Tawnya D. Peterson

Determining indicator taxa across spatial and seasonal gradients in the Columbia River coastal margin.

Bacterioplankton communities are deeply diverse and highly variable across space and time, but several recent studies demonstrate repeatable and predictable patterns in this diversity. We expanded on previous studies by determining patterns of variability in both individual taxa and bacterial communities across coastal environmental gradients. We surveyed bacterioplankton diversity across the Columbia River coastal margin, USA, using amplicon pyrosequencing of 16S rRNA genes from 596 water samples collected from 2007 to 2010. Our results showed seasonal shifts and annual reassembly of bacterioplankton communities in the freshwater-influenced Columbia River, estuary, and plume, and identified indicator taxa, including species from freshwater SAR11, Oceanospirillales, and Flavobacteria groups, that characterize the changing seasonal conditions in these environments. In the river and estuary, Actinobacteria and Betaproteobacteria indicator taxa correlated strongly with seasonal fluctuations in particulate organic carbon (ρ=−0.664) and residence time (ρ=0.512), respectively. In contrast, seasonal change in communities was not detected in the coastal ocean and varied more with the spatial variability of environmental factors including temperature and dissolved oxygen. Indicator taxa of coastal ocean environments included SAR406 and SUP05 taxa from the deep ocean, and Prochlorococcus and SAR11 taxa from the upper water column. We found that in the Columbia River coastal margin, freshwater-influenced environments were consistent and predictable, whereas coastal ocean community variability was difficult to interpret due to complex physical conditions. This study moves beyond beta-diversity patterns to focus on the occurrence of specific taxa and lends insight into the potential ecological roles these taxa have in coastal ocean environments.

Disinfection of Ballast Water with Iron Activated Persulfate

The treatment of ballast water carried onboard ships is critical to reduce the spread of nonindigenous aquatic organisms that potentially include noxious and harmful taxa. We tested the efficacy of persulfate (peroxydisulfate, S2O8(2-), PS) activated with zerovalent iron (Fe(0)) as a chemical biocide against two taxa of marine phytoplankton grown in bench-scale, batch cultures: the diatom, Pseudonitzshia delicatissima and the green alga, Dunaliella tertiolecta . After testing a range of PS concentrations (0-4 mM activated PS) and exposure times (1-7 days), we determined that a dosage of 4 mM of activated PS was required to inactivate cells from both species, as indicated by reduced or halted growth and a reduction in photosynthetic performance. Longer exposure times were required to fully inactivate D. tertiolecta (7 days) compared to P. delicatissima (5 days). Under these conditions, no recovery was observed upon placing cells from the exposed cultures into fresh media lacking biocide. The results demonstrate that activated PS is an effective chemical biocide against species of marine phytoplankton. The lack of harmful byproducts produced during application makes PS an attractive alternative to other biocides currently in use for ballast water treatments and merits further testing at a larger scale.

Impacts on phytoplankton biomass and productivity in the Pacific Northwest during the warm ocean conditions of 2005

[1] Delayed onset of the spring transition and upwellingfavorable winds in the Pacific Northwest during springsummer 2005 resulted in a positive temperature anomaly and a pronounced negative anomaly in surface phytoplankton biomass (chlorophyll a )a nd primary productivity. Compared to time periods before and after the warm water event, total biomass was reduced by ca. 50% along a hydrographic line extending seaward from Grays Harbor, WA (47 N), with a concomitant decrease of ca. 40% in surface and depth-integrated primary productivity. Associated with these declines in biomass and productivity was a change in mean phytoplankton size, with >50% of the nearshore assemblage less than 5m mi n size during the warm event, compared to <30% during more normal conditions. Unlike higher trophic levels, the phytoplankton rapidly recovered with the onset of upwelling, returning to more typical size structure, biomass, and productivity within one week of the onset of upwelling-favorable winds. Citation: Kudela, R. M., W. P. Cochlan, T. D. Peterson, and C. G. Trick (2006), Impacts on phytoplankton biomass and productivity in the Pacific Northwest during the warm ocean conditions of 2005, Geophys. Res. Lett., 33, L22S06, doi:10.1029/2006GL026772.

Red Waters of Myrionecta rubra are Biogeochemical Hotspots for the Columbia River Estuary with Impacts on Primary/Secondary Productions and Nutrient Cycles

The localized impact of blooms of the mixotrophic ciliate Myrionecta rubra in the Columbia River estuary during 2007–2010 was evaluated with biogeochemical, light microscopy, physiological, and molecular data. M. rubra affected surrounding estuarine nutrient cycles, as indicated by high and low concentrations of organic nutrients and inorganic nitrogen, respectively, associated with red waters. M. rubra blooms also altered the energy transfer pattern in patches of the estuarine water that contain the ciliate by creating areas characterized by high primary production and elevated levels of fresh autochthonous particulate organic matter, therefore shifting the trophic status in emergent red water areas of the estuary from net heterotrophy towards autotrophy. The pelagic estuarine bacterial community structure was unaffected by M. rubra abundance, but red waters of the ciliate do offer a possible link between autotrophic and heterotrophic processes since they were associated with elevated dissolved organic matter and showed a tendency for enhanced microbial secondary production. Taken together, these findings suggest that M. rubra red waters are biogeochemical hotspots of the Columbia River estuary.

Seasonal changes in bacterial and archaeal gene expression patterns across salinity gradients in the Columbia River coastal margin

Through their metabolic activities, microbial populations mediate the impact of high gradient regions on ecological function and productivity of the highly dynamic Columbia River coastal margin (CRCM). A 2226-probe oligonucleotide DNA microarray was developed to investigate expression patterns for microbial genes involved in nitrogen and carbon metabolism in the CRCM. Initial experiments with the environmental microarrays were directed toward validation of the platform and yielded high reproducibility in multiple tests. Bioinformatic and experimental validation also indicated that >85% of the microarray probes were specific for their corresponding target genes and for a few homologs within the same microbial family. The validated probe set was used to query gene expression responses by microbial assemblages to environmental variability. Sixty-four samples from the river, estuary, plume, and adjacent ocean were collected in different seasons and analyzed to correlate the measured variability in chemical, physical and biological water parameters to differences in global gene expression profiles. The method produced robust seasonal profiles corresponding to pre-freshet spring (April) and late summer (August). Overall relative gene expression was high in both seasons and was consistent with high microbial abundance measured by total RNA, heterotrophic bacterial production, and chlorophyll a. Both seasonal patterns involved large numbers of genes that were highly expressed relative to background, yet each produced very different gene expression profiles. April patterns revealed high differential gene expression in the coastal margin samples (estuary, plume and adjacent ocean) relative to freshwater, while little differential gene expression was observed along the river-to-ocean transition in August. Microbial gene expression profiles appeared to relate, in part, to seasonal differences in nutrient availability and potential resource competition. Furthermore, our results suggest that highly-active particle-attached microbiota in the Columbia River water column may perform dissimilatory nitrate reduction (both dentrification and DNRA) within anoxic particle microniches.

Infrastructure for collaborative science and societal applications in the Columbia River estuary

To meet societal needs, modern estuarine science needs to be interdisciplinary and collaborative, combine discovery with hypotheses testing, and be responsive to issues facing both regional and global stakeholders. Such an approach is best conducted with the benefit of data-rich environments, where information from sensors and models is openly accessible within convenient timeframes. Here, we introduce the operational infrastructure of one such data-rich environment, a collaboratory created to support (a) interdisciplinary research in the Columbia River estuary by the multi-institutional team of investigators of the Science and Technology Center for Coastal Margin Observation & Prediction and (b) the integration of scientific knowledge into regional decision making. Core components of the operational infrastructure are an observation network, a modeling system and a cyber-infrastructure, each of which is described. The observation network is anchored on an extensive array of long-term stations, many of them interdisciplinary, and is complemented by on-demand deployment of temporary stations and mobile platforms, often in coordinated field campaigns. The modeling system is based on finiteelement unstructured-grid codes and includes operational and process-oriented simulations of circulation, sediments and ecosystem processes. The flow of information is managed through a dedicated cyber-infrastructure, conversant with regional and national observing systems.

Discovery of a Katablepharis sp. in the Columbia River estuary that is abundant during the spring and bears a unique large ribosomal subunit sequence element.

Heterotrophic protists play significant roles in pelagic food webs as bacterivorous and herbivorous consumers. However, heterotrophic protists—unlike autotrophic ones—are often difficult to track since they tend to lack features such as photosynthetic pigments that allow for remote sensing or for bulk characterization. Difficulty in the identification of heterotrophic protists has often resulted in lumping them into broad groups, but there is a strong need to develop methods that increase the spatial and temporal resolution of observations applied to particular organisms in order to discover the drivers of population structure and ecological function. In surveys of small subunit rRNA, gene (SSU) sequences of microbial eukaryotes from the Columbia River to the Pacific Ocean, the heterotrophic flagellate Katablepharis sp. were found to dominate protist assemblages (including autotrophic and heterotrophic fractions) in the spring, prior to the freshet. We discovered a 332 base pair unique sequence element (USE) insertion in the large subunit rRNA gene (28S) that is not present in other katablepharids or in any other eukaryote. Using this USE, we were able to detect Katablepharis within mixed assemblages in river, estuarine, and oceanic samples and determine spatial and temporal patterns in absolute abundance through quantitative PCR and fluorescence in situ hybridization. Given their high abundance and repeatable temporal patterns of occurrence, we hypothesize that the Columbia River Estuary Katablepharis (Katablepharis CRE) plays an important role in estuarine biogeochemical and ecosystem function.

Observations of a Diatom Chytrid Parasite in the Lower Columbia River

Abstract Often overlooked, zoosporic fungal parasites of phytoplankton (‘chytrids’) are present in aquatic systems worldwide. Although extensive studies in lakes give insight into potential impacts of chytrid epidemics on phytoplankton blooms and organic matter cycling, the ecological significance of chytrid infections of phytoplankton is still poorly understood in lotic systems. Here, we report the first observations of chytrid parasites attached to multiple diatom species in the lower Columbia River. We isolated a chytrid parasite of the dominant spring bloom diatom, Asterionella formosa, and sequenced DNA from several regions within the ribosomal RNA gene. We also investigated the specificity of the A. formosa chytrid to host and non-host diatoms with isolated cultures and found no cross infection at the species level. In the Columbia River, alterations in the hydrograph following the installation of hydroelectric dams may have opened a niche for chytrid parasites. Greater retention times may allow diatoms to bloom and provide a prolonged interaction period whereby chytrid parasites are able to infect hosts. Future research is needed to assess the seasonality and severity of chytrid infections on diatoms in the lower Columbia River and to evaluate the potential role of zoosporic fungi in influencing the food web structure and biogeochemical cycling in river systems.

Enumeration of Parasitic Chytrid Zoospores in the Columbia River via Quantitative PCR

ABSTRACT Through lethal infection, fungal parasites of phytoplankton (“chytrids”) repackage organic material from the large, effectively inedible, colonial diatoms they infect into much smaller zoospores, which are easier for zooplankton to consume. However, their small size and lack of distinguishing morphological features render it difficult to distinguish zoospores from other small flagellates in mixed assemblages in the natural environment. In this study, we developed and tested a method to quantify chytrid zoospores in field studies using quantitative PCR (qPCR) targeting the internal transcribed spacer 2 (ITS2) region within the rRNA gene cluster. To achieve accurate quantification, the assay was designed to be highly specific for a parasite (Rhizophydium planktonicum) of the diatom Asterionella formosa; however, the approach is applicable to additional host-parasite systems. Parasitic zoospores were detected and quantified in the freshwater reaches of the lower Columbia River, as well as in the salt-influenced estuary and river plume. The coincidence between zoospore abundances and a prevalence of small zooplankton during blooms of large, colonial diatoms in the spring suggests that Columbia River zooplankton may be poised to benefit nutritionally from chytrid zoospores, thus providing a mechanism to retain organic carbon within the system and reduce losses to downstream export. We estimate that ∼15% of the carbon biomass tied up in blooms of the dominant diatom species is transformed into zoospores through the parasitic shunt during spring. IMPORTANCE The small size of the parasitic fungi that infect phytoplankton makes it difficult to identify and quantify them in natural systems. We developed and tested a method to quantify these organisms (chytrid zoospores) using a molecular technique that targets the internal transcribed spacer region within the rRNA gene cluster. Using this method, we quantified the abundance of the motile stage of a specific parasite in the freshwater and saltwater-influenced regions of the Columbia River in the U.S. Pacific Northwest. Parasitic chytrid zoospores were found to be present throughout the year and at higher abundances during the spring, when phytoplankton blooms occur. The presence of these organisms indicates not only that they may be responsible for the death of host phytoplankton cells but that they may also provide a readily available food source to small consumers (zooplankton) in the food web of the Columbia River.

Development of an economical, autonomous pHstat system for culturing phytoplankton under steady state or dynamic conditions

Laboratory investigations of physiological processes in phytoplankton require precise control of experimental conditions. Chemostats customized to control and maintain stable pH levels (pHstats) are ideally suited for investigations of the effects of pH on phytoplankton physiology, for example in context of ocean acidification. Here we designed and constructed a simple, flexible pHstat system and demonstrated its operational capabilities under laboratory culture conditions. In particular, the system is useful for simulating natural cyclic pH variability within aquatic ecosystems, such as diel fluctuations that result from metabolic activity or tidal mixing in estuaries. The pHstat system operates in two modes: (1) static/set point pH, which maintains pH at a constant level, or (2) dynamic pH, which generates regular, sinusoidal pH fluctuations by systematically varying pH according to user-defined parameters. The pHstat is self-regulating through the use of interchangeable electronically controlled reagent or gas-mediated pH-modification manifolds, both of which feature flow regulation by solenoid valves. Although effective pH control was achieved using both liquid reagent additions and gas-mediated methods, the liquid manifold exhibited tighter control (±0.03pH units) of the desired pH than the gas manifold (±0.10pH units). The precise control provided by this pHstat system, as well as its operational flexibility will facilitate studies that examine responses by marine microbiota to fluctuations in pH in aquatic ecosystems.