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Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Understanding the chromosome evolution and mechanisms of speciation can shed light on developing novel ecological-friendly vector control techniques. Sibling species of the An. gambiae complex provide an excellent model system for these topics. To understand the mechanisms of speciation, we investigated the cellular basis and phenotypes of hybrid male sterility in species crosses of the An. gambiae complex. By performing inter-species crosses of An. coluzzii/An. gambiae and An. merus lab strains, we found an asymmetric pattern of hybrid male sterility existed in sons from reciprocal interspecies crosses. Compared with pure species, hybrid males from crosses of ♀An. merus  ♂An. gambiae/An. coluzzii were normal in the morphology of male reproductive tracts; however, the testes of which that process the reductional meiotic division failed to produce primary spermatocytes and were accompanied with unpaired and insufficiently condensed chromosomes. As a result, primary spermatocytes undergo a mitosis-like anaphase division, producing nonmotile and malfunctional diploid sperm with two tails. However, individuals can mate with females normally and form the mating plug to induce the female monogamy. In contrast, hybrid males from the opposite crosses manifest severely underdeveloped reproductive tracts and a premeiotic arrest of germline stem cells in the testis, accompanied by a strong suppression of premeiotic and meiotic genes. In addition, hybrid males from this cross suffered from a shorter copulation time and failed to form mating plugs to induce female monogamous behaviors, albeit the expression of male accessory gland specific genes were similar between hybrids and pure species. To figure out chromosome evolution in the An. gambiae complex, we studied the molecular organization of heterochromatin and investigated the spatial organizations of autosomal regions of polytene chromosomes in soma and germline cells. We found that molecular composition of pericentrometric autosome and sex chromosome repetitive DNA differs among sibling species of An. gambiae complex with highly similarity between An. coluzzii and An. arabiensis. In addition, heterochromatin blocks of chromosomes have distinct compositions of satellite DNA sequences. Next, in order to address the relationship between inter-chromosomal (Chr-Chr) contacts and chromosome-nuclear envelope (Chr-NE) attachments during the development of the organism, we conducted microscopic analyses of the 3D organization of polytene chromosome in An. gambiae, An. coluzzii, and An. merus. Our quantitative study on chromosome territories in larval salivary gland cells and adult ovarian nurse cells showed that, compared with autosomal arms, the X chromosome has a significantly smaller volume and occupies more compact territories. The number of Chr-Chr contacts and the percentage of Chr-NE attachment were conserved among the species within the same cell type. Our data also demonstrated that there is a significantly and consistently inverse relationship between the frequencies of Chr–NE and Chr–Chr attachments on autosomes of two cell types in all tested species. / Doctor of Philosophy / Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted through the bites of infected females of a few Anopheles mosquito species. Despite being treatable and preventable, malaria is estimated to cause large numbers of deaths every year. Since 2015, the malaria elimination program has stalled largely due to increased insecticide resistance. Novel transgenic techniques have a huge potential in reducing malaria transmission more effectively. However, there are large concerns about the potential negative effects of releasing genetically modified mosquitoes, such as a possibility of accidental spread to non-target species with incomplete reproductive barriers and unpredicted ecological damage. Understanding the mechanisms of speciation about how reproductive isolation occurred and developed as well as chromosome evolution can not only empower the development of ecologically friendly vector control techniques but also improve our basic knowledge. To study mechanisms of speciation, we mated males and females from different closely related species in the Anopheles gambiae complex to investigate the fecundity of hybrid generations. Our study identified two different types of reproductive abnormalities leading to hybrid male sterility. Hybrid males from female An. merus and male An. gambiae or An. coluzzii have normal appearing testes and male accessary glands but the testes produce abnormal sperms, which cannot move and have two tails. Hybrid males from female An. gambiae or An. coluzzii and An. merus have severely underdeveloped testes and male accessary glands. The sperm producing process stops unusually very early in their tiny underdeveloped testes. We also investigated chromosome evolution in species of An. gambiae complex. We found that chromosomal parts containing repetitive DNA, the sequence in the genome not producing proteins, evolve rapidly in An. coluzzii, An. arabiensis, An. quadriannulatus, and An. merus. In contrast, chromosome territories of gene rich regions in giant polytene chromosomes from larval salivary gland cells and adult ovarian nurse cells of An. gambiae, An. coluzzii, and An. merus, were relatively conserved within the same cell type among different species. However, the chromosomal 3D distribution pattern is different among various cell types in these species.

Retrogenes are a group of functional genes produced by gene retroduplication events during evolution. It has been observed that many retrogenes have formed since the evolutionary divergence of Anopheles mosquitoes from the Aedes lineage as a result of developing heteromorphic sex chromosomes. It has been further observed that these retroduplications predominately occur from parent genes on the heteromorphic X chromosome to autosomes and have a predisposition to have enriched expression in testis. In order to investigate the nature of this male-biased expression in testis, we utilized bioinformatic techniques to identify retrotransposition events and assign them relative ages based on evolutionary branches of divergence. This list of parent genes and retrogenes were then analyzed and a total of twenty-five gene pairs were selected for further examination. Available gene expression data in the form of RNA-seq and DNA microarray were used in tandem with gene annotation data to computationally investigate gene pairs in An. coluzzii. These pairs were further investigated experimentally by means of RT-PCR conducted on dissected head, thorax, abdomen, and reproductive organs in both male and female Anopheles coluzzii Mopti strain. Testis and male accessory glands (MAGs) were also investigated by this method in An. coluzzii. Available expression data support previously observed testis enriched expression of retrogenes and provides evidence for the predominate expression of retrogenes occurring in postmeiotic cells suggesting retrogene involvement in sperm development. Experimental evidence revealed a small group of five retrogenes which exhibit the expected male-biased expression in male testis with little to no expression in female ovaries, although a shared expression in the heads of both sexes was observed. Of the five retrogenes, four carry out energy related functions involving mitochondria, suggesting contribution to energy requirements of developing sperm. Testis and MAG experiments in An. coluzzii revealed a predisposition for retrogenes to be expressed in testis while parent genes tended to have higher expression in MAGs, and this phenomenon is partially supported by DNA microarray expression data. Overall, these results suggest further investigation of retrogenes in An. coluzzii may reveal unique functions in male mosquito fertility that are exploitable in genetic approaches to mosquito control. / Master of Science in Life Sciences / Malaria is a potentially deadly disease which effects thousands of people every year. Malaria around the world is spread by multiple species of mosquitoes in a genus called Anopheles. Controlling the populations of these disease spreading mosquitoes is essential to preventing the spread of malaria. Current insecticide-based approaches used to stop mosquitoes are becoming less effective overtime as mosquitoes become resistant. A potential way to develop new techniques for mosquito control is through research involving mosquito reproductive genetics. Understanding the genes involved in how mosquitoes reproduce could improve future techniques designed to reduce or prevent mosquitos from reproducing. This research focuses on a group of genes called retrogenes which have formed over the evolution of these mosquitoes via the duplication from a separate parent gene. In mosquitoes these retrogenes are understood to be involved in male reproduction. The retrogenes involved in male mosquito reproduction could have important functions in male sexual reproduction and sterility. These important genes could be manipulated to interrupt whatever important functions these genes have in reproduction. In this research we first computationally identified retrogenes and their parent genes and categorized them by age. We then utilized available annotation and expression data to analyze the potential significance of retrogenes to male fertility and found that multiple retrogenes tended to be expressed during sperm development. Lastly, we conducted gene expression experiments using dissected head, thorax, abdomen, and reproductive organs in both male and female Anopheles mosquitoes. Results revealed unique patterns of expression that suggest male specific roles of five retrogenes in testes and head expression in both males and females suggesting a possible role in mating behavior. These results provide evidence that retrogenes do have functional roles in male fertility specifically related to the maturation and development of sperm.

My graduate work focused on the syntheses and pharmacology of species-selective aryl methylcarbamate acetylcholinesterase inhibitors to combat the malaria-transmitting mosquito, Anopheles gambiae. We identified six novel carbamates that demonstrated levels of target selectivity exceeding our project milestone of 100-fold. Among the C2-substituted phenylcarbamates examined (class II), 2'-(2- ethylbutoxy)phenyl N-methylcarbamate (9bd*) was extraordinarily selective (570-fold ± 72). The high level of selectivity observed for many of the class II carbamates was attributed to a helical displacement within the active site of An. gambiae acetylcholinesterase, able to accommodate carbamates with larger C2-substituted secondary β-branching side chains. Conversely, this type of side chain forms unfavorable interactions within the active site of human acetylcholinesterase. The C3-substituted carbamates (class I), such as terbam (9c), were less selective than many of the class II carbamates; however, class I carbamates related to terbam (9c) were highly toxic to An. gambiae. In particular, the contact toxicity measured for 9c (LC₅₀ = 0.037 mg/mL) was equal to the commonly used agricultural insecticide, propoxur (9a, LC₅₀ = 0.037 mg/mL). In total, seventy aryl carbamates were screened for their inhibition potency and contact toxicity towards An. gambiae. The common final step in all of these syntheses was the carbamoylation of a phenol, which normally proceeded in a 70 to 90% yield. Thirty seven novel carbamates are reported out of the seventy two prepared. Although sixteen of the phenols were commercially available, the others were prepared with known and adapted synthetic methodologies. The emerging structure-activity relationships led us to focus on the synthesis of 3-tert-alkylphenols (Class I) and 2-alkoxy or 2-alkylthio-substituted phenols (Class II). Three methods particularly stand out: First, we applied the methods of Tanaka to prepare 3-tert-alkylphenols wherein a methyl group was replaced by a trifluoromethyl group. Second, we adapted the methods of Tanaka to prepare 3-tert-alkylphenols that lack fluorine substitution. This method is competitive with the little known method of Reetz to convert aryl ketones to the corresponding 1,1-dimethylalkyl group and allows one to access electron rich tert-alkyl-substituted aromatics that are not accessible by the Friedel-Crafts alkylation (Friedel-Crafts restricted). Third, we found a convenient and high-yielding method for selective S-alkylation of 2-mercaptophenol. In addition to the synthesis of carbamates, the preparation of one hundred three intermediates, phenols, and electron rich tert-alkyl arenes are reported. / Ph. D.

In this project, an outgrowth and optical density (OD) measurement step was developed and validated for an established protocol for antibiotic treatment of Anopheles stephensi mosquitoes. This step allows the elimination of uncleared mosquitoes from experimental pools. Our findings suggest that, as previously demonstrated, antibiotic treatment reduced the mosquito microbiome to an extent, as evidenced by decreased bacterial growth observed through OD readings of our experimental samples. Additionally, qPCR analysis of 16S abundance provided quantitative data on bacterial abundance, which further supported the effectiveness of antibiotic treatment. Finally, a comparison of mosquito samples pooled randomly or with high OD individuals removed showed a significant improvement in bacterial clearance in samples with our refinement step included. The method established here can generate experimental samples for third-generation sequencing with minimal bacterial contamination for robust hypothesis testing. The addition of OD measurement is a rapid, cost-effective step that can potentially improve hypothesis testing on large pools of samples. This study contributes to our understanding of the impact of antibiotic treatment on mosquito microbiomes. Improvements in molecular and bench-level techniques can greatly contribute to our ability to gain insights into the role of microbes in mosquito biology.

Anopheles gambiae sensu lato mosquitoes are among the dominant malaria vectors in sub-Saharan Africa. However, not much is known about the oviposition behaviour of these species necessary for the development of malaria vector control strategies. With the aim of investigating cues associated with selected oviposition sites, artificial oviposition sites- ponds (soil mixed with water) were set-up in an open field at Mbita, Western Kenya in 2012 and 2013. Ponds were allowed to be colonized by wild An. gambiae s.l.. The numbers of Anopheles early instar larvae were counted and used as a proxy for oviposition preference. Water samples were then analysed for physicochemical, bacterial and chemical profiles. The bacterial profiles were analysed using denaturing gradient gel electrophoresis (DGGE) and the chemical profiles with gas chromatography-mass spectrometry (GC-MS). The detection of possible oviposition cues from oviposition substrates requires sensitive analytical methods. Volatiles detection was improved seven times. The detection of bacteria deoxyribonucleic acid (DNA) bands with DGGE was also improved to a minimum DNA concentration of 50 ng/µl. Results showed that ponds were colonized differently. Fresh ponds were preferred over slightly older ponds. Bacterial analysis revealed a low number of bacteria colony forming units (CFU) in preferred ponds. Some volatiles, including: 6,10-dimethyl-5,9-undecadien-2-one (geranylacetone) and 4-ethylbenzaldehyde, were associated with the oviposition preferred pond. In addition, low pH and high turbidity were associated with the ponds selected for oviposition. Finally, fungi isolated from the rhizomes of nut grass yielded a promising array of volatiles of which one is known to attract oviposition site seeking malaria mosquitoes. This finding opens the door for a cost effective and environmental friendly method of using fungi in an “attract and kill” strategy targeting malaria vectors. / Myggor i Anopheles gambiae sensu lato komplexet tillhör de myggor som är bäst på att sprida malaria parasiter i afrika söder om Sahara. Kunskapen om de här myggornas äggläggningsbeteende är begränsad. Den här kunskapen behövs för att kunna utveckla nya och förbättra tillgängliga malaria vektor kontroll metoder. Nya metoder som kan komplettera de som används idag (insecticides treated nets (ITNs) och indoor residual spraying (IRS)) behövs eftersom de metoderna har problem med resistensutveckling. Två studier utfördes på icipe fältstation i Mbita västra Kenya under 2012 och 2013 med målet att identifiera faktorer som påverkar myggornas äggläggningsbeteende. Baljor fyllda med en blandning av jord och vatten (äggläggningssubstrat) användes för att tillverka artificiella äggläggningsplatser som liknar de vattenpölar som de här myggarterna gärna lägger ägg i. Baljorna koloniserades av vilda myggor och antalet mygglarver som detekterades i baljorna jämfördes och användes som en proxy för äggläggningspreferens. Fysikaliska och kemiska parametrar mättes på jordvattenblandningarna i baljorna och prover togs för att analysera bakteriepopulationer med hjälp av denaturing gradient gel electrophoresis (DGGE) och flyktiga ämnen med hjälp av gas-kromatografi kopplat till mass-spektrometri (GC-MS). För att kunna detektera de låga halter av flyktiga ämnen och bakterier som fanns i de här proverna krävdes det känsliga metoder. Antalet flyktiga ämnen som kunde detekteras ökades sju gånger genom att tillsätta NaCl till vattenproverna innan doften insamlades och termisk desorption användes istället för lösningsmedels desorption. För att förbättra detektionsgränsen för bakterier amplifierades bakterie-DNA i två PCR reaktioner som sedan mixades och koncentrerades. Resultaten från fältstudierna med baljorna visade att de koloniserades olika av Anopheles myggorna. Baljor med nyblandat substrat innehöll dubbelt så många mygglarver som baljor med jord-vattensubstrat som åldrats under en längre tid. Lägre mängd bakterier, lägre pH och högre grumlighet var gemensamt för de baljor som myggorna föredrog. De flyktiga ämnen som detekterades i de olika baljor varierade mellan olika försök och inget ämne fanns med i alla upprepningar av ett försök. Trots det detekterades några ämnen oftare i de baljor som myggorna föredrog att lägga ägg jämfört med de med en mindre mängd mygglarver. De inkluderar geranylacetone och 4-ethylbenzaldehyde. Svampar isolerades från rotstockar av gräs som fanns i den jord som användes för att göra äggläggningssubstraten i fältstudierna. De flyktiga ämnen som avgavs från svampkulturerna analyserades. Bland annat så identifierades ett ämne som fungerar som en äggläggningsattrahent för An. gambiae s.l. myggor.Resultaten från den här avhandlingen kommer att kunna användas för att utveckla miljövänliga ”attract and kill” metoder för att kontrollera malaria myggor. / <p>QC 20160211</p>

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.

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.

Introdução - A malária é uma das principais doenças humanas do mundo e afeta principalmente as populações pobres em regiões tropicais e subtropicais onde as condições ambientais são favoráveis tanto para a proliferação dos agentes etiológicos como dos mosquitos vetores. No Brasil, An. darlingi é considerado vetor primário de plasmódios humanos. Devido à importância médica, esse inseto tem sido objeto de campanhas de controle populacional. No entanto, não foi considerada a ocorrência de microevolução em An. darlingi e consequentemente, a possibilidade de populações diferentes apresentarem características biológicas distintas. Objetivo Verificar a presença de variabilidade genética e morfológica em populações de An. darlingi no Brasil. Metodologia - Foram analisados e comparados 26 atributos da morfologia externa de ovos de nove populações, bem como a morfometria geométrica da forma alar de espécimes de 10 localidades diferentes de Anopheles darlingi. Além disso, foram empregadas sequências do gene mitocondrial citocromo oxidase subunidade I (COI) para analisar a estrutura populacional de An. darlingi. Resultados e Conclusão Apesar dos atributos dos ovos apresentarem variação, somente as amostras de Tocantins e Pará foram diferenciadas das demais populações. As variações nas estruturas externas dos ovos são provavelmente adaptativas, com influência de fatores ambientais, como temperatura, umidade e disponibilidade de alimento para as fêmeas. A comparação da morfometria geométrica da asa demonstrou que existe maior similaridade entre as populações da costa (estados do Espírito Santo , do cerrado (estados de Goiás e Tocantins), do norte do rio Amazonas (estados do Amazonas e Amapá), e do sul do rio Amazonas (estados de Mato Grosso e Pará). As análises de COI demonstraram diferenciação das populações da costa da Mata Atlântica em relação às demais populações do Brasil central, Amazônia e Mata Atlântica do Interior. O padrão da diversidade de haplótipos de COI, os testes de neutralidade e a distribuição de diferenças pareadas indicaram recente expansão demográfica. A distância geográfica, as ecorregiões e fatores ambietais podem provavelmente limitar a dispersão da espécie, tendo alguma influência sobre a estrutura populacional de An. darlingi / Introduction Malaria is a major human disease that primarily affects poor persons in tropical and subtropical regions where environmental conditions are favorable to the proliferation of the etiological agent as well as vector mosquitoes. In Brazil, Anopheles darlingi is considered a primary vector of human plasmodia and has been the target of control campaigns because of its medical importance. However, the occurrence of microevolution in An. darlingi was not considered and, consequently, the possibility of different populations exhibit distinct biological characteristics. Objective Verify the presence of morphological and genetic variability in Brazilian Anopheles darlingi populations. Methods Twenty six attributes of external egg morphology were analyzed and compared in nine populations. Similarly, the geometric morphometry of wing shape was evaluated in ten An. darlingi localities. In addition, the mitochondrial gene cytochrome oxidase subunit I (COI) was employed to analyze the population structure of An. darlingi. Results and Conclusion - Although, egg attributes showed variation, only samples from Tocantins and Pará were differentiated from other populations. Variation in external egg structure is arguably adaptive in light of environmental factors such as temperature, humidity and food availability for females. Comparisons of wing geometric morphometry indicated that there is greater similarity between Coastal (Espírito Santo and Rio de Janeiro states) and Inner (São Paulo and Paraná states) populations of the Atlantic Forest, and differentiation of those from (Goiás and Tocantins states) and regions both north (Amazonas and Amapá states) and south (Pará and Mato Grosso states) of the Amazon Region. Cytochrome oxidase I analyses indicated differentiation between coastal Atlantic Forest populations (Espírito Santo and Rio de Janeiro states) and those from the cerrado, the Amazon and the interior Atlantic Forest. The pattern of COI haplotype diversity, neutrality tests and distribution of pairwise differences suggest a recent demographic expansion. Geographical distance, ecoregions and potentially environmental constraints may limit the species´ dispersal and influence the population structure of An. darlingi