Endosymbiotic bacteria
in the clam Loripes lacteus:
Location, characterization and
aspects of symbiont regulation
Emmanuelle Pales Espinosa1, Arnaud Tanguy
Sophie Le Panse 4, François Lallier 2,3
Bassem Allam1 and Isabelle Boutet 2,3
SoMAS, Stony Brook University, Stony Brook, NY 11794
2 CNRS, UMR 7144, Station Biologique de Roscoff, 29682 Roscoff, France,
UPMC Université Paris 6, Station Biologique de Roscoff, 29682, Roscoff, France
4 CRBM FR2424, Station Biologique de Roscoff, 29682 Roscoff, France
1
3
2,3
Symbiosis
Close interaction between different species
The endosymbiotic theory:
Multicellular organisms would result from a
symbiosis between various sorts of bacteria
(Brinkman et al., 2002; Margulis, 1999; Moran, 2006)
Therefore an effort to better understand the
interactions of actual host/symbiont may
shed lights on co-evolution histories
Hawaiian
bobtail squid/Vibrio
Fungus/Algae/Cyanobacteria
Clown
Loripes
Algae/Coral
fish/Anemona
lacteus/Bacteria
(photo:
Wikipedia)
(photo:
Wikipedia)
(photo:
(photo:
(photo:
Maricopa
Pales
Wikipedia)
Espinosa
CC) et al.)
In this study, we used:
(Pales Espinosa et al., in prep.)
The association bivalve/endosymbiotic
bacteria
Contemporary techniques to
Determine symbiont(s) phylogeny
Investigate their distribution
Study symbiont dynamics
Initial acquisition, regulation
Small marine Lucinids
Reduced sediments of
seagrass bed
Harbor endosymbiotic
bacteria in bacteriocytes
Chemoautotrophic
sulphur-oxidizing
bacteria (Herry et al.,
Roscoff, France
1989; Johnson and
Fernandez, 2001)
1 cm
Methods
Transmission electron
microscopy (TEM)
DNA extraction
16S rRNA specific
primers (bacteria)
Amplification, cloning
and sequencing
Phylogenetic tools
(Bioedit, PhyML, Treeview ..)
Gill of Lucinids
(Thesis of Elizabeth N., 2012)
Microvilli
Bacteria located in
bacteriocytes
Endobacteria
Gram negative with
double membrane,
DNA strands, and
electron dense or
translucent granules
No bacterial division
observed
Bacteriocyte
Gill
Granules
DNA strands
Double membrane
Endobacterium
Lucinids
γ-Proteobacteria
L. lacteus
from Croatia
Clone 1-Roscoff
Solemyids
Bacteria found
in sediment
Clone 2-Roscoff
0.1
Methods
Fluorescence in situ
hybridization (FISH)
DNA extraction
16S rRNA specific
primers for the 2 clones
18S rRNA primers
Real Time PCR
(clam)
(Loram
et al., 2007; Fink, 2011)
In situ hybridization (FISH)
Gill of L. lacteus
(logarithmic scale)
Relative 16S rRNA copy numbers
Relative 16S rRNA copy numbers of bacterial
clones in Loripes lacteus
102
Gill
a
Mantle
Gonad
n = 10
Holm-Sidak post-hoc test, p < 0.01
101
100
10-1
10-2
10-3
a
b
c
10-4
*
b
*
10-5
Clone 1-Roscoff
c
*
Clone 2-Roscoff
Light and fluorescence micrographs of gill
in Loripes lacteus (probe clone 1-Roscoff)
Gill
Gill
Lateral zone
Ciliated
zone
Hematoxylin-eosin staining
of gill filaments
Lateral zone
Ciliated
zone
Fluorescence in situ hybridization
(FISH) images of gill filaments
Fluorescence micrographs of different
organs in Loripes lacteus
Gill
Mantle
Female gonad
Gill
Male gonad
Digestive tract
Methods
In vivo experiments
effect of starvation on endobacteria
re-acquisition assay after starvation
Real Time PCR
Food (algae)
Optimal
conditions
No food
Food, Gill extract, Fresh
sediment, Sodium sulfite
5 weeks
8 weeks
3 weeks
Real Time PCR
Relative 16S rRNA copy numbers of bacterial clones in
Loripes lacteus submitted to 8 weeks of starvation
n = 10
Holm-Sidak post-hoc test, p < 0.001
Clone 1-Roscoff
No food
(logarithmic scale)
Relative 16S rRNA copy numbers
Clone 2-Roscoff
102
a
Food
a
a
b
101
b
b
b
c
c
100
10-1
a
a
10-2
a
bc
c
10-3
Control
W1
W3
W5
bc
c
W8
bc
c
W1
W3
W5
W8
Assay of re-acquisition of symbionts
in Loripes lacteus after starvation
(logarithmic scale)
Relative 16S rRNA copy numbers
Clone 1-Roscoff
102
Clone 2-Roscoff
n = 10
Holm-Sidak post-hoc test, p < 0.001
a
b
b
101
100
10-1
10-2
10-3
a
a
Control
W5
No food
W8
a
Optimal
Conditions
W8
Bacteriocytes (Bc) in healthy (A) and starved (B) clams
A
B
Bc
Bc
MC
Transmission electron micrographs of Loripes lacteus gill
sections with endosymbiotic bacteria
Endobacteria
Fusion
Dense
vacuole
Dense
vacuole
Lytic degradation
Lytic degradation
Clone1-Roscoff likely to be a symbiont
Initial acquisition: probably via environment
(data, other lucinids, sampling period) but we can
not rule out vertical transmission
Regulation
No symbiont multiplication inside
bacteriocytes (probable control from host)
Farming: degradation of bacteria
Reacquisition: assay failed (Gros et al, 2012)
Aposymbiotic clam / depleted clams
RNAseq (transcriptome analysis)
Which genes and proteins are involved in
symbiont acquisition ? (recognition, phagocytosis..)
How L. lacteus control the symbionts?
What is the role of the bacteriocytes?
Regis Lasbleiz and Dr. Thierry Comtet
(Station Biologique de Roscoff, France)
Dr. Sebastien Duperron
(Université Pierre et Marie Curie, Paris)
Dr. Jean-Marc Pons
(Muséum National d'Histoire Naturelle, Paris)
National Science Foundation (IOS-1050596)
New York Sea Grant (R/XG-19)