Marine Biology (2005) 146: 869–875
DOI 10.1007/s00227-004-1489-1
R ES E AR C H A RT I C L E
Alexander V. Ereskovsky Æ Elizaveta Gonobobleva
Andrey Vishnyakov
Morphological evidence for vertical transmission of symbiotic bacteria
in the viviparous sponge Halisarca dujardini Johnston
(Porifera, Demospongiae, Halisarcida)
Received: 12 March 2004 / Accepted: 8 October 2004 / Published online: 26 November 2004

Springer-Verlag 2004
Abstract All stages of vertical transmission of symbiotic
bacteria, from the penetration into oocytes to the for-
mation of rhagon, were investigated in the White Sea
(Arctic) representatives of Halisarca dujardini Johnston
(Demospongiae). Small populations of free-living spe-
cific symbiotic bacteria inhabit the mesohyl of H. du-
jardini. They are represented by a single morphotype of
small spiral gram-positive bacteria. Vertical transmis-
sion of symbiotic bacteria between generations in
sponges may occur in different ways. In the case of H.
dujardini the bacteria penetrate into growing oocytes by
endocytosis. A part of the bacteria plays a trophic role
for oocytes and the other part remains undigested in
membrane-bound vacuoles within the cytoplasm. In
cleaving embryos bacteria are situated between the
blastomeres or in the vacuoles. In the blastula all bac-
teria are disposed in the blastocoel. The symbionts are
situated in intercellular spaces in free-swimming larvae
and during metamorphosis. Symbiotic bacteria do not
play any trophic role in the period of embryonic and
postembryonic development of H. dujardini. No signs of
destruction and digestion of bacteria were revealed at
any stage of development.
Introduction
One of the specific features of the sponges (Porifera) is
the presence of obligate species-specific endosymbiotic
photosynthesizing or nonautotrophic bacteria in their
body (Sarà and Vacelet 1973; Simpson 1984; Althoff
Communicated by O. Kinne, Oldendorf/Luhe
A. V. Ereskovsky (&) Æ E. Gonobobleva Æ A. Vishnyakov
Department of Embryology, Biological Faculty,
St. Petersburg State University,
Universitetskaja nab. 7/9, 199034 St. Petersburg, Russia
E-mail: aereskovsky@pisem.net
et al. 1998; Sarà et al. 1998; Lopes et al. 1999).
Researchers enumerating and characterizing sponge–
microbial interactions have shown that bacterial bio-
mass can reach over 50% in some marine sponges, with
corresponding phenomenally huge diversity of bacteria
(Wilkinson 1987; Fuerst et al. 1999). The number of
symbiont morphotypes in a sponge varies from one to
eight (Fuerst et al. 1999; Muricy et al. 1999). An
important function of symbiotic bacteria is considered
to be their involvement in the physiology of sponges by
recycling insoluble proteins and their participation in
structural rearrangement of the sponge mesohyl (Wil-
kinson et al. 1979). Antimicrobial compounds have been
isolated from bacteria collected from sponges, which
could indicate that these bacteria may also play a role in
the defense mechanism of these invertebrates (Stierle
and Stierle 1992; Shigemori et al. 1992; Jayatilake et al.
1996). Symbiotic bacteria can serve as a supplementary
nutritive source, the sponges either utilizing them by
direct phagocytosis or using the products of their
metabolism (Vacelet 1975, 1979; Vacelet et al. 1995).
Marine sponges are of profound interest to chemists
and biologists alike in view of their pharmaceutical po-
tential (Garson 1994; Schmitz 1994). The bioactive
chemicals may be sponge derived (Garson et al. 1998;
Uriz et al. 1996), of symbiotic origin (Unson et al. 1994;
Bewley et al. 1996), or isolated from culture of associ-
ated microorganisms (Shigemori et al. 1992; Kobayashi
et al. 1993).
How do bacteria penetrate into sponges? Two
hypotheses have been proposed: (1) the capture of bac-
teria by the adult sponge from water by filtrating activity
(Reiswig 1971; Pile et al. 1996), and (2) the transfer of
symbiotic bacteria from maternal sponge to the next
generation (vertical transmission) through eggs (in
oviparous sponges) or through larvae (in viviparous
sponges). The transfer of symbionts represents an
important step of sponge biology.
The possibility of vertical transmission of symbiotic
bacteria in the sponges was first proposed by Lévi and
870
Porte (1962). They demonstrated the presence of bacte-
ria inside the larvae of Oscarella lobularis (Homoscler-
omorpha). Lévi and Lévi (1976) first showed the vertical
transmission for oviparous sponge Chondrosia renifor-
mis. There are only two studies of viviparous sponges
with evidence of vertical transmission (Kaye and Rei-
swig 1991; Ereskovsky and Boury-Esnault 2002).
The cleavage, embryonic development, and ultra-
structure of the larva of Halisarca dujardini (Demo-
spongiae, Halisarcida) were described at the electron
microscopic level (Sizova and Ereskovsky 1997; Eres-
kovsky and Gonobobleva 2000; Ereskovsky 2002;
Gonobobleva and Ereskovsky 2004). It was shown that
symbiotic bacteria were present inside the embryos and
larvae.
The aim of the present research was to investigate the
full cycle of vertical transmission of symbiotic bacteria
from the oocyte to the rhagon in viviparous species H.
dujardini from the White Sea on the basis of scanning
(SEM) and transmission (TEM) electron microscopy.
Materials and methods
Reproducing specimens of Halisarca dujardini Johnston,
1842 were collected in the Chupa inlet, 3305¢E, 6615¢N
(the Kandalaksha Bay, the White Sea, the Arctic) from a
depth of 1.5–5 m in June–July 1999–2003. Embryonic
development of H. dujardini is synchronous both in
single sponges and in local populations (Ereskovsky
2000).
For electron microscopic investigations the sponges
were cut into cubes of about 1 mm. The samples were
prefixed in 1% OsO4 for 10 min and fixed in 2.5% glu-
taraldehyde in phosphate buffer at room temperature for
1 h. After fixation, sponge samples were washed in
phosphate buffer and postfixed in 1% OsO4 in phos-
Fig. 1 Symbiotic bacteria of
Halisarca dujardini: a TEM
micrograph of the symbiotic
bacteria (B) in the mesohyl and
inside a mesohylar nucleolated
(N) amoebocyte. Scale bar
2 lm. b TEM micrograph of
the symbiotic bacteria (B) in the
mesohyl of the sponge. Scale
bar 15 nm. c SEM micrograph
of symbiotic bacteria (B) inside
the larva. Scale bar 1 lm
phate buffer for 1 h. Samples were dehydrated through a
graded ethanol series and embedded in Epon-Araldite.
Semi-thin sections were stained with toluidine blue. Ul-
trathin sections were stained with uranyl-acetate and
lead citrate and observed under a JEM 100 CX TEM.
For SEM the specimens were fractured in liquid nitro-
gen, critical-point dried, sputter coated with gold pal-
ladium, and observed under a Hitachi S 570 SEM.
Results
Halisarca dujardini is a widespread Atlantic boreal spe-
cies, which inhabits very diverse shallow-water envi-
ronments with fluctuating conditions (Ereskovsky 1993,
1994). In the White Sea H. dujardini dwells at depths of
1.5–8 m. Its size varies from 5 to 40 mm in width and
from 2 to 6 mm in height. The body surface is smooth
and slimy. Oscules are small, one or several per indi-
vidual (Ereskovsky 1993). H. dujardini is a dioecious,
viviparous sponge. Maturation and cleavage in sponges
from the White Sea normally take place in late June to
early July (Ereskovsky 2000).
Symbiotic bacteria of H. dujardini are represented by
a single morphotype both in the mesohyl of adult
sponges and in all stages of embryonic development.
Bacteria are almost evenly distributed in the mesohyl
and do not form accumulations. Sometimes bacteria are
situated in vacuoles of archaeocytes. They have a curved
spiral form, characteristic of the group of spirills
(Fig. 1). Their length is about 0.45 lm, their thickness
0.18 lm, and the cell wall 0.05 lm thick. The features of
its composition allow one to consider them to be gram-
positive. No flagella or piles at the surface of the bac-
terial wall were revealed. The cytoplasm is heteroge-
neous. Its peripheral part has medium electronic density,
then comes a small electron-transparent part. The cen-

tral part of the cell is electron dense, possibly corre-
sponding to the nucleoid.
The oocytes are scattered in the mesohyl of the cho-
anosomal region (Fig. 2a). They are 80–90 lm in diame-
ter. The surface of oocytes is irregular, with numerous
pseudopodia. Distal parts of late oocytes contained the
phagosomes and yolk granules at different stages of
maturation. Before the onset of the maturation division
the oocyte is oval, about 129·105 lm, without any
pseudopodia. The nucleus is round, 29 lm in diameter,
and located in the middle of the egg. There is a prominent
nucleolus (8–12 lm in diameter). The yolk granules are
distributed evenly through the bulk of the egg (Fig. 2b).
During vitellogenesis of H. dujardini, symbiotic bac-
teria, characteristic of the mesohyl of adult sponges, are
incorporated in the oocyte. The bacteria were often
contained in vacuoles, their entry into the oocyte
occurring by endocytosis, which takes place on the
whole oocyte surface (Fig. 2c). Some phagosomes of the
oocyte contained bacteria at various stages of digestion,
strongly electron-opaque areas with fibrous structure
suggesting residual bacterial membranes (Fig. 2d).
The cleavage of H. dujardini is equal, asynchronous,
and has a polyaxial pattern (Ereskovsky 2002). Cleavage
results in formation of a coeloblastula (Fig. 3a, c). The
few internal cells of the prelarva are derived from mul-
tipolar migration of external cells. During early
embryogenesis symbiotic bacteria are present in the
space between the cells of the follicular envelope and the
blastomeres, between the blastomeres, and inside the
blastomeres. Inside the blastomeres the bacteria are al-
ways surrounded by a vacuole (Fig. 3b). At the eight-cell
stage the bacteria start to concentrate in the forming
blastocoel (Fig. 3b, d).
Larvae are ovoid, 125–130 lm in diameter, and
completely ciliated, although the cilia are sparse on the
Fig. 2 a Semi-thin micrograph
of oocyte during the stage of
vitellogenesis of H. dujardini.
N Nucleus. Scale bar 30 lm.
b Semi-thin micrograph of
mature egg. N Nucleus. Scale
bar 30 lm. c TEM micrograph
of symbiotic bacteria (B) inside
the vacuole of oocyte. Y yolk
granule. Scale bar 0.5 lm.
d TEM micrograph of
symbiotic bacteria (B) inside the
vacuole and digested bacteria
(Db) inside the phagosome (Ph)
of oocyte. Y Yolk granule.
Scale bar 0.5 lm
871
posterior pole (Fig. 4a). Within the population of H.
dujardini under study variability of larval morphotypes
was observed. Besides disphaerula, coeloblastula-like
and parenchymella-like types of larvae also occur
(Gonobobleva and Ereskovsky 2004).
After termination of an embryogenesis the bacteria
are retained in the central part of the larva and in the
intercellular space of basal parts of ciliated cells until
metamorphosis (Fig. 4b). Quantity of bacteria increases
during development. The symbionts inside blastomeres,
embryos, and larvae of H. dujardini show no morpho-
logical signs of digestion, many of them undergoing
division.
After attachment to the substrate, the anterior
hemisphere of a larva is spread along the substratum
and the posterior one keeps its shape (Fig. 4c). During
metamorphosis, the regularity of larval epithelium is
disturbed. Most ciliated cells sink into the postlarva
formed; the rest stay on the surface, differentiating into
exopinacocytes. In 3–6 h after settlement and attach-
ment, a larva is finally transformed into a cell aggregate
with a length of 230 lm. Then the cell differentiation
and the formation of the aquiferous system of a rhagon
occur. At the final stage of metamorphosis, an oscular
tube is formed and the aquiferous system begins to
function. During the metamorphosis bacteria are pres-
ent in extracellular spaces in different parts of the pupa
and the rhagon (Fig. 4d). The symbionts inside the
rhagon of H. dujardini show no morphological signs of
digestion, and many of them undergo division.
Discussion
Endosymbiotic associations are well documented in all
adult sponges. These relationships may involve pro-
872

Fig. 3 a SEM micrograph of

cleaving embryos of H.
dujardini. Bl Blastomeres,
F follicle. Scale bar 25 lm.
b TEM micrograph of
symbiotic bacteria (B) inside
and between blastomeres of
cleaving embryos. Bl
Blastomeres, Y yolk granules.
Scale bar 1 lm. c SEM
micrograph of a coeloblastula
of H. dujardini. Bl Blastomeres,
F follicle. Scale bar 25 lm.
d TEM micrograph of
symbiotic bacteria (B) in the
coeloblastula cavity. Bc Basal
parts of blastular cells, Y yolk
granules. Scale bar 5 lm

Fig. 4 The larva and the

metamorphic stages of H.
dujardini and symbiotic bacteria
inside these stages. a Semi-thin
micrograph of disphaerula
larva of H. dujardini. Ic Internal
cavity, Pp posterior pole. Scale
bar 50 lm. b TEM of symbiotic
bacteria (B) inside a larva. Lc
larval cells. Scale bar 2 lm.
c SEM micrograph of a
metamorphic larva of H.
dujardini. Ex Exopinacocytes.
Scale bar 50 lm. d TEM photo
of symbiotic bacteria (B) inside
a metamorphic larva. Ex
Exopinacocytes, C cells of
metamorphic larva, N nucleus.
Scale bar 2 lm

karyotic or eukaryotic organisms as well as photosyn-
thetic and nonautotrophic symbionts that may be inter-
cellular or extracellular or both (Simpson 1984; Rützler
1990). There are three broad categories of bacterial
symbiosis in sponge bodies: (1) small populations of the
same nonspecific bacteria as in the surrounding seawater;
(2) large mesohyl populations of symbiotic bacteria,
which appear to constitute a specific flora; (3) small
populations of specific symbiotic intracellular bacteria
(Vacelet 1975). For Halisarca dujardini a fourth broad

category of bacterial symbiosis can be added: small
populations of specific symbiotic extracellular bacteria.
Symbiotic bacteria can be associated with marine
sponges in three ways: most are free living in the mes-
ohyl; large aggregates occur in bacteriocytes; and a few
are present in digestive vacuoles (Wilkinson 1978a,
1978b). In H. dujardini symbiotic bacteria are free living
in the mesohyl.
Vertical transmission of symbiotic bacteria between
generations is an important feature of sponge biology
and reproduction. This transmission can occur in dif-
ferent ways. The time and method of penetration also
varies in bacteria from different sponge species. At
present four modes of incorporation of the symbiotic
bacteria from maternal mesohyl into the egg or the
embryos are known (Fig. 5):
1. Oocytes gain bacteria directly from the adult mesohyl
by phagocytosis (Fig. 5a). This has been shown for
oviparous sponges: Verongia cavernicola (Gallissian
and Vacelet 1976), Chondrilla nucula (Gaino 1980),
Erylus discophorus (Sciscioli et al. 1989), Stelletta
grubii (Sciscioli et al. 1991), Geodia cydonium (Scisc-
ioli et al. 1994), Tethya tenuisclera and T. seychell-
ensis (Gaino and Sarà 1994), Chondrilla australiensis
(Usher et al. 2001). Our study of H. dujardini has
demonstrated this pathway of incorporation of the
symbiotic bacteria for the viviparous Demospongiae
for the first time.
Fig. 5 Diagram of different incorporation modes of the symbiotic
bacteria from maternal mesohyl into the egg or embryos of
sponges. a Phagocytosis of bacteria (B) by oocyte directly from the
adult mesohyl. b Incorporation of bacteria from maternal follicle
bacteriocytes (Bc) in the embryo of Chondrosia reniformis.
c Transfer of bacteria (B) from parent to embryo along a mucous
umbilicus (U) in some Dictyoceratida. F Follicle. d Penetration of
symbiotic bacteria (B) in the space between follicle (F) and egg
before closing of the follicle in Homoscleromorpha
873
2. The embryo of Chondrosia reniformis incorporates
bacteria from maternal follicle cells (Fig. 5b; Lévi
and Lévi 1976).
3. In four species of Dictyoceratida, Spongia barbara, S.
cheisis, S. graminea, and Hippospongia lachne, bac-
teria are apparently transferred from parent to em-
bryo along a mucous umbilicus during development
(Fig. 5c; Kaye 1991; Kaye and Reiswig 1991).
4. In Homoscleromorpha penetration of symbiotic
bacteria through the follicle occurs during late
oogenesis in the space between follicle and egg before
closing of the follicle (Fig. 5d; Ereskovsky and Bou-
ry-Esnault 2002).
In an early TEM investigation of Halisarca dujardini
oogenesis bacteria were not shown inside the egg (Ai-
senstadt and Korotkova 1976). The authors postulated
that the yolk originates mainly from phagocytosis of
mesohylar cells, and partly from authosynthetical pro-
cesses. Nevertheless we have shown the bacteria in the
eggs of these species, both undigested inside the vacuoles
and digested inside the yolk granules, to be formed. It is
evident that the symbiotic bacteria participate in the
formation of reserve material for embryonic develop-
ment in the eggs of H. dujardini.
In H. dujardini the transposition of bacteria between
blastomeres up to the blastocoel is possible. Transport
of the vacuoles with bacteria to the blastocoel starts in
the period of polarization of blastomeres and compac-
tion of the embryo. This process may be controlled by
intercellular transport.
Presently, symbiotic bacteria have been found in
embryos or larvae of all classes of Porifera: Demo-
spongiae—Alectona millary, A. wallichii, and A. mesat-
lantica (Garrone 1974; Vacelet 1999), Hamigera
hamigera (Boury-Esnault 1976), Aplysina cavernicola
(Gallissian and Vacelet 1976), Hemectyon ferox (Reiswig
1976), Chondrosia reniformis (Lévi and Lévi 1976), Va-
celetia crypta (Vacelet 1979), Spongia barbara, S.
graminea, Hippospongia lachne (Kaye 1991; Kaye and
Reiswig 1991), Haliclona tubifera (Woollacott 1993),
Cladorhiza sp. (Vacelet et al. 1995, 1996), Halisarca du-
jardini (Sizova and Ereskovsky 1997; Ereskovsky and
Gonobobleva 2000), Chondrilla australiensis (Usher
et al. 2001), Swenzea zeai (Rützler et al. 2003), Ircinia
oros (Ereskovsky and Tokina 2004), different species of
Homoscleromorpha (Ereskovsky and Boury-Esnault
2002; Boury-Esnault et al. 2003), Calcarea—Grantia
compressa (Lufty 1957; Gallissian 1983), Sycon ciliatum
(Franzen 1988), Hexactinellida—Oopsacas minuta (Bo-
ury-Esnault et al. 1999).
Gallissian and Vacelet (1976) excluded a trophic
function of symbiotic bacteria in the egg of Verongia.
Sciscioli et al. (1994) accepted that the participation of
the bacteria in the formation of reserve material is
minimal in the oocyte of Geodia cydonium. According to
our results the symbiotic bacteria in Halisarca dujardini
can participate in the formation of reserve material only
during oogenesis. The function of symbiotic bacteria in
874
embryos, larvae, rhagons, and adult sponges of H. du-
jardini is so far unknown.
Acknowledgements The authors thank Dr. Jean Vacelet and Nicole
Boury-Esnault for helpful discussions, and Chantal Bézac for help
during the SEM preparation. This work was funded by grant IN-
TAS-YSC 02-4441, by the program Universities of Russia no.
07.01.017, and by grant RFBR no. 03-04-49773; A. Ereskovsky
received a special grant from the Federal Scientific Policy of Bel-
gium, intended to promote collaboration S&T with oriental and
central Europe.
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