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Long-term cultivation of the deep-sea clam Calyptogena okutanii: Changes in the abundance of chemoautotrophic symbiont, elemental sulfur, and mucus.

Abstract. Survival of deep-sea Calyptogena clams depends on organic carbon produced by symbiotic, sulfur-oxidizing, autotrophic bacteria present in the epithelial cells of the gill. To understand the mechanism underlying this symbiosis, the development of a long-term cultivation system is essential. We cultivated specimens of Calyptogena okutanii in an artificial chemosynthetic aquarium with a hydrogen sulfide ([H.sub.2]S) supply system provided by the sulfate reduction of dog food buried in the sediment. We studied morphological and histochemical changes in the clams' gills by immunohistochemical and energy-dispersive X-ray analyses. The freshly collected clams contained a high amount of elemental sulfur in the gill epithelial cells, as well as densely packed symbiotic bacteria. Neither elemental sulfur nor symbiotic bacteria was detected in any other organs except the ovaries, where symbiotic bacteria, but not sulfur, was detected. The longest survival of an individual clam in this aquarium was 151 days. In the 3 clams dissected on Days 57 and 91 of the experiment, no elemental sulfur was detected in the gills. The symbiotic bacteria content had significantly decreased by Day 57, and was absent by Day 91. For comparison, we also studied the deep-sea mussel Bathymodiolus septemdierum, which harbors a phylogenetically close, sulfur-oxidizing, symbiotic bacterium with similar sulfur oxidation pathways. Sulfur particles were not detected, even in the gills of the freshly collected mussels. We discuss the importance of the proportion of available [H.sub.2]S and oxygen to the bivalves for elemental sulfur accumulation. Storage of nontoxic elemental sulfur, an energy source, seems to be an adaptive strategy of C. okutanii.

Introduction

In deep-sea hydrothermal vents and seeps, symbiotic associations between invertebrates and chemoautotrophic bacteria are common (Cavanaugh et al., 1981; Felbeck et al., 1981). Deep-sea clams belonging to the genus Calyptogena are the dominant members of various deep-sea chemosynthetic communities, and harbor intracellular, symbiotic, and thioautotrophic (i.e., sulfur (S)-oxidizing) bacteria in the gill (Boss and Turner, 1980; Cavanaugh, 1983). This symbiosis is strict and essential for the host clam, and no Calyptogena clam lacking the symbiont has been reported. Calyptogena gills are large, each consisting of two pairs of plate-like demibranches that comprise many rows of filaments. Densely packed symbiotic bacteria are found in the epithelial cells of the inner zone of the gill filaments that are called bacteriocytes (Boss and Turner, 1980; FialaMedioni and Metivier, 1986; Morton, 1986). The bacteria are thought to be transmitted vertically through the female germ line (Endow and Ohta, 1990; Cary and Giovannoni, 1993).

We previously showed the distribution of symbiotic bacteria in the bacteriocytes of Calyptogena okutanii Kojima and Ohta 1997, using immunohistochemical staining with anti-Escherichia coli GroEL (anti-E. coli GroEL) polyclonal antibody and in situ hybridization (Hongo et ah, 2013; Nakamura et al., 2013). Highly viscoelastic, mucuscontaining N-acetyl-D-glucosamine (GlcNac) fills the interfilament spaces of the inner zone (Nakamura et al., 2013). In contrast to the large size of the gill, the gill ciliary groove, labial palps, and digestive tract are reduced in size, suggesting that the survival of C. okutanii largely depends on the nutrients derived from the bacterial symbionts (Boss and Turner, 1980; Fiala-Medioni and Metivier, 1986; Morton, 1986). It is thought that hydrogen sulfide ([H.sub.2]S) provides the main energy source for the S-oxidizing bacteria, and thiosulfate generated by the partial oxidation of [H.sub.2]S also functions as a subsidiary energy source (Childress et al., 1991, 1993). The clam is believed to take up [H.sub.2]S through its vascularized foot, which is inserted into the sediments or cracks around hydrothermal vents and transports [H.sub.2]S internally via the specialized S-binding zinc protein (Znprotein) present in its blood (Arp et al., 1984; Fisher, 1990; Childress et al., 1993; Zal et al., 2000). Oxygen ([O.sub.2]) is taken up from the surrounding water via the gills (Fisher et al., 1988).

Recent genome analyses of the symbiotic bacteria of the Calyptogena clams, Calyptogena okutanii and Calyptogena magnifica Boss & Turner, 1980, have shown that each is composed of a single species: Candidatus (Ca.) Vesicomyosocius okutanii strain HA (Vok) and Ca. Ruthia magnifica strain Cm (Rma), respectively (Kuwahara er al., 2007; Newton et al., 2007). While both symbionts have reduced genomes, each possesses gene sets for chemolithoautotrophic metabolisms such as S oxidation and carbon dioxide fixation (Kuwahara et al., 2007; Newton et al., 2007). Regarding S oxidation, both Vok and Rma contain genes for two pathways, the S-oxidizing multi-enzyme system (SOX) and the SOX-independent system (Kuwahara et al., 2007; Newton et al., 2007; Harada et al., 2009). Expression of the genes involved in the two pathways has been confirmed in Vok (Harada et al., 2009).

In contrast to molecular data on the symbiotic bacteria, very little is known about the actual S-oxidation process, the translocation of S compounds in host-symbiont interaction, and the regulation of S metabolism in different environments--essential information for an understanding of thioautotrophic symbiosis in the deep sea. To study these biological features, long-term cultivation of Calyptogena clams is necessary, but cultivation is extremely difficult. We previously carried out long-term cultivation of deep-sea animals, including C. okutanii in an artificial chemosynthetic aquarium, using dog food (Miyake et al., 2006, 2012). In that system, [H.sub.2]S was supplied via sulfate reduction of the decomposing dog food buried beneath the aquarium sediment. In the present study, we used the same artificial chemosynthetic aquarium system to cultivate and observe the survival of C. okutanii. We also looked for changes in the morphological and histochemical characteristics of their organs and apparent S content and symbiotic bacterial abundance in their gills. We provide what may be the first report of changes in S content and bacterial symbiont abundance during long-term cultivation of Calyptogena clams.

In addition, we studied the deep-sea mussel Bathymodiolus septemdierum Hashimoto and Okutani 1994, whose epithelial gill cells also harbor thioautotrophic bacteria, which are closely related to those of Calyptogena clams (Nishijima et al., 2010; Ikuta et al., 2016). From the observed data, we discuss the S-oxidation pathways as they relate to environmental changes and compare survival strategies in these two deep-sea bivalve species.

Materials and Methods

Sampling and cultivation of clams and mussels

Calyptogena clams were collected at a seep site off Hatsushima Island in Sagami Bay, Japan (35[degrees]00.9' N, 139[degrees] 13.4' E; depth: 852-854 m), using the remotely operated underwater vehicle (ROV), Hyper Dolphin, during two cruises aboard the R/V Natsushima (NT11-09, June 15-26, 2011; and NT13-07, April 2-13, 2013). At this site, two very morphologically similar sibling species, Calyptogena okutanii and Calyptogena soyoae Okutani, 1957, form a mixed colony (Harada et al., 2009). Because of the species' morphological similarity, identification can be made only by partial sequencing of the cytochrome c oxidase subunit 1 gene (col) after dissection or death (Harada et al., 2009). Some clams were dissected aboard the research vessel soon after sampling; only those clams identified as Calyptogena okutanii were used as freshly collected controls. Unidentified specimens (n = 95) of C. okutanii and/or C. soyoae that were obtained during the NT13-07 cruise were kept in a large tank (288 x 89 x 110 cm) (width x length x depth) with a muddy sediment area for generation of hydrogen sulfide ([H.sub.2]S) (59.5 x 88 x 32 cm) (width x length x depth). The tank was filled with 2.8 tonne of seawater, maintained at 4 [degrees]C with a pH of 7.4, and exhibited to the public at Enoshima Aquarium, Kanagawa, Japan (see fig. 8 of Miyake et al., 2012). Approximately 20 kg of dog food (Bitawan, Japan Pet Food Co., Tokyo, Japan) was buried in the muddy sediment area at a depth of 5-15 cm. Two clams (E01: shell length 5.56 cm, and E02: shell length 10.15 cm) were dissected on Day 57; a third clam (E03: shell length 8.2 cm) was dissected on Day 91. All three clams were identified as Calyptogena okutanii. Biological changes in the gills of these three clams were examined in comparison to the freshly sampled clams, using the methods described below. Unfortunately, the clam that survived the longest (151 days) could not be used for analysis due to its loss of freshness when discovered.

Specimens of the mussel species Bathymodiolus septemdierum were collected in Myojin Knoll, Ogasawara (32[degrees]06.3' N, 139[degrees]52.0' E; depth: 1342 m) during the June 2011 cruise aboard the R/V Natsushima (NT11-09). The mussels were dissected aboard the research vessel soon after collection.

Aquarium chemistry

Dissolved oxygen (DO) in seawater in the artificial chemosynthetic aquarium was measured using a Galvanic cell DO sensor (ID-150; Iijima Electric Co., Ltd., Gamagori, Japan). The concentration of [H.sub.2]S in the seawater and sediment was measured by the voltammetry method (Luther et al., 2001a). A potentiostat system (HZ-3000; HokutoDenko Corp., Atsugi, Japan) and a working, 35-cm-long microelectrode, supported by a glass tube 4 mm in diameter, were used for voltammetry measurement and analyses. Measurements were carried out every 5 cm, from 0 (the surface) to a depth of 30 cm, at 6 fixed points in the aquarium every 3 or 4 days for 43 days (between Days 55 and 97).

Immunohistological analysis

Following clam dissection, the internal organs were photographed, and the gills and ovaries were then fixed with 4% paraformaldehyde in seawater. The fixed gills and ovaries were either frozen-embedded in an optimal cutting temperature compound in liquid nitrogen and stored at -80 [degrees]C or paraffin-embedded after dehydration. Thin, 4-[micro]m-fhick sections were prepared. Conventional hematoxylin and eosin (HE) staining was performed on the paraffin-embedded sections. Immunostaining was performed on both the frozen-embedded and paraffin-embedded sections. To detect symbiotic bacteria, the anti-E. coli GroEL rabbit-polyclonal-antibody (Sigma-Aldrich Co., St. Louis, MO) and anti-rabbit-immunoglobulin goat antibody conjugated with Alexa Fluor 594 (Life Technologies Co., Bedford, MA) were used as the primary and secondary antibodies, respectively. DNA was stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich Co.). The lectin, wheat germ agglutinin (WGA), which was conjugated with Alexa Fluor 488 (Life Technologies Co.), was used for staining of mucus and some epithelial cells in the gills. A microscope equipped with phase-contrast and fluorescence optics (BZ-9000; Keyence Corp., Osaka, Japan) and a Nikon Optiphoto microscope (Nikon, Tokyo, Japan) were used for observation. Some fluorescence images were also made, using the haze reduction function of a Keyence fluorescence microscope to eliminate out-of-focus fluorescence blurring.

Energy-dispersive X-ray analysis (EDX)

To detect sulfur (S) in the gill, frozen-embedded or paraffin-embedded sections 8-[micro]m thick were collected on glass slides. After they were dried, these samples were coated with carbon. Morphological observations and mapping of the S element were conducted using a field emission scanning electron microscope (SEM) (JSM-6700F; JEOL, Ltd., Tokyo, Japan) equipped with an EDX system (JED 2300; JEOL, Ltd.) under an accelerating voltage of 15 kV, 0.55 nA.

Results

Aquarium chemistry

Throughout the cultivation period, the concentration of dissolved oxygen (DO) in the seawater of our artificial chemosynthetic aquarium was approximately 310 [micro]mol [1.sup.-1]. The seawater concentration of [H.sub.2]S was less than 0.37 [micro]mol [1.sup.-1] (i.e., below the detection limit). In the sediment at a depth of 15-30 cm, [H.sub.2]S was undetectable to 347 [micro]mol [l.sup.-1] in concentration (when undetectable, the concentration was defined as 0; the average ([+ or -] SD) concentration was 31 [+ or -]61 [micro]mol [1.sup.-1]).

Survival rates

In the cultivation of 95 unidentified Calyptogena okutanii and/or Calyptogena soyoae in the artificial chemosynthetic aquarium, the longest survival time was 151 days. Survival rates of the clams on Days 57 and 91 were approximately 47% and 31%, respectively.

Comparison of abundance of the symbiotic bacteria in the gills of freshly sampled and long-term cultivated Calyptogena okutanii

The gills of freshly collected C. okutanii specimens were thick and whitish-yellow (Fig. 1 A, B). Each gill was composed of two pairs of V-shaped, plate-like demibranches, known as the inner and outer demibranches (Fig. 1 A, B), which contained many sheet-like gill filaments. In a transverse section of the gill filament, the inner zone between the frontal zones was mainly occupied by bacteriocytes harboring many symbiotic bacteria. The frontal zones contained no bacteriocytes, but did contain asymbiotic, ciliated cells (Fig. 1 C, D, I-N). The gills of the 3 clams, which were dissected on Days 57 (E01, E02) and 91 (E03), were all very thin and reddish-brown in color (Fig. 1 E). The gill of Clam E01 looked thinner than that of Clam E02, and the gill of Clam E03 was clearly the thinnest of the three. Histological examination confirmed that the gill epithelial cells were thinner than those of the freshly collected clams (Fig. 1 D, F-H).

Immunostaining with anti-E. coli GroEL antibody of the bacteriocytes in the gills of the freshly collected clams revealed high densities of symbiotic bacteria (Fig. 1 I-N). In the frozen sections, many black particles were observed in the inner zone (Fig. 1 I, K), and seemed to be associated with symbiotic bacteria in the bacteriocytes (Fig. 1 I-L). However, the black particles were not found in the paraffinembedded sections, although many symbiotic bacteria were observed there (Fig. 1 M, N). In the three long-term cultivated clams, black particles were not seen, even in the frozen gill sections (Fig. 1 O, Q, S). Anti-E. coli GroEL antibody signals were weakly detected in the gill sections of Clams E01 and E02 (Fig. 1 P, R), and almost no signal was detected in the gill section of Clam E03 (Fig. 1 T).

Examination of sulfur in freshly collected, long-term cultivated Calyptogena okutanii and Bathymodiolus septemdierum

To determine the composition of the black particles, which were seen only in the frozen gill sections of the freshly collected clams, energy-dispersive X-ray (EDX) analysis was performed. High sulfur (S) content was detected in most cells of the inner zone in the frozen sections (Fig. 2 A-D), but was not observed in the paraffin-embedded sections (data not shown). EDX analysis showed no S in the frozen sections from the three long-term cultivated clams (Fig. 2 E-J).

In the ovarian follicular cells of the freshly collected clams, clear signals of symbiotic bacteria were detected by anti-E. coli GroEL antibody staining (Fig. 2 K, L); however, no signal of S was observed (Fig. 2 M, N). No signals of symbiotic bacteria or S were detected in the other organs of C. okutanii (data not shown).

The gills of freshly collected B. septemdierum specimens were brown (Fig. 2 O), and the frozen gill sections contained no black particles (Fig. 2 P). Symbiotic bacteria signals detected by staining with DAPI and anti-E. coli GroEL antibody were weak compared to those signals for C. okutanii (Fig. 2 Q, R). Sulfur was not detectable by EDX analysis in these samples (Fig. 2 S, T).

Comparison of mucus and epithelial cells by wheat germ agglutinin (WGA) staining in the gills of freshly sampled, long-term cultivated Calyptogena okutanii

To examine changes in mucus in the gills, we used a lectin, WGA, which binds to N-acetyl-D-glucosamine (GlcNac). WGA-binding mucus was abundant in the interfilament spaces of the gills in the inner zone of the freshly sampled clams (Fig. 3 A, B). A few interspersed WGA-positive cells were observed (Fig. 3 B). These were asymbiotic epithelial cells; WGA seemed to bind to their intracellular granules. During cultivation, WGA-stained mucus decreased and its appearance seemed to change (Fig. 3 C-F), although its localization in the interfilamental space of the gill's inner zone was similar to that of the freshly collected clams (Fig. 3 D). By Day 57, the mucus had decreased (Fig. 3 C-E) and was observed only on the surface of the gill epithelial cells that were in direct contact with seawater (Fig. 3 E). By Day 91, the mucus in Clam E03 seemed to be thinner and bubble-like in appearance (Fig. 3 F). In addition, we found an increase in WGA-positive, asymbiotic epithelial cells in the inner zone of the gills of the three long-term cultivated clams (Fig. 3 C-F).

Discussion

In the frozen sections of the freshly collected clams, the presence of symbiotic bacteria, black particles, and sulfur (S) was shown to be related (Figs. 1, 2). As elemental sulfur ([S.sup.0]) is insoluble in water but soluble in organic solvent (Hildebrand, 1929), it appeared that the black particles in the bacteriocytes of the frozen sections were made up of [S.sup.0], which was lost during dehydration and deparaffinization. We have observed thick, whitish-yellow gills in at least 200 freshly collected clams, including those in the present study. Similar black particles and anti-E. coli GroEL antibody signals have been observed in a pattern similar to that shown in Figure 1 I-L in the frozen gill sections of more than 20 freshly collected clams. These findings indicate that the naturally living, healthy clam has densely packed symbiotic cells and [S.sup.0] in the gill, which is thick and whitish-yellow.

During the long-term (five months) cultivation of Calyptogena okutanii and Calyptogena soyoae in an artificial chemosynthetic aquarium, we observed a loss of symbiotic bacteria and [S.sup.0] in the gills of C. okutanii (Figs. 1, 2). The present results strongly suggest that the high content of [S.sup.0] existing in the gill epithelial cells of freshly sampled clams was lost prior to Day 57 of cultivation. Although only three long-term cultured clams were analyzed in the present study, the drastic changes observed are thought to be caused by long cultivation in the artificial chemosynthetic aquarium. Globules of [S.sup.0] localize in the periplasmic spaces of symbiotic bacteria in three species of deep-sea chemosymbiotic bivalves: Lucinoma annulata, Stewartia floridana (previous name, Lucina floridana), and Calyptogena elongata (Vetter, 1985). Although the spatial resolution of the analysis was not high enough to determine whether [S.sup.0] was or was not inside the bacterial cells, [S.sup.0] did seem to be associated with symbiotic bacteria (Fig. 2). Because [H.sub.2]S is toxic to aerobic organisms, [S.sup.0], which is nontoxic and oxidizable, would be an advantageous form of S energy storage for this symbiotic clam-thioautotrophic bacteria system.

In the seawater above the clam colony site off Hatsushima Island (depth: 1115 m), dissolved oxygen (DO) and [H.sub.2]S concentrations were reported as 31-47 [micro]mol [1.sup.-1] and extremely low or undetectable, respectively (Hashimoto et al., 1995). Concentrations of [H.sub.2]S in the sediments were much higher, reaching 7 [micro]mol [1.sup.-1] at a depth of 9 cm (Masuzawa et al., 1993). In our artificial chemosynthetic aquarium, however, the seawater DO concentration was much higher (approximately 310 [micro]mol [1.sup.-1]). Hydrogen sulfide, which was below the detection limit in seawater and was undetectable (less than 0.37 [micro]mol [1.sup.-1] ) to 347 [[micro]mol 1.sup.-1] in the sediments, may have been in short supply when compared to the natural environment. Environmental chemical conditions, particularly concentrations of [O.sub.2], iron, and S, strongly affect the distribution of organisms in hydrothermal vents (Luther et al., 2001b). It is therefore likely that the results we obtained were influenced by the chemical composition of the aquarium system.

The changes noted in the histological and immunohistochemical features of the clam gills over the cultivation period showed a breakdown of the symbiotic relationship between host and symbiotic bacteria. If a clam that lives in a natural habitat with occasional sulfide deficiency has enough [S.sup.0] in the gill to act as a reservoir of reduced S, the numbers of symbiotic bacteria may be sustained by a balance between bacterial proliferation and degradation or loss. The Vok lacks the transporter genes to export nutrients, including amino acids, to the host cells. Host Calyptogena clams are therefore thought to obtain nutrients and extra energy by digesting Vok (Kuwahara et al., 2007). Apparent digestion of symbiotic bacteria also has been reported in Calyptogena, based on histological analysis and lysozyme activity (Fiala-Medioni et al., 1994). It may be noteworthy that the symbiont population in the gill decreases more rapidly in Bathymodiolus mussels than in Calyptogena clams (Kadar et al., 2005). In mussels, decrease of the symbiotic bacteria probably starts soon after collection, under conditions of unsuitable sulfide supply, because the mussels do not possess [S.sup.0] storage (Fig. 2T).

When [H.sub.2]S, the energy source in the environment, decreases, the S-oxidation pathway of the symbiont seems to revert to the stocked [S.sup.0] in the energy reservoir. The possible pathways of sulfide oxidation, based on genome analysis (Kuwahara et al., 2007; Newton et al., 2007; Harada et al., 2009), are schematically depicted in Figure 4. Although seven genes, soxXYZABCD, are known in the S-oxidizing multi-enzyme (SOX) system, the Vok has a SOX system lacking soxC and soxD genes (SOX-CD). In the complete SOX system, [H.sub.2]S, [S.sup.0], and thiosulfate are completely oxidized to sulfate. However, in the SOX-CD system, [S.sup.0] is not oxidized, thiosulfate is partially oxidized and divided into [S.sup.0] and sulfate, and [H.sub.2]S is oxidized to sulfate (Friedrich et al., 2001). The other SOX-independent system is the dissimilatory sulfite reductase (DSR) system containing dsr, adenosine phosphosulfate reductase (apr), adenosine triphosphate (ATP) sulfurylase (sat), and sulfide-quinone oxidoreductase (sqr) (Kuwahara et al., 2007; Newton et al., 2007). Elemental sulfur is probably generated in the clams from [H.sub.2]S by the sqr gene product and/or from thiosulfate by the SOX-CD system (Friedrich et al., 2001; Nakagawa and Takal., 2008). A previous study indicated that the symbionts of Calyptogena mainly use sulfide rather than thiosulfate as an energy source (Childress et al., 1991). The high content of [S.sup.0] in the gills of freshly sampled C. okutanii is, therefore, expected to be produced from [H.sub.2]S by the sqr gene product (Fig. 4).

Generation of [S.sup.0] might be affected by the proportion of [H.sub.2]S to [O.sub.2] in the clam supply system and/or the symbiotic bacterial cells. The oxidation processes from [H.sub.2]S to sulfate (A) and [S.sup.0] (B) are: (A) [H.sub.2]S + 2[O.sub.2] [right arrow] [H.sub.2]S[O.sub.4] + 622.3 kJ; and (B) [H.sub.2]S + 0.5[O.sub.2] [right arrow] [S.sup.0] + [H.sub.2]0 + 217.7 kJ. Reaction (A) needs two [O.sub.2] molecules for the oxidation of one molecule of [H.sub.2]S and generates higher energy, whereas reaction (B) needs only 0.5 molecule of [O.sub.2], but generates less energy. With an [O.sub.2]:[H.sub.2]S proportion higher than 2, the most efficient sulfide oxidation process would be by reaction (A). However, under conditions with higher [H.sub.2]S and lower [O.sub.2] availabilities (i.e., as in the sediments where the clam inserts its foot), [S.sup.0] would be accumulated by reaction (B). During long-term cultivation with low concentrations of [H.sub.2]S, it is expected that reaction (A) would predominate without accumulation of [S.sup.0]. Thereafter, the stocked S would be consumed by the sqr gene product (Fig. 4).

Environmental conditions in the artificial chemosynthetic aquaculture system of our study are thought to have prolonged the life of the Calyptogena clam under atmospheric pressure by up to five months, compared to an ordinary seawater aquarium without a sulfide supply system. However, cultivation under a higher concentration of sulfide might have given the clam a longer life span.

It is interesting that [S.sup.0] content in the gills of the freshly collected Bathymodiolus septemdierum was below the detectable level (Fig. 2T), a result consistent with a previous chemical analysis (Fisher et al., 1987). Accumulation of [S.sup.0] seems to be a distinct feature of the gills of Calyptogena clams. The recently reported genome sequence of the symbiotic bacterium of Bathymodiolus septemdierum (Ikuta et al., 2016) had the same gene compositions for two S-oxidation systems as those of C. okutanii, that is, the SOX-CD system and the SOX-independent DSR system (dsr-apr-sat). The marked difference in accumulation of [S.sup.0] between the two bivalves may be explained by their biological and ecological differences rather than by the differences in their genomes. Bathymodiolus inhabits the surface of deep-sea sediments and does not insert the foot into the sediment, suggesting that it takes up [H.sub.2]S from seawater, where the concentration is low. At a B. septemdierum colony site in Myojin Knoll off Ogasawara, the observed [H.sub.2]S concentration was less than 10 [micro]mol [1.sup.-1] (K. Inoue, The University of Tokyo, pers. comm.). This value was much lower than that which was recorded in the sediment habitat of C. okutanii (Masuzawa et al., 1993). Under such conditions, reaction (A) might be the dominant process, in which case there would be no accumulation of [S.sup.0]. Pruski et al. (2002) reported the unexpected presence of [S.sup.0] in the gill of Bathymodiolus azoricus from the mid-Atlantic ridge. This finding suggests that the mode of [S.sup.0] accumulation can be altered according to environmental factors, such as the proportion of [H.sub.2]S to [O.sub.2] concentration.

Bathymodiolus septemdierum seems to prefer an environment with lower [H.sub.2]S and higher [O.sub.2] than does Calyptogena okutanii. The nutritional dependency of Bathymodiolus mussels on the symbiont may be less than that of Calyptogena clams; filter feeding has been reported in Bathymodiolus thermophilus, but not in Calyptogena clams (Page et al., 1990, 1991). Hence, C. okutanii and B. septemdierum may have different strategies for S oxidation in the deep-sea seawater-sediment boundary environments.

We observed clear signals of symbiotic bacteria in the follicular cells of the ovary of C. okutanii, but it was unclear whether the signal was detected in the oocytes (Fig. 2L). This finding is consistent with previous reports (Endow and Ohta, 1990; Cary and Giovannoni, 1993). No S signal was detected in the ovary by EDX analysis (Fig. 2N), indicating that the metabolism of S by the symbiont in the ovary of C. okutanii differs from that of the gill.

In contrast to the decrease in symbiotic bacteria, an increase in asymbiotic, WGA-positive cells was observed in the gills during cultivation of C. okutanii (Fig. 3C-F). Further experiments are necessary to clarify the relationship between the decrease of the symbiont population and the increase of WGA-positive cells.

We are uncertain of the function of the WGA-binding mucus in the gill inner zone (Fig. 3A, B). It may play a defense barrier role similar to the mucus in the human intestine, where intestinal symbiotic bacteria also exist (Lievin-Le Moal and Servin, 2006; Linden et al., 2008). Although information on mucosal immunity in bivalves is very limited, knowledge of immune defense factors in the mucus of the mantle and gill, such as lysozyme, has been accumulating (Allam and Espinosa, 2015). The histochemical changes in mucus and the asymbiotic epithelial cells shown by WGA staining may have indicated the reduced health of C. okutanii following the long-term aquarium cultivation.

In conclusion, the present study indicates that the environmental proportion of [H.sub.2]S to [O.sub.2] concentration is important in deep-sea bivalves. Further improvements in artificial chemosynthetic aquaculture systems, including higher sulfide concentrations than were used in this study, may extend culturing periods for clams and ultimately achieve cultivation across generations.

Acknowledgments

The authors thank the captains and crew members of the R/V Natsushima and the operation team of the ROV Hyper Dolphin for helping to collect deep-sea biological samples. The authors also thank Dr. K. Fujikura, Dr. Y. Nakamura (JAMSTEC), Mr. S. Nemoto, Mr. T. Takeshima, Mr. T. Sakiyama, and Ms. M. Kitajima (Enoshima Aquarium) for earnest discussion. Dr. T. Miwa of JAMSTEC and Mr. Y. Shimokawa (Yokohama City University) are acknowledged for [H.sub.2]S analysis in the artificial chemosynthetic aquarium. We are grateful to Ms. Y. Hori (the executive director of Enoshima Aquarium) and Mr. K. Hori (the president of Enoshima Aquarium) for their support of our research.

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KAZUE OHISHI (1,[dagger]), MASAHIRO YAMAMOTO (2), AKIHIRO TAME (3), CHIHO KUSAKA (4), YUKIKO NAGAI (4), MAKOTO SUGIMURA (5), KOJI INOUE (6), KATSUYUKI UEMATSU (3), TAKAO YOSHIDA (4), TETSURO IKUTA (4), TAKASHI TOYOFUKU (4), AND TADASHI MARUYAMA (1,7,*)

(1) Marine Biodiversity Research Program, and (2) Department of Subsurface Geobiological Analysis and Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan; (3) Department of Technical Services, Marine Works Japan, Ltd., Oppama higashi-cho, Yokosuka, Kanagawa 237-0063, Japan; (4) Department of Marine Biodiversity Research, JAMSTEC, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan; (5) Enoshima Aquarium, 2-19-1 Katasekaigan, Fujisawa, Kanagawa 251-0035, Japan; (6) Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba 277-8564, Japan; and (7) Research and Development Center for Marine Biosciences, JAMSTEC, 2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan

Received 7 December 2015; accepted 4 April 2016.

(*) To whom correspondence should be addressed. E-mail: tadashim@jamstec.go.jp

([dagger]) Current address: Kazue Ohishi, Tokyo Polytechnic University, 1583 Iiyama, Atsugi, 243-0297, Japan.

Abbreviations: EDX, energy-dispersive X-ray analysis; DAPI. 4',6-diamidino-2-phenylindole dihydrochloride; Gal., galactose; GlcNac, iV-acetyl-D-glucosamine; Man, mannose; S, sulfur; SOX, sulfur-oxidizing multi-enzyme system; Vok, Candidatus (Ca.) Vesicomyosocius okutanii strain HA; WGA. wheat germ agglutinin.

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Author:Ohishi, Kazue; Yamamoto, Masahiro; Tame, Akihiro; Kusaka, Chiho; Nagai, Yukiko; Sugimura, Makoto; In
Publication:The Biological Bulletin
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Date:Jun 1, 2016
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