CONTENT - International Society of Zoological Sciences

S20 ICZ2008 - Abstracts
How to quantify endosymbionts in the gills of the
hydrothermal vent mussel Bathymodiolus.azoricus ? 3D-FISH
versus qPCR
Isabelle Boutet 1 , Arnaud Tanguy 1 , François H. Lallier 1 , Sébastien
Halary 2 , Sébastien Duperron 2 and Françoise Gaill 2
1 UPMC-Paris 6 & CNRS, UMR 7144 Adaptation et Diversité en
Milieu Marin, Station Biologique de Roscoff, 29680 Roscoff,
France
2 UPMC-Paris 6 & CNRS, UMR 7138 Systématique, Adaptation et
Evolution, 7 quai St Bernard, 75005 Paris, France
The mussel Bathymodiolus azoricus forms dense colonies around
the deep sea hydrothermal vents of the mid-Atlantic ridge. They
derive most if not all of their metabolic needs from the two types of
endosymbiotic chemoautotrophic bacteria they harbour in their gills.
The two types of gamma-proteobacteria, a methanotroph and a
thiotroph, are found in variable amounts in the bacteriocytes,
apparently as a function of methane and sulfide availability in the
mussel environment. Here we compare two methods aiming at
quantifying each type of bacteria. The first one (3D-FISH)
measures volumes occupied by each type of symbiont in
bacteriocyte sections from a vent mussel gill filament using
fluorescence in situ hybridization with 16S rRNA-based specific
probes coupled to three dimentional microscopy and image
analysis carried out by a dedicated software. The other method
(qPCR) uses the same probes and some others targeting specific
metabolic genes to measure the relative expression of these
genes in gill extracts. Qualitatively, the two methods give
congruent results and confirm the impact of local environmental
parameters on symbiont abundances.
A novel view on relationships between Lucinidae (Mollusca:
Bivalvia) and intracellular sulfur-oxidizing bacteria
Terry Brissac 1 , Olivier Gros 2 , Audrey Caro 3 and Hervé Merçot 1
1 UMR 7138 CNRS UPMC MNHN IRD “Systématique, Adaptation,
Evolution”, Equipe: Génétique & Evolution, UPMC, 7 quai St
Bernard, 75252 Paris cedex 05, France ; 2 UMR 7138 CNRS
UPMC MNHN IRD “Systématique, Adaptation, Evolution”, Equipe:
Symbiose, UAG, UFR SEN, Dpt de Biologie, BP592, 97159
Pointe-à-Pitre cedex, Guadeloupe, France ; 3 UMR-CNRS 5119
Laboratoire Ecosystèmes Lagunaires, CC 93, Université de
Montpellier II, 34095 Montpellier cedex 5, France
Associations between marine invertebrates and chemoautotroph
bacteria constitute a large field for the study of symbiotic
associations. In these interactions, transmission of the symbiont
must represent the cornerstone to permit the persistence of the
association through generations. Within Bivalvia, in some cases
like Solemyidae or Vesicomyidae, the vertical transmission mode
of the symbiont occurs without any ambiguity. However, within
Lucinidae, the transmission mode is described in the literature as
environmental, symbionts being acquired by the new host
generations from the environment. Nevertheless, the precise origin
of the symbiont which infects lucinids is still unknown, and we can
pose two hypotheses. Symbionts’ origin could be: (i) bacteria
released after multiplication in a bivalve host of the parental
generation, (i.e. environmental transmission), (ii) free-living
bacteria which multiply only in the environment; in which case we
would not have transmission, but merely recruitment of nearby
bacteria by the Lucinidae. Our observations show that after
internalization of symbiosis competent bacteria from the
environment, it appears that symbionts can not divide inside the
bacteriocytes even if genome replication stays always active (Caro
et al., 2007). Moreover, it seems that symbionts are not released
by their host and at last are destroyed via lysosomal degradation
process (Liberge et al., 2001). According to these observations the
environmental transmission hypothesis can be rejected, and this
relationship seems more correspond to a predation than a
mutualistic relationship. Moreover the association seems to be
advantageous only for the bivalve and constitutes a dead-end for
the bacteria.
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Lucinidae/Sulfur-oxidizing bacteria: Ancestral heritage or
environment-dependant association?
Terry Brissac 1 , Olivier Gros 2 and Hervé Merçot 1
1
UMR 7138 CNRS UPMC MNHN IRD “Systématique, Adaptation,
Evolution”, Equipe: Génétique & Evolution, UPMC, 7 quai St
2
Bernard, 75252 Paris cedex 05, France ; UMR 7138
“Systématique, Adaptation, Evolution”, Equipe: Symbiose, UAG,
UFR SEN, Dpt de Biologie, BP592, 97159 Pointe-à-Pitre cedex,
Guadeloupe, France
In symbiosis, a question which arises concerns the origin and the
evolutionary story of symbiotic couples: the two partners could
have co-evolved or not from an ancestral couple (Distel et al.,
1994). Analysis of diversity and host/symbionts phylogenies
comparison could give us some evidences to discriminate between
these two hypotheses. The genetic diversity of gill-endosymbionts
associated to Lucinidae was studied via sequencing of 16S rRNA.
In Distel et al. (1988; 1994) a bacterial species is specifically
associated to each host species. In a second study, 6 host species
harbor the same bacterial species (Durand & Gros, 1996), results
which permit us to pose the hypothesis that thes associations are
not strict and are constituted according to the bacterial species
present in the environment. The comparison of hosts and
symbionts phylogenies of other lucinids from Philippines, confirms
that there is no specificity of association between Lucinidae and
their symbionts. Considering that 16S rRNA being maybe too less
discriminative to point a bacterial diversity at an intra-specific level,
we develop at the laboratory a MLST (Multi Locus Sequence
Typing) analysis with different markers (dnaE, gyrB, rpoB, ITS,
aprA) and analyzed species used by Durand & Gros (1996).
Different bacterial haplotypes were retrieved, which one specific to
two host species sampled in superficial strata of sediments. This
specificity is independent from the geographic localization and
does not constitute a phylotype. So, we sought to know if there
exists an ecotype i.e. a bacterial strain only localized in superficial
strata of the sediments.
Effects of host starvation (Codakia, Bivalve, Lucinidae) on its
symbiotic population
Audrey Caro 1 , Marc Bouvy 1 and Olivier Gros 2
1 UMR-CNRS 5119 Laboratoire Ecosystèmes Lagunaires, CC 93,
Université. Montpellier II, 34095 Montpellier cedex 5, France
2 UMR 7138 CNRS UPMC MNHN IRD “Systématique, Adaptation,
Evolution” Equipe: Symbiose, UAG, UFR SEN, Dpt de Biologie,
BP592, 97159 Pointe-à-Pitre cedex Guadeloupe
The association between bivalves and chemoautotrophic
symbionts allows the host to fix carbon either by autotrophic or
heterotrophic pathways, namely a mixotrophic diet. Codakia
orbicularis, a tropical lucinid bivalve, lives in association with
sulfur-oxidizing bacteria, supporting the autotrophic pathway,
despite the presence of a reduced digestive tract which sustains
filter feeding nutrition. To evaluate the effect of host starvation on
the symbiotic population, long term experiment was designed,
maintaining freshly collected bivalves in artificial seawater during
several months. The modification in the symbiotic population
housed in the gills of the bivalve, namely in bacteriocytes, was
monitored by transmission electron microscopy (TEM) and
fluorescence in situ hybridization (FISH). A major consequence of
host starvation consists in a gradual decrease in the symbiotic
population. Quantification of the fluorescence FISH signal through
gill section showed that one third of the initial symbiotic population
disappears each month of starvation. TEM observations reveal,
according to the presence of lysosomes along with the
disappearance of bacteriocytes, that symbionts could be digested
by the host. The capability of Codakia to survive in starvation
conditions for long period seems to rely mainly on symbiont
digestion. These results support evidence that symbiont digestion
could represent an important part in host nutrition in natural
conditions.