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CTENIDIAL STRUCTURE AND THREE BACTERIAL SYMBIONT MORPHOTYPES IN ANODONTIA (EUANODONTIA) OVUM (REEVE, 1850) FROM THE GREAT BARRIER REEF, AUSTRALIA (BIVALVIA: LUCINIDAE) ALEXANDER D. BALL 1 , KEVIN J. PURDY 3, EMILY A. GLOVER 2 AND JOHN D. TAYLOR 2 1 Department of Mineralogy, The Natural History Museum, London SW7 5BD, UK; Department of Zoology, The Natural History Museum, London SW7 5BD, UK; and Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK 2 3 (Received 6 August 2008; accepted 30 January 2009) ABSTRACT The structure of the ctenidia of the Indo-West Pacific chemosymbiotic lucinid bivalve Anodontia (Euanodontia) ovum was investigated by electron microscopy. Ctenidial filaments are similar in general morphology to those described from other Lucinidae, with a ciliated zone, a short intermediary zone and a thick abfrontal zone composed largely of bacteriocytes separated by narrow intercalary cells. The bacteriocyte zones of adjacent filaments are fused in the distal part to form short cylindrical channels. The apices of intercalary cells project as cytoplasmic protrusions in the form of spiky tufts, with sheets and tendrils spreading over adjacent bacteriocytes. Compared with other lucinids A. ovum lacks abfrontal granule cells, mucocytes are infrequent and the bacteriocyte channels are short. Three morphotypes of symbiotic bacteria were detected, associated with the bacteriocyte zone of the ctenidial filaments: (1) all bacteriocytes contained abundant bacteria 3–5 mm long and 0.5–1.0 mm wide, enclosed in single vacuoles; (2) some bacteriocytes possessed spherical vesicles enclosing masses of smaller rod-shaped bacteria c. 1.0 mm long; (3) probable spirochaete bacteria, 8–10 mm long and 0.3 mm wide, were abundant within the apical cytoplasmic protrusions of the intercalary cells. Preliminary molecular analysis using 16S rRNA gene sequences has so far identified only one bacterial symbiont, from the gamma division of Proteobacteria grouping in a cluster of symbiotic thiotrophs. This symbiont of A. ovum is closely similar to a symbiont previously reported from the western Atlantic lucinid Anodontia schrammi (originally cited as A. philippiana). INTRODUCTION Chemosymbiosis between marine bivalves of the family Lucinidae and sulphide-oxidizing bacteria housed in their ctenidia is now well known from numerous studies (Giere, 1985; Dando et al., 1985; Dando, Southward & Southward, 1986; Reid & Brand, 1986; Distel & Felbeck, 1987; Fisher, 1990; Distel, 1998; Taylor & Glover, 2000, 2006). The association is likely to be obligate, as all studied species possess the symbionts, known for a few species to be acquired by environmental transmission from the surrounding sediment (Gros, Frenkiel & Mouëza, 1998; Gros, Durand, Frenkiel & Mouëza, 1998; Gros et al., 2003). Nevertheless, lucinids are functionally capable of particulate feeding (Duplessis et al., 2004). The ctenidia of Lucinidae comprise large, thickened, single demibranchs that are structurally modified to house the symbionts. Ctenidial structure has been investigated in detail for a variety of lucinids including: Phacoides pectinata (Frenkiel, Gros & Mouëza, 1996); Codakia orbicularis (Frenkiel & Mouëza, 1995; Gros, Liberge & Felbeck, 2003); Lucinoma borealis (Dando et al., 1986); Lucina pensylvanica (as Linga; Gros, Frenkiel & Mouëza, 1996); Lucinoma aequizonata (Distel & Felbeck, 1987); Parvilucina tenuisculpta (Reid & Brand, 1986); Parvilucina crenella (as P. multilineata; Giere, 1985); Divaricella quadrisulcata (Gros, Frenkiel & Felbeck, 2000); Parvilucina costata – (as Lucina (?Parvilucina); Giere, 1985); Loripes lucinalis (Herry, Diouris & Correspondence: J.D. Taylor; email: [email protected] Le Pennec, 1989); Lucinella divaricata (Herry & Le Pennec, 1987); Stewartia floridana (as Lucina; Fisher & Hand, 1984); Anodontia alba (Gros et al., 2003) and A. (Pegophysema) schrammi (as A. philippiana; Giere, 1985). Ctenidial structure appears generally similar in most Lucinidae, but significant differences have been recorded between species, for example the presence of abundant granule cells in taxa such as Codakia, Lucinoma and Divaricella that are absent in others. Other variable features include the presence of peroxisomes, abundance of mucocytes, length of the intermediary zone and length of the bacteriocyte channels, but there has been no systematic analysis of these characters. Investigations of ctenidial structure have largely concerned northern Atlantic or northeastern Pacific species and, excepting a brief account (Janssen, 1992), the more diverse faunas of Lucinidae in the tropical Indo-West Pacific region have hardly been studied. Identification of the bacterial symbionts of lucinids is again largely based on studies of western Atlantic, northeastern Pacific (Distel, Felbeck & Cavanaugh, 1994; Gros, Liberge & Felbeck, 2003; Caro et al., 2007) or eastern Mediterranean taxa (Duperron et al., 2007). The symbionts, identified by 16S rRNA gene-sequence analysis, are all Gamma-Proteobacteria and group together with sulphide-oxidizing symbionts from Solemyidae, Thyasiridae and siboglinid polychaetes (Stewart, Newton & Cavanaugh, 2005). To date, only single taxa of symbionts have been identified from each lucinid host, with the possible exception of the report by Rodionov & Yushin (1991). Significantly, Journal of Molluscan Studies (2009) 75: 175–185. Advance Access Publication: 14 March 2009 # The Author 2009. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved doi:10.1093/mollus/eyp009 A. D. BALL ET AL. Gros, Liberge & Felbeck (2003) showed that six species of Caribbean lucinids all harboured the same symbiont species. Recent molecular analyses of Lucinidae (Williams, Taylor & Glover, 2004; Taylor & Glover, 2005) have identified an ‘Anodontia group’ of species as a distinct major clade within the Lucinidae, separated from other lucinids by wellsupported long branches. Morphologically, they possess some distinctive anatomical structures, including the mantle septum and digitate mantle gills (Taylor & Glover, 2000; 2005). Species of Anodontia are found in the western Atlantic (2 species), Mediterranean and tropical eastern Atlantic (5 species), tropical eastern Pacific (2 species) and throughout the Indo-West Pacific region (16 species). They are distinguished by subspherical, smooth, white shells and the absence of hinge teeth. This relative paucity of characters has given rise to considerable taxonomic confusion so that species’ names have often been used rather indiscriminately (Taylor & Glover, 2005). In this paper we provide a detailed description of ctenidial morphology and the characters of bacterial symbionts in Anodontia (Euanodontia) ovum (Reeve, 1850) from the Great Barrier Reef, Australia. This species is remarkable among lucinids in possessing three different bacterial morphotypes associated with ctenidial cells. The species is widely distributed in coral-reef-associated habitats of the Indo-West Pacific Province from East Africa to Fiji (Taylor & Glover, 2005). General anatomy For general anatomy shells were cracked and animals fixed in 8% formalin solution in seawater and later transferred to 80% ethanol. Electron microscopy After collection the bivalves were dissected in seawater and pieces of ctenidial tissue were fixed in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer with 3.5% sodium chloride added to adjust osmolarity to that of seawater. For scanning electron microscopy (SEM), pieces of ctenidium were cut transversely and longitudinally with a razor blade, dehydrated through ascending concentrations of acetone and then criticalpoint dried. The dried pieces were then coated with gold at a thickness of 20 nm and examined with a Philips field emission XL30 SEM. For optical and transmission electron microscopy (TEM), pieces of gill were rinsed in buffer and post-fixed in 1% aqueous osmium tetroxide, again rinsed in buffer and dehydrated in ethanol. Samples were then embedded in mediumgrade TAAB resin, polymerized at 708C and sectioned using a Reichert Ultracut S ultramicrotome. For light microscopy, sections were cut at a thickness of 1 mm, stained with methylene blue and azure II, and mounted in DPX. For TEM, samples were sectioned at 70 nm, stained with alcoholic uranyl acetate, counterstained with Reynold’s lead citrate and then examined using a Hitachi H7100 electron microscope. MATERIAL AND METHODS Molecular analysis of gill bacterial symbionts Collection of specimens and habitat Freshly collected animals were preserved in 100% ethanol after opening the shell valves. The ethanol was changed after 24 h. Specimens of Anodontia ovum were collected in November 2000 at Lizard Island, in the northern part of the Great Barrier Reef. The bivalves occurred mainly in sheltered parts of the lagoon (148400 52.3800 S, 1458270 25.6400 E) in the vicinity of two small mangrove patches at Mangrove Beach and One Tree Coconut Beach. At both of these sites there is some seepage of fresh water. The substrate comprised medium-grade sand of mixed quartz and skeletal carbonate grains. Anodontia ovum was deeply burrowed at around 25 –30 cm into the sediment. It was most abundant between the prop roots of Rhizophora stylosa at the outer edge of the mangrove patches, but also occurred in more open sandy areas with a light covering of the seagrass Halophila. Anodontia ovum is one of a group of lucinid species inhabiting the lagoon of Lizard Island that includes Ctena bella (Conrad, 1837), Codakia tigerina (Linnaeus, 1758), Wallucina fijiensis (Smith, 1885), Chavania striata (Tokunaga, 1906), Fimbria fimbriata (Linnaeus, 1758) and Divaricella irpex (Smith, 1885). Coexisting with these is the small Solemyarina terraereginae Iredale, 1929, representing another bivalve family (Solemyidae) that possesses chemosymbionts (Kreuger & Cavanaugh, 1997; Taylor, Glover & Williams, 2008). DNA extraction from lucinid gill tissue DNA was extracted from the gill tissue of Anodontia ovum using the method of Durand et al. (1996) with an additional step to improve lysis of bacterial cells. In brief, the gill tissue (c. 5 mm by 3 mm section), which had been stored in absolute ethanol, was finely chopped with a sterile razor blade and then ground in liquid nitrogen to a fine powder. The powder was then suspended in 200 ml of lysis buffer (100 mM NaCl, 100 mM Tris – HCl, pH 8.0, 50 mM EDTA, pH 8.0, and 20 mg of RNase A) and incubated at 378C for 1 h. After incubation 100 ml of TES-K [2% SDS in TE (10 mM Tris, 1 mM EDTA, pH 8.0)] and 200 mg ml21 proteinase K were added and incubated overnight at 378C. An additional mechanical lysis step was included by adding 0.5 g of glass beads (0.1 mm diameter) and then bead-beating (Mikro-dismembrator U, B Braun Biotech International, Melsungen, Germany) three times at 2000 rpm for 30 s, with 30 s on ice in between. The sample was then spun in a microcentrifuge and the supernatant extracted twice with phenol/chloroform/IAA (25:24:1 v/v), once with chloroform/IAA (24:1 v/v) and then ethanol precipitated (Sambrook et al., 1989). The precipitate was resuspended in TE, pH 8.0, and the DNA was visualized by ethidium-bromide-stained agarose gel electrophoresis. Taxonomy Because of previous uncertainty over the nomenclature of Anodontia species we have taken care to compare the Lizard Island species with all available type material and found it to be morphologically very close to the syntype specimens of Lucina ovum Reeve, 1850 (BMNH reg. no. 1963195/1-2) with the type locality of Burias Island, Philippines (see Taylor & Glover, 2005, for review of systematics of Anodontia). For further reference we cite the GenBank numbers AJ581844 and AJ581879 for the 18S and 28S rRNA gene sequences for the Lizard Island material (Williams et al., 2004). PCR amplification and analysis of endosymbiotic bacterial 16S rDNA The 16S rRNA gene was amplified from the DNA extracted using the bacteria-specific primers Epsilon and 1541R (30 cycles of PCR – Embley, 1991; Purdy et al., 2003). The PCR product was cleaned using a QiaQuick spin-column (Qiagen) and AT cloned into pGem-T easy (Promega, UK) as described 176 CTENIDIAL SYMBIONTS IN ANODONTIA by the manufacturers. A total of 62 recombinants contained the correct-sized insert (c. 1500 bp) as determined by colony PCR using the vector-based primers M13f and R. PCR products from the colony PCR were digested using HaeIII and HhaI (Promega) and then analysed on a 4% w/v Nu-Sieve 3:1 (Flowgen, UK) ethidium-bromide-stained agarose gel in 1X TAE buffer. Restriction fragment length polymorphism (RFLP) patterns were grouped by eye and representatives of the different groups were fully sequenced using a Big Dye sequencing kit (Applied Biosystems) and run on an ABI 377 automatic sequencer (Sequencing Facility, Natural History Museum, London). Sequences have been deposited into Genbank (accession numbers EU983577 –EU983579). In order to determine if any Archaea were present in the Anodontia gill tissue amplification using archaea-specific primers using the hemi-nested approach of Munson et al. (1997) was attempted. The ctenidia of Anodontia ovum comprise single demibranchs that are dark purple-brown in colour. Individual filaments are c. 200 mm long and 25 –30 mm wide. As in all previously studied Lucinidae, each filament consists of three distinct zones; a distal ciliated zone, a narrow intermediary zone and a broad abfrontal, lateral or bacteriocyte zone (Fig. 1A). with a glycocalyx. The bacteriocytes are packed with bacteria contained in individual vacuoles. Generally, the bacteria are aligned with long axes normal to the apical surface, particularly in distal bacteriocytes. The dark cytoplasmic patches (Fig. 4A) closely resemble those identified as haemoglobin in Phacoides pectinatus (Frenkiel et al., 1996) but we made no specific tests to confirm this. Nuclei are basal and there are few organelles except for large lyzosomes (up to 8 mm in diameter) and scattered mitochondria, the latter more common in the basal parts of the cells. The basal margins of bacteriocytes are highly folded, abutting the central blood space of the ctenidial filament (Fig. 4A). The central blood space contains abundant haemocytes (Fig. 2C). Between the bacteriocytes are narrow intercalary cells that are wider apically. These cells lack intracellular bacteria (Figs 2C, 5D) and have basal nuclei with frequent mitochondria, Golgi bodies and SER. The apical margins of intercalary cells are often projected as spiky cytoplasmic protrusions much longer and less regular than the microvilli of the bacteriocytes (Fig. 5A– C). These apical protrusions frequently extend as thin sheets or tendrils over the surfaces of adjacent bacteriocytes (Fig. 5A–C). Most intercalary cells have spiruliform bacteria intertwined among the microvilli and spiky apical protrusions (see below). Infrequent mucocytes are the only other cell type present, usually located in the distal part of the lateral zone. Ciliated zone Bacterial symbionts The distal part of each filament consists of a ciliated zone with frontal, eulatero-frontal and lateral ciliary tracts (Fig. 1B, C). The cilated cells contain abundant mitochondria. We distinguish three morphotypes of bacterial symbiont in the ctenidial cells of A. ovum. The principal intracellular bacteria in the bacteriocytes are large, rod-shaped, 3–5 mm long and 0.5 –1.0 mm wide (Figs 2B– C, 4A). These are abundant in all bacteriocytes and occupy single vacuoles. They have electron-lucent periplasmic areas, the sites of probable sulphur deposits removed during specimen preparation (Vetter, 1985; Lechaire et al., 2008). A second, smaller type of intracellular bacterium is contained in aggregations within spherical vacuoles about 5 –7 mm in diameter (Fig. 4B–C). These are short and rod-shaped, about 1.0 mm long and 0.4– 0.5 mm wide. They are relatively infrequent but sometimes several bacterium-packed vacuoles occur within an individual bacteriocyte (Fig. 4C). The third type of bacterium recognized is extracellular and spiruliform. These bacteria are clearly seen in full length on SEM images and as sections by TEM (Fig. 6A– H). They are long, narrow (8–10 mm in length and 0.2–0.3 mm wide) and spirally twisted; the presence of axial filaments (Fig. 6E, G, H; af ) indicates these are probably spirochaetes. The spiral bacteria are abundant and have a strong association with the apical tufts of intercalary cells, in which they are embedded and intertwined, or on the surface between bacteriocytes (Fig. 6A–C) and never among the microvilli of the bacteriocytes. The spiral bacteria also occur within the microvilli of the cells of the intermediary zone that resemble intercalary cells (Figs 1D, 6F). RESULTS Intermediary zone, located between the ciliated zone and lateral zone This has one large electron-lucent cell with abundant mitochondria, resembling the ciliated cells but only partly ciliated at the distal edge. This cell is succeeded proximally and partly overlain by 3–4 cells resembling intercalary cells (below) with long apical processes, basal nuclei and prominent smooth endoplasmic reticulum (SER). These have abundant spiruliform bacteria amongst the apical processes (Fig. 1D). Lateral zone Most of the length of the ctenidial filaments is composed of the lateral or bacteriocyte zone, largely made up of sheets of bacteriocytes and intercalary cells with a central blood space between (Figs 1A, 2). In sections from an A. ovum with a shell length of 25.5 mm there are about 14 –16 bacteriocytes on either side of each filament (Fig. 1A). Just proximal to the intermediary zone, about 50 –75 mm of the lateral zones of adjacent filaments are fused to form cylindrical channels (bacteriocyte channels of Distel & Felbeck, 1987) with a central lumen 40– 50 mm in width (Fig. 3A, B). The channels are lined with bacteriocytes and intercalary cells, with planar sections showing six to eight bacteriocytes around the circumference of each channel (Fig. 3B). In surface views of the outer faces of filaments (Fig. 2A) the bacteriocytes are around 15– 20 mm long and 15 mm wide, with the apices dome-shaped and covered with microvilli. Separating the bacteriocytes and lying in narrow (2 –4 mm wide) channels are intercalary cells with spiky apical surfaces or with more irregular microvilli. In section (Figs 2C, 4A), bacteriocytes are around 12 – 15 mm in height with convex, microvilli-covered apical areas DNA extraction and PCR amplification of bacterial 16S genes DNA was extracted from the gill tissue of the Anodontia ovum and bacterial 16S rRNA genes were successfully amplified. Sixty-two clones were obtained and screened using RFLP analysis which indicated that there were two major sequence types (A and B), while a third sequence type (C) was detected once. Representatives of all the sequence types were fully sequenced and on alignment proved to be almost identical to each other with no more than 8 bp differences between any two sequences. The symbiont sequences analysed fall within 177 A. D. BALL ET AL. Figure 1. A. Section (SEM) through a ctenidium showing the basic organization of filaments. B. Outer ciliated zone of filaments (SEM). C. Section (TEM) through ciliated zone and part of intermediary zone. D. Section (TEM) of intermediary zone (imc1, imc2) between ciliated zone and first bacteriocyte (bc1) of lateral zone. Abbreviations: b, bacteria; bc1, first bacteriocyte; bch, bacteriocyte channels; cz, ciliated zone, elc, eulaterofrontal cilia; fc, frontal cilia; imc, intermediary cell; imc1, intermediary cell type 1; imc2, intermediary cell type 2; lc, lateral cilia, lz, lateral zone; sp, spiruliform bacteria. the gamma-subdivision of the Proteobacteria, in the cluster containing previously examined symbiotic thioautotrophs from the Lucinidae. They were very closely related to the gill symbiont of a previously studied Anodontia species from the western Atlantic (.99% similarity; Distel et al., 1994). This close association was confirmed by phylogenetic analysis which showed a strongly supported and very close relationship between symbionts from A. ovum and A. schrammi (data not shown). No amplification products were detected using archaea-specific primers. DISCUSSION The unusual feature of Anodontia ovum is the association of three morphological types of symbiotic bacteria with ctenidial cells in the lateral bacteriocyte zone. Multiple symbionts have not 178 CTENIDIAL SYMBIONTS IN ANODONTIA Figure 2. A. Surface view (SEM) of lateral zone showing bacteriocytes and intercalary cells. B. Section through bacteriocyte (SEM). C. Section (TEM) through lateral zone showing bacteriocytes and intercalary cells to either side of central blood space with amoebocytes. Abbreviations: am, amoebocyte; b, bacteria; bc, bacteriocyte; bs, blood space; ic, intercalary cell; ly, lyzosome; n, nucleus. been previously recognized in Lucinidae, although dual symbiosis is known in Bathymodiolus species (Duperron et al., 2007) and multiple symbionts have been detected in oligochaetes (Dubilier et al., 1999). A possible exception is the report of two bacterial types in Pillucina pisidium from eastern Russia (Rodionov & Yushin, 1991) where, in addition to abundant small rod bacteria, some cells contained large, thick-walled, spherical bacteria 7– 12 mm in diameter containing abundant 179 A. D. BALL ET AL. Figure 3. A. Transverse semi-thin section through distal part of ctenidial filaments showing bacteriocyte channels. B. Transverse section through a single bacteriocyte channel. Abbreviations: bch, bacteriocyte channel; bs, blood space. refractory globules. It seems possible that these latter ‘bacteria’ are actually a form of the spherical granules reported from other lucinids (see Taylor & Glover, 2006: Fig. 9). In A. ovum the prominent bacteria occupying single vacuoles in the bacteriocytes are similar in location and abundance to the chemautotrophic symbionts reported in other Lucinidae (e.g. Frenkiel & Mouëza, 1995; Gros, Liberge & Felbeck, 2003). Additionally, some bacteriocytes enclose spherical vacuoles containing clusters of much smaller bacteria. Such vacuoles and bacterial clusters have not been reported in any other lucinid and the significance of these is unknown. Finally, the spiruliform bacteria associated with intercalary cells have not reported in any other lucinid despite many SEM and TEM studies. However, similar and equally abundant spiral bacteria have been detected within the apices of intercalary cells of A. philippiana (Reeve, 1850) from Thailand (Taylor & Glover, unpublished observations). Duperron et al. (2007) reported a spirochaete molecular sequence in the deep-water Lucinoma kazani, but could not confirm that this was a symbiont rather than an environmental contaminant. Spirochaete bacteria have also been reported from molecular analyses of symbionts from gutless chemosymbiotic oligochaetes, but again have not been identified morphologically (Dubilier et al., 1999; Blazejak et al., 2005). The location of the spiral bacteria associated with the apical areas of the intercalary cells of A. ovum is similar to that of spiral, subcuticular, symbiotic bacteria associated within epidermal microvilli recorded from a variety of echinoderms (Kelly et al., 1995; Foret & Lawrence, 2001). For echinoderms, no conclusion has been reached concerning the role of these bacteria, but they possibly metabolize dissolved organic material and the host epidermal cells frequently phagocytose the bacteria (Kelly et al., 1995). Despite the fact that three different bacterial morphotypes can be seen in the gill images, only a single sequence was detected in the small clone library analysed here. These sequences were most closely related to the gill endosymbiont of Anodontia ‘philippiana’ (Distel et al., 1994) from Bermuda (this is actually Anodontia schrammi, see Taylor & Glover, 2005). This close relationship is somewhat surprising given the physical distance between the sampling sites (Australia and western Atlantic) and is indicative of the close evolutionary relationship between many lucinids and their bacterial endosymbionts (Distel et al., 1994). The identity of the remaining bacterial morphotypes is unknown. It is possible that the other small bacteria, massed in spherical vacuoles, are a morphotype of the larger sulphide-oxidizers. However, it is unlikely that the spirochaete-like bacteria seen intertwined within the intercalary cell apices are also a morphotype of the thioautotrophs, particularly as the TEM images in Figure 6E, G and H appear to show axial filaments that are a distinctive feature of the spirochaetes. A more concerted effort, including fluorescent in situ hybridization analysis of the symbionts, will be required to determine the identities of all of the bacterial morphotypes seen in the gills of these bivalves. In its major features the ctenidial structure of A. ovum is similar to that described for other Lucinidae. The livercoloured ctenidia and the dark bacteriocyte cytoplasm suggest the presence of haemoglobin, as in Phacoides pectinatus (Frenkiel et al., 1996) but tests are needed to confirm this. The intermediary zone of A. ovum is very short, with only one or two cells, usually a single electron-lucent cell that is partially overlain by one or two cells similar in morphology to intercalary cells. In other lucinids the intermediary zone can be much longer; for example, four electron-lucent cells in Divaricella quadrisulcata (Gros et al., 2000), or four lucent cells plus two types of secretory cell with granules in Lucina pensylvanica (Gros et al., 1996). A feature of many, if not all, lucinids is the presence of bacteriocyte channels in the lateral zone of the ctenidial filaments where adjacent filaments become fused to form cylindrical channels between the filaments (Distel & Felbeck, 1987). In Anodontia ovum bacteriocyte channels occupy only around 50 – 75 mm of the distal part of the filaments (about 25% of filament length) compared with most of the filament length in Lucinoma aequizonata (Distel & Felbeck, 1987) and Anodontia philippiana (Taylor & Glover, 2005: Fig. 5). The cylindrical 180 CTENIDIAL SYMBIONTS IN ANODONTIA Figure 4. A. Section (TEM) through a single bacteriocyte with convoluted basal margin and bacteria symbionts with electron-lucent periplasmic areas. B. Section through bacteriocyte (SEM) with vacuoles containing small bacteria as well as larger symbionts. C. Section of bacteriocyte (SEM) with large vacuole containing small bacteria. D. Section through part of a bacteriocyte (TEM) with large bacteria plus three vacuoles containing smaller bacteria. b, bacteria; bs, blood space; ly, lyzosome; mv, microvilli; sbv, vacuoles with small bacteria. channels allow a greater number of bacterioctyes to be accommodated compared with lamellar filaments and the resulting three-dimensional network of blood space may provide a more efficient circulatory system. The cylinders could also provide greater structural rigidity of the thickened filaments while still facilitating water flow. 181 A. D. BALL ET AL. Figure 5. A. Intercalary cell with apical tufts and protruding cytoplasmic sheet overlapping bacteriocyte surface (SEM). B. Intercalary cells with protruding cytoplasmic sheet (SEM). C. Intercalary cell with cytoplasmic sheet and protruding tendrils (SEM). D. Section (TEM) through intercalary cell between two bacteriocytes. E. Cytoplasmic extensions of intercalary cells partially overlapping surface of bacteriocytes (TEM). Abbreviations; b, bacteria; bc, bacteriocyte; cs, cytoplasmic sheet; ct, cytoplasmic tendril; g, Golgi body, ic, intercalary cell; m, mitochondria; mv, microvilli; n, nucleus; sp, spiruliform bacteria. Intercalary cells located between the bacteriocytes of A. ovum are remarkable for the apparent mobility of the apical cytoplasmic protrusions that vary between spiky tufts to sheet-like extensions and long tendrils that encroach over neighbouring bacteriocyte surfaces. In Divaricella quadrisulcata, Gros et al. (2000) recognized two types of intercalary cell, either with microvilli or with tufty apical extensions, but these may represent just different states of one cell type. Previously, Distel & Felbeck (1987) and Gros et al. (2000) proposed that the encroachment of intercalary cells over bacteriocyte apical 182 CTENIDIAL SYMBIONTS IN ANODONTIA Figure 6. A. Intercalary cell with spiruliform bacteria (SEM). B. Spiruliform bacteria embedded amongst microvilli of an intercalary cell (SEM). C. Spiruliform bacteria amongst apical tufts of an intercalary cell. D. Spiruliform bacterium on protrusive cytoplasmic sheet of intercalary cell. E. Section (TEM) of spiruliform bacteria with axial filament. F. Section (TEM) of spiruliform bacteria amongst apical tufts of intermediary zone cells G. Section (TEM) of spiruliform bacterium intertwined amongst apical protrusions of an intercalary cell. H. Spiruliform bacteria within apical area of intercalary cell. Abbreviation: af, axial filament. 183 A. D. BALL ET AL. CARO, A., GROS, O., GOT, P., DE WIT, R. & TROUSSELLIER, M. 2007. Characterization of the population of the sulfur-oxidising symbiont of Codakia orbicularis (Bivalvia, Lucinidae) by single-cell analyses. Applied and Environmental Microbiology, 73: 2101– 2109. DANDO, P.R., SOUTHWARD, A.J. & SOUTHWARD, E.C. 1986. Chemoautotrophic symbionts in the gills of the bivalve mollusc Lucinoma borealis and the sediment chemistry of its habitat. Proceedings of the Royal Society of London, Series B, 227: 227– 247. DANDO, P.R., SOUTHWARD, A.J., SOUTHWARD, E.C., TERWILLIGER, N.B. & TERWILLIGER, R.C. 1985. Sulphur-oxidising bacteria and haemoglobin in gills of the bivalve mollusc Myrtea spinifera. Marine Ecology Progress Series, 23: 85–98. DISTEL, D.L. 1998. Evolution of chemoautotrophic endosymbioses in bivalves. Bioscience, 48: 277–286. DISTEL, D.L. & FELBECK, H. 1987. Endosymbiosis in the lucinid clams Lucinoma aequizonata, Lucinoma annulata and Lucina floridana: a rexamination of the functional morphology of the gills as bacteria-bearing organs. Marine Biology, 96: 79–86. DISTEL, D.L., FELBECK, H. & CAVANAUGH, C. 1994. Evidence for phylogenetic congruence among sulfur-oxidisng chemautotrophic bacterial symbionts and their bivalve host. Journal of Molecular Evolution, 38: 533–542. DUBILIER, N., AMANN, R., ERSEUS, C., MUYZER, G., PARK, S.Y., GIERE, O. & CAVANAUGH, C.M. 1999. Phylogenetic diversity of bacterial endosymbionts in the gutless marine oligochete Olavius loisae (Annelida) Marine Ecology Progress Series, 178: 271– 280. DUPERRON, S., FIALA-MEDIONI, A., CAPRAIS, J.-C., OLU, K. & SIBUET, M. 2007. Evidence for chemautotrophic symbiosis in a Mediterranean cold seep clam (Bivalvia: Lucinidae): comparative sequence analysis of bacterial 16S rRNA, APS reductase and RubisCO genes. FEMS Microbiology Ecology, 59: 64–70. DUPLESSIS, M.R., DUFOUR, S.C., BLANKENSHIP, L.E., FELBECK, H. & YAYANOS, A.A. 2004. Anatomical and experimental evidence for particulate feeding in Lucinoma aequizonata and Parvilucina tenuisculpta (Bivalvia: Lucinidae) from the Santa Barbara Basin. Marine Biology, 145: 551–561. DURAND, P, GROS, O., FRENKIEL, L. & PRIEUR, D. 1996. Phylogenetic characterization of sulfur-oxidising bacterial endosymbionts in three tropical Lucinidae by 16S rDNA sequence analysis. Molecular Marine Biology and Biotechnology, 5: 37–42. EMBLEY, T.M. 1991. The linear PCR reaction: a simple and robust method for sequencing amplified rRNA genes. Letters in Applied Microbiology, 13: 171 –174. FISHER, C.R. 1990. Chemoautotrophic and methanotrophic symbioses in marine invertebrates. Reviews in Aquatic Sciences, 2: 399–436. FISHER, M.R. & HAND, S.C. 1984. Chemautotrophic symbionts in the bivalve Lucina floridana from seagrass beds. Biological Bulletin, 167: 445– 459. FORET, T.W. & LAWRENCE, J.M. 2001. Variation in abundance of subcuticular bacteria in Florida echinoderms. Symbiosis, 31: 309–322. FRENKIEL, L. & MOUËZA, M. 1995. Gill ultrastructure and symbiotic bacteria in Codakia orbicularis (Bivalvia, Lucinidae). Zoomorphology, 115: 51–61. FRENKIEL, L., GROS, O. & MOUËZA, M. 1996. Gill structure in Lucina pectinata (Bivalvia: Lucinidae) with reference to hemoglobin in bivalves with symbiotic sulphur-oxidising bacteria. Marine Biology, 125: 511– 524. GIERE, O. 1985. Structure and position of bacterial endosymbionts in the gill filaments of Lucinidae from Bermuda (Mollusca, Bivalvia). Zoomorphology, 105: 296– 301. GLOVER, E.A., TAYLOR, J.D. & WILLIAMS, S.T. 2008. Mangrove associated lucinid bivalves of the central Indo-West Pacific: review of the “Austriella” group with a new genus and species (Mollusca: Bivalvia: Lucinidae). Raffles Bulletin of Zoology, Supplement 18: 25–40. GROS, O., DURAND, P., FRENKIEL, L. & MOUËZA, M. 1998. Putative environmental transmission of sulfur-oxidising gill endosymbionts in four tropical lucinid bivalves, inhabiting various environments. FEMS Microbiology Letters, 160: 257– 262. surfaces may restrict or regulate the contact of bacteriocytes with sulphide-rich pallial seawater. Alternatively, we suggest that the intercalary extensions of sheets and tendrils function to keep the bacteriocyte apices clean, removing debris or surface bacteria by phagocytosis. In some lucinids a striking feature of the lateral zone is the presence of granule cells packed with spherical granules up to around 5 mm in diameter. Such granule cells are a prominent feature of Codakia orbicularis where they comprise a large proportion of the inner part of the lateral zone (Frenkiel & Mouëza, 1995; Gros, Liberge & Felbeck, 2003). Other lucinids with granule cells are Lucinoma species (Distel & Felbeck, 1987; Dando et al., 1986); Divaricella quadrisulcata (Gros et al., 2000); Indoaustriella species (Glover, Taylor & Williams, 2008), Ctena species and Lucinisca nassula (Taylor & Glover, 2006). Such granule cells are absent in Anodontia ovum as well as other Anodontia species such as A. alba (Gros et al., 2003), A. bullula (Meyer et al., 2008) and A. philippiana (J.D. Taylor & E.A. Glover, unpublished observations). The functional significance of the granules is not understood; in Codakia orbicularis they are cystine-rich (Frenkiel & Mouëza, 1995) and their presence in some lucinids and not others probably reflects physiological differences in sulphur metabolism between the taxa. Another feature of ctenidial filament structure that varies between different lucinid species is the abundance of mucocytes. These are scarce in A. ovum, Lucinoma borealis (Southward, 1986), Loripes lucinalis (Herry et al., 1989) and Divaricella quadrisulcata (Gros et al., 2000), and apparently absent in Myrtea spinifera (Dando et al., 1985). In contrast, they are common in Lucina pensylvanica (Gros et al., 1996) and abundant in Codakia orbicularis (Frenkiel & Mouëza, 1995). Evidence from fossils suggests a long history for symbiosis in Lucinidae, with the main adaptations to the chemosymbionts in terms of ctenidial structure probably evolving in the Palaeozoic and certainly by the Mesozoic (Taylor & Glover, 2000, 2006). The clade comprising most Anodontia species appeared in the early Eocene and is genetically distinct from other lucinids (Williams et al., 2004). As well as the distinctive shell form and other anatomical features (Taylor & Glover, 2005), the ctenidia lack the granule cells that are notably abundant in the filaments of other lucinids such as Codakia species and Lucinoma species and this may reflect differences in sulphide metabolism. Anodontia ovum appears to be alone among Lucinidae in possessing three morphotypes of bacterial symbiont associated with the bacteriocyte zone and these differ in size, shape and location. Further research is needed to characterize these symbionts and to investigate their role. ACKNOWLEDGEMENTS We thank Lyle Vail and the staff of the Lizard Island Research Station for facilities and advice. The Great Barrier Reef Marine Parks Authority is acknowledged for permission to collect bivalves at Lizard Island. Yuri Kantor kindly translated a Russian publication. E.G. and J.T. are grateful for continuing support from Phil Rainbow and the Department of Zoology, Natural History Museum, London. K.J.P. is Team Leader of MicroComXT a Marie Curie Excellence Team under EU FP6 (MEXT-CT-2005-024112). REFERENCES BLAZEJAK, A., ERSEUS, C., AMANN, R. & DUBILIER, N. 2005. Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru Margin. Applied and Environmental Microbiology, 71: 1553–1561. 184 CTENIDIAL SYMBIONTS IN ANODONTIA GROS, O., FRENKIEL, L. & MOUËZA, M. 1996. Gill ultrastructure and symbiotic bacteria in the tropical lucinid, Linga pensylvanica (Linné). Symbiosis, 20: 259 –280. GROS, O., FRENKIEL, L. & MOUËZA, M. 1998. Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae). Invertebrate Reproduction and Development, 34: 219–231. GROS, O., FRENKIEL, L. & FELBECK, H. 2000. Sulfur-oxidising endosymbioisis in Divaricella quadrisulcata (Bivalvia: Lucinidae): morphological, ultrastructural and phylogenetic analysis. Symbiosis, 29: 293– 317. GROS, O., LIBERGE, M. & FELBECK, H. 2003. Interspecific infection of aposymbiotic juveniles of Codakia orbicularis by various tropical lucinid gill-endosymbionts. Marine Biology, 142: 57– 66. GROS, O., LIBERGE, M., HEDDI, A., KHATCHADOURIAN, C. & FELBECK, H. 2003. Detection of the free-living forms of sulfide-oxidizing gill endosymbionts in the lucinid habitat (Thalassia testudinum environment). Applied and Environmental Microbiology, 69: 6264– 6267. HERRY, A. & LE PENNEC, M. 1987. Endosymbiotic bacteria in the gills of the littoral bivalve molluscs Thyasira flexuosa (Thyasiridae) and Lucinella divaricata (Lucinidae). Symbiosis, 4: 25–36. HERRY, A., DIOURIS, M. & LE PENNEC, M. 1989. Chemoautotrophic symbionts and translocation of fixed carbon from bacteria to host tissues in the littoral bivalve Loripes lucinalis (Lucinidae). Marine Biology, 101: 305–312. JANSSEN, H.H. 1992. Philippine bivalves and microorganisms: past research, present progress and a perspective for aquaculture. Philippine Scientist, 29: 5 –32. KELLY, M.S., BARKER, M.F., MCKENZIE, J.D. & POWELL, J. 1995. The incidence and morphology of subcuticular bacteria in the echinoderm fauna of New Zealand. Biological Bulletin, 189: 91–105. KREUGER, D.M. & CAVANAUGH, C.M. 1997. Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes. Applied and Environmental Microbiology, 63: 91– 98. LECHAIRE, J.-P., FRÉBOURG, G., GAILL, F. & GROS, O. 2008. In situ characterization of sulphur in gill-endosymbionts of the shallow water lucinid Codakia orbicularis (Linné, 1758) by high-pressure cryofixation and EFTEM microanalysis. Marine Biology, 154: 693 –700. MEYER, E., NILKERD, B., GLOVER, E.A. & TAYLOR, J.D. 2008. Ecological importance of chemoautotrophic lucinid bivalves in a peri-mangrove community in eastern Thailand. Raffles Bulletin of Zoology, Supplement 18: 41–55. MUNSON, M.A., NEDWELL, D.B. & EMBLEY, T.M. 1997. Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh. Applied and Environmental Microbiology, 63: 4729– 4733. PURDY, K.J., NEDWELL, D.B. & EMBLEY, T.M. 2003. Analysis of the sulphate-reducing bacterial and methanogenic archaeal populations in contrasting Antarctic sediments. Applied and Environmental Microbiology, 69: 3181–3191. REID, R.G.B. & BRAND, D.G. 1986. Sulfide-oxidising symbiosis in lucinaceans: implications for bivalve evolution. Veliger, 29: 3– 24. RODIONOV, I.A. & YUSHIN, V.V. 1991. Procaryotic symbionts in gill cells of the bivalve mollusc Pillucina pisidium. Biologiya Morya, 1991: 39– 46. [in Russian] SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. 1989. Molecular cloning: a laboratory manual. Edn 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. SOUTHWARD, E.C. 1986. Gill symbionts in thyasirids and other bivalve molluscs. Journal of the Marine Biological Association of the UK, 66: 889–914. STEWART, F.J., NEWTON, I.L.G. & CAVANAUGH, C.M. 2005. Chemosymbiotic endosymbioses: adaptations to oxic-anoxic interfaces. Trends in Microbiology, 13: 439 –448. TAYLOR, J.D. & GLOVER, E.A. 2000. Functional anatomy, chemosymbiosis and evolution of the Lucinidae. In: The evolutionary biology of the Bivalvia. (E.M. Harper, J.D. Taylor & J.A. Crame) eds Geological Society of London Special Publications, 177: 207–225. TAYLOR, J.D. & GLOVER, E.A. 2005. Cryptic diversity of chemosymbiotic bivalves: a systematic revision of worldwide Anodontia (Mollusca: Bivalvia: Lucinidae). Systematics and Biodiversity, 3: 281–338. TAYLOR, J.D. & GLOVER, E.A. 2006. Lucinidae (Bivalvia) – the most diverse group of chemosymbiotic molluscs. Zoological Journal of the Linnean Society, 148: 421–438. TAYLOR, J.D., GLOVER, E.A. & WILLIAMS, S.T. 2008. Ancient chemosymbiotic bivalves: systematics of Solemyidae from eastern and southern Australia (Mollusca: Bivalvia). Memoirs of the Queensland Museum—Nature, 54: 75–104. VETTER, R.D. 1985. Elemental sulphur in the gills of three species of clams containing chemautotrophic symbiotic bacteria: a possible inorganic storage compound. Marine Biology, 88: 33–42. WILLIAMS, S.T., TAYLOR, J.D. & GLOVER, E.A. 2004. Molecular phylogeny of the Lucinoidea (Bivalvia): non-monophyly and separate acquisition of bacterial chemosymbiosis. Journal of Molluscan Studies, 70: 187– 202. 185