Thesis (Ph. D.)--Michigan State University. Dept. of Entomology, 2006. / Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.

La lutte anti-vectorielle est une composante importante de la lutte contre les maladies à transmission vectorielle. La connaissance des populations vectorielles, tant dans leur composition spécifique que dans leur répartition spatio-temporelle, est fondamentale pour la conception des stratégies de lutte contre ce type de maladie.Cette thèse a modélisé la distribution spatiale des formes moléculaires M et S d’Anopheles gambiae s.s., des vecteurs majeurs du paludisme au Burkina Faso. La modélisation a été faite à partir de l’analyse d’une série d’observations portant à la fois sur les vecteurs et l’environnement. Elle procède d’une combinaison de concepts et méthodes de biogéographie avec des techniques et outils d’analyse spatiale, d’analyse des données et des systèmes d’informations géographiques.Nous retenons de ce travail de recherche que l’abondance de la forme moléculaire S d’Anopheles gambiae s.s., diminue au fur et à mesure que l’on évolue des régions humides du sud et du sud-ouest vers celles les plus arides du nord et du nord-est. Le NDVI, l’ETP, et l’insolation sont les facteurs les plus déterminants de sa distribution spatiale. Par contre, l’abondance de la forme moléculaire M d’Anopheles gambiae s.s. augmente des régions humides du sud et du sud-ouest vers celles plus arides du nord et du nord-est. L’altitude, la pression, l’insolation, la densité de végétation sont les facteurs les plus déterminants de cette répartition spatiale. De ces résultats, ont été dérivés deux modèles qui ont servi à l’élaboration des cartes de distribution des formes moléculaires M et S d’Anopheles gambiae s.s.Enfin, cette thèse révèle le rôle de l’approche géographique dans la réflexion sur les questions de santé et sa méthodologie pourrait être testée sur d’autres sites et pour d’autres vecteurs de maladies. Elle peut s’enrichir d’une analyse multi échelle et d’une modélisation de la variabilité temporelle. / The anti-vectorial fight is an important constituent for vectorial borne diseases control. The knowledge of the vectors populations as well as their specific composition and spatiotemporal distribution is fundamental for the conception of the strategies of fight against this type of disease.This thesis modeled the spatial distribution of the molecular forms M and S of Anopheles gambiae s.s., major vectors of malaria in Burkina Faso. The modeling was made from the analysis of a series of observations concerning both the vectors and the environment.It proceeds of a combination of concepts and methods of biogeography with techniques and tools of spatial analysis, data analysis and the geographical information systems.We retain of this research work that the abundance of the molecular form S of Anopheles gambiae s.s., decreases as we move from wet regions of the south and the southwest to those the driest of the north and the northeast. The NDVI, the ETP, and the sunshine are the most determining factors of its spatial distribution.In contrast, the abundance of the molecular form M of Anopheles gambiae s.s. increases from the wet regions of the South and the southwest to those drier of the north and the northeast. The temperatures, the pressure, the sunshine and the NDVI are the most determining factors of this spatial distribution.Of these results, were diverted two models which we use to elaborate the Maps of distribution of the molecular forms M and S of Anopheles gambiae s.s.This thesis highlights also the role of the geographical approach in the reflection on health issues and its methodology could be tested on the other sites and for the other vectors of diseases. This methodology could be improved by multi-scale analysis and of temporal variability modeling.

Thèse de doctorat : Sciences de la Vie et de la Santé. Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg : 2009. / Titre provenant de l'écran-titre. Bibliogr. 12 p.

Thesis (Ph.D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiv, 175 p.; also includes graphics. Includes bibliographical references (p. 160-175). Available online via OhioLINK's ETD Center

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Brigita Mányová Supervisor: PharmDr. Marie Vopršalová, CSc. Supervisor-specialist: PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: Development of insecticides inhibiting acetylcholineseterase Pest insects cause problems and damage all over the world. They are vectors of serious diseases such as malaria, dengue fever, yellow fever, Zika virus disease and chikungunya disease. They cause huge damage to agricultural crops and are annoying to everyday life in living spaces. The aim of this diploma thesis was in vitro testing of selected compounds from the group of bis-isoquinoline and bispyridinium acetylcholinesterase inhibitors as potential insecticides. Another goal was also to create relationships between structure and effect. The ability of these compounds to inhibit both human (hAChE) and fly acetylcholinesterase (MdAChE) was evaluated. The modified Ellman spectrophotometric method was used. The half inhibitory concentration (IC50) values were obtained for both enzymes and the selectivity indexes (SI) were then calculated. Compounds having IC50s in micromolar or nanomolar range and exhibiting selectivity for MdAChE were most desirable. During the testing of these inhibitors, three...

Mosquito borne diseases continue to be a big threat to human health worldwide. Despite using various vector control methods, we lose a great number of lives to this malicious disease in tropical and subtropical countries each year. Not surprisingly, mosquito is considered as the deadliest animal on the earth, because mortality rates from mosquito-vectored infections only lag behind other major diseases such as HIV and tuberculosis. Current approaches of vector control are mostly limited to using insecticidal bed nets, thus novel techniques are required to prevent a staggering loss to human health and quality of life. Advances in the genome sequencing in the past decade have helped to uncover numerous secrets of diverse genomes. The genome of malaria mosquito Anopheles gambiae was first sequenced in 2002 and since then has been updated to include additional scaffolds, their orientations and correction of mis-assemblies. Yet, the greatest challenge remains in assembling the heterochromatin regions, that are repeat rich part and contain relatively low-gene density. Although previously neglected by scientific studies due to its characteristic paucity of genes, heterochromatin is now recognized to be crucial for several processes such as cell viability, chromosome pairing, meiosis, longevity etc. It is therefore not surprising that heterochromatin comprises of a significant portion of the genome in many species. The efforts to analyze the genome of malaria mosquito in order to identify potential new leads for vector control warrant a better understanding of the heterochromatin. Mosquitoes diploid chromosome number equal 6. While autosomes 2 and 3 are submetacentric and present in both sexes, females are homogametic with XX and males are heterogametic with XY sex chromosomes. To achieve a better understanding of the Anopheles heterochromatin, we investigated heterochromatic region of the X chromosome. Despite one arm of the X chromosome being completely heterochromatic, few studies have investigated the molecular content of this region. Protocols were developed for performing fluorescent in situ hybridization (FISH) on mitotic X chromosomes in An. gambiae. Using cytogenetics and molecular biology techniques, we characterized the X chromosome heterochromatin in members of the An. gambiae complex. Specific satellite DNA and 18S ribosomal DNA probes (major components of heterochromatin) were mapped to X chromosomes enabling their differentiation and characterization in the An. gambiae complex. Microarray studies have highlighted the importance of X chromosome during investigation of nascent species An. gambiae and An. coluzzii. Here for the first time qualitative differences in heterochromatin in between nascent species are described. Cytogenetic idiograms are developed as to include the molecular and qualitative differences between the species of the An. gambiae complex. These idiograms are expected to provide a better resolution of the X chromosome heterochromatin for comparison in major malaria vectors, closing some of the gaps present due to poor sequencing of unassembled repeat rich regions in An. gambiae complex. The current understanding of Y chromosome for transgenic manipulation is poor and limited to very few genes. Due to its near total heterochromatic composition, it is the hardest part of the genome to assemble. In collaboration with other researchers, the Y chromosome content was characterized among sibling species of the An. gambiae complex. Our data revealed the swift changes the Y chromosome has undergone in a relatively short evolutionary time period. These include a rapid rate of turnover not only in heterochromatin but also in euchromatin. In addition to previously described repeats, a novel highly repetitive element called Zanzibar was discovered and mapped to the males of various Anopheles sibling species. Our data can form the basis for evolutionary studies in heterochromatin for male mosquitoes within the An. gambiae complex while also help identify novel targets to create successful transgenic male populations. Along with the X chromosome heterochromatin, to our knowledge this is the most extensive contribution to improve the understanding of mitotic chromosome heterochromatin in malaria mosquitoes. This study also investigated if epigenetics play role in mosquito development, fecundity and heterochromatin formation. DNA methylation, histone modifications and small noncoding RNAs are among the epigenetic mechanisms scrutinized in mammals. However, knowledge about epigenetic mechanisms and their effects is sparse in mosquitoes. A protocol for testing the various effects of epigenetics on different stages of malaria mosquito was developed. An epigenetic drug was utilized to probe the effects on immature and adult malaria mosquitoes. Different concentrations of DZNep, a histone methyltransferase inhibitor, were administered to An. coluzzii larvae. Total survivorship and pupation were compared for treated and untreated groups. The drug was also administered to adult blood feeding females to determine any effects on fecundity and egg morphology, revealing a negative association with an increase in drug concentration. A dose dependent decrease in SAH hydrolase concentration in An. coluzzii was also noticed. These results suggest epigenetics plays a critical role in mosquito pupation and ovarian development. Our work lays the groundwork for future investigations into the field of epigenetics in mosquitoes by revealing its effect on several important developmental stages in malaria mosquitoes. Although genomics and next-gen sequencing technology have come a long way in the last decade since the first Anopheles genome was sequenced, considerable gaps still exist in case characterization of heterochromatin function in an organism. Through our work, we have endeavored to elucidate a few of the major roles that heterochromatin may play in organization, evolution and adaptation of the malaria mosquitoes. / Ph. D.

MSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Malaria is on the increase in Mozambique since 2001 and impacts primarily on children < 5 years of age. Insecticide resistance in the malaria vector mosquitoes is on the increase in Mozambique and Africa and is cause for serious concern. Maragra sugar estate is situated in close proximity to the nKomati river floodplain in a rural area in Mozambique and requires intense irrigation for cane growing and as a result provides extensive breeding sites for An. funestus and other mosquitoes. In the areas surrounding the estate there are two important vectors of malaria, Anopheles funestus group and An. gambiae complex. There is intense malaria transmission in the areas surrounding the sugar estate and the last entomological study on the vectors in the Manhica area was done in 1998. It was becoming increasingly urgent to identify to species level the vectors in this area and to monitor the insecticide resistance status of these vectors. Due to leakage (theft) of insecticides and a change by the National Malaria Control Programme (NMCP) to an insecticide to which the predominant vector is resistant, an entomological survey was carried out in this area from January 2009 to March 2009 to ascertain by Polymerase chain reaction (PCR) what species of malaria vectors were present inside and outside of the Maragra vector control area, their population levels and their vectorial status in these two areas. Insecticide resistance studies by insecticide exposure and the synergist piperonyl butoxide (pbo) were carried out using the World Health Organisation (WHO) bioassay method on collected An. funestus mosquitoes. This was done to establish this species resistance status to the four classes of insecticides recommended by the WHO for malaria vector control. The collections of An. arabiensis and An. merus that were identified were too few to carry out insecticide resistance tests on these two species. Enzyme linked v immuno-sorbent assay (ELISA) tests were undertaken to establish the vectorial capacity of Anopheles funestus and An. gambiae complex in this area. The predominant malaria vector species in this area is An. funestus s.s., with the secondary vector being An. arabiensis. An. funestus has a high vectorial capacity in this area and found to have a Plasmodium falciparum sporozoite rate of 6.02%. This is an increase in the sporozoite rate of 1.2% from 1998 when the last survey in this regard was carried out. Coupled with this increase is an increase in the An. funestus populations in this area since this time. One An. gambiae complex sample was found to be positive but the species is not known as this particular sample did not amplify on PCR. Anopheles funestus is highly resistant to synthetic pyrethroids and exhibits a lower level of resistance to bendiocarb, a carbamate insecticide in use at Maragra sugar estate. The synergist pbo mediates the resistance mechanism in both these insecticides indicating that the metabolic resistance mechanism present in this mosquito is strongly mediated by monooxygenase detoxification. The role of the medical entomologist is increasingly necessary and important in the monitoring of this resistance phenomenon in malaria vector mosquitoes, as is the role of the vector control programme manager in implementing and managing vector control programmes. The implication of cane sugar farming and its impact on vector production and malaria transmission is discussed. Insecticide resistance and the change by the NMCP to a synthetic pyrethroid to which the predominant vector of malaria is resistant is discussed.

Malaria remains a serious public health challenge in the tropical world, with 584,000 deaths globally in 2013, of which 90% occurred in Africa, and mostly in pregnant women and children under the age of five. Anopheles gambiae (An. gambiae) is the principal malaria vector in Africa, where vector control measures involve the use of insecticides in the forms of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). The development of insecticides resistance mitigates these approaches. Glutathione (GSH) is widely distributed among all living organisms, and is associated with detoxification pathways, especially the Glutathione S-transferases (GSTs). Its direct involvement and relevance in insecticide resistance in An. gambiae has not been determined. Thus, this work examines the contribution of GSH, its biosynthetic genes (GCLM, GCLC) and their possible transcriptional regulator Nrf2 in insecticide resistance in An. gambiae sampled from agricultural setting (areas of intensive agriculture) and residential setting (domestic area). Bioinformatics analysis, W.H.O. adult susceptibility bioassays and molecular techniques were employed to investigate. Total RNA was first isolated from the adults An. gambiae mosquitoes raised from agricultural and residential field-caught larvae which had been either challenged or unchallenged with insecticides. Semi-quantitative RT-PCR using gel image densitometry was used to determine the expression levels of GCLM, GCLC genes and Nrf2. Bioinformatics’ results established the presence of putative AGAP010259 (AhR) and AGAP005300 (Nf2e1) transcription factor binding sites in An. gambiae GCLC and GCLM promoters in silico. An. gambiae s.l. studied here were highly resistant to DDT and permethrin but less resistant to bendiocarb. Both knockdown resistance (kdr) mutation variants L1014S and L1014F that confers resistance to pyrethroid insecticides were identified in both An. coluzzii and An. arabiensis sampled from northern Nigeria. The L1014F was much associated with An. coluzzii. A significant positive correlation (P=0.04) between the frequency of the L1014F point mutation and resistance to DDT and permethrin was observed. However, a weak or non-significant correlation (P=0.772) between the frequency of the L1014S point mutation and resistance was also found. L1014S and L1014F mutations co-occurred in both agricultural and residential settings with high frequencies. However, the frequencies of the two mutations were greater in the agricultural settings than in the residential settings. The levels of total, reduced and oxidized GSH were significantly higher in mosquitoes from agricultural sites than those from residential sites. Increased oxidized GSH levels appears to correlate with higher DDT resistance. The expression levels of GCLM, GCLC and Nrf2 were also significantly up-regulated in adults An. gambiae raised from agricultural and residential field-caught larvae when challenged with insecticide. However, there was higher constitutive expression of GCLM, GCLC and Nrf2 in mosquitoes from agricultural setting. The increased expression levels of these genes and also GSH levels in this population suggest their roles in the response and adaptation of An. gambiae to insecticide challenges. There exists the feasibility of using GSH status in An. gambiae to monitor adaptation and resistance to insecticides.

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.

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