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Adult and laIval midgut trypsins from the mosquito Aedes aegypli were isolated using benzamidine affinity chromatography. The cDNA of the late trypsin induced by the blood meal was isolated using an anti-trypsin monoclonal antibody, cloned and sequenced. The 862 bp sequence codes for a 257 amino acid protein, which is presumably a trypsin precursor, since the sequence of purified mosquito trypsin begins at residue 26, immediately following an arginine residue in the precursor. The amino terminal 25 amino acids in the precursor are composed of a putative 15 amino acid signal peptide and a 10 amino acid activation peptide. The activation peptide in the mosquito is different from that of vertebrate trypsinogens and suggests that activation takes place by tryptic cleavage. The deduced amino acid sequence is homologous to that of other trypsins in those residues around the catalytic triad, and in several residues which are found only in trypsins. However, the sequence of the specificity pocket in mosquito trypsin, KESPC, differs from that found in other trypsins, KDSC. The Asp is thought to bind the basic residue of the substrate, and the Glu in the mosquito trypsin may serve the same role. The changes in trypsin protein and mRNA levels following a blood meal indicate that an important component of the regulation of trypsin synthesis is at the transcriptional level. A genomic clone of "late trypsin" was isolated, mapped, and 1.2 kb of the upstream regulatory region were sequenced. The gene has no introns within the coding region. A TAT A box consensus sequence (TAT AAA) was found at position -31 from the 5' end of the mature mRNA. A cluster of five repeat sequences homologous to the GCN4 DNA binding site was found within 200 nucleotides upstream of the cap site. GCN4 is required for derepression mediated control of general amino acid biosynthesis in response to amino acid starvation in yeast. This suggests that a similar protein might regulate expression of the late trypsin gene in the mosquito. Southern blot analysis of genomic DNA suggests that the regions flanking the late trypsin coding region are polymorphic.

Submitted by Gizele Lima (gizele.lima@inpa.gov.br) on 2016-11-23T12:52:52Z No. of bitstreams: 2 Juliana Bruning Azevedo.pdf: 2565810 bytes, checksum: 1751c9233813fc98d009c5515f8a7e46 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-23T12:52:52Z (GMT). No. of bitstreams: 2 Juliana Bruning Azevedo.pdf: 2565810 bytes, checksum: 1751c9233813fc98d009c5515f8a7e46 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Aedes aegypti is considered the main vector of dengue virus, urban yellow fever and Chikungunya fever. Insects are susceptible to changes in environmental factors such as rainfall and temperature. Responsible for regulating population size and aspects of mosquito biology, such as larval growth, development time, body size, longevity, fertility and blood supply. The Intergovernmental Panel on Climate Change (IPCC) predicts an increase of 2° to 4° C in average global temperature over the next century. These climatic changes may result in significant changes in landscapes and ecological patterns of infectious diseases, and interfere directly in the development and behavior of A. aegypti. This study evaluated different aspects of vector biology as amount amount of ingested blood, fertility, egg laying and hatching of eggs and longevity, climate change scenarios, planned for the year 2100, intermediate and extreme scenarios. Among the predicted variables to undergo changes due to climate change, the temperature is primarily responsible for changing the biology and vector behavior. Concentrations of CO2, even three times the current concentration does not significantly affect the results obtained in this study. The amount of blood ingested by females raised in higher temperature was higher compared to females who lived at lower temperatures, longevity is low at temperatures above 32° C, living approximately 40 days and no longevity difference between sex. The fertility is greatly affected by temperature, being reduced by half in higher temperatures. The oviposition is impaired in temperatures above 32° C, but this effect was not observed in the outbreak. Mathematical models project an increase in the number of dengue cases and geographic expansion in vector distribution. However, our data indicate that, in future scenarios of climate change, some aspects of the biology of A. aegypti will be affected. With the above information, it was possible to better understand the biology of the vector and taking also into account climate change, serving as a basis for other studies. / O Aedes aegyptié considerado o principal vetor do vírus da Dengue, Febre amarela urbana e da Febre Chikungunya. São insetos suscetíveis à variação de fatores ambientaiscomo, quantidade de chuva e temperatura. Responsáveis porregular o tamanho populacional e aspectos da biologia do mosquito, como o crescimento larval, tempo de desenvolvimento, tamanho corporal,longevidade, fecundidade e alimentação sanguínea. O Painel Intergovernamental sobre Mudança do Clima (IPCC) prevê um aumento de 2° a 4° C na temperatura média global, durante o próximo século. Essas alterações climaticas poderão resultar em mudanças significativas nas paisagens e nos padrões ecológicos das doenças infecciosas, e interferir diretamente no desenvolvimento e comportamento do A. aegypti. Neste trabalho foram avaliados diferentes aspectos da biologia do vetor como, ingestão de sague, fecundidade, oviposição e eclosão de ovos e longevidade, sobre cenários de mudanças climáticas, previstas para o ano de 2100, cenários intermediários e extremos.Dentre as variáveis preditas para sofrem alterações devido a mudanças do clima, a temperatura é principal responsável por alterar a biologia e o comportamento do vetor. As concentrações de CO2, mesmo três vezes maior que a concentração atual, não influenciou significativamente os resultados obtidos neste estudo.A quantidade de sangue ingerida por fêmeas criadas em temperatura mais elevada foi maior quando comparadas com as fêmeas que viviam em temperaturas menores, a longevidade é menor em temperaturas superiores a 32°C, vivendo aproximadamente 40 dias e não houve diferença de longevidade entre os sexos. A fecundidade é extremamente afetada pela temperatura, sendo reduzida pela metade em temperaturas maiores. A oviposição é prejudicada em temperaturas superiores a 32°C, porém esse efeito não foi observado na eclosão.Modelos matemáticos projetam um aumento no número de casos de dengue, além de uma expansão geográfica na distribuição do vetor. No entanto, nossos dados indicam que, em cenários futuros de mudanças climáticas, alguns aspectos da biologia do A. aegypti serão afetados. Com as informações acima mencionadas, foi possível conhecer melhor a biologia do vetor e levando também em consideração as mudanças climáticas, servindo como base para outros estudos.

Muitos trabalhos são desenvolvidos para identificar substâncias ou microrganismos capazes de controlar a população de insetos importantes para a saúde pública, sendo a identificação de lesões histológicas um critério para compreender o mecanismo de ação e eficácia do produto. As análises histopatológicas são baseadas na comparação entre tecidos de indivíduos controle e indivíduos expostos. Entretanto, são raros os trabalhos que referenciam o estado dos tecidos sadios. Neste trabalho caracterizou-se morfo-histoquimicamente os principais tecidos sadios do sistema digestório de larvas em 3º a 4º ínstar de Aedes aegypti, mostrando diversas imagens das variações que podem ser esperadas, além de apresentar as técnicas de processamento. Para isto, um total de trinta larvas foram fixadas, desidratadas e incluídas em historesina. Cortes foram realizados e corados com HE para morfologia e azul do Nilo, azul bromofenol, azul de toluidina e PAS para a caracterização histoquímica. Como resultados, o intestino das larvas se mostrou um tubo reto, formado por uma camada simples de células cúbicas, moderadamente acidófilas, com núcleo central e uma borda em escova voltada para o lúmen. Foram registradas variações celulares, uma vez que as células epiteliais também são secretivas das enzimas digestivas. Cortes sequenciais foram importantes para definir a morfologia, uma vez que o ângulo e altura de corte podem influenciar na sua aparência. O PAS revelou a presença de muitos grânulos de glicogênio por todos os tecidos, inclusive nas vesículas secretivas. Estas ainda possuem muitas proteínas e foram coradas fracamente para lipídios. Não foram registrados eventos de metacromasia na cor vermelha com o azul de toluidina, entretanto os tecidos parecem se corar diferentemente do tegumento. / Many surveys are designed to identify substances or microorganisms capable of controlling the population of insects that are important to public health, and the identification of resulting histological injures is a criterion for understanding the mechanism of action and the efficacy of the product. The histopathological analyses are based on the comparison between tissues of control subjects and exposed subjects, but there are rare studies that mention the state of healthy tissues. This work characterized the main morpho-histochemically healthy tissues of the digestive system of larvae in the 3rd and the 4th instar of Aedes aegypti, showing several images of the variations that can be expected, as well as displaying processing techniques. For this, a total of thirty larvae were pinned, dehydrated and embedded in historesin. Sections were made and stained with hematoxylin for morphology and Blue Nile, bromophenol blue, toluidine blue and PAS staining for the immunohistochemical characterization. As a result, the intestines of the larvae showed an upright tube formed by a single layer of cuboidal cells, moderately acidophilic, with a central nucleus and a brush border facing the lumen. Cellular changes were recorded, since epithelial cells also secrete digestive enzymes. Sequential slices were important in defining the morphology, since the angle and height of the cut may affect its appearance. The PAS revealed the presence of many glycogen granules in all tissues, including the secretory vesicles. These still have protein and were weakly colored to lipids. There were no metachromasia events in red with toluidine blue, though the tissues seem to blush differently from the integument.

Submitted by Gizele Lima (gizele.lima@inpa.gov.br) on 2018-03-07T19:05:24Z No. of bitstreams: 2 Tese JOELMA 25 01 18-1.pdf: 2217088 bytes, checksum: 4076fe5d3e54507c6b7c0ba6a34ae6e0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-07T19:05:24Z (GMT). No. of bitstreams: 2 Tese JOELMA 25 01 18-1.pdf: 2217088 bytes, checksum: 4076fe5d3e54507c6b7c0ba6a34ae6e0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The diversity of Bacillus thuringiensis obtained from three Brazilian biomes with pathogenic action for Ae. aegypti, An. darlingi and Cx. quinquefasciatus. Aspects as characterization of the active strains and the importance of the cyt1Aa gene also were analyzed. / Estudou-se a diversidade de Bacillus thuringiensis obtidos de três biomas brasileiros com ação patogênica para larvas de Ae. aegypti, An. darlingi e Cx. quinquefasciatus. Aspectos como caracterização molecular das linhagens ativas e a importância do gene cyt1Aa também foram analisados.

Plagiorchis elegans is a typical digenean parasite that cycles through aquatic molluscs and insects as intermediate hosts. During emergence of P. elegans cercariae, infected snails moved to the top of the water column where they remained immobile for 2-3h. Consequently, the cercariae formed a dense cloud which dispersed slowly. The infectivity of cercariae was $<$20% upon emergence and peaked at 76% 4-6h later. This delay in reaching maximum infectivity may be an adaptation to prevent superinfection and the associated mortality of insect hosts. Cercariae transformed into metacercariae after penetrating Aedes aegypti larvae, the experimental insect host. Overall development of metacercariae, and excystment of infective metacercariae in vitro, was temperature dependent. However, there was an initial 8-hour period of obligatory host-parasite contact that was temperature independent. This may represent a period of major nutrient acquisition since young metacercariae were more active metabolically than older metacercariae, as measured by the in vitro uptake of $ sp3$H-glucosamine and $ sp3$H-leucine. Mosquitoes may have mechanisms to reduce losses of larvae to parasites. Oviposition by adult A. aegypti was reduced in waters that had previously contained P. elegans-infected larvae. We propose that this selective oviposition was due to the production of an oviposition deterrent compound produced by parasitized larvae that serves to reduce oviposition in sites detrimental to larval development.