Vibrio parahaemolyticus is a halophilic Gram-negative bacterium indigenous to estuarine and marine environments worldwide. A minority of strains are responsible for causing severe gastroenteritis in humans, often associated with the consumption of raw or undercooked molluscan bivalves, especially oysters. Pathogenic strains are normally detected by screening environmental samples for the presence of the virulence-associated thermostable direct hemolysin (tdh). In Washington State, the utility of tdh is questionable given the prevalence of tdh+ strains (25/28, 89.3%) among environmental multilocus sequence types and the recent emergence of two tdh- clinical sequence types. In light of this public health challenge, we are currently validating the utility of new candidate virulence markers, which were discovered via comparative genomic analysis of 13 clinical and 6 environmental strains. We identified a unique α-hemolysin (605 amino acids) in the two emergent tdh- sequence types having homology (83% identity) to the hlyA of the RTX toxin cluster in the uropathogenic E. coli 536. To investigate the utility of this α-hemolysin as a new virulence marker, PCR primers targeting this gene were validated against a collection of clinical and environmental strains isolated from WA State. To date, 11/97 (11.3%) of the strains tested harbored this gene. Notably, this gene was present in 5/5 (100%) of the strains representing the two tdh- clinical sequence types. Current efforts are focused on the validation of this candidate virulence marker against a larger strain collection as well as the investigation of additional candidate virulence markers. Future efforts will focus on whether this hemolysin represents a newly described mechanism of V. parahaemolyticus pathogenesis.

Different species within the bacterial genus Francisella are pathogenic in mammals. Recently, an emerging pathogen in fish, F. noatunensis, has been causing significant mortalities in tilapia and Atlantic cod. The Francisella Pathogenicity Island (FPI) is a gene dense region on the Francisella chromosome that encodes essential virulence factors for the mammalian pathogens F. tularensis and novicida. To determine if the functionality of virulence factors has been conserved for the genus, a gene knockout for the pathogenicity determinant protein A (pdpA) gene in the F. noatunensis FPI was generated as a potential vaccine candidate. PdpA has been shown to be essential for the ability of Francisella to cause disease for the human pathogen. The F. noatunensis knockout was generated via homologous recombination using a suicide vector containing an antibiotic cassette flanked by portions of pdpA. Since polarity can be an issue for knockouts in gene dense regions, two different pdpA mutants were generated in opposing polarity. It has recently been shown that F. noatunensis infection in zebrafish closely mimics Francisella infection in mammals implying conserved virulence strategies for the genus. Therefore, zebrafish were used to assess the level of attenuation for the F. noatunensis pdpA knockouts generated in our laboratory. In addition, iglC is another gene found in the FPI that results in attenuation after it has been inactivated in the human pathogen. Our laboratory has acquired an iglC mutant for F. noatunensis from a collaborator that has reduced virulence in tilapia and will serve as a reference for our pdpA knockout infections in zebrafish. The results from this study will show the physiological similarities within the genus relative to their phylogenetic relationships.

Diatoms are unicellular photosynthetic algae, or phytoplankton, and account for approximately 20% of global primary production. The diatom Pseudo-nitzschia can produce a neurotoxin called domoic acid (DA) that builds up in the tissues of shellfish when this diatom blooms. DA poisoning causes life-threatening conditions in mammals and humans when these shellfish are ingested. Parameters such as shellfish exposure length and bloom toxicity can be found by understanding what regulates diatom communities. One mechanism of bloom regulation that we know little about is that of viral infection, despite viruses being the most abundant predator in the ocean. My lab is isolating and characterizing viruses to develop a model system. After a toxic Pseudo-nitzschia bloom at Sunset Beach, Oregon was sampled in 2009, the first Pseudo-nitzschia infecting virus was isolated by infecting the host P. multiseries Clnn-16. This virus was named PmDNAV and was characterized by its burst size, latent period, host range, morphology, and nucleic acid content. I hypothesize that there are many different viruses that can infect diatoms of the genus Pseudo-nitzschia in addition to the PmDNAV. My project focuses on isolating and characterizing more viruses, in order to compare them to the PmDNAV. I have samples from 4 different blooms and 43 different hosts in culture to test these lysates on. I found that the host Clnn-16 dies when inoculated with lysates from the Sunset Beach sampling. I repeated this experiment to prove that my lysates could infect a new healthy host. I am working to prove that this death is due to a virus by looking for small pinpricks of light on a slide after staining the nucleic acids of the putative viruses. After that, I will make the virus clonal and work to characterize this virus, later repeating this process with as many viruses as possible.

Synechococcus is a unicellular cyanobacterium about 1µm in diameter. It is an abundant photosynthetic microorganism and a great contributor to the primary productivity of the Earth’s oceans. Synechococcus communities are composed of different phylogenetic clades that are believed to represent physiologically and genetically diverse populations. Previous data has shown two phylogenetically distinct populations of Synechococcus, clade I and clade IV, to be dominant in Puget Sound, with fluctuations in these populations during different seasons and regions. For my research, I will attempt to identify a clade IV strain. In an attempt to enhance conditions where clade IV will be most competitive, I have varied the culturing parameters such as using multiple media types. Filtered whole water samples containing mixed assemblages of Synechococcus were collected from Puget Sound and were initially cultured in natural Puget Sound seawater. These cultures were poured onto agar plates with sterilized natural Puget Sound water as well as plates with artificial seawater with different concentrations of AMP1 nutrients. The 16S rDNA of the isolates identified as clade IV will be sequenced by the use of PCR using primer sets that target specifically Synechococcus clade I or clade IV. The success, or failure, of amplification will indicate whether the Synechococcus DNA template was Synechococcus clade I or Synechococcus clade IV. Any Synechococcus DNA that results in a successful amplification using the Synechococcus clade IV primer set, I will send off to be sequenced. We have nutrient and physical measurements for these samples to help explain the environmental parameters that control distribution of these populations. With this information and ITS sequences I can compare Synechococcus clade IV populations within Puget Sound, and compare these with coastal populations that have been previously isolated. We seek to understand how these populations fluctuate seasonally and throughout different regions of the Sound.

Nitrogen, a limiting nutrient, controls oceanic primary production which directly affects the global carbon cycle. Consequently the nitrogen cycle has great implications for how the oceans will react to climate change. Oxygen Minimum Zones (OMZs) are areas of the ocean characterized by low oxygen levels and as areas of nitrogen removal. OMZs are expected to grow in the future, possibly increasing nitrogen removal from the ocean. Because nitrogen is requirement for primary productivity, the growth of OMZs has potential to slow the biological pump. The Eastern Tropical Pacific (ETNP) OMZ is the largest OMZ in the world, running from 0-25 ° N with a core reaching depths from 280m to 850m.  Anammox (anaerobic ammonium oxidizing) bacteria are of particular interest in the ETNP because their ability to convert fixed nitrogen (ammonium and nitrite) into the form of N₂ gas, which is biologically unavailable. Anammox bacteria produce N₂ autotrophically by oxidizing ammonium in low oxygen environments using nitrite as an electron acceptor. Understanding the presence of anammox bacteria through the water column is crucial to gaining a clear understanding of the nitrogen cycle. Presence/ absence data of anammox bacteria has been constructed using PCR with primers that target the anammox specific Hzo gene. Samples from 5 vertical profiles were collected while aboard the RV Thomas G. Thompson during the oceanography senior thesis cruise from March 16^th to 27^th. Samples were taken at every layer in the water column, including the oxycline, the OMZ core and bottom of the OMZ. During the cruise denitrification rates and oxygen concentrations were determined. Comparing denitrification rates to presence absence data of anammox bacteria will help uncover the role of anammox bacteria as denitrifyers in the ETNP.

Beginning with the industrial revolution, levels of pCO₂ in the oceans have increased which in turn decreases the pH of waters and creates a chemical change known as ocean acidification. There are many potential impacts that ocean acidification may have on marine ecosystems and biodiversity, yet little research has been conducted on the effects of low pH levels on fish, particularly temperate species. In this study, we evaluated how different pCO₂ levels affect the survivorship and behavior of larval lingcod (Ophiodon elongates) under starvation conditions. To accomplish this task, we reared lingcod eggs that we obtained from the Manchester NOAA hatchery and the resulting larvae under three different pCO₂ levels that correspond to mean present day (~550 ppm) and possible moderate and high future pCO₂ levels (1050 and 2159 ppm, respectively) in Puget Sound. For the survivorship experiments, we recorded daily mortalities, which allowed comparisons of the cumulative mortality curves and mean survival times among treatments. For the behavioral experiments, we scored several different behaviors to assess activity levels and group cohesion. Five fish were taken from each container in each treatment and placed in a 4cm tall enclosure where they were filmed for ten minutes. Initial results indicate that mortalities were highest under present day conditions (550 ppm) and lowest under predicted high future pCO₂ levels (2159 ppm). These findings suggest that low pH, high pCO₂ levels benefit some species of fish larvae, which is the opposite effect demonstrated in most published larval fish studies to date. We discuss our findings in the context of Puget Sound’s dynamic pH environment and highlight areas of future research on this important yet understudied topic.