DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 102: 137–148, 2012doi: 10.3354/dao02541

Published December 27

INTRODUCTION

The presence of pathogenic bacteria is inherent toany natural system. However, incidences of bacterialinfections found in marine organisms have steadilyincreased over the past 20 yr (Goreau et al. 1998,Ben-Haim et al. 2003, Green & Bruckner 2000,Cervino et al. 2001, Rosenberg et al. 2007). This trend

has been reported in finfish (Pulkkinen et al. 2010),crustaceans in aquaculture (Chiu et al. 2007, Rebou -ças et al. 2011) and various types of coral (Kushmaroet al. 2001, Smith et al. 1996, Goreau et al. 1998,Sutherland et al. 2004, Cervino et al. 2008). Condi-tions that may have influenced increases in water-borne pathogens may include increased rates ofhuman population growth and development in sensi-

© Inter-Research 2012 · www.int-res.com*Corresponding author. Email: hauffbri@msu.edu

Ulcerated yellow spot syndrome: implications ofaquaculture-related pathogens associated with soft

coral Sarcophyton ehrenbergi tissue lesions

James M. Cervino1,2, Briana Hauff2,3,*, Joshua A. Haslun3, Kathryn Winiarski-Cervino4, Michael Cavazos3, Pamela Lawther2,

Andrew M. Wier2, Konrad Hughen1, Kevin B. Strychar3

1Woods Hole Oceanographic Institution, Department of Marine Chemistry & Geochemistry, Woods Hole, Massachussetts 02543, USA

2Pace University, Department of Biological Sciences, New York, New York 10570, USA3Texas A&M University − Corpus Christi, Department of Life Sciences, Corpus Christi, Texas 78412, USA

4c/o Environmental Defense Fund, 257 Park Avenue South, New York, New York 10010, USA

ABSTRACT: We introduce a new marine syndrome called ulcerated yellow spot, affecting the softcoral Sarcophyton ehrenbergi. To identify bacteria associated with tissue lesions, tissue andmucus samples were taken during a 2009 Indo-Pacific research expedition near the WakatobiIsland chain, Indonesia. Polymerase chain reaction targeting the 16S rDNA gene indicated asso-ciations with the known fish-disease-causing bacterium Photobacterium damselae, as well as mul-tiple Vibrio species. Results indicate a shift toward decreasing diversity of bacteria in lesionedsamples. Photobacterium damselae ssp. piscicida, formerly known as Pasteurella piscicida, isknown as the causative agent of fish pasteurellosis and in this study, was isolated solely inlesioned tissues. Globally, fish pasteurellosis is one of the most damaging fish diseases in marineaquaculture. Vibrio alginolyticus, a putative pathogen associated with yellow band disease inscleractinian coral, was also isolated from lesioned tissues. Lesions appear to be inflicting damageon symbiotic zooxanthellae (Symbiodinium sp.), measurable by decreases in mitotic index, celldensity and photosynthetic efficiency. Mitotic index of zooxanthellae within infected tissue sam-ples was decreased by ~80%, while zooxanthellae densities were decreased by ~40% in lesionedtissue samples compared with healthy coral. These results provide evidence for the presence ofknown aquaculture pathogens in lesioned soft coral and may be a concern with respect to cross-species epizootics in the tropics.

KEY WORDS: Photobacterium damselae · Indonesia · Vibrio · Zooxanthellae · 16S rDNA · Lesion

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Dis Aquat Org 102: 137–148, 2012

tive coastal areas (Dorfman & Rosselot 2011) andincreased aquaculture activities along shorelines(Naylor et al. 2000). As the disease resistance of coralis thought to decrease with changing environmentalconditions (Harvell et al. 1999) and global aquacul-ture facilities are concurrently increasing in number,the potential for epizootics and novel emerging dis-eases increases (Garren et al. 2008, Garren et al.2009).

As aquaculture fish farming is based on the con-cept of raising monoculture crops at high density, thepotential for pathogenic bacteria to exploit theseorganisms is high. Bacterial infection is the maincause of disease-induced death of fish raised in aqua-culture (Johansen et al. 2011). For example, since1969 the bacterium Photobacterium damselae hasbeen consistently associated with aquaculture dis-eases in Japan, causing infections in yellowtail Seri-ola quinqueradiata (Ku suda & Ya ma oka 1972). How-ever, since the 1990s, this patho gen has spread,causing severe economic losses in aquaculture stocksin numerous countries, including France, Italy, Spain,Greece, Turkey, Portugal, Malta and, recently, Ma -lay sia, e.g. La Mesa et al. (2008).

The growth of the aquaculture industry has broughtconcern about the potential effects of aquaculturepathogens on surrounding populations as the designof many of these farms is open and allows for trans-mission to the neighbouring environment. However,the implications of aquaculture pathogens on sur-rounding natural populations has been difficult toclassify as the relationships between host, pathogenand the environment are complex and may be influ-enced by a myriad of factors (Hedrick 1998).

China, India and Indonesia represent ~70% of theworld aquaculture market as of 2007, with 60, 6 and3% of the market respectively (FAO 2007), while har-boring nearly 45% of the world’s coastal coral reefs(Spalding et al. 2001). The close proximity of coastalaquaculture facilities to coral reef habitats in theseregions, with the influences of stock escapement andeffluent discharge, amplifies the probability of coralinteracting with microbial organisms, both patho-genic and autochthonous species, potentially result-ing in coral mucus community shifts, infection andmortality.

As demonstrated by Garren et al. (2009), coralstransplanted adjacent to an aquaculture facility inBolinao, Philippines, and exposed to high concentra-tions of effluent experienced a shift in microbial pop-ulations, resulting in increases in bacterial groupsassociated with human and coral pathogens. Prior tothe development of the aquaculture facility, Garren

et al. (2009) reported a native population of corals inthis location that has since died. That study alsofound that after 21 d, the transplanted coral experi-enced yet another change in microbial communitiesback to profiles more similar to those seen prior totheir exposure to high effluent from the facility.Despite the finding of an initial shift to increasedpathogens, the short duration of the study precludedobserving causation of coral infection and the devel-opment of disease symptoms. However, it did showthat pathogenic bacteria derived from aquacultureeffluent have the capacity to interact with coralmucus and were a possible cause of death of the priornative population of coral.

Here we present a new coral syndrome calledulcerated yellow spot (UYS) affecting Sarcophytonehrenbergi, a soft coral (common name ‘leathercoral’). We choose to use the term ‘syndrome’ or ‘dis-ease-like syndrome’ to describe the complex of clini-cal symptoms (Dorland 1982) that we have identifiedwith this coral, as the term ‘syndrome’ is less readilyassociated with causation than the term ‘disease’ andwe have not performed any definitive causality ex -periments. Although diseases of scleractinian coralsare becoming more common (Rosenberg & Loya2004), infections on soft corals are rare (Williams etal. 2011). For example, a recent assessment of thePalmyra Atoll in the Central Pacific found disease insoft corals, compared to stony corals, to be the leastprevalent (<0.03% of all disease found) in all reefssurveyed (Williams et al. 2011). This study presents apreliminary analysis of various groups of marine bac-teria found on the surface of these soft corals, includ-ing healthy tissues and areas displaying yellow le -sions, using 16S rDNA gene sequencing for speciesidentification. We also present evidence of impairedzooxanthellae function within lesioned tissues indi-cated by reduced photosynthesis and decreased den-sities and mitotic indices. Finally, we discuss theprevalence of UYS throughout the study site, the sim-ilarities of bacteria found within lesions to thoseknown to cause diseases in aquaculture species, andimplications for the future of these corals and coralreefs.

MATERIALS AND METHODS

UYS was observed in the Wakatobi archipelago ofIndonesia (5° 18’ 34.47’’ S, 123° 35’ 08.31’’ E; Fig. 1).To assess the frequency of these lesions on Sarco-phyton ehrenbergi, lesioned versus healthy coralswere counted along transects (n = 3 per site) at 8

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study sites. Study sites were chosen based upon theemergence of UYS at sites visited during 2009where the disease was absent in 2004. The names ofeach site surveyed and their associated site numbersin the island chain were: (1) House Reef #1, (2)House Reef #2, (3) Barracuda, (4) Roma, (5) Zoo, (6)Conchita, (7) Table Coral City and (8) Turkey Beach(Fig. 1). Transect lines (50 × 1 m transects), ~3 mabove each reef-flat surface, were established andcorals were counted and photographed (Fig. 2) tohelp determine the frequency of soft coral colonieswith UYS lesions versus those coral colonies notinfected. Transects were begun at the shore-diveentry point and were continued at increasing depthand distance from the shore (final depth = 18 m).Colonies were photographed using an UnderwaterNikon RS (SLR system). Two-way ANOVA was per-formed with transect site and coral health (lesionedversus healthy regions) as factors and abundance asa response variable (R v. 2.10.1; The R Foundationfor Statistical Computing).

Pulse amplitude modulated fluorometry

Along each transect line, measurements of photo-synthetic efficiency were taken of lesioned andhealthy tissues of every Sarcophyton ehrenbergiencountered using a diving pulse amplitude modu-lated (PAM) fluorometer (Walz). Healthy and le sionedtissues were probed to obtain measurements of zoo-xanthellae overall quantum yield of photochemicalenergy conversion (i.e. dF/Fm’) as a function of sym-biont photosynthetic efficiency. Relative rate of elec-tron transport (ETR) was also measured to ensurethat dF/Fm’ (yield) readings were indicative of changesin photosynthetic incompetence, rather than fluctua-tions of environmental parameters. Measurementswere taken at each site and transect where healthyversus lesioned coral data were collected. A one-wayANOVA was used to determine the differences influorescent yield between the 2 conditions of health(R v. 2.10.1). We further analyzed the relationshipbetween other parameters measured by the DivingPAM — photosynthetically active radiation, ETR andyield — as well as depth, using multiple logistic re -gression, in order to determine which of these para -meters were best used to predict lesions. Varianceinflation factors were used to simplify the model witha cut-off value of 2.5. Data exploration was carriedout using scatterplots of the independent variablesand R2 coefficients.

Sample collection of Sarcophyton ehrenbergi

Coral tissue samples were collected for zooxanthel-lae analysis using SCUBA. For each targeted colony,one 2.5 cm2 tissue sample was taken using stainlesssteel scissors. One sample was taken within thelesion of the affected coral while the other samplewas taken from a separate healthy coral. At each survey site 6 corals were sampled, 3 lesioned and 3healthy. Individual tissue samples were placed in100 ml polyethylene bottles in filtered seawater(FSW). Samples were kept on ice in coolers duringtransit back to the laboratory on shore.

Mitotic index

In the laboratory, tissue was removed from all spec-imens using a Water Pik following the methods ofJohannes & Wiebe (1970). The liquid extract contain-ing tissue and zooxanthellae was homogenized andcentrifuged at 10 000 × g for 5 min in order to collect

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Fig. 1. Study sites, located on the eastern side of Tomia andOnemobaa Islands in the southeast of Indonesia, indicatedby the hatched area. Disease transects were carried out ateach of the 8 sites: (1) House Reef #1, (2) House Reef #2, (3)Barracuda, (4) Roma, (5) Zoo, (6) Conchita, (7) Table Coral

City and (8) Turkey Beach

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zooxanthellae. Supernatant was discarded, and thepellet was re-suspended in 1 ml of 10% gluteralde-hyde, 15% PBS, FSW solution and stored at 20°C.Zooxanthellae density and mitotic indices (MI%)were determined from each 2.5 cm tissue sample.These were determined by direct examination undera Zeiss ApoTome Fluorescence microscope at ×20 to×100 using a Neubauer hemocytometer followingWilkerson et al. (1988). Welch 2-sample t-tests wereused to determine differences in mitotic and zooxan-thellae cell abundances between the 2 conditions ofhealth (R v. 2.10.1).

Sample collection for bacterial isolation

Microbial analysis consisted of 6−10 ml sterilesyringe samples drawn from the surface layers oflesioned and healthy Sarcophyton ehrenbergi from3 dive locations using SCUBA. Sampling sites weredetermined based on accessibility and time con-straints, as each site had high levels of lesion occur-rence (Fig. 2). At each of the 3 dive sites, one ulcer-ated coral was chosen for surface mucus samples as

well as one healthy coral, thereby totalling 6 coralssampled for bacterial analysis. Although we realizethis is a fairly low replication number, constraintsregarding boat availability, time and supplies led toour sample sizes.

To minimize between-sample contaminations,each sample was taken by a diver wearing gloves,using a sterile, labelled syringe, and stored in indi-vidual plastic bags for transport to the surface. Sam-ples were taken within the ulcerated lesion and onhealthy tissue. Syringe samples were then trans-ferred to the surface and immediately inoculatedonto Glycerol Artificial Sea Water (GASW) (Smith &Hayasaka 1982) slants.

Due to restricted availability of a laboratory inIndonesia, samples were re-inoculated upon returnto the USA in liquid GASW medium for 16−24 h at26°C (the average water temperature during all sam-pling events). Each original sample (at concentrationC) was then serially diluted in 1 ml of liquid media in6−15 ml falcon tubes to a final concentration of 10−6C.The entire sample (1 ml) was then plated on GASWplates for dilutions of 10−6C and 10−5C and incubatedfor 16 to 24 h at 26°C.

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Fig. 2. Lesioned and healthy Sarcophyton ehrenbergi. (A) S. ehrenbergi displaying areas of yellow lesioned tissue. (B) Magni-fied image of ulcerated lesion. A healthy S. ehrenbergi is not included here but looks like the coral shown in (A) minus

the yellow tissue. (C,D) Examples of a healthy S. ehrenbergi

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Post incubation, single colonies were isolated viacolony morphology, including size, shape and color.Each individual colony was subsequently circled (i.e.on the back of the Petri dish), labelled and re-platedon GASW plates using a sterile toothpick. Overall,135 pure cultures were isolated, 74 originating fromhealthy tissue samples and 65 originating fromlesioned coral samples.

PCR of bacterial 16S rDNA gene

Bacterial isolates taken from each isolated colonywere amplified using 16S rDNA. The forward bacte-rial primer Bact-8F (5’-AGA GTT TGA TCC TGGCTC AG-3’) and the reverse bacterial primer Bact-1512R (5’-GGT TAC CTT GTT ACG ACT T-3’) wereused (Weisburg et al. 1991). Amplification mixturescontained 30 mM Tris-HCl, 50 mM KCl, 1.5 mMMgCl2, 200 µM (each) deoxynucleoside triphos-phates, 150 pmol of each primer and 5 U Taq poly-merase (Promega) in a final volume of 100 µl. Steriletoothpicks were used to transfer a small amount ofeach isolate to the PCR reaction tubes. A controlstrain of Vibrio alginolyticus (R-370) was used, aswell as negative controls containing no DNA tem-plates to ensure non-target sources of bacteria werenot amplified. PCR was conducted with an Eppen-dorf Mastercycler Personal. PCR amplification con-sisted of 2 min denaturation at 94°C, followed by 35cycles of 1 min denaturation at 94°C, a 1 min anneal-ing at 40°C, 1 min 30 s extension at 72°C with a final5 min extension at 72°C after completion of the lastcycle. Finally, 5 µl of the PCR reaction was visualizedon 1% agarose containing SYBR® Safe DNA gelstain (Invitrogen), with a UV light.

DNA sequencing

DNA was sequenced on a Beckman-CoulterDNA automated sequencer (Applied Biosystems).Sequence chromatograms of forward and reversesequences were edited and assembled usingSequencher 4.8 (Gene Codes). The sequences werethen aligned using BioEdit Sequence Alignment Edi-tor version 7.0.9.0 (Hall 1999) and ClustalX (Thomp-son et al. 1997). A maximum likelihood tree wasdeveloped using MEGA5 and edited in Fig Treev1.3.1. The tree was rooted using Enterovibriocoralii. Initial tree(s) for the heuristic search wereobtained automatically as follows. When the numberof common sites was <100 or less than one-fourth of

the total number of sites, the maximum parsimonymethod was used; otherwise the BIONJ (BiologicalNeighbor Joining) method with an MCL (MarkovLinkage Clustering) distance matrix was used. Thetree is drawn to scale, with branch lengths measuredin the number of substitutions per site. The analysisinvolved 45 nucleotide sequences. Codon positionsincluded were 1st, 2nd and 3rd noncoding. All posi-tions containing gaps and missing data were elimi-nated. There were a total of 628 positions in the finaldata set. Evolutionary analyses were conducted inMEGA5.

RESULTS

Transect data show a significantly higher fre-quency of lesioned Sarcophyton ehrenbergi com-pared with healthy coral colonies at all 8 sites except

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Fig. 3. Mean (±SE) abundance of healthy and lesioned coralalong 50 m transects at individual sites. Lesioned coral weresignificantly more prevalent at all sites except Site 7. Aster-isks represent a significant difference between lesioned andhealthy coral at a particular site (*p < 0.05, **p < 0.01, ***p <

0.001, ****p < 0.0001)

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for Site 7. However, at this site the mean abundanceof lesioned coral was still greater than that of healthyindividuals (Fig. 3). The mean abundance of lesionedS. ehrenbergi was more than double that of healthyS. ehrenbergi at nearly all sites (1, 2, 4, 6 and 8). At all8 sites, the size of yellow lesions affecting S. ehren-bergi was similar, with the average lesion size ~1 to2 cm. Lesions on individual coral did not seem to bespatially or temporally correlated. However, coralwith lesions seemed to be closer together, rather thanhaphazardly distributed throughout the reef.

Mean density of zooxanthellae in healthy tissueswas significantly (p < 0.0001) different from cell den-sities found in lesioned tissue (Fig. 4A), with densitiesin healthy tissue ~50% greater than those of lesioned

tissues. MI% was also significantly (p < 0.0001) dif-ferent in healthy tissues compared with lesioned tis-sues, with a 5-fold increase in mitotic cells fromlesioned to healthy tissue (Fig. 4B). During MI%counts, it was also noted that zooxanthellae fromlesioned tissue were in poorer physiological condi-tion than those from healthy tissue.

Results of PAM analysis also demonstrated a dis-ruption of zooxanthellae photosynthetic efficiency,with mean fluorescent yield (dF/Fm’) of lesioned tis-sue significantly lower (p < 0.0001) than that ofhealthy tissue (Fig. 5). ETRs were also significantlylower (p < 0.0001) in lesioned versus healthy tissue.Multiple logistic regression indicated yield to be sig-nificantly different between healthy and lesioned tis-sue. Predicted values from this model indicate that ayield of 0.3 will provide an ~95% probability of indi-cating non-healthy tissue. Regression analysis alsoindicated that depth and ETR add significantly to thedetermination of lesioned tissue; however, oddsratios for these variables were 1.289 and 1.031, re -spectively, and therefore less predictive than yield.ETRs <10 indicated an 80% chance of non-healthytissue. Shallower depths increased the probability offinding non-healthy tissue; however, within the rangeof depths sampled, a high degree of variability existed

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Fig. 4. (A) Mean (±SE) zooxanthellae density and (B) zoo-xanthellae mitotic index (MI%) for healthy and lesioned tis-sue samples taken from individual infected Sarcophyton sp.colonies. The means were calculated using all sites. Signifi-cant differences are reported with asterisks (****p < 0.0001)

Fig. 5. Mean (±SE) fluorescent yield of healthy versus lesioned corals along 50 m transects recorded at all sites(n = 666 healthy, n = 736 lesioned). Significant differences

are reported with asterisks (****p < 0.0001)

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with respect to lesioned tissue occurrence. We there-fore believe that depth does not play a large role interms of lesion frequency.

As described earlier, we isolated 135 bacterialcolonies. However, only 113 isolates could be se -

quenced successfully (51 from healthy and 62 fromlesioned coral tissues). Unsuccessful sequences werelikely due to poor quality DNA amplification duringPCR reactions. Results of 16S rDNA sequencingrevealed Vibrio (74%) and Pseudoalteromonas (18%)

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Fig. 6. (A) Bacterial strains isolated from healthy Sarcophyton ehrenbergi tissue samples. Strains with a small number of repli-cates were designated as ‘rare’ for figure clarity and separated into groups A and B for identification in the figure legend. Iso-lates in rare group A (all at 2%) are Vibrio sp. (H3), Vibrio sp. (PaD3.33f1), Vibrio sp. (PaH2.13D3), Vibrio sp. (PaH3.38), Vibriosp. (PaH2.18a1), Vibrio sp. (PaH2.18a2), Vibrionaceae bacterium (DS12), Vibrionaceae bacterium (WEED1), Photobacteriumjeanii, and Allomonas enterica. Isolates from rare group B (both at 2%) are Vibrio sp. (2ta18) and Vibrio sp. (05071F2). (B) Bac-terial strains isolated from lesioned S. ehrenbergi tissue samples. Isolates in rare group A, with their percentages, are Vibriosp. (BD8A) (3%), Vibrio sp. (CF4-11) (3%), Vibrio sp. (CN87) (2%), Vibrio sp. (K323) (2%), Vibrio sp. (PaH1.06a) (2%), Vibriosp. (PaH1.24) (3%), Vibrio sp. (PaH3.38) (3%), Vibrio sp. PaH3.39 (3%), andVibrio sp. (Y4tang) (3%). Isolates in rare group Bare Vibrio ponticus (5%), Vibrio natrigens (UST040801-014) (2%), Vibrio alginolyticus (1306) (2%), Vibrio harveyi (LB4) (2%),Photobacterium sp. (PaH1.37b) (5%), Photobacterium sp. (PaH1.03b) (5%), and Photobacterium sp. (PaD3.26) (3%). (C) Theevolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model. The tree withthe highest log likelihood (–3369.9) is shown. The percentage of trees in which the associated taxa clustered together is shown

next to the branches. *Strains isolated in lesion tissue sample

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as the predominant genera representedin cultures isolated from healthy tissue(Table 1, Fig. 6A,C). Single isolates ofPhotobacterium jeanii and Allomonasenterica were also present in healthy tis-sue samples. Other notable bacterialspecies represented in healthy Sarco-phyton ehrenbergi tissue include V. fur-nissi, V. campbelli, V. azureus and V.harveyi.

Vibrio spp. were also largely repre-sented (48%) in lesioned tissues; how-ever, most were different strains fromthose isolated from healthy tissues (Fig.6B,C, Table 1). Notably, Vibrio algi-nolyticus, a known coral disease-causingbacterium (Cervino et al. 2004, 2008),was present in lesioned samples. Unlikehealthy tissue, however, there was alarge presence of Photobacterium spp.(52%) in lesioned coral tissues. Four sep-arate strains of Photobacterium damse-lae were identified, as well as 2 strains ofP. damselae ssp. piscidia. Three otherstrains of Photobacterium spp. were alsoidentified, but not to the species level.Other notable bacteria identified inlesioned tissues include V. harveyi, V.natri e gens and V. ponticus.

Three Vibrio strains were isolated inboth healthy and lesioned coral tissuesamples (Fig. 6C, Table 1). These in -cluded V. harveyi (LB4), Vibrio sp.(BD8A) and Vibrio sp. (PaH3.38). Al -though present in both healthy andlesioned coral tissue samples, Vibrio spp.were found in different concentrations.For example, 4 isolates of V. harveyi(LB4) were identified in healthy tissues,whereas only one was found in lesionedcoral samples. Six isolates of Vibrio sp.(BD8A) were isolated in healthy coral tis-sue, while only 2 were found associatedwith lesioned coral tissue, and 2 Vibriosp. (PaH3.38) were found with lesionedand one with healthy coral tissue.

DISCUSSION

As reports of coral and marine diseasesand syndromes have increased over thepast 30 to 40 yr (Harvell et al. 1999,

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Name n GenBank Identity accession number (%)

Healthy samplesAllomonas enterica 1 HQ908698.1 99Photobacterium jeanii (R-21419) 1 GU065212.1 99Pseudoalteromonas sp. (cu2i-13) 3 JN594612.1 100Pseudoalteromonas sp. (SB-G1) 2 AB457053.1 98Pseudoalteromonas sp. (VS-1) 4 FJ497708.1 99Uncultured Vibrio sp. (2.15) 1 AM930481.1 99Uncultured Vibrio sp. (4.5) 4 AM930497.1 98Vibrio furnissii (35016 T) 1 X76336.1 99Vibrio azureus (F75109) 6 HQ908698 99Vibrio campbelli (CAIM 780) 1 FM204852.1 99V. campbelli (6-8PIN) 1 GQ203112.1 99Vibrio furnissi (M1) 3 EU204961.1 98Vibrio harveyi (AM11) 1 AB512470.1 99V. harveyi (04103) 1 AM422802.1 99V. harveyi (LB4)* 4 FJ952784.1 99Vibrio sp. (05071F2) 1 HQ449441.1 99Vibrio sp. (2ta18) 1 FJ952784.1 98Vibrio sp. (BD8A)* 6 EU372939.1 95Vibrio sp. (h3) 1 EF187010.1 98Vibrio sp. (PaD3.33f1) 1 GQ406671.1 99Vibrio sp. (PaH2.13D3) 1 GQ406760.1 99Vibrio sp. (PaH2.18a1) 1 GQ391955.1 99Vibrio sp. (PaH2.18a2) 1 GQ391956.1 99Vibrio sp. (PaH3.38)* 1 GQ406800.1 99Vibrionaceae bacterium (DS12) 1 EF584044.1 98Vibrionaceae bacterium (WEED1) 1 EF584103.1 99

Lesioned samplesPhotobacterium damselae (DFIL2.4) 1 FR873782 100P. damselae (F77068) 1 JF281756 100P. damselae (ST1) 14 GU228793.1 99P. damselae (UCP4) 1 DQ530294.1 99P. damselae ssp. piscidia (PN510) 6 AY1478601 99P. damselae ssp. piscidia (SWZX5) 1 EF643517.1 99Photobacterium sp. (PaD3.26) 2 GQ392013.1 98Photobacterium sp. (PaH1.03b) 3 GQ406683 99Photobacterium sp. (PaH1.37b) 3 GQ406722.1 99Vibrio alginolyticus (1306) 1 GU726845.1 99Vibrio harveyi (LB4)* 1 DQ146935.1 99Vibrio natriegens (UST040801-014) 1 EU834012.1 98Vibrio ponticus (292) 2 AJ630202.1 98V. ponticus (69) 1 NR02032.1 97Vibrio sp. (A975) 8 GU223593.1 99Vibrio sp. (BD8A)* 2 EU372939.1 95Vibrio sp. (CF4-11) 2 FJ169995.1 99Vibrio sp. (CN87) 1 EU413955.1 99Vibrio sp. (K323) 1 GU223600.1 99Vibrio sp. (PaH1.06a) 1 GQ406687.1 99Vibrio sp. (PaH1.24) 2 GQ406711.1 97Vibrio sp. (PaH3.38)* 2 GQ406800.1 99Vibrio sp. (PaH3.39) 2 GQ40680.1 99Vibrio sp. (Y4tang) 2 EF187013.1 99

Table 1. Complete list of bacterial strains isolated from healthy and lesionedSarcophyton ehrenbergi tissue samples. n: number of each strain isolated inhealthy tissue; identity: percentage identity to closest-match bacterial rep-resentative in GenBank database. Asterisks (*) indicate strains isolated in

healthy and lesioned tissue samples

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Porter et al. 2001, Sweatman et al. 2001), it has fre-quently been argued whether these diseases are‘novel’, have always been present but are increasingin incidence and severity, or have recently begun tobe noticed more readily as we become more aware oftheir presence (Ward & Lafferty 2004). Although UYSmay have been present on Sarcophyton ehrenbergicolonies prior to 2009, its frequency and intensity hadnot been large enough for documented observation,research and publication. It is also important to notethat at these locations our group identified no lesionsduring the initial 2004 research trip. In 2009 weobserved that all sites contained significantly (p <0.01) more S. ehrenbergi colonies with lesions thanwithout, except at Site 7 (Fig. 3). Based on sample sitelocations, this symptom seems to be spread evenlyalong the east coast of Tomia and Onemobaa Islands,part of the Wakatobi archipelago.

The cellular impairment mechanisms associatedwith diseases such as yellow band disease (YBD) areunusual when compared with those of other coralstressors. With YBD, the pathogenic strains of bac -teria associated with the disease (Vibrio spp.) are at-tacking the symbiotic algae in vivo (Cervino et al.2004, 2008, Roff et al. 2008) instead of resulting insymbiont expulsion, as is common during stress-re-lated coral bleaching (Gates et al. 1992, Strychar et al.2004). Other disease-causing Vibrio spp. have beenisolated from Mediterranean and Red Sea corals, andthese appear to attack the symbiotic zooxanthellaeliving in the tissues of gorgonians and reef builders(Ben-Haim et al. 1999). Results from our study indi-cate the possibility of ulcerated lesions on the surfaceof the coral that create a microbial mat, which maybe decreasing light penetration into the gastrodermwhere the zooxanthellae reside. This effect is nega-tively impacting zooxanthellae function via decreasesin zooxanthellae MI% (Fig. 4B) and photosyntheticefficiency observed in overall quantum yield of photochemical energy conversion (dF/Fm’) (Fig. 5).Zooxanthellae density also de creased significantly(Fig. 4A) from healthy to lesioned coral tissue (p <0.0001), although the mechanism behind this de-crease in density is not yet known (i.e. expulsion ver-sus degradation). Further, previous studies on coraldiseases have shown causative pathogens to produceextracellular toxins, which negatively impact thesymbiont (e.g. Banin et al. 2000). Although we did notdirectly address the potential production of bacterialtoxins, future studies of these isolates may benefitfrom such an investigation.

Results obtained from sequencing culturable bac-teria isolated from lesioned versus healthy coral tis-

sue suggest 2 different states of microbial associa-tions, regardless of low replication rates from tissuesampling. Healthy coral tissue was found to have agreater overall diversity of bacteria compared withlesioned coral tissue (Fig. 6A,B). Previous studies ofbacteria associated with diseased and healthy tissuein coral have shown a decrease in bacterial diversityas well as shifts towards Vibrio spp. dominance ininfected coral during periods of elevated tempera-tures (e.g. Ritchie 2006). Vibrio is a common genusfound in coral-associated microbial assemblages dur-ing healthy and disease states (Ritchie & Smith 2004,Bourne & Munn 2005, Ritchie 2006). Interpretation ofthese results is speculative as the methodologiesused here may have created competitive exclusion ofculturable microbial constituents and therefore maynot express the true degree of culturable bacterialdiversity between these 2 tissue conditions.

In addition to known species of non-pathogenicVibrio, previous studies have found Pseudo altero -monas sp. to be regularly associated with healthycoral (e.g. Rohwer et al. 2001), similar to our results.In the present study, Vibrio represented 48% of thetotal culturable species isolated from lesioned tissuesand 74% in healthy tissues. However, the species orstrain of Vibrio isolated in each tissue type was differ-ent, with those isolated in lesioned tissue being morereadily associated with pathogenicity and disease.

Multiple Vibrio species have been identified aspathogenic to coral, including but not limited to V.harveyi, V. alginolyticus, V. shiloii and V. coralliilyti-cus (Kushmaro et al. 1996, Ben-Haim et al. 2003,Cervino et al. 2004, Luna et al. 2010). As seen inTable 1, one strain of V. alginolyticus was identifiedin our samples from lesioned coral tissues. Vibrioalginolyticus has been previously implicated as amember of the putative YBD-causing consortium ofbacteria (Cervino et al. 2004, 2008) along with 4 othernovel Vibrio strains. V. alginolyticus isolates havealso been isolated from numerous aquaculture sitesworldwide, indicating that it may be the most ubiqui-tous Vibrio phenotype found in diseased crustaceans,marine mammals (Buck et al. 1991) and finfish (Col-well & Grimes 1984, Austin et al. 1993).

Along with Vibrio alginolyticus, several more notablebacterial species were isolated in our samples, in -cluding V. ponticus, Photobacterium damselae and P.damselae ssp. piscidia. V. ponticus has previouslybeen isolated from diseased and non-diseased condi-tions (Macián et al. 2004). Photobacterium spp. areGram-negative, halo philic bacteria found in the fam-ily Vibrionacea, are commonly found in marine sea-water and can be isolated from coastal waters and

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sediments all over the world (Rubin & Tilton 1975).This pathogen is known to cause systemic infectionin aquaculture fish leading to high rates of mortality(Botella et al. 2002, Pujalte et al. 2003). P. damselae isassociated with 2 different subspecies, ssp. damselae(formerly known as Vibrio damselae) and ssp. pisci-cida (formerly known as Pasteurella piscicida) (Gau-thier et al. 1995). Although both subspecies are con-sidered to be pathogenic in marine fish, it has beenshown that only ssp. piscicida causes infections withhigh rates of mortality (Botella et al. 2002, Pujalte etal. 2003). P. damselae ssp. piscicida is the causativeagent of pasteurellosis (also known as pseudotuber-culosis), known as one of the most significant fish dis-eases in marine aquaculture (Magariños et al. 1996).Romalde et al. (2002) has shown that this speciesbecomes pathogenic when sea surface temperaturesincrease above 18−20°C. Below this temperature,however, fish can carry the pathogen as a subclinicalinfection and may be carriers for extended time peri-ods (Romalde 2002). In our study these strains wereassociated exclusively with lesioned coral tissue.

Three species of Vibrio were found in both healthyand lesioned coral samples: Vibrio sp. (PaH3.38), V.harveyi (LB4) and Vibrio sp. (BD8A). Vibrio sp.(PaH3.38) was originally isolated from a healthyPseudopterogorgia americana mucus sample (Viz-caino et al. 2009), while V. harveyi (LB4) was isolatedfrom Montipora aequituberculata infected with whitesyndrome (Sussman et al. 2008) and Vibrio sp.(BD8A) was isolated from a diseased fish in an aqua-culture pond (Wang et al. 2010). With the exceptionof Vibrio sp. (PaH3.38), fewer numbers of the remain-ing strains were isolated from lesioned versushealthy coral tissue. Although there seems to be adifference in density of these bacteria betweenhealthy and lesioned tissues, isolation of the samebacterial strains, some of which may be associatedwith disease, may be a result of the beginning ofinfection, but not yet providing visual evidence of alesion. For example, a study by Ritchie (2006) indi-cated a population shift of Vibrio species on the sur-face mucus of corals in healthy versus diseased states(see also Ritchie & Smith 1997, Rohwer et al. 2001).

The goal of the molecular portion of this study wasnot to identify the causative pathogens of this syn-drome, but to preliminarily identify some of the bac-teria associated with lesioned and healthy tissues aswell as to describe the complex of symptoms associ-ated with these yellow ulcerated lesions. As themetho dologies used here (i.e. GASW medium,growth temperature and time, non-degenerate 16SPCR primers, etc.) are highly selective, interpretation

of these preliminary results is speculative. Here wealso defined a lesion as any functional or morpholog-ical abnormality in coral tissues (Thompson 1978).Accordingly, lesions may be caused by many thingsbesides bacterial infection, including but not limitedto predation, human diving-related injuries, nutri-tional deficiencies and exposure to pollutants (Work& Aeby 2006). Because the goal of our study was notto determine the etiology of these lesions, we cannotdirectly state that bacteria are the cause. However,our results do yield potential links between aqua -culture-associated pathogens and a new marine syndrome affecting Sarcophyton ehrenbergi in theWakatobi archipelago of Indonesia, as known aqua-culture pathogens were found associated solely withlesioned tissues. Our data also indicate that this syn-drome is causing inhibition of symbiotic algal photo-synthesis and replication, similar to the effects ofknown coral diseases (e.g. YBD) (Cervino et al. 2004,2008).

Although key species of bacteria have been identi-fied in this study, Fuhrman & Campbell (1998) sug-gest that, in a given marine sample, more than 95%of bacteria cannot be cultured. Considering this,future studies regarding this syndrome should con-sider metagenomic analysis of lesioned versushealthy mucus samples of Sarcophyton ehrenbergias well as determining background microbial com-munities via sampling of the surrounding sedimentand water column for a comparative analysis. Vari-ous metagenomic-sequencing techniques of environ-mental samples will allow detection of unculturablespecies, which is likely to be important whenattempting to assess the coral microbial microbiome(Wegley et al. 2007, Vega Thurber et al. 2009). It hasalso been suggested by Dinsdale et al. (2008) thattaxonomic evaluation of microbes associated with anenvironment through 16S rDNA sequencing is lessefficient at describing microbiome characteristicsthan functional profiling of microbes through fullgenome sequencing.

The links between coral infections, high organicmatter concentrations and changing microbial diver-sity over the past 30 yr appears to be exacerbated byhuman development, affecting sensitive tropicalcoastal environments. Finding and isolating identicalfish pathogens from both aquaculture farms and thesurfaces of corals would be strong evidence thatanthropogenic sources of pollution may be affectingcorals. From a pathology perspective, future re searchis needed to isolate identical fish pathogens at aqua-culture farms and compare them with isolated patho-gens found on corals, including re-infectivity studies

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that satisfy Koch’s postulates. Results from this studydemonstrate the need for rethinking the way humansutilize coastal zones all over the world in order tohelp control infectious marine diseases and promotea healthier and sustainable coastal environment.

Acknowledgements. The authors thank the Coastal Preser-vation Network, Restoration & Conservation LLC and TheGlobal Coral Reef Alliance for funding this research. Wealso thank Dr. Robert Trench, Angela Richards Donà, Dr.Esther Peters, Ryan Chabarria and Sharon J. Furiness forscientific discussions, feedback and help with figure prepa-rations. Finally, we give special thanks to the anonymousreviewers for improving the clarity of this manuscript.

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Editorial responsibility: Garriet Smith, Aiken, South Carolina, USA

Submitted: January 20, 2012; Accepted: October 10, 2012Proofs received from author(s): November 28, 2012

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