Journal of Chemical Ecology, Vol. 12, No. 1, 1986
CHEMICAL FRACTIONATION OF SHRIMP EXTRACTS INDUCING BOTTOM FOOD SEARCH BEHAVIOR IN COD (Gadus morhua L.)
O.F. ELLINGSEN and K.B. DOVING Institute of Biology, University of Oslo P.O. Box 1051, Blindern, N-0316 Oslo 3, Norway (Received March 22, 1985; accepted June 3, 1985)
Abstract--The bottom food search (BFS) feeding behavior in cod (Gadus morhua L.), has been used in a bioassay for chemical isolation of the feeding stimulant substances present in shrimp (Pandalus borealis). An aqueous methanol extract of ground shrimp was separated into acidic, neutral, and amphoteric/basic fractions by ion-exchange chromatography and into single components by preparative high-pressure liquid chromatography (HPLC). Of the isolated single components, the amino acid glycine was most potent, followed by alanine. Two unidentified substances were also highly potent. There was a synergistic effect between glycine, alanine, proline, and arginine. These four amino acids were more potent than the total amino acid pool found in the shrimp extract, indicating that there may be amino acids in this pool having an antagonistic effect.
Key Words--Shrimp, Pandalus borealis, cod, Gadus morhua, food search, behavior, amino acids. INTRODUCTION
The consumption of food in cod is composed of a series of distinct behavior patterns which add up to the total consumatory act. The behavior patterns involved in food search activity are orientation, approach, nibbling, biting, rushing, snapping, and swal!owing (Tilseth and Solemdal, 1977). It seems reasonable to assume that different sensory stimuli are responsible for eliciting parts or sequences of the behavior patterns. Evidence for this assumption has become more tenable when it comes to olfactory stimuli as D6ving and Selset (1980) showed that two distinct phases of the food search activity can be elicited via the olfactory system. These two behaviors are also observed in free-swimming cod (Brawn, 1969; Pawson, 1977). 155 0098-0331/86/0100-0155505.00/0 9 1986PlenumPublishingCorporation
156
ELLINGSEN AND D~VING
During the initial phase, called orienting reaction, the fish swims around making sharp turns to the side, and up and down in the water. During the second phase, a benthic food search is displayed as an arrest of swimming close tothe bottom, then the fish swims backwards using the pectoral fins and tail with the head down, trailing the barbel and the pelvic fin rays along the bottom. These behaviors are qualitatively different and can be elicited by electrical stimulation of the lateral and medial part of the lateral olfactory tract, respectively, demonstrating that the behaviors are released via different neural pathways. As the behaviors are elicited by different nervous pathways, it is reasonable to assume that the chemical stimuli must be different. In the present study we have focused on the chemical basis for eliciting one particular component of the food search behavior, viz., the bottom food search (BFS) behavior pattern. Following a new line of chemical isolation procedures, we have isolated fractions and components releasing this behavior pattern in the cod. Chemical analysis shows that several substances with similar physical/chemical properties are involved in releasing the BFS, among them are the amino acids alanine and glycine. Two components, as yet unidentified, are also potent agents releasing BFS behavior.
METHODS AND MATERIALS
Maintenance ofFish. Cod (Gadus morhua L.) were caught in fish pots at Drrbak 30 km south of Oslo and transported to the aquarium facilities at the University of Oslo. The fish were 25-35 cm long and had been kept in captivity in the observation tanks for one week to two months prior to experimentation. During the test period, feeding the fish and cleaning the aquaria were regularly done on Fridays after the experimental series. The tests were performed the five first days of the week. The test fish were calm at the time of testing. They showed no signs of alarm reaction and moved quietly around in the observation aquaria. Sick or aggressive fish were removed from experiments and replaced by healthy, nonaggresive fish. A total of 28 fish were used during the period of bioassay. Procedure. The cod were observed in four glass aquaria, 120 x 60 • 50 cm each, with a flow of 3 liters/min of seawater. The water temperature was 10 _+ 2~ Four cod marked with individual colored tags were placed in each aquarium. Test solutions were introduced at the bottom of the aquaria via two separate glass tubes (ID 1.5 ram). Teflon tubes of the same internal diameter as the glass capillaries provided connections between the delivery syringes and the glass tubes. The outlet from these glass tubes were hidden in the sand coveting the bottom. The dead volume of the delivery lines was 5 ml. The stimuli were presented from syringes placed in an adjustable infusion pump. The solutions were injected into the aquarium at a rate of 2 ml/min during a period of
SHRIMP EXTRACTS
157
10 rain. Stimulations were performed once a day. The test solutions were introduced in parallel with seawater blanks. Each test solution was given at five different concentrations. The aquarium seawater was used for dilution of all solutions to be tested in the bioassay. Behavior Patterns. A benthic food search is observed in the cod when the olfactory tract is stimulated electrically and also when the fish is searching for a food source (Brawn, 1969; Pawson, 1977). Initial activity consists of an abrupt cessation of forward swimming, the cod descending to the bottom and moving backwards with the barbel and rays of the pelvic fins trailing the bottom. Each backward swim over the tube outlet was counted as a bottom food search (BFS). The number of fish doing BFS and the total number of BFS counted for the fish during a stimulation period of 10 min were used as a measure of the potency of a test solution. Other parts of the food search behavior pattern were also noted but not included in the present study. These were: orienting reaction, in the sense used by Pavlov (1927); aggression, as described by Brawn (1961); snapping, a rapid opening and closing of the jaws; and bellying, the fish making a quick swim towards the bottorr~, turning to its side, flashing its belly and swimming along sideways close to the bottom. Extracts and Synthetic Solutions. Shrimp was ground twice in an electric grinder and then extracted with 3 vol of methanol-distilled water (75 : 25 v/v) by stirring at 800 rpm for 30 min (Franz Morat, Eisenbach, Hochschwarzwald, FRG, electrical stirrer, type R-16). The liquid phase was separated from the protein phase by vacuum filtration through a Whatman No. 1 filter. The filtrate was evaporated to dryness on a rotary evaporator at 40~ The residue was dissolved in distilled water to a "dry-weight" content of 100 g/liter. This solution (stock solution) was used for chemical analysis, fractionation, and bioassays. Synthetic solutions were prepared with distilled water so that the concentration of each individual component was the same as that measured in the stock solution. Chemicals. Homarine HC1 was obtained from Carl Roth KG, Karlsruhe, FGR, dimethylthetine HCt from ICN Pharmaceuticals, Inc., Plainview, New York. Other chemicals were from Sigma Chemicals, St. Louis. Characterization of Stimulants and Analytical Procedures. Analyses and characterization of the stimulants were conducted on the stock solution. Amino acid concentrations were measured on a Biotronic LC 5000 automatic amino acid analyzer. The concentration of trimethylamine was determined by a microdiffusion technique (Conway and Bryne, 1933). Trimethylamine oxide was determined as trimethylamine after reduction with titanium trichloride (HjorthHansen, 1952). Characterization and purification of stimulants are summarized in Figure 1. By ion-exchange chromatography, the compounds were separated into three fractions: (1) acidic compounds, adsorbed on the anion exchanger and eluted
ELLINGSEN AND D~VING
158
ACTIVESOLUTIONS
NON-ACTXV~OR WEAKLY ACTIVE SOLUTIONS
GROUND SHRIMPS ~ Extraction with aqueous methanol
I STOCK SOLUTION, 100 g/] I Removal of lipids with chloroform
J CHLOROFORM EXTRACT: q LIPIDS, 3,5% BY WEIGHT
[ LIPID-FREE EXTRACT
I Elution of adsorbed lI FRACTION I. $ompounds w~th 1M HC~ACIDIC COMPOUNDS, ~l 16% BY WEIGHT OF LIPID-FREE EXTRACT
I ichromatography ni~ exchange
m
!
[
ater effluent
I AMPHOTERIC, BASIC AND NEUTRAL COMPOUNDS
]
Cation exchange chromatography
Water effluent
Elution of adsorbed compounds with 4M HCI
IAMPHOTERIC FRACTION3 . AND BASIC
COMPOUNDS. 72% BY WEIGHT OF LIPID-FREE EXTRACT
i
Separation into single components by preparative HPLC (RP-18)
FRACTION 4. SEE FIG. 2
FRACTION2.
NEUTRAL COMPOUNDS.
Wl 12% BY WEIGHT OF
[LIPID-FREE EXTRACT
Jw [ SEE FIG. 5.2 FRACTION
I
I
FIG. 1. Flow chart indicating the procedure followed for fractionation of food-searchinducing compounds in extract from shrimp.
SHRIMP EXTRACTS
159
with excess 1 M HC1; (2) neutral compounds, eluted from the cation exchanger with excess water; and (3) amphoteric and basic compounds, eluted from the cation exchanger with excess 4 M HC1. Solutions with the same concentration as in the stock solution were made up from fractions 1, 2, and 3 and tested in bioassays. Test solutions made up from the stock solution and fraction 3 were also tested in equal concentrations. Traces of fats and pigments (ca. 3.5 % by weight) were removed with chloroform before chromatography. One volume of stock solution was extracted twice with two volumes chloroform. Defatted material (30 g) was dissolved in 50 ml distilled water, pH adjusted to 7.0. This solution was applied to an anion exchange column (Amberlite IRA-401, C1- form, 16-40 mesh, Koch-Light Laboratories, England, 2.5 x 100 cm). The column was washed with 1500 ml distilled water and adsorbed substances were eluted with excess 1 M HC1 (1500 ml). The effluent and washings were combined, concentrated, and dissolved in 50 ml distilled water. The pH was adjusted to 7.0, and the sample was applied to a cation-exchange column (Dowex 50-X8, H § form, 16-40 mesh, Dowex, Switzerland, 2.5 x 100 cm) and rinsed with 1000 ml distilled water. The adsorbed substances were eluted with excess 4 M HC1 (1500 ml). The eluate was concentrated, dissolved in buffer, and the pH was adjusted to 4.0 before separation into single components by preparative HPLC (PerkinElmer, Norwalk, Connecticut, Series 3B liquid chromatograph with a PerkinElmer column C-18 bounded phase, 10/~m particle size, 1.8 x 25 cm). The initial eluent was 0.05 M KH2PO4,pH 4.0, and the final eluent methanol. Buffer was used during the first 20 min, then a linear gradient of 0-100% final eluent was used over the next 45 min. Methanol was run as a final eluent for 20 min. The flow rate was kept constant at 2 ml/min. The eluate was monitored at 210 nm (Perkin-Elmer LC 75, spectrophotometric detector). The components separated by HPLC gave the chromatogram shown in Figure 2; they were collected in two fractions. Fraction 4 contained the substances giving rise to peaks 1-6, and fraction 5 contained all components eluted thereafter. Both fractions were tested in the bioassay at equal concentrations. Single components eluted in fraction 4 were also collected. Before testing, the buffer was removed by the following procedure: Water was removed on a rotary evaporator and the residue dried at 40 ~ to a constant weight. The dried, waterfree material was then dissolved in excess water-free methanol, and buffer was separated from the methanol soluble components by filtration through a Millipore filter, Millipore Corp., Bedford, Massachusetts (45-#m pore size). RESULTS
The bioassays were performed from September 1983 to September 1984. In all experiments, samples of stock solution were tested as a reference for other solutions applied to the fish (see Tables 2, 4, and 5). This control was panic-
Fraction 4
20
30
Fraction 5
5O
Retention time (mini
40
60
70
80
FiG. 2. Chmmatogmm showing the separation of amphotm~c/basic components, fraction 3, by preparative HPLC. Amount injected: 50 rag. The experimental conditions are given in the text.
I0
|
9
Z
r-' r
SHRIMP EXTRACTS
161
ulafly important as the behavior and threshold for eliciting BFS varied over the year. Comparison between Shrimp Extracts and Synthetic Solutions. The concentrations of amino acids, trimethylamine oxide, and trimethylamine found in the stock solution are given in Table 1. When a synthetic solution based on the concentration of amino acids found in the stock solution was prepared and tested, it was observed that the stock solution was 100-200 times as potent by weight as the synthetic solution (Tables 2A and B). The four amino acids (glycine, alanine, proline, and arginine) which were most abundant in the stock solution seemed more efficient in evoking the BFS behavior than the total amino acid pool (Tables 2B and C), and the results indicated that these four amino acids were only 10 times less potent than the stock solution (Tables 2A and C). Two amino acids, glycine and alanine alone, did not seem as potent as the four amino acids taken together, but were still more potent than the total amino acid pool (Tables 2B and D). When the four main amino acids were tested individually in equimolar concentrations (Table 3), glycine appeared to be most potent followed by alanine. A solution containing the amino acids with five carbon atoms, viz., valine, TABLE 1. CONCENTRATIONS OF AMINO ACIDS, TRIMETHYLAMINE, AND TRIMETHYLAMINE OXIDE IN STOCK SOLUTIONa
Concentration Substance Aspartic acid Threonine Serine
Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine Taurine Trimethylamine Trimethylamine oxide aDry weight content: 100 g/liter.
mmol/liter
g/liter
1.28 5.13 3.17 4.47 21.31 58.76 12.52 5.25 2.19 4.26 5.72 2.94 2.63 5.69 14.50 27.47 2.13 75.07
0.17 0.61 0.33 0.66 2.46 4.41 1.12 0.62 0.33 0.56 0.75 0.49 0.41 0.83 2.53 3.44 0.13 5.63
162
ELLINGSEN AND D•VING
TABLE 2. COMPARISON OF BFS ACTIVITY INDUCED BY SAMPLES OF STOCK SOLUTION AND SYNTHETIC SOLUTION OF AMINO ACIDS a
A. Stock solution,
Dilution factor
Concentration,
x 104
g/liter x 10 -4
Number of fish performing BFS
Number of BFS counted
1
100
16
40
2.5 5 10 15
40 20 10 5
13 10 7 2
31 20 18 5
16 !2 9 5 1
43 33 25 13 3
16 13 10 7
53 46 27 17
3
5
9 9 8 8 5
3i 30 27 33 10
B. Total amino acid pool,
x 102
O.5
4O 20 4 2 1
1
5 10 15 C. Glycine, alanine, proline, and arginine, • 10 3 0.5 1 2.5 5
g/liter X 10 -3 21.0 10.5 4.2 2.1
10
1.05
D. Glycine and alanine, 0.5 1 2.5 5 10
g/liter x 10 -2
x 10 3
g/liter x 10 3 11.0 5.5 2.2 1.1 0.55
"The amino acid concentrations are in the proportion found in the stock solution. The entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: September-December 1983. p r o l i n e , m e t h i o n i n e , a n d g l u t a m i c acid, i n d u c e d n o B F S a c t i v i t y w h e n t e s t e d at t h e s a m e c o n c e n t r a t i o n s as t h a t f o u n d in t h e s t o c k s o l u t i o n . A c o m b i n a t i o n of the basic amino acids lysine, arginine, and histidine, scored only a few BFS p a t t e r n s at t h e h i g h e s t c o n c e n t r a t i o n s u s e d ( 1 / 5 0 o f t h e c o n c e n t r a t i o n in s t o c k s o l u t i o n ) . T h e c o m b i n a t i o n o f m e t h i o n i n e , t h e f i v e - c a r b o n a m i n o acids, a n d t h e b a s i c a m i n o a c i d s i n d u c e d B F S a c t i v i t y at t h e s a m e rate as d i d t h e b a s i c a m i n o acids.
SHRIMP EXTRACTS
163
TABLE 3. COMPARISON OF BFS ACTIVITY INDUCED BY INDIVIDUAL SUBSTANCES TESTED IN EQUIMOLAR CONCENTRATIONSa
Substance Glycine
Alanine
Concentration (mol/liter)
Number of fish performing BFS
Number of BFS counted
10-2 10 3 10-4 10-5 10 2 10 3
10 7 6 2 6 4 3 0 6 4 0 0 3 0 0 0 5 0 0 0
19 9 8 6 18 11 9 0 14 8 0 0 4 0 0 0 9 0 0 0
t0 -4
Arginine
Proline
Dimethylthetine
10-5 10-2 10-3 10-4 10 5 10-2 10-3 10-4 10 5 10 2 10-3 10-4 10-5
aThe entries give the number of fish doing BFS and the total counts of BFS performed during the injection period. Concentrationsare in tool/liter of material injected. Test period: February-April 1984. Trimethylamine oxide and trimethylamine HC1 were tested in the same concentrations as found in the natural extract. They did not induce any BFS activity when tested alone. They did not affect the activity scores when tested in combination with the amino acids. Comparison between Chemical Fractions of Stock Solution. As seen from Figure 1, the weight ratio of the adsorbed compounds was 16 : 12 : 72 for fractions 1, 2, and 3, respectively. Only the acidic fraction (fraction 1) and the fraction containing amphoteric and basic components (fraction 3) scored significantly on the bioassay tests as demonstrated in Table 4. When the stock solution and fraction 3 were compared in equal concentrations, the latter was slightly more potent (Table 5). Further separation of fraction 3 by HPLC (Figure 2), yielded 60% by weight of the material in fraction 4, while components in fraction 5 accounted for 40%. No BFS was induced by fraction 5, while the activity induced by fraction 4 was virtually the same as the activity induced by fraction 3. Of the single components eluted in fraction 4, most of the activity was located in peaks
TABLE 4. COMPARISON OF B F S ACTIVITY INDUCED BY SAMPLES OF STOCK SOLUTION AND FRACTIONS 1, 2, AND 3 ISOLATED BY IoN-EXCHANGE CHROMATOGRAPHY a
Number of fish performing BFS
Number of BFS counted
16 11 10 7 4
65 36 20 15 10
B. Acidic fraction, 1 1.6 0.16 0.032
9 2 0
13 4 0
C. Neutral fraction, 2 1.2 0.12 0.024
3 2 0
4 3 0
16 12 8 5 3
40 28 15 8 4
Concentration (g/liter) A. Stock solution 10 1 0.5 0.1 0.01
D. Basic/amphoteric fraction, 3 7.2 0.72 0.144 0.072 0.0072
aThe entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: AugustSeptember 1984. TABLE 5. COMPARISON OF B F S ACTIVITY INDUCED BY STOCK SOLUTION (SS) AND BASIC/AMPHOTERIC FRACTION, 3 a
Sample
Concentration (g/liter)
Number of fish performing BFS
Number of BFS counted
SS 3 SS 3 SS 3 SS 3 SS 3
1 1 0.1 0.1 0.01 0.01 0.005 0.005 0.001 0.001
10 16 7 8 6 5 5 3 1 2
20 40 12 14 10 10 8 7 2 4
~The entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: May 1984.
SHRIMP EXTRACTS
165
1, 2, and 3. Peaks 4 and 5 were less active when tested in equal concentrations (1 g/liter). DISCUSSION
The chemicals emanating from prey induce feeding behavior in cod. In the present study we have shown that shrimp contain amino acids and substances with amphoteric/basic properties which are effective inducers of a particular part of the feeding behavior, viz., the BFS. We discuss the results of our experiments in light of previous studies on cod and attempts to isolate feeding substances for cod and other fish species. The methods developed for the chemical isolation procedure are specifically considered since they might be useful in forthcoming analyses. The four amino acids in the stock solution, glycine, alanine, proline, and arginine, appeared to be more potent than the total amino acid pool. These results demonstrate that the amino acid pool in a shrimp extract may contain amino acids which have an antagonistic effect on BFS induction in cod. The four main amino acids (glycine, alanine, proline, and arginine) act highly synergistically. This is demonstrated from the results presented in Table 2C and 3. At a total amino acid concentration of 10.5 • 10 -3, 46 BFS were performed by 13 fish when glycine, alanine, proline, and arginine were tested in combination (Table 2C). When the same four amino acids were tested individually at a concentration of 10 -4 mol/liter (ca. 10 -3 g/liter), glycine induced eight BFS (performed by six fish); alanine, nine BFS (performed by three fish); proline and arginine did not induce BFS at this concentration (Table 3). Of the individually tested amino acids, glycine appeared to be most potent, this observation being in agreement with Pawson (1977). Johnstone (1980) determined detection threshold values to nine amino acids in cod by a conditioning method and found that tyrosine was most potent, followed by cysteine, phenylalanine, and glycine. The difference between threshold values was small. Tilseth and Solemdal (1977) examined the preference of cod to a wide variety of natural baits derived from invertebrates and fish tissue. The relative effectiveness of 10 different natural baits in initiating biting of a bag indicated that unconditioned cod clearly responded best to extracts of tissue from crustaceans such as euphasids, shrimp, and deep-sea prawns. Attempts to characterize the effective components in aqueous extracts of crustacean-based baits indicated a reduction in activity at pH below 4 or above pH 10. On adjustment of pH to 11 in a diluted solution of the stock solution, we observed a similar reduction in activity. This treatment was associated with the release of dimethyl sulfide and a precipitate. Boiling of the same solution for 3 hr at pH 1 only resulted in a small decrease in food search activity. The fractionation procedure used in this work for isolating new food-search
166
ELLINGSENANDD~VING
stimulants, other than amino acids, was a modified procedure used for isolation of quaternary bases (Abe and Kaneda, 1975; Konusu and Hayashi, 1975; Hayashi et al., 1978). These authors used the O H - form of an anion exchanger. The anion exchanger has a higher selectivity for C1- than O H - ; the O H - form is therefore more effective in exchanging acidic compounds than the C1- form. In our preliminary studies, we also applied the lipid-free stock solution on a column with the anion exchanger in the O H - form. This procedure resulted in a highly alkaline effluent, and the release of dimethyl sulfide and a precipitate. When this sample was analyzed by HPLC after passing through the cation exchanger, the chromatogram was totally different from that seen in Figure 2. Peaks 2 and 3 did not appear. Therefore, the anion exchanger was used in the C1- form in the fractionation procedure. The activity associated with the acidic fraction (fraction 1) should be related to adsorbed amino acids and to nucleotides which also adsorb onto the C1- form of an anion exchanger. (Hayashi et al., 1978). The activity associated with the neutral fraction (fraction 2) was regarded as insignificant and was neglected in later work. Since the amphoteric/basic fraction (fraction 3) was the most potent of the three tested fractions and even had a higher activity by weight than the stock solution, further work on identification of food-search stimulants was mainly limited to this fraction. Most of the activity induced by single components eluted by HPLC was located in substances giving rise to peaks 1, 2, and 3 in Figure 2. The substance giving rise to peak 1 was identified as the amino acid glycine, possibly containing a small amount of alanine [identical IR and mass spectra with authentic glycine, NMR (60 MHz, D20): 6 = 3.6 ppm (s, CH2), 1.5 ppm (d, J = 3.5, CH 3 of alanine, very weak signal, shift position and coupling constant identical with authentic alanine)]. Peaks 2 and 3 are still unidentified. These components carry the very distinctive smell of shrimp and are very labile to alkali, releasing dimethyl sulfide on treatment with cold aqueous 1 M NaOH. The presence of dimethyl-fi-propiothetine (DMPT) in several species of multicellular algae is well documented (Challenger, 1959; Ishida, 1968; Noda and Horiguchi, 1975; Larher et al.0 1977). The decomposition of this substance by cold alkali, yielding dimethyl sulfide, is believed to be a specific reaction (Ackman and Dale, 1965). An indirect determination of DMPT, as dimethyl sulfide, is based on this assumption. Such analyses have been carried out for marine multicellular algae (Ackman et al., 1966), for Labrador cod (Ackman et al., 1967), for brackish water algae and baltic herring (Granroth and Huttula, 1976), and for antarctic krill (Tokunaga et al., 1977). From these data it would seem possible that either peak 2 or 3 might be DMPT. The IR spectra and NMR spectra [60 MHz, D20, signals at 6 = 3.8 ppm(s) and 3.3 ppm(s)] were not in accordance with pub, lished IR spectra (Ishida, 1968; Noda and Horiguchi, 1975) and NMR spectra (Ishida, 1968; Larher et al., 1977). Weak signals in the NMR spectrum at b =
SHRIMP EXTRACTS
167
3.0 ppm and 3.6 ppm might suggest the presence of DMPT as a minor component in peak 3. Due to the lack of verified structures, it is impossible to determine the concentration in the stock solution, although there are areas in HPLC comparable to glycine. The reduction in activity on alkali treatment of aqueous extracts of crustacean-based baits, as observed by Tilseth and Solemdal (1977) and in this work, obviously should be related to the alkali-labile components giving rise to peaks 2 and 3 in Figure 2. As a representative for thetines, dimethylthetine was tested in our bioassay. This compound does not occur in marine organisms, but it has been described as potent in inducing food consumption in juvenile Dover sole (Mackie et al., 1980). As demonstrated in Table 3, this compound even induced BFS when tested in our bioassay. Peak 4, observed as a shoulder on peak 3, has been identified as glycinebetaine [superimposable IR and mass spectra, NMR (60 MHz, D20): ~ = 3.3 (s, 9H, 3X N--CH3) and 4.2 ppm (s, 2H, CH2)]. The importance of glycinebetaine as a BFS inducer in cod is very doubtful. When glycine-betaine was tested in the same concentrations as the amino acids glycine, alanine, arginine, and proline, no BFS could be observed. The exact concentration of this compound is difficult to determine in our chromatographic system due to low concentration and lack of separation from peak 3. The concentration was determined to be approximately 0.05 g/liter in the stock solution. We were not able to show that glycine-betaine potentiated the stimulation power of amino acids when this component was included. Components 5 and 6 are still unidentified. Compared to the substances giving rise to peaks 1, 2, and 3, they induced only a very weak degree of BFS. The main peak in the chromatogram, peak 7, has identical retention time with homarine. No attempt has been made to identify this component by spectroscopy as neither the isolated component nor authentic homarine induced BFSactivity. The variability of response to the stock solution could be expected due to the cyclic feeding activity of cod found in the coastal zone. Experiments were made both during the most intense feeding period and during breeding season when feeding activity is low even among juvenile cod. Variability of the seawater at the aquarium facilities could also be responsible for the changes in behavioral activity although precautions were taken to keep the water quality constant. The ordinary control routines of the water quality were unaltered during the testing period.
Acknowledgments--The authors are grateful to amanuensis Karl E. Malterud, Institute of Pharmacy, University of Oslo, for recording of spectra and for valuable discussions, and to Gro Svendsen for skillful technical assistance. This work was supported financially by The Norwegian Fisheries Research Council, project 203.15.
168
ELLINGSEN AND DCVXNO REFERENCES
ABE, S., and KANEDA,T. 1975. Studies on the effect of marine products on cholesterol metabolism in rats. X. Isolation of B-homobetaine from oyster and betaine contents in oyster and scallop. Bull. Jpn. Soc. Sci. Fish. 41(4):467-471. ACKMAN,R.G., and DALE, J. 1965. Reactor for Determination of dimethyl-~-propiothetin in tissue of marine origin by gas-liquid chromatography. J. Fish. Res. Board Can. 22(4):875-883. ACKMAN, R.G., HINCLEY, J., and MAY, A.W. 1967. Dimethyl sulfide in Labrador cod. J. Fish. Res. Board Can. 24(2):457-461. ACKMAN,R.G., TOCHER, C. S., McLACHLAN,T. 1966. Dimethyl-~-propiothetin. Determination by reactor gas-liquid chromatography, occurrence in algae and implication on fisheries, pp. 235242, in Proc. V Int. Seaweed Symp., G. E. Young and J. L. McLaughlan (eds.), Pergamon Press, New York. BRAWN,V. M. 1961. Aggressive behavior in the cod (Gadus callarias L.) Behavior 18:107-147. BRAWN, V.M. 1969. Feeding behavior of cod (Gadus morhua). J. Fish. Res. Board. Can. 26(3):583-596. CHALLENGER, F. 1959. Aspects of the Organic Chemistry of Sulfur. Butterworths Scientific Publications, London. CONWAY,E.J., and BYRNE,A. 1933. An absorption apparatus for the microdetermination of certain volatile substances. I. The micro determination of ammonia. Biochem. J. 27:419-421. DovIN~, K.B., and SELSET, R. 1980. Behavior patterns in cod released by electrical stimulation of olfactory tract bundlets. Science 207:559-560. GRANROTH, B., and HUTTULA, T. 1976. Formation of dimethyl sulfide by braldsh water and possible implication for the flavor of baltic herring. Finn. Chem. Lett. 6:148-150. HAYASHI, T., YAMAGUCttI, K., and KONOSU, S. 1978. Studies on flavor components in boiled crabs. II. Nucleotids and organic bases in the extracts. Bull. Jpn. Soc. Sci. Fish. 44(12): 13571362. HJORTtt-HANSEN, S. 1952. M6thode pour le dosage de L 6xyde de trimrthylamine. Anal. Chim. Acta 6:438-441. ISHIDA, Y. 1968. Physiological studies on evolution of dimethyl sulfide from unicellular marine algae. Mem. Coll. Agric. Kyoto Univ. 94:47-82. JOHNSTONE, A.D.F. 1980. The detection of dissolved amino acids by the Atlantic cod; Gadus morhua L. J. Fish Biol. 17:219-230. KONOSU, S., and HAYASHI, T. 1975. Determination of r betaine and glycine betaine in some marine invertebrates. Bull. Jpn. Soc. Sci. Fish. 41(7):743-746. LARHER, F., HAMELIN,J., and STEWART, G.R. 1977. L' acide dimdthylsulfonium-3 propanoique de spartina anglica. Phytochemestry 16:2019-2020. MACKIE, A.M., ADRON,J.W., and GRANT, P.T. 1980. Chemical nature of feeding stimulants for the juvenile Dover sole, Solea solea (L.). J. Fish Biol. 16:701-708. NODA, H., and HORIGUCHI,Y. 1975. Studies on the flavor substances of "Nori", the dried Laver porphra tenera. I. Dimethyl sulfide and dimethyl-~-propiothetin. Bull. Jpn. Soc. Sci. Fish. 41(4):481-486. PAVLOV,J.P. 1927. Conditioned Reflexes. Oxford University Press, London. PAWSON, M.G. 1977. Analysis of a natural chemical attractant for whiting (Merlangus merlangus L.) and cod (Gadus morhua L.) using a behavioral bioassay. Comp. Biochem. Physiol. 56A:129-135. TILSETH, S., and SOLEMDAL,P. 1977. Methods for testing smell response in fish. Int. Con. Explor. Sea Gear Behav. Commission, B45. TOKUNAGA, T., IIDA, H., and NAKAMURA,K. 1977. Formation of dimethyl sulfide in antarctic krill, Euphausia superha. Bull. Jpn. Soc. Sci. Fish. 43(10):1209-1217.
CHEMICAL FRACTIONATION OF SHRIMP EXTRACTS INDUCING BOTTOM FOOD SEARCH BEHAVIOR IN COD (Gadus morhua L.)
O.F. ELLINGSEN and K.B. DOVING Institute of Biology, University of Oslo P.O. Box 1051, Blindern, N-0316 Oslo 3, Norway (Received March 22, 1985; accepted June 3, 1985)
Abstract--The bottom food search (BFS) feeding behavior in cod (Gadus morhua L.), has been used in a bioassay for chemical isolation of the feeding stimulant substances present in shrimp (Pandalus borealis). An aqueous methanol extract of ground shrimp was separated into acidic, neutral, and amphoteric/basic fractions by ion-exchange chromatography and into single components by preparative high-pressure liquid chromatography (HPLC). Of the isolated single components, the amino acid glycine was most potent, followed by alanine. Two unidentified substances were also highly potent. There was a synergistic effect between glycine, alanine, proline, and arginine. These four amino acids were more potent than the total amino acid pool found in the shrimp extract, indicating that there may be amino acids in this pool having an antagonistic effect.
Key Words--Shrimp, Pandalus borealis, cod, Gadus morhua, food search, behavior, amino acids. INTRODUCTION
The consumption of food in cod is composed of a series of distinct behavior patterns which add up to the total consumatory act. The behavior patterns involved in food search activity are orientation, approach, nibbling, biting, rushing, snapping, and swal!owing (Tilseth and Solemdal, 1977). It seems reasonable to assume that different sensory stimuli are responsible for eliciting parts or sequences of the behavior patterns. Evidence for this assumption has become more tenable when it comes to olfactory stimuli as D6ving and Selset (1980) showed that two distinct phases of the food search activity can be elicited via the olfactory system. These two behaviors are also observed in free-swimming cod (Brawn, 1969; Pawson, 1977). 155 0098-0331/86/0100-0155505.00/0 9 1986PlenumPublishingCorporation
156
ELLINGSEN AND D~VING
During the initial phase, called orienting reaction, the fish swims around making sharp turns to the side, and up and down in the water. During the second phase, a benthic food search is displayed as an arrest of swimming close tothe bottom, then the fish swims backwards using the pectoral fins and tail with the head down, trailing the barbel and the pelvic fin rays along the bottom. These behaviors are qualitatively different and can be elicited by electrical stimulation of the lateral and medial part of the lateral olfactory tract, respectively, demonstrating that the behaviors are released via different neural pathways. As the behaviors are elicited by different nervous pathways, it is reasonable to assume that the chemical stimuli must be different. In the present study we have focused on the chemical basis for eliciting one particular component of the food search behavior, viz., the bottom food search (BFS) behavior pattern. Following a new line of chemical isolation procedures, we have isolated fractions and components releasing this behavior pattern in the cod. Chemical analysis shows that several substances with similar physical/chemical properties are involved in releasing the BFS, among them are the amino acids alanine and glycine. Two components, as yet unidentified, are also potent agents releasing BFS behavior.
METHODS AND MATERIALS
Maintenance ofFish. Cod (Gadus morhua L.) were caught in fish pots at Drrbak 30 km south of Oslo and transported to the aquarium facilities at the University of Oslo. The fish were 25-35 cm long and had been kept in captivity in the observation tanks for one week to two months prior to experimentation. During the test period, feeding the fish and cleaning the aquaria were regularly done on Fridays after the experimental series. The tests were performed the five first days of the week. The test fish were calm at the time of testing. They showed no signs of alarm reaction and moved quietly around in the observation aquaria. Sick or aggressive fish were removed from experiments and replaced by healthy, nonaggresive fish. A total of 28 fish were used during the period of bioassay. Procedure. The cod were observed in four glass aquaria, 120 x 60 • 50 cm each, with a flow of 3 liters/min of seawater. The water temperature was 10 _+ 2~ Four cod marked with individual colored tags were placed in each aquarium. Test solutions were introduced at the bottom of the aquaria via two separate glass tubes (ID 1.5 ram). Teflon tubes of the same internal diameter as the glass capillaries provided connections between the delivery syringes and the glass tubes. The outlet from these glass tubes were hidden in the sand coveting the bottom. The dead volume of the delivery lines was 5 ml. The stimuli were presented from syringes placed in an adjustable infusion pump. The solutions were injected into the aquarium at a rate of 2 ml/min during a period of
SHRIMP EXTRACTS
157
10 rain. Stimulations were performed once a day. The test solutions were introduced in parallel with seawater blanks. Each test solution was given at five different concentrations. The aquarium seawater was used for dilution of all solutions to be tested in the bioassay. Behavior Patterns. A benthic food search is observed in the cod when the olfactory tract is stimulated electrically and also when the fish is searching for a food source (Brawn, 1969; Pawson, 1977). Initial activity consists of an abrupt cessation of forward swimming, the cod descending to the bottom and moving backwards with the barbel and rays of the pelvic fins trailing the bottom. Each backward swim over the tube outlet was counted as a bottom food search (BFS). The number of fish doing BFS and the total number of BFS counted for the fish during a stimulation period of 10 min were used as a measure of the potency of a test solution. Other parts of the food search behavior pattern were also noted but not included in the present study. These were: orienting reaction, in the sense used by Pavlov (1927); aggression, as described by Brawn (1961); snapping, a rapid opening and closing of the jaws; and bellying, the fish making a quick swim towards the bottorr~, turning to its side, flashing its belly and swimming along sideways close to the bottom. Extracts and Synthetic Solutions. Shrimp was ground twice in an electric grinder and then extracted with 3 vol of methanol-distilled water (75 : 25 v/v) by stirring at 800 rpm for 30 min (Franz Morat, Eisenbach, Hochschwarzwald, FRG, electrical stirrer, type R-16). The liquid phase was separated from the protein phase by vacuum filtration through a Whatman No. 1 filter. The filtrate was evaporated to dryness on a rotary evaporator at 40~ The residue was dissolved in distilled water to a "dry-weight" content of 100 g/liter. This solution (stock solution) was used for chemical analysis, fractionation, and bioassays. Synthetic solutions were prepared with distilled water so that the concentration of each individual component was the same as that measured in the stock solution. Chemicals. Homarine HC1 was obtained from Carl Roth KG, Karlsruhe, FGR, dimethylthetine HCt from ICN Pharmaceuticals, Inc., Plainview, New York. Other chemicals were from Sigma Chemicals, St. Louis. Characterization of Stimulants and Analytical Procedures. Analyses and characterization of the stimulants were conducted on the stock solution. Amino acid concentrations were measured on a Biotronic LC 5000 automatic amino acid analyzer. The concentration of trimethylamine was determined by a microdiffusion technique (Conway and Bryne, 1933). Trimethylamine oxide was determined as trimethylamine after reduction with titanium trichloride (HjorthHansen, 1952). Characterization and purification of stimulants are summarized in Figure 1. By ion-exchange chromatography, the compounds were separated into three fractions: (1) acidic compounds, adsorbed on the anion exchanger and eluted
ELLINGSEN AND D~VING
158
ACTIVESOLUTIONS
NON-ACTXV~OR WEAKLY ACTIVE SOLUTIONS
GROUND SHRIMPS ~ Extraction with aqueous methanol
I STOCK SOLUTION, 100 g/] I Removal of lipids with chloroform
J CHLOROFORM EXTRACT: q LIPIDS, 3,5% BY WEIGHT
[ LIPID-FREE EXTRACT
I Elution of adsorbed lI FRACTION I. $ompounds w~th 1M HC~ACIDIC COMPOUNDS, ~l 16% BY WEIGHT OF LIPID-FREE EXTRACT
I ichromatography ni~ exchange
m
!
[
ater effluent
I AMPHOTERIC, BASIC AND NEUTRAL COMPOUNDS
]
Cation exchange chromatography
Water effluent
Elution of adsorbed compounds with 4M HCI
IAMPHOTERIC FRACTION3 . AND BASIC
COMPOUNDS. 72% BY WEIGHT OF LIPID-FREE EXTRACT
i
Separation into single components by preparative HPLC (RP-18)
FRACTION 4. SEE FIG. 2
FRACTION2.
NEUTRAL COMPOUNDS.
Wl 12% BY WEIGHT OF
[LIPID-FREE EXTRACT
Jw [ SEE FIG. 5.2 FRACTION
I
I
FIG. 1. Flow chart indicating the procedure followed for fractionation of food-searchinducing compounds in extract from shrimp.
SHRIMP EXTRACTS
159
with excess 1 M HC1; (2) neutral compounds, eluted from the cation exchanger with excess water; and (3) amphoteric and basic compounds, eluted from the cation exchanger with excess 4 M HC1. Solutions with the same concentration as in the stock solution were made up from fractions 1, 2, and 3 and tested in bioassays. Test solutions made up from the stock solution and fraction 3 were also tested in equal concentrations. Traces of fats and pigments (ca. 3.5 % by weight) were removed with chloroform before chromatography. One volume of stock solution was extracted twice with two volumes chloroform. Defatted material (30 g) was dissolved in 50 ml distilled water, pH adjusted to 7.0. This solution was applied to an anion exchange column (Amberlite IRA-401, C1- form, 16-40 mesh, Koch-Light Laboratories, England, 2.5 x 100 cm). The column was washed with 1500 ml distilled water and adsorbed substances were eluted with excess 1 M HC1 (1500 ml). The effluent and washings were combined, concentrated, and dissolved in 50 ml distilled water. The pH was adjusted to 7.0, and the sample was applied to a cation-exchange column (Dowex 50-X8, H § form, 16-40 mesh, Dowex, Switzerland, 2.5 x 100 cm) and rinsed with 1000 ml distilled water. The adsorbed substances were eluted with excess 4 M HC1 (1500 ml). The eluate was concentrated, dissolved in buffer, and the pH was adjusted to 4.0 before separation into single components by preparative HPLC (PerkinElmer, Norwalk, Connecticut, Series 3B liquid chromatograph with a PerkinElmer column C-18 bounded phase, 10/~m particle size, 1.8 x 25 cm). The initial eluent was 0.05 M KH2PO4,pH 4.0, and the final eluent methanol. Buffer was used during the first 20 min, then a linear gradient of 0-100% final eluent was used over the next 45 min. Methanol was run as a final eluent for 20 min. The flow rate was kept constant at 2 ml/min. The eluate was monitored at 210 nm (Perkin-Elmer LC 75, spectrophotometric detector). The components separated by HPLC gave the chromatogram shown in Figure 2; they were collected in two fractions. Fraction 4 contained the substances giving rise to peaks 1-6, and fraction 5 contained all components eluted thereafter. Both fractions were tested in the bioassay at equal concentrations. Single components eluted in fraction 4 were also collected. Before testing, the buffer was removed by the following procedure: Water was removed on a rotary evaporator and the residue dried at 40 ~ to a constant weight. The dried, waterfree material was then dissolved in excess water-free methanol, and buffer was separated from the methanol soluble components by filtration through a Millipore filter, Millipore Corp., Bedford, Massachusetts (45-#m pore size). RESULTS
The bioassays were performed from September 1983 to September 1984. In all experiments, samples of stock solution were tested as a reference for other solutions applied to the fish (see Tables 2, 4, and 5). This control was panic-
Fraction 4
20
30
Fraction 5
5O
Retention time (mini
40
60
70
80
FiG. 2. Chmmatogmm showing the separation of amphotm~c/basic components, fraction 3, by preparative HPLC. Amount injected: 50 rag. The experimental conditions are given in the text.
I0
|
9
Z
r-' r
SHRIMP EXTRACTS
161
ulafly important as the behavior and threshold for eliciting BFS varied over the year. Comparison between Shrimp Extracts and Synthetic Solutions. The concentrations of amino acids, trimethylamine oxide, and trimethylamine found in the stock solution are given in Table 1. When a synthetic solution based on the concentration of amino acids found in the stock solution was prepared and tested, it was observed that the stock solution was 100-200 times as potent by weight as the synthetic solution (Tables 2A and B). The four amino acids (glycine, alanine, proline, and arginine) which were most abundant in the stock solution seemed more efficient in evoking the BFS behavior than the total amino acid pool (Tables 2B and C), and the results indicated that these four amino acids were only 10 times less potent than the stock solution (Tables 2A and C). Two amino acids, glycine and alanine alone, did not seem as potent as the four amino acids taken together, but were still more potent than the total amino acid pool (Tables 2B and D). When the four main amino acids were tested individually in equimolar concentrations (Table 3), glycine appeared to be most potent followed by alanine. A solution containing the amino acids with five carbon atoms, viz., valine, TABLE 1. CONCENTRATIONS OF AMINO ACIDS, TRIMETHYLAMINE, AND TRIMETHYLAMINE OXIDE IN STOCK SOLUTIONa
Concentration Substance Aspartic acid Threonine Serine
Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine Taurine Trimethylamine Trimethylamine oxide aDry weight content: 100 g/liter.
mmol/liter
g/liter
1.28 5.13 3.17 4.47 21.31 58.76 12.52 5.25 2.19 4.26 5.72 2.94 2.63 5.69 14.50 27.47 2.13 75.07
0.17 0.61 0.33 0.66 2.46 4.41 1.12 0.62 0.33 0.56 0.75 0.49 0.41 0.83 2.53 3.44 0.13 5.63
162
ELLINGSEN AND D•VING
TABLE 2. COMPARISON OF BFS ACTIVITY INDUCED BY SAMPLES OF STOCK SOLUTION AND SYNTHETIC SOLUTION OF AMINO ACIDS a
A. Stock solution,
Dilution factor
Concentration,
x 104
g/liter x 10 -4
Number of fish performing BFS
Number of BFS counted
1
100
16
40
2.5 5 10 15
40 20 10 5
13 10 7 2
31 20 18 5
16 !2 9 5 1
43 33 25 13 3
16 13 10 7
53 46 27 17
3
5
9 9 8 8 5
3i 30 27 33 10
B. Total amino acid pool,
x 102
O.5
4O 20 4 2 1
1
5 10 15 C. Glycine, alanine, proline, and arginine, • 10 3 0.5 1 2.5 5
g/liter X 10 -3 21.0 10.5 4.2 2.1
10
1.05
D. Glycine and alanine, 0.5 1 2.5 5 10
g/liter x 10 -2
x 10 3
g/liter x 10 3 11.0 5.5 2.2 1.1 0.55
"The amino acid concentrations are in the proportion found in the stock solution. The entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: September-December 1983. p r o l i n e , m e t h i o n i n e , a n d g l u t a m i c acid, i n d u c e d n o B F S a c t i v i t y w h e n t e s t e d at t h e s a m e c o n c e n t r a t i o n s as t h a t f o u n d in t h e s t o c k s o l u t i o n . A c o m b i n a t i o n of the basic amino acids lysine, arginine, and histidine, scored only a few BFS p a t t e r n s at t h e h i g h e s t c o n c e n t r a t i o n s u s e d ( 1 / 5 0 o f t h e c o n c e n t r a t i o n in s t o c k s o l u t i o n ) . T h e c o m b i n a t i o n o f m e t h i o n i n e , t h e f i v e - c a r b o n a m i n o acids, a n d t h e b a s i c a m i n o a c i d s i n d u c e d B F S a c t i v i t y at t h e s a m e rate as d i d t h e b a s i c a m i n o acids.
SHRIMP EXTRACTS
163
TABLE 3. COMPARISON OF BFS ACTIVITY INDUCED BY INDIVIDUAL SUBSTANCES TESTED IN EQUIMOLAR CONCENTRATIONSa
Substance Glycine
Alanine
Concentration (mol/liter)
Number of fish performing BFS
Number of BFS counted
10-2 10 3 10-4 10-5 10 2 10 3
10 7 6 2 6 4 3 0 6 4 0 0 3 0 0 0 5 0 0 0
19 9 8 6 18 11 9 0 14 8 0 0 4 0 0 0 9 0 0 0
t0 -4
Arginine
Proline
Dimethylthetine
10-5 10-2 10-3 10-4 10 5 10-2 10-3 10-4 10 5 10 2 10-3 10-4 10-5
aThe entries give the number of fish doing BFS and the total counts of BFS performed during the injection period. Concentrationsare in tool/liter of material injected. Test period: February-April 1984. Trimethylamine oxide and trimethylamine HC1 were tested in the same concentrations as found in the natural extract. They did not induce any BFS activity when tested alone. They did not affect the activity scores when tested in combination with the amino acids. Comparison between Chemical Fractions of Stock Solution. As seen from Figure 1, the weight ratio of the adsorbed compounds was 16 : 12 : 72 for fractions 1, 2, and 3, respectively. Only the acidic fraction (fraction 1) and the fraction containing amphoteric and basic components (fraction 3) scored significantly on the bioassay tests as demonstrated in Table 4. When the stock solution and fraction 3 were compared in equal concentrations, the latter was slightly more potent (Table 5). Further separation of fraction 3 by HPLC (Figure 2), yielded 60% by weight of the material in fraction 4, while components in fraction 5 accounted for 40%. No BFS was induced by fraction 5, while the activity induced by fraction 4 was virtually the same as the activity induced by fraction 3. Of the single components eluted in fraction 4, most of the activity was located in peaks
TABLE 4. COMPARISON OF B F S ACTIVITY INDUCED BY SAMPLES OF STOCK SOLUTION AND FRACTIONS 1, 2, AND 3 ISOLATED BY IoN-EXCHANGE CHROMATOGRAPHY a
Number of fish performing BFS
Number of BFS counted
16 11 10 7 4
65 36 20 15 10
B. Acidic fraction, 1 1.6 0.16 0.032
9 2 0
13 4 0
C. Neutral fraction, 2 1.2 0.12 0.024
3 2 0
4 3 0
16 12 8 5 3
40 28 15 8 4
Concentration (g/liter) A. Stock solution 10 1 0.5 0.1 0.01
D. Basic/amphoteric fraction, 3 7.2 0.72 0.144 0.072 0.0072
aThe entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: AugustSeptember 1984. TABLE 5. COMPARISON OF B F S ACTIVITY INDUCED BY STOCK SOLUTION (SS) AND BASIC/AMPHOTERIC FRACTION, 3 a
Sample
Concentration (g/liter)
Number of fish performing BFS
Number of BFS counted
SS 3 SS 3 SS 3 SS 3 SS 3
1 1 0.1 0.1 0.01 0.01 0.005 0.005 0.001 0.001
10 16 7 8 6 5 5 3 1 2
20 40 12 14 10 10 8 7 2 4
~The entries give the number of fish out of 16 doing BFS and the total counts of BFS performed during the injection period. Concentrations are in g/liter of material injected. Test period: May 1984.
SHRIMP EXTRACTS
165
1, 2, and 3. Peaks 4 and 5 were less active when tested in equal concentrations (1 g/liter). DISCUSSION
The chemicals emanating from prey induce feeding behavior in cod. In the present study we have shown that shrimp contain amino acids and substances with amphoteric/basic properties which are effective inducers of a particular part of the feeding behavior, viz., the BFS. We discuss the results of our experiments in light of previous studies on cod and attempts to isolate feeding substances for cod and other fish species. The methods developed for the chemical isolation procedure are specifically considered since they might be useful in forthcoming analyses. The four amino acids in the stock solution, glycine, alanine, proline, and arginine, appeared to be more potent than the total amino acid pool. These results demonstrate that the amino acid pool in a shrimp extract may contain amino acids which have an antagonistic effect on BFS induction in cod. The four main amino acids (glycine, alanine, proline, and arginine) act highly synergistically. This is demonstrated from the results presented in Table 2C and 3. At a total amino acid concentration of 10.5 • 10 -3, 46 BFS were performed by 13 fish when glycine, alanine, proline, and arginine were tested in combination (Table 2C). When the same four amino acids were tested individually at a concentration of 10 -4 mol/liter (ca. 10 -3 g/liter), glycine induced eight BFS (performed by six fish); alanine, nine BFS (performed by three fish); proline and arginine did not induce BFS at this concentration (Table 3). Of the individually tested amino acids, glycine appeared to be most potent, this observation being in agreement with Pawson (1977). Johnstone (1980) determined detection threshold values to nine amino acids in cod by a conditioning method and found that tyrosine was most potent, followed by cysteine, phenylalanine, and glycine. The difference between threshold values was small. Tilseth and Solemdal (1977) examined the preference of cod to a wide variety of natural baits derived from invertebrates and fish tissue. The relative effectiveness of 10 different natural baits in initiating biting of a bag indicated that unconditioned cod clearly responded best to extracts of tissue from crustaceans such as euphasids, shrimp, and deep-sea prawns. Attempts to characterize the effective components in aqueous extracts of crustacean-based baits indicated a reduction in activity at pH below 4 or above pH 10. On adjustment of pH to 11 in a diluted solution of the stock solution, we observed a similar reduction in activity. This treatment was associated with the release of dimethyl sulfide and a precipitate. Boiling of the same solution for 3 hr at pH 1 only resulted in a small decrease in food search activity. The fractionation procedure used in this work for isolating new food-search
166
ELLINGSENANDD~VING
stimulants, other than amino acids, was a modified procedure used for isolation of quaternary bases (Abe and Kaneda, 1975; Konusu and Hayashi, 1975; Hayashi et al., 1978). These authors used the O H - form of an anion exchanger. The anion exchanger has a higher selectivity for C1- than O H - ; the O H - form is therefore more effective in exchanging acidic compounds than the C1- form. In our preliminary studies, we also applied the lipid-free stock solution on a column with the anion exchanger in the O H - form. This procedure resulted in a highly alkaline effluent, and the release of dimethyl sulfide and a precipitate. When this sample was analyzed by HPLC after passing through the cation exchanger, the chromatogram was totally different from that seen in Figure 2. Peaks 2 and 3 did not appear. Therefore, the anion exchanger was used in the C1- form in the fractionation procedure. The activity associated with the acidic fraction (fraction 1) should be related to adsorbed amino acids and to nucleotides which also adsorb onto the C1- form of an anion exchanger. (Hayashi et al., 1978). The activity associated with the neutral fraction (fraction 2) was regarded as insignificant and was neglected in later work. Since the amphoteric/basic fraction (fraction 3) was the most potent of the three tested fractions and even had a higher activity by weight than the stock solution, further work on identification of food-search stimulants was mainly limited to this fraction. Most of the activity induced by single components eluted by HPLC was located in substances giving rise to peaks 1, 2, and 3 in Figure 2. The substance giving rise to peak 1 was identified as the amino acid glycine, possibly containing a small amount of alanine [identical IR and mass spectra with authentic glycine, NMR (60 MHz, D20): 6 = 3.6 ppm (s, CH2), 1.5 ppm (d, J = 3.5, CH 3 of alanine, very weak signal, shift position and coupling constant identical with authentic alanine)]. Peaks 2 and 3 are still unidentified. These components carry the very distinctive smell of shrimp and are very labile to alkali, releasing dimethyl sulfide on treatment with cold aqueous 1 M NaOH. The presence of dimethyl-fi-propiothetine (DMPT) in several species of multicellular algae is well documented (Challenger, 1959; Ishida, 1968; Noda and Horiguchi, 1975; Larher et al.0 1977). The decomposition of this substance by cold alkali, yielding dimethyl sulfide, is believed to be a specific reaction (Ackman and Dale, 1965). An indirect determination of DMPT, as dimethyl sulfide, is based on this assumption. Such analyses have been carried out for marine multicellular algae (Ackman et al., 1966), for Labrador cod (Ackman et al., 1967), for brackish water algae and baltic herring (Granroth and Huttula, 1976), and for antarctic krill (Tokunaga et al., 1977). From these data it would seem possible that either peak 2 or 3 might be DMPT. The IR spectra and NMR spectra [60 MHz, D20, signals at 6 = 3.8 ppm(s) and 3.3 ppm(s)] were not in accordance with pub, lished IR spectra (Ishida, 1968; Noda and Horiguchi, 1975) and NMR spectra (Ishida, 1968; Larher et al., 1977). Weak signals in the NMR spectrum at b =
SHRIMP EXTRACTS
167
3.0 ppm and 3.6 ppm might suggest the presence of DMPT as a minor component in peak 3. Due to the lack of verified structures, it is impossible to determine the concentration in the stock solution, although there are areas in HPLC comparable to glycine. The reduction in activity on alkali treatment of aqueous extracts of crustacean-based baits, as observed by Tilseth and Solemdal (1977) and in this work, obviously should be related to the alkali-labile components giving rise to peaks 2 and 3 in Figure 2. As a representative for thetines, dimethylthetine was tested in our bioassay. This compound does not occur in marine organisms, but it has been described as potent in inducing food consumption in juvenile Dover sole (Mackie et al., 1980). As demonstrated in Table 3, this compound even induced BFS when tested in our bioassay. Peak 4, observed as a shoulder on peak 3, has been identified as glycinebetaine [superimposable IR and mass spectra, NMR (60 MHz, D20): ~ = 3.3 (s, 9H, 3X N--CH3) and 4.2 ppm (s, 2H, CH2)]. The importance of glycinebetaine as a BFS inducer in cod is very doubtful. When glycine-betaine was tested in the same concentrations as the amino acids glycine, alanine, arginine, and proline, no BFS could be observed. The exact concentration of this compound is difficult to determine in our chromatographic system due to low concentration and lack of separation from peak 3. The concentration was determined to be approximately 0.05 g/liter in the stock solution. We were not able to show that glycine-betaine potentiated the stimulation power of amino acids when this component was included. Components 5 and 6 are still unidentified. Compared to the substances giving rise to peaks 1, 2, and 3, they induced only a very weak degree of BFS. The main peak in the chromatogram, peak 7, has identical retention time with homarine. No attempt has been made to identify this component by spectroscopy as neither the isolated component nor authentic homarine induced BFSactivity. The variability of response to the stock solution could be expected due to the cyclic feeding activity of cod found in the coastal zone. Experiments were made both during the most intense feeding period and during breeding season when feeding activity is low even among juvenile cod. Variability of the seawater at the aquarium facilities could also be responsible for the changes in behavioral activity although precautions were taken to keep the water quality constant. The ordinary control routines of the water quality were unaltered during the testing period.
Acknowledgments--The authors are grateful to amanuensis Karl E. Malterud, Institute of Pharmacy, University of Oslo, for recording of spectra and for valuable discussions, and to Gro Svendsen for skillful technical assistance. This work was supported financially by The Norwegian Fisheries Research Council, project 203.15.
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