Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.

A l'heure de la protéomique, la spectrométrie de masse s'est révélée un outil puissant<br />pour la recherche et l'identification des biomolécules à partir d'échantillons purifiés. Une<br />nouvelle ère s'ouvre, avec l'imagerie MALDI, permettant en plus la localisation de<br />biomolécules telles que les peptides, les protéines ou les lipides au sein des tissus. Des<br />développements cruciaux restent encore à réaliser pour améliorer les performances de cette<br />technologie. Dans ce contexte, nous nous sommes tout d'abord intéressés à la mise au point<br />de nouveaux protocoles adaptés à l'analyse directe et l'imagerie par spectromètrie de masse<br />de petits organismes en particulier la sangsue Hirudo medicinalis. Ce modèle est<br />particulièrement intéressant du point de vue des phénomènes de régénération nerveuse et nous<br />avons débuté des études sur les lipides pouvant y être impliqués. Le deuxième point abordé<br />est l'étude des apports de la métallisation pour la spectromètrie de masse. Tout d'abord un<br />dépôt métallique sur des lames histologiques permet à la fois une corrélation des informations<br />morphologiques obtenues en microscopie optique avec les images moléculaires d'IMS. La<br />métallisation de l'échantillon quand à elle, a permis de supprimer les décalages de pics vers<br />les plus hauts rapports m/z, d'obtenir des spectres MALDI de meilleures qualités et grâce à<br />une reproductibilité plus importante entre 2 spectres, de produire des images MALDI de plus<br />grandes qualités. Enfin, une partie des développements a été dédiée à la possibilité<br />d'améliorer la résolution de l'image grâce à l'utilisation d'un système permettant de diminuer<br />la zone accessible au laser

ABSTRACT Benthic foraminifers that host algal symbionts are similar to corals in that they rely on their algal endosymbionts for their energy needs, calcify prolifically, and are sensitive to changes in environmental conditions. They are abundant in the benthos of coastal coral-reef areas and are found throughout the tropical and subtropical regions. Pulse Amplitude Modulated (PAM) chlorophyll fluorometry and chlorophyll a extraction techniques were used to quantify and compare the photosynthetic responses of the benthic foraminiferal, Archaias angulatus and their isolated endosymbionts, Chlamydomonas hedleyi, to short-term changes in temperature. Maximum quantum efficiency (Fv/Fm) and rapid light curves (RLCs), from which relative electron transport rates (rETR) of photosystem II (PSII) were derived, were investigated over a thermal range from 4.4° to 33.9 °C in three experiments that were 7 to 31 days in duration. Typical mean yields (Fv/Fm for healthy holobionts (symbionts in hospite) were 0.6 - 0.7, and for isolated symbionts 0.5 - 0.6. Chronic photoinhibition, indicated by significant decreases in Fv/Fm, occurred at temperatures above 31.0°C; there was minimal reduction in efficiency in cooler treatments. The trends between holobiont and symbionts were very similar in all of the photophysiological parameters measured [yield, photoefficiency (<α>), ETRmax and minimum saturating irradiance (Ek)] and supported the temperature range findings in terms of the tolerance of the specimens in the low temperatures up to 31.0 °C. For all photochemical measurements assessed, the holobiont values tended to be somewhat higher than those for the symbionts, with the exception of Ek, possibly indicating a tight coupling in the host-symbiont response during photosynthesis. Chlorophyll a (<μ>g/foram) was negatively correlated with temperature (r = -0.37, p < 0.001) in Experiments 1 and 2. However, in all 3 experiments, chlorophyll a was variable, suggesting a high degree of individual variability in A. angulatus and the ability to acclimate. Some differences observed among treatments may be related to differences in seasons when the specimens were collected and in length of time in culture prior to experiments. Holobiont median rETR light curve trends and photophysiological derived parameters recorded median Ek ranges of ~100-150 <μ>mol photons m-2 s-1, observed ETRmax light intensities of ~200 <μ>mol photons m-2 s-1 and photoinhibition, induced by increasing irradiance intensities, which occurred > 500 <μ>mol photons m-2 s-1. These light curve trends and derived parameters generally supported previous photosynthesis O2 and CO2 gas production studies of A. angulatus. The differences in responses associated with acclimation should be considered in design of future experimental studies. This was the first known physiological study of the viable temperature range and photobiology of A. angulatus using chlorophyll fluorometry methods. Though commonly found in Caribbean and Atlantic waters ranging from 14.0 - 31.0 °C, these results indicate a wider thermal-tolerance range for A. angulatus than was previously known. Keywords: Foraminifera, Chlamydomonas sp., PAM fluorometry, photosynthesis, algal symbiosis

Understanding the factors influencing species ranges and dispersal are becoming increasingly important as climate change alters species distributions worldwide. If species are to persist, life-history strategies must rapidly evolve to accommodate shifting environments. This dissertation assesses the factors modulating dispersal in corals. First, I examined if there were any systematic differences in settlement between Indo-Pacific and Caribbean coral larvae that might explain Caribbean recruitment failures. No differences were observed, however I detected significant divergences in settlement cue preferences among coral species across both the Caribbean (Diploria strigosa, and Montastraea franksi) and the Indo-Pacific (Acropora tenuis, A. millepora, and Favia lizardensis), even for coral larvae from the same reef. Secondly, I established the extent of coral dispersal between remote reefs. I evaluated the genetic diversity and divergence across Micronesia for two coral species and investigated if these islands served as a connectivity corridor between the Indo-West-Pacific (Coral Triangle) and the Central Pacific. I found isolation-by-distance patterns whose strength depended on species, suggesting these corals are not panmictic across their ranges and that island stepping-stones facilitate gene flow to remote Pacific reefs. Next, I investigated genetic structure of symbionts in these same corals, to see if horizontally transmitted symbionts are less dispersive than their coral hosts. Symbiont genetic divergence between islands was an order of magnitude larger than host divergence and both host species and environment modulated symbiont composition. These results suggest that symbiont populations are host-specific and associating with local symbionts might be a mechanism for broadly dispersing corals to adapt locally. Lastly, I estimated heritable variation in dispersal-related traits in coral larvae. I observed strong heritable variation in gene expression, as well as parental effects on two phenotypic traits, settlement and fluorescence. I observed that patterns of differential expression in three-day-old larvae predicted variation in settlement and fluorescence two days later. Correlations between proteoglycan expression and settlement suggest that the larval extracellular matrix plays a role in settlement. Down-regulation of ribosomal proteins and differential expression of oxidative stress genes correlated with increasing fluorescence, possibly indicating reduced growth and increased stress. Overall, this dissertation contributes to our knowledge of factors affecting coral dispersal and the potential for evolution of dispersal-related traits. / text

A comparison of Elysia (=Tridachia) crispata (Mörch, 1863) from the Virgin Islands with elysiid slugs from the mangrove swamps and canals in the Florida Keys that have previously been identified as E. crispata reveals many differences in habitat, gross and microscopic anatomy, food preferences of juveniles, sources of symbiotic chloroplasts and their localization within the digestive tubules, radular morphology, and nucleic acid sequences of two genes. The differences between the two groups of slugs are such that the Florida Keys animals are considered to represent a new species, Elysia clarki. Elysia clarki feeds on siphonaceous algae, and intracellularly sequesters the chloroplasts, which actively photosynthesize for up to 4 months. We have determined the algal source of the chloroplasts in adult E. clarki from 2 populations in the Florida Keys, using molecular techniques, feeding experiments, and electron microscopy. Our results clearly demonstrate that adult E. clark i sequester chloroplasts from 7 different species of algae, representing two genera, of which 5 were identified; Penicillus lamourouxii, P. capitatus, Halimeda incrassata, H. monile, and Bryopsis pennata. In addition, chloroplasts from more than 1 species of algae are sequestered in the same digestive cell simultaneously. Phylogenetic analysis of rbcL sequences from the order Bryopsidales showed that E. clarki feeding was restricted to calcareous members of the family Udoteaceae and the family Bryopsidaceae. Feeding experiments were conducted, using individuals raised in the laboratory from egg masses laid by E. clarki adults which had been collected from Grassy Key, Florida, USA, and 29 species of macroalgae. For the first 14 d post-metamorphosis, juveniles ate only the thin filamentous coenocytes, Bryopsis plumosa or Derbesia tenuissima. Electron microscopy showed that the chloroplasts from both algae were sequestered intracellularly in juvenile slugs. Individuals offered any other macroalga, including the four calcareous species fed on by adults, did not feed on or incorporate any chloroplasts, and soon died. Juveniles switched from B. plumosa to P. capitatus at a length of ~ 1.0 cm, and fixed for microscopy 14 days later had intact intracellular chloroplasts from both algae.

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Cooperation is pervasive in nature but paradoxically also provides opportunity to cheaters. My dissertation involves the study of both cooperation and conflict in two species of Megalopta bees. Megalopta is a Neotropical genus of halictid bees whose biology is characterized by complex life cycles that can range from solitary to eusocial. These bees nest in dead wood and forage under dim light conditions. Megalopta’s nests are inhabited by an extensive array of organisms and each nest therefore constitutes a miniature ecosystem providing opportunities for cooperation and conflict, both within and between species. I first delineate the social structure of M. genalis and M. ecuadoria nests in several Panamanian populations and integrate the factors that play a role in the behavioral decisions of females when joining a social group or not. Within a kin-selection framework, I discuss how genetic relatedness plays a role in the formation of social nests. Second, I investigate the conflict between host bees and a congener social parasite, and I elucidate reproductive structures that are relevant for understanding the evolution of parasitism. Finally, I describe a cleaning mutualism between Megalopta bees and their mite associates. Bee-mite associations encompass a broad spectrum of interspecific interactions. Some bee-mites are thought to perform cleaning services for their hosts in exchange for suitable environments for reproduction and dispersal. Field observations and experimental manipulation reveal a significant correlation between the presence of mites and the absence of fungi inside the brood cells, as well as between the absence of mites and increased bee mortality. This study therefore provides evidence of the sanitary effect of mites in nests of Megalopta bees. This bee-mite association constitutes one of the few examples of terrestrial cleaning mutualisms. / text

Fungal communities of aquatic ecosystems are understudied, leaving major gaps in estimating global fungal biodiversity. In particular, little is known regarding the distinctiveness of (1) aquatic endophytes vs. those in terrestrial plants, and (2) waterborne fungi vs. those inhabiting aquatic plants or sediments. I assessed the diversity, composition, host affiliations, and geographic structure of culturable endophytes associated with aquatic angiosperms in reservoirs in northern Arizona, and their distinctiveness relative to waterborne- and sediment fungi. Endophytes were low in abundance yet extremely diverse. Communities differed significantly in composition among reservoirs and tissues. Aquatic endophytes were largely distinct from those in terrestrial plants, and from those in water and sediment, but did not demonstrate host specificity. This work reveals the uncommon diversity and distinctiveness of endophytes in aquatic plants, and provides a first quantitative estimation of endophytic associations in diverse, ecologically important, and economically relevant aquatic plants.

A hierarchical symbiotic framework for policy search with genetic programming (GP) is evaluated in two control-style temporal sequence learning domains. The symbiotic formulation assumes each policy takes the form of a cooperative team between multiple symbiont programs. An initial cycle of evolution establishes a diverse range of host behaviours with limited capability. The second cycle uses these initial policies as meta actions for reuse by symbiont programs. The relationship between development and ecology is explored by explicitly altering the interaction between learning agent and environment at fixed points throughout evolution. In both task domains, this developmental diversity significantly improves performance. Specifically, ecologies designed to promote good specialists in the first developmental phase and then good generalists result in much stronger organisms from the perspective of generalization ability and efficiency. Conversely, when there is no diversity in the interaction between task environment and policy learner, the resulting hierarchy is not as robust or general. The relative contribution from each cycle of evolution in the resulting hierarchical policies is measured from the perspective of multi-level selection. These multi-level policies are shown to be significantly better than the sum of contributing meta actions.

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.