Thesis (Ph. D.)--University of California, Dec. 1927. / Description based on print version record. Bibliography: p. 127-133.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 96-103). Also available for download via the World Wide Web; free to University of Oregon users.

The legacy of historical biogeography impacts many organisms and results in a wide range of character variation over a latitudinal gradient. The pitcher-plant mosquito Wyeomyia smithii is one such organism that demonstrates a wide range of phenotypic and genotypic variation over the entirety of its range from the Gulf Coast to Canada. A geographic cline established by the presence and recession of the Laurentide Ice Sheet is manifest in the narrow range of thermal tolerance exhibited by different populations and also in the differing propensity of bloodfeeding by these mosquitoes. These contemporary clines were analyzed by a variety of experimental methods ranging from year-long fitness assays, scanning electron microscopy, and RNA-sequencing to determine the patterns underlying the resulting evolutionary differences among established populations. This dissertation includes both unpublished and co-authored material.

Seeds of many grasses and legumes from the Canadian Prairies have dormancy which prevents the germination of viable seeds in otherwise favorable conditions. Plant-derived smoke can improve germination in dormant seeds. Seeds of eight grasses, two legumes, and Lactuca sativa were investigated for the effects of seed priming in aqueous smoke solutions on germination, seedling emergence, seedling growth, and standing crop. Aqueous smoke solutions were produced by bubbling smoke generated from the incomplete combustion of wheat straw (Triticum aestivum cv. Unity) or prairie hay (Festuca hallii) through distilled water. Seeds were primed for 24 h in darkness using serial dilutions (1/1000v/v, 1/100v/v, 1/10v/v and 1/1v/v) of the aqueous smoke solutions. After priming, seeds were dried at 20°C in darkness for 7d and placed in petri dishes containing filter paper, after which 5 mL of distilled water were applied. Seeds were incubated at 10/0°C or 25/15°C in 12h light/12h darkness or 24 h darkness for 49 d. Seeds were also primed using 1/100v/v aqueous smoke solutions of wheat straw or prairie hay and seeded in the field. Non-primed seeds and those primed in distilled water (0/1v/v) were used as controls. Within species, germination varied significantly (P≤0.05) among concentrations of aqueous solutions of smoke, smoke type, light, temperature, and their interactions. Total germination of Astragalus cicer, Trifolium ambiguum, Hesperostipa comata, Stipa viridula, and Pascopyrum smithii was not changed by priming seeds. Depending on light or temperature treatments, priming seeds of Agropyron dasystachyum, Elymus junceus, Dactylis glomerata, Elymus angustus, and Festuca hallii in aqueous smoke solutions improved germination by 16%, 20%, 32%, 49%, and 50%, respectively. Priming seeds in aqueous smoke solutions reduced the number of days to 50% germination for Trifolium ambiguum, Lactuca sativa, Festuca hallii, and Stipa viridula (2 d), Elymus junceus (3 d), Dactylis glomerata (4 d), Hesperostipa comata (10 d), and Pascopyrum smithii (15 d). Priming seeds in aqueous smoke solutions increased seedling lengths (combined hypocotyl and radicle lengths) for Elymus angustus and Hesperostipa comata by 28% and 100%, respectively, but it reduced seedling lengths of Lactuca sativa, Festuca hallii, and Trifolium ambiguum. Seedlings from seeds primed in aqueous smoke solutions generated from wheat straw were longer for Lactuca sativa (83%), Elymus angustus (52%), and Hesperostipa comata (36%) as compared with prairie hay, respectively. Priming seeds interacted with smoke type to increase seedling lengths for Pascopyrum smithii (92%), Elymus junceus (100%), and Agropyron dasystachyum (100%), but it reduced seedling lengths for Astralagus cicer (26%), Trifolium ambiguum (55%), and Dactylis glomerata (90%). Exposing seeds to aqueous smoke solutions partially substituted a light requirement for germination in Pascopyrum smithii, Festuca hallii, Hesperostipa comata, Dactylis glomerata, Agropyron dasystachyum, Stipa viridula, and Elymus junceus. Priming seeds in aqueous smoke solutions increased standing crop of Dactylis glomerata by 57%, but total seedling emergence and rate of emergence of seedlings in the field were not different (P>0.05) among priming treatments. Priming seeds in aqueous smoke solutions generated from wheat straw or prairie hay can stimulate germination in Agropyron dasystachyum, Dactylis glomerata, Elymus junceus, Elymus angustus, and, Festuca hallii.

Photoperiodism is the ability of plants and animals to utilize day length or night length to mitigate seasonal exigencies. The circadian clock allows organisms to organize daily demands. Both process are set by light, and for more than 80 years a functional relationship has been pursued. Previous experiments have revealed, through phenotypic expression, that the daily circadian clock and seasonal photoperiodic timer have evolved independently, yet molecular evidence is lacking. Herein, we use the mosquito, Wyeomyia smithii, to understand the relationship between the photoperiodic response, diapause, and the daily circadian clock. We measured variation in the formal properties of the core circadian clock over a latitudinal and altitudinal gradient which we compare to the critical photoperiod, a measure of diapause, over the same geographic gradient. We found that there is no correlation with any of the formal properties of the core circadian clock and critical photoperiod, indicating independent evolution.

L’étude du microbiote digestif a connu un regain d’intérêt au début des années 2000, avec l’avènement des techniques moléculaires. La culturomics a démontré sa complémentarité depuis 2010 en réduisant une partie des biais des méthodes moléculaires. Une revue sur les techniques d’étude du microbiote digestif et l’analyse du microbiote des sujets africains. Les études de métagénomique en Afrique ont révélé une augmentation de la biodiversité, en particulier des Spirochaetes et des Prevotella chez les africains par rapport aux occidentaux. Sur les 1162 bactéries isolées par culturomics, 476 n'étaient pas africaines, 445 étaient communes et 241 étaient d’origine africaine dont 68 nouvelles espèces. Pour ma participation au travail de culturomics, 102750 colonies testées par MALDI-TOF,ont permis d'identifier 377 espèces incluant 40 nouvelles espèces,17 nouveaux genres et 2 nouvelles familles.Ces nouvelles espèces ont été décrites par taxonogenomics ou new species announcement.Les archaea méthanogènes ont une prévalence de 97,4% pour M. smithii et associés à des pathologies comme l’abcès du cerveau,les parodontites etc. La culture est fastidieuse et nécessitait une source extérieure d’hydrogène. Sous enceinte anaérobie, nous avons cultivé avec succès M. smithii à partir d’un milieu de culture liquide inoculé d’échantillon de selle. L’isolement en culture pure a été un succès sur milieu gélosé en réalisant une coculture avec Bacteroides thetaiotaomicron. Nous avons aussi testé avec succès la coculture de M. smithii avec d’autres bactéries productrices d’hydrogène connues. Les tests de chromatographie en phase gazeuse montraient que ces souches produisaient de l’hydrogène. / The study of the digestive microbiota was a renewed interest in the early 2000s, with the advent of molecular techniques. The culturomics has demonstrated its complementarity since 2010 by reducing some of the biases of molecular methods. A review on the techniques of studying the digestive microbiota and the analysis of the microbiota of African subjects. Metagenomic studies in Africa have revealed an increase in biodiversity, especially Spirochaetes and Prevotella among Africans compared to Westerners. Of the 1162 bacteria isolated by culturomics, 476 were non-African, 445 were common, and 241 were of African origin, including 68 new species. For my participation in the work of culturomics, 102750 colonies tested by MALDI-TOF, identified 377 species including 40 new species, 17 new genera and 2 new families. These new species have been described by taxonogenomics or new species announcement.Methanogenic archaea have a prevalence of 97.4% for M. smithii and associated with pathologies such as brain abscess, periodontitis and so on. The cultivation is tedious and required an external source of hydrogen. Under anaerobic enclosure, we successfully cultivated M. smithii from a liquid culture medium inoculated with a stool sample. The isolation in pure culture was a success on agar medium by performing a coculture with Bacteroides thetaiotaomicron. We have also successfully tested the co-culture of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen.

Les Archaea methanogènes sont des organismes environnementaux ayant été également détectés dans certaines flores associées aux muqueuses des mammifères. Chez l’homme ces microorganismes ont été associés avec les muqueuses intestinale, vaginale et orale. Ces organismes sont des procaryotes anaérobies stricts et leurs conditions de culture restent fastidieuses et très mal connues. En effet, uniquement trois Archaea methanogènes ont été cultivées à partir de prélèvements humains, Methanobrevibater smithii et Methanosphaera stadtmanae à partir des selles puis Methanobrevibater oralis à partir de la plaque dentaire. Récemment l’ADN d’autres Archaea methanogènes et d’Archaea non-methanogènes a été détecté dans des selles humaines, y compris des séquences indiquant la présence d’espèces appartenant à un nouvel ordre de méthanogènes n’ayant aucun représentant cultivé. La connaissance actuelle sur la diversité de ces methanogènes chez l’homme et sur leurs effets potentiels sur la santé humaine est en grande partie basée sur les techniques de détection de l’ADN par PCR et métagénomique. Ces techniques fondées sur la détection de l’ADN ribosomal 16S et du gène mcrA codant la sous-unité alpha du methyl-coenzyme M reductase, une enzyme clé dans le processus de méthanogenèse, ont montré dans un premier temps que M. smithii était détecté chez moins de 50% des individus et M. stadtmanae chez 0-20 % seulement. Ces résultats étaient contradictoires avec le rôle de la méthanogenèse dans l’élimination des acides et d’autres produits du processus digestion, et nous avons émis l’hypothèse que ces résultats pouvaient ne pas refléter la quantité réelle des méthanogènes dans le tube digestif humain, suggérant la mise au point de nouvelles méthodes de détection moléculaire et de culture adaptées aux caractéristiques de ces organismes fastidieux. Dans ce travail, nous nous sommes fixés comme premier objectif de mettre au point une méthode moléculaire permettant de détecter M. smithii chez tous les individus testés et nous avons mis au point un protocole d’extraction et de détection d’ADN d’Archaea à partir des selles en se basant sur les génomes séquencés de M. smithii et M. stadtmanae. Ce protocole nous a permis de détecter M. smithii chez 95,5% des individus et M. stadtmanae chez 29,4% des individus. En ce basant sur ce protocole et moyennant une approche moléculaire basée sur une PCR universelle de l’ADN ribosomal 16S des méthanogènes, le séquençage et le clonage, nous avons également détecté chez 4% de la population, une séquence correspondant à un phylotype (FJ823135) ayant déjà été rapporté comme représentant un nouvel ordre de méthanogènes. A partir de là, nous avons choisi un prélèvement de selle susceptible de contenir le plus fort ratio de FJ823135/ M. smithii et nous avons réussi à isoler et à cultiver une nouvelle Archaea que nous avons nommé Methanomassiliicoccus luminyensis, premier représentant cultivé d’un nouvel ordre de méthanogènes et la quatrième Archaea cultivée chez l’homme. M. luminyensis et M. stadtmanae présentent des métabolismes similaires en réduisant le méthanol en méthane en utilisant l’hydrogène comme donneur d’électrons, cette observation nous a incité à tester l’addition de tungstate de sélénium, requis pour la croissance de M. luminyensis, dans une culture M. stadtmanae, et nous avons observé une accélération de la vitesse de croissance de M. stadtmanae par un facteur 3. Nous avons ensuite étudié la sensibilité des méthanogènes isolés chez l’homme aux antibiotiques et établi qu’ils sont seulement sensibles à des molécules efficaces contre les bactéries et les eucaryotes, ceci étant en accord avec leur position phylogénétique en tant qu’un des quatre domaines de la vie. [...] / Methanogenic Archaea are environmental organisms which have also been associated to mammals mucosa. In humans these microorganisms have been detected in the vaginal, intestinal and oral mucosa. These organisms are strict anaerobes and their culture conditions remains fastidious and poorly known. In fact only three methanogens have been isolated from human samples, both Methanobrevibater smithii and Methanosphaera stadtmanae from stool and Methanobrevibater oralis from dental plaque. Current knowledge on the diversity of methanogens in humans and their potential effects on human health were largely based on DNA detection methods as PCR and metagenomics. These techniques based on 16S rDNA and mcrA gene (encoding the alpha subunit of methyl coenzyme-M-reductase, a key enzyme in methanogenesis process) detection, showed that M. smithii was the most present in man and that the presence of M. stadtmanae was transient. Recently, the DNA of other methanogenic and non- methanogenic Archaea, has been detected in human feces, including sequences indicating the presence of non-cultured species belonging to potential new order of methanogens with no cultured representative. However, these studies detected M. smithii with variable prevalence in less than half of the tested individuals and no M. stadtmanae; such results does not confirm the paramount role of methanogenesis in preventing the accumulation of acids and other reaction end products during the digestion process, and can not reflect the actual amount of these two methanogens in the human digestive tract because of their specific association with the intestinal mucosa. Therefore, these studies pointed that the diversity of methanogens in humans has been underestimated suggesting the development of new molecular detection methods and cultural approaches adapted these fastidious organisms. In this work, we preset as first criteria, the detection of M. smithii in all tested individuals, therefore we developed an improved protocol for archaeal DNA extraction and detection from stool based on sequenced genomes of M. smithii and M. stadtmanae, this protocol allowed us to detect the first one DNA in 95.5% tested individuals and the second in a prevalence of 29.4%. Based on this protocol and through molecular approach based on universal amplification of methanogenic 16S rDNA, sequencing and cloning, we detected in 4% of the tested population, a sequence corresponding to a new phylotype (FJ823135) that has been previously reported and proposed as a representative of a new order of methanogens. From there, we chose one stool specimen susceptible to contain the highest amount of FJ823135 and successfully isolated Methanomassiliicoccus luminyensis B10T clone, the first cultured representative of a new order of methanogens and the fourth Archaea cultured in humans.This archaeon exhibited a similar type of metabolism to that of M. stadtmanae by oxidizing H2 and reducing methanol to methane but require tungstate-selenite, an element essential for its growth, this fact prompted us testing tungstate-selenite addition on M. stadtmanae growth and establishing that it was strongly stimulatory with a growth rate three times faster. We have thereafter studied the sensitivity of methanogens isolated from humans to antibiotics and established that they are susceptible only to molecules also effective against both Bacteria and Eucarya, in agreement with their phylogenetic location as a unique domain of life. The aim of the latter part of this work was to test the effectiveness of MALDI-TOF mass spectrometry identification of environmental and host-associated Archaea. The obtained data indicated that that MALDI-TOF-MS protein profiling is an efficient first-line step for the rapid phenotypic identification of cultured Archaea organisms including host-associated ones. [...]

The pitcher plant mosquito Wyeomyia smithii maintains a broad geographic range from the Gulf of Mexico to central Canada, and throughout its range is genetically and phenotypically variable, though fully interfertile. Many of the traits that vary across the broad range of this mosquito owe their diversity to selection on populations, which maximize fitness in the unique environment in which each populations finds itself. While a diversity of traits vary by latitude and merit the interest of evolutionary biologists, including critical photoperiod, voltinism, and thermal tolerance, of interest in the following thesis is the variation in blood-feeding propensity within this single species of mosquito. In no other mosquito species are some populations obligate non-biters while in other populations willingly hematophagous. This thesis explores the evolutionary transition from biting to non-biting in the pitcher plant mosquito at multiple levels of biological integration, starting first by establishing a heritable basis for the transition, then moving to the fitness and life historical consequences of both the natural system and of a line artificially selected in the lab. The latter half of this thesis moves on to probe the genetic architecture underlying the shift in phenotype and ends after examining the transition to non-biting at the level of the gene using an RNA-sequencing experiment. The results stemming from this thesis are thoroughly discussed: in short, we find that fitness differs between biting and non-biting populations, that complex genetic architectures underlie the transition to non-biting in nature, but not under artificial selection, and finally, that many candidate loci are differentially regulated in biting populations relative to non-biting populations and that these loci most often cluster with metabolic biological pathways.

The F420-dependent NADP reductase of Methanobrevibacter smithii has been partially purified employing a combination of affinity chromatography with Blue Sepharose (Cl-6B) and molecular sieve chromatography with Sephacryl S-200, The enzyme, which requires reduced F420 as an electron donor, has been purified over 145 fold with a recovery of 6%. A molecular weight of 120,00 for the native enzyme was determined by Sephacryl S-200 chromatography. A subunit molecular weight of 28,200 was determined by SDS-PAGE, indicating that the native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 45°C, with a pH optimum of 7.5. The NADP reductase had an apparent Km of 42 uM for reduced F420, and an apparent Km of 4l uM for NADP. The enzyme was stable in 0.05 M sodium phosphate buffer (plus 10 mM cysteine) at pH 7.0, when gassed with nitrogen or hydrogen and stored at 4°C.

L’analyse du microbiote oral et de son évolution séculaire se fait principalement à partir de l’analyse du tartre dentaire ancien des populations passées et du biofilm dentaire des populations modernes. Nous avons dans un premier temps fait le point des connaissances sur la paléomicrobiologie des bactéries et des archaea contenues dans le tartre dentaire et montré que les archaea faisaient partie du microbiote oral chez l'homme. Dans la deuxième partie, nous avons mis en évidence le répertoire des archaea méthanogènes vivant dans la cavité orale par la culture (une nouvelle espèce Methanobrevibacter massiliense, Methanobrevibacter smithii et Methanobrevibacter oralis). La prévalence de ces archaea était significativement plus élevée chez les patients atteints de parodontite que chez les personnes contrôles. Ensuite, nous avons développé une méthode de génotypage Multispacer Sequence Typing pour typer M. oralis et M. smithii et révélé différents génotypes. Enfin, nous avons analysé le répertoire des archaea méthanogènes dans des échantillons de tartre dentaire ancien datant du 14ème au 19ème siècle. La prévalence et la diversité des archaea méthanogènes dans la cavité orale ont diminué significativement au cours des sept derniers siècles. Des archaea méthanogènes ont été retrouvées dans 75% des prélèvements de tartre dentaire (Candidatus M. massiliense à 44,6%, M. oralis à 19,6%, Methanomassiliicoccus luminyensis-like à 12,5%, Candidatus Nitrososphaera evergladensis-like dans un prélèvement et Methanoculleus bourgensis dans un autre prélèvement). Un prélèvement de tartre positif pour Candidatus M. massiliense a été documenté par hybridation in situ en fluorescence. / The analyses of oral microbiome and its secular evolution mainly use dental calculus in past populations and dental plaque in modern populations. In our thesis, we initially reviewed the knowledge actual about bacteria and archaea paleomicrobiology of the dental calculus. The review disclosed that archaea taked part in the secular core-microbiota in past and modern populations. In the second work, we demonstrated the repertoire of methanogenic archaea currently living in the oral cavity using culture-based approach and succeeded in isolating for the first time a new species named Methanobrevibacter massiliense in addition to Methanobrevibacter smithii and Methanobrevibacter oralis from dental plaque in periodontitis patients. This work showed that the prevalence of methanogens was significantly higher in periodontitis patients than in controls. Some methanogenic archaea were involved in periodontitis. Then, we developed Multispacer Sequence Typing to evaluate M. oralis and M. smithii and revealed different genetic variants in these archaea. Finally, we examined the repertory of methanogenic archaea in ancient dental calculus dating from the 14th to the 19th century. The prevalence and diversity of methanogenic archaea in the oral cavity decreased significantly during the last seven centuries. Methanogenic archaea were found in 75% of dental calculis (Candidatus M. massiliense, 44.6%; M. oralis, 19.6%; Methanomassiliicoccus luminyensis-like, 12.5%; Candidatus Nitrososphaera evergladensis-like in one and Methanoculleus bourgensis in one specimen). One Candidatus M. massiliense dental calculus was further documented by fluorescent in situ hybridization.