KEIKO ENDOW and SUGURU OHTA Ocean Research Institute, University of Tokyo , Minamidal, Nakano-ku, Tokyo 164, Japan

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1 Nihon Biseibutsu Seitai Gakkaiho (Bulletin of Japanese Society of Microbial Ecology) Vol. 3, No. 2, 73-82, 1989 The Symbiotic Relationship between Bacteria and a Mesogastropod Snail, Alviniconcha hessleri, collected from Hydrothermal Vents of the Mariana Back-Arc Basin KEIKO ENDOW and SUGURU OHTA Ocean Research Institute, University of Tokyo , Minamidal, Nakano-ku, Tokyo 164, Japan Abstract: Additional, intracytoplasmic membrane-stacked bacterial symbionts were found to colonize the same bacteriocytes of a hydrothermal vent snail, Alviniconcha hessleri, along with previously found slender rod-shaped symbionts. These membrane-stacked bacteria (MSB) were observed only in a part of the bacteriocytes in gill sections examined. Electron microscopy revealed that the bacteriocytes of A. hessleri possessed phagocytic activity. The phagocytic incorporation of MSB by bacteriocytes, in addition to uneven distribution of these bacteria among gill filaments, strongly suggest that MSB were acquired by the bacteriocyte as guests from the external environment. Electron micrographs revealed an intermediate phase of intracellular and extracellular existence of both types of bacteria. This mode of occurrence can be explained by the compromise between the avoidance of self defense mechanisms of host cell and keeping intimate contact with their host. Phage-like particles (PLPs) were found in the slender rod-shaped symbionts of A. hessleri. This is the first observation of PLPs inside symbiotic chemoautotrophic bacteria. Key words: Symbiosis, chemoautotrophic bacteria, mollusc, hydrothermal vent Introduction A variety of microbes have found their habitats in the cells of other organisms. Endosymbiotic associations of bacteria with eukaryotic hosts are widespread in nature. Recently, chemoautotrophic and methylotrophic bacteria have been added to the collection of bacterial endosymbionts (Felbeck, 1981; Cavanaugh et al., 1981, 1987). The entry of nonpathogenic or nonparasitic bacteria into host cells largely relies on the phagocytic ability of the cells (Smith, 1979). This is the very reason why the great majority of hosts are phagotrophic feeders. In unicellular hosts, symbionts once established in a cell, can be rather easily transmitted to daughter cells through binary fission. In multicellular organisms, however, transmission of endosymbionts through gametes is very rare and, if present, maternal (Taylor, 1983). In mutualistic symbioses, multicellular hosts have evolved other effective means for transmission of symbionts from generation to generation (Buchner, 1965). In the symbioses of chemosynthetic bacteria with marine invertebrates, the situation appears to be common (Cavanaugh et al., 1981; Giere and Langheld, 1987; Gustafson and Reid, 1988). Besides the vertical (generation to generation) or horizontal (individual to individual) transmission of symbionts, acquirement of microbes from an environmental stock is also possible. De Burgh and Singla (1984) first found phagocytic activity in the gill epithelial cells of an exosymbiont-bearing hydrothermal vent limpet from the Juan de Fuca Ridge. Southward (1986) reported the phagocytic incorporation of exosymbiotic bacteria in the gill epithelial cells of several thyasirid bivalves. In both cases phagocytozed bacteria had rapidly undergone destruction by lysosome fusion, thus

2 74 ENDOW and OHTA stable endosymbiotic associations could not be established. A hydrothermal vent snail Alviniconcha hessleri from the Mariana Back-Arc Basin was demonstrated to harbor a kind of chemoautotrophic symbiont by preliminary transmission electron microscopic observation and enzymic studies (Stein et al., 1988). Based on further electron microscopic studies, we report here several new aspects of the symbiotic association between bacteria and a vent snail, including phagocytic incorporation of one of the bacterial symbiont by bacteriocytes and endurance (at least at present) of the symbionts 'imprisoned' in the host cells. Materials and Methods Specimens of vent snail Alviniconcha hessleri were collected with the submersible Alvin from hydrothermal vent fields at a water depth of around 3,650m during dives #1836 (April 27, 1987; 1810, 95'N, 14443, 20'E) and #1845 (May 6, 1987; 1812, 59'N, 14442, 43'E) (Hessler et al., 1988; Okutani and Ohta, 1988). Gills were dissected on board, and fixed with a mixed aldehyde fixative (0.5% paraformaldehyde, 2.0% glutaraldehyde in M cacodylate buffer at ph 7.4 containing 5.6% w/w sucrose) and stored in the first fixative at 4C for 1 month. Postfixation was performed on land with 1% osmium tetroxide in buffered sucrose Dehydration was performed in a graded ethanol series followed by propylene oxide and then embedded in Epon 812 (TAAB). Ultrathin sections were cut with a diamond knife and stained with uranyl acetate and lead citrate, and were examined with a JEOL 100CX transmission electron microscope (TEM). In all, three specimens were examined (the largest one was collected during dive #1845, and the remaining two were collected during dive #1836). Ruthenium red forms an electron dense precipitate which cannot penetrate into a diffusion barrier, therefore used for the demonstration of permeability barrier. In order to examine the internalization of symbionts, ruthenium red staining of gill tissues were performed with 30 ppm (final concentration) ruthenium red in 0.12 M buffered sodium chloride (0.067 M cacodylate buffer at ph 7.4) containing 1.67% osmium tetroxide at room temperature for 3 hours. Dehydration and embedding were performed in the same way as described above. Ultrathin sections for TEM observations were examined without electron staining. Results A low magnification electron micrograph of the gill filament of A. hessleri revealed a row of epithelial cells colonized by symbiotic slender rodshaped bacteria (RSB) (Fig. 1; see also in Stein et al., 1988). The bacteriocytes were fringed by well-developed microvilli. Many lysosome-like organelles were found in these bacteriocytes. A large part of these lysosome-like organelles were located at the basal part of the cells. Sometimes the fusion of lysosome membrane with peribacterial membrane(s) was found (Fig. 2). Besides the RSB, we found one more type of symbiont inside the bacteriocytes of the largest specimen examined (Fig 3). These newly-found symbionts, which possessed well-developed complex membrane stacks, were coccoids and/or stout rods with Gram-negative type cell walls (Fig. 4). The bacterial nature of these symbionts was apparent from: 1) the absence of internal membranebound organelles other than intracytoplasmic membrane stacks; 2) the presence of nonmembrane bound nuclear regions (Fig. 3, arrows); and 3) the possession of Gram-negative type cell walls (Fig. 4). Among the two types of symbionts, RSB were predominant. In a rough estimate, membranestacked bacteria (MSB) amounted to 10% or less of the symbiont population (counted on electron micrographs). RSB occurred in all of the bacteriocytes examined. On the other hand, MSB occupied only a part of the bacteriocytes in gill

3 The symbiotic relationship between bacteria and a mesogastropod snail 75 Fig. 1. Alviniconcha hessleri, Gill filament showing a row of bacteriocytes. bc: bacteriocyte; bs: blood space; l: lysosome-like organelle; mv: microvilli; n: nucleus. sections examined, though they always occurred along with RSB in the same cell and sometimes even coexisted in the same vacuole (Fig. 4). In rare occasions (in two gill sections), electron microscopy showed that the bacteriocytes of A. hessleri possessed phagocytic capacity (Fig. 5). We observed three bacteria phagocytozed by bacteriocytes of the vent snail. In these cases, all of the phagocytozed bacteria were intracytoplasmic membrane-stacked forms. Empty cavities suggesting exocytosis were also observed at the apices. Both types of symbionts seemed to be released, because both types of bacteria protruded into the cavities. No phagocytic incorporation of bacteria has been observed at the basal part of bacteriocytes. Both types of symbionts reproduce by transverse binary fission. Dividing forms were only rarely observed in both types of symbionts; 16 fission doublets per 404 MSB and 8 fission doublets per 427 RSB were counted on electron micrographs. Upon the calculation, we only counted the bacteria showing entire figures sectioned through the middle of the longitudinal axis of cells or at least nearly so. Statistical examination using a x2 test showed no difference between reproduction rates of both types of symbionts at the 5% significance level. On the other hand, statistical examination using two-tailed Fisher's exact probability test revealed that the reproduction rate of the MSB was higher than that of the RSB at the 5% significance level. Considerable numbers of both types of symbionts occurred in 'direct' contact with exterior by means of narrow duct(s) at the apical part of bacteriocyte (Fig. 6). In twenty out of eighty examples, more than two ducts were counted. These ducts were of some tens of nanometers in diameter as determined on electron micrographs (65nm+12nm in diameter; n=10; range 50-80

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5 The symbiotic relationship between bacteria and a mesogastropod snail 77 nm). Ultrathin sections of specimens which were stained with ruthenium red exhibited electron dense cytoplasmic membranes and microvilli (Fig. 7). In the apical part of the left cell in Fig. 7, darkly stained bacteria surrounded by darkly stained peribacterial membranes are evident (Fig. 7, arrow). Occasionally a small number of bacteria which were not darkly stained occurred in the apical part of the bacteriocyte. On the other hand, both kinds of symbionts remained unstained at the basal parts of the host cells. The bacteriocyte located lower right in Fig. 7 revealed the penetration of ruthenium red into the cell from a broken part of the cytoplasmic membrane. Considerable numbers of symbionts remained unstained in this broken cell (Fig. 7, double arrow). A number of dark phage-like particles (PLPs), polyhedral in shape of about 40 nm, occurred in the RSB (Fig. 8) residing in the bacteriocytes of two hydrothermal vent snails collected during dive #1836. Sometimes these PLPs were observed in secondary lysosome-like organelles (Fig. 9). In these cases, electron micrographs revealed that these PLPs possessed spikes. RSB housing these PLPs did not occur in a cluster but were scattered within and among bacteriocytes. In MSB, no structures resembling to phages have been found. However, electron microscopy revealed capsid-like particles (CLPs) adsorbed to the cell walls of MSB (Fig. 4, arrowheads). These CLPs did not possess spikes, and clearly differed from the PLPs inside RSB. Disseussion The bacteriocytes of A. hessleri harbored numerous Gram-negative RSB of sulfur oxidizing nature (Fig. 1; Stein et al., 1988). In addition to the RSB, we found another type of symbiont inhabiting the bacteriocytes of the same vent snail collected during dive #1845 (Figs. 3, 4, 7). These newly-found symbionts were Gram-negative coccoids or stout rods with complex intracytoplasmic membrane stacks (Figs. 3, 4). Other than cyanobacteria, complex intracytoplasmic membrane stacks are known to occur in very limited groups of bacteria, namely phototrophs, nitrifying bacteria and methylotrophs. Phototrophs were excluded, because specimens for this study were collected from a water depth of about 3,650m. The membrane stacks of the snail symbionts most resemble those of the type I methylotrophs. However, Stein did not find methane oxidizing activity in his test specimens (Stein et al., 1988). This discrepancy may imply that: 1) the intracytoplasmic membrane stacked symbionts of A. hessleri are nitrifying bacteria; 2) these bacteria are of methane oxidizing nature, but because of uneven distribution of these bacteria in gill tissue, Stein's test pieces contained only very small number of the MSB, and that the methane oxidizing activity was below the limit of detection; or 3) it is also possible that there exist no MSB at all in his test pieces. The distribution pattern of the RSB in A. hessleri is similar to those of the gill symbionts in vesicomyid and lucinid clams (Fiala-Medioni and Metivier, 1986; Distel and Felbeck, 1987). However, the distribution pattern of the MSB among gill filaments of the vent snail clearly differes from others. This unusual distribution pattern of MSB among bacteriocytes along with their possible uneven distribution among host individuals can be Figs Alviniconcha hessleri. 2. Vacuolar membranes surronding symbiotic bacteria fuse with a putative lysosome membrane. Arrows indicate the fusion of lysosome membrane with peribacterial membranes. 3. Two types of symbiont occur simultaneously in the same bacteriocyte of a vent snail. Arrows indicate non-membrane bound nuclear regions. 4. Additional symbiont with intracytoplasmic membrane stacks occurring along with slender rod-shaped bacteria in the same vacuole. Arrow indicates Gram-negative type cell membrane. Arrowheads indicate adsorbed capsid-like particles. 1: lysosome-like organelle; MSB: membrane-stacked bacteria; RSB: rod-shaped bacteria.

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7 The symbiotic relationship between bacteria and a mesogastropod snail 79 explained by acquisition of guest symbionts from the outer environment or by the multiplication of hidden symbionts. There also exists the possibility that the MSB are the older symbionts of A. hessleri, and we are looking at the elimination process of the older symbionts by newcomers. However, it is difficult to believe that this elimination process is now going way, because: 1) the multiplication rate of the RSB is very low (2%; calculated on electron micrographs), comparable or slightly less than that of MSB (4%; calculated as above), so it is unlikely that the newcomers are more vital and wilder than the older symbionts lost vitality during long intracellular life, thus overcoming the older symbionts through their high activity of multiplication; 2) since both types of symbionts coexisted in the same vacuole with no sign of deterioration, it is unlikely that the newcomers are harmful to the older symbionts by producing toxic (or inhibitory) compounds; and further 3) electron microscopy showed the phagocytic incorporation of MSB (here assumed to be older symbionts) but no phagocytic incorporation of RSB. If RSB were newcomers, the reverse should be observed. Because no phagocytic incorporation of bacteria from the basal part of bacteriocytes have been observed, it is unlikely that the symbionts migrated from other tissues and/or organs born on blood streams to enter into gill bacteriocytes to multiply. The possibility that the both types of bacteria found in A. hessleri belong to the same species and that variations in shape and structure represent different stages is also discarded, for no itermediate forms have been found in the gill sections examined and sulfur oxidizing activity has been detected in the gill tissue of A, hessleri (Stein et al., 1988). Based on the above facts, we consider that the MSB are guest symbionts coming into the bacteriocytes of A. hessleri from the exterior, being brought into the snail with water current introduced for respiration. Ruthenium red-stained sections of A, hessleri (Fig. 7) along with TEM observations of duct(s) (Fig. 6) show that both types of symbiont at the periphery of host cells live in 'direct' contact with external environment. On the other hand, most of the symbionts at the basal part of the cells seem to be fully enclosed. Unstained symbionts in the ruthenium red-penetrated cell strongly suggest the enclosure (Fig. 7). Fairly frequent fusion of putative lysosome membranes with peribacterial membranes (Fig. 2) also supports the internalization of symbionts within the host cells. In order for an endosymbiotic association to become stable, many problems must be solved by both sides of the symbiosis. Among these, it is essential for symbionts to effectively escape from lysosomal attack of host cells (Southward, 1986; Giere and Langheld, 1987). To keep away from areas of high lysosomal activity to areas of low or no digestive activity is one of the most simple way of settlements (Bannister, 1979; Giere and Langheld, 1987). Residing in the invagination pockets at the periphery of host cells may be a solution for keeping intimate relationship between them and their host, and at the same time evading the destruction by lysosomal enzymes. These bacteria inside the invagination pockets may well serve an endosymbiotic bacterial reserve of the vent snails. PLPs were found in some of the slender rodshaped symbionts of A. hessleri collected during dive #1836 (Fig. 8). It is the first observation, to our knowledge, of something like phages inside the symbiotic chemoautotrophic bacteria. Sometimes the particles were observed in secondary Figs Alviniconcha hessleri. 5. Phagocytic incorporation of intracytoplasmic membrane-stacked bacteria at the apical part of host cell. 6. Electron micrograph showing a duct connecting host cell membrane and peribacterial membrane. 7. Ruthenium red-stained gill section of A, hessleri showing the bacteria residing in the invagination pockets of host cell membranes at the periphery of the bacteriocyte. Arrow indicates darkly stained bacteria, while double arrows indicate the bacteria remained unstained inside vacuoles. MSB: membrane-stacked bacteria.

8 80 ENDOW and OHTA Figs Alviniconcha hessleri. 8. Phage-like particles inside the rod-shaped symbiont at the central part of the figure. 9. Phage-like particles occurring in a lysosome-like organelle. Arrow indicates the phage-like particle with spikes.

9 The symbiotic relationship between bacteria and a mesogastropod snail 81 lysosome-like organelles (Fig. 9). On the other hand, we found many CLPs attached to the surface of MSB, though, none of PLPs existed inside MSB. Phages or viruses cannot be regarded as symbionts. However, they may cause important, sometimes even decisive effect upon bacteria or eukaryotes. Sometimes viruses or plasmids play some unique role in symbiotic relationships. For example, root-nodule bacteria lacking their plasmid on which symbiotic genes are located cannot construct symbiotic association with their leguminous hosts (Truchet et al., 1985). Van Etten et al. (1982) suggested that viruses in zoochlorellae isolated from five sources of green hydra and protozoan Paramecium bursaria may play role in determining the acceptability of the zoochlorellae to the host. In order to clarify the nature of the PLPs, thorough investigations are needed. If these particles really were phages, analysis of such a three-level genetic system will be of special interest. Gills are known to be the site of molluscan gill chemosynthetic symbioses (Dando and Southward, 1986; Fisher and Childress, 1986; Fisher et al., 1987; Stein et al., 1988). The gill epithelial cells of some bivalves and gastropods has been shown to retain phagocytic activity not only for a restricted stage of ontogenesis but for a fairly expanded span of life (De Burgh and Singla, 1984; Southward, 1986; this paper). It is easy to see that the organisms retain phagocytic activity for a long time have good chances for acquisition of mixed population of microbes. In this respect, bivalves and/or gastropods (perhaps excluding carnivores) may offer good candidates for intracellular multiple symbiosis (=coexistence of plural endosymbionts within individual host organisms). Cavanaugh et al. (1987) reported two types of symbiont in the same bacteriocyte of the seepage mussel of the Florida Escarpment. Based on the co-occurrence of type I methylotrophic enzyme activities and type I intracytoplasmic membrane stacks of methylotrophs, they suggested that one of the mussel symbionts was a methane oxidizer. We also find two kinds of symbiont, oxidizer one is a sulfur (Stein et al., 1988) and the other possesses complex intracytoplasmic membrane stacks, in the same gill epithelial cell of the largest specimen of the hydrothermal vent snail. Whether these disymbiotic associations in which two different bacterial symbionts coexist in the same cell are maintained throughout generation(s) or not is, to date, unknown. It is not so common in nature that multicellular hosts harbor more than one kind of symbionts at the same time in the same cell. However, it would be expected that microbes with unique requirement for energy or nutrition, such as chemolithotroph, methylotrophs, etc., can possibly live together with vast range of organisms without severe competition. The vent snail may confer a good example for investigating the process of development of symbiotic association from the beginning of the establishment of an 'intracellular' (di)-symbiosis in the same cell or of the failure of establishment (di)-symbiosis. The specimens Acknowledgments of this study were kindly donated to us by Dr. Robert R. Hessler, Scripps Institution of Oceanography. We wish to thank Drs. H. Sakai and U. Simidu of Ocean Research Institute, University of Tokyo for their interest and encouragement throughout the work. References Bannister, L.H., The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms. Proc. R. Soc. Lond., B. 204, Buchner, P., Methods of transmission. In: Endosymbiosis of animals with plant microorganisms. pp lnterscience Publ., New York Cavanaugh, C.M., S.L. Gardiner, M.L. Jones, H.W. Jannasch and J.B. Waterbury, Prokaryotic cells in the hydrothermal vent tube worm Riftia pachyptila Jones: Possible chemoautotrophic of

10 82 ENDOW and OHTA symbionts. Science, 213, Cavanaugh, C.M., P.R. Levering, J.S. Maki, R. Mitchell and M.E. Lidstrom, Symbiosis of methylotrophic bacteria and deep-sea mussels. Nature, 325, Dando, P.R, and A.J. Southward, Chemoautotrophy in bivalve molluscs of the genus Thyasira. J. mar, biol. Ass. U.K., 66, De Burgh, M.E. and C.L. Singla, Bacterial colonization and endocytosis on the gill of a new limpet species from a hydrothermal vent. Mar. Biol., 84, 1-6. Distel, D.L. and H. Felbeck, Endosymbiosis in the lucinid clams Lucinoma aequizonata, Lucinoma annulata and Lucina floridana: a reexamination of the functional morphology of the gills as bacteria-bearing organs. Mar. Biol., 96, Felbeck, H., Chemoautotrophic potential of the hydrothermal vent tube worm, Riftia pachyptila Jones (Vestimentifera). Science, 213, Fiala-Medioni, A. and C. Metivier, Ultrastructure of the gill of the hydrothermal vent bivalve Calyptogena magn{fica, with a discussion of its nutrition. Mar. Biol., 90, Fisher, C.R. and J.J. Childress, 1986, Translocation of fixed carbon from symbiotic bacteria to host tissues in the gutless bivalve Solemya reidi. Mar. Biol., 93, Fisher, C.R., J.J. Childress, R.S. Oremland and R.R. Bidigare, The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels. Mar. Biol., 96, Giere, O. and C. Langheld, Structural organization, transfer and biological fate of endosymbiotic bacteria in gutless oligochaetes. Mar. Biol., 93, Gustafson, R.G. and R.G.B. Reid, Association of bacteria with larvae of the gutless protobranch bivalve Solemya reidi (Cryptodonta Solemyidae). Mar. Biol., 97, Hessler, R.,P. Lonsdale and J. Hawkins, Patterns on the ocean floor. New Scientist, 117, Okutani, T. and S. Ohta, A new gastropod mollusk associated with hydrothermal vents in the Mariana Back-Arc Basin, Western Pacific. Venus (Jap, Jour. Malac.), 47, 1-9. Smith, D.C., From extracellular to intracellular: the establishment of a symbiosis. Proc. R. Soc. Lond., B. 204, Southward, E.C., Gill symbionts in thyasirids and other bivalve molluscs. J. mar. biol. Ass. U. K., 66, Stein, J.L., S.C. Cary, R.R. Hessler, S. Ohta, R.D, Vetter, J.J. Childress and H. Felbeck, Chemoautotrophic symbiosis in a hydrothermal vent gastropod. Biol. Bull., 174, Taylor, F.J.R., 1983, Some eco-evolutionary aspects of intracellular symbiosis. In: Intracellular symbiosis (edited by K.W. Jeon) pp Academic Press, New York Truchet, G., F. Debelle, J. Vasse, B. Terzaghi, A.-M. Garnerone, C. Rosenberg, J. Batut, F. Maillet and J. Denarie, Identification of a Rhizobium meliloti psym2011 region controlling the host specificity of root hair curling and nodulation. J. Bacteriol., 164, Van Etten, J.L., R.H. Meints, D. Kuczmarski, D.E. Burbank and K. Lee, Viruses of symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis. Proc. Natl. Acad. Sci. USA, 79, (Received October 28, 1988-Accepted December 25, 1988)