Abstract

Traditionally marine food webs have been studied on the basis of size, assuming that larger organisms eat smaller ones, and that they eat everything in a given size range. This simplification has not stood the test of time, especially for the smallest microbial plankton. Not only do smaller organisms sometimes eat larger ones, there is growing evidence that chemical signaling and defenses are acting at these small scales. This project addresses the central premise that the oxidative stress response is an emergent property of phototrophic cellular systems, with implications for nearly every aspect of a phytoplankton cell's life in the upper ocean. Oxidative stress (OS), arising from numerous environmental stressors, can damage cells and lead to release of compounds suspected to be potent signals regulating protist behavior. Through chemical signaling, OS is hypothesized to govern relationships among environmental variability, phytoplankton condition, and protist predation. The study of these integrated processes has three overarching objectives: 1) Use light stress to create oxidatively stressed phytoplankton in the laboratory, and characterize the response using an array of fluorescent probes, biochemical measurements, and physiological assays. In addition, production and release of potential signal molecules will be quantified. 2) Examine protist predation and behavioral responses to oxidatively stressed phytoplankton and associated chemical signals. Responses will be investigated by means of manipulation experiments and resulting signal chemistry. 3) Investigate the prevalence of OS, its environmental correlates, and the microzooplankton predation response in a local embayment. Broader impacts will build directly on these experimental and observational activities. The project will support several graduate students and involve undergraduates through two programs at Shannon Point Marine Center, including one targeting under-represented minorities. Funds will support a significant collection of heterotrophic protists isolates. The fluorometer will be used in developing new units for at least 3 different Western Washington University marine-related courses. In addition, graduate students will use protist images as the basis for outreach activities aimed at the general public and K-12 students.

This research will help to elucidate some of the many ways in which the OS response can affect phytoplankton fitness, characterizing the position of key coastal species along an OS response spectrum. Findings will also inform the relatively new and exciting field of chemical signaling in planktonic communities. Finally, this study will help elucidate the links between environmental stress, phytoplankton response, and predation in planktonic ecosystems. These links relate to central issues in biological oceanography, including the predator-prey interactions that influence bloom demise, and the mechanisms by which protists feed selectively and thereby structure prey communities. The research is a cross-cutting endeavor that unites subjects usually studied in isolation through a novel conceptual framework. Outcomes have the potential to generate broadly applicable insights into the ecological and evolutionary regulation of this key trophic link in planktonic food webs.

Project Outcomes: This project investigated the interactions between environmental stress, chemical signaling, and predator-prey interactions within the communities of single-celled organisms that make up most of the ocean's plankton. Experimental studies showed that even closely related microalgal species have evolved contrasting responses to high light stress, showing that the light stress response is an important aspect of the ecological niche or habitat that can by occupied by a given species. Exposing microalgae or their protist consumers to high light stress can change how food webs function. Light stress in one algal species (Emiliania huxleyi,the most common and abundant coccolithophore) reduced predation on those cells, while other types of microalgae did not show this effect. Similarly, light stressing protist grazers (heterotrophic dinoflagellates) was detrimental to one species, but not to another. In summary, light stress can shape planktonic communities through species differences in tolerance, and by modulating predation rates. Some microalgal groups produce mineral shells or armor-like plates whose thickness depends on environmental conditions. These shells and plates have been hypothesized to act as defenses against predators. We found that thicker silica shells (frustules) on diatoms did not reduce predation by dinoflagellates, but did slow digestion. Similarly, the calcium carbonate plates on coccolithophores did not consistently reduce dinoflagellate predation rates, but might have affected digestion. In summary, mineral structures on microalgal cells did not deter feeding by predators, but could decrease their ability to digest the cells that they eat, reducing community-level predation and increasing the likelihood that these microalgal species will form blooms. We undertook extensive methods development and experimental study of hydrogen peroxide (H2O2) production by microalgae. H2O2 is known to be a signaling molecule in other biological systems, and can be produced in response to stress. All microalgal species tested, including eukaryotes and cyanobacteria, could produce H2O2 and most could also degrade this reactive oxygen species (ROS). In some cases, bacteria associated with the microalgal cultures could also produce H2O2, although little bacterial degradation was seen. Although microalgal production of ROS is often reported to be associated with high light stress in the scientific literature, we found that almost no previous studies controlled for abiotic photochemical ROS production. In our experiments, all high light-associated H2O2 production was due to abiotic photochemistry, with none attributable to the microalgal cells themselves. We found evidence that both H2O2 and another, sulfur-containing algal signaling molecule (dimethylsulfoniopropionate, or DMSP) are produced at higher per-cell levels when concentrations of algal cells are low. This suggests a potential quorum sensing strategy, whereby microalgal cells adjust output of signal molecules as a means of assessing community cell density and/or of maintaining a particular environmental concentration of the signal molecule. Development of the H2O2 measurement assay allowed us to investigate H2O2 production by a toxic microalga, the fish-killing raphidophyte Heterosigma akashiwo. It has long been hypothesized that H2O2 is the agent of toxicity for this species, although the data remain inconclusive. We found that H. akashiwo does produce moderate amounts of H2O2, with the per-cell production rates dependent on strain (i.e. isolate), and on salinity (with higher production rates at higher salinities). H2O2 production rates increase when H. akashiwo is in the presence of competitor microalgal species or predators. Adding catalase, an enzyme that degrades H2O2, to experimental systems reduced the toxicity of H. akashiwo to two different ciliate predators, demonstrating conclusively that H2O2 is, at least in part, responsible for the toxic effects of this harmful bloom-forming (HAB) species. In summary, H2O2 appears to act as a signal molecule for a wide range of microalgal species, which have the ability to dynamically regulate its concentration through variations in production and decomposition rate. Production of relatively high H2O2 concentrations makes Heterosigma akashiwo toxic to predators; this HAB species can dynamically regulate H2O2 production in response to environmental conditions and to the presence of competitor and predator species. Broader impacts include training of graduate and undergraduate students (3 Masters theses were supported) and maintenance of a protist culture collection that was shared with researchers nationally and internationally. One of the main dinoflagellate predator species used in the above studies has recently been sequenced by our collaborators at the University of British Columbia. With its phylogenetic position now established, a redescription is in process that will place the organism into a newly described genus, Deanodinium. Seven data sets describing the chemical composition, environmental stress responses, and chemical signal production of various microalgae were added to a public data repository (BCO-DMO). Last Modified: 12/14/2020 Submitted by: Suzanne L Strom