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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
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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
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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
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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
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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
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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.
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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
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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.
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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).
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