Characterization of geographically distinct bacterial communities associated with coral mucus produced by Acropora spp. and Porites spp.
ABSTRACT Acropora and Porites corals are important reef builders in the Indo-Pacific and Caribbean. Bacteria associated with mucus produced by Porites spp. and Acropora spp. from Caribbean (Punta Maroma, Mexico) and Indo-Pacific (Hoga and Sampela, Indonesia) reefs were determined. Analysis of pyrosequencing libraries showed that bacterial communities from Caribbean corals were significantly more diverse (H', 3.18 to 4.25) than their Indonesian counterparts (H', 2.54 to 3.25). Dominant taxa were Gammaproteobacteria, Alphaproteobacteria, Firmicutes, and Cyanobacteria, which varied in relative abundance between coral genera and region. Distinct coral host-specific communities were also found; for example, Clostridiales were dominant on Acropora spp. (at Hoga and the Mexican Caribbean) compared to Porites spp. and seawater. Within the Gammproteobacteria, Halomonas spp. dominated sequence libraries from Porites spp. (49%) and Acropora spp. (5.6%) from the Mexican Caribbean, compared to the corresponding Indonesian coral libraries (<2%). Interestingly, with the exception of Porites spp. from the Mexican Caribbean, there was also a ubiquity of Psychrobacter spp., which dominated Acropora and Porites libraries from Indonesia and Acropora libraries from the Caribbean. In conclusion, there was a dominance of Halomonas spp. (associated with Acropora and Porites [Mexican Caribbean]), Firmicutes (associated with Acropora [Mexican Caribbean] and with Acropora and Porites [Hoga]), and Cyanobacteria (associated with Acropora and Porites [Hoga] and Porites [Sampela]). This is also the first report describing geographically distinct Psychrobacter spp. associated with coral mucus. In addition, the predominance of Clostridiales associated with Acropora spp. provided additional evidence for coral host-specific microorganisms.
- [Show abstract] [Hide abstract]
ABSTRACT: Diazotrophic bacteria potentially play an important functional role in supplying fixed nitrogen to the coral holobiont, but the value of such a partnership depends on the stability of the association. Here we evaluate the composition of diazotroph assemblages associated with the coral Acropora millepora, throughout four seasons and at two reefs, an inshore and an offshore (mid-shelf) reef on the Great Barrier Reef, Australia. Amplicon pyrosequencing of the nifH gene revealed that diazotrophs are ubiquitous members of the bacterial community associated with A. millepora. Rhizobia (65% of the overall nifH sequences retrieved) and particularly Bradyrhizobia sp.-affiliated sequences (>50% of rhizobia sequences), dominated diazotrophic assemblages across all coral samples from the two sites throughout the year. In contrast to this consistency in the spatial and temporal patterns of occurrence of diazotroph assemblages, the overall coral-associated bacterial community, assessed through amplicon sequencing of the general bacterial 16S rRNA gene, differed between inshore and mid-shelf reef locations. Sequences associated with the Oceanospirillales family, particularly with Endozoicomonas sp., dominated bacterial communities associated with inshore corals. Although rhizobia represented a variable and generally small fraction of the overall bacterial community associated with Acropora millepora, consistency in the structure of these diazotrophic assemblages suggests they have a functional role in the coral holobiont.Environmental Microbiology 12/2013; · 6.24 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Bacteria living within the surface mucus layer of corals compete for nutrients and space. A number of stresses affect the outcome of this competition. The interactions between native microorganisms and opportunistic pathogens largely determine the coral holobiont's overall health and fitness. In this study, we tested the hypothesis that commensal bacteria isolated from the mucus layer of a healthy elkhorn coral, Acropora palmata, are capable of inhibition of opportunistic pathogens, Vibrio shiloi AK1 and Vibrio coralliilyticus. These vibrios are known to cause disease in corals and their virulence is temperature dependent. Elevated temperature (30 °C) increased the cell numbers of one commensal and both Vibrio pathogens in monocultures. We further tested the hypothesis that elevated temperature favors pathogenic organisms by simultaneously increasing the fitness of vibrios and decreasing the fitness of commensals by measuring growth of each species within a co-culture over the course of 1 week. In competition experiments between vibrios and commensals, the proportion of Vibrio spp. increased significantly under elevated temperature. We finished by investigating several temperature-dependent mechanisms that could influence co-culture differences via changes in competitive fitness. The ability of Vibrio spp. to utilize glycoproteins found in A. palmata mucus increased or remained stable when exposed to elevated temperature, while commensals' tended to decrease utilization. In both vibrios and commensals, protease activity increased at 30 °C, while chiA expression increased under elevated temperatures for Vibrio spp. These results provide insight into potential mechanisms through which elevated temperature may select for pathogenic bacterial dominance and lead to disease or a decrease in coral fitness.Microbial Ecology 12/2013; · 3.12 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Diverse sessile organisms inhabit the coral reef ecosystems, including corals, sponges, and sea anemones. In the past decades, scleractinian corals (Cnidaria, Anthozoa, Scleractinia) and their associated microorganisms have attracted much attention. Zoanthids (Cnidaria, Anthozoa, Zoanthidea) are commonly found in coral reefs. However, little is known about the community structure of zoanthid-associated microbiota. In this study, the microbial community associated with the zoanthid Palythoa australiae in the South China Sea was investigated by 454 pyrosequencing. As a result, 2,353 bacterial, 583 archaeal, and 36 eukaryotic microbial ribotypes were detected, respectively. A total of 22 bacterial phyla (16 formally described phyla and six candidate phyla) were recovered. Proteobacteria was the most abundant group, followed by Chloroflexi and Actinobacteria. High-abundance Rhizobiales and diverse Chloroflexi were observed in the bacterial community. The archaeal population was composed of Crenarchaeota and Euryarchaeota, with Marine Group I as the dominant lineage. In particular, Candidatus Nitrosopumilus dominated the archaeal community. Besides bacteria and archaea, the zoanthid harbored eukaryotic microorganisms including fungi and algae though their diversity was very low. This study provided the first insights into the microbial community associated with P. australiae by 454 pyrosequencing, consequently laid a basis for the understanding of the association of P. australiae-microbes symbioses.Microbial Ecology 02/2014; · 3.12 Impact Factor
Characterization of Geographically Distinct Bacterial Communities
Associated with Coral Mucus Produced by Acropora spp. and
B. A. McKew, A. J. Dumbrell, S. D. Daud, L. Hepburn, E. Thorpe, L. Mogensen, and C. Whitby
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, United Kingdom
andregion.Distinctcoralhost-specificcommunitieswerealsofound;forexample, Clostridiales weredominanton Acroporaspp.
(atHogaandtheMexicanCaribbean)comparedto Poritesspp.andseawater.Withinthe Gammproteobacteria,Halomonas spp.
therewasalsoaubiquityof Psychrobacter spp.,whichdominated AcroporaandPoriteslibrariesfromIndonesiaand Acropora
librariesfromtheCaribbean.Inconclusion,therewasadominanceof Halomonas spp.(associatedwith AcroporaandPorites
[MexicanCaribbean]), Firmicutes (associatedwith Acropora[MexicanCaribbean]andwith AcroporaandPorites[Hoga]),and
Cyanobacteria (associatedwith AcroporaandPorites[Hoga]andPorites[Sampela]).Thisisalsothefirstreportdescribinggeo-
graphicallydistinct Psychrobacter spp.associatedwithcoralmucus.Inaddition,thepredominanceof Clostridiales associated
nutrition (7), response to stress (12), and health and disease (6,
40). Coral bleaching and various coral diseases are increasing due
to changes in environmental conditions that result in either an
fore, understanding the microbial communities associated with
corals and how they vary in response to changing environmental
conditions is important for understanding the future health of
Previous studies that used both culture-dependent and cul-
ture-independent methods demonstrated that coral-associated
those dominating the surrounding reef water (4, 17, 39). Similar
cies from geographically different locations, and different bacte-
rial communities have been found on different coral species (38,
39). By using clone libraries and sequence analysis, Bourne and
Munn (4) found that the majority of clones recovered from the
coral tissue of Pocillopora damicornis were related to Gammapro-
teobacteria, while Alphaproteobacteria were dominant in the coral
mucus, thus further supporting the hypothesis that specific bac-
terium-coral associations exist.
Although many studies have supported the hypothesis that
corals harbor unique microbiota, inconsistencies across studies
have raised many questions about the specificity and dynamics of
associations between corals and microbes. One of the major lim-
ing methods do not allow for characterization of the microbial
community beyond the most dominant taxa (47). However, cur-
icroorganisms are important to coral reef ecosystems
through their roles in carbon/nitrogen cycling (43), coral
tection of rare taxa (45). These rare taxa remain largely unex-
plored, but they may be extremely important and may become
more dominant in response to environmental changes (47).
of bacterial communities across reef bioregions and environmen-
with a hemispherical shape and slow growth rates. As a result,
Porites tend to be longer lived, often for hundreds of years, and
grow to large sizes (37, 48). The Caribbean Porites asteroides may
grow up to 1 m but tends to form more numerous, smaller colo-
nies, while Porites lutea from Indonesia grows up to a few meters
pora formosa from Indonesia grows on average up to 1 m. Both
genera occur in shallow, tropical reef environments, reef slopes,
and in lagoons (48).
the bacterial community structures associated with the coral mu-
cus produced by Porites astreoides and Acropora palmata from
Received 8 December 2011 Accepted 26 April 2012
Published ahead of print 25 May 2012
Address correspondence to C. Whitby, email@example.com.
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
August 2012 Volume 78 Number 15 Applied and Environmental Microbiologyp. 5229–5237aem.asm.org
Mexican Caribbean reefs with those associated with the closely
related Porites lutea and Acropora formosa found in Indonesia
(southeast Sulawesi, Hoga and Sampela reefs). Such information
understanding of coral physiology, health, and ecosystems.
MATERIALS AND METHODS
from three regions on a colony, from triplicate living colonies) of Porites
astreoides and Acropora palmata at Punta Maroma, Mexican Caribbean
and Acropora formosa at Hoga (18 samples) and Sampela (18 samples),
mucus samples were filtered using 0.22-?m filters, and the filters were
stored at ?20°C. Overlying seawater (two 500-ml volumes) adjacent to
the coral colonies was also collected at a depth of 10 m, filtered through
0.22-?m filters, and stored at ?20°C. The water temperature during col-
lections at both sites was typically 28°C.
Total community DNA was extracted from the filtered seawater and mu-
cus samples (from pooled colony regions on each colony replicate) by
using a modified beadbeating method (26). Eubacterial 16S rRNA genes
were PCR amplified using the primers for positions 341 to 534 in Esche-
richia coli (Table 1) (28). PCRs were performed in a GeneAmp PCR sys-
(50-?l volumes): 1? buffer (Qiagen), 0.2 mM deoxynucleoside triphos-
phates (Fermentas), 0.4 ?M each primer, 2.5 U Taq DNA polymerase
(Qiagen), and approximately 25 ng of DNA (26). PCR cycling conditions
were as follows: 95°C for 5 min, followed by 30 cycles of 94°C for 1 min,
at 4°C. PCR products were analyzed using 1% (wt/vol) 1? TAE agarose
gels (40 mM Tris-acetate, 1.0 mM EDTA; pH 8.0), stained with ethidium
bromide (0.5 mg liter?1), and visualized under UV light by using the
Gel-Doc system (Bio-Rad). Denaturing gradient gel electrophoresis
silver stained (30).
Clone libraries. PCR products were obtained using the primers pA/
efficiency JM109 Escherichia coli cells (Promega) according to the manu-
a plasmid purification kit (Qiagen) according to the manufacturer’s in-
clones by Geneservice Ltd., Cambridge, United Kingdom. Partial se-
the GenBank database by using the Basic Local Alignment Search Tool
(BLAST) network service (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi)
(1). Sequences were aligned with sequences from GenBank by using the
RDP INFERNAL alignment tool (29). Analysis was performed using
PHYLIP 3.4 (16) with Jukes-Cantor DNA distance correction and neigh-
bor-joining methods (21, 42). Bootstrap analysis was based on 100 repli-
was performed using Treeview (WIN32; version 1.5.2) (31). For pyrose-
quencing, PCR products were obtained as described above using the
primers F341-GC and 534 R (Table 1) (28), except that the forward
primer had no GC clamp and a 5= modification with a 454 amplicon
adaptor followed by a unique 10-nucleotide barcode (2, 33). PCR prod-
ucts were quantified with a Nanodrop ND-1000 spectrophotometer, and
pooled sample was analyzed by pyrosequencing by the NERC Biomolec-
ular Analysis Facility.
Pyrosequence reads were analyzed using the QIIME pipeline and its
associated modules (8). All sequences were checked for the presence of
correct pyrosequencing adaptors, 10-bp barcodes, and taxon-specific
removed. In addition, sequences of ?150 bp or ?200 bp in read length,
sequences with low quality scores (?20), and sequences containing ho-
mopolymer inserts were also removed from further analysis. All pyrose-
FIG 1 Sampling sites. (A) Map of the Wakatobi National Park, Sulawesi, Indonesia, showing the Sampela and Hoga (Hoga buoy 2) reefs. (Reprinted from
reference 19 with permission of the publisher.) (B) Map showing Punta Maroma on the northeastern coast of the Yucatan Peninsula, Mexico.
TABLE 1 Summary of PCR primer sequences used in this study
aPrimer 341 contains a 40-nucleotide GC-rich sequence (GC clamp) for DGGE
McKew et al.
aem.asm.org Applied and Environmental Microbiology
quence reads were clustered into operational taxonomic units (OTUs) by
using the UClust algorithm (14). Representative sequences from each
OTU were identified using the RDP classifier, which assigns taxonomic
identities against the RDP database by using a naïve Bayesian classifier
(49). Finally, all singletons were removed before further analysis.
Statistical analysis. Similarity between DGGE profiles was calculated
using binary data indicating the presence of particular bands (Jaccard’s
index) and a hierarchal cluster analysis constructed using Primer E soft-
ing (NMDS) ordination of distance matrices calculated from the OTU
in composition between sites were assessed using permutation-based
multivariate analysis of variance (PERMANOVA), based on the distance
Geographic location (either the Mexican Caribbean, Sampela, or Hoga)
was not explored.
Species diversity (number and relative abundance of OTUs) was cal-
corals by using a simple randomization test, based on 10,000 randomiza-
tions (46). This randomization approach was also used to compare Jac-
card index results between coral species. The randomization approach
used treats the entire community as a single data set and is an absolute
statistical measure that does not require replication to produce probabil-
where full replication of sampled communities is impossible (25). All
analyses were conducted in the R statistical language version 2.7.2 and
using the R standard libraries and the community ecology analysis-spe-
cific package Vegan (version 2007; R Development Core Team).
mitted to GenBank and assigned accession numbers HQ456683 to
Community and phylogenetic analyses. Bacterial communities
from coral and seawater samples were analyzed by 16S rRNA
PCR-DGGE analysis (see Fig. S1 in the supplemental material).
There were clear differences in DGGE profiles between corals and
their geographical regions (see Fig. S1). Profiles were, however,
similar between colony replicates, and so the replicates were
pooled for detailed community analysis by pyrosequencing of the
16S rRNA gene. Libraries comprising a total of 9,353 sequences
with low quality scores [?20], and sequences containing ho-
cus samples produced by P. astreoides and A. palmata (Mexican
Caribbean) and P. lutea and A. formosa (Hoga and Sampela, In-
donesia) were analyzed (Table 2).
NMDS ordination revealed distinct microbial communities
associated with samples from geographically distinct regions (Ta-
ble 2; Fig. 2). PERMANOVA results supported the NMDS ordi-
nation and showed that the compositions of the bacterial assem-
blages were significantly different between geographic sites
(PERMANOVA, based on 10,000 randomizations; F1,8? 2.08;
P ? 0.03), but not between coral species and seawater samples
(F1,8? 0.58; P ? 0.88). The most noticeable result was that bac-
terial communities from the corals and seawater in the Mexican
Caribbean were clearly distinct from those from Indonesia (Fig.
2). Although across all sites there was no significant difference
between coral species in the composition of the associated bacte-
rial assemblages (see above), by focusing only on data from the
ter samples (simple pairwise randomization test based on 10,000
randomizations; Jaccard’s index, ?0.89; P ? 0.01 in all cases).
Clustering and classification (Table 2) and diversity analysis
(Table 2; Fig. 3A and B) of pyrosequencing libraries revealed geo-
graphically distinct coral-bacteria associations. In general, bacte-
diverse (H=, 3.18 to 4.25), followed by samples from Hoga (H=,
terial assemblages (H=, 2.54 to 2.64) (Fig. 3B). However, most
samples had similar levels of bacterial diversity, and only in bac-
terial assemblages from the Mexican Caribbean Acropora samples
were diversity levels significantly higher than those from other
sites or corals (simple pairwise randomization test based on
10,000 randomizations; ?H=, ?0.87; P ? 0.001 in all cases) (Fig.
3B). Analyses of diversity indices were supported by analysis of
rarefied species richness, which provided quantitatively similar
results but accounted for differences in sequencing intensities be-
tween samples (Fig. 3A).
Overall, Gammaproteobacteria dominated pyrosequencing li-
from Porites (76.1%) (from the Mexican Caribbean) (Table 2).
This was in contrast to Acropora and Porites libraries from Sam-
pela, which were dominated by Alphaproteobacteria (comprising
?54.3%). Bacterial communities that differed with coral genera
(from either Indonesia or the Caribbean) were also observed.
For example, within the Gammaproteobacteria, Halomonas spp.
(49%) and Alteromonadales (9.1%) were predominant in Porites
libraries (from the Mexican Caribbean), compared to Acropora at
the same location and the corresponding Indonesian corals
(?5.6%) (Table 2). Other differences in coral-associated bacteria
included Clostridiales, which were more predominant from Acro-
Mexican Caribbean, 7.2% of sequences were related to Clostridi-
ales from Acropora, compared to 0.2% from Porites libraries. In
compared to Acropora (6.4%) and the corresponding corals from
As with Indonesia, other distinct sequences were associated
with the corals found in the Mexican Caribbean (Table 2). For
example, there was a relative dominance of Firmicutes (24.1%),
primarily bacilli (16.7%), found in Acropora libraries, but Firmic-
utes were much rarer (?2.8%) in the Porites libraries from the
spp. were more dominant in the Mexican Caribbean Acropora
libraries (6.2%) than Porites (2.0%) and the corresponding Indo-
nesian coral libraries (?0.2%). In contrast, Sulfitobacter spp.
dominated both coral libraries from Sampela (comprising
?47.3%) compared to the corresponding coral libraries from
Hoga and the Mexican Caribbean (?3.2%).
Similarities were also found with the coral-associated bacterial
communities (from either Indonesia or the Caribbean) (Table 2).
ies (with the exception of Porites from the Mexican Caribbean).
Psychrobacter spp. were also more dominant in Acropora (63.4%)
and Porites (53.4%) from Hoga (compared to the corresponding
corals in Sampela [27.1% and 30.1%, respectively] and Acropora
[26.7%] from the Mexican Caribbean) (Table 2). Similarly, cya-
Bacterial Diversity Associated with Coral Mucus
August 2012 Volume 78 Number 15aem.asm.org 5231
nobacteria (28%) were also more dominant in Porites than in
libraries from Sampela (?5.2%) and the Mexican Caribbean
Not surprisingly, there were also differences observed in sea-
ribbean (Table 2). For example, Gammaproteobacteria (70.1%)
water from Sampela (23.6%) and the Mexican Caribbean (26.7%).
Conversely, Alphaproteobacteria (67.7%) were more dominant in
seawater from Sampela, Indonesia, than in seawater from Hoga
(20.0%) and the Mexican Caribbean (42.3%). In addition to Alpha-
and Gammaproteobacteria, seawater from the Mexican Caribbean
was dominated by Sphingomonadales (31.9%), Cyanobacteria
Clone libraries. In order to obtain almost-full-length 16S
rRNA gene sequences for phylogenetic analysis, clone libraries
were generated from each coral sample (Fig. 4A and B). Libraries
16S rRNA gene sequences (of ?500 bp) were obtained (distrib-
uted relatively evenly across all samples). Generally, there was
sequences from Acropora and Porites (at Hoga) predominantly
clusters associated with Psychrobacter spp. and Halomonas spp.
In addition to Gammaproteobacteria, one clone (HP7) from
Porites (Hoga) grouped with Synechococcus spp. within the Cya-
nobacteria, and two clones (HA11 and MA13) from Acropora
(from Hoga and the Mexican Caribbean, respectively) clustered
TABLE 2 Bacterial assemblages based on 16S rRNA pyrosequencing libraries from coral mucus and seawater
Phylum, class, or order
% of sequences (total n) in phylum, class, or order from sample areaa
Indonesia Mexican Caribbean
Fusobacteria0.6 1.200 0.20000
Rhodobacterales (not including Silicibacter spp. or
Oceanospirillales (not including Halomonas spp.)
Pseudomonadales (not including Acinetobacter
spp., Psychrobacter spp.)
20.059.0 54.367.728.3 14.043.2
aSamples were from Acropora formosa at Hoga (HA), Porites lutea at Hoga (HP), seawater at Hoga (HSW), Acropora formosa at Sampela (SA), Porites lutea at Sampela (SP),
seawater at Sampela (SSW), Acropora palmata at the Mexican Caribbean (MA), Porites astreoides at the Mexican Caribbean (MP), and seawater at the Mexican Caribbean (MSW).
Numbers in bold indicate that the group represents ?5% of the community.
McKew et al.
aem.asm.orgApplied and Environmental Microbiology
with an uncultured Gram-positive bacterium with strong boot-
dominant within the Alphaproteobacteria, followed by Gamma-
proteobacteria (Fig. 4A and B). Dominant sequences from Acro-
fitobacter spp., while dominant clone sequences from Porites
(Sampela) affiliated with Gammaproteobacteria, including Halo-
monas spp., Alteromonas spp., and Psychrobacter spp. In addition
to Gammaproteobacteria sequences, one clone (SP19) associated
with Porites from Sampela clustered with Synechococcus spp.
within the Cyanobacteria. Another clone (SP12) associated with
Porites from Sampela clustered with Exiguobacterium spp. within
In comparison to clones from Indonesian corals, the clone li-
braries from Acropora and Porites from the Mexican Caribbean
spanned several phyla, including Gamma- and Alphaproteobacte-
ria, Firmicutes, Altermonadales, and Actinobacteria (Fig. 4A and
ples from Indonesia, only three clones from Acropora (Mexican
Caribbean) clustered within the Gammaproteobacteria. In addi-
tion, two clones (MA2 and MA11) were closely related to Exiguo-
bacterium spp. within the Firmicutes with strong bootstrap sup-
port (100%) (Fig. 4B).
Gammaproteobacteria (specifically, Halomonas spp. and Al-
teromonas spp.) dominated the Porites library (Mexican Carib-
bean), followed by Alphaproteobacteria. We also found that no
clone sequences from the Porites library (Mexican Caribbean)
clustered with Psychrobacter spp. In addition, two clones (MA1
and MP1; from Acropora and Porites libraries, respectively) had
99% sequence identity to Dietzia spp. within the Actinobacteria,
coccus spp. with strong bootstrap support (100%).
Although clone libraries generally corroborated information
from pyrosequencing libraries, some differences were observed,
ing library from Porites for Sampela (54.3%), but only one clone
sequence (SP7) was found within the Alphaproteobacteria (Fig.
4B). Conversely, Vibrionales were rarer in the pyrosequencing li-
brary, while three clone sequences (HP6, HP10, and HP13) from
Porites (Hoga) grouped with Vibrio spp. with strong bootstrap
support (96%) (Fig. 4A).
The coral mucus samples yielded more diverse 16S rRNA pyrose-
found a high relative abundance of Gammaproteobacteria se-
quences associated with Porites from the Mexican Caribbean, fol-
Porites from Sampela. Similar findings have been previously re-
with Porites astreoides from Panama and Bermuda (39). Further-
more, a high relative abundance of Gammaproteobacteria in the
coral Montastrea cavernosa from the Mexican Caribbean has also
been found (17). In our study, within the Gammaproteobacteria
there was a predominance of sequences relating to Halomonas
spp. associated with Porites and Acropora from the Mexican Ca-
ribbean; in addition, this is the first study to report an abundance
of sequences relating to Psychrobacter spp. associated with both
Acropora spp. and Porites spp.
In addition to Gammaproteobacteria, another study found
Alphaproteobacteria to be the dominant microbial group within
the coral mucus of Pocillopora damicornis from the Great Barrier
Reef (4). More specifically, 36% of the clones were affiliated with
FIG 3 Bacterial community diversity based on the analysis of rarefied species
(OTU) richness (A) and the Shannon-Wiener diversity index (B). OTUs were
taxonomic identities using the RDP classifier. Samples are from Acropora
formosa at Hoga (HA), Porites lutea at Hoga (HP), seawater at Hoga (HSW),
Acropora formosa at Sampela (SA), Porites lutea at Sampela (SP), seawater at
Sampela (SSW), Acropora palmata at the Mexican Caribbean (MA), Porites
FIG 2 NMDS ordination of distance matrices calculated from the OTU
pyrosequence read matrix and using Jaccard’s index. Shown are the bacterial
communities associated with Acropora formosa at Hoga (HA), Porites lutea at
Hoga (HP), seawater at Hoga (HSW), Acropora formosa at Sampela (SA),
the Mexican Caribbean (MA), Porites astreoides at the Mexican Caribbean
(MP), and seawater at the Mexican Caribbean (MSW) based on the 454 pyro-
Bacterial Diversity Associated with Coral Mucus
August 2012 Volume 78 Number 15 aem.asm.org 5233
aem.asm.org Applied and Environmental Microbiology
In our study, Alphaproteobacteria also dominated microbial com-
munities associated with Acropora and Porites from Sampela and
Acropora and Porites from the Mexican Caribbean.
It was previously suggested that mucus of different coral spe-
cies enriches for different bacterial communities (12, 37). In our
study, differences in the library compositions in the coral mucus
of Porites spp. versus Acropora spp. were observed. A meta-
genomic analysis of the microbial community associated with the
teria were Proteobacteria (68%), followed by Firmicutes (10%),
Cyanobacteria (7%), and Actinobacteria (6%) (50), similar to the
findings in the present study. Interestingly, in the present study,
was recovered from Acropora from Hoga and the Mexican Carib-
bean (compared to Porites and seawater samples from the same
FIG 4 Phylogenetic analysis of the 16S rRNA gene sequences from selected clones. Included are type strains obtained from GenBank. Sequence analysis was
performed on common partial sequences (?500 bp) by using Jukes-Cantor DNA distance and neighbor-joining methods. Bootstrap values represent percent-
ages from 100 replicates of the data; percentages of ?80% are shown. Bar, 0.1 substitutions per nucleotide base. The 16S rRNA gene sequences from clones
clustered within the Gammaproteobacteria (A) and within the Alphaproteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Cyanobacteria, and uncultured
brown), Porites lutea at Sampela (SP; blue), Acropora palmata at the Mexican Caribbean (MA; purple), and Porites astreoides at the Mexican Caribbean (MP;
green). Unique clone identifiers are shown following the species names.
Bacterial Diversity Associated with Coral Mucus
August 2012 Volume 78 Number 15aem.asm.org 5235
the Clostridia spp. may play a role in the breakdown of complex
carbon compounds present in the mucus produced by Acropora,
although the available evidence remains inconclusive. Since clos-
tridia are generally obligate anaerobes, it is possible that oxygen
becomes depleted during complex carbon degradation, generat-
ing anaerobic or low-oxygen microniches within the thick mucus
and facilitating their proliferation.
It is known that Porites spp. produce more and denser mucus
than Acropora spp. (12), which may be attributable to its greater
tolerance to sedimentation (7, 27). Furthermore, metagenomic
studies of microbial communities associated with P. astreoides
have shown that coral-associated bacteria possess a large number
of genes for the uptake and processing of protein and sugars, re-
flecting the compounds found in coral mucus (50). However, it is
also entirely possible that bacteria associated with other corals
may possess similar genes.
The Hoga reef in this study has been designated a protected
area (10, 11). The Sampela reef is in an enclosed lagoon, buffered
from the Hoga-Kaledupa Channel by an outer reef wall and lo-
a population of ?1,500 people (3, 44). Due to continued human
activities, the Sampela reef has low light availability and high sed-
imentation rates (10). Specifically, Sampela sedimentation rates
mg cm?2day?1) (20). Consequently, corals at Sampela may be
more likely to be stressed and produce more mucus (27).
It has also been suggested that coral bleaching and coral dis-
eases may be more likely to occur in Hoga than Sampela due to
become infected than those from Sampela, where low light pene-
tration may account for the lack of Vibrio spp. Furthermore,
Porites spp. may produce a higher disease prevalence than Acro-
pora spp. (19). Sulfite-oxidizing bacteria have also been isolated
relating to Sulfitobacter spp. were found in corals from Sampela.
In the Mexican Caribbean, where the reefs are enclosed in a
Indonesian counterparts. Interestingly, two clone sequences, one
from Acropora and one from Porites, were closely related to Acti-
nobacteria. Similar findings have been obtained from the Red Sea
coral Fungia scutaria, whereas Actinobacteria were cultured from
the mucus of healthy corals (22, 23). In addition, our results re-
vealed that the bacterial assemblages associated with corals in the
Mexican Caribbean had significantly distinct compositions from
those from Indonesia, as well significantly distinct communities
specific effects structuring these bacterial assemblages, but also
isolation by distance effects due to the relative geographic separa-
tion of the main sites contributes. Thus, adding further support-
ing evidence for the hypothesis that both the dispersal limitation
and environmental gradients (biotic coral-host niche) affect the
structure microbial communities (13).
been identified within the coral holobiont (43). In our study, mi-
croorganisms closely affiliated with Synechococcus spp. (Cyano-
nitrogen to the coral host.
In conclusion, the microbes associated with mucus from Pori-
tes and Acropora spp. from the Mexican Caribbean and Indonesia
(Hoga and Sampela) reefs were determined. The pyrosequence
library composition associated with the mucus of Acropora spp.
and Porites spp. was more diverse in the Mexican Caribbean than
Indonesia. To our knowledge, this is also the first report describ-
mucus. We found that different coral species harbored different
bacterial sequences that were distinct from seawater, and some
bacteria-coral relationships appeared to be host specific, such as
for Clostridiales with Acropora spp. Since corals are increasingly
tion of coral-associated microbes and their interactions with the
coral host is essential in order to better understand the dynamics
of coral reef systems and their responses to environmental
We thank the University of Essex and the Society for General Microbiol-
ogy for funding this research.
We also thank UNAM (Mexico), in particular the station at Puerto
Morelos, and Paul Blanchon for facilitating fieldwork in Mexico. We also
thank Operation Wallacea and David Smith, University of Essex, for
facilitating the fieldwork at Hoga and Sampela.
1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local
alignment search tool. J. Mol. Biol. 215:403–410.
2. Alvarez LA, Exton DA, Timmis KN, Suggett DJ, McGenity TJ. 2009.
Characterization of marine isoprene-degrading communities. Environ.
3. Bell JJ, Smith DJ. 2004. Ecology of sponge assemblages (Porifera) in the
J. Mar. Biol. Assoc. UK 84:581–591.
4. Bourne DG, Munn CB. 2005. Diversity of bacteria associated with the
5. Boyett HV, Bourne DG, Willis BL. 2007. Elevated temperature and light
enhance progression and spread of black band disease on staghorn corals
of the Great Barrier Reef. Mar. Biol. 151:1711–1720.
6. Breitbart M, Bhagooli R, Griffin S, Johnston I, Rohwer F. 2005. Micro-
bial communities associated with skeletal tumors on Porites compressa.
FEMS Microbiol. Lett. 243:431–436.
7. Brown BE, Bythell JC. 2005. Perspectives on mucus secretion in reef
corals. Mar. Ecol. Prog. Ser. 296:291–309.
8. Caporaso JG, et al. 2010. QIIME allows analysis of high-throughput
community sequence data. Nat. Methods 7:335–336.
10. Crabbe MJC, Smith DJ. 2002. Comparison of two reef sites in the Waka-
tobi Marine National Park (SE Sulawesi, Indonesia). Using digital image
analysis. Coral Reefs 24:437–441.
11. Crabbe MJC, Karaviotis S, Smith DJ. 2004. Preliminary comparison of
Indonesia): estimated recruitment dates compared with Discovery Bay,
Jamaica. Bull. Mar. Sci. 74:469–476.
the mucus layers on living corals. Limnol. Oceanogr. 24:715–725.
13. Dumbrell AJ, Nelson M, Dytham C, Helgason T, Fitter AH. 2010.
community. ISME J. 4:337–345.
McKew et al.
aem.asm.orgApplied and Environmental Microbiology
14. Edgar RC. 2010. Search and clustering orders of magnitude faster than
BLAST. Bioinformatics 26:2460–2461.
15. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC. 1989. Isolation
and direct complete nucleotide determination of entire genes: character-
ization of a gene coding for 16S-ribosomal RNA. Nucleic Acids Res. 17:
16. Felsenstein J. 1989. PHYLIP: Phylogeny Inference package (version 3.2).
17. Frias-Lopez J, Zerkle AL, Bonheyo GT, Fouke BW. 2002. Partitioning of
and dead coral surfaces. Appl. Environ. Microbiol. 68:2214–2228.
18. Glynn PW. 1993. Coral reef bleaching: ecological perspectives. Coral
19. Haapkylä J, Seymour AS, Trebilco J, Smith DJ. 2007. Coral disease
prevalence and coral health in the Wakatobi Marine Park, southeast Su-
lawesi, Indonesia. J. Mar. Biol. Assoc. UK 87:1–12.
20. Haapkyla et al. 2009. Spatio-temporal coral disease dynamics in the
Wakatobi Marine National Park, South-East Sulawesi. Indones. Dis.
Aquat. Organ. 87:105–115.
Munro HN (ed), Mammalian protein metabolism. Academic Press, New
22. Lampert Y, Kelman D, Dubinsky Z, Nitzan Y, Hill RT. 2006. Diversity
of culturable bacteria in the mucus of the Red Sea coral Fungia scutaria.
FEMS Microbiol. Ecol. 58:99–108.
23. Lampert Y, et al. 2008. Phylogenetic diversity of bacteria associated with
the mucus of Red Sea corals. FEMS Microbiol. Ecol. 64:187–198.
24. Lozupone C, Hamady M, Knight R. 2006. UniFrac: an online tool for
comparing microbial community diversity in a phylogenetic context.
BMC Bioinformatics 7:371.
25. Mac ˇek I, et al. 2011. Local adaptation to soil hypoxia determines the
structure of an arbuscular mycorrhizal fungal community in natural CO2
springs. Appl. Environ. Microbiol. 77:4770–4777.
26. McKew BA, Coulon F, Osborn AM, Timmis KN, McGenity TJ. 2007.
Determining the identity and roles of oil-metabolizing marine bacteria
from the Thames estuary UK. Environ. Microbiol. 9:165–176.
27. Meikle P, Richards GN, Yellowlees D. 1988. Structural investigations on
the mucus from six species of coral. Mar. Biol. 99:187–193.
28. Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex
microbial populations by denaturing gradient gel electrophoresis analysis
Environ. Microbiol. 59:695–700.
29. Nawrocki EP, Eddy SR. 2007. Query-dependent banding (QDB) for
faster RNA similarity searches. PLoS Comput. Biol. 3:e56. doi:10.1371/
30. Nicol G, Tscherko D, Embley TM, Prosser JI. 2005. Primary succession
of soil Crenarchaeota across a receding glacier foreland. Environ. Micro-
31. Page RDM. 1996. TreeView: an application to display phylogenetic trees
on personal computers. Bioinformatics 12:357–358.
32. Pantos O, et al. 2003. The bacterial ecology of a plague-like disease
affecting the Caribbean coral Montastrea annularis. Environ. Microbiol.
33. Parameswaran P, et al. 2007. A pyrosequencing-tailored nucleotide bar-
code design unveils opportunities for large-scale sample multiplexing.
Nucleic Acids Res. 35:e130.
34. Remily ER, Richardson LL. 2006. Ecological physiology of a coral patho-
gen and the coral reef environment. Microb. Ecol. 51:345–352.
35. Riegl B, Bruckner A, Coles SL, Renaud P, Dodge RE. 2009. Coral reefs:
threats and conservation in an era of global change. Ann. N. Y. Acad. Sci.
36. Ritchie KB. 2006. Regulation of microbial populations by coral surface
mucus and mucus-associated bacteria. Mar. Ecol. Progr. Ser. 322:1–14.
Int. Coral Reef Symp. Smithsonian Tropical Research Institute, Balboa,
38. Rohwer F, Breitbart M, Jara J, Azam F, Knowlton N. 2001. Diversity of
bacteria associated with the Caribbean coral Montastraea franksi. Coral
39. Rohwer F, Seguritan V, Azam F, Knowlton N. 2002. Diversity and
distribution of coral-associated bacteria. Mar. Ecol. Progr. Ser. 243:1–10.
40. Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I. 2007.
The role of microorganisms in coral health, disease and evolution. Nat.
Rev. Microbiol. 5:355–362.
41. Ruìz-Renterìa F, van Tussenbroek BI, Jordán-Dahlgren E. 1998. Puerto
Caribbean coral reef, seagrass and mangrove sites. UNESCO, Paris,
reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.
43. Shashar N, Cohen Y, Loya Y, Sar N. 1994. Nitrogen-fixation (acetylene
reduction) in stony corals: evidence for coral-bacteria interactions. Mar.
Ecol. Progr. Ser. 111:259–264.
In Pretty J, et al (ed), Sage handbook on environment and society. Sage
Publications, London, England.
45. Sogin ML, et al. 2006. Microbial diversity in the deep sea and the under-
46. Solow AR. 1993. A simple test for change in community structure. J.
Anim. Ecol. 62:191–193.
47. Sunagawa S, Woodley CM, Medina M. 2010. Threatened corals provide
underexplored microbial habitats. PLoS One 5:1–6. doi:10.1371/
48. Wallace CC, Chen CA, Fukami H, Muir PR. 2007. Recognition of
separate genera within Acropora based on new morphological, reproduc-
tive and genetic evidence from Acropora togianensis, and elevation of the
subgenus Iso pora Studer, 1878 to genus (Scleractinia: Astrocoeniidae;
Acroporidae). Coral Reefs 26:231–239.
49. Wang Q, Garrity GM, Tiedje JM, Cole JR. 2007. Naïve Bayesian classifier
Appl. Environ. Microbiol. 73:5261–5267.
environments. Mar. Ecol. Progr. Ser. 267:159–171.
Bacterial Diversity Associated with Coral Mucus
August 2012 Volume 78 Number 15aem.asm.org 5237