THE CELL CYCLE OF SYMBIOTIC I. THE RELATIONSHIP BETWEEN HOST FEEDING AND ALGAL CELL GROWTH AND DIVISION

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1 J. Cell Sci. 77, (1985) 225 Printed in Great Britain Company of Biologists Limited 1985 THE CELL CYCLE OF SYMBIOTIC CHLORELLA I. THE RELATIONSHIP BETWEEN HOST FEEDING AND ALGAL CELL GROWTH AND DIVISION P. J. McAULEY* Department of Life Sciences, University of California at Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90024, U.SA. SUMMARY When green hydra were starved, cell division of the symbiotic algae within their digestive cells was inhibited, but algal cell growth, measured as increase in either mean volume or protein content per cell, was not. Therefore, control of algal division by the host digestive cells must be effected by direct inhibition of algal mitosis rather than by controlling algal cell growth. The number of algae per digestive cell increased slightly during starvation, eventually reaching a new stable level. A number of experiments demonstrated that although there was a relationship between host cell and algal mitosis, this was not causal: the apparent entrainment of algal mitosis to that of the host cells could be disrupted. Thus, there was a delay in algal but not host cell mitosis when hydra were fed after prolonged starvation, and algae repopulated starved hydra with lower than normal numbers of algae (reinfected aposymbionts or hydra transferred to light after growth in continuous darkness). Two experiments demonstrated a direct stimulation of algal cell division by host feeding. Relationships of algal and host cell mitosis to numbers olartemia digested per hydra were different, and in hydra fed extracted Anemia algal, but not host cell, mitosis was reduced in comparison to that in control hydra fed live shrimp. It is proposed that algal division may be dependent on a division factor, derived from host digestion of prey, whose supply is controlled by the host cells. Numbers of algae per cell would be regulated by competition for division factor, except at host cell mitosis, when the algae may have temporarily uncontrolled access to host pools of division factor. The identity of the division factor is not known, but presumably is a metabolite needed by both host cells and algae. INTRODUCTION The Chlorella algae symbiotic with green hydra are enclosed by individual vacuoles within the host endothelial digestive cells, and the cell division of the symbiotic algae appears to be closely associated with that of the host cell. Stimulation of host cell division by feeding, or in excised regenerating peduncles or heads, also stimulates algal division; conversely, algal and host cell division decrease when hydra are starved (McAuley, 1981a, 1982). The entrainment of algal cell division to that of the digestive cells has obvious implications regarding the problem of how digestive cells regulate numbers of algae to a constant level in constant conditions (Pardy & Muscatine, 1973; Pardy, 1974a,b; Muscatine & Pool, 1979; McAuley, 1981a,6; Douglas & Smith 1984), but the way in which algal division is stimulated or inhibited is not known. At Present address: Department of Agricultural Science, University of Oxford, Parks Road, Oxford OX1 3PF, U.K. Key words: Chlorella, green hydra, symbiosis, mitosis, cell cycle, division factor.

2 226 P. J. McAuley present, three different mechanisms have been proposed, although none is supported by direct evidence. Firstly, algal division may be controlled by a density-dependent inhibitor, either one whose production ceases when the host cell divides (McAuley, 1982), or one produced at the hypostome, since in certain circumstances excision of the head causes an increase in numbers of algae in digestive cells of the remaining gastric region or peduncle, and grafting heads to excised peduncles prevents this increase (Pardy & Heacox, 1976; Bossert & Slobodkin, 1983). However, there is no direct evidence for an inhibitor, and the observation that the numbers of algae per cell increase in excised regenerating heads as well as in excised peduncles (McAuley, 1981a) is difficult to reconcile with the hypothesis that the hypostome is the source of an inhibitor of algal division. Secondly, enrichment of hydra culture medium with inorganic nutrients (M + N solution) causes overgrowth of the host by the symbiotic algae in the Florida strain of green hydra, and it has been postulated that algal division may be regulated by availability of these nutrients within the host cell (Muscatine & Necklemann, 1981: Necklemann & Muscatine, 1983). However, supply of sulphate (Cook, 1976, 1980) and phosphate (Wilkerson, 1980) to the symbiotic algae does not appear to be limited by the host cells and the effects of addition of any single inorganic nutrient are difficult to reconcile with the effects of complete M + N. For instance, by itself sulphate causes an increase in numbers of algae per cell, but overgrowth of hydra by the algae is faster in M + N without sulphate than in complete M + N (Muscatine & Necklemann, 1981). Finally, it has been suggested that host cells regulate cell growth of symbiotic algae so that algal division is prevented. Pardy (1981) has shown that in a non-dividing population of symbiotic algae, in starved hydra, the mean cell size of the algae does not significantly increase over 5 days. Low ph causes release of large amounts of maltose by the algae (Cernichiari, Muscatine & Smith, 1969; Mews, 1980; Mews & Smith, 1982), and Douglas & Smith (1984) proposed that host cells may regulate the ph of the perialgal vacuole to a low level, diverting photosynthetically fixed carbon from cell growth. During host cell division vacuolar ph would rise, possibly because of alkalinization of host cytoplasm (Nuccitelli & Deamer, 1982), and algal maltose release would cease. Fixed carbon would become available for growth of algal cells and they would proceed through the cell cycle. However, no direct measurements of vacuolar ph have been made, and Douglas & Smith noted an increase in algal cell diameter during host starvation, contradicting the observation of Pardy (1981). Lack of evidence for any particular model of control of algal cell division has prompted a more rigorous examination in this paper of the way in which algal cell growth and division may be affected by host feeding, since this parameter can be varied in a number of ways and is known to be associated with an increase in algal mitosis (McAuley, 1982). Evidence that host feeding may directly stimulate algal cell division, perhaps through supply of metabolites that are otherwise limiting, is described and discussed.

3 MATERIALS AND METHODS Maintenance of Hydra Cell cycle of Chlorella in Hydra 227 Stock cultures of green and aposy mbiotic (from which the symbiotic algae had been removed by the photobleaching method of Pardy (1976)) Hydra viridissima of the European strain were grown in M solution (Muscatine & Lenhoff, 1965) at 18 C in an illuminated incubator. Mean irradiance was 2-0 X 10~ 5 Einsteins m"^" 1 ; photoperiod was 12 h light/12 h dark. Cultures were fed with freshly hatched nauplii of AftCTnjasa/ina(Loomis&Lenhoff, 1956) on each Monday, Wednesday and Friday. Estimation of numbers of algae per digestive cell Individual gastric regions of hydra (Pardy & Muscatine, 1973) were dissociated into cell suspensions by the maceration technique of David (1973) and examined by X400 interference contrast microscopy. Numbers of algae were counted in 30 randomly selected digestive cells. Estimation of algal cell volume Five hydra or gastric regions of hydra were homogenized in a glass microtissue homogenizer and the resulting suspension of algae was examined using X1000 interference contrast microscopy. Diameters of 50 randomly selected cells were measured using an occular micrometer. Volumes were computed from diameters by assuming the algae to be perfect spheres. Isolation of symbiotic algae Symbiotic algae were isolated from green hydra by homogenization of animals in a glass microtissue homogenizer at 4 C. Contaminating animal material was removed from the resulting suspension of intact algae by washing with 0-05% sodium dodecyl sulphate according to the protocol described by McAuley (1985). Estimation of algal protein and chlorophyll content To determine protein content of algal cells, samples were freeze-thawed and incubated with an equal volume of 0'4 M-NaOH for 1 h before protein was measured by the Lowry colormetric method (Lowry, Rosebrough, Farr & Randall, 1951). Bovine serum albumin (Fraction V, Sigma Chemical Co., St Louis, Miss.) was used as a standard. Chlorophyll content of algal cells was determined by extracting cells with 90% (v/v) methanol at 4 C in darkness for 24h and, after centrifugation, measuring absorbance of the supernatant at 650 nm and 665 nm. The amount of chlorophyll was determined from these measurements by using the equations of Holden (1965). Determination of algal and digestive cell mitotic indices Numbers of dividing algae or digestive cells per 1000 cells in homogenates or macerates of five gastric regions of hydra were determined as described by McAuley (1982). Fluorescent staining of algal nucleii Algae were collected by centrifugation of homogenates of hydra and extracted overnight in 90 % methanol. Fixed, extracted cells brought to distilled water through a methanol series were suspended in % acridine orange (Allied Chemical Co., New York) in 0-01 M-EDTA. Stained cells were examined directly by X1000 epifluorescence optics and 1000 cells were scored for the number of nucleii they contained. Algae pretreated with DNase showed little or no staining with acridine orange. RESULTS Effect of host starvation on algal cell growth and division When European green hydra were starved in the light, both the number of algae per

4 228 P. J. McAuley 120 r u 80 I 70 <- j i i i i 24 r- 22 I ' a o 18 d Days starved Fig. 1. A. Mean cell volume of algae in starved ( ) and fed (O O) hydra. Mean volumes were calculated from measurements of diameters of 50 algae in homogenates of gastric regions of five hydra. Each point represents the amalgamated mean ± S.E.M. of duplicate experiments. B. Number of algae per digestive cell in starved ( ) and fed (O O) hydra. Numbers of algae were counted in 30 digestive cells in each of three macerates of individual gastric regions. Each point represents the amalgamated mean ± S.E.M. of duplicate experiments. digestive cell and the mean volume of the algal cells increased (Fig. 1A,B). Douglas & Smith (1984) have also found that the number of algae per cell increased when European hydra were starved and a similar increase has been observed in starved Florida (Pool, 1976; Muscatine & Necklemann, 1981) but not in starved Jubilee (McAuley, 19816) green hydra. The increase measured here reached a stable level after about 10 days. Since most of the symbiotic algae divide into four daughter cells (Oschman, 1967) this increase represented division of about 10 % of the algal population above that associated with digestive cell division.

5 Cell cycle of Chlorella in Hydra 229 Mean algal cell volume increased over more or less the entire period of measurement, from about 85 /im 3 to 110/im 3 in 21 days. Douglas & Smith (1984) measured an increase of 10/im 3 in algal cell volume over a 10-day period of starvation. Increase in cell size was confirmed by measurements of protein content of algal cells from 1-day and 22-day starved hydra (Table 1), although there was no difference in chlorophyll content. In 22-day starved hydra, a greater proportion of the algae were within the size range of dividing algae (Fig. 2); that is, /im diameter (McAuley, unpublished results). However, measurements of the number of multinucleate algal cells in fed and starved hydra (Fig. 3) showed a fall in algal nuclear division within the first week of host starvation, and it remained at a low level thereafter, confirming previous observations that host starvation reduces the frequency of both digestive cell and algal mitosis (McAuley, 1982). Thus, although the algal cells were apparently able to synthesize protein and slowly increase in size during host starvation, nuclear and hence cell division was inhibited. The increase in number of algae per cell in starved hydra may have been due to a slight lag in the decrease in algal division with respect to that of the digestive cells. Table 1. Size, and protein and chlorophyll content of algae from European hydra starved for I or 22 days Days starved Algal volume (/*m 3 ) pg chlorophyll/alga pg protein/alga pg protein/nm ± 6-51* 1 69 ± 0-lot ±1-82f ± 4-36* 1 64 ±0-21f ±3-39f Values are means ± S.E.M. Diameters of 50 algae were measured in each of two homogenates of five hydra. fsix replicate samples of algae isolated from homogenates of hydra. Effect of host feeding on algal cell growth and division The following experiments describe situations in which algal mitosis is uncoupled from that of the host cell, and show that it may be directly stimulated by host feeding. Effect of feeding after prolonged starvation. Hydra starved for long periods contain more algae per cell than those cultured normally. To investigate whether these elevated numbers of algae were regulated to normal levels when starved hydra were fed, numbers of algae per digestive cell were determined in 10-day and 3-day (controls with normal numbers of algae per cell) starved hydra after a single feeding. The results (Fig. 4) appeared ambiguous, for although numbers of algae per cell in 10-day starved hydra fell below those of controls immediately after feeding, they showed a greater increase than in controls during the subsequent period of starvation. This was investigated further by measuring algal and digestive cell mitotic indices in 3-day and 10-day starved hydra immediately before and after a single feeding. Mitosis

6 230 P. jf. McAuley z 15 j 10, J I r J i Cell diameter (^m) Fig. 2. Distribution of algal cell diameters in 1-day ( ) and 22-day ( ) hydra; 50 cell diameters were measured in each of six replicate homogenates of gastric regions of five hydra. Bar indicates size of dividing algae. of algae and digestive cells increased approximately in step when 3-day starved controls were fed (Fig. 5), confirming previous results (McAuley, 1982). However, in 10-day starved hydra, although digestive cell mitosis increased normally after feeding, algal mitosis was delayed and then showed a peak value about twice as high as that of controls. These results suggested that the initial fall and subsequent increase in numbers of algae per cell after 10-day starved hydra were fed was due to uncoupling of the normally close association between digestive cell and algal mitosis. Effect of feeding on repopulation of digestive cells by algae. In aposymbiotic hydra recently reinfected with native algae, and in hydra grown in continuous darkness, numbers of algae per digestive cell are considerably lower than those in hydra from stock cultures (McAuley, 19816; McAuley & Smith, 1982). During repopulation of the digestive cells of these hydra (after infection or upon transfer to light), algal mitosis must be uncoupled from that of host cells to some extent. If host feeding directly stimulates algal division, then repopulation would proceed more quickly in fed than in starved hosts. To test this, aposymbionts were fed or starved after being injected with algae isolated from green hydra and numbers of algae per cell were determined (Fig. 6). There was a similar increase in numbers of algae per cell in both fed and starved reinfected aposymbionts during the first week, but thereafter there was a greater increase in numbers in fed aposymbionts. Since this experiment did not take into account the effect of dilution by host cell division, which would have been higher in fed than in starved hydra (McAuley, 1982), the actual stimulation of algal mitosis by

7 Cell cycle of Chlorella in Hydra 231 _ >. Days starved Fig. 3. Percentage of algae with two nucleii (A) and four nucleii (B) in starved ( ) and fed (O O) hydra. Each pointed represents the mean of duplicate experiments in which 1000 fixed, stained algae were scored for number of nucleii. 26 r 24 S 22.S? ' B Days after feeding 10 Fig. 4. Numbers of algae per digestive cell after 3-day (O O) and 10-day ( ) starved hydra were fed once on day 0. Each point is the amalgamated mean ± S.E.M. of duplicate experiments in which numbers of algae were counted in 30 digestive cells in each of three macerates of individual gastric regions.

8 232 P. J. McAuley Table 2. Effect of feeding on increase in algal number, algal cell size and chloropyll content of algae upon transition from continuous darkness to light Day 5 DayO Fed once Starved (1) Number of algae/digestive cell ± ± ±0-53 (2) Algal cell volume (/an 3 ) ± ±3-59 (3) Chlorophyll/algal cell (pg) 0-98 ± ± ± 011 (4) Number of algae/hydranth 7-55 ± ± ±0-79 (X 10" 4 ) All values are means ± S.E.M. (1) From six replicate macerates of gastric regions, in which numbers of algae were counted in 30 digestive cells. (2) From 50 measurements of algal cell diameter in each of two replicate homogenates of five standard hydra. (3) and (4) From six replicates, each of algae from 10 standard hydra. host feeding may have been higher than that measured simply as increase in numbers of algae per cell. In an attempt to take into account the diluting effect of host cell division, an experiment was devised to measure the effect of feeding or starvation on increase in numbers of algae after transfer of dark-grown hydra to the light. Numbers of algae per cell and numbers of algae per hydranth were measured in 'standard' hydra (each bearing one advanced bud) selected from cultures. Changes in algal cell volume and in chlorophyll content were also measured. The results (Table 2) showed that numbers of algae per cell increased in both fed and starved hydra. Increase in algal cell size (smaller in darkness than in light) was significantly greater in fed than in starved hydra, but algae from starved hydra contained more chlorophyll per cell. From these values alone, it is difficult to discern a general stimulatory effect of feeding on either cell growth or cell division of algae. However, when increase in numbers of algae per hydranth was measured, it was found to be very much greater in fed than in starved hydra. By multiplying mean algal cell volume and chlorophyll content by numbers of algae per hydranth, and comparing these values in fed and starved hydra, it was found that there was a greater increase in volume of algae and amount of chlorophyll per hydranth in fed hydra (Table 3). Direct observation of stimulation of algal division by host feeding. Since feeding stimulates digestive cell division (David & Campbell, 1972; McAuley, 1982), its apparent effects on algal cell growth and division may be due to indirect causes. For instance, algal cell division may be suppressed in non-dividing but not in dividing host cells. Accordingly, experiments were devised in which direct stimulation of algal mitosis by host feeding could be measured. Batches of hydra were fed different numbers of Artemia and mitosis of algae and digestive cells was measured after 24 h. If algal mitosis was dependent upon that of the digestive cells rather than on the amount of food (number of Artemia) available, then the relationships of algal and digestive cell mitotic indices to number of Artemia consumed should be identical. Mitotic indices of digestive cells and algae rose in

9 Cell cycle of Chlorella in Hydra r S 3-0 o Hours after feeding Fig. 5. Effect of a single feeding (on day 0) on digestive cell (A) and algal (B) mitosis in 3-day (O O) and 10-day ( ) starved hydra. Each point is the mean of duplicate experiments. parallel between 0-4 Artemia consumed (Fig. 7), but thereafter the algal mitotic index continued to rise linearly although that of digestive cells did not. The above experiment suggested that division of algae may be dependent, at least in part, upon substances derived from digestion of Artemia. To investigate this possibility, Artemia crudely extracted by heating at 70 C distilled water for 20min

10 234 P. J. McAuley 14 r 12 5 io "S 3 Z 8 11 Days after reinfection Fig. 6. Numbers of algae per digestive cell in aposymbiotic hydra starved (O O) or fed ( #) after reinfection with native algae. Each point is the amalgamated mean ± S.E.M. of duplicate experiments in which numbers of algae were counted in 30 digestive cells in each of three macerates of individual gastic regions. Table 3. Increase in total volume of algae and total chlorophyll content per hydranth Day 5 Day 0 Fed once Starved Algal volume/hydranth (/an 3 ) Chlorophyll/hydranth (pg) 3-48 X SX X X X x 10 6 Values derived from measurements (2), (3) and (4) in Table 2. (which removed about 50 % of the protein content) were fed to hydra, and algal and digestive cell mitosis was determined 24 h later (Table 4). Algal and digestive cell mitotic indices were higher in hydra fed either live or extracted Anemia than in starved controls. However, algal but not digestive cell mitosis was reduced in hydra fed extracted Artemia compared with those fed live Artemia. David & Campbell (1972) suggested that division of epithelial cells (including

11 Cell cycle of Chlorella in Hydra Number of Anemia digested per hydra Fig. 7. Effect of number of Artemia digested on the mitotic index of algae ( ) and digestive cells (O O) 24 h after capture of prey. Mitotic index was determined by scoring dividing forms per 1000 cells in macerates of five gastric regions. Each point represents a single determination. Table 4. Effect of feeding extracted Artemia on digestive cell and algal mitosis Mitotic index (%) Starved controls Fed 2 live Artemia Fed 2 extracted Artemia Algae 0-32 ± ± ±0-25 Digestive cells 0-62± ± ±0-41 Hydra previously starved for 3 days were fed 2 heated or live Artemia or were not fed (controls). Mitotic indices were measured after 24 h in five gastric regions per sample. Values are means ± S.E.M. of five replicate experiments. digestive cells) after feeding may be due to a response to a feeding stimulus, such as stretching of tissue by food in the gastric cavity, rather than uptake of metabolites, as epithelial cells spend most of their cycle in Gi. This may explain why digestive cell mitosis was unaffected when hydra were fed extracted Artemia, while algal mitosis may have been reduced because extraction removed a substance needed by the algae for cell division. Alternatively, extraction may have reduced the amount of a substance needed by both algae and digestive cells for mitosis, which the digestive cells were able to use more efficiently than the algae. DISCUSSION The cell cycle may be divided into a period of cell growth and one of nuclear and cell division (Mitchison, 1971). There is considerable evidence that timing of DNA synthesis and mitosis may be determined, at least in part, by growth of the cell in G\,

12 236 P.J.McAuley Prey host Pool of,.. division factor digestion 1 > Host cell metabolism I 1 I hostce " mitosis excess I I I > Algal metabolism-j Fig. 8. Suggested model of control of algal division. since microorganisms cannot proceed into S phase until a critical cell size is reached (Fantesef al. 1975; Mitchison, 1977; Nurse, 1980; Fantes & Nurse, 1981). While it has been suggested that division of symbiotic algae in digestive cells of green hydra may be controlled by inhibition of algal cell growth (Pardy, 1981; Douglas & Smith, 1984), experiments described in this paper showed that in light algal cell growth was independent of host cell division. In starved hydra, in which algal and host cell division is almost completely inhibited (McAuley, 1982), cell growth of algae was measurable but proceeded at a very slow rate, measured here and by Douglas & Smith (1984) as a mean increase in volume of approximately 1 /im 3 per cell day. The suggestion of Pardy (1981) that cell growth of symbiotic algae 'grinds to a halt' in starved hydra was based on a period of measurement (5 days) that was too short to show any significant increase in algal cell size. The slow growth of the algal cells may have been due to export of a large proportion of photosynthetically fixed carbon as maltose, but this is probably not the mechanism by which host cells inhibit algal division. During host starvation algae accumulate at sizes at which they normally divide, but nuclear and cell division does not take place. Therefore, algal division does not appear to be controlled by host cells preventing algae from reaching the critical size necessary for division, but by inhibition applied to some part of the cell cycle before nuclear division. A number of experiments suggested that host feeding may directly stimulate algal mitosis, but the simple idea that mitosis was initiated by a substance or substances directly produced by host digestion of prey was not supported by observations that numbers of algae could increase in digestive cells of starved hydra (Pool, 1976; Douglas & Smith, 1984; Fig. 1B, this paper) and in dividing digestive cells of excised, unfed, regenerating heads or peduncles (McAuley, 1981a, 1982). To explain these results, it is proposed that host cells may regulate access by algae to a pool or pools of metabolite(s) needed for cell division (division factor). This model of regulation is represented schematically in Fig. 8. Normally, only a limited amount of division factor reaches the algae via host cell metabolism (broken line). During host cell mitosis, however, the algae may have temporarily uncontrolled access to the pool of division factor (continuous line), which would explain the increase in algal division when host cell division is stimulated (McAuley, 1981a, 1982). At present, the

13 Cell cycle of Chlorella in Hydra 237 mechanism by which host cells control supply of division factor must remain speculative, but of possible relevance is the demonstration of a low molecular weight protein in the host fraction of the Cassiopea xamac/tana/zooxanthellae symbiosis, which inhibits uptake of alanine by the symbiotic algae (Carroll & Blanquet, 1984). Synthesis of a similar inhibitor in hydra cells would presumably be interrupted by cell division. The problem of why only a proportion of algae divide at host cell division may be explained by competition between algae for division factor. Since the pool size must be finite, algal division would become density-dependent after the algal population reached a certain size. In digestive cells with a depleted complement of algae, the algae would divide until the level imposed by competition was reached; in starved hydra the pool of division factor (replenished by host feeding) would decline, so that algal division and hence increase in algal numbers woud not continue indefinitely. The lag in algal mitosis observed after feeding 10-day starved hydra (in which pools of division factor would have become depleted) may represent the time needed to refill the pool, and algal mitosis was lower when supply was reduced by feeding hydra with extracted Artemia. This model agrees with the suggestion of Reisser, Meier & Kurmeier (1983) that the constant size of algal population observed in symbiotic associations grown in constant conditions is a result of an equilibrium reached between algal cell growth and division, and host cell growth and division, rather than limitation of the algal population to a specific proportion of the host cell volume. Changes in environmental parameters that would affect the components of this equilibrium, such as photoperiod or temperature, have been shown to cause changes in numbers of algae in hydra digestive cells (Pardy, 1974a; McAuley, 1980), while in the same environmental conditions different strains of symbiotic algae achieve different stable population levels in host cells (Mews & Smith, 1982; Reisser et al. 1983; Douglas & Smith, 1984). There is no evidence for the identity of the division factor. It need not be a complex compound and may simply be an intermediary metabolite used by the symbiotic algae and/or the host cell. Cook (1972) and Thorington & Margulis (1981) showed that substances derived from host digestive processes, including amino acids and nucleotide precursors, may pass into algal metabolic pools. Analysis of extracts of Artemia may provide indirect evidence about the nature of the division factor, as may comparison of the metabolic requirements of native Chlorella with those that infect hydra but appear to be regulated by ejection rather than suppression of division, such as NC64A (Douglas & Smith, 1984) and Fs (Rahat & Reich, 1984) strains. I am grateful to Professor L. Muscatine for providing support and encouragement at all stages of this work, and to Professor D. C. Smith, F.R.S. and Dr A. Douglas for suggesting improvements to drafts of this paper. This work was supported by a NATO/SERC Postdoctoral Fellowship. REFERENCES BOSSERT, P. & SLOBODKIN, L. B. (1983). The effect of fast, and regeneration in light vs dark, on regulation in the hydra-algal symbiosis. Biol. Bull. mar. biol. Lab., Woods Hole, 154,

14 238 P. J. McAuley CARROLL, S. & BLANQUET, R. S. (1984). Alanine uptake by isolated zooxanthellae of the mangrove jellyfish, Cassiopea xamachana. II. Inhibition by host homogenate fraction. Biol.Bull. mar. biol. Lab., Woods Hole, 166, CERNICHIARI, E., MUSCATINE, L. & SMITH, D. C. (1969). Maltose excretion by the symbiotic algae of Hydra viridis. Proc. R. Soc. B, 173, COOK, C. B. (1972). Benefit to symbiotic zooxanthellae from feeding by green hydra. Biol. Bull. mar. biol. Lab., Woods Hole, 142, COOK, C. B. (1976). Sulphate utilisation in green hydra. In Coelenteraie Ecology and Behaviour (ed. G. O. Mackie), pp London: Plenum Press. COOK, C. B. (1980). Sulfur metabolism in the green hydra symbiosis: The incorporation of sulfatesulfur by symbiotic and aposymbiotic Hydra viridis. In Endocytobiology (ed. W. Schwemmler & H. E. A. Schenk), pp Berlin, New York: Walter de Gruyter. DAVID, C. N. (1973). A quantitative method for maceration of hydra tissue. Wilhelm Roux Arch. EntviMech. Org. 171, DAVID, C. N. & CAMPBELL, D. (1972). Cell cycle kinetics and development of Hydra attenuata. I. Epithelial cells. J. Cell Sd. 11, DOUGLAS, A. & SMITH, D. C. (1984). The green hydra symbiosis. VIII. Mechanisms in symbiont regulation. Proc. R. Soc. Land. B, 221, FANTES, P. A., GRANT, W. D., PWTCHARD, R. H., SUDBERY, P. E. & WHEALS, A. E. (1975). The regulation of cell size and the control of mitosis. J. theor. Biol. 50, FANTES, P. A. & NURSE, P. (1981). Division timing: controls, models and mechanisms. inthecell Cycle (ed. P. C. L. John), pp Cambridge University Press. HOLDEN, M. (1965). Chlorophylls. In Chemistry and Biochemistry of Plant Pigments (ed. T. W. Goodwin), pp London, New York: Academic Press. LOOMIS, W. F. & LENHOFF, H. M. (1956). Growth and sexual differentiation of Hydra in mass culture. J. exp. Zool. 132, LOWRY, 0. H., ROSEBROUGH, N. J. FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Ckem. 193, MCAULEY, P. J. (1980). Variation and regulation in the green hydra symbiosis. Ph.D. thesis, University of Bristol. MCAULEY, P. J. (1981a). Control of cell division of the intracellular Chlorella symbionts in green hydra. J. Cell Sd. 47, MCAULEY, P. J. (19816). Ejection of algae in the green hydra symbiosis. J. exp. Zool. 127, MCAULEY, P. J. (1982). Temporal relationships of host cell and algal mitosis in the green hydra symbiosis. J. Cell Sd. 58, MCAULEY, P. J. (1985). Isolation of viable uncontaminated symbiotic Chlorella from green hydra. Limnol. Oceanogr. (in press). MCAULEY, P. J. & SMITH, D. C. (1982). The green hydra symbiosis. V. Stages in the intracellular recognition of algal symbionts by digestive cells. Proc. R. Soc. Lond. B, 216, MEWS, L. K. (1980). The green hydra symbiosis. III. The biotrophic transport of carbohydrate from alga to animal. Proc. R. Soc. Lond. B, 209, MEWS, L. K. & SMITH, D. C. (1982). The green hydra symbiosis. VI. What is the role of maltose transfer from alga to animal? Proc. R. Soc. Lond. B, 216, MITCHISON, J. M. (1971). The Biology of the Cell Cycle. Cambridge University Press. MITCHISON, J. M. (1977). The timing of cell cycle events. InMitosis: Facts and Questions (ed. M. Little, N. Paweletc, C. Petcelt, M. Ponstingl, D. Schroeter & H.-P. Zimmermann), pp Berlin, Heidelberg, New York: Springer-Verlag. MUSCATINE, L. & LENHOFF, H. M. (1965). Symbiosis of hydra and algae. I. Effects of some environmental cations on growth of symbiotic and aposymbiotic hydra. Biol. Bull. mar. biol. Lab., Woods Hole, 128, MUSCATINE, L. & NECKLEMANN, N. (1981). Regulation of numbers of algae in the Hydra-Chlorella symbiosis. Ber. dt. bot. Ges. 94, MUSCATINE, L. & POOL, R. (1979). Regulation of numbers of intracellular algae. Proc. R. Soc. Lond. B, 204, NECKLEMANN, N. & MUSCATINE, L. (1983). Regulatory mechanisms maintaining the Hydra-Chlorella symbiosis. Proc. R. Soc. Lond. B, 219,

15 Cell cycle of Chlorella in Hydra 239 NUCCITELLI, R. & DEAMER, D. W. (ed.)- (1982). Intracellular ph: Its Measurement, Regulation and Utilisation in Cellular Functions. Kroc Foundation Series, no. 15. New York: Alan R. Lisa, Inc. NURSE, P. (1980). Cell cycle control - both deterministic and probabilistic? Nature, Land. 286, OSCHMAN, J. L. (1967). Structure and reproduction of the algal symbionts of Hydra viridis. J. Phycol. 3, PARDY, R. L. (1974a). Some factors affecting the growth and distribution of the algal endosymbionts of Hydra viridis. Biol. Bull. mar. biol. Lab., Woods Hole, 147, PARDY, R. L. (19746). Regulation of the endosymbiotic algae in hydra by digestive cells and tissue growth. Am. Zool. 14, PARDY, R. L. (1976). The production of aposymbiotic hydra by the photodestruction of green hydra zoochlorellae. Biol. Bull. mar. biol. Lab., Woods Hole, 151, PARDY, R. L. (1981). Cell size distribution of green symbionts from Hydra viridis. Cytobios 32, PARDY, R. L. & HEACOX, A. E. (1976). Growth of algal symbionts in regenerating hydra. Nature, Lond. 260, PARDY, R. L. & MUSCATINE, L. (1973). Recognition of symbiotic algae by Hydra viridis. A quantitative study of the uptake of living algae by aposymbiotic H. viridis. Biol. Bull. mar. biol. Lab., Woods Hole, 145, POOL, R. (1976). Symbiosis of Chlorella and Chlorohydra viridissima. Ph.D. thesis, University of California. RAHAT, M. & REICH, U. (1984). Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp: Initiation of a stable symbiosis..? Cell Sci. 65, REISSER, W., MEIER, R. & KURMEIER, B. (1983). The regulation of the endosymbiotic algal population size in ciliate algae associations. An ecological model. In Endocytobiology II (ed. M. E. A. Schenk & W. Schwemmler), pp , Berlin, New York: Walter de Gruyter. THORINGTON, G. & MARGULIS, L. (1981). Hydra viridis: Transfer of metabolites between hydra and symbiotic algae. Biol. Bull mar. biol. Lab., Woods Hole, 160, WILKERSON, F. P. (1980). Symbionts involved in phosphate uptake by green hydra. In Endocytobiology (ed. W. Schwemmler&M. E. A. Schenk), pp , Berlin, New York: Walter de Gruyter. (Received 16 October Accepted, in revised form, 27 March 1985)

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