David A. Caron - US grants

This award establishes a microbial observatory at an ocean site located midway between Los Angeles and the University of Southern California's Wrigley Marine Science Center on Santa Catalina Island to discover and study previously undescribed microorganisms. Marine microorganisms play crucial roles in the biology and chemistry of the sea and thus have a strong influence on processes ranging from the development of toxic algal blooms to carbon cycling in the ecosystem, and thus ultimately on fisheries and other uses of the coastal ocean. The tiny size and lack of easily distinguishable morphological features, plus the difficulty and expense of cultivating microorganisms in the laboratory, have made it extremely difficult to identify most of these species until very recently. This situation has been particularly true of marine archaea, bacteria, and the smallest microalgae and protozoa. Therefore, important details are lacking regarding the kinds of microorganisms present in the water column, how they vary in time and space, and exactly what they might be doing. This project will apply newly developed molecular biological approaches that will permit us to discover and identify even the smallest microorganisms by their genetic characteristics, directly from field samples and without the need to grow them in laboratory culture. The different microbial types will also be quantified directly from field samples with state-of-the-art fluorescence probe technology, and a combined isotope-fluorescence probe technology will be applied to investigate the physiological characteristics of the dominant identified organisms within their mixed natural communities. Measurements will be taken biweekly for most of the 4-year project. Access to the study site will be facilitated by the USC Wrigley Institute for Environmental Studies which operates an oceanographic time series station with biweekly sampling, and also by boats traversing between LA and USC's marine laboratory twice weekly. The project will take full advantage of the physical, biological, and chemical data already being collected for the ocean time series. It is anticipated that this study will discover new species of microorganisms (based on previously unreported genetic signatures), indicate new approaches for bringing these newly discovered microbes into laboratory culture (based on phylogenetic analyses of their DNA sequences), and thus answer many questions concerning the composition and activities of one of the most abundant yet poorly understood groups of organisms on the face of the planet.

EIA-0121141
Requicha, Aristides
University of Southern California

ITR/SI+AP: Active Sensor Networks with Applications in Marine Microorganism Monitoring





The proposed research combines networking, distributed robotics, nanorobotics, and microbiology in an effort to develop and apply technology for the in-situ, real-time monitoring of microbial populations in aquatic environments, such as the ocean or water supply systems. The application context provides feedback from experiments with realistic systems, and this feedback is essential to the progress of the Information Technology (IT) research proposed here. This project addresses two key challenges for IT during this decade: moving from virtual to physical applications, and moving from macro to micro and nano.

The IT focus is on the study of Physically-Coupled Scalable Information Infrastructures (PCSIIs), which effectively "embed the internet". The sensors and actuators in the proposed PCSII must have small physical dimensions, comparable to those of the microorganisms to be monitored. They must be deployed in very large numbers to achieve the unprecedented spatial and temporal resolution necessary to investigate the causal relationships between environmental conditions and microorganisms. Control and coordination of a multitude of such devices of limited and heterogeneous capabilities raise major challenges for networking, distributed coordination and distributed algorithms. Sensing for detection and identification of microorganisms is another challenge, which will be tackled by using nanorobotic Scanning Probe Microscope technology.

Caron
0125437

Phototrophic and heterotrophic protists are ubiquitous in extreme cold-water environments where they are central to the production and utilization of energy and the cycling of elements. The dominance of protists in Antarctic food webs indicates major ecological and biogeochemical roles for these unicellular eukaryotes. Understanding the structure and diversity of these communities, and the adaptations that allow these assemblages to flourish near the lower limit of temperature in the ocean, is of fundamental importance to biological oceanography and to understanding the activities and evolution of life on our planet. The diversity of protistan assemblages has traditionally been studied using microscopy and morphological characterization. Due to the tedious nature of this approach and the inherent lack of taxonomic characters associated with most small protists, these approaches are inadequate for ecological studies of these communities. Molecular methods that utilize gene sequences for the identification and quantitation of naturally occurring protists offer a solution to this problem. This project will address issues of protistan community structure, population abundance, and adaptation to life in extreme cold through molecular and physiological studies on assemblages in the seawater and ice habitats of the Ross Sea, Antarctica. Research will focus primarily on species of phagotrophic protists (protozoa) which are ecologically important but for which no information currently exists. The work is designed to contribute new understanding concerning the biodiversity of protistan assemblages of coastal Antarctica, provide tools for ecological studies of these assemblages, and produce benchmark data on the basic physiological processes of protistan species in this extreme cold-water environment.

Protistan assemblages are essential components of food webs in the vast majority of aquatic ecosystems that have been examined to date. Despite the pivotal roles that these organisms play in carbon fixation, energy utilization and elemental cycling in marine ecosystems, little information exists on the presence, abundance and activities of these species in the deep ocean in general, and in particular at and around hydrothermal vents. The latter environments should be particularly conducive to the growth of phagotrophic protists in the deep ocean because biomass is greatly elevated at these sites and organic carbon availability in these regions is largely mediated by archaeal and bacterial assemblages. This research project will address fundamental questions regarding the species diversity, abundances of specific taxa, and trophic activities of protists within the deep ocean including hydrothermal vent areas. Specifically, the PIs will determine whether deep-sea communities harbor endemic assemblages of protists, establish the identity of the protists associated with these environments that are trophically active, and document the protistan taxa that dominate these assemblages in situ. They will examine the diversity of the protistan assemblages in the deep-sea away from the vents and in the overlying water column, and in microenvironments in and around hydrothermal vents and associated with dominant macrofauna of vents. Traditional (microscopy) and molecular biological (18S rDNA) approaches will be used to characterize abundance and diversity. Observations of the ingestion of fluorescently labeled prey will be used to establish trophically active protists in situ. They will also employ 18S rDNA clone libraries, cultures and microscopy-based observations to target specific taxa for the application of fluorescent in situ hybridization (FISH) to link morphological identity to commonly occurring (and trophically active) phylotypes. A culture collection of deep-sea protists will be established to further characterize protistan diversity and also to provide specimens for baseline physiological measurements in the laboratory. Growth rates will be examined in the laboratory using cultured protists grown under temperatures, pressures and water chemistries representative of deep-sea environments. Hydrothermal vent research is an excellent vehicle for incorporation into education at all levels ranging from elementary through graduate level. The information resulting from this research will be incorporated into undergraduate and graduate courses by the PIs, and will be featured on the Caron Lab Homepage. The PIs and the postdoctoral investigator supported by this project will participate directly in an ongoing teacher education program (Centers for Ocean Science Education Excellence; COSEE-West) that will reach middle and high school students, most of whom are Hispanic, African-American or other ethnic minorities and most of whom are economically disadvantaged. This will be accomplished through existing teacher enhancement and student enrichment activities that will incorporate this research into a learning experience that will enhance student awareness of environmental science, microbiology and the natural world. This work will be the center feature for the internationally acclaimed Extreme 2000 outreach program for secondary schools which has, over the last 5 years, directly engaged over 180,000 students throughout the US and 9 foreign countries. Contributions from this project will educate the public at every level about the deepsea, and the importance of microbes in forming and maintaining the biosphere.

Recent studies of marine ecosystems show conflicting evidence for trophic cascades, and in particular the relative strength of the crustacean zooplankton-phytoplankton link. The Ross Sea is a natural laboratory for investigating this apparent conflict. It is a site of seasonally high abundances of phytoplankton, characterized by regions of distinct phytoplankton taxa; the southcentral polynya is strongly dominated by the colony-forming prymnesiophyte Phaeocystis antarctica, while coastal regions of this sea are typically dominated by diatoms or flagellate species. Recent studies indicate that, while the south-central polynya exhibits a massive phytoplankton bloom, the poor food quality of P. antarctica for many crustacean zooplankton prevents direct utilization of much of this phytoplankton bloom. Rather, evidence suggests that indirect utilization of this production may be the primary mechanism by which carbon and energy become available to those higher trophic levels. Specifically, we hypothesize that nano and microzooplankton constitute an important food source for crustacean zooplankton (largely copepods and juvenile euphausiids) during the summer period in the Ross Sea where the phytoplankton assemblage is dominated by the prymnesiophyte. In turn, we also hypothesize that predation by copepods (and other Crustacea) controls and structures the species composition of these protistan assemblages. We will occupy stations in the south-central Ross Sea Polynya (RSP) and Terra Nova Bay (TNB) during austral summer to test these hypotheses. We hypothesize that the diatom species that dominate the phytoplankton assemblage in TNB constitute a direct source of nutrition to herbivorous/omnivorous zooplankton (relative to the situation in the south-central RSP). That is, the contribution of heterotrophic protists to crustacean diets will be reduced in TNB. Our research will address fundamental gaps in our knowledge of food web structure and trophic cascades, and provide better understanding of the flow of carbon and energy within the biological community of this perennially cold sea. The PIs will play active roles in public education (K-12) via curriculum development (on Antarctic biology) and teacher trainer activities in the Centers for Ocean Science Education Excellence (COSEE-West), an innovative, NSF-funded program centered at USC and UCLA.

Observing systems facilitate scientific studies by instrumenting the real world and collecting corresponding measurements, with the aim of detecting and tracking phenomena of interest. In this proposal, we focus on a class of observing systems which are (1) embedded into the environment, (2) consist of stationary and mobile sensors, and (3) react to collected observations by reconfiguring the system and adapting which observations are collected next, these are referred to as Reactive Observing Systems (ROS). The goal of ROS is to help scientists verify or falsify hypotheses with useful samples taken by the stationary and mobile units, as well as to analyze data autonomously to discover interesting trends or alarming conditions.
A wide range of critical environmental monitoring objectives in resource management, environmental protection, and public health all require distributed observing systems. This project will explore ROS in the context of a marine biology application, where the system monitors, e.g., water temperature and light as well as concentrations of micro-organisms and algae in a body of water. Using a hybrid network of stationary and mobile sensors, communicating both via wired and wireless links, the system collects fine-grained measurements of interesting information in near real-time. An example of the use of such a system is the rapid identification of micro-organisms to predict the onset of algal blooms. Such blooms can have devastating economic consequences.

Current technology precludes sampling all possibly relevant data. Therefore there is need to develop approaches for optimizing and controlling the set of samples to be taken at any given time, taking into consideration the application's objectives and system resource constraints. To support such an optimization and control process, a significant part of the framework must be dedicated to the development of models of data, and their automatic validation or adaptation. As part of the validation and adaptation process, the framework must also include a distributed support mechanism for locating data of interest. The methods to be pursued in the project include a multi-scale modeling framework for ROS, that allows applications to construct inter-related models of varying spatio-temporal scope based on collected data. Guided by the models, the reactive elements of the system predict where interesting data and phenomena are likely to be found. In the process of constructing models, the system actively seeks most useful data to improve both, the models and phenomenon detection and tracking. In a feedback cycle, this data acquisition is guided by previous, perhaps less precise, models. Thus, the system to be developed (AMBROsia) enables optimal collection of measurements in a manner that respects system resource constraints, yet improves the overall fidelity of phenomenon detection and tracking. Such a system will aid scientific research by facilitating the testing of scientific hypothesis. It will provide timely predictions of sampling needs, and tracking information for dynamic phenomena. Overall, AMBROSia will facilitate observation, detection, and tracking of scientific phenomena that were previous only partially (or not at all) observable and/or understood.

The availability of genome sequences provides the foundation for understanding how microorganisms function and live, and how they interact with their environments. Ocean microbes are thought to represent one of the greatest untapped sources of biodiversity. However, sub-disciplines studying marine prokaryote microbes, such as bacteria, are much further advanced than those studying more recently evolved unicellular eukaryotic life forms. Large and well-established prokaryote databases are available due to many recent concerted research efforts, while baseline genomic information for marine microscopic eukaryotes is far less advanced. Frequently used molecular methods to evaluate taxonomic diversity provide limited information on potential ecological roles or function of identified organisms. The program described here is designed to obtain large quantities of detailed DNA sequence data linked to known organisms. Data will be obtained for four previously understudied marine taxa from the Kingdoms Euglenozoa, Alveolata, Stramenopila and Cercozoa. These more detailed data will significantly increase genomic information on environmental microbial eukaryotes in the public domain and begin to allow detailed studies of relationships between and among prokaryote and eukaryote taxa. The sampling will take place at the University of Southern California's (USC) well-studied oceanographic time series station in the San Pedro channel, where the research team will have access to abundant biological and chemical data collected by other scientists working at the same site. This study holds tremendous promise to unlock an area of marine microbial research not well studied and will help to advance the state of knowledge within the field of marine microbiology and marine metagenomics.

In addition to basic research, the project will also have several significant broader educational impacts. Genomics is a rapidly developing field that is often misunderstood by the general population. The information resulting from the research will enhance teaching curriculum and will provide opportunities for both undergraduate and graduate level research. The research team will partner with USC Wrigley Institute for Environmental Studies (WIES) educators working to develop broad based educational outreach programs for K through grey audiences. The WEIS program is an extraordinary platform for education, and the work is ideally suited to enhance their existing programs. The research team will also participate directly in an ongoing teacher education enhancement program (the NSF funded Centers for Ocean Science Education Excellence; COSEE-West) run by the USC Wrigley Institute, UCLA and several informal science partners. The COSEE-West program has a proven track record for reaching middle and high school students in the Los Angeles Unified School District, most of whom are economically disadvantaged Hispanic, African-American or other ethnic minorities. In cooperation with COSEE-West, the research and education teams will develop modules to add to existing teacher enhancement and student enrichment projects. Finally, all data will be summarized on a dedicated San Pedro Ocean Time Series website with links from all team member websites.

The USC Microbial Observatory was established in 2000. The research focus of this observatory is an investigation of the microbial diversity and microbial community composition at a study site in the San Pedro Channel and Basin off the coast of southern California. The Channel area encompasses a diversity of coastal ocean habitats. The near-coast region borders one of the most highly urbanized areas of the country (greater Los Angeles) while open ocean waters impinge on the Channel Island archipelago that extends to within 30 km of the mainland. The San Pedro Basin is a deep-water environment (approximately 890 m) that exhibits very low oxygen concentration. The overarching objective of this project is the derivation of fundamental understanding of how microbial communities in the ocean are organized spatially (with depth) and temporally (at scales of months-to-years), and how environmental and biological factors shape this organization. The basic premise of the research is that "guilds" or "consortia" of microbial species exist that constitute functional subunits within the huge diversity of taxa that comprise planktonic microbial communities. The microbial species forming these guilds are functionally interdependent, and act as ecological units that replace one another in time and space as environmental conditions change. The program consists of monthly sampling at four depths in order to document the abundance, biomass and species composition of all planktonic microorganisms at the mid-channel sampling station. A variety of microscopical and molecular biological approaches are employed to examine archaeal, bacterial and microeukaryote (microalgal, protozoan, micrometazoan) diversity. The observatory is unique in that it entails an assessment of the complete spectrum of microorganisms (from viruses to the largest protists) in the water column. Genetic fingerprinting of the total microbial community is the primary tool for revealing the trophic roles and relationships among microbial taxa (predation, mutualism, commensalism, parasitism/infection), and to generate hypotheses on the interdependences among these species. Experimental studies involve manipulative food web experiments to test hypotheses concerning the relationships and interactions among the various microbial species. The data support extensive statistical analyses to identify relationships between microbial taxa, and with environmental parameters.
This research program strives to develop a fundamental understanding of the factors controlling the structure of microbial communities in aquatic ecosystems. Therefore, the results have far-reaching consequences for predicting biogeochemical processes mediated by microbial activities in nature. The project also incorporates a strong educational component aimed at reaching students ranging from elementary school children to graduate students. The information resulting from the research is incorporated into undergraduate and graduate courses taught by the principal investigators, and both types of students participate actively in the research. In addition, the principal investigators and graduate students supported by this project participate directly in an ongoing teacher education program (Centers for Ocean Science Education Excellence; COSEE-West) that reaches middle and high school students, many of whom are Hispanic, African-American or other ethnic minorities and most of whom are economically disadvantaged. This education/outreach goal is accomplished through an existing teacher enhancement and student enrichment program that incorporates the research from this microbial observatory into a learning experience that enhances student awareness of environmental science, microbiology and the natural world. The observatory principal investigators work is featured and publicly available on the internet as a part of the USC Microbial Observatory Website at http://www.usc.edu/microbialobservatory).

Intellectual Merit. Progressing ocean acidification and increasing sea surface temperature may significantly impact marine plankton community structure and community-level processes. Yet, our ability to predict specific responses is still limited because of the tremendous taxonomic complexity of microbial assemblages and the limitations of the methodological and experimental tools presently available to test specific hypotheses. Research to study community level effects due to a changing CO2/temperature regime often involve short-term field incubations that subject organisms to simulated 'greenhouse' conditions. A central question for understanding global climate change is whether the trends and patterns that are observed in communities during short-term manipulations can be extrapolated to the responses of fully acclimated plankton communities over decadal or longer timescales.

The specific objectives of this research program are: 1) to examine how protistan communities restructure in response to increased seawater CO2 concentrations and temperature in semi-continuous field incubation experiments, and 2) to evaluate if the dominant algal species that are isolated from either ambient or increased CO2 and temperature treatments in field experiments will re-establish dominance under the same conditions in acclimated laboratory culture competition studies. Changes in community structure of natural protistan assemblages in our experimental treatments will be followed using image-based methods (flow cytometry, FlowCAM and microscopy) in combination with state-of the art molecular tools (DNA fingerprinting). Molecular approaches have begun to reveal an incredible high diversity for marine microbes and stimulate debate in regard to the ubiquitous presence of a microbial 'Rare Biosphere'; that is, the presence of a huge number of species that are present at extremely small percentages of the total abundance of microbes, among a much smaller percentage of dominant ones. Little is known about the ecological significance of these rare species, and the investigators hypothesize that change in CO2 and temperature will select for some of these members that are inconspicuous under ambient conditions.

The unique aspect of this experimental approach is the combined use of field incubations that encompass entire natural microbial assemblages, with a series of laboratory culture competition trials that focus on the same groups of algae after extended acclimation, to evaluate the validity of short-term experiments that examine changing CO2 and temperature. First, field incubation experiments will be conducted to characterize changes in protistan community structure under ambient and future CO2/temperature regimes. Second, clonal algal strains will be isolated from dominant taxa in present day and greenhouse treatments, and cultivated for extended periods under their 'preferred' CO2/temperature conditions. Finally, mixtures of these acclimated strains will be competed against each other, to re-examine their responses to ambient and greenhouse conditions and compare them to the responses observed in the unacclimated field incubation experiments.

Broader Impacts. Two graduate students will make this project the focus of their Ph.D. research at USC, and undergraduate students will be involved in the field and laboratory work. Results from this research will be incorporated in lesson plans on microbial diversity and global climate change. Dissemination of data and results is planned on a project website. The PIs in this project also participate in an on-going, innovative, NSF-funded program (Centers for Ocean Science Education Excellence; COSEE-West) which focuses on personal involvement of faculty in a custom framework to allow an effective connection with K-12 teachers, thus improving math and science education in disadvantaged parts of Southern California.

"This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)."
Proposal #: 09-60163
PI(s): Sukhatme, Gaurav; Caron, David A.; Edwards, Katrina J.; Jones, Burton, H; Mitra, Urbashi
Institution: University of Southern California
Title: MRI-R2: Acq. of a Networked AUV-based Instrument for the Southern California Bight
Project Proposed:
This project, acquiring a networked instrument composed of two complementary Autonomous Underwater Vehicles (AUVs), supports an extensive program of research in robotics, underwater acoustic communication and networking, marine biology, oceanography, and biogeochemistry at the Center for Integrated Networked Aquatic PlatformS (CINAPS). These two AUVs add capability to those already in use by CINAPS. The work addresses two current key limitations: limited depth (depth no greater than 200m) and limited communication (only supports communication through the air, i.e., when the vehicles are not below the surface.) These instruments impact agendas in two main fields: research in the Southern California coastal marine ecosystem (in physical oceanography, geobiological oceanography, and microbial ecology), and research in robotics, communication, and networking.
The instrumentation consists of a Slocum Electric Glider and an EcoMapper-EP (Expandable Payload) Autonomous Underwater Vehicle. The Slocum glider, for use up to 1000m, was developed by in the early 1990's, and has become an integral tool of ocean science in this decade. It is driven entirely by a variable buoyancy system rather than active propulsion. The glider's wings convert the buoyancy-dependent vertical motion into forward velocity. Slocum gliders are truly autonomous, requiring a surface vessel only for deployment and recovery. On-board communications capabilities include a two-way RF modem, Iridium satellite and an ARGOS locator. With its onboard instrumentation, combined with its mobility and long-range capabilities, the glider provides continuous, near-real-time information, about the physics and biogeochemistry of the ocean. Designed specifically for water quality and bathymetry mapping applications, the Ecomapper for shallow use (up to 200m) is a unique AUV. Easily deployed by one person, it is able to perform a wide-area survey without a surface workboat or associated personnel. The EP class EcoMapper allows for additional ports for customized sensor integration and space for a second low-power CPU to support the additional sensors and software. This additional CPU enables mission adaptations to occur on-the-fly based on real-time sensor readings that are necessary when trying to detect and track dynamically changing oceanic features a range of important processes and associated questions can only be studied with the coupling of deep AUV operations (up to 1000m) with shallow (up to 200m). These include (a) the toxic effect of harmful algal blooms at greater depth, (b) the temporal and spatial variations of the low oxygen interface (deep basins can become hypoxic or anoxic due to isolation), and (c) fluxes of particulate material from urban runoff to the deeper sea that are discontinuous in both time and space; the spatial extent of which can be best resolved with autonomous vehicles that can maintain a presence over several events. To enable these studies, new algorithms for multi-AUV communication and control are necessary. The ability to coordinate vehicle trajectories and missions while submerged presents a current limitation. This is of particular concern in the Southern California Bight, the study site, a region with high maritime traffic where surfacing of the AUVs needs to be minimized. Used as a field instrument deployed in the Southern California Bight (SCB), the instrument is programmed and bench-tested in the Robotic Embedded Systems Laboratory on the USC main campus in LA. The instrument manager is supported by the PI;s grants. USC covers all operational and maintenance costs.
Broader Impacts:
The instrumentation impacts faculty research and student education, contributing to enable new science in diverse research projects and impacting the ongoing instruction at the institution and serves as a learning tool to develop student scientific proficiency (through existing courses and participation in faculty-led research). This acquisition plays an important role in the Southern California Bight Study planned for Spring 2010, coordinated by the Southern California Coastal Water Research Project (SCCWRP), a public agency focusing on the coastal ecosystems of Southern California. The PIs have close collaborative ties to this agency. Moreover, the networked and adaptive sensor systems provide important data relating to the climate and health of Southern California coastal ocean.
Undergraduate students are involved through REU site awards at USC. Since the PI runs the USC Computer Science department REU site for research in various areas of computer science, students in his lab often assist with data analysis and programming for the robotic boats. The EcoMapper vehicle, which is designed to be portable and deployable in shallow water, is particularly suitable for the next generation of REU students. Other PIs participate as faculty mentors in USC biochemistry REU site encouraging similar participation from undergraduate students in biology.

Bacteria, Archaea, and Protists dominate global elemental cycling and are immensely diverse genetically, taxonomically, and functionally. Yet the extent of marine microbial diversity, its patterns, and relationships among genetic, taxonomic, and functional diversity are very poorly characterized, even though the ocean covers 70% of the planet's surface. Among the least well known variables is the effect of human impacts on native marine microbial systems, although it is recognized that impacted systems are more prone to events like harmful algal blooms. Knowledge of these relationships and impacts are necessary to anticipate the responses of biota to global changes and feedback mechanisms that may alter the extents, rates, and even pathways of such changes. This project will expand upon an existing NSF-funded 10+-year monthly ocean time series (Microbial Observatory) that has focused on a single site midway between Los Angeles and Santa Catalina Island, to also include quarterly sampling adjacent to the impacted LA Harbor region to the barely-impacted Catalina coast. USC already runs facilities in LA Harbor and Catalina, with daily boats between (no cost). Measurements include (1) Genetic diversity: high throughput DNA sequences of "housekeeping" and functional genes. (2) Taxonomic diversity: high throughput tag sequences of small subunit ribosomal RNA genes, flow cytometry, automated image analysis (3) Functional Diversity: (a) Functional measurements (carbon fixation and respiration rates, microbial growth and grazing rates, cell size, morphology, and biomass variations), (b) distribution and expression of particular target functional genes involved with processes central to the cycles of carbon, nitrogen, and sulfur, (c) exploratory metatranscriptomics to explore functionalities that were not anticipated. (4) Integrating these: Multivariate statistical and network approaches including newly developed techniques (e.g. Bayesian networks to examine cause-effect relationships), and high speed computational approaches to assess the relationships among the genetic, taxonomic, and functional aspects of biodiversity observed. The PIs will also examine the collected data for signatures and specific effects (on organism identity and functions) associated with human impacted harbor site vs. the relatively pristine one.

Integration: The PIs will use network and time series analysis, along with other statistical tools to integrate "classical" microbial and oceanographic rate process measurements, flow cytometric and microscopic characterizations of communities, along with targeted as well as untargeted metagenomics and metatranscriptomics to relate genetic and taxonomic diversity with specific functions (at organismal, food web, and system levels). For example, they should be able to determine how different variants of particular taxa (e.g. at resolution levels ranging from what might be considered near the subspecies to genus levels) would differ in their association with particular measured functions, functional genes, or particular other taxa - or they might see how particular clusters of related organisms behave similarly or differently in their associations. This project offers an unprecedented and potentially transformative opportunity to combine and integrate measurements of genetic, taxonomic, and functional diversity along with direct measurements of system function in a well studied marine system that includes a gradient from one of the world's busiest harbors to a largely pristine ocean habitat. Far beyond just describing the distributions of organisms and functions (itself a necessary first step), they will specifically link spatial and temporal variations in a variety of functions with variations in genetic and taxonomic community composition.

Broader Impacts: The project will involve PhD training of participants as well as a direct connection to K-12 education via the COSEE teacher-training program at USC. Additionally, the PIs' labs routinely include undergraduate students from underrepresented minorities, who would participate. Outreach will include an interactive website and direct interaction with the public at the Wrigley Marine Science Center.

Planktonic marine microbial communities consist of a diverse collection of bacteria, archaea, viruses, protists (phytoplankton and protozoa) and small animals (metazoan). Collectively, these species are responsible for virtually all marine pelagic primary production where they form the basis of food webs and carry out a large fraction of respiratory processes. Microbial interactions include the traditional role of predation, but recent research recognizes the importance of parasitism, symbiosis and viral infection. Characterizing the response of pelagic microbial communities and processes to environmental influences is fundamental to understanding and modeling carbon flow and energy utilization in the ocean, but very few studies have attempted to study all of these assemblages in the same study. This project is comprised of long-term (monthly) and short-term (daily) sampling at the San Pedro Ocean Time-series (SPOT) site. Analysis of the resulting datasets investigates co-occurrence patterns of microbial taxa (e.g. protist-virus and protist-prokaryote interactions, both positive and negative) indicating which species consistently co-occur and potentially interact, followed by examination gene expression to help define the underlying mechanisms. This study augments 20 years of baseline studies of microbial abundance, diversity, rates at the site, and will enable detection of low-frequency changes in composition and potential ecological interactions among microbes, and their responses to changing environmental forcing factors. These responses have important consequences for higher trophic levels and ocean-atmosphere feedbacks. The broader impacts of this project include training graduate and undergraduate students, providing local high school student with summer lab experiences, and PI presentations at local K-12 schools, museums, aquaria and informal learning centers in the region. Additionally, the PIs advise at the local, county and state level regarding coastal marine water quality.

This research project is unique in that it is a holistic study (including all microbes from viruses to small metazoa) of microbial species diversity and ecological activities, carried out at the SPOT site off the coast of southern California. In studying all microbes simultaneously, this work aims to identify important ecological interactions among microbial species, and identify the basis(es) for those interactions. This research involves (1) extensive analyses of prokaryote (archaean and bacterial) and eukaryote (protistan and micro-metazoan) diversity via the sequencing of marker genes, (2) studies of whole-community gene expression by eukaryotes and prokaryotes in order to identify key functional characteristics of microorganismal groups and the detection of active viral infections, and (3) metagenomic analysis of viruses and bacteria to aid interpretation of transcriptomic analyses using genome-encoded information. The project includes exploratory metatranscriptomic analysis of poorly-understood aphotic and hypoxic-zone protists, to examine their stratification, functions and hypothesized prokaryotic symbioses.