Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

Oyster culturing
Oysters (Crassostrea virginica) were cultured as spat-on-shell at the Auburn University Shellfish Laboratory (AUSL) on Dauphin Island, AL starting in late May 2019 using standard techniques (Congrove et al. 2009). Oyster larvae were settled onto sun-bleached oyster shells to create spat-on-shell. After 3 days, when oyster spat were approximately 1.0 millimeters, they were exposed to either exudates from predatory blue crabs or empty cage controls in four flow-through holding tanks (length = 2.4 meters, width = 0.9 meters, water depth = 0.4 meters) supplied with unfiltered seawater pumped directly from the Gulf of Mexico. The number of spat per shell varied from approximately 5 – 40 and we elected to not alter the initial density to mimic natural settlement during the induction period. Oysters were suspended above the tank bottom in oyster aquaculture baskets (64 x 23 x 14 centimeters with 140 spat-covered per shells basket) to prevent sediment buildup from suffocating oysters. Seven oyster baskets were present in each tank (28 total).

Spat were exposed to blue crab predator cues by holding four live caged adult blue crabs (Callinectes sapidus) in two of the tanks (8 crabs total), whereas the remaining two tanks contained empty cages (control) to mimic conditions where oysters regularly experience predator cues or are limited in their exposure from cues. Water volumes and crab densities were informed from established procedures (Belgrad et al. 2021). Crabs in each tank were held in four separate cages (32 x 23 x 14 centimeters) to prevent crabs from consuming the experimental oysters or each other. Every crab was fed one adult oyster daily (approximately 5.0 centimeters in length) to maximize predator cue intensity as experimental oysters would be exposed to exudates from predators and damaged conspecifics. This ensured that oysters were exposed to the most natural set of cues indicative of a predation event, which produces a strong response in oysters (Scherer et al. 2016). Crabs were replaced during the experiment as needed due to mortality. Experimental oyster baskets were rotated around the crab cages daily to reduce differences in oyster growth due to proximity to predator cues, and no differences among cages were found. The induction period was 2 months.

Shell morphology measurements
We sampled subsets of oysters to confirm that our predator cue treatments were causing control and induced oysters to exhibit different shell morphologies. Two shells were taken from every basket and three live spat were chosen from each shell for measuring spat shell characteristics after two months (number of individuals = 84 for each cue exposure treatment; 56 shells and 168 spat total). Spat shell properties were assessed by measuring shell size and shell crushing force. Oysters are roughly round at these early life stages, and we measured the shell length from the umbo to the outer shell edge to the nearest 0.01 millimeters using digital calipers. We took care to only measure individuals that were not bounded by cohorts to reduce any confounding effects on growth due to space limitation.

The force required to break each spat shell was quantified by a penetrometer attached to a charge amplifier (Kistler force sensor 9203 and Kistler charge amplifier 5995). The sensor probe was placed in the center of the shell, perpendicular to the shell surface. Gentle, consistent pressure was applied until the shell cracked, and the maximum force applied by the sensor to break the shell (in Newtons) was recorded. We divided shell crushing force by shell length to produce a size-standardized metric of shell strength (i.e., standardized crushing force, Newtons per millimeter) because larger individuals naturally have a stronger shell as a byproduct of their larger size (shell thickness).

Oyster shells had their soft tissue removed and were placed in a drying oven for 48 hours at 70 degrees Celsius to obtain the shell dry weight.

BCO-DMO Processing Description:
- Rounded "standard_force" column to 2 decimal places

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.