Metatranscriptomic assembly, predicted open reading frames, counts, and annotation files from seawater samples obtained in the western North Atlantic Ocean. GitHub notebooks are located here: https://github.com/cnatalie/BATS. Assembly was created using the eukrhythmic pipeline: https://github.com/AlexanderLabWHOI/eukrhythmic merged_merged.fasta.gz = Final assembly, merged across 44 metatranscriptomes using 4 different assemblers merged.fasta.transdecoder.pep.zip = Open reading frames of final assembly, predicted by Transdecoder merged.fasta.transdecoder-estimated-taxonomy.out.zip = EUKulele-derived taxonomic annotations of ORFs using a combined EukProt, PhyloDB, and RefSeq reference database newtaxa.eukprot.merged.fasta.transdecoder-estimated-taxonomy.out.zip = similar to above, but manually curated mid-level taxonomy for supergroups of interest eggnog.emapper.annotations.zip = eggnog-mapper annotations of ORFs table.tab.zip = counts associated with ORFs (merged.fasta.transdecoder.pep) generated with Salmon TPM_table.tab.zip = community-wide TPM (normalized) counts associated with ORFs (merged.fasta.transdecoder.pep) generated with Salmon copiesperL.tab.zip = raw counts associated with ORFs (merged.fasta.transdecoder.pep) converted to copies per L using spiked-in RNA standard concentration (copies), standard reads mapped, and volume of seawater filtered assembly.table.tab.zip = counts associated with final assembly (merged_merged.fasta) generated with Salmon SamplesViewReportCLIO_AE1913merged_trans210506_updated220606exclusive.zip = Exclusive spectral counts associated with ORFs (merged.fasta.transdecoder.pep). Peptide-spectrum matches were performed using Sequest algorithm within IseNode Proteome Discoverer 2.2.0.388 (Thermo Fisher Scientific). Scaffold 5.1.2 (Proteome Software) was used for protein grouping and exclusive spectral counting. Note, the (+x) data has been removed from protein names, which indicates whether (and how many) proteins sharing peptides were designated into the same protein group. cds.length2.tab.zip = Length of proteins (ORFs) in nucleotide base pairs CTD.zip = CTD files from cruise AE1913

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.

We observed negative and positive delta O-18 and delta H-2 deviations from fluids collected at Kama.ehuakanaloa (previously known as L o.ihi) seamount relative to Pacific seawater from the same depth. Hydrothermal vents on the crater floor of Pele's Pit at Kama.ehuakanaloa, at a depth of 1320 m, had delta O-18 and delta H-2 values as low as 0.19% and 0.3%, respectively. Seawater collected within the caldera, within a zone 45 m above the crater floor, had delta O-18 and delta H-2 values as high as 1.15% and 6.5%, respectively. In comparison, Pacific seawater at 1200 m and 1400 m at nearby station ALOHA exhibit intermediate delta O-18 and delta H-2 values of 0.2% and 0.34%. The high delta O-18 and delta H-2 values observed in the caldera water-column may be explained by isotopic modification processes, including water-rock reactions. However, we did not observe a vent source with similar isotopic composition; this suggests that substantial hydrothermal flow is entering the caldera through unidentified sources, potentially individually small but collectively important. Further, the low d18O and d2H values of the crater floor vents cannot be readily explained by isotopic modification processes alone. We conclude the crater floor vents predominantly reflect the isotopic composition of the Pacific seawater entrained into the hydrothermal system. Our observations suggest recharging seawater is entrained from below >4500 m, in the zone of Pacific Bottom Water. These findings illustrate the heterogeneity of hydrothermal transport processes at a volcanic seamount and how seamount hydrothermal convection can provide a mechanism that may contribute to vertical ocean mixing by transporting deep bottom waters to intermediate ocean depths.

Single-celled microbial eukaryotes inhabit deep-sea hydrothermal vent environments and play critical ecological roles in the vent-associated microbial food web. 18S rRNA amplicon sequencing of diffuse venting fluids from four geographically- and geochemically-distinct hydrothermal vent fields was applied to investigate community diversity patterns among protistan assemblages. The four vent fields include Axial Seamount at the Juan de Fuca Ridge, Sea Cliff and Apollo at the Gorda Ridge, all in the NE Pacific Ocean, and Piccard and Von Damm at the Mid-Cayman Rise in the Caribbean Sea. We describe species diversity patterns with respect to hydrothermal vent field and sample type, identify putative vent endemic microbial eukaryotes, and test how vent fluid geochemistry may influence microbial community diversity. At a semi-global scale, microbial eukaryotic communities at deep-sea vents were composed of similar proportions of dinoflagellates, ciliates, Rhizaria, and stramenopiles. Individual vent fields supported distinct and highly diverse assemblages of protists that included potentially endemic or novel vent-associated strains. These findings represent a census of deep-sea hydrothermal vent protistan communities. Protistan diversity, which is shaped by the hydrothermal vent environment at a local scale, ultimately influences the vent-associated microbial food web and the broader deep-sea carbon cycle.

Microbial eukaryotes (or protists) in marine ecosystems are a link between primary producers and all higher trophic levels, and the rate at which heterotrophic protistan grazers consume microbial prey is a key mechanism for carbon transport and recycling in microbial food webs. At deep-sea hydrothermal vents, chemosynthetic bacteria and archaea form the base of a food web that functions in the absence of sunlight, but the role of protistan grazers in these highly productive ecosystems is largely unexplored. Here, we pair grazing experiments with a molecular survey to quantify protistan grazing and to characterize the composition of vent-associated protists in low-temperature diffuse venting fluids from Gorda Ridge in the northeast Pacific Ocean. Results reveal protists exert higher predation pressure at vents compared to the surrounding deep seawater environment and may account for consuming 28 to 62% of the daily stock of prokaryotic biomass within discharging hydrothermal vent fluids. The vent-associated protistan community was more species rich relative to the background deep sea, and patterns in the distribution and co-occurrence of vent microbes provide additional insights into potential predator-prey interactions. Ciliates, followed by dinoflagellates, Syndiniales, rhizaria, and stramenopiles, dominated the vent protistan community and included bacterivorous species, species known to host symbionts, and parasites. Our findings provide an estimate of protistan grazing pressure within hydrothermal vent food webs, highlighting the important role that diverse protistan communities play in deep-sea carbon cycling.

This manuscript reports on a robot called Clio that we developed to facilitate basin-scale studies of ocean microbial communities and their biochemistry, to better understand how marine microorganisms regulate ocean and Earth system environmental cycles. Clio is designed to facilitate global-scale studies of ocean biochemistry, to move vertically through the water column with high precision, and specifically to return sensor data and samples from large swaths of the ocean ranging in depths from the surface to 6,000 m. Clio is capable of flexible, precise vertical motion that few other ocean robots can perform, and none to our knowledge over this depth range. We tested Clio extensively over several years, six cruises, and 26 dives, it is now fully operational and this manuscript describes all that we did to convince ourselves this was so. In June 2019, it completed its first large-scale ocean survey, and for which this manuscript will be the first data presentation.

Vast and diverse microbial communities exist within the ocean. To better understand the global influence of these microorganisms on Earth’s climate, we developed a robot capable of sampling dissolved and particulate seawater biochemistry across ocean basins while still capturing the fine-scale biogeochemical processes therein. Carbon and other nutrients are acquired and released by marine microorganisms as they build and break down organic matter. The scale of the ocean makes these processes globally relevant and, at the same time, challenging to fully characterize. Microbial community composition and ocean biochemistry vary across multiple physical scales up to that of the ocean basins. Other autonomous underwater vehicles are optimized for moving continuously and, primarily, horizontally through the ocean. In contrast, Clio , the robot that we describe, is designed to efficiently and precisely move vertically through the ocean, drift laterally in a Lagrangian manner to better observe water masses, and integrate with research vessel operations to map large horizontal scales to a depth of 6000 meters. We present results that show how Clio conducts high-resolution sensor surveys and sample return missions, including a mapping of 1144 kilometers of the Sargasso Sea to a depth of 1000 meters. We further show how the samples obtain filtered biomass from seawater that enable genomic and proteomic measurements not possible through in situ sensing. These results demonstrate a robotic oceanography approach for global-scale surveys of ocean biochemistry.

Microbial aerobic oxidation is known to be a significant sink of marine methane (CH4), contributing to the relatively minor atmospheric release of this greenhouse gas over vast stretches of the ocean. However, the chemical kinetics of aerobic CH4 oxidation are not well established, making it difficult to predict and assess the extent that CH4 is oxidized in seawater following seafloor release. Here we investigate the kinetics of aerobic CH4 oxidation using mesocosm incubations of fresh seawater samples collected from seep fields in Hudson Canyon, U.S. Atlantic Margin and MC118, Gulf of Mexico to gain a fundamental chemical understanding of this CH4 sink. The goals of this investigation were to determine the response or lag time following CH4 release until more rapid oxidation begins, the reaction order, and the stoichiometry of reactants utilized (i.e., CH4, oxygen, nitrate, phosphate, trace metals) during CH4 oxidation. The results for both Hudson Canyon and MC118 environments show that CH4 oxidation rates sharply increased within less than one month following the CH4 inoculation of seawater. However, the exact temporal characteristics of this more rapid CH4 oxidation varied based on location, possibly dependent on the local circulation and biogeochemical conditions at the point of seawater collection. The data further suggest that methane oxidation behaves as a first-order kinetic process and that the reaction rate constant remains constant once rapid CH4 oxidation begins.

We present a stereo-camera-based 3D multiple-vehicle-tracking system that utilizes Kalman filtering to improve robustness. The objective of our system is to accurately predict locations and orientations of vehicles from stereo camera data. It consists of three modules: a 2D object detection network, 3D position extraction, and 3D object correlation/smoothing. The system approaches the 3D localization performance of LIDAR and significantly outperforms the state-of-the-art monocular vehicle tracking systems. The addition of Kalman filtering increases our system’s robustness to missed detections, and improves the recall of our detector. Kalman filtering improves the MAP score of 3D localization for moderately difficult vehicles by 7.7%, compared to our unfiltered baseline. Our system predicts the correct orientation of vehicles with 78% accuracy. Our code, as well as a video demo, is viewable here.

During aerobic oxidation of methane (CH4) in seawater, a process which mitigates atmospheric emissions, the C-12-isotopologue reacts with a slightly greater rate constant than the C-13-isotopologue, leaving the residual CH4 isotopically fractionated. Prior studies have attempted to exploit this systematic isotopic fractionation from methane oxidation to quantify the extent that a CH4 pool has been oxidized in seawater. However, cultivation-based studies have suggested that isotopic fractionation fundamentally changes as a microbial population blooms in response to an influx of reactive substrates. Using a systematic mesocosm incubation study with recently collected seawater, here we investigate the fundamental isotopic kinetics of aerobic CH4 oxidation during a microbial bloom. As detailed in a companion paper, seawater samples were collected from seep fields in Hudson Canyon, U.S. Atlantic Margin, and atop Woolsey Mound (also known as Sleeping Dragon) which is part of lease block MC118 in the northern Gulf of Mexico, and used in these investigations. The results from both Hudson Canyon and MC118 show that in these natural environments isotopic fraction for CH4 oxidation follows a first-order kinetic process. The results also show that the isotopic fractionation factor remains constant during this methanotrophic bloom once rapid CH4 oxidation begins and that the magnitude of the fractionation factor appears correlated with the first-order reaction rate constant. These findings greatly simplify the use of natural stable isotope changes in CH4 to assess the extent that CH4 is oxidized in seawater following seafloor release.

Rare earth elements have generally not been thought to have a biological role. However, recent work has demonstrated that the light REEs (LREEs: La, Ce, Pr, and Nd) are essential for at least some methanotrophs, being co-factors in the XoxF type of methanol dehydrogenase (MDH). We show here that dissolved LREEs were significantly removed in a submerged plume of methane-rich water during the Deepwater Horizon (DWH) well blowout. Furthermore, incubation experiments conducted with naturally methane-enriched waters from hydrocarbon seeps in the vicinity of the DWH wellhead also showed LREE removal concurrent with methane consumption. Metagenomic sequencing of incubation samples revealed that LREE-containing MDHs were present. Our field and laboratory observations provide further insight into the biochemical pathways of methanotrophy during the DWH blowout. Additionally, our results are the first observations of direct biological alteration of REE distributions in oceanic systems. In view of the ubiquity of LREE-containing MDHs in oceanic systems, our results suggest that biological uptake of LREEs is an overlooked aspect of the oceanic geochemistry of this group of elements previously thought to be biologically inactive and an unresolved factor in the flux of methane, a potent greenhouse gas, from the ocean.

Previous studies of microbially mediated methane oxidation in oceanic environments have examined the many different factors that control the rates of oxidation. However, there is debate on what factor(s) are limiting in these types of environments. These factors include the availability of methane, O2, trace metals, nutrients, the density of cell population, and the influence that CO2 production may have on pH. To look at this process in its entirety, we developed an automated mesocosm incubation system with a Dissolved Gas Analysis System (DGAS) coupled to a myriad of analytical tools to monitor chemical changes during methane oxidation. Here, we present new high temporal resolution techniques for investigating dissolved methane and carbon dioxide concentrations and stable isotopic dynamics during aqueous mesocosm and pure culture incubations. These techniques enable us to analyze the gases dissolved in solution and are nondestructive to both the liquid media and the analyzed gases enabling the investigation of a mesocosm or pure culture experiment in a completely closed system, if so desired.