Chemosensory Responses by the Heterotrophic Marine Dinoflagellate Crypthecodinium cohnii DONALD C. R. HAUSER AND M. LEVANDOWSKY S. H. HUTNER, L. CHUNOSOFF, AND J. S. HOLLWlTZ 1 Haskins Laboratories at Pace University, New York 10038, Biology Department, Pace University, New York 10038, and Biology Department, New York University, New York 10003
Abstract Chemosensory responses by the colorless inshore marine dinoflagellate Crypth ecodinium cohnii were observed in quadrant-divided Petri plates containing an agar layer + liquid overlay. A suspension of organisms in salt solution was poured onto this and allowed to stand 3 hr. A differential tendency of the ceils to become firmly attached or embedded in the substratum was observed when various substances were incorporated in the gel. A positive response (tendency to attach) occurred with: rV-L-fucose,dimethyl-~-propiothetin, betaine, sucrose, glycine, L-alanine, hemin, and fructose; negative response: formalin, glutathione, acid hydrolyzed agar, protamine SO4, L-glutamic acid, lactose, glutamine, taurine, L-aspartic acid, putrescine 2 HC1, choline citrate, choline bitartrate, K citrate, and choline HC1. ~/-Aminobutyricacid was negative or positive dependingon concentration. Dead or immotile cells did not become attached. The following compounds elicited no response: ~-D-fucose, dimethyl acetothetin chloride, cyclic AMP, and glucose.
Successful studies of responses by flagellate protozoa to chemical cues are relatively rare. Early workers [1-3] observed tactic or kinetic responses by some species to chemical gradients produced by diffusion from a point source, such as a microcapillary. Such techniques are quite useful for detecting bacterial responses [4, 5] and ciliate chemotaxis [6, 7]. However, the hydrodynamic interactions of larger and faster eukaryotes with each other and with their medium produce convective effects and tend to obscure responses to gradients; such techniques have been generally less successful with protozoa than with bacteria. For similar reasons, early reports of orientation of spermatozoa to capillary sources were severely criticized [8] as highly artifact-prone.
1Submitted in partial fulfillment of the requirements for the M.S. degree, New York University, by D. C. R. Hauser.
246 MICROBIAL ECOLOGY, Vol.1,246-254 (1975) 9 by Springer-VerlagNew York Inc.
Chemosensory Responses by Crypthecodinium cohnii
247
Working with the colorless marine dinoflagellate Crypthecodinium cohnfi we did not obtain reproducible responses to capillary sources despite many trials. We were convinced however on ecological grounds that chemosensory effects were likely, and eventually observed responses to chemical impregnation of agar surfaces. This is reminiscent of the "trap effect" observed with spermatozoa [9] and the settling behavior of invertebrate larvae [10].
Table 1.
Maintenance Medium (Weight/l O0 ml Medium}a, b
K3citrate H20
0.1
g
*KH2PO 4
0.01
g
*MgSO4 (anhyd.)
0.35
g
*KC1
0.08
g
*NaC1
3.0
g
*CaC03 (= Ca, 0.02 g)
0.05
g
NH4C1
0.008 g
DL-Alanine
0.01
g
Betaine HC1
0.01
g
Sucrose
0.1
g
Glucose
0.5
g
Nitrilotriacetic acid
2.0
mg
L-Glutamic acid
0.1
g
Folic acid
5.0
/./g
Glycerol
0.2
g
Thiamine HC1
0.05
mg
Biotin
0.2
//g
aTrace elements. Supplied from a dry mix [28] to yield (mg/100 ml of medium): Fe, 0.25; Zn, 0.2; Mo, 0.08; Cu, 0.016; V, 0.008; B, 0.004; Cr, 0.004; Ni, 0.004. pH adjusted to 6.0-6.2 with Tris. bSuspension medium: this consists of substances marked with * above.
248
Donald C. R. Hauser et al. Materials a n d M e t h o d s
Fe!sen quadrant-type culture dishes (A. H. Thomas Co. no. 34834271;inside diameter 18 cm, glass partitions 6 mm high; outside height 22 mm borosilicate glass) were washed with Alconox and rinsed 3X with tap and 4X with distilled water. Into each of two diagonally opposed quadrants, 9.0 ml of 1.5% agar (Ionagar No. 2, in U. S., Flow Laboratories, Rockville, MD) prepared with a mineral suspension medium (Table 1) was placed as control; the other two quadrants were filled with this preparation mixed with a candidate substance. The agar layers had the same height as the quadrant partitions. Crypthecodinium cohnii, Puerto Rico strain obtained from Dr. K. Gold, was grown in a defined liquid medium (Table 1). Stock cultures were kept on agar slants [11]. Both were maintained at room temperature. The liquid medium was used for our experiments. The inoculum for each series of experiments was grown in 30 ml of liquid medium in a Petri dish for 3 days. The Petri dish provided a large surface area, which insured sufficient air for optimal growth. A 1:10 dilution was then prepared with the suspension medium and allowed to stand for 3-4 hr. Twenty milliliters of suspension was pipetted onto each dish. After 3 hr the liquid was poured off and the excess was removed with a Pasteur pipette. The dish was flooded with Lugol's iodine and rinsed with 20-40 ml distilled water from a wash bottle. Care was taken that the stream was not directed toward any of the agar quadrants, but to the periphery allowing only a gentle flow of water over the center. The rinse with distilled water can be postponed for up to 24 hr, if the dishes are kept at 4-6~ An 80• dissecting microscope served to observe the stained cells imbedded in the agar; 20 sample points in each quadrant were counted, making a total of 80 sample points counted per dish. To eliminate side effects due to phototactic responses noted in preliminary work, experiments were under uniform vertical illumination from a fluorescent lamp 1 m above the dishes. All substances to be tested were adjusted to 6.2-6.5, the pH of both mineral adaptation and maintenance media. Sample points for counting were chosen away from the quadrant edges, but otherwise with approximately uniform distribution with no protocol of random coordinates. Resulting counts were taken as a measure of density of entrapped cells in a quadrant. Distributions of cells were highly skewed with variance many times the mean; this precluded standard statistical methods such as Student's t-test. Instead, we compared quadrants nonparametrically, using the sign test. Results D u r i n g t h e 3 h r t h a t t h e dinoflageUate s u s p e n s i o n was in c o n t a c t w i t h t h e agar q u a d r a n t s s o m e o f t h e active o r g a n i s m s b e c a m e e m b e d d e d or firmly a t t a c h e d to t h e surface a n d r e m a i n e d in place d u r i n g s u b s e q u e n t w a s h i n g a n d s t a i n i n g p r o c e d u r e s . O r g a n i s m s killed or i m m o b i l i z e d b y f o r m a l i n , i o d i n e , or p r o t a m i n e S 0 4 d o n o t e m b e d in o r a t t a c h to t h e agar a n d are w a s h e d o f f t h e p l a t e in t h e rinse. This e m b e d d i n g p h e n o m e n o n o c c u r r e d p r e f e r e n t i a l l y in q u a d r a n t s w i t h c e r t a i n o f t h e test s u b s t a n c e s a n d was t a k e n t o i n d i c a t e a c h e m o s e n s o r y r e s p o n s e b y t h e organisms.
Chemosensory Responses by Crypthecodinium cohnii
249
Clumped Distributions o f Cells. The variance of cell counts was in all cases greater than the mean, usually by an order of magnitude (Table 2). This indicates, according to statistical theory, that attached cells were not distributed in a random (Poisson) manner, but tended to form aggregates or clumps on the scale of counting. Response to Substances in the Agar. The main observations are summarized in Table 3. We assayed various amino acids, quaternary amines, and carbohydrates that have been reported as cues for other organisms as well as dimethyl-~-propio thetin which occurs in rotting brown algae [ 13]. Protamine S04 has been used to immobilize and attach protozoans to glass slides [14]. The negative effects of protamine S04 and formalin indicate that immobilized or dead cells do not remain on agar after rinsing, and that an active attachment or imbedding response must occur in positive cases. Statistical Significance. Where no statistically significant effect was seen, this may be due to use of nonparametric methods which do not use most of the quantitative information. If we take as null hypothesis that the distribution among quadrants is random with no chemoreceptive response, we are more likely to commit an error of type II (acceptance of a false null hypothesis at a given confidence level) than of type I (rejection of a true null hypothesis). Thus in the cases where a chemosensory effect is detected, our results may be viewed as a conservative interpretation of the data.
Table 2.
Clumping of Attached Cells in Typical Cases. Typical Results from 4 Experiments: 2 Agar Controls with Different Mean Densities o f A ttached Cells and Two with Tested Chemicals Attached cells per field Experiment
Substance
Mean
Variance
1
Agar control
17.3
150.0
2
Agar control
9.2
40.1
3
Trimethylamine HC1, 0.1%
10.3
196.0
5.3
47.3
Betaine 0.01%
30.3
157.0
Betaine (agar control)
16.7
47.6
Trimethylamine HC1 (agar control) 4
Donald C. R. Hauser et al.
250
Table 3. Responses Significant at the 0.05% Level or Better. Concentrations as g/lO0 ml. Tested were 10 - 1 , 10 - 2 , 10 - 3 , 10 - 4 , 10 - 5 , andSomet~mes 1 0 - 6 Except for Agar Hydrolysate, Tested at 2X these Concentrations Active c o n c e n t r a t i o n s
Positive response Dimethyl-/3-propiothetin ot-L-Fucose Hemin Betaine Sucrose Glycine L-Alanine Fructose
10101010101010I 0-
5.104.10s.104-10S_l 0 s -102 1
l 2 2 3 4 4
10102 X 1010101010101010101010-
610-1 2_10- 1 10-5-2 X 10- 1 5_10- l 6-10- 2 3_I 0 - 1 4 S_l 0 - 2 s _t 0 - 1 5 _1 0 - 2 s.10- 1 s _1 0 - 2 5 _1 0 - l 3.10- 1
Negative response Formalin *Glutathione **Agar h y d r o l y s a t e P r o t a m i n e SO 4 L-Glutamic acid Glutamine Lactose L-Aspartic acid Taurine Putrescine 2 HCI C h o l i n e citrate K citrate C h o l i n e HCI Choline bitartrate
Concentration dependent: G A B A positive " negative
10- s.10- 4 1 0 - 3_i 0 - 1
No response detected Dimethyl acetothetin chloride Glucose Cyclic A M P c~-D-Fucose Trimethylamine.HC1 *The r e d u c e d f o r m was used; h o w e v e r u n d e r e x p e r i m e n t a l c o n d i t i o n s ( e x p o s u r e to air in a h o t agar s o l u t i o n ) it was p r o b a b l y largely oxidized. * * P r e p a r e d b y a u t o c l a v i n g a 2% soln. of ionagar n o . 2 at pH 2 a n d filtering; f i l t r a t e was used.
Chemosensory Responses by Crypthecodinium cohnii
251
Discussion
Crypthecodium cohnii, absent in routine plankton samples, is obtained from enrichments of rotting Fucus or other seaweeds [15]. In such enrichments it often forms a dense bloom, and defined axenic media support heavy growth of some strains [16]. Species characteristic of transient habitats, where they often grow rapidly to great abundance, were termed fugitive species by Hutchinson [17], comparable to Winogradsky's [18] zymogenous species of soil microbes. For such, dispersal and location of the transient habitat is a major evolutionary problem. In the case of C. cohnii, passive dispersal can occur by the nearshore currents, which would from time to time bring the planktonic flagellate near the surface of the rotting seaweed. Under these circumstances an imbedding or attaching reaction to suitable chemical cues would clearly be adaptive. Several of the tested substances provide appropriate cues: dimethyl~.propiothetin, fucose, and betaine should indicate the presence of rotting seaweed; hemin is a relatively persistent product of cell break-down. Of the substances tested the following are known to be metabolized by various strains of C cohnii; dimethyl-~-propiothetin, betaine, glucose, sucrose, choline, glycine, and L-alanine. Some of these were positive, some negative, and some appeared inert as chemical cues, suggesting that the fact that a substance is utilized does not necessarily make it a good chemical cue for this habitat. Why did imbedded cells have a clumped distribution? This may denote a tendency of cells to imbed in aggregates, because of a natural attraction based again on chemoreception. Our experience is that dense cultures of C. cohnii are resistant to bacterial contamination, and it may be that such aggregates would better resist that inroads of competing bacteria, especially if currents prevent these from building up in the surrounding medium. We have occasionally observed motile cells forming aggregates in wet mount preparations; this is the subject of further investigation in our laboratory. Chemical cues are known for other aquatic organisms. Adler and colleagues [4] found at least eight different genetically determined receptors for various simple sugars and amino acids in E. coli, and positive responses to cellulose oligomers and prey exudates have been observed in bacterial predators of marine algae and a phycomycete [5]. Polytoma uvella responds positively to substrates such as acetate and butyrate, and negatively to others [3, 19]. Among invertebrate scavengers or detritus feeders, behavioral and electrophysiological studies have demonstrated responses to several amino acids, simple sugars, and quaternary amines in decapod crustaceans [20-22], cirripedes [23], and deposit-feeding snails [24]. Some authors have suggested an evolutionary link between receptor sensitivity to some of these compounds and postsynaptic sensitivity to neurotransmitters such as acetycholine and 7-aminobutyrate. Our
252
Donald C. R. Hauser et al.
data do not fit this pattern, and many of the invertebrate cues were inactive or negative for C. cohnii. We did find a positive response to the quaternary amine betaine, a component of rotting seaweeds, as well as a neurotransmitter. It is not clear that a recent model of a 3-point electrostatically charged molecular receptor site proposed for invertebrate receptors [25] would fit our observations. A possible ecological concordance is provided by the activity of dimethyl/~-propiothetin; some unicellular phytoplankters, including C cohnii itself, are rich in it [13]. Betaine, a stimulatory nutrient for C cohnii [16], is the nitrogen analog o f dimethyl-~-acetothetin, which appeared inert. Similar specificity is seen in the case of the two fucose isomers. Cyclic AMP is a cue for aggregation and prey detection in cellular slime molds and mediator of vertebr.ate hormones. No consistent response to cyclic AMP was found. The activity of 3,-aminobutyrate (GABA) is provocative because of the intense interest attending it as a possible major inhibitory transmitter [26]. One biosynthetic precursor of GABA is glutamate which was intensely negative. The positive activity of glycine is likewise provocative because of evidence for it being an inhibitory transmitter in the spinal cord (but not in the cerebral cortex [261). Thus, evolutionary relations of sensory responses of C cohnii to those of other organisms remain unclear. Their probable ecological role however may be plausibly argued. So, for the moment at least, we should probably be guided more by ecology than by evolution in trying to find behavioral responses of swimming protozoa to chemical cues. Yet we should keep in mind that some compounds which are neurotransmitters in metazoa occur with entirely unknown function in protozoa, catecholamines and indoleamines being intriguing examples [27]. Acknowledgments This work was partly supported by a grant by the S. and W. T. Golden foundation to S. H. H. and by an honorarium to D. C. R. H. by the New York State Museum and Science Service.
References 1.
Pfeffer, W. 1888. Ober chemotaktische Bewegungen yon Bakterien, FlageUaten und Volvocineen. Untersuch. Bot. Inst. 7"ffbingen 2: 1-46.
2.
Jennings, H. S. 1906. Behavior of the Lower Organisms. Columbia Univ. Press, New York.
Chemosensory Responses by Crypthecodinium cohnii
253
3.
Pringsheim, E. G., and Mainx, F. 1926. Untersuchungen zwischen chemotaktischer Reizwirkung und chemischer konstitution. Planta I: 621-50.
4.
Adler, J. 1973. Chemotaxis in Escherichia coli. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum Press, Baltimore.
5.
Chet, I., Fogel, S., and Mitchell, R. 1971. Chemical detection of microbial prey by bacterial predators. J. Bacteriol. 106: 863-877.
6.
Dryl, S. 1973. Chemotaxis in ciliate protozoa, p. 16-31. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum, New York.
7.
Nakatani, I. 1968. Chemotactic response of Paramecium candatum. J. Fac. Sci. Hokkaido Univ. 66: 553-555.
8.
Miller, R. L. 1973. Chemotaxis of animal spermatozoa. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum Press, New York.
9.
Rothchild, L. 1956. Fertilization. Methuen, London.
10.
Crisp, D. J. 1974. Factors Affecting the Settlement of Marine Invertebrate Larvae In: Chemoreception in Marine Organisms. P. T. Grant, editor. Academic Press, London.
11.
Keller, S. E., Hutner, S. H., and Keller, D. E. 1968. Rearing the colorless marine dinoflageUate Crypthecodinium cohnii for use as a biochemical tool. 3". l~'otozool. 15: 792-795.
12.
Siegel, S. 1956. Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill, New York.
13.
Kadota, H., and Ishida, Y. 1972. Production of volatile sulfur compounds by microorganisms. AnrL Rev. Microbiol. 12: 127-139.
14.
Marsot, P., and Couillard, P. 1973. The use ofprotamine-coated slides for immobilizing protozoa. J. Protozool. 20: 105-106.
15.
Javornicky, P. 1962. Two scarsely known genera of the class Dinophyceae: Bernardinium Chodat and Crypthecodinium Biecheler Preslia 3 4 : 9 8 - 1 1 3 .
16.
Provasoli, L., and Gold, K. 1962. Nutrition of the American strain of Gyrodinium cohnis Arch. Microbiol. 42: 196-203.
17.
Hutchinson, G. E. 1951. Copepodology for the ornithologist. Ecology 32: 571-577.
18.
Conn, H. J. 1948. The most abundant groups of bacteria in soil. Bacteriol. Rev. 12: 257-273.
19.
Links, J. 1956. Een hypothese over bet mechanisme van de (phobo-) chemotaxis. Dissertation, Univ. of Utrecht.
20.
Case, J. 1964. Properties of the dactyl chemorectors of Cancer antennarius and C. productus. Biol Bull. 127: 428-46.
21.
Laverack, M. S. 1963. Aspects of chemoreeeption in crustacea. Comp. Biochem. Physiol. 8: 141-151.
22.
Levandowsky, M., and Hodgson, E. S. 1965. Amino acid and amine receptors in lobsters. Comp. Phys. Bioch. 16: 159-161.
23.
Crisp, D. J. 1967. Chemoreception in cirripedes. Biol Bull. 133: 128-141.
24.
Carr, W. E. S. 1967. Chemoreception in the mud snail Nassarius obsoletus T. Biol. Bull. 133: 90-106.
25.
Parmentier, J., and Case, J. 1973. Structure-activity relationships of amino acid
254
Donald C. R. Hauser etal. receptor sites on an identifiable cell body in the brain of the land snail Helix aspersa. Comp. Bioch. Physiol. 35: 511-518.
26.
Snyder, S. H., Logan, W. J., Bennett, J. P., and Arregui, A. 1973. Amino acids as central nervous transmitters: Biochemical studies, p. 131-157. In: Chemical Approaches to brain Function. S. Ehrenpreis and I. J. Kopen, editors. Academic Press, New York.
27.
Hutner, S. H., Baker, H., Frank, O., and Cox, D. 1973. Tetrahymena as a nutritional pharmacological tool, p. 411-433. In: Biology of Tetrahymena. A. M. Elliot, editor. Dowden, Hutchinson & Ross, Stroudsburg, Pa.
28.
Hutner, S. H. 1972. Inorganic Nutrition. Annu. Rev. Microbiol 106: 863-867.
Abstract Chemosensory responses by the colorless inshore marine dinoflagellate Crypth ecodinium cohnii were observed in quadrant-divided Petri plates containing an agar layer + liquid overlay. A suspension of organisms in salt solution was poured onto this and allowed to stand 3 hr. A differential tendency of the ceils to become firmly attached or embedded in the substratum was observed when various substances were incorporated in the gel. A positive response (tendency to attach) occurred with: rV-L-fucose,dimethyl-~-propiothetin, betaine, sucrose, glycine, L-alanine, hemin, and fructose; negative response: formalin, glutathione, acid hydrolyzed agar, protamine SO4, L-glutamic acid, lactose, glutamine, taurine, L-aspartic acid, putrescine 2 HC1, choline citrate, choline bitartrate, K citrate, and choline HC1. ~/-Aminobutyricacid was negative or positive dependingon concentration. Dead or immotile cells did not become attached. The following compounds elicited no response: ~-D-fucose, dimethyl acetothetin chloride, cyclic AMP, and glucose.
Successful studies of responses by flagellate protozoa to chemical cues are relatively rare. Early workers [1-3] observed tactic or kinetic responses by some species to chemical gradients produced by diffusion from a point source, such as a microcapillary. Such techniques are quite useful for detecting bacterial responses [4, 5] and ciliate chemotaxis [6, 7]. However, the hydrodynamic interactions of larger and faster eukaryotes with each other and with their medium produce convective effects and tend to obscure responses to gradients; such techniques have been generally less successful with protozoa than with bacteria. For similar reasons, early reports of orientation of spermatozoa to capillary sources were severely criticized [8] as highly artifact-prone.
1Submitted in partial fulfillment of the requirements for the M.S. degree, New York University, by D. C. R. Hauser.
246 MICROBIAL ECOLOGY, Vol.1,246-254 (1975) 9 by Springer-VerlagNew York Inc.
Chemosensory Responses by Crypthecodinium cohnii
247
Working with the colorless marine dinoflagellate Crypthecodinium cohnfi we did not obtain reproducible responses to capillary sources despite many trials. We were convinced however on ecological grounds that chemosensory effects were likely, and eventually observed responses to chemical impregnation of agar surfaces. This is reminiscent of the "trap effect" observed with spermatozoa [9] and the settling behavior of invertebrate larvae [10].
Table 1.
Maintenance Medium (Weight/l O0 ml Medium}a, b
K3citrate H20
0.1
g
*KH2PO 4
0.01
g
*MgSO4 (anhyd.)
0.35
g
*KC1
0.08
g
*NaC1
3.0
g
*CaC03 (= Ca, 0.02 g)
0.05
g
NH4C1
0.008 g
DL-Alanine
0.01
g
Betaine HC1
0.01
g
Sucrose
0.1
g
Glucose
0.5
g
Nitrilotriacetic acid
2.0
mg
L-Glutamic acid
0.1
g
Folic acid
5.0
/./g
Glycerol
0.2
g
Thiamine HC1
0.05
mg
Biotin
0.2
//g
aTrace elements. Supplied from a dry mix [28] to yield (mg/100 ml of medium): Fe, 0.25; Zn, 0.2; Mo, 0.08; Cu, 0.016; V, 0.008; B, 0.004; Cr, 0.004; Ni, 0.004. pH adjusted to 6.0-6.2 with Tris. bSuspension medium: this consists of substances marked with * above.
248
Donald C. R. Hauser et al. Materials a n d M e t h o d s
Fe!sen quadrant-type culture dishes (A. H. Thomas Co. no. 34834271;inside diameter 18 cm, glass partitions 6 mm high; outside height 22 mm borosilicate glass) were washed with Alconox and rinsed 3X with tap and 4X with distilled water. Into each of two diagonally opposed quadrants, 9.0 ml of 1.5% agar (Ionagar No. 2, in U. S., Flow Laboratories, Rockville, MD) prepared with a mineral suspension medium (Table 1) was placed as control; the other two quadrants were filled with this preparation mixed with a candidate substance. The agar layers had the same height as the quadrant partitions. Crypthecodinium cohnii, Puerto Rico strain obtained from Dr. K. Gold, was grown in a defined liquid medium (Table 1). Stock cultures were kept on agar slants [11]. Both were maintained at room temperature. The liquid medium was used for our experiments. The inoculum for each series of experiments was grown in 30 ml of liquid medium in a Petri dish for 3 days. The Petri dish provided a large surface area, which insured sufficient air for optimal growth. A 1:10 dilution was then prepared with the suspension medium and allowed to stand for 3-4 hr. Twenty milliliters of suspension was pipetted onto each dish. After 3 hr the liquid was poured off and the excess was removed with a Pasteur pipette. The dish was flooded with Lugol's iodine and rinsed with 20-40 ml distilled water from a wash bottle. Care was taken that the stream was not directed toward any of the agar quadrants, but to the periphery allowing only a gentle flow of water over the center. The rinse with distilled water can be postponed for up to 24 hr, if the dishes are kept at 4-6~ An 80• dissecting microscope served to observe the stained cells imbedded in the agar; 20 sample points in each quadrant were counted, making a total of 80 sample points counted per dish. To eliminate side effects due to phototactic responses noted in preliminary work, experiments were under uniform vertical illumination from a fluorescent lamp 1 m above the dishes. All substances to be tested were adjusted to 6.2-6.5, the pH of both mineral adaptation and maintenance media. Sample points for counting were chosen away from the quadrant edges, but otherwise with approximately uniform distribution with no protocol of random coordinates. Resulting counts were taken as a measure of density of entrapped cells in a quadrant. Distributions of cells were highly skewed with variance many times the mean; this precluded standard statistical methods such as Student's t-test. Instead, we compared quadrants nonparametrically, using the sign test. Results D u r i n g t h e 3 h r t h a t t h e dinoflageUate s u s p e n s i o n was in c o n t a c t w i t h t h e agar q u a d r a n t s s o m e o f t h e active o r g a n i s m s b e c a m e e m b e d d e d or firmly a t t a c h e d to t h e surface a n d r e m a i n e d in place d u r i n g s u b s e q u e n t w a s h i n g a n d s t a i n i n g p r o c e d u r e s . O r g a n i s m s killed or i m m o b i l i z e d b y f o r m a l i n , i o d i n e , or p r o t a m i n e S 0 4 d o n o t e m b e d in o r a t t a c h to t h e agar a n d are w a s h e d o f f t h e p l a t e in t h e rinse. This e m b e d d i n g p h e n o m e n o n o c c u r r e d p r e f e r e n t i a l l y in q u a d r a n t s w i t h c e r t a i n o f t h e test s u b s t a n c e s a n d was t a k e n t o i n d i c a t e a c h e m o s e n s o r y r e s p o n s e b y t h e organisms.
Chemosensory Responses by Crypthecodinium cohnii
249
Clumped Distributions o f Cells. The variance of cell counts was in all cases greater than the mean, usually by an order of magnitude (Table 2). This indicates, according to statistical theory, that attached cells were not distributed in a random (Poisson) manner, but tended to form aggregates or clumps on the scale of counting. Response to Substances in the Agar. The main observations are summarized in Table 3. We assayed various amino acids, quaternary amines, and carbohydrates that have been reported as cues for other organisms as well as dimethyl-~-propio thetin which occurs in rotting brown algae [ 13]. Protamine S04 has been used to immobilize and attach protozoans to glass slides [14]. The negative effects of protamine S04 and formalin indicate that immobilized or dead cells do not remain on agar after rinsing, and that an active attachment or imbedding response must occur in positive cases. Statistical Significance. Where no statistically significant effect was seen, this may be due to use of nonparametric methods which do not use most of the quantitative information. If we take as null hypothesis that the distribution among quadrants is random with no chemoreceptive response, we are more likely to commit an error of type II (acceptance of a false null hypothesis at a given confidence level) than of type I (rejection of a true null hypothesis). Thus in the cases where a chemosensory effect is detected, our results may be viewed as a conservative interpretation of the data.
Table 2.
Clumping of Attached Cells in Typical Cases. Typical Results from 4 Experiments: 2 Agar Controls with Different Mean Densities o f A ttached Cells and Two with Tested Chemicals Attached cells per field Experiment
Substance
Mean
Variance
1
Agar control
17.3
150.0
2
Agar control
9.2
40.1
3
Trimethylamine HC1, 0.1%
10.3
196.0
5.3
47.3
Betaine 0.01%
30.3
157.0
Betaine (agar control)
16.7
47.6
Trimethylamine HC1 (agar control) 4
Donald C. R. Hauser et al.
250
Table 3. Responses Significant at the 0.05% Level or Better. Concentrations as g/lO0 ml. Tested were 10 - 1 , 10 - 2 , 10 - 3 , 10 - 4 , 10 - 5 , andSomet~mes 1 0 - 6 Except for Agar Hydrolysate, Tested at 2X these Concentrations Active c o n c e n t r a t i o n s
Positive response Dimethyl-/3-propiothetin ot-L-Fucose Hemin Betaine Sucrose Glycine L-Alanine Fructose
10101010101010I 0-
5.104.10s.104-10S_l 0 s -102 1
l 2 2 3 4 4
10102 X 1010101010101010101010-
610-1 2_10- 1 10-5-2 X 10- 1 5_10- l 6-10- 2 3_I 0 - 1 4 S_l 0 - 2 s _t 0 - 1 5 _1 0 - 2 s.10- 1 s _1 0 - 2 5 _1 0 - l 3.10- 1
Negative response Formalin *Glutathione **Agar h y d r o l y s a t e P r o t a m i n e SO 4 L-Glutamic acid Glutamine Lactose L-Aspartic acid Taurine Putrescine 2 HCI C h o l i n e citrate K citrate C h o l i n e HCI Choline bitartrate
Concentration dependent: G A B A positive " negative
10- s.10- 4 1 0 - 3_i 0 - 1
No response detected Dimethyl acetothetin chloride Glucose Cyclic A M P c~-D-Fucose Trimethylamine.HC1 *The r e d u c e d f o r m was used; h o w e v e r u n d e r e x p e r i m e n t a l c o n d i t i o n s ( e x p o s u r e to air in a h o t agar s o l u t i o n ) it was p r o b a b l y largely oxidized. * * P r e p a r e d b y a u t o c l a v i n g a 2% soln. of ionagar n o . 2 at pH 2 a n d filtering; f i l t r a t e was used.
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Discussion
Crypthecodium cohnii, absent in routine plankton samples, is obtained from enrichments of rotting Fucus or other seaweeds [15]. In such enrichments it often forms a dense bloom, and defined axenic media support heavy growth of some strains [16]. Species characteristic of transient habitats, where they often grow rapidly to great abundance, were termed fugitive species by Hutchinson [17], comparable to Winogradsky's [18] zymogenous species of soil microbes. For such, dispersal and location of the transient habitat is a major evolutionary problem. In the case of C. cohnii, passive dispersal can occur by the nearshore currents, which would from time to time bring the planktonic flagellate near the surface of the rotting seaweed. Under these circumstances an imbedding or attaching reaction to suitable chemical cues would clearly be adaptive. Several of the tested substances provide appropriate cues: dimethyl~.propiothetin, fucose, and betaine should indicate the presence of rotting seaweed; hemin is a relatively persistent product of cell break-down. Of the substances tested the following are known to be metabolized by various strains of C cohnii; dimethyl-~-propiothetin, betaine, glucose, sucrose, choline, glycine, and L-alanine. Some of these were positive, some negative, and some appeared inert as chemical cues, suggesting that the fact that a substance is utilized does not necessarily make it a good chemical cue for this habitat. Why did imbedded cells have a clumped distribution? This may denote a tendency of cells to imbed in aggregates, because of a natural attraction based again on chemoreception. Our experience is that dense cultures of C. cohnii are resistant to bacterial contamination, and it may be that such aggregates would better resist that inroads of competing bacteria, especially if currents prevent these from building up in the surrounding medium. We have occasionally observed motile cells forming aggregates in wet mount preparations; this is the subject of further investigation in our laboratory. Chemical cues are known for other aquatic organisms. Adler and colleagues [4] found at least eight different genetically determined receptors for various simple sugars and amino acids in E. coli, and positive responses to cellulose oligomers and prey exudates have been observed in bacterial predators of marine algae and a phycomycete [5]. Polytoma uvella responds positively to substrates such as acetate and butyrate, and negatively to others [3, 19]. Among invertebrate scavengers or detritus feeders, behavioral and electrophysiological studies have demonstrated responses to several amino acids, simple sugars, and quaternary amines in decapod crustaceans [20-22], cirripedes [23], and deposit-feeding snails [24]. Some authors have suggested an evolutionary link between receptor sensitivity to some of these compounds and postsynaptic sensitivity to neurotransmitters such as acetycholine and 7-aminobutyrate. Our
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data do not fit this pattern, and many of the invertebrate cues were inactive or negative for C. cohnii. We did find a positive response to the quaternary amine betaine, a component of rotting seaweeds, as well as a neurotransmitter. It is not clear that a recent model of a 3-point electrostatically charged molecular receptor site proposed for invertebrate receptors [25] would fit our observations. A possible ecological concordance is provided by the activity of dimethyl/~-propiothetin; some unicellular phytoplankters, including C cohnii itself, are rich in it [13]. Betaine, a stimulatory nutrient for C cohnii [16], is the nitrogen analog o f dimethyl-~-acetothetin, which appeared inert. Similar specificity is seen in the case of the two fucose isomers. Cyclic AMP is a cue for aggregation and prey detection in cellular slime molds and mediator of vertebr.ate hormones. No consistent response to cyclic AMP was found. The activity of 3,-aminobutyrate (GABA) is provocative because of the intense interest attending it as a possible major inhibitory transmitter [26]. One biosynthetic precursor of GABA is glutamate which was intensely negative. The positive activity of glycine is likewise provocative because of evidence for it being an inhibitory transmitter in the spinal cord (but not in the cerebral cortex [261). Thus, evolutionary relations of sensory responses of C cohnii to those of other organisms remain unclear. Their probable ecological role however may be plausibly argued. So, for the moment at least, we should probably be guided more by ecology than by evolution in trying to find behavioral responses of swimming protozoa to chemical cues. Yet we should keep in mind that some compounds which are neurotransmitters in metazoa occur with entirely unknown function in protozoa, catecholamines and indoleamines being intriguing examples [27]. Acknowledgments This work was partly supported by a grant by the S. and W. T. Golden foundation to S. H. H. and by an honorarium to D. C. R. H. by the New York State Museum and Science Service.
References 1.
Pfeffer, W. 1888. Ober chemotaktische Bewegungen yon Bakterien, FlageUaten und Volvocineen. Untersuch. Bot. Inst. 7"ffbingen 2: 1-46.
2.
Jennings, H. S. 1906. Behavior of the Lower Organisms. Columbia Univ. Press, New York.
Chemosensory Responses by Crypthecodinium cohnii
253
3.
Pringsheim, E. G., and Mainx, F. 1926. Untersuchungen zwischen chemotaktischer Reizwirkung und chemischer konstitution. Planta I: 621-50.
4.
Adler, J. 1973. Chemotaxis in Escherichia coli. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum Press, Baltimore.
5.
Chet, I., Fogel, S., and Mitchell, R. 1971. Chemical detection of microbial prey by bacterial predators. J. Bacteriol. 106: 863-877.
6.
Dryl, S. 1973. Chemotaxis in ciliate protozoa, p. 16-31. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum, New York.
7.
Nakatani, I. 1968. Chemotactic response of Paramecium candatum. J. Fac. Sci. Hokkaido Univ. 66: 553-555.
8.
Miller, R. L. 1973. Chemotaxis of animal spermatozoa. In: Behavior of Microorganisms. A. Perez-Miravete, coord. Plenum Press, New York.
9.
Rothchild, L. 1956. Fertilization. Methuen, London.
10.
Crisp, D. J. 1974. Factors Affecting the Settlement of Marine Invertebrate Larvae In: Chemoreception in Marine Organisms. P. T. Grant, editor. Academic Press, London.
11.
Keller, S. E., Hutner, S. H., and Keller, D. E. 1968. Rearing the colorless marine dinoflageUate Crypthecodinium cohnii for use as a biochemical tool. 3". l~'otozool. 15: 792-795.
12.
Siegel, S. 1956. Non-Parametric Statistics for the Behavioral Sciences. McGraw-Hill, New York.
13.
Kadota, H., and Ishida, Y. 1972. Production of volatile sulfur compounds by microorganisms. AnrL Rev. Microbiol. 12: 127-139.
14.
Marsot, P., and Couillard, P. 1973. The use ofprotamine-coated slides for immobilizing protozoa. J. Protozool. 20: 105-106.
15.
Javornicky, P. 1962. Two scarsely known genera of the class Dinophyceae: Bernardinium Chodat and Crypthecodinium Biecheler Preslia 3 4 : 9 8 - 1 1 3 .
16.
Provasoli, L., and Gold, K. 1962. Nutrition of the American strain of Gyrodinium cohnis Arch. Microbiol. 42: 196-203.
17.
Hutchinson, G. E. 1951. Copepodology for the ornithologist. Ecology 32: 571-577.
18.
Conn, H. J. 1948. The most abundant groups of bacteria in soil. Bacteriol. Rev. 12: 257-273.
19.
Links, J. 1956. Een hypothese over bet mechanisme van de (phobo-) chemotaxis. Dissertation, Univ. of Utrecht.
20.
Case, J. 1964. Properties of the dactyl chemorectors of Cancer antennarius and C. productus. Biol Bull. 127: 428-46.
21.
Laverack, M. S. 1963. Aspects of chemoreeeption in crustacea. Comp. Biochem. Physiol. 8: 141-151.
22.
Levandowsky, M., and Hodgson, E. S. 1965. Amino acid and amine receptors in lobsters. Comp. Phys. Bioch. 16: 159-161.
23.
Crisp, D. J. 1967. Chemoreception in cirripedes. Biol Bull. 133: 128-141.
24.
Carr, W. E. S. 1967. Chemoreception in the mud snail Nassarius obsoletus T. Biol. Bull. 133: 90-106.
25.
Parmentier, J., and Case, J. 1973. Structure-activity relationships of amino acid
254
Donald C. R. Hauser etal. receptor sites on an identifiable cell body in the brain of the land snail Helix aspersa. Comp. Bioch. Physiol. 35: 511-518.
26.
Snyder, S. H., Logan, W. J., Bennett, J. P., and Arregui, A. 1973. Amino acids as central nervous transmitters: Biochemical studies, p. 131-157. In: Chemical Approaches to brain Function. S. Ehrenpreis and I. J. Kopen, editors. Academic Press, New York.
27.
Hutner, S. H., Baker, H., Frank, O., and Cox, D. 1973. Tetrahymena as a nutritional pharmacological tool, p. 411-433. In: Biology of Tetrahymena. A. M. Elliot, editor. Dowden, Hutchinson & Ross, Stroudsburg, Pa.
28.
Hutner, S. H. 1972. Inorganic Nutrition. Annu. Rev. Microbiol 106: 863-867.
