Mcb150 Report-hydrothermal Vent Host Shrimp
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Transcript of Mcb150 Report-hydrothermal Vent Host Shrimp
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Inorganic carbon fixation by chemosyntheticectosymbionts and nutritional transfers to the
hydrothermal vent host-shrimp Rimicaris
exoculata
Julie Ponsard, Marie-Anne Cambon-Bonavita, MagaliZbinden, Gilles Lepoint, Andre´ Joassin, Laure Corbari,
Bruce Shillito, Lucile Durand, Vale´rie Cueff-Gauchardand Philippe Compe`re
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Ectosymbiosis
– a type of symbiosis in which one organism remainsoutside of the other organism
Epibiosis
– the colonization of a living surface by sessileanimals or plants, a facultative association of twoorganisms: the epibiont and the basibiont
Epibiont
– organisms that are attached to the surface of aliving substratum during the sessile phase of theirlife cycle
Basibiont
– lodges and constitutes a support for the epibiont
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Hydrothermal vent
A hydrothermal vent forms whenseawater meets hot magma
The cold seawater is heated by hotmagma and reemerges to form thevents.
Precipitating minerals can fall out of vent fluids to form “chimneys” andother formations on the sea floor.
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Introduction
R. exoculata is hypothesized to be theprimary consumer of chemoautotrophic
bacterial community in its gill chamber current hypotheses concerning the
epibiont‟s chemoautotrophy and the
mutualistic character of this associationneed to be tested
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gill chamber epibionts constitute adiversified community with variousmorphotypes, phylotypes andmetabolisms
– no study has directly demonstrated whichsubstrate(s) the bacteria oxidise to fueltheir „chemosynthetic‟ metabolism(s).
harbours a digestive bacterialcommunity whose role remainshypothetical (detoxification, nutritionand/or pathogen control)
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The goals of the study are to:
– Test inorganic carbon fixation by active
bacterial chemosynthetic metabolisms; and – Test the hypothesis of bacterial organic
carbon transfer (soluble molecules) to theshrimp tissues.
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Rimicaris exoculata
hydrothermal decapod Nutritional ectosymbiosis
dominates the megafauna of several deep-sea hydrothermal vent sites of the Mid- Atlantic Ridge
forms dense aggregates around active
chimneys harbours a luxuriant bacterial community
in its enlarged gill chamber
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Rimicaris exoculata
hydrothermal decapod Nutritional ectosymbiosis dominates the megafauna of several deep-
sea hydrothermal vent sites of the Mid- Atlantic Ridge
forms dense aggregates around activechimneys
harbours a luxuriant bacterial communityin its enlarged gill chamber
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How carbon is transferredfrom bacteria to shrimp?
direct transfer of dissolved molecules acrossthe shrimp integument, especially the gill
chamber lining – based on morphological observations (features
dedicated to bacteria farming, absence of scrapemarks on the biofilm)
the shrimp gets organic matter mainly fromits epibionts, rather than from grazing free-living bacteria associated with chimney walls – based on stable isotope signatures and essential
fatty acid composition
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Shrimp collection
pressurised
IPOCAMP
incubation
13C- and 14C-bicarbonate
incorporation
C-acetate and H-lysineincorporation
Stable isotopeand elementalanalysis
Measurement of incorporatedradioactivity
AutoradiographyStatisticalanalysis
Methods
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slurp-gun operated from themanned submersible Nautileat the Rainbow hydrothermalvent site (36114⁰N –33154⁰W,
2320m depth) during the „MoMARDREAM-Naut‟ cruise,
July 2007
IPOCAMP incubator at in situpressure (230 bars) andtemperature (15⁰C) for in
vivo experiments
tracer excess was washed off
in 0.22-mm-filtered SW: 15min for 13C and 1, 5 and 30s, 5 and 15 min for the other
experiments
the samples were rinsed
again and treated to removeunfixed tracers and topreserve the labelled organic
molecules beforequantification
freeze at -80⁰C after
collection or dissected intobody parts and tissues of
interest
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After sample collection
Frozen for autoradiography andscintillation analysis
Dissected into body parts & tissues of interest
– Regions colonized by the bacteria
– Exchange/absorption organs
Control shrimps
– Radioactivity level was undetectable to
very low in bacterial mats & shrimp tissues
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The dissected tissue samples wererinsed 3x in dH2O
Dehydration overnight at 60 ⁰C Dried samples were finely ground to
obtain a powder to vaporise in the mass
spectrometer.
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13C-bicarbonate incorporation
4 hrs incubation
10 hrs incubation
in filtered SWenriched with iron(100 µM FeCl2) or
thiosulphate (100 µMNa2S2O3)
in filtered SWcontrol without
iron
250-ml flasks
4shrimps
2 shrimps
1 g/L NaH13CO3 (98%)
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14C-bicarbonate incorporation
6 hrs incubation
6 hrs incubation
labelled with 80 µMNaH14CO3(4 µCi/ml)and dissolved in
SW enriched with
an electron donor(Fe2+ or Na2S2O3)at the same conc.
labelled with 80µM NaH14CO3(4
µCi/ml) anddissolved in SW
control
50-ml flasks
2 specimens
0.25 g/L NaH12CO3
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Rinse 4x in dH2O
Determine fresh weight Overnight digestion at 50-55C in glass
scintillation vials with 1ml quaternary
ammonium hydroxide (0.5N) in toluene Acidify for 2 hrs at room temp. by add‟n
of 250 µL acetic acid
– Removes remaining 14C-bicarbonate & CaCO3 present
Add scintillation liquid (10ml) to all
samples before reading
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1 hr incubation
1 hr incubation
20 µCi/ml3
H-lysine(0.2 µM)
10 µCi/mlsodium 14C-
acetate (0.2µM)
14C-acetate and 3H-lysine incorporation
Two sets of three to four shrimps50 ml SW
50 ml SW
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Where:•% at 13Cinc = atomic percentage of incorporated 13C;
•DWsam = dry weight of the sample,•%Cdw = carbon content expressed as a percentage of dryweight DWsam ,•%Cdw = weight of carbon in the sample,• W13Cinc = weight of 13C incorporated into the sample during
the experiment,•MMC = molar mass of carbon,•t = duration of the experiment, and•C inc = carbon incorporation rate expressed in moles of incorporated carbon per time unit and weight unit of total
carbon in the sample
Stable isotope & elementalanalysis
M t i t
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Measurement o incorporateradioactivity
The beta-radioactivity of the samples wasmeasured for 15 min in liquid phase in aBeckman LS 6500 Scintillation Counter
(effective resolution: 0.06 keV).
Moles of incorporated radioelement areknown from the measured number of
disintegrations per second (disintegrationsper second x 2 half-life in seconds/Avogadro‟snumber).
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Where:
xEinc is the number of moles of incorporatedradioelement
f is the ratio of radioactive to cold molecules in the
incubation medium FWsam is the fresh weight of the sample
p is the dry weight/fresh weight ratio measured oncontrol shrimps
%Cdw is the carbon content expressed as apercentage of sample dry weight
R inc is the total incorporation rate expressed in molesof incorporated molecules per time unit and weight of
total carbon in the sample
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Autoradiography
Histological localisation of radiotracers wasdone by autoradiography on frozen shrimpsincubated with a radiotracer.
To isolate shrimp segments, each frozen shrimp
was embedded in Tissue-Tek gel directly frozenin liquid nitrogen, and cut into segments 1 cmlong with a hacksaw in a cryostat at -20 ⁰C
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Segments of the cephalothorax (including MP andgills) and the abdomen were directly thawedovernight in fixative solution (2.5% glutaraldehyde)
Then decalcified in EDTA (0.2 M, pH 8), post-fixedin 1%OsO4 for 2 h, rinsed in distilled water (410min), dehydrated with ethanol, and embedded in
Steedman‟s wax (polyethylene glycol distearate 9:1;99%)
12 µ cross sections were obtained with a microtomeat three levels (MP, gills and abdominal muscles)
and deposited on gelatinised glass slides. They were rehydrated, washed in distilled water
and then coated with a photographic emulsion filmin the dark according to the classical wet method
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Microtome technique
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Statistical analysis
All data are reported as mean values ± s.d.
Outliers were eliminated with Dixon‟s Q test(P<0.05).
One-way analysis of variance (Kruskall –Wallis)
Dunn‟s Method performed with Sigma Plot
11.0 software to compare samples Dunn‟s multiple comparisons vs control group
method were used to test the realincorporation of 14C.
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Results
1.) 13C-bicarbonate experiments
a.) After a 10-h incubation, the 13C
uptake measured in the bacterial matswas approximately twice as high asafter 4 h.
b.) Incorporation rate was higher in themouth part of the shrimp.
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Results
2.) 14C-bicarbonate experiments
a.) High 14C incorporation rates were
observed in the bacterial mats of themouth part and brachiostegite biofilm.
b.) Most of the shrimp tissues analyzed
showed significant 14C incorporation,whatever the incubation conditions.
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Results
3.) 14C-acetate experiments
a.) Bacterial mats showed high rates,
indicating that acetate is a suitablecarbon source for these bacteria.
b.) High rates of 14C incorporation
were measured in the integument,especially in areas lining the gillchamber(OB and Gi).
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Results
4.) 3H-lysine experiments
a.) The highest rates were again found
in the bacterial mats (MP and BB),suggesting that these bacteria can uselysine, probably for protein synthesis.
b.) Significant rates were measured inthe gill chamber integument; internaltissues showed low labelling
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Results
5.) Autoradiographya.) 3H-lysine gave the best result with theshortest exposure time, followed by 14C-acetate and 14C-bicarbonateb.) The shrimp tegumental tissuesappeared densely labelled after incubationwith 3H-lysine or 14C acetate, but sections
of shrimps incubated with 14C-bicarbonaterequired very long exposure times beforethe label appeared
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5.) Autoradiography
c.) After 20 –30 days of exposure, gills
and muscles also appeared moderatelylabelled but neither the DT nor the HPshowed any measurable labelling.
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Discussion
1.) Bacteria identified
– Gill chamber epibiont population of R.
exoculata is dominated by gamma- andepsilonproteobacteria, and interaction issyntrophic.
– Epsilonproteobacteria oxidizes sulphur
compounds through the Sox pathway, andfix carbon through the reverse tricarboxylicacid cycle
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Discussion
– Gammaproteobacteria appears asfilamentous and rod-shaped morphotypes
that grow as sulfide oxidizers – Type-I methanotrophicgammaproteobacteria anddeltaproteobacteria have been identified
through aprA and hydrogenase genesequences
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Discussion
– Authors hypothesise that an internalsulphur cycle between sulfur-oxidizing
epsilon- and gammaproteobacteria and sulphate-reducingdeltaproteobacteria could take place inthe shrimp gill chamber.
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Discussion
2.) Shrimp-associated bacteria carry outchemosynthetic inorganic carbon fixationa) the carbon incorporation rates for 13C- and
14C-bicarbonate are in good agreementb) carbon fixation rate increases in thepresence of electron donors (S2O3 2 , Fe2þ)c) they are stable over time, as confirmed by
the time related increase in 13C incorporationd) autoradiographs of histological sectionsshow labelling of bacterial mats
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Discussion
3.) On the carbon fixation rate- The carbon fixation rates observed hereare similar to but somewhat higher thanthose observed by Polz et al. in 1998 withcarbon content of proteins being estimatedat 45%- The carbon fixation rates determined
here are much lower, however, than thoseobtained for other chemosyntheticsymbionts
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Discussion
4.) R. exoculata epibionts have an internal energystore
- The relatively high carbon fixation rateobserved suggests that epibionts have aninternal energy store that supplies autotrophicmetabolisms in the absence of an externalelectron donor
- Energy could be stored as iron polyphosphateand sulphur globules in thin filamentousbacteria such as gamma-ectosymbionts
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Discussion
4.) R. exoculata epibionts have an internalenergy store- globules were shown to be empty inspecimens maintained in a pressurisedaquarium without electron donors- This suggests that the enhanced carbonfixation observed might reflect a switch of
gamma/epsilonepibionts to an externalenergy source such as thiosulphate whichsupports autotrophic metabolism.
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Discussion
5.) Carbon transfer of bacteria to shrimp
- results demonstrate the transfer of carbon
fixed by bacteria to the host shrimp tissuesand the ability of shrimps to take up dissolvedorganic molecules across their integument.
- autoradiographs show that there are higher
radiotracer concentrations in the shrimptissues than in the incubation media
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Discussion
5.) Carbon transfer of bacteria to shrimp
– Findings confirm that R. exoculata depends
on its epibionts for its nutrition. – They thus strengthen the view that the
relationship between bacteria and shrimp ismutualistic.
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Conclusion
1.) Ectosymbionts of R. exoculata carryout chemosynthetic inorganic carbon
fixation; and2.) R. exoculata assimilates organic
products of its ectosymbiotic bacteria;
thus3.) Mutualistic relationship between
R. exoculata and ectosymbionts