AQUATIC BIOLOGYAquat Biol

Vol. 4: 289296, 2009doi: 10.3354/ab00102

Printed January 2009Published online January 27, 2009

INTRODUCTION

Coral diseases are now widely reported on reefsworldwide and are increasing in incidence and viru-lence, contributing to worldwide reef degradation(Harvell et al. 2002, Sutherland et al. 2004). The recentincrease in coral disease events has been linked toenvironmental stress and climate change (Aronson &Precht 2001, Harvell et al. 2002, Lesser et al. 2007).Both the increased coral host susceptibility (Lesser etal. 2007) and increased pathogenicity of microbialcommunities (Rosenberg & Ben-Haim 2002, Bruno et

al. 2007) have recently been proposed to be drivingthese incidents of disease. Given increased environ-mental stressors and the potential role of microbialsystems in driving disease in reef ecosystems, it isnecessary to investigate the causes and mechanismsthat link disease and environmental stress. Therefore itis important to accurately document the role of coralmicrobial communities in changing reef environments.The present study examined the in situ (within-tissue)bacterial community dynamics of reef-building coralsand how these change during stable and disturbedenvironmental conditions.

Inter-Research 2009 www.int-res.com*Email: tracy.ainsworth@jcu.edu.au

Bacterial communities closely associated with coraltissues vary under experimental and natural reef

conditions and thermal stress

T. D. Ainsworth*, O. Hoegh-Guldberg

Centre for Marine Studies and ARC Centre of Excellence for Coral Reefs Studies, University of Queensland, St. Lucia, Queensland 4072, Australia

ABSTRACT: The coral holobiont model highlights the integral role bacteria play in the health of reef-building corals. Documenting the natural diversity of bacterial communities within, and closely asso-ciated with, coral tissues provides information on the diversity, interaction and roles of bacteria to thefunction of reef-building corals. Fluorescence in situ hybridisation was used to visualise bacterialcommunities closely associated with the tissues of experimentally manipulated reef corals to deter-mine how tissue-associated coralbacterial interactions vary from normal associations in apparentlyhealthy reef corals, to those occurring in controlled and thermally stressed experimental conditions.Branches of 2 coral species of the Great Barrier Reef, Acropora aspera and Stylophora pistillata, werecollected from reefs adjacent to Heron Island and were maintained in controlled outdoor flow-through aquaria conditions. Following acclimation, the branches were stressed using elevated tem-peratures to investigate the in situ (within-tissue) bacterial community changes. In situ bacterial com-munity dynamics were found to vary not only due to maintenance within the aquaria conditions, butalso following coral bleaching. An aggregation of rod-shaped -proteobacteria was evident within thegastrodermis of corals regardless of health or bleaching status, consistent with aggregationsdescribed within other coral species. However, bacterial colonisation of the tissues occurred onlyfollowing the temperature-induced bleaching of the coral tissues. This study demonstrates that thenatural bacterial communities of corals are severely altered during stress associated with experi-mental and field conditions, which suggests a potential mechanism for the link between disease andstresses arising from global warming.

KEY WORDS: Coral Bacterial associations Coral bleaching

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OPENPEN ACCESSCCESS

Aquat Biol 4: 289296, 2009

Very little is known about in situ bacterial commu-nity dynamics within coral tissues. There is strong evi-dence that along with endosymbiotic dinoflagellates,bacterial communities play important roles in normalcoral physiology (Rohwer et al. 2002, Lesser et al. 2004).The occurrence of highly diverse bacterial communi-ties has been documented using culture-independentmethodologies such as DNA-based phylogenetic ana-lysis of consortia extracted from coral-symbiotic as-sociations. While many studies have suggested keyecological and physiological roles of these complexcoralbacterial associations, only a few, such as Lesseret al. (2004), have investigated these associationsspecifically within the different coral layers and coraltissues. As such, little is known about the microbialprocesses that occur within coral tissues or how thesechange during stress. Koren & Rosenberg (2006) haveproposed that coral microbial diversity is high withincoral tissues of the invasive Mediterranean coralOculina patagonica, while studies using in situ tech-niques have revealed diverse communities associatedspecifically with the endolithic layer within the coralskeleton of O. patagonica (Ainsworth et al. 2008). Thecoral surface mucus layer has also been demonstratedto harbour a large diversity of coral bacterial communi-ties (Ritchie & Smith 1997, review by Brown & Bythell2005, Ritchie 2006). Lesser et al. (2004) described anddemonstrated a cyanobacterial symbiont within thetissues of the Caribbean coral Montastrea cavernosaand, based on the presence of bacterial genes govern-ing nitrogen fixation, proposed these symbionts ashaving a key role in coral physiology. Determining thenature of coralbacterial associations in situ provides abasis on which to determine the physiological roleof bacterial communities within reef-building corals.Determining the endosymbiotic versus the ectosymbi-otic nature of different bacterial community membersmay also be extremely important in differentiatingmicrobial contributions to the coral holobiont model.

We set out to explore the in situ bacterial associationwithin the tissue layers of branching coral species andto investigate how coral-tissue bacterial associationsvary in situ during controlled and thermally stressedexperimental conditions. Understanding the normal,stable, and disturbed bacterial association with coraltissues provides a basis upon which to understand notonly the role of bacterial associations but also theirvariability in stable and disturbed conditions.

MATERIALS AND METHODS

Coral sample collection. Coral were collected onSCUBA and snorkel from the reef flat (1 m depth, Acro-pora aspera) and reef slope (up to 8 m depth, Stylo-

phora pistillata) areas of Heron Island, the southernGreat Barrier Reef (23.44 S, 151.91 E) during earlyDecember 2005. Coral branches were collected, heldin seawater and transported immediately. Healthy coralbranches of

Ainsworth & Hoegh-Guldberg: Bacterial communities closely associated with corals

perfrost Plus slides (Menzel). The tissue sections werethen stained using Harriss haematoxylin and eosin (withphyloxine B) (Sigma-Aldrich, HHS32 and HT110-1-32)and adjacent sections were used in fluorescence in situhybridisation (FISH).

FISH. Visualisation of bacterial communities associ-ated with coral tissues was conducted using FISH com-bined with spectral imaging (Ainsworth et al. 2006).Serial (4 m) sections of each sample collected wereused in the hybridisation with bacterial probes todetermine the presence of bacteria within the tissuesand determine if the bacteria were members of the-proteobacterial group and Vibrio genus. The probesused included: universal bacterial probe mix (EUB-mix), a -proteobacteria probe mix (GAM42A) anda Vibrio sp. (MV) (see Amann et al. 1990, 1995, 1996,2000, Manz et al. 1992, 2000, Daims et al. 1999,Moreno et al. 1999, Moter & Gobel 2000). The probes,Cy3-labelled oligonucleotide probes (Thermo Elec-tron), were used in a standardised FISH protocol(Manz et al. 2000). The hybridisation was conductedusing 35% formamide in hybridisation buffer (0.9 MNaCl, 0.01% sodium dodecyl sulphate [SDS], 0.01 MTris-HCl pH 7.2) for 1.5 h at 46C, followed by a 10 minwash in pre-warmed wash buffer (0.08 M NaCl, 0.01%SDS, 0.01 M Tris-HCl pH 7.2, 0.05 M EDTA). A ZeissMeta 510 confocal scanning laser microscope com-bined with spectral emissions profiling of tissue auto-fluorescence was used for visualisation of FISH-labelled bacterial communities in association withcoral tissues and the disease lesion.

Transmission electron microscopy. Samples of Acro-pora aspera (n = 3) and Stylophora pistillata (n = 3)from apparently healthy coral from reef conditionswere also preserved for transmission electron micro-scopy for investigation of in situ bacterial communitiesand bacterial morphology. Small fragments (0.2 to0.5 cm3) from each colony were immediately fixed in3% glutaraldehyde in 0.1 M cacodylate buffer. Samplepreparation used methods outlined in Le Tissier (1990),and decalcified in 20% EDTA over 2 wk at 4C.Sample grids with ultra-thin sections were viewed ina transmission electron microscope (JEOL 1010) atacceleration voltage 80 kV, and images taken usingthe Megaview III Soft Imaging system.

RESULTS

In situ bacterial populations associated with naturalcoral communities

The natural bacterial community associations withinthe tissues of reef-building corals were limited to bac-terial aggregates within the gastrodermis. Both Acro-

pora aspera and Stylophora pistillata were found tohave pleiomorphic bacterial aggregates within thegastrodermis (Fig. 1). This is consistent with previousreports (Peters et al. 1983, Peters 1984, Santavy &Peters 1997, Rohwer et al. 2002, Ainsworth et al.2007a,b) of similar bacterial communities associatedwith the gastrodermis tissue layer of both healthy anddiseased coral tissues (Table 1). Electron microscopy oftissues of both A. aspera and S. pistillata of the GreatBarrier Reef show apparently uniform rod-shapedmorphology of the 1 to 3 m bacteria within the aggre-gates, and this appears as the only morpho-type iden-tified within the aggregate regardless of aggregatesize (Fig. 1). Using FISH, these bacterial aggregateswere identified as members of the -proteobacteriagroup by selective binding of the GAM42A and EUB-mix probes (Fig. 1). A lack of binding with a Vibrio sp.-specific group probe (MV) suggests that these bacteriaare not members of the Vibrio genus. Aggregate sizewas found to vary from as little as 7 m to >80 m indiameter (Fig. 2). Based upon estimations of the aggre-gate volume (volume of ellipsoid = 1/6 length width depth), the bacterial density within a 5 mthick section of an aggregate ranged from ~120 bacte-ria within a single aggregate measuring 7 m long by5 m wide, up to ~2700 bacteria within a single aggre-gate measuring 80 m long by 10 m wide.

Bacterial community changes associated withexperimental conditions

Bacteria within the coral tissue layers in controlledand experimentally heated aquarium conditions wereboth found to be different to those associated withapparently healthy, naturally occurring, coral tissue-bacterial community associations. Bacterial popula-tions penetrated and proliferated within the outertissue layers (epithelium) in what appear to be mucuscells, and penetrated throughout the adjacent coralcells and tissue regions (Fig. 3AC). These bacterialinteractions within the coral epithelium tissue layerwere apparent in both the control and the thermallystressed conditions (Fig. 3AF), and occurred within3 d of being held in aquaria and experimental condi-tions (Table 2). These interactions were not found inapparently healthy, normal coral populations sampleddirectly from the reef without maintenance within theaquaria or experimental conditions. While this prolifer-ation did change the normal flora seen within the tis-sues of coral it did not appear to have any obviousshort-term health consequences to the corals main-tained in thermal experimental and control conditions.A complete bacterial colonisation or overgrowth of thecoral tissues was only evident after increasing thermal

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Fig. 1. Electron micrographs ofrod-shaped bacteria within thegastrodermis of Acropora asperaindicate the consistent morphologyof the populations in large ag-gregates of (A) ~60 m and (B)~25 m (diameter). (CE) Aggre-gates at greater magnification. agg:aggregate; ep: epithelium; ga: gas-trodermis; mg: mesoglea; n: nema-tocyst; zx: endosymbiotic dinofla-gellate. Scale bars (bottom right of

panels) = 1 m

Fig. 2. (AD) Size variability ofbacterial aggregates within coralgastrodermal tissue layers fromas small as 7 m (A) to 80 m(D) in length in Acropora asperaas identified using EUBmix, sim-ilar to that of Tabular Acroporaof the GBR (C). Location andmorphology is consistent withbranching Acroporid coral of theRed Sea (D). Blue: coral tissue;green: endosymbiotic dinoflagel-lates; red: bacteria; agg: aggre-gate; ep: epithelium; ga: gastro-dermis; zx: endosymbiotic dino-flagellates. Scale bars = 10 m

Ainsworth & Hoegh-Guldberg: Bacterial communities closely associated with corals

stress resulted in coral bleaching (after exposure to thebleaching threshold of 32C, and loss of the endosym-biotic dinoflagellates) (Fig. 4). Within the bleachedcoral tissue regions (as determined by loss of the asso-ciated endosymbiotic algae), mixed bacterial popula-tions were detected associated within the mesentarialfilaments, gastrodermis and epithelial layers. Thesame extent of proliferation of bacteria around theinternal/mesentarial layers did not occur in any othercoral tissues nor in corals maintained in control aquaria(Table 2).

DISCUSSION

Symbioses between corals and bacteria have beenwidely proposed in recent coral literature. Complex anddiverse coralbacterial associations have also been sug-gested to show species-, temporal- and spatial-specificity(Ritchie & Smith 1997, Rohwer et al. 2002). Thesecoralbacterial associations are proposed to occur in

several regions, including within the coral tissue layers,endolithic layer of the skeleton, and also coral surfacemucus layers (Ritchie & Smith 1997, review by Brown &Bythell 2005, Ritchie 2006, Ainsworth et al. 2006). Deter-mining the specific in situ bacterial associations thatoccur within the coral tissue layers, as opposed to withinthe coral surface mucus-layer, coral gut, and skeletalendolithic layers, is important in order to understand thevarious roles bacterial populations may play in coralphysiology. In the present study, we have described thestructure and dynamics of coral tissue-specific bacterialassociates under normal and stressed conditions. One ofthe important issues to arise here is that extreme caremust be taken when interpreting bacterial populationchanges in corals maintained in aquarium conditions.This echoes the observations of Kline et al. (2006), whofound major increases in the abundance of bacteriawhen corals were maintained in aquaria.

The normal bacterial populations within apparentlyhealthy coral tissues were identified as aggregates of1 m rod-shaped -proteobacteria. These aggregations

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Table 1. In situ bacterial aggregates in reef-building corals, including healthy, thermally stressed (T-S), white syndrome (WS) and white disease (WD) affected corals. GBR, Great Barrier Reef

Genus or Location Health -proteobacteria aggregates Sourcespecies status Gastrodermis Epithelium

Acropora aspera GBR Healthy Present Not present Ainsworth et al. (2006)GBR T-S Present Present studyGBR Bleached Present Present Present studyGBR Control aquaria Present Present Present study

Acropora sp. GBR WS Present Ainsworth et al. (2007a)Eilat Healthy Present Not present Ainsworth et al. (2007b)Eilat WS Present Not present Ainsworth et al. (2007b)Caribbean WD Present Peters (1984)

Stylophora pistilata GBR Healthy Present Not present Present studyGBR T-S Present Not present Present studyGBR Bleached Present Present studyGBR Aquaria Present Present Present studyEilat Healthy Present Not present Ainsworth et al. (2007b)Eilat WD Present Not present Ainsworth et al. (2007b)

Porities sp. Caribbean Present Rohwer et al. (2002)

Table 2. Acropora aspera. Identification of bacterial aggregates in the tissues of thermally stressed and control condition corals(the bleaching threshold for A. aspera on the reef flat was 32C). Ep: epithelium; Ga: gastrodermis. Bold: change in bacterialcommunities detected compared to that of prior samples. All corals were held for 5 d acclimation prior to the onset of the

experiment period

Temperature in Tissue Temperature in No. of days in Tissueheated aquaria (C) association control aquaria (C) control aquaria association

28 Ga 28 6 Ga30 Ga/Ep 28 7 Ga/Ep31 Ga/Ep 28 8 Ga/Ep32 Ga/Ep 28 9 Ga/Ep32 Ga/Ep 28 10 Ga/EpBleached Colonisation Unbleached 11 No colonisation

Aquat Biol 4: 289296, 2009

were observed within vacuoles or membrane-boundregions of the gastrodermis, similar to that of the peri-algal space of the coraldinoflagellate symbiosis. The-proteobacteria bacterial aggregates within the gastro-dermis were uniform in structure and located within thegastrodermis tissue layers, consistent with previous re-ports in other corals (Peters et al. 1983, Peters 1984, San-tavy & Peters 1997, Rohwer et al. 2002, Ainsworth et al.2007a,b) (Table 2). Specific coralbacterial symbioses

have previously been described andnitrogen-fixing cyanobacterial sym-bionts were found within the tissue lay-ers of Montastrea cavernosa (Lesser etal. 2004). Here, our data support previ-ous suggestions that the rod-shaped-proteobacterial population within thegastrodermis tissue layer may alsoprove to be a universal symbiont group(Peters 1984, Santavy & Peters 1997,Rohwer et al. 2002, Ainsworth et al.2006, 2007a,b). Previous descriptions ofthese include dense ovoid bacterialaggregates in corals of the Caribbean(Peters et al. 1983, Peters 1984, Santavy& Peters 1997) and a universally domi-nant PA1 ribotype from poritid corals(Rohwer et al. 2002). Further investiga-tion should be conducted to determinethe identity, function and role of thesewidespread and potentially symbioticbacteria.

Bacterial aggregates were foundonly within the gastrodermis layer ofcorals taken directly from the field.This, however, was not the case forcorals maintained in captivity. In bothcontrolled ambient conditions andthermally stressed conditions, the insitu bacterial association changedfrom the limited gastrodermis aggre-gates seen in corals collected fromthe natural reef environment, to bac-terial communities penetrating andproliferating within the outer tissuesof the epithelial layer. The bacterialpenetration of the epithelial layer ofcorals maintained in control aquariawas evident throughout the experi-mental period (a total of 11 d), dur-ing which time corals did not revealmacroscopically apparent adverse ef-fects such as tissue loss or whiteningof the tissues. These changes maybe a product of several conditions,including fragmentation of the tissues,

handling of the coral samples in preparation of exper-imental conditions, or a result of the altered andincreased bacterial loads of contained aquarium con-ditions compared to those normally experienced bythe corals in reef flat conditions (Kline et al. 2006).The coral mucus-associated microbial community hasalso been shown to undergo a significant shift duringmaintenance within aquarium conditions (Koopermanet al. 2007). It is important to determine if this shift in

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Fig. 3. Acropora aspera. Bacterial populations within epithelial tissue layers duringboth (AD) thermal stress, and (E,F) control conditions. Blue: coral tissue; green:endosymbiotic dinoflagellates; red: bacteria; agg: aggregate; ep: epithelium; ga:gastroderm; m: mucosal cell; zx: endosymbiotic dinoflagellates. Scale bars = 10 m

Ainsworth & Hoegh-Guldberg: Bacterial communities closely associated with corals

mucus-associated communities allows bacterial com-munities to penetrate and proliferate within coraltissues that would not normally occur. This mucus-associated community change, combined with theresults of the present study, have important ramifica-tions for the interpretation of culture-based bacterialisolation studies when applied to corals in experimen-tal scenarios.

CONCLUSIONS

These results highlight a series of important ques-tions that need to be addressed relating to ourunderstanding of the role of coral-bacterial associa-tions, the impact of experimental stressors in chang-ing normal associations, and the role of bacterialcommunities in colonisation of bleached coral tis-sue. In general, our understanding of in situ coral-bacterial community dynamics and endo- and ecto-bacterial symbioses remains in its infancy. Our studyprovides evidence of potential bacterial endosym-bionts that may play a role in the physiology andecology of corals. The details of these interactionsremain sketchy and should be the focus of futurework. Symbionts that are associated with the outerlayers, while not prominent in the present study,should not be ignored. It is clear overall, however,that there are significant and important featuresassociated with the bacterial communities of coralsthat require description before we fully understandthe linkages between stress, disease and the micro-bial flora of reef-building corals.

Acknowledgements. We thank Dr. S. Dove, Dr. O. Levy, andMs. R. Middlebrook for assistance during sample collection atHeron Island Research Station. We also thank the staff ofHeron Island Research Station and Mr. D. Harris and Dr. B.Leggat for assistance with the experimental set-up, designand sample collection, and Ms. D. Burgess. We thankthe ARC Centre of Excellence for Coral Reef Studies(www.coralcoe.org.au/) and the Coral Reef Targeted Re-search Program (www.gefcoral.org) for financial support ofthis research project.

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Fig. 4. Acropora aspera. Bacter-ial colonisation of mesentarialfilaments of following thermalstress and coral bleaching inregions with (A,B) no or fewendosymbiotic dinoflagellates,and (C) many endosymbioticdinoflagellates, but is not evi-dent in control conditions (D) orprior to bleaching (E,F). bac:bacteria; mf: mesentarial fila-ments; zx: endosymbiotic dino-flagellates. Scale bars = 20 m

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Editorial responsibility: Michael Khl, Helsingr, Denmark

Submitted: December 13, 2007; Accepted: September 30, 2008Proofs received from author(s): January 15, 2009

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