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 Tokyo1-15-1, 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 habitatsin the cells of other organisms. Endosymbiotic

associations of bacteria with eukaryotic hosts are

widespread in nature. Recently, chemoauto-trophic 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 bacte-

ria into host cells largely relies on the phagocytic

ability of the cells (Smith, 1979). This is the veryreason why the great majority of hosts are

phagotrophic feeders. In unicellular hosts,symbionts once established in a cell, can be rathereasily 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 bacte-

ria 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 anenvironmental stock is also possible. De Burgh

and Singla (1984) first found phagocytic activity inthe gill epithelial cells of an exosymbiont-bearing

hydrothermal vent limpet from the Juan de FucaRidge. Southward (1986) reported the phagocytic

incorporation of exosymbiotic bacteria in the gillepithelial cells of several thyasirid bivalves. Inboth cases phagocytozed bacteria had rapidly

undergone destruction by lysosome fusion, thus

74 ENDOW and OHTA

stable endosymbiotic associations could not be

established.

A hydrothermal vent snail Alviniconcha hesslerifrom the Mariana Back-Arc Basin was demonstrat-

ed to harbor a kind of chemoautotrophic symbiont

by preliminary transmission electron microscopicobservation and enzymic studies (Stein et al.,

1988). Based on further electron microscopic

studies, we report here several new aspects of thesymbiotic association between bacteria and a vent

snail, including phagocytic incorporation of one of

the bacterial symbiont by bacteriocytes and endur-ance (at least at present) of the symbionts 'im-

prisoned' in the host cells.

Materials and Methods

Specimens of vent snail Alviniconcha hessleriwere 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 amixed aldehyde fixative (0.5% paraformaldehyde,

2.0% glutaraldehyde in 0.075 M cacodylate bufferat pH 7.4 containing 5.6% w/w sucrose) and stored

in the first fixative at 4C for 1 month. Post-

fixation was performed on land with 1% osmiumtetroxide in buffered sucrose Dehydration was

performed in a graded ethanol series followed bypropylene oxide and then embedded in Epon 812

(TAAB). Ultrathin sections were cut with adiamond knife and stained with uranyl acetate andlead 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 theremaining two were collected during dive #1836).

Ruthenium red forms an electron dense precipi-

tate which cannot penetrate into a diffusion bar-rier, therefore used for the demonstration of perme-

ability barrier. In order to examine the internal-ization of symbionts, ruthenium red staining of gill

tissues were performed with 30 ppm (final concen-

tration) ruthenium red in 0.12 M buffered sodiumchloride (0.067 M cacodylate buffer at pH 7.4)

containing 1.67% osmium tetroxide at room tem-

perature for 3 hours. Dehydration and embed-ding 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 epith-elial cells colonized by symbiotic slender rod-shaped bacteria (RSB) (Fig. 1; see also in Stein et

al., 1988). The bacteriocytes were fringed bywell-developed microvilli. Many lysosome-like

organelles were found in these bacteriocytes. A

large part of these lysosome-like organelles werelocated at the basal part of the cells. Sometimes

the fusion of lysosome membrane with peribacter-

ial 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-foundsymbionts, which possessed well-developed com-

plex membrane stacks, were coccoids and/or stoutrods with Gram-negative type cell walls (Fig. 4).The bacterial nature of these symbionts was appar-

ent from: 1) the absence of internal membrane-

bound organelles other than intracytoplasmicmembrane stacks; 2) the presence of non-

membrane bound nuclear regions (Fig. 3, arrows);

and 3) the possession of Gram-negative type cellwalls (Fig. 4).

Among the two types of symbionts, RSB were

predominant. In a rough estimate, membrane-stacked bacteria (MSB) amounted to 10% or less of

the symbiont population (counted on electronmicrographs). RSB occurred in all of the bacter-iocytes examined. On the other hand, MSB oc-

cupied only a part of the bacteriocytes in gill

The symbiotic relationship between bacteria and a mesogastropod snail 75

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 intracytoplas-

mic 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 mid-

dle 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

Fig. 1. Alviniconcha hessleri, Gill filament showing a row of bacteriocytes. bc: bacteriocyte; bs: blood

space; l: lysosome-like organelle; mv: microvilli; n: nucleus.

76 ENDOW and OHTA

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 bacte-

ria which were not darkly stained occurred in the

apical part of the bacteriocyte. On the otherhand, both kinds of symbionts remained unstained

at the basal parts of the host cells. The bacter-iocyte located lower right in Fig. 7 revealed the

penetration of ruthenium red into the cell from abroken part of the cytoplasmic membrane. Con-

siderable numbers of symbionts remained un-

stained in this broken cell (Fig. 7, double arrow).A number of dark phage-like particles (PLPs),

polyhedral in shape of about 40 nm, occurred inthe RSB (Fig. 8) residing in the bacteriocytes of

two hydrothermal vent snails collected during dive

#1836. Sometimes these PLPs were observed insecondary lysosome-like organelles (Fig. 9). In

these cases, electron micrographs revealed that

these PLPs possessed spikes. RSB housing thesePLPs did not occur in a cluster but were scattered

within and among bacteriocytes.

In MSB, no structures resembling to phages havebeen 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 differedfrom the PLPs inside RSB.

Disseussion

The bacteriocytes of A. hessleri harbored numer-

ous Gram-negative RSB of sulfur oxidizing nature

(Fig. 1; Stein et al., 1988). In addition to theRSB, we found another type of symbiont inhabit-

ing the bacteriocytes of the same vent snail col-

lected during dive #1845 (Figs. 3, 4, 7). Thesenewly-found symbionts were Gram-negative coc-

coids or stout rods with complex intracytoplasmic

membrane stacks (Figs. 3, 4).

Other than cyanobacteria, complex intracyto-

plasmic membrane stacks are known to occur invery limited groups of bacteria, namely

phototrophs, nitrifying bacteria and meth-

ylotrophs. Phototrophs were excluded, becausespecimens for this study were collected from awater depth of about 3,650m. The membrane

stacks of the snail symbionts most resemble those

of the type I methylotrophs. However, Stein didnot find methane oxidizing activity in his test

specimens (Stein et al., 1988). This discrepancy

may imply that: 1) the intracytoplasmic mem-

brane stacked symbionts of A. hessleri are nitrify-ing bacteria; 2) these bacteria are of methane

oxidizing nature, but because of uneven distribu-

tion of these bacteria in gill tissue, Stein's test

pieces contained only very small number of theMSB, 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 testpieces.

The distribution pattern of the RSB in A. hess-

leri is similar to those of the gill symbionts in

vesicomyid and lucinid clams (Fiala-Medioni andMetivier, 1986; Distel and Felbeck, 1987). How-

ever, the distribution pattern of the MSB among

gill filaments of the vent snail clearly differes fromothers. This unusual distribution pattern of MSB

among bacteriocytes along with their possibleuneven distribution among host individuals can be

Figs. 2-4. 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.

78 ENDOW and OHTA

The symbiotic relationship between bacteria and a mesogastropod snail 79

explained by acquisition of guest symbionts fromthe outer environment or by the multiplication of

hidden symbionts. There also exists the possibil-

ity 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 elimina-

tion process is now going way, because: 1) themultiplication 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 aremore vital and wilder than the older symbionts lost

vitality during long intracellular life, thus over-

coming the older symbionts through their high

activity of multiplication; 2) since both types ofsymbionts coexisted in the same vacuole with no

sign of deterioration, it is unlikely that the new-

comers are harmful to the older symbionts by

producing toxic (or inhibitory) compounds; andfurther 3) electron microscopy showed the

phagocytic incorporation of MSB (here assumed tobe older symbionts) but no phagocytic incorpora-

tion of RSB. If RSB were newcomers, the reverseshould be observed. Because no phagocytic incor-

poration of bacteria from the basal part of bacter-iocytes have been observed, it is unlikely that the

symbionts migrated from other tissues and/ororgans born on blood streams to enter into gill

bacteriocytes to multiply. The possibility that the

both types of bacteria found in A. hessleri belongto 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 sulfuroxidizing 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 bacter-

iocytes of A. hessleri from the exterior, beingbrought 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 withexternal environment. On the other hand, most of

the symbionts at the basal part of the cells seem tobe fully enclosed. Unstained symbionts in the

ruthenium red-penetrated cell strongly suggest the

enclosure (Fig. 7). Fairly frequent fusion of puta-

tive lysosome membranes with peribacterial mem-branes (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 byboth 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 fromareas 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 Langh-

eld, 1987). Residing in the invagination pocketsat 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 bacteriainside the invagination pockets may well serve an

endosymbiotic bacterial reserve of the vent snails.PLPs were found in some of the slender rod-

shaped 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. Sometimesthe particles were observed in secondary

Figs. 5-7. Alviniconcha hessleri. 5. Phagocytic incorporation of intracytoplasmic membrane-stacked bacte-

ria 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.

80 ENDOW and OHTA

Figs. 8-9. 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.

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 oreukaryotes. Sometimes viruses or plasmids play

some unique role in symbiotic relationships. Forexample, root-nodule bacteria lacking their plas-

mid on which symbiotic genes are located cannot

construct symbiotic association with their legumi-nous 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 indetermining the acceptability of the zoochlorellae

to the host.In order to clarify the nature of the PLPs, thor-

ough 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 arestricted 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 ofmixed population of microbes. In this respect,

bivalves and/or gastropods (perhaps excluding

carnivores) may offer good candidates for intracel-lular 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 seepagemussel of the Florida Escarpment. Based on theco-occurrence of type I methylotrophic enzyme

activities and type I intracytoplasmic membranestacks of methylotrophs, they suggested that one of

the mussel symbionts was a methane oxidizer.

We also find two kinds of symbiont, one is a sulfur

oxidizer (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 di-

symbiotic associations in which two different bac-

terial 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 of

(di)-symbiosis.

Acknowledgments

The specimens 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 encour-

agement throughout the work.

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(Received October 28, 1988-Accepted December 25,1988)