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Phytoplankton communities residing in the open ocean, the largest habitat on Earth, play a key role in global primary production. Through their influence on nutrient supply to the euphotic zone, open-ocean eddies impact the magnitude of primary production and its spatial and temporal distributions. It is important to gain a deeper understanding of the microbial ecology of marine ecosystems under the influence of eddy physics with the aid of advanced technologies. In March and April 2018, we deployed autonomous underwater and surface vehicles in a cyclonic eddy in the North Pacific Subtropical Gyre to investigate the variability of the microbial community in the deep chlorophyll maximum (DCM) layer. One long-range autonomous underwater vehicle (LRAUV) carrying a third-generation Environmental Sample Processor (3G-ESP) autonomously tracked and sampled the DCM layer for four days without surfacing. The sampling LRAUV's vertical position in the DCM layer was maintained by locking onto the isotherm corresponding to the chlorophyll peak. The vehicle ran on tight circles while drifting with the eddy current. This mode of operation enabled a quasi-Lagrangian time series focused on sampling the temporal variation of the DCM population. A companion LRAUV surveyed a cylindrical volume around the sampling LRAUV to monitor spatial and temporal variation in contextual water column properties. The simultaneous sampling and mapping enabled observation of DCM microbial community in its natural frame of reference.

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.

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.

An ecologically and economically disruptive harmful algal bloom (HAB) affected much of the northeast Pacific margin in 2015, during a prolonged oceanic warm anomaly. Caused by diatoms of the genus Pseudo-nitzschia, this HAB produced the highest particulate concentrations of the biotoxin domoic acid (DA) ever recorded in Monterey Bay, California. Bloom inception followed strong upwelling during the spring transition, which introduced nutrients and eliminated the warm anomaly locally. Subsequently, moderate and intermittent upwelling created favorable conditions for growth and accumulation of HAB biomass, which was dominated by a highly toxigenic species, P. australis. High cellular DA concentrations were associated with available nitrogen for DA synthesis coincident with silicate exhaustion. This nutrient influence resulted from two factors: (1) disproportionate depletion of silicate in upwelling source waters during the warm anomaly, the most severe depletion observed in 24 years, and (2) silicate uptake by the dense diatom 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., Design Type(s)transcription profiling design • sequence assembly objective • biodiversity assessment objectiveMeasurement Type(s)transcription profiling assay • marine metagenome • microbial communityTechnology Type(s)RNA sequencing • DNA sequencing • amplicon sequencingFactor Type(s)assay protocol • temporal_instantSample Characteristic(s)marine metagenome • Monterey Bay • ocean biome Machine-accessible metadata file describing the reported data (ISA-Tab format)

The overarching logistical challenge in microbial oceanography is acquiring enough samples to provide meaningful scientific interpretation. The number of samples collected during ship expeditions is limited by weather, time on station, and budget. Here we describe a robotic, autonomous vehicle platform equipped with a unique sampling instrument that mitigates some of these constraints. In a joint cruise on the R/V Falkor, the Monterey Bay Aquarium Research Institute and the University of Hawaii deployed two of these vehicles in a mesoscale eddy north of the island of Maui. One vehicle collected contextual measurements while circling a freely drifting sampling vehicle. On the sampling vehicle we implemented several behaviors, including sampling every three hours for a 4-day underwater drift while maintaining position within the deep chlorophyll maximum layer (~100m). Results demonstrate the ability to remain with features of interest and point to an exciting future of long-term, directed, persistent sampling.

The Columbia River has a dynamic and fast flushing estuary impacted by strong advection and mixing of riverine and oceanic waters, and high but variable loads of suspended particulate matter. Transient, but recurring water and nutrient fluxes from end-members impart strong spatial and temporal gradients, contributing to microbiological hotspots that play important ecological roles in the estuary. Investigations of corresponding microbiota require precisely timed samples that are contextualized by physical and biogeochemical data. To accomplish this, we embedded a robotic microbial sampler (Environmental Sample Processor, ESP) within the operations of an interdisciplinary observation and prediction system (Science and Technology University Research Network, SATURN; www.stccmop.org/saturn). Autonomous, adaptively sampled water collection by the ESP was implemented based on environmental conditions assessed from SATURN physical and biogeochemical sensors. Water was pumped from multiple depths to sensors and the ESP on dry land. If water met user-defined parameters, ESP sampling was automatically initiated. This strategy was tested during three deployments in summer 2013, during which operational tools for analysis and visualization were used to formulate well-constrained mission plans by providing estimates of the intensity and timing of oxygen-depleted ocean water intrusion and estuarine turbidity maxima. This allowed the effective characterization of the impact of these events on selected estuarine microbiota.

An observational study was performed in the central Southern California Bight in Spring 2010 to understand the relationship between seasonal spring phytoplankton blooms and coastal processes that included nutrient input from upwelling, wastewater effluent plumes, and other processes. Multi-month Webb Slocum glider deployments combined with MBARI environmental sample processors (ESPs), weekly pier sampling, and ocean color data provided a multidimensional characterization of the development and evolution of harmful algal blooms (HABs). Results from the glider and ESP observations demonstrated that blooms of toxic Pseudo-nitzschia sp. can develop offshore and subsurface prior to their manifestation in the surface layer and/or near the coast. A significant outbreak and surface manifestation of the blooms coincided with periods of upwelling, or other processes that caused shallowing of the pycnocline and subsurface chlorophyll maximum. Our results indicate that subsurface populations can be an important source for “seeding” surface Pseudo-nitzschia HAB events in southern California.

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.

Here we present achievements and challenges associated with the inaugural freshwater deployment of the Environmental Sampler Processor (ESP) in the western Lake Erie. We discuss scientific advances in the detection methodology related to utilizing the ESP for monitoring toxic cHABs and establishing the ability to provide water resource managers with early warning of impending toxicity.

Euro NCAP continually examines and modifies its protocols to encourage the automotive industry to improve vehicles and the safety of occupants. The aim of this study is to identify the key differences between the past and current Euro NCAP protocols regarding side impact, in order to apply optimized countermeasures for fulfilling the new requirements in a previously designed vehicle. Outcomes from a possible Euro NCAP increase in crash speed from 50 km/h to 60 km/h were evaluated. The current protocol introduces the dummy WorldSID 50th percentile and changes in the barrier. The changes in the dummy lead to a reduction in the distance between the occupant and the door panel, and the current geometry of the barrier causes the car’s structure, in particular the sill, to be additionally loaded. An increase in mass of the current barrier causes an increase in energy absorption, thus presenting different deformation patterns. With the updated protocol, the most critical anatomical areas observed in this research were the pelvis and the shoulder. Countermeasures in this study were especially focused on decreasing the load on these anatomical structures. The evaluated countermeasures were: - Sill and B-pillar geometric adaptation to better perform against the AE-MDB - Reduced stiffness of the door panel at the occupant impact location - Optimized door beam Finite Element Method (FEM) tools were used as the basis of this research, including validated and correlated models with experimental full car tests. Initially, the research started with FEM testing of the current vehicle according to both past and current Euro NCAP protocols. Pelvic biomechanical values reached the lower performance limits when testing the AE-MDB barrier at 50 km/h. This new test has an especial impact in midsized cars in terms of pelvis loading, leading the pubic and sacroiliac forces to increase in 38.6% and 25.8% respectively, in the vehicle studied. After the application of the structural countermeasures, the sacroiliac force decreased by 24.9% and the pubic force decreased by 32.5%. Changes in the Euro NCAP protocol and regulations are always a challenge for design departments. In this research the differences between Euro NCAP side protocols were analyzed and some potential countermeasures were highlighted.

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.

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.

A wording error in a sentence in the abstract misrepresented a main finding of the research. The sentence should read: 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. The sentence has now been corrected. The supplementary file published with the paper was missing figure legends. The correct file is now provided.