Osedax are gutless siboglinid worms that thrive on vertebrate bones lying on the ocean floor, mainly those of whales. The posterior body of female Osedax penetrates into the bone forming extensions known as ‘roots’, which host heterotrophic symbiotic bacteria in bacteriocytes beneath the epidermis. The Osedax root epithelium presumably absorbs bone collagen and/or lipids, which are metabolized by the symbiotic bacteria that in turn serve for Osedax's nutrition. Here, we show that Osedax roots express extremely high amounts of vacuolar-H+-ATPase (VHA), which is located in the apical membrane and in cytoplasmic vesicles of root and ovisac epithelial cells. The enzyme carbonic anhydrase (CA), which catalyses the hydration of CO2 into H+ and HCO3−, is also expressed in roots and throughout Osedax body. These results suggest Osedax roots have massive acid-secreting capacity via VHA, fuelled by H+ derived from the CA-catalysed hydration of CO2 produced by aerobic metabolism. We propose the secreted acid dissolves the bone carbonate matrix to then allow the absorption of bone-derived nutrients across the skin. In an exciting example of convergent evolution, this model for acid secretion is remarkably similar to mammalian osteoclast cells. However, while osteoclasts dissolve bone for repairing and remodelling, the Osedax root epithelium secretes acid to dissolve foreign bone to access nutrients.
Osedax is a genus of peculiar siboglinid worms that were originally discovered living on whale bones on the deep-ocean floor , and subsequently found to also survive on human-deployed cow and fish bones [2,3]. Female Osedax lack a mouth and a gut, and presumably feed by absorbing bone nutrients across extensive ramifications at the posterior end of their body known as ‘roots’ [1,4] (figure 1). The Osedax roots host endosymbiotic aerobic heterotrophic bacteria of the order Oceanospirillales in specialized bacteriocytes. The symbionts appear to be involved in metabolizing bone-derived complex organic compounds and in turn are digested by the host worm [1,4]. This contrasts with most other siboglinids, which obtain their nutrients via endosymbiotic thiotrophic (sulfur-oxidizing) bacteria. Osedax males are significantly smaller than females, ranging from 0.3 to 1 mm in length, and, depending on the species, hundreds of ‘dwarf’ males may inhabit the tube of a single female with the only purpose of producing sperm to fertilize her eggs. The males lack symbiotic bacteria and seem to rely on yolk reserves only for sperm production [1,5,6].
In order to access bone nutrients, female Osedax must first dissolve the inorganic bone matrix, which largely consists of calcium phosphate in the form of hydroxyapatite . Other invertebrate animals bore into calcium carbonate structures using a combination of chemical and mechanical methods; for example, some sponges excavate into corals by secreting acid or enzymes followed by removal of etched chips of calcium carbonate [8,9]; some snails drill through shells of prey gastropods, bivalves or barnacles using a combination of acid/enzymes and radulae action [10,11]; and a range of polychaetes secrete calcium-chelating mucus containing acidic mucopolysaccharides to burrow into lime rocks, aided by removal of loosened sediments by chaetae [12,13].
The epithelium of Osedax roots comprises cells displaying typical acid-secreting features, such as an extensive ruffled apical membrane and abundant mitochondria in the apical region [14,15]. Since Osedax do not have any obvious bioabrasive structures, Katz et al. [14,15] suggested Osedax bored into bones by secreting acid. This possibility was also discussed by Higgs et al. , who additionally found acidic mucopolysaccharides in the mucus of the root tissue and proposed that this material is important in the boring mechanism by acting as a chelating agent. However, the specific mechanisms by which Osedax secretes acid remain unknown.
In addition to dissolving the bone, Osedax must absorb nutrients, presumably collagen and possibly lipids [4,17], from the very material they erode into. The dual ‘drilling and feeding’ function does not occur in the boring invertebrate animals mentioned earlier, but it is analogous to the mechanism for bone resorption in osteoclast cells of vertebrate animals . The potential similarities between marine boring organisms and osteoclasts have previously been recognized for sponges  and snails , and also Osedax .
Osteoclasts dissolve the bone carbonate matrix by secreting acid using vacuolar-H+-ATPases (VHA) . This holoenzyme comprises multiple subunits, and it uses energy derived from ATP hydrolysis to pump H+ against the concentration gradient (reviewed in ). VHAs are multifunctional enzymes since they also function in acid/base regulation, vesicular transport, endosomal acidification and energizing ion and amino acid transport (reviewed in [20–22]).
In this study, we demonstrate that root epithelial cells express very high amounts of VHA, indicating massive capacity for acid secretion. We also establish that the enzyme carbonic anhydrase (CA) is also abundantly expressed in Osedax roots, suggesting the secreted acid is derived from CO2 produced by aerobic metabolism at the roots. This bioeroding mechanism is essentially identical to the one used by osteoclasts [19,23]. However, while osteoclasts secrete acid for bone remodelling, Osedax secretes acid as part of a unique feeding mechanism that allows them to thrive on an otherwise difficult to access resource.
2. Material and methods
Female Osedax spp. (O. ‘yellow collar’, O. frankpressi, O. roseus, O. ‘orange collar’, and O. rubiplumus; ) were obtained from whale or cow bones at depths of 382 (whale), 633 m (whale), 1820 m (whale and cow) and 2893 m (whale and cow) in the Monterey Bay Canyon, California . The remotely operated submersibles Tiburon, Ventana and Doc Ricketts were used both for deploying the cow bones and for collecting all bones. Soon after reaching the surface, Osedax specimens were dissected from bones and fixed for light microscopy (3% glutaraldehyde in 0.2 mol l−1 cacodylate buffer with 10% w/v sucrose, pH 7.4), immunofluorescence (2% paraformaldehyde in 0.1 M phosphate buffer with 10% w/v sucrose, pH 7.8) or transmission electron microscopy (TEM) (3% glutaraldehyde in 0.1 mol l−1 phosphate buffer with 10% w/v sucrose, pH 7.4). After fixation, specimens were rinsed several times in buffer and stored at 4°C in buffer or 70 per cent ethanol. Some pieces of bone containing Osedax were decalcified using RDO Gold (Apex Engineering Products Corporation) and then sliced with a scalpel to show the bone/‘root’ interface. Additional specimens were flash-frozen in liquid N2 for immunoblotting.
(b) TEM and light microscopy
Specimens were post-fixed for 80 min in 1 per cent osmium tetroxide in 0.1 mol l−1 phosphate buffer with 10 per cent w/v sucrose, pH 7.4 after several buffer rinses, dehydrated in a graded ethanol series, and embedded in Spurr's epoxy resin. Semi-thin (1 μm) and ultra-thin (80 nm) sections were cut with a Leica Ultracut S microtome. Semi-thin sections were stained with toluidine blue and examined with a Leica DMR compound microscope. Ultra-thin sections were stained with uranyl acetate and lead citrate and viewed with a Philips CM100 TEM.
Polyclonal antibodies against two VHA subunits were used for immunofluorescence and immunoblots. Polyclonal antibodies against the epitope ‘AREEVPGRRGFPGY’ in the VHA B subunit were produced in rabbit. BLAST searches of various databases reveal this epitope is conserved in VHA B from all animals examined, including several annelid worms. Preliminary transcriptomic analyses demonstrate this epitope is 100 per cent conserved in VHA B from Osedax frankpressi (H. Tresguerres 2012, unpublished observation). The antibodies specifically recognize VHA B from fish, shark and mollusc tissues by Western blot, as well as ‘VHA-rich cells’ in shark gills by immunohistochemistry .
Antibodies against mouse VHA A were a kind gift from Dr Dennis Brown (Harvard University, MA, USA). These antibodies recognize the epitope ‘MQNAFRSLED’ , which is more than 80 per cent conserved from cnidarians to mammals. These antibodies specifically cross-react with VHA A from a wide range of species, including mammals , fish  and mollusc tissues (M. Tresguerres 2012, personal observation).
Antibodies against human CA2 were purchased from Rockland Inc. (Gilbertsville, PA, USA). These antibodies specifically detect an approximately 30 KDa protein by immunoblotting in mammals, fishes  and molluscs (M.T. 2012, personal observation).
Some samples were dehydrated in an increasing ethanol series, embedded in paraffin and sectioned at 5 µm. Other samples were cryosectioned at 5–10 µm after embedding in Optimal Cutting Temperature compound. Both methods yielded identical results. Sections were attached to glass slides, rehydrated and processed for immunofluorescence as previously described . After blocking non-specific binding sites (2% normal goat serum in 10 mM PBS, pH 7.4), sections were incubated overnight at 4°C with antibodies against VHA or CA2 (3 μg ml−1 in PBS). Secondary Alexa-fluor goat anti-rabbit antibodies (Life Technologies Inc.; 1 : 500 dilution) were applied for 1 h at room temperature. Nuclei were stained with Hoechst v. 33 342 (1 μg ml−1 in phosphate buffered saline (PBS), 5 min at room temperature). In between steps, sections were profusely rinsed in PBS.
Immunofluorescence was detected using an epifluorescence microscope (Zeiss AxioObserver Z1) connected to a metal halide lamp and appropriate filters. Localization of VHA in the membrane was confirmed using structured illumination (Zeiss Apotome2) and confocal microscopy (Zeiss LSM-700).
Digital images were adjusted, for brightness and contrast only, using Zeiss Axiovision software and Adobe Photoshop.
Osedax females were either dissected into ‘roots/ovisac’, and ‘trunk/palps’ and then frozen or dissected into these fractions from frozen whole specimens. These were then homogenized in 1 : 10 w/v ice-cold buffer (250 mM sucrose, 1 mM EDTA, 30 mM Tris, protease inhibitor cocktail (Sigma), pH 7.4). Debris and nuclei were removed by a low-speed centrifugation step (3000g, 10 min, 4°C); an aliquot of the supernatant was separated as ‘crude homogenate’, the reminder was centrifuged (22 000g, 30 min, 4°C) to obtain a ‘membrane-enriched fraction’ (pellet) and a ‘soluble fraction’ (supernatant). Protein concentration in each sample was quantified by the Bradford method. VHA abundance was determined in ‘crude homogenates’, CA2 abundance was determined in supernatants. Equivalent amounts of total protein (approx. 20 μg) were separated in 10 per cent polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Following blocking (5% skimmed milk in 0.5 M Tris-buffered saline with 0.1% Triton X-100 (TBS-T), 30 min at room temperature), PVDF membranes were incubated with primary antibodies against VHA A (1 : 1000), VHA B (1 : 1000) or CAII (1 : 3333) with gentle rocking at 4°C overnight. After three washes (15 min each) in TBS-T, PVDF membranes were incubated with HRP-linked goat anti-rabbit secondary antibodies (1 h, room temperature), washed (three times, 15 min). Immunolabelled protein bands were developed by incubation in Luminol solution, and visualized and quantified in a BioRad Chemidoc Imaging system.
(a) General morphology
As shown in figure 1a (also see [2,4,30]), whalebones can sustain a large Osedax population. The anterior portion of the body of female Osedax, consisting of the upper trunk and the four palps (‘gills’), is exposed to sea water (figure 1a,b). However, the trunk and palps can retract into the mucous tube secreted by the animal, and even partially into the excavated cavity in the bone, probably to avoid predators. The female Osedax lower trunk, ovisac and roots lie in a cavity excavated in the bone (figure 1b). The roots may branch out to create an extensive network inside the bone, although the shape varies from female to female, such that there may be bulbous lobes extending from the ovisac or thin elongate roots (figure 1c). Roots comprise epithelial cells, muscle cells, bacteriocytes containing symbiotic bacteria, non-symbiotic cells, and a network of blood vessels and capillaries. The green-coloured tissue corresponds to epidermis and bacteriocytes [1,15]. In the ovisac and roots, the major dorsal and ventral blood vessels of the trunk branch into several larger vessels, some of which reach the tip of the roots (figure 1d). Larger blood vessels and epithelial cells are distinguishable at relatively low magnification (figure 1b,d). A sheath of material, which is similar to and continuous with the external mucus tube, covers the ovisac and much of the roots (figure 1b,c). The Osedax roots are lined with a typical ion-transporting epithelium consisting of apical microvilli and abundant mitochondria, as well as numerous electron-dense and electron-lucent vacuoles (figure 1e,f). This matches previous ultrastructural descriptions for other Osedax species [14,15].
(b) VHA abundance and localization
The anti-VHA B antibodies specifically immunodetected VHA B in Osedax tissues, based on the size of the band (approx. 54 KDa) and on lack of bands in controls (omission of primary antibodies and preabsorption of anti-VHA B antibodies with antigen peptide). Quantitative Western blots demonstrated VHA B is 6.2-fold more abundant in Osedax ‘roots/ovisac’ compared with ‘trunk/palps’ (p < 0.05, paired t-test, n = 5; figure 2a). Anti-VHA A antibodies yielded similar results (not shown).
Immunofluorescence revealed VHA B is abundantly present in epithelial cells of the roots and lower trunk, both in putative cytoplasmic vesicles and in the cell apical membrane (figure 2c–f). There is an abrupt transition between cells expressing abundant VHA in the lower trunk region and cells without detectable VHA immunosignal in the upper trunk (figure 2f); this region presumably delimits the epithelial cells that actively secrete acid. VHA B immunostaining was also detected in Osedax palp cells. However, since the signal in palps was much dimmer compared with roots and ovisac much longer exposure times were required in order to visualize the VHA immunofluorescent signal. VHA B intracellular localization in palps is also different as it is absent from the apical membrane and seems preferentially located in cytoplasmic vesicles and in the basolateral (‘blood-side’) membrane of cells (figure 2g). Anti-VHA A antibodies yielded identical results (not shown).
(c) CA abundance and localization
The anti-human CA2 antibodies specifically immunodetected an approximately 35 KDa band in Osedax tissues (figure 3a) which was not present in preabsorption controls. Although roots had higher abundance of CA2-like protein compared with the main trunk and palps, no statistically significant differences were found between body regions.
Immunolabelling of Osedax sections matched the immunoblot results, as CA2 signal was present throughout the worm (figure 3b). However, some areas of the roots, blood vessel cells, muscle cells and palp cells (figure 3c–f) demonstrated more intense staining. CA2 intracellular localization was generally cytoplasmic, although it appeared stronger in the apical region of root cells (figure 3c) and in apical and basolateral regions of palp cells (figure 3f).
Osedax feeding is a two-step challenge: bone demineralization and nutrient absorption. Confirming the previous hypothesis that Osedax dissolves the bone by secreting acid [14–16], we found that VHA is abundantly present at the root epithelium, thus solving the first step of the supposed feeding mechanism.
Female Osedax are clearly specialized for generating acid and drilling into bone (figure 4). We propose the mechanism of bone demineralization involves: (i) transport of oxygen from sea water to the roots via the extensive vascular system; (ii) production of CO2 by aerobic metabolism in root epithelial cells; (iii) hydration of CO2 into H+ and HCO3− by intracellular CA; (iv) secretion of H+ via VHA across the apical membrane directly onto the bone surface, and absorption of HCO3− into blood by as yet unknown mechanisms; (v) transport of HCO3− in blood to the palps; and (vi) excretion to sea water by cells expressing basolateral VHA and CA.
The most likely source of acid in Osedax root epithelial cells is hydration of metabolic CO2 by CA. This would result in both H+ and HCO3−, which have to be differentially transported across apical and basolateral membranes, respectively. The acid is secreted by VHAs directly onto the bone surface to dissolve bone hydroxyapatite, thus exposing trapped collagen. Osedax root epithelial cells have abundant mitochondria and extensive apical microvilli. These cells also express high levels of VHA throughout the cell and directly in the apical membrane, as well as intracellular CA2-like proteins. These features are typical of other cells specialized for acid secretion, such as mammalian kidney α-intercalated cells, clear cells in the epididymis, osteoclasts and mitochondrion-rich cells in turtle and amphibian urinary bladder, frog skin and insect salivary gland (reviewed in ).
Hydration of CO2 in epithelial cells also produces equimolar amounts of HCO3−, which must be removed from the epidermal cells to maintain an elevated CO2 hydration rate and avoid acid/base homeostasis disturbances. Because secretion of HCO3− across the apical membrane into the bone cavity would titrate the secreted H+, thus slowing down bone dissolution, we propose that HCO3− must instead be secreted across the basolateral membrane. After making their way across muscle layers and bacteriocytes, the absorbed HCO3− ions must reach the coelom and blood to eventually be secreted to the surrounding sea water across the palps. The network of blood capillaries and larger vessels (figure 1b–d) ensures efficient removal of HCO3− as well as oxygen supply. The large amounts of cytoplasmic CA2-like present throughout the worm may function (together with extracellular CAs and membrane ion transporters) to bring CO2/HCO3− to the palps. In a mirror image of the ion-transporting processes that take place in root epithelial cells, the branchial cellular mechanism for HCO3− secretion seems to rely on cytoplasmic CA, and basolateral (instead of apical) VHAs. This mechanism resembles other specialized HCO3−-secreting, H+-absorbing cells such as gill cells from hagfish [31,32], shark [26,33–35] and trout , and β-intercalated cells from mammalian kidney [21,37,38].
Osedax roots are often partially covered by a sheath that is continuous with the tube that protects the worm's trunk. For Osedax mucofloris this has been reported as consisting of mucus containing acidic mucopolysaccharides . This sheath was also noted here in our examination of some Osedax females, though it did not appear to be continuous all the way to the end of the roots (figure 1b). While it has been speculated that the mucous sheath helps dissolve the bone by acting as a chelating agent , it could have at least three other, not mutually exclusive, functions. First, it could prevent damage of Osedax skin by soaking up excess acid. Second, it could act to direct acid to the site of most active erosion. Third, it could help prevent regions of the bone matrix around the worm from dissolving, thus stabilizing the position of the worm in the bone. In any case, figure 1b–d shows the Osedax epidermis can be in direct contact with the bone (except for a thin area analogous to the resorption lacuna of osteoclasts [19,23]), demonstrating direct secretion of H+ onto the bone can occur.
A well-developed respiratory system  and high capacity for O2/CO2/HCO3− transport in fluids are probable preadaptations for the Osedax unique ‘dissolving and feeding’ mechanism, as these features are also present in other siboglinid worms (reviewed in ). For example, Riftia pachyptila uses VHAs and CAs in the palps to secrete H+ and absorb CO2 (in addition to O2 and H2S) from hydrothermal vent fluids, followed by transport of HCO3− in blood and coelomic fluid to the trophosome where chemoautotrophic symbiotic bacteria generate organic compounds [41–45]. In fact, [HCO3−] in Riftia blood and coelomic fluid can reach over 30 mM . While Vestimentifera that live at hydrothermal vents, such as Riftia , take up sulfide via their anterior plume, seep-dwelling taxa such as Lamellibrachia use a single posterior extension of the tube (called a ‘root’) to reach deep into the sediment to take up sulfide from the interstitial water [47,48]. Similarly, the open posterior end of the tubes of Frenulata allows them to access sulfide from the interstitial water of the anoxic sediment their posterior body is buried in [49,50]. Compared with vent vestimentiferans such as Riftia, the H+ and HCO3− fluxes are reversed in Osedax, however they may be similar in Osedax, seep vestimentiferans such as Lamellibrachia and possibly in frenulates. Altogether, this evidence suggests that our proposed mechanism for acid secretion across the roots has specialized from pre-existing mechanisms that relied on VHA and CA for other physiological functions.
Lamellibrachia release the majority of SO42− and H+, both byproducts of the metabolism of their symbiotic bacteria, into the sediment via their posterior trunk and opisthosoma that is contained within the ‘root’. The sulfate can be recycled by being reduced to sulfide again, thus sustaining large tubeworm aggregations for long periods. The release of H+ may enhance sulfide production and help limit carbonate precipitation onto their tubes [51–53]. Both SO42− and H+ excretion across the Lamellibrachia root seems to follow passive facilitated diffusion , but the specific ion transporters still need to be identified. Additionally, active H+ secretion would possibly allow Lamellibrachia to initially penetrate through the carbonate rock, just as Osedax dissolve the mineral part of the bone. Clearly, the ‘roots’ in Lamellibrachia and Osedax serve a similar function in nutrient access and supply. However, whether or not VHA is involved in proton excretion in the Lamellibrachia root region, as in Osedax roots and whether or not Lamellibrachia and Osedax roots correspond to homologous body regions remains to be determined.
We are currently conducting experiments to elucidate the second step of the proposed feeding mechanism, which must start by absorption of bone-derived nutrients across the root epithelium. Osedax root tissue has robust collagenolytic activity and high concentration of Zn2+ (the metal cofactor for many collagenases) , suggesting Osedax secretes collagenases to degrade bone collagen at the root-bone interface followed by absorption of smaller organic molecules. However, it has not yet been determined whether the collagenolytic activity is derived from the host, the endosymbionts or both. The absorption of nutrients could take place via at least two mechanisms: amino acid transporters (as described across skin and gill epithelia of many marine invertebrates [54–57] and hagfish ), and transcytosis of macromolecules (as in osteoclasts ). Similar to previous reports [14,15], our TEM images show abundant vesicles in the Osedax root epithelium; however, we cannot establish whether they are transcytotic.
As host worm tissues are enriched in fatty acids with a distinct bacterial signature, Goffredi et al.  suggested at least part of the nutrients are transported to the symbionts, which eventually transfer metabolites back to the host. Also, Katz et al.  found evidence for bacteriocyte degradation in the ovisac region compared with the more distal roots, suggesting the worm eventually digests bacteriocytes for nutrition. However, other researchers have doubted that Osedax females host enough endosymbiotic bacteria to sustain all of their metabolic needs, though they did not provide an explanation for how Osedax might otherwise acquire nutrition . We suggest that if Osedax females do not fully rely on bacterial symbionts, further nutrition could be acquired by direct absorption of bone amino acids, as described earlier, followed by digestion without the intermediate bacterial step.
Based on the worldwide distribution of Osedax and its utilization of mammal, bird, fish and reptile bones as substrates, they are probably substantial contributors to the recycling of vertebrate organic matter in oceanic ecosystems. In a remarkable case of convergent evolution, the main features of Osedax ‘dissolving and feeding’ mechanism resemble the mechanism for bone resorption in mammalian osteoclasts [18,19]. However, while osteoclasts are single (though multinucleate) cells present in internal fluids that regulate bone remodelling and blood acid/base homeostasis, the Osedax root system has an epithelium specialized for feeding. Understanding the process of bone demineralization in Osedax is only the first step in understanding this unusual feeding mode. Investigation of nutrient uptake and transport, as well as the nutritional relationships between the host and endosymbiotic bacteria in this symbiosis, will expand our knowledge on novel modes of nutrition.
This research was supported by SIO funds to G.W.R. and M.T. S.K. was supported by an Erwin Schrödinger postdoctoral fellowship (Austrian Science Fund project no. J3149-B17). Thanks to Dr Tufan Gokirmak (SIO) for his assistance with confocal microscopy. Special thanks to Dr Bob Vrijenhoek (Monterey Bay Aquarium and Research Institute) for inviting G.W.R. on many cruises to collect Osedax, and to Dr Shana Goffredi (Occidental College) for supplying some frozen specimens.
- Received March 11, 2013.
- Accepted April 10, 2013.
- © 2013 The Author(s) Published by the Royal Society. All rights reserved.