2727_C04.fm Page 95 Wednesday, June 30, 2004 12:00 PM 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: joerg.ott@univie.ac.at 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) 0-8493-2727-X/04/$0.00+$1.50 Oceanography and Marine Biology: An Annual Review 2004, 42, 95–118 © R N Gibson, R J A Atkinson, and J D M Gordon, Editors © 2005 by CRC Press LLC 95 2727_C04.fm Page 96 Wednesday, June 30, 2004 12:00 PM 96 J Ott, M Bright & S Bulgheresi 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 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) © 2005 by CRC Press LLC 2727_C04.fm Page 97 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 97 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 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 © 2005 by CRC Press LLC 2727_C04.fm Page 98 Wednesday, June 30, 2004 12:00 PM 98 J Ott, M Bright & S Bulgheresi 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 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, 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 © 2005 by CRC Press LLC 2727_C04.fm Page 99 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 99 Figure Light microscopy (LM) micrograph of living specimen of Kentrophoros fistulosus; scale bar = 500 mm Figure 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 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 SEM micrograph of contracted colony showing several microzooids (mi) and one macrozooid (ma) covered by symbionts; scale bar = 50 mm © 2005 by CRC Press LLC 2727_C04.fm Page 100 Wednesday, June 30, 2004 12:00 PM 100 J Ott, M Bright & S Bulgheresi 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 hours the first branch is formed, and within 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 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 mm to about 15 mm (Figure 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 © 2005 by CRC Press LLC 2727_C04.fm Page 101 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 101 Figure 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 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 © 2005 by CRC Press LLC 2727_C04.fm Page 102 Wednesday, June 30, 2004 12:00 PM 102 J Ott, M Bright & S Bulgheresi Figure 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 SEM of shrimp appendage; scale bar = 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 and maxilliped 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 © 2005 by CRC Press LLC 2727_C04.fm Page 103 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 103 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 ¥ 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 © 2005 by CRC Press LLC 2727_C04.fm Page 104 Wednesday, June 30, 2004 12:00 PM 104 J Ott, M Bright & S Bulgheresi Figure Kentrophoros fistulosus with rod (r) and spirochaetes (s) on the dorsal body side; scale bar = 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 = 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 © 2005 by CRC Press LLC 2727_C04.fm Page 105 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 105 niveum show a high rate of oxygen uptake of about 450 nl of O2 mm–2 colony surface Within 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) © 2005 by CRC Press LLC 2727_C04.fm Page 106 Wednesday, June 30, 2004 12:00 PM 106 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 © 2005 by CRC Press LLC 2727_C04.fm Page 107 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 107 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) © 2005 by CRC Press LLC 2727_C04.fm Page 108 Wednesday, June 30, 2004 12:00 PM 108 J Ott, M Bright & S Bulgheresi 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 (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 = 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 = 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 = m © 2005 by CRC Press LLC 2727_C04.fm Page 109 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 109 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 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 © 2005 by CRC Press LLC 2727_C04.fm Page 110 Wednesday, June 30, 2004 12:00 PM 110 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 ¥ 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 © 2005 by CRC Press LLC 2727_C04.fm Page 111 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 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 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 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) © 2005 by CRC Press LLC 2727_C04.fm Page 112 Wednesday, June 30, 2004 12:00 PM 112 J Ott, M Bright & S Bulgheresi 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 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 ¥ 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 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 References Alayse-Danet, A.M., Desbruyères, D & Gaill, F 1987 The possible nutritional or detoxification role of the epibiotic bacteria of alvinellid polychaetes: review of current data Symbiosis 4, 51–62 Alayse-Danet, A.M., Gaill, F & Desbruyères, D 1986 In situ bicarbonate uptake by bacteria-Alvinella associations Pubblicazioni della Stazione Zoologica di Napoli I Marine Ecology 7, 233–240 Allen, C.E 1958 On the basic form and adaptations to the habitat in Lucinaceae (Eulamellibranchia) Philosophical Transactions of the Royal Society of London Biological Sciences 241, 421–484 Allen, C.E., Copley, J.T & Tyler, P.A 2001 Lipid partitioning in the hydrothermal vent shrimp Rimicaris exoculata Pubblicazioni della Stazione Zoologica di Napoli I Marine Ecology 22, 241–253 © 2005 by CRC Press LLC 2727_C04.fm Page 113 Wednesday, June 30, 2004 12:00 PM Marine Microbial Thiotrophic Ectosymbioses 113 Allen, C.E., Copley, J.T., Tyler, P.A & Varney, M 1998 Lipid profiles of hydrothermal vent shrimps Cahiers de Biologie Marine 39, 229–231 Alt, J.C 1995 Subsea floor processes in mid-ocean ridge hydrothermal systems In Sea-Floor Hydrothermal Systems: Physical, Chemical, Biological and Geological Interactions, S.E Humphris et al (eds) Washington, DC: American Geophysical Union, Geophysical Monograph 91, 85–114 Bauer-Nebelsick, M., Bardele, C.F & Ott, J.A 1996a Redescription of Zoothamnium niveum (Hemprich & Ehrenberg, 1831) Ehrenberg 1838 (Oligohymenophora, Peritrichida), a ciliate with ectosymbiotic, chemoautotrophic bacteria European Journal of Protistology 32, 18–30 Bauer-Nebelsick, M., Bardele, C.F & Ott, J.A 1996b Electron microscopic studies on Zoothamnium niveum (Hemprich & Ehrenberg, 1831) Ehrenberg 1838 (Oligohymenophora, Peritrichida), a ciliate with ectosymbiotic, chemoautotrophic bacteria European Journal of Protistology 32, 202–215 Bauer-Nebelsick, M., Blumer, M., Urbancik, W & Ott, J.A 1995 The glandular sensory organ of Desmodoridae (Nematoda): ultrastructure and phylogenetic implications Invertebrate Biology 114, 211–219 Bernhard, J.M., Buck, K.R., Farmer, M.A & Bowser, S.S 2000 The Santa Barbara Basin is a symbiosis oasis Nature 403, 77–80 Bright, M 2002 Life strategies of thiotrophic ectosymbioses In The Vienna School of Marine Biology: A Tribute to Jörg Ott, M Bright et al (eds) Wien: Facultas Universitätsverlag, pp 19–32 Bright, M., Arndt, C., Keckeis, H & Felbeck, H 2003 A temperature-tolerant interstitial worm with associated epibiotic bacteria from the shallow water fumaroles of Deception Island, Antarctica Deep-Sea Research II 50, 1859–1871 Buck, K.R., Barry, J.P & Simpson, A.G.B 2000 Monterey Bay cold seep biota: Euglenozoa with chemoautotrophic bacterial epibionts European Journal of Protistology 36, 117–126 Campbell, J.B & Cary, C.S 2001 Characterization of a novel spirochete associated with the hydrothermal vent polychaete annelid, Alvinella pompejana Applied Environmental Microbiology 67, 110–117 Carney, R.S 1994 Consideration of the oasis analogy for chemosynthetic communities at Gulf of Mexico hydrocarbon vents Geo-Marine Letters 14, 149–159 Cary, 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Composition and distribution of macro- and meiobenthos around sub-littoral hydrothermal vents in the Bay of Plenty, New Zealand New Zealand Journal of Marine and Freshwater Research 27, 40 7? ?41 8 Kampfer,... at deep-sea vents and seeps In Deep-Sea and Extreme Shallow-Water Habitats: Affinities and Adaptations, F Uiblein et al (eds) Vienna: Austrian Academy of Sciences Press, Biosystematics and Ecology... 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 and maxilliped