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Olfactory responses from amarine elasmobranch, theatlantic stingray, DasyatissabinaWayne L. Silver aa Department of Biological Science, Florida StateUniversity, Tallahassee, Florida, 32306Version of record first published: 22 Jan 2009.

To cite this article: Wayne L. Silver (1979): Olfactory responses from a marineelasmobranch, the atlantic stingray, Dasyatis sabina , Marine Behaviour andPhysiology, 6:4, 297-305

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Mar. Behav. Physiol., 1979, Vol. 6, pp. 297-3050091-181X/79/0604-0297$04.50/0© 1979 Gordon and Breach Science Publishers, Inc.Printed in Great Britain

Olfactory Responses from a

Marine Elasmobranch, the

Atlantic Stingray,

Dasyatis sabina

WAYNE L. SILVER

Department of Biological Science, Florida State University, Tallahassee,Florida 32306

(Received May 9, 1979)

Underwater electro-olfactogram (EOG) responses were recorded from the olfactory organof the Atlantic stingray, Dasyatis sabina. Amino acids were shown to be effective stimuliwith thresholds determined to be between 10-6 and 10-8 M. The five most stimulatorycompounds tested were l-glutamic acid γ methylester, l-ethionine, l-serine, l-glutamicacid, and l-methionine. The D-isomer of alanine was much less stimulatory than the l-isomer. The EOG response magnitude increased exponentially with logarithmic increase(spanning 4-5 log units) of stimulus concentration. Due to shunting of signals by the highlyconductive seawater, response magnitudes to 10 - 3 M l-alanine averaged only 0.2 mV,approximately 40 to 50 times less than the magnitude seen at the same concentration in afreshwater fish.

INTRODUCTION

Elasmobranchs have long been reputed to possess an extremely acute

sense of smell. This reputation is based partly on anecdote and partly on

their highly prominent olfactory system (Hodgson et ah, 1967), although

recent evidence suggests that the "olfactory brain" in elasmobranchs is

not as extensive as once thought (see Northcutt, 1978). Behavioral studies

have demonstrated that the olfactory system plays an important role in the

feeding behavior of sharks (Sheldon, 1911; Parker, 1914; Tester, 1963;

Hobson, 1963; Dijkgraff, 1975). The importance of olfaction in ray feeding

behavior has also been suggested (Bateson, 1890; Steven, 1932; Nicol, 1967).

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The behavior of many teleosts is strongly influenced by amino acids(Hashimoto et al, 1968; Tucker and Suzuki, 1972; Carr and Chaney, 1976;Carr et al, 1977). Hodgson et al. (1967) suggested that amino acids and aminesare also potential stimuli for sharks and may be chemical cues for theinitiation of feeding behavior. Tester (1963) tested the effectiveness of L-and D-serine as a shark repellent based on the observation that L-serineinduced an avoidance reaction in adult salmon on their spawning migra-tion (Idler et al, 1956). L-serine but not D-serine had a repellent effect onsharks.

The extreme sensitivity of the olfactory system of freshwater teleosts toamino acids has been well established electrophysiologically (Sutterlin andSutterlin, 1971; Suzuki and Tucker, 1971; Hara, 1972, 1973; Hara, et al,1973; Belghaug and Daving, 1977; Goh and Tamura, 1978; Caprio, 1978).Thresholds were estimated to be as low as 10~8 M for the most effectivestimuli. Electrophysiological examinations of the olfactory receptor pro-perties of marine fish including elasmobranchs have been few, partly becauseof the difficulties in electrical recording due to shunting by the seawater.D0ving and Holmberg (1974) reported positive going potential changesfrom the excised olfactory organ of the Atlantic hagfish. In a preliminaryreport Silver et al. (1976) were able to record from the olfactory organsof the Atlantic stingray and sea catfish by using the underwater electro-olfactogram (EOG).

The present study with the underwater EOG was initiated to examine inmore detail the olfactory responses to some amino acids in a marine elasmo-branch, the Atlantic stingray, Dasyatis sabina.

MATERIALS AND METHODS

Specimens of the Atlantic stingray, Dasyatis sabina, were obtained from theGulf of Mexico either by seine or by hook. They were transported to thelaboratory in large styrofoam or fibreglass containers and placed in all-glassaquaria containing Gulf seawater. All the seawater used in the experimentscame from the Gulf. The rays were held no longer than two weeks beforethey were used. The fish were anesthetized with MS222 (tricaine-methanesulphonate 1: 10,000) and positioned in a plexiglass holder. Aerated seawatercontaining MS222 was perfused through the mouth and over the gillsthroughout the experiment. The spiracles were plugged with pieces of spongeto force water over the gills. The flaps covering the olfactory lamellae wereretracted with hemostats and a continuous flow of seawater was directedonto the lamellae by a combination of glassware and Teflon tubing. Stimuliwere dissolved in seawater and introduced into the stimulus injection port

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OLFACTORY RESPONSES OF THE STINGRAY 299

with disposable Pasteur pipettes. A volume of 1 cc of the stimulus wasinjected into the port. Stimuli (12 L-amino acids, D-alanine, and L-glutamicacid y methyl ester) were diluted 50% upon reaching the olfactory organ asdetermined by photodensitometry of dye solution. Stock solutions weremade up weekly and fresh dilutions were made daily during the experiment.

The EOG was recorded with calomel electrodes via Ringer-agar filledcapillary pipettes. The active electrode was place in the flowing seawaterjust above the olfactory lamellae and directly over the midline. The referenceelectrode was placed in the mouth or contralateral nasal capsule. The animalwas grounded with an alligator clip attached to its tail. Electrical activity wasamplified by a direct-coupled amplifier, displayed on an oscilloscope, andpermanently recorded with a heated stylus recorder.

RESULTS

Amino acid stimuli elicited a negative, slowly adapting, change in potentialfrom the stingray olfactory organ, as illustrated in Figure 1. Average responsesto 1.0 mM L-alanine were about 0.2 mV as compared to 8 mV for the fresh-water eel.

1 min

0.1 mV

FIGURE 1 EOG response from stingray olfactory organ to 10""* M L-alanine. Notenegative is upward.

The relative responses to 12 L-amino acids, D-alanine, and L-glutamic acidy methyl ester at 10~4 M are shown in Table I. The ordering is based on theresponse to L-alanine, the standard stimulus. Of the stimuli tested, L-glutamicacid y methyl ester produced the largest response at 138% relative to L-alanine 100%. Records of responses to various amino acids are presented inFigure 2.

A typical concentration-response series for L-alanine is shown in Figure 3.The interstimulus interval was 2 minutes at the lowest concentration andincreased to 10 minutes at the highest concentration. There is an exponentialincrease of response magnitude (R) with logarithmic increase in stimulusconcentration (C), R — &(10)"IogC. This result signifies a power function

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300 W. L. SILVER

TABLE I

Relative olfactory effectiveness of some amino acids at 10 -* M

Compound

L-glutamic acid y methyl esterL-ethionineL-serineL-glutamic acidL-methionineL-alanineL-glutamineL-cysteineL-a-amino butyric acidL-arginineL-phenylalanineL-tyrosineGlycineL-proIineControlD-alanine

% Stimulatory effectiveness(mean±S.D.)

138±4115±7112±14111±16111±9100 STD68 ±761 ±225447 ±2142±1734±1330±1927±168±27±3

Number offish tested

2558

11118714538242

l-Alanine D-AUniie Clycine l-Serine

It I»I

tailL-Alaiini t-CyttliH IMitkiniM 1-EttiMiM

jO.1l*

In i.L-Gfiitamiu Ltlitiaie hit Ulitimic Acid

> Mettyl Estir

FIGURE 2 EOG responses to several amino acids at 10~*M. The records are fromthree different preparations. L-alanine is the standard in each case.

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OLFACTORY RESPONSES OF THE STINGRAY 301

with linear increase of concentration, i.e., R = kC. The exponent, n, wasdetermined from the slope of a least squares linear regression line fitted to thepoints in a log-log plot, log R — n log C+log K, and for the curve shown inFigure 4 was 0.370. For L-alanine the average exponent (slope) was 0.309+

Control10~5M

in-4-5*JV

10"4M 10"3«

g.t**

tail.

FIGURE 3 Typical EOG responses from the stingray olfactory organ at seven differentconcentrations of L-alanine. Control is response to seawater alone.

0 r

r: _i _

COoLU

to• o

_J

- 2

LOG E0G=.37 LOG C0NC.+.59

R=.99

.I

-7 -6 -5 -4

LOG MOLftR CONCENTRRTION

- 3

FIGURE 4 EOG response-concentration curve for L-alanine. Dotted line shows inter-section of linear regression line with control response yielding threshold value.

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302 W. L. SILVER

0.056 (N — 4). Exponents for L-methionine and L-glutamic acid y methylester were 0.270+0.050 (N = 3) and 0.369 (N = 1) respectively.

Thresholds were determined by the intersection of the regression line inthe log-log plot with the control response value (Figure 4). The averagethreshold to L-alanine was io~7-8 ± 0-7 M (N — 4). For L-glutamic acidy methylester the threshold was 10~6-7 M (N — 1) and for L-methionine, 10~7-4±0-2 M(N = 3).

DISCUSSION

The olfactory organs of the Atlantic stingray, Dasyatis sabina, are containedin paired, open nasal capsules which lie on the ventral sides of the headbetween the tip of the snout and the mouth. The nasal capsules are connectedto the mouth by the nasolabial grooves. A flap covering the capsule formsthe incurrent and excurrent openings. The olfactory organ contains twoseries of lamellae called a rosette. Each lamella contains secondary folds.The current of water through the nasal capsule is induced by the ordinarymovements of respiration and the forward movement of the ray. In addition,observations with fine charcoal granules demonstrated the presence ofpropulsive cilia which propel particles between the lamellae.

Because of the difficulty involved in recording neural responses from theolfactory receptors of marine fish due to the shunting of signals by the seawater, the underwater EOG (Silver et ah, 1976) was employed to test thesensitivity of the stingray olfactory receptors and the relative stimulatoryeffectiveness of some amino acids. However, in some preparations themagnitudes of the responses were very small (microvolts) and could hardlybe distinguished from baseline.

The shape of the EOG produced by amino acid stimulation, i.e. the initialrise (slow compared to the eel or catfish) followed by the very slow return tobaseline (Figure 1) might be explained by the geometry of the stimulationsetup. The nasal capsule is deep and bowl shaped and the stimulus solutionmay not have been flushed cleanly after introduction into the capsule.Instead, the capsule may have acted like an exponential diluter, the stimulushaving been gradually removed causing a long gradual decline to baselinelevels.

Hodgson et al. (1967) tested the responses of sharks to amino acids,using both behavioral observations and the EEG recorded from the forebrain.They used L- and o-methionine, L- and D-serine, glycine, glutamic acid, andcysteine (the chirality of glu and cysh was not specified). All were said to bestimulatory, but no ordering was presented. No differences were observed forresponses to the L- and D-isomers. In the present experiment the L-isomer of

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OLFACTORY RESPONSES OF THE STINGRAY 303

alanine was much more stimulatory than the D-isomer (100% to 7%).The greater potency of the L-isomer has been demonstrated for many otherfishes (Sutterlin and Sutterlin, 1971; Suzuki and Tucker, 1971; Hara, 19721973; Hara et ah, 1973; Goh and Tamura, 1978; Caprio, 1978).

The most notable differences in the sequence of amino acid effectivenessbetween the stingray and other fishes is the positioning of L-glutamic acidand L-glutamine. L-glutamic acid is considerably more stimulatory thanL-glutamine (111 % to 69 %) to the ray while the opposite appears to be truefor most other fishes. Hodgson and Mathewson (1978), recording sharkEEG responses, reported that glutamic acid and glycine were the mosteffective of the pure amino acids tested. Glutamic acid is also known to bethe most efficacious stimulant for several marine crustaceans (Case et al.,1960; Case and Gwilliam, 1961).

The (o ester, L-glutamic acid y methyl ester, was by far the most stimulatoryof the compounds tested. This was also true for the channel catfish (Caprio,1978). Caprio suggested that the enhanced efficacy of this compound may bedue to the removal of the side chain charge or the increase in the length of theamino acid. For the stingray the former does not appear to apply sinceL-glutamine is much less stimulatory than L-glutamic acid. Increasing thelength of the amino acid may play a role in the increased potency as is alsoseen in the relative responses to L-ethionine and L-methionine.

The control response, i.e. the response to seawater alone, had an effective-ness of 8% relative to L-alanine 100%. A response to control stimulus hasbeen reported in several of the electrophysiological studies of fish olfaction.Goh and Tamura (1978) suggested that the control response may be due totemperature differences between the injected stimulus and the flowingwater. However, Suzuki and Tucker (1971) and Caprio (1978) ruled out thepossibility of either thermal or mechanical stimulation and concluded thatthe control response was due to low level chemical contamination.

Although the EOG is a relatively simple technique for studying olfactoryresponses in marine fish, caution should be observed in relating it with theneural response. Tucker and Suzuki (1972) demonstrated for the whitecatfish, and Silver and Tucker (1977) for the American eel, that the EOG isnot linearly related to the neural response. The EOG is best described as anexponential function of the neural response. Although both the EOG andneural concentration-response curves are described by power functions, theEOG exponent is twice as large as the neural exponent for the eel (Silver andTucker, 1977) and channel catfish (Caprio, 1978). Silver and Tucker (1977)also reported for the eel that thresholds determined with the EOG are alog unit higher than thresholds obtained with the neural response, althoughTucker and Suzuki (1972) noticed no difference between the two for thewhite catfish.

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304 W. L. SILVER

For the stingray, as has been reported for many other fish, the concentra-tion-response curve obtained from the olfactory receptors is best describedby a power function. In addition, thresholds determined from the intersectionof the equivalent straight line on a log-log plot with the control responsevalue are similar to those reported for other fish. Despite its large olfactoryapparatus, the stingray does not appear, electrophysiologically at least, to beany more sensitive to amino acids than fish with less prominent olfactorysystems, but since behavioral thresholds are often lower than electro-physiological thresholds it is probable that olfactory sensitivity is lower thanreported here.

Acknowledgements

I am very grateful to Drs. D. Tucker and J. Caprio for their help and advice throughoutthis study. This study was supported by grants from the National Institutes of Health,Numbers NS-08814 and NS-05258.

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