Table of Contents

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

Sample Collection

In this experiment, plankton were treated as two groups: those exposed to microbial biofilms (2024) and those not (2022). To collect biofilm, “Biofilm Colonizers,” sections of PVC piping covered in stainless steel mesh on either side and attached with metal band clamps, were deployed on the benthos by HOV Alvin on sites with high diffuse flow (confirmed by temperature probe reading higher than ambient temperatures) and left to be colonized by vent biofilm-forming bacteria for 10 d before recovery. For the 2024 (biofilm) observations, segments of colonized mesh were cut to size using flame-sterilized tweezers and scissors, secured in the custom aluminum inserts, and placed in the bottom of the plankton observation chamber. This design was intended to test response to a substratum-bound cue assuming a tactile trigger, but the biofilm was flocculent and became suspended when the chamber was filled, likely bringing it into contact with larvae throughout the observation pool. For the 2022 (no biofilm) observations, the insert was not used.

Larvae were collected using a McLane Laboratories large-volume water pump (WTS-LV) with a filter assembly to capture larvae onto a large (19 cm diameter) filter of 63 μm mesh. The filter system was designed with an expanded head space and thick insulation to maximize retrieval of live specimens (Beaulieu et al. 2009). The pump was mounted on a lander to facilitate manual positioning via HOV Alvin. The lander was placed at about 10 m distance from the active vent site, and the inlet was positioned at 1.5 m above the seafloor, where the water temperature was 2°C and pressure was 252 bar. The pump was programmed to run at a rate of 25 L per minute for 21 h to end just before release from the seafloor to maintain larval health, capturing samples of roughly 31,000 L. The lander ascended to the surface at a rate of approximately 45 m per minute. Due to the warm surface waters of the eastern tropical Pacific, care had to be taken to recover the sample quickly, as prolonged exposure to elevated temperature can damage the larvae. At this site, our CTD data showed that temperature was below 5°C at depths greater than 1000 m, reached 10°C at about 300 m, and was above 25°C at the surface. After the pump was recovered and secured on deck, the pump filter assembly was immediately placed in chilled seawater (4°C). Gastropod larvae were sorted manually from the samples, on ice under dissecting microscopes, for 1–2 h, and identified by trained experts. All available gastropod larvae (150–400 μm) and 6–12 copepods were placed in the plankton observation chamber. Copepods, being larger and more active swimmers, were added as a visual aid to ensure the camera remained in focus and conditions in the chamber remained habitable. The chamber was topped off with filtered bottom seawater, sealed, and fastened to the mount to prepare for recording behavior. In 2022, one of six recovered pump samples yielded actively swimming gastropod larvae (pump 8), whereas in 2024, one of seven pump samples yielded swimming larvae (pump 5). In 2022, three gastropod larvae were introduced into the chamber (Lepetodrilus sp., Laeviphitus sp., and “unknown”), and all revived sufficiently to swim actively in the chamber. In 2024, after the biofilm insert was placed in the chamber, nine gastropod larvae were introduced (identified with certainty Lepetodrilus sp. and Peltospira sp., and provisionally Laeviphitus sp., Echinopelta sp., Clypeosectus sp. and “unknown”); two of these individuals revived, but it was not possible to distinguish which ones.

Recording set-up

Deep sea larvae were loaded and sealed in a high-pressure chamber within two hours of surfacing. The chamber was kept in a dark 4 degree (Celsius) cold room for duration of the experiments. Bottom pressure (252 bar) was slowly reached over the course of 15 min inside the chamber.  Once at pressure, pump flowrate was kept at 0.05-0.2 milliliters per minute (mL/min). Videos were recorded as TIFF stacks using a frame rate of 30-60 fps. Occasionally, the chamber was manually flipped to attempt to stimulate larvae into swimming. Experiments continued until larvae stopped swimming, between 2-3 hrs. Recording was paused, breaking up footage into "Sessions" due to limited writing speed of our data storage equipment  and followed by waiting periods of up to 3 minutes while memory was written to disk. "Sessions" were broken up into "partitions" whenever the camera frame of reference was moved, or if the chamber was flipped during recording. 

Differences between cruises

AT50-06 (2022): Camera resolution was limited to 944x950 and chamber viewport was partially in view at 798 pixels per centimeter. Pressure chamber and camera were secured but detached one another. Framerate was set to 60 frames per seconds and sessions were limited to 3 minutes. Chamber was flipped a total of four times.

AT50-20 (2024):  Camera resolution was set to the full 2048x2048 pixels, putting the whole chamber viewport in view at 563 pixels per centimeter. Pressure chamber was secured on a custom 3d-printed mount, attached to an aluminum railed that attached the mounted camera, keeping centered and fixed on the chamber. Framerate was lowered to 30 frames per second, and sessions were extended to last 30 minutes. Chamber was flipped a total of 2 times. Gastropods were exposed to vent-specific microbial biofilm for the duration of this experiment.

Particle tracking (generating vtracks files)

Custom two-dimensional particle tracking scripts on MATLAB (version 2023a) were used to process the TIFF stacks and extract tracking data.  After removing a mean background image, tracked particles, treated as weighted centroids, were filtered for minimum intensity, minimum size, and maximum speed. Instantaneous vertical velocity was calculated using a 7-point differencing stencil and a gaussian filter as in Oullette et al. (2006). 

Swim metrics (generating swim metrics file)

Larval tracks were identified manually by watching the videos with particle labelling overlay generated by the tracking scripts. Tracks of larvae were assigned into behavior categories (downhelix, downswim, sink, uphelix, upswim, and meander) after watching the resulting overlaid tracking videos and observing the larva's behavior. Larval behavior tracks of interest were entered into a formatted spreadsheet. If a single track contained multiple behaviors, the track was split into multiple spreadsheet entries with different start and end frames. This spreadsheet was then fed into another MATLAB script to extract the swim metrics from the vtracks files that were used to create our descriptive table.

- Opened "swim_metrics_datatable_ZunigaMouretetal_L&Om.xlsx" in Excel and formatted all decimals to "#.##############################" to capture all numbers in decimals
- Exported file from Excel as a CSV, "swim_metrics_datatable_ZunigaMouretetal_L&Om.csv"
- Imported "swim_metrics_datatable_ZunigaMouretetal_L&Om.csv" into the BCO-DMO system
- Combined the pump, session, partition, and track number ids into a "trackID" parameter that should correspond with the ID used in the .mat files
- Created a "matFilename" parameter that connects the rows to the .mat file in the dataset supplemental attachment
- Exported file as "982238_v1_swim_metrics_vent_gastropod_larvae.csv"

Organism LSIDs added from matches at the World Register of Marine Species (WoRMS) on 2025-10-18. All species names in metadata are exact matches.

Lepetodrilus (urn:lsid:marinespecies.org:taxname:180907)
Peltospira (urn:lsid:marinespecies.org:taxname:180916)
Laeviphitus (urn:lsid:marinespecies.org:taxname:137922)
Echinopelta (Echinopelta fistulosa) (urn:lsid:marinespecies.org:taxname:449957)
Clypeosectus (Clypeosectus delectus) (urn:lsid:marinespecies.org:taxname:449949)

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.