Marine Chemical Ecology - Chapter 14 pptx

Only relatively recently have electrophysiological and biochemical characterizations of chemore-ceptors for environmental chemical signals been accomplished, and the molecular characteri

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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

C Social Interactions 471

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

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

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

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464 Marine Chemical Ecology

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

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

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

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

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

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Contributions of Marine Chemical Ecology to Chemosensory Neurobiology 465

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

III CHEMORECEPTION IN BACTERIA

Chemically mediated behavior in bacteria was first noted by Engelmann in 1881 and Pfeffer in

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

Chemotaxis is perhaps the best studied bacterial responses to environmental chemical signals

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

by means of receptors that recognized the chemical structures of these signals This work was then

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

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

transduction mechanisms that are both qualitatively and quantitatively different than those

Bacteria also use chemical signals to communicate with each other The observation that the

densities were low led to the identification of bacterial metabolites that have become termed

are acylated homoserine lactones that are synthesized by the bacteria and released into the

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

1884 (see Paoni et al

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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,

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

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

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,

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

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beat and causes the cells to turn sharply The linkage of receptor occupation with membrane potential

its electrochemical gradient into the cells, resulting in a transitory increase in concentration to as

axonemal machinery results in a directional change in ciliary beat

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

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

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

Subsequently, it was shown that representatives of every class of cnidarian exhibit a feeding response

other amino acids, and the quaternary ammonium compound betaine being the most typical initiators

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

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

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

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2 Physiological, Biochemical, and Molecular Studies

of the animal Subsequently, a number of studies have been performed to biochemically evaluate

feeding behavior

Although electrophysiological and biochemical studies of chemoreception in other aquatic invertebrates demonstrated similar support for chemoreceptor-mediated detection of environmental

relatively large size and accessibility of the chemosensory organs of these animals have led to their

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

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

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

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

Ache and coworkers demonstrated that both cyclic nucleotides and inositol phosphates

cells contain various protein subunits that would be necessary for signal detection by

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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

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,

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

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

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

fractions of catfish taste epithelium, and Brand, Bryant, Kalinoski and colleagues comprehensively

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

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

phenom-ena such as light, substrate surface texture, and hydrostatic pressure can be the

In some cases, the nature of the chemical signal is known as well Larvae of the tube worm

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respond metamorphically to material from adults of this species (a peptide of about 1000 Da); this

with a molecular weight of about 1000 Da that is present on the surface of this alga appears to be

agariciid corals are induced to metamorphose by sulfated polysaccharides found at the surfaces of

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

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

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

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

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

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

of these experiments, as exemplified by the finding that a brief period of larval exposure to elevated

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

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(baclofen), to characterize the interactions of settlement-inducing compounds with larval abalone They demonstrated the existence of reversible binding of this metamorphosis-inducing compound

sites) of 15 fmoles/larva It was further shown that this binding could be competed for by

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

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

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

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

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

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

phosphorylated by PKC enhance responses to metamorphic signals remain unknown

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

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

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In addition, they can deliver, via their urine, chemical signals that can indicate to nearby conspecifics

substances, some of which are released by members of their own species and others that may

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

This pheromonal phenomenon is taken advantage of by workers in the lobster fishery, who use live

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

Using a comprehensive series of behavioral, biochemical, and molecular biological experiments,

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

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

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

The goldfish has been established as a model system for the study of chemically mediated

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

have been consistent with receptor mediation of the behavioral responses to these environmentally encountered chemical signals

olfactory epithelium In an attempt to elucidate the molecular basis of pheromone recognition, Cao

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