Dataset: Dose response evaluation of oyster shell strengthening in response to homarine and trigonelline

Diploid oyster spat were purchased from the Auburn University Shellfish Laboratory and settled onto 4.5 cm × 4.5 cm marble tiles. For one week after settlement, spat on tiles were caged and kept in 1250 L mesocosms with natural flowing seawater from Mobile Bay at a flow rate of 20 L/min. We then ensured each tile had at least 15 oyster spat and used high-density polyethylene fishing line to tie tiles back-to-back. Four tile pairs were placed in each aquarium, ensuring that every tile pair was upright to maintain good water flow around the spat. An intact, sun-bleached adult oyster shell was also placed into each aquarium for spat tile pairs to lean on so that they maintained an upright position. Aquaria were filled with 2 L of natural seawater (with the exception of the predator water control, which received 1.5 L seawater + 0.5 L predator water) that had settled for at least 3 days to remove sediment particles. Seawater was supplemented with either Instant Ocean salt or deionized water to reach 20 ppt (± 2 ppt). Each aquarium contained filtered air bubblers for oxygenation. Aquaria were covered with lids to reduce evaporation and stored outside under a covered pavilion in a water bath containing ambient flow-through seawater to regulate temperature. Spat were fed Instant Algae Shellfish Diet 1800 (Reed Mariculture). At the start of the experiment, spat were fed 0.5 mL twice daily, but we increased this amount to 1 mL twice daily as spat grew larger. Complete water changes and aquarium cleanings were performed twice weekly, and 1 mL of predator chemical cues (i.e., homarine, trigonelline, or homarine + trigonelline at the correct assigned dose) were added to the aquaria immediately after water changes. 

Stock solutions for homarine (7.4 mM) (Santa Cruz Biotechnology Company), trigonelline (6.6 mM) (Toronto Research Chemicals), and homarine + trigonelline were prepared in LCMS grade water, aliquoted to avoid freezing and thawing the solutions more than once, and stored at -80 °C. Serial dilutions for each cue were prepared in deionized water the same day they were added to their respective aquaria (Table S2 in Supplemental File PDF). Concentrations were determined by calculating half-log steps encompassing the natural concentrations of homarine and trigonelline found in the urine of blue crabs fed an oyster diet (SI Appendix, Fig. S1 in Roney et al. (submitted)). On the day of cue addition, serial dilutions were performed with deionized water and using a micropipette (for volumes under 5 mL) and 10 mL graduated cylinder (for volumes greater than 5 mL). Mixtures were vortexed for 10 seconds and manually agitated for 10 seconds before continuing with the serial dilution. Dilutions for treatments of individual compounds and the homarine + trigonelline treatments were prepared at the same half-log concentrations (Table S2 in Supplemental File PDF). The same stock solutions were used to prepare both the individual compound and homarine + trigonelline treatments. All serial dilutions were done in tandem. Once chemical mixtures were made, they were stored in glass bottles, and 1 mL of each solution was pipetted into the appropriate experimental aquaria with a clean pipette tip. Chemical solutions were added after water changes, which were performed twice weekly. This experiment also included a seawater control group, which received the same care (without cue addition) as all other experimental aquaria.

The experiment was conducted for 56 days (8 weeks), and at the completion of the experiment tile pairs were removed from their jars, measured, and crushed according to the same methodology from the predator cue bioassay (See related dataset: https://www.bco-dmo.org/dataset/883945). There were four replicate aquaria per concentration, and within these replicate aquaria, we took an average of 17 crushed oysters. Replicates with high mortality (less than 6 spat alive) were excluded from statistical analyses. In total, 14 aquaria from the homarine dose array, 16 aquaria from the trigonelline dose array, and 17 aquaria from the trigonelline + homarine dose array were included in the statistical analysis.

Instruments:
Individual spat width was measured for each crushed oyster to 0.01 mm using a Vernier digital caliper. The force required to crush oysters was measured using a Kistler 5995 charge amplifier and Kistler 9207 force sensor following standard protocol (Robinson et al., 2014). 

Problems/Issues:
Only live oysters were crushed for the experiment. If an oyster was crushed and found to be dead (no soft tissue within shell), that was noted on the data file to be excluded from the analysis. Due to this, some treatments had fewer replicates than others, but this was accounted for in data analysis (see methods above).