ocimum selloi Benth. (Lamieceae), também conhecida como "alfavaca", é uma planta herbácea que ocorre no Brasil. O óleo essencial obtido das suas folhas tem sido usado como aditivo aromatizante em alimentos, produtos odontológicos e como ingrediente de fragrâncias. Na medicina popular brasileira, o O. selloi tem sido amplamente empregado como remédio antiinflamatório e antiespasmódico e, também, para tratar diarréia. O principal objetivo deste estudo foi avaliar a atividade repelente de mosquitos e o potencial de irritação dérmica do óleo de O. selloi em voluntários. Além disso, foram obtidos também dados sobre o perfil toxicológico (toxicidade aguda oral data e mutagenicidade no teste de Ames), e sobre as propriedades físico-químicas e constituintes químicas e constituintes químicos do óleo essencial do O. selloi coletado em Ponta Grossa, estado do Paraná. O óleo essencial foi obtido por hidrodestilação de folhas de O. selloi. Os constituintes químicos foram determinados por Cromatoia em Camada Delgada (CCD) e GC-MS/MSD. Os principais constituintes do óleo volátil analisado por GC-MS/MSD foram o estragol (55,38 por cento) e o trans-anetol (34,23 por cento). O óleo de O. selloi foi agudamente tóxico e letal para camundongos (fêmeas mais suscetíveis que machos) em doses iguais ou superiores a 1500 mg/kg de peso corporal. Não foi observada atividade mutagênica em ensaios in vitro com as linhagens de Salmonella thyphimurium TA97a, TA98 e TA100, sem e com adição de mistura S9. Não foi constatada irritação dérmica no antebraço de 30 voluntários expostos ao O. selloi por 4 horas. No estudo de campo em que seis voluntários (cada indivíduo é seu próprio controle) foram expostos Anopheles brasiliensis por 30 minutos o O. selloi (solução 10 por cento p/v em etanol) reduziu drasticamente a freqüência de picadas em relação ao etanol (redução de 88 por cento, P=0,01) demonstrando que é um repelente eficaz.

Made available in DSpace on 2016-04-12T12:46:43Z (GMT). No. of bitstreams: 2 jordi_ribas_ioc_dout_2015.pdf: 5425058 bytes, checksum: 28ae0ad564677b8019b24ede40155b12 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A malária se constitui numa das principais endemias em populações indígenas na Amazônia. Porém, fatores modeladores da dinâmica da transmissão são ainda pouco entendidos, tais como os determinantes da sua heterogeneidade espacial e da ocorrência de surtos sazonais de elevadas taxas de morbimortalidade. Neste estudo, buscamos determinar a fauna, aspectos ecológicos e taxa de infecção plasmodial de anofelinos em três áreas indígenas Yanomami (Parafuri, Toototobi e Marari). Considerando as três áreas, as espécies mais abundantes foram An. oswaldoi s.l. e An. darlingi, que juntas corresponderam a 59,5% dos 6.333 anofelinos adultos capturados. No Toototobi, as espécies mais frequentes foram An. oswaldoi s.l. (47,3%) e An. intermedius (31,8%), em Parafuri foram An. oswaldoi s.l. (48,7%) e An. darlingi (42,5%) e, em Marari An. nuneztovari s.l. (54,9%) e An. darlingi (21,6%). A maioria dos anofelinos foi coletada fora das habitações e o pico horário de picadas variou entre crepuscular (An. oswaldoi s.l.) e próximo à meia noite (An. darlingi). Foram processados 2.014 e 461 anofelinos para a detecção de esporozoítos dentre os coletados nos Polo Base Marari e Toototobi, respectivamente. Sete An. darlingi foram positivos para P. vivax, com taxas de infecção de 1,63%, 1,52% e 2,02% nas comunidades de Alapusi, Gasolina e Taibrapa, respectivamente. As taxas de inoculação entomológica correspondentes nestas comunidades foram de 65,4, 58,6 e 48,3 picadas infetantes por pessoa por ano no peridomicílio. Identificamos 160 criadouros, sendo 54 em Toototobi, 53 em Parafuri e 53 em Marari e propusemos uma nova classificação de criadouros naturais em oito tipos, baseada principalmente na associação com os pulsos de inundação, sazonalidade e grau de exposição ao sol Adicionalmente, utilizamos uma nova metodologia para amostrar as formas imaturas, baseada na quantificação da Área Efetiva de Criação (AEC), definida em (m2). Apresentamos também o Modelo de Sanfona, para explicar as flutuações do raio de ocorrência das espécies de anofelinos por estações do ano e sua dispersão em áreas associadas a rios de baixa ordem. A abundância dos tipos de AEC variou muito entre aldeias, assim como as flutuações deste parâmetro entre as diferentes estações do ano. Os três Polos Base estão localizados próximos aos rios de baixa ordem, que se caracterizam por apresentarem flutuações periódicas dos níveis de descarga dos rios, Pulsos de Inundação (PI) de caráter polimodal, de curta duração e imprevisíveis. Foram coletadas 6.295 larvas de anofelinos, das quais 1.399 (22,2%) eram An. darlingi, sendo que 96,9% desta espécie foram coletados em lagos associados aos rios (LAR). An. darlingi foi a espécie mais abundante nos criadouros em Parafuri (35%), sendo praticamente ausente em Toototobi, onde o An. oswaldoi s.l. (12,5%) foi a espécie mais abundante. An. darlingi apresentou maiores densidades em criadouros expostos ao sol e associados aos PI. Em contraste, An. costai-like predominou em criadouros temporários e não associados aos rios. Os nossos dados mostraram uma marcada heterogeneidade na ocorrência e densidades das espécies de anofelinos entre as comunidades, que explicaria a complexidade da dinâmica da transmissão da malária nestas áreas / Malaria is a major endemic disease in indigenous populations in the Amazon . However, modulating factors of the t ransmission dynamics are poorly understood, such as the determinants influencing its spatial hetereogeneity and the occur rence of seasonal outbreaks associated with high rates of morbidity and mortality . In the present study, we intend to determine the fau nal composition, ecological aspects and infection rates of the anophelines occurring in three Yanomami areas (Parafuri, Toototobi and Marari) of Brazil. We collected a total of 6, 333 adult anophelines and the most abundant species, considering the three a reas, were An. o swaldoi s.l. and An. d arlingi , which together accounted for 59 . 5% of the total collected. In Toototobi, t he most frequent species were An. oswaldoi s.l. (47 . 3%) and An. intermedius (31. 8%) , in Parafuri were An. oswaldoi s.l. (48. 7%) and An. darlingi (42. 5%) and in Marari were An. nuneztovari s.l. (54. 9%) and An. darlingi (21. 6%). Most anophelines were collected outside the indigenous huts and the biting activity peak differed between crepuscular for An. oswaldoi s.l. to near midnight for An . darlingi . A total of 2, 014 anophelines from Marari were processed for sporozoites detection and 461 from Toototobi. We found 7 An. darlingi infected with P. vivax in Mara ri, with infectivity rates of 1.63%, 1. 52% and 2. 02% in Alapusi, Gasolina and Taibra pa communities respectively. Based in the peridomiciliary collections, the Entomological Inoculation Rates (EIR) calculate d for these communities were 65.4, 58.6 and 48. 3 infective bites per person per year respectively. We identified 160 breeding sites; 5 4 in Toototobi, 53 in Parafuri and 53 in Marari and w e proposed a new larval habitat nomenclature divided in 8 types, based mainly in the association with river flood pulses, seasonality and degree of sun exposure. Also we used a new methodology to sample immature forms, based on the quantification of the Effective Breeding Area (EBA), set in (m 2 ). We also present the Squeezebox Model, in order to explain the fluctuations of the occurrence range of anopheline species in each season and how they disperse in areas a ssociated with low - order rivers . We found high variability of EBA types between communities and different seasons. All three Yanomami areas are located close to low - order rivers, which are characterized by fluctuations of the river discharge or Floo d Pulses (FP) that are polymodal, short and unpredictable . We collected a total of 6,295 anophe line larvae, of which 1,399 (22. 2%) were An. d arlingi with 96.9% of this species collected in lakes associated with the river (LAR) . An. darlingi was the most ab undant species in Parafuri (35%) being virtually absent in Toototobi, where An . oswaldoi s.l. predominated (12. 5%). An. darlingi showed higher densities in breeding sites exposed to the sun and associated with flood pulses. In contrast, An. costai - like, pr edominated in larval habitats that were temporary and not associated with river flood pulses. Our data showed a marked heterogeneity in the occurrence and density of Anopheles species, which would explain the complexity of the dynamics of malaria transmiss ion in these areas.