Eastern oysters were spawned at the Auburn University Shellfish Laboratory in Dauphin Island, AL, USA and juveniles were raised in a nursery system at Dauphin Island Sea Lab, Dauphin Island, AL, USA. Data collection took place at The College of New Jersey, Ewing, NJ, USA.
Oyster Culturing:
The oyster spat used in this experiment were preserved from a previous study on shell strength of predator-induced spat, which confirmed that exposure to predator cues resulted in stronger shells, measured as the amount of force (N) required to break shells standardized to size (Belgrad et al. 2021). Oysters were spawned and cultured at Auburn University Shellfish Laboratory on Dauphin Island, AL, USA, in May 2019. Oyster larvae were settled onto sun-bleached oyster shell where they metamorphosized into spat-on-shell and were housed in four flow-through holding tanks (2.4 meters x 0.9 meters) filled to a water depth of 0.4 meters (m) and flowing at a rate of 36.9 liters per minute (L/min) with natural seawater from Mobile Bay, AL, USA. Oyster spat-on-shell were randomly placed in seven oyster aquaculture baskets (~140 adult shells per basket, 20,000 spat/tank) spaced evenly along the length of the tank and suspended within the tanks to avoid sediment smothering the spat (28 baskets, 80,000 spat total). Two of the holding tanks were kept with only oysters to serve as controls (non-induced), whereas the two treatment tanks also each held four caged live adult blue crabs (Callinectes sapidus, Rathbun) to add predator cues to the tanks (induced). These crabs were fed one adult oyster (~5.0 centimeters in length) daily and were replaced with healthy, fresh-caught crabs at least biweekly. Oyster cages were rotated daily around crab cages to reduce differences in growth due to proximity to cue sources or water intake. A subset of spat-on-shell were removed from each tank after four weeks and eight weeks of culturing under these conditions and stored in 70% ethanol until March 2023 when shell structure analysis began.
Sample Preparation:
Oyster spat-on-shell were removed from ethanol solution in March 2023. A total of 5 left (top) shell valves from each nursery tank per induction state and age group (n = 10 per induction state and age group; 40 valves total) were carefully removed from individual spat and any soft tissue was removed using forceps and scalpel. The separated shell valves sat in 100% ethanol overnight to assist with removal of tissue remains, then were rinsed with DI water, dried at room temperature for 2-3 hours and finished drying in a low-temperature vacuum oven at 45 degrees Celsius (°C) and 25 millimeters of mercury (Hg mm) for 2 hours. The shell valves were then mounted and polished following standard techniques (Prezant et al. 2022). Each valve was placed in a 32-millimeter (mm) mounting cup, with the ventral edge of the shell affixed to the base of the cup using a coil mounting clip that had been glued to the bottom of the cup. This was left to dry overnight, before mounting with Bisphenol A Epichlorohydrin epoxy and hardener mixed in a 10:3 ratio (Allied High Tech). After the epoxy hardened for 24 hours, the mounted valves were removed from their mounting cups and ground to a plane that was visually approximated near the center of the shell running along the longest axis from anterior to posterior end. The shell valves were then polished to 0.04 micrometers (µm) using polycrystalline diamond solution and colloidal silica suspension (Allied High Tech).
Panoramic images of each shell sample were taken under polarized light using a reflected light microscope (Zeiss Axioscope.A1 with a Zeiss, AxioCam 105 color camera), with the analyzer set to 10°. Panoramas were constructed using imaging software (Zeiss Zen 3.8). Four-week-old spat valves were imaged under a 5x objective, and eight-week-old spat valves were imaged under a 2.5x objective.
Determining Shell Microhardness:
Microhardness testing was performed with a Mitutoyo HM-200 microhardness testing machine. Hardness was measured as an average of 10 indents per shell layer conducted on each shell sample, spaced approximately evenly across the middle third region of the shell (200 to 700 µm apart, depending on the size of each shell). All indents were performed with a 5 g load with a 5-second approach time, 5-second dwelling time, and 5-second unload time. An image of each indent was taken using a Zeiss Axiocam 305 color microscope and the diagonals of each indent (µm) were measured using Zeiss Zen 3.6 Blue edition software. The Vickers Microhardness Number (VHN) was determined as 1.8543 F/d2, where F is force (kilograms (kg)) and d is diagonal length (mm), as described by the American Society for Testing and Materials (ASTM Designation C1327-15R19, 2019). During image analysis, we also recorded the number of cracks and the length of the longest crack emanating from each Vickers microhardness indentation. Crack length (µm) was measured by placing a circle at the centermost point of the Vickers microhardness indentation and measuring circle radius to the end of the longest crack whose source could be traced back to the indentation (Anstis et al. 1981; Baldassarri et al. 2008).
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