Possession Sound as a part of the larger Puget Sound is located between the Whidbey and Snohomish county shoreline. This location has significant influence from both freshwater and nutrient inflow from the Snohomish River as well as saline water resulting from tidal exchange. Students at the Ocean Research College Academy (ORCA), an early college program at Everett Community College, have been conducting baseline estuarine monitoring since 2007. The Washington State Department of Ecology has been aerially monitoring phytoplankton blooms and maintains a long-term collection of nutrient data throughout Puget Sound. According to this data set there has been a nutrient shift that is hypothesized to impact diatom abundance. This project combines the ORCA data set of plankton levels and nutrient concentrations to see if a similar shift is occurring in Possession Sound. Additionally, temperature variation is analyzed. Two hypotheses combine to identify special and temporal distribution of nutrients and plankton abundance. First, when silicate levels decline, diatomaceous phytoplankton are limited by this reduction. Second, the deep water location further from the river will have less variation in temperature, less nutrient availability and subsequently less phytoplankton abundance and diversity. The location nearest to the mouth of the Snohomish Riveris predicted to experience dramatic temperature variation and higher seasonal nutrient levels. The conclusions from this work will influence future plankton enumeration protocols at ORCA.

Through their use of fertilizer, humans have critically impacted the nitrogen cycle, which must be understood in terms of addition and loss. This research focused on one aspect of the nitrogen cycle; the loss of nitrogen from the ocean that occurs in suboxic waters. Nitrogen is added to the ocean reservoir through river inputs, nitrogen fixation, and deposition from the air. Nitrogen losses ultimately strip nitrogen out of the ocean through annamox and denitrification. Oxygen minimum zones (OMZs) are responsible for 50% of oceanic nitrogen loss. One of the 3 largest OMZs where anammox (ANerobic AMMonium Oxidation), and denitrification occurs is in the Eastern Tropic South Pacific (ETSP) where I utilized a new method of targeted proteomics. I quantified the abundance of four enzymes key in the loss of nitrogen from the oceans. N2O reductase catalyzes the critical step of denitrification. Hydrazine hydrolase & hydroxylamine oxidoreductase are two enzymes necessary for the anammox reaction and cytochrome c nitrite reductase (nrfA), an enzyme specific to the DNRA pathway (where DNRA stands for Dissimilatory Nitrate Reduction to Ammonium). My overarching question was “Are there trends in the abundances of the enzymes over space within the ETSP?” I collected samples during the summer of 2013 on the R/V Nathaniel Palmer. Tryptic (a proteoenzyme that cleaves peptide chains after specific amino acids) digest of the samples were injected on a Liquid Chromatography Mass spectrometry and quantified. The data are being analyzed for spatial trends, focusing on a near shore to distant from shore transect. Of the 26 samples analyzed, 15 had detectable anammox proteins, and 6 had detectable denitrification proteins. None of the proteins for DNRA gave usable data. I will discuss quantities and correlations of enzymes over space within the ETSP and connect it to the local and global nitrogen budget.

 The ocean is the largest and arguably the most important ecosystem on the planet. Within the ocean a successful and widespread group of algae, the diatoms, are major contributors to the global carbon cycle with primary production rates rivaling the terrestrial rainforests. Yet in many areas of the open ocean, low concentrations of bioavailable iron limit the growth and photosynthesis of diatoms and other phytoplankton. Marine phytoplankton have evolved several adaptive strategies to compensate for the scarcity of this metal. Here we present a comparative analysis of the molecular repertoire utilized by diatoms to respond to variable iron concentrations. Utilizing sequence data from over 75 diatom transcriptomes, we have identified homologues for key iron metabolism genes. We are using this data in conjunction with environmental metatranscriptomes to map the distribution of these genes in the environment. Here we will demonstrate a selection of our findings. We show that the gene coding for ferritin, the iron storage protein, is expressed in ancient and derived diatom groups and forms a monophyletic clade, sharing a common ancestor with marine cyanobacteria. Several species of diatoms co-express two unique ferritin paralogs, and these show differential transcription in the environment. We also show widespread constitutive expression of the electron transfer protein flavodoxin, which substitutes iron-using ferredoxin under iron limitation. Iron responsive Clade II flavodoxin is expressed by members of coastal and off-shore diatom assemblies. We are now able to examine these and other iron-related genes with a higher taxonomic resolution and utilize them as environmental biomarkers.

Pseudo-nitzschia is a cosmopolitan genus of marine diatoms known for its production of the neurotoxin domoic acid (DA) and resultant harmful algal blooms. The production of DA varies across species; of approximately 37 defined Pseudo-nitzschia species, at least 14 have produced DA in laboratory studies. Previous work based on ribosomal RNA sequences suggested the genus Pseudo-nitzschia could be divided into two major clades, or groups made up of an ancestor and its descendants. With the discovery of new species and identification of cryptic species complexes, the structure of these clades has eroded. We sought to integrate phylogeny, morphology and physiology of Pseudo-nitzschia, by re-examining their phylogeny, using both ribosomal LSU sequences and the rbcL gene, which encodes the large subunit of the carbon fixation enzyme, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). Trees based on rbcL sequences provided higher resolution to resolve the branching orders of Pseudo-nitzschia species. By mapping physiological characteristics such as cell size, frustule morphology, pigment content and DA production onto the tree we can interpret the evolutionary history of these features and species distributions in the context of our presumed clades.

The cosmopolitan pennate diatom Pseudo-nitzschia is notable for production of the neurotoxin domoic acid. Recently, a virus was isolated on Pseudo-nitzschia multiseries that has a broad host range, infecting many genera of diatoms. This is contrary to previous reports of very specific host ranges amongst diatom viruses, and suggests a nested infection network whereby specialist viruses infect only the most permissive hosts, while generalist viruses can also infect hosts that are well-defended. To explore the infection patterns of Pseudo-nitzschia and its viruses, new Pseudo-nitzschia strains were isolated monthly at a coastal and estuarine location in the Pacific Northwest for one year. Over 100 new strains of Pseudo-nitzschia were identified by sequencing the ITS region. These new strains are then challenged with environmental viral communities that were sampled at each location concurrently. To date, 28 of 47 strains tested are susceptible to infection by at least one viral community. Three strains are lysed by the majority of viral communities, suggesting we have identified permissive hosts. This analysis will continue until all currently isolated strains are challenged. Seasonally, May and July contained viruses that infected the widest range of strains, suggesting either high concentration of generalist viruses in those months, or high concentration of both specialist and generalists together. Understanding the seasonal structure of these infection networks will inform future virus isolation attempts and provide insight into a key component of these diatom-virus interactions.

Most living systems have complex bacterial communities (microbiomes) associated with them that are essential to the host's wellbeing. Microbiomes provide their host with nutrients, protection against pathogens and stress, and influence host immunity. Microbiomes can consist of hundreds of different bacterial species making them difficult to study. However, the algal species Chrysochromulina sp. (referred to as P3) has a small microbiome consisting of less than 10 species, making it a prime candidate for studying interactions between a host and its microbiome. Chrysochromulina sp. is also of interest as a source of biofuels since it produces a variety of fatty acids, and it belongs to an algal taxa that plays a major role in global carbon sequestration. I hypothesize that the microbiome of P3 allows P3 to withstand periods of stress. I tested this by growing P3 and an axenic (bacteria-free) culture known as P5.5 in high salt medium, a known stressor, and determined cell counts by flow cytrometry and bacterial colony counts by culturing. I found the P3 grew better than P5.5, indicating the bacteria help the alga survive high salt conditions. We cultured and identified 8 different bacterial species associated with P3 by sequencing the 16S rDNA, which encodes ribosomal RNA and is unique to each species. Next I will determine which bacterial species protect P3 from high salt. I will further determine the protective role of the microbiome by growing P3 and P5.5 under other stress conditions (e.g. low nitrogen, low pH, low light, and temperature shifts) to understand how the microbiome protects Chrysochromulina sp. during periods of environmental challenge. This will allow insight into how to optimize Chrysochomulina sp. growth for biofuel production and the effects of global warming on this alga.

Half of all carbon fixation on the planet is performed by phytoplankton, making these photosynthetic marine microbes essential to the global carbon cycle. It is therefore critical that we understand the factors affecting phytoplankton dynamics to predict the responses of the communities to future climate changes. While we have learned a great deal about phytoplankton growth, little is known about the factors that influence phytoplankton mortality. Studies that have focused on phytoplankton mortality rates have relied on incubations in a bottle, a labor-intensive approach that is limited in its broad-scale applicability. Our approach utilizes SeaFlow, a novel flow cytometer that can continuously measure the abundance and size distribution of phytoplankton in situ, from which cell division rates and net population loss rates are estimated using a size-structured population model. This approach essentially eliminates the bottle effects and allows for greater spatial and temporal resolution. Our goal is to monitor cell growth and loss processes of cryptophyte Rhodomonas prey populations during a bloom of a dinoflagellate predator in the Columbia River estuary, using SeaFlow. We first estimated cell growth of the cryptophyte during a time course experiment in the lab to validate our model. We then monitored the dynamics of cryptophyte populations in the Columbia River estuary, and have found that cell growth and loss processes varied widely during tidal cycles. Our findings shed new light into the biological and physical interactions that drive the dynamics of cryptophyte populations in the Columbia River estuary, and our approach will be able to be applied to previously gathered field data.

In the marine environment, hypoxia is defined as: a level of dissolved oxygen (DO) that has fallen below 2.0 mg O₂/L. This level is approximately 20% of a system that is at oxygen saturation. Should the DO reach 0% it is described as “anoxic”. Due to anthropogenic influences, incidences of hypoxia and anoxia in coastal marine environments are increasing globally. In Hood Canal, a sub-estuary of Puget Sound, Washington, research is underway to define and quantify the effects of hypoxia on zooplankton distribution and community structure. One copepod, Calanus pacificus, is considered to be a keystone species in this community due to its prominence in the food chain. My research has been to determine the physiological tolerances of Calanus to dissolved oxygen then compare with data from samples taken in Hood Canal to determine whether there is evidence that Calanus avoids areas with low DO. To identify oxygen tolerances, DO in seawater was manipulated in the laboratory by bubbling with N₂ gas, then 24-hr survival of copepods was tested at several DO levels. In the field, Calanus abundance was estimated from depth-stratified plankton samples taken during day and night, in regions and at depths, of high and low DO. Laboratory experiments showed that Calanus were adversely affected by low DO with a sharp decrease in survival from 87% survival at 1.44 mg O₂/L to 13% at 0.96 mg O₂/L. My research relates these findings to distributions observed in the field data. Substantial decreases in Calanus populations due to increased hypoxia in Hood Canal could cause large changes in the food web. Further research into Calanus’ behavioral reactions to these conditions could help understand, and even predict these changes.