- Email: [email protected]
Comparison of the olfactory response to amino acids in rainbow trout, brook trout, and whitefish
Comp. Biochem. Physiol., 1973, Vol. 4SA, pp. 969 to 977. Pergamon Press. Printed in Great Britain
COMPARISON OF THE OLFACTORY AMINO ACIDS IN RAINBOW TROUT, AND WHITEFISH
RESPONSE TO BROOK TROUT,
TOSHIAKI
and
J. HARA,
Y. M. CAROLINA
LAW
B. R. HOBDEN
Fisheries Research Board of Canada, Freshwater Institute, Winnipeg, Manitoba, Canada R3T 2N6 (Received
26 October 1972)
Abstract-l. The olfactory apparatus and the bulbar electrical responses induced by nasal infusion of amino acid solutions were compared among three salmonid species, rainbow trout, brook trout and whitefish. 2. Morphology of the olfactory organ was similar in all species examined, except that the peripherial olfactory organ of whitefish was slightly smaller. 3. The threshold concentration for the most effective amino acids ranged from low7 to 10-s M in rainbow and brook trout and was higher in whitefish. 4. Olfactory bulbar response spectra to amino acids were similar in all species. However, correlation was slightly higher between rainbow and brook trout (r = 0.89) than between rainbow trout and whitefish (r = 0.82). INTRODUCTION
PREVIOUS studies indicate rather specific effectiveness of certain amino acids for eliciting the electrical response in the olfactory bulb of rainbow trout and Pacific salmon (Hara, 1972, 1973). The studies on the olfactory receptor responses to amino acids in Atlantic salmon (Sutterlin & Sutterlin, 1971) and in catfish (Suzuki & Tucker, 1971) also support this view. However, specific significance of free amino acids in olfactorily mediated behaviour of fish remains to be determined. The present experiments were undertaken to compare morphology of the olfactory organ and the electrical response spectra of the olfactory bulb in three salmonid species, each of which has different feeding habits and habitat. An attempt was made to correlate the bulbar response spectrum of rainbow trout with those of the other two species to find out any similarity between them. MATERIALS
AND METHODS
Rainbow trout (Salmo gairdneri) and brook trout (Salvelinus fontinalis) were obtained from a local hatchery. Whitefish (Coregonus clupeuformis), which had been planted from a hatchery, were taken from a lake. They were all held in large tanks for several months and were transferred to glass aquaria, supplied with a continuous flow of dechlorinated water (temp. 13” + l”C), for acclimation at least 3 weeks prior to use. For anatomical studies fish were put in 10% formalin solution for several days, then the olfactory apparatus was examined. For histological studies, excised olfactory rosettes were fixed in Bouin or formalin solution and stained with haematoxylin-eosin. 969
970
TOSHIAKIJ. HARA, Y. M. CAROLINALAW ANDB. R. HOBDEN
Operative procedures of the fish and recording techniques of the electrical response from the olfactory bulb have been fully described previously (Hara, 1972,1973 ; Hara & Law, 1972; Hara et al., 1973).
RESULTS
Morphology of the olfactory apparatus In all species examined, anterior and posterior nostrils separated by a nasal bridge (skin flap) are located anterodorsal to the eye as in most other fish. In whitefish both nostrils are separated by two flaps, anterior and posterior. In the nasal sac there is an almost circular olfactory rosette where the sensory epithelium lodges. It consists of a rostrocaudally oriented median depression from which varying numbers of lamellae radiate. As shown in Table 1, the size of the olfactory rosette and the number of the lamellae of whitefish were smaller compared with TABLE ~--SIZE OF OLFACTORYAPPARATUS AND NUMBEROF PRIMARYOLFACTORY LAMELL~ (LENGTHIN mm.) No. of primary olfactory lamellae Species
Body length
Nasal sac
Olfactory nerve
Olfactory bulb
Left
Right
Salmo gairdneri
197 206 184 207
3.4 3.5 4.4 5.0
x x x x
4.6 4.6 4~8 4.6
8.0 8.0 7.0 9.6
1.7 x 1.2 x 1.1 x 2.2x
2.5 3.2 2.2 2.8
15 16 14 16
16 16 14 18
Salvelinus fontinalis
173 157 149 170
4.4 3.3 3.2 4.2
x x x x
4.8 3.8 3.9 4.0
7.0 6.0 6.0 6.0
1*2x 2.5 1.2x2.0 1.4 x 2.3 1.3 x2.4
14 12 12 13
13 12 12 12
Coregonus
187 172 203 172
1.5 2.4 4.0 2.4
x x x x
1.1 2.9 4.4 3.2
8.4 7.5 9.0 6.4
1.3x1.4 1.5 x 2.3 1.6x2.5 1.6x2.0
13 13 16 12
12 14 14 12
clupeaformis
those of the other two species of similar body length. Each lamella is slightly different in size. A secondary folding of the lamellae was observed in all species
examined. In some small fish, however, no lamella has this secondary folding. Histological sections of the lamellae shows no indication of sensory cells on the secondary lamellae (Fig. 1). The olfactory nerve, the olfactory bulb and other brain structures are essentially similar both in pattern and size. These are summarized in Fig. 2 and Table 1.
FIG. 1. Cross-sections of olfactory rosettes, showing development of the secondary olfactory lamellae. 1, primary olfactory lamellae of small brook trout (about 15 cm). No secondary lamella is developed. Most of the primary lamella is covered with sensory epithelium except for the top part. 2, cross-section of olfactory lamellae of whitefish. secondary lamellae are well developed. Notice nonsensory cells and goblet cells on top of ,both primary and secondary lamellae. 3, secondary olfactory lamellae are shown in higher magnification. pl, primary lamella; sl, secondary lamella.
OLFACTORY
RESPONSE
TO
AMINO
ACIDS
IN
FISH
971
.OR
FIG. 2. Schematic drawing of brain and olfactory organ of whitefish, dorsal view. Their sizes are shown in Table 1. CE, cerebellum; MC, mesencephalon; OB, olfactory bulb; ON, olfactory nerve; OR, olfactory rosette; TC, telencephalon.
Electrical responses from the olfactory bulb
Figure 3 illustrates typical responses from the olfactory bulb of whitefish when the nares were stimulated with hand rinse (a forefinger dipped in 100 ml water for 10 set), L-serine, isoserine, and phenylalanine, all at 1O-4 M. In rainbow and brook trout, the lowest estimated threshold concentrations for most effective amino acids are usually between lo-’ and 10-s M, though lower thresholds had been observed. However, it was much higher in whitefish. In Fig. 4, percentage response to different concentrations of L-serine are compared among the three species. Responses to a hand rinse, known to be an effective olfactory stimulant to fish (cf., Hara, 1970, 1971), generally exceed those to lo4 M L-serine in both rainbow and brook trout-140 and 150 per cent of those to lOA L-serine, respectively. In whitefish, however, the response was nearly of the same magnitude
972
TOSHIAKI J. HARA, T. M. CAROLINALAW ANDB. R. HOBDEN
FIG. 3. Typical responses recorded from the olfactory bulb of whitefish when the nares were stimulated with hand-rinse (A), 1O-4 M L-serine (B), low4 M isoserine (C), and 10m4 M phenylalanine (D). The upper tracing (a) of each pair is the integrated responses of the lower (b). Period of stimulation and time scale (each division = 1 set) are shown at the bottom. IOO-
o-
I
-!
I
-6 -5 log molarconcentration
I
-4
FIG. 4. Percentage responses to different concentrations of L-serine in rainbow trout ( * ), brook trout ( x ), and whitefish (A).Magnitudes of the response in each fish species is represented as a percentage of the response to 10e4 M L-serine.
as that to 1O-4 M L-serine. The effectiveness of n-isomers was always less than that of the L-isomers in all three species. A comparison of the stimulator-y effectiveness of twenty-four of the most effective amino acids tested between brook trout and whitefish is shown in Fig. 5. All chemicals were tested at the same concentration of 10m4 M. The relative effectiveness of each chemical is represented as a percentage of the response to the standard L-serine. It is clearly noticed that responses of whitefish to amino acids are generally much smaller than those of brook trout except for the six most effective amino acids, i.e. L-glutamine, L-methionine, L-alanine, homoserine, L-serine and L-cystine.
OLFACTORY RESPONSE TO AMINO ACIDS IN FISH
973
Homoser L-met L-glu L-ala L-asp L-cys L-leu L-thr L- ser L- VOI L - org
GUY L -his L-lys L-cys-cys lsoleu GABA lsoser L-tyr D-ala L-try D-ser L -phe /3-ola
FIG. 5. Comparison of the stimulatory effectiveness of twenty-four of the most effective amino acids tested between brook trout (white bars) and whitefish (black bars). All chemicals were tested at 10m4 M. Relative effectiveness of each chemical is represented as a percentage of the response to the standard L-serine (obliquelined). To compare these response spectra with the one previously obtained in rainbow fit was computed for each pair of the three trout (Hara, 1973), a least-squares groups. As shown in Fig. 6, a highly significant correlation was found between any
974
TOSHIAKIJ.HARA,Y.
M.
CAROLINA
LAW AND B.R.
HOBDEN
IFJO.
.
y=0*78xitl*36 r=089
0
I 50
0
I 100
I 150
Brooktrovt FIG. 6 (a) 150. l l
. . /
l.
.^A IUUl
.
l
l
/
.
. l
l
+ 21.48 Y '=0.78x f 60.82
l
I
I
50
100
Whitefish FIG. 6 (b)
I
150
975
OLFACTORY RESPONSE TO AMINO ACIDS IN FISH
.
1
100
50
I
150
Whitefish
FIG. 6 (c) FIG. 6. Relation of the olfactory bulbar electrical responses to amino acids between rainbow trout and brook trout (a), rainbow trout and whitefish (b) and brook trout and whitefish (c). Each dot indicates the mean of three to eight or more tests in each species. Least-squares fit was computed for each pair. 7 = correlation coefficient.
pair of the three species. The highest correlation was between rainbow and brook trout (correlation coefficient, r = 089). Correlation coefficients between rainbow trout and whitefish, and between brook trout and whitefish were 0.82 and 083, respectively. DISCUSSION
Different types of olfactory rosettes in teleosts have been described (Holl, 1965). In all the species examined the olfactory rosettes were almost circular in shape. The number of primary olfactory lamellae is a function of size, and not of age in Pacific salmon (Pfeiffer, 1963) and Baltic sea trout (Bertmar, 1972). The present observations generally agree with these reports. As shown in Table 1, the size of the nasal sac and the number of the primary olfactory lamellae of whitefish are slightly smaller compared with those of the other two species. Histological examination shows that the secondary lamellae are well developed in whitefish. It is not likely, therefore, that the higher threshold of the bulbar electrical response, as observed in whitefish, is the result of a lower degree of development in the secondary olfactory lamellae. The secondary folding of the primary lamellae certainly results
976
TOSHIAKIJ. HARA, Y. M. CAROLINALAW ANDB. R. HOBDEN
in an increase in the surface area of the olfactory epithelium, though most of this increase can be referred to as the indifferent epithelium (Bertmar, 1972). Nevertheless, folding of the lamellae increases effective use of the space in the olfactory chamber, which, in turn, seems to result in an increase in the total olfactory capacity. Results from the present experiments on electrical responses of the olfactory bulb of brook trout and whitefish to amino acids are in good agreement with earlier reports (Hara, 1973) that there are several highly effective amino acids and that the effectiveness of n-isomers is always less than that of L-isomers. These results suggest that highly effective amino acids may play an important role in the life of these fish species. However, no such behavioral evidence has been available so far, except the fact that the amino acid r.-serine is one of the active components of mammalian skin extract and is known to be a repellent to migratory Pacific salmon (Brett & MacKinnon, 19.54; Idler et al., 1956). In this connection, whitefish which were less responsive to a hand rinse generally showed lower electrical responses to all amino acids tested, Satou (1972, personal communication) recorded relatively small electrical responses from the olfactory bulb of carp, Cyprinus carpio, when stimulated with solutions of L-glutamine, L-methionine, and L-cysteine at rather higher concentrations, 5 x 10~~ to 10~~ M. No or little response was induced with other amino acids tested. This lower sensitivity to amino acids in carp is in agreement with its lower responsiveness to food extract (Satou, 1971). Hashimoto et al. (1968) and Konosu et al. (1968) fractionated the extract of a clam which evoked exploratory and feeding response in the Japanese eel, Anguilla japonica, and found that the amino acids, taurine, aspartic acid, serine, threonine, glutamic acid, glycine, and alanine, were the main components responsible for the activity, while the other constituents, such as nucleotides, organic acids and quarternary ammonium bases, made little contribution. They suggested that the stimulating activity of the extract is attributable mainly to the synergistic or additive interaction of these amino acids, since each of these amino acids was not effective when tested individually. Thus, it seems likely that certain free amino acids or their appropriate mixtures may play a fundamental role in behaviour, such as food selection, predator-prey interactions and migration, in some species of fish. Chemical and physiological characterization of interactions between the olfactory receptors and natural odorous substances pertinent to olfactorily mediated fish behaviour will be required. Acknowledgements-We thank Dr. J. G. Eales, Department of Zoology, University of Manitoba, for critical reading of the manuscript, and Mr. S. T. Zettler and Miss C. L. Boyce for photography and drafting. REFERENCES BERTMARG. (1972) Secondary folding of olfactory organ in young and adult sea trout. Actu zool. 53, 113-l 20. BRETT J. R. & MACKINNOND. (1954) Some aspects of olfactory perception in migrating adult coho and spring salmon. J. Fish. lies. Bd Can. 11, 310-318.
OLFACTORYRESPONSETO AMINOACIDSIN FISH
977
HAM T. J. (1970) An electrophysiological
basis for olfactory discrimination in homing salmon: a review. r. Fish. Res. Bd Can. 27, 565-586. HARA T. J. (1971) Chemoreception. In Fish Physiology vol. 5 (Edited by HOAR W. S. & BANnALL D. J.), pp. 79-120. Academic Press, New York. HARAT. J. (1973) Electrical responses of the olfactory bulb of Pacific salmon, Oncorhynchus nerka and 0. kisutch. J. Fish. Res. Bd Can. 29, 1351-1355. HARA T. J. (1972) Olfactory responses to amino acids in rainbow trout, Salmo gairdneri. Con@. Biochem. Physiol. (In press) HARA T. J. & LAW Y. M. CAROLINA(1972) Adaptation of the olfactory bulbar response in fish. Brain Res. 47, 259-261. HARAT. J., LAW Y. M. C. & VAN DER VEEN E. (1973) A stimulatory apparatus for studying the olfactory activity in fishes. J. Fish. Res. Bd Can. (In press) HASHIMOTOY., KONOSU S., FUSETANI N. & NOSE T. (1968) Attractants for eels in the extracts of short-necked clam-I. Survey of constituents eliciting feeding behaviour by the omission test. Bull.Jap. Sot. Sci. Fish. 34, 78-83. HOLL A. (1965) Vergleichende morphologische und histologische Untersuchungen am Geruchsorgan der Knochenfische. 2. Morph. Okol. Tiere 54, 707-782. IDLER D. E., FAGERLUNDU. H. & MAYOH H. (1956) Olfactory perception in migrating salmon J. gen. Physiol. 39, 889-892. KONOSU S., FUSETANI N., NOSE T. & HASHIMOTOY. (1968) Attractants for eels in the extracts of short-necked clam-II. Survey of constituents eliciting feeding behavior by fractionation of the extracts. Bull. rap. Sot. Sci. Fish. 34, 84-87. PFEIFFER W. (1963) The morphology of the olfactory organ of the Pacific salmon (Oncorhynthus). Can. J. Zool. 41, 1233-1236. SATOU M. (1971) Electrophysiological study of the olfactory system in fish-I. Bulbar responses with special reference to adaptation in the carp, Cyprinus carpio L. J. Fat. Sci. Univ. Tokyo Sec. IV 12, 183-218. SUTTERLIN A. M. & SUTTERLIN N. (1971) Electrical responses of the olfactory epithelium of Atlantic salmon (Salmo salar). J. Fish. Res. Bd Can. 28, 565-572. SUZUKI N. & TUCKER D. (1971) Amino acids as olfactory stimuli in freshwater catfish, Ictalurus catus (Linn.). Comp. Biochem. Physiol. 4QA, 399-404. Key Word Index-Olfaction; fontinalis; Coregonus clupeaformis.
amino acids;
fish smell;
Salmo gairdneri;
Salvelinus
COMPARISON OF THE OLFACTORY AMINO ACIDS IN RAINBOW TROUT, AND WHITEFISH
RESPONSE TO BROOK TROUT,
TOSHIAKI
and
J. HARA,
Y. M. CAROLINA
LAW
B. R. HOBDEN
Fisheries Research Board of Canada, Freshwater Institute, Winnipeg, Manitoba, Canada R3T 2N6 (Received
26 October 1972)
Abstract-l. The olfactory apparatus and the bulbar electrical responses induced by nasal infusion of amino acid solutions were compared among three salmonid species, rainbow trout, brook trout and whitefish. 2. Morphology of the olfactory organ was similar in all species examined, except that the peripherial olfactory organ of whitefish was slightly smaller. 3. The threshold concentration for the most effective amino acids ranged from low7 to 10-s M in rainbow and brook trout and was higher in whitefish. 4. Olfactory bulbar response spectra to amino acids were similar in all species. However, correlation was slightly higher between rainbow and brook trout (r = 0.89) than between rainbow trout and whitefish (r = 0.82). INTRODUCTION
PREVIOUS studies indicate rather specific effectiveness of certain amino acids for eliciting the electrical response in the olfactory bulb of rainbow trout and Pacific salmon (Hara, 1972, 1973). The studies on the olfactory receptor responses to amino acids in Atlantic salmon (Sutterlin & Sutterlin, 1971) and in catfish (Suzuki & Tucker, 1971) also support this view. However, specific significance of free amino acids in olfactorily mediated behaviour of fish remains to be determined. The present experiments were undertaken to compare morphology of the olfactory organ and the electrical response spectra of the olfactory bulb in three salmonid species, each of which has different feeding habits and habitat. An attempt was made to correlate the bulbar response spectrum of rainbow trout with those of the other two species to find out any similarity between them. MATERIALS
AND METHODS
Rainbow trout (Salmo gairdneri) and brook trout (Salvelinus fontinalis) were obtained from a local hatchery. Whitefish (Coregonus clupeuformis), which had been planted from a hatchery, were taken from a lake. They were all held in large tanks for several months and were transferred to glass aquaria, supplied with a continuous flow of dechlorinated water (temp. 13” + l”C), for acclimation at least 3 weeks prior to use. For anatomical studies fish were put in 10% formalin solution for several days, then the olfactory apparatus was examined. For histological studies, excised olfactory rosettes were fixed in Bouin or formalin solution and stained with haematoxylin-eosin. 969
970
TOSHIAKIJ. HARA, Y. M. CAROLINALAW ANDB. R. HOBDEN
Operative procedures of the fish and recording techniques of the electrical response from the olfactory bulb have been fully described previously (Hara, 1972,1973 ; Hara & Law, 1972; Hara et al., 1973).
RESULTS
Morphology of the olfactory apparatus In all species examined, anterior and posterior nostrils separated by a nasal bridge (skin flap) are located anterodorsal to the eye as in most other fish. In whitefish both nostrils are separated by two flaps, anterior and posterior. In the nasal sac there is an almost circular olfactory rosette where the sensory epithelium lodges. It consists of a rostrocaudally oriented median depression from which varying numbers of lamellae radiate. As shown in Table 1, the size of the olfactory rosette and the number of the lamellae of whitefish were smaller compared with TABLE ~--SIZE OF OLFACTORYAPPARATUS AND NUMBEROF PRIMARYOLFACTORY LAMELL~ (LENGTHIN mm.) No. of primary olfactory lamellae Species
Body length
Nasal sac
Olfactory nerve
Olfactory bulb
Left
Right
Salmo gairdneri
197 206 184 207
3.4 3.5 4.4 5.0
x x x x
4.6 4.6 4~8 4.6
8.0 8.0 7.0 9.6
1.7 x 1.2 x 1.1 x 2.2x
2.5 3.2 2.2 2.8
15 16 14 16
16 16 14 18
Salvelinus fontinalis
173 157 149 170
4.4 3.3 3.2 4.2
x x x x
4.8 3.8 3.9 4.0
7.0 6.0 6.0 6.0
1*2x 2.5 1.2x2.0 1.4 x 2.3 1.3 x2.4
14 12 12 13
13 12 12 12
Coregonus
187 172 203 172
1.5 2.4 4.0 2.4
x x x x
1.1 2.9 4.4 3.2
8.4 7.5 9.0 6.4
1.3x1.4 1.5 x 2.3 1.6x2.5 1.6x2.0
13 13 16 12
12 14 14 12
clupeaformis
those of the other two species of similar body length. Each lamella is slightly different in size. A secondary folding of the lamellae was observed in all species
examined. In some small fish, however, no lamella has this secondary folding. Histological sections of the lamellae shows no indication of sensory cells on the secondary lamellae (Fig. 1). The olfactory nerve, the olfactory bulb and other brain structures are essentially similar both in pattern and size. These are summarized in Fig. 2 and Table 1.
FIG. 1. Cross-sections of olfactory rosettes, showing development of the secondary olfactory lamellae. 1, primary olfactory lamellae of small brook trout (about 15 cm). No secondary lamella is developed. Most of the primary lamella is covered with sensory epithelium except for the top part. 2, cross-section of olfactory lamellae of whitefish. secondary lamellae are well developed. Notice nonsensory cells and goblet cells on top of ,both primary and secondary lamellae. 3, secondary olfactory lamellae are shown in higher magnification. pl, primary lamella; sl, secondary lamella.
OLFACTORY
RESPONSE
TO
AMINO
ACIDS
IN
FISH
971
.OR
FIG. 2. Schematic drawing of brain and olfactory organ of whitefish, dorsal view. Their sizes are shown in Table 1. CE, cerebellum; MC, mesencephalon; OB, olfactory bulb; ON, olfactory nerve; OR, olfactory rosette; TC, telencephalon.
Electrical responses from the olfactory bulb
Figure 3 illustrates typical responses from the olfactory bulb of whitefish when the nares were stimulated with hand rinse (a forefinger dipped in 100 ml water for 10 set), L-serine, isoserine, and phenylalanine, all at 1O-4 M. In rainbow and brook trout, the lowest estimated threshold concentrations for most effective amino acids are usually between lo-’ and 10-s M, though lower thresholds had been observed. However, it was much higher in whitefish. In Fig. 4, percentage response to different concentrations of L-serine are compared among the three species. Responses to a hand rinse, known to be an effective olfactory stimulant to fish (cf., Hara, 1970, 1971), generally exceed those to lo4 M L-serine in both rainbow and brook trout-140 and 150 per cent of those to lOA L-serine, respectively. In whitefish, however, the response was nearly of the same magnitude
972
TOSHIAKI J. HARA, T. M. CAROLINALAW ANDB. R. HOBDEN
FIG. 3. Typical responses recorded from the olfactory bulb of whitefish when the nares were stimulated with hand-rinse (A), 1O-4 M L-serine (B), low4 M isoserine (C), and 10m4 M phenylalanine (D). The upper tracing (a) of each pair is the integrated responses of the lower (b). Period of stimulation and time scale (each division = 1 set) are shown at the bottom. IOO-
o-
I
-!
I
-6 -5 log molarconcentration
I
-4
FIG. 4. Percentage responses to different concentrations of L-serine in rainbow trout ( * ), brook trout ( x ), and whitefish (A).Magnitudes of the response in each fish species is represented as a percentage of the response to 10e4 M L-serine.
as that to 1O-4 M L-serine. The effectiveness of n-isomers was always less than that of the L-isomers in all three species. A comparison of the stimulator-y effectiveness of twenty-four of the most effective amino acids tested between brook trout and whitefish is shown in Fig. 5. All chemicals were tested at the same concentration of 10m4 M. The relative effectiveness of each chemical is represented as a percentage of the response to the standard L-serine. It is clearly noticed that responses of whitefish to amino acids are generally much smaller than those of brook trout except for the six most effective amino acids, i.e. L-glutamine, L-methionine, L-alanine, homoserine, L-serine and L-cystine.
OLFACTORY RESPONSE TO AMINO ACIDS IN FISH
973
Homoser L-met L-glu L-ala L-asp L-cys L-leu L-thr L- ser L- VOI L - org
GUY L -his L-lys L-cys-cys lsoleu GABA lsoser L-tyr D-ala L-try D-ser L -phe /3-ola
FIG. 5. Comparison of the stimulatory effectiveness of twenty-four of the most effective amino acids tested between brook trout (white bars) and whitefish (black bars). All chemicals were tested at 10m4 M. Relative effectiveness of each chemical is represented as a percentage of the response to the standard L-serine (obliquelined). To compare these response spectra with the one previously obtained in rainbow fit was computed for each pair of the three trout (Hara, 1973), a least-squares groups. As shown in Fig. 6, a highly significant correlation was found between any
974
TOSHIAKIJ.HARA,Y.
M.
CAROLINA
LAW AND B.R.
HOBDEN
IFJO.
.
y=0*78xitl*36 r=089
0
I 50
0
I 100
I 150
Brooktrovt FIG. 6 (a) 150. l l
. . /
l.
.^A IUUl
.
l
l
/
.
. l
l
+ 21.48 Y '=0.78x f 60.82
l
I
I
50
100
Whitefish FIG. 6 (b)
I
150
975
OLFACTORY RESPONSE TO AMINO ACIDS IN FISH
.
1
100
50
I
150
Whitefish
FIG. 6 (c) FIG. 6. Relation of the olfactory bulbar electrical responses to amino acids between rainbow trout and brook trout (a), rainbow trout and whitefish (b) and brook trout and whitefish (c). Each dot indicates the mean of three to eight or more tests in each species. Least-squares fit was computed for each pair. 7 = correlation coefficient.
pair of the three species. The highest correlation was between rainbow and brook trout (correlation coefficient, r = 089). Correlation coefficients between rainbow trout and whitefish, and between brook trout and whitefish were 0.82 and 083, respectively. DISCUSSION
Different types of olfactory rosettes in teleosts have been described (Holl, 1965). In all the species examined the olfactory rosettes were almost circular in shape. The number of primary olfactory lamellae is a function of size, and not of age in Pacific salmon (Pfeiffer, 1963) and Baltic sea trout (Bertmar, 1972). The present observations generally agree with these reports. As shown in Table 1, the size of the nasal sac and the number of the primary olfactory lamellae of whitefish are slightly smaller compared with those of the other two species. Histological examination shows that the secondary lamellae are well developed in whitefish. It is not likely, therefore, that the higher threshold of the bulbar electrical response, as observed in whitefish, is the result of a lower degree of development in the secondary olfactory lamellae. The secondary folding of the primary lamellae certainly results
976
TOSHIAKIJ. HARA, Y. M. CAROLINALAW ANDB. R. HOBDEN
in an increase in the surface area of the olfactory epithelium, though most of this increase can be referred to as the indifferent epithelium (Bertmar, 1972). Nevertheless, folding of the lamellae increases effective use of the space in the olfactory chamber, which, in turn, seems to result in an increase in the total olfactory capacity. Results from the present experiments on electrical responses of the olfactory bulb of brook trout and whitefish to amino acids are in good agreement with earlier reports (Hara, 1973) that there are several highly effective amino acids and that the effectiveness of n-isomers is always less than that of L-isomers. These results suggest that highly effective amino acids may play an important role in the life of these fish species. However, no such behavioral evidence has been available so far, except the fact that the amino acid r.-serine is one of the active components of mammalian skin extract and is known to be a repellent to migratory Pacific salmon (Brett & MacKinnon, 19.54; Idler et al., 1956). In this connection, whitefish which were less responsive to a hand rinse generally showed lower electrical responses to all amino acids tested, Satou (1972, personal communication) recorded relatively small electrical responses from the olfactory bulb of carp, Cyprinus carpio, when stimulated with solutions of L-glutamine, L-methionine, and L-cysteine at rather higher concentrations, 5 x 10~~ to 10~~ M. No or little response was induced with other amino acids tested. This lower sensitivity to amino acids in carp is in agreement with its lower responsiveness to food extract (Satou, 1971). Hashimoto et al. (1968) and Konosu et al. (1968) fractionated the extract of a clam which evoked exploratory and feeding response in the Japanese eel, Anguilla japonica, and found that the amino acids, taurine, aspartic acid, serine, threonine, glutamic acid, glycine, and alanine, were the main components responsible for the activity, while the other constituents, such as nucleotides, organic acids and quarternary ammonium bases, made little contribution. They suggested that the stimulating activity of the extract is attributable mainly to the synergistic or additive interaction of these amino acids, since each of these amino acids was not effective when tested individually. Thus, it seems likely that certain free amino acids or their appropriate mixtures may play a fundamental role in behaviour, such as food selection, predator-prey interactions and migration, in some species of fish. Chemical and physiological characterization of interactions between the olfactory receptors and natural odorous substances pertinent to olfactorily mediated fish behaviour will be required. Acknowledgements-We thank Dr. J. G. Eales, Department of Zoology, University of Manitoba, for critical reading of the manuscript, and Mr. S. T. Zettler and Miss C. L. Boyce for photography and drafting. REFERENCES BERTMARG. (1972) Secondary folding of olfactory organ in young and adult sea trout. Actu zool. 53, 113-l 20. BRETT J. R. & MACKINNOND. (1954) Some aspects of olfactory perception in migrating adult coho and spring salmon. J. Fish. lies. Bd Can. 11, 310-318.
OLFACTORYRESPONSETO AMINOACIDSIN FISH
977
HAM T. J. (1970) An electrophysiological
basis for olfactory discrimination in homing salmon: a review. r. Fish. Res. Bd Can. 27, 565-586. HARA T. J. (1971) Chemoreception. In Fish Physiology vol. 5 (Edited by HOAR W. S. & BANnALL D. J.), pp. 79-120. Academic Press, New York. HARAT. J. (1973) Electrical responses of the olfactory bulb of Pacific salmon, Oncorhynchus nerka and 0. kisutch. J. Fish. Res. Bd Can. 29, 1351-1355. HARA T. J. (1972) Olfactory responses to amino acids in rainbow trout, Salmo gairdneri. Con@. Biochem. Physiol. (In press) HARA T. J. & LAW Y. M. CAROLINA(1972) Adaptation of the olfactory bulbar response in fish. Brain Res. 47, 259-261. HARAT. J., LAW Y. M. C. & VAN DER VEEN E. (1973) A stimulatory apparatus for studying the olfactory activity in fishes. J. Fish. Res. Bd Can. (In press) HASHIMOTOY., KONOSU S., FUSETANI N. & NOSE T. (1968) Attractants for eels in the extracts of short-necked clam-I. Survey of constituents eliciting feeding behaviour by the omission test. Bull.Jap. Sot. Sci. Fish. 34, 78-83. HOLL A. (1965) Vergleichende morphologische und histologische Untersuchungen am Geruchsorgan der Knochenfische. 2. Morph. Okol. Tiere 54, 707-782. IDLER D. E., FAGERLUNDU. H. & MAYOH H. (1956) Olfactory perception in migrating salmon J. gen. Physiol. 39, 889-892. KONOSU S., FUSETANI N., NOSE T. & HASHIMOTOY. (1968) Attractants for eels in the extracts of short-necked clam-II. Survey of constituents eliciting feeding behavior by fractionation of the extracts. Bull. rap. Sot. Sci. Fish. 34, 84-87. PFEIFFER W. (1963) The morphology of the olfactory organ of the Pacific salmon (Oncorhynthus). Can. J. Zool. 41, 1233-1236. SATOU M. (1971) Electrophysiological study of the olfactory system in fish-I. Bulbar responses with special reference to adaptation in the carp, Cyprinus carpio L. J. Fat. Sci. Univ. Tokyo Sec. IV 12, 183-218. SUTTERLIN A. M. & SUTTERLIN N. (1971) Electrical responses of the olfactory epithelium of Atlantic salmon (Salmo salar). J. Fish. Res. Bd Can. 28, 565-572. SUZUKI N. & TUCKER D. (1971) Amino acids as olfactory stimuli in freshwater catfish, Ictalurus catus (Linn.). Comp. Biochem. Physiol. 4QA, 399-404. Key Word Index-Olfaction; fontinalis; Coregonus clupeaformis.
amino acids;
fish smell;
Salmo gairdneri;
Salvelinus