Table of Contents

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

See "Related Datasets" section for results of other predator cue bioassay experiments.

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 predator chemical cues were added to the aquaria immediately after water changes. 

Blue crabs were collected from crab pots near Dauphin Island, AL and stored in the same facility and conditions as described for the predator cue bioassay in 2020 (See related dataset: https://www.bco-dmo.org/dataset/883945). Urine collection methods remained the same as for previous experiments (2020 predator cue bioassay) except urine was pooled from all crabs for this experiment. All crabs for this experiment were fed an oyster diet (i.e., one adult oyster (~6-7 cm in length) twice weekly). Crab were kept in aquaria for a one week acclimation period before beginning a urine extraction regimen. Urine was collected twice weekly from 22 crabs that ranged 13 – 18 cm in size and each crab produced 1.31 ± 0.18 mL. Urine dose concentrations were determined based on the average concentrations of homarine and trigonelline quantified in blue crab urine in the 2020 predator cue bioassay (homarine, 13 ± 21 µM; trigonelline, 3.6 ± 6.9 µM). It was assumed that 1 mL of pooled blue crab urine contained these concentrations, and doses were adjusted by half-log steps until approximated concentrations of homarine and trigonelline spanned four orders of magnitude (Table S3 in Supplemental File PDF). Though initial experimental design was based on an assumed concentration of homarine and trigonelline, all urine doses were reported as urine volume divided by the total volume of seawater within an experimental aquaria. For the six lowest doses, 1 mL aliquots of blue crab urine were prepared via serial dilution. The two highest doses received 5.00 mL and 1.25 mL aliquots of pure urine respectively. All aliquots were frozen at –80 °C to be used on the day of cue addition.

 The preparation and maintenance of the urine dose response experiment was identical to the previous homarine and trigonelline dose response experiment. The experiment was conducted for 56 days (8 weeks) from 08 Aug. 2022 – 03 Oct., 2022 and at the completion of the experiment tile pairs were removed from their jars, measured, and crushed according to previous methodology (Robinson et al. 2014). There were five replicate aquaria per concentration, and within these replicate aquaria, we took an average of 32 crushed oysters. 

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: None.

Data were entered and stored in Microsoft 365 Excel for Windows Version 2011. No processing (removals, transformations, etc) occurred on this data. 

BCO-DMO Data Manager Processing Notes:
* Sheet 1 of file "BC Urine Dose Response Oyster Crushing Data.xlsx" was imported into the BCO-DMO data system with values "NA" interpreted as a missing data identifier. 
** Missing data values are displayed differently based on the file format you download.  They are blank in csv files, "NaN" in MatLab files, etc.
* stand_crushing_force rounded to two decimal places as indicated as the correct precision in provided metadata.

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.