Cephalopod bacterial associations: characterization and isolation of the symbiotic complex in the Accessory Nidamental Glands

2007·4.4 MB·English

by  Delphine Pichon

Boll. Malacol., 43 (1-8): 96-102 (2007)
Cephalopod bacterial associations: characterization
and isolation of the symbiotic complex
in the Accessory Nidamental Glands
Delphine Pichón, Isabelle Domart-Coulon & Renata Boucher-Rodoni
DMPA-BOME- USM401 Abstract
/UMR 5178. Muséum Accessory mdamental glands (ANGs) in the reproductive system of female Myopsidae and Sepioidae (Ce-
Nationald'Histoire phalopoda) host a dense bacterial community in their tubules. Bacteria associated to the ANGs were cha-
Naturelle. racterized morphologically and molecularly by complementary approaches: culture dependent (isolation of
55, rue Buffon, 75005
bacterial strains) and culture independent (direct tissue extraction, sequence comparison of gene for 16S
Paris- France,
[email protected] rRNAand fluorescent insitu hybridization). Populations ofalpha- and gamma-proteobacteria, Gram-positi-
ve bacteria, and Cytophaga-Flavobacteria-Bacteroides were detected in five families of European, Asian
and Australian decabrachian cephalopods. In order to obtain bacterial strains difficult to propagate artifi-
cially from the host, we have established co-cultures of Sepia officinalis ANG cells with their native bacte-
ria. Cellswere maintained viable over one month and the co-cultures contained bacterial strainswhich co-
uld not be cultured in classic microbiological media. Cephalopod cell culture can be used as a newtool to
study bacteria-host interactions and their role in the physiologyofeach partner.
Riassunto
Le ghiandole nidamentali accessorie (ANGs) del sistema riproduttivo delle femmine delle famiglie Myopsi-
dae e Sepioidae (Cephalopoda) ospitano nei loro tubuli una densa comunità batterica. I batteri associati al-
le ANGs sono stati caratterizzati morfologicamente e molecolarmente mediante approcci complementari:
coltura dipendenti (isolamento di ceppi batterici) e coltura dipendenti (estrazione diretta dal tessuto, con-
fronto della sequenza del gene 16S rRNA e ibridizzazione in situ a fluorescenza). Sono state scoperte po-
polazioni di proteobatteri-alfa e gamma, batteri Gram-positivi, e di Cytophaga-Flavobacteria-Bacteroides in
cinque famiglie di cefalopodi decabrachiati dell'Europa, dell'Asia e dell'Australia. Al fine di ottenere ceppi
batterici difficili da propagare artificialmente dall'ospite, si sono fondate co-colture di cellule ANG di Sepia
officinalis con i loro batteri originari. Le cellule sono state mantenute vitali per più di un mese e le loro co-
colture contenevano ceppi batterici che non potrebbero essere tenuti in coltura con i classici metodi micro-
biologi. La coltura delle cellule di cefalopodi può essere usata come un nuovo strumento di studiotra le in-
terazioni batteri-ospite e il loro ruolo nella fisiologia di ogni partner.
Keywords
Cephalopods. Bacterial symbionts. 16S rDNA. Bacterial isolation. Co-culture.
introduction bacteria, Gram-positive bacteria and Cytophaga-Flavo-
bacteria-Bacteroides are present in different cephalopod
Symbiotic relationships between invertebrates and bac- accessorynidamental glands.
teria are ubiquitous in marine environments. These Accessory nidamental glands may have a role in egg
symbioses are described as binary involving just one protection since these bacteria are found associated
type of microorganism (Cary et al, 1993; McFall-Ngai, with loliginid egg cases (Kaufman et al, 1998; Barbieri
1999), as involving a complex and diversebacterial con- et al, 2001; Pichón et al, 2005a), but this hypothesis still
sortium. In cephalopod accessory nidamental glands awaitsconfirmation.
(ANGs) however, we have previously shown the exis- In this project, we used a comparative approach to
tence of a complex consortium of bacteria (Barbieri et characterize the bacterial community present in the
al., 2001; Grigionietal., 2000; Pichónetal.,2005a). ANGs of cephalopod decabrachians. Diversity of the
The female reproductive system of myopsids, sepioids bacterial consortium was revealed both by a culture in-
and spirulids possesses a pair of accessory nidamental dependent method (sequencing of the bacterial 16S ri-
glands, colorless in juveniles and deep orange at sexual bosomal RNAsubunit gene (16S rDNA) and fluorescent
maturity, implicated in egg laying. Tubules of these in situ hybridization) and a cultivation approach (both
glands are filled with a dense bacterial community re- microbiological and cell-culturebased).
sponsible for the orange coloration of the glands at sex-
ual maturity (Bloodgood et al, 1977). Bacterial consor- Material and methods
tium present in these ANGs has been described in my-
opsids (Barbieri etal, 2001, Kaufman etal, 1998, Pichón Specimen collection
et al, 2005a) and in sepioids (Grigioni et al, 2000,
Pichón, 2005). Specifically alpha- and gamma-proteo- Females belonging to five families ofcephalopods were
collected from European seawater (English Channel bacteria (GAM42A: 5'-GCCTTCCCACATCGTTT-3’)
and Mediterranean Sea), from Taiwan, Andaman Sea (Manz et al, 1992). Stringency conditions were evaluat-
and east coast ofAustralia (Table 1). All tissue samples ed with a gradient of formamide (10-50%). Thirty per-
(ANGs, embryo, yolk and egg cases) were obtained by cent formamide gave the optimal images (high specific
aseptic dissection, freshly dilacerated to analyze the fluorescent reaction, and low background fluorescence)
cultivable bacterial fraction or preserved in 100% and was chosen for all hybridizations. Control sections
ethanolprior tomolecularanalysis. were hybridized without a probe to check for back-
ground autofluorescence.
Histological analysis Hybridization were performed in 8 gl hybridization
mM
buffer (0.9M NaCl, 20 Tris/HCl, 20% or 30% N-N-
ANGs and eggs were fixed and dehydrated in 100% dimethylformamide, 0.01%SDS) and 2 gl of probe (25
ethanol, embedded into paraffin, and sectioned for his- ng.gl-1 labeled with fluorescein or Cy3) during 90min
tology (7gm). at 48°C. Washing lasted 20 min at 48°C (1.02M NaCl, 20
Gram reactivity was tested on histological sections of mM Tris/HCl pH 7.2, 10 mM EDTA pH8, 0.01% SDS). Cephalopod
all individuals and bacterial smears following Gerhard Ten microliter of a 0.0001% solution of 4',6-diamino-2'-
etal. (1994) protocol. phenylindole (DAPI) was applied; samples were subse-
Fluorescent in situ hybridization (Amann et al, 1990; quently incubated for 10 min, then rinsed with distilled bacterial
Hahn et al, 1992; Zarda et al., 1997; Grigioni et al., 2000) water and air dried. Samples were mounted with
was performed on bacterial smears and on histological Cityfluor immersion oil solution (Chemical Laboratory,
associations:
sectionswithprobesspecifictoeukaryoticcells (EUK502: The University Canterbury, England) and immediately
5'-ACCAGACTTGCCCTCC-3') (Amann et al. 1990), to observed with an epifluorescence microscope, equipp-
Bacteria (EUB338: 5'-GCTGCCTCCCGTAGGAGT-3') ed with a high-pressure mercury bulb using filter sets
(Amann etal, 1990), to alpha-proteobacteria (ALF1B: 5'- (Leica) for fluorescein (480/40, 527/30), for Cy3 characterization
CGTTCGYTCTGAGCCAG-3'), and to gamma-proteo- (535/50, 610/75) and for DAPI (340-380/425nm).
and
Cephalopod family Species Localization ANGs Eggs
isolation
Sepiidae Sepiaelegans MediterraneanSea +
of
Sepiaofficinalis MediterraneanSeaand EnglishChannel + + the
Sepiaorbignyana MediterraneanSea +
symbiotic
Sepiaesculenta Taiwan +
Sepiarecurvirostra Taiwan + complex
Sepiaaculeata Taiwan + In
the
Sepiamadokai Taiwan +
Sepiamestus AndamanSea + Accessory
Sepiolidae Sepiettaneglecta MediterraneanSea +
Sepiettaobscura MediterraneanSea +
Nidamental
Sepiolarondeleti MediterraneanSea +
Euprymnahyllebergii AndamanSea + Glands
Sepiadariidae Sepiadariumkochii Australia + +
Loliginidae Loligovulgaris EnglishChannel + +
Loligoforbesi EnglishChannel +
Photololigochinensis Taiwan +
Photololigoduvaucelii Taiwan +
Photololigoedulis Taiwan +
Loliolusbeka Taiwan +
Loliolusuyii Taiwan +
Sepioteuthislessoniana AustraliaandTaiwan + +
Idiosepiidae Idiosepiuspygmaeus AustraliaandAndamanSea + +
Tab. 1. Natureandorigin ofspecimens. ANGs: AccessoryNidamental glands.
Tab. 1. Naturaed originedei campioni. ANGs: GhiandoleNidamentall accessorie. 97
Bacterial isolation and growth conditions Co-culture
For the isolation of pure bacterial strains, ANGs from Whole GNAs were rinsed for 90 min at room tempera-
live mature females belonging to five cephalopod ture in 25 ml ofmodified Locke's solution (pH 7.5) with
species (Sepia officinalis, S. orbignyana, Sepiola rondeleti, penicilline (500 U/ml), streptomycine (500 gg/ml) and
Sepiadarium kochii and Sepioteuthis lessoniana) were dis- bacitracine (250 U/ml) to limitcontaminationby surface
sected under aseptic conditions, and washed twice in bacteria. Tissue was then digested twice for 15 min at
sterile water. Tissue homogenates (100 pi) were spread 25°C in modified Locke's solution (pH 7.5) containing
on culture media: Marine Agar (DIFCO 2216, 2% NaCl) trypsin (0.2%). Resulting suspensions were pooled and
and DSMZ 308 Vibrio Medium (10 g Tryptone, 10 g dissociated cells and native bacteria were washed once
NaCl, 4 g MgC12*6H20, 1 g KC1, per liter distilled wa- in calcium and magnesium free seawater (CaMgFSW):
tteor,72pFhIi7n.5o)r.dTehretoploabtteasinwearceolilnecctuiboanteofdcautlt2i5v°aCblfeorba4c8teh- 1/42.05mglNaTCrils/-0H.C3l80g.5KCM1/p0H.58g.0Npae2rSO5040/0m.l125ofgdNeiaoHniCz0ed3
Delphine ria. Each distinct colony morphotypes was re-isolated H 0 [28]. Cephalopod cells were enriched from the dis-
2
in pure culture on Marine Agar. Phenotypic traits were sociated suspension by differential density sedimenta-
Pichón, used on Marine Agar to identify common phenotypes tion over a 2% sucrose cushion in CaMgFSW (10 min,
(Krieg & Holt 1984, Holt et al. 1994). Representative for 200 g centrifugation at room temperature). Pellet frac-
Isabelle each phenotypes group were selected for 16S rRNA se- tions enriched in cephalopod cells were then plated in
quenceanalysis. co-cultures. They were rinsed once in CaMgFSW and
transferred to plastic culture dishes and Lab-tek cham-
DNA bers (Sigma-Aldrich, St Quentin Fallavier, France), pre-
Domart-Coulon extraction
coated with collagen 0.01% to promote adherence of
Total DNAwas extracted fromANGs using the DNeasy cephalopod cells, in 1.5 ml of the following culture
& Tissue Kit (Qiagen), following the specific protocol for medium without antibiotics: supplemented DMEM
Renata bacteria for all specimens studied. For bacterial strains, (Gibco 41965, Invitrogen, Cergy-Pontoise, France, sup-
DNA was extracted from colonies picked and boiled in plemented with 10.8 g NaCl/0.18 g KC1/0.6 g CaCl2-
50 gl ofsterilewater, prior to 16S rRNAamplification. 2H20/5.1 g MgCl2-6H2O/0.5 g Na2S04/ 2.38 g Hepes
pH 7.8 per 500 ml) halfdiluted withArtificial Sea Water
Boucher-Rodonl
Amplification of the 16S rRNA gene M(1g4Clg2-N6aHC2lO//00..358 gg NKaC21S/O0.4/703.1g2 gCaCNla2-H2CH0203//52..2328 gg
PCR was conducted with dNTP (0.2 rnM) (Eurogentec), Hepes pH 7.8 per500 ml ofdeionized H20). FinalpH of
primers (10 gM each), Taq polymerase (2.5 U) and the medium was 7.8. Co-cultures were incubated at
mM mM 18°C with medium addition once every week. Cephalo-
Buffer (lOrnM Tris-HCl, 15 MgC12, 500 Cl)
(A.T.G.C. Biotechnologie) in a GeneAmp Thermocycler pod cell viabilitywas determined on a haemocytometer
(Perkin Elmer) with a denaturation step at 94°C for 5 with the trypan blue exclusion assay and the metabolic
min, followed by 32 cycles of 94°C (30 s), 55°C (30 s), activity of the co-culture was checked with the colori-
and 72°C (1 min) and a final elongation step at 72°C for metricMTTreductionassay.
7 min. Universal prokaryote primers were used: 27F- Supernatants from co-cultures (1.5 ml) were sampled at
G13A8G5TRTpTaGirAT(rCeCspTeGctGiCveTlCyAE-sc3h'eriachnida pcoolsiitpioosniti1o3n859 : 55’'-- 7DaNnAdw28asdaeyxst.raAcftteedrfcernotmritfhuegabtaicotenraital15pe0l0l0etgs,foarnd5 mgienn.e,
CGGTGTGTRCAAGGCCC-31 which produced m:ost coding for 16SrRNAwassequenced.
),
the entire 16S rDNA (ca 1400bp). Each PCR product
wasvisualizedby 1.5%agarosegel electrophoresis.
Results
Purified PCR products (QIAquick PCR Purification Kit,
Qiagen Inc.) were cloned by insertion into plasmid vec- Histological results
tor PCR 2.1 TOPO TA Cloning (Invitrogen) following
the instructions of the manufacturer. Minipreps of the Gram staining and DAPI staining of histological sec-
clones were sequenced using M13(-20) and M13R tions ofANGs demonstrated that allANG tubules were
primers (5'-GTAAAACGACGGCCAGT-3' and 5’- filled with bacteria (Figure 1 a). Fluorescent in situ hy-
CAGGAAACAGCTATGAG-3'). All sequenced clones bridization detected a diverse consortium of long and
were analyzed for the presence of chimeras using the thin rod and coccoid shaped alpha-proteobacteria,
Chimera Check program (version 2.7) (Maidak et al. small rod and coccoid shaped gamma-proteobacteria
1997) of the Ribosomal Database Project (RDP-II) and andlargecoccoid-shaped grampositivebacteria.
the Bellerophon program (Huberetal., 2004). Sequences Similar morphotypes were present in loliginids, sepiids,
suspected of being chimeric were not included in fur- sepiolids, sepiadarids and idiosepiids, but quantitative
ther analyses. Similarity between sequences was calcu- differences were observed in the abundance of each
lated without gap penalties. Different sequences were bacterial group. Gram-positive bacteria were very
arbitrarily clustered into operational taxonomic units abundant in sepiids, sepiolids and sepiadarids. Among
(OTU) with similarities > 97%, which defined the genus Gram-negative bacteria, hybridization with specific
98 ofthebacteria (Stackebrandt and Goebel, 1994). probes revealed that gamma-proteobacteria were pre-
Cephalopod
bacterial
associations:
characterization
and
isolation
of
the
symbiotic
Fig. 1. a. DAPI staining ofhistological section ofSepia elegansANGs; b. DAPI staining of histological section ofa partofLoligovulgarisegg showing
embryonictissueand membranes; c.ANG cellsand associated bacteriaco-culture, d.fluorescentinsituhybridizationofcellsand bacteria in co-culture complex
after 28 dayswith eukaryotic probe EUK502*fluorescein and eubacteria probe EUB338*Cy3 b. bacteria, c. eukaryotic cell; cy: cytoplasm of cephalo- in
podcell; im: internal membraneofegg cases; em: external membranesofegg cases; nu: eukaryoticnucleus.
the
Fig. 1. a. Colorazione DAPI di una sezione Istologica di ANGs di Sepia elegans', b. Colorazione DAPI di una sezione istologica di una parte di uovo di
Loligovulgarische mostratessutoe membrane, c.celluleANG elaco-coltura del batteri associati, d. ibridizzazioneinsituafluorescenza dellecellulee
del batteri In co-coltura dopo 28giorni con la sonda eucarlotlcafluoroscelna EUK502* e la sonda eubatterica EUB338*Cy3 b. batteri, c. celluleeuca- Accessory
riotiche; cy: citoplasma dellecelluledi cefalopode; im: menbrana internadellecapsuleovigere; nu: nucleoeucarlota.
Nidamental
dominant in loliginids and idiosepiids, whereas alpha- (Table 2). 80% of strains isolated on artificial media be-
proteobacteria were predominant in the other families. longed to the gamma-proteobacteria group (Table 3).
No differences were observed in relation to the geogra- Using a culture independentapproach, directextraction Glands
phyofthehost. of the DNA from the ANGs followed by bacterial
Histological sections of eggs confirmed the presence of 16SrDNA sequencing confirmed the presence of the
similarbacterial morphotypes surrounding theenvelopes same bacterial groups as isolated in pure cultures, and
oftheeggcasesofsepioidsandmyopsids (Fig. 1 b). allowed us to identify in the tissue three more groups:
Roseobacter (alpha-proteobacteria), sulfur-oxidizing sym-
biont (gamma-proteobacteria) and Gram positive bacte-
Bacterial diversity
ria (Table 2). Compared to the microbiological ap-
Using a culture dependant, microbiological approach, a proach, there was a shift in the relative abundance of
total of 54 strains were isolated on artificial media from each bacterial group revealed by the molecular ap-
the ANGs of seven cephalopod species (sepioids and proach: 79% of clones belonged to the alpha-proteobac-
myopsids). Gram reactivity and physiological charac- teria group, and only 6% to the gamma-proteobacteria
teristics led to identification of 6 bacterial groups. group (Table 3).
16rRNA gene sequencing of representative isolates for Compared to the ANGs of mature females, similar bac-
each group revealed alpha-proteobacteria Agrobacte- terial diversity was observed in the egg cases of some
(
rium, Rhodobacter), gamma-proteobacteria (Vibrio, She- species as evidenced by direct 16SrRNA sequencing
zvanella and Pseudoalteromonas) and Cytophaga group andbybacterial isolation on artificial media. Percentage 99
Alphaproteobacteria Gammaproteobacteria
Group Hostspecies
Ag Ro Rb Ps Sh V Sos Cfb G
Sepiidae Sepiaaculeata C c C c
Sepiaelegans C c C c c
Sepiaesculenta C c C c
Sepiamadokai C C c
Sepiamestus C C
Sepiaofficinalis CI c CI CI CI CI CI CI
Sepiaorbignyana C c c I
Delphine Sepia recurvirostra c c c
Sepiolidae Sepiettaneglecta C c c
Pichón,
Sepiettaobscura C c c I c
Isabelle Sepiola rondeleti c c c I c
Euprymnahyllebergii c c c c c
Sepiadaridae Sepiadariumkochii c c I CI
Domart-Coulon
Loliginidae Loligoforbesi c c
& Loligopealei c c I 1 I I
Loligovulgaris c c c c
Renata
Loliolusbeka c c c
Loliolus uyii c c
Boucher-Rodoni
Photololigochinensis c c c c
Photololigoduvaucelii c c c c
Photololigoedulis c c c c
Sepioteuthislessoniana c c I I
Idiosepiidae Idiosepiuspygmaeus c c c
Tab. 2. ANG bacterial diversity. C: clones; I: isolates; A:Agrobacterium; Ro: Roseobacter; Rb: Rhodobacter; Ps: Pseudoalteromonas; Sh:Shewanella;V:
Vibrio Sos: Sulfur-Oxidizing-Symbiont; Cfb: Cytophaga-Flavobacteria-Bacteroidetes; G: Gram Positive bacteria. Thedataforloliginidsarefrom Barbieri
;
etal., 2001 and Pichón etal., 2005a.
Tab. 2. ANG diversità bactérica. C: cloni; I: isolati; A: Agrobacterium; Ro: Roseobacter; Rb: Rhodobacter; Ps: Pseudoalteromonas; Sh: Shewanella; V:
Vibrio; Sos: Simbionti Zulfo-Osidanti-; Cfb: Cytophaga-Flavobacteria-Bacteroidetes; G: batteri Gram Positivi. I dati per i loliginidi sono stati tratti da
Barbierietal., 2001 ePichónetal., 2005a.
of sequence similarity between common bacterial ratio of 2/3) (Fig. 1 c). Hybridization observations
strains fromANGs and from egg caseswas in the range (FISH with eubacterial probe) revealed proximity of
of90%to 100%. bacteria with the surface of cephalopod cells (Fig. 1 d).
ANG cells isolated with their native bacteria and main- Bacteria were also motile in the supernatants and sur-
tained in vitro remained viable and adherent to the col- rounding thecells,andwereable toformcolonieswhen
lagen-coated substrate over a month. Glandular cell re-isolated on marine agarand incubated for24h to48h.
typewaspredominant in co-culture, whichwaspolygo- Similar bacterial morphotypes in the co-cultures as in
nal, 12 pm in diameter, with voluminous nucleus (N/C the histological sections were observed: coccoid-
Cytophaga-
Alpha- Gamma- Gram-positive
Approach Flavobacteria-
proteobacteria proteobacteria bacteria
Bacteroides
Directsequencing 79% 6% 6% 9%
—
Bacterialisolation 18% 80% 2%
— —
Co-Culture 43% 57%
Tab. 3. Bacterial diversityevidenced intheANGswiththedifferentapproaches.
100 Tab. 3. Diversità battericheevidenziatecon l'ANGscon diversi metodi.
1
shaped, long and thin rod-shaped and small rod- cephalopod accessory nidamental glands is extremely
shaped. Only gram negative bacteria were present in diverse, as opposed to the luminous organ symbiotic
the co-cultures. Bacterial 16SrRNA analyses revealed system in the sepiolid Euprymna scolopes, which invol-
presence of alpha-proteobacteria Roseobacter and Rho- ves just one symbiont species Vibrio fischeri (Nyholm
(
dobacter and gamma- proteobacteria Shewanella and and McFall-Ngai, 2004). So the difficulty of physiologi-
) (
Vibrio) (Table 3). cal studiesintheANGs is thecomplexityofthissystem.
Percentages of sequence similarity between bacterial Future studies will investigate the role of each partner,
strains detected in the ANGs and in the co-culture at 7 especially the physiological role of the bacteria in the
days and 28 days were 97 to 99.5%, and the co-cultures ANGs and in the egg cases. Several alpha-proteobacte-
remained metabolically active over a month as revealed ria (Roseobacter, Rhodobacter) present in theANGs of im-
by themaintenanceofthe MTTreduction assay. mature and mature females are known to produce caro-
tenoids (Gonzalesand Moran, 1997; Hiraishietal, 1995).
Discussion During sexual maturation, they may be responsible for
the orange coloration of the glands under physiological Cephalopod
Bacterial isolation and direct bacterial DNA extraction control by thehost (Pichón et al, 2005b). The role ofthis
from tissue confirmed presence of 4 bacterial phyla in coloration is still unclear, it may ensure UV protection
theANGs ofall cephalopod species studied. Theclosely of the reproductive system, and/or promote sexual at- bacterial
related bacterial subgroups were present both in imma- traction of the males. The production ofcarotenoids ap-
ture and mature ANGs, and in egg cases. Detection pears to be an activity stimulated by hormone secretion
associations:
both in the ANGs and in the egg cases can suggest that by the optical glands (Bloodgood, 1977; Van den Bran-
some bacteria may be transmitted to the eggs (Barbieri den et al, 1978). The role of hormones in carotenoids
et al, 2001; Pichón et al, 2005a). But more studies are production by bacterial strains needs further investiga-
necessary to investigate the hypothesis ofvertical trans- tion. characterization
mission. The potential implication of these bacteria in Whatever the environment, whatever the developmen-
eggprotection alsoneeds tobe further investigated. tal stageof the female, the diversity ofthebacterial con-
Complementary approaches were used, which were de- sortium hosted in the accessory nidamental glands is and
pendant or not dependant on propagation on artificial conserved, which suggest an important role for the host
isolation
media (microbiology) in order to alleviate bias towards physiology.
specific groups. Isolation of bacterial strains detected of
essentially gamma-proteobacteria, whereas the direct Acknowledgments the
extraction identified preferentially alpha-proteobacte-
ria. These results confirm and explain the preliminary The authors wish to thank: Dr. C.C. Lu and Dr M. Nor- symbiotic
results obtained for myopsids and sepioids (Barbieri et man for their help during collection of specimens, and
al., 2001; Grigioni et al, 2000; Pichón et al., 2005a). M. Pernice for his assistance. The technical help was complex
Indeed the microbiological approach used for Loligo performed by M. Martin. The authors thank the «So-
in
pealei detected mostly gamma-proteobacteria, whereas ciété Franqaise de Malacologie» for subvention the par-
the
the molecular approach used for both Loligo pealei and ticipation to the IV International Congress of the Eu-
Sepia officinalis detected mostly alpha-proteobacteria. ropeanMalacologicalSocieties. Accessory
We have developed a new approach for co-culture of
ANGs cells with their associated bacteria (Pernice et al,
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