Successful harmful algal bloom (HAB) prediction and monitoring employs a variety of observational and research strategies. We add to the existing suite of tools for detecting toxigenic Pseudo-nitzschia diatoms by developing a novel digital PCR (dPCR) assay targeting a key gene of the domoic acid biosynthetic pathway, dabA. Sequence alignments and synthetic gene fragments of dabA genes from Pseudo-nitzschia australis, P. multiseries, P. multistriata, and P. seriata, along with the closely-related red-algal genes kabA and radA, were used to design a dPCR assay and assess its specificity. This dPCR assay was demonstrated to be specific for dabA and can quantify concentrations >1 copy µL-1 in a reaction. The biogeography of published dabA genes suggests that the assay may be useful globally for target species. When screening a culture collection of Pseudo-nitzschia isolated from coastal California, the dPCR assay detected dabA genes from three of the eleven species tested. In samples collected during field campaigns in 2022 off Santa Barbara, CA and in 2023 in Monterey Bay, CA during toxic HAB events, Pseudo-nitzschia dabA genes were only detected in Monterey Bay samples. While the dPCR assay does not capture all dabA diversity, it is specific and provides a targeted means to assess the genetic potential of a toxic HAB event by dominant and potent toxin-producing Pseudo-nitzschia species in coastal California. Because dabA expression is a key indicator of DA production, using the assay to quantify gene transcription could speed the acquisition of data needed to forecast HABs before toxin is detected.

Freshwater cyanobacterial harmful algal blooms (CHABs) are a well-known global public health threat. Monitoring and early detection of CHAB toxins are currently accomplished using labor-intensive sampling techniques and subsequent shore-based analyses, with results typically reported 24-48 h after sample collection. We have developed and implemented an uncrewed, autonomous mobile sampler-analytical system capable of conducting targeted in situ toxin measurements in < 2 h. A surface plasmon resonance (SPR) instrument was combined with the environmental sample processor (ESP) to fully automate detection and quantification of particle-associated cyanobacterial microcystins (pMC). This sensor-sampler system was integrated with a long-range autonomous underwater vehicle (LRAUV) and deployed in western Lake Erie for field trials in the summer of 2021. The LRAUV was remotely piloted to acquire samples at selected locations within and adjacent to a CHAB. Sixteen pMC measurements ranging from 0.09 to 0.55 mu g/L lake water were obtained over a 14-day period without recovery of the LRAUV. The SPR/ESP/LRAUV system complements existing satellite, aerial, and manual sampling CHAB survey techniques, and could be used to enhance predictive models that underpin bloom and toxicity forecasts. This system is also extensible to detection of other algal toxins in freshwater and marine environments, with its near real-time assessment of bloom toxin levels potentially offering additional socioeconomic benefits and public health protection in a variety of settings.

Cyanobacterial harmful algal blooms (CyanoHABs) in the Great Lakes pose risks to residential drinking water use, fisheries, and recreation. Active mitigation of these risks requires rapid detection of CyanoHABs and quantification of the toxins they produce. Here, we present a method of using a long-range autonomous underwater vehicle (LRAUV) equipped with a 3rd-generation Environmental Sample Processor (3G-ESP) to search for and adaptively sample areas of high chlorophyll potentially representative of CyanoHAB biomass. In August 2021, this method was used in western Lake Erie. The experiment highlighted the effectiveness of the LRAUV autonomous search-and-sample methodology, and demonstrated how an interdisciplinary team located in different states virtually coordinated LRAUV operations and directed sampling activities via Internet connectivity using shared, web-based situational awareness tools. The advancements made provide a foundation for future work to increase LRAUV autonomy and adaptiveness for CyanoHAB studies and monitoring in both freshwater and marine settings.

Ocean microbes are the foundation of marine food webs, regulating carbon cycling and ecosystem dynamics. How they proliferate, die, move, and interact is regulated by physical, chemical, and biological factors that are dynamic and challenging to quantify in the natural environment. A significant limitation in many marine field studies is the inability to continuously sample the ever-changing ocean environment over space and time. In this study, we integrated spatiotemporal and multi-omic sample collection in an intensive sampling effort of phytoplankton ecology in Monterey Bay, California during the spring of 2021. Sampling methods coupled: (1) manual shipboard CTD sampling, (2) autonomous sampling using a Long-Range Autonomous Underwater Vehicle (LRAUV) equipped with an Environmental Sampling Processor (ESP), and (3) high-resolution physical measurements by an autonomous vertical profiler (Wirewalker). Sampling occurred as upwelling waned alongside declining domoic acid (DA) and low abundances of toxigenic Pseudo-nitzschia . Conditions needed to spark a widespread and toxic Pseudo-nitzschia bloom were absent, yet low-level DA was driven by similar mechanisms to those causing elevated DA. Three DA biosynthetic intermediate molecules were reported in the environment for the first time. Both shipboard and ESP sampling approaches identified DA biosynthetic gene expression at frontal zones. DA and expression of dabA , the gene encoding the first committed step of DA biosynthesis, were higher in association with recently upwelled water that supplied nutrients for growth and DA biosynthesis. Detection of subtle variations in dab gene expression in response to environmental variation provide a window into the ecological dynamics underpinning major toxic events. Graphical Abstract

Biomolecular analyses are used to investigate the dynamics of cyanobacterial harmful algal blooms (cyanoHABs), with samples collected during monitoring often analyzed by qPCR and sometimes amplicon and metagenomic sequencing. However, cyanoHAB research and monitoring programs face operational constraints due to the reliance on human resources for sample collections. To address this impediment, a third-generation Environmental Sample Processor (3G ESP) integrated with a long-range autonomous underwater vehicle (LRAUV) was tested during seasonal blooms of Microcystis in western Lake Erie (WLE) in 2018 and 2019. The LRAUV-3G ESP successfully performed flexible, autonomous sampling across a wide range of cyanoHAB conditions, and results indicated equivalency between autonomous and manual methods. No significant differences were found between LRAUV-3G ESP and manual sample collection and handling methods in the 12 parameters tested. Analyzed parameters included concentrations of total cyanobacteria and microcystin toxin gene via qPCR; relative abundances of bacterial amplicon sequence variants (ASVs) from 16S rRNA gene amplicon sequencing; and community diversity measures from both 16S amplicon and metagenomic sequencing. The LRAUV-3G ESP provided additional sampling capacity and revealed differences between field seasons for bacterial taxa and concentrations of total cyanobacteria and microcystin toxin gene. Metagenomic analysis of multiple microcystin toxin genes corroborated the use of the mcyE gene as a proxy for the genomic potential of WLE cyanoHABs to produce microcystin. Overall, this study provides support for the use of autonomous ‘omics capability in WLE to help expand the spatial and temporal coverage of cyanoHAB monitoring operations.

Logging data are measurements of physical properties of the formation surrounding a borehole, acquired in situ after completion of coring (wireline logging) or during drilling (Logging-While-Drilling, LWD). The range of data (resistivity, gamma radiation, velocity, density, borehole images,…) in any hole depends on the scientific objectives and operational constraints.

Environmental DNA (eDNA) can be used to identify macroorganisms and describe biodiversity, and thus has promise to supplement biological monitoring in marine ecosystems. Despite this promise, scaling sample acquisition to the spatial and temporal scales needed for effective monitoring would require prohibitively large investments in time and human resources. To address this challenge, we evaluated the efficacy of an autonomous eDNA sampling system and compare results obtained to traditional eDNA sampling methods. The autonomous sampling instrument consisted of the Environmental Sample Processor (ESP) coupled to an autonomous underwater vehicle (AUV). We tested equivalency between the ESP and traditional eDNA sampling techniques by comparing the quantification of eDNA across a broad range of taxa, from microbes (SAR11), phytoplankton (Pseudo-nitzschia spp.), and invertebrates (krill: Euphausia pacifica) to vertebrates (anchovy: Engraulis mordax). No significant differences in eDNA densities were observed between the two sample collection and filtration methods. eDNA filters collected by the ESP were preserved and stable for 21 days, the typical deployment length of the instrumentation. Finally, we demonstrated the unique capabilities of an autonomous, mobile ESP during a deployment near Monterey Bay, CA, by remotely and repeatedly sampling a water mass over 12 h. The development of a mobile ESP demonstrates the promise of utilizing eDNA measurements to observe complex biological processes in the ocean absent a human presence.

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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.

Roughly 25 years ago, "ecogenomic sensors" were conceived of as autonomous devices that would be used to apply molecular analytical techniques below the sea surface as one part of a futuristic, integrated ocean observing system. The Environmental Sample Processor (ESP) was built to address that idea-an instrument to help define both the technological and operational elements that underlie the ecogenomic sensor concept. Over time, the ESP emerged as a working example of that class of instrument, enabling the application of DNA probe and protein arrays as well as use of the quantitative polymerase chain reaction (qPCR) technique to assess the presence and abundance of a wide range of organisms, specific genes, and metabolites. The ESP is also used to preserve samples for a variety of laboratory tests not yet possible to carry out in situ (e.g., DNA sequencing). The instrument has been deployed on a variety of platforms, including coastal moorings, piers, an open ocean drifter, research vessels, a shallow water benthic lander, and a 4,000 m rated "elevator" designed for use on deep-sea cabled observatories. A new version of the ESP is currently being developed for use aboard an autonomous underwater vehicle. This article traces the evolution of the ESP from its conception to present-day status.

Despite years of research into microbial activity at diffuse flow hydrothermal vents, the extent of microbial niche diversity in these settings is not known. To better understand the relationship between microbial activity and the associated physical and geochemical conditions, we obtained co-registered metatranscriptomic and geochemical data from a variety of different fluid regimes within the ASHES vent field on the Juan de Fuca Ridge. Microbial activity in the majority of the cool and warm fluids sampled was dominated by a population of Gammaproteobacteria (likely sulfur oxidizers) that appear to thrive in a variety of chemically distinct fluids. Only the warmest, most hydrothermally-influenced flows were dominated by active populations of canonically vent-endemic Epsilonproteobacteria. These data suggest that the Gammaproteobacteria collected during this study may be generalists, capable of thriving over a broader range of geochemical conditions than the Epsilonproteobacteria. Notably, the apparent metabolic activity of the Gammaproteobacteria—particularly carbon fixation—in the seawater found between discrete fluid flows (the intra-field water) suggests that this area within the Axial caldera is a highly productive, and previously overlooked, habitat. By extension, our findings suggest that analogous, diffuse flow fields may be similarly productive and thus constitute a very important and underappreciated aspect of deep-sea biogeochemical cycling that is occurring at the global scale.

The documented presence of the pesticide DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) and its metabolites DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), and DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane), collectively referred to as ΣDDT, in sediments from the Monterey Submarine Canyon, Monterey Fan Channel and Monterey Fan indicates that during the present sea-level highstand the Monterey Fan has been an active depositional system. The presence of ΣDDT in marine sediment is an indication of the recent arrival of terrigenous fine-grained sediment because DDT was only applied on land in California between 1944 and 1972, and is being transported to the ocean in the fine-grained sediment fraction of rivers. ΣDDT was found in 81 out of 82 cores collected in water depths deeper than 3000m in the Monterey Fan Channel and in the Monterey Fan, within a geographical extent of 250km to the south and 210km to the west of the Monterey Canyon head, with surface concentrations averaging 6 and 3ngg−1, in the upper and lower Monterey Fan, respectively. This is the first documentation of ΣDDT presence in ocean sediments below 3000m water depth worldwide. The estimated total amount of ΣDDT in the fan is consistent with the amount of ΣDDT discharged by the Salinas and Pajaro Rivers since DDT started being used in California, suggesting that current fine-grained sediment discharge from these rivers has been accumulating in the fan. The ratio DDE/ΣDDT, an indication of the extent of DDT degradation, provides a first-order proxy for the relative age of the sediments. DDT is found along the thalweg of the Monterey Fan channel as far as 215km from the canyon head, indicating recent fine-grained sediment transport along the channel. In contrast, only DDE was detected in sediments on the fan, which suggests that the fan does not accumulate fine-grained sediments at a constant rate.

Episodic 13C depletions in the carbon isotopic composition of benthic foraminiferal tests preserved in the stratigraphic record have been interpreted as an active incorporation of methane-derived carbon. Understanding the extent to which these isotope excursions reflect basin-wide fluxes of methane carbon to bottom waters versus a local supply of methane carbon within the sediments in which benthic foraminifera live, or a postmortem diagenetic imprint is critical to the interpretation of δ13C paleoceanographic proxies. Here we evaluate the impact of chemical gradients measured in pore waters adjacent to active methane vents on carbon assimilation by living benthic foraminifera and show that those living near methane vents do not assimilate the distinctly 13C depleted methane-derived dissolved inorganic carbon into their tests from the pore water in which they were found. Our observations can be explained by the recently articulated physiological limits imposed on deep-sea fauna by low-oxygen and high-pCO2 environments. Understanding the importance of the different processes involved in the observed disequilibrium between the carbon isotopic composition of the benthic forams and the pore waters where they were found has important implications on the reliability of carbon isotopic composition of benthic foraminifera for paleoceanographic reconstructions. In particular, the observation on the inhospitability of these environments for benthic foraminifera at least for reproduction and growth raises the issue on the overprint either in the late adult stages of foraminifera that grew in a different neighboring environment or during early diagenesis in these geochemically active environments.

Hydrothermal vents are hotspots of microbial primary productivity often described as" windows into the subsurface biosphere." High temperature vents have received the majority of research attention, but cooler diffuse flows are as, if not more, important a source of heat and chemicals to the overlying ocean. We studied patterns of in situ gene expression and co-registered geochemistry in order to 1) describe the diversity and physiological poise of active microbial communities that span thermal and geochemical gradients from active diffuse flow to background vent field seawater, and 2) determine to what extent seawater or subsurface microbes were active throughout this environment. Analyses of multiple metatranscriptomes from 5 geochemically distinct sites (some from samples preserved in situ) show that proximate diffuse flows showed strikingly different transcription profiles. Specifically, caldera background …

While submarine canyons are the major conduits through which sediments are transported from the continents out into the deep sea, the time it takes for sediment to pass down through a submarine canyon system is poorly constrained. Here we report on the first study to couple optically stimulated luminescence (OSL) ages of quartz sand deposits and accelerator mass spectrometry 14 C ages measured on benthic foraminifera to examine the timing of sediment transport through the axial channel of Monterey Submarine Canyon and Fan, offshore California. The OSL ages date the timing of sediment entry into the canyon head while the 14 C ages of benthic foraminifera record the deposition of hemipelagic sediments that bound the sand horizons. We use both single-grain and small (∼2 mm area) single-aliquot regeneration approaches on vibracore samples from fining-upward sequences at various water depths to demonstrate relatively rapid, decadal-scale sand transport to at least 1.1 km depth and more variable decadal- to millennial-scale transport to a least 3.5 km depth on the fan. Significant differences between the time sand was last exposed at the canyon head (OSL age) and the timing of deposition of the sand (from 14 C ages of benthic foraminifera in bracketing hemipelagic sediments) are interpreted as indicating that the sand does not pass through the entire canyon instantly in large individual events, but rather moves multiple times before emerging onto the fan. The increased spread in single-grain OSL dates with water depth provides evidence of mixing and temporary storage of sediment as it moves through the canyon system. The ages also indicate that the frequency of sediment transport events decreases with distance down the canyon channel system. The amalgamated sands near the canyon head yield OSL ages that are consistent with a sub-decadal recurrence frequency while the fining-upward sand sequences on the fan indicate that the channel is still experiencing events with a 150–250 year recurrence frequency out to 3.5 km water depths.

The traditional ocean chemical explanation for the emergence of suboxia is that once O-2 levels decline to about 10 mu mol kg(-1) then onset of NO reduction occurs. This piece of ocean chemical lore is well founded in observations and is typically phrased as a microbial choice and not as an obligate requirement. The argument based on O-2 levels alone could also be phrased as being dependent on an equivalent amount of NO3- that would yield the same energy gain. This description is based on the availability of the electron acceptor: but the oxidation reactions are usually written out as free energy yield per mole of organic matter, thus not addressing the oxidant availability constraint invoked by ocean scientists. Here we show that the argument can be phrased simply as competing rate processes dependent on the free energy yield ratio per amount of electron acceptor obtained, and thus the [NO3-] : [0(2)] molar ratio is the critical variable. The rate at which a microbe can acquire either O-2 or NO3- to carry out the oxidation reactions is dependent on both the concentration in the bulk ocean, and on the diffusivity within the microbial external molecular boundary layer. From the free energy yield calculations combined with the similar to 25% greater diffusivity of the O-2 molecule we find that the equivalent energy yield occurs at a ratio of about 3.8 NO3- O-2 for a typical Redfield ratio reaction, consistent with an ocean where NO; reduction onset occurs at about 10 mu mol O-2:40 mu mol NO3-. and the reactions then proceed in parallel along a line of this slope until the next energy barrier is approached. Within highly localized microbial consortia intensely reducing pockets may occur in a bulk ocean containing finite low O-2 levels: and the local flux of reduced species from strongly reducing shelf sediments will perturb the large scale water column relationship. But all localized reactions drive towards maximal energy gain from their immediate diffusive surroundings, thus the ocean macroscopic chemical fields quite well approximate the net efficiency and operational mode of the ensemble microbial engine. (c) 2014 Elsevier Ltd. All rights reserved.

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.

The sulfate-methane transition (SMT) zone is a diagenetic transition within anoxic marine sediments created by the metabolic activity of a consortium of sulfate-reducing bacteria and methane-oxidizing Archaea. As interstitial dissolved sulfate is consumed by microbially mediated sulfate reduction of sedimentary organic matter (SOM) and anaerobic oxidation of methane (AOM) large enrichments of S-34 occur in the interstitial sulfate pool. These isotopic enrichments are transmitted to the dissolved sulfide pool (Sigma HS-) and subsequently into sulfide minerals (So, similar to FeS, FeS2).We investigate the sulfur isotopic composition of pore-water sulfate and sulfide minerals at three sites underlain by gas hydrates at the Blake Ridge. The isotopic composition of sulfate-sulfur is most positive at the SMT showing maximum values of +29.1, 49.6, 51.6 parts per thousand VCDT at each of the respective sites. delta S-34 values of bulk sulfide minerals tend to be more enriched in S-34 at and below the SMT ranging from -12.7 to +23.6 parts per thousand, corresponding to enrichments of 26.7-62.4 parts per thousand relative to the mean value of -38.8 parts per thousand in the sulfate reduction zone. Both enhanced delivery of methane to the SMT, and non-steady-state sedimentation appear necessary to create large S-34 enrichments in sulfide minerals. Similar associations of AOM and large delta S-34 enrichments (>0 parts per thousand) occur in other gas hydrate terranes (Cascadia margin) but their exact origin is equivocal at present. An analysis of delta S-34 data from freshwater and marine sedimentary environments reveals that S-34 enrichments within sulfide minerals occur under a range of conditions, but are statistically associated with AOM and systems not limited by dissolved interstitial iron.In methane-rich sediments, methane delivery to the SMT increases the role of AOM in sulfate depletion that impacts the formation and isotopic composition of authigenic sulfide minerals. We hypothesize that under certain diagenetic conditions large S-34 enrichments within sulfide minerals in the geologic record potentially identify: (1) the former occurrence of AOM (2) present-day and "fossil" locations of the sulfate-methane transition zone; and (3) a diagenetic terrane, today characteristic of deep-water, methane-rich, marine sediments conducive to gas hydrate formation. Thus, S-34-enriched sulfide minerals preserved in modern and ancient continental-margin sediments may allow for the identification of AOM-related processes that occur in methane-rich sediments. (C) 2013 Elsevier Ltd. All rights reserved.

Diverse copper-containing membrane-bound monooxygenase-encoding sequences (Cu-MMOs) have recently been described from the marine environment, suggesting widespread potential for oxidation of reduced substrates. Here, we used the well-defined oxygen and methane gradients associated with the Costa Rican oxygen minimum zone (OMZ) to gain insight into the physico-chemical parameters influencing the distribution and abundance of Cu-MMO-encoding marine microorganisms. Two Methylococcales-related Cu-MMO-encoding lineages, termed groups OPU1 and OPU3, demonstrated differences in their relative abundance, with both pmoA and candidate 16S rRNA genes correlating significantly with reduced environmental oxygen concentrations and depth. In contrast, a newly identified Cu-MMO-encoding lineage, Group C, was primarily associated with the oxygenated euphotic zone. An updated phylogenetic analysis including these sequences, a marine pxmABC gene cluster, ethylene-utilizing Cu-MMO-encoding lineages and previously reported planktonic Cu-MMOs (Groups W, X, Z and O) demonstrates the breadth of diversity of Cu-MMO-encoding marine microorganisms. Groups C and X affiliated phylogenetically with ethane- and ethylene-oxidizing Cu-MMOs, Groups W and O affiliated phylogenetically with the recently described Cu-MMO 'pXMO', and Group Z clustered with Cu-MMOs recovered from soils. Collectively, these data demonstrate widespread genetic potential in ocean waters for the oxidation of small, reduced molecules and advance our understanding of the microorganisms involved in methane cycling in the OMZ environment.

A deep ocean robotic platform capable of in situ microbial identification and quantification at depths to 4000 m has been developed. The platform is a free benthic lander, containing a low pressure microbial instrument and deep ocean sampling systems that can monitor and collect raw seawater from multiple sources, then decompress the raw seawater for processing by the detection instrument. This has enabled the autonomous collection, processing, and archiving of microbes from multiple sites. This allows for direct, time correlated comparison of different microbial populations along with the seawater physical and chemical composition.