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



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



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.



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



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)



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.



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



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



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



Shrimp collection

pressurised

IPOCAMP

incubation

13C- and 14C-bicarbonate

incorporation

C-acetate and H-lysineincorporation

Stable isotopeand elementalanalysis

Measurement of incorporatedradioactivity

AutoradiographyStatisticalanalysis

Methods



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



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



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.



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%)



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



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



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



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



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).



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



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



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



Microtome technique



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.



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.



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.



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).



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



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



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.



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



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



Discussion

– Authors hypothesise that an internalsulphur cycle between sulfur-oxidizing

epsilon- and gammaproteobacteria and sulphate-reducingdeltaproteobacteria could take place inthe shrimp gill chamber.



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



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



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



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.



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



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.



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

mcb150 report-hydrothermal vent host shrimp

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