J. Cell Sci. 86, 273-286 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
273
ALGAL ENDOSYMBIOSIS IN BROWN HYDRA:
HOST/SYMBIONT SPECIFICITY
M. RAHAT AND V. REICH
Department of Zoology, The Hebrew University ofJerusalem, Jerusalem 91904, Israel
SUMMARY
Host/symbiont specificity has been investigated in non-symbiotic and aposymbiotic brown and
green hydra infected with various free-living and symbiotic species and strains of Chlorella and
Chlorococcum. Morphology and infrastructure of the symbioses obtained have been compared.
Aposymbiotic Swiss Hydra viridis and Japanese H. magnipapillata served as controls.
In two strains of H. attenuata stable hereditary symbioses were obtained with Chlorococcum
isolated from H. magnipapillata. In one strain of H. vulgaris, in H, oligactis and in aposymbiotic
H. viridis chlorococci persisted for more than a week. Eight species of free-living Chlorococcum, 10
symbiotic and 10 free-living strains of Chlorella disappeared from the brown hydra within 1-2
days.
In H. magnipapillata there was a graded distribution of chlorococci along the polyps. In
hypostomal cells there were >30 algae/cell while in endodermal cells of the mid-section or
peduncle <10 algae/cell were found. In H. attenuata the algal distribution was irregular, there
were up to five chlorocci/cell, and up to 20 cells/hydra hosted algae.
In the dark most cells of Chlorococcum disappeared from H. magnipapillata and aposymbiotic
hydra were obtained. Chlorococcum is thus an obligate phototroph, and host-dependent heterotrophy is not required for the preservation of a symbiosis. The few chlorococci that survived in the
dark seem to belong to a less-demanding physiological strain.
In variance with known ChlorellaJH. viridis endosymbioses the chlorococci in H. magnipapillata and H. attenuata were tightly enveloped in the vacuolar membrane of the hosting cells
with no visible perialgal space. Chlorococcum reproduced in these vacuoles and up to eight
daughter cells were found within the same vacuole.
We suggest that the graded or scant distribution of chlorococci in the various brown hydra, their
inability to live in H. viridis and the inability of the various chlorellae to live in brown hydra are the
result of differences in nutrients available to the algae in the respective hosting cells.
We conclude that host/symbiont specificity and the various forms of interrelations we show in
green and brown hydra with chlorococci and chlorellae are based on nutritional-ecological factors.
These interrelations demonstrate successive stages in the evolution of stable obligatoric symbioses
from chance encounters of preadapted potential cosymbionts.
INTRODUCTION
Two major groups have been recognized in the genus Hydra. One group
comprises the green 'viridissima' hydra that host endosymbiotic algae in their
digestive cells, and the second group are the 'brown' non-symbiotic hydra such as
H. vulgaris or H. attenuata that were known to be found in nature without algal
endosymbionts (Campbell, 1983).
Goetsch (1924, 1926, cited by Kanaev, 1952), claimed that he found in his
laboratory H. attenuata infected with a free-living 'Chlorella magna', and that he
Key words: algae, Chlorella, Chlorococcum, Hydra, symbiosis.
274
M. Rahat and V. Reich
could infect these hydra with other chlorellae. Such infected brown hydra have not
been reported since, and later attempts to repeat these experiments and infect brown
hydra with chlorellae have failed (Pardy & Muscatine, 1973; Muscatine et al. 1975;
Jolley & Smith, 1980; Rahat, 1985a).
To date, only Chlorella spp., of symbiotic or free-living origin, have been reported
to form stable symbioses in the green hydra (Park et al. 1967; Muscatine et al. 1975;
Jolley & Smith, 1980; Rahat & Reich, 1984, 1985a). It has been claimed for the
brown hydra that they cannot phagocytose green algae or host them to form stable
symbioses (Pardy & Muscatine, 1973; Jolley & Smith, 1980; Rahat, 1985a).
The Japanese//, magnipapillata has been classified with the 'vulgaris' group of the
non-symbiotic brown hydra (Campbell, 1983), but has recently been shown by us to
host in its endodermal cells unicellular green algae of the genus Chlorococcum (Rahat
& Reich, 19856).
Extensive data have been acquired on the Chlorella/H. viridis endosymbioses
(e.g. see Parker al 1967; Pool, 1979; Jolley & Smith, 1980; Meints & Pardy, 1980;
McNeil ef al. 1981; McAuley & Smith, 1982; Rahat, 19856; Rahat & Reich, 1985a).
It is thus of special interest to compare the specificity and interrelations of the 'old'
and 'new' cosymbionts.
We describe here the infection of green and brown aposymbiotic and nonsymbiotic hydra with chlorellae and chlorococci, various degrees of prolonged
persistence of algae in some aposymbiotic and non-symbiotic species of hydra and the
consequent formation of stable algal endosymbioses in H. attenuata. We describe
in detail some of the host/symbiont nutritional and ultrastructural spatial interrelations.
MATERIALS AND METHODS
Solutions used
M solution: a buffered salt solution resembling pond water (Lenhoff & Brown, 1970), used for
growing hydra. We added Phenol Red to this medium to facilitate the visual monitoring of the pH
in this medium (Rahat & Reich, 1983a). BBM+: Bolds Basal Medium (Bischoff & Bold, 1963),
with an addition of organic nutrients (Rahat & Reich, 1985a), used to grow the algae in vitro.
Antibiotics mixture: lOO^gml" 1 of each of penicillin, streptomycin, neomycin and rifampicin
(Rahat & Reich, 19836), used to obtain axenic hydra.
Hydra used
The Japanese H. magnipapillata (Sugiyama& Fujisawa, 1977; Sugiyama, 1983; Rahat & Reich,
19856), a South African and an Australian strain of H. attenuata were used together with several
strains of European hydra. The latter were H. oligactis, two strains of H. vulgaris and aposymbiotic hydra of a Swiss strain of H. viridis (Rahat et al. 1979). The European and Australian
hydra were originally obtained from Professor P. Tardent, Institute of Zoology, University of
Zurich, Switzerland, and have been cultured in our laboratory for several years. Three strains of
green H. viridis were used: Swiss, European and a Jerusalem strain isolated here from an
aquarium.
The hydra were grown in M solution at 20 (±2)°C, under continuous illumination of 2500 lux
(6500lux for H. magnipapillata), and fed three times a week with freshly hatched larvae of
Artemia sp.
Algal endosymbiosis in brown hydra
275
Preparation of aposymbiotic H. magnipapillata
One single bud was cut from a dark-grown polyp and verified by light and ultraviolet microscopy
to be aposymbiotic. This bud and its descendants were fed six times a week to repletion. Within
3 weeks a clone of about 100 aposymbiotic H. magnipapillata was obtained.
Isolation of Chlorococcum for infection
About 50 green H. magnipapillata were incubated for 48 h in a mixture of antibiotics to render
them bacteria-free (Rahat & Reich, 19836). The hydra were then washed out of the antibiotics and
placed separately into sterile test tubes containing 5 ml of BBM+ solution, one hydra in each test
tube. In this medium Chlorococcum slowly 'leaked' out of the hydra to the bottom of the test tube.
After a few days the algae were collected with a pipette, washed by repeated centrifugation and
resuspended in M solution. These algae were used to infect the various species of hydra. Similarly
isolated Chlorococcum were used to initiate in vitro cultures of these algae, to be described
separately. In later experiments, in vitro cultured Chlorococcum were successfully used for
infections.
Infection of hydra with Chlorococcum and Chlorella
Larvae of Artemia spp. were used as a vector to infect the various hydra, respectively, with
symbiotic Chlorococcum isolated from H. magnipapillata, with eight species of free-living
Chlorococcum, with 16 strains of in vitro cultured symbiotic and non-symbiotic chlorellae (Rahat
& Reich, 1985a), and with symbiotic chlorellae freshly isolated by homogenization of three strains
of green H. viridis, i.e. Swiss, European and Jerusalem strains. Four- to five-day-old larvae were
mixed in M solution with a suspension of the respective algae, and incubated for 20—30 min. When
the gut of the Artemia turned green, they were fed to the hydra. The algae were thus phagocytosed
together with food particles. Compared to in vitro cultured Chlorococcum, chlorococci separated
from freshly homogenized H. magnipapillata gave less satisfactory infections, as nematocytes
present in such preparations prevented the Artemia from ingesting many algae.
The infected hydra were fed two to three times a week. Examination of the hydra for infecting
algae was best done after several days of starvation.
Light and electron microscopy
For microscopic examination of their cells, hydra were macerated according to David (1973).
Photomicrography (Figs 1, 2) was done by Nomarski Differential Interference Microscopy on
Technical Pan film.
For electron microscopy (Figs 3, 4, 5), hydra were starved for 8 days to eliminate disturbing
lipid globules from their tissue. The hydra were then fixed for 1 h in a solution containing 4 %
glutaraldehyde in 0-1 M-cacodylate buffer at pH 7-4, rinsed in the same buffer and postfixed for 1 h
in 1 % OsO^ and 0-1 M-cacodylate buffer, rinsed again and left overnight in cacodylate buffer at
4°C. The hydra were then stained for 30 min in the dark with 2 % uranyl acetate, rinsed in distilled
water, dehydrated through a graded ethanol series and embedded in epoxy resin (Spuir, 1969).
Some sections were stained again with uranyl acetate.
RESULTS
Infection experiments
As a control for all infection experiments, aposymbiotic H. viridis and H. magnipapillata were reinfected, respectively, with Chlorella and Chlorococcum originally
isolated from these hydra, and algal persistence was verified.
Chlorellae freshly isolated from three strains of green H. viridis and 16 strains of
chlorellae grown in vitro in our laboratory disappeared from H. magnipapillata and
the other brown hydra within 1-2 days, although eight of the strains grown in vitiv
form stable symbioses with H. viridis (Rahat & Reich, 1985a).
276
M. Rahat and V. Reich
Chlorococci from H. magnipapillata formed stable symbioses with two strains of
H. attenuata that retained these algae for almost a year, at the time of writing this
paper, and passed the algae to buds through many generations. In one strain of
H. vulgaris, in H. oligactis and in the Swiss strain of aposymbiotic H. viridis
chlorococci could be found for up to 6 days after infection. No persistent infections
were obtained with any of the free-living species of Chlorococcum. Table 1 summarizes these results.
Identification of Chlorococcum
Chlorococcum has been isolated from H. magnipapillata and grown in vitro (to be
described in a separate publication). Comparison of microscopic morphology of the
isolated Chlorococcum with eight species of free-living Chlorococcum generously
supplied by the Cambridge Culture Centre for Algae and Protozoa, confirmed the
endosymbiotic algae to be a Chlorococcum sp. (Starr, 1955; Bold & Parker, 1962).
Populations of Chlorococcum in H. magnipapillata and H. attenuata
In given batches of hydra simultaneously infected with Chlorococcum, some hydra
retained more algae than others, and in some very few or no algae were found.
H. magnipapillata grown at the light intensity used (6500 lux) were green to the
naked eye, and the hypostome could be seen to be dark green (Fig. 1). At lower
Table 1. Infection, persistence of algae and formation of stable symbioses in brown
and green hydra with free-living and symbiotic Chlorella and Chlorococcum
Infecting algae
Infected
hydra*
H.
H.
H.
H.
H.
H.
H.
magnipapillata (Jap)
attenuata (Afr)
attenuata (Aus)
vulgaris (Eur)
vulgaris (Eur)
oligactis (Eur)
viridis (Eur)
Symbiotic
Chlorella}
Free-living
Chlorella%
Symbiotic
Chlorococcum^
Free-living
Chlorococcum^
—
—
—
—
—
—
++
—
—
—
—
—
++
+
+
+—
—
+—
+—
—
—
—
—
—
—
—
( —) All infecting algae disappear within 3 days; ( + —) some algae persist in cells of hydra
for more than 6 days; (+) symbiosis, infecting algae reproducing and passed on to buds;
(+ + ) symbiosis, hydra green to the naked eye.
•Geographical origins of the various hydra are shown in parentheses: Jap, Japan; Afr, South
Africa; Aus, Australia; Eur, Europe.
f Eight free-living strains of in vitro cultured Chlorella that form stable symbiosis with Swiss
aposymbiotic H. viridis (Rahat & Reich, 1985a), and symbiotic chlorellae freshly isolated from
three strains of this hydra.
\ Nine free-living strains of Chlorella that do not persist in aposymbiotic Swiss H. viridis (Rahat
& Reich, 1985a).
§/« vitro cultured Chlorococcum spp. isolated from//, magnipapillata.
\ Eight species of free-living Chlorococcum obtained from the Cambridge Algal Collection (see
Materials and Methods).
Algal endosymbiosis in brown hydra
111
•A,
Abbreviations used for all figures: c, chloroplast; cc, Chlorococcum, cm, hydra-cell
membrane; cw, algal cell wall; ect, ectoderm; end, endoderm; gl, gland cell of hydra;
mil, mitochondria of hydra; n, nucleus; st, starch grains.
Fig. 1. Photomicrograph of: A, thick cross-section from upper third of H. magnipapillata showing Chlorococcum spp. in the endoderm. Starch grains in algae are seen as
white dots. B. Chlorococcum in cells of macerated H. magnipapillata; left, cells from
hypostome of hydra packed with many chlorococci; right, cells from lower part of hydra
hosting a few algae only. Scale: 10 Jim between bars.
278
M. Rahat and V. Reich
light intensities the number of algae/hydra and number of 'green' polyps in the
population decreased. If grown in the dark for prolonged periods most algae were
lost from the hydra, but in some hydra a few algae remained. When the hydra were
returned to intense light these algae reproduced and the hydra greened again. From
H. magmpaptllata that lost all their symbiotic algae in the dark we obtained an
aposymbiotic strain.
Some polyps of the Australian strain of H. attenuata turned completely green
after infection with symbiotic chlorococci, but others were pale to the naked eye
although they hosted many single or clusters of Chlowcoccum for at least a year at this
writing (Fig. 2). Judged from colour changes in the symbiotic H. attenuata the
Fig. 2. Photomicrograph of thick mid-section and cells of H. attenuata showing
Chlorococcum spp. in endodermal cells. Starch grains are seen as white dots in algae.
Scale: 10^m between bars.
Algal endosymbiosis in brown hydra
279
number of chlorococci/hydra varied in time although growth conditions were
constant.
Distribution of Chlorococcum in tissue and cells
Like Chlorella in H. viridis, the endosymbiotic algae in H. magnipapillata and
H. attenuata were hosted in the endodermal cells, but were located both distal and
proximal to the cell nucleus (Figs 1,2).
In H. magnipapillata there was a distinct quantitatively graded distribution of
Chlorococcum along the polyp. Most algae were located in the hypostomal cells, there
were less in the mid-section and very few in the peduncle and tentacles. The
quantitative differential distribution of the endosymbiotic algae was reflected in the
number of algae in cells from the upper and lower parts of the hydra. In hypostomal
cells there were >30 algae/cell while in some endodermal cells from the peduncle
there were <10 or none at all (Fig. 1). In H. magnipapillata bisected transversely,
the number of algae increased in the tip regenerating the new hypostome.
In the polyps and gastroderm of H. attenuata distribution of chlorococci was
irregular, and algae could be found both proximally and distally to the cell's nucleus
(Fig- 2).
An average number of Chlorococcum /cell or hydra cannot be given because of the
great variation (between 0 and 30) in number of these algae in the digestive cells of
the hydra, and in different polyps in a population.
The vacuolar habitat
The host/symbiont spatial interrelations in H. magnipapillata and H. attenuata
were similar. Symbiotic chlorococci were closely enveloped in the vacuolar membrane of the host cell with no visible perialgal space (Figs 3, 4). In H. viridis
similarly prepared for electron microscopy as a control, there was such a space (see
also Rahat & Reich, 1984).
Chlorococcum reproduced inside the vacuoles of the hydra by cell fission (Figs 3, 4),
and up to eight daughter cells were sometimes found within the same vacuole. The
cell wall of the symbiotic Chlorococcum was rather thick compared to that of endosymbiotic chlorellae found in H. viridis (Rahat & Reich, 1984), showed a 'wavy' rim
and sometimes a thickening of the cell wall (Fig. 5).
DISCUSSION
What is it that enables some algae to form symbioses in hydra while others do not,
and why do some hydra host algae while others do not?
To examine these questions critically we have to define our terms and summarize
known data. We should distinguish between: (1) infection of hydra by algae that are
rejected or digested within 1—2 days. (2) Persistence of algae in the hydra that can
last for more than a week. (3) Stable hereditary algal/hydra symbioses. In the latter
case the algae reproduce in the cells of the hydra and are passed on to subsequent cell
Fig. 3. Electron micrograph of Chlorococcum spp. in endoderm of: A, H. magnipapillata
(X4600); B, H. attenuata (XS200). Note dividing algal cells.
Algal endosymbiosis in brx/wn hydra
Fig. 4. Electron micrograph of: A, Chlorococcum spp. dividing in cell of//, attenuata
(X8000); B, detail of A (x 17 000).
212
M. Rahat and V. Reich
and polyp generations. Host/symbiont coevolution may then lead from a facultative
to an obligate endosymbiosis.
Our data from present and former studies show that: (1) all hydra we examined
phagocytosed algae together with their prey. (2) Symbiotic and some free-living
cm
Fig. 5. Electron micrograph of: A, Chlorococcum spp. in cell of H. magnipapillata
(X13 500); B, detail of algal cell wall and hydra cell membrane (X53 000).
Algal endosymbiosis in brown hydra
283
chlorellae can form symbioses with various strains of H. viridis (Rahat & Reich,
1985a), but they do not persist in brown symbiotic or non-symbiotic hydra. (3) The
Japanese brown H. magnipapillata hosts symbiotic Chlorococcum (Rahat & Reich,
19856). (4) Symbiotic Chlorococcum, but not free-living chlorococci, persist for
prolonged periods in cells of green and brown hydra and some may form symbioses
in 'non-symbiotic' brown hydra (Table 1). (5). In contrast to symbiotic chlorellae
the symbiotic Chlorococcum is an obligate phototroph. It might survive in the dark
but will not reproduce there. In the light it will also grow in vitro.
Whatever alleged identity algae might have in order to be recognized as endosymbionts (Muscatine et al. 1975; Muscatine, 1982), it is certainly contained in algae
freshly isolated from H. viridis or H. magnipapillata. These algae however do not
form reciprocal symbioses. In H. viridis symbiotic Chlorococcum could survive for
several days, while chlorellae disappeared from H. magnipapillata within 1-2 days.
There must thus be some factors other than an algal 'identity' recognized by hydra
that determine their ability to form symbioses.
Execution of the claimed barriers to colonization by algae of the hydra cell, i.e.
refusal to take up certain algae and rejection of algae taken up, or their digestion, are
accomplished by the hydra cells within 1-2 days. Phagocytosed algae that persist in
the cells of hydra for more than a few days have passed the above barriers. Such
successful algae, however, may later disappear from their host due to their inability
to reproduce in the intravacuolar environment or to compete with the reproduction
rate of the host cell (Rahat, 1985a,b).
We have recently shown correlations between algal growth requirements and their
ability to form symbioses in hydra (Rahat & Reich, 1985a). We interpret the present
respective data (Table 1), to show that the vacuolar environments in H. viridis and
H. magnipapillata cannot satisfy the nutritional requirements of the algae that
cannot form reciprocal symbioses. A histochemical study of nutrients present in the
respective cells of hydra might explain the different numbers of chlorococci they
host (Figs 1,2). We conclude that host/symbiont specificity in hydra is based on
ecological—nutritional factors (Rahat & Reich, 1986).
In H. viridis chlorellae are hosted in spacious vacuoles (Muscatine et al. 1975;
Rahat & Reich, 1984, 1985a). The close contact of Chlorococcum with the host-cell
membrane and cytoplasm in H. magnipapillata and H. attenuata (Figs 3, 4, 5)
indicates different host/symbiont inter-relations. Any interchange of nutrients would
be direct, without the mediation of a perialgal vacuolar medium.
The conspicuous thick cell wall found in the symbiotic Chlorococcum (Figs 4, 5)
might be correlated with its ability to live as an endosymbiont. However, some other
species of this genus that we have examined have a thick cell wall (Bold & Parker,
1962), but did not form symbioses in our hydra.
We tried to investigate the intravacuolar environment in hydra by correlating
growth in vivo with that in vitro (Rahat & Reich, 1985a). We assumed that whatever
is required for growth in vitro is available to the algae growing symbiotically in vivo.
When aposymbiotic H. viridis are used for such a study, there is always the
possibility that the hydra might be infected by an unwanted contaminating strain of
284
M. Rahat and V. Reich
chlorellae (Rahat & Reich, 1984). Furthermore, the morphology of many strains or
species of Chlorella that grow both in vivo and in vitro is almost identical and some
cannot be told apart when inside a hosting cell (Rahat, 1985a). For such a study
chlorococci should be far superior to chlorellae. Of the nine species examined by us,
only Chlorococcum from H. magnipapillata has been found to infect hydra, and its
morphology clearly differs from other chlorococci or chlorellae. A detailed study of
in vitro growth requirements of Chlorococcum will thus unmistakenly inform us
about the nutritional environment in the hydra cell vacuole.
Green Chlorococcum-hostingH. magnipapillata were collected by Dr T. Sugiyama
at two sites in Japan (personal communication). The symbiotic Chlorococcum grows
in vitro and is probably also free-living at the collection sites. This Chlorococcum,
formed in our laboratory, symbioses with Australian and African strains of
H. attenuata, but hydra infected with this alga have not been reported outside
Japan. The symbiotic Chlorococcum might thus be endemic to Japan and the 'failure
of opportunity' should be considered a factor in the distribution of some algae/hydra
symbioses.
From the inability of symbiotic chlorococci to grow in the dark we may conclude
that they do not obtain from their host cells all the required nutrients and that the
supply by the host of such nutrients is not essential for the formation or maintenance
of a stable symbiosis.
As in any other population, variation and mutations also occur in algal populations
(Hochberg et al. 1972). The few algae that are retained in H. magnipapillata even
after prolonged growth in the dark must be a less-demanding variant of the
Chlorococcum populations. The presence of such variants among chlorococci from
H. magnipapillata is shown by the different numbers of algae in infected hydra and
of infected hydra in a population. Similarly, during our attempts to culture in vitro
Chlorococcum freshly isolated from H. magnipapillata, better growth was obtained
on subsequent transfers, showing a selection of variants more adapted to growth
in vitro. The latter variant could still reinfect H. magnipapillata.
The 'new' symbioses we describe in the present study may not survive competition
in nature (Rahat, 1985a), but they show that it is not 'recognition' that enables algae
to be hosted in hydra, but rather a preadapted ability to live in the intravacuolar
environment. Persistence in the hydra of a preadapted variant from a given infecting
population of algae, and the selection of algae suitable for coevolution towards the
formation of a stable symbiosis, are then the means by which chance encounters
evolve into stable symbioses.
Evolutionary steps that might lead from a 'free' life to an obligatory endosymbiotic
existence are demonstrated by the following: (1) some free-living strains and
species of chlorellae and chlorococci cannot grow in hydra. (2) Some chlorellae,
and Chlorococcum from H. magnipapillata, grow both in vitro and in vivo.
(3) Chlorococcum in H. magnipapillata being an obligate phototroph, does not get
all the required nutrients from its host. (4) Some native chlorellae from H. viridis
also persist in the dark, thus getting all the required nutrients from their host, and
cannot be grown in vitro.
Algal endosymbiosis in brown hydra
285
On the basis of our data and to answer the questions we posed in this discussion we
propose the 'Test Tube Hypothesis': algae ingested into the vacuole of a hydra cell
are subjected to a selection similar to that occurring in any abiotic habitat in nature or
in medium in a test tube. Algae that are preadapted to live and reproduce in the given
environment or medium will survive. If their rate of reproduction equals or surpasses
that of the hosting cell they will remain as successful colonizers, exposed to mutual
selective coevolution towards the formation of a stable endosymbiosis.
We thank Mr E. Hatab for his assistance with electron microscopy.
REFERENCES
BISCHOFF, H. W. & BOLD, H. C. (1963). Phycological Studies, vol. 4. University of Texas
publication no. 6318.
BOLD, H. C. & PARKER, B. C. (1962). Some supplementary attributes in the classification of
Chlorococcum species. Arch. Mikrobiol. 42, 267-288.
CAMPBELL, R. D. (1983). Identifying hydra species. In Hydra: Research Methods (ed. H. M.
Lehoff), pp.19-28. New York: Plenum Press.
DAVID, C. N. (1973). A quantitative method for the maceration of hydra tissue. Wilhelm Roux
Arch. EntwMech. Org. 171, 259-268.
HOCHBERG, A., PIMSTEIN, R. & RAHAT, M. (1972)'. Properties of an ethionine-resistant (ER)
mutant of Ochromonas danica.J. Protozool. 19, 66-69.
JOLLEY, E. & SMITH, D. C. (1980). The green hydra symbiosis. II. The biology of the
establishment of the association. Proc. R. Soc. Land. B 207, 311-333.
KANAEV, I. I. (1952). Hydra: Essays on the Biology of Fresh Water Polyps (translated by E. J.
Burrows & H. M. Lenhoff). Edited and published by H. M. Lenhoff, 1969, University of
California, Irvine CA 92717.
LENHOFF, H. M. & BROWN, R. D. (1970). Mass culture of hydra: An improved method and its
application to other aquatic invertebrates. Lab. Anim. 4, 139-154.
MCAULEY, P. J. & SMITH, D. C. (1982). The green hydra symbiosis. VII. Conservation of the host
cell habitat by the symbiotic algae. Proc. R. Soc. Land. B 216, 415-426.
MCNEIL, P. L., HOHMAN, T . & MUSCATINE, L. (1981). Mechanisms of nutritive endocytosis.
II. The effect of charged agents on phagocytic recognition of digestive cells, jf. Cell Set. 52,
243-269.
MEINTS, R. H. & PARDY, R. L. (1980). Quantitative demonstration of cell surface involvement in a
plant-animal symbiosis: Lectin inhibition of reassociation. J. Cell Sci. 43, 239-251.
MUSCATINE, L. (1982). Establishment of photosynthetic eukaryotes as endosymbionts in animal
cells. In On the Origin of Chloroplasts (ed. J. A. Schiff), pp. 77-92. Amsterdam: Elsevier-North
Holland.
MUSCATINE, L., COOK, C. B., PARDY, R. L. & POOL, R. R. (1975). Uptake recognition and
maintenance of symbiotic chlorellae by Hydra viridis. Symp. Soc. exp. Biol. 29, 175-203.
PARDY, R. L. & MUSCATINE, L. (1973). Recognition of symbiotic algae by Hydra virdis. A
quantitative study of the uptake of living algae by aposymbiotic H. viridis. Biol. Bull. mar. Lab.,
Woods Hole 145, 565-579.
PARK, H. D., GREENBLATT, C. L., MATTERN, C. F. T. & MERRIL, C. R. (1967). Some
relationships between Chlorohydra, its endosymbionts and some other chlorophyllous forms.
y. exp. Zool. 164, 141-162.
POOL, R. R. (1979). The role of antigenic determinants in the recognition of potential algal
symbionts by cells of Chlorohydra. J. Cell Sci. 35, 367-379.
RAHAT, M. (1985a). Competition between chlorellae in chimeric infections of Hydra viridis: the
evolution of a stable symbiosis. J . Cell Sci. 77, 87-92.
RAHAT, M. (19856). Origin and evolution of symbiosis in green hydra. Archs Sci., Geneve 38,
385-399.
RAHAT, M. & REICH, V. (1983a). Visual monitoring of pH with phenol-red. In Hydra: Research
Methods (ed. H. M. Lenhoff), pp. 35-37. New York: Plenum Press.
286
M. Rahat and V. Reich
RAHAT, M. & REICH, V. (19836). Preparing axenic hydra. In Hydra: Research Methods (ed. H. M.
Lenhoff), pp. 79-83. New York: Plenum Press.
RAHAT, M. & REICH, V. (1984). Intracellular infection of aposymbiotic Hydra viridis by a freeliving Chlorella sp.: Initiation of a stable symbiosis. J. Cell Set. 65, 265-277.
RAHAT, M. & REICH, V. (1985a). Correlations between characteristics of some free-living Chlorella
sp. and their ability to form stable symbioses with Hydra viridis. J. Cell Sa. 74, 257-266.
RAHAT, M. & REICH, V. (19856). A new alga/hydra symbiosis: Hydra magnipapillata of the
'nonsymbiotic' Vulgaris group hosts a Chlorococcum-Yikt alga. Symbiosis 1, 177-184.
RAHAT, M. & REICH, V. (1986). Algal/hydra symbiosis: a topic in ecology. (1986). In
Environmental
Quality
and Ecosystem
Stability,
vol. I l l (ed. Z. D u b i n s k y ) . Israel: Bar Ilan
University Press.
RAHAT, M., ZELDES, D. & REICH, V. (1979). Photoproducts of chloramphenicol: their cytotoxic
effects on Hydra viridis and its symbiotic algae. Comp. Biochem. Physiol. 63c, 27-30.
SPURR, A. R. (1969). A low viscosity epoxy resin embedding medium for electron microscopy.
J.Ultrastruct.Res. 26, 31-43.
STARR, R. C. (1955). A comparative study of Chlorococcum meneghini and other spherical zoospore
producing genera of the Chlorococcales. Indiana Univ. Publ. Sci. Ser. 20.
SUGIYAMA, T. (1983). Isolating hydra mutants by sexual inbreeding. inHydra: Research Methods
(ed. H. M. Lenhoff), pp. 211-221. New York: Plenum Press.
SUGIYAMA, T. & FUJISAWA, T. (1977). Genetic analysis of developmental mechanisms in hydra.
I. Sexual reproduction of Hydra magnipapillata and isolation of mutants. Dev. Growth Differ.
19, 187-200.
(Received 1 May 1986 -Accepted, in revised form, 27 August 1986)