Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection.

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 µm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Effects of nutrient enrichment on surface microbial community gene expression in the oligotrophic North Pacific Subtropical Gyre.

Marine microbial communities are critical for biogeochemical cycles and the productivity of ocean ecosystems. Primary productivity in the surface ocean is constrained by nutrients which are supplied, in part, by mixing with deeper water. Little is known about the time scales, frequency, or impact of mixing on microbial communities. We combined in situ sampling using the Environmental Sample Processor and a small-scale mixing experiment with lower euphotic zone water to determine how individual populations respond to mixing. Transcriptional responses were measured using the MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories) microarray, which targets all three domains of life and viruses. The experiment showed that mixing substantially affects photosynthetic taxa as expected, but surprisingly also showed that populations respond differently to unfiltered deep water which contains particles (organisms and detritus) compared to filtered deep water that only contains nutrients and viruses, pointing to the impact of biological interactions associated with these events. Comparison between experimental and in situ population transcription patterns indicated that manipulated populations can serve as analogs for natural populations, and that natural populations may be frequently or continuously responding to nutrients from deeper waters. Finally, this study also shows that the microarray approach, which is complementary to metatranscriptomic sequencing, is useful for determining the physiological status of in situ microbial communities.

Simultaneous monitoring of faecal indicators and harmful algae using an in-situ autonomous sensor.

UNLABELLED: Faecal indicator bacteria (FIB) and harmful algal blooms (HABs) threaten the health and the economy of coastal communities worldwide. Emerging automated sampling technologies combined with molecular analytical techniques could enable rapid detection of micro-organisms in-situ, thereby improving resource management and public health decision-making. We evaluated this concept using a robotic device, the Environmental Sample Processor (ESP). The ESP automates in-situ sample collection, nucleic acid extraction and molecular analyses. Here, the ESP measured and reported concentrations of FIB (Enterococcus spp.), a microbial source-tracking marker (human-specific Bacteriodales) and a HAB species (Psuedo-nitzschia spp.) over a 45-day deployment on the Santa Cruz Municipal Wharf (Santa Cruz, CA, USA). Both FIB and HABs were enumerated from single in-situ collected water samples. The in-situ qPCR efficiencies ranged from 86% to 105%, while the limit of quantifications during the deployment was 10 copies reaction(-1) . No differences were observed in the concentrations of enterococci, the human-specific marker in Bacteroidales spp., and P. australis between in-situ collected sample and traditional hand sampling methods (P > 0·05). Analytical results were Internet-accessible within hours of sample collection, demonstrating the feasibility of same-day public notification of current water quality conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents the first report of in-situ qPCR enumeration of both faecal indicators and harmful algal species in coastal marine waters. We utilize a robotic device for in-situ quantification of enterococci, the human-specific marker in Bacteriodales and Pseudo-nitzschia spp. from the same water samples collected and processed in-situ. The results demonstrate that rapid, in-situ monitoring can be utilized to identify and quantify multiple health-relevant micro-organisms important in water quality monitoring and that this monitoring can be used to inform same-day notifications.

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.

Detection of stable pre-rRNA in toxigenic Pseudo-nitzschia species.

Nucleotide sequence analysis of ribosomal DNA (rDNA) spacer regions is useful for taxonomic comparisons of closely related microorganisms. These regions have been less useful for routine microbial identification and detection, partly because rRNA precursors (pre-rRNAs) in microbial cells are assumed to be too labile to be detectable by high-throughput probe hybridization methods. We characterized the sequence diversity and physiological stability of pre-rRNA in the toxigenic marine diatoms Pseudo-nitzschia australis, P. multiseries, and P. pungens. As with nucleotide sequences of the first internal transcribed spacer (ITS1) reported previously, sequences of ITS2 and the 5' external transcribed spacer (ETS1) exhibited considerable divergence among these species, including large insertions-deletions detectable by PCR-based spacer length analysis. In slot blot hybridization assays on RNA extracted from lysates of Pseudo-nitzschia cells, oligonucleotide probes directed to pre-rRNA spacers generated much stronger signals than did complementary probes directed to the coding strands of the rDNAs, indicating that the pre-rRNA-targeted probes detected multicopy transcripts. A group of probes directed to a discrete 90-base region within the ITS1 pre-rRNA gave no detectable signal, suggesting that this region is degraded early in the rRNA maturation pathway. Other pre-rRNA regions were always detectable and, in marked contrast to prokaryotic systems analyzed in this manner, were stable and abundant in both actively dividing and nondividing cells. Long, multilabeled RNA probes, which would exhibit considerable cross-reactivity if directed to mature rRNA sequences, detected species-specific pre-rRNA sequences from as few as 1,000 cells. Pre-rRNA is a potentially useful molecular target for detecting and identifying Pseudo-nitzschia species and possibly other unicellular eukaryotes as well.

Ribosomal DNA sequences discriminate among toxic and non-toxic Pseudonitzschia species.

Cultured isolates of Pseudonitzschia australis Frenguelli, P. delicatissima (Cleve) Heiden, P. americana (Hasle) Fryxell, P. pungens (Grunow) Hasle, and P. pungens f. multiseries (Hasle) Hasle from Monterey Bay, California, were compared on the basis of their large-subunit ribosomal RNA gene (LsrDNA). Pseudonitzschia australis, P. pungens f. multiseries, and P. delicatissima were previously shown to produce the neurotoxin domoic acid; the remaining isolates are considered non-toxic. For each isolate approximately 800 base pairs of LsrDNA, encompassing both evolutionarily conserved and evolutionarily variable regions of the molecule, were amplified using the polymerase chain reaction (PCR) and sequenced. Phylogenetic trees generated by parsimony analysis of aligned sequences afford a preliminary view of the organisms genetic relationships. Species defined by morphological criteria are also distinguishable by LsrDNA sequence. Organisms known or suspected to produce domoic acid cluster at different termini on the phylogenetic tree. Two genetically distinct strains of P. australis and P. pungens were identified. Development of a restriction fragment length polymorphism (RFLP) assay of the LsrDNA is described. The RFLP assay discriminates each species, including distinguished strains of P. australis and P. pungens. The restriction test provides a rapid and convenient method for screening isolates' LsrDNA, facilitating further tests of the apparent positive correlation between Pseudonitzschia species' ribosomal gene signatures, morphology, and capacity to produce domoic acid.