11th ICRS Abstract book - Nova Southeastern University

Oral Mini-Symposium 5: Functional Biology of Corals and Coral Symbiosis: Molecular Biology, Cell Biology and Physiology
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Mechanisms Of Coral Bleaching And Cell Death Under Thermal Stress
Badrun NESA* 1 , Michio HIDAKA 2
1 Department of Marine and Environmental Science, Graduate School of Engeneering and
Science, University of The Ryukyus, Nishihara, Okinawa 903-0213, Japan, Nishihara,
Japan, 2 Chemistry, Biology and Marine Science, Faculty of Science, University of the
Ryukyus, Nishihara, Okinawa 903-0213, Japan, Nishihara, Japan
We have established an experimental system to study the response of coral cells to stress
treatment using coral cell aggregates (tissue balls). Dissociated coral cells aggregate to
form spherical bodies, which rotate by ciliary movement. These spherical bodies (tissue
balls) stop their rotation and become disintegrated when exposed to stress. The objective
of this study was to test the hypothesis that zooxanthellae become a burden for coral
hosts under stressful conditions and to study cell death mechanisms using tissue balls as
experimental system. Tissue balls prepared from dissociated cells of Pavona divaricata
and Fungia sp. were exposed to elevated (31 0 C) and control temperature (25 0 C) under
normal light (35 µmol m -2 s -1 ). The relationship between the survival time and
zooxanthella density of tissue balls were recorded. Cell death mechanisms were
investigated using a Comet Assay (single cell gel electrophoresis), which can detect DNA
damage in individual target cells. There was a negative correlation between the survival
time and zooxanthella density of tissue balls at 31 o C, while no significant correlation
between these parameters was found at 25 o C. The present results support the hypothesis
that zooxanthellae become a burden for host corals under high temperature stress and
suggest that zooxanthellae produce harmful substances under stress condition.
Antioxidants extended the survival time of tissue balls at high temperature in some cases.
This suggests that zooxanthellae produced active oxygen species under the stress
condition. Apoptotic death of coral cells was detected in tissue balls exposed to high
temperature stress using comet assay. This study also showed that tissue balls provide us
a good experimental system to study the effect of stress and various chemical reagents on
corals cells.
Key words: Coral, apoptosis, comet assay, bleaching
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Characterizing Bleaching Responses in Corals Exposed To Dcmu, Copper, And
Elevated Temperature Using Pam Fluorimetry And Gene Expression Profiling.
Amy ANDERSON* 1 , Alexander VENN 2 , Ross JONES 2 , Michael MORGAN 1
1 Berry College, Mount Berry, GA, 2 Bermuda Institute of Ocean Sciences, Ferry Reach, St
George's, Bermuda
The photosynthetic inhibitor DCMU, the heavy metal copper, and heat stress, are all
individually capable of inducing bleaching (the dissociation of the coral-algal symbiosis)
in hard corals. Whether this is by the same or different cellular or physiological
mechanism is presently unknown. In this study, small branches of the hard coral
Madracis mirabilis were exposed to various concentrations (0, 10, 30, 100, 300 ppb)
of DCMU, or copper, or to different temperatures (28°C or 32°C) for 72 h. Pulse
Amplitude Modulated (PAM) chlorophyll fluorimetry was used to characterize effect of
these treatments on the photosynthetic capacity of the symbiotic dinoflaglleates in the
tissues (in hospite). These analyses were combined with Representational Difference
Analysis (RDA) to amplify differentially expressed genes associated with each treatment.
Sixty-six genes were isolated from corals exposed to 300 ppb DCMU and a subset of
these genes appears to differentially expressed. Genomic and Proteomic database
searches reveal many of the RDA products have significant homology to proteins of
functional relevance. Expression profiles for each putatively differentially expressed gene
were established by probing for targeted transcripts within RNA samples from each
stressor treatment. A number of genes showed up-regulation at different concentration of
DCMU. Copper exposures also produced varied expression profiles for the genes
investigated. In contrast, most of the genes exhibited decreased expression as temperature
increased. The specificity of responses varied between genes as well as between
concentrations used for an individual stressor exposure. The expression profiles
generated in this study represent a new and informative way to characterize of how corals
respond to different environmentally relevant stressors as well as being a useful tool for
examining the molecular mechanism associated with coral bleaching.
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Differential Stability Of The Photosynthetic Membrane Of Symbiotic Dinoflagellates in
Response To Elevated Temperature
Erika M. DÍAZ-ALMEYDA 1 , Roberto IGLESIAS-PRIETO 2 , Patricia E. THOMÉ* 2
1 Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología, Universidad
Nacional Autónoma de México, Cancún, Quintana Roo, Mexico, 2 Unidad Académica Puerto
Morelos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de
México, Puerto Morelos, Quintana Roo, Mexico
Coral bleaching has been correlated with small increments of sea surface temperature of local
summer averages. This phenomenon is initiated with the uncoupling of light harvesting and
photosynthesis and may result in expulsion or death of the symbiont and eventually death of the
host. Not all coral species are equally susceptible to elevated temperature. Using charge
separation efficiency of photosystem II (Fv/Fm) after brief exposures (5 minutes) to high
temperatures, we assessed the fluidity of the photosynthetic membrane of cultured and freshly
isolated symbiotic dinoflagellates. The melting temperatures of the thylakoid membrane in 5
cultures grown at 24°C, show differences of 4.5°C between the most temperature-sensitive
Symbiodinium microadriaticum and the temperature resistant Symbiodinium sp. (clade D1a)
suggesting a strong genetic component. To explore temperature acclimation responses, these
two symbionts were grown at 31°C. Results indicate changes in melting temperatures as well as
changes in lipid composition of the photosynthetic membrane, suggesting limited acclimation to
high temperature.
The photosynthetic membrane from freshly isolated dinoflagellates of the Madracis auretenra
were found to be more temperature resistant than those isolated from Montastrea faveolata,
suggesting that the stability of the photosynthetic membrane is an important component in
determining susceptibility to coral bleaching.
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The Role of Oxidative DNA Damage and Repair in Cnidarian-Dinoflagellate Symbiosis
Breakdown
Joshua MEISEL* 1 , Ruth REEF 2 , Mauricio RODRIGUEZ-LANETTY 2 , Sophie DOVE 2 , Ove
HOEGH-GULDBERG 2
1 Centre for Marine Studies, University of Queensland, Highgate Hill, Australia, 2 Centre for
Marine Studies, University of Queensland, St. Lucia, Australia
In an effort to further understand the cellular and molecular processes underlying cnidariandinoflagellate
symbiosis breakdown, this study investigates the role of oxidative DNA damage
and repair in coral bleaching. In the presence of high light and heat, compromised
dinoflagellate photosystems can generate reactive oxygen species (ROS) that damage both host
and symbiont cellular components and may ultimately initiate a bleaching response. This study
focuses on the oxidative damage ROS inflict on the genomes of the Scleractinian coral
Acropora aspera and its endosymbiont Symbiodinum sp. and what mechanisms exist to combat
this damage. Acropora branches collected from the Heron Island reef flat were mounted into
racks and subjected to a gradual 20-day heating regime, peaking at 32 degrees, and symbiosis
breakdown was quantified through dinoflagellate counts and dark-adapted photosynthetic
yields. DNA was extracted from both host and symbiont at 8 time points throughout the
bleaching course and probed for the oxidative DNA base lesion 8-hydroxyguanine using a
competitive ELISA assay. To further understand the role of oxidative stress in this process,
experiments were repeated in the presence of DCMU (a photosynthesis-inhibitor that generates
ROS), catalase (an anti-oxidant enzyme that neutralizes hydrogen peroxide), 3-amino-1,2,4triazole
(a catalase-inhibitor), and hydrogen peroxide. To investigate pathways that may
operate in repairing this damage, the OGG1 protein (which excises 8-hydroxyguanine lesions)
was cloned from Acropora and will be used in qPCR studies to monitor oxidative DNA repair.
This is the first study to quantify oxidative DNA damage in the cnidarian-dinoflagellate system
and may provide insight into how oxidative stress, DNA damage, and genomic instability
initiate symbiosis breakdown and coral bleaching.
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