Mar Biol (2009) 156:1625–1632

DOI 10.1007/s00227-009-1198-x

ORIGINAL PAPER

Bacterial symbionts as an additional cytological marker for identiWcation of sponges without a skeleton

Andrey E. Vishnyakov · Alexander V. Ereskovsky

Received: 13 November 2008 / Accepted: 3 April 2009 / Published online: 18 April 2009© Springer-Verlag 2009

Abstract Symbiotic bacteria from six Oscarella species(adults and embryos) collected in the Mediterranean Sea(O. lobularis, O. tuberculata, O. imperialis, O. microlo-bata, O. viridis) and the Sea of Japan (O. malakhovi) wereinvestigated by scanning electron microscopy and transmis-sion electron microscopy. In most cases, symbionts arerather numerous. Each sponge species has a deWnite set ofbacterial morphological types. All bacteria are extracellu-lar. Symbionts occupy the mesohyl of adult sponges orintercellular space in embryos and are often in contact withmesohylar Wlaments or cells. Bacteria of some morphotypeshave characteristic blebs. Most symbionts are gram-nega-tive, and two types of bacteria have traits of Archaea andone type of bacteria is similar to Planctomycetes. Data onmorphology of bacterial symbionts can be a good addi-tional character for identiWcation of Oscarella species,which have no skeleton.

Introduction

Bacterial symbionts occur in many animal groups. Associa-tion with one or more species of bacterial symbionts isobligatory for members of the phylum Porifera (AlthoVet al. 1998; Sarà et al. 1998; Lopez et al. 1999). Spongescontain large numbers of bacteria (for review, see ImhoVand Stöhr 2003; Hentschel et al. 2006). The populationconsists mostly of extracellular bacteria that lie in the mes-ohyl matrix and are physically separated from the seawaterby pinacoderm.

These symbionts belong to about half of recognized bac-terial phyla and both major archaeal lineages (Hentschelet al. 2006; Taylor et al. 2007). It is a distinctive feature ofsymbiosis in sponges in comparison to well-studied othersymbiosis, in which one-host-one-symbiont variants ofassociations are usually recorded (Schmitt et al. 2007).According to 16S rRNA gene analysis, there are some bac-terial phyla, representatives of which are most frequentlyidentiWed as sponge-associated microorganisms (Hentschelet al. 2006; Taylor et al. 2007). In addition, the bacterialsymbiont diversity can be divided into specialists, spongeassociates, and generalists depending on their occurrence inseawater and sponges as hosts (Taylor et al. 2004). In theoverwhelming majority of these studies, taxonomically dis-tantly related sponge species were chosen (Wilkinson 1984;Hentschel et al. 2002; Taylor et al. 2007). DiVerent spongespecies from one genus can possess various bacteria mor-photypes (Boury-Esnault et al. 1992; Muricy et al. 1996,1999; Hentschel et al. 2001; Muricy and Pearse 2004; Eres-kovsky 2006). Nevertheless, there is very little comparativedata on symbiont diversity in sponges belonging to onetaxon (Erpenbeck et al. 2002).

The Homoscleromorpha contains one family, the Plaki-nidae Schulze, 1880 with seven genera (Muricy and Diaz

Communicated by Ulrich Sommer.

A. E. Vishnyakov (&) · A. V. EreskovskyDepartment of Invertebrate Zoology, Biological Faculty, St. Petersburg State University, 7/9 Universitetskaya nab, 199034 St. Petersburg, Russiae-mail: vishnyakov@hotmail.com

A. V. EreskovskyCentre d’Océanologie de Marseille, Station marine d’Endoume, Aix-Marseille Université, CNRS UMR 6540-DIMAR, rue de la Batterie des Lions, 13007 Marseille, France

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2002). The genus Oscarella Vosmaer, 1884 is representedby eight valid species. Most species of Oscarella have beendescribed from the Mediterranean (Boury-Esnault et al.1992; Muricy et al. 1996), there are also two Indo-PaciWcspecies (Bergquist and Kelly 2004) and two from the NorthPaciWc (Muricy and Pearse 2004; Ereskovsky 2006).

Cytological studies have been recently undertaken onMediterranean species from the Homoscleromorpha, suchas the genera Oscarella, Corticium, Plakina, Pseudocorti-cium (Boury-Esnault et al. 1992, 1995; Muricy et al. 1996,1999; Ereskovsky and Boury-Esnault 2002; de Caralt et al.2007; Riesgo et al. 2007). Some ultrastructure features,

such as sponge cell and microbial symbiont morphologywere used as diagnostic characters in their taxonomy.

Determination of Oscarella species with incrustinglobate body but without skeleton and spicules is fre-quently diYcult and demands detailed studies of theirWne structure (Muricy and Diaz 2002). In this paper, wedescribe in detail the ultrastructure of bacterial symbi-onts of six Oscarella species and show that the symbi-onts can be used as additional markers for these spongespecies.

Materials and methods

Symbiotic bacteria were investigated in embryos andadult sponges in Wve Oscarella species from the westernpart of the Mediterranean Sea: O. lobularis (Schmidt1862), O. tuberculata (Schmidt 1868), O. imperialisMuricy et al. 1996, O. microlobata Muricy et al. 1996,O. viridis Muricy et al. 1996, and one species—O. mala-khovi Ereskovsky 2006 from the western part of theJapan Sea.

The sponges were collected by SCUBA diving fromJune to August 1999 and August to September 2007, in thewestern Mediterranean Sea, at depths of 5–25 m (Fig. 1;Table 1), in August of 2006 in the Japan Sea at a depth of1–5 m. Pieces of each individual were Wxed in situ orimmediately after collection.

For transmission electron microscopy (TEM) andscanning electron microscopy (SEM), pieces of spongeswere Wxed in 2.5% glutaraldehyde in a mixture of 0.4 Mcacodylate buVer and seawater (1:4:5; 1,120 mOsm),post-Wxed in 2% OsO4 in seawater and dehydratedthrough a graded ethanol series. For SEM, specimenswere fractured in liquid nitrogen, critical-point-dried,sputter-coated with gold–palladium, and observed undera Hitachi S570 SEM. For TEM, the pieces were embed-ded in Araldite. Thin sections, stained with uranyl ace-tate and lead citrate, were observed under a Zeiss-1000TEM and LEO 910.

Fig. 1 Map of the north-west Mediterranean coast showing the sites ofcollection of the studied species. a Details of the Provence coast. Thenumbers refer to the sites of collection

Table 1 Stations and sampling depths of Oscarella species collected in the north-west Mediterranean. The numbers refer to the map (Fig. 1)

Station/species Oscarella lobularis O. tuberculata O. microlobata O. viridis O. imperialis

Endoume cave (1) 4–7 m 4–7 m – – –

Maire island (2) 13–15 m 13–15 m – – –

Jarre island (3) 20–24 m 7–9 m – – 22–25 m

Jarre cave (3) – 12–13 m 12–13 m 12–13 m –

Riou archipelago (4) 22–25 m 8–10 m – – –

La Vesse (5) 10–15 m 8–13 m – – –

3PP Cave (6) – – – – –

Medes islands (7) 15–17 m 13–17 m – – –

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Results

Target species characteristics

Sponges of the genus Oscarella only grow on rocky bot-toms. In the Mediterranean, which exhibits a high diversityof homoscleromorph sponges, Oscarella species are dwell-ers in sciaphilic hard substratum communities, such as thecoralligenous, semi-dark and dark submarine caves, and arefound from the Gibraltar Strait to the Eastern Mediterra-nean (Ereskovsky et al. 2009a). They are mainly located inshallow waters from 4 to 35 m, making the sampling aswell as in situ monitoring easy.

Oscarella lobularis (Fig. 2a) is thinly encrusting tolobate, from white to deep purple and sometimes blue. It isdistributed from shallow waters down to 300 m, in thecoralligenous community and at the entrance of caves.O. lobularis is one of the most common and abundanthomoscleromorphs in Mediterranean, conditioning speciWcfacies in some places. As other species of the same genus,O. lobularis seems to be a strong competitor for space,overgrowing massive sponges, sea fans and bryozoans(Ereskovsky et al. 2009b).

Oscarella tuberculata (Fig. 2b) is the “sister species” ofO. lobularis. O. tuberculata is also thinly encrusting tolobate, but its color is highly variable (yellow, green, blue,and sometimes pink). Its consistency is more cartilaginousthan O. lobularis, it harbors a particular type of vacuolarcell, which also allows distinguishing both “sister” species.It is also common in shallow coralligenous community.This species predominantly inhabits spotlit rocky habitat.Nevertheless, O. tuberculata has been found inside caves atdemi-obscure conditions.

Oscarella imperialis (Fig. 2c) is lobate thinly encrustingup to 2–20 mm thick. Colour is yellowish-white. Consistency

is soft like in O. lobularis. O. imperialis is an extremely rarespecies. It inhabits vertical calcareous wall at the depthbetween 15 and 25 m epizoic over a gorgonian. This speciesshares its habitat with O. lobularis and O. tuberculata.

Oscarella microlobata (Fig. 2d) and O. viridis (Fig. 2e)are sciophilous, cave-dwelling species. Both species wereonly registered in one or two Mediterranean underwatercaves (Ereskovsky et al. 2009a). For this research, we col-lected O. microlobata and O. viridis from sciaphilic darkzones of Jarre cave between 30 and 50 m from the entranceat 13–15 m depth. O. microlobata is a thinly encrustingsponge with a thickness of 3–8 mm. Colour is light brownto yellowish-brown. It is soft and fragile in consistence.O. viridis is also a thinly encrusting lobate sponge up to1–5 mm thick. Colour is light green. This sponge isextremely soft and fragile in consistency.

Oscarella malakhovi (Fig. 2f) inhabits the shell ofbivalve Crenomytilus grayanus, or on the lower side ofstones and rocks in the Gulf of the Peter the Great, VostokBay (42°54�N–132°38�W) at 0.4–4 m depth. As all Osca-rella, this sponge is thinly encrusting (1–2 mm thick) withlumpy, microlobate surface. It has pinky-beige to yellowcolor and soft, slimy consistency (Ereskovsky 2006).

Symbiotic bacteria description

In each species studied, adult sponges and their embryoshad the same set of symbiotic bacteria.

Symbionts of O. lobularis

Symbiotic bacteria of O. lobularis belong to three morpho-types (types a, b, and c). They have distinct characters butthe type of their bacterial wall is diYcult to deWne. Bacteriaof type a (Figs. 3, 4) are ovoid, about 0.8 �m in diameter

Fig. 2 In situ images of investigated Oscarella species. a O. lobularis, b O. tuberculata, c O. imperialis, d O. microlobata, e O. viridis, f O. malakhovi. Scale bars a 5 cm, b 4 cm, c 1 cm, d 2 cm, e 2 cm, f 2 cm

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and 1.5 �m in length. Cytoplasm consists of a central nucle-oid zone with a network of Wlaments and 1–2 dark granules,and a peripheral electron-opaque layer 60 nm thick. It is notquite clear whether the cell wall has one or two membranes.Over the membranes, there is a space, about 80 nm thick,with short, radially arranged Wlaments. We cannot be certainwhether these bacteria have a double membrane cell wallwith an S-layer, as in Archaea, or they are gram-positive.Symbionts of type b are rod-like, about 2.2 �m in length and0.6 �m in diameter (Figs. 3, 5). A remarkable feature ofthese bacteria is the presence of dark dots on the outer cellmembrane. They might be markers of some special interac-

tions with Wlaments and cells of sponge mesohyl. The nucle-oid is Wlamentous. Both types of symbionts are in closecontact with collagen Wlaments of mesohyl. Symbiotic bac-teria of type c are very small in diameter, 0.2 �m, and about2.0 �m in length (Figs. 3, 6). Membranes of the gram-nega-tive cell wall are tightly adjacent to each other. Nucleoidlooks like a Wlamentous network with a central narrow core.

Symbionts of O. tuberculata

Oscarella tuberculata has symbionts of two morphotypes(types a and b), which are in close contact with mesohyl

Figs. 3–14 Bacterial symbi-onts (types a, b, c) of Oscarella lobularis (SEM). Scale bar 0.5 �m. Fig. 4 Symbiotic bacte-ria (type a) of Oscarella lobularis (TEM). N nucleoid, Ps suggested S-layer of Archaea. Scale bar 0.5 �m. Fig. 5 Symbi-otic bacterium (type b) of Osca-rella lobularis (TEM). Dd dark dots on the outer cell membrane, N nucleoid. Scale bar 0.5 �m. Fig. 6 Symbiotic bacteria (type c) of Oscarella lobularis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 7 Bacterial symbionts (only type a) in Oscarella tuberculata mesohyl (SEM). Scale bar 0.5 �m. Fig. 8 Symbiotic bacterium (type a) of Oscarella tuberculata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 9 Symbiotic bacterium (type b) of Oscarella tuberculata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 10 Bacterial symbi-onts (types a, b, c, d) of Oscarella microlobata (SEM). Scale bar 0.5 �m. Fig. 11 Symbiotic bac-terium (type a) of Oscarella microlobata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 12 Symbiotic bacterium (type b) of Oscarella microlobata (TEM). N nucleoid, Sl suggested S-layer of Archaea. Scale bar 0.5 �m. Fig. 13 Symbiotic bacterium (type c) of Oscarella microlobata (TEM). Bb blebs, CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 14 Symbiotic bacteria (type d) of Oscarella microlobata (TEM). Bb blebs, CW cell wall, N nucleoid. Scale bar 0.5 �m

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Figs. 15–25 Fig. 15 Bacterial symbionts (types a, b) of Osca-rella imperialis (SEM). Scale bar 0.5 �m. Fig. 16 Symbiotic bacterium (type a) of Oscarella imperialis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 17 Symbiotic bacterium (type b) of Oscarella imperialis (TEM). CW cell wall, N nucle-oid. Scale bar 0.1 �m. Fig. 18 Symbiotic bacteria (type c) of Oscarella imperialis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 19 Bacterial symbionts (types a, b, c) of Oscarella viridis (SEM). Scale bar 0.5 �m. Fig. 20 Symbiotic bacterium (type a) of Oscarella viridis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 21 Symbiotic bacterium (type b) of Oscarella viridis (TEM). CW cell wall, N nucle-oid. Scale bar 0.5 �m. Fig. 22 Symbiotic bacterium (type c) of Oscarella viridis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m. Fig. 23 Bacterial symbi-onts (types a, b, c) of Oscarella malakhovi (SEM). Scale bar 0.5 �m. Fig. 24 Symbiotic bac-terium (type a) of Oscarella malakhovi (TEM). N nucleoid, Pp paryphoplasm. Scale bar 0.5 �m. Fig. 25 Symbiotic bac-terium (type b) of Oscarella malakhovi (TEM). CW cell wall, N nucleoid. Scale bar 0.5 �m

Figs. 26–29 Fig. 26 Symbiotic bacteria (types a, b, c) of Oscarella lobularis (TEM). Scale bar 0.5 �m. Fig. 27 Symbiotic bacteria (type a) of Oscarella tuberculata (TEM). Scale bar 0.5 �m. Fig. 28 Symbiotic bacteria (types a, b, c) of Oscarella viridis (TEM). Scale bar 0.5 �m. Fig. 29 Symbiotic bacteria (type a, b) of Oscarella malakhovi (TEM) Scale bar 0.5 �m

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Wlaments. Bacteria of the Wrst type (type a) (Figs. 7, 8) arerod-like, about 1.5 �m in length and 0.3 �m in diameter,with a gram-negative bacterial wall, which, in most cases,is poorly visible. A thin layer of cytoplasm surrounds anucleoid zone with a network of Wlaments. Bacteria of thesecond type (type b) (Fig. 9) are not numerous and have aspirilla-like body, about 0.3 �m in diameter and 2.0 �m inlength. The bacteria have a cell wall with two membranesand a Wlamentous nucleoid. The cytoplasm layer is thickerthan in the bacteria of the type a.

Symbionts of O. microlobata

Symbiotic bacteria of O. microlobata are usually sur-rounded by some space without mesohyl Wlaments. Theybelong to four morphotypes (types a, b, c, and d) with diVer-ent size characteristics and internal structure (Fig. 10). Thelargest bacteria (type a) (Fig. 11) are oval, about 0.9–1.0 �min diameter and 2.0 �m in length. The cell wall is mostprobably gram-negative, with a periplasmic space of highelectron density. A large volume of the nucleoid spaceincludes a network of Wlaments and electron-opaque bodies,similar in electron density to the peripheral cytoplasm. Insome cases, there is a thin wavy extracellular layer near thecell. The type b bacteria (Fig. 12), 0.6–0.8 �m in diameterand about 2.5 �m in length, have an unusual cell wall. Athick extracellular layer over gram-negative bacterial wallseems to be similar to the S-layer typical of Archaea andsome gram-positive bacteria. Intracellular volume includesa spacious network of nucleoid Wlaments and a small layerof the peripheral electron-opaque cytoplasm. Bacteria of thetypes c and d (Figs. 13, 14) are rod-like, their size is 0.4 and0.2 �m in diameter and 4.0 and 2.8 �m in length, respec-tively. An internal structure of their nucleoid and cytoplasmis similar to that of type b. However, both types have thegram-negative cell wall, which carries solitary blebs.

Symbionts of O. imperialis

Oscarella imperialis has symbiotic bacteria of three morpho-types (types a, b, and c). Bacteria of type a are elliptical, 0.6by 1.5 �m (Figs. 15, 16). A thin and dense layer of cytoplasmspreads under the gram-negative bacterial wall. A nucleoidregion is Wlamentous. Symbionts of morphological type b are0.3 �m in diameter and about 1.5 �m in length (Figs. 15, 17).Gram-negative cell wall, a homogenous cytoplasmic layer ofan average electron density, and a Wlamentous nucleoid zoneare characteristics of these bacteria. The third morphotype ofsymbionts, type c, was observed only once and includesbacteria that have a rod-like hexahedral shape, 0.75 �m longby 0.2 �m wide (Fig. 18). They diVer from the other bacteriaby a very electron-dense cytoplasm and a two part nucleoid.The cell wall is gram-negative.

Symbionts of O. viridis

Oscarella viridis has three morphological types of bacterialsymbionts. Biggest and ovoid bacteria (type a) are about1.2 �m in length and 0.6 �m in diameter (Figs. 19, 20).A layer of dense cytoplasm and gram-negative cell wallsurround the spacious nucleoid region. Symbionts of thistype rarely occur in TEM images. In contrast to this, symbi-onts of type b and c are most numerous. The cells of type blook like short rods with diameter 0.4–0.5 �m and maxi-mum length about 2.0–2.5 �m (Figs. 19, 21). A layer ofextracellular substance over double membrane cell wall issimilar to type b symbionts of O. microlobata. In somecells, the outline of this layer is dense enough to be con-fused with the membrane structure. The peripheral cyto-plasm is dark and the nucleoid region has a network ofWlaments. Oval (0.15–0.25 �m in diameter) electron-trans-parent inclusions occur in the cytoplasm. The symbionts ofthe third morphotype (type c) are long and narrow rods with0.2–0.22 �m in diameter and about 2.0 �m in length(Figs. 19, 22). The outer membrane of the gram-negativecell wall has blebs and sometimes is wavy. The contents ofthe cytoplasm are dark close to the cytoplasmic membraneand there is a globular network of Wlaments in the nucleoidregion.

Symbionts of O. malakhovi

Oscarella malakhovi has two morphotypes of symbioticbacteria (types a and b) (Figs. 23, 24, 25). Bacteria of typea (Figs. 23, 24) are more numerous. They are about 1.8 �min length, 0.5 �m in diameter in the middle part of the celland 0.8 �m in diameter near the distal ends. Below thegram-negative cell wall, there is a vast transparent spacewith some Wlaments. The space is similar to paryphoplasmof Planctomycetes, according to Fuerst (2005). Thicknessof the space is very variable. In some images, this space hasan invagination to the central electron dense part of the cell,which is surrounded by a membrane. The Wlamentous net-work of the nucleoid is irregular, with thick elements in thecentre and thin Wlaments closer to periphery. Bacteria oftype b (Fig. 23, 25) have an elongated, slightly curvedshape. Their cell diameter is about 0.25 �m, the length isabout 1.0 �m. The cell wall consists of two membranes(gram-negative bacteria). Thick nucleoid Wlaments form aregular voluminous structure with a thin periphery of thedark cytoplasm.

Discussion

Modern consideration of sponges as organisms in perma-nent and obligatory symbiotic association with bacteria

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opens new perspectives in investigation of symbiosis ascommensal microbe–host interaction (Schmitt et al. 2007)and in practical utilization of these animals (Hentschel et al.2006). Stability of this association suggests an intimaterelationship between the host and the symbionts, so that thelatter can be regarded along with the host’s own cells(Weisz et al. 2007). Therefore, bacteria might be consid-ered as a good additional species marker for some of thesponges without a skeleton.

The taxonomy of sponges is mainly based on the charac-ters of the skeleton, Wbres and spicules. The identiWcationof Oscarella at the species level is diYcult because speciesof this genus have no skeleton, and histological charactersare homogeneous. The diVerences among species aremostly in external traits: color, consistency, and aspect ofthe surface (Boury-Esnault et al. 1992; Muricy et al. 1996;Muricy and Diaz 2002; Bergquist and Kelly 2004; Muricyand Pearse 2004; Ereskovsky 2006). But these characterdescriptions can be highly subjective. At the same time,cytological characters, such as the types of cells with inclu-sions present and symbiotic bacteria morphology, are veryimportant for Oscarella species identiWcation (Muricy et al.1996; Muricy 1999; Muricy and Pearse 2004; Ereskovsky2006).

Our research supplements and somewhat corrects earlierdata on symbionts from Oscarella species. In O. tubercu-lata, we found a new bacterial morphotype—type b, whichis much more infrequent than the other one. Symbiont mor-photypes a and b of O. microlobata correspond to types aand b of previous description, but we are inclined to con-sider morphotype c, described by Muricy et al. (1996) as avariant of morphotype a. Bacteria of morphotypes c and dare similar to morphotypes e and f in the description ofMuricy et al. (1996), correspondingly, with one addition:morphotype c (e in: Muricy et al. 1996) is rod-like. Unfor-tunately, our SEM and TEM images did not show vermi-form bacteria of type d. Probably, they are rather rare, as isthe morphotype b of O. tuberculata. From three symbiontmorphotypes of O. imperialis described in our work, thetype b is similar to the only symbiont morphotype in theprevious description (Muricy et al. 1996). In O. lobularis,we also found two new symbiont morphotypes in additionto the one described early by Boury-Esnault et al. (1992).The fact of similarity of symbionts of type b in O. viridisand O. microlobata (and may be type c and d, correspond-ingly, too) rouse the interest because both of these spongespecies were found only in one cave in the MediterraneanSea near Marseille as neighbors. It can suggest a parallelevolution of these two species from one ancestor, whichalready had at least a part of representatives of symbioticcommunity characteristic of modern species.

O. malakhovi, a sponge from the Japan Sea (Ereskovsky2006), is a second species of genus Oscarella in PaciWc

Ocean described after O. carmela (Muricy and Pearse2004). Symbiotic bacteria of these two species are com-pletely diVerent and easily distinguishable according totheir morphological traits.

The morphological investigations using TEM and SEMusually provide a starting point for research, calling for fur-ther molecular identiWcation with rRNA group-speciWc oli-gonucleotides. Nevertheless, they are suYcient to show, forinstance, in O. microlobata (morphotype b) and probably inO. lobularis (morphotype a) and O. viridis (morphotype b)the presence of a bacterial morphotype with a cell wall sim-ilar to that of Archaea. Our data extend the earlier list ofsponge species with these kinds of symbionts (Preston et al.1996; Webster et al. 2001; Margot et al. 2002). Symbiontsof one of the morphotypes of O. malakhovi from the JapanSea (morphotype a) closely resemble Planctomycetes, bac-teria with a nucleoid enclosed by membrane, recently foundin other sponges (Fuerst et al. 1998; Fieseler et al. 2004;Fuerst 2005).

Among all bacterial morphotypes of Oscarella only twoof them, b in O. tuberculata and c in O. imperialis, can beconsidered as very rare or may be sporadic bacteria in thesponges. Other symbiotic bacteria are constant componentof mesohyl in studied sponges. Well-irrigated tissues ofthese sponges have a low density of symbiotic microorgan-isms (Figs. 26, 27, 28, 29). This correlation was describedearlier (Vacelet and Donadey 1977). However, in our studythe number of morphological types of bacteria in majorityof Oscarella species studied can be more, than one, asreported by Vacelet and Donadey (1977). It is obvious thatthe proportion of symbionts of diVerent morphotypes inevery species can depend on the environment, seasons andso on. This aspect suggests a further study.

Host–symbiont system of some Oscarella species fromdiVerent regions of the World Ocean can be a convenientmodel for a comparative study of coevolution processesand correlation between variants of symbiotic associationand environment factors.

Acknowledgments The authors are grateful to Chantal Bézac, Cen-tre d’Océanologie de Marseille, France and Daria Tokina, ZoologicalInstitute RAS, St. Petersburg, Russia, for technical assistance, ThierryPerez, Roland Graille and Pierre Chevaldonné for diving assistance.Financial support for this work was provided by grants RFBR NN 07-04-01097, 06-04-48660 and European Marie Curie Mobility program(fellowship of A. Ereskovsky, MIF1-CT-2006-040065).

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