Introduction

Bacterial symbionts occur in many animal groups. Association with one or more species of bacterial symbionts is obligatory for members of the phylum Porifera (Althoff et al. 1998; Sarà et al. 1998; Lopez et al. 1999). Sponges contain large numbers of bacteria (for review, see Imhoff and Stöhr 2003; Hentschel et al. 2006). The population consists mostly of extracellular bacteria that lie in the mesohyl matrix and are physically separated from the seawater by pinacoderm.

These symbionts belong to about half of recognized bacterial phyla and both major archaeal lineages (Hentschel et al. 2006; Taylor et al. 2007). It is a distinctive feature of symbiosis in sponges in comparison to well-studied other symbiosis, in which one-host-one-symbiont variants of associations are usually recorded (Schmitt et al. 2007). According to 16S rRNA gene analysis, there are some bacterial phyla, representatives of which are most frequently identified as sponge-associated microorganisms (Hentschel et al. 2006; Taylor et al. 2007). In addition, the bacterial symbiont diversity can be divided into specialists, sponge associates, and generalists depending on their occurrence in seawater and sponges as hosts (Taylor et al. 2004). In the overwhelming majority of these studies, taxonomically distantly related sponge species were chosen (Wilkinson 1984; Hentschel et al. 2002; Taylor et al. 2007). Different sponge species from one genus can possess various bacteria morphotypes (Boury-Esnault et al. 1992; Muricy et al. 1996, 1999; Hentschel et al. 2001; Muricy and Pearse 2004; Ereskovsky 2006). Nevertheless, there is very little comparative data on symbiont diversity in sponges belonging to one taxon (Erpenbeck et al. 2002).

The Homoscleromorpha contains one family, the Plakinidae Schulze, 1880 with seven genera (Muricy and Diaz 2002). The genus Oscarella Vosmaer, 1884 is represented by eight valid species. Most species of Oscarella have been described from the Mediterranean (Boury-Esnault et al. 1992; Muricy et al. 1996), there are also two Indo-Pacific species (Bergquist and Kelly 2004) and two from the North Pacific (Muricy and Pearse 2004; Ereskovsky 2006).

Cytological studies have been recently undertaken on Mediterranean species from the Homoscleromorpha, such as the genera Oscarella, Corticium, Plakina, Pseudocorticium (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 morphology were used as diagnostic characters in their taxonomy.

Determination of Oscarella species with incrusting lobate body but without skeleton and spicules is frequently difficult and demands detailed studies of their fine structure (Muricy and Diaz 2002). In this paper, we describe in detail the ultrastructure of bacterial symbionts of six Oscarella species and show that the symbionts can be used as additional markers for these sponge species.

Materials and methods

Symbiotic bacteria were investigated in embryos and adult sponges in five Oscarella species from the western part of the Mediterranean Sea: O. lobularis (Schmidt 1862), O. tuberculata (Schmidt 1868), O. imperialis Muricy et al. 1996, O. microlobata Muricy et al. 1996, O. viridis Muricy et al. 1996, and one species—O. malakhovi Ereskovsky 2006 from the western part of the Japan Sea.

The sponges were collected by SCUBA diving from June to August 1999 and August to September 2007, in the western Mediterranean Sea, at depths of 5–25 m (Fig. 1; Table 1), in August of 2006 in the Japan Sea at a depth of 1–5 m. Pieces of each individual were fixed in situ or immediately after collection.

Fig. 1
figure 1

Map of the north-west Mediterranean coast showing the sites of collection of the studied species. a Details of the Provence coast. The numbers refer to the sites of collection

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Table 1 Stations and sampling depths of Oscarella species collected in the north-west Mediterranean. The numbers refer to the map (Fig. 1)
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For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), pieces of sponges were fixed in 2.5% glutaraldehyde in a mixture of 0.4 M cacodylate buffer and seawater (1:4:5; 1,120 mOsm), post-fixed in 2% OsO4 in seawater and dehydrated through a graded ethanol series. For SEM, specimens were fractured in liquid nitrogen, critical-point-dried, sputter-coated with gold–palladium, and observed under a Hitachi S570 SEM. For TEM, the pieces were embedded in Araldite. Thin sections, stained with uranyl acetate and lead citrate, were observed under a Zeiss-1000 TEM and LEO 910.

Results

Target species characteristics

Sponges of the genus Oscarella only grow on rocky bottoms. In the Mediterranean, which exhibits a high diversity of homoscleromorph sponges, Oscarella species are dwellers in sciaphilic hard substratum communities, such as the coralligenous, semi-dark and dark submarine caves, and are found from the Gibraltar Strait to the Eastern Mediterranean (Ereskovsky et al. 2009a). They are mainly located in shallow waters from 4 to 35 m, making the sampling as well as in situ monitoring easy.

Oscarella lobularis (Fig. 2a) is thinly encrusting to lobate, from white to deep purple and sometimes blue. It is distributed from shallow waters down to 300 m, in the coralligenous community and at the entrance of caves. O. lobularis is one of the most common and abundant homoscleromorphs in Mediterranean, conditioning specific facies 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).

Fig. 2
figure 2

In situ images of investigated Oscarella species. aO. lobularis, bO. tuberculata, cO. imperialis, dO. microlobata, eO. viridis, fO. 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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Oscarella tuberculata (Fig. 2b) is the “sister species” of O. lobularis. O. tuberculata is also thinly encrusting to lobate, but its color is highly variable (yellow, green, blue, and sometimes pink). Its consistency is more cartilaginous than O. lobularis, it harbors a particular type of vacuolar cell, 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 at demi-obscure conditions.

Oscarella imperialis (Fig. 2c) is lobate thinly encrusting up to 2–20 mm thick. Colour is yellowish-white. Consistency is soft like in O. lobularis. O. imperialis is an extremely rare species. It inhabits vertical calcareous wall at the depth between 15 and 25 m epizoic over a gorgonian. This species shares its habitat with O. lobularis and O. tuberculata.

Oscarella microlobata (Fig. 2d) and O. viridis (Fig. 2e) are sciophilous, cave-dwelling species. Both species were only registered in one or two Mediterranean underwater caves (Ereskovsky et al. 2009a). For this research, we collected O. microlobata and O. viridis from sciaphilic dark zones of Jarre cave between 30 and 50 m from the entrance at 13–15 m depth. O. microlobata is a thinly encrusting sponge with a thickness of 3–8 mm. Colour is light brown to yellowish-brown. It is soft and fragile in consistence. O. viridis is also a thinly encrusting lobate sponge up to 1–5 mm thick. Colour is light green. This sponge is extremely soft and fragile in consistency.

Oscarella malakhovi (Fig. 2f) inhabits the shell of bivalve Crenomytilus grayanus, or on the lower side of stones and rocks in the Gulf of the Peter the Great, Vostok Bay (42°54′N–132°38′W) at 0.4–4 m depth. As all Oscarella, this sponge is thinly encrusting (1–2 mm thick) with lumpy, microlobate surface. It has pinky-beige to yellow color and soft, slimy consistency (Ereskovsky 2006).

Symbiotic bacteria description

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

Symbionts of O. lobularis

Symbiotic bacteria of O. lobularis belong to three morphotypes (types a, b, and c). They have distinct characters but the type of their bacterial wall is difficult to define. Bacteria of type a (Figs. 3, 4) are ovoid, about 0.8 μm in diameter and 1.5 μm in length. Cytoplasm consists of a central nucleoid zone with a network of filaments and 1–2 dark granules, and a peripheral electron-opaque layer 60 nm thick. It is not quite 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 filaments. We cannot be certain whether these bacteria have a double membrane cell wall with an S-layer, as in Archaea, or they are gram-positive. Symbionts of type b are rod-like, about 2.2 μm in length and 0.6 μm in diameter (Figs. 3, 5). A remarkable feature of these bacteria is the presence of dark dots on the outer cell membrane. They might be markers of some special interactions with filaments and cells of sponge mesohyl. The nucleoid is filamentous. Both types of symbionts are in close contact with collagen filaments of mesohyl. Symbiotic bacteria of type c are very small in diameter, 0.2 μm, and about 2.0 μm in length (Figs. 3, 6). Membranes of the gram-negative cell wall are tightly adjacent to each other. Nucleoid looks like a filamentous network with a central narrow core.

Figs. 3–14
figure 3-14

Bacterial symbionts (types a, b, c) of Oscarella lobularis (SEM). Scale bar 0.5 μm. Fig. 4 Symbiotic bacteria (type a) of Oscarella lobularis (TEM). N nucleoid, Ps suggested S-layer of Archaea. Scale bar 0.5 μm. Fig. 5 Symbiotic bacterium (type b) of Oscarella lobularis (TEM). Dd dark dots on the outer cell membrane, N nucleoid. Scale bar 0.5 μm. Fig. 6 Symbiotic bacteria (type c) of Oscarella lobularis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 7 Bacterial symbionts (only type a) in Oscarella tuberculata mesohyl (SEM). Scale bar 0.5 μm. Fig. 8 Symbiotic bacterium (type a) of Oscarella tuberculata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 9 Symbiotic bacterium (type b) of Oscarella tuberculata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 10 Bacterial symbionts (types a, b, c, d) of Oscarella microlobata (SEM). Scale bar 0.5 μm. Fig. 11 Symbiotic bacterium (type a) of Oscarella microlobata (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 12 Symbiotic bacterium (type b) of Oscarella microlobata (TEM). N nucleoid, Sl suggested S-layer of Archaea. Scale bar 0.5 μm. Fig. 13 Symbiotic bacterium (type c) of Oscarella microlobata (TEM). Bb blebs, CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 14 Symbiotic bacteria (type d) of Oscarella microlobata (TEM). Bb blebs, CW cell wall, N nucleoid. Scale bar 0.5 μm

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Figs. 15–25
figure 15-25

Fig. 15 Bacterial symbionts (types a, b) of Oscarella imperialis (SEM). Scale bar 0.5 μm. Fig. 16 Symbiotic bacterium (type a) of Oscarella imperialis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 17 Symbiotic bacterium (type b) of Oscarella imperialis (TEM). CW cell wall, N nucleoid. Scale bar 0.1 μm. Fig. 18 Symbiotic bacteria (type c) of Oscarella imperialis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 19 Bacterial symbionts (types a, b, c) of Oscarella viridis (SEM). Scale bar 0.5 μm. Fig. 20 Symbiotic bacterium (type a) of Oscarella viridis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 21 Symbiotic bacterium (type b) of Oscarella viridis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 22 Symbiotic bacterium (type c) of Oscarella viridis (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm. Fig. 23 Bacterial symbionts (types a, b, c) of Oscarella malakhovi (SEM). Scale bar 0.5 μm. Fig. 24 Symbiotic bacterium (type a) of Oscarella malakhovi (TEM). N nucleoid, Pp paryphoplasm. Scale bar 0.5 μm. Fig. 25 Symbiotic bacterium (type b) of Oscarella malakhovi (TEM). CW cell wall, N nucleoid. Scale bar 0.5 μm

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Figs. 26–29
figure 26-29

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

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Symbionts of O. tuberculata

Oscarella tuberculata has symbionts of two morphotypes (types a and b), which are in close contact with mesohyl filaments. Bacteria of the first type (type a) (Figs. 7, 8) are rod-like, about 1.5 μm in length and 0.3 μm in diameter, with a gram-negative bacterial wall, which, in most cases, is poorly visible. A thin layer of cytoplasm surrounds a nucleoid zone with a network of filaments. Bacteria of the second type (type b) (Fig. 9) are not numerous and have a spirilla-like body, about 0.3 μm in diameter and 2.0 μm in length. The bacteria have a cell wall with two membranes and a filamentous nucleoid. The cytoplasm layer is thicker than in the bacteria of the type a.

Symbionts of O. microlobata

Symbiotic bacteria of O. microlobata are usually surrounded by some space without mesohyl filaments. They belong to four morphotypes (types a, b, c, and d) with different size characteristics and internal structure (Fig. 10). The largest bacteria (type a) (Fig. 11) are oval, about 0.9–1.0 μm in diameter and 2.0 μm in length. The cell wall is most probably gram-negative, with a periplasmic space of high electron density. A large volume of the nucleoid space includes a network of filaments and electron-opaque bodies, similar in electron density to the peripheral cytoplasm. In some cases, there is a thin wavy extracellular layer near the cell. The type b bacteria (Fig. 12), 0.6–0.8 μm in diameter and about 2.5 μm in length, have an unusual cell wall. A thick extracellular layer over gram-negative bacterial wall seems to be similar to the S-layer typical of Archaea and some gram-positive bacteria. Intracellular volume includes a spacious network of nucleoid filaments and a small layer of the peripheral electron-opaque cytoplasm. Bacteria of the types c and d (Figs. 13, 14) are rod-like, their size is 0.4 and 0.2 μm in diameter and 4.0 and 2.8 μm in length, respectively. An internal structure of their nucleoid and cytoplasm is similar to that of type b. However, both types have the gram-negative cell wall, which carries solitary blebs.

Symbionts of O. imperialis

Oscarella imperialis has symbiotic bacteria of three morphotypes (types a, b, and c). Bacteria of type a are elliptical, 0.6 by 1.5 μm (Figs. 15, 16). A thin and dense layer of cytoplasm spreads under the gram-negative bacterial wall. A nucleoid region is filamentous. Symbionts of morphological type b are 0.3 μm in diameter and about 1.5 μm in length (Figs. 15, 17). Gram-negative cell wall, a homogenous cytoplasmic layer of an average electron density, and a filamentous nucleoid zone are characteristics of these bacteria. The third morphotype of symbionts, type c, was observed only once and includes bacteria that have a rod-like hexahedral shape, 0.75 μm long by 0.2 μm wide (Fig. 18). They differ from the other bacteria by 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 bacterial symbionts. Biggest and ovoid bacteria (type a) are about 1.2 μm in length and 0.6 μm in diameter (Figs. 19, 20). A layer of dense cytoplasm and gram-negative cell wall surround the spacious nucleoid region. Symbionts of this type rarely occur in TEM images. In contrast to this, symbionts of type b and c are most numerous. The cells of type b look like short rods with diameter 0.4–0.5 μm and maximum length about 2.0–2.5 μm (Figs. 19, 21). A layer of extracellular substance over double membrane cell wall is similar to type b symbionts of O. microlobata. In some cells, the outline of this layer is dense enough to be confused with the membrane structure. The peripheral cytoplasm is dark and the nucleoid region has a network of filaments. Oval (0.15–0.25 μm in diameter) electron-transparent inclusions occur in the cytoplasm. The symbionts of the third morphotype (type c) are long and narrow rods with 0.2–0.22 μm in diameter and about 2.0 μm in length (Figs. 19, 22). The outer membrane of the gram-negative cell wall has blebs and sometimes is wavy. The contents of the cytoplasm are dark close to the cytoplasmic membrane and there is a globular network of filaments in the nucleoid region.

Symbionts of O. malakhovi

Oscarella malakhovi has two morphotypes of symbiotic bacteria (types a and b) (Figs. 23, 24, 25). Bacteria of type a (Figs. 23, 24) are more numerous. They are about 1.8 μm in length, 0.5 μm in diameter in the middle part of the cell and 0.8 μm in diameter near the distal ends. Below the gram-negative cell wall, there is a vast transparent space with some filaments. The space is similar to paryphoplasm of Planctomycetes, according to Fuerst (2005). Thickness of the space is very variable. In some images, this space has an invagination to the central electron dense part of the cell, which is surrounded by a membrane. The filamentous network of the nucleoid is irregular, with thick elements in the centre and thin filaments closer to periphery. Bacteria of type b (Fig. 23, 25) have an elongated, slightly curved shape. Their cell diameter is about 0.25 μm, the length is about 1.0 μm. The cell wall consists of two membranes (gram-negative bacteria). Thick nucleoid filaments form a regular voluminous structure with a thin periphery of the dark cytoplasm.

Discussion

Modern consideration of sponges as organisms in permanent and obligatory symbiotic association with bacteria opens new perspectives in investigation of symbiosis as commensal microbe–host interaction (Schmitt et al. 2007) and in practical utilization of these animals (Hentschel et al. 2006). Stability of this association suggests an intimate relationship between the host and the symbionts, so that the latter can be regarded along with the host’s own cells (Weisz et al. 2007). Therefore, bacteria might be considered as a good additional species marker for some of the sponges without a skeleton.

The taxonomy of sponges is mainly based on the characters of the skeleton, fibres and spicules. The identification of Oscarella at the species level is difficult because species of this genus have no skeleton, and histological characters are homogeneous. The differences among species are mostly in external traits: color, consistency, and aspect of the surface (Boury-Esnault et al. 1992; Muricy et al. 1996; Muricy and Diaz 2002; Bergquist and Kelly 2004; Muricy and Pearse 2004; Ereskovsky 2006). But these character descriptions can be highly subjective. At the same time, cytological characters, such as the types of cells with inclusions present and symbiotic bacteria morphology, are very important for Oscarella species identification (Muricy et al. 1996; Muricy 1999; Muricy and Pearse 2004; Ereskovsky 2006).

Our research supplements and somewhat corrects earlier data on symbionts from Oscarella species. In O. tuberculata, we found a new bacterial morphotype—type b, which is much more infrequent than the other one. Symbiont morphotypes a and b of O. microlobata correspond to types a and b of previous description, but we are inclined to consider morphotype c, described by Muricy et al. (1996) as a variant of morphotype a. Bacteria of morphotypes c and d are similar to morphotypes e and f in the description of Muricy et al. (1996), correspondingly, with one addition: morphotype c (e in: Muricy et al. 1996) is rod-like. Unfortunately, our SEM and TEM images did not show vermiform bacteria of type d. Probably, they are rather rare, as is the morphotype b of O. tuberculata. From three symbiont morphotypes of O. imperialis described in our work, the type b is similar to the only symbiont morphotype in the previous description (Muricy et al. 1996). In O. lobularis, we also found two new symbiont morphotypes in addition to the one described early by Boury-Esnault et al. (1992). The fact of similarity of symbionts of type b in O. viridis and O. microlobata (and may be type c and d, correspondingly, too) rouse the interest because both of these sponge species were found only in one cave in the Mediterranean Sea near Marseille as neighbors. It can suggest a parallel evolution of these two species from one ancestor, which already had at least a part of representatives of symbiotic community characteristic of modern species.

O. malakhovi, a sponge from the Japan Sea (Ereskovsky 2006), is a second species of genus Oscarella in Pacific Ocean described after O. carmela (Muricy and Pearse 2004). Symbiotic bacteria of these two species are completely different and easily distinguishable according to their morphological traits.

The morphological investigations using TEM and SEM usually provide a starting point for research, calling for further molecular identification with rRNA group-specific oligonucleotides. Nevertheless, they are sufficient to show, for instance, in O. microlobata (morphotype b) and probably in O. lobularis (morphotype a) and O. viridis (morphotype b) the presence of a bacterial morphotype with a cell wall similar to that of Archaea. Our data extend the earlier list of sponge species with these kinds of symbionts (Preston et al. 1996; Webster et al. 2001; Margot et al. 2002). Symbionts of one of the morphotypes of O. malakhovi from the Japan Sea (morphotype a) closely resemble Planctomycetes, bacteria with a nucleoid enclosed by membrane, recently found in other sponges (Fuerst et al. 1998; Fieseler et al. 2004; Fuerst 2005).

Among all bacterial morphotypes of Oscarella only two of them, b in O. tuberculata and c in O. imperialis, can be considered as very rare or may be sporadic bacteria in the sponges. Other symbiotic bacteria are constant component of mesohyl in studied sponges. Well-irrigated tissues of these sponges have a low density of symbiotic microorganisms (Figs. 26, 27, 28, 29). This correlation was described earlier (Vacelet and Donadey 1977). However, in our study the number of morphological types of bacteria in majority of Oscarella species studied can be more, than one, as reported by Vacelet and Donadey (1977). It is obvious that the proportion of symbionts of different morphotypes in every species can depend on the environment, seasons and so on. This aspect suggests a further study.

Host–symbiont system of some Oscarella species from different regions of the World Ocean can be a convenient model for a comparative study of coevolution processes and correlation between variants of symbiotic association and environment factors.