Specificity of Associations between Bacteria and the Coral Pocilloporameandrina during Early Development

Amy Apprill,a,b* Heather Q. Marlow,c* Mark Q. Martindale,c and Michael S. Rappéa

Hawaii Institute of Marine Biology, SOEST, University of Hawaii, Kaneohe, Hawaii, USAa; Department of Oceanography, SOEST, University of Hawaii, Honolulu, Hawaii,USAb; and Kewalo Marine Laboratory, Pacific Biosciences Research Center, University of Hawaii, Honolulu, Hawaii, USAc

Relationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study,the specificity of bacteria associating with the coral Pocillopora meandrina was investigated by exposing coral embryos to vari-ous strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location ofincorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of the Alpha-proteobacteria, a Pseudoalteromonas species of the Gammaproteobacteria, and a Synechococcus species of the Cyanobacteria phy-lum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacte-rial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may havebeen externally associated from the outset of the experiment. Microscopic examination of P. meandrina planulae by fluores-cence in situ hybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the den-sities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains of Pseudoalteromonas andRoseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade.Planulae exposed to sterile seawater or strains of the SAR11 clade and Synechococcus did not show evidence of prominent bacte-rial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence ofinternally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific micro-organisms may be an important factor in the establishment of coral-bacterial relationships.

Adiverse assemblage of endosymbiotic dinoflagellates (zoox-anthellae), endolithic algae, fungi, bacteria, archaea, and vi-

ruses reside within the distinctive skeletal, tissue, and mucus hab-itats of scleractinian corals (7, 9, 47, 48, 52, 63). This complexcommunity of organisms, referred to as the coral holobiont, ishypothesized to work together to maintain the healthy function-ing of individual coral colonies (22, 49). A current challenge incoral biological research is to understand how the different com-ponents of the coral holobiont function, both independently andcollectively, to create a healthy, functioning colony. The study ofmicroorganisms (defined here as bacteria and archaea) associatedwith corals has been greatly advanced by the use of cultivation-independent approaches, and numerous studies have now high-lighted the abundance and diversity of microbes associated withadult corals (48, 54). These microorganisms are hypothesized toplay a role in the transfer of potentially sparse nutrients to thecoral organism (31, 62) and the production of probiotic com-pounds (19, 44).

Cultivation-independent studies are often performed on ho-mogenized coral samples, making it difficult to assign specifichabitats to the identified microorganisms within the biochemi-cally diverse mucus, tissue, and skeletal microenvironments of thecoral (17, 25). These microhabitats provide unique niches that areundoubtedly favorable to particular microbial functions and,therefore, may harbor specific assemblages of microorganisms.The application of fluorescence in situ hybridization (FISH) tech-niques to examine the proximity of microorganisms to the coral’scellular architecture provides one means to assess how specificmicrobial taxa physically associate with the coral and offers aframework for examining taxon-specific microbial interactionswithin the holobiont. To date, only a few studies have localized

individual microbial cells within the coral holobiont using FISHtechniques (2–5, 13).

Relationships with specific bacterial taxa have now been dem-onstrated for several species of coral over various geographicranges (33, 49). For example, the Gammaproteobacteria speciesPA1 was shown to be widely distributed with the coral Poritesastreoides—a relationship maintained over thousands of kilome-ters (49). However, the mechanisms that corals utilize to preservespecific associations with microorganisms are not well under-stood. Many invertebrates maternally transmit microbial symbi-onts to their offspring in order to ensure the longevity of theassociation, and this mechanism provides opportunities forcoevolution and codiversification of the hosts and their symbionts(41). Horizontal transmission of microorganisms from the envi-ronment is also a well-utilized mechanism for acquiring symbi-onts (20, 39, 45). These relationships are also frequently speciesspecific and require the availability of symbionts from the envi-ronment and for both partners to be actively involved in the sym-biont incorporation process (39). Additionally, the accessibility of

Received 18 April 2012 Accepted 8 August 2012

Published ahead of print 17 August 2012

Address correspondence to Michael S. Rappé, rappe@hawaii.edu.

* Present address: Amy Apprill, Woods Hole Oceanographic Institution, WoodsHole, Massachusetts, USA; Heather Q. Marlow, European Molecular BiologyLaboratory, Heidelberg, Germany.

This article is SOEST contribution 8732 and HIMB contribution 1514.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01232-12

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certain symbionts from the environment may be reliant on par-ticular conditions.

The onset of association between microorganisms and coralswas recently examined throughout the early developmental cycleof the coral Pocillopora meandrina using a variety of moleculartechniques, including FISH (6). In that study, microorganismswere not detected internally within the tissues of eggs or embryosup to 51 h old, but bacteria (primarily belonging to the Roseobac-ter clade of Alphaproteobacteria) were found within the tissues ofplanulae (free-swimming larval corals possessing a simplifiedbody plan including a mouth and ectoderm [outer] and endo-derm [inner] tissues) that were at least 79 h old. Based on thedetection of the specific Roseobacter lineage in the surroundingseawater and observations that the number of Roseobacter cellsincorporated into P. meandrina tissues increased with the densityof microorganisms they were reared with, it was hypothesized thatthe bacteria originated from seawater (6).

In an effort to expand our previous observations, we sought tofurther examine the specificity of bacterial associations with de-veloping Pocillopora meandrina. We exposed developing embryosto dilute seawater cultures of marine microorganisms that wereisolated from seawater of a coral reef environment and from anadult coral. A rRNA gene-based approach was utilized in conjunc-tion with fluorescence microscopy to determine whether and howthe cultured microorganisms associated with 170-h-old planulae.Additionally, electron microscopy was utilized to examine thespecific location of microbial cells in relation to the larval coraltissues. The ability to rear P. meandrina with different strains ofmicroorganisms provides a unique system to examine the speci-ficity and localization of microorganisms incorporated intoplanular tissues.

MATERIALS AND METHODSExposure of P. meandrina embryos and larvae to bacterial cultures. On21 April 2008, fragments of healthy P. meandrina colonies were removedfrom the reef flat in Kaneohe Bay, Oahu, HI (21°27.345=N, 157°46.961=W)and held in seawater aquaria for 2 days prior to spawning on the morningof 23 April 2008. Eggs from 10 colonies were combined, fertilized withsperm for 30 min, and subsequently rinsed using seawater sterilized by themethod outlined below. A subsample of 4-h-old embryos was rinsed withsterile seawater and preserved for microscopy by fixation with 3.7% para-formaldehyde in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) for 6 h at 4°C, followed by fivewashes in phosphate-buffered saline for 5 min each at room temperature.The embryos were subsequently dehydrated in 100% ethanol and frozento �20°C.

The remaining embryos were placed in individual 100-mm-diameterpetri dishes containing raw seawater, sterile seawater, or individual bac-terial strains as treatments (Table 1). The bacterial strains utilized in this

study are single-strain laboratory cultures that represent some of the ma-jor groups of planktonic bacteria present in Kaneohe Bay seawater (M. S.Rappé and M. L. Brandon, unpublished data), as well as an isolate from anadult coral (Table 1). Water for the sterile-seawater treatment was col-lected from the surface of Kaneohe Bay 4 days prior to spawning andsterilized by tangential flow filtration (TFF) using a Millipore Pellicon 2mini-TFF unit equipped with a 30-kDa-cutoff regenerated-cellulose filtercassette (Millipore Corp., Billerica, MA). The sterility of this water wasconfirmed by microscopy daily. The raw (nonsterile)-seawater treatmentwas prepared by coarsely filtering Kaneohe Bay surface seawater througha 1.6-�m-pore-sized GF/A filter (Whatman International Ltd., Kent,United Kingdom) in order to exclude zooplankton and larger organisms.

The experimental methods are described in detail in the subsequentparagraphs. Briefly, bacterial treatments were established by growing bac-terial strains in liquid batch culture and subsequently incubating diluted(1 �104 or 5 �104 bacterial cells ml�1) bacterial cells with rinsed coralplanulae. After 24 h of incubation, planulae were removed, rinsed, andincubated with the same bacterial strain, freshly diluted. After repeatingthe treatment again at 48 h and incubating the planulae for another 24-hperiod (T � 72 h), free-living bacterial cells were counted via microscopy(described below) and a subsample collected for community structurecharacterization. At 170 h, planulae were rinsed and collected for commu-nity structure characterization and microscopy analysis, and subsamples offree-living bacterial cells were again collected for characterization of bacterialcommunity structure. This characterization was performed in order to assesswhether the bacterial community was altered following incubation with coralembryos.

Axenic cultures of each strain were grown in specific media: Roseo-bacter strain HIMB1 and SAR11 strain HIMB4 were grown in low-nutri-ent medium (sterile seawater amended with ammonia and phosphate),Synechococcus strain HIMB12 was grown in low-nutrient mediumamended with dilute carbon additions (D-glucose, D-ribose, pyruvate,succinate, ethanol, glycerol and N-acetylglucosamine, each at 0.001% fi-nal concentration), and Pseudoalteromonas strain HIMB1276 was grownin R2A medium with seawater as the base (Sigma-Aldrich, St. Louis MO).Cells were grown in 12-h light/dark cycles at 30°C. Every 24 h, 30-mltreatments of each bacterial strain were prepared by diluting growingcultures 10� to 1,000� (e.g., adding 1 to 0.004 ml of culture to 30 mltotal) into sterile seawater (Table 1). Cellular abundances in axenic bac-terial cultures were monitored daily by fixing aliquots (1 ml) with filtered,buffered formaldehyde to a final concentration of ca. 2% for 15 min in thedark at room temperature. The cells were subsequently filtered onto 25-mm-diameter, 0.2-�m polycarbonate membranes (GE Osmonics, Inc.,Minnetonka, MN) and stained with the DNA-binding dye DAPI (4=,6=-diamidino-2-phenylindole) at a final concentration of 5 �g ml�1 in 15%formamide solution (0.9 M NaCl, 20 mM Tris-HCl [pH 7.4], 15% form-amide, 0.01% sodium dodecyl sulfate [SDS]) for 10 min at room temper-ature, followed by two 5-min washes in 15% formamide solution. Themembranes were air dried and mounted on microscope slides, and cellswere enumerated using a Leica DM5000B phase contrast/fluorescencemicroscope (Leica Microsystems, Wetzlar, Germany) with a 100� objec-tive under UV light.

TABLE 1 Summary of treatments applied to developing P. meandrina embryos and larvae

Treatment Source

Cell density (cells ml�1) in medium at:

Each 24-h addition 72 ha

Roseobacter strain HIMB1 Isolated from surface seawater of Kaneohe Bay, Oahu, HI 1.00 � 104 1.43 � 105

SAR11 strain HIMB4 Isolated from surface seawater of Kaneohe Bay, Oahu, HI 5.00 � 104 4.80 � 105

Synechococcus strain HIMB12 Isolated from surface seawater of Kaneohe Bay, Oahu, HI 5.00 � 104 5.18 � 105

Pseudoalteromonas strain HIMB1276 Isolated from tissues of adult colony of the coral Pocillopora damicornis 5.00 � 104 1.89 � 106

Sterile seawater Filtered (TFF, 30-kDa cutoff) surface seawater from Kaneohe Bay, HI �102 (detection limit) 9.00 � 102

Raw seawater Filtered (1.6-�m pore size) surface seawater from Kaneohe Bay, HI 4.30 � 105 1.00 � 107

a Growth determined 72 h after start of experiment but 24 h after addition of a fresh axenic bacterial culture or seawater treatment.

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Planulae were rinsed daily using 40-�m-mesh-size cell strainers(Fisher Scientific, Pittsburg, PA) and sterile seawater and placed into newpetri dishes containing fresh treatments. Treatments were initially repli-cated in triplicate, but after high embryo mortality during the first 24 h,replicates were subsequently combined to ensure that adequate materialwas available for the duration of the experiment. After 72 h (i.e., followingthree 24-h treatments), the abundance of microbial cells present in thetreatment water exposed to the planulae was determined by microscopicenumeration as described above. Additionally, after 72 and 170 h (i.e.,following three and seven 24-h treatments, respectively), subsamples of 30to 50 ml of treatment water were filtered onto 13-mm-diameter, 0.2-�m-pore-size polyethersulfone membrane filters (Supor 200; Pall Gelman,Inc., Ann Arbor, MI). Filters were stored at �80°C in DNA lysis buffer (20mM Tris-HCl [pH 8.0], 2 mM EDTA [pH 8.0], 1.2% [vol/vol] TritonX-100) for subsequent DNA extraction (56).

After 170 h, 100 to 200 planulae per treatment were preserved formicroscopy and frozen to �20°C as described above, and 50 to 100 plan-ulae per treatment were preserved in 250 �l of lysis buffer and frozen to�80°C for subsequent DNA extraction.

T-RFLP of bacterial SSU rRNA genes. DNA was extracted using theDNeasy tissue kit (Qiagen, Inc., Valencia, CA) with modifications (8) andquantified using the PicoGreen fluorescent assay (Invitrogen Corp.,Carlsbad, CA) on a SpectraMax M2 plate reader (Molecular DevicesCorp., Sunnyvale, CA). For terminal restriction fragment length poly-morphism (T-RFLP) analysis (34), bacterial small-subunit (SSU) rRNAgenes were amplified via PCR using oligonucleotide primers 27F-B-FAM(5=-AGRGTTYGATYMTGGCTCAG-3=) and 519R-VIC (5=-GWATTACCGCGGCKGCTG-3=), with “FAM” and “VIC” indicating 5=-end labelingwith FAM or VIC fluorochromes, respectively. Each 50-�l PCR mixturecontained 2 U of Sahara enzyme (Bioline USA, Inc., Taunton, MA), 1�Sahara reaction buffer, 2 mM Sahara MgCl2, 200 �M each deoxynucleo-side triphosphates (dNTPs), 200 nM each primer, and 100 ng of templategenomic DNA (up to 1 �g for samples that did not amplify at lowerconcentrations). After an initial denaturation step at 95°C for 5 min, thereaction conditions were as follows: 29 cycles of 95°C denaturation for 30s, 55°C annealing for 1 min, and 72°C extension for 2 min, concludingwith an extension at 72°C for 20 min. The reactions were performed in aMyCycler personal thermal cycler (Bio-Rad Laboratories, Hercules, CA).Products were purified using the QIAquick PCR purification kit (Qiagen,Inc.) and subsequently restricted in a 10-�l reaction mixture containing100 ng of purified amplification product, 2 �g of bovine serum albumin(BSA), 1� enzymatic reaction buffer, and 5 units of HaeIII restrictionendonuclease (10 units �l�1; Promega, Madison, WI) for 7 h at 37°C.Restriction digests were purified using the QIAquick nucleotide removalkit (Qiagen, Inc.), and 30 ng �l�1 of each product was subsequently elec-trophoresed on an ABI 3100 genetic analyzer (Applied Biosystems, FosterCity, CA). Operational taxonomic units (OTUs) were identified as indi-vidual T-RF peaks from both the forward and reverse labeled primer forfragments between 33 and 550 bp in length. To account for small differ-ences in the amount of DNA loaded on the ABI 3100, data were normal-ized by excluding peaks that contributed �0.05% of the total peak area foreach sample (46).

FISH. Planulae were examined using fluorescence in situ hybridiza-tion (FISH) with an oligonucleotide probe suite targeting the SSU rRNAof all bacteria (40) and an oligonucleotide probe targeting the SSU rRNAof members of the Roseobacter clade (10). In order to prepare samples forhybridization, planulae were fixed and subsequently permeabilized andsecondarily fixed for hybridization as previously described (6). For hy-bridization reactions using the general bacterial probe suite, stored sam-ples were first washed for 10 min at room temperature in 15% formamidehybridization solution (0.9 M NaCl, 20 mM Tris-HCl [pH 7.4], 15%formamide, 0.01% SDS), followed by incubation in fresh formamide hy-bridization solution for 30 min at 37°C. A suite of five Cy3-labeled probes(EUB-27R, EUB-338Rpl, EUB-700R, EUB-700Ral, and EUB-1522R) (40)were each added at 2 ng �l�1 in 15% formamide hybridization solution

and hybridized to the fixed planulae at 37°C for 14 h in the dark. Negative-control samples were incubated with nonsense oligonucleotide 338F-Cy3(40), added at 2 and 10 ng �l�1, as well as a no-probe control, under thesame conditions. After hybridization, the samples were washed two timesfor 10 min at 50°C in 0.15 M NaCl hybridization wash (150 mM NaCl, 20mM Tris-HCl [pH 7.4], 6 mM EDTA, 0.01% SDS). Planulae were alsoexamined for the presence of members of the Roseobacter clade using theCy3-labeled oligonucleotide probe Roseo536R (10). Sample preparationand hybridization reactions were performed as described above with thefollowing exceptions: hybridizations were performed in a 35% formamidehybridization solution (0.9 M NaCl, 20 mM Tris-HCl [pH 7.4], 35%formamide, 0.01% SDS) at 37°C, and the posthybridization washes wereperformed at 52°C in a 0.07 M NaCl hybridization wash (70 mM NaCl, 20mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.01% SDS). Control samples lack-ing oligonucleotide probe were prepared using the same conditions.

After being washed, samples were dehydrated in 100% isopropanol for5 min at room temperature and placed in a solution containing one partbenzyl alcohol to two parts benzyl benzoate. The planulae were mountedon slides in this solution. Sample visualization was performed with a ZeissLSM 510 confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Ger-many) and a 63� objective. Ten specimens were examined from eachtreatment, and representative specimens were imaged into optical stacksand processed using Zeiss LSM Image Browser version 4.2.0.121 (CarlZeiss, Inc.). The maximal densities of probe-hybridized cells were com-puted from images by counting the number of cells visible in a 10- by 10-by 1-�m area in the x, y, and z dimensions, respectively. Due to rapidfading of probe-conferred fluorescence, only small regions were targetedfor counting. Multiple slices within an optical stack were counted if theywere at least 2 �m apart. Bacterial cells were considered internalizedwithin the coral if surrounded by the autofluorescent coral tissues undermagnification.

Postprocessing of confocal stack images was conducted using Volocitysoftware (Improvision, Coventry, United Kingdom). An intensity render-ing was uniformly applied to all images.

Electron microscopy. One hundred 70-h-old planulae exposed to theraw-seawater treatment were analyzed for the presence of microbial cellsvia transmission electron microscopy. Samples were fixed in 2.5% glutar-aldehyde in 0.1 M sodium cacodylate with 0.32 M sucrose (pH 7.4) for 48h at 4°C, washed with 0.1 M sodium cacodylate with 0.44 M sucrose, andpostfixed with 1% osmium tetroxide in 0.1 M sodium cacodylate. Afterdehydration in 100% ethanol, samples were embedded in LX-112 epoxyresin (Ladd Research Industries, Burlington, VT). Ultrathin sections (60to 80 nm) were cut on an Ultracut E ultramicrotome (Reichert-Jung/LeicaMicrosystems, Wetzlar, Germany), mounted onto copper grids, andviewed at 100 kV accelerating voltage on a LEO 912 energy-filtered trans-mission electron microscope (Zeiss, Oberkochen, Germany). Images weretaken with a slow-scan, fast-transfer, charge-coupled-device camera(ProScan, Impala Park, South Africa).

RESULTSMicrobes in treatment media. An increase in free-living micro-bial cells was observed in all treatments during exposure to devel-oping P. meandrina (Table 1). After 72 h of exposure to the devel-oping coral, (but only 24 h after the addition of a fresh strain),cultured strain treatments primarily contained cell morphologiesconsistent with those of the respective treatment microorganisms;however, other cell morphologies were also observed via micros-copy (data not shown). After 72 h, sterile-seawater treatments alsocontained free-living microbial cells possessing a variety of cellu-lar morphologies (Table 1).

Bacterial community structure analysis. Using SSU rRNAgene T-RFLP, a comparison of bacterial communities associatedwith 170-h-old P. meandrina planulae incubated with differentbacterial strains revealed that the planulae were not solely associ-

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ated with the specific forward and reverse T-RF pair of the bacte-rial strain they were exposed to but were instead associated with amixed bacterial community (Fig. 1). Bacterial communities asso-ciated with planulae exposed to axenic strains of Roseobacterclade strain HIMB1 and Pseudoalteromonas strain HIMB1276possessed the signature T-RF patterns of each isolate (Fig. 1),strongly suggesting that these bacteria physically associated withor attached to the planulae. However, the planulae-associated bac-terial communities resulting from these two treatments also con-tained several other T-RFs that did not originate from the axenicbacterial strains used as treatments (Fig. 1). In contrast, the bac-terial communities associated with planulae exposed to axenicstrains of SAR11 strain HIMB4 and Synechococcus strain HIMB12did not possess the T-RFs characteristic of their respective treat-ment strains (limit of detection, �0.5% relative abundance), in-dicating that these isolates did not physically associate with P.meandrina planulae (Fig. 1). Instead, the planulae-associated bac-terial communities resulting from these two treatments werecomprised of several different T-RFs (Fig. 1).

Prior to the addition of planulae, the sterile-seawater treatmentalone failed to yield an SSU rRNA gene PCR amplicon. However,the 170-h-old planulae reared in sterile seawater were associatedwith several T-RFs, including F-34, F-191, and R-91 (where F andR indicate forward and reverse and the number is the fragmentlength in base pairs) (Fig. 1). Planulae reared in raw seawater wereassociated with the T-RFs F-191, R-91, and R-144 (among others),which were not abundant members of the raw-seawater treatmentprior to the addition of planulae (Fig. 1).

Some bacterial community members were found to be ubiqui-tously associated with 170-h-old planulae regardless of treatment(Fig. 1). In particular, bacteria represented by T-RFs F-34, F-191,F-225, R-91, and R-115 were shared between the planulae from alltreatments. These T-RF signatures include cells previously identi-fied as belonging to the Roseobacter clade of Alphaproteobacteria

(F-34 and R-91) and Gammaproteobacteria Pseudoalteromonasspp. (F-34 and R-115) (6). Planktonic microbial communitiesgrowing in the media collected following 72 or 170 h of planulaeexposure also harbored many of these T-RFs (Fig. 1).

Localization of Bacteria and Roseobacter clade cells. Irre-spective of treatment, bacterial cells were found to be associatedwith 60% of the P. meandrina planulae when visualized with thebacterial probe suite via FISH after 170 h of treatment exposure.Planulae exposed to Roseobacter strain HIMB1, Pseudoalteromo-nas strain HIMB1276, and raw seawater possessed the most con-sistent associations and the highest densities of bacteria (Table 2and Fig. 2). Planulae exposed to Pseudoalteromonas strainHIMB1276 harbored bacterial cells that were primarily localizedto the external edge and outer ectoderm (Fig. 2c). Bacterial cellswere also associated with the ectodermal tissues of planulae ex-posed to Roseobacter strain HIMB1 (Fig. 2a) and with planulaeheld in the raw-seawater treatments (Fig. 2e). Only 2/10 of plan-ulae reared in the presence of Synechococcus strain HIMB12,SAR11 strain HIMB4, or the sterile-water treatment containedvisibly associated bacterial cells; those that did harbored a lowdensity of cells in ectodermal tissues (Table 2; also see Fig. S1 in thesupplemental material).

Hybridization experiments with a probe specific for the Roseo-bacter clade of Alphaproteobacteria revealed that cells of this phy-logenetic lineage appeared to be most consistently and abundantlyassociated with 170-h-old P. meandrina planulae exposed to eitherRoseobacter strain HIMB1 or raw seawater (Table 2 and Fig. 3).Planulae exposed to SAR11 strain HIMB4, Synechococcus strainHIMB12, and Pseudoalteromonas strain HIMB1276 were occa-sionally (2 of 10 planula each) associated with a low density ofRoseobacter clade cells, which were located in the ectoderm (Ta-ble 2; also see Fig. S1 in the supplemental material). Nine of 10planulae exposed to the sterile-seawater treatments contained novisible Roseobacter clade cells (Table 2).

FIG 1 Heat map depicting the relative abundance of bacterial T-RFs from 170-h-old P. meandrina planulae and the medium treatment water after two different24-h bacterial culture exposures (72 and 170 h). Percentages in the T-RFLP profiles, shown in the color key, are from combined forward and reverse profiles. Alsoshown are the T-RFLP profiles for all bacterial culture and seawater treatments at T � 0.

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No internally or externally associated microbial cells could bedetected in FISH assays targeting all Bacteria and members of theRoseobacter clade in 4-h-old P. meandrina embryos (see Fig. S2 inthe supplemental material).

Detection of planula-associated microorganisms in electronmicrographs. Planulae exposed to raw seawater as the treatmentwere examined via transmission electron microscopy, which re-vealed spherical cells in the outer ectodermal tissue and surround-

TABLE 2 Summary of FISH results on 170-h-old P. meandrina planulae

Treatment

Bacterial probe suite Roseobacter clade probe

Probe-positiveplanulaea Locationb

Maximum density(cells �m�3)c

Probe-positiveplanulaea Locationb

Maximum density(cells �m�3)c

Roseobacter strain HIMB1 10/10 Ectoderm 0.06–0.10 10/10 Ectoderm 0.02–0.05SAR11 strain HIMB4 2/10 Ectoderm 0–0.03 2/10 Ectoderm 0.01–0.02Synechococcus strain HIMB12 2/10 Ectoderm 0.02–0.03 2/10 Ectoderm 0–0.01Pseudoalteromonas strain HIMB1276 10/10 External, ectoderm 0.04–0.07 2/10 Ectoderm 0.01–0.02Sterile seawater 2/10 Ectoderm 0–0.03 1/10 Ectoderm 0–0.04Raw seawater 10/10 Ectoderm, endoderm 0.04–0.06 10/10 Ectoderm 0.04–0.05a Proportion of planulae that positively hybridized with each probe assay out of the total number inspected.b Most commonly observed location of positively hybridized cells within the planulae.c Maximum density of positively hybridized cells within the planulae.

FIG 2 Confocal epifluorescence micrographs of 170-h-old P. meandrina planulae reared in the presence of Roseobacter clade strain HIMB1 (a, b), Pseudo-alteromonas strain HIMB1276 (c, d), and raw seawater (e, f). Bacterial cells were observed in planulae hybridized with the bacterial probe suite (indicated byarrows in panels a, c, and e) but not in control samples exposed to a nonsense control probe (b, d, f). Coral tissues and zooxanthellae (representative cells indicatedwith “z”) are illuminated due to autofluorescence in all images. Gland cells and/or nematocysts (g/n) are prominent in samples due to nonspecific hybridization.The ectodermal (ec) and endodermal (en) differentiation is drawn for reference.

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ing the mucus glands that ranged in diameter from 0.4 to 0.7 �mand were consistent with the size and shape of prokaryotic cells(Fig. 4a to c). In addition, aggregations of virus-like particles(VLPs) were also detected in the outer ectoderm. The VLPs rangedin size from 40 to 70 nm in diameter (Fig. 4d to f). While theprokaryotic cells and VLPs were both located in the ectoderm,they were not in close proximity to one another and, thus, did notappear specifically associated.

DISCUSSIONSpecificity in developing coral-bacterial relationships. The re-sults of this study provide experimental evidence supporting thehypothesis that taxon specificity plays a role in the onset of rela-tionships between bacteria and developing corals. Differenceswere visible in the location and density of bacterial cells incorpo-rated into P. meandrina planulae that were exposed to bacteriathat differ broadly in phylogenetic affiliation. Incubation with rawseawater, Roseobacter clade strain HIMB1, and Pseudoalteromo-nas strain HIMB1276 resulted in planulae containing prominent,internally associated microbial cells, while planulae incubatedwith sterile seawater, Synechococcus strain HIMB12, and SAR11strain HIMB4 did not. These results suggest that the establishment

of this intimate association is not passive and that the planula,bacterium, or both play an active role. Specific host-microbialrelationships have been documented for a variety of organisms(38, 39), including associations between corals and Symbiodinium(1, 29, 50, 53). Corals possess an innate immune system whichfunctions primarily in self- or nonself-recognition (36). Thus,some mechanism of recognition must exist in order to determinewhich bacterial cells can cross the coral epithelium. Proteins in-volved in pattern recognition may play a role in facilitating specificbacterial cells to cross into the epithelium of P. meandrina (26, 27),and further investigation of these mechanisms is important forunderstanding the initiation of any coral-microbial relationship.

Planulae exposed to Roseobacter clade strain HIMB1 formedthe most prominent internal bacterial associations, which wassomewhat surprising due to the planktonic lifestyle of this isolate.However, marine roseobacters represent a diverse group of bacte-ria that possess a variety of physiologies and have been previouslyfound in internal associations with developing P. meandrina (6)and Porites astreoides (51). Members of the Roseobacter clade alsofrequently form relationships with adult corals (14, 16, 21, 23, 24,30, 33, 48, 54, 55), but it is unclear how this specificity translatesinto homogeneous or heterogeneous roles within the coral holo-

FIG 3 Confocal epifluorescence micrographs of 170-h-old P. meandrina planulae hybridized with a probe specific for cells of the Roseobacter clade. Planulaereared in the presence of Roseobacter clade strain HIMB1 (a) and raw seawater (b) are shown. Cells of the Roseobacter clade are indicated with arrows. The linedifferentiates the ectodermal (ec) and endodermal (en) tissues.

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biont. Members of this important group of marine bacteria areknown to perform a variety of distinct functions (12, 37), includ-ing the consumption of the organic sulfur metabolite dimethyl-sulfoniopropionate, which is abundantly secreted by coral zoox-anthellae (11, 18), and the production of probiotic compounds(33, 49).

Pseudoalteromonas strain HIMB1276 also formed a prominentassociation with P. meandrina planulae, although its T-RF patternwas never identified in planulae incubated in raw seawater. Inter-estingly, it was isolated from the coral Pocillopora damicornis,which belongs to the same genus of coral as utilized in this study.Thus, this isolate appears to harbor a host-associated lifestyle.Planulae exposed to Pseudoalteromonas strain HIMB1276 primar-ily harbored cells in the outermost edge of the ectoderm, and it ispossible that these cells played a direct or indirect role in the set-tlement or adhesion processes of the planulae. Microbial biofilmson benthic substrates can attract and/or promote settlement ofcoral larvae, presumably through the production of chemical sig-nals (42, 60, 61). In fact, a strain of Pseudoalteromonas appears tobe involved in the metamorphosis of an acroporid coral (42). Dur-ing the course of our experiment, planulae exposed to Pseudo-alteromonas strain HIMB1276 tended to settle onto the surfaceof the petri dish (data not shown). Future studies are necessaryto investigate the specific role of Pseudoalteromonas strainHIMB1276 and other members of the Pseudoalteromonas genuswith developing corals and whether or not these associations per-sist in adults. Investigations examining their role in the settlementprocess, such as their ability to produce or detect chemical signals,may be particularly worthwhile.

The SAR11 strain HIMB4 and Synechococcus strain HIMB12formed less prominent internal associations with P. meandrinaplanulae and probably possess properties that are not suitable fora host environment. For example, the SAR11 strain HIMB4 is a

planktonic organism which is particularly prominent in oligotro-phic waters (40). In contrast, Synechococcus is a diverse group ofbacteria containing species with planktonic and host-associatedlifestyles. Cyanobacteria, but not specifically Synechococcus, havebeen known to internally associate with corals (28, 32). The Syn-echococcus strain utilized in this study (HIMB12) is planktonic,was isolated from a coastal ecosystem, and is not known to be hostassociated.

Widespread Roseobacter associations with developing P.meandrina organisms. After 170 h of development, P. meandrinaplanulae exposed to all treatments were associated with cells of theRoseobacter clade, suggesting that roseobacters may be particu-larly adept at forming associations with developing P. meandrinaorganisms. Because the associations were more prominent whenroseobacters were concentrated in the seawater or media, it ispossible that quorum sensing via microbe-secreted autoinducermolecules plays a role in the establishment of these relationships.This mechanism is utilized by microbes in other host associations,including pathogens (43, 65). The origin of the Roseobacter cladecells in other treatments besides that of Roseobacter strain HIMB1is not yet known but is of considerable interest. These cells poten-tially originate from surface (mucus) associations with the em-bryos and are later incorporated into the ectodermal tissues dur-ing development. While Roseobacter and other bacterial cellswere not observed to be externally or internally associated with4-h embryos, it is possible that external associations may be dis-rupted while preparing samples for FISH. In addition, it is alsofeasible that the Roseobacter clade cells originated from the free-living bacterioplankton community and were not removed duringthe rinsing process prior to establishing the treatments. Despitedaily water changes of sterile-seawater medium, the sterile-seawa-ter treatment showed evidence of a small community of microbialcells after 72 h (with sterile water changes every 24 h) of exposure

FIG 4 Transmission electron micrographs of prokaryotic cells and virus-like particles (VLPs) within the ectodermal tissues of 170-h-old P. meandrina planulae.(a to c) Single prokaryotic cells sized 0.4 to 0.7 �m in diameter are located in the ectodermal tissue near the exterior (ex) edge of the planulae. (d) A cluster of VLPs(each VLP sized 40 to 70 nm) near the exterior edge. (e) VLPs near a nucleus (n). (f) Multiple clusters of VLPs near the exterior. Scale bars are 500 nm for panelsa to d and 1 �m for panels e and f.

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to the planulae. Both of these scenarios may explain why, follow-ing planula exposure, the free-living bacterial communities oftencontained multiple community members that were similar de-spite differences in the treatments. The planulae were not treatedwith antibiotics prior to incubation with the various microbialstrains and seawater treatments, which might have been useful toremove loosely associated bacterial cells and obtain barren em-bryos.

Roseobacters were highly associated with the raw-seawater-exposed P. meandrina planulae in a previous study, but the cells inthat study were primarily members of the Jannaschia genus (6).While the SSU rRNA gene T-RF pattern representing the genusJannaschia (F-191 and R-178) was detected in the planulae rearedwith Roseobacter clade strain HIMB1 and Pseudoalteromonasstrain HIMB1276, it was not detected within the bacterial com-munity associated with planulae reared in the raw-seawater treat-ment. However, the T-RF pattern broadly associated with the ma-jority of members of the Roseobacter clade (F-34 and R-91/-92)was prominently associated. Thus, this study provides support forthe hypothesis that members of the Roseobacter clade are impor-tant for developing corals. Because the T-RFLP analysis methodused here provides relatively low taxonomic resolution and is notable to differentiate between most subclades within the Roseobac-ter clade, additional analysis of the identity of the specific commu-nity members is needed to further examine the specificity of theRoseobacter clade cells associating with P. meandrina planulae.While it should be noted that the potential for cross-contamina-tion was present due to the employment of a member of the Ro-seobacter clade possessing the T-RF pattern F-34 and R-91 as atreatment (HIMB1), this scenario is highly unlikely because theexperimental setup limited the opportunity for contaminating ex-posure.

Virus-like particles associated with treatment-exposed plan-ulae. The electron micrographs provide further evidence forprokaryotic cells in P. meandrina planulae and present the firstobservations of VLPs associated with coral planulae. The planulae-associated VLPs were similar in size and shape to those uncoveredin the tissues of healthy adult corals (47). Metagenomic analyses ofcorals have also validated the association of phage and eukaryoticviruses with healthy corals (35, 59, 62). Viruses are thought to playa role in disease manifestation (15, 57, 64), but they may alsobenefit corals (58). The role of viruses in healthy adult and devel-oping corals requires further research to better understand theirpotential beneficial effects or threats for the coral holobiont.

Summary. In this study, we found that the taxonomic compo-sition of free-living, planktonic bacteria influences the frequencyand nature of bacterial cell incorporation by P. meandrina planu-lae. We provide additional evidence that the Roseobacter clade ofmarine Alphaproteobacteria appear particularly adept at formingintimate relationships with developing P. meandrina, making itimportant to understand how these particular bacteria influencethe development, settlement, or health of the coral. Field studiesinvestigating the nature of microorganisms associating with de-veloping corals will undoubtedly be useful to verify laboratory-based results. Additionally, assessing the persistence of associationbetween microorganisms and developing corals with time is nec-essary to address whether the relationship is opportunistic ormore intimate in nature. Finally, we anticipate that continuedinvestigation of how developing P. meandrina organisms respondto altered communities of bacterioplankton will lead to important

insights regarding the ability of corals to develop, metamorphose,and endure as adult colonies in a changing marine environment.

ACKNOWLEDGMENTS

We thank P. Jokiel for the use of aquaria, G. Carter and M. Hagedorn forsampling assistance, M. Miller for assistance with the bacterial cultures,and T. Carvalho (UH Biological Electron Microscope Facility) for assis-tance with electron microscopy.

This study was supported by funding from a research partnershipbetween the Northwestern Hawaiian Island Coral Reef Ecosystem Reserveand the Hawaii Institute of Marine Biology (NMSP MOA 2005-008/66882) and by a grant from the National Science Foundation (OCE-0928806) to M.S.R.

Coral collections were conducted under the state of Hawaii’s Depart-ment of Land and Natural Resources special activity permit no. 2008-99.

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