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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)