Dataset: Mortality metrics from minimally-lethal genotyping with eastern oysters (Crassostrea virginica) conducted in 2022

Juvenile oysters ranging from 30mm to 60mm were sourced from the Aquaculture Genetics and Breeding Technology Center (ABC) at the Virginia Institute of Marine Science (VIMS). Each oyster was cleaned using a 1% bleach solution, air-dried for an hour, and labeled with a shellfish tag (Hallprint FPN 8x4 mm) using coral epoxy (Instant Ocean HoldFast® Epoxy Stick) (Figure 1A). Shell width and length were measured for each oyster before the experiment (Kraeuter et al., 2007). To store the oysters before the experiment, the oysters were stored at 5°C for up to 48 hours before experimentation. After each trial, the oysters were transferred to sea tables (flow-through tanks filled with minimally filtered seawater from the adjacent York River), and mortality was monitored daily for 11 days. Daily mortality checks were conducted where each oyster was removed from the sea table briefly to check for shell gapes, which indicated loss of adductor muscle function and death.

Treatment groups: We planned nine treatments to determine which protocol would have the best DNA yield with the lowest mortality (Figure 1 of Leung et al. 2026 (in press)). Each treatment had at least 20 individual oysters. Groups of 10 oysters per each treatment were placed into separate trays in the sea tables. The treatments and short names are described in Table 1 of Leung et al. 2026 (in press).

1. Control Groups: We established two control groups in our experiment: one to monitor mortality and the other to quantify the amount of DNA extracted:
2. Mortality Control Groups: We had two groups to control for mortality. Mortality_Control_1: At the beginning of the experiment, we labeled 30 individuals using coral epoxy and placed them into the sea tables. Mortality_Control_2: At the end of our trials, we added another 30 individuals to the sea tables that were stored in the refrigerator for two days. This was to monitor any effects of being in the refrigerator, controlling for the longest time of oyster refrigeration.
3. DNA_Control: To establish a baseline for DNA quantification and quality, we sampled mantle, adductor, and gill tissues from 30 individuals and placed them into 2 mL O-ring vials with 95% molecular-grade ethanol. We stored the samples temporarily in a -20℃ freezer before transferring them to a -80℃ freezer.

Accessing the oyster tissue within the shell: Two methods were used to enter the oyster shell: creating a notch in the shell or relaxing the oyster. Creating a notch allowed for the extraction of fluid that contains genetic material (e.g., hemolymph or EPF), while relaxing the oyster allowed for tissue swabbing or direct sampling of the tissue.

1. Notch Protocol: A variable-speed dremel was used to make a small, V-shaped notch in the oyster shell, just large enough for the needle (BD 23G PrecisionGlide Needle) to fit in at different angles. The size of the notch depended on how much new shell an oyster had recently produced, but was typically a few mm in size. The notch was created on the right valve of the oyster, approximately two-thirds to the top of the shell. The needle, attached to a syringe (BD 1mL Syringe), was used to extract fluid as described below for the Hemolymph Protocol and the EPF Protocol.
2. Relaxation Protocol: For the relaxation protocol, 2 L of 1µm filtered seawater was mixed with 100g of Epsom salt until all the salt dissolved following protocol by Proestou et al. (2025). A maximum of ten oysters were placed in each replicate 2L container for 16 hours before the cell sampling (see Treatment Groups for experimental design). Oysters were fully submerged in the Epsom salt solution and held at room temperature. During relaxation, containers were aerated using air stones and airlines to maintain oxygen levels.

Collection of genetic material

Cell collection for notched oysters: Cells were collected from notched oysters by sampling hemolymph or EPF fluids. For the preservation of fluids, we split the samples and placed half in liquid nitrogen and the other half in FTA cards (see below):

1. Hemolymph Protocol: We inserted the needle through the notch diagonally down into the adductor muscle, which we identified through increased resistance. We collected 125 to 250 µL of hemolymph.
2. EPF Protocol: We inserted the needle into the notch until it reached the mantle cavity of the shell and collected 80 to 500 µL of EPF from each oyster. If necessary, we removed and reinserted the needle at multiple angles for small oysters to obtain a minimum fluid amount of 80 µL.

Cell collection for relaxed oysters: Cells were collected from relaxed oysters by taking a mantle biopsy or swabbing the mantle tissue.

1. Mantle Biopsy Protocol: We used forceps and scissors to remove a small piece (~10-20mg) of the mantle wrapped around the adductor muscle. We sampled the distal edge of the mantle tissue, approximately 2-3mm away from the visible edge of the adductor muscle. After each sample was acquired, we sterilized the forceps and scissors using an ethanol lamp.
2. Swab Protocol: Using an OmniSwap (Qiagen; WB100035), we swabbed in a circular motion, starting at the top layer of the mantle and moving down to the mantle around the adductor, then to the mantle’s bottom layer, and then reversing this path. The motion was repeated five times for each sampled individual.

Preservation of genetic material: We used three preservation protocols for the genetic samples: ethanol, flash-freezing, and FTA cards:

1. Ethanol Protocol: The mantle sampled from the relaxed oysters was preserved with 95% molecular-grade ethanol. It was temporarily stored in a -20℃ freezer and then transferred to a -80℃ freezer.
2. Flash-Freeze Protocol: Liquid nitrogen was used to flash-freeze half of each EPF or hemolymph sample from the notched oysters. After the samples were flash-frozen, they were stored in a -80℃ freezer. Additionally, the swabs used on the relaxed oysters were flash-frozen and then stored in a -80℃ freezer.
3. FTA Cards Protocol: The remaining half of the EPF and hemolymph samples were stored on QIAcard FTA Classic Cards (Qiagen; WB120305). Following the manufacturer's instructions, we put up to 125 µL of fluid on each circle in the FTA card. FTA cards were stored in WhirlPak bags with color-indicating silica beads (desiccants that absorb moisture and change color to indicate saturation) following the manufacturer’s recommendations.