Dataset: 16S rRNA V4 sequence metadata from Apostichopus californicus-associated flavivirus experiment under suboxic conditions and organic matter amendment

Forty-two specimens of Apostichopus californicus (urn:lsid:marinespecies.org:taxname:529363) were collected by SCUBA divers in Thimbleberry Bay (57.0297N, 135.2283W), near Sitka, Alaska on 10 November 2021 and transported together in plastic tubs to the lab at the University of Alaska Southeast (Japonski Island, Sitka). Specimens were immediately weighed, photographed, and placed into individual mesh containers within 7 x 1200 L outdoor mesocosms (6 specimens per mesocosm) filled with seawater from the nearby Sitka Channel. Two mesocosms served as controls (no amendment), 4 mesocosms were subject to daily organic matter (20 µM) amendment, and 1 mesocosm was continuously sparged with N₂ (Airgas, medical grade; the other 6 mesocosms were bubbled continuously with air). Seawater was subject to 50% volume water change daily and specimens were not fed during captivity. Mesocosms were covered while not sampled (i.e., they were light limited). We selected two organic matter substrates (glucose and peptone) based on their ability to stimulate microbial activity in prior work in addition to two common constituents of dissolved organic matter in coastal environments (N-acetylglucosamine and fucose + rhamnose). We monitored dissolved oxygen levels in each mesocosm using continuous submersible HOBO loggers.

Surface swabs of each individual were collected daily by rubbing a Puritan polyester sterile swab over a ~ 1 cm² area of epidermis. Swabs were cut using clean scissors to remove the polyester tip and placed into 2 mL cryovials containing RNALater. Tube feet samples were collected from each specimen daily using disposable plastic forceps, and feet were placed immediately into cryovials containing RNALater. A 5mm biopsy punch of the dorsal body wall was collected from half of the individuals in each mesocosm at 0, 1, 3, and 6 d, which were then preserved in RNALater. All specimens were photographed and weighed daily. All RNALater preserved samples were frozen at -80°C and transported in liquid N₂ to the laboratory at Cornell University. Animal carcasses were frozen at -20°C and transported on blue ice to Cornell.

Surface swabs were collected from 42 animals over the course of 7 d (sampled on t = 0 d, 1 d, 3 d, 5 d, and 7 d) and frozen at -20°C until further processing. DNA was extracted from frozen swabs using Zymo Quick-DNA Fungal/Bacterial kits (Zymo Research) as per the manufacturer’s protocol. Bacterial communities in sample extracts were identified using dual-barcoded PCR (polymerase chain reaction) amplification and sequencing of the V4 region of the 16S rRNA gene. Each 40 µL PCR reaction comprised 1X PCR master mix (One-Taq Quick-Load 2x Master Mix with Standard; New England Biolabs), 0.125 µM of each barcoded primer (515f; 5’-GTG YCA GCM GCC GCG GTA A-3’ and 806r; 5’-GGA CTA CNV GGG TWT CTA AT-3’), and 2 uL of template (swab extract). 16S rRNA amplicons were pooled at equimolar concentrations using SequalPrep Normalization Plate kit (Invitrogen) and sequenced on one lane of Illumina MiSeq (2 x 250 paired end) at the Cornell University Biotechnology Research Center. 16S rRNA amplicon sequences were submitted to NCBI (BioProject accession number PRJNA947521, see Related Datasets).