The delineation of desmid species was traditionally based on purely morphological features. However, a frequent misinterpretation of morphological variability in desmids has led in the past to extensive taxonomical confusion within this important group of green algae which complicates the interpretation of their biodiversity in freshwater ecology, biogeography and biomonitoring. Consequently, I focused in this thesis predominantly on a previously neglected issue, the application of polyphasic approaches in the species-level taxonomy of desmids. In the most studies, a combination of both traditional morphological and modern molecular phylogenetic and geometric morphometric methods has been used to evaluate the taxonomy of selected desmid species, particularly representatives of the morphologically complex genera Micrasterias and Xanthidium. In two papers, I used the combination of traditional morphological and autecological data to clear up the taxonomy of several morphologically less prominent desmid taxa. Generally, the results of the thesis demonstrated that the way we recently see the diversity and distribution of desmids should be thoroughly changed. The real species diversity is mostly distinctly finer than that estimated by classical morphological taxonomy, often corresponds to varieties of...

Der obligat intrazelluläre Parasit Toxoplasma gondii ist der Erreger der Toxoplasmose, und dient zugleich als wichtiger Modellorganismus für weitere Human- und Tierpathogene, wie z.B. Plasmodium oder Eimeria. Die Vermehrung von T. gondii erfordert eine effiziente Biosynthese von Phospholipiden für die Herstellung neuer Membranen, was durch die de novo Synthese durch den Parasiten, und/oder den Import von Lipiden aus der umgebenden Wirtszelle gewährleistet werden kann. Während der Parasit zahlreiche Möglichkeiten für Synthese oder Import von PtdEtn und PtdSer verwendet, scheint die Biosynthese des abundantesten Membranlipids PtdCho auschließlich über den CDP-Cholin Weg zu erfolgen. Dieser erstreckt sich in T. gondii über 3 zelluläre Kompartimente, mit einer cytosolischen Cholin-Kinase (TgCK), einer im Zellkern lokalisierenden Cholin-Cytidylyltransferase (TgCCT) und einer Cholin-Phosphotransferase (TgCPT) im ER. Anders als die substrat-spezifische Ethanolamin-Kinase (TgEK), kann TgCK neben Cholin außerdem Ethanolamin phosphorylieren. TgCK zeigt eine geringe Affinität zu Cholin (Km ~0.77 mM), während eine verkürzte TgCK (TgCKS), welcher eine als Signalpeptid vorhergesagte N-terminale Sequenz (20 Aminosäuren) fehlt, eine etwa 3-fach höhere Aktivität aufweist (Km ~0.26 mM). Während jedoch die Wildtyp-TgCK cytosolische Cluster in Toxoplasma bildet, zeigt die verkürzte TgCK eine gleichmäßigere cytosolische Lokalisierung. Wir schlussfolgern daraus, dass der hydrophobe N-Terminus nicht notwendig ist für eine funktionale TgCK, sondern eine strukturelle Funktion bei der Protein-Lokalisierung hat. Eine konitionelle Mutante, in welcher der TgCK Promoter gegen den Tetracyclin-regulierbaren Promoter pTetO7Sag4 ausgetauscht wurde (Deltatgcki), zeigt erstaunlicherweise normales Wachstum und PtdCho Biosynthese. Die TgCK Aktivität und die daraus resultierende PtdCho Synthese sind nur zu ~30% regulierbar. Unsere Ergebnisse deuten auf die Verwendung eines alternativen Startcodons bzw. Promoters hin, welcher zur Expression einer verkürzten (~53-kDa) aber vermutlich aktiven Cholin Kinase führt, wodurch der Verlust der TgCK (~70-kDa) kompensiert wird. Der konditionelle Knockout von TgCCT, dem regulatorischen Enzym des CDP-Cholin Wegs, hatte einen 50%igen Wachstumsdefekt zur Folge. Diese Studie zeigt eine erstaunliche Flexibilität des Parasiten bezüglich seiner Membranzusammensetzung, und bestätigt zugleich die Annahme, dass PtdCho nicht von der Wirtszelle importiert werden kann. Diese Anpassungsfähigkeit stellt einen möglichen Faktor dar, der es T. gondii erlaubt sich in einem breiten Spektrum von Wirten zu vermehren. / Toxoplasma gondii is an obligate intracellular apicomplexan parasite that causes life-threatening disease in neonates and in immunocompromised people. Successful replication of Toxoplasma requires substantial membrane biogenesis, which must be satisfied irrespective of the host-cell milieu. Like in other eukaryotes, the two most abundant phospholipids in the T. gondii membrane are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). Bioinformatics and precursor labeling analyses confirm their synthesis via the CDP-choline and CDP-ethanolamine pathway, respectively. This work shows that the 3-step CDP-choline pathway, involving the activities of TgCK, TgCCT and TgCPT, localizes to the cytosol, nucleus and ER membrane, respectively. The initial reaction is catalyzed by a dual-specificity choline kinase (TgCK, ~70-kDa), capable of phosphorylating choline as well as ethanolamine. The purified full-length TgCK displayed a low affinity for choline (Km ~0.77 mM). TgCK harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. The displacement of the TgCK promoter in a conditional mutant of T. gondii (deltatgcki) attenuated the enzyme expression by ~80%. Unexpectedly, the ?tgcki mutant was not impaired in intracellular growth, and exhibited a normal PtdCho biogenesis. To recompense for the loss of full-length TgCK, the mutant appears to make use of an alternative promoter and/or start codon, resulting in the expression of a shorter but active TgCK isoform identified by the anti-TgCK antiserum, which correlated with its persistent choline kinase activity. Accordingly, the ?tgcki showed an expected incorporation of choline into PtdCho, and susceptibility to dimethylethanolamine (a choline analog). Interestingly, the conditional mutant displayed a regular growth in off state despite a 25% decline in PtdCho content, which suggests a compositional flexibility in T. gondii membranes and insignificant salvage of host-derived PtdCho. The two-step conditional mutagenesis of TgCCT, which caused a reduced growth rate to about 50%, further substantiated this finding. The enzymatic activity of TgCCT and its role in PtdCho synthesis remain to be proven, however. Taken together, the results demonstrate that the CDP-route is likely essential in T. gondii. The competitive inhibition of choline kinase to block the parasite replication appears a potential therapeutic application.The work also reveals a remarkably adaptable membrane biogenesis in T. gondii, which may underly the evolution of Toxoplasma as a promiscuous pathogen.

A especiação de isolados clínicos de Aspergillus ganhou relevância nos últimos anos devido à descrição de espécies crípticas resistentes aos derivados azólicos. A identificação morfológica convencional não é capaz de discriminar as espécies de Aspergillus e o sequenciamento de DNA é uma técnica pouco adaptada a laboratórios clínicos. A espectrometria de massas por ionização/dessorção a laser auxiliada por matriz tempo-devoo (EM MALDI-TOF) é uma metodologia emergente que vem sendo explorada com intuito de fornecer identificação rápida e acurada de microrganismos, incluindo os fungos filamentosos de importância clínica. Entretanto, há poucos estudos avaliando a plataforma VITEK MS® para a identificação de espécies de Aspergillus. O presente estudo teve como objetivo fornecer dados adicionais sobre a performance dos sistemas do VITEK MS® e suas bibliotecas de espectros de referências (ER) In Vitro Diagnostics (IVD) e Research Use Only (RUO) para identificar as espécies de Aspergillus de relevância clínica. Uma biblioteca de ER in-house também foi construída e avaliada. Um total de 106 organismos foram avaliados por EM, incluindo 47 cepas provenientes das coleções de fungos do Westerdijk Fungal Biodiversity Institute (Holanda) e do LEMIUNIFESP, seis isolados ambientais (IMT-USP) e 53 isolados cínicos do HC-FMUSP. Foram utilizados dois protocolos de extração proteica, um recomendado pelo fabricante e outro empregando meio de cultura líquido para os isolados/cepas com baixa esporulação. Trinta e cinco organismos foram selecionadas para construir os ERs, e os 71 restantes foram usados para avaliação de desempenho das bibliotecas IVD, RUO e RUO+in-house. Entre os 71 organismos analisados, 91,5%, 84,5% e 100% tiveram identificação correta de gênero pelos bibliotecas IVD, RUO e RUO+in-house, respectivamente. Enquanto para identificação de espécie, as bibliotecas IVD, RUO e RUO+in-house mostraram 83,1%, 77,4% e 90,1% de identificações de espécie, respectivamente. Para as 16 espécies crípticas de Aspergillus analisadas, a identificação foi correta em 31,2%, 18,7% e 62,5%, pelos sistemas IVD, RUO e RUO+in-house, respectivamente. Entre as espécies crípticas resistentes aos derivados azólicos, o sistema IVD forneceu identificação correta para as espécies Aspergillus lentulus, Aspergillus calidoustus e Aspergillus sydowii. Entretanto, Aspergillus fumigatiaffinis e Neosartorya pseudofischeri foram erroneamente identificadas como Aspergillus fumigatus pelo sistema IVD. A biblioteca in-house demonstrou melhor performance, mas espécies filogeneticamente próximas como A. fumigatiaffinis e A. lentulus tiveram identificações cruzadas. Concluímos que o VITEK® MS demonstrou boa performance para a identificação das espécies de Aspergillus, porém para algumas espécies crípticas, há necessidade de melhoria das bibliotecas de espectro de referência comercializadas. Algumas espécies crípticas filogeneticamente relacionadas apresentaram espectros similares e são de difícil diferenciação por EM MALDI, mesmo com a construção de uma biblioteca de ER in-house com vários representantes de cada espécie / Aspergillus spp. identification has become more relevant in clinical practice since azoleresistant cryptic species have emerged in the last years. Conventional morphologic identification is not able to discriminate Aspergillus species and DNA sequencing is not feasible for clinical laboratories. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is an emergent technology that has been explored to provide fast and accurate identification of microorganisms, including clinically relevant molds. However, only a few studies have explored the platform VITEK MS® for the identification of Aspergillus species. The present study aimed to provide additional data regarding the performance of the In Vitro Diagnostics (IVD) and Research Use Only (RUO) systems for the identification of Aspergillus species, including azoleresistant ones, and also to construct and to evaluate an in-house reference spectrum library. A total of 106 organisms were evaluated by MS, including 47 Aspergillus strains (Westerdijk Fungal Biodiversity Institute and LEMI-UNIFESP collections), six environmental (IMT-USP) and 53 clinical isolates (HC-FMUSP). Two protein extraction protocols were used, one recommended by the manufacturer and another with liquid broth for the organisms with poor sporulation. Thirty-five organisms were selected to construct the in-house reference spectrum library and the remaining 71 were used for the performance evaluation. Correct genus identification was provided in 91.5%, 84.5% and 100% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Correct species identification was provided in 83.1%, 77.4% and 90.1% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the 16 Aspergillus cryptic species, correct identification was provided in 31.5%, 18.7% and 62.5% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the azole-resistant cryptic species, the IVD system provided correct identification for Aspergillus lentulus, Aspergillus calidoustus and Aspergillus sydowii. However, Aspergillus fumigatiaffinis and Neosartorya pseudofischeri were misidentified as Aspergillus fumigatus by the IVD system. The in-house library had better performance for the identification of Aspergillus cryptic species, but closely related taxa may be difficult to have correct differentiation by MALDI-TOF MS. In conclusion, VITEK® MS showed good performance for the identification of Aspergillus species and some azoleresistant species. However, a more robust reference spectrum library including more representatives of azole-resistant cryptic species may be necessary to achieve better identification performance of closely related taxa

Enquanto muitas espécies de mosquitos são conhecidas por sua importância epidemiológica, a capacidade vetorial de algumas ainda não é clara. A identificação taxonômica, bem como a sexagem dos espécimes de Culicidae são essenciais para qualquer estudo ou ação, entretanto muitas vezes caracteres-chave estão danificados ou são restritos a uma fase de vida do animal. Em espécies crípticas, a identificação dos espécimes também pode ser prejudicada, o que dificulta, sobretudo, a compreensão da atividade vetorial em mosquitos. A morfometria geométrica alar, uma técnica barata e precisa para identificação de sutis dissimilaridades morfológicas, pode contribuir para a resolução destes tipos de problemas. O grupo de pesquisa MosquitoLab aplica esta técnica há mais de 10 anos e acumulou milhares imagens de asas com seus respectivos metadados. Estas informações, se organizadas como banco de dados relacional, podem permitir a sistematização do uso, consulta e armazenamento dos dados, além de viabilizar novos estudos e servir também como base para aplicações de identificação automática de mosquitos. Portanto, os objetivos deste trabalho foram avaliar a utilidade da morfometria geométrica alar para a resolução de dois problemas biológicos: dimorfismo sexual e espécies crípticas, e desenvolver um protótipo de plataforma para armazenamento de dados biológicos relacionados à morfometria de asa, por meio de um banco de dados relacional e um sistema web nomeado WingBank. Com base na técnica de morfometria geométrica alar, dois estudos de caso foram realizados: a) avaliação do dimorfismo sexual de 10 espécies de mosquitos de interesse médico e, b) diferenciação morfológica alar de Anopheles strodei s.s. e Anopheles arthuri s.l. com base em dois diferentes conjuntos de pontos anatômicos. Para construção do protótipo do WingBank uma equipe multidisciplinar realizou levantamento de requisitos, modelagem e criação do banco de dados relacional, e implementação de uma plataforma web. Os resultados referentes ao dimorfismo sexual alar apresentaram significativa diferenciação entre os sexos e padrões específicos de forma alar em todas as espécies estudadas. Os pontos anatômicos alares mais variáveis foram os das regiões proximal e distal das veias mediana e radial. Fêmeas apresentaram asas significativamente mais largas e curtas do que os machos. Diferenciação morfológica alar entre as espécies crípticas avaliadas foi observada em ambos os conjuntos de dados (18 e 22 pontos), sendo que no conjunto de 22 pontos com alometria, foi mais evidente. Os pontos anatômicos mais variáveis nas análises com os conjuntos de 18 e 22 pontos foram 1, 2 e 17, e 1, 2, 19, respectivamente. Finalmente, o protótipo WingBank foi implementado com dados referentes a 77 espécies pertencentes a 15 gêneros de Culicidae. Ao todo foram catalogados 13.287 registros de asas, dos quais 2.138 já estão disponibilizados a partir do presente trabalho, para uso de terceiros. Globalmente, este é o maior banco de dados de asas de Culicidae de que temos conhecimento. / While many species of mosquitoes are known for their epidemiological importance, the vector capacity of some is still unclear. Taxonomic identification and sexing of Culicidae specimens are essential for any study or action; however, often key characters are damaged or restricted to a stage of life of the animal. In cryptic species, the identification of specimens can also be damaged, which makes it difficult to understand the vector activity in mosquitoes. Geometric morphometry, an cheap and precise technique for identifying subtle morphological dissimilarities, may contribute to the resolution of these types of problems. The MosquitoLab research group has applied this technique for more than 10 years and has accumulated thousands of wing images with their metadata. This information, if organized as a relational database, may allow the systematization of the use, consultation and storage of the data, besides making possible new studies and lso serve as a basis for applications of automatic identification of mosquitoes. Therefore, the aims of this work were to evaluate the usefulness of the geometric morphometry to solve two biological problems: sexual dimorphism and cryptic species, and to develop a prototype of plataform for the storage of biological data related to wing morphometry, by means of a relational database and a web system named \"WingBank\". Based on the technique of wing geometric morphometry, two case studies were carried out: a) evaluation of the sexual dimorphism of 10 species of mosquitoes of medical importance and b) wing morphological differentiation of Anopheles strodei s.s. and Anopheles arthuri s.l. based on two different sets of landmarks. In order to build the WingBank prototype a multidisciplinary team performed requirements survey, modeling and creation of the relational database, and implemented a web platform. The results regarding wing sexual dimorphism showed significant differentiation between the sexes and specific patterns of wing shape in all species studied. The most variable landmarks were those of the proximal and distal regions of the medial and radial veins. Females showed slightly and significantly wider and shorter wings than males. Wing morphological differentiation between An. strodei s.s. and An. arthuri s.l. was observed in both sets of data (18 and 22 landmarks), but in the set of 22 with allometry, it was more evident. The most variable landmarks in the analyzes with the sets of 18 and 22 landmarks were 1, 2 and 17, and 1, 2, 19, respectively. Finally, the WingBank prototype was implemented with data referring to 77 species belonging to 15 genera of Culicidae. In all, 13,287 wing records were cataloged, of which 2,138 are from the present work, already available for use by third parties. Globally, as far as we know, this is the largest database of Culicidae wings.

O fitoplâncton é composto por uma gama de organismos fotossintetizantes de diversas origens evolutivas. São os principais produtores primários dos oceanos embora sua biodiversidade seja muito pouco estudada, principalmente as frações do nano e picoplâncton. A composição florística dessas duas frações do fitoplâncton marinho é praticamente desconhecida nas águas brasileiras, entretanto diversos trabalhos ecológicos e oceanográficos relatam sua importância, pois são os mais abundantes nas regiões marinhas. O presente estudo teve como objetivo isolar espécies de microalgas, com ênfase nos fitoflagelados, a partir de amostras coletadas de Cananéia, Ubatuba e Santos, a fim de aumentar o conhecimento acerca da composição taxonômica desses organismos e permitir estudos futuros, envolvendo análises moleculares, ultraestruturais e fisiológicas. Para isso, foram usadas várias técnicas tradicionais de isolamento, como cultura mista, micropipetagem e diluições sucessivas, plaqueamento em ágar e diluição seriada. Foram isolados 49 cepas das classes Cyanophyceae, Trebouxiophyceae, Cryptophyceae, Bacillariophyceae, Prasinophyceae, Nephroselmidophyceae, Chrysophyceae e outros não identificados. Análises pigmentares foram realizadas em especial para o grupo das Cryptophyceae para confirmar a identificação em nível genérico. Vários dos táxons isolados representam novos registros para o estado de São Paulo e para a costa do Brasil. Os resultados do presente estudo são uma boa partida para a inclusão filogenética dos organismos isolados na costa do Brasil no contexto mundial, auxiliando a desvendar a biodiversidade críptica de espécies até o momento desconhecidas para as regiões estudadas / Phytoplankton comprises a range of photosynthetic organisms with different evolutionary origins. They are the main primary producers in the oceans but its biodiversity is poorly studied, especially on the nano and picoplankton fractions. Although the floristic composition of these two fractions of the marine phytoplankton is virtually unknown in Brazilian waters, several studies have reported the oceanographic and ecological importance of these groups, which are the most abundant in marine environment. This study aimed to isolate microalgae species with emphasis on \"phytoflagellates\" from samples collected in Cananéia, Ubatuba and Santos in order to increase the knowledge about their taxonomic composition and allow future studies involving molecular, ultrastructural and physiological analysis. For this, several traditional single-cell isolation techniques were used such as enrichment culture, micropipette, agar plating and serial dilutions. There were isolated 49 strains of Cyanophyceae, Trebouxiophyceae, Cryptophyceae, Bacillariophyceae, Prasinophyceae, Nephroselmidophyceae, Chrysophyceae classes and others unidentified. Pigment analyzes were performed specially on Cryptophyceae group in order to confirm their identification at generic level. Several of the isolated taxa are new records for the state of São Paulo and the coast of Brazil. The findings of this study enable the phylogenetic inclusion of organisms isolated from the Brazilian coast in a world context, helping to unravel the cryptic biodiversity of organisms hitherto unknown for the studied regions

A infecção de trato respiratório superior em gatos é uma afecção muito frequente em indivíduos que vivem em abrigos, com elevada morbidade e em alguns casos, fatal. O herpesvírus felino tipo1 (FHV-1) e a Chlamydophila felis estão entre os principais causadores. O FHV-1 ocasiona quadros de espirros, secreção nasal e alterações oculares como conjuntivite. A C. felis é responsável pelos piores casos de conjuntivite e apresenta um plasmídeo críptico como possível fator de virulência. A presença dos retrovírus da leucemia felina (FeLV) e/ou imunodeficiência dos felinos (FIV) debilita a função do sistema imunológico, causando imunossupressão e consequentemente aumento no índice de morbidade e mortalidade. Neste trabalho foram avaliados quatro abrigos, três gatis particulares não-comercias (um localizado em Osasco/SP e outros dois São Paulo/SP). Os gatis possuiam alta densidade populacional e a procedência dos gatos alojados era desconhecida. A detecção de FHV-1, como de C. felis e de três genes do plasmídeo criptico foram realizadas por PCR em amostras de mucosa oral e de conjuntiva ocular de ambos os olhos obtidas com swabs de algodão, secos e estéreis. Amostras de sangue foram coletadas para a detecção do FIV e FeLV por meio de teste imunoenzimático. O sintomas clínicos dos animais foram classificados de 1 a 4, sendo 4 atribuído àqueles que apresentavam pior sintomatologia. A ocorrência de FIV e FeLV no 1° gatil foi de 4,63% e 3,70%, no 2° gatil foi de 0% e 6,45%, enquanto que no 3° gatil foi 75% e 0% respectivamente, estes vírus não foram detectados no 4° gatil. FHV-1 foi observado em 61,11% dos gatos no 1° gatil; 90,32% no 2° gatil, 100% no 3° gatil e em 89,74% dos animais do 4° gatil. No 1° gatil, 7,41% das amostras apresentavam C. felis, no 2° gatil, 58,06%; no 4° gatil, 23,08%; enquanto que no 3° gatil o agente não foi detectado. Dentre as amostras positivas para C. felis, os genes do plasmídeo críptico foram detectados; no 1o gatil o gene 1 estava presente em 62,50% das amostras, o gene 2 e 3 em 75%, para o 2° gatil obteve-se 61,11% de positividade para os genes 1 e 2 e 55,56% para o gene 3; no 4° gatil o gene 1 e 3 estavam presentes em 77,78% das amostras, o gene 2 em 55,56%. Os óbitos relatados no período do estudo foram de animais classificados com sintomas 3 ou 4 e positivos para C. felis e para o plasmídeo críptico. No presente trabalho foi observada uma elevada ocorrência de C. felis e de seu plasmídeo críptico, apesar da baixa ocorrência de FIV e FeLV nos gatis. / The infection of upper respiratory disease in cats is very common in individuals that living in shelters, with high morbidity, and in some cases, fatal. The feline herpesvirus type 1 (FHV- 1) and Chlamydophila felis are agents the main causes. The FHV- 1 causes sneezing, nasal discharge and ocular abnormalities such as conjunctivitis. The C. felis is responsible for the worst cases of conjunctivitis and features a cryptic plasmid as a possible virulence factor. The presence of the feline leukemia virus (FeLV) and/or vírus of feline immunodeficiency (FIV) weaken the function of the immune system, causing immunosuppression and therefore increased morbidity and mortality. This study evaluated four shelters, three catteries private non-commercial (one located in Osasco/SP and two in São Paulo/SP). Catteries possessed high population density and cats housed origin was unknown. The detection of FHV- 1 as three genes of the C. felis and cryptic plasmid was performed by PCR in oral mucosa and the ocular conjunctiva of both eyes obtained with cotton swabs, dried and sterile. Blood samples were collected for the detection of FeLV and FIV by enzyme immunoassay. The clinical symptoms of animals were classified from 1 to 4 , with 4 assigned to worst symptoms. The presence of the FIV and FeLV was in the first cattery 4.63% and 3.70%, in the second cattery was 0% and 6.45%, while in the third cattery was 75% and 0%, respectively, these viruses do not were detected in the 4th cattery. FHV- 1 was observed in 61.11 % of the cats in the first cattery; 90.32 % in the second cattery 100 % in the third and 89.74% of the animals of the fourth cattery. In the first cattery, 7.41% of the samples had C. felis, the second cattery, 58.06 %, in the fourth cattery, 23.08%, while the third cattery the agent was not detected. Among the samples positive for C. felis genes were detected cryptic plasmid; in the first cattery, the first gene was present in 62.50%, gene 2 and 3 in 75% of the samples; for the second cattery was obtained 61.11 % positive for 1 and 2 genes and 55.56 % to the third gene; in fourth cattery the first and the third genes were present at 77.78% of the samples in the second gene was in 55.56%. The deaths reported during the study period were classified in animals with symptoms 3 or 4 and positive for C. felis and the cryptic plasmid. In this study we observed a high incidence of C. felis and the cryptic plasmid, despite the low occurrence of FIV and FeLV in catteries.

Entre as doenças de maior impacto nos vinhedos paulistas, encontram-se o míldio (Plasmopara viticola) e a ferrugem (Phakopsora euvitis). Os objetivos deste trabalho foram: i. caracterizar a estrutura genética populacional e identificar espécies crípticas de P. viticola; ii. avaliar o progresso temporal de míldio da videira, o efeito da doença na produção das plantas e verificar a contribuição da reprodução sexuada e assexuada de P. viticola na epidemia da doença e; iii. avaliar o progresso temporal de ferrugem da videira. Foram coletados 516 isolados de P. viticola em 11 municípios de São Paulo. Os isolados foram genotipados com 7 marcadores microssatélites. Para a identificação de espécies crípticas de P. viticola, uma subamostra de 130 isolados foi analisada pela técnica CAPS (Cleaved Amplified Polymorphic Sequence), e 94 isolados foram sequenciados para a região ITS1. O progresso temporal do míldio e da ferrugem da videira foram avaliados em 3 ciclos de produção (2014/15, 2015/16 e 2016/17) nos sistemas de condução em espaldeira (ESP), Y descoberto (YD) e Y sob cobertura plástica (YC). A área experimental, localizada em Piracicaba-SP, foi constituída por plantas da variedade Niágara Rosada. As variáveis analisadas foram fenologia, número de folhas por ramo, incidência e severidade das doenças. Para verificar o efeito do míldio na produção das plantas, avaliaram-se a massa média do cacho, a produção por planta, o teor de sólidos solúveis totais (SST) e a acidez titulável (AT) dos frutos. A área abaixo da curva (AAC) foi calculada para o número de folhas por ramo, a incidência e a severidade de míldio e de ferrugem. Os valores de massa do fruto, produção por planta, SST e AT, e os resultados da AAC para as variáveis analisadas, foram submetidos a análise de variância e as médias comparadas pelo teste de Tukey (P<0,05). Na safra 2015/16, 166 isolados de P. viticola foram coletados em três diferentes momentos da epidemia, os quais foram genotipados com os 7 microssatélites mencionados anteriormente. P. viticola clado aestivalis foi a única espécie críptica identificada no Estado de São Paulo. Dentre os 516 isolados analisados, 55 genótipos multilocos (MLG) foram identificados. Quatro MLGs dominantes representaram 65,7% dos genótipos observados. Para o míldio da videira, em 2 das 3 safras avaliadas observou-se desfolha precoce das plantas doentes. A severidade média máxima de míldio observada foi de 14,3% na ESP e 30,9% no YD. Para esses valores de severidade, a doença causou a redução na produção por planta (0,7 a 0,9 kg por planta) e na massa do cacho (67 a 78 g). Em nenhuma das safras houve efeito da doença no teor de SST e na AT. Maiores valores de AAC da severidade de míldio foram verificados no YD em comparação à espaldeira, sugerindo que o sistema em YD favorece o progresso da doença. No YC, a redução da incidência de míldio foi de até 93,7% em relação aos sistemas descobertos, indicando que o sistema em Y associado ao cultivo protegido foi uma ferramenta eficiente no controle da doença. O monitoramento de genótipos de P. viticola na safra 2015/16 revelou que a epidemia foi resultante, predominantemente, de infecções clonais múltiplas causadas por um único genótipo. Queda prematura de folhas em decorrência da epidemia de ferrugem foi observada para os sistemas em YC e YD com valores de severidade inferiores a 5,6%. Em função da desfolha, verificaram-se alterações na fenologia das plantas nos estádios fenológicos finais de desenvolvimento. O progresso da incidência de ferrugem foi mais rápido nos sistemas em YC e YD em relação a ESP. Diferentemente das epidemias de míldio da videira, o uso da cobertura plástica não foi suficiente para o controle satisfatório da ferrugem. / Downy mildew (Plasmopara viticola) and rust (Phakopsora euvitis) are important diseases on vineyards located in São Paulo State. The aims of this study were: i. characterize the population genetic structure and identify cryptic species of P. viticola; ii. evaluate the temporal progress of grapevine downy mildew, the disease effect on yield, and verify the contribution of sexual and asexual reproduction of P. viticola on disease epidemic and; iii. evaluate the temporal progress of grapevine rust. A total of 516 isolates of P. viticola were collected from 11 locations of São Paulo State. The isolates were genotyped with 7 microsatellite markers. To identify cryptic species of P. viticola, a subsample of 130 isolates were analized by Cleaved Amplified Polymorphic Sequence (CAPS), and 94 isolates were sequenced for ITS1 region. Temporal progress of downy mildew and rust were evaluated in 3 growing seasons (2014/15, 2015/16 and 2015/16) on vertical trellis (VT), Y-trellis without plastic cover (YWP), and Y-trellis with plastic cover (YPC). The experimental area, located in Piracicaba-SP, was composed by plants of the variety Niagara Rosada. The variables analyzed were phenology, number of leaves per shoot, disease incidence and disease severity. To verify the effect of downy mildew on yield were evaluated bunch weight, yield per plant, total soluble solids content (TSS) and titratable acidity (TA) of the fruits. Area under the curve (AUC) was calculated for number of leaves per shoot, downy mildew and rust incidence and downy mildew and rust severity. The values of bunch weight, yield per plant, TSS and TA, and AUC results for the analyzed variables, were submitted to variance analysis and the mean values were compared by Tukey test (P<0.05). In the growing season of 2015/16, 166 isolates of P. viticola were collected at three different moments of downy mildew epidemic. The isolates were genotyped with the 7 microsatellites previously mentioned. P. viticola clado aestivalis was the only cryptic species identified in São Paulo State. Among the 516 isolates analyzed, 55 multilocus genotypes (MLG) were identified. Four dominant MLGs represented 65.7% of the observed genotypes. For downy mildew, 2 of the 3 growing seasons evaluated were observed early defoliation of the diseased plants. The maximum average values of disease severity observed were 14.3% in ESP and 30.9% in YD. For these disease severity values, the disease reduced yield per plant (0.7 to 0.9 kg per plant) and bunch weight (67 to 78 g). No effect of downy mildew was verified on TSS and TA in none of the growing seasons. Higher AUC values for downy mildew severity were observed in YWP compared to the VT, suggesting that YWP favors the disease progress. On the YPC, incidence of downy mildew was reduced by up to 93.7% compared to the uncovered systems, indicating that Y-trellis associated with plastic cover was an efficient tool to control the disease. The monitoring of genotypes of P. viticola in the 2015/16 growing season revealed that disease epidemic was drived predominantly by multiple clonal infections caused by a single genotype. Early defoliation due to grape rust epidemic was observed on YWP and YPC with disease severity lower than 5.6%. Due to defoliation caused by rust modification on plant phenology were verified in the final stages of plant development. The progress of rust incidence was faster in YWP and YPC than in VT. Unlikely grape downy mildew epidemics, the use of plastic cover was not sufficient to effectivly control grape rust.

Resultados anteriores sugerem que existem diferenças biológicas entre espécimens de Anopheles oswaldoi capturados no Estado do Acre e os do Estado de Rondônia, Brasil. Esta espécie tem sido apontada como um importante vetor de malária em localidades do Peru e Acre. Entretanto, em Rondônia, somente um número pequeno de A. oswaldoi alimentados em pacientes com malária desenvolveram infecção nas glândulas salivares. Além disso, há suspeita de que espécimens identificados como A. oswaldoi, capturados em áreas abertas em Costa Marques, Rondônia, são na verdade A. konderi, e que A. oswaldoi sensu stricto estaria restrito a áreas de florestas. Estes dados, juntamente com as dificuldades encontradas na identificação de anofelinos do grupo Oswaldoi, baseadas em critérios morfológicos, sugerem que espécimens de A. oswaldoi são membros de um complexo de espécies crípticas. A distinção de espécies crípticas de insetos vetores é de grande importância, já que diferentes membros em um complexo podem exibir diferenças na ecologia, capacidade vetorial e resposta a medidas de controle. Análise de sequências de DNA, particularmente da região do ITS2 do cistron do DNA ribossômico, tem sido usada como um caracter diagnóstico em alguns grupos de espécies crípticas, tornando-se um instrumento para estudos taxonômicos e filogenéticos. A primeira parte deste estudo teve o objetivo de determinar as diferenças encontradas nas sequências de ITS2 de espécimens de A. oswaldoi capturados em várias localidades da América do Sul. As regiões dos ITS2 destes anofelinos foram amplificadas usando oligonucleotídeos iniciadores conservados das regiões 5.8S e 28S e os produtos de PCR foram clonados e sequenciados. Os ITS2 de todos os mosquitos capturados tiveram tamanho aproximado de 350 nucleotídeos, com aproximadamente 53% de conteúdo de GC. Análise do alinhamento destas sequências, mostrou similaridade variando entre 87% e 100%, e a análise de uma árvore de similaridade, neighbor-joining, produzida com p-distance usando as diferenças nas sequências de ITS2, separou estes espécimens em quatro grupos. Um deles está provavelmente relacionado ao A. oswaldoi sensu stricto, e um outro pode estar relacionado à espécie A. konderi. Os outros dois grupos podem corresponder a espécies cuja identificação morfológica permanece para ser esclarecida no complexo A. oswaldoi. Estes dados são evidências de que espécimens de A. oswaldoi estão incluídos em um complexo de espécies crípticas e que identificação por DNA pode resolver estas questões taxonômicas. A. konderi tem sido considerado uma sinonímia de A. oswaldoi. Embora estágios adultos e imaturos destas espécies de anofelinos apresentem características morfológicas idênticas, aspectos encontrados na genitália masculina podem distinguir estes taxa. A segunda parte deste estudo foi conduzida com o objetivo de comparar a susceptibilidade de A. oswaldoi s.s. e de A. konderi à infecção com Plasmodium vivax. A susceptibilidade foi baseada na proporção de mosquitos com oocistos e esporozoítos. Os anofelinos foram capturados no Acre e em Rondônia para obtenção de progênies F1. Após a emergência dos adultos, as genitálias masculinas dos mosquitos de cada progênie foram dissecadas e examinadas. Todas progênies originadas dos mosquitos capturados em Rondônia corresponderam a A. konderi, enquanto que cerca de 85,0% das progênies do Acre eram A. oswaldoi s.s.. Estas progênies F1 de A. oswaldoi s.s., A. konderi e A. darlingi foram alimentadas simultaneamente com sangue infectado com P. vivax. Estes mosquitos foram dissecados 10-12 dias após infecção e examinados para verificação da presença de oocistos e esporozoítos. Tanto A. oswaldoi s.s. como A. konderi apresentaram oocistos nos tratos digestivos, entretanto, a porcentagem de tratos digestivos positivos para oocistos foi maior em A. oswaldoi s.s. (13,8%) do que em A. konderi (3,3%). Esporozoítos foram encontrados somente nas glândulas salivares de A. oswaldoi s.s., com 6,9% de positividade. As taxas de infecção nos controles A. darlingi foram de 22,5% a 30,0%, para ambos oocistos e esporozoítos. Estes resultados indicam que A. oswaldoi s.s. pode transmitir P. vivax e sugerem que esta espécie é mais susceptível que A. konderi. Embora A. oswaldoi s.s. seja uma espécie exofílica e zoofílica, este anofelino pode estar envolvido na transmissão da malária humana como parece estar ocorrendo no Estado do Acre. / Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

<p>Multiple mating by females is common in nature. Yet, the evolution and maintenance of polyandry remains a bit of an evolutionary puzzle. It was my aim in this thesis to reach a greater understanding of this phenomenon as well as to investigate the consequences of polyandry on the evolution of reproductive divergence in insects. In an extensive meta analysis addressing the direct effects of multiple mating on female fitness in insects, I found that insects gain from multiple matings in terms of increased lifetime offspring production. In species without nuptial feeding, increased mating rate leads to decreased female lifespan and my results strongly support the existence of an intermediate optimal female mating rate. However, results from an experimental study where I examined the relationship between female fitness and mating rate in the bean weevil (Callosobruchus maculatus) showed that female fitness was maximized at two alternative mating rates, indicating that some species may exhibit a more complex relationship between the costs and benefits of mating. In the meta analysis on species with nuptial feeding, I found only positive effects of increased mating rate and the puzzle is rather what constrains the actual mating rates of females in these groups.</p><p>Sexual selection is a very potent driver of rapid evolutionary change in reproductive characters. Most research has focussed on precopulatory sexual selection, but in promiscuous species sexual selection continues after copulation and variance in male fertilization success gives rise to postcopulatory sexual selection. In this thesis I found that three allopatric populations of the red flour beetle (Tribolium castaneum) have diverged in traits related to reproduction. Male genotype affected all aspects of female reproduction, but more interestingly, males and females interacted in their effect on offspring production and reproductive rate, showing that the divergence was due to the evolution of both male and female reproductive traits.</p><p>When studying postcopulatory sexual selection, sperm competition has been put forward as the main source of variance in fertilization success. The results from a set of double-mating experiments, using the same populations of flour beetles, provided strong evidence that cryptic female choice is also important in generating variance in male fertilization success. I found not only main effects of female genotype on male fertilization success but also male-female interactions which provide more unambiguous evidence for cryptic female choice. Finally, I attempted to uncover which male signals-female receptors are involved in the reproductive divergence observed in the Tribolium populations. In a double-mating experiment I manipulated female perception of two male reproductive signals, copulatory courtship and cuticular hydrocarbons, and the results indicate that, within populations, both signals are sexually selected. However, only male cuticular hydrocarbons seem to be involved in the reproductive divergence between the populations. </p><p>In conclusion, multiple mating by female insects can be understood solely in terms of direct fitness benefits resulting from increased offspring production. I have shown that postcopulatory sexual selection can lead to rapid divergence in reproductive traits related to mating and that cryptic female choice plays an important role in this divergence.</p>

<p>Harmony and cooperation was for long believed to dominate sexual interactions. This view slowly started to change 25 years ago and is today replaced with a view where males and females act based on what is best from a costs-benefits perspective. When sex specific costs and benefits differ, concerning reproductive decision influenced by both sexes, sexual conflict will occur. The basis for discordant reproductive interests between the sexes is that males produce many small gametes, while females’ produce few and large gametes. One result of this difference is that the optimal mating rate differs between the sexes. Males, with their many small sperm, maximize their reproductive output by mating with many females, while females often do best by not mating more frequently than to fertilize their eggs, since mating often entails a cost. Sexual conflict over mating is thus an important factor shaping the interactions between the sexes. In this thesis I study this and related conflicts between the sexes, using mathematical models, fruit flies and comparative methods. Mathematical modelling was used to explore how males and females may coevolve under sexual conflict over mating. This model shows that sexual conflict over mating results in the evolution of costly female mate choice, in terms high resistance to matings, and costly exaggerated male sexual traits, aimed to manipulate females into mating. A key assumption in this model is that males which females find attractive also are more harmful to females. This assumption was tested by housing fruit fly females with either attractive or unattractive males. Females kept with attractive males were courted and mated more, and suffered a 16 percent reduction in lifetime offspring production. In another study I measured genetic variation in two antagonistic male traits used to compete over females; offence - a male’s ability to acquire new mates and supplant stored sperm, and defence - a male’s ability to induce fidelity in his mates and prevent sperm displacement when remating occurs. Independent additive genetic variation and positive selection gradients were found for both these traits, indicating an ongoing arms race between these male antagonistic traits. This arms race also had a negative impact on females, since high values of offence compromised female fitness. Genetic variation in female ability to withstand male harm was also tested for and found, indicating that females evolve counter adaptations to reduce the effect of harmful male traits. Finally, the proposed link between sexual conflict and speciation was tested. Theory suggests that perpetual sexual arms races will cause allopatric populations to evolve along different evolutionary trajectories, resulting in speciation. This theory was tested using comparative methods by contrasting the number of extant species in taxa with high and low opportunity for sexual conflict. The study showed that taxa with high opportunity for sexual conflict, on average, has four times as many species as those with low opportunity, supporting that sexual conflict is a key process in speciation.</p>