The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs through their chemical senses

Neuroscience Institute and Department of Biology, Georgia State University, Atlanta, United States; Department of Ocean Science, Tokyo University of Marine Science and Technology, Japan
Animal Behaviour 01/2010; DOI: 10.1016/j.anbehav.2010.04.003

ABSTRACT Sea hares release an ink secretion composed of purple ink and white opaline as a potential chemical defence against predators. The aim of our study was to identify deterrent molecules in the ink of Aplysia californica against an allopatric generalist crustacean predator, the blue crab Callinectes sapidus, and to define the mechanisms of action of the deterrents against crabs. We used two behavioural assays, a squirting assay and an ingestion assay, to show that ink is highly effective and that opaline is moderately effective in suppressing feeding of crabs. Results with reversibly blinded crabs demonstrate that the deterrence is mediated through the crabs’ chemical senses. We used bioassay-guided fractionation to identify the purple molecules aplysioviolin and phycoerythrobilin as a major and minor deterrent, respectively, in ink against crabs. These molecules derive from a light-harvesting protein in the photosynthetic system of dietary algae. This is the first demonstration of an animal converting a photosynthetic pigment into a chemical deterrent. Mixing opaline and ink enzymatically produces hydrogen peroxide, which also functions as a chemical deterrent against crabs. Our results and those of other studies show that sea hares use a diversity of molecules in their skin, mucus and ink secretion to chemically defend themselves against their potential predators. Aplysioviolin, phycoerythrobilin and hydrogen peroxide also exist in ink secretion of Aplysia dactylomela, a sea hare sympatric to blue crabs, and thus we posit that these molecules are potentially effective in ecologically relevant predator–prey interactions and need to be scrutinized in more ecologically relevant experiments.

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    ABSTRACT: Chemical defenses are used by many organisms to avoid predation, and these defenses may function by stimulating predators' chemosensory systems. Our study examined detection mechanisms for components of defensive ink of sea hares, Aplysia californica, by predatory sea catfish, Ariopsis felis. Behavioral analyses show aplysioviolin and phycoerythrobilin are detected intra-orally and by barbels and are deterrent at concentrations as low as 0.1% full strength. We performed electrophysiological recordings from the facial-trigeminal nerve complex innervating the maxillary barbel and tested aplysioviolin, phycoerythrobilin, amino acids, and bile salts in cross-adaptation experiments. Amino acids and bile salts are known stimulatory compounds for teleost taste systems. Our results show aplysioviolin and phycoerythrobilin are equally stimulatory and completely cross-adapt to each other's responses. Adaptation to aplysioviolin or phycoerythrobilin reduced but did not eliminate responses to amino acids or bile salts. Adaptation to amino acids or bile salts incompletely reduced responses to aplysioviolin or phycoerythrobilin. The fact that cross-adaptations with aplysioviolin and phycoerythrobilin were not completely reciprocal indicates there are amino acid and bile salt sensitive fibers insensitive to aplysioviolin and phycoerythrobilin. These results indicate two gustatory pathways for aplysioviolin and phycoerythrobilin: one independent of amino acids and bile salts and another shared with some amino acids.
    Journal of Comparative Physiology 12/2011; 198(4):283-94. · 1.86 Impact Factor
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    ABSTRACT: Many animals release secretions in defense against predators. Some marine molluscs, including cephalopods (squid, octopus, cuttlefish) and gastropods (sea hares), release a colored ink secretion. Observational evidence supports the idea that inking is a defensive behavior that protects cephalopods from predators by forming a visual smokescreen or visual mimic (pseudomorph). Another possible function of cephalopod ink is to act against the chemical senses of predators either as a deterrent or distracting food mimic (phagomimic). Experimental tests of both hypotheses are lacking for cephalopods. In our study, we tested the hypothesis that squid use ink as a defense against attacks by predatory fish by performing three sets of experiments to examine the behavior of juvenile French grunts, Haemulon flavolineatum, toward ink from Caribbean reef squid, Sepioteuthis sepioidea. In the first set of experiments, a pseudomorph assay, in which ink was presented between a fish and a piece of food, assessed effects of ink on the approach and capture phase of a predator's attack. This showed that a pseudomorph of squid ink hindered the attack by significantly delaying food capture as well as evoking significantly more avoidance of or biting at the pseudomorph compared to a control pseudomorph of carboxymethylcellulose. A pseudomorph of carboxymethylcellulose plus food color to simulate the color of squid ink had a similar effect to the squid ink pseudomorph. In a second set of experiments, a disc assay, in which ink was added to meat-flavored paper discs, examined ink's effect on the consumption of food, simulating ink's protective effect if a squid and its ink are taken into a predator's mouth. This showed that squid ink added to meat-flavored discs significantly changed handling of the discs and increased, though non-significantly, their rejection. The same food color as used in the pseudomorph assay, when added to meat-flavored discs, significantly affected handling and rejection of the discs, showing that the food color itself, intended as a control, is unpalatable. In the third set of experiments, the disc assay was used to show that ink did not increase the acceptance of unflavored (i.e. without meat) discs, a result suggesting that ink is not a phagomimic. Our study presents the first experimental results supporting the hypothesis that inking protects squid against predatory fish, and that it acts during both the capture and consummatory phases of attacks: during the capture phase through visual and/or chemical effects against predators, and during the consummatory phase through unpalatable chemicals.
    Journal of Experimental Marine Biology and Ecology. 01/2010;
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    ABSTRACT: Chemicals are a frequent means whereby organisms defend themselves against predators, competitors, parasites, microbes, and other potentially harmful organisms. Much progress has been made in understanding how a phylogenetic diversity of organisms living in a variety of environments uses chemical defenses. Chief among these advances is determining the molecular identity of defensive chemicals and the roles they play in shaping interactions between individuals. Some progress has been made in deciphering the molecular, cellular, and systems level mechanisms underlying these interactions, as well as how these interactions can lead to structuring of communities and even ecosystems. The neuroecological approach unifies practices and principles from these diverse disciplines and at all scales as it attempts to explain in a single conceptual framework the abundances of organisms and the distributions of species within natural habitats. This article explores the neuroecology of chemical defenses with a focus on aquatic organisms and environments. We review the concept of molecules of keystone significance, including examples of how saxitoxin and tetrodotoxin can shape the organization and dynamics of marine and riparian communities, respectively. We also describe the current status and future directions of a topic of interest to our research group-the use of ink by marine molluscs, especially sea hares, in their defense. We describe a diversity of molecules and mechanisms mediating the protective effects of sea hares' ink, including use as chemical defenses against predators and as alarm cues toward conspecifics, and postulate that some defensive molecules may function as molecules of keystone significance. Finally, we propose future directions for studying the neuroecology of the chemical defenses of sea hares and their molluscan relatives, the cephalopods.
    Integrative and Comparative Biology 06/2011; 51(5):771-80. · 3.02 Impact Factor


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