Thesis--Université de Paris Sud, Centre d'Orsay, 1977. / Summary in French and English. Includes bibliographical references (p. 225-228).

Master of Science / Department of Biology / Rollie Clem / Apoptosis, or programmed cell death, is a crucial conserved process among organisms for deleting damaged unwanted cells, as well as for development and viral defense, and plays an important role in multiple diseases. Too much apoptosis may lead to Alzheimer’s disease, and too little may result in cancer. Therefore, the ability to understand this process is essential for improved medical knowledge today. Apoptosis has been explored in a number of species and pathways seem relatively conserved among most, with unique aspects contained in each, but little is known about apoptosis in mosquitoes. Improved knowledge and growing interest concerning apoptosis in mosquitoes is necessary considering the vast health effects seen across the globe as a result of diseases transferred by the mosquito vector. The Dengue virus mosquito vector Aedes aegypti was the focus here. A new player named defense repressor 1 was discovered in Drosophila melanogaster (DmDnr1), shown to play a role in apoptosis, and the homolog discovered in A. aegypti (AeDnr1). Silencing Dmdnr1 resulted in cells sensitized to apoptosis but was not enough to induce spontaneous apoptosis. In contrast, silencing Aednr1 in the A. aegypti cell line, Aag2, led to spontaneously induced apoptosis. This showed the importance of AeDnr1 as a member of the apoptotic pathway in this species. Epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the initiator caspase Dronc and the effector caspase CASPS8, whereas apoptosis induced by silencing the inhibitor of apoptosis, Aeiap1, also requires Dronc but acts through the effector caspase CASPS7. Further epistasis experiments showed that apoptosis induced by silencing Aednr1 requires the IAP antagonist Mx, but not IMP. This showed for the first time a gene regulating upstream of an IAP antagonist. Biochemical studies showed that AeDnr1 regulates active CASPS8 but not CASPS7, and interacts with Mx and CASPS8 but not AeDronc, CASPS7 nor AeIAP1. Studies also showed Mx competes effectively with CASPS8 but not CASPS7 for AeIAP1 binding, and IMP competes effectively with CASPS7 but not CASPS8 for AeIAP1 binding. An improved apoptosis pathway for the mosquito A. aegypti emerged involving a potential feedback loop with explanations for the upstream IAP antagonist preference as well as the downstream effector caspase preference resulting from apoptosis induced by Aednr1 silencing. Through the discussed research, multiple unique findings resulted. Studying the mosquito model will allow us to find certain gene relations that are more difficult to uncover in the Drosophila model. Because Dnr1 is found in most systems, this improved pathway may shed light not only on a potential role of Dnr1 in apoptosis in insects but higher organisms as well.

No description available.

published_or_final_version / Nursing Studies / Master / Master of Nursing

A new strategy to fight mosquito-borne disease is based on infections of the maternally-transmitted, intracellular bacterium Wolbachia pipientis. Estimates predict that Wolbachia infects nearly half of all insect species, as well as other arthropods and some nematodes. Wolbachia manipulates the reproduction of its host to promote infection, most commonly causing a form of conditional sterility known as cytoplasmic incompatibility. Generally, Wolbachia infections are benign and do not inflict significant costs upon its host. However, studies demonstrate that some infections are associated with substantial costs to its host. These same infections can also induce pathogen interference and decrease vector competency of important disease vectors. Theory predicts that organisms that incur costs relative to conspecifics are less competitive and their competitive exclusion is expected. In the case of Wolbachia, the bacterium can influence reproduction such that phenotypes with lower fitness may still reach fixation in natural populations. In this dissertation, I describe theoretical and empirical experiments that aim to understand the invasion and stability of Wolbachia infections that impose costs on their host. Particular attention is paid to immature insect lifestages, which have been previously marginalized. These results are discussed in relation to ongoing vector control strategies that would use Wolbachia to manipulate vector populations. Specifically, I discuss the cost of novel Wolbachia infections in Aedespolynesiensis, which decreases larval survival and overall fitness relative to wild-type mosquitoes. Then, a theoretical framework was developed to determine the significance of reductions in larval viability in relation to the population replacement disease control strategy. Further theoretical studies determined that Wolbachia infections, once established, resist re-invasion by uninfected individuals despite relatively high costs associated with infection so long as the infection produces reproductive manipulations. Additional studies determined that larvae hatched from old eggs experience reduced survival in mosquito strains with novel Wolbachia infections when compared to the wild-type. To validate the theoretical studies, model predictions were tested empirically to determine the importance of the larval viability. Finally, a COPAS PLUS machine was evaluated and its role in understanding early larval development in mosquitoes is discussed. The importance of integrated research in disease control is highlighted.

<p>Mosquitoes are found in extremely large numbers in the lower parts of the River Dalälven. In the year 2000 the mosquito nuisance was especially high, resulting in foundation of the Biological mosquito control project. Since 2001 mosquito larvae are controlled by using a biological pesticide BTI (Bacillus thuringiensis ssp israelensis). The mosquito fauna in the area is dominated by flood water mosquitoes, a group of mosquitoes that are very aggressive and form new generations of mosquitoes during every single flooding event during the summer. To be able to efficiently control the mosquitoes it is essential to know the extension and locality of the flooding. A flooding event is evaluated by how high the water level will reach and at which time the top of the hydrograph is accruing. There is a need for a tool for short time prediction of flooding events in the Nedre Dalälven region. In this study a hydraulic model in the software HEC-RAS has been used, here called HEC-RAS NEDA. The hydraulic model came up with good results when predicting water surface levels. The validation process made from figures of a period with large amplitude in water levels showed that the modelled water level was within ± 0,05 m from the observed water stands. It is a stable model that can handle 100-year flows with quick changes in the flow. The correspondence is good concerning the water levels and it functions well when studying the top of the hydrograph. HEC-RAS NEDA is an accurate and easy to manage tool for prediction of flooding in the lake of Bysjön, Österviken and Färnebofjärden. Modelled results have been compared with the model made by Swedish meteorological and hydrological institute on the request of Räddningsverket. The results from the two models shows large discrepancies, probably because of low accuracy data entered into the model from Räddningsverket. Räddningsverket has used the Geographical Sweden Data height data bank´and I used the height data from a laser scanned digital terrain model. Area studies have also been made according to how the area of the water surface changes with the water stand. These studies show that during the flood in year 2000, 55 km2 was flooded based on laserscanning in Färnebofjärden.</p> / <p>Mygg förekommer i extremt stora antal i området kring nedre delen av Dalälven. År 2000 var myggplågan särskilt stor vilket medförde att projekt Biologisk Myggkontroll bildades. Myggbekämpning har bedrivits i Nedre Dalälven sedan 2001 med ett biologiskt bekämpningsmedel, Bacillus thuringiensis ssp israelensis (BTI). Områdets myggfauna domineras av så kallade översvämningsmygg, en grupp stickmyggor som är mycket aggressiva och bildar nya generationer efter varje enskild översvämning under sommaren. För en effektiv bekämpning av mygglarverna krävs att man vet översvämningens omfattning och lokalisering. En översvämning bedöms med avseende på hur högt vattenståndet når samt vid vilken tidpunkt hydrografens toppvärde inträffar. Det finns ett behov av ett verktyg som kan utföra korttidsprognoser av översvämningar i Nedre Dalälven. I denna studie har en hydraulisk modell i programvaran HEC-RAS använts, här kallad för HEC-RAS NEDA. Den hydrauliska modellen fungerar bra till att användas för prediktering av vattenstånd i Nedre Dalälven. Valideringen av modellen mot värden under en testperiod med stor amplitud i vattenståndet visade att modellerade vattenstånd var inom ± 0,05 m från de uppmätta värdena. Modellen är stabil och klarar av att hantera 100-årsflöden med snabba flödesfluktuationer. HEC-RAS NEDA är ett noggrant och lätthanterligt predikteringsverktyg för översvämningar i Bysjön, Österviken och Färnebofjärden. Överensstämmelsen är god gällande nivån av vattenståndet och den fungerar bra för att studera hydrografens topp temporärt. Modellerade resultat har jämförts med karteringar som Sveriges meteorologiska och hydrologiska institut har gjort på Räddningsverkets begäran. Resultaten skiljer sig betydligt mellan HEC-RAS NEDA och Räddningsverkets modell, troligen på grund av att Räddningsverket har stora svagheter i indata till modellen. Räddningsverket har använt Geografiska Sverige Data-Höjddata och modellen HEC-RAS NEDA är framtagen med höjddata från en digital terrängmodell baserad på laserscanning. Areella studier i GIS över hur arean av vattenspegeln förändras med vattenståndet visar att Färnebofjärden under augusti år 2000 lade 55 km2 under vatten.</p>

West Nile virus (WNV) is a mosquito-borne virus that infects many bird species. Examination of American crows and house sparrows from the Winnipeg region confirmed that WNV levels were at least 1000 times higher in crows than sparrows. No species differences were observed in the level of transcripts encoding a putative WNV receptor, β3 integrin. Differences in mosquito vector competence can be due to differences in the ability of WNV to enter mosquito cells. Using RNAi techniques, the role of two clathrin coat adaptor proteins in facilitating WNV infections in mosquito cells was examined, and the findings suggest that these proteins may act as resistance factors in Aedes aegypti, and as susceptibility factors in Culex quinquefasciatus. These findings will contribute to our understanding of the molecular basis of vector competence in different mosquitoes, and may help us determine whether other species could serve as potential vectors of this health-threatening virus. / October 2015

Les interactions hôtes-parasites sont classiquement vues comme un équilibre dynamique entre seulement deux partenaires ; cependant, ce paradigme a progressivement changé ces dernières années avec la découverte de nombreux hôtes naturellement co-infectés par de multiples parasites. L'étude des co-infections s'avère particulièrement pertinente lorsque l'hôte est un vecteur de maladies, dans la mesure où les infections multiples peuvent avoir d'importantes conséquences sur la transmission d'un parasite, à la fois à des échelles écologiques et évolutives. Wolbachia pipientis est la bactérie endosymbiotique la plus commune chez les insectes, et de fait, suscite un intérêt particulier pour comprendre le rôle des co-infections sur l'issue des infections parasitaires. Afin de déterminer si l'infection naturelle par Wolbachia affecte la capacité des moustiques à transmettre la malaria, nous avons étudié une triade qui partage une histoire évolutive commune : le parasite de la malaria aviaire P. relictum SGS1, son vecteur naturel Cx. pipiens, et un ensemble de souches wPip de Wolbachia naturellement présentes chez ce moustique. Nous nous sommes tout d'abord intéressé à l'impact de différents types de wPip sur la prévalence et la diversité des parasites responsables de la malaria aviaires dans les populations naturelles de Cx. pipiens de la région de Montpellier. Puis, par l'utilisation de différentes lignées isogéniques de moustiques, infectés ou non par Wolbachia, nous avons étudiés l'impact de la présence du symbiote sur différents traits d'histoire de vie du moustique essentiels pour la transmission de Plasmodium. Nous montrons ainsi que Wolbachia profite à Cx. pipiens, mais également à Plasmodium: elle améliore plusieurs traits d'histoire de vie des moustiques, tels que leur longévité et leur fécondité, leur tolérance à l'infection par P. relictum, et facilite l'infection par ce parasite à la fois qualitativement et quantitativement (i.e. elle augmente à la fois la prévalence et l'intensité de l'infection). Bien que les mécanismes impliqués dans cette interaction tripartite restent encore inconnus, ces résultats suggèrent que Wolbachia peut avoir d'importantes implications sur la transmission de la malaria dans la nature. De plus, ces résultats suggèrent la nécessité de réévaluer l'utilisation de Wolbachia comme moyen de lutte contre les pathogènes et soulignent la nécessité de mieux comprendre les interactions multipartites. / In recent years, there has been a shift in the one host one parasite paradigm with the realization that, in the field, most hosts are co-infected with multiple parasites. Coinfections are particularly relevant when the host is a vector of diseases, because multiple infections can have drastic consequences for parasite transmission at both the ecological and evolutionary time scales. Wolbachia pipientis is the most common parasitic microorganism in insects and as such it is of special interest for understanding the role of coinfections in the outcome of parasite infections. This thesis investigates whether a natural Wolbachia infection can alter the quality of mosquitoes as vectors of malaria. To address this issue, we used a Wolbachia-mosquito-Plasmodium triad with a common evolutionary history. Our experimental system consists in the avian malaria parasite P. relictum SGS1 and its natural vector, the mosquito Cx. pipiens, which naturally harbours several wPip Wolbachia strains. First, we investigated the impact of different wPip groups on the prevalence and diversity on avian malaria in natural populations of Cx. pipiens mosquitoes in the Montpellier region. Second, using different isogenic laboratory mosquito strains harboring or not Wolbachia, we investigated the impact of the presence of Wolbachia on several mosquito and Plasmodium life history traits relevant for malaria transmission. We show that Wolbachia benefits both Cx. pipiens and Plasmodium: it enhances several mosquito life history traits, such as longevity and fecundity, increases their tolerance to P. relictum (i.e. compensates for a Plasmodium-induced mortality) and facilitates P. relictum infection both qualitatively (increases infection prevalence) and quantitatively (increases infection intensity). Although the mechanisms involved in the mosquito-Wolbachia-Plasmodium interaction remain elusive, these results suggest that Wolbachia may have important implications on the transmission of malaria in nature. This is consistent with the high prevalence and diversity of avian malaria parasites found in natural populations of Cx. pipiens. Further, these results suggest the need to reassess the use of Wolbachia as a way to fight pathogens and highlight the need to better understand parasite multipartite interactions.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Each year, over 500 million people are infected with mosquito-borne diseases, including malaria, yellow fever and dengue fever, which cause several million deaths, and long-term disability and suffering. This dissertation focused on the mosquito Aedes aegypti, a vector for dengue virus and yellow fever virus. Since Sindbis virus (SINV) is an arthropod-borne virus (arbovirus) that is vectored by A. aegypti and is well characterized at the molecular level, the SINV - A. aegypti model was used to determine whether apoptosis plays a role in the control of vector competency. In Chapter 2, the effects of inducing or inhibiting apoptosis on SINV replication were tested in mosquito cells. It was observed that recombinant SINVs expressing pro-apoptotic genes caused extensive apoptosis in mosquito cells, with decreased virus production after the cells underwent apoptosis. Infection of mosquito cells with SINV expressing the caspase inhibitor P35 inhibited actinomycin D-induced apoptosis, but had no observable effects on virus replication. This study was the first to test directly whether inducing or inhibiting apoptosis affects arbovirus replication in mosquito cells. Chapter 3 examined the effects of silencing apoptosis regulatory genes on SINV replication and dissemination in A. aegypti. Genes which either positively or negatively regulate apoptosis were silenced by RNA interference in mosquitoes, which were then infected with a recombinant SINV expressing green fluorescent protein (GFP). Reciprocal effects were observed on both the occurrence and intensity of expression of GFP in various tissues. These results suggest that systemic apoptosis positively influences SINV replication in A. aegypti. This was the first direct study to explore the role of apoptosis in determining mosquito vector competence for arboviruses. Finally, in Chapter 4, the mechanisms of apoptosis were explored in A. aegypti. Overexpression of IAP antagonists caused extensive cell death in mosquito cells, while silencing the expression of IAP antagonists attenuated apoptosis. The results showed that the IAP binding motif (IBM) of IAP antagonists was critical for their binding to AeIAP1. The IAP antagonists released initiator and effector caspases from AeIAP1 by competing for the binding sites and caused caspase-dependent apoptosis. These findings imply that the mechanisms of IAP antagonists regulating apoptosis are conserved between mosquitoes and the model insect where apoptosis has been mainly studied, Drosophila melanogaster.

Les moustiques sont les principaux vecteurs incriminés dans la transmission d’agents pathogènes à l’homme. L’identification précise des espèces de moustiques est importante pour distinguer les espèces vectrices des non vectrices. La détermination de l’origine du repas sanguin des moustiques vecteurs est indispensable dans la compréhension du comportement des espèces vectrices. Nous avons mise à jour la littérature actuelle sur la faune Culicidienne du Mali. Ainsi, nous avons listé 106 espèces de moustiques actuellement enregistrée au Mali dont 28 Anophelinae et 78 Culicinae. Nous avons ensuite évalué l’efficacité du MALDI-TOF MS à identifier des moustiques collectés au Mali et déterminer leur source de repas sanguin. Nous avons confirmé la robustesse du MALDI-TOF MS à identifier un grand nombre de sang d’animaux. Nous avons artificiellement gorgé des femelles de An. gambiae et An. coluzzii sur différents types de sang d’animaux. Nous avons obtenu 100% d'identification correcte du repas de sang pour les spécimens collectés 1h à 24h après le gorgement. Ensuite nous avons expérimentalement gorgés An. gambiae, An. coluzzii et Ae. albopictus sur des repas de sang successif et mixte par MALDI-TOF MS. Nos résultats révèlent que le MALDI-TOF MS est tout à fait capable d’identifier le repas mixte. Mais en ce qui concerne le repas successif seul le dernier repas de sang est identifié. Enfin nous avons utilisé la culturomique et le MALDI-TOF pour l’étude du microbiote digestif de moustiques collectés sur le terrain au Mali et à Marseille. Cette approche a révélé une grande diversité du microbiote digestif des moustiques An. gambiae, Ae. albopictus et Cx. quinquefasciatus. / Mosquitoes are the main vectors involved in the transmission of pathogens to humans. Accurate identification of mosquito species is crucial to distinguish between vector and non-vector species. The mosquito blood meal determination is fundamental in understanding the behavior of vector species. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. Then, we evaluated the effectiveness of MALDI-TOF MS for identified mosquitoes collected in Mali and to determine their blood meal source. The results obtained show the ability of MALDI-TOF MS to identify mosquitoes collected in Mali and their source of blood meal. Subsequently, we were able to confirm the robustness of MALDI-TOF MS to identify other animal blood samples. We artificially engorged Anopheles gambiae and Anopheles coluzzii on eight animal bloods samples. We obtained 100% correct identification of the blood source for samples taken 1 to 24 hours after feeding. Then, we experimentally engorged An. gambiae, An. coluzzii and Ae. albopictus on successive and mixed blood meals using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. In addition we used MALDI-TOF and culturomics for the microbiota study of the mosquito collected in the field, notably in Marseille and Mali. The culturomics approach revealed a great diversity of the digestive microbiota of the An. gambiae, Ae. albopictus and Cx. quinquefasciatus mosquitoes.