Chemical encoding of risk perception and predator detection among estuarine invertebrates Remington X. Poulina,b, Serge Lavoieb,c, Katherine Siegeld, David A. Gaula, Marc J. Weissburgb,c, and Julia Kubaneka,b,c,e,1 aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332; bAquatic Chemical Ecology Center, Georgia Institute of Technology, Atlanta, GA 30332; cSchool of Biological Sciences, Georgia Institute of Technology, Atlanta, GA 30332; dSchool of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332; and eInstitute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved November 29, 2017 (received for review August 8, 2017) An effective strategy for prey to survive in habitats rich in predators aquatic systems, the receptors that mediate chemoreception and is to avoid being noticed. Thus, prey are under selection pressure to recognition of these cues, and the specificity to these cues, necessi- recognize predators and adjust their behavior, which can impact tating further research. numerous community-wide interactions. Many animals in murky One explanation for the lack of characterized waterborne cues is and turbulent aquatic environments rely on waterborne chemical that purification methodologies for highly water-soluble compounds cues. Previous research showed that the mud crab, Panopeus herb- have been unsatisfactory, especially when working with seawater stii, recognizes the predatory blue crab, Callinectus sapidus,viaacue samples that contain substantial quantities of inorganic salts. Stan- in blue crab urine. This cue is strongest if blue crabs recently preyed dard sampling methods for airborne chemical cues such as head- upon mud crabs. Subsequently, mud crabs suppress their foraging space analysis for volatile insect pheromones (20) are unavailable activity, reducing predation by blue crabs. Using NMR spectroscopy- for nonvolatile waterborne cues. Many waterborne cues are present and mass spectrometry-based metabolomics, chemical variation in in very low concentrations, unstable to handling in the laboratory, urine from blue crabs fed different diets was related to prey behav- and produced by organisms that either are not abundant or not ior. We identified the urinary metabolites trigonelline and homarine readily cultivated in the laboratory. It may be difficult to identify in as components of the cue that mud crabs use to detect blue crabs, which tissues of an organism these cues are produced or stored, and with concentrations of each metabolite dependent on the blue when this is possible the signaling molecules may occur as part of a crab’s diet. At concentrations found naturally in blue crab urine, complex mixture with many other, irrelevant metabolites. trigonelline and homarine, alone as well as in a mixture, alerted Traditionally, chemists have applied a process of bioassay- mud crabs to the presence of blue crabs, leading to decreased guided fractionation to purify and then characterize biologically foraging by mud crabs. Risk perception by waterborne cues has active compounds. However, this multistep approach often leads been widely observed by ecologists, but the molecular nature of to decomposition of labile cues and exclusion of multicomponent these cues has not been previously identified. Metabolomics pro- cues, while simultaneously requiring substantial quantities of the vides an opportunity to study waterborne cues where other ap- chemical cue mixture for biological testing after each chemical proaches have historically failed, advancing our understanding of separation step (21). Recent advances in NMR spectroscopy the chemical nature of a wide range of ecological interactions. and MS metabolomics allow for fast, efficient, and cost-effective blue crab | chemical ecology | metabolomics | nonconsumptive effects | Significance predation Chemical cues are essential to marine life, particularly for athering and interpreting information from the environment ’ detecting predators. Despite decades of research, almost Gis imperative to organisms ability to recognize food, mates, nothing is known of the molecular nature of these waterborne predators, and appropriate habitat. Many aquatic species rely cues. This prevents us from assessing environmental variation on chemical cues in environments where auditory, visual, and and impacts of these cues and from understanding and ma- mechanosensory mechanisms are often compromised (1–3). nipulating predator–prey signaling pathways. Leveraging nat- Significant efforts have been made to understand chemical de- ural chemical variation in the urine of a predatory crab using fenses and feeding deterrents (4–7) as many have potential metabolomics, the chemical profiles of urine from crabs fed medicinal applications (8–10); however, waterborne cues remain different diets were revealed to be predictive of their fear- almost completely unidentified. inducing potency. This pattern led us to identify the major The ability to sense and recognize predators remotely is partic- constituents of the chemical cue used by mud crab prey to ularly important for organisms because it allows for the production detect and avoid their predator. This investigation serves as a of behavioral, morphological, or life historical adjustments to min- blueprint for investigating the molecular nature of these imize predation (11). Although significant evidence exists for community-structuring waterborne cues. widespread chemical detection of predators and alarm cues among conspecifics in the marine environment (12–14), little is Author contributions: R.X.P., S.L., M.J.W., and J.K. designed research; R.X.P., S.L., K.S., and D.A.G. performed research; R.X.P., S.L., D.A.G., and J.K. analyzed data; and R.X.P., S.L., known about the molecular nature of the cues involved. These M.J.W., and J.K. wrote the paper. cues are of particular importance as they routinely produce eco- The authors declare no conflict of interest. logically significant nonconsumptive effects, that is, altered species This article is a PNAS Direct Submission. interactions beyond the effects of lost prey by cause of consumption due to changes in the morphology, behavior, or life history of prey Published under the PNAS license. Data deposition: Metabolomic data for this project are publicly available on the Biological (15). Nonconsumptive effects havebeendemonstratedinaquatic and Chemical Oceanography Data Management Office database (https://www.bco-dmo.org/ (16–18) and terrestrial environments (19)andaresuggestedtohave project/565703). even greater capacity to structure communities than the direct effects 1To whom correspondence should be addressed. Email: [email protected]. of consuming prey (15). Due to the lack of a molecular understanding This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of these cue systems, little is known about how cues disperse in 1073/pnas.1713901115/-/DCSupplemental. 662–667 | PNAS | January 23, 2018 | vol. 115 | no. 4 www.pnas.org/cgi/doi/10.1073/pnas.1713901115 Downloaded by guest on October 2, 2021 profiling of complex mixtures containing waterborne cues, Table 1. Metabolites identified from blue crab urine by whose chemical variation can be leveraged to correlate the NMR spectroscopy-based PCA model (n = 9–10 urine samples for presence or abundance of particular compounds within the each diet) mixture with biological potency of the mixture. Recently, a novel Concentration in urine of blue crabs fed microalgal mate attraction pheromone was identified using MS- mud crabs based metabolomics despite pheromone concentrations in the nanomolar range (22). Metabolite Absolute, μM Relative to oyster diet Previous research showed that the mud crab, Panopeus herb- Trigonelline (1)90 1.8× stii, detects its predator, the blue crab, Callinectus sapidus, using Pyrimidine (2) 72 1.6× unknown metabolites released in the urine of blue crabs (23). Lactate (3) 250 1.3× Mud crabs reacted differently to urine from blue crabs fed mud Carnitine (4) 160 1.2× crabs vs. other prey. Using NMR- and MS-based metabolomics Choline (5) 110 1.2× we aimed to leverage the chemical variation in urine from blue Threonine (6) 160 1.1× crabs fed different diets to identify the component(s) of the cue Creatinine (7) 62 0.72× mixture that mud crabs use to recognize blue crabs. This study Trimethylamine (8) —* >1.0× provides a roadmap to identify complex waterborne cues that can Methyl glutarate (9) —* <1.0× be used to further our understanding of chemically mediated Acetate (10) —* <1.0× interactions in the marine environment. Creatine (11) —* <1.0× N-methylhistidine (12) 99 0.75× Results Alanine (13) 180 0.50× Mud Crabs Perceive Risk via Concentration Differences in Predator Urinary Metabolites. When we employed 1H NMR-based metab- *Concentration could not be determined due to NMR spectral overlap of olomics comparing the chemical profiles of urine from blue crabs diagnostic protons. fed mud crabs vs. oysters, principal component analysis (PCA) revealed a single principal component accounting for 19.5% of the identifications (SI Appendix,Fig.S6andTableS1). Thus, whereas total variation among urine samples which differentiated urine of A individual metabolites pinpointed via PCA were not purified from blue crabs fed the two diets (Fig. 1 ). An orthogonalized partial blue crab urine, their structures were confidently assigned,