Les arbovirus (virus transmis par des arthropodes) sont à l'origine de maladies humaines telles que la dengue, le chikungunya ou encore le Zika. Le moustique Aedes aegypti, est le vecteur majeur de ces trois arbovirus. La faible efficacité des méthodes de contrôle des populations de moustiques, principalement réalisées au moyen d'insecticides chimiques ouvre un champ de développement de nouvelles approches en lutte antivectorielle. Le moustique, hôte vecteur, contrôle la réplication virale en limitant les réponses immunitaires antivirales. La machinerie RNA interférence (RNAi) est la voie jouant un rôle majeur dans l'immunité antivirale chez le moustique. Alors que le rôle des deux voies, siRNA (" small interfering RNA ") et piRNA (" piwi-interfering RNA "), est de mieux en mieux compris dans les réactions antivirales du vecteur, peu de connaissances sont disponibles à ce jour en ce qui concernent les interactions entre la voie miRNA (" micro RNA ") et les arbovirus. Ainsi, nous proposons une analyse détaillée des mécanismes par lesquels les miARN tentent de réguler la réplication virale chez le moustique. Dans la première partie de la thèse, nous avons effectué une analyse génomique pour identifier les miRNAs pouvant interagir chez Ae. aegypti avec divers lignées/génotypes des virus chikungunya (CHIKV), de dengue (DENV) et de Zika. Avec l'aide d'outils de prédiction faisant appel à divers algorithmes, plusieurs sites de liaison de miARN avec différents lignées/génotypes de chaque arbovirus ont été identifiés. Nous avons ensuite sélectionné les miARN pouvant cibler plus d'un arbovirus et nécessitant un faible seuil d'énergie lors de la formation des complexes entre l'ARNm. / Mosquito-borne arboviruses cause some of the world’s most devastating diseases and are responsible for recent dengue, chikungunya and Zika pandemics. The yellow-fever mosquito. Aedes aegypti, plays an important role in the transmission of all three viruses. The ineffectiveness of chemical control methods targeting Ae. aegypti makes urgent the need for novel vector-based approaches for controlling these diseases. Mosquitoes control arbovirus replication by triggering immune responses. RNAi machinery is the most significant pathway playing a role on antiviral immunity. Although the role of exogenous siRNA and piRNA pathways in mosquito antiviral immunity is increasingly better understood, there is still little knowledge regarding interactions between the mosquito cellular miRNA pathway and arboviruses. Thus further analysis of mechanisms by which miRNAs may regulate arbovirus replication in mosquitoes is pivotal. In the first part of the thesis, we carried out genomic analysis to identify Ae. aegypti miRNAs that potentially interact with various lineages and genotypes of chikungunya (CHIKV), dengue (DENV) and Zika viruses. By using prediction tools with distinct algorithms, several miRNA binding sites were commonly found within different genotypes/and or lineages of each arbovirus. We further analyzed the miRNAs that could target more than one arbovirus and required a low energy threshold to form miRNA-vRNA (viral RNA) complexes and predicted potential RNA structures using RNAhybrid software. Thus, we predicted miRNA candidates that might participate in regulating arboviral replication in Ae. aegypti. In the second part of the thesis, we developed a miRNA-based approach that results in a dual resistance phenotype in mosquitoes to dengue serotype 3 (DENV-3) and chikungunya (CHIKV) viruses for stopping arboviruses spreading within urban cycles. The target viruses are from two distinct arboviral families and the antiviral mechanism is designed to function through the endogenous miRNA pathway in infected mosquitoes. Ten artificial antiviral 4 miRNAs capable of targeting ~97% of all published strains were designed based on derived consensus sequences of CHIKV and DENV-3. The antiviral miRNA constructs were placed under control of either an Aedes PolyUbiquitin (PUb) or Carboxypeptidase A (AeCPA) gene promoter triggering respectively expression ubiquitously in the transgenic mosquitoes or more locally in the midgut epithelial cells following a blood meal. Challenge experiments using viruses added in blood meals showed subsequent reductions in viral transmission efficiency in the saliva of transgenic mosquitoes as a result of lowered infection rate and dissemination efficiency. Several components of mosquito fitness, including larval development time, larval/pupal mortality, adult lifespan, sex ratio, and male mating competitiveness, were examined: transgenic mosquitoes with the PUb promoter showed minor fitness costs at all developing stages whereas those based on AeCPA exhibited a high fitness cost. Further development of these strains with gene editing tools could make them candidates for releases in population replacement strategies for sustainable control of multiple arbovirus diseases.

<p>Meteorological&nbsp; conditions&nbsp; have&nbsp; a&nbsp; significant influence on the time of occurrence, abundance and activity of the mosquito vector. In the current context of climate change, it is of great importance to assess the&nbsp; impact&nbsp; of&nbsp; shifts&nbsp; in&nbsp; climatic&nbsp; conditions&nbsp; on&nbsp; the suitability for the establishment and annual activity of&nbsp; the&nbsp; vector&nbsp; species.&nbsp; Moreover,&nbsp; changes&nbsp; in&nbsp; the variability&nbsp; of&nbsp; meteorological&nbsp; elements&nbsp; and&nbsp; their extremes&nbsp; can&nbsp; generate&nbsp; unexpected&nbsp; changes&nbsp; in&nbsp; the mosquito&nbsp; vector&nbsp; population&nbsp; which&nbsp; in&nbsp; turn&nbsp; have&nbsp; an&nbsp; important effect on human health. One of the ways to put&nbsp; these&nbsp; causes&nbsp; and&nbsp; effects&nbsp; into&nbsp; perspective&nbsp; is&nbsp; to simulate the activity of the vector within a processbased framework which allows for the analysis of the contribution of individual factors on the different life stages of the vector. Such analysis is presented by use of&nbsp; sophisticated&nbsp; dynamical&nbsp; models&nbsp; simulating&nbsp; the characteristics of the biological population, forced by observed&nbsp; meteorological&nbsp; data,&nbsp; capturing&nbsp; the&nbsp; localmicro-environment&nbsp; of&nbsp; the&nbsp; vector&nbsp; habitat,&nbsp; and validated by the observed entomology.Numerical&nbsp; models&nbsp; are&nbsp; being&nbsp; developed&nbsp; to&nbsp; model vector&nbsp; population&nbsp; dynamics&nbsp; and&nbsp; the&nbsp; expected circulation of the virus within a closed system. Two modelling&nbsp; approaches&nbsp; are&nbsp; standardly&nbsp; applied&nbsp; to modelling vector population dynamics: Mechanistic and&nbsp; Stochastic.&nbsp; The&nbsp; advantage&nbsp; of&nbsp; mechanistic&nbsp; over<br />statistical&nbsp; models&nbsp; is&nbsp; that&nbsp; they&nbsp; can&nbsp; provide&nbsp; a deterministic&nbsp; framework&nbsp; allowing&nbsp; for&nbsp; the&nbsp; isolated evaluation of each input parameter and their effect on the modelled system. Mechanistic dynamical models are used to describe the biophysical processes or part<br />of&nbsp; the&nbsp; process&nbsp; as&nbsp; a&nbsp; response&nbsp; to&nbsp; changes&nbsp; in&nbsp; the meteorological conditions.<br />The&nbsp; work&nbsp; carried&nbsp; out&nbsp; in&nbsp; this&nbsp; thesis&nbsp; can&nbsp; be summarized as follows: (i) Analysis of the association between&nbsp; the&nbsp; most&nbsp; important&nbsp; abiotic&nbsp; drivers influencing the population dynamics, annual activity and&nbsp; time&nbsp; of&nbsp; occurrence&nbsp; of Culex&nbsp; pipiens and Aedes aegypti;&nbsp; (ii)&nbsp; Identifying&nbsp; the&nbsp; most&nbsp; important&nbsp; climatic factors and model settings as a function of climatic characteristics of the study region; (iii) Modelling the vector population dynamics and stability analysis of the&nbsp; dynamical&nbsp; system&nbsp; (iv)&nbsp; Analysis&nbsp; of&nbsp; different verification techniques and implications in terms of model&nbsp; application;&nbsp; (v)&nbsp; Feasibility&nbsp; analysis&nbsp; of<br />improving&nbsp; the&nbsp; model&nbsp; with&nbsp; a&nbsp; Land-Surface Parametrization scheme and short-range forecasting of pest population dynamics.</p> / <p>Meteorolo&scaron;ki&nbsp; uslovi&nbsp; bitno&nbsp; utiču&nbsp; na&nbsp; vreme&nbsp; pojave, brojnost&nbsp; vektora&nbsp; i&nbsp; njihovu&nbsp; aktivnost.&nbsp; U&nbsp; uslovima evidentnih promene klime, od ogromne je važnosti sagledati&nbsp; uticaj&nbsp; očekivanih&nbsp; promena&nbsp; klime&nbsp; na pogodnost&nbsp; uslova&nbsp; na&nbsp; pojavu&nbsp; izabranih&nbsp; vektora.<br />Takodje, značajne promene kolebanja meteorolo&scaron;kih elemenata&nbsp; u&nbsp; odnosu&nbsp; na&nbsp; vi&scaron;egodi&scaron;nji&nbsp; prosek&nbsp; i&nbsp; sve če&scaron;će pojave nepovoljnih vremenskih prilika dovode do neočekivanog pona&scaron;anja populacije komarca &scaron;to značajno&nbsp; utiče&nbsp; na&nbsp; kvalitet&nbsp; života&nbsp; i&nbsp; zdravlje&nbsp; ljudi. Jedini&nbsp; način&nbsp; da&nbsp; se&nbsp; sagledaju&nbsp; uzroci&nbsp; i&nbsp; posledice navedenih pojava zasniva se na simulaciji aktivnosti i&nbsp; brojnosti&nbsp; vektora&nbsp; uz&nbsp; mogućnost&nbsp; testiranja&nbsp; uticaja svakog&nbsp; pojedinačnog&nbsp; faktora.&nbsp; Ovu&nbsp; mogućnost pružaju samo visoko sofistikovani dinamički modeli koju su pro&scaron;li proces kalibracije i validacije zasnovan<br />na izmerenim vrednostima meteorolo&scaron;kih elemenata i karakteristika biolo&scaron;ke populacije.<br />Sofistikovani&nbsp; modeli&nbsp; za&nbsp; simulaciju&nbsp; dinamike populacije vektora i očekivane cirkulacije vektorskih transmisivnih bolesti se koriste sa ciljem modeliranja potencijalnog rizika od zaraze i epidemije. Modeli zasimulaciju dinamike vektora mogu da se podele na dve&nbsp; glavne&nbsp; grupe:&nbsp; Mehanističke&nbsp; i&nbsp; Statističke. Prednost&nbsp; mehanističkih&nbsp; modela&nbsp; nad&nbsp; statističkim&nbsp; je &scaron;to&nbsp; mogu&nbsp; da&nbsp; se&nbsp; koriste&nbsp; za&nbsp; evaluaciju&nbsp; uticaja izolovanog&nbsp; faktora&nbsp; na&nbsp; dinamički&nbsp; sistem&nbsp; i odgovarajuće promene brojnosti unutar svake faze u razvoju&nbsp; vektora.&nbsp; Mehanistički&nbsp; dinamički&nbsp; sistemi&nbsp; se koriste&nbsp; kako&nbsp; bi&nbsp; se&nbsp; opisao&nbsp; mehanizam&nbsp; biofizičkog procesa&nbsp; ili&nbsp; dela&nbsp; procesa&nbsp; u&nbsp; zavisnosti&nbsp; od&nbsp; forsirajuće veličine.<br />Predmet&nbsp; istraživanja&nbsp; u&nbsp; ovom&nbsp; radu&nbsp; jeste identifikovanje&nbsp; najznačajnijih&nbsp; biolo&scaron;kih&nbsp; i&nbsp; fizičkih procesa&nbsp; kao&nbsp; i&nbsp; odgovarajućih&nbsp; faktora&nbsp; koji&nbsp; utiču&nbsp; na brojnost i aktivnost vektora roda Aedes i Culex. Ciljevi istraživanja mogu da se sumiraju na sledeći način: (i)<br />analiza najznačajnijih meteorolo&scaron;kih parametara koji utiču na vreme pojave, brojnost i aktivnost vektora Aedes&nbsp; i&nbsp; Culex&nbsp; roda;&nbsp; (ii)&nbsp; definisanje&nbsp; najznačajnijih klimatskih&nbsp; faktora&nbsp; i&nbsp; stepena&nbsp; osetljivosti&nbsp; procesa&nbsp; na njih; (iii) modeliranje dinamike populacije vektora i analiza&nbsp; stabilnosti&nbsp; dinamičkog&nbsp; sistema;&nbsp;&nbsp; (iv) verifikacija&nbsp; i&nbsp; analiza&nbsp; metoda&nbsp; verifikacije&nbsp; i&nbsp; validacije dinamičkog&nbsp; modela;&nbsp; (v)&nbsp; kratkoročna&nbsp; prognoza dinamike&nbsp; populacije&nbsp; komarca&nbsp; i&nbsp; formulacija hidrolo&scaron;kog modula upotrebom SURFEX povr&scaron;inske<br />&scaron;eme sa ECOCLIMAP fiziogeografskim podacima.</p>

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Made available in DSpace on 2016-01-26T12:51:53Z (GMT). No. of bitstreams: 1 margarethdibo_tese.pdf: 1810718 bytes, checksum: 4df01c13c55a541f2c4aff4668fbacd7 (MD5) Previous issue date: 2004-03-17 / Fundação de Amparo a Pesquisa do Estado de São Paulo / The strategy to contain the vector of Dengue fever in the state of São Paulo is centered around a reduction in breeding places and by chemical controls. To obtain better results in the reduction of the vectorial density it is important to incorporate other measures in the routine approach. Egg traps are considered to be preventative and control measures of Aedes aegypti, however, there are no studies that define the favorite egg-laying locations in residences. The utilization of an infusion of Panicum maximum in traps increases its attractiveness to vector females and increases the number of eggs collected. This study aims to contribute to techniques by using traps to evaluate the number of weeks this infusion remains attractive to females. More importantly, it aims to assess the egg-laying behavior of the Aedes aegypti and Aedes albopictus populations in Mirassol, a town situated in the region of São José do Rio Preto, Brazil. Traps with egg-collecting paddle were installed in 200 houses in 20 blocks giving a total of 10 houses to each block. Half of the blocks were chosen at random for the installation of traps baited with the infusion and in the other half, traps with ordinary water were installed. Two egg-traps were placed inside the house (bedroom and living room) and two outside (one under cover and the other in the open). Weekly visits were made from September to December 2002, to analyze all the traps, collecting the existing sticks and leaving new ones. In the laboratory the viable and nonviable eggs on the sticks were counted. Positive sticks were considered to be those on which eggs were found and these were correlated with the type of trap, infusion or water, the location of the trap within the house and the number of weeks after the start of the trial. A sample of these sticks was placed in water, to hatch the larvae with the aim of identifying the species. The mean numbers of eggs and larvae of the Aedes aegypti and Aedes albopictus were calculated and associated with the location within the house, the type of trap used, infusion or water, and the week number. Analyzing the number of positive sticks, and the mean number of eggs on the traps, it was evidenced that the traps with infusion were more attractive/stimulating for egg-laying for females of the genera Aedes than those with water in the first three weeks of exposition. The uncovered outdoor traps were the most favored location for egg-laying during the entire study period; in second place were the outdoor ones under cover. High values of the Spearman correlation coefficients and p-values less than 0.004 demonstrated a strong correlation between the positivity and the mean number of eggs, indicating that one variable predicts the other. From the analysis of the hatched larvae, Aedes aegypti were identified on 99.6% (CI 95%: 99.04 99.84) of the sticks and Aedes albopictus on 1.8% (CI 95%: 1.19 2.70) of them. In Mirassol the probability of finding eggs of Aedes aegypti on any one stick under these circumstances is greater than 99%. / A estratégia para controle do vetor do dengue no estado de São Paulo está centrada na redução de criadouros e ações de controle químico. Para a obtenção de melhores resultados na redução da densidade vetorial é importante que se incorpore outras medidas de uso rotineiro. Armadilhas de oviposição são consideradas medidas de vigilância e controle de Aedes aegypti, entretanto não existem estudos sobre locais do domicílio preferidos para oviposição. A utilização de infusão de Panicum maximum em armadilhas propicia maior atração às fêmeas do vetor e coleta de maior quantidade de ovos. Através deste estudo, procurou-se subsidiar as técnicas para o uso de armadilhas, avaliar durante quantas semanas a referida infusão manteve sua atração sobre as fêmeas e, principalmente, avaliar o comportamento de oviposição de populações de Aedes aegypti em Mírassol, cidade situada na região de São José do Rio Preto. Instalaram-se armadilhas de oviposição em 200 casas, divididas em 20 quadras, distribuindo-as em 10 casas por quadra. Foram sorteadas 50% das quadras para instalação de armadilhas iscadas com infusão, e nas demais se instalou armadilhas com água. Duas armadilhas foram colocadas no intradomicílio (quarto e sala) e duas no peridomicílio (uma sob cobertura e outra ao relento). Foram feitas visitas semanais entre setembro e dezembro de 2002, para a análise de todas as armadilhas, e substituição das palhetas. Em laboratório, contaram-se ovos viáveis e inviáveis existentes nas palhetas. A presença de ovos na palheta significou a sua positividade, a qual foi verificada quanto à presença ou não de infusão, local de instalação na casa e número de semanas. Uma amostra destas palhetas foi colocada em água, para eclosão de larvas, visando à identificação específica. Os números médios de ovos e de larvas Aedes aegypti e Aedes albopictus foram quantificados segundo o posicionamento no domicílio, presença de armadilha iscada com infusão ou água e o número de semanas. Analisando-se Nota de Resumo as positividades e os números médios de ovos das armadilhas, obteve-se que as armadilhas iscadas com infusão mostraram-se mais atraentes/estimulantes para oviposição pelas fêmeas do gênero Aedes que as com água, nas três primeiras semanas de exposição. O relento foi o local com maior preferência para oviposição durante todo o estudo; em segundo lugar encontrou-se o coberto. Elevados valores dos coeficientes de correlação de Spearman e valores de p inferiores a 0,004 mostraram forte correlação entre positividade e média de ovos, indicando que uma variável prediz a outra. A partir das análises das larvas eclodidas, identificou-se a presença de Aedes aegypti em 99,6% (IC 95%: 99,04 -99,84) das palhetas e de Aedes albopictus em 1,8% (1C95%: 1,19 - 2,70) delas. Em Mirassol, a probabilidade de se encontrar ovos de Aedes aegypti em uma palheta sob estes condições é superior a 99%.

Zika (ZIKV), La Crosse (LACV), and Cache Valley (CVV) viruses are mosquito-vectored diseases that cause significant morbidity and mortality in humans and animals. Transmission of these viruses are dependent on numerous factors including vector competence and the effects of mosquito-virus interactions. We conducted vector competence studies of local Aedes and Culex mosquitoes for ZIKV and CVV, and found that all Aedes mosquitoes were competent for CVV and only Aedes albopictus and Aedes japonicus were competent for ZIKV. Vector competence for CVV was dose-dependent, where mosquitoes orally infected with high titers developed higher transmission rates. We also found that vector competence for ZIKV was limited by midgut and salivary gland barriers. Second, we looked at the effects of LACV and ZIKV infection on repellent response in Aedes mosquitoes and found that infected mosquitoes were refractory to low concentrations of DEET, picaridin, and PMD. Increasing concentrations of the repellents to ≥10% was able to increase percent protection (%p) against infected and uninfected mosquitoes. Lastly, we determined the effects of ZIKV and LACV infection on oxidative stress and glutathione-S-transferase (GST) activity in Aedes albopictus. Virus infection had no effect on oxidative stress, but GST activity was significantly different for mosquitoes 3-days post-exposure. We found that oxidative stress levels and GST activity had an inverse relationship for infected and uninfected mosquitoes, where oxidative stress decreased and GST activity increased over the 10-day test period. This indicates that GSTs may aid in controlling byproducts of oxidative stress. The results from this entire study identified competent vectors for emerging arboviruses and demonstrated the behavioral and physiological effects of virus infection in the mosquito vector. / Doctor of Philosophy / Zika (ZIKV), La Crosse (LACV), and Cache Valley (CVV) viruses are transmitted by mosquitoes and can make humans and animals very sick. There are many biological factors that determine if a mosquito can transmit a virus and these viruses can change the biology of a mosquito. We conducted laboratory studies to see if Aedes and Culex mosquitoes can transmit ZIKV and CVV. We found that all Aedes mosquitoes were able to transmit CVV and only the Asian tiger mosquito and Asian rock pool mosquito were able to transmit ZIKV. Mosquitoes infected with high amounts of CVV developed higher transmission rates. We also found that transmission of ZIKV was limited by barriers in the mosquito midgut and salivary glands. Second, we looked at the effects of LACV and ZIKV infection on how Aedes mosquitoes respond to repellents and found that infected mosquitoes were less sensitive to low concentrations of DEET, picaridin, and PMD. Increasing concentrations of the repellents to 10% or higher was able to provide adequate protection against infected and uninfected mosquitoes. Lastly, we determined the effects of ZIKV and LACV infection on oxidative stress and glutathione-S-transferase (GST) activity in the Asian tiger mosquito. Virus infection did not change oxidative stress, but GST activity was higher in infected mosquitoes tested after 3 days after infection. We found that oxidative stress decreased and GST activity increased over the 10-day test period. This indicates that GSTs may help control damaging products from oxidative stress. The results from this entire study identified what mosquitoes were able to transmit emerging mosquito-borne viruses and demonstrated the biological effects of virus infection in the mosquitoes.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Stephen Higgs / The genetic composition of arboviruses is a critical determinant of viral infectivity and the capacity for virus dissemination in arthropod vectors. Due to concerns related to a hypothetical potential for loss of attenuation, the supression of vector infection and dissemination is a critical component for the rationale-based design of live-attenuated flavivirus vaccine candidates. The yellow fever virus (YFV) 17D vaccine virus is not only attenuated in vertebrates, but also has low infectivity for Aedes agypti mosquitoes and since it does not disseminate, it is not transmissible. Using a reverse genetics system, the mutations present in the envelope protein YFV 17D virus were characterized in Ae. aegypti to determine the role of mutations in limiting the viral infectivity and dissemination capacity. This knowledge would contribute to the rational design of live attenuated vaccines with the desirable phenotype of being nontransmissible by arthropod vectors. The upper lateral portion of the YFV 17D envelope (E) protein domain III (EDIII) habors the T380R mutation in the FG loop. Experiments demonstrated that the T380R mutation was associated with the viral infectivity phenotype for mosquitoes, but did not influence dissemination into the secondary tissues. The G52R mutation in the molecular hinge region that is located between E protein domains I (EDI) and II, significantly reduced viral infectivity for mosquitoes. In contrast, when cloned into the Asibi wildtype virus genetic backbone, the T173I mutation in the loop structure between the G0 and H0 β- strands did not attenuate viral infection and dissemination. The double mutant virus containing both the G52R and T173I mutations in the E protein, showed a similar attenuated reduced infectivity to the single G52R mutant. The M299I mutation in the linker region between EDI and EDIII resulted in a significantly lower viral infectivity at the initial phase of viral infection at 7 days post-infection in Ae. aegypti. In conclusion, the characterization on four mutations in the YFV 17D vaccine E protein have demonstrated three genetic loci, that can influence the process of YFV infection in Ae. aegypti. These results provide new knowledge and understanding which may have broad applications for the rationale design of safe flavivirus vaccines, via targeting genetic loci and introducing specific mutations that preclude infection of, and transmission by arthropod vectors.

Female mosquitoes of the species, Aedes aegypti (yellow fever mosquito, Diptera), blood feed for oogenesis. Therefore, mosquitoes are exposed to high iron loads and possibly blood-borne pathogens. We are interested in studying iron metabolism in A. aegypti to find methods for controlling mosquito populations, and thereby reduce human exposure to these pathogens. First, we found that the expression of the Aedes ferritin light chain homologue (LCH) is up-regulated by blood feeding. Ferritin LCH and heavy chain homologue (HCH) genes are closely clustered together and both mRNA transcripts increase with iron and oxidative stress (H2O2 and hemin). Second, we show A. aegypti larval cells synthesize and secrete ferritin in response to iron. Cytoplasmic ferritin is maximal at low levels of iron, consists of a specific subunit composition and reflects cytoplasmic iron levels. Secreted ferritin increases in linear relationship to increasing iron dose and is composed of different subunits than cytoplasmic ferritin. HCH and LCH transcripts increase with increasing cytoplasmic iron suggesting transcriptional control of ferritin synthesis. We previously reported that the mosquito HCH mRNA has an iron responsive element (IRE), but LCH mRNA does not have a canonical IRE. We show that iron regulatory protein 1 (IRP1)/IRE binding activity declines in response to increasing cytoplasmic iron levels. These data would indicate that HCH synthesis is controlled at transcription and translation. Third, we report that A. aegypti larval cell cytoplasmic iron concentration does not change temporally with iron treatment. However, membrane iron levels increase with iron over time. Iron temporally up-regulates both HCH and LCH mRNA. Ferritin secretion increases with time in response to iron and reflects that most of the intracellular ferritin is found in the membrane fraction. Membrane ferritin has the same subunit composition as cytoplasmic ferritin. Finally, membrane ferritin is found in both non-iron and iron-treated cells. This suggests a mechanism to store iron from a blood meal in membrane ferritin. These results indicate Aedes ferritin could act as an antioxidant and holoferritin secretion is likely the mechanism whereby mosquito cells protect against iron overload and, thus reduce the intracellular potential for iron-mediated oxidative stress during blood feeding.

Aedes aegypti Linnaeus, 1762 es una especie de Diptero que pertenece a la familia Culicidae, subgénero Stegomyia Theobald, 1901. La investigación túvo como objetivos: Realizar vigilancia epidemiológica de Aedes aegypti en el distrito de Villa El Salvador, determinar indicadores entomológicos: Índice Aédico (IA), Índice de Recipientes (IR) e Índice de Breteau (IB) y si existía diferencias significativas entre ellos. Para ellos se realizó un diseño de estudio donde se incluye el protocolo establecido por el Ministerio de Salud para el estudio de la localidad, visita a las viviendas y colección de muestras. Al comparar las fluctuaciones de los Índices Entomológicos (IE): Índice Aédico (IA), Índice de Recipientes (IR) e Índice de Breteau (IB), por sectores y meses se observó que no existieron diferencias estadísticamente significativas. Solo se halló foco positivos de Aedes aegypti durante los meses de Marzo y Abril en los sectores 2 y 3 respectivamente. Aun cuando se focalizó el análisis para los dos meses y sectores, estadísticamente no se encontraron diferencias significativas ni riesgo entomológico; esto último de acuerdo a los porcentaje establecidos en la estratificación de riesgo entomológico.Aedes aegypti Linnaeus, 1762 is a species of Diptera that belongs to the Culicidae family, subgenus Stegomyia Theobald, 1901. This study aimed to: perform a surveillance of Aedes aegypti in the district of Villa El Salvador, determine the entomological indicators: Aédico Index (AI), Container Index (RI) and Breteau Index (BI) and if there were significant differences between them. For those purposes, it a study design that includes the established Ministry of Health protocol for the study of the town, home visitings and sample collection. When comparing the fluctuations of the Entomological indices (EI): Aédico Index (AI), Container Index (RI) and Breteau Index (BI), by sector and months, no statistically significant differences were observed. The only positive focus for Aedes aegypti was found during the months of March and April in sectors two and three, respectively. Although further analysis was focused for the two months and sectors mentiones, no statistically significant differences or entomological risk was found, the latter according to the percentage specified in entomological risk stratification

Aedes aegypti is one of the most important mosquito vectors of human disease, transmitting dengue, Chikungunya and yellow fever viruses. New control methods are much needed for this species and an ability to skew sex ratios toward males would be very useful for a number of strategies. In this study, male-specific chromosomal regions were searched for in Ae. aegypti using novel approaches such as microdissection of chromosomes. The sex determination pathway of Ae. aegypti was also explored, using Drosophila melanogaster as a model, to find candidate genes that could be used to induce male biased sex ratios. The transformer-2 (tra-2) gene is necessary for sexual differentiation of females in D. melanogaster, but its role remains unknown in mosquitoes. A homolog of tra-2 was identified in Ae. aegypti. Rapid Amplification of cDNA ends (RACE) experiments were conducted to characterize this gene in Ae. aegypti. The Ae. aegypti tra-2 gene, as for D. melanogaster, was highly variable in transcription due to alternative splicing and alternative polyadenylation, with 9 different variants identified. RNA interference (RNAi) was then used to determine if knockdown of all variants of Ae. aegypti tra-2 can be achieved and if it would cause gender switching in individuals, initially by means of direct injection of double stranded RNA, and then progressing to germline transformation. A construct designed to produce tra-2 dsRNA was injected into Ae. aegypti embryos and integrated into the Ae. aegypti genome. The transgenic population showed up to 100% male bias in single pair crosses. The male bias effect could still be seen in the heterozygous population. The results suggest that knockdown of tra-2 could provide a useful tool for sex ratio distortion as part of the development of novel control methods for Ae. aegypti.

A família dos culicídeos é diversa e possui muitas espécies com alta relevância epidemiológica e que transmitem os patógenos causadores de doenças como dengue, febre amarela e malária. Os imaturos se criam exclusivamente na água e nos mais diversos tipos de criadouros, podendo ser influenciados por fatores biológicos e fatores físico-químicos, que incluem pH, salinidade, condutividade, sólidos totais dissolvidos, entre outros. Entender as variações das condições dos criadouros é importante para entender e esclarecer como esses fatores agem no desenvolvimento dos imaturos, na sua fisiologia e nas relações interespecíficas, que influenciam o desenvolvimento larval e a sobrevivência dos mosquitos. Portanto, o objetivo principal desse trabalho foi investigar a influência de fatores físico-químicos e do tipo de criadouro sobre a ocorrência das espécies de culicídeos em dois remanescentes de Mata Atlântica no município de São Paulo. As coletas dos imaturos ocorreram em três diferentes pontos no Parque Estadual da Cantareira e quatro diferentes pontos na Área de Proteção Ambiental Capivari-Monos, remanescentes de Mata Atlântica inseridos na Grande São Paulo, durante vinte e sete meses para as coletas de diversidade geral e doze meses para as coletas de diversidade juntamente com fatores físico químicos da água. Os imaturos foram coletados em bromélias epífitas e de solo, bambus, poças d\'água no solo e em pedras, ocos de árvore, lagos e recipientes artificiais. As análises dos dados foram feitas através do cálculo do coeficiente de variação das variáveis para os tipos de criadouros, Análise Restrita de Proximidade (CAP), Análise de Correspondência Canônica (CCA) e boxplots sem outliers. Os resultados indicaram que há uma diferença estatisticamente significante na composição de espécies em função dos diferentes tipos de criadouros e que o pH possui influência sobre a ocorrência das espécies, tanto para a variável tipo de criadouro quanto para situações em que se retira essa variável. As espécies com importância epidemiológica coletadas foram: Aedes aegypti, Aedes albopictus, Anopheles cruzii, Haemagogus leucocelaenus, Psorophora ferox e Sabethes purpureus, com ocorrência das duas espécies de Aedes e Haemagogus no mesmo criadouro. Esses resultados são importantes para reforçar a necessidade do monitoramento de criadouros das espécies vetoras de patógenos e para um melhor entendimento da ecologia das espécies. / The Culicidae family is diverse and has many species of high epidemiological relevance that transmit the pathogens that cause diseases such as dengue fever, yellow fever and malaria. The immatures are created exclusively in the water and in the most diverse types of breeding places, being able to be influenced by biological factors and physicochemical factors, that include pH, salinity, conductivity, total solids dissolved, among others. Understanding variations in breeding conditions is important in understanding and clarifying how these factors affect the development of immatures, their physiology, and interspecific relationships that influence larval development and mosquito survival. Therefore, the main objective of this work was to investigate the influence of physical-chemical factors and breeding type on the occurrence of Culicidae species in two remnants of Atlantic Forest in the city of São Paulo. Immature collections occurred at three different points in the Cantareira State Park and four different points in the Capivari-Monos Environmental Protection Area, remnants of the Mata Atlântica, located in Greater São Paulo, during twenty-seven months for collections of general diversity and twelve months for collections of diversity along with physical chemical factors of water. The immatures were collected in epiphytic and soil bromeliads, bamboos, pools of water in the soil and stones, tree hollows, lakes and artificial containers. Data analysis was done by calculating the coefficient of variation of the variables for breeding types, Restricted Proximity Analysis (CAP), Canonical Correspondence Analysis (CCA) and boxplots without outliers. The results indicated that there is a statistically significant difference in species composition as a function of the diverse types of breeding sites and that pH has an influence on the occurrence of species, both for the type of breeder variable and for situations in which this variable is removed. The species with epidemiological importance were: Aedes aegypti, Aedes albopictus, Anopheles cruzii, Haemagogus leucocelaenus, Psorophora ferox and Sabethes purpureus, with occurrence of both Aedes and Haemagogus species in the same breeding ground. These results are important to reinforce the need to monitor breeding sites of pathogen vector species and to better understand species ecology.