Oceanography and Marine Biology: An Annual Review (Volume 42) - Chapter 4 potx

Marine Microbial Thiotrophic Ectosymbioses 97Cold seeps Wherever the sea sediments are subjected to pressure, pore fluid is squeezed from the intersticesand seeps from the surface.. Sinc

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Oceanography and Marine Biology: An Annual Review 2004, 42, 95–118

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 Proteo-bacteria Whereas some of the better-known symbioses are highly evolved endosymbioses, the moreprimitive ectosymbioses are less well known The sulphur-oxidising chemolithotrophic nature ofthe bacteria and their nutritive importance to the eukaryote host have been demonstrated for theciliates Kentrophoros spp and Zoothamnium niveum, the nematode subfamily Stilbonematinae, andthe carid shrimp Rimicaris exoculata For a number of other regular bacteria–eukaryote associations,such a symbiotic relationship has been hypothesised based on ecological, morphological, physio-logical 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 hydrothermalvents has led to the discovery of an unexpected diversity of microbe/animal relationships in avariety 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) havespurred 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 water 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 thethiotrophic symbioses play in an extremely food-limited setting In shallow water the predominance

shallow-of photoautotrophic production restricts thiotrophic symbioses to a more cryptic existence

To date symbioses with sulphur-oxidising chemolithoautotrophic bacteria have been recordedfor protists (ciliates and probably also flagellates) and seven animal phyla (Platyhelminthes, Nem-atoda, Echiurida, Annelida, Mollusca, Arthropoda, and Echinodermata) With the exception ofPlatyhelminthes, Echiurida, and Echinodermata, the development of thiotrophic symbioses hasoccurred more than once in each phylum

The diversity of the microbial symbionts is as high as that of the hosts, and although they allbelong 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 pods, the vestigial gut in Astomonema southwardorum, or under the cuticle between epidermis cells

gastro-of Oligochaeta (Fisher 1996, Giere 1996)

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96 J Ott, M Bright & S Bulgheresi

In many cases, however, they appear ectosymbiotically, attached to the body surface of theireukaryote host, such as in flagellates, ciliates, Nematoda (Stilbonematinae), and in certain Annelida(Alvinellidae) and Arthropoda (Ott 1996, Polz & Cavanaugh 1996) This review summarises ourknowledge 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 environmentand physiology of the bacterial partner The animal hosts appear less modified than is the case inendosymbioses, and in most cases, the relationship to nonsymbiotic relatives can be traced withconfidence Rarely is a particular ectosymbiosis characteristic for taxa higher than genera Althoughmorphological modifications in conjunction with the symbiotic way of life are present in practicallyall ectosymbioses, they never reach the extent found in endosymbioses In some cases the partnersmay be separated and kept alive at least for some time These characteristics allow us to makeinferences on how these symbioses originated, something that is extremely difficult to do in highlyevolved 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 beacquired in each generation, most probably from the free-living bacterial community in the respec-tive habitat The mechanisms of recognition, attachment, and internalisation of the microbial partnerare still unclear

of the rift valley or seeps through sediments Mineral precipitates may form chimneys dozens ofmetres high, from which the hydrothermal fluid emanates as black clouds coloured by precipitatingmetal sulphides (Goldfarb et al 1983)

Chemolithoautotrophic bacteria and Archaea already grow in the chemical gradients within theconduits in the basaltic rocks (Karl et al 1980) Where the hydrothermal fluid is injected into theoxygen-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 hydrothermalvents, which solely depends on the production of the chemolithoautotrophic microbes (Van Dover2000) Whereas many animals are suspension feeders or simply graze the microbial turf, otherslive 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 survivalstrategies here include associations either with mobile animals that may follow gradients or withlarge sessile organisms that provide the necessary milieu for the bacteria Both endo- and ecto-symbioses are found in these bizarre environments The most prominent examples are the vesti-mentiferan 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 inconjunction 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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Marine Microbial Thiotrophic Ectosymbioses 97

Cold seeps

Wherever the sea sediments are subjected to pressure, pore fluid is squeezed from the intersticesand seeps from the surface This seepage may occur under a variety of tectonic forces: in accretionprisms formed during subduction of one lithospheric plate under another, at the margin of mudvolcanoes, in places where salt diapirs rise through continental margin sediments, or in connectionwith hydrocarbon seeps The expelled fluids differ chemically from the surrounding sea water Theyare anoxic and may contain methane or sulphide, hydrocarbons, or high salt concentrations, butnot 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 detectionlimit 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 withinthe 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, marshand mangrove sediments, over essentially all of the shelf sediments, to dysoxic basins and upper-slope sediments It occurs under an oxic surface layer of variable thickness, ranging from a fewmillimetres to several centimetres, from which it is separated by a chemocline – the redox potentialdiscontinuity layer (RPD) (Fenchel & Riedl 1970) Within the RPD, electron acceptors for theoxidation of organic material change in sequence from oxygen to nitrate, ferric iron, manganeseand 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 oxidationprocess for carbon fixation (Jørgensen 1989) Since sediment layers containing sulphide may beseparated from those containing the best electron acceptor (oxygen) by several millimetres tocentimetres, 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 observedfor hot vents and cold seeps, bacteria have succeeded in finding hosts that provide them with bothoxygen 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 (catenulidflatworms, phallodrilid oligochaetes, nematodes of the genus Astomonema) (Giere 1996) or asectosymbionts (ciliates, stilbonematid nematodes) (Ott 1996) Recently, a number of flagellatesliving in the soft sediment of a dysoxic basin have been described to harbour potentially chemo-autotrophic 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 mayaccumulate (Fenchel 1970, Mann 1976) The decomposition of this organic matter creates sulphidichabitats of various spatial and temporal extents Mangrove peat is a relatively stable substratumhaving internal sulphide concentrations of up to 4 mM (McKee 1993) Diffusion of sulphide intothe 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 macrophyte2727_C04.fm Page 97 Wednesday, June 30, 2004 12:00 PM

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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 andcaves among rocks

Thiotrophic ectosymbioses have been reported from mangrove peat and decomposing sea grassand 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 RPDseveral centimetres beneath the surface (Fenchel & Finlay 1989) Soon after the description of thefirst 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 bythe 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 flattenedshape is interpreted as an adaptation to provide ample space for the bacterial symbionts; it increasesthe surface-to-volume ratio by a factor of 6–7 compared with other similarly sized ciliates Theventral (or right) side bears cilia arranged in longitudinal rows The cells have only a vestigialcytostome (Foissner 1995) Rod-shaped bacteria occupy the unciliated dorsal (or left) side of thecell, 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 theoxic–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 berandomly distributed in a sulphide gradient in the absence of oxygen (Fenchel & Finlay 1989).Under experimental conditions and under the assumption that the ectosymbiotic bacteria con-stitute its sole food, Kentrophoros was calculated to have a doubling time of 18 h at roomtemperature, which is low compared with similarly sized ciliates This difference was attributed tosuboptimal culture conditions (Fenchel & Finlay 1989)

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Figure 1 Light microscopy (LM) micrograph of living specimen of Kentrophoros fistulosus; scale bar = 500

dorsal side and cilia on the ventral side; scale bar = 20 m m (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 m m Figure 4 SEM micrograph of contracted colony showing several microzooids (mi) and one macrozooid (ma) covered by symbionts; scale bar = 50 m m.

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Large colonies sprout secondary fans and may have about 200 branches bearing up to 20 feedingmicrozooids each, adding up to over 3000 microzooids The tips of the stalk and of still-growingbranches bear nonfeeding, terminal zooids that divide by unequal longitudinal fission On somebranches the proximal zooid develops into a globular nonfeeding macrozooid that eventuallydetaches as a motile swarmer Except for the noncontractile basal part of the stalk, branches andzooids 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 betweensulphide and oxygen are developed within a few millimetres The ciliary action of the microzooidseffectively mixes sulphidic and oxic water (Vopel et al 2001, 2002) In addition, the coloniescontract into the sulphidic boundary layer and subsequently expand again into the surroundingoxygen-containing water (Ott et al 1998) The life cycle of Z niveum has been studied throughseveral generations in the laboratory Using sulphide as a cue, the swarmers of Z niveum activelyseek and colonise patches in the environment with high sulphide flux, such as areas where the peatsurface has been recently disturbed Upon settling, each swarmer changes into a terminal zooidthat produces a stalk and starts to divide into microzooids and terminal zooids, which in turn growinto branches After about 4 hours the first branch is formed, and within 4 days maximum size isreached During the period of exponential growth on the second and third days, the terminal zooidsdivide approximately once every hour (own unpublished data) The colonies continue to live to amean 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 Swarmersmay settle at the same spot or colonise a new sulphide patch The growth data obtained underlaboratory conditions fit those observed on the peat wall in the field Z niveum is usually found ingroups of a few to several hundred colonies Small groups typically consist of young colonies andnewly 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 relatedgenera of free-living nematodes within the family Desmodoridae (Chromadoria, Adenophorea),classified as the subfamily Stilbonematinae Originally thought to be parts of the worm (Greeff1869), fungal spores (Chitwood 1936) or epibiotic cyanobacteria (Gerlach 1950, Wieser 1959),they have been finally identified as sulphide-oxidising chemoautotrophic bacteria that coat thenematode surface in a species-specific pattern (Ott et al 1991)

Stilbonematinae occur in all intertidal and subtidal porous sediments where an oxidised surfacelayer overlies a deeper, reduced, sulphidic body of sediments They are most abundant in and nearthe RPD (Ott & Novak 1989) Highest abundance and diversity are found in tropical calcareoussands 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 accumulatingamong 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 majoroceans, 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 arehighly diverse (Urbancik et al 1996a,b) Unifying characters are the weak or absent buccal armatureand the special construction of the foregut (pharynx), where muscles appear to be concentrated inthe anterior-most part and the remainder is mostly glandular (Hoschitz et al 2001) These similar-ities could be interpreted to reflect convergent evolution due to the symbiotic lifestyle and thespecialisation on a single food item The monophyly of the Stilbonematinae, however, is clearly2727_C04.fm Page 100 Wednesday, June 30, 2004 12:00 PM

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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 forseveral 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 largerspecies may be kept in sand buckets and even in dishes with sea water for many days up to severalweeks, but neither moulting nor egg laying has been observed here They probably grow slowlyand 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 andsluggish 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 originallyplaced 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 onsurface-living bacteria (Van Dover et al 1989) or suspension feeders (Jannasch et al 1991), theconspicuous and regular epigrowth of bacteria on the mouthparts and the inner surface of the

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 m m. Figure 6 SEM micrograph of Eubostrichus dianae (Stilbonematinae) with nonseptate filaments (f); scale bar = 100 m m.

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modified carapace pointed to a symbiotic lifestyle (Gebruk et al 1993) The carapace enclosesthe anterior body almost completely, forming voluminous chambers on either side There is norostrum and the first and second antennae are stout and strong The exopodites of maxilla 2 andmaxilliped 1 are greatly enlarged and densely covered with plumose setae (bacteriophores), whichalso 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 oflight emanating from vent chimneys (Van Dover et al 1988, Van Dover & Fry 1994) This enablesthe shrimp to find the vents from a distance A smooth cornea replaces the lenses of the compoundeye, the photoreceptors in the fused retina are large with enlarged photosensitive regions, and theeye 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 sensillalocated 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 mering water”; some cling to the rock, forming layers several specimens thick and some move

“shim-in and out of the thermal plumes When dislodged by turbulence they rapidly move back to thechimneys They ingest sulphide particles, attached bacteria and the bacteria growing on theircuticles They are among the most important primary consumers and their ectosymbiotic thio-trophic 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) Juvenileshrimps 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

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.)

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these substances Such storage compounds are used during settlement and metamorphosis, whilethe 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 chemoautotrophicnature has been inferred on various grounds such as colour, ultrastructure, presence of ribulose-1,5-biphosphate carboxylase (RuBisCo, the enzyme necessary for carbon fixation), uptake oflabelled inorganic carbon, presence of sulphur-oxidation enzymes and ecological data from thehabitat

Kentrophoros

According to Fenchel & Finlay (1989) the symbiotic bacteria are rod shaped, about 3.6 mm longand 0.8 mm wide They appear brown to black in transmitted light due to sulphur inclusions thatare contained in membrane-bound vesicles that occupy a large part of the cell volume Among thecell organelles are probably carboxysomes, which contain RuBisCo The rods are arranged per-pendicular to the ciliate surface and show an unusual longitudinal division (Figure 9) They areembedded 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 thatwas equivalent to a doubling time of 5.3 h The reduction of benzyl viologen in the presence ofsulphide and uptake of 35S from labelled sulphide were indicative of sulphide oxidation The bacteriaare tightly packed with a density of 0.75 bacteria mm–2 of ciliate surface For a 170-mm-long ciliatethis amounts to 4500 bacteria with a total volume of 7650 mm3; this is roughly equivalent to thevolume of the host or half of the volume of the symbiotic consortium While K fasciolata onlycontained one type of bacterium in transmission electron microscopy (TEM) sections, K fistulosus

also showed ectosymbiotic spirochaetes (Figure 9) (Foissner 1995) and K latus intracellularprokaryotes of unknown function (Raikov 1974)

Zoothamnium

The bacteria found on stalk, branches, terminal zooids, and macrozooids are rod shaped, 1.4 mmlong 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 rodsalso cover the basal (proximal) parts of the microzooids Toward the peristomal disc the bacteriagradually 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 incontact with the microzooid surface Especially when the microzooids are contracted the cocciseem to form more than one layer on the ciliate The cocci have been observed to detach when theciliates are active and become entrained in the feeding currents created by the paroral and adoralmembranelles 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 whenthey 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

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Figure 9 Kentrophoros fistulosus with rod (r) and spirochaetes (s) on the dorsal body side; scale bar = 5 m m (Courtesy of W Foissner.) Figure 10Zoothamnium niveum with cocci (c) on oral and rods (r) on aboral parts of the microzooids; scale bar = 10 m m. Figure 11 Stilbonema sp with cocci (c); scale bar = 10

m m. Figure 12 Laxus oneistus with rods (r); arrow points to dividing rod; scale bar = 5 m m. Figure 13

Eubostrichus parasitiferus with nonseptate filaments (f) attached to the host’s cuticle in a spiral pattern; scale bar = 10 m m. Figure 14 Rimicaris exoculata with septate filaments (f) and rods (r); scale bar = 50 m m (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

carboxy-somes (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

membrane-bound 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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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

e-proteobacteria 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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