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Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

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Alveolate (ciliates and dinoflagellates) grazers are integral components of the marine food web and must therefore be able to sense a range of mechanical and chemical signals produced by prey and predators, integrating them via signal transduction mechanisms to respond with effective prey capture and predator evasion behaviors. However, the sensory biology of alveolate grazers is poorly understood. Using novel techniques that combine electrophysiological measurements and high-speed videomicroscopy, we investigated the sensory biology of Favella sp., a model alveolate grazer, in the context of its trophic ecology. Favella sp. produced frequent rhythmic depolarizations (∼500 ms long) that caused backward swimming and are responsible for endogenous swimming patterns relevant to foraging. Contact of both prey cells and non-prey polystyrene microspheres at the cilia produced immediate mechanostimulated depolarizations (∼500 ms long) that caused backward swimming, and likely underlie aggregative swimming patterns of Favella sp. in response to patches of prey. Contact of particles at the peristomal cavity that were not suitable for ingestion resulted in depolarizations after a lag of ∼600 ms, allowing time for particles to be processed before rejection. Ingestion of preferred prey particles was accompanied by transient hyperpolarizations (∼1 s) that likely regulate this step of the feeding process. Predation attempts by the copepod Acartia tonsa elicited fast (∼20 ms) animal-like action potentials accompanied by rapid contraction of the cell to avoid predation. We have shown that the sensory mechanisms of Favella sp. are finely tuned to the type, location, and intensity of stimuli from prey and predators.

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We studied the protist grazers of Boiling Springs Lake (BSL), an acid geothermal feature in Lassen Volcanic National Park, using a combination of culture and genetic approaches. The major predator in BSL is a vahlkampfiid ameba closely related (95% 18S+ITS rRNA identity) to Tetramitus thermacidophilus, a heterolobose ameboflagellate recently isolated from volcanic geothermal acidic sites in Europe and Russia, as well as an uncultured heterolobosean from the nearby Iron Mountain acid mine drainage site. Tetramitus thermacidophilus strain BSL is capable of surviving the physical extremes of BSL, with optimal growth at 38-50 °C and pH 2-5. This bacterivore also ingested conidiospores of the ascomycete Phialophora sp., but ultrastructural observations reveal the latter may not be readily digested, and conidia were not separable from the ameoboflagellate culture, suggesting a possible symbiosis. DGGE fingerprint transects studies showed the organism is restricted to near-lake environs, and we detected an average of ~500 viable cysts/cm(3) sediment on the shoreline. Other grazing protists were isolated from lakeshore environments, including the lobose amebae Acanthamoeba sp. and Hartmannella sp., and the kinetoplastid flagellate Bodo sp., but none could tolerate both low pH and high temperature. These appear to be restricted to cooler near lake geothermal features, which also contain other potential grazer morphotypes observed but not successfully cultured, including ciliates, euglenids, testate amebae, and possible cercozoans. We compare the food web of BSL with other acidic or geothermal sites, and discuss the impact of protists in this unique environment.

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Planktonic alveolates (ciliates and dinoflagellates), key trophic links in marine planktonic communities, exhibit complex behaviors that are underappreciated by microbiologists and ecologists. Furthermore, the physiological mechanisms underlying these behaviors are still poorly understood except in a few freshwater model ciliates, which are significantly different in cell structure and behavior than marine planktonic species. Here, we argue for an interdisciplinary research approach to connect physiological mechanisms with population-level outcomes of behaviors. Presenting the tintinnid ciliate Favella as a model alveolate, we review its population ecology, behavior, and cellular/molecular biology in the context of sensory biology and synthesize past research and current findings to construct a conceptual model describing the sensory biology of Favella. We discuss how emerging genomic information and new technical methods for integrating research across different levels of biological organization are paving the way for rapid advance. These research approaches will yield a deeper understanding of the role that planktonic alveolates may play in biogeochemical cycles, and how they may respond to future ocean conditions.

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Boiling Springs Lake (BSL) is an oligotrophic, acidic geothermal feature where even very low levels of microbial heterotrophic production still exceed autotrophy. To test whether allochthonous leaf litter (LL) inputs fuel this excess, we quantified leaf litterfall, leaching and decomposition kinetics, and measured the impact of organic amendments on production, germination and cell growth, using pyrosequencing to track changes in microbial community composition. Coniferous leaves in BSL exhibited high mass loss rates during leaching and decomposition, likely due to a combination of chemical hydrolysis and contributions of both introduced and endemic microbes. We measured very low in situ (3)H-thymidine incorporation over hours by the dominant chemolithotroph Acidimicrobium (13-65 µg C L(-1) day(-1)), which was inhibited by simple C sources (acetate, glucose). Longer term incubations with additions of 0.01-0.02% complex C/N sources induced germination of the Firmicute Alicyclobacillus within 1-2 days, as well as growth of Acetobacteraceae after 3-4 days. LL additions yielded the opposite successional patterns of these r-selected heterotrophs, boosting production to 30-150 µg C L(-1) day(-1). Growth and germination studies suggest both prokaryotes and fungi likely consume allochthonous organics, and might be novel sources of lignocellulose-degrading enzymes. A model of BSL's C budget supports the hypothesis that allochthonous inputs fuel seasonal microbial heterotrophy, but that dissolved organic C sources greatly exceed direct LL inputs.

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We describe the construction of temperature-light gradient incubator with a novel material: a thermally-conductive graphite foam that is lightweight, chemically resistant, economically competitive with metal, and much cheaper to fabricate. We combined this material with a variable-intensity LED light array to construct a low-cost light-temperature gradient incubator, and demonstrate its use for studies of microbial growth, enrichment, and isolation.

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Systemic acquired resistance (SAR) is an inducible systemic plant defense against a broad spectrum of plant pathogens, with the potential to secrete antimicrobial compounds into the soil. However, its impact on rhizosphere bacteria is not known. In this study, we examined fingerprints of bacterial communities in the rhizosphere of the model plant Arabidopsis thaliana to determine the effect of SAR on bacterial community structure and diversity. We compared Arabidopsis mutants that are constitutive and non-inducible for SAR and verified SAR activation by measuring pathogenesis-related protein activity via a beta-glucoronidase (GUS) reporter construct driven by the beta-1-3 glucanase promoter. We used terminal restriction fragment length polymorphism (T-RFLP) analysis of MspI- and HaeIII-digested 16S rDNA to estimate bacterial rhizosphere community diversity, with Lactobacillus sp. added as internal controls. T-RFLP analysis showed a clear rhizosphere effect on community structure, and diversity analysis of both rhizosphere and bulk soil operational taxonomic units (as defined by terminal restriction fragments) using richness, Shannon-Weiner, and Simpson's diversity indices and evenness confirmed that the presence of Arabidopsis roots significantly altered bacterial communities. This effect of altered soil microbial community structure by plants was also seen upon multivariate cluster analysis of the terminal restriction fragments. We also found visible differences in the rhizosphere community fingerprints of different Arabidopsis SAR mutants; however, there was no clear decrease of rhizosphere diversity because of constitutive SAR expression. Our study suggests that SAR can alter rhizosphere bacterial communities, opening the door to further understanding and application of inducible plant defense as a driving force in structuring soil bacterial assemblages.

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We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devil's Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85 degrees C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68 degrees C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%-99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68 degrees C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassen's acidic hydrothermal features may harbor novel protists.

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