Dataset: V3-V4 16S rRNA sequence accessions from whole larvae and juveniles of Gigantidas childressi and co-located water samples
Deployment: TN391

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.