Este trabalho consistiu em observações de campo e de laboratório, sendo que a primeira foi desenvolvida na localidade da Fazenda Folha Larga, município de Cananéia, Estado de São Paulo. As capturas foram feitas nos ambientes extra e peridomiciliares com isca humana em horário de pico máximo de atividade hematófaga de Anopheles (Kerteszia) cruzii. Resultados preliminares obtidos em campo objetivaram determinar a paridade, enquanto que sob condições laboratoriais procurou-se conhecer a duração do ciclo gonotrófico, ambos considerados parâmetros implicados na determinação da capacidade vetora de An. cruzii. Assim pois, das 631 fêmeas dissecadas, 90,49 por cento revelaram-se nulíparas e somente 9,50 por cento uníparas, mostrando que a maior parte da população era constituída de indivíduos jovens e sem haver diferença significativa quanto a proporção de fêmeas uníparas nos diversos ambientes. No laboratório trabalhou-se com 1.158 larvas dessa espécie, oriundas de aproximadamente 600 bromélias. O período de desenvolvimento larvário foi de aproximadamente 40 dias. Desse material, 283 fêmeas foram alimentadas com sangue humano, sendo que 50 por cento conseguiu o seu desenvolvimento folicular completo com um único repasto sangüíneo. Outras 25 fêmeas, não receberam alimentação sangüínea e, quando dissecadas, todas apresentaram o seu primeiro folículo no estágio II de Christophers e Mer, provavelmente pela falta de estímulo sangüíneo. Além do mais, determinou-se que o tempo médio entre a alimentação sangüínea e a oviposição foi de 6 a 7 dias. Em vista disso, as observações preliminares, possibilitam considerar esta espécie com razoável capacidade vetora, apesar da baixa longevidade, pois esta é provavelmente, compensada pela alta densidade. / This study consisted on field and laboratory observations. The former was developed at Folha Larga Farm., Cananéia County, São Paulo State. Collections were made in extra and peridomiciliary environments with human bait during the maximum peak of Anopheles cruzii blood feeding activity. Preliminary results obtained in the field, led to the determination of parity, and those developed in the laboratory led to the determination of the gonotrophic cycle length, both considered as involved parameters in the determination of vector capacity. From 631 females dissected, 90.49 per cent were nulliparous, and 9.0 per cent uniparous, showing that the greater part of the population was composed of young females. No significant difference between the proportion of uniparous females in the extra and peridomiciliary environment was found. At the laboratory 1,158 larvae o f An.cruzii from 600 bromelia were examined. The period of larval development was about 40 days. From this material, 283 females were fed with human blood, 50 per cent of them reached complete follicular development witb a single bloodmeal, showing the first follicular in the Christophers and Mer\'s stages ll, probably because of the lack of blood stimulus. Besides, was determined that the average time between bloodmeal and oviposition was 6 to 7 days. As a result of these preliminary observations was possible to consider this species as a vector with positive capacity, inspite of its low longevity, that could be probably compensated by its high density.

Submitted by Dominick Jesus (dominickdejesus@hotmail.com) on 2016-02-04T19:28:16Z No. of bitstreams: 2 Tese_Caroline Dantas de Oliveira.pdf: 3375525 bytes, checksum: 445c62c608ddf4ddcea75b2dbfd50231 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-02-04T19:28:16Z (GMT). No. of bitstreams: 2 Tese_Caroline Dantas de Oliveira.pdf: 3375525 bytes, checksum: 445c62c608ddf4ddcea75b2dbfd50231 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-02-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The mosquito Anopheles darlingi is the main vector of human malaria in Brazil. Nevertheless, little information exists on the gonotrophic cycle of this important vector, whose knowledge is important for an effecient control strategy. Therefore, this work studied the oogenesis in this mosquito species. Molecular, biologic and morphological aspects of oogenesis, before and after the blood meal (BM), were investigated in female A. darlingi collected at the suburb of Manaus and Coari city to study the. Before the BM, ovaries of female A. darlingi are small and contain few lipid vesicles. At this stage the expression of vitellogenin (Vg) gene is absent. After the BM, there were marked internal and external changes in the ovaries due to storage of yolk in the oocytes. Small granules of yolk are already observed at 3h post BM, although the expression of Vg gene was only detected at 6h post BM. The expression of Vg and their storage in the ovaries are more evident after 24 hours of BM, remaining constant between 36 to 48 hours post BM. The final dimensions of the egg are reached at 72 hours post BM when Vg gene expression decreased. Oviposition occurred at 96 hours post BM when the expression of Vg is absent. In the analyses of the frequence of blood meals taken by female A. darlingi, 80% ingested two blood meals within 24h and 75% fed three times in a period of three days, showing the ability of this species to feed more than once during a single gonotrophic cycle. An apparent increase in Vg gene expression was noticed in females taking multiple BMs. / O mosquito Anopheles darlingi é o principal vetor da malária humana no Brasil. Apesar disso, existem poucas informações sobre o ciclo gonotrófico desse importante vetor, cujo conhecimento é importante para uma efeciente estratégia de controle. Por esse motivo, neste trabalho essa espécie é alvo do estudo da ovogênese. Para tanto, fêmeas de A. darlingi foram coletadas na periferia da cidade de Manaus e Coari para estudar os aspectos biológicos, morfológicos e moleculares do processo da ovogênese antes e após o repasto sanguíneo (RS). Antes do repasto sanguíneo os ovários das fêmeas de A. darlingi são pequenos e contêm algumas vesículas de lipídios. Nesta fase o mRNA do gene da vitelogenina (Vg) é ausente. Após o RS, ocorrem acentuadas mudanças internas e externas nos ovários devido à estocagem das vitelinas nos ovócitos. Pequenos grânulos de vitelinas são observados a partir de 3h após o RS, embora a expressão do gene Vg só é detectável a partir de 6h. A expressão e a estocagem da Vg é mais evidente após 24h do RS, mantendo-se constante entre 36h e 48h após o RS. As dimensões definitivas do ovo são alcançadas após 72h do RS quando a expressão do gene Vg diminui. A oviposição ocorreu após 96h do RS quando a expressão de Vg é ausente. Nas análises de múltiplos repastos sanguíneos, 80% das fêmeas de A. darlingi tiveram dois repastos sanguíneos dentro de 24h e 75% delas alimentaram três vezes num período de três dias, mostrando a capacidade dessa espécie de se alimentar por mais de uma vez durante um único ciclo gonotrófico. Nestas fêmeas foi verificado um aumento aparente na expressão do gene Vg de acordo com um número maior de alimentações sanguíneas.

A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.

Die Tropenkrankheit Malaria, wird durch eine Infektion mit einzelligen Parasiten der Gattung Plasmodium verursacht und durch den Stich der weiblichen Anopheles-Mücke von Mensch zu Mensch verbreitet. Dabei kann eine erfolgreiche Übertragung des Parasiten auf den Menschen nur dann stattfinden, wenn der Parasit seine sexuelle Entwicklungsphase im Mitteldarm der Mücke erfolgreich durchläuft. Ziel dieser Arbeit war es daher, die Wechselwirkungen des Malariaparasiten im Mitteldarm der Mücke in Hinblick auf die Identifizierung möglicher neuer transmissionsblockierender Strategien zu untersuchen. Der Zweck von transmissionsblockierende Strategien ist es, der Verbreitung der Malaria durch die Mücke entgegenzuwirken, indem die Entwicklung des Parasiten in der Mücke unterbunden und dadurch der Lebenszyklus des Parasiten unterbrochen wird. Der Schwerpunkt der vorliegenden Arbeit lag auf insgesamt drei Aspekten. Der erste Aspekt der Arbeit befasste sich mit der Wechselwirkung zwischen dem Para-siten und der mikrobiellen Darmflora der Mücke. Dabei sollte der mögliche Einfluss des Parasiten auf die Darmflora untersucht werden und weiterführend die potentielle Verwendung von Darmbakterien als Vehikel für die Herstellung paratransgener Mücken erforscht werden. Vergleichende16S-rRNA- und DGGE-Analysen an der Darmflora des asiatischen Malariavektors Anopheles stephensi zeigten eine deutliche Reduktion der mikrobiellen Diversität während der Entwicklung vom Ei zur adulten Mücke. Zudem konnte das gram-negative Bakterium Elizabethkingia meningoseptica, das sich stadien- und generationsübergreifend verbreitet, als dominante Darmspezies bei im Labor aufgezogenen weiblichen und männlichen An. stephensi festgestellt werden. Die Dominanz von E. meningoseptica wurde zudem nicht durch die Aufnahme von infiziertem Blut oder einer veränderten Nahrung beeinflusst. Für die Studien wurde sowohl der humanpathogene Parasit P. falciparum als auch der Nagermalariaerreger P. berghei verwendet. Weiterführende Versuche zeigten, dass Extrakte von E. meningoseptica antibakterielle, antifungale und antiplasmodiale Aktivitäten aufwiesen, die ein möglicher Grund für die Dominanz dieser Spezies im Mitteldarm des Vektors waren. Isolate von E. meningoseptica sind im Labor kultivierbar; dadurch stellt das Bakterium einen potentiellen Kandidaten zur Generierung von paratransgenen Anopheles-Mücken dar. Ein zweites Ziel dieser Arbeit war es, mögliche Unterschiede in der Genexpression von P. falciparum darzustellen, die in den ersten 30 Minuten nach dessen Übertragung auf die Mücke erfolgen. Dies hatte zum einen zum Zweck, die durch den Wirtswechsel hervorgerufenen Genregulationen besser zu verstehen, und bot zum anderen die Möglichkeit, neue Proteine zu identifizieren, die als potentielle transmissionsblockierende Ziele genutzt werden können. Mittels supression substractive hybridization (SSH) konnten insgesamt 126 Gene identifiziert werden, deren Expression sich während der Gametogenese verändert. Die identifizierten Gene konnten einer Vielzahl von putativen Funktionen wie zum Beispiel in der Signaltransduktion (17,5%), im Zellzyklus (14,3%) oder im Zytoskelett (8,7%) zugeordnet werden. Des Weiteren wurden 7,9% der Gene eine Funktion in der Proteastase und 6,4% in metabolischen Prozessen zugeordnet. 12,7% der Gene kodierten für zelloberflächenassoziierte Proteine. 11,9% der Gene hatten anderen Funktionen, während 20% der Gene keine putative Funktion zugeordnet werden konnte. Etwa 40% der identifizierten Genprodukte waren bisher nicht in Proteomstudien nachgewiesen worden. In weiterführenden Analysen wurden 34 Gene aus jeder ontologischen Gruppe ausgewählt und deren Expressionsveränderung per quantitativer real time RT-PCR im Detail untersucht. Für 29 Gene konnte dabei eine Transkriptexpression in Gametozyten nachgewiesen werden. Zudem wiesen 20 Gene eine erhöhte Expression in Gametozyten im Vergleich asexuellen Stadien auf. Insgesamt zeigten 8 Gene besonders hohe Transkriptlevel in aktivierten Gametozyten, was auf eine Funktion dieser Proteine während der Übertragung des Parasiten auf die Mücke hindeutet und diese somit potentielle Angriffspunkte für transmissionsblockierende Strategien darstellen könnten. Im letzten Teil dieser Arbeit stand die Untersuchung verschiedener antimikrobieller Substanzen in Bezug auf ihre transmissionsblockierenden Eigenschaften im Vordergrund. Die Substanzen waren entweder direkt aus der Hämolymphe verschiedener Insekten isoliert oder rekombinant in transgenem Tabak exprimiert worden. Dabei wurden die rekombinanten Peptide so ausgewählt, dass sie entweder gegen die Mitteldarmstadien des Parasiten wirken oder mückenspezifische Rezeptoren blockieren, die der Parasit für seine weitere Entwicklung benötigt. Dabei konnte gezeigt werden, dass das antimikrobielle Molekül Harmonin, ein Abwehrmolekül aus der Hämolymphe des asiatischen Marienkäfers Harmonia axyridis, antiplasmodiale als auch transmissions-blockierende Eigenschaften besitzt. Harmonin stellt daher eine potentielle Leitstruktur für die Entwicklung neuer Malariawirkstoffe dar / Malaria is a vector-borne disease caused by the protozoan parasite of the genus Plasmodium and it is transmitted from human to human by female Anopheles mosquitoes during a blood meal. For malaria transmission to occur, the malaria parasite must undergo a crucial developmental sexual phase inside the mosquito midgut. In this study, we sought to investigate the interplay of the malaria parasite in the mosquito midgut with regard to the identification of novel types of transmission blocking intervention strategies. These strategies are aimed at reducing the spread of malaria by blocking the development of the mosquito midgut-specific stages of Plasmodium. We focused on three aspects. The first aspect was to investigate the interplay between mosquito midgut bacteria and malaria parasites in order to determine the potential influence of malaria parasites on the composition of the mosquito gut microbiota and also determine midgut bacteria which could be exploited as vehicles for the generation of paratransgenic Anopheles mosquitoes. We analyzed the microbial diversity of gut bacteria of the Asian malaria vector Anopheles stephensi during development and under different feeding regimes, including feeds on malaria parasite-infected blood, using the human pathogenic P. falciparum as well as the rodent malaria model P. berghei. 16S rRNA and DGGE analyses demonstrated a reduction in the microbial diversity during mosquito development from egg to adult and identified the gram-negative bacterium Elizabethkingia meningoseptica as the dominant species in the midgut of laboratory-reared male and female mosquitoes. E. meningoseptica is transmitted between generations and its predominance in the mosquito midgut was not altered by diet, when the gut microbiota was compared between sugar-fed and blood-fed female mosquitoes. Furthermore, feeds on blood infected with malaria parasites did not impact the presence of E. men-ingoseptica in the gut. Interestingly, extracts from E. meningoseptica exhibited antibacterial, antifungal and antiplasmodial activities, which may account for its dominance in the midgut of the malaria vector. Isolates of E. meningoseptica were cultivable, making the bacterium a potential candidate vehicle for the generation of paratransgenic Anopheles mosquitoes. The second aspect of this thesis was to determine transcriptome changes that occur during the first half hour following transmission of P. falciparum to the mosquito vector in order to better understand gene regulation mechanisms important for the change of hosts and determine novel proteins which could be exploited in malaria transmission blocking interventions. We initially used suppression subtractive hybridization (SSH) to compare mRNA levels of P. falciparum gametocytes before and 30 min fol-lowing activation. We identified a total of 126 genes for which transcript expression changed during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or motor complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were genes encoding for cell surface associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. We further selected a subset of 34 genes from all the above ontology groups and analyzed the transcript changes during gametogenesis in detail by quantitative realtime RT-PCR. Of these, 29 genes were expressed in gametocytes, and for 20 genes transcript expres-sion in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of eight genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito which could be exploited in malaria transmission blocking strategies. The last aspect of this thesis was to determine the transmission blocking effect of a range of antimicrobial molecules as transmission blocking agents. The molecules were either isolated from insect hemolymph or recombinantly expressed in tobacco and designed to act either directly on the mosquito midgut stages or cover receptors on mosquito tissues like the midgut epithelium which the parasite would need for transit. We were able to show an antiplasmodial and transmission blocking effect of the anti-microbial molecule harmonine, a defense compound isolated from the hemolymph of the Asian ladybug Harmonia axyridis. Harmonine thus represents a potential lead structure for the development of novel antimalarials.

<p>The use of transformed bacteria to stop or kill disease-causing agents in the gut of vector insects is called paratransgenics. Two of the major steps in creating a paratransgenic <i>Anopheles</i> mosquito, unable to spread the<i> Plasmodium</i> parasites that cause malaria, are to find a bacterium suitable for the purpose and a way to introduce the transformed bacterium into mosquitoes in the field. In this project, bacteria associated with malaria mosquitoes have been identified by phylogenetic analysis of their 16S rRNA genes. First, the midgut flora of field-caught <i>Anopheles</i> mosquitoes was examined using two pathways, one culture dependent and one culture independent. Second, six bacterial species from an<i> An. gambiae </i>laboratory colony, and third, ten isolates from <i>Anopheles</i> oviposition sites have been identified. Altogether, 32 bacterial species, representing 16 families, seven classes and four phyla were identified. Interestingly, several of them are related to bacteria known to be symbionts in other insects. Two possible ways of introducing bacteria into mosquitoes in the field in a paratransgenic approach were investigated in a laboratory setting. It was shown that sugar solutions with or without bacteria are equally attractive to <i>An. gambiae</i> mosquitoes and that the mosquitoes were able to take up bacteria from the water they emerged from. These results show that it may be possible to use sugar-baits and oviposition sites for distribution of genetically modified bacteria in the field. To facilitate the distribution of the modified bacteria mosquito attractants should be used. We investigated whether the bacterial isolates identified in this project produce attractants affecting mosquito sugar-feeding or oviposition site selection. While no responses were observed from the mosquitoes towards bacteria-containing sugar solutions, seven of the 19 isolates examined mediated positive oviposition responses. In total, 13 putative oviposition attractants were identified among the volatiles emitted by the attractive bacteria.</p>

The use of transformed bacteria to stop or kill disease-causing agents in the gut of vector insects is called paratransgenics. Two of the major steps in creating a paratransgenic Anopheles mosquito, unable to spread the Plasmodium parasites that cause malaria, are to find a bacterium suitable for the purpose and a way to introduce the transformed bacterium into mosquitoes in the field. In this project, bacteria associated with malaria mosquitoes have been identified by phylogenetic analysis of their 16S rRNA genes. First, the midgut flora of field-caught Anopheles mosquitoes was examined using two pathways, one culture dependent and one culture independent. Second, six bacterial species from an An. gambiae laboratory colony, and third, ten isolates from Anopheles oviposition sites have been identified. Altogether, 32 bacterial species, representing 16 families, seven classes and four phyla were identified. Interestingly, several of them are related to bacteria known to be symbionts in other insects. Two possible ways of introducing bacteria into mosquitoes in the field in a paratransgenic approach were investigated in a laboratory setting. It was shown that sugar solutions with or without bacteria are equally attractive to An. gambiae mosquitoes and that the mosquitoes were able to take up bacteria from the water they emerged from. These results show that it may be possible to use sugar-baits and oviposition sites for distribution of genetically modified bacteria in the field. To facilitate the distribution of the modified bacteria mosquito attractants should be used. We investigated whether the bacterial isolates identified in this project produce attractants affecting mosquito sugar-feeding or oviposition site selection. While no responses were observed from the mosquitoes towards bacteria-containing sugar solutions, seven of the 19 isolates examined mediated positive oviposition responses. In total, 13 putative oviposition attractants were identified among the volatiles emitted by the attractive bacteria.

In order to reduce malaria prevalence worldwide, a better understanding of parasite transmission and the effect of drug resistance is needed. The effect of drug resistance on malaria transmission has been examined for some drugs, but not for mitochondrial inhibitors such as atovaquone and the current basis of malaria therapy, artemisinin. Therefore, the goal of this study was to produce gametocytes, the life cycle stage that transmits from mosquito to human, in several different drug resistant patient isolates as well as to determine the effect of drug resistance on gametocyte development and transmission. Previous studies have shown that the mutation that confers resistance to atovaquone, a common antimalarial, occurs de novo after treatment and transmission of this resistance is not seen in the field. Therefore, to determine whether or not the resistance mutation can be transmitted, mosquito-feeding experiments were conducted using atovaquone resistant parasites and resulting oocyst DNA was analyzed. In addition to these atovaquone studies, artemisinin resistant gametocytes were also grown in vitro and drug pressure was added to determine if resistance mechanisms affect gametocyte development. This study is the first examine gametocyte development in these resistant strains and the first to report that transmission of the atovaquone resistant mutation may be possible. However, data is currently inconclusive on the effect of artemisinin resistance on gametocyte development.

Opportunistic feeding behaviour and partial exophily make An. arabiensis much more difficult to control by indoor residual spraying than any other vector in the Afro-tropical region. The persistent malaria outbreaks in Zimbabwe despite decades of indoor house spraying prompted this investigation into the role of An. arabiensis in malaria transmission and assessment of the possible impact of this control measure. The study was conducted in the malaria endemic districts of Binga and Gokwe. An. gambiae complex mosquitoes were collected from artificial outdoor resting sites, and from human dwellings by i) daytime hut searches, ii) pyrethrum spray catches and iii) exit window traps. Mosquito components were processed to enable: i) the distinction of An. arabiensis from An. quadriannulatus and An. merus on the basis of the pale band at the junction of the hind leg 3/4 tarsomeres; ii) species identification and scoring of inversion polymorphism on the basis of the X chromosome and autosomes respectively; iii) the determination of blood meal sources using the Ouchterlony precipitin test; and iv) identification of An. gambiae s.l. using polymerase chain reaction (PCR) and enzyme electrophoresis techniques. Entomological assessment of residual spraying included determining: the vector resting densities indoors and outdoors, bioassay and insecticides susceptibility tests. Data were also collected on hut profiles, knowledge-attitudes-practices surveys, and household malaria prevalence . surveys. An. arabiensis and An. quadriannulatus were found in sympatry in Binga and Gokwe, and in addition, An. merus was found in Gokwe. Most species identifications were made using PCR; which was found to have 7.5% and 41.6% levels of error for An. arabiensis and An. quadriannulatus respectively, using the cytogenetic technique as benchmark. The pale band technique yielded > 80% correct identification for An. arabiensis but the extent of overlap in the pale band lengths between An. arabiensis and An. quadriannulatus renders the method unsuitable for distinguishing these two species. Inversions 2Rb and 3Ra were found floating in An. arabiensis, with 60% frequency in the former. The Wright's F statistic value of -0.0416 indicated an excess of heterozygotes, and a state of panmixis in the vector population. No significant differences were observed between 2Rb karyotypes in host choice. Human blood indices among indoor (0.82), exit trap (0.98) and outdoor resting (0.30) specimens suggested exophilic behaviour. This was corroborated by the high fed:gravid ratios of 6.8: 1 and 11.6: 1 in sprayed and non-sprayed dwellings respectively. This was worsened by a high feeder-survivor index (FSI) of 93 % among exit trap specimens. The susceptibility to deltamethrin coupled with residual efficacy nine weeks post-spray indicated the suitability of the insecticide. Rural dwellings were suitably built for spraying but had no mosquito proofing. Personal protective measures are hardly known; sleeping outdoors occurs in Siabuwa. While An. arabiensis bites humans indoors the partial exophily it exhibits is a threat to indoor residual insecticide spraying. An integrated malaria control approach is recommended. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Transgenic mosquitoes are beneficial for the design and implementation of various pathogen control programs. However, low and variable expression of transgenes caused by position effects is a hindrance to the characterization and effective use of transgenes in mosquito species. The use of insulator sequences to flank transgenes may have the ability to overcome position effects caused by the genomic environment surrounding the insertion site. CTCF is a multifunctional protein, conserved from humans to Drosophila. Its role as an enhancer blocker in the Drosophila bithorax complex and its proximal binding to other insulator proteins on Drosophila chromosomes makes it a good candidate for identifying insulator sequences throughout the mosquito genome that may be used to improve mosquito transgenesis. Its multi-functionality as a transcription factor and genome organizer also makes CTCF worthy of investigation for an improved understanding of the regulation of the mosquito genome. This study uses chromatin immunoprecipitation with an An. gambiae CTCF antibody followed by Illumina deep sequencing (ChIP-Seq) to identify regions of CTCF binding throughout the An. gambiae genome. A subset of the CTCF binding site peaks was validated using ChIP-PCR. Another subset of this data set, including the ChIP-PCR validated peaks, was input into the motif finding tool, AlignACE, in order to identify a CTCF binding site consensus. Four motifs were identified, none of which were found in more than 11.9% of the ChIP-Seq data set. These results lead us to conclude that An. gambiae CTCF binds to a wider variety of sequences compared to Drosophila CTCF. This work also includes a comparison of the expression profiles of the dipteran insulator proteins, Su(Hw) and CP190, with that of CTCF across multiple life stages in Ae. aegypti. The results of this study suggest the possibility of genomic colocalization, as has been recently discovered in Drosophila. The identification of CTCF binding site peaks throughout the An. gambiae genome provides a large data set of potential insulator sequences that may be used to improve mosquito transgenesis, and provide a new model for the study of CTCF function in a species with medical significance.

Insecticide-treated nets (ITN) are an effective tool to reduce the burden of malaria in Africa. To address questions concerning the sustained impact of ITN in the face of changing transmission intensity and spreading insecticide resistance a series of four entomological investigations was undertaken in Tanzania. / First, a longitudinal survey of vector populations at different altitudes was conducted in the Hai District, Kilimanjaro Region, Tanzania. Entomological parameters relevant to malaria transmission were measured. Vector densities and entomological inoculation rates decreased dramatically with increasing altitude. Seasonal variations in the host preference and resting behaviour of Anopheles arabiensismay influence malaria transmission and control. / Second, the insecticide susceptibility status of vector populations from agricultural sites in the Kilimanjaro Region was evaluated. Malaria vectors from six sites were highly susceptible to diagnostic concentrations of pyrethroid insecticides and DDT in susceptibility tests. Permethrin tolerance in populations of An. arabiensis from an irrigated rice-growing area was not directly related to agricultural use of insecticides. Synergist bioassays indicated the contribution of a metabolic oxidase-based mechanism to the observed resistance while the knockdown resistance (kdr) mechanism was not detected. Close monitoring of resistance development in this area is needed. / Third, the biological efficacy of ITN and the insecticide susceptibility status of mosquito populations from areas with long-term use of ITN were investigated. Data were compiled from national surveys and long-term ITN projects to examine trends in resistance development in Tanzania. Anopheles species were highly susceptible to ITN and diagnostic concentrations of pyrethroid insecticides, while 'nuisance-biting' Culex quinquefasciatus displayed low susceptibility to these compounds. Low efficacy of ITN against C. quinquefasciatus has implications for user acceptance of ITN. / Finally, a novel method for kdr genotyping was developed and evaluated. Sequence specific oligonucleotide probes (SSOP) were used to identify kdr mutations in Anopheles gambiae s.l. in a high-throughput enzyme-linked immunosorbent assay (ELISA) format. Evaluation of the method on field-collected specimens detected the leucine-phenylalanine kdr mutation in two individual An. arabiensis, raising implications for the geographic spread of kdr-based resistance. The novel SSOP-ELISA method is a useful tool for high-throughput screening allowing sensitive detection of insecticide resistance.