Recovery and identification of Pseudo-nitzschia (Bacillariophyceae) frustules from natural samples acquired using the environmental sample processor.

Many species within the diatom genus Pseudo-nitzschia are difficult to distinguish without applying molecular analytical or microscopy-based methods. DNA, antibody and lectin probes have previously been used to provide rapid and specific detection of species and strains in complex field assemblages. Recently, however, well-documented cryptic genetic diversity within the group has confounded results of DNA probe tests in particular. Moreover, the number of species descriptions within the genus continues to increase, as do insights into toxin production by both new and previously described species. Therefore, a combination of classical morphological techniques and modern molecular methodologies is needed to resolve ecophysiological traits of Pseudo-nitzschia species. Here, we present an approach to recover and identify frustules from sample collection filters used for toxin analysis onboard the Environmental Sample Processor (ESP), an in situ sample collection and analytical platform. This approach provides a new and powerful tool for correlating species presence with toxin detected remotely and in situ by the ESP, and has the potential to be applied broadly to other sampling configurations. This new technique will contribute to a better understanding of naturally occurring Pseudo-nitzschia community structure with respect to observed domoic acid outbreaks.

Autonomous application of quantitative PCR in the deep sea: in situ surveys of aerobic methanotrophs using the deep-sea environmental sample processor.

Recent advances in ocean observing systems and genomic technologies have led to the development of the deep-sea environmental sample processor (D-ESP). The D-ESP filters particulates from seawater at depths up to 4000 m and applies a variety of molecular assays to the particulates, including quantitative PCR (qPCR), to identify particular organisms and genes in situ. Preserved samples enable laboratory-based validation of in situ results and expanded studies of genomic diversity and gene expression. Tests of the D-ESP at a methane-rich mound in the Santa Monica Basin centered on detection of 16S rRNA and particulate methane monooxygenase (pmoA) genes for two putative aerobic methanotrophs. Comparison of in situ qPCR results with laboratory-based assays of preserved samples demonstrates the D-ESP generated high-quality qPCR data while operating autonomously on the seafloor. Levels of 16S rRNA and pmoA cDNA detected in preserved samples are consistent with an active community of aerobic methanotrophs near the methane-rich mound. These findings are substantiated at low methane sites off Point Conception and in Monterey Bay where target genes are at or below detection limits. Successful deployment of the D-ESP is a major step toward developing autonomous systems to facilitate a wide range of marine microbiological investigations.

Robotic environmental DNA bio-surveillance of freshwater health.

Autonomous water sampling technologies may help to overcome the human resource challenges of monitoring biological threats to rivers over long time periods and across large geographic areas. The Monterey Bay Aquarium Research Institute has pioneered a robotic Environmental Sample Processor (ESP) that overcomes some of the constraints associated with traditional sampling since it can automate water sample filtration and preservation of the captured material. The ESP was originally developed for marine environment applications. Here we evaluated whether the ESP can provide reliable, timely information on environmental (e)DNA detections of human and fish pathogens and introduced fishes at U.S. Geological Survey streamgage sites in freshwater rivers. We compared eDNA collected via ESP at high frequency (e.g., every 3 h) with manual eDNA collections collected at lower frequency (e.g., weekly). We found that water samples filtered and preserved by ESPs successfully detected the DNA of human pathogens, fish pathogens and introduced fishes. Both ESP and manually collected samples provided similar information about target DNA presence. We suggest that the greatest current benefit of the ESP is the cost savings of high frequency, bio-surveillance at remote or hard to access sites. The full potential of robotic technologies like the ESP will be realized when they can more easily execute in situ analyses of water samples and rapidly transmit results to decision-makers.

Remote, autonomous real-time monitoring of environmental DNA from commercial fish.

Environmental DNA (eDNA) is increasingly used for monitoring marine organisms; however, offshore sampling and time lag from sampling to results remain problematic. In order to overcome these challenges a robotic sampler, a 2nd generation Environmental Sample Processor (ESP), was tested for autonomous analysis of eDNA from four commercial fish species in a 4.5 million liter mesocosm. The ESP enabled in situ analysis, consisting of water collection, filtration, DNA extraction and qPCR analysis, which allowed for real-time remote reporting and archival sample collection, consisting of water collection, filtration and chemical preservation followed by post-deployment laboratory analysis. The results demonstrate that the 2G ESP was able to consistently detect and quantify target molecules from the most abundant species (Atlantic mackerel) both in real-time and from the archived samples. In contrast, detection of low abundant species was challenged by both biological and technical aspects coupled to the ecology of eDNA and the 2G ESP instrumentation. Comparison of the in situ analysis and archival samples demonstrated variance, which potentially was linked to diel migration patterns of the Atlantic mackerel. The study demonstrates strong potential for remote autonomous in situ monitoring which open new possibilities for the field of eDNA and marine monitoring.

Near real-time, autonomous detection of marine bacterioplankton on a coastal mooring in Monterey Bay, California, using rRNA-targeted DNA probes.

A sandwich hybridization assay (SHA) was developed to detect 16S rRNAs indicative of phylogenetically distinct groups of marine bacterioplankton in a 96-well plate format as well as low-density arrays printed on a membrane support. The arrays were used in a field-deployable instrument, the Environmental Sample Processor (ESP). The SHA employs a chaotropic buffer for both cell homogenization and hybridization, thus target sequences are captured directly from crude homogenates. Capture probes for seven of nine different bacterioplankton clades examined reacted specifically when challenged with target and non-target 16S rRNAs derived from in vitro transcribed 16S rRNA genes cloned from natural samples. Detection limits were between 0.10-1.98 and 4.43- 12.54 fmole ml(-1) homogenate for the 96-well plate and array SHA respectively. Arrays printed with five of the bacterioplankton-specific capture probes were deployed on the ESP in Monterey Bay, CA, twice in 2006 for a total of 25 days and also utilized in a laboratory time series study. Groups detected included marine alphaproteobacteria, SAR11, marine cyanobacteria, marine group I crenarchaea, and marine group II euryarchaea. To our knowledge this represents the first report of remote in situ DNA probe-based detection of marine bacterioplankton.

Microbial metagenomes and metatranscriptomes during a coastal phytoplankton bloom.

Metagenomic and metatranscriptomic time-series data covering a 52-day period in the fall of 2016 provide an inventory of bacterial and archaeal community genes, transcripts, and taxonomy during an intense dinoflagellate bloom in Monterey Bay, CA, USA. The dataset comprises 84 metagenomes (0.8 terabases), 82 metatranscriptomes (1.1 terabases), and 88 16S rRNA amplicon libraries from samples collected on 41 dates. The dataset also includes 88 18S rRNA amplicon libraries, characterizing the taxonomy of the eukaryotic community during the bloom. Accompanying the sequence data are chemical and biological measurements associated with each sample. These datasets will facilitate studies of the structure and function of marine bacterial communities during episodic phytoplankton blooms.

Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay: Perspectives from targeted and adaptive sampling.

Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo-nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.

Sandwich hybridization probes for the detection of Pseudo-nitzschia (Bacillariophyceae) species: An update to existing probes and a description of new probes.

New sandwich hybridization assay (SHA) probes for detecting Pseudo-nitzschia species (P. arenysensis, P. fraudulenta, P. hasleana, P. pungens) are presented, along with updated cross-reactivity information on historical probes (SHA and FISH; fluorescence in situ hybridization) targeting P. australis and P. multiseries. Pseudo-nitzschia species are a cosmopolitan group of diatoms that produce varying levels of domoic acid (DA), a neurotoxin that can accumulate in finfish and shellfish and transfer throughout the food web. Consumption of infected food sources can lead to illness in humans (amnesic shellfish poisoning; ASP) and marine wildlife (domoic acid poisoning; DAP). The threat of human illness, along with economic loss from fishery closures has resulted in the implementation of monitoring protocols and intensive ecological studies. SHA probes have been instrumental in some of these efforts, as the technique performs well in complex heterogeneous sample matrices and has been adapted to benchtop and deployable (Environmental Sample Processor) platforms. The expanded probe set will enhance future efforts towards understanding spatial, temporal and successional patterns in species during bloom and non-bloom periods.

Molecular detection of marine invertebrate larvae.

The ecological patterns of many invertebrate larvae remain an ongoing mystery, in large part owing to the difficult task of detecting them in the water column. The development of nucleic-acid-based technology has the potential to resolve this issue by direct identification and monitoring of embryonic and larval forms in situ. We report herein on the successful development and application of nucleic-acid-based sandwich hybridization assays that detect barnacles using rRNA-targeted probes with both group-(order Thoracica) and species-(Balanus glandula) specificity. Primary results include the determination of target 18S rRNA sequences and the construction of "capture" probes for detection of larvae using hybridization techniques. In addition, we modified existing protocols for whole cell hybridization of invertebrate larvae as confirmation of the sandwich hybridization results. We used both hybridization techniques successfully in the laboratory on a plankton time series collected over 3 months, as well as a week-long in situ deployment of the technique in Monterey Bay, CA. The adaptability of this technology promises to be further applicable to various organisms and could be used to enhance our understanding of larval presence in the world's oceans.

Single-taxon field measurements of bacterial gene regulation controlling DMSP fate.

The 'bacterial switch' is a proposed regulatory point in the global sulfur cycle that routes dimethylsulfoniopropionate (DMSP) to two fundamentally different fates in seawater through genes encoding either the cleavage or demethylation pathway, and affects the flux of volatile sulfur from ocean surface waters to the atmosphere. Yet which ecological or physiological factors might control the bacterial switch remains a topic of considerable debate. Here we report the first field observations of dynamic changes in expression of DMSP pathway genes by a single marine bacterial species in its natural environment. Detection of taxon-specific gene expression in Roseobacter species HTCC2255 during a month-long deployment of an autonomous ocean sensor in Monterey Bay, CA captured in situ regulation of the first gene in each DMSP pathway (dddP and dmdA) that corresponded with shifts in the taxonomy of the phytoplankton community. Expression of the demethylation pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the cleavage pathway was greater in the presence of a mixed diatom and dinoflagellate community [corrected].These field data fit the prevailing hypothesis for bacterial DMSP gene regulation based on bacterial sulfur demand, but also suggest a modification involving oxidative stress response, evidenced as upregulation of catalase via katG, when DMSP is demethylated.

Ocean microbes. Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages.

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.

Ecogenomic sensor reveals controls on N2-fixing microorganisms in the North Pacific Ocean.

Nitrogen-fixing microorganisms (diazotrophs) are keystone species that reduce atmospheric dinitrogen (N2) gas to fixed nitrogen (N), thereby accounting for much of N-based new production annually in the oligotrophic North Pacific. However, current approaches to study N2 fixation provide relatively limited spatiotemporal sampling resolution; hence, little is known about the ecological controls on these microorganisms or the scales over which they change. In the present study, we used a drifting robotic gene sensor to obtain high-resolution data on the distributions and abundances of N2-fixing populations over small spatiotemporal scales. The resulting measurements demonstrate that concentrations of N2 fixers can be highly variable, changing in abundance by nearly three orders of magnitude in less than 2 days and 30 km. Concurrent shipboard measurements and long-term time-series sampling uncovered a striking and previously unrecognized correlation between phosphate, which is undergoing long-term change in the region, and N2-fixing cyanobacterial abundances. These results underscore the value of high-resolution sampling and its applications for modeling the effects of global change.

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.

Underwater application of quantitative PCR on an ocean mooring.

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.