Profiling multiomic biomarkers in bulk and in situ provides critical information which enables basic research and clinical applications. Unfortunately, most existing profiling methods are limited due to either low multiplexing, sensitivity, costs, or assay complexity. This thesis aims to develop two core technologies that address 1) bulk profiling issues with sensitivity and low throughput as well as 2) in situ profiling issues with low multiplexing capabilities, costs, and limited throughput. To address the first issue, this work introduces a novel liquid biopsy approach that utilizes a platform technology called Integrated Comprehensive Droplet Digital Detection (IC3D). This integrated approach combines microfluidic droplet partitioning technology, fluorescent multiplexed PCR chemistry, and our own unique and rapid particle counting technology to deliver ultrasensitive and ultrafast detection of colorectal cancer-specific genomic biomarkers from minimally processed blood samples. To address the second issue, this work introduces a new spatial multi-omics technology termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) that integrates a) in situ labeling of molecular markers (e.g. mRNA, proteins) in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, and b) spectra and time-resolved fluorescence imaging and analysis to enable rapid, high-throughput, and cost-effective spatial profiling of multi-omics biomarkers. By utilizing both time and intensity domains for labeling and imaging, this technology seeks to discriminate a vast repertoire of lifetime and spectral components simultaneously within the same pixel or image of a sample to enable highly increased multiplexing capabilities with standard optical systems. Overall, these two technologies represent simple, versatile, and scalable tools which enable more rapid, sensitive, and/or multiplexed protein/transcriptomic analysis.
The natural world is an extraordinary source of diverse organisms and natural product compounds. The use of natural products and their derivatives has been a recurring theme in the discovery and development of new therapeutics. The marine world is vast and contains incredible biological and chemical diversity. Therefore, the oceans have the potential for new discoveries of compounds with medicinal applications. One group of marine organism that has been shown to be a prolific producer of bioactive compounds is the cyanobacteria. This work focuses on the study of new compounds from marine cyanobacteria for their chemical diversity and biological activity. The work in this dissertation focuses on the discovery and characterization of compounds from two different species of cyanobacteria, Leptolyngbya sp. and Moorena producens. These were collected from American Samoa and Puerto Rico, respectively. The characterization of these compounds was achieved through the integration of a variety of techniques including mass spectrometry, advanced NMR technology and genomic information. This analysis led to the characterization of a new ionophore from the Leptolygbya sp. The collection of Moorena producens afforded new analogues of the bioactive compound, curacin A. 2D NMR was an essential aspect in these studies. Therefore, the optimization of different NMR techniques for faster data acquisition was studied in this dissertation for its applications on lower concentrations of natural products. This work highlights the novelty of compounds that can be discovered by the application of the multiple techniques. Additionally, it demonstrates the reduction in time that is possible with new techniques for the experimental acquisition of NMR data. Finally, this dissertation adds to the body of knowledge of the chemistry that originates from the world’s oceans.
Marine aerosols play a large role in the Earth’s climate by cooling via interaction with energy from the sun and altering the chemical and physical properties of clouds. The dissolved organic matter at the ocean surface, where sea spray aerosols and marine gases can be generated, is formed by the microbial loop by circulating nutrients and the ingestion of organisms like phytoplankton or bacteria – with additional inputs from terrestrial sources. The colored fraction of this organic matter, known as marine chromophoric dissolved organic matter, is a subject of considerable interest due to its ability to photosensitize nearby molecules. This indirect photochemical mechanism in the marine environment is not well understood. This dissertation first investigates the composition and properties of this fraction by conducting both simple model experiments in the laboratory and larger experiments such as the use of an indoor ocean-atmosphere facility. The ability to bridge the gap between these two types of study provides this thesis an excellent opportunity to answer various questions regarding the importance of understanding the role of heterogeneous chemistry and photochemistry in our surrounding environment. Lastly, this dissertation applies a similar perspective on photochemistry to explore the multiphase chemistry relevant to indoor environments. Humans spend 20 hours a day on average inside buildings, and while atmospheric pollution has been thoroughly studied, the pollution indoors is widely unknown and unregulated. Inspired by experiments conducted in a real home, various experimental model systems were investigated regarding indoor surfaces. The ultimate goal of the thesis being, to provide insight into the many vital heterogeneous and multiphase processes currently undiscovered in environmental chemistry community.
The exceptional concentration of marine biodiversity in the Coral Triangle is among the best-known biogeographic patterns in the ocean. Marine biodiversity peaks in the islands of Eastern part of Indonesia and the Philippines, the heart of the Coral Triangle, and significantly decreases moving away from this global biodiversity hotspot. However, data supporting this pattern largely come from fishes, corals and larger metazoans, and exclude smaller organisms that comprise the majority of marine biodiversity. This study utilized Autonomous Reef Monitoring Structure (ARMS) and DNA metabarcoding to examine biodiversity patterns of marine communities across Indonesia, the largest and most biologically diverse region of the Coral Triangle. In Chapter 1, I examine eukaryote biodiversity patterns of marine communities across Indonesia. Results demonstrate that smaller cryptofauna display similar biodiversity patterns to larger metazoans; the most diverse parts of Indonesia had more diversity per unit area, and greater heterogeneity and beta diversity across all spatial scales, individual ARMS, reefs, or regions. The results show that processes shaping biodiversity hotspots appear consistent in marine and terrestrial ecosystems, and across size and spatial scales. In Chapter 2, I examine patterns marine bacterial diversity across Indonesia, comparing microbial diversity to eukaryotic and metazoan diversity from ARMS. Results showed strong regional differentiation in microbial communities. Microbial diversity tracked eukaryote and metazoan diversity, and displayed a significant pattern of isolation by distance, strongly indicating that associations with larger eukaryotes and physical limitations to dispersal differentiate microbial communities in the Coral Triangle. These results are counter to the hypothesis that “everything is everywhere, but the environment selects”, and provide novel insights into the processes shaping marine microbial diversity in the world’s most diverse marine ecosystem. In Chapter 3, I re-examine data from Chapter 1 to determine how strategies for marine ecosystem monitoring in Indonesia could be developed to yield the best results for the least cost, allowing resource managers to harness the power of metabarcoding to better monitor this region’s biodiversity. Comparisons of cytochrome oxidase 1 (COI) and 18S rRNA metabarcoding data across three separate organismal size classes recovered from ARMS indicate that metabarcoding the 100 �m size fraction with COI captures the largest amount of diversity at the highest resolution. Results indicate that metabarcoding the 100 �m size fraction with COI provides the most accurate and economical approach to monitoring diversity in megadiverse regions where limited research investment precludes sequencing multiple size fractions with multiple metabarcoding markers. Combined, the results of this thesis demonstrate the power of ARMS and metabarcoding for the study and monitoring of marine biodiversity, providing new tools for the study and management of the exceptional marine biodiversity of the Coral Triangle.
Coral coverage reduction of up to 90% became a barometer of planetary health in the last three decades. As a result, coral scientists anticipate coral extinction with catastrophic effects on life in the oceans and propose direct interventions to rehabilitate ecological functions and extend corals’ lives. Some scholars have offered a critical approach to coral restoration’s naturalization of corporate forms of responsibility (Moore 2018) and controversies among coral scientists about what coral restoration can achieve (Braverman 2018). My dissertation draws upon twenty months of immersive study as a volunteer for the Center of Research, Education, and Recreation (CEINER) in the Rosario archipelago (part of the Corals of Rosario and San Bernardo Nature Reserve). CEINER simultaneously works on coral restoration and the assisted reproduction of endangered fish species. In addition, I volunteered for other coral restoration and reef checks in Isla Fuerte, Santa Marta, Taganga, and San Andrés. I also attended and presented posters and papers at international conferences on conservation biology, coral science, and ecological restoration. My work with scientists and other residents and visitors in the Rosario archipelago has pushed my analysis beyond extinction’s recognition to consider the migration of coral and fish further and deeper in the ocean. Using the word “migrations,” I intend to reframe the terms of destruction from planetary accounts to elusive ecologies—for both scientists and artisanal fishers. I explore different and co-incidental sea compositions, undecidable temporal horizons in coral reproductive urgencies, and more ways through which various islanders grow coral and fish in this sea. My research conceptually integrates and advances discussions surrounding extinction, managerial juridical frameworks, and environmental and animal studies. Throughout my dissertation, I build a vocabulary to think and imagine affective sea ecologies and unlikely, partial, and strategic collaborations among coral restoration scientists and other islanders.
Fish and fishers are affected by the environmental conditions they experience throughout their lives, from daily, annual to decadal time scales. Currently, the oceans are changing fast, as global warming increases the temperature of the water and reduces oxygen levels within it. However, there is still an important knowledge gap about how these shifting conditions influence wild populations of fish, especially in the early life stages of tropical species inhabiting mangrove lagoons or for adult fishes dwelling in the deep ocean. In this dissertation, we use the chronological and chemical properties of otoliths – calcified structures within the inner ear of fish – to investigate how temperature correlates with fish growth, to improve our understanding of their populations, and to develop proxies for hypoxia exposure in deep-sea fishes. Chapter 1 asks how the water temperature inside mangrove lagoons regulates the first year of growth for yellow snappers in the Gulf of California. We found that these animals grow faster in warmer waters until they experience a thermal threshold (~ 32° C) beyond which their growth rate is reduced. Chapter 2 tests the effects of extrinsic (water chemistry and temperature) and intrinsic (growth rate and taxonomy) factors on otolith chemistry. Using distinct species from Galápagos (yellow snapper and sailfin grouper) and the same species (yellow snapper) between Galápagos and the Gulf of California, we observed that extrinsic factors seem to be more important than intrinsic factors as influences on otolith microchemistry. Chapter 3 examines the population structure of yellow snappers in the Gulf of California and Galápagos mangroves by using otolith microchemistry and genetic analyses in tandem. These methodologies were complementary and helped to elucidate a source-sink metapopulation structure for Galápagos snappers, and a self-recruitment scenario for the Gulf snappers, with important implications for the mangrove management at these ecosystems. Chapter 4 explores the use of fish as mobile monitors of hypoxic conditions in Oxygen Minimum Zones (OMZs). Surprisingly, fishes with distinct life-history traits (longevity and thermal history) and from different OMZs (NE Pacific and SE Atlantic), but exposed to comparable low oxygen conditions, exhibited high similarity in their otolith chemistry. These findings may provide a baseline for tracking the ongoing expansion of OMZs. Lastly, Chapter 5 inquires how fishers’ local ecological knowledge (LEK) in the Galápagos Archipelago can help to elucidate the effects of climate variability on fish. We observed that LEK is in line with the scientific literature regarding distributional shifts in marine species and anomalous weather conditions during strong El Niño years.
Marine protected areas (MPAs) have been established worldwide to protect coastal ecosystems and the species inhabiting them. However, it is difficult to quantify whether these areas are adequately protecting the targeted species. Current monitoring methods, such as diver surveys, allow fish species to be identified in situ, but are known to alter fish presence and behavior. Other methods, such as acoustic telemetry, are relatively invasive, requiring the implantation of a transmitter tag into the fish. Additionally, both these approaches are laborious and expensive, relying on good weather and a talented pool of fisherman and divers. Methods that are non-invasive, such as passive acoustics, offer good spatial and temporal coverage, but ascribing specific calls or sounds to the species creating them is difficult, particularly for fishes. Camera deployments allow for in situ observations of behavior, diversity and frequency of occurrence of a wide variety of animals but are often hindered by low-light and battery limitations. Here, I developed passive acoustic and optical imaging tools to study sound-producing fish that allow improved performance over contemporary methods. These tools were used to study chorusing fish in protected kelp forests along the southern California coast. First, an autonomous Wave Glider was equipped with a passive acoustic recorder to map the distribution of five soniferous fish spawning aggregations. The fish choruses started near sunset and ended before sunrise, and were almost exclusively recorded offshore of kelp forests. Second, a low-cost underwater optical imaging system that utilizes a consumer-grade camera to capture high-quality images in low-light aquatic habitats without artificial lighting was designed and developed. The system was used to captured >1,500 images per day over 14 days, which revealed biologically important behaviors as well as daily patterns of presence/absence. Lastly, an underwater controlled source of known position was used to improve an acoustic localization algorithm to track fish to a resolution of a few meters. The fish remained outside of the MPA while vocalizing. This work demonstrates the promise of these tools to non-invasively monitor animal behavior, biodiversity and frequency of occurrence in MPAs as well as other nearshore areas.
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.
Coastal regions lie along a dual ecotone: the boundary of terrestrial and marine ecosystems and at the convergence of fresh and saltwater aquatic ecosystems. These ecotones are biogeochemically active regions that are stimulated by the supply and transport of organic material by way of their aquatic linkages. My dissertation addresses questions of organic matter and nutrient supply, retention, and transformation in the coastal regions of the Santa Barbara Channel with a focus on maintaining sufficiently high nitrogen concentrations to support primary production of kelp forests during low nutrient periods. To examine fluctuations of nitrogen concentrations in nearshore marine waters, and their relationships with physical and biological factors, I conducted intensive sampling for ammonium concentrations during the summer season and found a distinct periodicity in concentrations throughout the full water column in relationship to the tidal cycle. To determine if permeable marine sediment is a source of dissolved inorganic nitrogen to the overlying water column, I conducted a multi-year series of nutrient flux measurements using flow-through sediment bioreactors containing sediment collected near kelp forests and found that they are a source of ammonium and total dissolved nitrogen during the summer season. To investigate organic matter supply to marine sediment, I analyzed coastal sediment samples for evidence of terrestrial organic matter input before, during, and after a period with considerable rainfall that followed a 5 year drought, and I found evidence in both stream and marine sediment of terrestrial organic matter inputs becoming increasingly varied and less degraded over time. Using a Santa Barbara Coastal LTER dataset, I examined carbon and nitrogen in giant kelp tissue to evaluate patterns in nutritional content as they relate to changes in seawater temperature and larger oceanographic indices. I found that the nutritional content of giant kelp tissue collected in the Santa Barbara Channel has declined over the past 17 years, and this decline is correlated with increasing seawater temperatures and fluctuations of the North Pacific Gyre Oscillation index.
Ionocytes are specialized epithelial cells that excrete or absorb ions across an epithelium to regulate ionic, osmotic and acid-base levels in internal fluids. These ionocytes perform a wide range of functions (e.g. osmoregulation, pH regulation, and calcification) across various organs (e.g. gill, skin, inner ear). As atmospheric CO2 levels rise and oceanic pH levels fall, teleosts may increase their investment on ionocytes to survive in future ocean conditions. But generally speaking, the gill, skin, and inner ear ionocytes within marine teleost are not well characterized. This dissertation contains research spanning five southern Californian teleosts: the Blacksmith Chromis punctipinnis, the Yellowfin Tuna Thunnus albacares, the White Seabass Atractoscion nobilis, the Pacific Mackerel Scomber japonicus, and the Splitnose Rockfish Sebastes diploproa. In Chapter II, I investigated the individual and group behavioral responses of the Blacksmith, a temperate damselfish, after exposure to CO2-induced low-pH conditions. In Chapter III, I describe a novel technique used to quantify skin ionocytes in larval fishes. In Chapter IV, I applied the Chapter III’s technique to document developmental patterns in the skin and gill ionocytes of larval Yellowfin Tuna. In Chapter V, I investigated larval White Seabass response to hypercapnia by monitoring oxygen consumption rate and quantifying ionocyte abundance and relative ionocyte area across development. In Chapter VI, I characterized two types of inner ear ionocytes responsible for otolith calcification in the Pacific Mackerel. In Chapter VII, I investigated whether future CO2 /pH conditions would affect the gill and inner ear ionocytes of Splitnose Rockfish. Altogether, this work across the multiple teleosts demonstrates that ionocytes 1) have the plasticity to respond to external pH stress, 2) are sufficient to maintain internal homeostasis despite significant differences in CO2/pH levels, and 3) differ greatly in protein, morphology, and function depending on the tissue in question.