Reduced access to atmospheric oxygen greatly increased early instar Aedes aegypti mortality. Data suggest that death was by drowning mainly because slow swimming speed and low visual acuity delayed larvae from finding access to air. Late instar larvae experienced developmental delays when food depletion in the immediate vicinity of the breathing site forced them to forage farther away. As the distance between feeding and breathing sites increased, feeding efficiency of fourth instars may have declined enough to reduce their pupation success and to cause increased pupal mortality, reduced adult emergence and sex ratio skewed in favour of males among emerging functional adults. Thus reduced access to atmospheric oxygen at the pre-imago level may reduce the vectorial capacity of emerging adult population.

No description available.

BACKGROUND:The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.RESULTS:We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.CONCLUSIONS:These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

Master of Science / Division of Biology / Rollie J. Clem / Caspases are a conserved family of cysteine proteases that play important roles in apoptosis and innate immunity as well as other cellular processes. Eleven caspase genes have been annotated in the mosquito Aedes aegypti. Amongst these, previous studies have demonstrated functional roles for AeDronc, CASPS7 and CASPS8 in the Ae. aegypti apoptosis pathway, while CASPS18 and CASPS19 have also been functionally characterized. A previous study from our research group showed that AeIAP1 has preferential binding for CASPS7 compared to CASPS8. In this study, it was confirmed that AeIAP1 has a higher capacity to inhibit CASPS7 than CASPS8. Furthermore, five of the remaining Ae. aegypti caspases, namely CASPS15, CASPS16, CASPS17, CASPS20 and CASPS21, were characterized. An attempt was made to classify these caspases as initiator or effector caspases, based on factors such as the length of their prodomain, sequence similarity to known Drosophila initiator and effector caspases, and their substrate specificity. The functions of these caspases in apoptosis was examined in the Ae. aegypti cell line Aag2, by using RNA interference to reduce their expression and test the effect on apoptosis. Recombinant CASPS16, 17, 20 and 21 were produced in bacteria and the abilities of these recombinant proteins to cleave different caspase substrates were examined. From the resulting data, it was concluded that CASPS17 and CASPS21 are likely to be effector caspases since they preferred a effector caspase substrate. When considering the prodomain length, CASPS17 has a short prodomain, but CASPS21 has a long prodomain, which is normally associated with initiator caspases. CASPS20 did not show preference for any specific substrate and has a short prodomain. Since it did not have a specific preference of substrate, it is likely to be an effector caspase based on prodomain length. CASPS16 showed a slightly higher preference for the initiator caspase substrate WEHD, and has a long prodomain. Based on these results, CASPS16 is likely an initiator caspase. To examine the potential roles of CASPS15, 16, 17, 20 and 21 in apoptosis, their expression in Aag2 cells was knocked down using RNA interference. Successful knockdown was verified by qRT-PCR. After silencing specific caspases, the cells were exposed to two different apoptotic stimuli, ultraviolet radiation (UV) or the RNA synthesis inhibitor actinomycin D (ActD). Following apoptotic treatment, apoptosis was measured by two methods; caspase activity was measured using an effector caspase substrate, and phosphatidyl serine exposure on the outer leaflet of the plasma membrane, which occurs in apoptotic cells, was measured by Annexin V staining and flow cytometry. In cells where CASPS15, 16, 17, 20 or 21 had been knocked down and the cells were then treated with UV or ActD, it was observed that effector caspase activity and Annexin V staining were both significantly lower than in UV- or ActD-treated cells that had received control double-stranded RNA. Together these results suggest that all of these caspases are involved in apoptosis in Aag2 cells. This study serves as a starting point for further research on Ae. aegypti caspases and their roles in specific cellular processes.

Introducción: El Dengue es una enfermedad infecciosa, que constituye un problema de salud pública a nivel nacional e internacional, por el incremento del número de casos y que aumentan los casos de morbi-mortalidad en las personas. Objetivo:Determinar el efecto de una intervención educativa en enfermería en el incremento del nivel de conocimientos y mejora de prácticas preventivas contra el Dengue en los pobladores adultos de la Asociación Los Claveles – Lurín 2015. Metodología: Estudio de enfoque cuantitativo, tipo aplicativo, de método experimental y de diseño pre – experimental, con una muestra de 30 pobladores adultos de la Asociación “Los Claveles – Lurín”, de selección muestral no probabilístico por conveniencia; la técnica que se utilizó para obtener la recolección de datos fue la encuesta y la guía de observación, como instrumento se utilizó dos cuestionarios estructurados sobre conocimientos y prácticas preventivas con preguntas de opción múltiple; en la intervención educativa se aplicó primero el pre-test, seguido por 2 sesiones educativas y por talleres demostrativos sobre el lavado, tapado hermético de los envases de agua y la conservación de floreros, y posterior a ello se aplicó el post-test; y por último se utilizó una guía de observación para las prácticas preventivas. Para el análisis de datos se utilizó el programa SPSS versión 21. Resultados: El nivel de conocimiento antes de la intervención educativa fue un nivel medio 77% (23) y después de la intervención educativa fue de alto el 90% (27) y 10% (3) nivel medio dado el valor de T de Student -12,1791 (P=0,00000); y las prácticas preventivas antes de la intervención educativa eran inadecuadas 57% (17) y despúes adecuadas 80% (24) e inadecuadas 20% (6) con un valor de T de Student de -11,9721 (P=0,00000).Conclusión:La aplicación de la intervención educativa tuvo un efecto significativo en el incremento de conocimiento y prácticas preventivas contra el Dengue en los pobladores.

Ascogregarina taiwanensis e Ascogregarina culicis são protozoários que parasitam Aedes albopictus e Aedes aegypti, respectivamente, e são conhecidos por interferir fisiologicamente em seus hospedeiros. Alguns estudos têm relatado a distribuição destes protozoários em diferentes regiões do globo, entretanto, no Brasil, relatos destes protozoários são raros. O objetivo deste trabalho foi verificar a presença de Ascogregarina spp. em Ae. albopictus e Ae. aegypti no estado de Santa Catarina e a possível influência de seu parasitismo aliado aos inseticidas temephos e ao óleo de neem Azadirachta indica. Os resultados revelaram a presença de A. taiwanensis em larvas de Ae. albopictus nas cidades de Capivari de Baixo, Laguna e Gravatal. Somente em Tubarão foram encontradas as duas espécies de mosquitos albergando este protozoário. Com relação aos bioensaios químicos onde se testou insetos parasitados (+) e não parasitados (-) por A. taiwanensis, as CL50 foram 0.025 mg/L (+) e 0.063 mg/L (-) para temephos e 0,815 mg/L (+) e 1.812 mg/L (-) para óleo de A. indica. Os resultados demonstram que houve diferença significativa entre os valores de mortalidade em relação ao grupo controle com P<0.001. Divergindo dos relatos na literatura cientifica, observou-se infecção por A. taiwanensis em Ae. aegypti, porém, a transmissão vertical em laboratório foi observada somente em Ae. albopictus.Este é o primeiro registro de A. taiwanensis para Santa Catarina e o terceiro para o Brasil. A utilização dessa espécie de gregarina associada a inseticidas poderia ser um método interessante para futuros estudos de controle de Ae. aegypti. / Ascogregarina taiwanensis and Ascogregarina culicis are protozoa parasites of Aedes albopictus and Aedes aegypti, respectively and these parasites are able to interfere physiologically in their hosts. Some studies have reported the distribution of these protozoa in different parts of the world, however in Brazil, reports about these protozoa are rare. The aim of this study was to verify the presence of Ascogregarina spp. in Ae. albopictus and Ae. aegypti in Santa Catarina and the possible influence of its parasitism with insecticide temephos and the Azadirachta indica oil. The results demonstrated the presence of A. taiwanensis in Ae. albopictus larvae in the cities of Capivari de Baixo, Gravatal and Laguna. Only in the city of Tubarao the two species of mosquitoes have been found harbouring Ascogregarina taiwanensis. Regarding to bioassays evaluating parasite (+) and non-parasited (-) mosquitoes by A. taiwanensis, the LC50 0.063 mgL (+) and 0.025 mgL (-) for temephos and 0.815 mgL(+) and 1,812 mgL (-) for A. indica oil. The results show that there was significant difference between the mortality values comparing to the control group with P<0.001. Diverging from scientific literature reports, infection of this gregarine was observed, both in field and laboratory reared Ae. aegypti populations. However, the vertical transmission of A. taiwanensis could be observed only in Ae. albopictus. This is the first record of A. taiwanensis in Santa Catarina and the third in Brazil. The use of this species of gregarine in association with insecticides this could be an interesting method for future studies about control of Ae. aegypti.

Made available in DSpace on 2015-04-22T22:06:15Z (GMT). No. of bitstreams: 1 Ivana Cristina Lopes da Cunha.pdf: 2287821 bytes, checksum: b8be5da98e942cd7c40cc08eb0d09841 (MD5) Previous issue date: 2009-05-27 / The most important vector of Dengue virus is Aedes aegypti, an originally African mosquito species. The presence of this vector in the Americas dates back to the 17th-18th centuries; it is thought to have been first introduced into Brazil by the beginning of the 20th century. However, the process of invasion of the continent by Ae. aegypti remains poorly understood, and the relationships between the dynamics of vector introduction/establishment/expansion and dengue epidemiological trends have not been thoroughly assessed. Here we test a series of hypotheses regarding the origins, number, and spatial and temporal dynamics of the invasion of the Americas by Ae. aegypti. Key predictions were tested using a database composed of over 3000 mitochondrial ND4 gene sequences. This database, which was compiled and completed in the course of the project, contains sequences from specimens collected in five regions of the Americas (from the United States to southern Brazil), in Africa, and in Asia. Analyses covered the following subjects: (i) genetic diversity; (ii) spatial patterns of haplotype occurrence; (iii) genealogical and phylogenetic relationships; (iv) population genetic structuring; and (v) historical demography. Results suggest two major, probably old events of Ae. aegypti introduction into the Americas. Both involved the early arrival of moderately divergent African populations to the Caribbean and North-Mesoamerica. One of these lineages dispersed to Venezuela and spread southwards in two invasion waves. The first wave reached northern Amazonia, where some sub-populations became isolated; we suggest that the spread of these vectors was involved in the first American dengue pandemic (1824-1828). The second, much more recent wave resulted in the colonization of most of South America by this lineage. In contrast, the second major lineage reached South America by the Brazilian Southeastern region, and dispersed northwards during the second pandemic (1845-1851); the persistence of this lineage in Brazil suggests that xi eradication campaigns were never completely successful. The secondary encounter of the descendants of both major lineages gave rise to the often-reported pattern of high genetic diversity. The data suggest that passive vector dispersal and the effects of control interventions on local populations produce a pattern of strong genetic structuring. The recent evolution of dengue epidemiological patterns in Brazil suggests that health sector reform and decentralization in the 1990s limited the efficacy of control interventions. We finally suggest how the results of studies on vector genetics can be incorporated into the design of better control-surveillance strategies; they can help identify more invasive or more diverse vector populations, or help define critical locations for entomological surveillance and control. Our data show how these interventions should be pursued even in localities already infested by the vector. We have developed, making use of data on population genetic variability, a comprehensive proposal on the process of invasion of the Americas by Ae. aegypti, and tentatively established the correspondence between the patterns of genetic diversity of this vector species and the spatial and temporal dynamics of dengue epidemiology in Brazil / O principal vetor do vírus Dengue é o Aedes aegypti, uma espécie originária da África. A presença do vetor nas Américas data dos séculos XVII-XVIII; a primeira introdução no Brasil aconteceu, provavelmente, no início do século XIX. Contudo, o processo de invasão do continente por Ae. aegypti ainda é mal compreendido, e as relações entre as dinâmicas de introdução/estabelecimento/expansão do vetor e as tendências epidemiológicas da doença não têm sido avaliadas de forma detalhada. Neste trabalho testamos uma série de hipóteses sobre as origens, número e dinâmicas espaciais e temporais da invasão das Américas por Ae. aegypti. As predições-chave foram testadas utilizando uma base de mais de 3000 seqüências de um fragmento do gene ND4 mitocondrial. Esta base, compilada e completada durante o desenvolvimento deste projeto, contém dados de espécimes de cinco regiões das Américas (desde os Estados Unidos até o Sul do Brasil), da África e da Ásia. As análises incluíram os seguintes aspectos: (i) diversidade genética; (ii) padrões espaciais de ocorrência de diferentes haplótipos; (iii) relações genealógicas e filogenéticas; (iv) estruturação genética populacional; e (v) demografia histórica. Os resultados sugerem dois eventos principais, provavelmente antigos, de introdução de Ae. aegypti nas Américas. Ambos envolveram a chegada inicial de populações africanas moderadamente diferenciadas à região do Caribe/Norte-Mesoamérica. Uma destas linhagens se dispersou até a Venezuela e avançou para o sul em duas ondas. A primeira onda alcançou o norte da Amazônia, onde algumas sub-populações ficaram isoladas; sugerimos que a dispersão destes vetores foi a responsável pela primeira pandemia americana de dengue (1824-1828). A segunda onda, muito mais recente, resultou na colonização de grande parte da América do Sul. A segunda linhagem, pelo contrário, alcançou o continente Sul-americano pelo Sudeste do Brasil, e se dispersou em direção norte durante a segunda pandemia (1845- ix 1851); a persistência desta linhagem no país sugere que as campanhas de erradicação nunca alcançaram seu objetivo. O encontro secundário dos descendentes das duas linhagens principais é responsável pelo padrão repetidamente reportado de alta diversidade genética das populações locais. Os dados sugerem que a dispersão passiva do vetor e os efeitos das ações de controle geram um padrão de forte estruturação genética. Em conjunto, os dados genéticos e a evolução recente do perfil epidemiológico da dengue no Brasil indicam que a efetividade das intervenções de controle se viu comprometida pelo processo de descentralização do sistema de saúde nos anos 1990. Finalmente, mostramos como os resultados das análises genéticas podem ajudar no desenho de melhores estratégias de controle e vigilância; eles podem ajudar a identificar sub-populações mais invasivas ou mais diversas do vetor, ou a definir pontos críticos para o controle-vigilância entomológicos. Os dados sugerem que estas intervenções serão importantes inclusive em localidades já colonizadas pelo vetor. Em definitiva, temos desenvolvido, utilizando dados sobre variabilidade genética populacional, uma proposta capaz de dar conta do processo de invasão das Américas por A. aegypti e estabelecido as possíveis correspondências entre os padrões de diversidade genética desta espécie de vetor e as dinâmicas espaciais e temporais da epidemiologia da dengue no Brasil.

This study investigated some functional and biochemical characteristics of a bacteriolytic protein in the salivary glands of adult Aedes aecupti. A method for the isolation of this protein from other mosquito salivary gland components is also described. Based on some of its biochemical properties, this bacteriolytic protein can be classified as a lysozyme. This protein is strongly-linked to mosquito sugarfeeding activity because there is a statistically significant (P < 0.05) increase in the levels of lytic activity six hours before mosquitoes start to sugar feed. By its bacteriolytic action, it may function as a protective mechanism against bacteria-contaminated sugar meals. Preliminary work suggests that mosquitoes exposed to lyophilized Micrococcus lysodeikticus in their sugar meal respond by increasing the lytic activity in their salivary glands. The levels of bacteriolytic activity are apparently not affected by bloodfeeding. In the absence of feeding, as in teneral and bloodfed mosquitoes, salivary bacteriolytic activity increases to a maximum, then levels off. This suggests a regulation of the synthesis of this salivary protein that is independent of the feeding state of the adult mosquito. A combination of centrifugation, polyacrylamide gel electrophoresis (non-denaturing and denaturing), cation exchange chromatography and gel filtration, was used to isolate the protein from other mosquito salivary gland components. This salivary protein is lysozyme-like in several aspects: 1) it lyses bacterial cell walls of M. lysodeikticus, 2) it is a basic protein with a pI between 7.47 and 8.89, 3) it is thermostable at low pH, and loses its activity at high pH, and 4) it is composed of one polypeptide chain. Its molecular weight is twice that of hen egg white lysozyme. This salivary bacteriolytic protein is the first insect exocrine lysozyme to be characterized. / Graduation date: 1992

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.

Abstract Background. Vector borne diseases such as dengue fever and malaria are spread through hematophagous insects. Aedes aegypti is a species of mosquito that transmits dengue and chikungunya in Asia. In Lao PDR the estimated direct and indirect cost of dengue fever alone is 5 million USD. Even though research and innovations in the field of vaccines are moving forward there are yet no effective treatments for these diseases. Vector control methods are in place to suppress the Ae. aegypti population but there are still more than 100,000 cases annually. However, insecticide resistance, mosquito behavioral changes, high costs and health issues make todays measures inadequate. An effective measure is to decrease the mosquito-human contact by applying topical repellents. Aims. This study investigates plants used traditionally for repelling hematophagous insects in Laos, with the aim of finding means to empower local communities to create their own repellents. Methods. After interviewing local communities in Laos and reviewing literature, 24 candidate species were compiled. Lemongrass (Cymbopogon citratus) and ginger (Zingiber officinale) were hydro-distilled to extract essential oils. These oils were then analyzed through GS-MS to understand their chemical composition. Finally the essential oils were formulated with soybean oil to pilot a topical repellent that was tested in vivo on Ae. aegypti under controlled conditions. Results. The formulations elicited about 60 minutes of full protection but when combined, a possible additive effect was noted, prolonging the efficacy by nearly 50%. The main constituents of C. citratus are neral (34.77%) and geranial (56.44%) while, in the more complex, Z. officinale the main components are β-Linalool (9.84%), Geranial (14.44%) and Zingiberene (14.43%). Discussion and conclusions. Botanical repellents are a viable, cheap and sustainable solution of repelling hematophagous disease vectors. The mixture of ginger and lemongrass oil can be further improved in formulation by stabilizing it, and thus prolonging the protection. Increasing yield using alternative means of extracting the essential oils would also make these oils more feasible for commercial production.