J. Cell Sci. 77, 87-92 (1985) 87Printed in Great Britain © The Company of Biologists Limited 1985

COMPETITION BETWEEN CHLORELLAE IN

CHIMERIC INFECTIONS OF HYDRA VIRIDIS: THE

EVOLUTION OF A STABLE SYMBIOSIS

M.RAHATDepartment of Zoology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

SUMMARY

Aposymbiotic polyps of Hydra viridis were infected with one or two of the following strains ofChlorella: the native strain obtained from green H. viridis, and the originally non-symbiotic strainsFs and 21 l/8p cultured in vitro. Larvae of Anemia served as infecting vectors.

Chimeric infections were obtained with two different Chlorella strains cohabiting in the same cellsand polyps. In time, the chimeric infections disappeared and mixed populations of Hydra wereformed with different strains of Chlorella in different polyps.

We suggest that the Chlorella/Hydra symbiosis was initiated originally by an infection ofpreadapted hydra by preadapted chlorellae. Through intracellular interalgal competition and com-petition between dissimilar infected cells and polyps, the present-day stable symbiosis has evolved.

INTRODUCTION

The green fresh-water Hydra viridis hosts in its digestive cells symbiotic unicellularalgae of the genus Chlorella. Morphological and physiological aspects of this sym-biosis have been studied (Park, Greenblatt, Mattern & Merril, 1967; Oshman, 1967;Pardy, 1976; Cantor & Rahat, 1982; Rahat & Reich, 1983), but only speculationscould be made as to the process by which this symbiosis originated in nature (Smith,1980; Cook, 1981).

Recently, we have shown that aposymbiotic polyps of H. viridis can be infectedwith several strains of free-living chlorellae that are apparently preadapted to live ina nutrient rich environment (Rahat & Reich, 1984, 1985) such as the cell of a hydra(Cook, 1972; Thorington & Margulis, 1981).

Thus we obtained a model to imitate nature and in which to investigate experiment-ally the formation of a new symbiosis through infection of aposymbiotic specimens ofH. viridis with different strains of Chlorella.

In this study we present the first report of a chimeric infection in which chlorellaeof two different strains cohabit in the same polyp and cell.

We conclude that interalgal competition in the cells of infected H. viridis, com-petition between dissimilar infected cells in the hydra, and competition betweenpolyps hosting different chlorellae determine which strain remains as the solesymbiont in//, viridis.

Key words: Chlorella, Hydra viridis, symbiosis.

M.Rahat

MATERIALS AND METHODS

Stock cultures

Swiss symbiotic H. viridis (Ssh), aposymbionts (Sah) derived from the same strain (Rahat,Zeldes & Reich, 1979) and a non-symbiotic brown hydra were used in all our experiments. Threestrains of Chlorella were used, the originally non-symbiotic Fs and 211/8p grown in vitrv (Rahat& Reich, 1985), and the native Ss (Swiss symbiont) obtained by homogenization from Ssh.

Strain 211 /8p was chosen as it differs in form from the other two strains and can be distinguishedfrom them even when inhabiting the same cell (Fig. 1).

Hydra and chlorellae were cultured as described before (Rahat & Reich, 1985).

Infection of hydra with chlorellae

Larvae of Anemia sp. fed with chlorellae were used as vectors to infect the hydra (Rahat & Reich,1984). For double infections, e.g. Fs and 211/8p, the respective chlorellae were fed separately to4 to 5-day-old Artemia. The hydra were then offered larvae containing the respective chlorellae (twoto three larvae of each).

In this manner we infected Ssh, Sah and hydra already infected by Fs (SFsh) or 21 l/8p(S211/8ph).

For detailed examination of infection, the hydra were macerated (David, 1973) and the occurrenceof the different strains of chlorellae was determined using interference and fluorescence microscopy.

RESULTS

Irregular and uncontrolled infections

As we reported for Fs (Rahat & Reich, 1984), the number of 211/8p chlorellae in

cells of hydra and their distribution along the polyps were inconsistent and fluctuated

with time. In the same polyp some cells contained up to 30 chlorellae while adjacent

cells contained a few only or none at all. Some polyps were completely green while

others had irregular patches, and there was no consistency in the proportions of more-

and less-infected hydra in a given population.

The 21 l/8p chlorellae, like Fs, reproduced rapidly in the cells of hydra and surplus

algae were continuously expelled from the cells into the coelenteron.

No constant quantitative parameters, e.g. number of chlorellae per hydra cell or

polyp, could thus be determined in hydra infected with 211/8p.

All chlorellae infecting brown hydra were eliminated in 1-2 days.

Chimeric infections in cells and polyps

Following double infections, i.e. Ss + 211/8p and Fs + 21l/8p, chimeric infec-

tions were formed (Fig. 1); chlorellae of two different strains being present in the

same cells, and dissimilar infected cells comprising the same polyp (Table 1). Mixed

populations resulted, i.e. some hydra contained Ss or Fs and others 211/8p.

Fig. 1. Chimeric infections in cells of macerated H. viridis, polyps infected with one ortwo different strains of Chlorella sp. A. Cell with native (Ss) symbionts. B. TWO adjacentcells from the same hydra, containing different chlorellae. c. Two different chlorellae inthe same cell. D. Cell containing Fs chlorellae at its apex. E. TWO different chlorellae inthe same cell. F. Cell containing 21l/8p chlorellae. Scale, 10 fan between bars. (Notedifferent magnification for c.)

Free-living chlorellae in H. viridis m

1A

!*£•!*

Fig. 1

Tab

le 1

. Com

petit

ion

of c

him

eric

sym

bios

es i

n H

. vir

idis

Str

ains

of

Chl

orel

la s

p.

Dig

esti

ve c

ells

,d

for

infe

ctio

n Q

uant

itat

ive

esti

mat

ion

of t

he d

iffe

rent

T

yp

e of

hos

t fr

ee o

f st

rain

s of

chl

orel

lae

in c

ells

of

mac

erat

ed

Res

ulti

np s

vmbi

oses

2-3

-

u.

Exp

. no

. hy

dra*

ch

lore

llae

t S

s F

s 21

1/8p

hy

dra

2-3

wee

ks a

fter

inf

ecti

on1

mon

ths

afte

r in

fect

ion

1 S

ah

All

+ +

S~

>>

>2

11

/8~

>>

Ss;

211/

8p

Ssh

2 S

ah

All

+ +

Fs>

>>

211

/8p

SF

&

3 S

sh

Non

e +

Ss o

nly

Ssh

4 S

21 1

/8ph

S

ome

+ S

s>>

>21

1/8p

>>

Ss;

21

1/8

p S

sh

5 S

211 /

8ph

Som

e +

211/

8p>

>>

Fs>

>21

1

/8p

; F

s S

211/

8p>

>>

Fsh

$ f?

6 S

Fsh

S

ome

+ F

s>>

>21

1/8

p>>

Fs;

21

1/8

p S

Fsh

'Sah

, ap

osym

biot

ic h

ydra

of

the

Swis

s st

rain

. S

sh,

Swis

s sy

mbi

otic

hyd

ra c

onta

inin

g th

e na

tive

sym

bion

ts,

Ss.

S21

1/8p

h,

Sah

inf

ecte

d w

ith

211/

8p a

nd c

onta

inin

g th

is s

trai

n on

ly.

SF

sh,

Sah

infe

cted

wit

h F

s an

d co

ntai

ning

this

str

ain

only

. t I

n S

ah a

ll di

gest

ive

cell

s ar

e av

aila

ble

for

alga

l in

fect

ions

. In

Ssh

all

dige

stiv

e ce

lls a

re o

ccup

ied

by t

he n

ativ

e S

s sy

mbi

onts

. In

hyd

ra c

onta

inin

g F

s or

211

/8p,

onl

y so

me

of t

he c

ells

are

infe

cted

, an

d m

any

dige

stiv

e ce

lls

are

stil

l av

aila

ble

for

new

inf

ecti

ons.

1 S

~>

>>

21

1/8p

>>

Ss;

21

1/8p

: th

e m

ajor

ity o

f th

e ce

lls

cont

ain

Ss

only

, so

me

cont

ain

211/

8p,

and

a fe

w c

onta

in c

hlor

ella

e of

bot

h st

rain

s..

$ S21

1/8p

>>

>F

sh:

hydr

a ho

stin

g bo

th 2

11/8

p an

d F

s, t

he f

orm

er b

eing

by

far

mor

e ab

unda

nt th

an t

he la

tter

.

Free-living chlorellae in H. viridis 91

Competition and preservation of territory

In time (weeks or months) the chimeric infections disappeared, first from the cellsand then from the polyps, and homogeneously infected cells were predominant amongthe hydra. Similarly in mixed populations, in a short time, only one strain could befound in each hydra (Table 1).

When Sah was infected with Ss or Fs together with 21 l/8p, the latter strain eventu-ally disappeared and Ssh or SFsh populations were obtained (Table 1, exp. 1,2).When hydra already containing a different strain were infected with Ss or Fs chlorellae(exp. 3, 5, 6), the 'settled' chlorellae apparently had some advantage and maintainedtheir territory (McAuley & Smith, 1982). Ss, however, seemed to be more successfuland they displaced 21l/8p from their intracellular habitat (exp. 4).

DISCUSSION

The inconsistent number of algal symbionts per cell, and the irregular distributionpattern of the 21 l/8p and Fs infections in hydra, as compared to that of the native Sssymbiotic chlorellae, probably indicate the absence of the long period of coadaptationthat the latter had in Ssh. However, both strains formed stable symbioses withH. viridis. No symbioses were formed between brown hydra and these strains, or withfive other strains that do infect//, viridis (Rahat & Reich, 1985).

We must thus assume that like the preadaptation required of the chlorellae to enablethem to live in hydra, a preadaptation is also required of the hydra. We have noinformation as to what these preadaptations might be.

The digestive cells of hydra were found to be open to double infections (Fig. 1),being host to different chlorellae for weeks. As in any abiotic habitat, these chlorellaeprobably also competed for the intracellular habitat. We do not know yet what charac-teristics enabled one to displace the other (Table 1).

Hydra are known to replace their cells every 2-3 weeks (Campbell, 1967). Thus,when different digestive cells of the same.polyp were infected, respectively, withdifferent strains of chlorellae, the strain that enhanced proliferation of its host cellswould apparently soon occupy the whole polyp.

From former studies we know that hydra containing various 'new' symbionts allhave a lower budding rate than Ss-containing Ssh, and some have less buds than theothers. In a population of dissimilar infected hydra the effect of the respective algalsymbionts on budding would certainly affect the long-time survival of a given infec-tion in that population.

We conclude that competition between algae invading a cell of hydra, towards theestablishment of a stable symbiosis in the host species, occurs at the intracellular -interalgal, cellular and polyp levels. We should distinguish between an infectionformed in a cell of hydra that lasts the life of that cell, a symbiosis in a polyp that laststhe life of that polyp, and a perpetuating symbiosis in the population of a given speciesof hydra. It is only the last symbionts that would in time coevolve with the host.

On the basis of our results we may now reconstruct the formation and evolution of

92 M. Rahat

the present-day stable Clorella/Hydra symbiosis as it might have happened in nature.Preying on filter-feeding crustaceans, the digestive cells of hydra became infected withseveral strains of free-living algae. Some strains of Chlorella, preadapted to reproducein a nutrient-rich environment, survived in cells of preadapted hydra. Throughinteralgal competition inside these cells, competition between cells of the hydra con-taining different chlorellae, and competition between differently infected polyps, onestrain of chlorellae survived with its host species. In time, through coevolution, thepresent-day stable symbiosis that is obligatory for the chlorella was formed. Furthercurrently occurring 'foreign' infections are constantly eliminated from the hydra, inwhich the settled chlorellae preserve their territory.

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(Received 12 December 1984 -Accepted 14 February 1985)