Christopher A. Scholin

Mortality of sea lions along the central California coast linked to a toxic diatom bloom.

Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.

IDENTIFICATION AND ENUMERATION OF CULTURED AND WILD PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) USING SPECIES‐SPECIFIC LSU rRNA‐TARGETED FLUORESCENT PROBES AND FILTER‐BASED WHOLE CELL HYBRIDIZATION

Efforts to understand the ecologic and environmental parameters that govern harmful algal blooms (HABs) require rapid and specific identification of causative species. Traditional methods of species identification using light and electron microscopy are useful in this regard but are also time consuming, making routine analysis of a large number of samples difficult. Techniques that speed and ease the detection of HAB species as they occur in natural populations are therefore desirable. In this paper, we continue efforts to develop species‐specific large subunit ribosomal RNA (LSU rRNA)‐targeted fluorescent DNA probes for a variety of Pseudo‐nitzschia H. Peragallo species, a group of marine pennate diatoms that includes representatives linked to production of domoic acid and amnesic shellfish poisoning (ASP). A custom filter tube and filtration manifold that has utility for both whole cell (in situ) hybridization as well as preparing samples for scanning electron microscopy (SEM) is described. Filter‐based whole cell hybridization was used to identify a variety of newly isolated Pseudo‐nitzschia clones, and probe results were confirmed using SEM. Some isolates of P. pungens (Grunow) Hasle exhibited variable (intraclonal) reactivity toward the P. pungens‐specific probe. Three isolates of P. subpacifica (Hasle) Hasle were found to cross‐react with probes designed for P. fraudulenta (Cleve) Hasle and P. heimii Manguin. Four isolates did not react with any species‐specific probes; this group comprised three distinct morphotypes whose fine‐scale morphologic features did not agree with published descriptions of Pseudo‐nitzschia species. Evaluation of the filter method using cultured cells added to natural (whole water) samples indicated quantitative recovery of target species. Confirming results of probe assays using SEM was difficult when the target species was less than 104 cells·L−1 in the presence of greater than 106 cells·L−1 of other nontarget diatom species. A variety of Pseudo‐nitzschia, including P. australis Frenguelli, P. fraudulenta, P. heimii, P. pseudodelicatissima (Hasle) Hasle, P. pungens, P. multiseries (Hasle) Hasle, and Nitzschia americana Hasle, were identified using whole cell hybridization in a variety of field samples containing mixed assemblages of plankton, and these results were confirmed using SEM. The filter tube method of applying probes was used onboard ship for near real‐time identification and enumeration of a variety of Pseudo‐nitzschia species.

Identification of cultured Pseudo-nitzschia (Bacillariophyceae) using species-specific LSU rRNA-targeted fluorescent probes

Some, but not all, marine pennate diatoms of the genus Pseudo‐nitzschia H. Peragallo are associated with the production of domoic acid, a naturally occurring amino acid responsible for amnesic shellfish poisoning. Distinguishing between potentially toxic and nontoxic representatives of this genus is time‐consuming and difficult because it demands scanning electron microscopy of cleaned frustules. The objective of this work is to speed and ease identification of these organisms by using whole‐cell (in situ) hybridization and species‐specific large‐subunit ribosomal RNA (LSU rRNA)‐targeted oligonucleotide probes. Toward that end, cultures of P. australis Frenguelli, P. pungens (Grunow) Hasle, P. multiseries (Hasle) Hasle, P. fraudulenta (P. T. Cleve) Heiden, P. heimii Manguin, P. delicatissima (P. T. Cleve) Heiden, P. pseudo‐delicatissima (Hasle) Hasle, and P. americana (Hasle) Fryxell were screened with a suite of 15 putative species‐specific probes. Of those, a subset of eight probes was found that distinguished each species tested. In addition, Pseudo‐nitzschia chloroplasts were labeled with a probe directed against a eubacterial‐conserved sequence. Identification of new cultures based on their reactivity toward a set of probes agreed with species designations as defined by morphological criteria. Whole‐cell hybridization is a rapid, simple, and cost‐effective technique for discriminating among cultured Pseudo‐nitzschia species.

DNA PROBES AND A RECEPTOR‐BINDING ASSAY FOR DETECTION OF PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) SPECIES AND DOMOIC ACID ACTIVITY IN CULTURED AND NATURAL SAMPLES

Large‐subunit ribosomal RNA‐targeted probes for Pseudo‐nitzschia australis Frenguelli, P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, and P. pungens (Grunow) Hasle were applied to cultured and natural samples using whole‐cell and sandwich hybridization. Testing of the latter method is emphasized here, and technique refinements that took place during 1996–1997 are documented. Application of the sandwich hybridization test showed that the signal intensity obtained for a given number of target cells remained constant as batch cultures of these organisms progressed from active through stationary growth phases. This suggests that cellular rRNA content for each target species remained relatively stable despite changes in growth state. Application of whole‐cell and sandwich hybridization assays to natural samples showed that both methods could be used to detect wild P. australis, P. pseudodelicatissima, and to a lesser degree P. multiseries, but detection of P. pungens was prone to error. A receptor‐binding assay for domoic acid (DA) enabled detection of this toxin activity associated with a particulate fraction of the plankton and provided a context in which to view results of the rRNA probe tests. In one case, the probe for P. australis cross‐reacted with P. cf. delicatissima. The sample that contained the latter species also contained a low amount of DA activity. Under certain field conditions, results of whole‐cell and sandwich hybridization tests disagreed. Detailed analysis of selected field samples illustrates how such situations arose. Collectively, the rRNA probe and toxin analyses suggest that manifestation of DA in the environment is possible in the absence of readily recognizable intact cells.

Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages

Up and down go the cyanobacteria Plankton move together in strikingly coordinated daily patterns, sinking at night to avoid being eaten and rising to the surface in daylight to photosynthesize. Otteson et al. found similar activity patterns in even the smallest of planktonic organisms, such as photosynthetic bacteria (see the Perspective by Armbrust). Because its hard to take regular samples in the open ocean, the authors built a robotic sampler and set it adrift for several days in the mid-Pacific. The captured bacteria showed immediate responses to changes in light, temperature, and salinity in ways that could affect the oceans carbon and nitrogen cycles. Science, this issue p. 207; see also p. 134 Multispecies’ daily waves of gene transcription are observed in open ocean microplankton. [Also see Perspective by Armbrust] Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.

Pattern and synchrony of gene expression among sympatric marine microbial populations

Significance Microbial communities regulate the cycling of energy and matter in the environment, yet how they respond to environmental change is not well-known. We describe here a day in the life of wild planktonic microbial species using robotic sampling coupled with genome-wide gene expression analysis. Our results showed that closely related populations, as well as very different bacterial and archaeal species, displayed remarkably similar time-variable synchronous patterns of gene expression over 2 d. Our results suggest that specific environmental cues may elicit cross-species coordination of gene expression among diverse microbial groups, potentially enabling multispecies coupling of metabolic activity. Planktonic marine microbes live in dynamic habitats that demand rapid sensing and response to periodic as well as stochastic environmental change. The kinetics, regularity, and specificity of microbial responses in situ, however, are not well-described. We report here simultaneous multitaxon genome-wide transcriptome profiling in a naturally occurring picoplankton community. An in situ robotic sampler using a Lagrangian sampling strategy enabled continuous tracking and repeated sampling of coherent microbial populations over 2 d. Subsequent RNA sequencing analyses yielded genome-wide transcriptome profiles of eukaryotic (Ostreococcus) and bacterial (Synechococcus) photosynthetic picoplankton as well as proteorhodopsin-containing heterotrophs, including Pelagibacter, SAR86-cluster Gammaproteobacteria, and marine Euryarchaea. The photosynthetic picoplankton exhibited strong diel rhythms over thousands of gene transcripts that were remarkably consistent with diel cycling observed in laboratory pure cultures. In contrast, the heterotrophs did not cycle diurnally. Instead, heterotrophic picoplankton populations exhibited cross-species synchronous, tightly regulated, temporally variable patterns of gene expression for many genes, particularly those genes associated with growth and nutrient acquisition. This multitaxon, population-wide gene regulation seemed to reflect sporadic, short-term, reversible responses to high-frequency environmental variability. Although the timing of the environmental responses among different heterotrophic species seemed synchronous, the specific metabolic genes that were expressed varied from taxon to taxon. In aggregate, these results provide insights into the kinetics, diversity, and functional patterns of microbial community response to environmental change. Our results also suggest a means by which complex multispecies metabolic processes could be coordinated, facilitating the regulation of matter and energy processing in a dynamically changing environment.

PSEUDO-NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN LOUISIANA COASTAL WATERS: MOLECULAR PROBE FIELD TRIALS, GENETIC VARIABILITY, AND DOMOIC ACID ANALYSES

An 18‐month field survey of the Pseudo‐nitzschia population present in Louisiana coastal waters was conducted comparing species abundance estimates by novel fluorescent molecular probes (16S large subunit rDNA oligonucleotide sequences) with traditional electron and differential‐interference light microscopy. While the probe and microscopic analyses agreed on the presence or absence of four common Pseudo‐nitzschia species (P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, and P. pungens (Grunow) Hasle in 66% of the samples analyzed, the probes gave conflicting results with the microscopic methods in the remaining 34% of the samples. The majority of the discrepancies appear to be because of genetic variation within the Pseudo‐nitzschia population, especially in P. pseudodelicatissima, indicating that the Monterey Bay Pseudo‐nitzschia spp. may not be appropriate reference strains for distinguishing Louisiana Pseudo‐nitzschia spp. Additionally, P. pseudodelicatissima has been associated with domoic acid (DA) activity in three field samples, at levels up to 22 times higher than the highest value given inother published reports of DA production by this species. The contemporaneous existence of multiple strains of P. pseudodelicatissima (toxic and non‐toxic) presents new challenges to the study of the ecophysiology and population dynamics of this bloom‐forming species.

Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.

On detection of pseudo‐nitzschia (bacillariophyceae) species using whole cell hybridization: sample fixation and stability

Some species within the genus Pseudo‐nitzschia H. Peragallo are associated with production of domoic acid, the agent responsible for amnesic shellfish poisoning (ASP). Identification and enumeration of particular Pseudo‐nitzschia in natural populations is often difficult and time consuming because of the need for detailed morphological observations, which often require scanning or transmission electron microscopy. In earlier publications we described the development of large subunit ribosomal RNA (LSU rRNA)‐targeted fluorescent DNA probes for discriminating among a variety of Pseudo‐nitzschia species collected from Monterey Bay, California. Probes are applied using whole cell hybridization and a custom filtration manifold, enabling rapid identification and quantification of target species in cultured as well as field samples. In this work we compared a variety of preservation techniques and assessed the stability of stored samples with respect to their reactivity towards the probes. Of the preservatives tested, a saline ethanol‐based treatment gave the best results in terms of probes yielding a bright and uniform cell label. Culture samples treated with this fixative continued to react well with the probes for at least 6 weeks post‐fixation whether stored in the preservative or dried post‐preservation, with samples being kept at either room temperature or −20° C. Likewise, field samples containing a variety of diatoms and dinoflagellate species stored in the saline ethanol solution at room temperature were also stable for at least 4–6 weeks, reacting brilliantly towards a positive control probe. After prolonged storage, however, cell reactivity towards the probes diminished dramatically. Post‐hybridization, samples stored at 4° C were found to retain their fluorescence for at least 1 week. These results indicate a wider window of opportunity for Pseudo‐nitzschia analysis using whole cell hybridization than previously reported. Sample collection, preservation, and probing protocols optimized for Pseudo‐nitzschia are also applicable to a wide range of phytoplankton species. The time required to execute the whole cell hybridization protocol was reduced by premixing probe with hybridization buffer. The premixed probe solutions as well as fixative and wash solutions are all stable at room temperature for at least 6 weeks. Application of two different species‐specific probes, each labeled with a different fluorochrome, allowed detection of two species on a single filter. The latter could be adopted in the future to increase the rate of sample processing and decrease the cost of sample analysis.

Microbial community transcriptional networks are conserved in three domains at ocean basin scales

Significance Microbes drive biogeochemical cycles across the globe, collectively playing a central role in shaping the biosphere. Despite their immense importance, the in situ activities of communities of microbes, in particular uncultivated lineages of “microbial dark matter,” remain poorly elucidated. In this study, we report that common temporal and ecological dynamics underpin disparate marine microbial communities, providing the first evidence that trans-Pacific diurnal transcriptional patterns in these communities may regulate ecological and biogeochemical processes across the ocean. In total, our findings indicate a remarkable regularity in the timing of community-wide activity in the ocean, and suggest that global patterns of a variety of biogeochemical transformations may be temporally predictable and governed by structured ecological determinants. Planktonic microbial communities in the ocean are typically dominated by several cosmopolitan clades of Bacteria, Archaea, and Eukarya characterized by their ribosomal RNA gene phylogenies and genomic features. Although the environments these communities inhabit range from coastal to open ocean waters, how the biological dynamics vary between such disparate habitats is not well known. To gain insight into the differential activities of microbial populations inhabiting different oceanic provinces we compared the daily metatranscriptome profiles of related microbial populations inhabiting surface waters of both a coastal California upwelling region (CC) as well as the oligotrophic North Pacific Subtropical Gyre (NPSG). Transcriptional networks revealed that the dominant photoautotrophic microbes in each environment (Ostreococcus in CC, Prochlorococcus in NPSG) were central determinants of overall community transcriptome dynamics. Furthermore, heterotrophic bacterial clades common to both ecosystems (SAR11, SAR116, SAR86, SAR406, and Roseobacter) displayed conserved, genome-wide inter- and intrataxon transcriptional patterns and diel cycles. Populations of SAR11 and SAR86 clades in particular exhibited tightly coordinated transcriptional patterns in both coastal and pelagic ecosystems, suggesting that specific biological interactions between these groups are widespread in nature. Our results identify common diurnally oscillating behaviors among diverse planktonic microbial species regardless of habitat, suggesting that highly conserved temporally phased biotic interactions are ubiquitous among planktonic microbial communities worldwide.