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Eur. J. Phycol. (2002), 37 : 523–530. # 2002 British Phycological Society
DOI : 10.1017\S0967026202003955 Printed in the United Kingdom
523
In situ identification and localization of bacteria associated with
Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by
electron and confocal microscopy
E L S A A L V E R C A1 *, I S A B E L L E C . B I E G A L A2 *, G A B R I E L L E M . K E N N A W A Y3 ,
J A N E L E W I S3 A N D S U S A N A F R A N C A1
" LME, Instituto Nacional de SauT de Dr Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon codex, Portugal
# Station Biologique de Roscoff, CNRS UPR 9042, UniversiteT Pierre et Marie Curie, Place Georges Teissier, BP 74,
29682 Roscoff Cedex, France
$ Phytosciences Research Group, School of Biosciences, University of Westminster, 115 New Cavendish Street,
London W1M 8JS, UK
(Received 10 March 2002 ; accepted 2 August 2002)
The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been
described but the bacterial flora associated with this species has not been characterized. In this study, new results of
transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide
probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been
confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the
betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the
cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside
the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of
intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of
dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have
occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of
bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained
by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.
Key words : confocal microscopy, dinoflagellates, Gyrodinium instriatum, in situ hybridization, intracellular bacteria,
oligonucleotide probes, TEM, ultrastructure
Introduction
Bacteria and dinoflagellates are important members
of the coastal plankton and their metabolism has a
major input to pelagic energy flow and nutrient
cycling through the water column (Cole, 1982 ;
Doucette, 1995). Like other protozoa, both nonphotosynthetic and photosynthetic dinoflagellates
can actively prey on bacteria and other protozoa
(Schnepf & Elbra$ chter, 1992 ; Jacobson & Anderson, 1996 ; Uchida et al., 1997 ; Skovgaard, 2000).
Besides trophic interactions between the two organisms, bacteria and dinoflagellates can also develop
close associations. The occurrence of bacteria-like
particles within cells of cultured dinoflagellates was
first described by Silva (1962). Subsequent transmission electron microscopy (TEM) studies proCorrespondence to : E. Alverca. Fax j351 21 7526400. e-mail
elsa.alverca!insa.min-saude.pt
* These two authors have contributed equally to this work.
vided further evidence of the presence of bacterialike structures within several heterotrophic and
photosynthetic dinoflagellate species (Gold & Pollingher, 1971 ; Silva, 1978 ; Lucas, 1982 ; Rausch de
Traubenberg et al., 1995 ; Doucette et al., 1998 ;
Lewis et al., 2001). Generally these relationships
have been postulated to be ‘ symbiotic ’ as the two
organisms coexist without apparent symptoms and
in some cases the association is maintained for a
long time. Additionally, it has been suggested that
bacteria associated with algal cells may play a direct
or indirect role in phycotoxin production (Silva,
1982 b ; Kodama et al., 1990 ; Franca et al., 1995 ;
Gallacher et al., 1997 ; To$ be et al., 2001).
Gyrodinium instriatum Freudenthal & Lee is an
athecate photosynthetic dinoflagellate, which is
often abundant in seawater and lagoons or inlets
along the Portuguese coast. TEM observations of
this species showed the occurrence of numerous
intracellular bacteria both in the cytoplasm and in
Manz et al. (1996)
Manz et al. (1992)
Manz et al. (1992)
Materials and methods
Culture conditions
HRP, horseradish peroxidase ; ISH, in situ hybridization.
aEscherichia coli numbering of the ribosomal RNA operon.
50
16S (319–336)
TGGTCCGTGTCTCAGTAC
Cytophaga-Flavobacterium
cluster of CFB phylum
CF319a
50
23S (1027–1043)
GCCTTCCCACATCGTTT
Gammaproteobacteria
GCCTTCCCACTTCGTTT
GAM42a
50
23S (1027–1043)
HRP
HRP
–
50
36
36
GCTGCCTCCCGTAGGAGT
CGTTCG(C\T)TCTGAGCCA
CGTTC(A\C)TTCTGAGCCA
Eubacteria
Alphaproteobacteria
Mitochondria that have one
mismatch with ALF1b
Betaproteobacteria
EUB338R
ALF1b
ALF1b
competitor
BET42a
16S (338–355)
16S (20–35)
16S (20–35)
% formamide
in ISH buffer
Targeta site
(rRNA positions)
Sequence (5h-3h) of probe
Specificity
Probe
Table 1. Oligonucleotide probes used
the nucleoplasm (Silva, 1982 a). However, this
was not the case for all clonal cultures isolated
from several natural populations, as some clones
were found without endonuclear bacteria (Silva &
Franca, 1985).
To further our understanding of the interactions
between dinoflagellates and their associated bacteria the identification of intracellular and free
bacteria is essential. Identification of bacterial
groups in marine environments has been made
possible in the past decade by the application of
molecular techniques (Giovannoni et al., 1990).
Bacterial associations with dinoflagellates have
often been probed with destructive techniques, involving DNA extraction and sequence analysis
(Doucette & Tricks, 1995 ; Seibold et al., 2001 ; Hold
et al., 2001 a, To$ be et al., 2001). Recently, Biegala
et al. (2002) used non-destructive techniques, i.e.
whole cell hybridization of 16S rRNA-targeted
oligonucleotide probes in conjunction with confocal microscopy, to identify attached or intracellular bacteria within dinoflagellates. One of the
major advantages of the latter technique was to
allow rapid identification and three-dimensional
localization of bacteria within photosynthetic host
cells.
The aim of the present study was to localize and
identify at class level the free and intracellular
bacteria associated with a strain of Gyrodinium
instriatum maintained in laboratory culture for
18 years.
HRP or not when used as
competitor
HRP or not when used as
competitor
HRP
Amann et al. (1990)
Modified from Manz et al. (1992)
Simon et al. (unpublished)
524
HRP-labelled
Reference
E. Alverca et al.
The strain of Gyrodinium instriatum (LME 184, Lisbon,
Portugal) used in this study was isolated in 1982 from Sto
Andre! ’s Lagoon, Portugal (Silva & Franca, 1985). The
culture was maintained in a mixture of Provasoli’s media
ASP1jASP2 (1 : 1) (Provasoli, 1963) until 1998, when
Guillard’s medium f\2 was used (Sigma-Aldrich ; Guillard & Ryther, 1962). The culture was maintained at
19–21 mC on a 14 : 10 light : dark cycle.
Cell fixation
Cells were typically harvested for fixation in mid- and
late-exponential growth phase. For ultrastructural studies, a sample of the dinoflagellate culture was pre-fixed
in 0n1 % glutaraldehyde (Merck) for 1 h at room temperature. Cells were then concentrated by centrifugation
and fixed for 1 h at room temperature with 4 % paraformaldehyde (Merck), 3 % glutaraldehyde (Karnowsky,
1965), in 0n1 M PIPES buffer (piperazine N,Nh-bis 2ethanosulphonic acid, Sigma-Aldrich) with 14 % sucrose
and rinsed twice in 0n1 M PIPES buffer with 14 % sucrose.
The secondary fixative was made up of two solutions :
(1) 1 % OsO (EMS) made up in water to which 3 %
%
potassium ferricyanide w : v (Sigma-Aldrich) was added
and (2) 6 % sodium iodate (Sigma-Aldrich) made up in
0n1 M PIPES buffer. Equal volumes of the two solutions
Bacteria associated with G. instriatum
525
Figs 1–6. Transmission electron micrographs of Gyrodinium instriatum. Fig. 1. Clusters of bacteria (arrows) in the
cytoplasm (C) and dividing bacteria (arrowhead). Fig. 2. Intranuclear bacterial clusters in the nucleus (N) periphery. Note
isolated bacteria (arrow) among the chromosomes (Chr). Fig. 3. DNA fibrils (arrow) extending from the dinoflagellate
chromosome (Chr) to the bacteria (Ba). Fig. 4. Bacteria being released from the nucleus (N) to the cytoplasm (C) (arrow).
Fig. 5. Dividing bacteria (arrowhead) inside the nucleus (N). Fig. 6. Degrading bacteria (arrow) in cytoplasmic vacuole.
Scale bars represent 1 µm.
were combined (A. Page, personal communication).
Samples were left overnight at room temperature or
at 4 mC. Cells were rinsed in water, dehydrated in an
ethanol series and embedded in Spurr resin (Agar
Scientific ; Spurr, 1969).
For fluorescence in situ hybridization (FISH), cells
were fixed with 1 % paraformaldehyde in PBS buffer
(phosphate-buffered saline) at 4 mC for 24 h, collected on
inorganic filter membranes and dehydrated in an ethanol
series (Amann, 1995). The filters were then stored in the
dark at room temperature to await FISH experiments.
Probes
Oligonucleotide probes (listed in Table 1) were obtained
from Interactiva (St. Malo, France) and labelled with
HRP (Horseradish Peroxidase) (Roche Diagnostics) as
described by Urdea et al. (1988) and Amann et al. (1992).
Fluorescence in situ hybridization
The protocol used for TSA-FISH (tyramide signal
amplification–fluorescence in situ hybridization) was
E. Alverca et al.
according to Biegala et al. (2002). The hybridized cells
immobilized on filters were kept at 4 mC in the dark.
Confocal observations were made within 2 weeks of
preparation without significant loss of fluorescence.
Microscopy
Ultrathin sections were cut on an LKB 8800 Ultratome
III microtome (Bromma, Sweden) with a Diatome diamond knife (Bienne, Switzerland) and counterstained
with uranyl acetate and lead citrate (Reynolds, 1963).
Stained grids were examined on a Philips EM 301 TEM
(Eindhoven, The Netherlands).
Fluorescence images were acquired with a confocal
laser scanning microscope (CLSM) Fluoview (Olympus
Optical, Tokyo, Japan) equipped with an argon–krypton
laser (643-OLYM-A03 Omnichrome, Melles Griot,
Carlsbad, CA, USA) and a pulsed laser (Mira 900,
Coherent, Santa Clara, CA, USA).
Results
TEM
TEM observations revealed the presence of numerous intracellular bacteria in the majority of the
cell sections, both in the cytoplasm (Fig. 1) and in
the nucleus (Figs 2, 3). Endonuclear bacterial
structures were observed dispersed throughout the
nucleoplasm, aggregated in dense clusters, less
frequently isolated between the chromosomes and
usually surrounded by a transparent halo (Fig. 2).
Endonuclear bacteria were mainly observed at the
periphery of the nucleus adjacent to the nuclear
envelope, while the chromosomes were aggregated
in a more central region of the nucleus (Fig. 2). Few
bacteria established close connections with the
chromosomes. When such cases occurred DNA
fibrils could be seen extended towards the bacteria
through an electron-transparent zone (Fig. 3).
Occasionally, invaginations of the nuclear envelope
were observed enclosing single bacteria or clusters
of bacteria that appeared to be about to be released
into the cytoplasm by constriction of the nuclear
membrane (Fig. 4). Actively dividing bacteria were
observed in the nucleus (Fig. 5) and more rarely in
the cytoplasm (Fig. 1). Bacteria in the cytoplasm
were organized in clusters surrounded by a host
membrane or, alternatively, isolated among the
dinoflagellate organelles and separated from them
by a transparent region (Fig. 1). Furthermore, some
sections showed the presence of cytoplasmic
vacuoles containing electron-dense masses and concentric multilamellar structures (Fig. 6).
Fluorescence in situ hybridization
Endocytoplasmic and endonucleoplasmic bacteria
were successfully labelled with the EUB338R probe
(specific for the Bacteria domain) in all Gyrodinium
instriatum cells examined (Fig. 7). Hybridized bac-
526
teria were abundant in the nucleus and cytoplasm
and were present either singly or in clusters. Bacteria
present in dividing dinoflagellates were also strongly
labelled with this probe (Fig. 8 a). Hybridization
with probes (Table 1) specific for gram-negative
alpha-, beta- and gammaproteobacteria and Cytophaga-Flavobacterium-Bacteroides (CFB) showed
that endonucleoplasmic and endocytoplasmic bacteria were mainly labelled by the BET42a probe
(Figs 9 a, 10 a, 11 a, 12 a). Endonuclear betaproteobacteria were mostly observed at the nuclear periphery, although a few were located between the
dinoflagellate chromosomes (Fig. 10 a). No intracellular alpha- and gammaproteobacteria were
detected in any of the cells examined (Figs 9 a, 11 a).
Some dinoflagellates showed intense endocytoplasmic fluorescence when hybridized with the
probe targeting CFB, although no endonuclear
labelling was observed. Bacteria labelled with the
CF319a probe were associated in large clusters
(15i5 µm ; Fig. 12 a). All extracellular bacteria
were labelled with the EUB338R probe (data not
shown), and most belonged to the beta- and
gammaproteobacteria. Very few extracellular bacteria were labelled with the CF319a probe and none
were labelled with the alphaproteobacteria.
Discussion
The presence of intracellular bacteria in G. instriatum has been described at a morphological level in
numerous studies (Silva, 1982 a, b ; Silva & Franca,
1985). However, the present work identifies for the
first time the bacterial flora associated with this
dinoflagellate using whole-cell hybridization of 16S
rRNA oligonucleotide probes. Among the different
classes of eubacteria probed, representatives of
betaproteobacteria were found to be the dominant
microorganisms intracellularly and extracellularly.
Furthermore, they were the only group of bacteria
identified within the nucleoplasm of G. instriatum.
This result is surprising as endosymbiotic bacteria
of eukaryotic organisms usually belong to the alpha
and gammaproteobacteria (Garrity, 2001). Nevertheless, symbiotic relationships of betaproteobacteria with some groups of insects have been described by Du et al. (1994), Fukatsu & Nikoh (2000)
and Von Dohlen et al. (2001). To our knowledge,
the present study reports for the first time the
presence of intracellular betaproteobacteria in dinoflagellates or other free-living protists (Fokin et al.,
1996 ; Fritsche et al., 1999 ; Seibold et al., 2001 ;
Schweikert & Meyer, 2001). Betaproteobacteria
have been considered rare in marine ecosystems but
dominate freshwater environments (Glo$ ckner et al.,
1999). Nevertheless, betaproteobacteria have recently been observed at high concentrations in
marine sediments (Nold et al., 2000).
Bacteria associated with G. instriatum
527
Figs 7–12. Confocal laser scanning microscope 0n7 µm optical sections through Gyrodinium instriatum cells immobilized
on a filter. Figs 7 a, 8 a, 9 a, 10 a, 11 a, 12 a excited at 488 nm and hybridized with specific probes (Table 1) labelled with
fluorescein using TSA-FISH technique. Figs 7 b, 8 b, 9 b, 10 b, 11 b, 12 b excited at 380 nm and stained with DAPI. Fig. 7 a.
Intracellular bacteria in the cytoplasm (C) and in the nucleus (N) labelled with the EUB338-HRP probe, present both
isolated and in clusters. Fig. 8 a. Dividing cell showing the same distribution of labelled intracellular eubacteria as observed
in Fig. 7 a. Fig. 9 a. Optical section of a cell with no intracellular bacteria labelled with the ALF1b-HRP probe. Fig. 10 a.
Three dinoflagellate cells with endocytoplasmic and endonuclear bacteria (arrows) labelled with the BET42a-HRP probe.
Fig. 11 a. Optical section of two dinoflagellates with no intracellular bacteria labelled with GAM42a probe. Fig. 12 a. Large
clusters of endocytoplasmic bacteria labelled with the CF319a-HRP probe. Scale bars represent 20 µm.
Marine bacterioplankton is dominated by alphaand gammaproteobacteria as well as CFB (Glo$ ckner et al., 1999 ; Cottrell & Kirchman, 2000 ;
Hagstro$ m et al., 2000 ; Riemann et al., 2000), which
occur either as free-living organisms or attached to
phytoplankton. These three classes of bacteria have
already been found associated with different species
of dinoflagellates (Lafay et al., 1995 ; Hold et al.,
2001 a). A recent study (Hold et al., 2001 b) indicated
that the majority of the bacteria associated with
four different dinoflagellate clonal cultures were
alphaproteobacteria, and fluctuations in abundance
were observed at different phases of the growth
cycle. The absence of alphaproteobacteria either in
the culture medium or associated with this clone of
G. instriatum is therefore surprising. There are
several possible explanations for this discrepancy.
Firstly the results produced in the present study
may reflect the bacterial community present at the
time of sampling for hybridization experiments.
Secondly, the dominance of alphaproteobacteria in
dinoflagellate cultures, as previously mentioned by
other authors, might not reflect their real abundance
in nature. All these studies used polymerase chain
reaction (PCR)-based techniques, which are known
to preferentially amplify a particular genus or group
of target organisms. And finally, in natural environments alphaproteobacteria have been described
attached to live or detrital particles less frequently
than CFB and gammaproteobacteria (Riemann et
al., 2000 ; Cottrell & Kirchman, 2000). It is thus
probable that when G. instriatum cells were isolated
for the establishment of this clonal culture, no
alphaproteobacteria were attached to the dinoflagellates. In contrast, CFB, gamma and betaproteobacteria may have been recovered from nature in
the clonal culture.
In a few G. instriatum cells, strong cytoplasmic
colonization by CFB was observed. Mixotrophy is a
nutritional strategy quite common among different
E. Alverca et al.
dinoflagellate species (Hansen, 1991 ; Schnepf &
Elbra$ chter, 1992 ; Jacobson & Anderson, 1996 ;
Skovgaard, 2000), including G. instriatum (Uchida
et al., 1997). Therefore, it is possible that the
observed cytoplasmic CFB were taken up as prey
and maintained as isolated clusters in the cytoplasm.
However, these bacteria were rare as free-living
organisms in the culture medium, making this
hypothesis unlikely. Additionally, the FISH signal
observed was very strong, which would not be the
case if the target cells were being digested. It seems
more reasonable to assume that the large CFB
clusters observed corresponded to colonization of
senescent or dead dinoflagellate cells by this group
of bacteria. This bacterial strategy is common
within the CFB group, which have been shown to
develop after the collapse of a phytoplankton bloom
(Riemann et al., 2000). Additionally, from pureculture studies, it is known that members of this
class are able to aerobically degrade a large spectrum of substrates ranging from various proteins,
carbohydrates, pesticides and insecticides to complex macromolecules (Bernardet et al., 1996).
Although the latter hypothesis is the most probable,
an intimate association between these two organisms, such as a symbiosis or parasitism, cannot be
ruled out. It has recently been reported that members of the CFB group establish symbiotic relationships with other protozoa such as amoebae (Horn
et al., 2001). The nature of the relationship between
G. instriatum cells and bacteria from the CFB is
unknown, but it seems quite different from the one
established with betaproteobacteria, both in the
number of dinoflagellate cells colonized (it represents only 2 % of the observed cells versus 100 % of
cells with betaproteobacteria) and in the intracellular location of the two bacterial types.
The presence of endonuclear bacteria in dinoflagellates is rare, and to our knowledge has been
reported in a limited number of species (Silva, 1978 ;
Silva & Franca, 1985). The origin of the endonuclear
betaproteobacteria in G. instriatum is not fully
understood. It can be hypothesized that under
certain environmental conditions, such as low nutrient levels, free-living bacteria may be taken up as
prey, with those colonizing the nucleus avoiding
digestion in the cytoplasm. This route of nuclear
infection has been previously suggested by Go$ rtz
(1986) to explain the origin of endonucleobiosis
in ciliates. Nuclear colonization by prokaryotic
microbes is commonly observed in protozoa such as
euglenoids (Leedale, 1969) and ciliates (Go$ rtz et al.,
1989 ; Kawai & Fujishima, 2000). Intranuclear
bacteria may have advantages over those that
colonize the cytoplasm because : (1) they ensure
stability of the association by homogeneous distribution to daughter cells during cell division – in
G. instriatum the nuclear envelope remains intact
528
during mitosis and (2) nuclear bacteria could benefit
from the nuclear pools of metabolites, renewed at
each host division (Go$ rtz, 1986).
TEM observations showed dividing bacteria
within the nucleus of dinoflagellate cells (Fig. 5),
without apparent prejudice to the host’s normal
growth, which is similar to the growth rate of clonal
cultures without endocellular bacteria (data not
shown). Similar TEM observations have been made
on this species by Silva & Franca (1985), who
suggested the maintenance of a stable equilibrium
between the two organisms. As described by these
authors, a decrease in the host’s division rates may
lead to an increase in intranuclear bacteria, some of
which are subsequently expelled into the cytoplasm
via vesicles pinched off from the nuclear membrane,
as a reaction of the host. Supporting this hypothesis
are TEM observations showing nuclear envelope
constrictions enclosing bacteria (Silva & Franca
1985, figures therein and Fig. 4 in this study),
indicating that the nucleus in this G. instriatum
clone may act as a reservoir of bacteria which will
be either ‘ expelled ’ or digested in the cytoplasm
according to the host’s needs (Silva, 1982 b).
Some endonuclear bacteria were observed dispersed among or closely apposed to dinoflagellate
chromosomes through host DNA fibrils. Similar
observations have been made in ciliates, and it has
been suggested that certain bacterial endonucleobionts digest host chromatin (Go$ rtz, 1986). Gortz
further suggested that in a densely colonized nucleus, the destruction of the chromatin would be
significant and probably fatal to the host. The G.
instriatum clone under study has been successfully
maintained in culture without loss of its endonuclear
bacteria for a long period. Therefore, it seems
unlikely that G. instriatum possesses endonuclear
bacteria that feed directly on chromatin, unless it
has an adequate chromatin repair mechanism ensuring cell viability.
The transfer of prokaryotic DNA to a eukaryote
has been previously reported (Taylor, 1979). The
presence of abundant intranuclear bacteria and the
close connection established by some with dinoflagellate chromosomes may lead to speculation
about an eventual uptake of prokaryotic DNA, or
its export and translation by the host. Besides its
evolutionary implications, it could act as a mechanism through which endonuclear bacteria induce
the production of special metabolites by the host
cell. This aspect may be of particular relevance in
harmful algal species.
The microscopic study of Gyrodinium instriatum
cells confirmed the maintenance of the association
of this dinoflagellate with intracellular bacteria over
a period of 18 years. As the cells were not cultured
axenically, the possibility exists that during this
period bacteria were taken up and released from\
Bacteria associated with G. instriatum
into the medium. However, the long-term presence of intranuclear bacteria suggests a symbiotic
relation between the dinoflagellates and these
bacteria.
Further studies on interactions between dinoflagellates and bacteria are necessary to improve
understanding of the way both organisms survive
and benefit from the association. Since one of the
sources of genetic novelty has been identified as
being the repeated fusion of bacterial endosymbionts with host cells (Taylor, 1979), these studies
may help to give a better insight into evolutionary
processes in the Dinophyceae. Sequencing of bacterial ribosomal genes and the design of probes
should be pursued and applied to both cultured and
natural samples, thereby enlarging the limited information available about the natural bacterial flora
associated with dinoflagellates. Although toxicity
tests were not part of this study, this species has
previously been described as being toxic (Silva,
1982 b). Therefore, an approach combining the
techniques used here with toxin detection testing
could add much relevant information regarding the
involvement of bacteria in the phenomenon of
harmful algal blooms (HAB).
Acknowledgements
We gratefully acknowledge Dr J. F. Lennon for
assistance with confocal microscopy and Dr D.
Vaulot for helpful discussion and critical reading of
the manuscript. The EC Project FAIR CT96-1558
supported this work.
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