Growth of endosymbiotic algae in the green hydra, Hydra viridissima

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1 Growth of endosymbiotic algae in the green hydra, Hydra viridissima KENNETH DUNN* Department of Ecology and Evolution, State University of Sew York, Stony Iiwok, Seio York, SY 11794, USA * Present address: Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA Summary Stable endosymbiosis depends upon balanced growth of the symbionts. In green hydra, coincident patterns of host and algal mitotic index suggest that coordinated reproduction provides for balanced growth. However, when hydra shrink during food shortage, the population of endosymbiotic algae in a green hydra must likewise decline in size. Thus far, no mechanism of reducing the size of the endosymbiont population has been described. Algal mitosis was found here to be stimulated by host feeding and clumped in its distribution among host cells, supporting the notion of some degree of control of algal mitosis exercised at the level of the host cell. However, comparisons of the rates of algal mitosis with the realized rates of algal population growth show that substantial numbers of algae disappear from hydra, in numbers in excess even of those necessary to accommodate host shrinkage. Only a small proportion of these lost algae was found to be expelled by hydra. Microscopic observations of the cells of macerated hydra show evidence of algal disintegration in nearly % of the digestive epithelial cells of regularly fed hydra. Coincidence of remnants of algal cells and food-derived materials within the same vacuoles suggests that algae are digested by host cells. Key words: symbiosis, hydra, Chlorella. Introduction Coordinated symbiont growth is a requirement of any stable endosymbiosis. Such coordinated growth is found in green hydra, in which green algae live inside host digestive epithelial cells. For any given set of conditions, the average number of algae per host cell varies closely around a fixed value, typically between ten and twenty (Pardy, 1974; Muscatine & Pool, 1979; McAuley, 1981a,b; Muscatine & Neckelmann, 1981; McAuley & Smith, 1982). Hydra tissue growth is the net result of cell mitosis and cell death, primarily among the epithelial cells (Marcum & Campbell, 1978; Takano et al. 1980). The nutritional state of the hydra determines the balance of these opposing processes, so that the combined amount of tissue of a parent and its buds is dynamic, growing after a meal and shrinking during protracted intervals between meals (Kelty & Cook, 1976; Otto & Campbell, 1977; Bosch & David, 1984). As green hydra maintain a reasonably constant density of algae within their tissue, the endosymbiont population must obviously grow and shrink in harmony with the host. Journal of Cell Science 88, (1987) Printed in Great Britain The Company of Biologists Limited 1987 The potential growth rate of the endosymbiont population is much higher than that of the hydra (Jolley & Smith, 1978), but there are at least three possible ways that the host may restrain the growth of its endosymbiont population. The host may restrict algal division rates to suit host growth rate, the host may expel excess algae, or the host may digest excess algae. On the basis of the coincidence of host and algal mitotic activity (McAuley, 1981a, 1982, 1985, 1986; Bossert & Dunn, 1986), it is believed that the regulation of algal number per host cell is achieved through the inhibition of algal mitosis except during host cell division (Reisser et al. 1983; Douglas & Smith, 1984; McAuley, 1985, 1986). However, when hydra epithelial cell number decreases during periods of food limitation (Otto & Campbell, 1977; Bosch & David, 1984), the fate of the endosymbiotic algae inside those lost host cells is not clear. There is no published evidence of significant expulsion of algae by hydra under normal conditions, although hydra may be experimentally induced to expel algae (Pardy, 1976; McAuley, 19816; Muscatine & 571

2 Neckclmann, 1981; Steele & Smith, 1981; Neckelmann & Muscatine, 1983). Nor is it generally believed that hydra digest their algae (Neckelmann & Muscatine, 1983; Douglas & Smith, 1984). In natural, undisturbed green hydra, disintegrating algae have rarely been observed (Oschman, 1967; O'Brien, 1982) and lysosomal enzymes have never been found in pcrialgal vacuoles (O'Brien, 1982; McNeil & McAuley, 1984). When introduced into the gut of aposymbiotic hydra, native endosymbiotic algae are avidly phagocytosed by host digestive cells, but the algae then actively prevent the fusion of their enclosing phagosome with host lysosomes (McNeil et al. 1981; Hohman et al. 1982;"o'Brien, 1982; McNeil & McAuley, 1984). By contrast, phagosomes of inappropriate algae, dead algae and food material are fused with host lysosomes and their contents digested by host enzymes. In this study, comparisons of the patterns of mitotic index and net growth of the algal population in hydra showed that large numbers of endosymbionts were lost by hydra. The algae showed substantial rates of mitosis, even as their populations declined in size. The numbers of algae lost by these hydra thus exceeded even those amounts necessary to accommodate host shrinkage. The numbers of algae expelled by hydra were found to be negligible. Cytological evidence presented here suggests that under certain normal circumstances, hydra digest symbiotic algae. Materials and methods Experimental organisms The Carolina strain of Hydra vindissima was obtained from the Carolina Biological Supply Co. The European strain was obtained from Dr P. J. McAuley. Culture conditions Hydra stock cultures were maintained in a controlled temperature chamber at 17 C under continuous or diurnal illumination (14 h light, 10 h dark) of between 15 and 25 microe m~ 2 s~' in M solution (Muscatine & Lenhoff, 1965). They were fed to repletion every Monday, Wednesday and Friday for a period of several months before the start of each experiment. Culture dishes were rinsed 2h and 10 h after feeding, and were scrubbed once a week. Growth study experimental design The effect of feeding on the growth of host and algae was monitored by sampling identically treated groups of hydra at various times after feeding. For each experiment five samples of thirty hydra apiece were assembled randomly from a pool of standard hydra (hydra with one fully formed bud) prior to feeding. From these five samples one was then collected at each of the time points just prior to, and 12, 24, 36 and 48 h following experimental feeding. Experimental hydra were fed either one Artemia nauplius apiece (two experiments) or ad lib. (three experiments). Two control experiments were also conducted on unfed hydra. For each time point, hydra samples were homogenized in a glass tissue homogenizer at 0 C and algae separated from host tissue by three rounds of centrifugation (at 630g) and resuspension of the algal pellet in 0 C M solution. Host and algal fractions were then frozen for later analyses. According to counts of algal cells in each fraction, an average of 1-3% (±0-15%, S.E.M.) of the algae were included in the host fraction. Photosynthesis and carbon translocation data for these hydra are presented in a separate report (Dunn, unpublished). Algal growth measurement Algal growth rate was established from haemocytometer counts and from measurements of algal population mitotic index. Between four and eight haemocytometer counts were taken of both the host and the algal fractions of a particular sample observed under 400X epifluorescence. Mitotic index was quantified as the percentage incidence of tetraspores in a sample of between 1000 and 10 algal cells observed under X1000 magnification. A tetraspore was operationally defined as a cell divided into four daughter cells still enclosed in the spore mother cell wall. All samples were scored blind. Algal expulsion measurements To determine the rate of algal expulsion by unfed hydra, culture medium samples were collected along with particular experimental hydra samples. Expulsion by fed hydra was measured in separate duplicate experiments in which the medium surrounding a single set of experimentally fed hydra was collected every 12 h. Sample cell densities were maximized either by minimizing the amount of medium surrounding hydra or by concentrating the algae of the medium sample into a minimal volume through centrifugation at 630 g\ Equivalent results were obtained by either method. Numbers of algae in these medium samples were then established from four to eight haemocytometer counts. Medium samples included vigorous rinses of the hydra and thus the counts included any free, but adherent algae. The medium sample counts were assumed to include more than 95 % of the intact algae expelled by the hydra, since in a separate study spontaneous lysis of algae in M solution was found to be less than 5 % in 12 h, the maximum amount of time any expelled algal cell would be exposed to the medium in all but one time point in the expulsion experiments. Hydra digestive cell observations Hydra were dissociated by the maceration method of David (1973) into a suspension of intact single cells. This suspension was placed on a slide, dried, mounted and observed under phase-contrast and epifluorescent microscopy. Photomicrographs were made using a Zeiss phase/epifluorescent microscope with filters for fluorescein fluorescence at X970 magnification. Protein determination Triplicate protein determinations were made of host and algal fractions by the method of Lowry et al. (1951) using bovine serum albumin as a standard. 572 K. Dunn

3 Statistical analyses Statistical tests were conducted as described by Sokal & Rohlf (1981). Results Regulation of algal number by restriction of algal mitosis The endosymbionts show a baseline level of mitotic activity in the absence of food that increases shortly after host feeding (Fig. 1). It has previously been shown that this increase corresponds to an increase in host mitosis that results in host growth following feeding (McAuley, 1982, 1985, 1986; Bossert & Dunn, 1986). If algal mitosis is keyed by certain events in the cell cycle of the surrounding host cell, one would expect to find that algal tetraspores are not randomly distributed among host cells, but rather clustered in particular host cells that 'permit' algal mitosis. In fact, the distribution of tetraspores in a sample of 200 host cells differed significantly from a random Poisson expectation: a tetraspore is more likely to be found in a cell containing another tetraspore than chance would predict Hours after feeding Fig. 2. Mean numbers of algae (±S.E.M.) per hydra counted within hydra tissue over time after feeding (open symbols) and cumulative mean numbers of algae per hydra collected from hydra culture medium over time after feeding (filled symbols). ( ) fed ad lib.; (A) fed one Artemia; (<^>) unfed. 48 Hours after feeding Fig. 1. Mean percentage (±S.E.M.) of algal cells observed to be in the tetraspore stage as a function of time after feeding. ( ) fed ad lib.; (A) fed one Anemia; (O) unfed. (P<0-025, C-test). To the extent that algal mitosis is regulated by the host, it seems to be regulated at the level of the host cell. When one compares the actual growth rates of the algal populations (Fig. 2) with their corresponding mitotic rates, however, one finds that mitotic control is not sufficient to explain the dynamics of the algal population within the host tissue. In hydra fed a single Artemia, the endosymbiont population declined by around 10% during the two days following feeding. The algae of hydra fed ad lib., however, increased in number by more than 19% during that time. Host protein likewise decreased by 11 % after a meal of one Artemia and grew by 28% when fed ad lib. (Fig. 3). The growing algal population, however, had an average mitotic index that was no higher than that of the declining algal population (1-7 ±0-4% vs. 19 ±0-2%, respectively). Furthermore, intervals of especially high growth do not correlate with intervals of especially high mitotic index. A serious problem for a model of algal regulation strictly through mitotic restriction is that it does not Groivth of algae in green hydra 573

4 the 48 h following a meal of a single Artemia and less than 2% of the average number of algae lost by hydra between 12 and 24 h following ad lib. feeding. Note that these are percentages of the net losses of algae, ignoring algal reproduction, and so are upper limit estimates of the significance of expulsion. Hours after feeding Fig. 3. Mean protein contents (±S.E.M.) per hydra of host fractions (open symbols) and algal fractions (filled symbols) as a function of time after feeding ad lib. (squares) or on a singleartemia (triangles). account for the disappearance of algae. In several examples in Fig. 2, significant declines in the algal population occur despite a substantial frequency of dividing algal cells. Ignoring the addition of cells to the algal populations through cell reproduction, and considering just the net losses of algae, the number of lost algae is substantial. At a minimum, 37 % of the initial algal population was lost during a given 48-h interval in an unfed hydra (a significant decline, P<0-001, / test). A minimum of 10 % of the original algal population was lost in the 48 h following a meal of one Artemia (a significant decline, P< 0-025, / test). The algal population of hydra fed ad lib. grew by approximately 20 % in 48 h (a significant increase, P<0-001, t test), but experienced a 10% drop between 12 and 24 h after feeding (a significant decline, P< 0005, / test). Regulation of algal number by expulsion of excess algae Expulsion was a minor sink for algae in these experiments (see Fig. 2). Only 8 % of the algae lost by unfed hydra were found in their medium. Expulsion accounts for only 6 % of the average number of algae lost during Regulation of algal number by intracellular digestion of excess algae Dissociated cell suspensions from starved and recently fed hydra which had been maintained in either continuous light or in a diurnal photoperiod were scored for the incidence of algal disintegration inside host digestive cells. Remnants of algae in various obvious stages of disintegration were identified by their green colour, red fluorescence and location inside vacuoles (see Fig. 4). The algal origin of these particles is corroborated by their restriction to the digestive cells of green hydra and their absence from any cells of aposymbionts or brown hydra. They are not associated with host feeding. Table 1 shows that between 44 and % of a regularly fed hydra's digestive cells contained remnants of disintegrating algae. If each incidence of a host cell with algal debris reflects the disintegration of a single algal cell, then these frequencies mean that almost 3 % of the algal population of these hydra was found to be in some state of disintegration. The data in Table 1 show no significant effect of either photoperiod or feeding on the incidence of disintegrating algae when hydra are regularly fed, but after 7 weeks of starvation, the incidence of disintegrating algae significantly increased (P< 0-005, G-test). Despite the fact that the starving hydra were obviously shrinking, and their endosymbionts declining in number, the algae appear to have been reproducing at an appreciable rate, with a mitotic index of 0-9% (±0-4%). Table 1 also shows how host cells containing disintegrating algae are distributed according to location in the tentacles, main body column, bud and peduncle. It appears that algal disintegration may be more marked in some parts of the hydra's body than in others. The vacuoles enclosing algal remnants are clearly defined under phase-contrast or fluorescence optics. Although their characteristically apical location and large size is similar to that of secondary lysosomes, it cannot be determined if algae are degraded lysosomally without electron microscopic localization of lysosomal enzymes within the perialgal vacuole. It appears, however, that much of the algal disintegration occurs in vacuoles involved in the processing of food. Two thirds of the vacuoles containing algal remnants also contain food particles shortly after feeding (see Table 2). 574 K. Dunn

5 Estimated rates of algal disintegration While the net declines in the algal population are substantial, actual rates of algal disintegration must be somewhat higher to offset algal reproduction. Using certain conservative assumptions, calculations presented in the Appendix yield minimum estimates of the magnitude of algal reproduction as well as algal disintegration. According to these calculations, during the Fig. 4. Photomicrographs of a hydra digestive epithelial cell with intact algae clustered at the cell base and algal remnants within a vacuole at the cell apex. A. Phase contrast illumination: algal debris appears as the phase dense material within a clear, phase lucent vacuole. B. Epifluorescent illumination: algal debris is amorphous and dimly fiuorescing relative to the spherical intact algae. Bar, 10[im. Table 1. Incidence of algal remnants in hydra host cells Condition Number host cells observed Percentage with algal remnants Algae per host cell Continuous light 0 h overall 24 li peduncle 24 h gastric 24 h tentacle 24 h bud 24 h overall 0 h + 24 h overall ± 6 % 30 ±6% 44 ±8% 58 ±6% 48 ± 8 % 49 ±2% 49 ±2% 21-1 ± ± ± ± ± ± ±0-9 Diurnal light 0 h overall 4 h overall 0 h + 4 h overall 7 wks overall ±6% 46 ± 6 % 45 ± 3 % 84 ± 8 % European strain, continuous light 24 h overall 1 25 ±4% The incidence of remnants of algal cells inside vacuoles of host cells at various times after feeding, in various regions of the body, in hydra raised in continuous and diurnal light regimes. Values given arc means plus or minus a standard error of the mean. Oh samples were scored prior to the Monday feeding, at which time the hydra had not eaten for 72 h. Growth of algae in green hydra 575

6 Table 2. Incidence of orange particles and algal remnants in hvdra host cells Orange particles Alfjal remnants Both in same vacuolc Time after feeding Oh 4h 7 wks Oh 4h 7 wks Oh 4h 7 wks Number host cells observed Percent cells with particles The incidence of orange particles interpreted as carotene derived from Artemia food (see Krinsky & Lenhoff, 1965), algal remnants and the coincidence of both types of particles within the same host vacnole in hydra before feeding, 4h following feeding and after 7 weeks of starvation. Oh samples were scored prior to the Monday feeding, at which time the hydra had not eaten for 72 h. While the incidence of green particles is not affected by feeding (l > > 0-5, G test), the incidence of orange particles, and the incidence of vacuoles containing both green and orange particles together, more than doubles following feeding (/ J <0-005, G tests). Orange particles can be found in digestive cell vacuoles of all types of hydra maintained on a diet of Artemia. These particles virtually disappear after protracted starvation. two-day period of observation at least 15 % of the algae of hydra fed a single Artemia disintegrated each day, while unfed hydra lost at least 21 % of their algae daily (sec Table 3). As explained in the appendix, these calculations derive from the conservative assumption that no algal disintegration occurred in hydra fed ad lib. Relaxing this assumption will yield higher estimates of disintegration rates in all three feeding conditions. Discussion Data presented here for the Carolina strain of green hydra show that some degree of control of algal mitosis occurs at the level of the host digestive cell. The feeding stimulus had the quality of a trigger, stimulating a fixed amount of algal division regardless of the size of the meal. In contrast, in the only other study relating the rate of algal division to the size of the host's meal, McAuley (1985) found a positive relationship between host meal size and the frequency of algal mitosis in the European strain. Bossert & Dunn (1986) speculated that the morphogens responsible for hydra size (Schaller et al. 1977) might, through their mitogenic actions (Schaller, 1976(7,6; Bossert, unpublished), interact with algal mitosis in such a way that small hydra, such as the European strain, exercise effective mitotic control of their algae, whereas larger hydra, such as the Carolina strain, must depend upon additional means of regulating algal numbers. In a comparison of three strains of green hydra, we found that the coordination between host and algal mitotic index broke down as hydra size increased. The comparisons of algal mitotic indexes with algal population growth rates presented here corroborate the interpretation that there is more to algal density regulation than host restriction of algal mitosis in large strains, such as the Carolina strain. Significant numbers of algae were lost by the Carolina strain in this study. Very few of the lost algae were found free in the hydra medium and it is clear that expulsion is normally a minor sink for endosymbiotic algae, one that does little to explain the observed losses. Most of the algae lost by hydra may have been lost through intracellular digestion. Large numbers of algal cells in every stage of disintegration were found inside vacuoles of host digestive cells. Several factors suggest that these vacuoles are digestive in nature, although their identification as lysosomes awaits detection of lysosomal enzyme activity. No evidence of lysosomal enzyme activity has previously been demonstrated in perialgal vacuoles of either the Florida or the European strains (O'Brien, 1982; McNeil & McAuley, 1984). Algal disintegration is much more prominent in this study than in previous studies (see Introduction). This may be due to differences between hydra strains. Bossert & Dunn (1986) suggested that digestion of endosymbiotic algae by the host may be more marked in large strains of hydra, such as the Carolina strain, Table 3. Estimated rates of algal reproduction and disintegration Number of Artemia fed Original algal pop. size (XI0 3 ) 48 h average mitotic index Specific algal repro. rate (h -1 ) Final algal pop. size (XlO" 3 ) Specific algal disintegration rate (h' 1 ) % cells lost per dav ad lib Algal population sizes are expressed per hydra. The specific algal reproductive rate is given by the equation, r= ln(l + «/)/'j, where /,i = 11 6h, ii = 3 and/is the 48h average algal mitotic index. The specific algal disintegration rate is calculated as described in the Appendix. Note that the rate of algal disintegration in hydra fed ad lib. was defined to equal zero in order to calculate an upper limit to /,i. These are thus minimum estimates of the rates of algal disintegration. 576 K. Dunn

7 whereas all research to date on algal digestion has been conducted on the smaller European and Florida strains. Algal disintegration appears to be less pronounced in the European strain than in the Carolina strain; Table 1 shows that digestive cells with vacuoles containing algal debris occur half as often in the European strain as in the Carolina strain (P< 0-005, G- test). The mechanism by which a host cell might select algae for digestion is not clear. It is possible that algae targeted for digestion by a given cell may not arise from the resident population of that cell, but rather may be phagocytosed, and thereby targeted for digestion, through a form of epithelial cell autophagy observed in brown hydra. McConnell (1931) and, more recently, Bosch & David (1984) have observed the phagocytosis of epithelial cells by other epithelial cells. Bosch & David suggested that excess cell production in poorly fed hydra is eliminated through phagocytosis of epithelial cells by other epithelial cells. While epithelial cell autophagy is yet to be demonstrated in green hydra, the results presented here are consistent with this mechanism of algal digestion. First, Bosch & David found that poorly fed hydra lose epithelial cells at approximately the same rate as I found green hydra to lose algae (20 % vs. 15 % to 21 % daily). Second, while the number of algal cells in a hydra drops during starvation (Fig. 2), the number of algae per host cell increases (Muscatine & Neckelmann, 1981; Douglas & Smith, 1984; McAuley, 1985a); most of the lost algae must be lost along with their host cells. Finally, Bosch & David found that starvation increased both the incidence of epithelial autophagic vacuoles and the estimated rates of epithelial cell loss, just as I found it increased both the incidence of vacuoles containing algal debris and the estimated rates of algal cell loss. This research was supported by research grants from the National Science Foundation, the Mellon Foundation and the Hudson River Foundation to Dr L. B. Slobodkin, and a grant from Sigma Xi to Dr Kenneth Dunn. I am grateful to Linda Graziadei and Farida Vasi for technical assistance and I thank John McDonald, Dr L. B. Slobodkin, Linda Graziadei, Dr Harvard Lyman and an anonymous reviewer for suggested improvements in the manuscript. I also thank Dr Leonard Muscatine for his encouragement and guidance during a nine month stay in his laboratory at UCLA. Appendix Calculation of the rales of algal reproduction and algal disintegration The frequency of mitosis, as measured here, can be used to calculate the rate of algal reproduction if one knows the duration of the tetraspore stage. This parameter has not yet been measured for endosymbiotic algae. To generate an upper limit estimate for / d, we may use the modified equation of McDuff & Chisholm (1982): where tj is the duration of the tetraspore stage, // is one less than the number of daughter cells (in this case, n = 3),/is the average mitotic index during the interval of interest and /' is the specific reproductive rate. If we make the conservative assumption in the ad lib. feeding case, where algal growth was highest, that no algal mortality occurred, then the reproductive rate equals the population growth rate. In the ad lib. feeding experiments: r = (lruv, - lnivo)// = h"', where JVQ and A', are the sizes of the algal population at the beginning of the interval and after / h, respectively. From the first equation, then: / d =ll-6h. Taking / cl to be constant and substituting it into the first equation, the rates of algal reproduction were calculated for unfed hydra and hydra fed one Anemia from their corresponding average mitotic indexes. If the per capita probability of algal disintegration is constant, algal growth can be approximated from the equation: where D is the specific rate of disintegration. For intervals of time as short as those considered here, the proportion of the algal population disintegrating is approximately: or (A' 0 e"-a' t )/A' 0 e rl, 1-e -Dt If la is constant across different feeding conditions, these calculations will yield conservative estimates of the magnitude of algal disintegration h is an upper limit to the duration of algal mitosis, as larger values of fj yield algal reproductive rates too low to account for the observed growth of the algal population in hydra fed ad lib. If <j is shorter than this upper limit, the calculated rates of algal reproduction and algal disintegration will be correspondingly higher at all three host feeding levels. References BOSCH, T. C. G. & DAVID, C. N. (1984). Growth regulation in Hydra: relationship between epithelial cell cycle length and growth rate. Devi Biol. 104, Growth of algae in green hydra 577

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