Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Collection:
Meroplankton larvae and benthopelagic invertebrates were collected using DeepZoo, a novel autonomous sampler developed at Woods Hole Oceanographic Institution's Autonomous Vehicles and Sensor Technologies Innovation Hub (WHOI AVAST). DeepZoo is a cost-effective, lightweight (6.4 kg), all-depth device currently at the working prototype stage. It integrates two systems: (1) sample collection, composed of a lid with an oil-filled 10 RPM gear motor (ServoCity, USA), a 63-µm Nitex mesh net, and a T200 thruster (Blue Robotics, USA) to drive water flow; and (2) system control, housed in titanium and containing electronics, including a 14.8 V, 10 Ah rechargeable lithium-polymer battery (Blue Robotics). Pre-programmed in Arduino IDE 2.3.6 (Arduino 2024), DeepZoo runs autonomously, controlling motor and thruster operations (i.e., timing and speed) according to dive plans. The thruster operates at a specific rate, defined by a pulse-width modulation (PWM) signal, which ranges from 1500 to 1900 microseconds. The two systems are contained within a lightweight PVC frame with SpeedRail connectors and a wire-mesh base (91.4 × 27.9 × 30.5 cm).

DeepZoo was deployed three times in a horizontal configuration, once on the MISO Lander and two on the HOV Alvin science basket. On the MISO lander, DeepZoo was deployed in parallel with a large-volume plankton pump (McLane WTS-LV50, Falmouth, MA, USA). Before sampling, HOV Alvin repositioned the lander to be within meters of YBW-Shimmering Forest. Both systems were sampled over the same 21.5-hour period. This sampling design makes the two systems comparable, where the key difference is that the pump is situated 1.5 m above the seafloor and samples vertically. On HOV Alvin, DeepZoo was mounted on the starboard side of the basket with the mouth facing forward. During these two deployments, DeepZoo sampled while HOV Alvin was moving on the seafloor, including during transit to and from a station. Compared to the lander, DeepZoo sampled for a shorter period at a higher thrust rate.

For the named vent sites in this dataset, we used the most recent benchmarked, georeferenced positions from Table 1 in Wu et al. (2022). Bottom depth for positions near 9 50’ N was determined in QGIS using bathymetry acquired in 2018, 2019, and 2021 (Parnell-Turner et al. 2022).

Shipboard sample processing:
Upon recovery, the net section of DeepZoo was placed into a 5-gallon bucket with chilled, filtered seawater (CFS; 0.5-micron) and brought to the cold room (4°C). The net was rinsed with CFS and then poured over a 63-micron sieve. The sieve was washed with 95% non-denatured ethanol into a 50 mL Falcon tube. 

Laboratory sorting and morphological identification:
Samples were poured over nested 250- and 63-micron sieves and sorted in dishes with 96% ethanol under a Leica S9i stereo microscope at magnifications up to 55x. Meroplankton larvae and benthopelagic invertebrates were transferred to a 6-well plate with 96% ethanol. 

Morphological identifications followed the identifications set by the “EPR pump time series” dataset (986309; see Related Datasets section below). Individuals were identified to the lowest taxonomic level as morphotypes, with an emphasis on larval gastropods (Mills et al., 2009). Gastropods were identified by Susan Mills and Johanna Weston. All morphotypes of meroplankton larvae and benthopelagic invertebrates (e.g., amphipods) were enumerated and placed into taxon-specific containers. The presence or absence of “possibly benthic forams” was denoted. Holoplanktonic taxa (e.g., planktonic copepods and chaetognaths) were excluded from counts but denoted for their presence or absence.  

A subset of 35 individuals spanning morphotypes were selected for the DNA barcoding of the cytochrome c oxidase subunit I (COI) region (target ~650 bp) using the HotShot method (Truett et al., 2000). Each individual was placed into a 0.5 mL PCR tube with 10 μL of HotShot lysis reagent (25 mM NaOH, 0.2 mM EDTA). Lysis reactions were incubated at 95°C for 30 min, then cooled to 4°C for 15 min. Subsequently, 10 μL of the neutralization reagent (40 mM Tris-HCl) was added, the mixture was spun down, and it was incubated at 4°C for 10 min. PCR amplification was performed as described by Meyer-Kaiser et al. (2025) with the COI primers jgLCO1490 [5’-TITCIACIAAYCAYAARGAYATTGG-3’] and jgHCO2198 [3’-TAIACYTCIGGRTGICCRAARAAYCA-5’] (Geller et al., 2013) and Promega Go Taq™ Master Mix. PCR products were visualized using a blueGel™ electrophoresis system (miniPCR). All products with banding were sent to Sequegen Inc. (Worcester, MA) for silica-glass purification and Sanger sequencing. 

PCR products and clean sequences were generated from 15 individuals. Electropherograms were manually inspected and trimmed in MEGA 11 (Tamura et al., 2021). Any ambiguous base calls were denoted by 'N'. Sequences were translated to assess for the presence of stop codons. Sequences were compared against existing published sequences in the Barcode of Life Database (BOLD v5, Ratnasingham & Hebert, 2007) and in GenBank using the BLASTn algorithm (Camacho et al., 2009). Sequences with >97% identity were considered species-level matches, while those that matched >93% to published records were considered a genus-level match. Sequences with >80% match to published records were assigned to the family or superfamily level based on morphological identification and tree placement in BOLDv5. Genetic sequences are submitted to GenBank (accession numbers: PX280612-PX280626).  

The scientificName determination came from the combination of the morphotype and genetic evidence, when available, to match the lowest-level identification to the World Register of Marine Species (WoRMS) taxonomic database. For this dataset, the verbatimIdentifications include the 64 morphotypes found across the “EPR pump time series” (https://www.bco-dmo.org/dataset/986309), allowing for direct relative abundance and community composition comparison. In addition to the shared 64 morphotypes, eight morphotypes were included. Six of the eight morphotypes represent lower taxonomic levels identified (e.g., amphipods to Ventiella sulfuris) or new morphotypes not seen in the pump dataset (e.g., an unknown gastropod bubble). The other two of eight morphotypes, chaetognaths and copepods, were denoted in this dataset as presence/absence.

Funding Acknowledgement
Support for this work and the generation of these data was provided by the U.S. National Science Foundation Division of Ocean Sciences; the Rinehart Initiative for “Access to the Sea” at Woods Hole Oceanographic Institution; and the Cecil H. and Ida M. Green Technology Innovation Award II Fund and the Alfred M. Zeien Endowed Fund for Innovative Ocean Research at Woods Hole Oceanographic Institution.

The data was processed by populating hand-generated notes and counts into this long-format table that was formed on the foundation of the BCO-DMO dataset  “EPR pump time series” (https://www.bco-dmo.org/dataset/986309). The  event columns information was manually pulled from cruise report data.

This dataset is provided as a long-format, comma-separated variable (csv) file that joins left/right 2 additionally provided csv files. Leftmost columns match to the provided Darwin Core Occurrence extension table; rightmost columns match to the provided Darwin Core Event core table. The occurrence columns of the data table include taxonomic standardization for the count.

- Loaded "EPR_DeepZoo_BCODMO_submitted_20251106.csv" into the BCO-DMO system treating "NA" and blank values as missing
- Extracted latitude and longitude coordinates from the footprintWKT column using regex
- Extracted geometry type (LINE or MULTIPOINT) from the footprintWKT column using regex
- Added cruise ID of AT50-33
- Exported file as "991014_v1_deepzoo_epr.csv"

NSF Award Abstract:
Over four decades of research have shown that tiny free-swimming offspring of the unique inhabitants of hydrothermal vents can disperse effectively between their specialized habitats. Yet, we know almost nothing about how these larval animals complete the journey by locating and settling down in suitable locations. This question remains one of the key unresolved puzzles in the ecology of the deep sea and is becoming increasingly important to solve as hydrothermal vents are becoming threatened by human impacts. The investigators suggest that the films of bacteria that first form at vents are good signposts for settlement of larvae because they indicate that the hydrothermal vents are suitable for life. This project uses a combined program of field experiments, cutting-edge molecular biology techniques, and shipboard experiments with hydrothermal-vent larvae and cultured bacterial films. The project also connects undergraduate research interns at a primarily undergraduate institution (Western Washington University) with undergraduate research interns at two research institutions (Rutgers and Woods Hole Oceanographic Institution) while working on the project at sea together. Finally, the team is producing a science-in-action documentary filled with ocean science and exploration intended for television distribution and museum screenings. The investigators are using footage of the deep-sea vents, shipboard and diving operations, and laboratory work to create a documentary that highlights the foundation of scientific research—hypothesis-driven research, the application of the scientific method, and the importance of critical thinking—all in the framework of the study of an exciting, but threatened, ecosystem.

Hydrothermal vents are particularly tractable systems in which to study questions about the roles of biofilms in larval settlement because biofilms at vents are relatively low-complexity; vent animals are strictly dependent on vent microbes, often through symbiotic partnerships acquired after settlement; and environmental variations are present within the range of a common larval pool. Moreover, decades of research on settlement in model organisms give us good insight into biofilm cues; there is solid foundational understanding about colonization patterns at vents; we now have excellent tools to collect, identify, and culture vent larvae and microbes; and modern environmental "-omics" techniques are a good tool to characterize biological cues produced by biofilms. The project provides an unprecedented, quantitative look into the role of microbial biofilms in structuring larval settlement at hydrothermal vents, achieved only through the close collaboration of microbial and larval ecologists. The combined field program of short-term settlement experiments, microbial "-omics" work, and subsequent shipboard settlement experiments allows the investigative team to use field experiments to statistically model the factors that best predict larval settlement in the field, then test those predictions with shipboard experiments that decouple covarying conditions. This extensive characterization of putative larval settlement cues and their relationship to colonization success in heterogeneous vent habitat niches will contribute to a broader understanding of colonization success across diverse marine ecosystems. Understanding the role that the initial settlement of larvae plays in the recovery and resilience of hydrothermal-vent ecosystems is critical to developing informed management plans for deep-sea mining.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Award Abstract:
This project is investigating a newly discovered community of animals and microbes near deep-sea hydrothermal vents that appears to inhabit only cool, inactive sulfide features. The main objectives are to determine what species live on these features, whether they are new to science, and how they function in the community. The discovery of this novel community, which may be fueled by production of resident microbes, is likely to change the way we think about inactive vents and their contribution to deep-sea biodiversity and productivity. This project has broad impact in four different areas: 1) Informing policy for sustainable use (mining) of inactive sulfides; 2) Contributing to global data systems and the NSF-funded repository at BCO-DMO to make our data available for research use at other temporal, spatial, and taxonomic scales; 3) Increasing public scientific literacy by enhancing K-12 education in the sciences at Memorial Junior High in Eagle Pass TX with about 98% Hispanic and 2% Native American students and a high number of English Language Learners and migrants; and 4) Developing a diverse workforce by engaging students from under-represented and marginalized groups into undergraduate intern programs.

Hydrothermal venting of heated, reduced fluids from the seafloor occurs globally at plate tectonic boundaries and mid-plate hotspots and has been the subject of vigorous geological, chemical and biological research. However, this venting is ultimately transient, leaving behind only the sulfide mineral-rich deposits after the fluid flow stops. This project investigates the organisms living on these lesser studied inactive sulfide features in order to understand their ecology and associations with the mineral substratum. Recent discoveries indicate that some microbial and animal species inhabiting inactive sulfides are not found elsewhere in the marine environment, suggesting the sulfides serve as a unique habitat, distinct from other seafloor topographic features. The main project objectives are to characterize the species and functional diversity of the inactive sulfide ecosystem across all three domains of life (eukaryotic, bacterial, and archaeal), determine which animal species are endemic or predominantly associated with inactive sulfides, and explore the biological and geological characteristics governing those associations. The investigators are conducting field studies between 9-10 degrees N on the East Pacific Rise at sites within the axial summit trough as well as at recently discovered off-axis sites away from modern day venting features. The discovery of this novel community of organisms inhabiting inactive sulfide features at hydrothermal vent fields, fueled by resident chemolithotrophic microorganisms, is likely to change the way we think about the role of these ecosystems in deep-sea biodiversity and productivity.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.