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Marine Chemosynthetic Symbioses

Marine Chemosynthetic Symbioses

Prokaryotes (2006) 1:475–507 DOI: 10.1007/0-387-30741-9_18

CHAPTER 2.5 Marine Marine Chemosynthetic Symbioses

Marine Chemosynthetic Symbioses

COLLEEN M. CAVANAUGH, ZOE P. MCKINESS, IRENE L.G. NEWTON AND FRANK J. STEWART

Introduction ide or methane for carbon fixation and utiliza- tion. On the basis of these unique biosynthetic and marine often coexist in capacities, notably the ability to synthesize C3 symbioses that significantly influence the ecol- compounds from C1 compounds, we refer collec- ogy, and of both partners. tively to these bacterial symbionts as “chemosyn- De Bary (1879) defined as “the living thetic.” together of differently named organisms,” imply- Given the sulfide-rich habitats in which ing that the term encompasses both positive (e.g., chemoautotrophic symbioses occur, researchers mutualism) and negative (e.g., parasitism) asso- infer that the bacterial symbionts oxidize ciations. Many researchers now view symbiotic reduced inorganic compounds to obtain interactions as those that persist over the major- energy and reducing power for autotrophic car- ity of the lifespan of the organisms involved and bon fixation. While some (such as that provide benefits to each partner beyond those in the protobranchs velum and S. those obtained in the absence of association. This reidi; Cavanaugh, 1983; Anderson et al., 1987) µ chapter describes such symbioses, specifically utilize (S2O3 ), an intermediate in sul- those between marine and fide oxidation, hydrogen sulfide is inferred to be hosts and chemosynthetic bacterial symbionts. the preferred energy source in a variety of sym- These bacteria, which cluster primarily within bioses (see review in Van Dover, 2000). But for the (Fig. 1), are chemo- many symbioses the actual energy source has not or . In both chemo- been identified definitively; rather, only an autotrophic and methanotrophic symbioses, the autotrophic metabolism has been confirmed. hosts, through an astonishing array of physio- Indeed, chemosynthetic bacteria utilizing other logical and behavioral adaptations, provide the energy sources (e.g., hydrogen or ) symbiont access to the substrates (i.e., electron could also serve similar nutritional roles in donors and acceptors) necessary for the genera- symbiotic associations. In this review, bacterial tion of energy and bacterial . In symbionts that have been shown to use reduced µ µ2 µ o exchange, a portion of the carbon fixed by the sulfur compounds (H2S, HS , S , S2O3 , S ) for symbiont is used, either directly or indirectly, for energy metabolism are referred to as thio- energy and biosynthesis. These symbioses autotrophs, while the more general term thereby increase the metabolic capabilities, and “chemoautotroph” is used to describe symbionts therefore the number of ecological niches, of for which data supporting autotrophic CO2 fixa- both the host and the bacterial symbiont. tion exist but for which the lithotrophic energy In those symbioses for which the electron source is unknown. donor has been explicitly identified, sulfide and other inorganic reduced sulfur compounds History (e.g., thiosulfate) fuel energy generation by the chemoautotrophic symbionts, serving as elec- The discovery of deep- hydrothermal vents tron sources for oxidative phosphorylation. In and the flourishing associated with these symbioses, the ATP produced in electron them significantly advanced scientific under- transport fuels autotrophic CO2 fixation via the standing of chemosynthetic symbioses. Oceanog- . In contrast, bacteria in marine raphers in the research submersible Alvin invertebrate- symbioses use meth- discovered hydrothermal vents along the Gal- ane (CH4) as an energy, electron, and carbon apagos Rift in 1977. In stark contrast to percep- source. Unlike their protist or metazoan hosts, tions of the deep as a cold, food-limited chemoautotrophs and methanotrophs share the habitat incapable of supporting substantial bio- ability to use reduced inorganic compounds or mass, hydrothermal vents are oases, charac- methane for energy generation and carbon diox- terized by high concentrations of free-living

476 C.M. Cavanaugh et al. CHAPTER 2.5

Chemoautotrophic symbionts: symbionts Vesicomyid clam symbionts Solemyid clam symbionts Lucinid clam symbionts Thyasirid clam symbionts Tubeworm symbionts Oligochaete symbionts Escherichia coli symbionts Coxiella burnetii Solemva reidi symbiont Methanotrophic symbionts GAMMA JTB254 Epsilon symbionts Achromatium oxaliferum 86 54 leukodermatus symbiont 100 algarvensis gamma symbiont 65 sp symbiont Olavius loisae gamma symbiont 82 Solemva relum symbiont Solemva occidentalis symbiont Lucinella nassula symbiont Codakia orbicularis symbiont 97 98 Lucina floridana symbiont Chemoautotrophic 57 Codakia costata symbiont 68 Lucinoma annulata symbiont symbionts Lucinoma aequizonata symbiont 94 100 Ridgeia piscesae symbiont Rifna packyptria symbiont 100 Escorpia spicata symbiont Lamembrachia colmnua symbiont 93 64 Anodontia phillipiana symbiont 70 Solemya terraeregina symbiont 69 Thyasira flexuosa symbiont Solemya pusillu symbiont Lucina pectinata symbiont 80 Pseudomonas fluorescens 100 Pseudomonas putida 88 Pseudomonas lundensis 81 Pseudomonas fragi 100 Marinobacter sp CAB Marinobacter aquaeolei Oceanospirillum kriegii Thiothrix eikelboomii 100 52 98 Thiothrix unzii Thiothrix nivea Thiothrix ramosa Bathymodiolus japonicus symbiont 54 Methanotrophic 100 Bathymodiolus childressi symbiont Bathymodiolus platifrons symbiont symbionts 100 Bathymodiolus puteoserpentis symbiont 2 100 76 Methylobacter sp BB5.1 53 Methylococcus luteus 60 Methylococcus capsulatus BARH 61 Methyllobacter whittenburyi 100 Methylomonas aurantiaca Methylomonas rubra Methylococcus thermophilus GAMMA JTB35 100 Thiomicrospira sp. 82 Thiomicrospira crunogena 82 Thiomicrospira L12 100 Thiomicrospira milos T2 Thiomicrospira milos T1 69 100 Thiomicrospira thyasirae Thiomicrospira pelophila Maorithyas hadalis symbiont 1 Juan de Fuca mussel symbiont 55 Bathymodiolus septemdierum symbiont 52 70 Bathymodiolus thermophilus symbiont Bathymodiolus puteoserpentis symbiont 1 Bathymodiolus aff. brevior symbiont 62 Calyptogena magnifica symbiont Chemoautotrophic 100 Calyptogena fossajaponica symbiont 82 Calyptogena phaseoliformis symbiont symbionts Calyptogena FL sp. symbiont 94 Calyptogena pacifica symbiont 86 100 Vesicomya lepta symbiont 87 Vesicomya chordata symbiont 100 Calyptogena elongata symbiont 100 Calyptogena kilmeri symbiont Vesicomya gigas symbiont Ectenagena extenta symbiont Maorithyas hadalis symbiont 2 100 Halothiobacillus kellyi Halothiobacillus neapolitanus Non-gamma Olavius loisae alpha symbiont oligochaete Olavius loisae spirochete symbiont 100 delta symbiont symbionts Desulfosarcina variabilis 100 84 epibiont APB13B Alvinella pompejana epibiont APG44B Epsilon 87 Alvinella pompejana epibiont APG5B epibionts Rimicaris exoculata symbiont 100 Alvinella pompejana epibiont APG56B 100 Sulfurospirillum sp 18.1 Sulfurospirillum Am-N

Fig. 1. Phylogeny showing the strict consensus of 46 trees obtained via parsimony analyses of 16S rRNA sequences (1456 bp) from symbiotic and free-living bacteria. Results greater than 50% from a 500 replicate bootstrap analysis are reported above respective branches. Chemosynthetic symbiont taxa are color-coded (see key on Figure).

CHAPTER 2.5 Marine Chemosynthetic Symbioses 477

source for R. pachyptila, implying a role for chemoautotrophy in tubeworm metabolism. Following confirmation of a chemosynthetic endosymbiosis within the giant tubeworm, researchers questioned the putative reliance on filter feeding by other vent . Ulti- mately, anatomical, enzymological, and isotopic analyses revealed the presence of sulfur- oxidizing bacterial symbionts either within the tissues (endosymbiotic) or attached to the sur- faces (episymbiotic) of most vent taxa, including vesicomyid clams, mytilid , , and worms (Cavanaugh, 1994; Nelson and Fisher, 1995a; Van Dover, 2000). Recognizing the ubiquity of chemoau- totrophic symbioses at hydrothermal vents, researchers searched for and discovered similar symbiotic associations in other marine habitats, including coastal and subtidal reducing sedi- ments (e.g., Felbeck et al., 1981b; Southward et al., 1981; Southward, 1982; Cavanaugh, 1983; Giere, 1985; Bauer-Nebelsick et al., 1996), brine and hydrocarbon seeps (Sibuet and Olu, 1998), and whale (Bennet et al., 1994; Smith and Baco, 2003), thereby extending the host Fig. 2. Symbiont-containing host organisms from hydrother- taxa to include solemyid and lucinid bivalves, mal vents and cold seeps. (A) Calyptogena magnifica shell; pogonophoran tubeworms, echinoids and cili- courtesy of Emilio Jorge Power. (B) Filamentous bacteria on ates. In addition, methanotrophic bacteria were sulfide deposits from the East Pacific Rise. (C) Bathymodio- detected in a marine (Vacelet et al., lus childressi from the Gulf of Mexico; courtesy of the 1995), a pogonophoran tubeworm (Schmaljo- National Oceanic and Atmospheric Administration. (D) at the East Pacific Rise. hann and Flügel, 1987), and in vent and seep mussels, sometimes co-occurring with sulfur- oxidizing chemoautotrophs in a “dual symbiosis” microorganisms and dense aggregations of inver- (Childress et al., 1986; Cavanaugh et al., 1987; tebrates (Lonsdale, 1977; Grassle, 1985; Van Cavanaugh, 1993). Dover, 2000). Researchers first argued that vent invertebrates achieved high densities by filtering Habitat Chemistry organic matter, which was presumably trans- ported to vent sites in hydrothermally-driven The seemingly disparate ecological niches where convection cells (Lonsdale, 1977). A second these symbioses are found all possess a chemical hypothesis suggested that the invertebrate com- gradient, or chemocline, which chemosynthetic munity fed directly on locally dense populations bacteria exploit for energy production. Chemo- of free-living chemoautotrophic bacteria clines form where reduced, high-energy com- (Lonsdale, 1977; Corliss et al., 1979). pounds such as sulfide or methane (typically However, studies of the giant tubeworm, Riftia produced in anoxic habitats, including vent fluids pachyptila (Fig. 2), whose lack of and gut and ) underlie an oxic . precludes suspension feeding, suggested that Chemosynthetic microorganisms must bridge sulfide-oxidizing endosymbiotic bacteria might the oxic-anoxic interface to access both the contribute substantially to the vent food web. reduced compounds (e.g., H2S) used as an energy Cavanaugh et al. (1981) proposed that symbiotic source and the to which electrons are chemosynthetic bacteria occurred in R. pachyp- shuttled in aerobic energy metabolism. tila. Microscopic and biochemical evidence The source of reduced compounds used in indicated Gram-negative bacteria were present, chemoautotrophic energy metabolism differs packed within the tubeworm among habitats. In marine sediments microbial µ2 (Cavanaugh, 1981; Fig. 3), a highly vascularized reduction (in which SO4 is used as an in the tubeworm trunk in which activities electron acceptor during the oxidation of organic of involved in sulfide oxidation and car- matter) dominates, and the sulfide utilized by bon fixation were also detected (Felbeck, 1981a). thioautotrophic symbioses (e.g., involving lucinid In addition, Rau (1981) used stable isotope clams or solemyid protobranchs) in these habi- signatures to show a nonphotosynthetic carbon tats is of biogenic origin. In contrast, sulfide at

478 C.M. Cavanaugh et al. CHAPTER 2.5

Fig. 3. Riftia pachyptila Jones (A–C: A D Galapagos Rift; D, E: 21°N, East Pacific Rise). (A) Photograph show- ing elemental sulfur (arrows) scattered throughout trophosome; courtesy of M. L. Jones. (B) Scanning electron micrograph showing lobules b of trophosome; arrow indicates area of C (below) where surface was removed to reveal symbionts within trophosome. (C) Same, higher magnification, showing symbionts within trophosome; note spherical cells as well as rod-shaped cells (small arrows); large arrows indicate likely B host membranes. (D) Cross- section of portion of trophosome lobule showing variable fine structure b m of symbionts, including membrane- bound vesicles in many cells; all symbionts contained within mem- brane-bound vacuoles, either singly or in groups of two or more; arrow: divid- ing bacterium; b: bacteria; m: mito- tc chondria; tc: trunk coelomic cavity. (E) Same, higher magnification, show- C E ing cell envelope of symbiont (resem- bling that of Gram-negative bacteria), pm intracytoplasmic vesicles, and peribac- terial membrane; v: vesicle; cm: sym- om biont cytoplasmic membrane; om: symbiont outer membrane; pm: perib- cm acterial membrane. Scale bars: A, 1 µm; B, 250 µm; C, 10 µm; D, 3 µm; and E, 0.2 µm. From Cavanaugh (1985), with permission.

v

hydrothermal vents is produced by the geother- 750–6500 µmol/kg of Fe (Elderfield and Schultz, mal reduction of sulfate and by the 1996). As it exits the seafloor and mixes with the interaction between geothermally heated water ambient bottom oxygenated seawater (pH, ca. 8; and sulfur-containing rocks (e.g., basalt; Alt, temperature = 1.8°C; [O2], ca. 110 µM), metallic 1995; Elderfield and Schultz, 1996; Rouxel et al., sulfides precipitate out resulting in “black 2004). Seawater that percolates into the develop- smokers” (reviewed in Elderfield and Schultz, ing crust becomes heated and reacts with oceanic 1996). Vent organisms are typically found clus- basalt, becoming enriched with metals and sul- tered around more diffuse or low flow vents, fide and charged with volcanic gases such as which are caused by ambient seawater mixing in methane and . Heated vent water the shallow subsurface with vent fluid. These then exits with concentrations of reduced com- vents are characterized by a higher pH (ca. 6), pounds orders of magnitude higher than in lower temperatures (1.8 to ca. 40°C), and, conse- ambient seawater. Hydrothermal effluent is hot quently, lower concentrations of reduced chemi- (temperatures up to 350–400°C), acidic (pH ~ 3), cals (Van Dover, 2000). anoxic, and can contain 3–12 mmol/kg of H2S, The relative acidity of vent fluid (black smok- 25–100 µmol/kg of CH4, and 0.05–1 mmol/kg of ers: pH, ca. 3; diffuse flow vents: pH, ca. 6) sig- H2, as well as 360–1140 µmol/kg of Mn and nificantly impacts the concentration of inorganic

CHAPTER 2.5 Marine Chemosynthetic Symbioses 479 chemicals available to chemosynthetic symbio- of stable isotope signatures of symbiont- ses. For example, carbon dioxide (CO2), bicar- containing and symbiont-free host , pro- µ µ2 bonate (HCO3 ), and carbonate (CO3 ), the vide valuable insight into the trophic dynamics three distinct chemical of the dissolved of symbiont-based communities. Molecular inorganic carbon (DIC) pool, vary in relative approaches, such as polymerase chain reaction abundance depending on pH; pKa values for (PCR)-based gene probing, 16S rRNA gene µ these compounds are 6.4 for CO2 : HCO3 and analysis, and fluorescent in situ hybridization µ µ2 10.3 for HCO3 : CO3 at 25°C (Stumm and (), are increasingly used to characterize the Morgan, 1996). Thus CO2, which diffuses freely systematic relationships of symbiont and host across biological membranes and is the DIC spe- species (e.g., Distel et al., 1995; Peek et al., 1998; cies fixed by chemoautotrophic symbionts utiliz- Dubilier et al., 1999) and the metabolism and ing the Calvin cycle, is readily available at vents gene flow of the bacterial symbionts (Robinson (Cavanaugh and Robinson, 1996; Goffredi et al., et al., 1998; Lee et al., 1999; Millikan et al., 1999; 1997b). In addition, sulfide exists at three levels Podar et al., 2002). Molecular techniques have µ µ2 of dissociation (H2S, HS , and S ) depending on also been used to detect symbiont transmission µ pH, with pKa values of 7.0 for H2S : HS and 12.9 modes (Cary and Giovannoni, 1993a; Krueger, for HSµ : Sµ2 at 25°C (Stumm and Morgan, 1996). 1996a) as well as symbiont abundance (Polz and Therefore, in the relatively acidic vent fluids Cavanaugh, 1995). sulfide occurs predominantly as H2S. In such µ µ2 effluent, total sulfide (H2S, HS , and S ) concen- Summary tration correlates positively with temperature (Johnson et al., 1988); conversely, higher temper- This chapter reviews symbiotic associations atures (>30°C) may facilitate reactions between between chemosynthetic bacteria and marine sulfide and other dissolved elements (such as invertebrate and protist hosts. A bias toward iron) that reduce free sulfide availability (Luther symbioses between chemoautotrophic bacteria et al., 2001). The chemical environment (i.e., and invertebrates is evident, + concentrations of CO2, O2, H2S, CH4, H , and primarily because our knowledge of marine bac- dissolved metals) is therefore expected to signif- terial symbioses stems largely from studies of icantly influence the ecology and evolution of vent fauna done in the 27 years following the chemosynthetic symbioses. discovery of these unique organisms. But despite this impressive body of research, much about Methods for Studying these marine symbioses remains to be revealed. Chemosynthetic Symbioses In conjunction with several earlier reviews that provide a thorough and thoughtful treatment of To date, the bacteria involved in these symbiotic symbioses occurring at hydrothermal vents and associations have not yet been isolated and cold seeps (Fisher, 1990; Felbeck and Distel, grown in pure culture—perhaps because the 1991; Childress and Fisher, 1992; Cavanaugh, unique environment encountered in situ by 1994; Nelson and Fisher, 1995a), the following chemosynthetic symbionts has not been recre- chapter presents an overview of the ecology, ated or because a reduced , characteristic physiology and evolution of chemosynthetic of many endosymbionts, has precluded growth symbioses. outside of the host. Symbiotic bacteria are there- fore studied indirectly, using methods to assess their physiology, ecology and phylogeny within Host Diversity the context of the intact symbiosis. Traditionally, researchers identify chemosynthetic symbioses Chemosynthetic bacteria are known to associ- using a combination of microscopy (light, confo- ate with a diversity of invertebrate hosts (six cal, scanning and transmission electron), which phyla), as well as with (Table 1 provides visual information on the location, mor- and references therein). To date, the majority phology and ultrastructure of symbionts, and of the symbionts characterized via 16S rRNA assays, which detect and quantify the phylogenetic analyses fall within the Gamma- activity of key involved in chemoau- division (Fig. 1; discussed fur- totrophic (e.g., ribulose 1,5-bisphosphate car- ther below). The intimacy of these associations boxylase-oxygenase) or methanotrophic (e.g., varies among taxa. The bacterial partners may methanol dehydrogenase) metabolism. In addi- be episymbionts living on the surface of the tion, tracing the incorporation of radiolabeled host (e.g., on shrimp, , , lim- substrates (e.g., carbon dioxide, methane and pets and ; Figs. 4−6), or endosymbionts nitrogen species) within the host helps define the living either extracellularly within host tissue physiology of the host-bacteria partnership. Such (e.g., in oligochaetes; Fig. 7) or in specialized physiological assays, in conjunction with analyses host cells and organs (e.g., in bivalves and

480 C.M. Cavanaugh et al. CHAPTER 2.5 References isher and Childress, 1986 isher and Childress, enchel and Finlay, 1989 enchel and Finlay, olz et al., 1992 olz et al., Stein et al., 1988 Stein et al., Fisher et al., 1988 et al., Fisher Endow and Ohta, 1989 Endow and Ohta, F 1989 Conway et al., 1985 Schweimanns and Felbeck, 1987 Herry and Le Pennec, Rau, 1981 Rau, 1986 Childress et al., 1987 Cavanaugh et al., 1992 Cavanaugh et al., Ott et al., 1998 Ott et al., 2000 Buck et al., F P Symbiont type and/or methanotroph Methanotroph 1996 1995, et al., Vacelet Chemoautotroph 1983 Cavanaugh, ChemoautotrophChemoautotroph 1985 Giere, 1986 Dando and Southward, Chemoautotroph 1983 Cavanaugh, Chemoautotroph Chemoautotroph 1996 et al., Bauer-Nebelsick Chemoautotroph 1990 Schiemer et al., b c c c Habitat cold seeps cold seeps cold seeps cold seeps vents, cold seeps vents, swamp sediments hydrothermal vents, a . Location Epibiotic Reducing Intracellular Reducing sediments, IntracellularIntracellular Reducing sediments, Hydrothermal Extracellular Hydrothermal Intracellular Hydrothermal vents Chemoautotroph Intracellular Reducing sediments, Intracellular Epibiotic Cold seeps, Extracellular Cold seeps tissue Cuticle Gills Gills Gills Gills NA NA Gills Symbiont-containing name Common Clam Clam Mussel Ciliate Sponge Clam amily Lucinidae amily Thyasiridae amily Clam amily amily Provannidae Snail amily F F F F F F amily Subclass Subclass Pteriomorphia F Subfamily Nematode Subclass Protobranchia able 1. List of invertebrate taxa hosting chemoautotrophic or methanotrophic bacterial symbionts T Group Ciliata Class Porifera Class Demospongiae Nemata Class

CHAPTER 2.5 Marine Chemosynthetic Symbioses 481 1993). , ilier et al., 2001). ilier et al., References elbeck, 1981 elbeck, elbeck et al., 1983 elbeck et al., olz and Cavanaugh, 1995 olz and Cavanaugh, 1999 olz et al., emara et al., 1993 emara et al., ox et al., 2002 ox et al., de Burgh and Singla, 1984 de Burgh and Singla, Van Dover et al., 1988 Dover et al., Van Desbruyeres et al., 1983, 1985 1983, Desbruyeres et al., F 2004 Bates et al., Cary et al., 2003 Cary et al., F 1981 Southward et al., 1987 Brooks et al., 1987 Schmaljohann and Flügel, 1988 Southward and Southward, 1989 de Burgh et al., P P F 1985 1981, Giere, 1987 Giere and Langheld, T 1998 Brigmon and de Ridder, e f they are listed as separate groups for identification , Symbiont type rom cold seeps in the Gulf of Mexico, Sagami Bay, and Sagami Bay, rom cold seeps in the Gulf of Mexico, Chemoautotroph Chemoautotroph 1981 Cavanaugh et al., 1999). Annelida emara et al., 1993; Brigmon and de Ridder, 1998). Brigmon and de Ridder, 1993; emara et al., Habitat vents, cold seeps, vents, reducing sediments, fjords , but the symbiosis has not been characterized (Metivier and Voncosel but the symbiosis has not been characterized (Metivier and , 1998). Location Extracellular Coralline sands Extracellular Reducing sediments Chemoautotroph Epibiotic Hydrothermal vents Chemoautotroph Epibiotic Hydrothermal vents Chemoautotroph Epibiotic Hydrothermal vents Chemoautotroph c, and lucinid and thyasirid clams have been collected f c, physiological, enzymatic, and molecular data. enzymatic, physiological, tissue surface Gut Trophosome Intracellular Deep-sea hydrothermal Gills Dorsal Carapace Symbiont-containing like on the basis of morphology, physiology, and immunological assays (T physiology, like on the basis of morphology, has been described from the Lau Basin hydrothermal vents , name Common Thiothrix- has been shown to have an additional symbiont which is a sulfur reducing bacterium and member of the delta Proteobacteria (Dub

Sea urchin Worm Acharax alinae Acharax Olavius algarvensis has been shown to host an alpha proteobacterium and a spirochete as well as a chemoautotroph (Dubilier et al.,

d amily Tubeworm amily Lepetodrilidae Limpet amily Alvinellidae Alvinocarididaeamily Shrimp F F F F ) Subfamily Oligochaete Subcuticular hough it is now accepted that the pogonophoran and vestimentiferan worms are not separate phyla but members of he oligochaete his symbiont has been described as A solemyid protobranch, A solemyid protobranch, T Chemosynthetic status of symbionts inferred from ultrastructural, T Solemyid clams have been collected from cold seeps in the eastern Pacifi T Annelida (Vestimentifera and (Vestimentifera Class Echinodermata Class Echinoidea Group Class Polychaeta Arthropoda Class Crustacea not applicable. NA, Abbreviation: a b c d e Olavius loisae f purposes. Barbados Prism, though the presence of symbionts has not been formally described (Sibuet and Olu, Barbados Prism,

482 C.M. Cavanaugh et al. CHAPTER 2.5

A B

10 µm 100 µm

C D

µ 1000 µm 100 m

E F

5 µm 1 µm

G H

1 µm 10 µm

Fig. 4. Scanning electron micrographs showing the morphological diversity of ectosymbiotic bacteria on the colonial ciliate Zoothamnium niveum (A, B), the shrimp Rimicaris exoculata (C, D), and the nematodes within the subfamily Stilbonema- tinae (E-H). (A) Entire ciliate colony with zooids attached to a common stem, and (B) bacterial epigrowth on an individual zooid. (C) Shrimp appendage covered by dense arrays of filamentous bacteria, and (D) detail of the hair-like bacterial covering. Epigrowth on different species of nematodes showing (E) irregular epigrowth of two morphological types on Robbea sp., (F) coccoid bacteria forming multilayers on Stibonema sp., (G) upright standing, longitudinally dividing rods on Laxus oneistus, and (H) dense array of nonseptate filaments that can reach up to 100 mm in length on Eubostrichus dianae. From Polz et al. (2000), with permission. vestimentiferan tubeworms; Figs. 2, 3, and 8). fit from these intimate associations. Indeed, In intracellular endosymbioses the symbionts as in the giant vent tubeworms, the digestive are housed within specialized host cells called system of many -containing “bacteriocytes” and are contained within a host- is either reduced (e.g., in derived membrane bound vacuole (Cavanaugh, coastal solemyid protobranchs) or absent alto- 1983; Cavanaugh, 1994; Fisher, 1990). Host gether (e.g., in oligochaetes and vestimentiferan morphology clearly suggests a nutritional bene- and pogonophoran tubeworms), consistent with

CHAPTER 2.5 Marine Chemosynthetic Symbioses 483 host dependence on the symbiont for part or all In mollusk symbioses the bacteria occur only in of its nutrition. the gills; bacteria have been found within All of the members of the tubeworm epithelial cells of solemyid protobranchs Siboglinidae examined to date, including the ves- (Cavanaugh, 1983; Krueger et al., 1996b; Figs. timentiferan and the smaller pogonophoran tubeworms, contain intracellular symbionts. Most of these symbionts are inferred to be A chemoautotrophic, but methanotrophs have CU been found in one host species (Siboglinum poseidoni; Schmaljohann and Flügel, 1987). The vent tubeworm Riftia pachyptila and other vesti- mentiferan and pogonophoran tubeworm spe- cies possess a unique morphological adaptation to accommodate their symbionts. Tubeworm bacteria reside within a lobular and highly vas- cularized organ (the trophosome) that occupies S most of the tubeworm trunk and functions spe- m cifically to house bacteria (Cavanaugh, 1981; C Felbeck, 1981a; Figs. 3 and 9). The symbiosis is obligate for these worms, as they are mouthless and gutless as adults and depend on their inter- nal bacteria for their nutrition. Chemosynthetic symbioses are widespread within the Mollusca and have been detected in five bivalve and two gastropod families (Table 1).

B

CU

n bs es

b Fig. 7. Inanidrilus leukodermatus. (A) Light micrograph of a 10 µm cross section of an oligochaete worm. (B) Transmission elec- tron micrograph of symbiont-containing region just below Fig. 5. Lepetodrilus fucensis. Transverse section of gill tissue the cuticle. Note smaller and larger symbiont morphotypes from the hydrothermal vent limpet showing episymbiotic fil- (smaller and larger arrows, respectively). c, coelomic cavity; amentous bacteria partially embedded in the host epithelium. m, muscle tissue; s, symbiont-containing region between cuti- b, bacteria; es, extracellular space; n, nucleus; bs, space. cle and ; cu, cuticle. Scale bars: A, 20 µm; B, 2 µm. Courtesy of Amanda Bates. From Dubilier et al. (1995), with permission.

AB

Fig. 6. Eubostrichus cf. parasitiferus. Scanning electron micrographs showing the symbiotic bacteria on the surface of the nematode. (A) Anterior (bottom) and posterior (top) end with symbionts arranged in a characteristic helix. (B) Higher magnification. Bacteria are attached with both ends to the worm’s cuticle. Note the increasing length of the cells from proximal to distal along the worm’s surface. Scale bars: A, 20 µm; B, 2 µm. From Polz et al. (1992), with permission. 484 C.M. Cavanaugh et al. CHAPTER 2.5

Fig. 8. Calyptogena magnifica Boss A mv and Turner (21°N East Pacific Rise). ni (A) Transmission electron micro- graph of slightly oblique transverse section of gill filament, showing coccoid-shaped bacteria within gill bacteriocyte and intercalary cells lacking symbionts; b: bacteria; mv: microvilli (of both cell-types); nb: b nucleus of bacteriocyte; ni: nucleus of intercalary cell. (B) Same, higher magnification, transverse section of coccoid-shaped symbionts, showing cell ultrastructure typical of Gram- negative bacteria and peribacterial membrane (arrow). Scale bars: A, 5 µm; B, 0.25 µm. From Cavanaugh (1985), with permission.

B nb

A B

b Fig. 9. Escarpia spicata Jones (San Clemente Fault). (A) Transmission electron micrograph, portion of tro- phosome lobule showing numerous coccoid- to ovoid-shaped bacterial nb symbionts, some of which appear intracellular. (B) Same, higher magnification, showing bacterial cell envelope (resembling that of Gram- negative bacteria) and intracytoplas- mic membrane-bound vesicles; arrow: peribacterial membrane; b: bacteria; nb nb: nucleus of bacteriocyte. Scale bars: A, 10 µm; B, 1 µm. From Cavanaugh (1985), with permission.

10–13), lucinid clams (Cavanaugh, 1983; Felbeck, totrophic symbionts have been detected only in 1983a), thyasirid clams (Felbeck et al., 1981b; members of the subfamily Bathymodiolinae, Cavanaugh, 1983; Arp et al., 1984), vesicomyid which are found exclusively in the deep-sea. clams (Boss and Turner, 1980; Rau, 1981; Arp et Further, dual symbioses involving both methan- al., 1984; Fig. 8), mytilid mussels (Fiala-Médioni, otrophs and chemoautotrophs are restricted to 1984; Le Pennec and Hily, 1984; Figs. 2 and 14), species of deep-sea bathymodioline mussels col- and provannid gastropods (Stein et al., 1988; lected from methane seeps and hydrothermal Windoffer and Giere, 1997). Within certain vents (Cavanaugh, 1994; Nelson and Fisher, mollusk families (e.g., Solemyidae, Lucinidae 1995a; Van Dover, 2000; Fig. 14). and Thyasiridae), all species examined form Chemosynthetic bacteria also occur as episym- symbioses with chemoautotrophic bacteria. In bionts on marine invertebrates (Table 1; Fig. 4). other families, such as the Mytilidae, chemoau- These symbionts include the Epsilonproteo- CHAPTER 2.5 Marine Chemosynthetic Symbioses 485

Fig. 10. Solemya sp. (right hand) collected from deep-sea vent sites (2380 m depth) along the subduction zone off Ore- gon, and (left hand) collected from subtidal reducing sediments (<1 m depth, mean low tide) of Massa- chusetts eelgrass beds. Photo courtesy of Dr. Ruth D. Turner.

Fig. 11. Solemya velum. Characteris- tic Y-shaped burrows dug by the coastal protobranch clam to bridge the oxic-anoxic interface and access both reduced sulfur (from below) and oxygen (from above). From Stanley (1970), with permission. PLATE 3. SOLEMYA

A mv

i b

Fig. 12. Solemya borealis. (A) Transverse section of gill filaments showing intracellular rod-shaped c bacteria (arrows, rectangle). Bacterio- cytes are confined to the region prox- imal to the ciliated edge of the gill and bl are flanked by symbiont-free interca- lary cells that appear to comprise the microvillar surface of the gill filament. Light micrograph. b: bacteriocyte ci nucleus; c: ciliated ; i: intercalary cell nucleus; bl: blood B space; ci: cilia; mv: microvilli. (B) Higher magnification of symbionts showing cell ultrastructure typical of Gram-negative. Inset: Detail of sym- biont cell envelope and peribacterial membrane. p: peribacterial mem- cm brane; cm: ; om: outer om membrane. Scale bars: A, 20 µm; B, 1 µm; inset, 0.05 µm. From Conway p et al. (1992b), with permission. 486 C.M. Cavanaugh et al. CHAPTER 2.5

Fig. 13. Solemya velum. (A) Trans- AB m mission electron micrograph, trans- ni verse section of gill filament, showing rod-shaped bacteria within gill bacte- nb riocyte and intercalary cells lacking symbionts; b: bacteria; mv: microvilli; nb: nucleus of bacteriocyte; ni: nucleus of intercalary cell. (B) Same, higher magnification, transverse section of rod-shaped bacterium, showing cell ultrastructure typical of b Gram-negative bacteria and peri- bacterial membrane (arrows). Scale mv bars: A, 3 µm; B, 0.2 µm. From Cavanaugh (1985), with permission.

A CB

I S

B

Fig. 14. Bathymodiolus puteoserpentis. Transverse section of Mid-Atlantic Ridge (MAR) mytilid gill filament, showing symbiont-containing gill epithelial cells (bacteriocytes). (A) n l Diagram of gill filament. Bacteriocytes are confined to the region proximal to the ciliated border of the gill. Small box shows positions of Figs. 14B. B: symbiont-containing bacte- riocyte region; and C: symbiont-free ciliated region. (B) Transmission electron micrograph. Large and small sym- bionts (large and small arrows, respectively) are located in the apical region of the cells, while nuclei and lysosomal residual bodies occupy the region closest to the blood sinus. Note centrally stacked intracytoplasmic membranes in large symbionts. 1, Lysosomal residual body; n, bacteriocyte nucleus; and s, blood sinus. Scale bar: 5 µm. From Distel et S al. (1995), with permission.

bacteria that cover the cuticle of Rimicaris and the surfaces of alvinellid shrimp, dominant members of the metazoan (Desbruyères et al., 1985; Cary et al., 1997). fauna at vents on the Mid-Atlantic Ridge (MAR; Chemosynthetic episymbionts also associate Polz et al., 1998) and the Central Indian Ridge with nematodes (Weiser, 1959; Ott et al., 1991; (CIR; Van Dover et al., 2001; Van Dover, 2002a), Polz et al., 1992; Polz et al., 1994) and ciliates CHAPTER 2.5 Marine Chemosynthetic Symbioses 487

(e.g., Fenchel and Finlay, 1989; Bauer-Nebelsick ferences in the lifecycle stage and metabolism et al., 1996). In addition, methanotrophic epi- among symbiont cells (Bright et al., 2000). symbionts have been found living on a deep-sea sponge (Vacelet et al., 1995; Vacelet et al., 1996). Symbiont Phylogeny Vent limpet-bacteria associations seem to be an intermediate between epi- and endosymbioses; While chemoautotrophic symbionts have consis- bacteria exist partially embedded in the limpet tently evaded culture, the suite of cellular and gill epidermis and may be endocytosed or fed on molecular methods used to characterize these by the host (de Burgh and Singla, 1984; Bates bacteria has revealed startling evolutionary et al., 2004; Fig. 5). Some epibiont communities, trends. Investigators have successfully sequenced like those residing on the Rimicaris shrimp and 16S rRNA from symbiont-containing tis- the nematode Laxus sp., are dominated by a sue and subsequently confirmed the symbiont single phylotype (Polz et al., 1994; Polz and origin of these sequences via hybridization with Cavanaugh, 1995), while others are quite diverse symbiont-specific probes. In contrast to the wide (Polz et al., 1999; Campbell et al., 2003). But diversity of host taxa involved in these symbio- given that morphological plasticity often belies ses, chemosynthetic symbionts cluster primarily the phylogenetic identity of symbionts (Polz et within a single bacterial division, the Gamma- al., 1999; Giere and Krieger, 2001), symbiont proteobacteria, on the basis of 16S rRNA diversity estimates are only appropriate when gene sequences (Distel and Cavanaugh, 1994; putative symbiont phylotypes are confirmed Dubilier et al., 1999; McKiness, 2004). Such anal- using hybridization methods (e.g., FISH). yses have also shown that most host species typ- ically form a relationship with a unique symbiont phylotype. But this clearly is not always the case. For example, while strain level variation may Symbiont Diversity occur, vestimentiferan tubeworms belonging to Morphology and Ultrastructure the genera Riftia, Tevnia, and Oasisia appear to share a single, or very similar, symbiont phylo- Symbiont morphology varies among functional type based on 16S rRNA gene sequences types (chemoautotroph vs. methanotroph), (Feldman et al., 1997; Laue and Nelson, 1997; Di among phylotypes within the same functional Meo et al., 2000; Nelson and Fisher, 2000; group, and among individuals in a population of McMullin et al., 2003), as do some species of a single phylotype. The symbionts all have a tropical lucinid clams (Durand and Gros, 1996a; Gram-negative cell envelope but range from Durand et al., 1996b). small (ca. 0.25 µm diameter) coccoid endosym- Recently, McKiness (2004) reported phyloge- bionts within mussel gills (Cavanaugh, 1985; netic analyses of 16S rRNA gene sequences from Dubilier et al., 1998) to large (>10 µm length) chemosynthetic symbionts within the Gamma- rod-shaped and filamentous episymbionts on proteobacteria. This study represented the most vent shrimp (Hentschel et al., 1993b; Polz and comprehensive analysis of chemoautotrophic Cavanaugh, 1995; Fig. 4). Some bathymodioline symbionts to date. It included 39 symbiont mussels host two metabolically distinct sym- sequences and over 30 sequences from free-living bionts: small (<0.5 µm) chemoautotrophs and bacteria representatives of chemoautotrophs, larger (1.5–2.0 µm) methanotrophic bacteria methanotrophs, and marine bacterioplankton. possessing stacked intracytoplasmic membranes, Here, an expanded phylogenetic analysis is pre- which are typical of Type I methanotrophs (e.g., sented that includes the Childress et al., 1986; Cavanaugh et al., 1987; symbionts of shrimp and alvinellid worms (Fig. Cavanaugh et al., 1992; Fiala-Médioni et al., 1). This consensus tree illustrates the strong level 2002; Pimenov et al., 2002; Fig. 14). Morpholog- of resolution afforded by the 16S rRNA gene ical diversity can also occur throughout mono- and shows that almost all of the chemosynthetic specific populations within a single host . symbionts for which sequence data are available For example, populations of sulfur-oxidizing cluster into two main . The first chemoautotrophic symbionts in the tubeworm includes symbionts of lucinid and thyasirid Riftia pachyptila contain distinct morphotypes clams, solemyid protobranchs, tubeworms, nem- that vary in abundance depending on location atodes, and oligochaetes, and the second clade within the trophosome lobule (Bright et al., includes the mytilid mussel and vesicomyid clam 2000); small, rod-shaped bacteria occur primarily symbionts. in the innermost zone of the lobule nearest the Though the first clade as a whole has relatively host’s axial blood vessel, while small and large low bootstrap support, smaller clusters within cocci (1.6–10.7 µm diameter) occupy zones the first clade are strongly supported. For exam- nearer the periphery of the trophosome (Bright ple, the monophyletic cluster of nematode and et al., 2000). Such variability may relate to dif- oligochaete symbionts has 100% bootstrap sup- 488 C.M. Cavanaugh et al. CHAPTER 2.5 port. Similarly, the vestimentiferan tubeworm The symbionts of the thyasirid clam Maorhith- symbiont clade also has high bootstrap support, yas hadalis occupy unique positions in this phy- corroborating prior evidence that these worms logenetic framework. On the basis of 16S rRNA share a single or very similar symbiont phylo- sequence data and in situ hybridization, Fujiwara type, which is consistent with environmental et al. (2001) described two different symbionts transmission of symbionts (Feldman et al., 1997; within this clam. One of the symbionts shows Laue and Nelson, 1997; Di Meo et al., 2000; evolutionary relatedness to the bathymodioline Nelson and Fisher, 2000; McMullin et al., 2003; mussel and vesicomyid clam symbionts, occur- see the section Ecology and Evolution in this ring basal to the clade containing these bacteria, Chapter). In contrast, the clam symbionts exhibit and the other symbiont clusters with the free- a more complicated relationship. The lucinid living Thiomicrospira spp. This free-living clam symbionts form a paraphyletic group; some “symbiont” phylotype, however, may be a con- are sister to the tubeworm symbionts while oth- taminant. Difficulties with in situ hybridization ers group with thyasirid and solemyid symbionts. have precluded attempts to describe the micro- The solemyid symbionts show similarly compli- distribution of the two symbiont types (Fujiwara cated relationships, as they are polyphyletic and and Uematsu, 2002), bringing into question the scattered throughout the first clade. Solemya phylogenetic identity of the clam symbionts. velum and S. occidentalis symbionts cluster with The phylogenetic positions of the methan- the nematode and oligochaete symbionts, while otrophic endosymbionts and the filamentous S. terraeregina and S. pusilla symbionts cluster epibionts are shown also in Fig. 1. The methan- with lucinid and thyasirid clam symbionts. Thus, otrophic symbionts characterized to date all this disjointed distribution does not suggest belong to the Gammaproteobacteria, forming a cospeciation between host taxa and symbionts in clade with 100% bootstrap support. The sister this first clade and indicates that there were mul- group of this clade consists of free-living Type I tiple initiations of symbiosis within the solemyid methanotrophs (Methylococcus, Methylobacter and lucinid clams. and Methylomonas spp.). Given their mono- The position of the S. reidi symbiont within phyly, the mussel symbionts apparently arose this first main group is curious; this symbiont falls from a common ancestor. But the question of at the base of this first main group, clustering whether these symbionts subsequently cospeci- with an intracellular pathogen, Coxiella burnetti, ated with their hosts remains unanswered. and an environmental clone from a Japanese The episymbionts in this analysis include the , Gamma JTB254 (discussed further sulfur-oxidizing Epsilonproteobacteria found on below). This symbiont sequence has held a basal the Mid-Atlantic Ridge shrimp Rimicaris exocu- position in other analyses (see Bayesian analysis lata and the eastern Pacific polychaete Alvinella in McKiness, 2004) and provokes questions con- pompejana. Interestingly, the shrimp epibiont cerning the of symbiosis in protobranch clusters with the polychaete epibionts despite the bivalves. As additional sequences become avail- fact that R. exoculata occurs on the Mid-Atlantic able, it will be necessary to reassess the position Ridge while alvinellid polychaetes inhabit vents of the S. reidi symbiont with respect to other in the eastern Pacific . chemoautotrophic symbionts. Free-living microorganisms can potentially The second clade of symbionts, which includes provide insight into the ancestral form of endo- the mytilid mussel and vesicomyid clam sym- symbionts. For instance, the evolution of insect bionts from vents and cold seeps, shows 100% endosymbionts (e.g., Wolbachia and Buchnera bootstrap support (support for the mussel and spp.) is commonly studied by comparative anal- vesicomyid clades being 70% and 99%, respec- yses with free-living, closely related microbes tively). The symbiont from the Central Indian (Wernegreen, 2002; Moran, 2003). But until Ridge mussel, Bathymodiolus aff. brevior, falls at recently, the chemoautotrophic symbiont clades the base of the vesicomyid clam symbionts, with have not included any free-living bacteria. Fig- 82% bootstrap support. In contrast to the first ure 1 includes two species of bacteria that are symbiont clade (Fig. 1, top), evidence suggests not chemoautotrophic symbionts (Coxiella bur- that an ancestral symbiont initiated symbioses netti and Achromatium oxaliferum) and an envi- with both the vesicomyid clams and the bathy- ronmental clone (JTB254). All three of these modioline mussels and predated the split sequences fall within the first symbiont clade. between these bivalve lineages. On the basis of Coxiella burnetii is an intracellular pathogenic 16S rRNA gene sequence data, the divergence bacterium (Woldehiwet, 2004) and the Gamma of the clam and mussel symbionts has been dated JTB254 clone was recovered from a deep-sea to 125–300 million years ago (Mya). This is cor- cold seep in the Trench (Li et al., 1999). roborated by the record, which dates the They both fall in a cluster with the S. reidi sym- bathymodioline mussel hosts to 150 Mya and the biont. Both C. burnetii and the S. reidi symbionts vesicomyid clams to 100 Mya (Distel, 1998). are able to maintain an intracellular existence in CHAPTER 2.5 Marine Chemosynthetic Symbioses 489 eukaryotic hosts. Coxiella burnetii, however, is shrimp, sea urchins, and ciliates (Felbeck et al., capable of growth in animal cell lines (e.g., 1981b; Cavanaugh, 1983; Cavanaugh et al., 1988; Woldehiwet, 2004) and pathogenically infects a Polz et al., 1992; Johnson et al., 1994; Nelson wide range of hosts (Niemczuk and Kondracki, et al., 1995b; Bauer-Nebelsick et al., 1996; 2004; Watanabe, 2004; Woldehiwet, 2004). In Krieger et al., 2000; Elsaied et al., 2002; Fiala- addition, A. oxaliferum, a freshwater sulfur- Médioni et al., 2002). While RubisCO has not oxidizing bacterium, falls out with the nematode been detected in the alvinellid epibionts, genes and oligochaete symbionts clade. Achromatium encoding citrate lyase, a key enzyme of the oxaliferum occurs in freshwater sediments along reductive tricarboxylic acid (TCA) cycle, the zone where it has access to sulfide and recently have been detected via analyses of oxygen (Head et al., 1996; Glockner et al., 1999; symbiont DNA sequences, suggesting that the Gray et al., 1999). As cultivation methods Epsilonproteobacteria symbionts of alvinellid improve and sequences are added to the 16 worms fix carbon via this pathway (Campbell rRNA gene database, other free-living bacteria et al., 2003). that are closely related to symbionts will likely Enzymes involved in chemosynthetic energy be identified. Indeed, recent studies incorporat- generation have also been used to characterize ing 16S rRNA gene sequences from unidentified these symbionts. Although the sulfur metabolism environmental clones into phylogenetic analyses enzymes are not unique to sulfur oxidation, of free-living and symbiotic bacteria suggest that certain enzymes such as ATP sulfurylase, when chemosynthetic symbionts may in fact resolve detected in high activities, have been used to into three distinct clades (N. Dubilier, personal infer sulfur-based chemolithotrophy (Felbeck, communication; Duperron et al., 2004). Free- 1981a; Fisher et al., 1993b; Laue and Nelson, living relatives of chemosynthetic symbionts 1994). As methane monoxygenase, the enzyme should reveal much about the ecological and that catalyzes the first step in the oxidation of evolutionary constraints on the symbiont as well methane in aerobic methanotrophs, is notori- as about the potential for gene loss during the ously labile (Prior and Dalton, 1985; Cavanaugh, transition from the free-living to the symbiotic 1993), methanol dehydrogenase (MeDH), the state. enzyme that catalyzes the second oxidation step (i.e., methanol to formaldehyde) and is known to occur only in methylotrophs, has been used Symbiont Characterization extensively to diagnose methanotrophy. MeDH has been detected in gill extracts of mussels host- Enzyme Activities Researchers routinely dem- ing methanotrophs or both methanotrophs and onstrate chemoautotrophy or methanotrophy in thioautotrophs (Cavanaugh et al., 1992; Fisher et symbionts by the activity or presence of diagnos- al., 1993b; Robinson et al., 1998; Fiala-Médioni tic enzymes. Indeed, given the inability to culture et al., 2002; Pimenov et al., 2002; Barry et al., chemoautotrophic symbionts, detection of such 2002) and in a deep-sea sponge (Vacelet et al., enzymes is often the only evidence used to 1996). Such enzymatic evidence strongly infer symbiont metabolism. This characterization suggests methanotrophy, particularly when often involves physiological assays using tissue coupled with ultrastructural observations show- or purified extract, immunodetection, or ing symbionts with the complex intracytoplasmic PCR-based gene probing. Such studies were membranes that are characteristic of Type I initially conducted on the tubeworm Riftia methanotrophs. pachyptila. For example, Felbeck (1981a) assayed tubeworm trophosome tissue for the activities of key enzymes of the Calvin cycle, the Stable Isotope Signatures CO2-fixing enzyme, ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO), and phos- Carbon Isotopes In addition to enzymology, sta- phoribulose kinase (PRK) as well as enzymes ble isotope data provided some of the first evi- associated with the oxidation of reduced inor- dence in support of chemoautotrophy in marine ganic sulfur compounds. The activity or presence invertebrate-bacteria symbioses (e.g., Rau and of RubisCO has subsequently been used to diag- Hedges, 1979; Spiro et al., 1986) and continue to nose symbiont autotrophy in a diversity of host be useful in assessing symbiont metabolism and species, including all of the chemoautotroph- tracking energy and carbon transfer in chemo- harboring invertebrates listed in Table 1 (exclud- synthetic symbioses (Colaco et al., 2002; Levin ing alvinellid polychaetes; see below): shallow and Michener, 2002; Van Dover, 2002a; water solemyid and lucinid bivalves, vent and Robinson et al., 2003; Scott et al., 2004). Because seep tubeworms and bivalves (including mussels enzymes involved in distinct carbon fixation hosting both methanotrophic and chemoau- pathways discriminate differently against the use totrophic symbionts), nematodes, oligochetes, of the heavier carbon isotope (13C), the stable 490 C.M. Cavanaugh et al. CHAPTER 2.5 carbon isotope ratio comparing 13C to 12C (δ13C) the fossil lucinid hosted chemosynthetic sym- can be used to help distinguish different bionts ca. 120,000 ya (CoBabe, 1991). Thus, sta- autotrophic metabolisms. For example, whereas ble carbon isotope analysis may be an effective δ13C values of marine typically tool for tracing the evolution of chemosynthetic vary between µ18‰ and µ28‰ (Fry and Sherr, symbioses in the fossil record. 1984; Gearing et al., 1984; Goericke et al., 1994), One of the main factors affecting stable carbon derived chemosynthetically at vents is carbon isotope signatures of chemoautotroph- either considerably lighter (enriched in 12C), invertebrate symbioses appears to be the form of with δ13C values from µ27‰ to µ35‰, or heavier RubisCO used by the symbionts. While δ13C (depleted in 12C), with values from µ9‰ to values for vent bivalves hosting sulfide-oxidizing µ16‰ (Childress and Fisher, 1992; Robinson symbionts cluster between µ27‰ and µ35‰ and Cavanaugh, 1995; Robinson et al., 2003). and resemble values for free-living chemoau- Depending on the source of methane, symbioses totrophic bacteria, values for vent tubeworms, between mussels and methanotrophic bacteria shrimp episymbionts, and many free living bac- may be even more depleted in 13C, with δ13C terial mats at vents are significantly heavier, ranging from µ37‰ to µ78‰ (Cavanaugh, 1993; ranging from µ9‰ to µ16‰ (Childress and Nelson and Fisher, 1995a; Barry et al., 2002). Fisher, 1992; Van Dover and Fry, 1994; Robinson Because consumers generally retain the car- and Cavanaugh, 1995; Cavanaugh and Robinson, bon isotopic signature of their food (i.e., “you are 1996; Robinson et al., 2003). The difference what you eat”; DeNiro and Epstein, 1979), com- between these groups relates to the form of 13 parisons between δ C signatures of symbiont- RubisCO used to fix CO2 by the symbionts, with containing host tissue and symbiont-free host form I RubisCO occurring in most members of tissue can be used to study the transfer of sym- the isotopically lighter group and form II in all biont-derived carbon to the host. For example, members of the heavier tubeworm group δ13C values (µ30.8‰ to µ35.8‰) in symbiont- (Robinson and Cavanaugh, 1995). Corro- containing gill tissue from the western Pacific borating this hypothesis, Robinson et al. (2003) vent mussel Bathymodiolus brevior were signifi- showed that the kinetic isotope effect (ε value), 13 13 cantly lower than values from symbiont-free foot the relative rate of CO2 to CO2 fixation tissue, suggesting that B. brevior supplements (12k/13k) and a measure of discrimination against its diet via filter feeding on photosynthetically 13C by the purified RubisCO enzyme in vitro, is derived carbon (Dubilier et al., 1998). In con- significantly lower for form II RubisCO from trast, other studies show a high dependence on Riftia pachyptila symbionts (ε = 19.5‰) than for symbiont carbon by host species, including the form I enzyme (ε = 22–30‰). Such variation coastal solemyid protobranchs (Fisher and may have arisen from evolution under differing Childress, 1986; Conway and Capuzzo, 1991), concentrations of CO2 and O2 (Robinson et al., thyasirid clams (Dando and Spiro, 1993; Fiala- 2003). Médioni et al., 1993), and vestimentiferan tube- But isotopic discrimination by RubisCO does worms (Kennicutt et al., 1992), as well as suggest not fully account for 13C-enrichment in these differences in the contribution of methanotrophs symbioses. For example, to explain the discrep- and chemoautotrophs to host carbon in mussels ancy in R. pachyptila biomass δ13C values, Scott containing dual symbioses (Cavanaugh, 1993; (2003) used a mass balance model to show that Trask and Van Dover, 1999; Fiala-Médioni et al., steep gradients in [CO2] among symbiont, host, 2002; Yamanaka et al., 2003). and environmental pools may drive 13C enrich- Stable carbon isotope signatures have also ment. RubisCO, which occurs in the symbiont 12 been used to detect chemosynthetic symbioses , preferentially fixes CO2, leaving 13 in fossil bivalves of the clam family Lucinidae, CO2 behind. If fixation is rapid, CO2 equilibra- whose extant members all host chemosynthetic tion between the isotopically lighter host cyto- symbionts. CoBabe (1991), by determining the plasm and the isotopically heavier symbiont δ13C values of organic matrix material extracted cytoplasm cannot occur, causing RubisCO to 13 from lucinid dating to ca. 120,000 ya, draw from a more enriched CO2 pool and showed that fossilized lucinid (Epilucina sp.) accounting for the relatively heavy δ13C of tube- shells (δ13C = µ25‰) were about 5‰ lighter than worm biomass. Further, stable carbon isotope values from other bivalves collected in the same values are also affected by the δ13C of the envi- deposit (Pt. Loma, CA). Also, the organic matter ronmental carbon pool (Fisher, 1995; Colaco et of lucinid fossils was similar to that from modern al., 2002). Scott et al. (2004) demonstrated that samples, implying that the fossil organic matrix the “light” δ13C values of the symbionts of the did not decay or change significantly over time. coastal protobranch clam Solemya velum are These results, along with strong evidence show- explained not only by the kinetic isotope effect ing that shell δ13C values are reasonable proxies of symbiont form I RubisCO (ε = 24.5‰) but 13 for tissue values in extant species, suggest that also by the δ C value of the CO2 in the . CHAPTER 2.5 Marine Chemosynthetic Symbioses 491

Similarly, the source of methane production Similarly, δ15N values, because they vary pre- can significantly impact the isotopic signature dictably and largely between producer and con- of methanotroph symbioses (Fisher, 1995; sumer trophic levels (increase of ca. 3.4 per MacAvoy et al., 2002). The δ13C of methane var- level), are particularly useful markers for study- ies considerably depending on whether it is pro- ing aquatic food web interactions (Minagawa 13 15 duced thermogenically (δ C of CH4 >µ45‰) or and Wada, 1984). In general, δ N values of 13 biologically by (δ C of CH4 chemoautotrophic organisms are significantly <µ60‰; Lilley et al., 1993; Fisher, 1995), and this lighter (<0‰; Van Dover and Fry, 1994) than variability is reflected in the δ13C values of values for photosynthetic organisms (>6‰; see chemosynthetic symbioses (Fisher, 1995). There- Michener and Schell [1994] and Fisher [1995]). fore, in instances where the δ13C of the source Researchers have used this discrepancy and the methane is unknown, conclusions about the con- predictable fractionation of 15N to tribution of methanotrophic symbionts to host show host reliance on symbiont-derived organic δ13C values should be interpreted with caution matter in a number of symbioses including the (Fisher, 1995). In addition, interpreting δ13C sig- coastal clams Solemya velum and S. borealis may be especially problematic for dual (Conway et al., 1989; Conway et al., 1992b) and symbioses in which both methanotrophic and in vent mussels from the Mid-Atlantic Ridge thioautotrophic symbionts co-occur in the same (MAR) and the Galapagos Rift (Fisher et al., host cell. In these symbioses the δ13C values of 1988; Trask and Van Dover, 1999). In addition, the symbionts and the host reflect a mix of meth- δ15N values have been used extensively in anotrophic and thioautotrophic metabolism conjunction with δ13C values to show the flow (Fisher, 1995). These signatures are potentially of chemosynthetically derived organic matter confounded in instances when the thioau- through vent food webs, including those on the totrophic symbionts use the CO2 respired by the MAR (Vereshchaka et al., 2000; Colaco et al., methanotrophs, resulting in a second discrimina- 2002), the Central Indian Ridge (Van Dover, tion against an already light pool of CO2 and an 2002a), and the Galapagos Rift (Fisher et al., anomalously light tissue δ13C value (Fisher, 1994). As with δ13C data, δ15N values vary con- 1993a). siderably among sites; δ15N may depend in part Thus, while δ13C values often provide the first on the δ15N of the dissolved inorganic nitrogen evidence that chemoautotrophic or methan- (DIN) pool, the proportions and δ15N values of + µ2 µ otrophic symbioses occur in certain animal spe- different components (NH4 , NO3 , NO2 , and cies, researchers must recognize that δ13C is urea) in the DIN pool (Waser et al., 1998; Colaco inherently responsive to physical, environmental et al., 2002), the uptake kinetics of different DIN and enzymatic factors. Stable carbon isotope sig- assimilation pathways (Waser et al., 1998; natures therefore should not be used apart from Krueger, 1996a), and, as shown for vent shrimp other corroborating evidence (e.g., physiological (Vereshchaka et al., 2000) and mussels (Trask and enzyme activity assays and genetic charac- and Van Dover, 1999), the ontogenetic stage of terization) to identify carbon fixation pathways the host. Therefore, as noted above with stable or methane oxidation in chemosynthetic symbi- carbon isotopes, in the absence of additional oses (Fisher, 1995; Scott, 2003; Scott et al., 2004). enzymatic, genetic and environmental data, cau- tion must be used when comparing δ15N values from different habitats and species. Sulfur and Nitrogen Isotopes In addition to carbon isotopes, stable isotopes of sulfur and nitrogen are also used to study sources and metabolism of these elements in symbioses. The Ecophysiology extent to which different sources of reduced sulfur—geothermal production in vent fluid Symbioses between chemosynthetic bacteria and or microbial sulfate reduction in bottom sedi- marine invertebrates must acquire all of the sub- ment—support thioautotrophic metabolism has strates necessary for chemosynthetic meta- been inferred from the δ34S value of biological bolism: reduced sulfur or methane, oxygen, samples. Such analyses revealed hydrothermally dissolved inorganic carbon (DIC, as CO2 or derived sulfide as the dominant sulfide source for CH4), and other nutrients (e.g., nitrogen and deep-sea vent symbioses (Fry et al., 1983; phosphorus) for use in biosynthesis. In particu- Yamanaka et al., 2003). In contrast, symbiotic lar, to support energy generation, these symbio- bacteria within a shallow water vestimentiferan ses must obtain substrates from both oxic and tubeworm, Lamellibrachia satsuma (Miura et al., anoxic environments. To meet these demands, 2002), and the protobranch, Solemya velum the host-symbiont association relies on special- (Conway et al., 1989), rely predominantly on sul- ized , physiology and behavior. fide derived from microbial sulfate reduction. These adaptations are best studied in thioau- 492 C.M. Cavanaugh et al. CHAPTER 2.5 totrophic endosymbioses and are discussed pri- Table 2. Adaptations of thioautotrophs and methanotrophs marily within this context below. for at oxic-anoxic interfaces.a Adaptation Example Spanning the Oxic-Anoxic Interface Attachment Thiothrix , chemotaxis Beggiatoa Thioploca Access to both oxygen and reduced chemicals Elemental sulfur deposition Beggiatoa Thiothrix is necessary for aerobic respiration by chemo- and sulfur storage Thiomargarita Thioploca synthetic symbionts. Specifically, thioautotrophs Create own interface Thiovulum shuttle electrons from reduced sulfur (e.g., Filamentous sulfur production Arcobacter sp. sulfide) to a terminal electron acceptor during Resting Methanotrophs oxidative phosphorylation, generating a proton Associate with Thioautotroph and gradient that drives ATP synthesis. Though some Methanotroph symbionts thioautotrophic symbionts (such as those in the aFrom Anthony (1982), Jørgensen and Postgate (1982), tubeworm Riftia pachyptila [Hentschel and Cavanaugh (1985), Schulz et al. (1999), and Wirsen et al. Felbeck, 1993a] and the clam Lucinoma aequizo- (2002). nata [Hentschel et al., 1993b]) may use nitrate as an electron acceptor during periods of anoxia, most thioautotrophic symbionts typically use formation by methanotrophs; Table 2 and refer- molecular oxygen for respiration. Similarly, ences therein). methanotrophs must obtain oxygen for respira- Symbiosis thus may be viewed as an adapta- tion as well as methane for both energy gen- tion to simultaneously obtain sulfide (or meth- eration (via methane oxidation) and carbon ane) and oxygen from anoxic-oxic interfaces, assimilation (Anthony, 1982). allowing thioautotroph or methanotroph sym- This dual requirement for oxygen and bionts, via association with a eukaryotic host, to reduced compounds poses unique problems for circumvent many of the problems of sulfide thioautotrophs and methanotrophs. First, these acquisition (Cavanaugh, 1985). Similarly to free- organisms must obtain energy substrates living sulfur bacteria, thioautotrophic symbioses from mutually exclusive environments—oxygen use specialized behavioral, anatomical or physi- is absent or at very low levels in the anoxic zones ological mechanisms, either to spatially or tem- from which sulfide or methane is typically porally bridge sulfidic and oxic zones or to obtained. Second, sulfide, the predominant simultaneously sequester sulfide and oxygen energy source for thioautotrophy, spontaneously (Cavanaugh, 1994; Fisher, 1996; Polz et al., 2000). reacts with oxygen to form less-reduced sulfur For instance, the cold seep vestimentiferan tube- 0 µ2 µ2 compounds (S , S2O3 , or SO4 ; Zhang and worm Lamellibrachia cf. luymesi acquires oxy- Millero, 1993), thereby decreasing the availabil- gen via its anterior plume while extending a ity of substrates for thioautotrophy. Though such posterior section of its tube (the root) deep into abiotic oxidation may be several orders of mag- the sediment to acquire sulfide (Julian et al., nitude slower than biological sulfide oxidation 1999; Freytag et al., 2001). Similar burrowing tac- (Millero et al., 1987; Johnson et al., 1988), thio- tics occur in some species of symbiont-containing autotrophic symbioses must still compete with thyasirid clams, which possess a superextensile oxygen for free sulfide. Also, in habitats contain- foot (up to 30 times the length of the shell) that ing both sulfide and methane, abiotic oxidation burrows into the sediment to access hydrogen of sulfide may limit the oxygen available for sulfide (Dufour and Felbeck, 2003), and in pro- methanotrophy. These limitations force free- tobranchs of the Solemya, which dig Y- living thioautotrophs and methanotrophs into shaped burrows in reducing sediments to allow microaerophilic zones at the interface, or simultaneous pumping of oxygenated water from chemocline, between oxic (e.g., water column) above and sulfide-rich pore water from below and anoxic (e.g., vent fluid and sediment pore (Stanley, 1970; Cavanaugh, 1983; Fig. 11). Also, water) habitats. Such free-living bacteria demon- shrimp, nematodes and oligochaetes migrate strate unique mechanisms to support life at the vertically along the oxygen-sulfide gradient or oxic-anoxic interface; these adaptations may be between separate oxic and anoxic zones, thereby behavioral (e.g., tracking the chemocline via enabling their symbionts to simultaneously gliding by Beggiatoa), anatomical (e.g., keeping access both energy substrates or to store reduced cells in the chemocline via “veil” formation by sulfur compounds for later oxidation (Polz et al., Thiovulum or creation of a filamentous sulfur 2000). matrix by Arcobacter), biochemical (e.g., inter- The vent tubeworm Riftia pachyptila possesses nal or external sulfur deposition that serves as a remarkable biochemical adaptation to simulta- an electron source or sink when sulfide or neously acquire sulfide and oxygen. R. pachyptila oxygen is limiting, as by Beggiatoa and Arco- produces coelomic and vascular bacter), or developmental (e.g., resting stage that, in contrast to most invertebrate and verte- CHAPTER 2.5 Marine Chemosynthetic Symbioses 493 brate hemoglobins, can bind oxygen in the pres- as the energy source in symbiotic carbon fixation. ence of sulfide (Arp et al., 1985; Arp et al., 1987; Subsequently, researchers have demonstrated Childress et al., 1991; Zal et al., 1996). R. pachyp- mitochondrial sulfide oxidation across a wide tila appears to preferentially take up HSµ from range of organisms, including polychaete worms, the surrounding fluid, despite a large H2S gradi- clams, fishes and chickens (Grieshaber and ent from tubeworm blood to the environment Volkel, 1998; Yong and Searcy, 2001). These data (Goffredi et al., 1997a). The HSµ diffuses across lend credence to the hypothesis that mitochon- the plume of the worm (Goffredi et al., 1997a) dria evolved from sulfide-oxidizing endosymbi- and then binds reversibly and independently of otic bacteria (Searcy, 1992). O2 at two free residues, each located on Readers should consult several additional a distinct globin type (Zal et al., 1997; Zal et al., reviews (e.g., Cavanaugh, 1994; Fisher, 1996; Polz 1998; Bailly et al., 2002). These residues are well et al., 2000) for a more extensive discussion of conserved in both symbiont-containing and the remarkable adaptations used by chemoau- symbiont-free from sulfidic environ- totrophic symbioses to sequester both oxygen ments but are absent in annelids from sulfide- and reduced chemicals across oxic-anoxic zones. free habitats (Bailly et al., 2002; Bailly et al., 2003). Bailly et al. (2003) suggest that the sulfide Carbon Uptake and Transport binding function may have been lost via positive selection, if the sulfide-binding cysteine residues In addition to oxygen and reduced sulfur com- react disadvantageously with other blood com- pounds, thioautotrophic symbionts utilizing the ponents in the absence of sulfide. Calvin cycle require CO2 for autotrophic carbon Extracellular hemoglobins that simulta- fixation. Acquisition of CO2 is not trivial given neously bind sulfide and oxygen are absent in that relative concentrations of the three distinct µ µ2 most other marine invertebrates that host sul- chemical species (CO2, HCO3 and CO3 ) in the fide-oxidizing symbionts (Weber and Vinogra- dissolved inorganic carbon (DIC) pool can vary dov, 2001); such organisms have evolved other considerably depending on pH (pKa of 6.4 for µ mechanisms for regulating sulfide toxicity and CO2 : HCO3 at 25°C; see the section Habitat delivery. For instance, the vesicomyid clam Chemistry in this Chapter). In general, the µ Calyptogena magnifica synthesizes a di-globular, majority of DIC in seawater (pH ~ 8.0) is HCO3 . non-heme molecule that readily binds free sul- But at vents the typically lower pH of the mixed fide within the blood serum, perhaps via zinc vent fluid and ambient bottom water generates residues (Arp et al., 1984; Zal et al., 2000). Also, higher concentrations of CO2, giving organisms several thioautotroph-containing species, includ- that use the Calvin cycle a distinct advantage. ing the vent mussel Bathymodiolus thermophilus The tubeworm Riftia pachyptila provides an and the coastal clam Solemya velum, appear to interesting model in which to study the uptake mediate detoxification in part by storage of sul- and transport of DIC. Goffredi et al. (1997b) fur in amino acids (e.g., taurine and thiotaurine; demonstrated that for R. pachyptila, pH plays an Conway and Capuzzo, 1992a; Pruski et al., 2000a; important role in DIC uptake. The acidity of dif- Joyner et al., 2003; Pruski and Fiala-Médioni, fuse vent fluid (pH ca. 6) around tubeworms 2003). Indeed, thiotaurine may be used effec- ensures that CO2 (pKa of 6.1 at in situ tempera- tively as a of thioautotrophic symbio- ture and pressure of ca. 10°C and 101.3 kPa; ses (Pruski et al., 2000b). Dickson and Millero, 1987) is the dominant DIC Other host organisms, including some bivalve form in the vent environment. This contrasts with mollusks, apparently avoid sulfide toxicity via the vascular fluid of the worm, which has an alka- mitochondrial oxidation of sulfide. Powell and line pH of 7.1–7.5, apparently because of the Somero (1986) first demonstrated mitochondrial action of H+-ATPases (Goffredi et al., 1999; sulfide oxidation in the coastal protobranch S. Goffredi and Childress, 2001; Girguis et al., reidi. The authors showed that mitochondria iso- 2002). The alkaline pH inside Riftia results in µ lated from the gill and foot of S. reidi exhibit rapid conversion of CO2 to HCO3 , which, ADP-stimulated oxygen uptake and ATP syn- because of its negative charge, cannot diffuse out thesis following the addition of sulfide. On the of the worm; this in effect creates a bicarbonate basis of the effects of cytochrome and reduced “trap” (Childress et al., 1993). Thus, a gradient nicotinamide adenine dinucleotide (NADH) oxi- of higher external [CO2] to lower internal [CO2] dase inhibitors, electrons from sulfide oxidation develops across the tubeworm plume and drives appear to enter the respiratory chain at cyto- of DIC into the blood (Childress et al., chrome c in S. reidi mitochondria (Powell and 1993; Goffredi et al., 1997b; Scott, 2003). Follow- Somero, 1986). Further characterization of this ing diffusion into the plume, DIC (as CO2 and 35 µ system using S showed that sulfide is oxidized HCO3 ) is transported by the vascular system to exclusively to thiosulfate (O’Brien and Vetter, the symbiont-containing trophosome. Here, car- 1990), a nontoxic intermediate that can function bonic anhydrase, the enzyme that reversibly con- 494 C.M. Cavanaugh et al. CHAPTER 2.5

µ verts CO2 into HCO3 in both prokaryotes and eral other organic acids and were excreted µ eukaryotes, may play a role in converting HCO3 by purified suspensions of R. pachyptila sym- into CO2, the DIC species used by RubisCO bionts, suggesting that these simple organic com- (Kochevar and Childress, 1996; De Cian et al., pounds might be important intermediates in the 2003a; De Cian et al., 2003b). As discussed above transfer of fixed carbon from symbionts to host. for Riftia, DIC incorporation into symbiont bio- Corroborating these data, Bright et al. (2000), mass occurs via CO2 fixation by a form II using labeling analysis, showed that the RubisCO of the Calvin-Benson cycle. Rapid CO2 bulk of organic carbon assimilated into R. pac- fixation rates create steep internal [CO2] gradi- hyptila tissue is first released by metabolically ents between symbiont and host cytoplasm that active bacteria at the center of a trophosome may, in combination with the relatively low dis- lobule. However, these authors also showed that crimination of form II RubisCO against 13C, a smaller fraction of host carbon is obtained by result in a 13C-enriched signature of symbiont of bacterial cells at the lobule periph- and host biomass (Robinson et al., 2003; Scott, ery (Bright et al., 2000). This evidence for diges- 2003). tion is supported by prior studies showing In chemosynthetic endosymbioses the host degenerative stages of bacteria within the R. benefits by obtaining part or all of its nutrition pachyptila trophosome (Bosch and Grassé, 1984; from the symbiont, via two potential transfer Hand, 1987). In addition, relatively high mechanisms: the host may assimilate - lysozyme activity in Riftia tissue further suggests ically fixed carbon that has been released by the that digestion of symbionts plays a role in tube- symbiont and translocated to host cells in the worm nutrition (Boetius and Felbeck, 1995). form of soluble organic molecules, or the host may directly digest bacterial cells. Radiotracer Nitrogen analysis and microscopy have proven particu- larly useful in studying host nutrition. For exam- The partners in a symbiosis must also acquire all ple, Fisher and Childress (1986) showed a rapid of the other macro- and micronutrients, particu- (within hours) appearance of radiolabeled car- larly nitrogen and phosphorus, for use in the bon in the symbiont-free tissues of the host clam biosynthesis of organic compounds. Currently, Solemya reidi following exposure to 14C-labeled very little is known about how various forms bicarbonate, suggesting release of fixed carbon (inorganic and organic) of phosphorus are trans- by the symbiont population. In contrast, a ferred to and among different pools within slow (1–5 days) transfer of labeled organic chemoautotrophic endosymbioses. Most studies carbon from methanotroph-containing tissue to have focused on nitrogen metabolism, using a symbiont-free tissue of a seep mussel exposed combination of enzyme characterizations and to 14C-labeled methane was inferred to be due to physiological experiments to elucidate nitrogen 14 µ2 initial CH4 incorporation by the symbionts with assimilation pathways. Nitrate (NO3 ), which is host digestion of symbionts occurring later abundant at vents (in situ concentrations of (Fisher and Childress, 1992). Electron micro- ~40 µM; Johnson et al., 1988), appears to be the scopy showing symbionts being degraded in the predominant nitrogen source for vent symbioses. basal region of bacteriocytes in other methane- For example, Lee et al. (1999) demonstrated the based and dual chemoautotroph-methanotroph activity of nitrate reductase, a bacterial enzyme mussel symbioses supports this interpretation involved in converting nitrate to ammonia for (Cavanaugh et al., 1992; Barry et al., 2002), as either assimilatory or respiratory purposes, in does the detection of lysosomal enzymes in the the vent tubeworms Riftia pachyptila and Tevnia gills of the vent bivalves Calyptogena magnifica jerichonana and the mussel Bathymodiolus and Bathymodiolus thermophilus (Fiala-Médioni thermophilus. In addition, the ammonia assimi- et al., 1994; Boetius and Felbeck, 1995) and the lation enzymes glutamine synthetase (GS) and shallow water clam Lucinoma aequizonata glutamate dehydrogenase (GDH) were detected (Boetius and Felbeck, 1995). in these symbioses, and almost all GS activity in In the R. pachyptila tubeworm symbiosis, the symbiont-containing tissue was shown to be due transfer of carbon from symbiont to host appears to enzyme produced by the bacterial symbiont to occur via both translocation and digestion and not the host (Lee et al., 1999). Supporting (Bright et al., 2000). Felbeck (1985) and Felbeck these data, physiological experiments on R. pac- and Turner (1995) documented a rapid (within hyptila kept in pressurized chambers showed seconds) appearance of labeled succinate and that the symbiont population reduces nitrate to malate in trophosome tissue and in vascular and ammonia not for respiratory purposes but for coelomic blood following exposure of whole incorporation into both symbiont and host worms (in pressure vessels) and plumes to 14C- biomass (Girguis et al., 2000). However, in R. bicarbonate. Subsequently, Felbeck and Jarchow pachyptila, high GS activity also occured in (1998) showed that succinate, malate, and sev- symbiont-free branchial plume tissue, suggesting CHAPTER 2.5 Marine Chemosynthetic Symbioses 495 that the host may also be involved in assimilation later- and Cenozoic) and suggests an of ammonia from the vent environment (Minic alternative hypothesis to vent taxa as living rel- et al., 2001). But further enzymatic characteriza- ics: vents were recently populated from shallow tion of Riftia tissues demonstrated that the tube- seeps or whale falls (Van Dover et al., 2002b; worm depends on its symbionts for the de novo Hurtado, 2002). Indeed, the communities most synthesis of pyrimidine nucleotides (Minic et al., similar to those of vents occur at seeps. Com- 2001) as well as for the biosynthesis of poly- pared to the spatially and temporally patchy amines (Minic and Herve, 2003), suggesting that distribution of vent fossils (with most being the trophosome is a primary site for nitrogen concentrated in the and rocks assimilation and metabolism. of the Ural mountains), seep fossils are ubiqui- In contrast, in the thioautotrophic symbiosis tous (Little and Vrijenhoek, 2003). At least 50, involving the shallow-water clam Solemya reidi, and perhaps as many as 200, fossilized seep sites inorganic nitrogen is readily assimilated in the dating from the Devonian to the Pleistocene form of ammonia (Lee and Childress, 1994), have been uncovered. These specimens are much which is abundant in the shallow water, nutrient- better preserved than most vent fossils and rich habitats of the clam (e.g., sewage outfalls). include extant vent taxa not yet uncovered at Ammonia incorporation rates are highest in the fossil vent sites (e.g., vesicomyids, thyasirids, symbiont-containing gill tissue, and the sulfur- mytilids and solemyids; Little and Vrijenhoek, containing amino acid taurine appears to be a 2003). This greater diversity supports the seeps- major end product of ammonia assimilation (Lee to-vents hypothesis. However, opponents argue et al., 1997). The mechanisms by which chemo- that the vent fossil record has been greatly synthetic symbionts, particularly those contained affected by high dissolution within the cells of invertebrate hosts (such as rates (Little and Vrijenhoek, 2003). Solemya and Riftia), acquire all of the other While the discrepancy between the evolution- macro- and micronutrients for biosynthesis have ary histories suggested by the fossil and molecu- yet to be characterized. lar evidence needs to be resolved, it must also be stressed that these data are not evidence for chemosynthetic symbioses. In a unique study, CoBabe (1991) was able to deduce a chemosyn- Ecology and Evolution thetic symbiosis by analyzing the organic matrix History from fossil lucinid shells using stable carbon iso- topes. This result is encouraging and suggests Prior to the use of molecular techniques, that both the age of these organisms and their researchers considered vent taxa to be relic spe- symbiosis can be addressed using current cies. These organisms, whose strange morpholo- methods. gies suggest a primitive state, purportedly survived past events due to the rela- Organism Interactions tive isolation of vents from the photic zone (McArthur and Tunnicliffe, 1998). This percep- In the relatively featureless and nutrient poor tion of vents as ancient ecosystems is supported , vent and seep environments are eco- by the fossil record, which shows that over 80% logical oases (Laubier, 1989). Initially, free-living of vent species are found only at vent sites chemoautotrophic bacteria were hypothesized to (Tunnicliffe, 1991; Tunnicliffe, 1992; Little et al., provide the bulk of primary production in these 1997; Little and Vrijenhoek, 2003) and that the communities (Lonsdale, 1977). Indeed, at some oldest vent site dates to the Silurian (~430 Mya; vent sites, suspended bacteria or bacteria in Little et al., 2004). But the fossil record for vents surface-attached mats are a large food source for is relatively poor. There are only 19 known fos- higher trophic levels (Humes and Lutz, 1994; silized vent sites on the planet, perhaps because Van Dover, 2000). But the dominant strategy for calcium carbonate structures dissolve relatively the major vent and seep fauna is symbiosis with quickly in vent fluids (Hunt, 1992; Kennish and chemoautotrophic bacteria (Cavanaugh, 1994), Lutz, 1999). Also, studying vent fauna evolution and these symbioses significantly influence the based on the morphological characters of fossils ecology of the nonsymbiotic community. Not is problematic if much of the specimen has only are chemosynthetic symbioses a major and degraded or if the preserved character is plastic stable source of organic carbon (Sarrazin and or isomorphic. In particular, vestimentiferan Juniper, 1999), but as biogenic structures, they tubeworms are known for the phenotypic plas- also provide living space for a diversity of species ticity of their tubes (Southward et al., 1995; in an otherwise two-dimensional landscape of Black et al., 1998). basalt or sediment (Bergquist et al., 2003). For In contrast, molecular evidence suggests that example, the tubes of chemosynthetic vestimen- vent taxa evolved more recently (22–150 Mya; tiferans support mussels, sponges and limpets, 496 C.M. Cavanaugh et al. CHAPTER 2.5 many of which host their own chemosynthetic larger and more genetically heterogeneous than symbionts (Yamamoto et al., 2002; Bergquist et populations transmitted vertically. Comparisons al., 2003; Bates et al., 2004). of 16S rRNA gene evolution between free-living Vent symbioses may also significantly impact bacteria, in which significant recombination the free-living bacterial community by providing occurs (Dykhuizen and Green, 1991; Levin and increased surface area for attachment. Free- Bergstrom, 2000), and symbiotic chemosynthetic living bacteria that cluster phylogenetically with bacteria revealed unexpected differences in rates known chemoautotrophic and heterotrophic of evolution depending on mode of transmission groups have been isolated from tubeworm sur- (Peek et al., 1998). While chemoautotrophic, faces (Lopez-Garcia et al., 2002; Yamamoto et maternally transmitted endosymbionts did al., 2002). On the Mid-Atlantic Ridge, a single exhibit rapid evolutionary rates, consistent with phylotype of shrimp episymbionts, which appear their small population sizes, environmentally to be transmitted among hosts via the environ- transmitted symbionts evolved more slowly than ment, represented over 60% of the free-living their free-living counterparts (Peek et al., 1998). bacteria (Polz and Cavanaugh, 1995). This sug- The authors suggest that this slower rate of evo- gests that the host inoculates inanimate surfaces lution could be caused by purifying selection in continuously, increasing the probability of a large population. These results, however, were symbiont attachment relative to the free-living based on one gene across many lineages; a true community (Polz and Cavanaugh, 1995). Such genomic analysis of evolution in chemosynthetic environmental inoculation may also occur in endosymbionts is necessary to extend these tubeworm and lucinid clam symbioses, in which findings. the symbionts also appear to be transmitted Because chemosynthetic symbionts have yet environmentally (Durand and Gros, 1996a; to be cultured and their hosts are difficult to Durand et al., 1996b; Di Meo et al., 2000; Nelson maintain in the laboratory, the transmission and Fisher, 2000; McMullin et al., 2003). strategy of a symbiosis has been inferred by phy- logenetic analysis or PCR-based detection of bacteria in host reproductive tissues or gametes. Transmission Strategies and If the symbionts are maternally transmitted and Effects on Symbiosis the symbioses stable, congruence of host and symbiont phylogenies should occur (e.g., Chen et The transmission strategy of a symbiosis reveals al., 1999; Thao et al., 2000; Degnan et al., 2004) much about the evolutionary dynamics between and bacterial symbionts should be found in ova- host and symbiont. Symbiont transmission can ries or oviducts of the host. Using these tech- occur environmentally (through a free-living niques, vertical transmission has been proposed population of symbiotic bacteria), horizontally for the solemyid protobranchs (Cary, 1994; (between contemporary organisms sharing the Krueger et al., 1996b) and vesicomyid clams same habitat), or vertically (from parent to (Endow and Ohta, 1990; Cary and Giovannoni, offspring). Vertically transmitted endosymbionts 1993a; Peek et al., 1998; Hurtado et al., 2003). are effectively disconnected from their free- Interestingly, although bacteria have been living counterparts. These symbionts experience detected via PCR in the gonads of female hosts, elevated rates of mutation and fixation of slightly this does not necessarily imply direct bacterial deleterious alleles because of genetic drift endocytic localization in host . Indeed, in (Wernegreen, 2002). For the most part, these Solemya reidi, the internal contents of do evolutionary effects are due to a vastly different not contain bacteria, and instead the transmis- selective regime inside the host and a severely sion mechanism is thought to occur via ingestion; depreciated population size (Ohta, 1973); endo- the larvae ingest the bacteria, which are then symbionts undergo a population bottleneck engulfed by hemocytes in the larval perivisceral upon host colonization and another upon cavity and transported to the developing gill transmission (Mira and Moran, 2002). But the (Gustafson and Reid, 1988). The oligochaetes asexuality and lack of recombination in endo- also exhibit an interesting mechanism of vertical symbionts exacerbate these genetic problems transmission. During oviposition, the eggs through what is known as “Muller’s rachet” appear to be infected with the symbiotic bacteria (Muller, 1964; Moran, 1996). In Muller’s ratchet, via the adult’s genital pad (Giere and Langheld, wildtype recombinants cannot be introduced 1987). During the development of the larvae, into the endosymbiont population (Moran and many of the bacteria exist intracellularly, but as Baumann, 1994; Dale et al., 2003); genetic drift the animal matures, the symbionts take their pri- therefore occurs quickly, and the population can- marily extracellular form. not recover after fixation of deleterious alleles. However, of the putatively vertically transmit- In contrast, symbiont populations that are ted symbioses, only associations involving vesi- environmentally transmitted are effectively comyid clams show phylogenetic congruence CHAPTER 2.5 Marine Chemosynthetic Symbioses 497 between host and symbiont (Peek et al., 1998; et al., 2003a; DeChaine et al., 2004). In addition, Hurtado et al., 2003). Cospeciation does not McKiness (2004) provided the first assessment of appear to have occurred in the solemyid proto- cospeciation between symbiont and host in branchs (Durand et al., 1996b; Krueger and Bathymodiolus mussels, analyzing molecular Cavanaugh, 1997) or in the mytilid mussels data for both methanotrophic and chemoau- (McKiness, 2004). When evaluating phylogenetic totrophic symbionts and testing phylogenetic congruence, however, other factors that influ- congruence with the hosts. The results showed ence a phylogenetic reconstruction, such as geo- weak support for vertical transmission of the graphic constraints, must be taken into account. chemoautotrophic symbionts but provided no Also, robust phylogenies with adequate taxa evidence for vertical transmission of the sampling for both host and symbiont are neces- methanotrophs. sary; incomplete phylogenies may be hindering analyses of the solemyid and mytilid symbioses. Biogeography and Population Genetics Lack of PCR-based evidence and phyloge- netic incongruence has been used to infer an The view of hydrothermal vents as deep-sea environmental mode of transmission for several islands frames questions of vent biogeography of the chemosynthetic symbioses. For instance, and population genetics. Compared to the rela- the lucinid clams exhibit environmental trans- tively uniform and stable environment of the mission (Durand and Gros, 1996a; Gros et al., abyssal deep sea, hydrothermal vents are ephem- 1996; Gros et al., 1998; Gros et al., 2003a; Gros eral, dynamic and geographically fragmented. A et al., 2003b). Researchers have even been able chain of vents along a mid-ocean ridge resembles to exchange symbionts between lucinid species a chain of islands in an archipelago. However, without affecting the development of the juve- genetic data for many vent species do not cleanly nile animal (Gros et al., 2003a). In addition, vent fit an “island” or “stepping-stone” model of bio- tubeworms appear to acquire their symbionts geography (Vrijenhoek et al., 1998). Some host from the environment (Distel and Cavanaugh, taxa do exhibit a decline in gene flow with 1994; Feldman et al., 1997; Laue and Nelson, increasing distance between sites (Black et al., 1997; Di Meo et al., 2000; Nelson and Fisher, 1994), as a stepping-stone model would predict 2000; McMullin et al., 2003), as evidenced in part (Kimura and Weiss, 1964), while others show a by the presence of functional genes for sensing more widespread gene flux (Karl et al., 1996) or and responding to the environment as well as a appear to encounter barriers to dispersal other flagellin gene in the Riftia symbiont (Hughes et than distance (Black et al., 1998). These differ- al., 1997; Hughes et al., 1998; Millikan et al., ences should be resolved with a greater under- 1999). Indeed the 16S rRNA phylotype has been standing of the major variables affecting vent detected in vent environments via both PCR and biogeography, including larval development and in situ hybridization, suggesting the vent tube- dispersal, symbiont distribution, oceanic flow, worm symbionts are environmentally transmit- and past and current bathymetry. This section ted (Harmer et al., 2004). While environmental focuses predominantly on host biogeography transmission of tubeworm symbionts seems to be because research on the population genetics and a potentially risky strategy, given the stochastic biogeography of bacterial symbionts is lacking. nature of environmental transmission and the Understanding host population dynamics, how- complete dependence of the adult tubeworms on ever, does provide valuable insight into the dis- their symbionts for nutrition, detection of “wild” tribution of the chemosynthetic symbionts to symbionts in conjunction with the phylogenetic which most vent fauna are tightly linked. evidence supports environmental transmission in Vent habitats are highly ephemeral and sensi- this species. tive to variations in tectonic activity, hydrother- The mechanism of transmission for the mytilid mal inputs, and geologic events. Consequently, mussels remains largely unresolved; on the basis the persistence of vent organisms, which are pre- of varying evidence, researchers have suggested dominantly sessile as adults, depends on success- both vertical and environmental transmission. ful larval dispersal to new sites. The dispersal Vertical transmission in Bathymodiolus thermo- strategy of larvae can significantly impact the philus, the thioautotroph-hosting mussel, was biogeography of the adult organism (Lalou and suggested in 1993, but evidence supporting this Brichet, 1982; Fustec et al., 1987). On the basis report is not yet published (Cary and of laboratory studies and comparisons with shal- Giovannoni, 1993a). In contrast, a recent study low water species, researchers infer that some based on genetic and ultrastructural data of the vent larvae are planktotrophic while others are chemoautotrophic symbionts of B. azoricus, a lecithotrophic (Lutz et al., 1980; Turner et al., MAR mussel hosting both thioautotrophs and 1985; Young et al., 1996; Marsh et al., 2001). methanotrophs, indicated environmental acqui- Although both forms are pelagic, planktotrophic sition of the chemoautotrophic symbionts (Won larvae are positively buoyant and feed in the 498 C.M. Cavanaugh et al. CHAPTER 2.5 water column while lecithotrophic larvae are primarily NNW and SSE along the axis (Marsh negatively buoyant and nonfeeding (Poulin et al., et al., 2001) may facilitate dispersal of larval B. 2001). Larvae of the large vesicomyid clam thermophilus, contributing to the homogeneity Calyptogena magnifica typify a planktotrophic observed along the EPR. However, there is some dispersal strategy successfully exploiting the vent genetic structure in the EPR mytilid populations; plume to carry them many kilometers (Pradillon the westward currents across the ridge axis at et al., 2001; Mullineaux et al., 2002). Although 15°N and the Easter Microplate are obstacles for planktotrophic larvae risk being carried off the planktotrophic larvae. At 15°N, a westward cur- ridge axis by cross currents, C. magnifica appar- rent flows across the ridge axis, partially isolating ently encounters no significant barriers to dis- the 17°S population from the other northern persal across the equator on the East Pacific Rise populations. Further south, at the Easter Micro- (EPR; Karl et al., 1996). Conversely, leci- plate, mussel populations are severely divergent thotrophic larvae are less affected by cross cur- (Won et al., 2003). Although morphologically rents but, because they are non-feeding, have indistinguishable, mussels north and south of the relatively short larval stages and therefore lim- Microplate are genetically distinct (Won et al., ited time for dispersal. For example, in labora- 2003). The Easter Microplate therefore appears tory studies, larval Riftia pachyptila exhibit a to be a significant topographic obstacle for larval lecithotrophic strategy, surviving a maximum of dispersal. Such a feature can produce cross-axis 38 days (Marsh et al., 2001). Assuming flow rates currents, like those at 15°N, that may sweep characteristic of EPR currents, this interval sug- bathymodiolid larvae (which are positively gests a maximum dispersal distance of 100 km buoyant) off the ridge axis (Fujio and Imasato, (Marsh et al., 2001); however, the in situ dis- 1991; Mullineaux et al., 1995). The degree to persal distance is unknown given that Riftia which such barriers also impact the genetic larvae have not been detected in the wild. diversity and biogeography of chemosynthetic The geology and tectonic activity associated symbiont populations remains an open question. with mid-ocean ridges also impact the biogeog- raphy of vent organisms. For instance, Iceland, an active site of crust formation, rises out of the Summary ocean along the northern MAR, forming a bar- rier that prevents dispersal along the ridge axis Scientific understanding of chemosynthetic sym- (Tyler and Young, 2003). Given that Iceland bioses continues to expand. The spectacular dis- interrupted the MAR approximately 55 Mya, the covery of hydrothermal vents highlighted the ridge axis north of Iceland constitutes one of the importance of chemosynthetic bacteria both in most isolated vent systems on the planet, per- food webs and in symbioses with eukaryotes and haps representing a new biogeographic province provided the impetus to examine less exotic (Bilyard and Carey, 1980; Dunton, 1992; Svavar- environments for such associations. As other sson et al., 1993). Similar dispersal barriers are oxic-anoxic environments (e.g., freshwater) and seen among vent fields abutting the Azores Rise the invertebrates and protists that inhabit them in the Atlantic (Tyler and Young, 2003) and also are explored, new symbioses will undoubtedly be evident between the EPR and the Northeast discovered. Further, chemosynthetic bacteria Pacific vent fields (Tunnicliffe, 1988; Tunnicliffe that use other sources of energy (e.g., hydrogen) and Fowler, 1996). These barriers are insur- may be found in similar associations. mountable and may provide the conditions for Current studies of these fascinating symbioses allopatric speciation of both host and symbiont. involve a range of experimental and diagnostic Research on EPR bathymodiolid mussels and tools, including physiological assays in special- their symbionts provides a good example of how ized growth chambers, enzyme characterizations, larval dispersal strategy, current regime, and immunodetections, and stable isotope analyses. bathymetry interact to structure biogeography Increasingly, molecular techniques, such as PCR- (Lonsdale, 1977; Corliss et al., 1979). Except at based gene probing, FISH, and 16 rRNA phylo- Northern Pacific sites, which are separated from genetic analysis, are used to complement the EPR by the North America landmass, vent traditional methods. These studies provide valu- communities on the Pacific ridge axis appear able insight into the population dynamics, relatively uniform. For example, the mussel evolutionary history, and carbon and nutrient Bathymodiolus thermophilus, which undergoes a metabolism of symbionts. In addition, projects planktotrophic larval stage, occurs over a dis- are currently underway to sequence the tance of 4900 km (from 13°N to 32°S) on the of some of the chemosynthetic symbionts EPR. Mussel populations along 13°N and 11°S described in this review (e.g., symbionts of Riftia are genetically indistinguishable, indicating no pachyptila and Solemya velum). Genomic ana- population subdivision (Craddock et al., 1995; lysis, in conjunction with new technologies to Won et al., 2003). Deep ocean currents that flow manipulate symbioses under in situ conditions CHAPTER 2.5 Marine Chemosynthetic Symbioses 499

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