11th ICRS Abstract book - Nova Southeastern University

7.187
Characterizing Surface Microorganism Diversity In Healthy And Diseased
Gorgonia Ventalina With Universal 16S Rdna Primers.
Jason MACRANDER* 1
1 University of Puerto Rico-Rio Piedras, San Juan
Over the past three decades there has been an increase in the severity of coral diseases.
Throughout the Caribbean Gorgonia ventalina are suffering from a disease called
Asprogillosis. Recent discoveries in coral microbiology have emphasized great diversity
in coral surface-dwelling microorganisms and their possible roles in pathogen defense.
The diversity of microorganisms on corals may allow for rapid adaptation and selection
for the most beneficial microbial communities in the surface mucopolysaccharide layer.
The aim of this study is to characterize diversity of surface dwelling-microorganisms
found on G. ventalina using non-culture PCR methods. Tissue samples were collected
from an equal number of healthy and diseased specimens from 3 coral reefs in waters
surrounding Puerto Rico. The initial screen for bacterial communities was derived using
16S rDNA primers in PCR. The sequences were subjected to BLAST and compared with
species (or closest related species) found in the NCBI sequence database. The initial
screening indicates tremendous diversity of bacteria present on G. ventalina surface.
Currently I am comparing derived bacterial communities from these results between
healthy and diseased specimens. These results may aid in the identification of bacterial
communities which are prevalent in healthy and diseased G. ventalina in waters
surrounding Puerto Rico. I am also using microsatellites in genotyping coral specimens to
see if there is a correlation between relatedness and bacterial communities. In the future I
hope to use additional molecular markers (AFLPs) to reinforce genotyping and explore
possible sources of innate immunity to assist our understanding of coral disease
resistance. Results from this study will aid in our understanding of coral defense against
disease and serve as a model for other species.
7.188
Ultrastructural And Histological Analysis Of Dark Spot Syndrome (Dss) in
siderastrea Siderea And agaricia Agaricites
D. Abigail RENEGAR* 1 , Patricia L. BLACKWELDER 1,2 , Deborah J. GOCHFELD 3 ,
Alison L. MOULDING 1
1 National Coral Reef Institute, Nova Southeastern University Oceanographic Center,
Dania, FL, 2 RSMAS, University of Miami, Miami, 3 National Center for Natural Products
Research, University of Mississippi, University, MS
Dark Spot Syndrome (DSS) typically manifests in scleractinian corals as lesions of
varying color, size, shape and location that can result in tissue death and skeletal changes.
A causative agent has not yet been identified. The objective of this study was
histological and ultrastructural investigation of the cellular and skeletal characteristics of
DSS affected and healthy Siderastrea siderea and Agaricia agaricites. The greater
resolution possible with transmission (TEM) and scanning (SEM) electron microscopy
can reveal microbial activity and initial tissue changes not resolvable utilizing histology.
Preliminary results indicate that DSS affected tissue has less integrity, with increasing
cellular degradation and vacuolization. The zooxanthellae population was in decline,
characterized by abnormal or necrotic cells with internal organelle disruption and debris.
A high concentration of electron dense inclusions, believed to be zymogen granules
based on morphology and staining properties, was concentrated in the calicodermis and
adjacent gastrodermal layer. Numerous fungal cells, dimensionally and morphologically
consistent with the genus Aspergillus, were observed directly beneath the tissue in close
proximity to the calicodermis in all of the DSS affected samples. Skeletal changes
observed included darkened areas of skeleton both directly beneath dark spots in the
tissue and some distance beneath the coral surface. Further histological and
ultrastructural analyses are currently being performed.
Poster Mini-Symposium 7: Diseases on Coral Reefs
7.189
Characterization of Microbial Communities Associated with Black Band Diseased Corals
by Targeting the 16S rRNA and DsrAB Genes
Raju SEKAR 1 , Longin KACZMARSKY 1 , Laurie RICHARDSON* 1
1 Department of Biological Sciences, Florida International University, Miami, FL
Black band disease (BBD) is a polymicrobial disease affecting both scleractinian and gorgonian
corals on a worldwide basis. The causative agent for BBD has not yet been identified although
several bacteria and cyanobacteria have been proposed as pathogens. We have been using 16S
rRNA gene analysis to characterize microbial communities associated with healthy surface
mucupolysaccharide (SML) and BBD microbial mats of various coral hosts such as
Sidereastrea siderea, Diploria strigosa and D. clivosa collected from Bahamas, Florida Keys
and St. Croix. Our earlier studies showed that the microorganisms associated with BBD were
very different with higher microbial diversity compared to those in the SML. Seven clone
libraries constructed from BBD samples of S. siderea from these regions showed the αproteobacteria
to be abundant in all the clone libraries followed by the γ- and δ-proteobacteria,
bacteroidetes and firmicutes. The clone libraries constructed using BBD samples of the second
coral host D. strigosa, showed a very different pattern of microbial communities. The clone
libraries were dominated by ε-proteobacteria and α-proteobacteria followed by γ-proteobacteria,
firmicutes and δ-proteobacteria. A clone library constructed from the third, host D. clivosa,
showed that the α-proteobacteria were dominant followed by firmicutes, cytophagaflavobacterium,
and γ-proteobacteria. The microbial diversity varied highly with coral hosts and
regions. Many sequences of bacteria involved in the sulfur cycle were observed in the clone
libraries. Earlier studies discussed the involvement of sulfate-reducing bacteria (SRB) and
sulfide-oxidizing bacteria (SOB) in BBD pathogenesis. We are currently analyzing BBD
samples from various host species by targeting the dissimilative sulfite-reductase (DsrAB) gene
to assess the diversity of SRB and SOB, and are using fluorescent in situ hybridization (FISH)
to determine the abundance and distribution of these bacteria in BBD.
7.190
Molecular Analysis Of Bacteria Associated With Healthy And Yellow-Band Diseased
Montastraea Faveolata
Joel THURMOND* 1 , Weil ERNESTO 2 , Kim RITCHIE 1
1 Mote Marine Laboratory, Sarasota, FL, 2 University of Puerto Rico, Mayaguez
Caribbean Yellow Band Disease (YBD) is a blotching and band disease that affects three
species in the major reef-building coral genus Montastraea in the wider Caribbean. The
microbial community associated with corals is dynamic and adapts to varying states of health,
stress and disease. Although Vibrios are implicated in disease in this coral, assessment of
microbial community shifts from healthy to diseased states have not been addressed using
culture independent methods. In this study we used both denaturing gradient gel electrophoresis
(DGGE) and terminal restriction fragment length polymorphisms (T-RFLP) to assess the
microbial community structure of healthy and diseased Montastraea faveolata colonies.
Genomic DNA was extracted from the surface mucus and tissue of five YBD colonies, five
healthy regions of the same diseased colonies, and five entirely healthy colonies collected from
a coral reef in Puerto Rico. The T-RFLP method was performed using restriction enzymes Cfo-
I, Msp-I and Rsa-I and elucidates community structure differences between healthy and YBDdiseased
corals. In addition, a nested PCR-DGGE method was performed using primers specific
to different bacterial groups. Analyses indicate the PCR-DGGE method is able to reveal
specific profiles within the microbial community. In one example, a sulfate-reducing bacterial
population shows increasing diversity from the healthy to diseased state.
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