Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
  • Problem Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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 (Fig. S1). 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 (Fig. S1, Anstis et al. 1981; Baldassarri et al. 2008).

Shell images and crack measurements were collected in Zeiss Zen 3.6 Blue edition software and data was recorded in Microsoft Excel. As outlined in the ASTM standard (ASTM Designation C1327–15R19 2019), microhardness tests were removed from the analysis if the indent was kite-shaped, with one diagonal more than 10% larger than the other, or were indented into an unreliable position on the shell, such as between prisms or within an unacceptable range (less than 1x the average diagonal length) from the edge.

currently being processed

NSF Award Abstract:
Many prey species use chemicals released in predator urine to detect imminent danger and respond appropriately, but the identity of these ‘molecules of fear’ remains largely unknown. This proposal examines whether prey detect different estuarine predators using the same chemical or whether the identity of the chemical signals varies. Experiments focus on common and important estuarine prey, mud crabs and oysters, and their predators including fishes, crustaceans and marine snails. Bioactive molecules are being collected from predators and prey and characterized. The goal is to determine if there are predictive relationships between either the composition of prey flesh or the predator taxon and the signal molecule. Understanding the molecular nature of these cues can determine if there are general rules governing likely signal molecules. Once identified, investigators will have the ability to precisely manipulate or control these molecules in ecological or other types of studies. Oysters are critical to estuarine health, and they are important social, cultural and economic resources. Broader impacts of the project include training of undergraduate and graduate students from diverse backgrounds and working with aquaculture facilities and conservation managers to improve growth and survival of oysters. One response to predator cues involves creating stronger shells to deter predation. Determining the identity of cues used by oysters to detect predators can provide management options to produce oysters that either grow faster or are more resistant to predators. Project personnel is working with oystermen to increase yields of farmed oysters by managing chemical cues.

For marine prey, waterborne chemical cues are important sources of information regarding the threat of predation, thus, modulating non-consumptive effects of predation in many systems. Often such cues are produced when the predators consume the flesh of that prey. In nearly all cases, the specific bioactive molecules responsible for modulating these interactions are unknown, raising the question whether there is a universal molecule of fear that prey respond to. Thus, the focus of the project is to determine the generality of fear-inducing metabolites released by predators and prey in estuarine food webs. The project combines metabolomics analysis of diet-derived urinary metabolites with bioassays to identify the bioactive molecules producing responses in two prey species from different taxonomic groups and trophic levels (oysters, mud crabs). Metabolites are sampled from three types of predators, fish, gastropods or crustaceans. This project aims to: 1) identify bioactive molecules produced by several common estuarine predators from different taxa; 2) compare cues from predators that induce defenses in prey vs. changes in prey behavior; and 3) contrast the identities and effects of predator-released cues with fear-inducing molecules from injured conspecifics. By identifying and contrasting the effects of waterborne molecules that induce prey responses from six predators and injured prey, this project is yielding insights into the mechanisms that mediate non-lethal predator effects, while addressing long-standing questions related to predator-prey interactions. In addition to the search of a universal molecule of fear, the experiments are exploring the role of complementary and distinct chemical information on the specificity of prey responses to different types of predators.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.