11th ICRS Abstract book - Nova Southeastern University

Poster Mini-Symposium 5: Functional Biology of Corals and Coral Symbiosis: Molecular Biology, Cell Biology and Physiology
5.116
Thermal Stress On Two Mediterranean Gorgonians: eunicella Singulari And
eunicella Cavolinii. Consequences Of The Symbiosis On Heat Stress Resistance.
Pierre-Laurent MERLE* 1 , Thamilla ZAMOUM 1 , Anthony BERTUCCI 2 , Marie
GEMINI 1 , Denis ALLEMAND 2 , Paola FURLA 1
1 ECOMERS, Univ. Nice-Sophia Antipolis, NICE, France, 2 Centre Scientifique de
Monaco, Monaco, Monaco
Global climate change has deep impacts not only on tropical marine invertebrates, but
also on many tempered species. In particular, the critical heat waves recorded in the late
1999 and 2003 summers coincided with massive death events locally observed among the
Mediterranean gorgonians, such as the symbiotic Eunicella singulari, and the nonsymbiotic
Eunicella cavolinii. This work aimed to study the early effects of strong and
short hyperthermia on these two Eunicella species. Several biological and oxidative stress
markers were analyzed (catalase activities, HSP expression, protein peroxidation and
ubiquitination levels) to compare the respective responses of these two gorgonian species
and to test whether zooxanthellae endosymbiosis offers higher resistance or sensitivity to
thermal stress. Increasing seawater temperature from 18°C (control) to 28°C (intense
short-term hyperthermia) provoked fast tissue necrosis in both species, but with a 2-day
delay for the symbiotic one, which did not presented any bleaching induction. This work,
which first validates the use of several stress biomarkers on these temperate gorgonians,
also pointed out high inter-individual variabilities. Although E.cavolinii had high antioxidative
basal defense level, heat stress induced significant protein ubiquitination. On
the other hand, E. singularis had reduced anti-oxidative basal defense level, albeit
showed more plastic response, which can partly explain their better tolerance to
hyperthermia. These studies, carried out to better understand the physiological impact of
thermal stress on temperate gorgonians, are a part of a wider program concerning the
Mediterranean gorgonian conservation (www.medchange.org).
5.117
Photobleaching Mechanism in Symbiotic symbiodinium Spp. Under Heat Stress
Shunichi TAKAHASHI* 1 , Spencer WHITNEY 2 , Shigeru ITOH 3 , Tadashi
MARUYAMA 4 , Murray BADGER 1
1 ARC Center of Excellence in Plant Energy Biology, Research School of Biological
Sciences, The Australian National University, Canberra, Australia, 2 Research School of
Biological Sciences, The Australian National University, Canberra, Australia, 3 Division
of Material Science (Physics), Nagoya University, Nagoya, Japan, 4 Japan Agency for
Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan
Coral bleaching, caused by heat stress, is associated with the light-induced loss of
photosynthetic pigments in symbiotic dinoflagellate algae (Symbiodinium spp.), however
the molecular mechanisms responsible for pigment loss are poorly understood. Here we
show that moderate heat stress primarily causes photobleaching through inhibition of the
de novo synthesis of intrinsic light harvesting antennae (chlorophyll a-chlorophyll c2peridinin-protein
complexes; acpPC) in cultured Symbiodinium algae and that two Clade
A Symbiodinium species showing different thermal sensitivities of photobleaching also
show differential sensitivity of this key protein synthesis process. Photoinhibition of
photosystem II (PSII) and subsequent photobleaching were observed at temperatures
exceeding 31°C in cultured Symbiodinium CS-73 cells grown at 25°C to 34°C but not in
cultures of the more thermally tolerant control Symbiodinium species OTcH-1. We found
that bleaching in CS-73 is associated with loss of acpPC that is a major antennae protein
in Symbiodinium. In addition, its thermally induced loss is light dependent, does not
coincide directly with PSII photoinhibition and is not caused by stimulated degradation of
acpPC. In cells treated at 34°C over 24 h the steady state acpPC mRNA pool was
modestly reduced ~30% while the corresponding synthesis rate of acpPC was diminished
by more than 80%. Our results suggest photobleaching in Symbiodinium is
consequentially linked to the relative susceptibility of PSII to photoinhibition during
thermal stress and occurs, at least partially, due to loss of acpPC via undefined
mechanisms(s) that hamper the de novo synthesis of acpPC primarily at the translational
processing step.
5.118
Preservation of Coral Samples in DMSO-Salt Buffer (SSDE) is Superior to Ethanol
Zoltan SZABO* 1 , Marc W CREPEAU 1 , Robert J TOONEN 1
1 Hawaii Institute of Marine Biology, Kaneohe, HI
DNA-based techniques form the basis of much of the recent global effort to quantify coral
diversity and connectivity. 95% ethanol (EtOH) is the most commonly used preservative of
coral samples, however transportation and storage can be a problem due to the flammable
nature of EtOH. About 2 years ago, we started collecting coral samples in both a buffer
consisting of 20% DMSO, 0.25M EDTA pH 8.0 saturated with NaCl (SSDE) and 95% EtOH,
and used these paired samples for our study here. We compared DNA quality from
representatives of both scleractinian and octocorals and found that DNA extraction from SSDEpreserved
samples produced significantly more high-molecular weight DNA relative to
degraded (low-molecular weight) DNA than extractions from the paired EtOH-preserved
samples of the same colony. In some EtOH-preserved samples (e.g., Pocillopora, Carijoa)
virtually no high-molecular weight DNA was visible on agarose gels. Additionally, our
preliminary data suggest that higher quality DNA will provide more copies of intact DNA
molecules available for amplification, resulting in fewer misincorporated nucleotides in PCR
products. Based on these results, we expect to see less DNA sequence variation from DMSO
preserved samples than from the paired EtOH preserved ones. Experiments are under way to
compare the matched DMSO/EtOH samples for sequence variation in PCR applications, and
will be presented at the conference. Coral DNA is notoriously difficult to work with, and we
hypothesize that some of this reputation may result from poor success with EtOH-preserved
coral samples. We have found that switching from EtOH to SSDE buffer in the field tends to
yield much higher quality DNA in the lab across a broad range of hard and soft coral species,
and that SSDE preservation results in far fewer problems with subsequent PCR applications of
preserved coral samples.
5.119
Ratiometric Imaging Of Gastrodermal Lipid Body in Cnidaria-Dinoflagellate
Endosymbiosis
Yi-Jun LUO* 1 , Hui-Ju HUANG 2 , Li-Hsueh WANG 1,2 , Wan-Nan UANG 3 , Lee-Shing FANG 4 ,
Chii-Shiarng CHEN 1,2
1 Institute of Marine Biotechnology, National Dong Hwa University, Pingtung, Taiwan, 2 Coral
Research Center, National Museum of Marine Biology and Aquarium, Pingtung, Taiwan,
3 Biological Science and Technology, I-Sou University, Kaohsiung, Taiwan, 4 Cheng Shiu
University, Kaohsiung, Taiwan
Cnidaria-dinoflagellate endosymbiosis is the phenomenon that an autotrophic symbiont lives
inside the endodermal cell of animal host. The molecular mechanism that regulates this
association remains unclear. Using quantitative microscopy, we now provide evidence that the
dynamic lipid change in endodermal “lipid body” (LB) reflects the symbiotic status between the
host cell and its symbiont in the hermatypic coral Euphyllia glabrescens. By ratiometric
imaging with a solvatochromic fluorescent lysochrome, nile red (9-diethylamino-5Hbenzo[
]phenoxazine-5-one), we show that the in situ distribution of polar versus non-polar
lipids (i.e. “red” over “green” fluorescence of nile red, or R/G ratio) of LB or retained
symbionts in living endodermal cells can be analyzed. R/G ratio in individual LB increases
during the bleaching process, indicating a retardation of non-polar lipid accumulation in
endodermal cells. On the other hand, non-polar lipids accumulation inside the symbiont results
in gradual decreases of R/G ratio. Interestingly, patterns of R/G ratio shift in symbionts are
different between bleaching and starvation processes. In the later, little lipid accumulation in
symbionts and results in no R/G ratio shift. These results suggest that a membrane lipid
trafficking must underlie to regulate the endosymbiotic association between the host cells and
symbionts.
287