Repopulation of a sea anemone with symbiotic dinoflagellates: Analysis by in vivo fluorescence

J. Exp. Mar. Biol. Ed., 170 (1993) 145-158 0 1993 Elsevier Science Publishers B.V. All rights reserved

JEMBE

145 0022-0981/93/$06.00

01990

Repopulation of a sea anemone with symbiotic dinoflagellates: Analysis by in vivo fluorescence T. Berner”, G. Baghdasarianb and L. Muscatineb “Departmentof Life Science. Bar Ilan University. Ramat Gan, Israel; b Department of Biology, University of California. Los Angeles. California, USA (Received

6 January

1993; revision received

18 February

1993; accepted

10 March

1993)

Abstract: Individuals in a population of aposymbiotic Aiptasia pulchella Carlgren were each inoculated with homologous rooxanthellae. The rate of repopulation of the anemones (i.e. the in situ growth rate of the zooxanthellae) was determined non-destructively from the mean in vivo fluorescence per anemone over 19 days. As zooxanthellae cell density increased, chlorophyll a per cell increased, but fluorescence per cell decreased, probably as a result of self-shading. The emergent relationship between in vivo fluorescence and number of zooxanthellae was linear over the range of cell densities investigated. The-specific growth rate during exponential growth was 0.4.day _’ between days 7 and 15. As repopulation approached saturation (ca. 0.5 x lo6 cells per mg animal soluble protein) at about 19 days, the growth rate decreased and approached the steady state growth rate of about 0.02.day-’ of normal symbiotic anemones. Rates of repopulation of A. pulchella by freshly isolated and cultured homologous zooxanthellae were virtually identical. Key words: Symbiosis;

Zooxanthellae;

Aiptasia; Repopulation:

Fluorescence

Endosymbiosis of dinoflagellates (“zooxanthellae”) and marine cnidarians is perpetuated in various ways. Aside from asexual reproduction, these symbioses are sustained through succeeding generations either through maternally inherited zooxanthellae (closed system), or by acquisition of zooxanthellae from the environment by larvae or adults (open system) (Trench, 1987). Although there is modest information on transmission of zooxanthellae via eggs (see references in Trench, 1987), relatively little is known of major features of repopulation of “open system” cnidarians. Such features include the primary loci of uptake of zooxanthellae, the in situ growth rate of zooxanthellae during repopulation, and the manner by which all of the potential host cells are repopulated by a small inoculum of zooxanthellae. In the most comprehensive studies to date, Colley & Trench (1983, 1985) and Fitt & Trench (1983) investigated aspects of repopulation of the scyphistoma larva of the jellyfish Cassiopeia xamachana with freshly isolated homologous zooxanthellae. The larvae are naturally free of zooxanthellae and will not strobilate unless repopulated de novo (Sugiura, 1964). Initially, modest numbers of zooxanthellae, free of contamCorrespondence CA 90024. USA.

address:

L. Muscatine,

Department

of Biology,

University

of California,

Los Angeles,

146

T.BERNERETAL

mating host tissue, are phagocytosed by endoderm cells. The number per scyphistoma declines for 2-3 days and then increases, reaching maximum densities after about 14 days. In contrast to these scyphozoan larvae, much less is known about the repopulation of aposymbiotic adult cnidarians. Some marine cnidarians, such as the corals Astrangia danae and Occulina dzQ7iisafrom the Atlantic coast of N. America, and Madracis decactis from Discovery Bay, Jamaica, are often aposymbiotic (Muscatine et al., 1979). Other species become aposymbiotic after exposure to abnormally high or low temperature (Jokiel & Coles, 1990; Muscatine et al., 1991). Sea anemones, such as the temperate zone intertidal AnthopZeura elegantissima, are often aposymbiotic as a result of dwelling in shade or exposure to high temperature (Buchsbaum, 1968). These naturallyoccurring aposymbiotic marine cnidarians, and those produced experimentally in the laboratory, provide a convenient source of animals for experimental investigation of repopulation. Growth rates of zooxanthellae during repopulation are most commonly estimated by counting cells in host animal homogenates, in tentacles sampled over time, or in individual host endodermal cells after maceration of host tissues. For example, Trench (1971) and Schoenberg & Trench (1980) introduced homologous zooxanthellae into aposymbiotic sea anemones (Anthopleura elegantissima; Aiptasia tagetes). By sampling tentacles and estimating the area1 density of zooxanthellae, they were able to establish the time course of repopulation and the attainment of a steady state population density. Hoegh-Guldberg & Smith (1989) and Szmant & Gassmann (1990) followed repopulation of bleached corals by counting zooxanthellae in samples of tissue taken at intervals. Meints & Pardy (1980) measured fluorescence of extracted chlorophyll to characterize repopulation of green hydra. All of these quantitative studies required partial or total destruction of the animals. In this paper we describe a non-destructive in vivo fluorescence technique to determine the in situ growth rate of zooxanthellae during repopulation of the sea anemone Aiptasia pulchella Carlgren. The use of in vivo fluorescence as a measure of algal biomass is based on the assumption that the ratio of fluorescence to extractable chlorophyll a is constant. However, the ratio may vary with season, space, time of day, species, and irradiance history (Loftus & Seliger, 1975). By calibrating in vivo fluorescence with known numbers of zooxanthellae, we demonstrate that the in vivo technique is feasible and enables the investigator to rapidly quantify population density of zooxanthellae in individual sea anemones during repopulation. MATERIALSAND

EXPERIMENTALANIMALS Aiptasiapulchella

Hawaii Institute

AND

METHODS

MAINTENANCE

was collected from a depth of 1 m on Checker Reef adjacent to the of Marine Biology (HIMB) Coconut Island, Hawaii and transported

REPOPULATION

to the University diameter

OF AIPTASIA

of California,

PULCHELLA

Los Angeles.

Anemones

glass bowls in 300 ml filtered seawater.

Dual Program

incubator

147

WITH ZOOXANTHELLAE

were maintained

in 10.5 cm

The bowls were kept in a Precision

at 24 & 1 “C at an irradiance

of 40 pmol photonam-‘es-’

on

a 12-h light: 12-h dark cycle. They were fed twice weekly on Artemia sp. nauplii and rinsed a few hours later to remove uningested food. Aposymbiotic anemones were obtained by chilling the native symbiotic anemones to 4 ’ C for 4 h in darkness and then maintaining them at 24 o C in darkness (Steen & Muscatine, 1987). Although chilling evokes loss of 399% of the zooxanthellae in a few days, some zooxanthellae persist after this treatment. These cells are difficult to detect by light microscopy as they have a very low chlorophyll content, but they are easily detected by epifluorescence microscopy. Specific cell density of zooxanthellae in our A. pulchella (Hawaiian strain) is about 0.5 x lo6 cells per mg animal soluble protein. This is somewhat less than the value of 1.0-2.6 x lo6 reported by Muller-Parker (1984, 1985). Specific density of residual zooxanthellae detected after one month in darkness was 8.2 x lo3 + 6.3 x lo3 cells per mg animal soluble protein (n = 3). As zooxanthellae are lost from chilled anemones by detachment and release of host cells (Gates et al., 1992), the aposymbiotic anemones were maintained in darkness and fed twice weekly for 3 wk before being used in repopulation experiments. Aposymbiotic anemones attained specific cell densities comparable to those in native anemones when provided with zooxanthellae and maintained in the light. ISOLATION

OF ZOOXANTHELLAE

Zooxanthellae were isolated from A. pulchella by homogenization and centrifugation as described by Steen (1987). Isolated cells were washed, resuspended in filtered seawater, and adjusted to a final concentration of lo6 per ml. INOCULATION

OF ZOOXANTHELLAE

INTO

APOSYMBIOTIC

ANEMONES

Forty-one aposymbiotic anemones were selected at random and allowed to attach to the bottom of a Petri dish. The anemones ranged in size from 0.8 to 10.4 mg animal soluble protein, determined when each anemone was sacrificed. A suspension of freshly isolated zooxanthellae was drawn to a microinjection apparatus (Pardy & the pharynx of each anemone, and up expelled into the coelenteron. Inoculated and maintained on the 12-h light: 12-h anemones, similarly maintained but not ous repopulation. MEASUREMENT

OF IN VIVO

into a tapered glass microelectrode tube fitted Muscatine, 1973). The tube was inserted into to 10 ~1 of the suspension (z lo4 cells) was anemones were then returned to the incubator dark cycle. Sixteen additional aposymbiotic inoculated, served as controls for spontane-

FLUORESCENCE

After egesting unincorporated zooxanthellae, usually within 18 h, each inoculated anemone was transferred to its own individual test tube (Kimax 10 x 75 mm) containing

T. BERNER

148

ET AL.

3 ml seawater. Each anemone was then allowed to attach to the end of a short solid glass rod (8 x 15 mm) resting on end at the bottom of the tube. The rod served as a pedestal which positioned the anemone at the level of the aperture leading to the photomultiplier after the test tube was inserted into the fluorometer. Care was taken to use anemones whose expanded height and width would not exceed the dimensions of the aperture (5 x 30 mm). Tubes with anemones were maintained in the incubator under the usual photoperiod during the observation period. Anemones were fed on Artemia nauplii twice weekly. Timing of feeding was adjusted so that fluorescence measurements could be made 24 h after feeding. The seawater in the tubes was changed daily and the walls of the test tubes were cleaned with a cotton swab. In vivo fluorescence was determined at 2-day intervals after inoculation. Test tubes containing individual anemones were dark-adapted for 45 min prior to being inserted into a lluorometer (Turner Model 112) fitted with C/S 5-60 excitation and C/S 2-64 emission filters, and a neutral density filter (value = 1.00). The door factor was 3 x . A test tube containing filtered seawater served as a blank. Measurements were taken between 0800 and 1000 to minimize any error introduced by die1 variations in fluorescence. Care was taken to record fluorescence only when anemones were expanded, as the areal density of zooxanthellae increases signi~cantly when anemones contract, causing a significant decrease in fluorescence. The relative fluorescence of each anemone was measured at four positions by rotating each tube 90” about its vertical axis, and the mean fluorescence per individual anemone was recorded. Because only 41 measurements (i.e. the last measurement before sacrificing the anemone) could be based on total soluble animal protein, repopulation was expressed generally as mean fluorescence per anemone, and in a few instances as specific fluorescence (i.e. mean fluorescence per mg anemone soluble protein). CALIBRATION

OF IN VIVO FLUORESCENCE

OF WHOLE

ANEMONES

AND

NUMBER

OF

ZOOXANTHELLAE

At each sampling interval, after the mean fluorescence of each anemone was recorded, up to 8 anemones were each homogenized in a Teflon-glass homogenizer (10 ml) in a known volume of seawater. Samples of homogenate were taken for determination of number of zooxanthellae and soluble animal protein. A curve was constructed from the empirical observations on relative fluorescence and corresponding zooxanthellae density. The curve was then used to derive zooxanthellae cell number in those anemones whose relative fluorescence was recorded at several intervals before being sacrificed. ANALYTICAL

METHODS

The number of zooxanthellae per anemone was determined from cell counts of homogenate using a hemacytometer (Spencer Bright Line).

REPOPULATION

OF AIPTASIA

PULCHELLA

WITH ZOOXANTHELLAE

149

Total soluble animal protein was measured by the spectrophotometric method of Whitaker & Granum (1980). Each anemone was homogenized and the total homogenate treated for 30 min with 0.1% sodium dodecyl sulfate. Algae and insoluble particulates were removed by centrifugation and the supernatant containing soluble animal protein was sampled for absolute measurement. Chlorophyll cl and c2 in freshly isolated zooxanthellae was measured by the method of Jeffrey & Humphrey (1975). Some anemones were photographed during repopulation with a Zeiss 3.5mm camera fitted to a Wild M5 dissecting microscope.

RESULTS

KINETICS

OF REPOPULATION

After inoculation with homologous zooxanthellae, the mean fluorescence per anemone in the population of anemones increased over a 19-day observation period (Table I). The standard error also increased, and ranged from about 8 to 20% of the mean. During this time spontaneous repopulation by uninoculated controls was minimal (see Fig. 4). To gain insight into factors affecting in vivo fluorescence, we measured Chl a and c2 per zooxanthellae cell in selected anemones during repopulation. Figure 1 shows that Chl a per cell is barely detectable at low cell densities, but increases to about 2.6 pgcell -’ at high cell densities, a value comparable to that in normal symbiotic anemones. As the Chl u per cell in the inoculum is at the normal high level, those zooxanthellae which infect the anemones apparently undergo an initial decrease in Chl a per cell followed by an increase as cell numbers increase. The point at the upper left of the graph may

TABLE 1 Mean ( & SE of the mean) relative fluorescence of Aiptasia ~uichel~~ during repopulation. Day

Fluorescence

n

2 5 7 9 11 13 15 17 19

2.2920.19 4.54 k 0.37 5.78 * 0.57 7.06 I: 0.77 11.66 * 1.51 13.36 k 2.28 20.98 _+2.72 22.59 + 4.64 25.34 + 4.52

41 39 32 26 18 15 12 5 4

Total observations = 192

T. BERNER

6

ET AL.

6

10

12

14

16

16

20

Number of zooxanthellae x 1O4 Chlorophyll u per cell in zooxanthellae during repopulation of Aiptasia pulchell~ (y = 0.08 + 0.125 x rz = 0.79; n = 13). Outlier at upper left omitted from regression; see text for explanation).

a

0.6

l

0.4

0-l

I

0

I

20

I

I

40

I

I

60

I

I

80

I

I

100

I

I

120

Number of zooxanthellae x 1O4 Fig. 2. In vivo fluorescence

per zooxanthella ( x 104) vs. mean number of zooxanthellae during repopulation of Aiptasia pulchella.

( x 104) per anemone

;

REPOPULATION

represent

OF AIPTASIA

cells still at control

per cell was detected

PULCHELLA

levels of Chl a per cell. No significant

during

repopulation

151

WITH ZOOXANTHELLAE

change in Chl c2

(Chl c2 per cell = 2.56 + [O. 12 zooxanthel-

lae x 104]; r* = 0.22). To determine

how the increase

in Chl a per cell during repopulation

might affect the

in vivo fluorescence signal, we computed fluorescence per lo4 zooxanthellae in anemones as a function of the increase in number of zooxanthellae. Figure 2 shows that as the number of zooxanthellae per anemone increases to about 20 x 104, fluorescence per lo4 zooxanthellae decreases about lo-fold. Further increase in numbers of zooxanthellae causes only a slight decrease in fluorescence per lo4 cells. Taken together, these data (Figs. 1 and 2) suggest that although increased Chl a per cell could potentially augment in vivo fluorescence, any such augmentation is apparently offset as cell numbers increase. It would appear that self-shading of the more densely packed cells either reduces fluorescence excitation, increases quenching (i.e. self-absorption) or both. Empirical data relating in vivo fluorescence and number of zooxanthellae are shown in Fig. 3. The non-zero intercept of the linear regression could be due, in part, either to the fluorescence of residual algae in “aposymbiotic” anemones, or low levels of fluorescence by the animal tissue or both. Figure 4 shows the increase in mean number of zooxanthellae per anemone over time during repopulation, as determined from 41 empirical measurements of number of 40

35

8

25

E s ?!

20

2 LL

15

I

0

I

I

20

I

I

40

I

I

60

I

I

80

I

I

100

I

/

4

120

Number of zooxanthellae x 1O4 Fig. 3. Mean in vivo fluorescence per anemone vs. number of zooxanthellae ( x 104) during repopulation of Aiptasia pulchella. Each point represents the mean of four replicate fluorescence measurements per anemone taken before sacrificing the anemone and counting numbers of zooxanthellae. (y= 2.47 + 0.29 x ; r* = 0.89; n = 41).

T.

BERNERET AL.

100

I/ I

60 -j 90

/ 1'

/

50

40

I

10 0

/ 0

/ 2

/

/ 4

1

/ 6

I

, 8

I

I 10

I

I 12

/

I $4

I

I 16

b

1 16

! 20

Time (days) Fig. 4. Number of zooxanthellae per anemone derived, in part, from mean in viva fluorescence per anemone during repopulation of Aipfasia pulchella aAer a single injection of homologous zooxanthellae (0). Vertical bars represent standard error of the mean (see Table I for n values). (u), Uninoculated aposymbiotic anemones (n = 16).

zooxanthellae per anemone, and 151 measurements using the calibration curve in Fig. 3. The mean specific growth rate p, where p = l/n (dn/dt), during exponential growth between days 9 and 15, is 0.40.day-‘. At day 19 the specific growth rate decreases to 0.035.day-’ as it approaches the steady state of 0.005-0.025.day-’ for laboratory-bred symbiotic anemone populations (Wilkerson et al., 1983, Muller-Parker, 1984, 1985). The experiment was terminated on day 19 as the specific cell density of the repopulated anemones (0.475 x lo6 it 1.29 x lo6 zooxanthellae per mg total soluble normal protein: n=4) approached symbiotic anemones that of (0.498 x lo6 & 1.07 x 106; n = 10). Uninoculated controls exposed to identical temperature and photope~od exhibited very low rates of spontaneous repopulation. The specific growth rate between days 9 and 15 was estimated to be O.O8*day-‘. THE

EFFECT

THELLAE

OF INOCULATION

ON RATE

WITH

FRESHLY

ISOLATED

VS. CULTURED

ZOOXAN-

OF REPOPULATION

Colley & Trench (1983, 1985) observed that zooxanthellae isolated from the scyphistoma larva of Cassiopeia xamachana are contaminated by remnants of the host cell

REPOPULATION

OF AIPTASIA

PULCHELLA

WITH

153

ZOOXANTHELLAE

OI,,,,,,,,,,,,,,,,,,, 0

2

4

6

a

10

12

14

16

18

20

Time (days) Fig. 5. Mean in vivo fluorescence per anemone during repopulation of Aiptasia pulchella (Java Clone) after a single injection of homologous zooxanthellae, either freshly isolated (0) (v = 1.39 + 0.11 x ; r2 = 0.97), or cultured (+) (y= 1.42 + 0.106 x; I’= 0.96). Uninoculated, (0) (J.= 0.79- 0.015 x ; r2= 0.26; n= 16).

vacuolar membrane. These remnants significantly affected the kinetics of uptake and sequestration of the zooxanthellae. To determine if the characteristics of repopulation of A. pulchella might be influenced by the source of inoculum of homologous zooxanthellae, we injected 120-day aposymbiotic anemones with either freshly isolated zooxantlrellae

and or with homologous

cultured

zooxanthellae.

In this experiment

we used

A. pulchella from Java (collected

and maintained by 0. Hoegh-Guldberg, Department of Biology, UCLA) as it was also the source of the cultured zooxanthellae (courtesy of V. Weis, Department of Biology, UCLA). Figure 5 shows that the rate of repopulation of aposymbiotic anemones, as determined by in vivo fluorescence, was similar in both cases. The specific growth rate was less than half that of A. pulchella from Hawaii (p = 0.16.day-‘; days 9-16). Uninoculated controls showed no evidence of spontaneous repopulation, and in some cases exhibited negative growth rates.

SITES

OF REPOPULATION

To gain some insight into initial sites of repopulation, a few anemones were sectioned longitudinally with a razor blade 3 days after inoculation. Microscopic examination

154

T. BERNER ET AL.

Fig. 6. (A) ~i~zus~~~~~c~e~~, initially aposymb~otic, 3 days after injection of homologous zooxanthellae into the coelenteron, showing (at arrows) relative dark short tentacles at base of tentacle crown due to relatively high areai density of repopulating zooxanthellae (30 x ). (B) The contrast in zooxanthellae area1 density in short vs. long tentacles is still evident after 15 days (60 x ).

revealed that, in the column, zooxanthellae were concentrated in the digestive zones of the mesenteries (not figured), while in the tentacles, zooxanthellae were concentrated in the short tentacles, as judged subjectively from areal population density (Fig. 6A). Higher area1 density of zooxanthellae in short tentacles was still evident 15 days after inoculation (Fig. 6B).

DISCUSSION

The results of this investigation show that whole animal in vivo fluorescence may be used to quantify in situ growth rate and population density of zooxanthellae in the sea anemones, Aiptusiapukhella. The in vivo method offers the advantage of non-destructive repetitive observations on the same individuals over time. The technique is constrained only by a limitation on size of the animal (< 30 mm expanded height). The method requires initial calibration of in vivo fluorescence with known numbers of zooxanthellae. The caveats governing interpretation of in vivo fluorescence measurements (Loftus & Seliger, 1975) are justified for A. pulcheila, as Chl a per cell increases and fluorescence per cell decreases with increasing cell density. However, as these changes are opposite in trend, the relationship between relative fluorescence and number of zooxanthellae emerges as linear, at least for the range of cell densities used in these experiments. The rate of uptake of inoculated zooxanthellae is rapid. We estimate that anemo-

REPOPULATION

OF AIPTASIA

PULCHELLA

nes are given from 3 to 10 ~1 of a suspension anemone

is therefore

unincorporated

presented

zooxanthellae

155

WITH ZOOXANTHELLAE

containing

lo6 zooxanthellae

with 3000 to 10000 cells. As anemones after 5-6 h, either the capacity

for uptake

per ml. Each often egest of zooxan-

thellae saturates at a relatively low density (< lo4 per anemone), or many of the zooxanthellae in the inoculum are unsuitable for retention. Schoenberg & Trench (1980) injected scyphistoma larvae with zooxanthellae mixed with an aqueous extract ofArtemiu sp. and observed egestion of zooxanthellae for up to 18 h after feeding. Thus, the manner in which the zooxanthellae are initially provided could affect the time course of both uptake and egestion. Nevertheless, rapid uptake and saturation of uptake sites is typical of those symbiotic cnidarians such as Hydra viridis (Pardy & Muscatine, 1973) Aiptasiu tugetes (Schoenberg & Trench, 1980) and scyphistoma larvae of Cussiopeia xamachana (Colley & Trench, 1983; Fitt & Trench, 1983) in which repopulation has been studied quantitatively. KINETICS

OF REPOPULATION

AND

IN SITU GROWTH

RATES

OF ZOOXANTHELLAE

Growth of zooxanthellae can be detected by in vivo anemone fluorescence two days after inoculation. The specific growth rate of 0.40.day-’ is a mean value for the population of anemones, and is among the highest yet reported for in situ growth. It is comparable to the value of 0.38.day-’ calculated from the data of Schoenberg & Trench (1980; p. 450, Fig. 1) on repopulation of Aiptasia tagetes tentacles. Their data show no lag period but, like ours, reach steady state levels in 18-20 days. Our data are also comparable to the growth rate of 0.39.day-‘, calculated from mitotic index data, for growth of low density populations of zooxanthellae in the nudibranch Pteraeolidae iunthina from temperature zone waters of Australia (Hoegh-Guldberg et al., 1986). The rate is higher than the growth rate of A. pulchella algae in culture (0.270.23.day-‘; Chang et al., 1983; 0.33*day-‘; G. Muller-Parker, pers. comm.), and somewhat higher than the in vitro growth rates of the free-living dinoflagellate Zooxanthellue sp. (0.36.day-‘; Loeblich & Sherley, 1979) and the symbiotic dinoflagellate from the zoanthid Zoanthus sociatus (0.34.day-‘; Domotor & D’Elia, 1984). Comparison with these data is interesting but complicated by differences in h-radiance and nutritional histories of the various algae. Steady state growth rates of zooxanthellae in fully repopulated anemones and in native controls (cf. Wilkerson et al., 1983; Muller-Parker, 1984, 1985) are an order of magnitude lower than those manifested during repopulation. It would appear that the steady state growth rate is limited by some as yet unknown population densitydependent factor. Maximum cell density in cultures of algae is cell size-dependent, with self-shading being the most probable feature governing the density maximum, but physiological contraints (e.g. nutrient limitation) cannot yet be ruled out (Agusti et al., 1987; Agusti & Kalff, 1989). Aposymbiotic anemones frequently contain some residual zooxanthellae which persist in darkness, and contribute to repopulation under favorable conditions. However,

T.BERNER

156

the rate of repopulation detectable observation

of uninoculated

ET AL

controls

in others (Fig. 4), and even negative period. The reason

was very low in some anemones,

not

(Fig. 5) in still others over the 19-day

for these low and negative

growth rates is unknown.

They may be related to the fact that, unlike freshly isolated zooxanthellae, residual algae have a very low chlorophyll content which may affect their growth potential.

EFFECTOFFRESHLYISOLATED

VS.CULTUREDALGAE

ONREPOPULATION

Trench (1988) summarized the characteristics of uptake of symbiotic dinoflagellates by cells of invertebrate hosts, noting that zooxanthellae freshly isolated from Cussiopeiu larvae are taken up rapidly by cells of the larvae because they are still enveloped by host cell vacuolar membrane (Colley & Trench, 1983, 1985). When the contaminating membranes were removed, or when cultured homologous zooxanthellae were introduced, the rate of phagocytosis was low. The host cell vacuolar membrane was thought to facilitate the recognition of self by the host phagocytic cells. Although we did not investigate rates of phagocytosis, the inoculation of A. pulchella with freshly isolated or cultured homologous zooxanthellae resulted in similar rates of growth during repopulation. This suggests that rate of repopulation was not affected by the source or treatment of homologous zooxanthellae.

SITEOFREPOPULATION

Zooxanthellae taken up by aposymbiotic anemones are abundant in the digestive zones of the mesenteries. This is not surprising, as the cells of this tissue are avidly phagocytic. Even in normal symbiotic anemones the digestive cells of the mesenteries contain abundant zooxanthellae. Many of these zooxanthellae are mo~hologic~ly aberrant. The fate of such cells, including those initially taken up by aposymbiotic anemones, or their potential for repopulation is not known. The early appearance of zooxanthellae in tentacles (Fig. 6A,B) following inoculation suggests that the cells of the tentacle endoderm also phagocytose zooxanthellae. Why they should be concentrated in short tentacles

is not yet understood.

The manner by which all of the potentially inhabitable cells of the endoderm become repopulated from a small inoculum is not yet known. Host cells with zooxanthellae might divide at a faster rate and outgrow neighbo~ng algae-free cells. Alternatively, as zooxanthellae grow and divide, some might be exocytosed by host cells and then immediately phagocytosed by neighboring algae-free cells.

ACKNOWLEDGEMENTS

This study was supported by a research grant from the National Science Foundation (OCE-8723090) to L. M. and a Fellowship from the Ministry of Education, Israel,

REPOPULATION

OF AIPTASIA

PULCHELLA

157

WITH ZOOXANTHELLAE

to T.B. We thank 0. Hoegh-Guldberg for A.pulchella (Java clone), V. Weis for cultures of zooxanthellae from A. pulchella (Java clone), and G. Muller-Parker for critical review of a draft of the manuscript. REFERENCES Agusti, S. & J. Kalff, 1989. The influence density and biomass. Agusti.

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