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

Veligers, pediveligers, juveniles, and adult mussels of Gigantidas childressi were collected using the remotely-operated vehicle (ROV) Jason II and automated underwater vehicle (AUV) Sentry (National Deep Submergence Facility, Woods Hole Oceanographic Institution) onboard the R/V Thomas G. Thompson (University of Washington) during cruise TN391. Samples were collected from Mississippi Canyon 853 (28° 7.37’ N, 89° 8.42’ W) in the Gulf of Mexico at a depth of 1070 meters on June 16th, 2021 (Jason dive J2-1337 and Sentry dive S595P). Adult Gigantidas childressi are morphologically distinguishable. Gigantidas childressi adults were sampled with ROV Jason and recovered to the surface in insulated bioboxes. Pediveligers (swimming larvae with a developed foot that are competent to undergo metamorphosis) and juveniles (metamorphosed) of G. childressi were collected from the interstices of mussel beds with the ROV Jason suction sampler and within samples of adults. Veligers (pre-competent swimming larvae without a developed foot) were collected in the water column at an altitude of 5 meters above bottom with AUV Sentry fitted with the SyPRID plankton sampler with 150-micron mesh nets (Billings et al. 2016). A paired 2-liter (L) water sample was also taken at an altitude of 1.5 meters above the bottom at the time of fauna collection with two 4L Niskin bottles equipped on ROV Jason.

Plankton samples recovered by the AUV Sentry SyPRID sampler were immediately rinsed from the collectors with cold 0.3-micrometer (um) filtered seawater (FSW) into canisters of chilled FSW. Larvae and juveniles recovered from ROV Jason suction and scoop samples were retained on a 253 um mesh sieve and resuspended in cold FSW. All live plankton and juveniles were maintained below 8 degrees Celsius (°C) (ambient temperature) during subsequent processing. Live G. childressi larvae were immediately sorted from the samples under a dissecting microscope, imaged on a compound light microscope, then preserved individually in 0.5 milliliters (mL) centrifuge tubes with 95% molecular grade ethanol. Snips of gill tissue from adult mussels were dissected aseptically on the ship and stored at -80°C in 2 mL cryovials. Background water samples were collected into sterilized plastic canisters, stored at ambient seafloor temperature (8°C), and processed immediately after recovery. Each sample was vacuum filtered onto a 0.2 um polycarbonate filter. Filters were placed into sterile 2-mL centrifuge tubes and stored at -80 °C until further processing.

All sample processing for DNA sequencing was conducted at Shannon Point Marine Center in Anacortes, Washington. DNA was extracted from individual larvae, juveniles, and samples of adult gill tissue (2.5 milligrams) using the Nucleospin Tissue XS kit (Machery-Nagel) following the manufacturer's instructions. DNA was extracted from filters using the Fast DNA Spin Kit for Soil (MP Bio) according to the manufacturer's instructions. The concentration of extracted DNA was quantified using a Qubit fluorometer (2.0).

For microbiome community composition, amplification, library preparation, and sequencing of the V3-V4 regions of the 16S rRNA gene was conducted by Exact Scientific (Ferndale, WA). Library preparation was performed following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (15044223B). DNA was amplified using the following bacterial-specific primers: Forward: 5’ TACGGGNGGCWGCAG, Reverse: 5’ GACTACHVGGGTATCTAATCC (S-DBact-0341-b-S-17/S-D-Bact-0785-a-A-2; Klindworth et al. 2013). PCR conditions for 16S rRNA amplicons were 95 °C for 3 minutes, with 25 cycles at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 5 minutes and a holding temperature of 4°C. Multiplexing indices and Illumina overhang adapters were attached with a second limited-cycle PCR step using the NEXTERA® XT Index Kit. Resulting PCR products were purified after each step with Ampure XP Beads™, 80% EtOH, and 10 mM Tris pH 8.5. Libraries were then normalized, pooled, and sequenced on an Illumina MiSeq platform using 600 cycle v3 chemistry.

Processing and analysis of Illumina sequences followed the QIIME2 computational pipeline for the V3/V4 region of the 16S rRNA gene as in Carrier et al. (2021). Illumina reads with quality information were processed using QIIME 2 (ver. 2021.8; Bolyen et al., 2019). Adapters and primers were removed and forward and reverse reads were paired with VSEARCH (Rognes et al. 2016). Paired sequences were filtered by a minimum quality score of 25 and denoised with Deblur (Amir et al. 2017). Features were analyzed as amplicon sequence variants (ASVs) and were assigned taxonomy using the latest version of the SILVA (ver. 138.1) bacterial database.

- Imported original file "BeaverAndArellano2024_SRA.csv" into the BCO-DMO system.
- Added columns for collection latitude, longitude, date, and depth.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved final file as "940537_v1_v3-v4_16s_rrna_sequences_g_childressi.csv"

NSF Award Abstract:
Ever since hydrothermal vents and methane seeps were first discovered in the deep ocean more than 40 years ago, scientists have wondered how these isolated communities, fully dependent on underwater "islands" of toxic chemicals, are first colonized by organisms, and how the populations of these specialized animals are exchanged and maintained. These fundamental processes depend on the transport of babies (larvae) by the ocean currents, yet because the larvae are microscopic and diluted in the vastness of the ocean, it is very difficult to determine where and how they drift. This project uses an autonomous underwater vehicle to collect larvae from precise regions of the water column. Larval traps on the bottom and chemical analyses of larval shells will also be used to determine the depths where larvae swim. These findings will provide realistic estimates for mathematical models that show how biology interacts with ocean currents to predict which methane seeps will be colonized by larvae originating at different depths. A detailed knowledge of larval dispersal is needed for conservation and management of the deep sea. Without such information, we cannot know the best placement of marine protected areas, nor can we facilitate the reestablishment of communities impacted by deep-sea mining, drilling, or other human activities. This project will provide hands-on at-sea training for college students to learn the rapidly vanishing skills needed for studies of larvae and embryos in their natural habitats. Learning opportunities will also be available to individuals of all ages through new, interactive exhibits on deep-sea biology and larval ecology produced for small museums and aquaria on the coasts of Oregon, Washington and North Carolina.

Reliable estimates of connectivity among metapopulations are increasingly important in marine conservation biology, ecology and phylogeography, yet biological parameters for biophysical models in the deep sea remain largely unavailable. The movements of deep-sea vent and seep larvae among islands of habitat suitable for chemosynthesis have been inferred from current patterns using numerical modeling, but virtually all such models have used untested assumptions about biological parameters that should have large impacts on the predictions. This project seeks to fill in the missing biological parameters while developing better models for predicting the dispersal patterns of methane seep animals living in the Gulf of Mexico and on the Western Atlantic Margin. Despite the existence of similar seeps at similar depths on two sides of the Florida peninsula, the Western Atlantic seeps support only a subset of the species found in the Gulf of Mexico. It is hypothesized that the ability of larvae to disperse through the relatively shallow waters of the Florida Straits depends on an interaction between the adult spawning depth and the dispersal depth of the larvae. Dispersal depth, in turn, will be influenced by larval flotation rates, swimming behaviors, feeding requirements, and ontogenetic migration patterns during the planktonic period. The recently developed SyPRID sampler deployed on AUV Sentry will be used to collect larvae from precise depth strata in the water column, including layers very near the ocean floor. Larval traps deployed on the bottom at three depths in each region will be used in conjunction with the plankton collections to determine what proportion of larvae are demersal. Comparisons of stable oxygen isotopes between larval and juvenile mollusk shells will provide information on the temperatures (and therefore depths) that larvae develop, and geochemical analyses of larval and juvenile shells will determine whether larval cohorts mix among depth strata. Ocean circulation and particle transport modeling incorporating realistic biological parameters will be used to predict the movements of larvae around the Florida Peninsula for various spawning depths and seasons.

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.