Chemical induction of colony formation in a green alga (Scenedesmus acutus) by grazers (Daphnia)

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1 Limnol. Oceanogr., 39(7), 1994, , by the American Society of Limnology and Oceanography, Inc. Chemical induction of colony formation in a green alga (Scenedesmus acutus) by grazers (Daphnia) Winfried Lampert, Karl Otto Rothhaupt, and Eric von Elert Max Planck Institute for Limnology, Postfach 165, PlSn, Germany Abstract The green alga, Scenedesmus acutus, grows in culture in unicellular form, but it forms colonies (coenobia) when exposed for 48 h to a chemical released by the grazer Daphnia magna. The colony-forming response can be evoked only in growing cells. The Daphnia factor affects colony size but not algal growth rate. The minimum concentration of Daphnia factor that induces colony formation is equivalent to a Daphnia biomass of 0.5 mg dry wt liter-* in the culture medium. Actively feeding daphniids induce a stronger response than starved ones. Homogenized Scenedesmus, homogenized Daphnia, ammonium, and urea are not effective. The Daphnia factor is a nonvolatile, organic substance of small molecular mass (< 500 Da). It is moderately lipophilic, heat stable, ph-resistant in a range from 1 to 12, and not affected by treatment with Pronase E. The chemical activity is not lost when the substance is dried but disappears during incineration. Colony formation can be interpreted as a grazing defense mechanism. The phenotypic response may have evolved because of the tradeoff between higher sinking rates and grazing resistance of colonial forms. Phytoplankton can be extremely variable, as species are composed of many different clones that can replace each other under differing environmental conditions (Wood and Leatham 1992). Clones established from single cells can also change their morphology when grown under laboratory conditions; thus, they are phenotypically plastic. The green algal genus Scenedesmus is known to be notoriously phenotypically flexible (Trainor 199 1). Individual strains of various Scenedesmus species can grow as unicells or can form colonies (coenobia) of four or eight cells. The cells can also vary with respect to the number and size of spines. Trainor (1992) claimed that the phenotypic change in Scenedesmus is an ordered sequence of ecomorphs that can be defined as cyclomorphosis (sensu Black and Slobodkin 1987) driven by environmental factors. Various abiotic factors (nutrients, ph) and the age of the culture affect colony size (Egan and Trainor 1989), but temperature is particularly effective in controlling Scenedesmus phenotypes. Unicells are predominant at warm temperatures, Acknowledgments We are indebted to Ellen van Donk and Dag Hessen who stimulated this work and encouraged us to pursue this problem. We also thank Maren Volquardsen and Heinke ClauBen for help with the algal tests, Heinke Buhtz for cell counts, and Nancy Zehrbach for linguistic corrections of the manuscript while colonies dominate at low temperatures (1 OOC) (Trainor 1993a). Recently, Hessen and van Donk (1993) discovered that colony formation and spine enforcement in Scenedesmus subspicatus can be induced by a biotic factor-a dissolved chemical released from Daphnia magna. Under the influence of a Daphnia factor, unicell Scenedesmus formed eight-cell colonies within 3-5 d. Hessen and van Donk (1993) suspected that large, spine-armored colonies may be protected from grazers, and they were able to demonstrate that Daphnia had lower grazing rates on colonies compared to single cells. The study of Hessen and van Donk (1993) is particularly exciting as it not only demonstrates the phenotypic development of a defensive mechanism in this alga but also shows that the algal response is mediated by a chemical stimulus released by the grazer (kairomone). Numerous such responses have recently been described for zooplankton (Larsson and Dodson 1993), and they have stimulated a growing interest in the theory of phenotypic plasticity. Phenotypic changes may be of great importance in phytoplankton-zooplankton interactions. These ideas caused us to repeat Hessen and van Donk s experiment with a different Scenedesmus species in a more quantitative and statistically rigorous way. Our aim was to test whether colony formation occurs at concentrations that are realistic for natural zooplank-

2 1544 Lampert et al. ton abundances and to determine how the chemical activity of the Daphnia factor is modified. The experiments were also designed to test whether a tradeoff between colony formation and algal growth rate can be detected. Finally, we performed tests to begin to characterize the chemical nature of the colony-inducing factor. Material and methods Scenedesmus acutus Meyen has been cultured in chemostats in our laboratory for many years (Lampert et al. 1988) with a modified, purely inorganic Chu 12 medium (Miiller 1972) at a growth rate of 0.7 d-l. The algal population consists mainly of single cells (mean equivalent spherical diameter ESD, 5.9 pm; mean volume, 108 pm3). On average, 1.7 cells form an aggregate and the cells do not have spines under these culture conditions. The chemostats are not axenic, but bacterial biomass is negligibly small. Tests were run in batch cultures in loo-ml cellulose-plug-stoppered Erlenmeyer flasks containing 50 ml of medium. In a standard biotest, each flask contained 45 ml of fresh Chu medium, 3 ml of algal inoculum, and either another 2 ml of Chu medium (controls) or 2 ml of test water (treatment). The initial algal concentration was x lo5 cells ml-l. Flasks were incubated in a temperature-controlled (22 C) and illuminated cabinet on a rotating shaking table (80 rpm). Continuous light from above was provided by fluorescent lamps (photon supply rate, 250 pmol m-2 s-l). Standard incubation time was 48 h, although this was occasionally varied for special tests. The amount of test water was also increased in some tests, but the algal inoculum was always 3 ml. All treatments and controls were run in triplicate. Daphnia magna Straus from a strain maintained in our laboratory for many years was cultured in 1.5-liter jars in membrane-filtered lake water from mesotrophic Schiihsee with Scenedesmus as food. To produce the standard Daphnia factor, we kept 40 adult daphniids (total dry mass, - 10 mg) in 200 ml of filtered lake water with sufficient Scenedesmus food. Since we did not find differences in the chemical activity of the water after 12, 24, and 36 h of Daphnia incubation, we used 24 h as the standard treatment. Water from the Daphnia jar was passed through a 0.1 -pm membrane filter before it was added to the algal cultures. The effect of starved Daphnia was tested in some experiments in which we incubated daphniids as in the standard procedure but did not feed them. Initial and final algal densities and particle size distributions were measured in a CASY particle analyzer (l OO-pm capillary). The size range from 3.5- to 15.5-pm ESD (270 channels) was analyzed. Depending on algal density, we diluted the samples 1 : 10 or 1 : 100 and measured 4 x 0.2 ml or 4 x 0.4 ml to count between 5,000 and 10,000 particles. A subsample of each treatment was preserved in Lugol s fixative and the number of cells per colony was determined for aggregates in the inverted microscope. Algal growth rates were calculated from the initial and final samples using both algal volume and cell numbers. Because the particle counter determines the number of aggregates rather than the number of individual cells, we calculated the mean individual cell volume from the mean particle volume (determined by CASY) and the mean number of cells per aggregate (determined by microscopic counts). The total algal volume (pm3 ml- ) was then divided by the individual cell volume (pm3) to calculate the number of cells ml - l. We gained some information about the chemical nature of the agent by subjecting sterile-filtered Daphnia water to various treatments before using it in the standard test procedure to see whether the chemical activity was still present. These treatments needed some time and the biotest could only be started the next day. A subsample of the untreated, filtered chemically active water was therefore stored at room temperature for the time the handling of the treatments required and was then stored in a refrigerator overnight, as were the treated samples. This subsample was used as a positive control to ensure that the agent did not lose its chemical activity during storage. Molecular size was determined by ultrafiltration with a YC05 membrane, which retains molecules >500 Da. Twenty milliliters of chemically active water were filtered, and the membrane was rinsed with another 20 ml of Chu medium before the membrane was dry. Just before the membrane was dry again, the

3 supernatant was diluted with 40 ml of Chu medium. Both filtrate and supernatant were adjusted to ph 8. We adjusted 20 ml of chemically active wa- s ter to ph 1.0 with 25% HCl and heated it to g 60 C in a closed vessel for 30 min. After cool- Z ing to room temperature, we readjusted the ph to 8.0 with 2 N NaOH. The same procedure.e = was carried out at ph 12 and 60 C. The chemically active water was dried in a s: 2 rotating evaporator at 40 C under a vacuum. o- o 2 _ The residues were redissolved in the same amount of ultrapure water. In a separate treatment, the dry residues were heated for 5 min in the flame of a Bunsen burner to destroy organic substances. Lipophily of the substance was tested by sol- Daphnia induces algal colonies Cells / Colony id-phase extraction of chemically active water. 1.0 A Cl8 cartridge was rinsed with 10 ml of meth- 1 - I anol and conditioned with 50 ml of ultrapure I water. We ran 20 ml of chemically active water g I through the cartridge and adjusted the water passed to ph 8. The cartridge was extracted with 10 ml of methanol, the solvent was evaporated at 40 C under a vacuum, and the residues were redissolved in 20 ml of Chu medium. To test for a proteinaceous substance, we incubated 20 ml of the chemically active water with 5 ml of 165 mm phosphate buffer (ph 7.5) and 1 ml of Pronase-E solution (Sigma, 2 mg ml-l, 33 mm phosphate buffer, ph 7) for 0.0 I I I 5 h at room temperature. Following the enzyme treatment, we adjusted samples to ph 8.0 and diluted them to 40 ml. Controls were prepared in exactly the same way but with 1 ml of water instead of the enzyme Volume Fig. 1. Effect of 2 ml of filtrate from a Daphnia culture (200 liter- ) added to a 50-ml Scenedesmus culture after 48 h. Different measures for colony formation from the same experiment. Upper panel-distribution of the number of cells forming a single aggregate in controls (white bars) and treatments (shaded bars). Error bars represent 1 SD (n = 3). Lower panel-size distributions of particles as determined in the particle counter. Broken line, controls; solid line, treatments. The abscissa represents 270 Results Colony formation - The addition of filtered Daphnia water to the Scenedesmus cultures resulted in a dramatic increase in the number measurements (channels). Each curve is the mean of three of cells per colony. For example, 2 ml of water replicates smoothed by running averages of five. from a culture of 200 Daphnia liter- (final concn equivalent to 2 mg dry wt liter - l) caused Cell aggregation is reflected in the distribu- ~50% of the cells to remain in eight-celled tion of particle sizes (Fig. 1). Small particles, coenobia; only 10% remained single cells. The dominant in the controls, disappeared in the opposite was observed in the controls (Fig. 1). treatments, while large particles increased. This figure is similar to the results of Hessen Control particles were slightly larger (mean, and van Donk (1993) with S. subspicatus, al- 145 l.cm3) than at the beginning of the incuthough no spines are induced in our strain of bation (mean, 108 pm3), but treatments were S. acutus. considerably larger (mean, 356 pm3). Note that \

4 1546 Lampert et al l 0 Table 1. Multiple-range ANOVA for mean particle volumes (+ 1 SE, n = 3) from Exp. A. Effect of varying numbers of Daphnia in the incubation water (2 ml added to 48 ml of algal culture). 2 x denotes a second addition of 2 ml of incubation water after 24 h. Daphnia homogenate is equivalent to 200 liter-l. Overall ANOVA: 1;9,20 = 156.1; P < Asterisks sharing the same vertical column indicate treatments that are not significantly different at the 95% level (Tukey s test). 100 I I I I I I Cells per aggregate Fig. 2. Relationship between the average number of cells per aggregate (determined microscopically) and the mean volume per particle (determined by particle counter) in treatments of various strengths. Mean particle Treatment volume Homogeneous groups Control 145.3(2.3) * Daphnia homogenate 149.7(1.9) * Daphnia liter-l (4.9) * (4.1) * (4.1) * (0.9) * 50(2x) 174.3(4.5) * * (5.6) * (8.0) * 200(2x) 357.3(16.3) * the 270 channels of the particle counter are not equally spaced on the x-axis when volume is plotted (distances increase to the right); hence, mean particle volume cannot be estimated directly from this curve. Cells per colony were counted for 16 treatments with three replicates each, and the mean number of cells per colony was compared with the mean particle volume as determined with the CASY. Both parameters are highly correlated (Fig. 2). Thus, mean particle volumes could be used for statistical comparison of the treatments. The regression of the 48 measurements plotted in Fig. 2 is log(mean particle volume) = x log(cells aggr.-l) (r2 = 0.868). The slope is < 1, which indicates a decreasing individual cell size in treatments with aggregated cells. The mechanism of colony formation is probably not an aggregation of already existing single cells. More likely, dividing cells remain adhered when they leave the parent cells (Trainor 1993b). Colony formation took place only in growing algal cultures in full medium. Daphnia culture water had no effect on nonmultiplying cells in nutrient-poor medium (e.g. in lake water from Schiihsee without additional nutrients). Algal growth rates -Differing treatments affected colony size but not algal growth rates (i.e. the Daphnia factor controls only the adhesion of cells, not their production). Growth rates calculated from total algal volume were higher than growth rates calculated from cell numbers. Hence, the individual cell volume increased when Scenedesmus was grown in the biotest (static cultures) compared to the chemostat. Variability of growth rates between treatments and across experiments was low. Coefficients of variance were 55%. For example, mean (&SD) growth rates of all treatments of experiment A (listed in Table 1) are 1.35kO.04 d- f or volume and d-l for cell numbers (n = 10). We calculated a oneway ANOVA for four concentrations of Daphnia factor (0, 50, 100, and 200 liter-l) and the controls. There was no significant difference for volume-based growth rates (F4,10 = 2.50; P = 0.110). The average mean growth rate was 1.363kO.016 d-l (n = 5). On the basis of cell numbers, the ANOVA indicated a significant difference between treatments (F4,10 = 9.48; P = 0.002). A post-hoc test (Tukey) showed that this difference was due to a single treatment. However, the deviation was minimal (< lo%), and the ANOVA was significant only because of the small within-group variability. The mean growth rate was d-l (n = 5).

5 Daphnia induces algal colonies 1547 Table 2. Multiple-range ANOVA for mean particle Table 3. Multiple-range ANOVA for mean particle volumes (+ 1 SE, n = 3) from Exp. B. Effect of varying volumes ( f 1 SE; n = 3) from Exp. C. Effect of ammonium numbers of fed and starved Daphnia in the incubation addition (final concn), varying incubation times of Duphwater (2 ml added to 48 ml of algal culture). Boiled niu, and addition of varying amounts of incubation water indicates that the incubation water was heated to 100 C to algal cultures. ST denotes starved during incubation. before adding it to the algal culture. Overall ANOVA: F,,,6 Overall ANOVA: F,,, 8 = 80.9; P < Asterisks as = 39.5; P < Asterisks as in Table 1. in Table 1. Treatment Control Algal homogenate Daphnia liter- 50, starved 50, fed 100, starved 100, fed 200, fed 200, fed, boiled Mean particle volume Homogeneous groups 130.7(2.3) * 135.3(1.9) * * 134.7(0.7) * * 177.6(7.4) * 151.3(7.3) * * 173.7(9.4) * * 284.3( 18.9) * 255.3(11.3) * Mean particle Treatment volume Homogeneous groups Control 158.3(4.5) * Ammonium-N 0.1 mg liter (1.7) * 1.0 mg liter-l 163.0(5.0) * 200 Daphnia liter-l 2 ml (12 h) (6.9) * 2 ml (24 h) 328.7( 18.8) * 2 ml (48 h) 346.0( 10.7) * 2 ml (48 h, ST) 250.3(7.3) * 5 ml (48 h) 366.3(19.3) * 10 ml (48 h) 490.0(23.1) * Although the five experiments (A-E) were spread out over 2 months (18 March-l 3 May), differences in the growth rates of the controls were negligible for all of them. A one-way ANOVA for volume-based growth rates of the controls of four experiments (no initial value available for Exp. D) yielded significant differences (F3,8 = 67.7; P < 0.001). Tukey s test showed two homogeneous groups (AB and CE). However, the differences were small, and the average mean growth rate (1.321kO.072 d-l; n = 4) was similar to the values obtained in Exp. A. Even the controls of additional experiments performed several months later (unpubl. data) did not differ from these values; hence, the biotest proved to be reliable. Induction strength -Absolute values of mean particle volume can only be compared within experiments where a single source of Daphnia factor was used. Small differences in the size of the daphniids, their food availability, or other conditions of the Daphnia cultures may have affected concentration and activity of the colony-inducing factor and have caused differences between otherwise identical treatments in succeeding experiments. The induction of colonies depends on the concentration of Daphnia factor (Table 1). When the incubation water of 200 Daphnia liter-l was serially diluted, the effect disappeared at 50 liter- I. Fifty Daphnia liter- l seems to be the threshold concentration under our experimental conditions, as it still produced a significant effect in Exp. B (Table 2). The ad- dition of a second portion of incubation water after 24 h of algal growth increased the mean particle size in the 50 liter - l treatment but not in the undiluted samples. However, this does not mean that the maximum effect is obtained at 200 liter-l because the mean particle volume can be further increased if 5 or 10 ml of incubation water (200 liter-l) are supplied instead of 2 ml (Table 3). The colony-inducing factor must be released from live Daphnia because homogenate of daphniids equivalent to a concentration of 200 liter-l had no effect (Table 1). Actively feeding Daphnia produced more of the colony-inducing factor than starved ones (Tables 2,3), although the effect was significant only at the highest concentration (200 liter-l). However, the colony-inducing factor is probably not a constituent of the Scenedesmus cells themselves, because algal homogenate was not effective (Table 2). Table 3 shows the effect of varying incubation times of Daphnia. The culture medium was nearly saturated with Daphnia factor after 24 h. Incubation of the daphniids for 48 or 72 h yielded only a small (insignificant) increase in colony formation. Hence, we incubated Daphnia for 24 h in all other experiments. Nature of the colony-inducing factor - During incubation, daphniids excrete ammonium, so we suspected that colony formation might be induced by ammonium. At 200 Daphnia

6 1548 Lampert et al. Table 4. Multiple-range ANOVA for mean particle volumes (+ 1 SE; n = 3) from Exp. D. Effect of biochemical treatments of incubation water before addition to algal cultures. Positive control: addition of untreated incubation water. Overall ANOVA: F6,14 = 62.5; P < Asterisks as in Table 1. Treatment Negative Positive control control Ultrafiltration Supernatant Filtrate C18, run through ph 1, 60 C ph 12, 60 C Mean particle volume Homogeneous groups 138.7(3.8) * 339.7(5.4) * 176.7(9.1) * 359.0(21.O) * 169.7(9.2) * 363.7(18.1) * 315.0(11.5) * g 0.8 E < 0.6 > z g t: E E! 0.2 a, Volume Fig. 3. Test of the effect of ultrafiltration (500 Da) on the Daphnia factor. Particle distributions as in Fig. 1. Broken line-negative control (no Daphnia factor); thin linepositive control (untreated Daphnia factor); thick linetreated Daphnia incubation water. Upper panel-filtrate. Lower panel-supernatant. The chemically active substance is in the filtrate (i.e. ~500 Da). liter-l, the incubation water contained 0.18 mg NH4-N liter-l. Thus, the addition of 2 ml of incubation water increased the ammonium-n concentration in the algal cultures by - 15 pg liter-l. Ammonium concentrations of 100 hg N liter-l and 1 mg N liter-l, however, had no effect on colony formation (Table 3). Negative results were also obtained with urea at concentrations of 0.8, 8.0, and 80 pg liter-l. We attempted to destroy the Daphnia factor relative to positive (untreated) controls. Figure 3 demonstrates the procedure with results of the ultrafiltration (500 Da). After ultrafiltra- tion of incubation water, the filtrate induced a response identical to the positive control, while the supernatant was no longer chemically active (Table 4). Hence, the inducing factor must have a molecular mass < 500 Da. Most of the chemical activity disappeared when the water was passed through a Cl8 solidphase adsorption cartridge (Table 4), but it could not be recovered by desorption of the cartridge with methanol (Table 5). Treatment with Pronase E did not destroy the chemical activity of the colony-inducing factor. Daphnia incubation water treated with the protease induced a mean particle size (& SD) of 264k10.6 pm3 and 3.9kO.2 cells aggregate - l; controls with protease had only 108.3k2.1 pm3 and cells aggregate-l (n = 3). The colony-inducing factor is heat stable and ph insensitive. Boiling the incubation water for a short time resulted in no significant loss of chemical activity (Table 2). Further tests showed that the factor is probably a nonvolatile organic substance, as it could be dried and resuspended without losing the chemical activity but was destroyed by incineration (Table 5). Discussion In the green alga Scenedesmus (Chlorococtales, Chlorophyta), cells divide inside a mother cell and leave through an opening in the cell wall (Van den Hoek et al. 1993). The daughter cells may either separate to form unicells or stay together in colonies (coenobia). Colony

7 Daphnia induces algal colonies 1549 formation is evidently a phenotypic trait controlled by environmental factors. Although there may be differences in genotype x environment interactions (Wood and Leatham 1992), phenotypic change in colony size occurs in several species of Scenedesmus (Trainor 1993b). Hessen and van Donk (1993) were the first to show that a chemical factor released by Daphnia was able to induce colonies in the spine-armored S. subspicatus. We found an identical response in the nonarmored S. acutus. Hence, we hypothesize that the potential for grazer-induced colony formation is also widespread in the genus. The direct response of Scenedesmus to the (chemical) presence of a grazer suggests an analogy with the morphological changes in zooplankton in response to kairomones released by their predators (Larsson and Dodson 1993). These morphological changes are supposed to be protective when predator densities vary in space and time (Dodson 1989). Most grazers are size selective, so colony formation may be a grazer defense. Hessen and van Donk (1993) found, in fact, lower grazing rates for relatively small D. magna when the share of colonies in the algal food was high. We performed grazing experiments with small (1.0 mm) and large (2.5 mm) D. magna using a dual-label approach (Lampert and Taylor 1985) with mixtures of induced and noninduced cells that had been altematively labeled with 14C or 32P. We also measured filtering rates by using separate 14C-labeled algal treatments. Contrary to Hessen and van Donk (1993), we did not detect differences in the uptake of unicells and colonies. With our present knowledge, we can only speculate on the reasons for this discrepancy. Daphnia is a rather nonselective filter-feeder but has an upper limit of particle sizes that it can ingest (Lampert 1987). Hessen and van Donk (1993) assumed that spined colonies of S. subspicatus were larger than the maximum size that could be ingested by a 1.75-mm daphniid. Our strain of S. acutus does not have spines, and it may therefore be ingestible even if it forms colonies. Because the maximum size of ingestible particles depends on the size of the daphniid (Bums 1968), a reduction of feeding rates can probably be seen only in individuals that are smaller than the ones we tested. Since our Scenedesmus strain does not seem to gain an Table 5. As Table 4, but from Exp. E. Overall ANO- VA: J qo = 80.2; P < Mean Particle Homogeneous Treatment volume groups Negative control 135.0(2.1) * Positive control 229.0(6.1) * Dried 208.3(6.2) * Incinerated 141.3(3.5) * C18, methanol eluate 149.7(4.8) * advantage from colony formation, we can assume that the colony-inducing factor evokes a general physiological response independent of actual cell size or degree of spine formation. If colony formation is only phenotypically induced when a chemical signals the presence of grazers, one might expect costs associated with colonial growth (Dodson 1989). These costs do not seem to be associated with algal growth rate (any growth rate reductions must be very small), because we could not detect differences between treatments and controls for volume-specific growth rates and cell multiplication rates. The same was observed by Hessen and van Donk (1993). However, large colonies must have higher sinking rates (Reynolds 1984). It is interesting that Trainor (1993a) found colony formation at cold temperatures and unicells at warm temperatures. Due to the higher viscosity of the water, the size effect on the sinking rate may be tolerable at low but not at high temperatures. The tradeoff between sinking and grazer resistance would favor a direct phenotypic response to the presence of grazers, as would be expected in a variable environment (Schlichting 1989). In warm water, when sinking losses are important, colonies will be formed only when mortality through grazing is high. Until now, grazer-induced colony formation has been demonstrated only in the laboratory. To prove its adaptive value, the mechanism must be demonstrated in the field. The minimum concentration of the colony-inducing factor found to be chemically active in our experiments was equivalent to a Daphnia biomass of -0.5 mg dry wt liter-l. Such biomasses are not uncommon during times of heavy grazing (Lampert 1988). The critical concentration in the field may even be lower when algae have more than 48 h to react. Hence, it is likely that the mechanism works

8 1550 Lampert et al. under natural conditions. Careful analysis of phytoplankton and zooplankton field samples and transplantation experiments may provide more evidence. Trainor (1992) cited several transplantation experiments that resulted in phenotypic changes in Scenedesmus. For example, unicells of S. armatus formed colonies after 10 d of incubation in the field according to Trainor and Roskosky (1967). At that time, they did not suspect the presence of a chemical signal in the field, but carefully designed experiments can now test the hypothesis, especially if the substance can be identified. The phenotypic response of Scenedesmus is the first experimental proof that there is a chemical signal from grazers that induces morphological changes in phytoplankton, although it has been suspected (Lynch 1980). Other phytoplankton may have similar responses. Further studies will show whether the response is unique to chlorococcal algae (or even Scenedesmus) due to their special development or whether it is typical for other algae as well. References BLACK, R., AND L. SLOBODKIN What is cyclomorphosis? Freshwater Biol. 18: BURNS, C. W The relationship between body-size of filter-feeding Cladocera and the maximum size of particles ingested. Limnol. Oceanogr. 13: DODSON, S Predator-induced reaction norms. Bioscience 39: EGAN, P. F., AND F. R. TRAINOR The role of unicells in the polymorphic Scenedesmus urmutus (Chlorophyceae). J. Phycol. 25: HESSEN, D. O., AND E. VAN DONK Morphological changes in Scenedesmus induced by substances released from Daphnia. Arch. Hydrobiol. 127: LAMPERT, W Feeding and nutrition in Daphnia. Mem. 1st. Ital. Idrobiol. 45: The relationship between zooplankton biomass and grazing: A review. Limnologica 19: 1 l , R. D. SCHMITT, AND P. MUCK Vertical migration of freshwater zooplankton: Test of some hypotheses predicting a metabolic advantage. Bull. Mar. Sci. 43: AND B. E. TA~OR Zooplankton grazing in a eutrophic lake: Implications of vertical migration. Ecology 66: LARSSON, P., AND S. DODSON Chemical communication in planktonic animals. Arch. Hydrobiol. 129: LYNCH, M Aphunizomenon blooms: Alternate control and cultivation by Daphnia pulex. Am. Sot. Limnol. Oceanogr. Spec. Symp. 3: New England. MILLER, H Wachstum und Phosphatbedarf von Nitzschia uctinustroides (Lemm.) v. Goor in statischer und homokontinuierlicher Kultur unter Phosphatlimitierung. Arch. Hydrobiol. Suppl. 38, p REYNOLDS, C. S The ecology of freshwater phytoplankton. Cambridge. S~HLI~HTKNG, C. D Phenotypic integration and environmental change. Bioscience 39: TRAINOR, F. R Discovering the various eco- morphs of Scenedesmus. The end of a taxonomic era. Arch. Protistenkd. 139: 125-l Cyclomorphosis in Scenedesmus urmutus (Chlorophyta): An ordered sequence of ecomorph development. J. Phycol. 28: ~. Cyclomorphosis in Scenedesmus subspicutus (Chlorococcales, Chlorophyta): Stimulation of colony development at low temperature. Phycologia 32: Cyclomorphosis in Scenedesmus subspicutus R. Chod. Cell behavior during the unicellcolony transformation of a phenotypically plastic organism. Arch. Protistenkd. 143: , AND F. ROSKOSKY Spine pattern in several clones of a Scenedesmus. Trans. Am. Microsc. Sot. 86: VANDENHOEK,C.,D.G.MANN,ANDH. M. JAHNS Algae. Cambridge. WOOD, A. M., AND T. LEATHAM The species concept in phytoplankton ecology. J. Phycol. 28: Submitted: 31 January 1994 Accepted: 19 April 1994 Amended: 25 May 1994