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MARINE MICROBIAL THIOTROPHIC ECTOSYMBIOSES
J. OTT,* M. BRIGHT & S. BULGHERESI
Institute of Ecology and Conservation Biology, University of Vienna,
Althanstrasse 14, A-1090 Vienna, Austria
*E-mail: [email protected]
Abstract A high diversity of thiotrophic symbioses is found in sulphide-rich marine habitats,
involving several phyla of protists and invertebrates, as well as several subdivisions of the Proteobacteria. Whereas some of the better-known symbioses are highly evolved endosymbioses, the more
primitive ectosymbioses are less well known. The sulphur-oxidising chemolithotrophic nature of
the bacteria and their nutritive importance to the eukaryote host have been demonstrated for the
ciliates Kentrophoros spp. and Zoothamnium niveum, the nematode subfamily Stilbonematinae, and
the carid shrimp Rimicaris exoculata. For a number of other regular bacteria–eukaryote associations,
such a symbiotic relationship has been hypothesised based on ecological, morphological, physiological or molecular data, but is still inconclusive.
The diversity of thiotrophic symbioses
The interest in thiotrophic symbioses awakened by the discovery of the deep-sea hydrothermal
vents has led to the discovery of an unexpected diversity of microbe/animal relationships in a
variety of habitats from the intertidal zone to the deep sea (Cavanaugh 1985, Fisher 1990, Nelson
& Fisher 1995). The fascination of food chains that operate without sunlight and the opportunity
to find clues about the origin of life on this and probably other celestial bodies (Farmer 1998) have
spurred research on hot vents and cold seeps in the deep sea and on continental slopes. In the wake
of these expensive endeavours, research has been conducted in more easily accessible shallowwater sulphidic habitats and has revealed a comparable variety of symbiotic relationships (Ott 1996,
Giere 1992). The deep-sea communities are unrivalled with regard to the importance that the
thiotrophic symbioses play in an extremely food-limited setting. In shallow water the predominance
of photoautotrophic production restricts thiotrophic symbioses to a more cryptic existence.
To date symbioses with sulphur-oxidising chemolithoautotrophic bacteria have been recorded
for protists (ciliates and probably also flagellates) and seven animal phyla (Platyhelminthes, Nematoda, Echiurida, Annelida, Mollusca, Arthropoda, and Echinodermata). With the exception of
Platyhelminthes, Echiurida, and Echinodermata, the development of thiotrophic symbioses has
occurred more than once in each phylum.
The diversity of the microbial symbionts is as high as that of the hosts, and although they all
belong to the Proteobacteria, there are representatives of the g-, e-, and a-subgroups. They occur
as endosymbionts intracellularly in special organs such as the trophosome of the Vestimentifera
(Siboglinidae, Annelida) and similar organs in Catenulida (Platyhelminthes) and the nematode
Astomonema jenneri, or within organs of other functions, such as the gills of bivalves and gastropods, the vestigial gut in Astomonema southwardorum, or under the cuticle between epidermis cells
of Oligochaeta (Fisher 1996, Giere 1996).
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Oceanography and Marine Biology: An Annual Review 2004, 42, 95–118
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In many cases, however, they appear ectosymbiotically, attached to the body surface of their
eukaryote host, such as in flagellates, ciliates, Nematoda (Stilbonematinae), and in certain Annelida
(Alvinellidae) and Arthropoda (Ott 1996, Polz & Cavanaugh 1996). This review summarises our
knowledge about these ectosymbioses.
Ectosymbioses differ from endosymbioses in many respects: the microbes are largely exposed
to ambient conditions, although the eukaryote host is responsible for the position within gradients
of environmental variables. Moreover, substances produced by the host may modify the environment
and physiology of the bacterial partner. The animal hosts appear less modified than is the case in
endosymbioses, and in most cases, the relationship to nonsymbiotic relatives can be traced with
confidence. Rarely is a particular ectosymbiosis characteristic for taxa higher than genera. Although
morphological modifications in conjunction with the symbiotic way of life are present in practically
all ectosymbioses, they never reach the extent found in endosymbioses. In some cases the partners
may be separated and kept alive at least for some time. These characteristics allow us to make
inferences on how these symbioses originated, something that is extremely difficult to do in highly
evolved endosymbioses.
In many thiotrophic symbioses where the method of transmission has been clarified, there is
no evidence of vertical transmission from parents to offspring. Apparently, the symbionts must be
acquired in each generation, most probably from the free-living bacterial community in the respective habitat. The mechanisms of recognition, attachment, and internalisation of the microbial partner
are still unclear.
Thiobiotic habitats
Hydrothermal Vents
Hydrothermal springs are found in all oceans along the central rift valley of mid-oceanic ridges.
Here, sea water percolates through the newly formed crust several kilometres deep. When it comes
in contact and reacts with hot rocks near the underlying magma chamber it undergoes profound
chemical changes (Alt 1995, Von Damm 1995). Most importantly sulphate is reduced to sulphide,
the water becomes anoxic and the concentrations of heavy metals increase dramatically. The heated
and chemically altered fluid then rises and flows warm (a few degrees above ambient deepwater
temperatures) to extremely hot (350–400˚C) from cracks in the basaltic rocks covering the floor
of the rift valley or seeps through sediments. Mineral precipitates may form chimneys dozens of
metres high, from which the hydrothermal fluid emanates as black clouds coloured by precipitating
metal sulphides (Goldfarb et al. 1983).
Chemolithoautotrophic bacteria and Archaea already grow in the chemical gradients within the
conduits in the basaltic rocks (Karl et al. 1980). Where the hydrothermal fluid is injected into the
oxygen-containing, cold, deep-sea water a profusion of microbial production occurs on the surface
of rocks and sediments. Most spectacular, however, is the animal life around the hydrothermal
vents, which solely depends on the production of the chemolithoautotrophic microbes (Van Dover
2000). Whereas many animals are suspension feeders or simply graze the microbial turf, others
live in symbiosis with special kinds of bacteria. Hydrothermal vents are unpredictable environments,
where fluid flows may vary on short temporal scales (Johnson et al. 1988). Successful survival
strategies here include associations either with mobile animals that may follow gradients or with
large sessile organisms that provide the necessary milieu for the bacteria. Both endo- and ectosymbioses are found in these bizarre environments. The most prominent examples are the vestimentiferan tube worms, bivalves such as Calyptogena spp. and Bathymodiolus spp., and the shrimp
Rimicaris spp.
Hydrothermal vents are not restricted to the deep sea but also occur in shallow water in
conjunction with volcanism. None of the few shallow hydrothermal vents studied so far, however,
showed such a highly specialised fauna (Dando et al. 1995).
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Cold seeps
Wherever the sea sediments are subjected to pressure, pore fluid is squeezed from the interstices
and seeps from the surface. This seepage may occur under a variety of tectonic forces: in accretion
prisms formed during subduction of one lithospheric plate under another, at the margin of mud
volcanoes, in places where salt diapirs rise through continental margin sediments, or in connection
with hydrocarbon seeps. The expelled fluids differ chemically from the surrounding sea water. They
are anoxic and may contain methane or sulphide, hydrocarbons, or high salt concentrations, but
not the high heavy metal concentrations typical for hydrothermal vents (Suess et al. 1987).
Flow speeds are generally low but steady and sulphide concentrations are often at the detection
limit near the sediment surface. In contrast to the vents, the sulphide here is of biological origin,
essentially having been produced by microbial sulphate reduction (Carney 1994).
Symbiotic biota associated with cold seeps include vestimentiferan and frenulate tube worms,
clams and mussels among the macrofauna, and stilbonematid nematodes among the meiofauna.
Some of the mussels contain both thiotrophic and methanotrophic endosymbionts, sometimes within
the same bacteriocyte (Fisher 1990). Methanotrophs are also found in the frenulate Siboglinum
poseidoni (Schmaljohann & Flügel 1987).
Shallow sheltered sediments
This is by far the largest thiotrophic habitat. It extends from intertidal sand and mudflats, marsh
and mangrove sediments, over essentially all of the shelf sediments, to dysoxic basins and upperslope sediments. It occurs under an oxic surface layer of variable thickness, ranging from a few
millimetres to several centimetres, from which it is separated by a chemocline – the redox potential
discontinuity layer (RPD) (Fenchel & Riedl 1970). Within the RPD, electron acceptors for the
oxidation of organic material change in sequence from oxygen to nitrate, ferric iron, manganese
and sulphate. Bacterial sulphate reduction produces sulphide in the deeper layers. Upward diffusion
of sulphide leads to its oxidation and a variety of microbes use the free energy of this oxidation
process for carbon fixation (Jørgensen 1989). Since sediment layers containing sulphide may be
separated from those containing the best electron acceptor (oxygen) by several millimetres to
centimetres, microorganisms are at a disadvantage when the sulphide/oxygen gradient is weak.
Some of the larger sulphur bacteria such as Beggiatoa and especially the giant spaghetti bacterium
Thioploca are mobile enough to bridge the gap (Gallardo 1977). Similar to what has been observed
for hot vents and cold seeps, bacteria have succeeded in finding hosts that provide them with both
oxygen and sulphide. Among the macrofauna, several families of bivalves, such as the Lucinidae,
Thyasiridae, and Solemyidae, have species containing sulphur-oxidising chemoautotrophic bacteria
in their gills (Allen 1958, Southward 1986). In those sediments with interstitial spaces, a variety
of protists and meiofauna animals have symbiotic bacteria, either as endosymbionts (catenulid
flatworms, phallodrilid oligochaetes, nematodes of the genus Astomonema) (Giere 1996) or as
ectosymbionts (ciliates, stilbonematid nematodes) (Ott 1996). Recently, a number of flagellates
living in the soft sediment of a dysoxic basin have been described to harbour potentially chemoautotrophic ectosymbionts (Buck et al. 2000, Bernhard et al. 2000).
Macrophyte debris
In shallow waters the debris originating from marsh and mangrove plants, algae, or sea grass may
accumulate (Fenchel 1970, Mann 1976). The decomposition of this organic matter creates sulphidic
habitats of various spatial and temporal extents. Mangrove peat is a relatively stable substratum
having internal sulphide concentrations of up to 4 mM (McKee 1993). Diffusion of sulphide into
the overlying water creates a few-millimetre-thick sulphidic boundary layer, whereby sulphide flux
is highest in recently disturbed patches on the peat surface (Ott et al. 1998). Loose macrophyte
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debris is much less stable and predictable than mangrove peat. It may, however, repeatedly collect
in defined places such as depressions in the vicinity of algae or sea grass stands or in crevices and
caves among rocks.
Thiotrophic ectosymbioses have been reported from mangrove peat and decomposing sea grass
and algae. In all cases the host is a sedentary peritrich ciliate belonging to the genus Zoothamnium.
Hosts
Ciliates
Kentrophoros
About 20 species of the ciliate genus Kentrophoros inhabit sheltered marine sands having an RPD
several centimetres beneath the surface (Fenchel & Finlay 1989). Soon after the description of the
first species (Sauerbrey 1928) it was recognised that the dorsal (left) surface of the cells is covered
by rod-shaped bacteria containing sulphur granules (Kahl 1935). Raikov (1971, 1974) suggested
a chemolithoautotrophic nature of the bacteria and showed that the bacteria are phagocytised by
the ciliate.
Specimens of Kentrophoros are ribbon shaped or tubularly involuted and, in the case of K.
fistulosus, can be up to 3 mm long, but are only 2–3 mm thick (Figure 1). The extremely flattened
shape is interpreted as an adaptation to provide ample space for the bacterial symbionts; it increases
the surface-to-volume ratio by a factor of 6–7 compared with other similarly sized ciliates. The
ventral (or right) side bears cilia arranged in longitudinal rows. The cells have only a vestigial
cytostome (Foissner 1995). Rod-shaped bacteria occupy the unciliated dorsal (or left) side of the
cell, which may be tubularly involuted in the central region (Figure 2).
The ciliates glide sluggishly between the sand grains. In the sediment they concentrate in the
oxic–anoxic chemocline. In an artificial oxygen gradient they aggregate around 5% saturation,
avoiding high oxygen tensions. The ciliates, however, do not react to sulphide but appear to be
randomly distributed in a sulphide gradient in the absence of oxygen (Fenchel & Finlay 1989).
Under experimental conditions and under the assumption that the ectosymbiotic bacteria constitute its sole food, Kentrophoros was calculated to have a doubling time of 18 h at room
temperature, which is low compared with similarly sized ciliates. This difference was attributed to
suboptimal culture conditions (Fenchel & Finlay 1989).
Zoothamnium
The sedentary colonial peritrich ciliate Zoothamnium niveum (Hemprich & Ehrenberg 1831,
Ehrenberg 1838) was originally described from the Red Sea. Although the authors were struck
by its white appearance, they could not attribute it to the presence of bacteria. The ciliate has
been redescribed by Bauer-Nebelsick et al. (1996a) based on material collected from mangrove
peat in the Belize Barrier Reef system. There it grows on vertical to overhanging walls of tidal
channels, and lagoons cut into mangrove peat of backreef islands. Since then, Zoothamnium
niveum has been found in the western Mediterranean near Calvi, Corsica, where it grows on
decomposing subtidal accumulations of leaf debris and adjacent rocks near stands of the large
Mediterranean sea grass Posidonia oceanica. Other reports of large, white Zoothamnium colonies come from the Florida Keys (Bauer-Nebelsick et al. 1996a), Lanzarote (Canary Islands)
(Wirtz & Debelius 2003), Elba (own unpublished observations), Giglio (T. Pillen, personal
communication), and Greece (G. Scatolin, personal communication). The up to 15-mm-long
colonies are feather-shaped fans with alternating branches growing from a central stalk (Figure
3). Stalk and branches contain a contractile spasmoneme that allows the colony to contract
rapidly. Colony contraction occurs spontaneously or upon disturbance.
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Figure 1 Light microscopy (LM) micrograph of living specimen of Kentrophoros fistulosus; scale bar = 500
mm. Figure 2 Scanning electron microscopy (SEM) micrograph of midbody region with rods (r) on the
dorsal side and cilia on the ventral side; scale bar = 20 mm. (Courtesy of W. Foissner.)
Figure 3 LM micrograph of Zoothamnium niveum colony with stalk (s) and terminal zooid (t) on its tip and
alternate branches with microzooids (mi) and macrozooids (ma); scale bar = 100 mm. Figure 4 SEM
micrograph of contracted colony showing several microzooids (mi) and one macrozooid (ma) covered by
symbionts; scale bar = 50 mm.
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Large colonies sprout secondary fans and may have about 200 branches bearing up to 20 feeding
microzooids each, adding up to over 3000 microzooids. The tips of the stalk and of still-growing
branches bear nonfeeding, terminal zooids that divide by unequal longitudinal fission. On some
branches the proximal zooid develops into a globular nonfeeding macrozooid that eventually
detaches as a motile swarmer. Except for the noncontractile basal part of the stalk, branches and
zooids are densely covered by bacteria (Figure 4) (Bauer-Nebelsick et al. 1996a). Zoothamnium
niveum occurs on the surfaces of macrophyte debris and peat where sharp gradients between
sulphide and oxygen are developed within a few millimetres. The ciliary action of the microzooids
effectively mixes sulphidic and oxic water (Vopel et al. 2001, 2002). In addition, the colonies
contract into the sulphidic boundary layer and subsequently expand again into the surrounding
oxygen-containing water (Ott et al. 1998). The life cycle of Z. niveum has been studied through
several generations in the laboratory. Using sulphide as a cue, the swarmers of Z. niveum actively
seek and colonise patches in the environment with high sulphide flux, such as areas where the peat
surface has been recently disturbed. Upon settling, each swarmer changes into a terminal zooid
that produces a stalk and starts to divide into microzooids and terminal zooids, which in turn grow
into branches. After about 4 hours the first branch is formed, and within 4 days maximum size is
reached. During the period of exponential growth on the second and third days, the terminal zooids
divide approximately once every hour (own unpublished data). The colonies continue to live to a
mean age of 7 days, showing loss of microzooids from proximal branches as signs of senescence.
Starting with a colony size of about 10 branches, macrozooids are produced and released. Swarmers
may settle at the same spot or colonise a new sulphide patch. The growth data obtained under
laboratory conditions fit those observed on the peat wall in the field. Z. niveum is usually found in
groups of a few to several hundred colonies. Small groups typically consist of young colonies and
newly settled swarmers, large groups mainly of senescent colonies. A patch exists for approximately
20 days, as has been determined for a population at Twin Cayes, Belize (Ott et al. 1998).
Invertebrates
Nematoda (Stilbonematinae)
Ectosymbiotic chemoautotrophic bacteria have been reported for a group of eight closely related
genera of free-living nematodes within the family Desmodoridae (Chromadoria, Adenophorea),
classified as the subfamily Stilbonematinae. Originally thought to be parts of the worm (Greeff
1869), fungal spores (Chitwood 1936) or epibiotic cyanobacteria (Gerlach 1950, Wieser 1959),
they have been finally identified as sulphide-oxidising chemoautotrophic bacteria that coat the
nematode surface in a species-specific pattern (Ott et al. 1991).
Stilbonematinae occur in all intertidal and subtidal porous sediments where an oxidised surface
layer overlies a deeper, reduced, sulphidic body of sediments. They are most abundant in and near
the RPD (Ott & Novak 1989). Highest abundance and diversity are found in tropical calcareous
sands. Special habitats include continental slope brine seeps (Jensen 1986), the shallow-water vents
in the Bay of Plenty (New Zealand) (Kamenev et al. 1993), and the reduced sediments accumulating
among the roots of the surf grass Phyllospadix spp. and in mussel banks on the wave-beaten U.S.
West Coast (own unpublished observations). Stilbonematinae have been reported from all major
oceans, the Mediterranean, the Red Sea, the Caribbean and the North Sea.
Adult sizes of the elongated cylindrical worms range from less than 2 mm to about 15 mm
(Figure 5 and Figure 6). The external appearance and the construction of the worm cuticle are
highly diverse (Urbancik et al. 1996a,b). Unifying characters are the weak or absent buccal armature
and the special construction of the foregut (pharynx), where muscles appear to be concentrated in
the anterior-most part and the remainder is mostly glandular (Hoschitz et al. 2001). These similarities could be interpreted to reflect convergent evolution due to the symbiotic lifestyle and the
specialisation on a single food item. The monophyly of the Stilbonematinae, however, is clearly
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Figure 5 SEM micrograph of Laxus cosmopolitus (Stilbonematinae) with rod-shaped symbionts; arrow points
to symbiont-free area showing rows of setae; scale bar = 100 mm. Figure 6 SEM micrograph of Eubostrichus
dianae (Stilbonematinae) with nonseptate filaments (f); scale bar = 100 mm.
supported by the presence of a unique glandular sense organ in the epidermis (Nebelsick et al.
1992, Bauer-Nebelsick et al. 1995) and it forms a distinct clade within the Desmodoridae according
to both morphological and molecular (18S rDNA sequence) characters (Kampfer et al. 1998).
The worms move sluggishly through the sediments and often coil up and remain stationary for
several hours. Riemann et al. (2003) even propose a hemisessile life strategy for Leptonemella spp.
from intertidal sediments in the North Sea.
No representative of the Stilbonematinae has been cultivated in the laboratory so far. The larger
species may be kept in sand buckets and even in dishes with sea water for many days up to several
weeks, but neither moulting nor egg laying has been observed here. They probably grow slowly
and have long intermoult periods, high life expectancy and few offspring. Juveniles are rare (Ott
et al. 1995) and moulting stages have only occasionally been found in field samples. The slow and
sluggish lifestyle fits with a basal metabolism that is among the lowest ever measured in nematodes
(Schiemer et al. 1990).
Crustacea (Rimicaris)
A number of decapod carid shrimps regularly occur at hydrothermal vents. They were originally
placed into the family Bresiliidae but later a separate family, Alvinocarididae, was proposed
(Christoffersen 1986). While species of the genus Alvinocaris are widespread scavengers, members
of the genus Rimicaris have only been reported at Mid-Atlantic Ridge hydrothermal sites (R.
exoculata; Williams & Rona 1986) and recently at a vent field on the Central Indian Ridge (R.
kairei; Watabe & Hashimoto 2002). A second Atlantic species, R. aurantiaca (Martin et al. 1997)
proved to be juveniles of R. exoculata (Shank et al. 1998).
The well-studied species R. exoculata occurs in enormous densities of up to 50,000 specimens
m–2 on solid surfaces where hydrothermal fluids emanate (Segonzac et al. 1993). Adult R.
exoculata are 40-to 60-mm-long whitish shrimps (Figure 7). Originally thought to be grazers on
surface-living bacteria (Van Dover et al. 1989) or suspension feeders (Jannasch et al. 1991), the
conspicuous and regular epigrowth of bacteria on the mouthparts and the inner surface of the
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Figure 7 LM micrograph of ventral and dorsal sides of Rimicaris exoculata specimens, approximately 40–60
mm in length. (Courtesy of M. Segonzac and D. Desbruyères.) Figure 8 SEM of shrimp appendage; scale
bar = 1 mm. (Courtesy of M.F. Polz.)
modified carapace pointed to a symbiotic lifestyle (Gebruk et al. 1993). The carapace encloses
the anterior body almost completely, forming voluminous chambers on either side. There is no
rostrum and the first and second antennae are stout and strong. The exopodites of maxilla 2 and
maxilliped 1 are greatly enlarged and densely covered with plumose setae (bacteriophores), which
also occur on the proximal parts of the thoracic legs (Figure 8) (Gebruk et al. 1993, Segonzac
et al. 1993, Casanova et al. 1993).
The eyestalks are fused to form a large dorsal eye believed to be able to detect low levels of
light emanating from vent chimneys (Van Dover et al. 1988, Van Dover & Fry 1994). This enables
the shrimp to find the vents from a distance. A smooth cornea replaces the lenses of the compound
eye, the photoreceptors in the fused retina are large with enlarged photosensitive regions, and the
eye is underlain by a thick layer of white cells scattering light upwards (O’Neill et al. 1995, Nuckley
et al. 1996, Chamberlain 2000). These modifications apparently sacrifice imaging ability in order
to increase visual sensitivity. At close range the shrimp may additionally be guided by sensilla
located on the second antennae, which show a concentration-dependent response to sulphide
(Renninger et al. 1995).
The shrimps form dense feeding swarms around hydrothermal chimneys and areas of “shimmering water”; some cling to the rock, forming layers several specimens thick and some move
in and out of the thermal plumes. When dislodged by turbulence they rapidly move back to the
chimneys. They ingest sulphide particles, attached bacteria and the bacteria growing on their
cuticles. They are among the most important primary consumers and their ectosymbiotic thiotrophic microbes are the dominant primary producers at certain sites (Van Dover 2002). In turn,
Rimicaris is an important food for larger megafauna, such as macrourid and zoarcid fishes
(Geistdoerfer 1994).
The shrimp have small eggs and planktotrophic larvae (Ramirez Llodra et al. 2000). Juvenile
shrimps have been found in midwater, where they spend an unknown period of time (Herring 1998).
They are characterised by high amounts of wax esters as lipid reserves (Pond et al. 1977a, Allen
et al. 1998, 2001). The fatty acid composition of these lipids points to a photosynthetic origin of
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these substances. Such storage compounds are used during settlement and metamorphosis, while
the shrimp develops the structures necessary to support the flora of ectosymbionts in adults (Gebruk
et al. 2000).
Microbial symbionts
To date all microbial symbionts have resisted cultivation. The phylogenetic relationship of some
of them to known bacteria was determined by 16S rRNA gene sequencing. The chemoautotrophic
nature has been inferred on various grounds such as colour, ultrastructure, presence of ribulose1,5-biphosphate carboxylase (RuBisCo, the enzyme necessary for carbon fixation), uptake of
labelled inorganic carbon, presence of sulphur-oxidation enzymes and ecological data from the
habitat.
Kentrophoros
According to Fenchel & Finlay (1989) the symbiotic bacteria are rod shaped, about 3.6 mm long
and 0.8 mm wide. They appear brown to black in transmitted light due to sulphur inclusions that
are contained in membrane-bound vesicles that occupy a large part of the cell volume. Among the
cell organelles are probably carboxysomes, which contain RuBisCo. The rods are arranged perpendicular to the ciliate surface and show an unusual longitudinal division (Figure 9). They are
embedded in a thick mucus layer produced by the ciliate that probably also covers the ciliated side
(Foissner 1995). 14C incubations followed by autoradiography showed a carbon uptake rate that
was equivalent to a doubling time of 5.3 h. The reduction of benzyl viologen in the presence of
sulphide and uptake of 35S from labelled sulphide were indicative of sulphide oxidation. The bacteria
are tightly packed with a density of 0.75 bacteria mm–2 of ciliate surface. For a 170-mm-long ciliate
this amounts to 4500 bacteria with a total volume of 7650 mm3; this is roughly equivalent to the
volume of the host or half of the volume of the symbiotic consortium. While K. fasciolata only
contained one type of bacterium in transmission electron microscopy (TEM) sections, K. fistulosus
also showed ectosymbiotic spirochaetes (Figure 9) (Foissner 1995) and K. latus intracellular
prokaryotes of unknown function (Raikov 1974).
Zoothamnium
The bacteria found on stalk, branches, terminal zooids, and macrozooids are rod shaped, 1.4 mm
long and 0.4 mm wide. They are attached along their longitudinal axis and are regularly arranged,
resembling knitting patterns (Bauer-Nebelsick et al. 1996a). They completely cover the surface in
a single layer except for the adhesive disc and the basal noncontractile part of the stalk. The rods
also cover the basal (proximal) parts of the microzooids. Toward the peristomal disc the bacteria
gradually change in shape, becoming more coccoid to slightly dumbbell shaped and growing larger
(1.9 ¥ 1 mm) (Figure 10). Their arrangement becomes irregular and not all cells appear to be in
contact with the microzooid surface. Especially when the microzooids are contracted the cocci
seem to form more than one layer on the ciliate. The cocci have been observed to detach when the
ciliates are active and become entrained in the feeding currents created by the paroral and adoral
membranelles. Both extreme morphotypes are assumed to belong to the same species and represent
a complex bacterial life cycle (rods/cocci coupled; Bright 2002). Both rods and cocci divide when
they reach sizes of 2.2 and 2.6 mm, respectively.
According to the 16S rRNA gene sequence, the bacteria belong to the g-proteobacteria (Molnar
et al. 2000).
The bacteria appear dark in transmitted light and pure white in incident light. When kept in
seawater without supply of sulphide, the bacteria pale and eventually detach. Freshly collected Z.
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Figure 9 Kentrophoros fistulosus with rod (r) and spirochaetes (s) on the dorsal body side; scale bar = 5 mm.
(Courtesy of W. Foissner.) Figure 10 Zoothamnium niveum with cocci (c) on oral and rods (r) on aboral
parts of the microzooids; scale bar = 10 mm. Figure 11 Stilbonema sp. with cocci (c); scale bar = 10
mm. Figure 12 Laxus oneistus with rods (r); arrow points to dividing rod; scale bar = 5 mm. Figure 13
Eubostrichus parasitiferus with nonseptate filaments (f) attached to the host’s cuticle in a spiral pattern; scale
bar = 10 mm. Figure 14 Rimicaris exoculata with septate filaments (f) and rods (r); scale bar = 50 mm.
(Courtesy of M.F. Polz) All figures are SEM micrographs.
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niveum show a high rate of oxygen uptake of about 450 nl of O2 mm–2 colony surface. Within 4 h
this drops to a sustained rate of 140–180 nl mm–2. Incubation of colonies, which have been kept
in normoxic sea water for 24 h, in 100 mM sulphide resulted in a significant increase in respiration
rate followed again by a subsequent decrease (Ott et al. 1998). The outer layer of the trilaminar
cell envelope undulates in a manner similar to that in free-living thiobacilli. The cells contain large
(diameter of 0.5 mm), electron-translucent, membrane-bound vesicles, which are indicative for
elemental sulphur storage. Smaller (0.1 mm), electron-dense inclusions are interpreted as carboxysomes (Bauer-Nebelsick et al. 1996b), and RuBisCo has been found in the bacteria (H. Felbeck,
personal communication). These physiological and morphological data, together with the ecological
conditions, are strong evidence for a sulphide-oxidising chemoautotrophic nature of the symbionts.
Density of bacteria is approximately 1.5 cells mm–2 for rods and 0.5 cells mm–2 for cocci. Since
the bacteria-covered surface of a microzooid is 1900–2000 mm2, it supports 1900–2000 bacteria
(assuming equal areas colonised by each morphotype). At an estimated volume of a microzooid of
5700–6000 mm3 and a bacterial volume (75% rods, 25% cocci) of 800–840 mm3, the microbial
symbionts amount to 12.1–12.3% of the volume of the symbiotic consortium. On stalks and
branches the respective percentages are even lower, ranging between 5% on thinner and 2.5% on
thicker parts.
In old colonies, white filamentous bacteria grow on basal parts of the stalk and branches together
with a diverse epigrowth of stalked bacteria and diatoms. This irregular fouling starts from the
basal noncontractile part of the stalk and gradually extends to those parts of the stalk and branches
where the symbiotic bacteria and microzooids have been lost (Bauer-Nebelsick et al. 1996a).
Stilbonematinae
A high diversity of ectosymbiotic bacteria is found within the Stilbonematinae. Form and size range
from small (1–2 mm) cocci (Figure 11) through 2- to 5-mm-long rods (Figure 12) to nonseptate
filaments of up to 100 mm in length (Figure 13 and Figure 14) containing approximately 50 nucleoids
(DAPI staining; own unpublished observations). They appear dark brown to almost black in
transmitted light and pure white in incident light due to sulphur inclusions contained in membranebound vesicles.
Their arrangement on nematode cuticles may be genera or even species specific. In most cases
they cover the whole body, leaving only the anterior-most part (head) and the tip of the tail free.
In species of the nematode genus Eubostrichus the worm is entirely covered by bacterial filaments.
In one species of the genus Laxus, L. oneistus, and in a yet undescribed species of the genus
Catanema the bacterial coat starts a few 100 mm–1 mm posterior to the head at a defined level,
where the diameter of the worm’s body decreases to accommodate the thickness of the bacterial
coat (Figure 12). Several layers of cocci embedded in a gelatinous matrix surrounding the host’s
body are typically found in species of the genera Stilbonema (Figure 11) and Leptonemella.
Monolayers of rods are found in Laxus, Catanema, and some Leptonemella and Robbea species;
in the latter genus two layers of rods in different orientations are present. In Laxus oneistus, L.
cosmopolitus, and Catanema sp. the rods are arranged perpendicular to the worm cuticle and divide
by longitudinal fission, a situation reminiscent of that in Kentrophoros. According to the 16S rRNA
sequence the bacteria of Laxus oneistus belong to the g-proteobacteria (Polz et al. 1994).
Uptake of 14C-bicarbonate (Schiemer et al. 1990) and the presence of RuBisCo (Polz et al.
1992) indicate an autotrophic nature of the bacteria. Uptake of 35S-sulphide (Powell et al. 1979),
the presence of sulphur metabolism key enzymes (ATP sulphurylase, sulphite-oxidase), and high
amounts of elemental sulphur (Polz et al. 1992) have been demonstrated. The ultrastructure of the
bacteria shows sulphur granules and possibly carboxysomes. Furthermore, d13C values of the
symbiotic consortium were –24.9 to –27.5, which is similar to animals with sulphur-oxidising
endosymbionts or thiobacilli (Ott et al. 1991).
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J. Ott, M. Bright & S. Bulgheresi
An 8-mm-long Stilbonema majum with a diameter of 60 mm covered by a mucus sheath of 7.5
mm thickness and containing 10 layers of 1.3 ¥ 0.6 mm cocci carries about 21 ¥ 106 bacteria. This
makes up 22% of the volume of the symbiotic consortium. In Laxus oneistus the density of the
upright rods is approximately 3.5 cells mm–2. A 9-mm-long male with a diameter of 50 mm and an
8-mm-long bacterial coat is covered by 4.5 ¥ 106 rods (size 2.1 ¥ 0.6 mm each). This represents
12.3% of the consortium volume.
In the genus Eubostrichus, two types of arrangement of the bacteria are found: in E. parasitiferus
and several similar undescribed species the bacteria are crescent-shaped nonseptate filaments, 0.6
¥ 30 mm in size, that are attached to the cuticle with both ends oriented parallel to the worm’s
longitudinal axis. About 80 bacteria are arranged in a spiral fashion around the circumference of
each worm, giving it the appearance of a rope. In cross section the bacteria appear to form several
layers, when, in fact, all bacteria are in contact with the worm surface. A 3-mm-long E. parasitiferus
with a diameter of 20 mm carries about 8000 bacteria, which make up only 7% of the volume of
the symbiotic consortium, despite their spectacular appearance. In E. dianae the bacteria form up
to 120-mm-long and 0.4-mm-thick filaments, which are attached to the cuticle by one end (Figure
6) and form a dense fur-like coat that in live worms appears nicely groomed. With a size similar
to that of E. parasitiferus, E. dianae carries 40–60 ¥ 103 filaments, contributing substantially
(36–44%) to the consortium volume. The dense microbial coat is colonised by additional bacterial
epibionts (Polz et al. 1999a).
Rimicaris
Three morphological types of bacteria have been described from Rimicaris by various authors (Van
Dover et al. 1988, Casanova et al. 1993, Gebruk et al. 1993, Polz & Cavanaugh 1995): rods with
a diameter from 0.2–0.4 mm and 0.5–3 mm in length and two kinds of filaments, a rare form with
a diameter of 0.2–0.5 mm and a common larger form with a diameter of 0.8–3 mm (Figure 14).
Several septate filaments grow from a common basal attachment disc and, in the large form, may
attain a length of 1.5 mm. The filaments consist of cylindrical cells of approximately the same
length as their diameter. These bacteria densely cover the inner surface of the extended carapace
and also cover the bacteriophores on the enlarged exopodites of the second maxilla and first
maxilliped and on the bases of the thoracic appendages. The rods co-occur with the filaments and
are especially abundant in juveniles. They are attached along their whole length to the cuticle of
the shrimp.
Using a 16S rRNA-specific fluorescent hybridisation probe, Polz & Cavanaugh (1995)
demonstrated that all three morphotypes belong to the same phylotype of e-proteobacteria. A
number of parasites, but also sulphur bacteria such as Thiovolum sp., are found among the eproteobacteria. Recently, bacteria related to Rimicaris symbionts have been detected with
molecular methods in marine anoxic water and sediments (Madrid et al. 2001, Lee et al. 2001).
Elemental sulphur within the cells and RuBisCo activity (Gebruk et al. 1993) strongly suggest
a thiotrophic nature of the bacteria. Polz & Cavanaugh (1995) estimate that an average shrimp
may carry 8.5 ¥ 106 bacteria.
Mutual benefits
The consensus is that the above symbioses are largely nutritional. On one hand, the bacteria provide
organic matter from their own primary chemoautotrophic production. On the other hand, the
eukaryote partner facilitates access to reduced sulphur compounds and electron acceptors, which
may be separated in space and time. Since sulphide is toxic for aerobic metazoans, the idea has
been proposed that the bacteria may act as a detoxification mechanism, oxidising sulphide into
elemental sulphur and finally to sulphate (Somero et al. 1989). Powell (1989) argued that in
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meiofauna, due to the small size of the animals, sulphur detoxification could not work and they
should be sulphide insensitive. In fact, high concentrations of thioles can be found in the tissues
of Stilbonematinae (Hentschel et al. 1999). A certain detoxification may nevertheless be provided,
because the bacterial symbionts increase heat tolerance in Stilbonematinae in the presence of
simultaneous sulphide stress (Ott 1995). Sulphide detoxification probably occurs in sulphideoxidising bodies in the gills of Rimicaris (Compere et al. 2002) without any involvement of the
ectosymbionts.
Nutrition
The limited success in culturing thiotrophic symbioses has precluded direct evidence that the
animals feed on the symbiotic bacteria and may grow with them as their sole food. The presence
of bacteria in feeding and digestive vacuoles has been reported for Kentrophoros (Raikov 1971,
1974, Fenchel & Finlay 1989) and Zoothamnium niveum (Bauer-Nebelsick et al. 1996b) and in the
gut lumen in several species of Stilbonematinae (Wieser 1959, Ott & Novak 1989, Riemann et al.
2003). In all cases the bacteria have the distinct morphology and fine structure of the respective
ectosymbionts and dominate the content of the digestive tract.
Transfer of labelled carbon from symbionts to host has been shown for Z. niveum by Rinke
(2002) in pulse and chase experiments. Most evidence for a nutritional interaction comes from
studies of natural tracers in food chains, such as fatty acids and stable isotopes. High proportions
of n-4 fatty acids, which are indicative for a bacterial origin, have been found in tissues of Rimicaris,
whereas in the closely related genera Alvinocaris and Mirocaris, n-3 fatty acids characteristic for
photosynthetically derived carbon are more abundant (Pond et al. 1997b,c). Muscles of Rimicaris
contain n-7 fatty acids, which are closer in d13C (–13‰) to the ectosymbionts (–12‰) than to
bacteria scraped from hydrothermal chimneys (–21‰) (Rieley et al. 1999). As a relic of its early
planktotrophic life, however, Rimicaris contains high levels of polyunsaturated fatty acids in storage
compounds (wax esters) in reproductive tissues. These fatty acids are thought to be important for
reproduction in Crustacea (Pond et al. 2000, Allen et al. 2001). Gebruk et al. (1993) report d13C
values for various tissues ranging from –10.5 to –12.5‰, which is similar to values reported for
other hydrothermal vent animals. Stilbonematinae have d13C of –25.9‰ without and –24.9 to
–27.5‰ with their symbionts, whereas nonsymbiotic nematodes and detritus from the same habitat
show values of –10.3 and –10.5‰, respectively (Ott et al. 1991).
In some cases the biomass of the bacterial symbionts dominates the food availability in the
environment (Gebruk et al. 1993, Van Dover 2002). Polz & Cavanaugh (1995) estimated that at a
density of 25,000 shrimp m–2 the number of symbiotic bacteria attached to Rimicaris would be 2.1
¥ 1011, which is almost three orders of magnitude higher than the 4.9 ¥ 108 bacteria attached to a
square meter of sulphide chimney surface.
Dependence on a special resource is implied by morphological changes in feeding structures,
such as the near disappearance of a functional mouth in Kentrophoros (Foissner 1995) or the
reduction in dentition and the weakening and rearrangement of pharynx musculature in the Stilbonematinae (Hoschitz et al. 2001).
Fenchel & Finlay (1989) calculated that the bacterial production in Kentrophoros allows a
doubling time of the ciliate of 18 h, which is low for a ciliate of this size, but would allow the
protozoan to grow with the symbionts as a unique food source. No such data are available for the
other symbioses. Zoothamnium niveum, however, can maintain the high growth speed to reach the
large size only in the presence of the bacteria. The growth rate and maximum size reached by
aposymbiotic colonies reared from macrozooids that had lost their bacteria is only about 10% of
those of symbiotic colonies. Stilbonematinae have an extremely low metabolism (Schiemer et al.
1990) and could probably be easily sustained on the production of their bacteria. Note also that
the bacteria appear to divide more rapidly on the margin of presumed feeding patches on the
nematode cuticle (Polz et al. 1992).
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Access to sulphide and electron acceptors
The main benefit for the bacteria is apparently that the association with a mobile host allows them
to exploit redox gradients that would otherwise not be available for their growth. The sharpness of
the gradient and the distances covered by the carrier host vary greatly (Figure 15 to Figure 17).
Zoothamnium niveum exploits the sharpest gradient, which develops within a boundary layer
between a sulphide source and ambient water on the surface of macrophyte debris accumulations
(Figure 15). This boundary layer is only a few millimetres thick but oxygen concentrations drop
from near saturation to virtually zero while sulphide concentrations increase from undetectable to
several hundred micromolar (Ott et al. 1998, Vopel et al. 2001). The ciliates effectively mix sulphidic
and oxygen-containing water and create high current speeds (up to 11 mm s–1) over the zooid
surfaces. Rapid contractions with speeds up to 520 mm s–1 exchange water along the colony, whereas
slow expansions (<1 mm s–1) drag sulphidic water along (Vopel et al. 2001, 2002). The rapid
contractions may also be necessary to loosen the bacteria and make them accessible for feeding
(Vopel et al. 2002).
Kentrophoros and Stilbonematinae live in a redox gradient that extends over several centimetres
in porous sediments (Figure 16). Both move at slow speeds, crossing the chemocline repeatedly
within a day. While Kentrophoros orients along the oxygen gradient (Fenchel & Finlay 1989), the
stilbonematid Laxus oneistus shows a more complex behaviour. At oxygen values near saturation
the worms are inactive, whereas decreasing oxygen concentration increases their activity and leads
to a positive geotactic movement. Encounters with sulphide concentrations of >300 mM make the
worms turn around and crawl in the direction of the surface. In this ping-pong fashion, worms
alternately visit deeper sulphidic layers and surface layers with abundant electron acceptors (oxygen,
nitrate) (Ott et al. 1991). Schiemer et al. (1990) showed that the symbiotic bacteria can take
advantage of this behaviour by storing reduced sulphur compounds and oxidising them upon
availability of electron acceptors.
Rimicaris is the most mobile carrier host, moving rapidly in and out of hydrothermal fluid
plumes where sulphidic and oxic water mixes (Figure 17). In addition, the ventilation of the chamber
formed by the enlarged carapace with the large scaphognathites is thought to create a favourable
environment for chemoautotrophic growth. Polz et al. (1999b, 2000) have put forward an interesting
Figure 15 Model of access to sulphide and oxygen in ectosymbioses. Zoothamnium niveum growing on peat
wall has access to sulphide leaking from the peat when contracted and access to oxygen from the overlying
sea water when expanded; scale bar = 1 mm. Figure 16 Model of access to sulphide and oxygen in
ectosymbioses. Interstitial Kentrophoros spp. and Stilbonematinae migrate between sulphidic deeper sediment
layers and oxic surface layers; scale bar = 1 cm. Figure 17 Model of access to sulphide and oxygen in
ectosymbioses. Rimicaris exoculata swims in and out of sulphidic hydrothermal fluid and oxic ambient deepsea water; scale bar = 1 m.
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hypothesis for the predominance of the symbiotic bacterial phylotype in the free-living community
attached to the sulphidic rocks, where it contributes to >60% of the bacterial nucleic acids recovered
from scrapings (Polz & Cavanaugh 1996). The constant inoculum through bacteria falling off the
shrimps provides additional positive feedback to the free-living population of the symbiotic bacteria,
helping it to rapidly reach high cell numbers and to outcompete other bacterial species. In turn,
the abundance and stability of the free-living stock increase the chance that newly arrived juveniles
or freshly moulted individuals will pick up the right symbiont.
Maintenance and evolution of thiotrophic ectosymbioses
A vertical transmission to subsequent generations has only been described in the ciliates. When a
Kentrophoros cell divides, both daughter cells get their share of the symbiotic bacteria (W. Foissner,
personal communication). In Zoothamnium the motile macrozooids (swarmers) are covered with
rods when they detach (Bauer-Nebelsick et al. 1996a), with a typical swarmer of 150 mm in diameter
carrying about 90,000 bacteria. During colony growth after settlement, all surfaces, except for the
basal 300–500 mm of the stalk, remain covered by the bacterial coat, which keeps pace with the
production of new surfaces by the terminal zooids.
In the nematodes there is no evidence of vertical transmission from parents to offspring.
Nevertheless, even very small (stage 1) juveniles have been observed to carry a complete microbial
coat (own unpublished observations). Since all ectosymbionts are attached to the worm cuticle,
they are shed when the host moults. In the nematodes this usually occurs four times in a life cycle.
Moulting stages of several species have been observed where all bacteria are left behind on the
exuvia (Wieser 1959, own unpublished observations). Recolonisation, however, must be a rapid
process, since aposymbiotic Stilbonematinae are rarely found in field collections. These facts argue
in favour of an environmental transmission with immediate colonisation of newly hatched and rapid
recolonisation of moulted worms. In the case of Laxus oneistus mannose-binding lectins expressed
on its cuticle could enable it to specifically select bacteria from the surrounding sediment
(Nussbaumer et al. 2004).
Larvae and juveniles of Rimicaris do not carry the symbiotic bacteria and feed on photosynthetic
microplankton (Pond et al. 1997b, Dixon et al. 1998). When returning to the vents they must acquire
the bacteria to populate the growing morphological structures that support the symbiotic population
of adult shrimp. The mechanism of this process is unknown. As in nematodes, the shrimps must
moult in order to grow and therefore repeatedly lose the symbionts during their life. The high
specificity of the symbiotic association suggests a precise recognition mechanism as postulated for
the Stilbonematinae.
Comparison with close relatives allows inferences about the evolution of the symbioses. There
are a number of prerequisites for the formation of such close associations. The ancestors of today’s
partners must have lived in the same environment, and loose, nonobligate relationships must have
preceded the tight and specific bonds of today. Morphological and physiological characters must
have preadapted the future partners for the ability to make and maintain contact, and behavioural
traits were necessary to select one of the many possible partners.
The most complete line of evidence exists for the Stilbonematinae symbiosis. Species of the
nematode family Desmodoridae are characteristic for oxygen-poor layers of marine sediment. In
fact, the species Spirinia gnaigeri has the lowest weight-specific respiration rate of all marine freeliving nematodes (Schiemer et al. 1990). Occurring in or close to the RPD, they share the habitat
with sulphur-oxidising chemoautotrophic bacteria, on which they probably feed together with other
bacteria occurring in the sand. Furthermore, desmodorids are frequently fouled by a diverse
assemblage of microorganisms, including a variety of bacterial morphotypes, stalked diatoms, and
suctorians (Ott 1996). This is in contrast to most other nematode taxa, which rarely show microbial
epigrowth. We may expect that sulphur bacteria were among this fortuitous microbial cover on the
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J. Ott, M. Bright & S. Bulgheresi
ancestors of the Stilbonematinae. Moreover, behavioural traits such as migration through the
chemocline — possibly to feed on dissolved organic matter (Riemann et al. 1990) — no doubt
selected for the sulphur bacteria because they not only tolerated but benefited from alternate
exposure to sulphide and oxygen. In the case of weak or unstable chemoclines, the association
with a mobile host may more than compensate for the grazing loss to the worms.
The Zoothamnium niveum symbiosis may have a similar history. Members of the large genus
Zoothamnium occur in a great variety of marine and limnic habitats, including those low in oxygen.
Zoothamnium alternans, a close relative of Z. niveum, is regularly found in the same habitat.
Zoothamnium species are notorious for microbial fouling. For the association between Z. pelagicum
and cyanobacteria, a symbiotic relationship has even been suggested (Laval-Peuto & Rassoulzadegan 1988). The spontaneous contractions of the colonies, which are typical for many sessile peritrich
ciliates, and the feeding currents that mix sulphidic and oxic water provided the selective force for
the association with sulphur bacteria.
The Alvinocarididae exhibit several modes of nutrition. While Alvinocaris appears to be an
unspecialised scavenger and predator, Chorocaris feeds on a mixed diet and has already modified
mouthparts overgrown with bacteria, although not to the extent of Rimicaris, which presumably is
an obligate bacteriovore. The high attractiveness of chemoautotrophic primary production in the
otherwise food-limited deep sea may have brought the ancestors of Rimicaris and its symbiont
together.
Suspected symbioses
In addition to the symbioses described above, a great variety of associations between protists or
invertebrates and ectosymbiotic bacteria have been described from marine sulphidic habitats. In
most of these cases the nature of the bacteria and their function in the symbiosis are unknown.
Among the protists, epibiotic bacteria in a regular arrangement suggesting a symbiotic association have been described from flagellates and several ciliates. Euglenozoans from a Monterey
Bay cold seep and the dysoxic Santa Barbara Basin (California) are densely covered by rod-shaped
bacteria. Putative sulphur vesicles in the bacteria and the presence of sulphide in the sediment
suggest a sulphur-oxidising metabolism for the symbionts. No evidence for ingestion of the
bacteria by the protists has been found (Buck et al. 2000, Bernhard et al. 2000). Several cases of
ciliates belonging to the genera Parablepharisma, Metopus, Caenomorpha, and Sonderia having
ectobiotic bacteria have been reported from anoxic and sulphidic sediments (Fenchel et al. 1977).
The bacteria are in all cases curved rods and probably utilise products of the ciliates’ fermentative
metabolism. Density varies from 1,000 to 100,000 bacteria per ciliate. In the ciliate Geleia fossata
from a tidal flat on the U.S. East Coast, short Gram-negative rods are positioned in and along the
ciliated grooves. They are embedded in deep cell membrane invaginations and some seem to be
enclosed in membrane-bound vesicles. There is no indication that the bacteria are chemolithoautotrophs as in the closely related genus Kentrophoros, and their low density and biomass (2–10
¥ 103 per ciliate, amounting to only a few percent of the ciliate biovolume) make a nutritive
dependence unlikely. Several other related ciliates belonging to the genera Tracheloraphis,
Paraspathidium, Loxophyllum, and Cyclidium from the same habitat showed scattered ectobiotic
bacteria, indicating that associations with bacteria are probably widespread in marine sediments
(Epstein et al. 1998).
Among the invertebrates, a yet undescribed flatworm belonging to the Monocelidae (Proseriata,
Platyhelminthes) dominates the meiofauna of sediments at a temperature of 30–40˚C under the
influence of shallow-water hydrothermal vents on Deception Island, Antarctica. The outer surface
of the worm is covered by a single morphotype of straight to slightly curved rods (0.7 ¥ 2 mm)
embedded in mucus at the level of the cilia tips. There is no indication for a chemoautotrophic
nature of the bacteria because tests for RuBisCo were negative. Ectobiotic bacteria have been
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111
occasionally reported for a number of free-living marine flatworms, but no indication of specificity
and persistence has been given. The uniformity of the bacteria and the fact that all specimens
carried the same dense coat suggest a symbiotic relationship of an as yet unknown function in the
Antarctic monocelid (Bright et al. 2003).
Similarly, the small bacteria that apparently represent a single morphotype covering the entire
surface of the annelid Xenonerilla bactericola (Nerillidae) from the dysoxic Santa Barbara Basin
off California are suspected to be symbionts. The nature of this symbiosis also remains unresolved
(Müller et al. 2001). In the same habitat the nematode Desmodora masira regularly has epibiotic
bacteria (Bernhard et al. 2000) a feature that is characteristic for many species of the family
Desmodoridae, to which the Stilbonematinae also belong (Ott 1996).
In the best-studied case, the hydrothermal vent annelid Alvinella pompejana, three morphotypes of bacteria occur on epidermal expansions and cuticular protrusions on the intersegmental
spaces. The cylindro-conical expansions, which may be 1 cm long, are peculiar to Alvinella. They
are glandular, not cuticularised, and are covered by two types of filamentous bacteria. Two
phylotypes of e-proteobacteria have been identified (Haddad et al. 1995, Cary et al. 1997). Whereas
one of these is also a major component of the free-living bacterial community, the other is
exclusively found on Alvinella and on the inside of its tube. Although uptake of inorganic carbon
and RuBisCo activity have been reported (Alayse-Danet et al. 1986) their low levels make an
important contribution of autotrophs unlikely. The presence of bisulphate reductase genes in
bacteria suggests that they are anaerobic sulphate reducers (Cottrell & Cary 1999) that probably
play a role in the formation of the typical “white smokers” associated with the presence of
Alvinella spp. Recently, abundant spirochetes (Campbell & Cary 2001) and a vibrio (Raguenes
et al. 1997) that probably are heterotrophs have been detected among the ectosymbionts by
molecular methods.
It is unclear to what extent A. pompejana feeds on its episymbiotic bacteria. Stable isotope
data point to a bacterial food source (Desbruyàres et al. 1983) but behavioural observation and gut
content analysis suggest that grazing on the tube may be the usual mode of nutrition in this worm.
The modifications found on those segments of Alvinella that carry bacteria are evidence for a close
relationship between the microbes and their host. The specific nature of the association — be it
nutritional or related to sulphide detoxification — has yet to be elucidated (Alayse-Danet et al.
1987). The biology of A. pompejana, which is an early coloniser of vent chimneys and has a
remarkably high temperature tolerance, has been exhaustively summarised by Desbruyàres et al.
(1998).
The priapulid Halicryptus spinulosus has a modified outer cuticle layer forming minute ridges
that greatly enlarge the cuticular surface. The crevices formed by these ridges are densely populated
by bacteria of three distinct morphological types embedded in mucus in a characteristic arrangement. Cluster-forming bacteria deep in the crevices precipitate iron probably as Fe-sulphide.
Oxidation with iron may bind small amounts of sulphide, and this may help the worm to survive
intertidal exposure until metabolic adaptations take over. The bacteria may be of potential nutritional
significance. No indication, however, of transepidermal transport was observed (Oeschger & Janssen
1991, Oeschger & Schmaljohann 1988).
Among the Crustacea, stalked barnacles (“Lau A,” Scalpellomorpha, Neolepadinae) from hydrothermal vents have dense, elongated cirral setae that are covered by white bacterial filaments. The
filaments have a diameter of 1.15 mm, are composed of cells approximately 1 mm long, and may
be up to 220 mm long. The mandibles appear modified compared with other stalked vent barnacles,
probably to facilitate combing of bacteria from the cirri. Except for the white colour, no other
indication of a sulphur metabolism of the bacteria is given. Ecological observations showing the
barnacles extending their cirri into diffuse hydrothermal flow could be taken to indicate a chemoautotrophic nature of the bacteria (Southward & Newman 1998).
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Summary and outlook
The importance of the thiotrophic ectosymbioses varies greatly between the different cases. Fenchel
& Finlay (1989) calculated that the biomass of bacteria associated with specimens of Kentrophoros
in their sediments is only 3.7 ¥ 10–4 g m–2 compared with a biomass of free-living sulphur-oxidising
bacteria of 5–20 g m–2. It may therefore be merely “an exotic phenomenon which makes only a
symbolic contribution” (Fenchel & Finlay 1989). In Zoothamnium niveum it may be important at
a microspatial scale: with an estimated weight of the bacteria on an average colony of 1000
microzooids of 1 mg, the symbionts would contribute only 1.2 mg m–2 (assuming 1200 colonies;
Ott et al. 1998). Because the colonies are highly aggregated, this value increases 100-fold when
only the patches are taken into account (assuming 100 colonies on an area of 10 cm2). In tropical
calcareous sands, where Stilbonematinae are a dominant element of the meiofauna (Ott & Novak
1989), the weight of the symbiotic bacteria may be in the same order of magnitude as that of freeliving sulphur bacteria. At a density of about 300 ¥ 103 worms m–2 (consisting of equal numbers
of Laxus oneistus and Stilbonema majum, carrying on average half of the bacteria calculated for
an adult worm), the combined weight of the symbiotic bacteria would amount to 0.7–0.8 g. In the
case of Rimicaris the ectosymbionts are (with estimated 2.1–4.2 ¥ 1011 bacterial cells m–2) significantly more abundant than the sulphur-oxidising bacteria attached to the chimney surface (approximately 4.9 ¥ 108 m–2), but also dominate the water close to the chimney, which contains about 5
¥ 108 bacteria l–1.
Sulphide symbioses are apparently a frequent outcome of the various associations of protists
and invertebrates with bacteria. The most common type of association leading to ectosymbioses is
fouling of surfaces, which originally involved a diversity of microbes, microalgae, and protists.
Evidence for this evolutionary intermediate stage is the many cases of irregular epigrowth reported
from ciliates, including relatives of Kentrophoros and Zoothamnium niveum, and nematodes that
are closely related to the Stilbonematinae. These fouled organisms may be regarded as models for
the ancestors of extant symbioses and apparently lack the ability to keep their surface free of
epibionts, which at high densities may become a nuisance (Ott 1996). This imperfection, however,
was a necessary precondition for the evolution of a successful mutualistic interaction. There is little
evidence pertaining to the age of thiotrophic symbioses. Most of the above-described hosts do not
fossilise well enough to leave an indication of bacterial symbioses. Only in the arthropods is there
some evidence that a microbial and possibly thiotrophic symbiosis existed in the Ordovician olenid
trilobites. These now extinct animals lived on reduced sediments, probably in dysoxic and sulphidic
bottom water. Their extended carapace has been interpreted as an incubation chamber for sulphur
bacteria, similar to that of Rimicaris (Fortey 2000).
The spectacular endosymbioses of the Vestimentifera and the large clams and mussels at hot
vents have directed much attention to these conspicuous animals. Until the discovery of the
Rimicaris symbiosis the study of ectosymbioses had not been pursued with the same effort. There
is, however, still much to discover both in shallow water and in the deep sea that will shed new
light on the fascinating functioning and evolution of thiotrophic symbioses.
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