Prochlorococcus and Synechococcus, both unicellular marine cyanobacteria, are the most abundant photosynthetic organisms on the planet. They typically range from 0.4-0.8 µm and 0.8-1.5 µm respectively in size. Prochlorococcus is limited to the subtropical and tropical oligotrophic oceans, particularly between 40ºN and 40ºS, while Synechococcus has more of a cosmopolitan distribution. However, in regions where these two bacteria co-exist, Prochlorococcus outnumbers Synechococcus. Both bacteria are found in the euphotic zone, but Synechococcus prefers to be in well lit waters while Prochlorococcus can extend as deep as 200 meters in the water column. Furthermore, Prochlorococcus can be broken down into two distinct light physiologies, high and low light adapted. From these physiologies, Prochlorococcus is subdivided into six ecotypes and Synechococcus into 14 different clades based on their genetic difference. The distribution of both of these organisms in the ocean is not yet completely understood, however Prochlorococcus has a more uniform, semi-predictable distribution than Synechococcus. On three different cruises, two in the North Pacific and one in the South Atlantic, samples were collected to determine the spatial extent of these organisms. Quantitative polymerase chain reaction (qPCR), which allows for the quantification of these submicroscopic ecotypes by using the internal transcribed spacer (ITS) of rDNA, was performed on these samples. Preliminary data shows that in the South Atlantic and south of Hawaii, Prochlorococcus MIT9312 was the dominant high light ecotype and its distribution tightly followed temperature trends within the water column. In the North Pacific, Prochlorococcus MED4 was found in high abundance as far north as 42ºN while MIT9312 only extended to 41ºN in very low concentrations. The distribution of Synechococcus is much more complex than Prochlorococcus. Analyzing these organisms’ distribution under different nutrient and temperature settings will provide crucial insight to their global distribution and patterns.

Diatoms, a type of phytoplankton, are microscopic organisms that live in the world’s oceans where sufficient sunlight and nutrients for photosynthesis is available. They require distinct nutrients to maximize their growth and photosynthetic ability, such as nitrogen (N). When available N is limited, diatom cells become stressed, changing their growth rates and gene expressions. In response to this stimuli, they may up- or down-regulate important genes. Such gene regulations can be determined using a tiling array, a type of microarray that uses short fragments as probes to cover the entire genome. This approach was used to test the gene expression of nutrient-replete and limited cultures of Thalassiosira pseudonana, the first diatom with a sequenced genome. Following these experiments, we chose Fragilariopsis cylindrus and Pseudo-nitzschia multiseries to investigate the effects of N-starvation on a diverse group of diatoms. In silico analysis conducted on F. cylindrus, P. multiseries and T. pseudonana identified numerous conserved genes among the three species with significant up- and down- regulation in T. pseudonana during N-limitation. Based on these results, we chose two genes of interest for further analysis. We designed primers to target and amplify UNKNOWN 4888 and CHR18 in both F. cylindrus and P. multiseries under limiting and control conditions. Changes in gene transcript accumulation were measured using quantitative PCR (qPCR). N-limited cultures of F. cylindrus and P. multiseries showed a decrease in growth rates. In F. cylindrus, UNKNOWN 4888 was down-regulated, which is consistent with our hypothesis and the pattern seen in T. pseudonana. CHR 18 showed no expression change, a result we did not expect and which was not seen in T. pseudonana. This result may be impacted by variability within the biological replicates. The gene regulations of P. multiseries are currently under investigation and will be compared to F. cylindrus and T. pseudonana.

Toxins produced by harmful algal blooms (HABs) in the ocean can affect human health and local economies. During the summer upwelling season off the coast of Washington state, blooms of the Pseudo-nitzschia phytoplankton species can produce the toxin domoic acid. Closures of the razor clam fishery occur when these toxic blooms are transported to the beach by coastal currents. Physical processes along the coast of Washington are a crucial factor in predicting the occurrence of these local HAB events. This study aims to develop a better understanding of the Juan de Fuca eddy, a site where Pseudo-nitzschia become toxic in the Pacific Northwest. The physical oceanographic processes that occur off the west coast of the United States are characterized by a southward surface current, the California Current, and a deep northward subsurface current, the California Undercurrent. These two water masses have different nutrient concentrations as well as species composition and therefore influence ecological systems in different ways. Off the coasts of Washington and British Columbia, physical processes are complicated by interactions with submarine canyons and freshwater sources, which contribute to the formation of the Juan de Fuca eddy where southern and northern water masses merge. This study uses data from the Ecology and Oceanography of Harmful Algae Blooms program in the Pacific Northwest (ECOHAB-PNW) to better understand this region’s unique oceanographic dynamics. Profiles of temperature and salinity from ECOHAB-PNW surveys are used to calculate geostrophic velocity along the coast and create objective maps of water properties. The relative strength of the coastal currents and the presence of different water masses is compared 1) during different parts of the summer upwelling season, and 2) during different years. This analysis allows us to evaluate seasonal and interannual variability off the Washington coast and guide the development of realistic models of the coast’s circulation patterns.

Iron is a major limiting nutrient in the ocean and phytoplankton must be able to cope with intermittent pulses of iron followed by long periods of time without it. Certain pennate diatoms can handle this limitation because they have ferritin proteins that regulate iron uptake and storage. It has been shown that transcript abundance of the ferritin gene (FTN) in the open-ocean diatom Pseudo-nitzchia granii is regulated by iron availability, and this relationship could be used as a potential molecular indicator of the iron status of phytoplankton in the ocean. This is valuable because there are many complex forms of iron in the ocean which make chemical measurements of iron complicated. Currently, we are examining the changes in FTN gene expression in laboratory cultures of Pseudo-nitzchia granii grown under a variety of conditions. We have developed primers for FTN and tested these primers on environmental DNA collected from two stations in the Northeast Pacific that are at either ends of a natural iron gradient. To determine how growth conditions might affect FTN gene expression, we are also culturing Fe-limited and Fe-replete P. granii under a matrix of three different light conditions and two macronutrient starvation (nitrate and silicic acid) conditions using trace metal clean techniques. Future work will include quantification of FTN RNA transcripts using real-time quantitative PCR. Ultimately, our goal is to create a molecular tool, the Pseudo-nitzchia Ferritin Index (PFI) which will help elucidate the iron status of phytoplankton in their natural environment.

Viruses are the most abundant biological entities in the ocean. Though minute in size, they are thought to be responsible for controlling large-scale processes such as bloom and bust oscillations of algal blooms, lateral gene transfer, and global carbon and nutrient cycles. Viruses are both abundant and diverse, for they infect every life form on the planet and exhibit host selectivity at the species or strain level. Isolating and characterizing marine viruses is an integral step in understanding how viruses mediate communities in the sea. Pseudo-nitzschia is a cosmopolitan diatom genus that is particularly common in the Pacific Northwest. These phytoplankton are of particular importance because some species are capable of producing domoic acid, a neurotoxin. During blooms this compound bioaccumulates and can be deadly to humans, marine birds and mammals. Although it has been hypothesized that viruses play an integral role in phytoplankton ecology, only six diatom viruses have been characterized, none of which infect Pseudo-nitzschia. Water samples were taken during three Pseudo-nitzschia blooms in the Pacific Northwest in the summer of 2009. Single cells or chains of Pseudo-nitzschia were isolated, propagated in culture, and then identified at the species level. Molecular techniques were used for identification because Pseudo-nitzschia are indistinguishable by light microscopy alone. DNA extractions and polymerase chain reactions (PCR) were used to amplify a region of the genome called the intergenic transcribed spacer (ITS) for each isolate. This region has a high degree of variability across a genus and can thus be used to differentiate between species. A database of known Pseudo-nitzschia ITS sequences was then used for identification. These identified isolates will ultimately be used as hosts during virus isolation experiments. Because of viral host specificity, knowing the species of Pseudo-nitzschia lab cultures is essential for future experimentation.

Organophosphate insecticides (OPs) are widely used pesticides in the United States and are often found in surface waters as mixtures of many different types of OPs. OPs act biologically to inhibit acetylcholinesterase (AChE), a key degrader of the neurotransmitter acetylcholine (ACh). In teleost fish, ACh is the responsible for processes such as muscle movement and regulation of heart rate. Currently, the ecological impacts of OPs, and the regulations on usage, either do not consider mixtures or consider OPs found in mixtures to be acting independently of one another, resulting in an additive effect on AChE activity. Our lab has shown previously that binary combinations of the OPs diazinon or chlorpyrifos with malathion produce a synergistic effect on AChE activity in juvenile coho salmon (Oncorhyncus kisutch), resulting in greater AChE inhibition than predicted by dose addition. Here, we examine the effects of binary combinations of six additional OPs each with malathion using larval zebrafish (Danio rerio) as a model for AChE inhibition in fish species. We exposed 72 hpf zebrafish to a range of concentrations for each OP for 24 hours and measured AChE activity. A nonlinear regression was fit to the data to establish a dose response curve, from which the EC₅₀ was calculated. Zebrafish were then exposed in the same way to binary mixtures of an OP with malathion at a cumulative 1.0 EC₅₀. Exposure to mixtures of ethoprop with malathion and methyl parathion with malathion resulted in greater inhibition than the expected 50%. The synergistic effects seen with specific OP combinations imply that certain combinations of OPs in the environment are a cause for concern, especially with regards to predicting the health of fish species in the Puget Sound, including threatened Pacific salmon. Zebrafish appear to be a suitable high-throughput model for toxicological testing.