Research paper on Archae

8/10/2019 Research paper on Archae

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ORIGINAL ARTICLE

Archaea in metazoan diets: implications for foodwebs and biogeochemical cycling

Andrew R Thurber1, Lisa A Levin1, Victoria J Orphan2 and Jeffrey J Marlow21Integrative Oceanography Division, Scripps Institution of Oceanography, University of California,San Diego, La Jolla, CA, USA and 2Division of Geological and Planetary Sciences, California Instituteof Technology, Pasadena, CA, USA

Although the importance of trophic linkages, including top-down forcing, on energy flow andecosystem productivity is recognized, the influence of metazoan grazing on Archaea and thebiogeochemical processes that they mediate is unknown. Here, we test if: (1) Archaea provide a foodsource sufficient to allow metazoan fauna to complete their life cycle; (2) neutral lipid biomarkers(including crocetane) can be used to identify Archaea consumers; and (3) archaeal aggregates are adietary source for methane seep metazoans. In the laboratory, we demonstrated that a dorvilleid

polychaete,Ophryotrocha labronica, can complete its life cycle on two strains of Euryarchaeota withthe same growth rate as when fed bacterial and eukaryotic food. Archaea were therefore confirmedas a digestible and nutritious food source sufficient to sustain metazoan populations. Both strainsof Euryarchaeota used as food sources had unique lipids that were not incorporated intoO. labronicatissues. At methane seeps, sulfate-reducing bacteria that form aggregations and livesyntrophically with anaerobic-methane oxidizing Archaea contain isotopically and structurallyunique fatty acids (FAs). These biomarkers were incorporated into tissues of an endolithofaunaldorvilleid polychaete species from Costa Rica (mean bulk d13C 924%; polar lipids 116%)documenting consumption of archaeal-bacterial aggregates. FA composition of additional soft-sediment methane seep species from Oregon and California provided evidence that consumption ofarchaeal-bacterial aggregates is widespread at methane seeps. This work is the first to show thatArchaea are consumed by heterotrophic metazoans, a trophic process we coin as archivory.The ISME Journal(2012) 6, 16021612; doi:10.1038/ismej.2012.16; published online 8 March 2012Subject Category: microbial ecosystem impactsKeywords: anaerobic methane oxidation; archivory; authigenic carbonate; biomarker;

microbialmetazoan interactions; cold seep

Introduction

Trophic interactions represent the most importantclass of feedback phenomena on Earth (Worm andDuffy, 2003), but we have yet to include one of themost abundant forms of life in the oceans, Archaea(Karner et al., 2001;Franciset al., 2005;Lippet al.,2008), into our understanding of these trophicrelationships. Archaea perform key ecosystem ser-vices, including nitrification (Beman and Francis,2006), anaerobic methane oxidation (Treude et al.,

2003) and chemosynthetic production (Wuchteret al., 2003;Boetius and Suess, 2004;Herndl et al.,2005). These services are likely impacted by grazingpressure, a top-down force that can cause changesin productivity and nutrient cycling (McNaughton,1985; Belovsky and Slade, 2000). Integrating Ar-chaea into our understanding of marinetrophic

relationships may provide fundamental informationabout mechanisms that control archaeal-drivenbiogeochemical processes. Yet, Archaea are knownto prevail in low-energy systems and grow slowly(Valentine, 2007); thus, it is unclear if Archaea canprovide a food source that supports persistentmetazoan populations. In addition, highly-stablecell membranes of Archaea (van de Vossenberget al., 1998) may provide protection from digestionwhile failing to provide essential fatty acids (FAs)that most Metazoa need for growth and reproduc-

tion. Here, we demonstrate that (1) Archaea is a foodsource that can provide the nutrients necessary for ametazoan to complete its life cycle and (2) archaealbacterial syntrophic aggregates are a primary foodsource for a family of methane-seep annelids, thusidentifying a novel relationship between Archaeaand Metazoa.

Methane-seep ecosystems contain abundantmethanotrophic Archaea that have a unique carbonisotopic and lipid signature. Aggregates of anaero-bic-methane oxidizing Archaea (ANMEs) andsulfate-reducing d-proteobacteria (SRBs) consumethe majority of methane released from deep-sea

Received 29 July 2011; revised 2 February 2012; accepted 2February 2012; published online 8 March 2012

Correspondence: AR Thurber. Current address: College of Earth,Ocean, and Atmospheric Sciences, Oregon State University, 104COAS Administration Building, Corvallis, OR 97331-5503, USA.E-mail:[email protected]

The ISME Journal (2012) 6, 16021612

&2012 International Society for Microbial Ecology All rights reserved 1751-7362/12

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reservoirs through the anaerobic oxidation ofmethane (AOM;Boetius et al., 2000;Orphan et al.,2001b, 2002; Reeburg, 2007). Methane often has adistinct 13C to 12C ratio that is depleted in 13Crelative to 12C (Whiticar, 1998d13C notation isexplained within). In turn, ANME consortia aredefined by similar highly negative d13C values,

ranging between 30% and 100% for archaealcells and 15% to 70% for associated symbioticSRB (Orphanet al., 2002;Houseet al., 2009). Thesevalues are often distinct from those of photosyn-thetic production, which normally has a d13C valueof15% to25% (Fry and Sherr, 1994). In additionto their unique cell carbon-isotopic composition,both ANMEs and SRBs possess unique membranelipids, which also exhibit a highly negative carbon-isotopic composition. ANME archaeal lipids haved13C values between 128% to 61% and arecomposed of repeating isoprene units with or with-out an ether-linked glycerol head (Elvertet al., 1999;Boetius et al., 2000;Hinrichs et al., 2000;Niemannand Elvert, 2008). SRB within these consortia haveFAs largely composed of 16:1(n5), cyc17:0w5,6,and/or a15:0 and 18:1(n7) FAs with d13C valuesbetween 112% and 65% (Elvert et al., 2003;Blumenberget al., 2004;Niemann and Elvert, 2008).These aggregates can make up to 80% of themicrobial biomass in methane seep sediments(Boetius et al., 2000) and act as deep-sea engineersby creating hard substrate in the form of authigeniccarbonates (Greinertet al., 2001;Luffet al., 2004). Inaddition, ANME aggregates routinely occur in thetop four centimeters of sediment (Boetius et al.,2000;Elvert et al., 2003;Knittel et al., 2003;House

et al., 2009), placing them in contact with metazoangrazers, including polychaetes of the familyDorvilleidae (Levin et al., 2003).

The 13C-depleted Archaea at methane seepsprovide a model system to explore archivory, a termwe define here to mean the consumption of Archaea.The carbon-isotopic signature of heterotrophic or-ganisms is derived from the isotopic signature of theprimary producer they or their prey feed upon(DeNeiro and Epstien, 1978). Within methane seeps,dorvilleids have been reported to exhibit extremelynegative d13C values (for example, 90.6% in theGulf of Alaska; Levin and Mendoza, 2007). Theseisotopic values potentially reflect the consumption

of ANME aggregates (Levin and Michener, 2002).Yet, bacterial methanotrophs also have a depleted13C, methane-derived isotopic composition (Elvertet al., 2000;Werne et al., 2002) and chemoautotro-phic producers, including sulfide-oxidizing bacte-ria, can incorporate a 13C-depleted isotopic signaturefrom a 13C-depleted DIC pool (Fisher, 1990). Thus, aconsumer with an extreme 13C depletion need notdirectly consume methanotrophic Archaea.

Grazer lipids are partially derived from their diet(Dalsgaardet al., 2003), providing a tool for trackingmicrobial consumption and potentially archi-vory. This phenomenon is best known for FAs, a

component of both eukaryotic and bacterial cellmembranes that can provide a quantitative measureof food web linkages (Iverson, 2009). In seepsettings, key FA biomarkers include 16:1(n7) and18:1(n7) that are abundant in sulfide-oxidizingbacteria (McCaffreyet al., 1989), the aforementioned16:1(n5) and cyc17w5,6 that are abundant in

sulfate-reducing bacteria (Elvert et al ., 2003;Blumenberg et al ., 2004), and 16:1(n6) and16:1(n8) that are indicative of type I aerobicbacterial methanotrophs (Bowman et al ., 1991).Composition of photosynthetic production can beidentified by polyunsaturated fatty acids, including22:6(n3) and 20:5(n3); (reviewed in Dalsgaardet al., 2003). Archaeal lipid biomarkers are diagnos-tic yet their incorporation into metazoan tissues hasyet to be documented, limiting their utility in food-web studies.

Owing to the ubiquity of dorvilleids in Archaea-fueled methane seep sediments and their 13C-depleted carbon-isotopic composition (Levin andMichener, 2002; Levin et al., 2003; Levin, 2005;Levin and Mendoza, 2007; Menot et al., 2010; Rittet al., 2010), we chose to use this polychaete familyto study archivory. Dorvilleids are tolerant to sulfidestress allowing them to numerically dominate themacrofauna in microbial-mat covered sediments atseeps (Levinet al., 2003;Levin, 2005), a habitat thathas high ANME abundance (Boetius and Suess,2004). To begin testing if Archaea are a viable andutilized food source, we combined laboratory-basedfeeding assays with trophic studies in soft-sedimentand authigenic-carbonate seep habitats to addressthe following questions: (1) Can metazoans survive

and reproduce on a diet exclusively of Archaea? (2)Does an archaeal diet manifest itself in consumerlipid signatures? (3) Is there evidence for archaealconsumption by metazoans in natural populations?

Materials and methods

Sample collection and preparationTo test if archaeal biomass provides sufficient nutri-tion for sustaining metazoan populations and resultsin a unique lipid pattern within archaeal consumers,we raised a species of dorvilleid polychaete onArchaea within the laboratory. The shallow water

dorvilleid, Ophryotrocha labronica, was raisedon monocultures of two types of halophilic Eury-archaea, Halobacterium salinarium and Haloferaxvolcanii, and its growth rate over a 4448 dayperiod was compared with the same species fedmonocultures of bacterial (Bacillus subtilis, a gram-positive Firmicute, or Photobacterium profundum,a gram-negative g-proteobacterium) or eukaryoticfood sources (Oryza sp., rice, or Spinacia oleracea,spinach). Individuals of O. labronica were raisedon either one of the two-archaeal sources or thebacterium B. subtilis for neutral lipid comparison.Between 11 and 14 feeding trials were run with each

Archivory by metazoansAR Thurber et al

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food source during which egg mass deposition wasnoted if it occurred (see Supplementary Materialsfor additional information).

In situ archivory was studied using specimensand substrate from three deep-sea, methane-rich locations with high abundances of Archaea:authigenic carbonates precipitated on Mound 12,

Costa Rica (8155.80N 84118.80W; 1000 m) and soft-sediment seep habitats at Eel River, California(40147.10N 124135.680W; 490520 m) and at HydrateRidge, Oregon (44140.10N 125105.80W; 580890 m).Authigenic carbonate rocks were collected fromCosta Rica during RV Atlantis cruise 1544 (rockL2; 21 February 8 March, 2009) and 1559 (rockE3; 612 January, 2010). The rocks were recoveredfrom active seep areas by the submersible DSRV Alvinand placed into insulated boxes for transport to theship. Carbonates were broken open using a chiseland endolithofauna were removed. We focused ona single undescribed dorvilleid polychaete specieswithin the genusDorvillea, which was found livingwithin the rocks. Soft-sediment seep habitats offCalifornia and Oregon containing a high abundanceof methanotrophic Archaea (Orphan et al., 2001b;Elvert et al., 2003; Knittel et al., 2003;Boetius andSuess, 2004) were sampled during R/V Atlantiscruises 157 (1327 July, 2006) and 1511 (26September10 October, 2006) using the DSRV Alvin.Sediment was collected using push cores or scoopsfrom clam beds and microbial mats. Sedimentsamples were sieved with a 300-mm sieve andinfauna were sorted live, yielding five species ofdorvilleid polychaetes for biomarker analysis(Ophryotrocha maciolekae, Ophryotrocha platyke-

phale, Parougia oregonensis, and an undescribedspecies of each Parougia and Exallopus). Allindividuals analyzed were allowed to evacuate theirguts overnight in 25-mm filtered sea water and frozenat 80 1C. To document the availability of ANMEaggregates to rock-dwelling dorvilleids, the micro-bial community present within a subsample of rockE3 was identified using FA profile analysis (mod-ified from Lewis et al., 2000; see SupplementaryMaterial for additional methods), 16S rRNA analysisand epifluorescence microscopy protocols describedinOrphanet al. (2001a).

Lipid analysesLipid profiles of archaeal food sources,O. labronicafed those food sources, and dorvilleids collectedin the field were measured to assess whetherarchaeal lipids can function as archivory biomarkers.Key archaeal lipids, including hydroxyarchaeol,archaeol, crocetane and pentamethylicosane (PMI)are present in the neutral lipid fraction (Elvertet al.1999; Hinrichs et al. 1999; Thiel et al., 1999; Obaet al., 2006) and if present within the tissue ofmetazoans that consume Archaea, would provide apowerful tool to identify Archaea. FAs of field-collected individuals and rock E3 were extracted

using a one-step extractiontransesterification. Thistechnique was chosen due to its effectiveness atextracting low-biomass (o0.2 mg) samples. Thisallowed us to avoid pooling individuals acrosssamples and thus have true replication in all butone of the species collected. Owing to the smallbiomass of most dorvilleids, gas chromatography-

mass spectrometry (GCMS) analysis, which ishighly sensitive but less quantitative than GCFID,was employed. Thus, whereas internal comparisonswithin this study are valid, absolute concentrationsare not quantitatively comparable to values gener-ated by other instruments.

Carbon isotopic analysesIn all, three types of isotopic analysis wereperformed on field-collected samples to trackmethane-derived carbon, potentially indicative ofarchivory: d13C Bulk, d13C Bulk-Lipid, and d13C

Compound-Specific. d13

C Bulk is the carbon-isotopic composition of the non-lipid extracteddorvilleid biomass. d13C Bulk-Lipid involved sepa-rate analyses of polar and neutral lipids withinDorvillea sp. from rock L2. Biomarkers that aredirectly incorporated from methanotrophic Archaeashould have a very 13C-depleted carbon isotopicsignature, thus this analysis identifies whether thepolar or neutral fraction contains Archaea-derivedcarbon. d13C Compound-Specific was used toidentify the isotopic composition of each of theFAs present within both rock E3 microorganismsand theDorvilleasp. that inhabited it. This allowedfine-scale identification of which FAs were present

within the food source (the rock) and were incorpo-rated intoDorvillea sp. tissues.

Results

Archaea as a food sourceLaboratory growth and fecundity experiments withO. labronica demonstrated that Archaea providesufficient nutrition for this species to close its lifecycle. Ophryotrocha labronica grew from 0.2 mm(post hatching) to B1.110.4 mm (adult size) in aslittle as 12 days on monospecific archaeal diets ofboth H. salinarium (n 13, separate egg broods,

which we heretofore refer to as a separate cohorts)and H. volcanii (n 11, separate cohorts). Meangrowth rate of O. labronica over a 4447-dayperiod did not vary as a function of food source(Figure 1; F5,29 1.36, P 0.27). After 4558 days,three cohorts fed H. salinarium produced eggmasses and one cohort fed H. volcaniiproduced anegg mass. This reproductive success was similar tothe cohorts fed the other two domains of life over asimilar 4548-day period. The two bacterial foodsources, B. subtilis and P. profundum supportedproduction of three and one egg mass by 14 and12 separate cohorts, respectively. The 12 cohorts


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of O. labronica fed Oryza sp. or S. oleracea, bothresulted in deposition of a single egg mass.

Neutral lipid biosignatures in laboratory-rearedspecimensArchaeal food sources did not generate a distinctiveneutral lipid pattern in O. labronica. Both strains ofEuryarchaeota had a neutral lipid composition thatincluded squalene, dihydrosqualene and tetrahy-

drosqualene in addition to a few other isoprene-based molecules (Table 1). The worms fed these twoarchaeal species did not incorporate these mole-cules, but instead had a neutral lipid profileconsisting largely of cholesterol and other sterols.With the exception of b-sitosterol, these samecompounds were found in O. labronica fed thebacterium B. subtilis (Table 1). None of the neutrallipids in Archaea-fed O. labronica tissues werearchaeal-specific, despite the fact that this speciescould grow to reproduction on an Archaea mono-culture. We note that although other researchershave found archaeol in both of the Archaea that wehave used here (Qiuet al., 2000;Stiehlet al., 2005),

we did not resolve these compounds within ourcultures and thus our findings do not include intactpolar lipids.

ANME aggregates in authigenic carbonatesArchaea available to dorvilleids at methane seepsoften occur as ANME aggregates. In the interior ofcarbonate rock E3, microbial aggregates resemblingpreviously described consortia of ANME and SRBwere observed (Figure 2;Orphan et al., 2002). PCR-based 16S rRNA gene analysis of DNA recoveredfrom rock E3 revealed an archaeal assemblage that T

able1Percentneutrallipidcompositionofcold-seepdorvilleidpolychaete

speciesandOphrotrochalabronicafedEuryarchaeainthelaboratory

Chemicalname

Formula

M.W.Dorvillea

sp.

O.

labronica

fedH.volcanii

O.

labronica

fedH.salinarium

O.

labronica

fedB.subtilis

Parougia

oregonensis

Exallopus

spp.

Ophryotrocha

maciolekae

Ophryotrocha

platykephale

H.volcanii

H.salinarium

Cholesterol

C27H46O

458

41.5

69.1

77.2

81.6

71.6

87.2

82.8

81.5

Desmosterol

C27H44O

456

27.1

9.6

12.5

Tr

3.5

7.8

13.7

Brassicasterol

C28H46O

470

25.3

8.6

Tr

ND

12.8

4.3

4.7

Cholestanol

C29H52O

488

1.9

Other

6.3

4.6

b-sitosterol

C29H50O

486

6.1

a

12.7

ND

Tr

9.2

2.9

ND

Squalene

410

6

0.3

3.1

Dihydrosqualene

412

3

0.7

4.3

Tetrahydrosqualene

414

7.4

81.2

Phytol

ND

ND

ND

ND

ND

ND

ND

ND

0.7

11.3

Phytane

370

ND

ND

ND

ND

ND

ND

ND

ND

1.1

Farnesane

294

ND

ND

ND

ND

ND

ND

ND

ND

2.5

Abbreviations:M.W.,molecularweightofTMSderivative;ND,notdetectedatcon

centrationanalyzed;Tr,trace.

Archaeaareidentifiedinboldandonly

compoundsthatweidentifiedarereported

.Otherindicatesthatitsmassspectracouldnotbeconclusivelyidentified.

aIndicateprobablyidentity.

Figure 1 Mean daily growth rate of Ophryotrocha lobronica

in the laboratory over a 4448-day period as a function of foodsource. Error bars 1 s.e.


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was dominated by methanotrophic ANME-2 (68%),with a lower percentage of ANME-1 (10%) phylo-types (following the classification ofOrphan et al.,

2001b). Rock E3 was largely composed of two FAs,cyc17:0 and 16:1(n5), each comprised more than20% percent of the total FA composition of the rock(Figure 3). The isotopic signatures of these two FAswere extremely depleted in 13C, with d13C values of115% and110%, respectively, providing furtherevidence that they are derived from SRBs that

participate in AOM (sensu Elvert et al., 2003). Inaddition, branched 15-carbon FAs and 18:1(n7),which are common in ANME-1/SRB aggregates(Elvertet al., 2003;Blumenberget al., 2004), formeda combined 23.8% of the FAs present within therock. This FA profile supports the findings ofthe 16S rRNA gene sequences that indicated thatthe majority of the microbial community within therock was comprised of ANME-2 consortia with asmaller subset belonging to ANME-1-associatedSRB. Furthermore, crocetane, an archaeal lipid,had a d13C value of 122% that clearly indicateda methane-derived energy source for the Archaeapresent. Both 16:0 and 18:0 were more enriched in13C than the other FAs present. Although these datawere corrected for concurrently run blanks, tracecontaminants from the sample processing wereapparent in both of these saturated FAs and thustheir isotopic values are unlikely to represent the

Figure 2 Micrograph of ANMESRB aggregate from inside rockE3 stained with DAPI. Scale bar 15mm.

Figure 3 Carbon isotopic compositions of fatty acids (FAs) and FA distribution within (upper panel) carbonate rock E3 and (lowerpanel) Dorvillea sp. from Costa Rica. Left yaxis and bars are percentage of FA and right yaxis and points are isotopic composition.Asterisks indicate that isotopic composition was potentially largely impacted by contaminants (see text for more details). Value for thisomitted FA ranged between 104% to47%. 18:2 and 20:1(n11) were left off this figure as they were o2% in only one individual ofDorvilleasp. See text for explanation ofde novosynthesis identification.


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isotopic composition of those FAs from the sample.There were no contaminats in the other FAsanalyzed. Diagnostic FA biomarkers for aerobicbacterial methanotrophs 16:1(n6) and 16:1(n8)(Bowman et al., 1991) were not recovered fromcarbonate rock E3.

SRB/ANMEs in the diet of dorvilleidsA combination of stable-carbon isotope and FAanalysis indicate that the carbonate-associatedDorvillea sp. at the Costa Rica seeps consumedmethanotrophic ANME/SRB aggregates as theirmain food source. Among the most 13C depletedmetazoans on record, the d13C bulk signatures ofDorvillea sp. were as low as 101% (from authi-genic carbonate rock L2) and within rock E3 thisspecies had a mean d13C of 91.73.5 (s.e.) %(n 10). TheDorvilleasp. lipid profile included FAslikely derived from ANME-associated SRBs (Fig-

ure 3). These FAs included 16:1(n5), whichcomposed 1.41.8% of the total FAs present withinDorvillea sp. from rocks E3 and L2. The FA18:1(n5) also formed between 3.3% and 5.3% of

the Dorvilleas FA profile. This FA can be synthe-sized by eukaryotes if they are provided with16:1(n5), but was not present within rock E3. Bothof these (n5) FAs had isotopic signatures within3% of that of the sulfate-reducing bacteria (n5) FA(Figure 3). This indicates that these FAs werederived from the carbonate AOM consortia.

To test if consumption of the AOM consortia is anevolutionary oddity limited to Costa Rica carbonatesor a common occurrence within methane-seepenvironments, we then examined five additionaldorvilleid polychaete species from NE Pacificmethane seep sediments. The d13C bulk composi-tion of these species varied between 19.5% and57.9% indicating use of a diversity of food sources,including potentially photosynthetic and AOM-derived organic matter. To understand the relation-ship between carbon isotopic signature and FAcomposition of the worms, a regression of percent(n5) FA on d13C was performed. As with thecarbonate endolithofauna, a subset of these dorvil-leids possessed (n5) FAs (Table 2). The distribu-tion of these FAs was not uniform among thespecies; instead the greater the composition of

Table 2 Bulk carbon isotopic composition and fatty acid (FA) percent of total FA profile of each dorvilleid polychaete species includedin analysis

Exallopusspp. Ophryotrochamaciolekae

Ophryotrochaplatykelphale

Parougiaoregonensis

Parougiasp. Dorvillieasp.

n 5 3 11 2 1 2d13C 45.03.5 24.50.6 26.22.4 52.6 39.4 91.5

14:0 2.3

0.4 3.5

0.9 1.5

0.4 2.4 0.916:1(n7) 11.71.3 4.51.9 5.61.1 2.4 3.3 1.916:1(n6) 0.60.2 0.10.1 0.116:1(n5) 3.11.3 0.20.1 0.6 0.8 1.616:0 16.01.8 25.65.2 20.23.4 11.7 11.5 11.018:2n6c 1.60.3 0.40.4 1.60.4 0.3 0.518:1n9t 0.40.4 1.21.2 0.30.318:1n9c 7.05.8 8.26.9 2.50.4 1.0 0.8 1.418:2n6t 1.61.6 1.40.7 0.40.418:1(n7) 27.86.7 7.43.7 22.03.6 48.5 48.3 39.918:2b 6.92.0 0.30.3 6.41.2 3.318:1(n5) 1.30.5 0.20.2 1.3 1.4 4.418:0 3.91.9 20.39.3 9.42.4 2.7 2.5 3.2cyc19 0.90.3 0.720:4n6 0.60.2 2.51.4 1.00.3 0.6 2.3 1.420:5n3 7.61.8 13.85.4 9.41.7 7.8 21.6 18.820:2 0.90.9 3.01.5 3.00.9 0.8

20:1(n13) 2.90.8 2.22.2 1.10.8 3.8 5.6 6.920:1(n9)a 0.90.920:1(n7) 1.30.6 0.90.5 0.6 1.1 0.720:1a 0.40.4 0.70.7 0.80.6 3.220:3 0.40.4 1.70.620:0 0.50.5 1.90.9 0.50.3 3.021:0 0.10.022:0 0.50.3 3.323:0 0.40.4 3.22.5 3.924:1 0.30.324:0 0.30.3

Isotopic data are provided for either a fraction of the individual extracted for lipids or from individuals within the same core or scoop sample; s.e.is given. Only data are presented for samples that corresponded to an isotopic measure and only FAs are presented that made up at least 1% ofany one sample.aIndicates that double-bond position was estimated from retention time rather than DMDS adduct formation.


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(n5) FAs the more negative the d13C value(Figure 4). This simple relationship between percent(n5) and d13C explained 50% of the varianceobserved in the carbon isotopic composition ofthese species (F1,20 20.4,Po0.01; R

2 50.5):

d13C 3:516 :1n 5 18 :1n5 28:2 1

Removal of one outlier appeared to have a largeinfluence on this regression (FA composition of10.1% (n5) FAs) and increased the negative slopeof the regression from 3.5 to 5.6 but had littleeffect on the variance explained (F1,19 17.1,

po0.01; R2

47.4). Thus, we chose to retain alldata. Inclusion of the (n7) FAs, common in sulfide-oxidizing bacteria (McCaffreyet al., 1989), improvedthe fit of the model only by 1.5%. A regression ofisotope signature against solely percent (n7) FAs,explained only 20% of the variance (F1,20 5.30,P 0.03;R2 20.9). There was a small percentage of16:1(n6) FA present in three of the dorvilleidspecies, which are FAs indicative of aerobic metha-notrophy, yet these were a minor component of thelipid profile (Table 2).

Neutral lipids of species that consume SRB/ANMEaggregatesThe neutral lipids within the Costa Rica seepdorvilleid species were isotopically distinct fromarchaeal lipids and had a sterol composition thatwas uniform among the species analyzed from CostaRica, NE Pacific and the laboratory (Table 1). Thecarbonate-associated individual with a bulk d13C of101%, had a polar-lipid d13C of 116% and aneutral lipid d13C of68%. This indicated that themost 13C-depleted carbon, and thus biomarkers frommethanotroph biomass, was incorporated into thepolar-lipid fraction rather than the neutral lipidswhen viewed in bulk. Although the neutral-lipid

fraction also showed clear incorporation ofmethane-derived carbon, this isotopic compositionis far removed from the lipid signature of the SRB(here, d13C 110%; Figure 3), the archaeal lipidcomposition (d13C 122%) or the dorvilleid iso-topic composition (d13C Bulk 924%) recov-ered from rock E3. In addition, the neutral-lipid

composition of this species,Dorvilleasp., and all ofthe other dorvilleid species did not include croce-tane.

Discussion

Ingestion or digestionThe FAs present within the Dorvillea sp. hadisotopic signatures that were o100% (except fora 20:0 FA that was 95%), clearly indicatingincorporation of methane-derived carbon (Figure 3).Of special note, two polyunsaturated fatty acids,20:5(n3) and 20:4(n6), commonly used as in-dicators of phytoplanktonic production (Dalsgaardet al., 2003), had d13C values between 103% and109%, indicating that they were formedin situ byconsumed methane-fueled bacteria or synthesizedde novo by the dorvilleids. The possibility thatannelids may be able to synthesize these FAs hasbeen previously considered; three species thatbelong to the family Siboglinidae, which lack bothmouth and anus and live off chemoautotrophicenergy from endosymbionts, all possessed theseFAs, and in one instance they had polyunsaturatedfatty acids with a d13C of72% (Pond et al., 2002;Losekannet al., 2008).

Dorvilleids possess chitonized jaws that provide amechanism to harvest ANME aggregates off ofcarbonate rocks. The effectiveness of this feedingstrategy is likely aided by the large size of ANMEaggregations hosted in carbonate rocks (up to 15 mmin diameter; Figure 2, compared with averagediameters of 37 mm for ANME aggregations insediments; House et al., 2009;Orphan et al., 2009)and is analogous to gastropod use of their radula toharvest epilithic algae in intertidal habitats. How-ever, the fate of ANME/SRB aggregates after theaggregate is consumed may be explained by twopotential hypotheses: (1) dorvilleids may consumethe aggregate but only digest the sulfate-reducing

bacteria and excrete the archaeal component, poten-tially alive or (2) dorvilleids digest the aggregates enmasse and thus their diet is composed of bothArchaea and SRB. Either of these scenarios wouldimpact the symbiotic relationship of the aggregateand the rate of AOM but to different extents.Previous studies have not found d13C SRB biomassto be more 13C depleted than 70% (Orphan et al.,2002; House et al., 2009), thus the incredibly 13C-depleted isotopic composition of the endolithofau-nal Dorvillea sp. supports the idea that archaealbiomass is indeed digested, as was observed in thelaboratory feeding trials.

Figure 4 Relationship between carbon isotopic composition andsum of 16:1(n5) and 18:1(n5) FAs present within five speciesof polychaete from Eel River and Hydrate Ridge. Solid line is alinear regression, including all dorvilleid polychaetes in whichboth isotopic and FA data are available within a single sample(R2 50.5). Dotted line indicates linear regression if the point tothe far right is removed (R2=47.4). Symbols indicate species.


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More information can be gained about these twohypotheses from the FA type and isotopic composi-tion. Eukaryotes have a suite of enzymes that allowthem to form or modify dietary-derived FAs. Inaddition to being able to elongate FAs, eukaryotescan also synthesize a diversity of unsaturated (n7)and (n9) FAs and a variety of 20:1 FAs (Kattner and

Hagen, 1995). Yet, knowledge to date suggests that(n5) FAs are not synthesized by eukaryotes(MacAvoy et al., 2003). Thus, the FA profile ofDorvillea sp. is as expected from a species thatsynthesizes almost all of its FAs except for the (n5)FAs provided by SRB. Elongation of dietary lipidoccurs through the addition of acetate. AssumingDorvillea sp. forms 18:1(n5) from 16:1(n5), wecan apply a carbon mass-balance approach tocalculate the d13C of acetate used by Dorvillea sp.to gain insight into its carbon source. This results inan estimate ofd13Cacetate 109% and 128% usedbyDorvilleasp. from rocks L2 and E3, respectively.Within rock E3, the only compound that had anisotopic composition that was close to 128% wasthe archaeal lipid crocetane (d13C 122%). Thissuggests that Dorvillea sp. at Costa Rica waselongating its FAs using archaeal-lipid derivedcarbon. As no 18:1(n5) was found in the rocksand two-carbon additions to dietary-derived FAsfollows established metazoan FA biosynthesispathways, the general assumptions behind thismass-balance calculation are likely appropriate.However, this does assume that there is noenzymatic fractionation, or specifically selection ofthe more 13C depleted 16:1(n-5) FAs that are thenconverted to 18:1(n-5) or selection of the most 13C

depleted acetate molecules used for this elongation;enzymatic carbon selectivity could preferentiallyselect 12C molecules for use during the elongationprocess. Furthermore, a pool of available acetateoccurs within the sediment and can also be quite 13Cdepleted. Few seep samples have had d13Cacetateanalyzed, but within Black Sea seep sediments thissource of acetate was found to be as negative asd13Cacetate 85% (Heueret al., 2006). Thus, the acetateused by Dorvillea sp. to elongate its FAs may be acombination of acetate derived from its diet and acetatepresent within the dissolved organic carbon poolwithin the rock.

Archaeal biomarkersNone of the archaeal lipids that we identified in thisstudy were preserved unmodified in those taxa thatconsumed Archaea, and neutral lipid compositionappeared independent of diet. These later findingsdo not appear to be entirely unique to dorvilleids;Capitella sp.I also augments its dietary-derivedsterols through biosynthesis (Marsh et al., 1990).Although beyond the scope of this study, whose aimwas to identify if Archaea biomass as a whole wassufficient to sustain metazoans populations, we cangain some insight into the catabolic pathway of

Archaea based on our isotopic results. Squalene, alipid provided by both archaeal food sources fromthe laboratory study, is a de novo formed precursorto most sterols synthesized by metazoans (Kanazawa,2001). Pentamethylicosane and Crocetane, two archaealhydrocarbons, have a similar structure to squaleneyet differ by saturation state. The incorporation

of any of these hydrocarbons into sterol synthe-tic pathways by archaeal consumers may benefitspecies that live in low-energy systems. However,this pathway seems unlikely as the bulk neutral-lipid isotopic composition for Dorvillea sp. was68%, a value far removed from crocetanes d13Cof 122%. Although we did not measure theisotopic composition of other archaeal lipids fromthe rock, we can use the isotopic values ofStadnitskaia et al. (2008) who measured the d13Cof archaeal lipids as 97% to 98% within theauthigenic carbonates at Mound 11, a nearby CostaRican methane seeps, as a potential isotopic valuefor larger archaeal lipids. Alternatively, we can usecrocetanes isotopic composition as a proxy for theisotopic composition of archaeal lipids in rock E3.Previous research has found that archaeal lipids areat most 12% enriched in 13C compared withcrocetane, if not more depleted (Niemann andElvert, 2008). Therefore, we can estimate that thed13C value of the archaeal lipids in this rock areo110%, a value far removed from the neutral lipidisotopic signature measured for Dorvillea sp. Assuch, it is unlikely that the neutral lipid valuereflects direct incorporation of archaeal lipids.

A caveat for the extraction technique used toanalyze neutral lipids is that it may have co-

extracted carbohydrates and/or proteins, contami-nants that may mask archaeal input into the neutrallipid class. Any contaminants extracted in this lipidclass, if 13C enriched, would impact our conclusionthat the neutral lipids do not contain archaeal-derived lipids beyond potential, and unresolved,trace amounts. The bulk isotopic composition of thecarbon withinDorvilleasp. was also much more 13Cdepleted than the neutral lipids that were extractedfrom it. As proteins and to a lesser extent carbohy-drates are often much more abundant in annelidsthan lipids (Blackstocket al. 1982) this bulk isotopiccomposition likely reflects the carbon isotopiccomposition of the potential contaminants. There-

fore, even if we did co-extract non-neutral lipids,potentially masking 13C-depleted sterols or unre-solved compounds, these contaminants wouldlikely skew our neutral lipid signature more nega-tive rather than positive.

The d13C value of the neutral lipids may beexplained by the worm deriving this carbon fromnon-lipid sources of either SRB or ANME aggregates,which are enriched in 13C compared with their lipids,or by the uptake of sterols from the environment,potentially reflecting recalcitrant pools of carbon.As sediments containing active AOM aggregates alsohave 13C-enriched sterols that are far removed from


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the 13C-depleated values of the AOM aggregates(Niemann and Elvert, 2008), uptake of these 13C-enriched sterol pools could impact the sterol neutrallipid isotopic composition. What is clear is thatwhen analyzing the neutral lipids of these species,no clear biomarkers are apparent. Further analysisshould examine larger compounds (including intact

polar lipids, and specifically targeting Archaeol andsn-2-hydroxyarchaeol as well at tetraethers), althoughthe neutral lipid isotopic composition measured heresuggests they are unlikely to be abundant.

Archaea in food websArchaea are a ubiquitous domain of life, and inaddition to their previously recognized importance,we have shown that they are capable of supportinggrowth and reproduction of a heterotrophic metazo-an. Symbiotic relationships are known betweenArchaea and ciliates (van Hoeket al., 2000), sponges(Preston et al., 1996) and even terrestrial ruminants(Joblin, 2005), defining a key role for Archaea insupporting eukaryotes through symbioses. It isunlikely that archaeal symbioses were present inDorvillea sp. as attempts to PCR amplify archaeal16S rRNA from this species were unsuccessful (SGoffredi pers. com.), and, as with all the dorvilleidswithin this study, Dorvillea sp. had a well-devel-oped digestive tract complete with mouth and anus.Thus, this family of polychaete provides the firstevidence for the ecological role of free-living Archaeaas a food source for heterotrophic metazoans. Furtherresearch may identify the specific catabolic pathwaysby which the different archaeal cellular constituents

are digested and used by consumers.ANME are thought to be the terminal sink ofmethane throughout the worlds oceans (Reeburg,2007) yet the impact of metazoan grazing on ANMEsis unknown. Because of aerobic methanotrophicbacteria and ANMEs metabolic activities, a majorityof methane emitted at seeps is consumed within thesediment before release into the water column(Sommer et al ., 2006; Reeburg, 2007). Here, wedemonstrate that the anaerobic part of this sedimentfilter is subject to grazing by metazoans at threeseparate seep locations, with unknown ramifica-tions for the biogeochemical cycling of methanewithin these seeps. Observation of this phenomenon

within authigenic carbonates, habitats that arewidespread on margins, opens a new avenue of Ccycling investigation. The incredibly 13C-depletedcarbon isotopic composition of the endolithofaunalpolychaete within this study highlights the un-known but clearly active role of carbonate-asso-ciated biota in the global methane cycle.

Acknowledgements

We are indebted to the Captains, Crew, Alvin group andScience parties from RV Atlantis legs 15-9; 15-11; 15-44;

and 15-59. Guillermo Mendoza, Jennifer Gonzalez and DrsBrigitte Ebbe, Ken Halanych, Ray Lee and Greg Rouse allhelped tremendously at sea with sorting and identificationof the dorvilleid species. Dr Shana Goffredi kindlychecked for archaeal symbionts within the Costa RicanDorvilleid. Dr William Gerwick provided access to theanalytical instrumentation necessary to carry out thisresearch and Cameron Coates, Jo Nunnery and Tak

Suyama were always present to help trouble shootanalytical problems. Two anonymous reviewers, and DrsG Rouse and Lihini Aluwihare provided helpful com-ments on an earlier version of this manuscript. Thisresearch was supported by NSF Grants OCE 0425317, OCE0826254 and OCE-0939557, and grant UAF-050141 fromthe West Coast National Undersea Research Center to LALevin, OCE-0939559 and OCE-0825791 grants to VJOrphan and a UC Marine Council award to W Gerwickand LA Levin, in addition to the Michael M MullinMemorial fellowship, Sidney E Frank FoundationFellowship, UC Marine Council CEQI Fellowshipand the graduate office of Scripps Institution of Oceano-graphys support of AR Thurber.

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