463 Contributions of Marine Chemical Ecology to Chemosensory Neurobiology Henry G. Trapido-Rosenthal CONTENTS I. Introduction 463 II. The Nature of Chemical Signals in the Marine Environment 464 III. Chemoreception in Bacteria 465 IV. Chemoreception in Eukaryotic Microorganisms 466 V. Chemoreception in Multicellular Organisms 467 A. Feeding 467 1. Behavioral Observations and Studies 467 2. Physiological, Biochemical, and Molecular Studies 468 B. Larval Development 469 1. Behavioral Observations and Studies 469 2. Physiological, Biochemical, and Molecular Studies 470 C. Social Interactions 471 1. Behavioral Observations and Studies 471 2. Physiological, Biochemical, and Molecular Studies 472 VI. Conclusions 473 Acknowledgments 473 References 473 I. INTRODUCTION The concept of specific receptors for bioactive chemical substances originated around the turn of the last century, as a consequence of Langley’s studies on the actions of plant alkaloids on animal tissues. In analyzing the results of his studies of the effects of nicotine and curare on the contraction of vertebrate skeletal muscle, he maintained that those substances must be interacting, not directly with the contractile machinery of the tissue, but rather with “receptive substance” of the muscle. 1 Langley came to the general conclusion that for bioactive molecules to effect specific actions, they must interact with specific entities; these have come to be known as receptors. The receptor concept was used to explain the effects of substances such as hormones, neurotransmitters, drugs, and poisons on cells within multicellular organisms. 2 The concept of receptor-mediated responses to waterborne environmental chemical signals was postulated by Haldane 3 in 1954, based on these developing concepts of receptor-mediated communication between the component cells of metazoan organisms. In the subsequent 20 years, it was not uncommon for chemical ecologists to hypothesize that observed chemically stimulated behaviors were mediated by specific chemoreceptors. In a 14 9064_ch14/fm Page 463 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC 464 Marine Chemical Ecology 1974 article on chemoreception in the marine environment, Laverack 4 reviewed studies of organ- ismal and cellular responses to environmental chemical signals and concluded that mediation by chemoreceptors was the most parsimonious way of accounting for these responses. However, experimental demonstration of the existence of such receptors had not yet been achieved, and Laverack used the existing knowledge of neurotransmitter receptors as a heuristic device to dem- onstrate to his readers how signal detection and transduction might operate in chemoreceptor cells. Only relatively recently have electrophysiological and biochemical characterizations of chemore- ceptors for environmental chemical signals been accomplished, and the molecular characterization of such receptors is just beginning. This chapter reviews the past five decades of work devoted to the study of chemoreceptors in aquatic organisms. Since, during this period of time, various aspects of this subject have been subjected to review by other writers, a temporal bias towards more recent work will be detected. It is the hope of this author that this bias will be overcome by directing the reader to the important reviews of work in marine chemoreception that precede this one. II. THE NATURE OF CHEMICAL SIGNALS IN THE MARINE ENVIRONMENT The chemical signals encountered by organisms in marine and other aquatic environments can be conceptually distributed among four categories. They can be chemically characterized as being either primary metabolites (roughly defined as substances used in the basic metabolic processes of organisms) or secondary metabolites (substances constructed by the condensation of primary metabolites into more complex structures, and which can be used as chemical signals that regulate both intracellular and intercellular processes), with the distinction between these two classifications being somewhat arbitrary and dependent on the interests and definitions of the classifier. 5,6 Sub- stances can be more positively characterized according to the way in which they are presented, or made accessible, to a detecting organism. An organism can detect the signal molecule either in the three-dimensional space of solution or on the two-dimensional space of a solid surface. A comprehensive review of the nature of chemical signals that are encountered by organisms in aquatic environments is presented by Carr. 7 In his review, Carr points out that many of the substances that we know to be important chemical signals in the marine environment are, in fact, also potent neuroactive agents, and their neuroactive properties are initiated by specific interactions with receptors. Thus, our understanding of the chemical nature of many of the substances that serve as signal molecules in the marine environment and occupants of cell-surface receptors in the internal environment of metazoans has led to the creation and testing of hypotheses concerning the receptor- mediated nature of cellular and organismal responses to environmentally important chemical signals encountered in marine and aquatic environments. Nevertheless, there has been some controversy on the subject of whether or not substances that are indeed neuroactive in the context of a multicellular organism’s central nervous system are in fact likely to be chemical signals in certain different contexts of an organism’s external environment; 8,9 these controversies have likewise contributed to the scientific investigation of the molecular mechanisms underlying the detection of environmental chemical signals. Among the substances that serve as chemical signals in both internal and external aqueous environments are nucleotides (such as AMP, ADP, and ATP), amino acids (such as glycine, glutamate, arginine, and taurine), and peptides (of which an astronomically large variety can exist due to the vast combinatorial possibilities that just the standard 20 protein-forming amino acids afford — 20 n , with the exponent n indicating the chain length of a peptide). In both internal and external environments, nucleotides and amino acids are typically presented to a receptor in solution while peptides can be presented either in the three-dimensional context of solution, or the two- dimensional context of solid-phase attachment to a surface. Depending on the particular identity and sequence of component amino acid residues, individual peptide molecules can also have higher 9064_ch14/fm Page 464 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 465 signal-to-noise ratios 10 and longer lifetimes as signal molecules than the smaller amino acids or nucleotides, due to slower biotic (enzymatic degradation and uptake) and abiotic (removal from solution by adsorption to colloids) clearance mechanisms; such increased residence time in the environment can be a distinct advantage in a chemical signaling system. 11 III. CHEMORECEPTION IN BACTERIA Chemically mediated behavior in bacteria was first noted by Engelmann in 1881 and Pfeffer in 12 ). At the time that he wrote his 1969 article entitled “Chemoreceptors in Bacteria,” Adler 13 maintained that answers to the questions of how bacteria detect and respond to chemical signals was still almost entirely unknown. In the decades since then, a great deal of progress has been made in determining the mechanisms by which bacterial responses to environ- mental chemicals are initiated and executed. Although much of this progress has been made using the “lab rat” bacteria Escherichia coli and Salmonella typhimurium , the results are thought to be broadly applicable to most bacterial species. 12 Chemotaxis is perhaps the best studied bacterial responses to environmental chemical signals (see Chapter 12, this volume). Bacteria typically move up concentration gradients of nutrient molecules and down concentration gradients of noxious compounds. Berg and Brown 14 showed that directionality is conferred by alteration of two behaviors, one a straight-ahead swimming behavior and the other a direction-changing tumbling behavior. When moving in a desired direction (up a concentration gradient of a nutrient, for example), swimming is rarely interrupted by tumbling episodes. When moving in a direction interpreted as undesirable, tumbling becomes more frequent; after each tumble, swimming begins anew, and, since tumbling results in a random reorientation of the bacterium, the chances are good that the new direction will be away from the source of the noxious chemical. Using behavioral and genetic assays, Adler 13 concluded that detection of chemical signals was by means of receptors that recognized the chemical structures of these signals. This work was then expanded by Koshland and others 15–17 to directly measure the interaction of various nutrient sugars and amino acids with their respective receptors. The molecular mechanisms by which enteric bacteria respond to occupation of chemoreceptors have been worked out in substantial detail by combining the techniques of classical genetics, molecular genetics, and biochemistry. Upon occupation of a receptor by an appropriate ligand, conformational changes in the structure of the receptor transmit information to the cell’s interior by altering the activities of enzymes that affect the methylation and phosphorylation states of proteins involved in the signal detection and signal transduction processes. 18 Of particular impor- tance among these chemotaxis (Che) proteins is Che-Y, the phosphorylation state of which governs the direction of rotation of the bacterial flagellum and, thus, determines whether the cell is swimming or tumbling. Other Che proteins are involved with adaptation, directly affecting the ability of the receptors to interact with their ligands. 19 Marine bacteria can respond more rapidly than enteric bacteria to environmentally encountered chemical signals, 20 suggesting that signal detection and transduction mechanisms that are both qualitatively and quantitatively different than those charac- terized in E. coli await elucidation. Bacteria also use chemical signals to communicate with each other. The observation that the marine bacteria Vibrio fischeri were brightly luminescent at high population densities but dim when densities were low led to the identification of bacterial metabolites that have become termed quorum-sensing factors. The quorum-sensing factors of marine Vibrio sp. that regulate luminescence are acylated homoserine lactones that are synthesized by the bacteria and released into the sur- rounding medium. Work by Bassler and colleagues 21 has shown that the quorum-sensing factor is detected by a transmembrane receptor-transducer molecule that has both kinase and phosphatase activities. When unoccupied, the receptor’s kinase activities bring about both autophosphorylation and the phosphorylation of a series of response regulator proteins; when phosphorylated, these 9064_ch14/fm Page 465 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC 1884 (see Paoni et al. 466 Marine Chemical Ecology proteins repress the transcription of the operon that codes for the proteins involved in light production (luciferase and the enzymes that synthesize the substrate from which luciferase generates light). When the receptor is occupied, it becomes a phosphatase; phosphate groups are removed from the response regulation enzymes, which lose their repressor functions, permitting the transcription of the genes in the light-generating operon, with the ultimate result of bacterial luminescence. A growing number of bacterial quorum-sensing factors are now being discovered. These include not only a number of variations on the homoserine lactone theme, but also a variety of peptides, as well as specific cocktails of amino acids. 22 They appear to be used to measure population densities of other, perhaps competing, bacteria, as well as of conspecifics, and many of them clearly function as regulators of transcriptional activity. The development, organization, and functional maintenance of bacterial biofilms appears to be mediated, in large part, by the generation, release, and detection of quorum-sensing factors. 23 Thus, the ability to detect and respond to these environmentally encountered chemical signals clearly has not only tremendous adaptive value for bacteria, but will be of fundamental importance to our understanding of the many biofilm-based communities that are important components of marine ecosystems. IV. CHEMORECEPTION IN EUKARYOTIC MICROORGANISMS In eukaryotic microorganisms, as in bacteria, detection and evaluation of environmental chemical signals, as well as responses to those signals, are all accomplished by the same cell. This enables the tight coupling of behavioral data with biochemical, physiological, and molecular investigations into the cellular and molecular mechanisms involved in chemoreception. A number of model systems, including the single-celled gametes of various multicellular organisms, slime molds, yeast, and paramecia, have been used in such studies. The latter well demonstrates the research value of eukaryotic microorganisms for chemosensory research and will be touched on here. Paramecium tetraurelia is a diploid eukaryotic unicellular organism that alters its swimming behavior when it encounters certain environmental chemicals. Like bacteria, paramecia move towards attractants and away from irritants by altering the ratios of turning behavior to swimming behavior. However, many of the molecular mechanisms by which these behavioral changes are brought about are significantly different. By reducing the amount of turning, paramecia move towards a number of compounds such as lactate, acetate, folate, cyclic AMP (cAMP), or the excreted bacterial metabolite biotin; these substances can be considered either of direct nutritional value or of informational value, as indicators of the presence of nutritional resources. By increasing the amount of turning, they move away from irritants such as quinidine-HCl. By combining series of electrophysiological, biochemical, and molecular experiments, Van Houten and colleagues 24–28 have made a great deal of progress in elucidating the molecular mechanisms that underlie the cellular (and in this case organismal) response to environmental chemical signals. The responses are typically initiated by the specific interaction of the environmentally encoun- tered chemical with receptors that are deployed on the cell surface. Radiolabeled biotin, for example, interacts with a structurally selective receptor with an estimated affinity (as represented by the K D ) of 400 µ M; this compares with a behavioral EC 50 for this substance of 300 µ M. Compounds that are structurally similar to biotin can compete for the binding of the radiolabeled molecule, whereas compounds that are structurally different cannot. Upon occupation of biotin receptors, the cell membrane becomes hyperpolarized. This hyper- polarization causes an increase in the posteriorly directed beating of the cell’s propulsive cilia, and the cells move smoothly up the concentration gradient. Importantly, the hyperpolarization also decreases the likelihood of membrane depolarization, and if mild, slows the ciliary beat frequency and slows the cells, while if large, brings about a calcium action potential that reverses the ciliary 9064_ch14/fm Page 466 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 467 beat and causes the cells to turn sharply. The linkage of receptor occupation with membrane potential and ciliary motion appears to be, in part, an ATP-powered Ca ++ pump that resides in the plasma membrane; when active, it serves to extrude Ca ++ from the cells, thus maintaining a low intracellular concentration (in the vicinity of 10 –8 M) of this cation. During action potentials, Ca ++ floods down its electrochemical gradient into the cells, resulting in a transitory increase in concentration to as high as 10 –6 M in the motile cilia; at this concentration, the interaction of Ca ++ with the cilia’s axonemal machinery results in a directional change in ciliary beat. An example of molecularly mediated social interaction is provided by the ciliate Euplotes raikovi . In this organism, mating is coordinated by a family of water-soluble peptide pheromones of modular construction, with highly conserved residues and regions (that, importantly, either consist of or include six cysteine residues that provide these peptides with three intramolecular disulfide bonds) mixed with variable regions that are presumed to provide a given pheromone its functional specificity. 29 Although receptors for these pheromones remain to be elucidated, biochemical, molec- ular, and behavioral data are consistent with the hypothesis that the cellular and organismal actions of these molecules are initiated by means of interaction with specific receptor molecules. V. CHEMORECEPTION IN MULTICELLULAR ORGANISMS The organismal division of labor that resulted from the development of multicellularity brought about behavioral repertoires that, by the standards of single-celled life forms, can be considered complex. The study of the organismal, cellular, and molecular ways in which environmentally encountered chemical signals influence behaviors associated with feeding, development, and social interactions has made important contributions to our understanding of chemoreception. A. F EEDING 1. Behavioral Observations and Studies Chemically initiated feeding behavior has long been observed and studied in a large variety of marine organisms. A well-studied example in an evolutionarily ancient metazoan that links this behavior to chemoreceptors has been the study of the responses of cnidarians to particular chemicals. Loomis 30 reported that reduced glutathione (GSH) initiated feeding behavior in Hydra littoralis . Subsequently, it was shown that representatives of every class of cnidarian exhibit a feeding response when exposed to one of a few small compounds, 31 with GSH, the amino acid proline, a variety of other amino acids, and the quaternary ammonium compound betaine being the most typical initiators of the behavior. 32 The apparent specificity led to the conclusion that the observed behaviors were likely to be receptor mediated. 33 Similar observations have been made, and similar conclusions drawn, with members of many other metazoan phyla including annelids, molluscs, and echinoderms (reviewed by Lenhoff and Lindstedt 34 ), arthropods (reviewed by Ache 35 ), and vertebrates (reviewed by Sorensen and Caprio 36 ). In addition to feeding attractants, behavioral observations have made it clear that many organ- isms are deterred from eating certain other plants and animals, and this deterrence is often chemical in nature. Whereas feeding attractants are often small molecules such as nucleotides, sugars, and amino acids that are components of important metabolic pathways and can be considered primary metabolites, feeding deterrents are often somewhat larger, more complex molecules that play no obvious role in basic metabolic pathways and, as mentioned earlier, are termed secondary metab- olites. 37 Some feeding deterrents function by interacting with the consuming organism’s peripheral chemosensory systems, and chemoreceptors are implicated in subsequent behavioral responses, but many function in a different manner entirely, by affecting one aspect or another of the physiology of the consuming organism (see also Chapter 11, this volume). 38 9064_ch14/fm Page 467 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC 468 Marine Chemical Ecology 2. Physiological, Biochemical, and Molecular Studies Building upon the behavioral observations of glutathione-initiated feeding in Hydra , Rushforth and colleagues 39,40 demonstrated the effects of this chemical on the electrophysiological activity of the animal. Subsequently, a number of studies have been performed to biochemically evaluate the nature of the interaction of this feeding stimulant with this organism. Bellis et al. 41 demonstrated a reversible interaction of glutathione with Hydra plasma membrane preparations that was char- acterized by a K D of 3.4 µ M, a value in close agreement with the EC 50 of glutathione-induced feeding behavior. Although electrophysiological and biochemical studies of chemoreception in other aquatic invertebrates demonstrated similar support for chemoreceptor-mediated detection of environmental chemical signals associated with feeding (e.g., Croll 42 ), the bulk of this sort of data has been collected from crustaceans (the lobsters Panulirus argus , P. californicus , and Homarus americanus , the crayfish Austropotamobius torrentium , and various crabs, such as Callinectes sapidus ). The relatively large size and accessibility of the chemosensory organs of these animals have led to their use as model systems to study the cellular electrophysiology of chemoreception. 43–46 The same attributes make them attractive organisms for biochemical and molecular studies. 47–49 Anatomically, the chemosensory cells of these animals share a unifying set of characteristics: they are bipolar neurons with ciliated dendrites closely apposed to the environment and axons that project into the central nervous system from a peripherally located cell body. This is a cellular bodyplan that is characteristic of chemosensory cells from a broad range of metazoan phyla, so much that has been learned by the study of crustacean chemosensory neurophysiology has been of heuristic value to the understanding of chemoreception in other organisms. Knowing that crustacea respond to the amino acids, nucleotides, and other compounds present in the food odors that stimulate feeding behavior in these animals, a number of researchers began studying the electrophysiological responses of crustacean chemosensory cells to these chemicals. Ache and colleagues 43,50–53 demonstrated that chemosensory cells responded to various amino acids and nucleotides. The structural specificities exhibited by receptor cells for stimulatory compounds were consistent with the hypothesis that the compounds were interacting with cell-surface receptors. In many cases, the structure–activity relationships were strikingly similar to the structure–activity relationships that had been described for internal receptors for these compounds. These similarities led to a restating of the Haldane hypothesis that there is an important evolutionary link between chemoreceptors that monitor the chemical composition of the external environment and those that monitor the chemical composition of the internal (but extracellular) environment of metazoans. 54 Derby and colleagues designed studies to characterize the interaction of amino acid and nucleotides with putative lobster olfactory receptors for these substances. They prepared plasma membrane fractions from the chemosensory dendrite-rich sensilla of the spiny lobster, and dem- onstrated specific, saturable, and reversible binding of the sulfonic amino acid taurine and the adenine nucleotide AMP to this material; 55,56 ultrastructural localization of binding sites on the dendritic membrane for AMP were also demonstrated. 57 In subsequent studies combining electro- physiological and biochemical experiments, multiple receptor types for L -glutamic acid were char- acterized. 58 Separate binding sites for L -alanine and D -alanine were characterized by Michel et al. 59 The interactions of mixtures of amino acid and nucleotides with receptors for individual amino acids have also been characterized and shown to bear close relationships to the inhibitory effects of mixtures upon electrophysiological and behavioral responses to individual amino acid and nucleotide odorants. 60,61 Ache and coworkers demonstrated that both cyclic nucleotides and inositol phosphates mediate the transduction of environmental chemical signals by the olfactory neurons of P. argus . 62–65 Both biochemical and molecular biological techniques have shown that the receptor cells contain various G-protein subunits that would be necessary for signal detection by G- protein-associated chemoreceptors. 48,49,66–69 In combination with electrophysiological studies, 9064_ch14/fm Page 468 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 469 these techniques have demonstrated the existence of an ensemble of ion channels in these cells, the opening and closing of which are mediated by the second messengers that are generated by the occupancy of receptors by odorants. It is interesting to note that individual receptor cells can be depolarized by one amino acid and hyperpolarized by another. 70 These results indicate that both cAMP- and IP 3 -gated channels are present in a single neuron. More importantly, these results are consistent with the hypothesis that in this marine invertebrate, single olfactory neurons can express more than one receptor. This is in apparent contrast with the situation in vertebrates, where it appears that an individual receptor cell deploys only a single chemoreceptor. 70–72 The demonstration of G-protein-mediated signal transduction of amino acid signals suggests that the chemoreceptors of the lobster olfactory organ for these substances are of the seven transmembrane-segment, G-protein-coupled (GPC) variety. Although the lobster olfactory organ contains mRNA transcripts, the sequences of which bear reasonable homology to GPC receptors from other organisms that are presumed to be chemosensory, the functional demonstration that these transcripts code for chemosensory receptors in the lobster has not yet been achieved. Electrophysiological studies of the smell and taste systems of fish have likewise demonstrated chemoreceptor cells that are responsive, with varying degrees of specificity, to the amino acids known to elicit feeding behavior. 73–75 In addition, a number of fish have receptor cells that respond to bile acids, amphipathic steroid compounds that are used as digestive detergents and that can be released into the environment in substantial quantities. Responses can exhibit both exquisite specificity for the structure of a bile acid, and extreme sensitivity, as best exemplified by the sea lamprey. 76,77 Membrane preparations of fish olfactory and gustatory organs have been used to test the hypothesis that receptors for odorants and tastants are resident in these membranes. Scientists at the Monell Chemical Senses Center have published an extensive series of papers on the biochemical characterization of trout and catfish receptors for amino acids. Krueger and Cagan 78 demonstrated a structurally specific, reversible interaction of the amino acid L -alanine with plasma membrane fractions of catfish taste epithelium, and Brand, Bryant, Kalinoski and colleagues comprehensively characterized a catfish taste receptor for L -arginine. 79–82 These scientists further reported the presence of G-proteins in catfish olfactory cilia, 83,84 with cyclic AMP (cAMP) being implicated as a second messenger involved in the transduction of amino acids binding to olfactory receptors. 85 Lo et al. 86 have shown that bile acids bring about increases in intracellular second messengers in the olfactory system of salmon, and they hypothesized that this second-messenger generation is, at least in part, receptor mediated. Recently, the cloning and functional expression of a goldfish odorant receptor that specifically interacts with basic amino acids has been achieved; analysis of the sequence of nucleotides that codes for this receptor demonstrates that it is a member of the G-protein-coupled family of receptors. 87 B. L ARVAL D EVELOPMENT 1. Behavioral Observations and Studies For many marine organisms, a larval period is an evolutionarily important component of the life cycle. In many case, the developmental transition from the larval stage to the juvenile stage is initiated by an appropriate environmental signal. Upon detection of this signal, appropriate internal developmental processes will be triggered or released; if the signal is not detected, larvae remain in a state of developmental arrest. 88 Behavioral observations have indicated that, although phenom- ena such as light, substrate surface texture, and hydrostatic pressure can be the metamorphosis- inducing trigger for selected species, 89 more frequently a trigger of a chemical nature has been implicated. 88–94 In some cases, the nature of the chemical signal is known as well. Larvae of the tube worm Phragmatopoma californica undergo metamorphosis in response to a proteinaceous substance present in the tubes built by conspecific adults. 95 Larvae of the sand dollar Dendraster excentricus 9064_ch14/fm Page 469 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC 470 Marine Chemical Ecology respond metamorphically to material from adults of this species (a peptide of about 1000 Da); this material can be found in the sand beds occupied by adults. 92 Larvae of the opisthobranch mollusc Phestilla sibogae are induced to metamorphose by a low molecular weight (300 to 500 Da) water- soluble material that is released from the coral Porites compressa ; this coral is fed upon by the adult form of this nudibranch. 91,96,97 Recently metamorphosed juvenile red abalone ( Haliotis rufescens ) are typically found on rocks encrusted with the red alga Lithothamnium californicum ; 98 a peptide with a molecular weight of about 1000 Da that is present on the surface of this alga appears to be the molecule that induces larval abalone to metamorphose to the juvenile state. 93,99 Red algae also provide the chemical cues that induce larvae of the conch Strombus gigas to undergo metamorpho- sis, 100 and larvae of the sea urchin Holopneusteus purpurascens are induced to metamorphose by a water-soluble complex of the red algal metabolites floridiside and isethionic acid. 101 Larvae of agariciid corals are induced to metamorphose by sulfated polysaccharides found at the surfaces of tropical species of corraline red algae. 102 Oyster larvae can be induced to settle by small, soluble peptides containing C-terminal arginine residues; 103 both adult conspecifics and microbial biofilms found in association with the adults could serve as the source of these inducing peptides. 104 Variations in peptide amino acid composition leads to alterations in the efficacy of a molecule as an inducer; the resulting structure–activity relationships strongly suggest interaction with specific chemorecep- tors. 105 The ability of specific exogenous compounds to initiate and modulate larval metamorphosis has led many students of this developmental phenomenon to implicate larval chemoreceptor mole- cules, deployed at the environment-facing surfaces of chemosensory cells, as key components that serve as an interface between the larval nervous system and the marine environment. 2. Physiological, Biochemical, and Molecular Studies The small size and challenging anatomy of molluscan larvae have made electrophysiological studies of chemically induced settlement and metamorphosis considerably more difficult than similar studies of the effects of feeding stimulants on the olfactory neurons of adult crustaceans. Compounds that induce the larvae of the abalone Haliotis rufescens to settle and metamorphose affect the firing of the motile ciliated velar cells that, in aggregate, comprise the swimming organ of the veliger. 106 However, this phenomenon appears to be mediated by the larval nervous system rather than by the inducing molecule itself — the velar cells are not themselves sensing metamorphosis-inducing chemicals in the environment. Arkett et al. 107 showed that cells on the propodium of the larval nudibranch Onchidoris bilamellata were depolarized by exposure to barnacle-derived compounds that induce these larvae to settle and undergo metamorphosis. Another approach to electrophysio- logical studies of chemically induced larval settlement and metamorphosis has been to focus on neurons one or more synapses away from the actual chemosensory neurons; Leise and Hadfield 108 demonstrated that cells in the central ganglia of Ilyanassa obsoleta larvae alter their firing patterns in response to compounds that induce the metamorphosis of these larvae. The results of these studies infer, as did behavioral assays, the existence of chemosensory cells with receptors for inducing molecules. In a series of imaginative experiments combining electrophysiological principals with behav- ioral observations, Yool (née Baloun) and colleagues subjected competent larvae from a number of marine genera to treatment with artificial seawaters containing ionic additions, substitutions, or deletions designed to either bring about or prevent the depolarization of neurons. 109,110 The results of these experiments, as exemplified by the finding that a brief period of larval exposure to elevated concentrations of K + would induce metamorphosis, were consistent with the hypothesis that the depolarization of larval neurons, perhaps but not necessarily chemosensory neurons, was a necessary step between the encountering of a metamorphosis-inducing environmental cue and the subsequent behavioral and developmental metamorphic events. There are few reports of direct biochemical characterization of larval chemoreceptors. Following the finding that abalone larvae could be induced to metamorphose by γ-aminobutyric acid (GABA) 9064_ch14/fm Page 470 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 471 as well as by peptidic materials extracted from the red alga Lithothamnium californicum, 111,112 Trapido-Rosenthal and Morse 113 used a radiolabeled GABA analog, β-chlorophenyl GABA (baclofen), to characterize the interactions of settlement-inducing compounds with larval abalone. They demonstrated the existence of reversible binding of this metamorphosis-inducing compound to larvae, an interaction that is characterized by a K D (the concentration of inducer at which half of the binding sites are occupied) on the order of 1 µM and a B max (the total number of binding sites) of 15 fmoles/larva. It was further shown that this binding could be competed for by prepa- rations of morphogenic peptides isolated from L. californicum. 114 These binding sites disappear from larvae at the time of metamorphosis, when the larvae shed both their velar cilia and the cilia of their apical tuft, to which chemosensory functions have been attributed on anatomical grounds. 115–117 In further experiments, Baxter and Morse 118,119 isolated velar and apical tuft cilia from competent abalone larvae and demonstrated that the cilia in these preparations specifically and reversibly bound the diamino acid lysine, a substance which itself is nonmorphogenic but which dramatically modulates the effectiveness of both algal and amino acid morphogens. 120 The transduction of the chemical signals that initiate and regulate metamorphosis has been investigated using imaginative combinations of a variety of techniques. The above-mentioned demonstrations by Yool and colleagues 109,110 (that conditions that bring about neuronal depolariza- tion induce abalone larvae to undergo metamorphosis) clearly corroborate the hypothesis that transmembrane ion fluxes are obligatory components of metamorphic responses. Results from experiments with tetraethylammonium, a membrane-impermeant blocker of chloride ion channels, in which the presence of this compound in seawater prevents larvae from responding to metamorphic signals, suggest that depolarization of cells exposed to the environment are necessary for the initiation of metamorphosis. However, Hadfield and colleagues 121 have demonstrated that the selective ablation of putative environment-contacting chemosensory cells does not prevent Phestilla larvae from undergoing metamorphosis when subjected to depolarizing concentrations of potassium or cesium ions; these results make it clear that depolarizations of cells one or more synapses downstream from the chemoreceptor cells are also required for metamorphosis. The modulatory effect of lysine on the induction of abalone metamorphosis by GABA or by appropriate algal peptides is mediated by receptors located on larval cilia. 118–120 When these recep- tors are occupied by an appropriate ligand, signal transduction is brought about by the interaction of the receptor–ligand complex with G-proteins; this interaction in turn activates a second messenger cascade involving phospholipase C and protein kinase C (PKC). 122 The ways in which the proteins phosphorylated by PKC enhance responses to metamorphic signals remain unknown. C. SOCIAL INTERACTIONS 1. BEHAVIORAL OBSERVATIONS AND STUDIES As important to an organism as eating and developing is staying alive. Detection of chemicals emanating from potential predators, or from the dead or damaged prey of these predators, can lead to a behavioral response that removes an animal from the predator’s environment. Some of the chemicals that induce escape responses are identical to compounds that, in a different context, serve as feeding deterrents. Thus, the starfish saponins that are feeding deterrents to animals that prey upon starfish warn molluscs that would be preyed upon by the starfish that they are in a dangerous environment. In other cases, an organism that is molested by a predator will release a compound that will, if detected by its conspecifics, induce an escape or avoidance behavior. An example of this is the release of navenones into the slime trail produced by an aggravated specimen of the nudibranch Navanax inermis; conspecifics detecting this signal will turn off of this trail. 123 Another example is the release of anthopleurines into the water by a damaged specimen of the anemone Anthopleura elegantissima; detection of this compound by nearby conspecifics will induce them to contract into a conformation less vulnerable to predatory damage. 124 Crustaceans can recognize 9064_ch14/fm Page 471 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC 472 Marine Chemical Ecology chemical cues emanating from predators, at which point they will engage in avoidance behavior. 125 In addition, they can deliver, via their urine, chemical signals that can indicate to nearby conspecifics the presence of a nearby stressor such as a predator. 126,127 Fish likewise recognize a variety of alarm substances, some of which are released by members of their own species and others that may emanate from other organisms, and signify a predatorial presence. 128–130 Crustaceans are known to use pheromones in behavioral contexts other than avoidance, includ- ing reproduction and social interactions. For example, at least one spiny lobster, the California spiny lobster Panulirus interruptus, has been shown to be attracted to the odor of conspecifics. 131 This pheromonal phenomenon is taken advantage of by workers in the lobster fishery, who use live lobsters as bait in their pots. 132 However, relatively little research has been performed on the topic of aggregation pheromones, including the nature of the signal and its sensory reception. Pheromonal chemical signals are also involved in the establishment and maintenance of social hierarchies in crustaceans. 133,134 Using a comprehensive series of behavioral, biochemical, and molecular biological experiments, Painter and colleagues 135 have identified and chemically characterized an aggregation pheromone, which they named attractin, from the opisthobranch mollusc Aplysia californica. This molecule, a glycosylated 58-residue peptide, is produced by the albumin gland and released into the environment with the material that this gland adds to the animal’s egg cordons. There is a striking structural similarity between Aplysia attractin and the peptide pheromones of the ciliate protozoan Euplotes. 29 Future research may reveal that molecules such as these, which have the possibilities of mixing highly conserved domains with variable domains, may well be used as pheromones by a number of marine organisms. Fish provide numerous examples of other chemically mediated social behaviors. A dramatic example is the ability of many fish to “home” or return to a particular geographic location, most typically the site of their nativity. The chemical signals used in homing behavior have not been comprehensively identified but are thought to include both molecules of plant origin that are characteristic of the natal site as well as odorants, including bile acids, that derive from conspecific fish. 136,137 Reproductive behavior in fish is a phenomenon that is synchronized by chemical means. 138–140 A fraction of the steroids that are involved in the internal development of oocytes are released by females into the environment, where they are encountered by males — detection of this steroid induces internal hormonal changes in males that bring about enhanced sperm production. At a later time in the reproductive cycle, prostaglandins in the female that are associated with the follicular rupture of mature egg cells are released. Upon detection of the appropriate prostaglandin, males begin mating behaviors that culminate in the release of gametes by both sexes. 2. Physiological, Biochemical, and Molecular Studies The goldfish has been established as a model system for the study of chemically mediated reproductive phenomena in aquatic vertebrates. Sorensen and colleagues 139,141–145 have performed extensive studies of the electrophysiological responses of the olfactory systems of males to the pheromonal steroids (preovulatory signals that prime males for subsequent sexual activity) and prostaglandins (released into the environment after ovulation, the function of which remains to be completely elucidated). Their results have shown that goldfish have receptor cells for steroids that are highly specific and sensitive, with minute changes in molecular structure resulting in one hundred-fold increases in nanomolar threshold concentrations. 139,145 The results of this work have been consistent with receptor mediation of the behavioral responses to these environmentally encountered chemical signals. Likewise, in goldfish, Rosenblum et al. 146 have characterized the interaction of the steroidal pheromone 17α,β-dihydroxy-4-pregnen-3-one to a plasma membrane isolated from the animal’s olfactory epithelium. In an attempt to elucidate the molecular basis of pheromone recognition, Cao 9064_ch14/fm Page 472 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC [...]... specificity of male goldfish to the preovulatory pheromone 17α,20β-dihydroxy-4-pregnen-3-one, J Comp Physiol A, 166, 373, 1990 140 Bjerselius, R and Olsén, K H., A study of the olfactory sensitivity of crucian carp (Carassius carassius) and goldfish (Carassius auratus) to 17α,20β-dihydroxy-4-pregnen-3-one and prostaglandin F2α, Chem Senses, 18, 427, 1993 141 Sorensen, P W., Hormones, pheromones, and chemoreception,... 2821 58 Burgess, M F and Derby, C D., Two novel types of L-glutamate receptors with affinities for NMDA and L-cysteine in the olfactory organ of the Caribbean spiny lobster Panulirus argus, Brain Res., 771, 292, 1997 © 2001 by CRC Press LLC 9064_ch14/fm Page 476 Tuesday, April 24, 2001 5:25 AM 476 Marine Chemical Ecology 59 Michel, W C., Trapido-Rosenthal, H G., Chao, E T., and Wachowiak, M., Stereoselective... of Marine Chemical Ecology to Chemosensory Neurobiology 477 83 Huque, T and Bruch, R C., Odorant- and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia, Biochem Biophys Res Commun., 137, 36, 1986 84 Bruch, R C and Kalinoski, D L., Interaction of GTP-binding regulatory proteins with chemosensory receptors, J Biol Chem., 262, 2401, 1987 85 Bruch, R C and Teeter, J H., Second-messenger... 1984 94 Pawlik, J R., Chemical ecology of the settlement of benthic marine invertebrates, Oceanogr Mar Biol Annu Rev., 30, 273, 1992 95 Jensen, R A and Morse, D E., Chemically induced metamorphosis of polychaete larvae in both the laboratory and ocean environment, J Chem Ecol., 16, 911, 1990 96 Hadfield, M G., Chemical interactions in larval settling of a marine gastropod, in Marine Natural Products... compounds on marine invertebrate larvae, Bull Mar Sci., 46, 512, 1990 © 2001 by CRC Press LLC 9064_ch14/fm Page 474 Tuesday, April 24, 2001 5:25 AM 474 Marine Chemical Ecology 9 Morse, D E., Recent progress in larval settlement and metamorphosis: closing the gaps between molecular biology and ecology, Bull Mar Sci., 46, 465, 1990 10 Rittschof, D and Bonaventura, J., Macromolecular cues in marine systems,... 1994 144 Sorensen, P W., Hara, T J., and Stacey, N E., Sex pheromones selectively stimulate the medial olfactory tracts of male goldfish, Brain Res., 558, 343, 1991 145 Sorensen, P W., Scott, A P., Stacey, N E., and Bowdin, L., Sulfated 17α,20β-dihydroxy-4-pregnen3-one functions as a potent and specific olfactory stimulant with pheromonal actions in the goldfish, Gen Comp Endocrinol., 100, 128, 1995 146 ... Endocrinol., 100, 128, 1995 146 Rosenblum, P M., Sorensen, P W., Stacey, N.E., and Peter, R E., Binding of the steroidal pheromone 17α, 20β-dihydroxy-4-pregnen-3-one to goldfish (Carassius auratus) olfactory epithelium membrane preparations, Chem Senses, 16, 143 , 1991 147 Cao, Y., Oh, B C., and Stryer, L., Cloning and localization of two multigene receptor families in goldfish olfactory epithelium, Proc... life and life-span extension in a marine mollusc, Science, 248, 356, 1990 89 Crisp, D J., Factors influencing the settlement of marine invertebrate larvae, in Chemoreception in Marine Organisms, Grant, P T and Mackie, A M., Eds., Academic Press, New York, 1974, 177 90 Chia, F S., Perspectives: settlement and metamorphosis of marine invertebrate larvae, in Settlement and Metamorphosis of Marine Invertebrate... invertebrates with special emphasis on the feeding behavior of coelenterates, in Chemoreception in the Marine Environment, Grant, P T and Mackie, A M., Eds., Academic Press, London, UK, 1974, 143 © 2001 by CRC Press LLC 9064_ch14/fm Page 475 Tuesday, April 24, 2001 5:25 AM Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 475 35 Ache, B W., Chemoreception and thermoreception, in The... 1991 103 Zimmer-Faust, R K and Tamburri, M N., Chemical identity and ecological implications of a waterborne, larval settlement cue, Limnol Oceanogr., 39, 1075, 1994 104 Tamburri, M N., Zimmer-Faust, R K., and Tamplin, M L., Natural sources and properties of chemical inducers mediating settlement of oyster larvae: a re-examination, Biol Bull., 183, 327, 1992 © 2001 by CRC Press LLC 9064_ch14/fm Page 478 . environmentally encountered chemical signals. Likewise, in goldfish, Rosenblum et al. 146 have characterized the interaction of the steroidal pheromone 17α,β-dihydroxy-4-pregnen-3-one to a plasma membrane. LLC 464 Marine Chemical Ecology 1974 article on chemoreception in the marine environment, Laverack 4 reviewed studies of organ- ismal and cellular responses to environmental chemical. higher 9064_ch14/fm Page 464 Tuesday, April 24, 2001 5:25 AM © 2001 by CRC Press LLC Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 465 signal-to-noise ratios 10