Dataset: Dynamics of the Apostichopus californicus-associated flavivirus under suboxic conditions and organic matter amendment in mesocosm experiment

Forty-two specimens of Apostichopus californicus (urn:lsid:marinespecies.org:taxname:529363) were collected by SCUBA divers in Thimbleberry Bay, 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.

Quantitative reverse transcriptase PCR (qRT-PCR) using primers PcaFV_NS5_F3 (5’- CCA GCC ATG GAT GAG TAA TG-3’) and PcaFV_NS5_R3 (5’- GCT GAA CTG CTC CTG AAA CC-3’), and probe PcaFV_NS5_Pr3 (5’-[FAM]CAC GAA TGT ACG GCA ACG GAC G[TAMRA]-3’) was used to determine PcaFV load within individual tissue RNA extracts. qRT-PCR reactions were performed in duplicate for each specimen and compared against duplicate reactions of oligonucleotide standards spanning the amplicon region (from 10² to 10⁷ copies µl^-1) and using 4 negative controls (nuclease free H₂O only). PCR reactions (20 µL) contained 1 X Luna Universal Probe One-Step Reaction Mix (New England Biolabs), 1X Luna WarmStart RT Enzyme Mix (New England Biolabs), 8 µmol each of primers and probes, and 1 µL of template RNA. Reactions were thermal cycled in an ABI StepOne qPCR machine. Reactions were initially held at 55°C for 10 min, followed by an initial denaturation step at 95°C for 1 min. Reactions were then subject to 45 cycles of denature at 95°C and anneal at 56°C, with fluorescence data collected after the annealing step. Linearity of standards was checked during downstream analysis of qPCR data.

Relative microbial activity was measured in each mesocosm and from the surface of 2 specimens per mesocosm by bromodeoxyuridine (BrDU) incorporation . Five 2 mL water samples were retrieved from each mesocosm by pipette and placed into 2 ml cryovials. Water samples (5 replicates) were collected from the surface of each of two haphazardly chosen specimens by pressing the pipette tip against the dorsal epidermis and slowly retrieving sample, which was then placed into 2 mL cryovials. Water samples were inoculated with 0.04 nmol BrDU and incubated in the mesocosm for 8 h. Incubations were then retrieved, and flash frozen in liquid N₂.

A standard for comparison between BrDU blots was made by amending 50 mL of water collected from an aquarium with 0.04 nmol BrDU. A single sheet of chromatography paper was placed in a Tupperware container soaked with 6X SSC Buffer with a nylon transfer membrane on top and allowed to soak for 10 minutes. The nylon transfer filter was then mounted in a slot blot manifold. Samples (0.5 mL) were pipetted into slot wells, where each membrane included 6 standards, then vacuum filtered through the membrane. The membrane was placed sample side down onto a stack of 3 sheets of chromatography paper with the lysis solution, then immediately turned sample side up and incubated for 10 min. This was repeated in the same fashion for neutralization solution. The membrane was then placed sample-side down on a piece chromatography paper soaked in FixDenat for 15 s, followed by 30 min incubation on the FixDenat stack. The membrane was placed between two sheets of chromatography paper and baked at 80°C for ~1 h.

The dried membrane was placed in a plastic tray, 25 mL Blocking Solution was added, and the apparatus incubated on a rotating incubator at 30 rev min^-1 for 1 h. After this time, the blocking solution was poured off and 6 mL of Antibody Incubation Solution was added to the tray, which was placed on a rotating incubator at 30 rev min^-1 for 3 h. The antibody incubation solution was poured off and 25 mL wash solution was added, which was incubated on rotating incubator for 5 min. The wash step was repeated. After washing, the wash solution was poured off, and 25 mL of maleic acid buffer was added and incubated on rotating incubator for 5 min. This maleic acid buffer treatment then was repeated. The nylon transfer membrane was placed in a plastic tray, sample side up, to which 4 mL of the Supersignal West Femto was added and incubated for 2 minutes. The membrane was visualized in a BioRad ChemiDoc system at high sensitivity and 4 x 4 binning with an exposure time of 600 s.

Protein content was measured in body wall tissues using RIPA buffer extraction and Bradford reagent spectrophotometry. Two biopsy punch (5 mm) tissue samples were harvested from frozen animal carcasses and placed into 2 mL microcentrifuge tubes. RIPA buffer (2 mL; 150mM NaCl, 10 mM Tris, 1 mM EDTA, 1% Triton X-100, 0.01% sodium dodecyl sulfate, and 0.01% sodium deoxycholate) was added to each sample, and samples were homogenized in a mortar and pestle for 2 min or until the tissue was completely homogenized. Samples were incubated at room temperature in a shaking incubator for 2 h. Following this, tubes were centrifuged at 16,000 x g at 4°C for 20 min, and supernatant removed into a new microcentrifuge tube. Protein quantity was determined against a bovine serum albumin standard. Bradford reagent (1500 µL) was added to 30 µL sample, allowed to incubate at room temperature for 15 min, then absorbance at 595 nm measured on a spectrophotometer.

Lipid content was assessed. Two biopsy punch (5 mm) tissue samples were harvested from animal carcass dorsal body wall and placed into 15 mL centrifuge tubes. Tissue samples were weighed, amended with 12 mL chloroform:methanol, then homogenized in a mortar and pestle until the tissue had completely disintegrated and resembled a paste. The homogenates were centrifuged at 3,000 x g for 10 min in a benchtop centrifuge and the solvent fraction removed into fresh centrifuge tubes. Three microcentrifuge tubes per sample were weighed, and then 2 mL of the solvent fraction from each tissue sample homogenate was placed into the tubes. Tubes were then placed into a rotating vacuum desiccator until they were dry. Sample tubes were then re-weighed, and mass difference calculated on aggregate for all replicate tubes per sample as a percentage of wet weight.