Phytoplankton and bacteria play a crucial role in the maintenance and stability of aquatic ecosystems. Together, they facilitate nutrient cycling and gas exchange, supporting the nutritional needs of higher trophic-level species. Recent molecular biology advancements have highlighted species-specific associations between the two groups. In some cases, these associations are mutualistic, benefiting the algal host. In exchange for carbon, it was reported that specific bacteria can provide vitamins and hormones and increase the bioavailability of limited nutrients to their phytoplankton host. These exchanges aid in sustaining the algal host in stressful environmental conditions. This study uses omics tools and co-culture experiments to explore the associations between diatoms, a phytoplankton group with ecological and industrial significance, and their microbiome. We aimed to leverage the knowledge of phytoplankton-bacteria interactions to optimize the utilization of diatom in biotechnological applications in Chapters 1 and 2. Diatoms can accumulate valuable biomolecules that can be utilized in fuel and food industries if produced in high biomass and a cost-effective manner. This work focused on studying diatom-microbiome interactions in large-scale cultivations. In Chapter 1, We reported a significant role of cultivation conditions in shaping the microbial community and the presence of specific bacteria that can be candidates for microbiome manipulation efforts to increase biomass. In Chapter 2, we reported that microbiome manipulation could increase algal biomass and the cost-effectiveness of algal farming as a sustainable alternative to traditional food and energy sources.Further, Chapter 3 presents compelling evidence of the species-specific modulation and response of two diatom species to natural microbial communities, highlighting the benefits of the modulated microbiomes. The results of this study hold substantial promise for advancing our understanding of diatom-microbe interactions in their natural habitats. The field of phytoplankton-bacteria associations offers numerous avenues for identifying ecologically significant associations, which, in turn, facilitate a deeper understanding of the underlying dynamics governing these interactions. Moreover, such research holds immense potential in developing and optimizing novel energy and food sources, which is becoming increasingly crucial in the current scenario. The work presented herein represents a noteworthy contribution towards enhancing our knowledge of these complex associations.

Single-celled marine protists belonging to the genus Thraustochytrium (class Labyrinthulomycetes) are of biotechnological interest due to their high production of polyunsaturated fatty acids (PUFAs). They produce particularly high amounts of omega-3 PUFAs making them an attractive source of omega-3 PUFAs as opposed to oil sourced from fish. Preliminary genomic information, laboratory growth on various carbon sources, effect on diatom (Cyclotella cryptica) growth in co-culture, and ultrastructural characteristics of the marine protist Thraustochytrium sp. LLF1b is presented in this work.

Ambient light intensity is a critical factor affecting species distribution, including the sea anemone, Anthopleura sola. In coastal waters, ambient light varies greatly with geographical span and within one location, for instance with increasing depth of the intertidal zone or within crevices of rocks in tidepools. A. sola inhabits diverse microhabitats within the subtidal and intertidal zones that are subject to differing light intensities, making it important to understand the effects of solar radiation on the sea anemone distribution in relation to its endosymbiotic zooxanthellae. A particularity of A. sola is it’s brightly fluorescent, which could be related to the presence of the species in the supratidal, well exposed to intense sunlight, in which case the fluorescence could be acting as a photoprotective mechanism. In this case, however, the species is also reported to cover itself with pieces of debris, thus mimicking exposure to shade rather than sunlight. Here, we postulated that the green fluorescent proteins (GFPs) of A. sola act as photoenhancement to redirect light to fuel photosynthesis for their endosymbiotic zooxanthellae. This was tested by investigating the native auto-fluorescence of A. sola in response to changes in low intensity of ambient light (to mimic shade), and to simultaneously monitor the photochemical efficiency of photosystem II of the zooxanthellae during a light controlled and flow through 106-day laboratory aquarium experiment. The experiment included an 82-day exposure period with three levels of ambient light intensities: 100% (control), 53%, and 22%, and a 24-day recovery period without light shading (100% of ambient light intensity). This research combines measurements of light intensity in the field and an experimental aquarium setting. Image analysis and an underwater fluorometer demonstrated changes in fluorescence intensity of sea anemones and the photosynthetic efficiency of zooxanthellae in response to light changes. Only the lowest light condition (22% of ambient light intensity) caused an increase in green fluorescence intensity, which suggests photoenhancement of GFPs. The endosymbiotic zooxanthellae chlorophyll maintained high efficiency of photosystem II in all light conditions and the zooxanthellae density remained unchanged; therefore, they seemed to not be affected by different light shading. The photoenhancement of A. sola’s GFPs in this experiment could explain A. sola’s ability to live and adapt to low light conditions in the intertidal and particularly the subtidal zone, explaining one factor affecting their geographical and local distribution.

Mesopelagic fishes are of utmost importance to the health of global oceanic ecosystems. These fishes comprise the largest known marine biomass, and are a vital source of food many economically important fish and marine mammals. Further, they serve as a major component of the biological carbon pump, moving food items through the water column via diel migration. Though significant effort has gone into understanding species diversity, and positioning in marine food webs, the microbial component of these animals remains poorly characterized. Symbiotic microbial populations associated with the gastrointestinal (GI) tract assist the host with nutrient uptake, digestion, defense against pathogenic microbes, but can also be detrimental to host health as parasites or pathogens. This study investigates the normal intestinal microbiota of wild mesopelagic fish to determine community diversity and distribution across multiple host families with varying migratory and dietary lifestyles. The results presented here indicate that midwater fish have unique microbial communities from fish of other pelagic zones, where, for example, mesopelagic hosts show enrichment in Betaproteobacteria and Gammaproteobacteria microbial classes. Within mesopelagic fish families, major differences were seen: Gonostomatidae are enriched in three Mycoplasma taxa, Stomiidae had highest alpha diversity, Melamphaidae had the highest abundance of Planctomycetes, and Myctophidae gut communities were enriched in Betaproteobacteria. This study brings new understanding to the microbial ecology of the mesopelagic, and demonstrates that despite sharing space in the water column, mesopelagic fishes contain different microbial communities. Clearly, life history traits must be considered in addition to environment in order to better understand the mesopelagic ecosystem.

Mesopelagic fishes are of utmost importance to the health of global oceanic ecosystems. These fishes comprise the largest known marine biomass, and are a vital source of food many economically important fish and marine mammals. Further, they serve as a major component of the biological carbon pump, moving food items through the water column via diel migration. Though significant effort has gone into understanding species diversity, and positioning in marine food webs, the microbial component of these animals remains poorly characterized. Symbiotic microbial populations associated with the gastrointestinal (GI) tract assist the host with nutrient uptake, digestion, defense against pathogenic microbes, but can also be detrimental to host health as parasites or pathogens. This study investigates the normal intestinal microbiota of wild mesopelagic fish to determine community diversity and distribution across multiple host families with varying migratory and dietary lifestyles. The results presented here indicate that midwater fish have unique microbial communities from fish of other pelagic zones, where, for example, mesopelagic hosts show enrichment in Betaproteobacteria and Gammaproteobacteria microbial classes. Within mesopelagic fish families, major differences were seen: Gonostomatidae are enriched in three Mycoplasma taxa, Stomiidae had highest alpha diversity, Melamphaidae had the highest abundance of Planctomycetes, and Myctophidae gut communities were enriched in Betaproteobacteria. This study brings new understanding to the microbial ecology of the mesopelagic, and demonstrates that despite sharing space in the water column, mesopelagic fishes contain different microbial communities. Clearly, life history traits must be considered in addition to environment in order to better understand the mesopelagic ecosystem.

The fish gill is a vital organ for fish health involved with gas exchange, nitrogen excretion and salt balance. The fish gill is also a chemically diverse environment which hosts a distinct microbiome, yet little is known about its constituents. Here, we describe the nature of the symbiosis between Pacific chub mackerel Scomber japonicus gills and a putative novel species, Shewanella sp., using a combination of whole genome sequencing (WGS) of isolated strains and a broad meta-analysis of 16S rRNA gene amplicon data derived from other marine fish species. It was found that three closely related amplicon sequence variants (ASVs) constituted a median of 30% of the gill microbiome. The successful cultivation of representative isolates matching these environmental ASVs were recovered from mackerel gill samples and whole genomes for these strains were assembled and analyzed. The taxonomy of isolated Shewanella spp. strains was established using a phylogenomic approach. Comparative genomics of diverse Shewanella species highlighted an enrichment of virulence factors in the gill associated Shewanella strains as well as notable loss of function in polyunsaturated fatty acid production, a hallmark characteristic conserved in most members of the genus Shewanella. Finally, meta-analysis of fish gill amplicon data suggests that Shewanella spp. may associate with the gills of forage fishes and provides evidence of trophic transmission to predators of forage fishes.

The majority of vertebrate species diversity are within fish. Marine fish occupy a diverse array of ecological niches including a wide range of salinity tolerance, oxygen tolerance, temperature, depth, desiccation, and light. Fish also have adapted a range of biological traits including varying trophic level, morphology, swimming performance, and reproduction. The microbiome, the total aggregation of microscopic organisms including fungi, bacteria, archaea, and viruses in a specified environment, has largely been studied in mammals, particularly humans from which many associations to disease and health have been demonstrated. Fish microbiome research has largely focused on the gut environment from freshwater captive populations including farmed carp, tilapia, and catfish with marine studies primarily limited to food fish such as salmon. The goal of this dissertation was to develop and apply microbiome tools including sampling methods, DNA extraction, and library preparation (16S and WGSS, whole genome shotgun sequencing) which could be deployed to study a wide range of questions surrounding the parameters which influence the fish mucosal microbiome. With these set of tools, I have asked 1) how do intentional anthropogenic impacts to the water column (organic fertilizer) influence fish gastrointestinal communities, 2) how body sites differ in mucosal communities and changes across environmental gradients, 3) feasibility of developing a model marine fish to use in microbiome experiments to mimic tuna, 4) how the hatchery built environment influences fish mucosal microbiota. My dissertation can be summarized by several key findings. First, the mucosal environments of fish are highly differentiated in that the gill, skin, and digesta communities from the same species of fish are colonized by a large range of phylogenetically diverse microbes. In a freshwater system, organic inputs do influence the fish gut communities but indirectly through nutrient changes. In a wild marine fish, body sites are impacted by different environmental gradients with external body sites like the gill and skin most influenced by temporally variable environmental conditions including sea water temperature. In both freshwater and marine indoor hatchery systems, the built environment plays a critical role in influencing or being influenced by the fish mucosal microbiome.

Post translational modifications such as ubiquitin and the Small Ubiquitin-Like Modifier (SUMO) regulate numerous cellular processes and are evolutionarily conserved in all eukaryotes. Studies on these post translational modifications often require the enrichment of their endogenous conjugates prior to analysis because the modified proteins exist transiently and are low in abundance. Current approaches for isolating these modified proteins contain several unaddressed issues that question the reliability of these approaches. Remarkably, it has been recently demonstrated that the catalytically inactive form of the SUMO protease Ulp1 can be used as a purification tool to purify SUMOylated proteins from yeast cell extracts. In the first chapter of this thesis, we used yeast surface display-based directed evolution to engineer the binding affinity of Ulp1 to further improve the purification strategy. In the second part of this thesis, we identified CthUbp15 as the ubiquitin protease most suited for directed evolution in order to create a similar purification tool for ubiquitin studies.

Cardiolipin, the signature phospholipid of mitochondria, is crucial for mitochondrial function and architecture in the heart. Defects in cardiolipin remodeling processes and metabolism lead to cardiomyopathy, as seen in patients with Barth Syndrome (BTHS). Tafazzin is a major acyltransferase in CL remodeling that transfers linoleoyl groups to monolyso-CL until the final symmetric acyl composition is achieved. Mutations in Taz that result in BTHS cause inefficient transacylation between phosphoplipid-lysophospholipids, leading to cardiomyopathy. However, little is known as to the detailed molecular mechanisms by which Taz deficiency and consequent CL absnormalities lead to the progression of cardiomyopathy. In our present study, we found that loss of Tafazzin results in increased levels of cardiac stress markers, Anf and Bnp. Histological analysis demonstrated that mice with Taz deletions had thinner left ventricular walls and dilated left ventricular chambers relative to the control mice. Echocardiography analysis also showed that loss of Tafazzin causes decreased cardiac function. Together, these observations suggest that deletion of Tafazzin in cardiomyocytes results in dilated cardiomyopathy as seen in Barth Syndrome.

Biological membranes are highly dynamic complex structures exquisitely tuned to alterations in the state of the physical environment. Increased hydrostatic pressure and reduced temperature elicit similar physical influences on the phase and fluidity properties of biological membranes. As hydrostatic pressure is increased, or temperature is decreased, membranes undergo a reversible change from a fluid disordered state to a non-fluid ordered state resulting in membrane supraoptimal viscosity or phase transition. These deleterious consequences result to a large extent from the close packing of phospholipid fatty acyl chains within the membrane bilayer. In the absence of corrective membrane restructuring events to alleviate this perturbation, a host of membrane localized essential processes are prone to inactivation. Consequently, preservation of a suitable membrane dynamic state is of paramount importance in allowing an organism to adapt accordingly to prevailing environmental conditions.

In this dissertation, the relative importance of unsaturated fatty acids (UF As) to growth at high pressure and low temperature has been evaluated employing the piezophilic deep-sea bacterium Photobacterium profundum strain SS9. SS9 modulates the levels of numerous fatty acid species in response to pressure and temperature change resulting in enhanced levels of UF As at high pressure and low temperature. Analysis of SS9 mutant strains exhibiting altered fatty acid profiles have revealed particular classes of UFA species important for growth at high pressure and low temperature. Furthermore, molecular genetic analyses of genes responsible for UFA synthesis have identified multiple genes required for pressure-responsive UFA production. The fatty acid biosynthetic condensing enzymes FabF (KAS II) and FabB (KAS I) are required for high pressure regulation of 18: 1 synthesis and growth at elevated pressure. In addition, the genes responsible for omega-3 polyunsaturated fatty acid (PUF A) synthesis in SS9 have been characterized. PUF A levels undergo both pressure- and temperature-dependent modulations yet they are not required for growth under these conditions. Lastly, the involvement of the global transcription factor CRP is evidenced to influence UFA synthesis in SS9. Overall, these studies present a comprehensive analysis of membrane-based adaptations important for growth and survival under elevated pressure and low temperature conditions.

Ecological communities have been shown to vary in some predictable ways through space, time, and along environmental gradients, suggesting there may be underlying “rules” in ecology which govern these patterns and processes. However, nearly all our knowledge of these trends comes from studies of large eukaryotes such as plants and animals, and we know very little about how these eukaryotic patterns compare to those of bacteria and archaea, the most dominant life forms on the planet. The goal of this dissertation is to determine whether ecological patterns that are evident in plants and animals are also applicable to host-associated microbes. First, I investigated whether these microbes exhibit large-scale spatial trends in diversity and community composition that are concordant with those of their eukaryotic hosts. Specifically, I investigated changes in community composition across a marine biogeographic boundary and changes in diversity along a latitudinal transect. Results showed that microbiome compositions varied significantly between geographic sites, but that the identity of the host species played a greater role than geography in determining community composition. Further, microbes associated with the California mussel, Mytilus californianus, did not show a traditional latitudinal diversity gradient, and latitudinal diversity patterns varied based on microbial group and host body site. Next, I investigated whether host-associated microbial communities vary over time and in response to environmental change in similar ways to their eukaryotic hosts. I found that over an 11-year period, and in response to environmental change, microbial communities of the bean clam, Donax gouldii, significantly differed in composition, but not in richness. Further, I found that microbes did not regularly diverge in concordance with their intertidal gastropod hosts in the ~3.5 million years since the closure of the Isthmus of Panama, though this was dependent on the host taxa and the body site from which the microbes were collected. Finally, I investigated whether current methodologies for determining the core microbiome are guided by ecological and evolutionary principles and identify critical areas for future research. Overall, this dissertation shows that large-scale patterns in host-associated microbial taxa are often context-dependent and distinct from those of their hosts.