Understanding the Stress Response
of Corals and Symbiodinium in
a rapidly changing environment
Proceedings
May 10 June 3 2005
Unidad Académica Puerto Morelos,
Instituto de Ciencias del
Mar y Limnología, UNAM
Mexico
Proceeding editor: Ove Hoegh-Guldberg
Chair, Targeted Research Group on
Coral Bleaching and Related Ecological Factors
(Bleaching Working Group)
www.gefcoral.org
1
Workshop 1 in a series coordinated by
Targeted Research Group on Coral Bleaching
and Related Ecological Factors
2
CONTENTS
Introduction to workshop................................................................................... 6
Acknowledgements ............................................................................................ 8
Introduction to PAM fluorometry.................................................................... 10
Peter Ralph........................................................................................ 10
The Photosynthetic Apparatus of Symbiodinium........................................... 12
Roberto Iglesias-Prieto ...................................................................... 12
Multiple scattering on coral skeleton enhances light absorption by symbiotic
algae.............................................................................................................. 15
Susana Enríquez, Eugenio R. Méndez1, & Roberto Iglesias-Prieto. . 15
An overview of the interpretation and current use of chlorophyll fluorescence
to understand coral bleaching ....................................................................... 18
Mark E. Warner1 ................................................................................ 18
Diel Cycling of Nitrogen Fixation in Corals with Symbiotic Cyanobacteria.... 21
Michael P. Lesser1, Luisa I. Falcón2, Aimé Rodríguez-Román3,
Susana Enríquez3, Ove Hoegh-Guldberg4, and Roberto Iglesias-
Prieto3................................................................................................ 21
Host pigments, photosynthetic efficiency and thermal stress........................ 25
Sophie Dove1, Carli Lovell1, Maoz Fine1, Susana Enríquez2, Roberto
Iglesias-Prieto2, Kenneth Anthony3 and Ove Hoegh-Guldberg1. ....... 25
Imaging-PAM: Operation and Possibilities .................................................... 29
Ross Hill1 and Karin E. Ulstrup1......................................................... 29
Seasonal fluctuations in the physiology of Stylophora pistillata .................... 32
Gidon Winters1, Sven Beer1* and Yossi Loya2 ................................... 32
The cellular mechanism of coral bleaching ................................................... 36
Daniel Tchernov 1,3 , L Haramaty1, T. S. Bibby1, Max Y.
Gorbunov1,and Paul G. Falkowski1,2.................................................. 36
Theme 2: Diversity, flexibility, stability, physiology of Symbiodinium and
the associated ecological ramifications (May 15-17)..................................... 37
The diversity, specificity and flexibility of Symbiodinium symbioses. ............ 38
Ove Hoegh-Guldberg ........................................................................ 38
Roberto Iglesias-Prieto ...................................................................... 43
Diversity and specificity of Symbiodinium ..................................................... 46
William K. Fitt..................................................................................... 46
Symbiodinium systematics: molecular markers and the techniques
appropriate for eco-evolutionary investigations............................................. 48
Todd C. LaJeunesse ......................................................................... 48
Coral bleaching as an exaptation that can promote rapid and beneficial
change in algal symbiont communities.......................................................... 50
Andrew C. Baker1, 2............................................................................ 50
Modeling the Adaptive Bleaching Hypothesis ............................................... 53
John R. Ware..................................................................................... 53
Dynamics of cnidarian-Symbiodinium symbioses: Thoughts on flexibility,
stability and ontogeny of the symbiosis......................................................... 56
Mary Alice Coffroth, A. R. Hannes, J. Holmberg, N. L. Kirk, C. L.
Lewis, D. M. Poland........................................................................... 56
Can the highly variable ITS2-region uncover ecologically relevant patterns in
the distribution and persistence of Symbiodinium sp. in Pocilloporid corals? 59
Eugenia Sampayo, Ove Hoegh-Guldberg and Sophie Dove ............ 59
Symbiodinium ITS-2 sequences: inter- or intraspecific data? Implications for
the detection of ecological & biogeographic patterns.................................... 62
Adrienne M. Romanski1 and Andrew C. Baker 2,3.............................. 62
Symbiont shuffling represents a trade-off for modern reef-builders .............. 65
3
Madeleine van Oppen1, David Abrego1,2, Angela Little1,2, Jos Mieog1,3,
Ray Berkelmans1 and Bette Willis2.................................................... 65
"Checks and Balances" in the identification of Symbiodinium diversity ........ 67
Scott R. Santos.................................................................................. 67
Transcriptome analysis of a cnidarian dinoflagellate mutualism reveals
complex modulation of host gene expression ............................................... 70
Mauricio Rodriguez-Lanetty, Wendy Phillips, and Virginia M. Weis .. 70
Theme 3: Exploration of the Coral and Symbiodinium genomes (May 19-21,
2005) ................................................................................................................... 75
Progress in coral genomics ........................................................................... 76
David J Miller, L Grasso, D Hayward, P Maxwell, J Maindonald, S
Rudd, U Technau, EE Ball................................................................. 76
A microarray approach to understanding stress responses and the functional
biology of corals ............................................................................................ 77
Madeleine van Oppen1, Andrew Negri1, David J. Miller2 ................... 77
Coral Reef Genomics: A Genome-wide Approach to the Study of Coral
Symbiosis ...................................................................................................... 79
Jodie Schwarz1, P. Brokstein1, C. Lewis2, C. Manohar3, D. Nelson3, A.
Szmant4, M.A. Coffroth2, M. Medina1................................................. 79
Targeted Functional Genomics of Coral Stress ............................................ 80
Theresa Seron, Karen Konzen and Mikhail Matz .............................. 80
Using Molecular Markers A Cautionary Tale.............................................. 83
Ruth D. Gates, Amy M. Apprill and Benjamin R. Wheeler................. 83
Distinct differences in a Symbiodinium EST library compared to other
dinoflagellates ............................................................................................... 86
William Leggat1, Ove Hoegh-Guldberg1, Sophie Dove1, David
Yellowlees2 ........................................................................................ 86
How does the Symbiodinium EST database add to our knowledge of
zooxanthellae and their metabolism?............................................................ 89
Theme 4: Targeted Research Working Group joint field methods (May 22) 91
Tracking coral populations through time ....................................................... 92
Rob van Woesik1 & Yossi Loya2........................................................ 92
Field methods to detect change in the coral communities of south eastern
Mexico ........................................................................................................... 99
Rob van Woesik1, Jessica Gilner1 and Eric Jordan-Dahlgren2 .......... 99
Theme 5: Integrated research on coral bleaching and disease (May 24-26)
.......................................................................................................................... 102
Ove Hoegh-Guldberg1, Michael P. Lesser2 and Roberto Iglesias
Prieto3 .................................................. 103
Bacterial bleaching of corals ....................................................................... 107
Eugene Rosenberg.......................................................................... 107
Experimental analysis of bacterial ecology of bleaching and disease......... 110
John Bythell1 and Olga Pantos2....................................................... 110
Post-mortem Microbial Communities on Dead Corals: Implications for Nutrient
Cycling?....................................................................................................... 111
Ron Johnstone, Mark Davey and Glen Holmes. ............................. 111
PAM fluorometry and Symbiodinium stress. ............................................... 114
Roberto Iglesias-Prieto .................................................................... 114
Oxidative Stress, Bleaching, and Coral Disease......................................... 117
Michael P. Lesser ............................................................................ 117
Seafan epizootic and resistence to Aspergillosis. ....................................... 120
Drew Harvell. ................................................................................... 120
Ecology, Physiology and Cell Biology of `White Syndrome' on the Great
Barrier Reef ................................................................................................. 121
4
Roff J1, Ainsworth TD1,2, Kvennefors EC1, Henderson M1,2, Blackall LL
1,2, Fine M 1,3, Hoegh-Guldberg O1................................................... 121
Contact details of workshop participants..................................................... 127
5
Introduction to workshop
The inaugural workshop for the World Bank/GEF Targeted Research Group on Coral
Bleaching and Related Ecological Factors (BWG) was held from May 10 - June 3
2005 in Puerto Morelos, Mexico. This workshop is the first of a series designed to
galvanize the international scientific community around problems, gaps and solutions
with respect to the global issue of coral bleaching and related ecological disturbances
to coral reefs. The Bleaching Working Group (BWG) was originally founded by the
Intergovernmental Oceanographic Commission (IOC) of UNESCO in April of 2001.
The group was then incorporated as one of the six targeted working groups within the
World Bank/Global Environment Facility (GEF) Coral Reef Targeted Research
Program coordinated by the University of Queensland. The BWG's initial efforts
included the investigation of whether there are specific indicators for coral bleaching
and its effects on coral reefs. Subsequently, it expanded its mandate to examine
specific physiological mechanisms for coral bleaching as well as the local ecological
factors that cause bleaching and its after-effects (e.g. coral recovery), and
differences between direct human stresses and those related to climate change.
The current members of the working group are:
· Ove Hoegh-Guldberg, Australia (Chair) · Roberto
Iglesias-Prieto,
Mexico
· Yossi Loya, Israel, (Co-Chair)
· Ruth Gates, USA
· William K. Fitt, USA
· Michael Lesser, USA
· John Bythell, UK
· Ron Johnstone, Australia
· Robert van Woesik, USA
· Tim McClanahan, Kenya
· David Obura, Kenya
· Christian Wild, Germany
The Mexican workshop included several themes which involved invitations to leading
researchers to participate in discussions and experimental components.
· May 10-13: Pulse Amplitude Modulation Fluorescence and the Stress Biology of
Reef-Building Corals
(coordinated by Roberto Iglesias-Prieto and Peter Ralph)
· May 15-17: Diversity, flexibility, stability, physiology of Symbiodinium and the
associated ecological ramifications. (coordinated by Ove Hoegh-
Guldberg and William K. Fitt)
· May 19-21: Exploration of the Coral and Symbiodinium genomes
(coordinated by William Leggat, Sophie Dove, David Yellowlees)
· May 22: Coral Reef Targeted Research Working Group joint field methods
(coordinated by Robert van Woesik)
· May 24-26: Integrated research on coral bleaching and disease
(coordinated by John Bythell, Drew Harvell)
After reviewing progress in each of these theme areas, participants focused on key
issues that arose from the research and addressing preconceived ideas in the of the
reef management community. The attendance of local scholars and students at
these international workshops, aided in the dissemination of the latest insights into
the phenomenon of coral bleaching and related ecological impacts to the coastal
community of the Mexican Yucatan coast. The research projects that were
undertaken during the workshop are currently being published in the reviewed
scientific literature. These proceedings include all scheduled papers. Please note
6
that papers by several seminars by special invitees such as Professor Michael Kuhl
and Mr Tom Oliver are regrettably not captured by these proceedings.
Ove Hoegh-Guldberg
Workshop coordinator and
Chair, Bleaching Working Group
Coral Reef Targeted Research Project
7
Acknowledgements
The Bleaching Working Group is very grateful to Dr. Adolfo Gracia, Director of the
Instituto de Ciencias del Mar y Limnologia at Universidad Nacional Autonoma de
Mexico and Dr. Brigitta van Tussenbroek, Head of UNAM's Unidad Académica
Puerto Morelos (UAPM) for hosting this workshop at their facility in Puerto Morelos.
We are particularly grateful for the enormous efforts spent by Dr. Roberto Iglesias-
Prieto, Dr. Susanna Enriquez, Dr. Anja Banaszak, Ms Aime Rodríguez Román, Mr.
Xavier Hernández Pech and many others at the Unidad Académica Puerto Morelos
(UAPM). There is clearly no way that this important workshop would have been able
to prosper without these efforts. We are also grateful to the township of Puerto
Morelos for their hospitality during the workshop. Lastly, we are grateful to the World
Bank - Global Environment Facility Coral Reef Targeted Research Project and the
University of Queensland for providing funding which enabled more than 80
participants to travel to and be accommodated during the meeting.
Participants of the workshop on "Understanding the Stress Response of
Corals and Symbiodinium in a rapidly changing environment", May 17 2005.
8
Theme 1: Pulse Amplitude Modulation Fluorometry and the
Stress Biology of Reef-Building Corals (May 11-13)
Pulse Amplitude Modulation (PAM) Fluorometry has become a key technique for the
investigatation of changes to the photosynthetic physiology of the dinoflagellate
symbionts of reef-building corals, Symbiodinium sp. Dr Roberto Iglesias-Prieto
(Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
UNAM) and Dr Peter Ralph (University of Technology, Sydney) coordinated a 3 day
workshop focused on the use of PAM fluorometry to dectect and monitor stress in
corals and Symbiodinium. A series of papers reviewing key aspects of the method
were presented. This review of the technique was followed by hands on training
sessions for researchers intending to use PAM fluorometry in their research. After
the practical training session, the PAM fluorometry workshop concluded with a
discussion of new technological developments in the field of PAM flourometry and
the limitations of the method. The focused session on PAM fluorometry was well
attended and involved over 40 participants.
Workshop participants in intense discussion following
presentations. From left to right are Daniel Tchernov, Roberto
Iglesias-Prieto, Tom Oliver, Mark Warner and Peter Ralph.
Sophie Dove and Susanna Enriquez discuss the finer points of light
capture and photosynthesis by reef-building corals.
9
Introduction to PAM fluorometry
Peter Ralph
Department of Environmental Sciences, University of Technology, Sydney
Cnr Westbourne Street & Pacific Highway, GORE HILL NSW 2065
To ensure that all attendees of the Bleaching Working Group Workshop were
conversant with the techniques of chlorophyll a fluorescence and specifically Pulse
Amplitude Modulation (PAM) fluorometry, a seminar was presented covering the
basics of this technology.
Several examples of fluorescent materials, materials that absorb photons and re-emit
them at a longer wavelength, were discussed. The concept of fluorescence was then
considered as it applies to the PSII reaction centre when a chlorophyll molecule is
excited by irradiance of either blue or red wavelengths. The amount of fluorescence
will vary as a result of the condition of the operational components of the
photosynthetic machinery. Following dark-adaptation, the minimum fluorescence
value (Fo) of the sample can be measured. If the sample is then exposed to enough
light so all the photocentres are full of photons (closed) the maximum amount of
fluorescence (Fm) can be measured. The difference between these two extreme
values is the variable fluorescence (Fv). Fv/Fm provides a measure of PSII
photochemical efficiency.
Energy absorbed by chlorophyll a can either be used for photochemistry, re-emitted it
at a longer wavelength as fluorescence or dissipated as heat (non-photochemical
quenching). The health of the photosystems defines the relative proportions of
energy directed through each of these competing pathways. The rate of electron
transport is directly influenced by limitations along the electron transport chain.
Quantum yield of PSII is linked to photosynthetic activity and under some conditions
is roughly proportional to oxygen production (or CO2 uptake); however this
relationship rarely holds up at elevated irradiances due to a range of competing
processes including photorespiration. Electron transport is influenced by the redox
state of the several critical components of PSII; primary electron acceptor (QA), the
secondary electron acceptor (QB), the plastoquinone pool (PQ), PSI activity and the
oxygen evolving complex.
The operational aspects of the PAM fluorometer were discussed including; light
sources, lock-in amplifier, and fibre optics. Firstly, the PAM fluorometry principle is
based on a 3 µs pulse of light that is synchronized to a lock-in amplifier. This allows
effective quantum yield determinations to be performed in sun light, as the lock-in
amplifier removes all signal not associated with the lock-in signal. The light sources
available include measuring light (< 0.4 µmol photon m-2s-1), actinic light (used to
drive photosynthesis, 1-2000 µmol photon m-2s-1), saturation pulse (> 6000 µmol
photon m-2s-1) and far-red light (used for stimulating PSI, 730 nm). When the PAM
fluorometer is being optimized for a new tissue/species it is important to set the
saturating pulse width and intensity to get reproducible data. The measuring light
needs to be set low enough to prevent activation of the photosystem, whilst still being
able to measure sufficient fluorescence to make a measurement. A range of fibre
optics are available for use with several models of PAM flurometer. , Two fibres that
are relevant to coral research include the 8 mm Diving-PAM fibre useful for
10
assessment of tissue type or whole colonies, whereas the microfibre (50-100 mm
fibre) as linked to the Microfibre-PAM is more appropriate for assessment of the
photosynthetic condition of microscale habitats (polyp and coenosarc scale). Plastic
fibre-optics have a higher attenuation, so you'll always have a lower signal than with
a glass fibre; however the cost difference is substantial. Imaging-PAM now provides
a high-resolution assessment of the spatially complex regions of corals. It is
important to remember that once the optical geometry has been set and the
fluorometer has been adjusted with the off-set to zero then the fluorescence signal
needs to be > 130 and < 1000 units for the best quantum yield estimates. The digital
gain can be adjusted to increase the fluorescence signal; however the noise also
increases, so it doesn't increase precision. To ensure published data can be
independently evaluated, I recommend that all data set are published with a single
line of data about the PAM settings, this will allow others to attempt to replicate the
experiment. For a "typical" coral, the "typical" Diving-PAM settings would be
measuring light 8, saturating intensity 8, saturating width 0.6s, gain 2 and damping 2.
The biophysical condition of the PSII reaction centre was discussed in relation to
maximum and minimum fluorescence. Minimum fluorescence occurs when the PSII
reaction centres are fully open. Maximum fluorescence occurs when the PSII
reaction centres are closed. A decrease in Fm' (light adapted maximum
fluorescence) is usually linked with non-photochemistry. The following formulae can
be used to assess the relative condition of the photosynthetic apparatus. Maximum
quantum yield requires the coral to be dark-adapted for at least 10 min (caution that
anaerobic conditions can develop), while effective quantum yield can be measured in
ambient light.
Maximum quantum yield = (Fm-Fo)/Fm = Fv/Fm
Effective quantum yield = (Fm'-F)/Fm' = F/Fm'
A rapid light curve is a tool for assessing the capacity of the photosynthetic tissue
when exposed to series of rapidly (10 s) changing light climates. A RLC is not a
photosynthesis irradiance (P-E) light curve, as the tissue does not reach steady state
during each incubation. A RLC should not be interpreted as a P-E curve. RLC work
best where ambient irradiance is rapidly fluctuating. RLC estimate the relative
electron transport rate (rETR) at each of the irradiances. The utility of the rapid light
curve was discussed amongst several of the delegates. rETR = F/Fm' x PAR,
where effective quantum yield is multiplied by the irradiance. This is a relative
estimate of electron transport. Maximum relative electron transport rate as defined as
the highest rate of ETR once the curve is fitted to an exponential decay function.
Quenching analysis was described and not discussed. This form of fluorescence
analysis has utility when considering the protection and recovery aspects of
photoinactivation and/or down regulation of coral.
11
The Photosynthetic Apparatus of
Symbiodinium.
Roberto Iglesias-Prieto
Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún QR
77500, México.
Invertebrates symbiotic with dinoflagellates in the genus Symbiodinum are among
the most important primary producers in coral reefs. In these environments they are
responsible not only for the high gross production, but also for the construction and
maintenance of the reef structure. Despite the importance of this symbiosis, our
knowledge about the biochemical organization of the photosynthetic apparatus of
dinoflagellates is still incomplete. This gap in our understanding is particularly
important in the context of the study of the photobiology of thermal stress. We have
commonly relied on the use of green plant models to make interpretations of
chlorophyll a fluorescence kinetic data from Symbiodinium. Although in some cases
these models can explain the experimental observations, caution should be taken
before ascribing photosynthetic phenomenon to Symbiodinium that have not been
characterized empirically in dinoflagellates, such as the induction of state transitions
and their putative role in photo-protection.
Chromophores
The functions of photosynthetic pigments are to capture photons, transfer excitation
energy to the reaction centers where primary photochemistry takes place, and, in
some cases, provide photo-protection), The light harvesting function in
dinoflagellates is performed by Chl c2 and peridinin, in addition to Chlorophyll a (Chl
a. Chlorophylls are porphyrin derivatives that form a cyclic tetrapyrrol with a chelated
Mg atom ligated at the center of the macrocycle (Scheer 1991). The spectral
characteristics of these molecules depend on the side groups attached to the
macrocycle. In contrast with other chromophyte algae, dinoflagellates contain only
Chl c2, and their diagnostic carotenoid, peridinin. This carotenoid is capable of
transfering excitation energy to Chl a
with efficiencies close to 100%, and
therefore plays a major role in light
collection. All functional
photosynthetic pigments are non-
covalently bound to specific proteins
forming Chl protein complexes.
The function of the protein moiety is
to orient and space the chorophores
to assure the the excitation eneregy
is efficiently transfered to the
reaction centers. Functionally, Chl-
protein complexes are divided into Fig 1. Schematic representation of the
light harvesting complexes or photosynthetic apparatus of Symbiodinium.
antennae and their reaction centers.
12
Light harvesting complexes
Dinoflagellates posses a unique light harvesting apparatus composed of the water-
soluble PCP (Peridin-Chl a-Protein) and a transmembrane system called acpPC (Chl
a-Chl c2-Peridin Protein Complexes). PCP was one of the first light-harvesting
complexes to be isolated and it is one of the best characterized, including a detailed
structure based on X-ray crystalography (Hofmann et al. 1996). Native PCP shows
apparent molecular masses between 35 to 39 kD. Analyses of the apoproteins
indicate that they can occur as either monomers of about 31-35 kD or as
homodimers of 14-15.5 kD. Immunological characterization of the quaternary
structure of PCP taken from different species of Symbiodinium showed that some
species contain only the monomeric or the dimeric form whereas others
simultaneously presented both forms. PCP apoproteins are encoded by a family of
nuclear genes. Chromophore analyses of PCP indicate the presence of variable Chl
a: peridinin stoichiometries ranging form 2:8 to 2:12 (Iglesias-Prieto 1996).
The use of density gradient centrifugation to fractionate thylakoid membranes
solubilized with glycosidic surfactants allowed the isolation of three distinct fractions
comprising up to 75% of the cellular Chl a content, maintaining efficient energy
transfer. The vast majority the accessory pigments dinoflagellates are bound to are in
the intrinsic acpPC (Iglesias-Prieto et al. 1993) These complexes have Chl a:Chl
c2:peridin with a molar ratio of 7:4:12, and contain the majority of the xanthophylls
involved in photo-protection. The content of these xanthophylls is variable depending
on the prevailing light conditions (Iglesias-Prieto & Trench 1997).
Reaction centers
The other two fractions isolated include a yellow band that may to be related to
photosystem II (PSII), but shows inefficient energy transfer and a photosystem I (PSI)
enriched fraction. The PSI-enriched fraction contains very little amounts of Chl c2 and
peridinin. This fraction exhibits spectroscopic and kinetic properties similar to PSI
isolated from green plants although there are significant differences. In Symbiodinium
the low temperature florescence emission spectrum shows a shoulder at 709 nm
instead of the characteristic peak at 730, and polyclonal antibodies raised against the
PSI core of green plant fail to recognize any polypeptide in this fraction. Despite
many efforts, the core of PSII in dinoflagellates has not been isolated and
characterized although antibodies specific to the core polypetides from green plants
cross-react with Symbiodinium preparations (Warner et al. 1999).
Conclusions and future directions
During the last 10 years very little progress has been made regarding the study of the
structure of the photosynthetic apparatus of dinoflagellates. This information is
needed if we are to describe the initial responses of these organisms to thermal and
light stress. The combined use of modern genomic approaches with traditional
biochemical techniques can result in significant progress in our understanding of the
function and regulation of the photosynthetic apparatus Symbiodinium under diverse
environmental scenarios in the near future.
References
Hofmann E, Wrench PM, Sharples FP, Hiller RG, Welte W, Diederichs K (1996)
Structural basis of light harvesting by carotenoids: peridinin-chlorophyll-protein
from Amphidinium carterae. Science 272:1788-1791
13
Iglesias-Prieto R (1996) Biochemical and spectroscopic properties of the light-
harvesting apparatus of dinoflagellates. In: Chaudhry BR, Agrawal SB (eds)
Cytology, Genetics & Molecular Biology of Algae. SPB Academic Publishing,
Amsterdam, p 301-322
Iglesias-Prieto R, Govind NS, Trench RK (1993) Isolation and Characterization of
three membrane-bound chlorophyll-protein complexes from four dinoflagellate
species. Philosophical Transactions of the Royal Society of London B. 340:381-
392
Iglesias-Prieto R, Trench RK (1997) Acclimation and adaptation to irradiance in
symbiotic dinoflagellates. II. Responses of chlorophyll protein complexes to
different light regimes. Marine Biology 130:23-33
Scheer H (1991) Structure and ocurrence of chlorophylls. In: Scheer H (ed)
Chlorphylls. CRC Press, Boca Raton, p 3-30
Warner ME, Fitt WK, Schmidt GW (1999) Damage to photosystem II in symbiotic
dinoflagellates: a determinant of coral bleaching. Proceedings of the National
Academy of Sciences USA 96:8007-8012
14
Multiple scattering on coral skeleton enhances
light absorption by symbiotic algae
Susana Enríquez, Eugenio R. Méndez1, & Roberto Iglesias-Prieto.
Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún QR
77500, México; 1. Departamento de Óptica, División de Física Aplicada, Centro de
Investigación Científica y Educación Superior de Ensenada, Km 107 carretera
Tijuana-Ensenada, Ensenada BC 22860, México.
New examination of the optical properties of intact corals
The evolutionary success of scleractinian corals as reef-builders relied on the
formation of mutualistic symbioses with photosynthetic dinoflagellates. Algal
photosynthesis provides nutritional advantages to scleractinians, as the translocation
of photosynthates may account for their entire metabolic needs, while promoting
rapid calcification. Therefore, symbiotic reef-building corals depend heavily on the
efficiency with which they collect solar energy. In spite of the significant progress that
coral photobiology has achieved over the last 20 years, the optical properties of intact
corals have not been directly assessed yet. A major obstacle for the study of the
optical properties of intact corals is the complexity of the coralline structure,
consisting of coelenterate tissue, endosymbiotic algae and the intricate geometries of
the aragonite coral skeleton. We provide in this work measurements of the
absorption spectra of intact corals for the first time. We selected the Caribbean
scleractinian Porites branneri, because the morphological characteristics of this
species permits the preparation of even and thin coral laminae (thickness = 3 ± 0.1
mm) with homogeneous pigmentation suitable for spectroscopic analyses. During a
natural bleaching event we collected specimens exhibiting a broad variation in
symbiont and chlorophyll a content per unit of surface area (Chl a density), which
allowed us to assess the effect of such variability on the optical properties of P.
branneri.
Methodology employed here
Absorption spectra of the coral laminae were recorded between 380 and 750 nm with
1 nm resolution, with an Aminco DW2 (USA) spectrophotometer controlled by an
OLIS (USA) data collection system. Skeleton laminae were used as reference. The
light beams of the spectrophotometer were baffled with black tape apertures to match
the exact dimensions of individual samples. Reflectance spectra of corals and
skeletons were measured between 400 and 750 nm with 1 nm resolution using a
4800S Lifetime spectrofluorometer (SLM-Aminco, USA) equipped with a red sensitive
photo-multiplier tube (R955, Hamamatsu, Japan). The use of thin laminae allowed us
to obtain high quality absorption spectra of intact coral surfaces. As a result of
bleaching, we had available a series of absorption spectra of specimens whose Chl a
density varied from 3.3 mg m-2 to 102,1 mg m-2. The observed 30-fold variation in Chl
a density resulted in an approximately 5-fold variation in coral absorptance.
Measurements of light absorption on transmission mode are not only laborious, but
can be difficult to implement with corals of other morphologies. We propose as an
alternative technique for estimating coral absorption, the determination of reflectance
spectra, since the inferred absorption spectrum compared well with those obtained in
transmission mode.
15
Variation in light absorption properties as a function of coral pigment content
Determination of absorptance as a function of the variation in pigment content showed
that the light-harvesting capacity of P. branneri decreases abruptly only for Chl a
density below 20 mg m-2, remaining practically constant for values above this
threshold. These results differ from former estimations based on filtered coral slurries(1-
4).
To quantify the variations in pigment light-absorption efficiency, we estimated the
changes in the chlorophyll a specific absorption coefficient (a*) as a function of Chl a
density. The analysis of this variation indicates that the values of a* estimated for
intact corals are between 2 and 5 times higher than those estimated from
suspensions of freshly isolated symbionts with similar pigment density. On the other
hand, the a* values obtained in our study for a suspension of freshly isolated
symbionts are consistent with measurements of the absorption of filtered blastates(1-
4), and with values reported for phytoplankton(5). The increase in the absorption
efficiency may be understood through simple physical considerations. In simplified
form, a coral structure may be visualized as a thin layer of small, pigmented particles,
above one dimensional surface of coral skeleton. As the illumination reaches the
pigment layer a fraction of the incident light is absorbed ( (i)
abs ). Part of the light
transmitted through the layer is backscattered by the skeleton and passes again
( (s)
abs
) as diffuse light through the layer of pigment, increasing thus the capacity of
light absorption by the particles. This theoretical model concludes that a flat
scattering surface can enhance the absorption of the particle by a factor of up to 3
(for a non absorbing surface,
(i)
(s)
(i)
abs= abs
+ abs
= (1+2R)abs ). The model
becomes theoretically intractable when the particle is placed inside a concavity or is
exposed to several reflective coral skeleton surfaces. Nevertheless, it concludes that
light absorption by the particle could be amplified by a factor much higher that 3.
The comparison done in the North Queensland Tropical Museum (Townsville,
Australia) on 56 coral skeletons of Favide spp, and 18 spp of other massive
taxonomic families, reveled a large variability among species in the variation of the
maximum enhancement factor. We found values from a minimum of 2.9 showed by
Caulastrea curvata to a maximum of 8.3 showed by Cyphastrea japonica. It is
noteworthy that the three Porites spp from the Indo-Pacific examined showed similar
values than the maximum value estimated in this work for Porites branneri. We
concluded that coral skeletons are efficient bulk scatterers allowing to enhancing the
capacity of light absorption by algal pigments through the diffusive propagation of
light over longer optical paths. Multiple scattering by the coral skeleton provides
diffuse and homogeneous light fields for the symbionts reducing pigment self-
shading.
Biological and ecological implications
The biological and ecological implications of the optical properties of the intact coral
structure revealed by this study are diverse. We concluded that: a) the light fields
within coral tissue are not predictable from the water column light attenuation
descriptions, and are very dependent on pigment density; b) the study of
photoacclimatization of different species of corals and the propagation of the thermal
stress needs to be revised from this new perspective; c) the modulation of the
internal light field by the coral skeleton may be an important driving force in the
evolution of symbiotic scleractinian corals; and d) determinations of the minimum
quantum requirements for symbiotic scleractinian corals need to be re-assesed. Our
results indicate that symbiotic corals are one of the most efficient solar energy
collectors in nature. These organisms are capable of harvesting the same amount of
incident radiation as the leaf of a terrestrial plant with six times less pigment density.
16
References
1- Dubinsky, Z., Falkowski, P.G., Porter, J.W., and Muscatine L. 1984. Proc. R. Soc.
Lond. B. 222: 203-214.
2- Dubinsky, Z., Stambler, N., Ben-Zion, M., McClauskey L.R., Muscatine L., and
Falkowski P.G. 1990. Proc. R. Soc. Lond. B. 239: 231-246.
3- Wyman, K.D., Dubinsky, Z., Porter, J.W., and Falskowski, P.G. 1987. Mar. Biol.
96: 283-292.
4- Lesser, M.P., Mazel, C., Phinney, D. and Yentsch, C.S. 2000. Limnol. Oceanogr.
45: 76-86.
5- Morel, A., and Bricaud A. 1981. Deep Sea Res. 28: 1375-1393.
6- Enríquez, S., Méndez, E.R., and Iglesias-Prieto R. 2005. Limnol. Oceanogr. 50:
1025-1032.
17
An overview of the interpretation and current
use of chlorophyll fluorescence to understand
coral bleaching
Mark E. Warner1
1. University of Delaware, College of Marine Studies, 700 Pilottown Rd. Lewes,
Delaware, 19958, USA.
As the thermal sensitivity of some zooxanthellae is seen as one of the primary
causes of coral bleaching, many laboratories have undertaken intensive studies in
the photobiology of these symbiotic dinoflagellates to better understand how
photosynthetic processes may be affected by excessive thermal and light exposure.
Several methods of measuring active chlorophyll fluorescence to infer photosynthetic
function are becoming widely used in coral biology and for assessing photosystem
stress during experimental or natural bleaching in particular. The saturation pulse
method commonly used with the pulse amplitude modulation (PAM) fluorometer was
introduced to the field of coral biology almost a decade ago, and has become a
common tool for many reef biologists investigating coral bleaching. While the method
and the data that it can quickly generate has proven quite beneficial in extending our
fundamental understanding of the photobiology of zooxanthellae symbioses, it is not
without certain caveats that should be addressed in light of current efforts to better
understand the biochemical and cellular pathways involved in coral bleaching.
Importance of experimental design and key parameters
While core proteins and pigments within the reaction centers of photosystem I and II
(PSI & PSII hereafter) are highly conserved across all known photosynthetic
eukaryotes, there are many differences at the level of light harvesting complex
structure and function which can and do affect commonly used fluorescence
parameters. It is prudent to note that the much of the theoretical groundwork that
forms the basis of using PAM fluorometry extends from work with higher plants which
can represent a significant departure from the physiological variability one is likely to
encounter in working with algal groups (including the dinoflagellates) that extend from
the red algal lineage. A second important point is that there are many differences in
current designs of coral bleaching experiments utilizing PAM fluorometry such that
results should be evaluated in light of the scale of the design itself. It is important to
view current results in relation to the ecological relevance of the experimental design
versus the degree of physiological reduction. The field of chlorophyll fluorescence
contains a semantic minefield of terminology that can be an unnecessary source of
confusion when comparing different bleaching experiments, and one should strive to
follow previously published guidelines for correct use of such terms (see Kromkamp
and Forster 2003). Some of the more common parameters in use today are the dark
acclimated quantum yield of PSII (Fv/Fm), the effective quantum yield of PSII
(F/Fm'), photochemical (qP) and nonphotochemical fluorescence quenching (qN
and NPQ), and electron transport rate (ETR).
Fv/Fm and F/Fm' are two of the most common parameters used for rapidly
assessing the status of PSII in zooxanthellae within corals, as they are easy to
measure and have a long history of use in plant and phytoplankton biology for
detecting photoinhibition. Corals exposed to thermal stress as well as those sampled
during natural bleaching events have shown a significant loss in Fv/Fm compared to
corals held in non-bleaching conditions which typically precedes detection of any loss
18
in zooxanthellae density. Declines in Fv/Fm are correlated with the loss of PSII D1
protein content in some cases (Warner et al. 1999; Lesser and Farrell 2004), thereby
enforcing the idea of thermal stress exacerbating a pathway of cellular damage seen
in light enhanced photoinhibition studies. However, more work is needed to fully
establish if this correlation holds across different species of zooxanthellae. For
example, some zooxanthellae could possibly show a loss in PSII activity while
degradation of reaction center proteins is impeded. Likewise, an important point is
that one should be careful to delineate stress related loss of Fv/Fm with possible
seasonal declines in Fv/Fm that largely reflect photoacclimation as opposed to
chronic cellular stress. Similarly, one can use the effective quantum yield (F/Fm') or
PSII capacity in the light acclimated state as a proxy for stress, so long as one has
an understanding of the typical patterns of diurnal decline and recovery due simply to
increased levels of light energy dissipation during daylight hours (i.e. enhanced
quenching of the fluorescence signal due to non-photochemical pathways such as
xanthophyll cycling) in their organism of interest. Recent work has shown that
analysis of F/Fm' can also indicate when a bleaching experimental design may be
inducing more artificial chronic stress than would be seen in the field (see Franklin et
al. 2004, Fig.1, for a good example). A second point of caution is that F/Fm' is
dependent on the previous light history of the alga and one should take care in
interpreting results for experiments that involve acute light shocks that may not
replicate physical conditions typically seen in the field. In this same vein, a
fluorescence induction curve conducted with a light intensity significantly higher than
that used in the experimental treatment can yield large decreases in F/Fm' which
reflect an artefact of the light intensity used for the induction curve rather than how a
coral may have processed light under the experimental conditions. Of the quenching
parameters typically measured, photochemical quenching (qP) is typically the
hardest to measure under field conditions. This difficulty is due to the fact that the
equations traditionally used to calculate this variable rely on accurate measurement
of any quenching of the initial fluorescence signal (Fo), which can happen quite
frequently in zooxanthellae (personal observation). Proper assessment of qP
requires the ability to rapidly darken a sample and apply a pulse of far-red light to re-
oxidize PSII traps. Such protocols are not available on current submersible
instrumentation (e.g. the diving PAM), and alternative equations developed with
higher plants to circumvent the need to know Fo for qP calculation have proven to be
less accurate for corals (personal observation).
Other parameters, such as excitation pressure over PSII (sensu Iglesias-Prieto et al.
2004) and NPQ have shown to provide a good proxy for potential thermal stress in
some corals. The central idea to this point is that some thermally sensitive
zooxanthellae may have less capacity to dissipate excess energy than others, and
their homeostatic level of excitation pressure is higher than that of other
zooxanthellae that show greater thermal resistance. On the other hand, more work is
currently underway to better understand how (or if) any compensatory electron
turnover at PSII may also play a role in explaining how some corals with elevated
NPQ or excitation pressure can maintain PSII function during thermal stress.
Lastly, electron transport rate or ETR has been used heavily to infer changes in
photosynthetic activity in general and during thermal and/or light stress. While
plotting ETR vs. irradiance is a common practice one should take great caution in
interpreting such data using traditional photosynthesis to irradiance terminology and
concepts, as they are not synonymous in many cases. For example, one should not
assume that maximal ETR is representative of maximal photosynthetic rate (Pmax) as
measured by other methods (e.g. respirometry). Comparisons of gross
photosynthesis and ETR in several groups of algae have shown that these two
variables co-vary only within a range of light intensities and that they can significantly
19
depart from each other at higher levels of light (Geel et al. 1997). Such an effect is
most likely due to non-assimilatory electron flow through PSII such as that due to
Mehler activity, which can change between different algae or under different physical
conditions. Likewise, current evidence suggests that coral absorptance can change
substantially during bleaching (Enriquez et al. 2005), thus rendering any
measurement of "relative" ETR that does not account for such absorptance changes
highly suspect to gross error.
References
Enriquez S, Mendez ER, Iglesias-Prieto R (2005) Limnol Oceanogr 50: 1025-1032
Franklin DJ, Hoegh-Guldberg P, Jones RJ, Berges JA (2004) Mar Ecol Prog Ser 272:
117-130
Geel C, Versluis W, Snel JFH (1997) Photosynth Res 51: 61-70
Iglesias-Prieto R, Beltran VH, LaJeunesse TC, Reyes-Bonilla H, Thome PE (2004)
Proc Royal Soc London 271: 1757-1763
Kromkamp J, Forster RM (2003) Eur J Phycol 38: 103-112
Lesser MP, Farrell JH (2004) Coral Reefs 23: 367-377
Warner ME, Fitt WK, Schmidt GW (1999) Proc Nat Acad Sci 96: 80078012
20
Diel Cycling of Nitrogen Fixation in Corals with
Symbiotic Cyanobacteria
Michael P. Lesser1, Luisa I. Falcón2, Aimé Rodríguez-Román3, Susana
Enríquez3, Ove Hoegh-Guldberg4, and Roberto Iglesias-Prieto3
1 Department of Zoology and Center for Marine Biology, University of New
Hampshire, Durham, New Hampshire 03824, USA; 2 Instituto de Ecología,
Universidad Nacional Autónoma de México, Circuito Exterior s/n Ciudad
Universitaria, CP 04510 México, D. F. México, 3 Unidad Académica Puerto Morelos,
Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de
México, Cancún QR 77500, México, 4 University of Queensland, Centre for Marine
Sciences, 4072, St. Lucia, Queensland, Australia
Corals in mutualistic symbiosis with endosymbiotic dinoflagellates (zooxanthellae)
are essential components of the ecological diversity of tropical coral reefs.
Zooxanthellate corals also exist in an environment where inorganic nitrogen limits the
growth and abundance of zooxanthellae in hospite1-3. Many colonies of the
Caribbean coral, Montastraea cavernosa, contain endosymbiotic cyanobacteria4.
These cyanobacteria co-exist with the zooxanthellae and express the nitrogen fixing
enzyme nitrogenase4. Here we show that the percentage of colonies containing
symbiotic cyanobacteria increases with increasing depth, and that measurements of
nitrogen fixation show a diel pattern with the highest rates of nitrogen fixation in the
early morning and evening. No nitrogen fixation was measurable in non-symbiotic
con-specifics. The 15N stable isotope data show a strong nitrogen fixation signal in
the zooxanthellae fraction of corals with cyanobacterial symbionts suggesting that
zooxanthellae use fixed nitrogen products. The timing of nitrogen fixation avoids
maximum periods of photosynthesis to avoid severe hyperoxia, and nitrogen fixation
does not occur when the coral experiences hypoxia or anoxia. These cyanobacteria
require low oxygen tensions to support the respiratory processes that provide the
energy required to fix nitrogen. Nitrogen fixation in these corals provides an
important supplemental source of a limiting element for this novel microbial
consortium.
Figure 1. Green-brown (left) and red morphs of Montastraea cavernosa under Blue
light and viewed through an orange filter set. Photo: O. Hoegh-Guldberg
21
Figure 2 Percent abundance of
Montastraea cavernosa colonies
containing endosymbiotic
cyanobacteria at different depths
around Lee Stocking Island, Bahamas.
Significant differences were observed
with depth using a contingency table
analysis; Likelihood ratio, 2 =18.17
P=0.006, Pearson, 2 =14.065
P=0.03. Post-hoc multiple comparison
testing showed that the population of
orange M. cavernosa were significantly
greater deeper (15 m) compared to
shallow depths ( 12 m).
The presence of cyanobacterial symbionts that can fix nitrogen in
zooxanthellate corals represents not just a novel microbial consortium of
photosynthetic eukaryotes and prokaryotes, but one that challenges the long-
term paradigm that nitrogen is limiting in corals. Our observations indicate that
several species of scleractinian corals in the Caribbean and on the Great
Barrier Reef have individuals harboring stable, non-pathogenic populations of
endosymbiotic cyanobacteria. If the occurrence of this consortium in other
species of scleractinian corals residing in oligotrophic tropical waters is more
common than previously believed, it could provide an important supplemental
source of a limiting element for zooxanthellate corals. These inputs of new
nitrogen from endosymbiotic cyanobacteria in corals not only has important
implications for our current understanding of the role of nitrogen as a limiting
and regulatory element in these associations, but also requires that we re-
examine the role corals play in the nitrogen budgets of coral reefs. Because
corals release large quantities of dissolved organic material containing high
concentrations of both organic and inorganic sources of nitrogen the implication
of corals as nitrogen fixes consortiums for the biogeochemical fluxes of nitrogen
in carbonate sands and pore waters of coral reefs are potentially very large.
22
Figure 3. a) Nitrogen fixation (acetylene reduction) brown/green (N=3) versus orange
(N=3) colonies of M. cavernosa. There were significant effects of colony type
(ANOVA: P<0.0001), time (ANOVA: P<0.0001), and the interaction of colony type
and time (ANOVA: P<0.0001), Post-hoc multiple comparisons show that there were
significant effects of time (SNK: P<0.05) between colony types. b) Stable 15N
isotope results for brown/green (N=3) versus orange (N=3) colonies of M. cavernosa.
There were no significant effects of colony for the animal fraction but a significant
effect (ANOVA: P=0.036) of colony was observed for the zooxanthellae fraction. c)
Cellular DNA content measured as relative fluorescence of Picogreen staining on
isolated zooxanthellae from brown/green (N=3) and ornage (N=3) colonies of M.
cavernosa. Solid line through distribution is smoothed curve for easier visualization.
d) Net photosynthesis-irradiance (oxygen flux) curves for brown/green (N=3) versus
orange (N=3) colonies of M. cavernosa. There were significant differences in
maximum productivity (ANOVA: P<0.05, brown/green; 3.63 ± 0.22 [SE] µmol O2 cm-
2 h-1, orange; 2.53 ± 0.29 [SE]), and in calculated rates of respiration (ANOVA:
P<0.05, brown/green; -0.871 ± 0.125 [SE] µmol O2 cm-2 h-1, orange; -0.543 ± 0.157
[SE]), but not the 13 light-limited portion of the fitted curves. The insert is the
phycoerythrin emission from the fluorescence induction on orange colonies with and
without exposure to the herbicide DCMU. An approximately 22% increase in
phycoerythrin emission was observed when exposed to DCMU.
23
Figure 4. a) The relationship between time (min) and depth (m) during which nitrogen
fixation can take place in Montastraea cavernosa with endosymbiotic cyanobacteria.
The open circles represent predictions using the data from the fitted P-I curve. The
closed squares are the simulation results using the empirical respiration rates, and
the closed circles represent the simulation results under conditions where we allowed
nitrogen fixation to take place at the same irradiances experienced in situ by our
coral samples. b) Using the percent abundance data of Montastraea cavernosa with
endosymbiotic cyanobacteria and the simulation data of the percentage of daylight
irradiances that are below the compensation point (± 10%) there is a significant
functional relationship (ANOVA: P<0.05, y=0.32139 (x) 0.54562) that predicts
increasing abundances of these corals with increasing depth.
24
Host pigments, photosynthetic efficiency and
thermal stress
Sophie Dove1, Carli Lovell1, Maoz Fine1, Susana Enríquez2, Roberto
Iglesias-Prieto2, Kenneth Anthony3 and Ove Hoegh-Guldberg1.
1. Centre for Marine Studies, University of Queensland, St Lucia 4067 Australia; 2.
Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún QR
77500, México; 3. School of Marine Biology and Aquaculture, James Cook University
Townsville 4811 Australia.
Scleractinian corals provide the calcium carbonate matrix of coral reefs due to
efficient photosynthesis by endosymbiotic dinoflagellates. The ability of these
dinoflagellates to harvest solar energy from within the host tissue is essential for, yet
dangerous to, the success of scleractinian corals as over-energization of the
photosynthetic units results in the formation of reactive oxygen species (ROS), which
cause cellular damage. We have investigated the hypothesis that host pigments may
act as an extension of the dinoflagellate photosynthetic pigments to absorb light for
utilization by phtosythesis that Dinoflagellate or pigmentation responds over a period
of days to months to changes in photon flux density (PFD) by reciprocally altering
chlorophyll (Chl) and carotenoid pools (Iglesias-Prieto & Trench, 1997). In high PFD
environments the carotenoid pool, especially the xanthophyll pool, is increased
enabling the quenching of harvested energy to heat and the direct quenching of
ROS. There are conflicting reports in the literature over whether host pigments,
similar to carotenoids, act photoprotectively. Dove (2004) argues for the
photoproctective nature of purple-blue non-fluorescent pigments. While Mazel et al.
(2003) argue the opposite for fluorescent green "GFP-homologs" that showed no
depth stratification.
Montipora monastriata from Wistari Reef (GBR, Australia) at a depth of 3-5 m occur
predominantly as purple-blue, tan or brown morph in the open; and green, brown or
red morph under the overhangs of the spur and grove formation. Tan morphs contain
host pigments, pocilloporin and a putative green GFP-homolog; blue morphs contain
pocilloporin, and green morphs contain the putative green GFP-homolog. We
investigated whether the colour morphs were a response to PFD; whether
photosynthetic flux measured as oxygen flux differed for the different morphs and
how these rates related to the light absorption capacity of the endosymbionts within
the specific host environments.
Effect of changing photon flux on host pigments
Similar to the changes in the carotenoid pool in high PFD, host pigments (pocilloporin
and the putative green GFP homolog) respond to 2 months of increased PFD by
increasing expression levels relative to total protein. Green cave dwelling morphs
increased pocilloporin, but not "green" GFP levels that were already elevated under
low PFD (Fig. 1). These results support a photoprotective role for these pigments,
whilst allow for a lack of depth stratification observed for green-fluorescent GFPs.
25
Figure 1. Effect of transplanting Montipora monasteriata between high (unshaded) and low
(shaded) light fields on host pigments. Absorption spectrum of (A) pocilloporin (B) putative
green GFP. Transplantation of blue and tan morph from high to low PFD (C,D); of green, red
and brown morphs from low to high PFD (E,F). *, p<0.05.
Effect of host pigmentation (Chl concentration and skeletal structure) on
oxygen flux and absorption capacity by endosymbiotic algal pigments
The rate of maximum photosynthesis (Pmax) is typically lower for low PFD (LL) versus
high PFD (HL) plant and algae (Nigoyi 1999). In line with this observation, cave-
dwelling M. monasteriata had lower Pmax than open dwelling blue morphs (Fig. 2AB).
Paradoxically, tan-HL morphs however had Pmax that were not significantly different
from the cave dwelling morphs, and at least half the value of blue-HL morphs.
Oxygen flux measurements were made in January of 2002 at the onset of a major
GBR bleaching (max. temp attained = 30 oC). Recently, Enriquez et al. (2005) have
shown that 675 nm light absorption by algae in symbiosis can be estimated from
reflectance measurement of coral surfaces. The specific absorption coefficient a* of
Chl a increases exponentially with drastic reductions in Chl a. We calculated a* for
Chl a concentrations resulting from control and 32oC heating for 6 h for blue.-HL-
pocilloporin containing morphs and brown-HL and brown-LL - lacking host
pigmentation morphs. The studied showed that this exponential rise occurred faster
at higher Chl a densities for morphs expressing pocilloporin (Fig. 2D). Light
enhancement within host tissue (as pigmentation nears zero) is due to multiple
scattering by diffuse skeletal surfaces (Enríquez et al. 2005). Potentially, certain
skeletal morphs are more predisposed to turn purple or blue (express host pigments)
in response to a loss in algal pigmentation than others. Host pigments may function
photo-protectively and facilitate algal photosynthesis under elevated internal PFD by
preventing overload of the photsystem reaction centres. The mechanisms by which
pocilloporin (and other pigmented GFPs) achieve this feat are yet to be determined,
yet are likely to include "optical dampening" by host pigments of UVR and/or PAR.
There is no paradox over Pmax in Tan and blue open-dwelling morphs: the difference
between Pmax blue-HL and tan-HL can be assigned to the difference in Chl a density
and hence the light fields directly experienced by algae in symbiosis (Fig. 2 B-D).
26
Figure 2. Typical mid-day light fields (A); maximum rate of photosynthesis; boxed text shows
ratio of photosynthesis to respiration (B); areal algal cell densities; boxed text shows
corresponding Chl a density (C); estimate of algal light absorption (a*) relative to Chl a density
for different colour morphs of Montipora monasteriata. , blue-HL morphs a* = 0.09 ± 0.01 x
e-0.013±0.002 x [Chl a], r2=0.83, p< 0.01; , brown-HL morph a* = 0.06 ± 0.005 x e-0.0092±0.001 x [Chl a],
r2=0.85, p< 0.01, , brown-LL morph, no relationship.
Conclusions and future directions
Dove et al. (in press) showed that heating M. monasteriata to 32oC for 6 h, not only
decreased the Chl concentration, but also the relative xanthophyll and pocilloporin
pools. Dove (2004) showed that pocilloporin-rich morphs of Acropora aspera were
photosynthetically more able to handle increases in PFD than pocilloporin-poor
morphs, but that after prolonged exposure to 32-33oC, this result was reversed and
coincided with elevated mortality. Corals obviously exist were water temperatures
frequently exceed 33oC in the summer: are these corals capable of expressing
pocilloporin and/or do they take on skeletal morphologies that minimise internal light
enhancement?
References
DOVE, S. G. 2004. Scleractinian corals with photoprotective host pigments are
hypersensitive to thermal bleaching. Mar. Ecol. Progr. Ser. 272: 99-116.
DOVE, S. G., J-C. ORTIZ, S. ENRÍQUEZ, M. FINE, P. FISHER, R. IGLESIAS-PRIETO, D.
THORNHILL, AND O. HOEGH-GULDBERG. in press. Response of holosymbiont
27
pigments from the scleractinian coral Montipora monasteriata to short term heat
stress. Limnol. Oceanogr.
ENRÍQUEZ, S., E. R. MÉNDEZ , R. IGLESIAS-PRIETO . 2005. Multiple scattering on coral
skeletons enhances light absorption by symbiotic algae. Limnol. Oceanogr. 50:
1025-1032.
IGLESIAS-PRIETO, R, AND R. K. TRENCH. 1997. Acclimation and adaptation to
irradiance in symbiotic dinoflagellates. II. Response of chlorophyll-protein
complexes to different photon-flux densities. Mar. Biol. 130: 23-33.
MAZEL, C., M. STRAND, M.P. LESSER, M. CROSBY, B. COLES, AND A. NEVIS. 2003.
Multispectral fluorescence laser line scan imaging of coral reefs. Limnol.
Oceanogr. 48: 522-534.
NIYOGI, K. K. 1999. Photoprotection revisited. Genetic and molecular approaches.
Ann. Rev. Plant Physiol. Plant Mol. Biol. 50: 333-359.
28
Imaging-PAM: Operation and Possibilities
Ross Hill1 and Karin E. Ulstrup1
1 Institute for Water and Environmental Resource Management and Department of
Environmental Sciences, University of Technology, Sydney, Westbourne St, Gore
Hill, NSW 2065, Australia.
High resolution imaging of variable chlorophyll a fluorescence emissions was used to
identify 2-dimensional heterogeneity of photosynthetic activity across the surface of
corals. In comparison to earlier studies of fluorescence analysis (Ralph et al. 2002),
the Imaging-PAM enables greater accuracy by allowing different tissues to be better
defined and by providing many more data points within a given time (Hill et al. 2004;
Ralph et al. 2005). The resolution of the instrument provides detail down to 100 µm
and the area imaged can be controlled by the user. The standard Imaging-PAM
measures an area of 3.5 x 4.5 cm and the Maxi-Imaging-PAM measures 10 x 13 cm.
A micro-head attachment is also available for more detailed, fine scale investigations.
An added component to the apparatus is a 96 well plate (imaged under the Maxi-
Imaging-PAM) which has applications for ecotoxicological studies. This instrument
contains a ring of blue, red and near-infra-red (NIR) LED's and a CCD camera for
fluorescence detection. A new feature provided by this instrument enables the
measurement of absorptivity = 1-(Red/NIR).
Photosynthetic responses of coral tissue to light
Images of fluorescence emission indicated that the photosynthetic activity of
coenosarc and polyp tissues responded differently to changing light and diel
fluctuations in Acropora nobilis, Goniastrea australiensis, and Pavona decussata.
Fig. 1 shows variable chlorophyll a fluorescence images of A. nobilis under 295 µmol
photons m-2 s-1.
Absorp. Ft Fm' EQY PS NPQ
32 mm
Fig. 1: Chl a fluorescence images of A. nobilis showing PAR absorptivity, Ft, Fm', effective
quantum yield (EQY), relative photosynthesis rate (PS) and non-photochemical quenching
(NPQ). Colour scale shown.
Diel fluctuations in Fv/Fm revealed that different tissue types showed varying degrees
of downregulation/photoinhibition spatially and temporally. Upon exposure to
experimentally controlled high light conditions (1000 µmol photons m-2 s-1),
downregulation of photosynthesis occurred as well as higher NPQ within the polyps
of G. australiensis and on the polyp walls and coenosarc of A. nobilis.
Studying coral bleaching with the Imaging-PAM
In Pocillopora damicornis, A. nobilis and Cyphastrea serailia the Imaging-PAM was
used to map the impact of bleaching stress. The effect of bleaching conditions (33°C
29
and 280 µmol photons m-2 s-1) was studied over a period of 8 h. Marked changes in
fluorescence parameters were observed for all three species. Although a decline in
EQY was observed, P. damicornis showed no visual signs of bleaching on the
Imaging-PAM after this time. In A. nobilis and C. serailia, visual signs of bleaching
over the 8 h period were accompanied by marked changes in Ft (light-adapted
fluorescence), NPQ and EQY. These changes were most noticeable over the first 5
h. The most sensitive species was A. nobilis, which after 8 h at 33°C had reached an
EQY value of almost zero across its whole surface (Fig. 2). Differential bleaching
responses between polyps and coenosarc tissue were found in P. damicornis, but
not in A. nobilis and C. serailia. Spatial variability of photosynthetic performance from
the tip to the distal parts was revealed in one species of branching coral, A. nobilis.
Fig. 2: EQY
of A. nobilis
over 8 h of
exposure to
bleaching
conditions.
Colour scale
shown.
Control 1 h 2 h 3 h 4 h 5 h 6 h 7 h 8 h
Photosynthetic performance of zooxanthellae within animal tissue affected by
Porites Ulcerative White Spot (PUWS) Syndrome
The Imaging-PAM was also used to map the
photosynthetic gradient across syndrome
F /F
v
m
lesions. In this case, Porites Ulcerative White
Spot (PUWS) Syndrome was imaged (Fig. 3).
Several variable chlorophyll a fluorescence
parameters (Fo, Fm and EQY at high
irradiances (596 µmol photons m-2 s-1)
F
F
showed distinct gradients across the o
m
syndrome lesion. This suggests that in the
case of PUWS, photosynthetic performance
of zooxanthellae is affected and that
fluorometry may be a useful tool to assess the
health of the symbionts associated with coral
EQY (596
EQY (5 )
96
EQY (5)
EQY (5
syndromes in general.
Fig. 3: Photograph and variable chlorophyll a
fluorescence of Porites sp. showing Fv/Fm, Fo, Fm,
EQY at 596 and 5 µmol photons m-2 s-1.
Conclusions and future directions
The Imaging-PAM allows for a range of photosynthetic parameters to be measured
across a 2-dimentional photosynthetic surface and also provides the means to
measure absorptivity. The results of these experiments indicate that stress-induced
photosynthetic responses are rarely continuous across a coral surface and that
variations exist between the various tissue types. As a result, it is emphasised that it
is unwise to extrapolate single-point measurements to a whole colony.
30
References
Hill R, Schreiber U, Gademann R, Larkum AWD, Kühl M, Ralph PJ (2004) Spatial
heterogeneity of photosynthesis and the effect of temperature-induced bleaching
conditions in three species of corals. Marine Biology 144, 633-640.
Ralph PJ, Gademann R, Larkum AWD, Kühl M (2002) Spatial heterogeneity in active
chlorophyll fluorescence and PSII activity of coral tissues. Marine Biology 141,
639-646.
Ralph PJ, Schreiber U, Gademann R, Kühl M, Larkum AWD (2005) Coral
photobiology studied with a new imaging pulse amplitude modulated fluorometer.
Journal of Phycology 41, 335-342.
31
Seasonal fluctuations in the physiology of
Stylophora pistillata
Gidon Winters1, Sven Beer1* and Yossi Loya2
1Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel;
Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel
Seasonal fluctuations in the maximal quantum yield of photosynthetic electron flow
through photosystem II (Fv/Fm) as measured in situ were found to occur naturally in
zooxanthellae of non-bleaching colonies of the branching coral Stylophora pistillata
growing at 5, 10 and 20m. These fluctuations correlated stronger with changes in
irradiance than changes in seawater temperature. Seasonal fluctuations were also
found in the chlorophyll a density, which was due mostly to seasonal changes in
zooxanthellae density. Results show that during the summer of a "non-bleaching
year", corals will loose 80% of their zooxanthellae (in comparison with zooxanthellae
densities measured during the winter) the equivalent of zooxanthellae loss measured
in the Caribbean during the 1998 mass bleaching event (Warner et al. 2002).
Underwater photographs taken monthly reveal the dramatic colour changes corals go
through even during "non-bleaching" years. These results shed some light on the
issue of what is "real" bleaching as apposed to seasonal changes. Possibly, what is
termed mass bleaching should be seen as taking this normal (seasonal) paling of
coral colour one notch forwards. For future PAM fluorometry based studies, it is
suggested that, in order to correlate Fv/Fm values with anthropologically caused
stresses, (a) Fv/Fm measurements be performed in situ under natural conditions and
(b) natural seasonal fluctuations in Fv/Fm be taken into account when using this
parameter for diagnosing coral bleaching. It is further suggested that high irradiances
may cause decreased Fv/Fm values at least as much as, if not more than, high
temperatures.
Figures
Fig.1. a) Study site and b) experimental set up: Specially made plastic holder for the
Diving-PAM's probe, allowing for repetitive measurements to be performed keeping
the same angle (69o) and distance (1 cm) between the sample and the PAM's probe
32
) 0.75
5m
Fm
10m
I
(
F
v/
0.7
20m
PSI
d of 0.65
el
yi
u
m
0.6
ant
qu
al 0.55
p
t
i
m
O
0.5
04
04
04
04
0
4
r
-
04
r
-
0
4
l
-
0
4
g
-
0
4
p-
t
-
0
4
v
-
0
4
c
-
0
4
J
a
n-
e
b-
a
y-
F
Ju
Ma
Ap
M
J
u
n-
Au
Se
Oc
No
De
Month (2004-5)
Fig. 2. Seasonal variations in Fv/Fm in Stylophora pistillata (n=5, ±SE) growing at 5,
10 and 20m
1200
29
Max diel global radiation
)
Max diel water temperature
-2
28
o C)
m 1000
W
27
a
t
u
r
e (
p
er
a
t
i
o
n (
26
a
di 800
t
e
m
a
l
r
a
t
er
l
ob
25
g
e
a
w
el
s
600
el
24
m di
di
mu
u
m
xi
23
m
400
xi
Ma
Ma
22
200
21
04
04
04
04
0
4
05
n/
/
04
/
04
a
r
/
04
c
t
/
04
n/
Ja
e
b/
p
r
/
04
a
y
/
04
e
p/
ec/
F
u
g
/
04
M
A
Jul
M
Jun/
A
S
O
Nov
D
Ja
Month (2004)
Fig. 3. Seasonal variations in global diel maximum global radiation (left y-axis) and
diel maximum seawater temperature (right y-axis) for the year 2004.
33
)
0.66
F
m
a
0.64
S
I
I
(
F
v/
P 0.62
e
l
d of
yi 0.6
r=0.16
um
nt
0.58
a
l
qua
0.56
i
m
Opt
0.54
20
21
22
23
24
25
26
27
28
Monthly average water temperature (oC)
) 0.66
m
F
0.64
S
I
I
(
F
v/
b
0.62
P
r=0.85
0.6
e
l
d of
yi
0.58
u
a
n
t
u
m
0.56
a
l
q
0.54
p
t
i
m
O
500
600
700
800
900
1000
Monthly average maximum diel radiation (W m-2)
Fig. 4 Correlations between monthly Fv/Fm measurements of Stylophora pistillata
(n=5) growing at 5m and monthly average of a) seawater temperature (oC) and b)
diel maximum global radiation (W m-2)
34
3
2.5
i
t
y
)2
2
l
ae dens
l
s
/
cm
1.5
ce6
anthel
0
1
(1
z
oox
0.5
0
04
5
5
l
-
0
4
0
4
04
05
0
5
05
t
-
0
4
v
-
0
4
c
-
0
4
-
0
5
a
r-0
J
un-
Ju
ug-
ep-
eb-
p
r-0
A
S
Oc
No
De
J
an-
F
M
A
May
J
un-
Month (2004-5)
Fig. 5. Seasonal dynamics in zooxanthellae density of Stylophora pistillata (n=5-6,
±SE) growing at 5m
Fig. 6. Photographs following the same branch of Stylophora pistillata growing at 5m
through out the year.
35
The cellular mechanism of coral bleaching
Daniel Tchernov 1,3 , L Haramaty1, T. S. Bibby1, Max Y. Gorbunov1,and
Paul G. Falkowski1,2
1 Environmental Biophysics and Molecular Ecology Program, Institute of Marine and
Coastal Sciences, Rutgers University, 71 Dudley Road New Brunswick, NJ
08901,USA; §Department of Geological Sciences, Rutgers University, Wright
Geological Laboratory, 610 Taylor Road, Piscataway, NJ 08854, USA; Ą The
Interuniversity Institute of Eilat, P.O.B 469, Eilat 88103, Israel
The phenomenon of mass coral bleaching has developed into a major concern
because of the coral reef's key ecological role as a major habitat for the most diverse
community in the marine realm and its key economic function in numerous
economies. Coral bleaching is induced by positive anomalous temperatures in
surface waters of 1.5 to 2 şC. However, not all reefs or corals within a reef are
equally susceptible to elevated temperature stress. Here we wish to report that
thylakoid membrane (TM) lipid composition in the algal symbiont plays a key role in
determining the symbiosis' susceptibility to thermal stress. However, there seems to
be no correlation between phylogenetic assignment of the symbiotic algal type and
TM lipid composition. In addition, we show that apoptosis, triggered by the production
of reactive oxygen species (ROS) in the symbiotic algae (zooxanthellae) that reside
within host animal cells, can induce an apoptotic caspase cascade in the host animal
that leads to expulsion of the algae and can also lead to death of animal. The
bleaching process can be experimentally manipulated by the addition of extracellular
ROS and caspase inhibitors. This mechanistic explanation is of major importance in
enabling a comprehensive understanding of bleaching on a biochemical and
molecular level. Our findings might also reflect on the interpretation of ecological and
evolutionary processes that are currently observed in coral reefs world wide.
Tolerant
Tolerant
26oC
32oC
A meltdown
of biological
membranes
Sensitive
Sensitive
26oC
32oC
A meltdown
of biological
membranes
Sensitive
Sensitive
S. pistillata
S. pistillata
26oC
32oC
36
Theme 2: Diversity, flexibility, stability,
physiology of Symbiodinium and the associated
ecological ramifications (May 15-17)
Dinoflagellates in the genus Symbiodinium are the principal endosymbionts of reef-
building corals as well as animal hosts from several other phyla. Understanding the
forces that have driven the evolution and distribution of these organisms has the
potential to provide important insights into the response of corals to past and present
environmental change. This workshop theme aimed to achieve greater synthesis of
the large number of studies that have focused on the diversity and specificity within
the genus Symbiodinium. This resulting discussion focused on the phylogeny,
specificity, biology and flexibility of Symbiodinium symbioses particularly where our
current understanding is of the ability or not of coral hosts to adapt rapidly to climate
based stresses by switching Symbiodinium partners. One of the key ambitions of
this discussion was to produce a consensus statement that will help guide future
research and project the current state of our understanding of the field. This was
achieved and appears at the end of this section of the workshop document.
Discussion conveners/coordinators:
Ove Hoegh-Guldberg (oveh@uq.edu.au, University of Queensland)
William K. Fitt (fitt@sparrow.ecology.uga.edu, University of Georgia)
Participants:
David Abrego.; Mebrahtu Ateweberhan; Andrew Baker; Ania Banaszak; ; Merideth
Bailey; Ranjeet Bhagooli; John Bythell; Mary Alice Coffroth; Jeffry Deckenback;
Sophie Dove; , Susanne Enriquez; Wil iam K Fitt,; Ruth Gates; Jessica Gilner; ,
Reia Guppy; Ross Hil ; Ove Hoegh-Guldberg; Glenn Holmes; Roberto Iglesias-
Prieto, Amita Jatkar, Ron Johnstone; Dusty Kemp, Paulina Kaniewska, Robert
Kinzie III; Baraka Kuguru, Mauricio Lanetty-Rodriguez; Todd LaJuenesse; Bill
Leggat; Michael Lesser; Mikail Matz; Mackenzie Manning; David Miller; Nancy
Muehllehner, Michael Kuhl; Adrienne Romanski; Peter Ralph; Hector Reyes; Jez
Roff, Romanski, Adrienne; Scott Santos; Roee Segal; Shenkar, Noa; Eugenia
Sampayo; Daniel Tchernov, Karin Ulstrup; Madeleine van Oppen; Shakil Visram;
Robert Van Woesik; Mark Warner; John Ware; Linda Wegley, Gidon Winter, David
Yellowlees, and Assaf Zevoluni
Goals of workshop component:
The goal of the special discussion was to assess our current state of understanding
of Symbiodinium symbioses in terms of:
1. Genetic diversity and taxonomy of Symbiodinium.
2. The cell biology of Symbiodinium symbiosis (initiation, selectivity, recognition)
3. The ecological & physiological benefits of harboring different symbionts.
4. Stability of Symbiodinium-host (=holobiont) combinations in time and space.
5. Flexibility by hosts in terms of establishing or swapping genotypes of
Symbiodinium.
37
The diversity, specificity and flexibility of
Symbiodinium symbioses.
Ove Hoegh-Guldberg
Centre for Marine Studies, University of Queensland, St Lucia 4072 QLD Australia
The dinoflagellate symbioses involving reef building corals are amongst the most
spectacular associations between animals and photosynthetic organisms. The
principal symbionts of corals (as well as organisms from at least five phyla) belong to
the genus Symbiodinium (Freudenthal 1962). In most cases, Symbiodinium spp. live
endosymbiotically within host cells with the notable exception of some molluscs,
where they are found extracellularly. Within their host invertebrates, Symbiodinium
spp. photosynthesize at rates comparable to free-living dinoflagellates but pass over
90% of the newly fixed carbon to their hosts. In exchange, Symbiodinium receives
access to the normally limiting inorganic nitrogen and phosphorous. In the case of
symbiotic Scleractinian corals, these dinoflagellate symbionts power metabolic needs
and provide the energy required for the precipitation of enormous quantities of
calcium carbonate. The precipitated calcium carbonate in turn forms the primary
framework of coral reefs, which is habitat for hundreds of thousands of species.
These highly productive structures also protect coastlines in many parts of the world
and provide subsistence and livelihood for several hundred mil ion people.
Understanding the taxonomy, biology and evolution of Symbiodiniu is
m
particularly
important given their central functional role within coral reefs. Until recently,
however, our understanding of these organisms has been limited, primarily due to the
cryptic diversity of Symbiodinium and the more ecological focus on coral reef studies
over the past 50 years. Combined, this situation has left us without a good
understanding of how the association between Symbiodinium and its hosts is
established, or how flexible the association is with respect to its symbiotic partners.
Both of these questions have come to the forefront recently within the question of
how coral reefs will respond to global climate change. On one hand, the recently
discovered diversity within the genus Symbiodinium is seen as evidence of enormous
flexibility in ecological time scales and as evidence that corals will adapt quickly to
climate change. On the other hand, the diversity is being interpreted as evidence
that host and symbiont are relatively faithful to each other, and that changes in the
partnership only occur at deeper, more evolutionary time scales. Given the
importance of the issue and the diversity of opinion within the field, focusing the
workshop on this issue is particularly important. In this paper, I will set out some of
the background to the issues which will be taken up by the workshop participants in
later papers.
The cryptic diversity of Symbiodinium
y
S mbiodinium was originally described from the symbiont of the jellyfish Cassiopeia
xamachana by Freudenthal (1962) and is now recognized as a member of the family
Gymnodiniaceae (Class Dinophyceae, Order Gymnodiniales; Freudenthal 1962,
Trench 1987). Suspicion that Symbiodinium microadriaticum was in fact a number of
species began with Robert Trench and his students in the late 1970s. Their studies
eventual y revealed large differences between cultured Symbiodinium from different
hosts. This include difference
d
s in isozymes, morphology and the ability of
Symbiodinium to establish symbioses with different host species (Schoenberg &
Trench 1980 a, b, c). Symbiodinium from various hosts also differed in the numbers
of condensed DNA bodies (Blank & Trench 1985; Trench & Blank 1987; Blank &
Huss 1989), photophysiology (Chang et al. 1983) and in their fatty acids and sterol
38
composition (Blank & Trench 1985). Analysis of the small subunit
o
rib somal RNA
gene (18S) supported these differences and revealed three major grou
s)
pings (clade
designated A, B and C (Rowan & Powers, 1991a). Subsequent work revealed that
Symbiodinium phylogeny included at least eight highly divergent lineages or clades
(A through H) on the basis of ribosomal DNA from the nucleus (18S and 28S rDNA)
and chloroplast (cpDNA; LaJeunesse 2001; Pawlowski et al. 2001;
l
Santos et a .
2002; Baker 2003; Pochon et al. 2004; LaJeunesse 2005).
These major clades are found in most oceans although Clade C appears to be the
most common genetic variety in the Indo-Pacific while Clades A and B share
dominance with Clade C in the Caribbean. The majority of coral colonies appear
dominated by a single type of Symbiodinium (Baker 2003; LaJeunesse 2005).
Cnidarian hosts, however, sometimes contain several clades (Rowan and Knowlton
1995; Rowan et al 1997; Loh et al 1997; Lewis and Coffroth 2004; Little et al. 2004).
Within colonies, these divergent types of Symbiodinium may occupy different
microhabitats within a host (Rowan et al 1997) or different depths (Baker et al. 1997).
Investigation of Symbiodinium using higher resolution markers such as internal
transcribed spacer regions (ITS 2, LaJeunesse 2001; ITS 1, Van Oppen et al. 2001),
microsatellites and flanking sequences (Santos et al. 2004), and DNA fingerprinting
(Goulet and Coffroth 2003) have subsequently revealed even greater complexity
within the rDNA defined clades of the genus Symbiodinium. Using ITS 2,
LaJeunesse, van Oppen and coworkers have uncovered distinct genotypes that vary
with host genera. These patterns indicate that co-evolution of host and symbiont has
occurred given that the distribution of these sub-cladal lineages is not random among
coral hosts. For example, C15 is distinctive of Porites while C3 is characteristic of
Acropora and other host species across the vast areas of the Pacific Ocean
(LaJeunesse et al. 2004). More recent work shows that ITS lineages within the major
clades of Symbiodinium also vary with depth and are likely to represent functionally
distinct genotypes or species of Symbiodinium (Iglesias-Prieto et al. 2004;
LaJeunesse 2005). As was seen with the major clades of Symbiodinium (A H),
cnidarians appear capable of hosting several varieties of ITS genotypes, which may
vary according to environmental conditions and host ontogeny. These issues will be
discussed further below. Recent work is also indicating that care must be taken with
respect to intragenomic variation as regards the interpretation of ITS patterns. In this
case, single celled PCR amplifications have revealed that cells may contain ITS
copies that classify out as several different clades or ITS genotypes (Van Oppen et
al, in press; Gates personal communication). Dominant patterns, however, may still
hold although the reality and abundance of multicladal assemblages is now open to
some question.
The establishment of coral-Symbiodinium symbioses
Despite its importance, we know very little about how the symbiosis between
Symbiodinium and its hosts is established. Work by Virginia Weis and others is
using perspectives from analogous symbioses like Hydra-Chlorella to develop a
better understanding of the cellular events that lead up to the establishment of coral-
Symbiodinium associations in species such as Fungia. In this case, the observation
of specific phagocytotic processes that engulf algal cells (e.g. McNeil 1981) and the
inhibition of phagosome-lysosome fusion (e.g. Hohman et al 1982) give clues to the
complex set of processes that are likely involved in the recognition and incorporation
of potential symbionts into coral host cells. Experiments similar to those in Hydra
that focused on the cellular events are only in the various earliest stages. A host of
other studies are now using molecular tools to investigate how the diversity of
associates within corals changes as the association matures. In this regard, there
39
appears to be some very interesting patterns in which the initial uptake of
Symbiodinium appears relatively unspecific but which narrows with maturation of the
colony (Little et al. 2004; Gomez 2005). This narrowing phase involves the eventual
dominance of 1-2 genotypes, which may or may not involve the retention of some of
the original diversity at background levels within the tissues of the adult corals. It is
clear we need to understand more about these steps in the establishment of a
mature symbiosis. Questions such as to whether there is a functional significance to
having particular varieties of Symbiodinium in the early stages of a symbiosis need
answering.
The establishment of the association between Symbiodinium and coral hosts might
be analogous to a potential guest hoping to enter a house (Figure 1). To get in the
front door, the guest might need to possess a key, one which is commonly available
to the particular group (in this case, the genus Symbiodinium). This might be the
right lectins on their membrane surface that result in a particle or alga being
exocytosed. Once through the front door, however, the group of guests only have
access to the lobby of the house. To go any further, the guests need to possess a
second key. In actuality, this might be the ability to avoid lysosomal attack by having
the right surface molecules to avoid recognition as foreign particle. Through this
door, only a select few guests can enter the corridor. But as before, the guests
require a third key. This might be in
reality the ability to integrate
metabolically with the host, allowing
Cell division
Kitchen
the passage of substrates through the
synchrony
double membrane in which the alga
B
now lives. Through this door, a small
subset of the original guests will pass
Metabolic
Corridor
into, say, the kitchen. At this point,
integration
integration of host and symbiont via
A B
cell signalling processes prevent
Avoid
Symbiodinium from dividing out of
Lobby
lysosomal
step with the host cell might be the
fusion
critical next step. And so on. Under
A B D
this model, the narrowing of diversity
might be due to the relative
Front door
Exocytosis
proliferation of the appropriate type of
Symbiodinium (B in Figure 1) over
A B C D E F
others, given it posses all the "keys". Figure 1. Hypothetical sequence of locks
Those that gain access to only the and keys involved in the establishment of a
lobby on the other hand, may be left in
symbiosis between Symbiodinium and a
a "semi-symbiosis" within this space. coral host. Letters are akin to genetic
This may explain the recent varieties of Symbiodinium.
observation of so-cal ed "moonlighting" Symbiodinium in many hosts.
These cellular events are likely to be highly selective, as one would expect in any
cellular process in which one cell goes to live within another. Studies that have
followed the genotypes of Symbiodinium within corals over time (in and out of
bleaching events) have revealed enormous stability within ecological time scales
(Gomez 2005; Stat 2005). Experimental manipulations have revealed in a few
instances (e.g. Baker 2001) that the relative proportions of different genotypes of
Symbiodinium may change. However, the definitive proof that new combinations are
indeed novel remains to be established (Hoegh-Guldberg et al. 2002).
40
Flexibility of coral-Symbiodinium assemblages over short time scales?
The mass bleaching of corals is one of the most dramatic changes to take place on a
coral reef. In these events, the symbiotic association breaks down as conditions
place the association under stress. In the case of recent global episodes, mass
bleaching is caused by warmer than normal sea temperatures (Hoegh-Guldberg
1999). Buddemeier and Fautin (1993) proposed the interesting idea that bleaching
may represent a strategy by which hosts may exchange their symbionts for ones that
have higher thermal tolerances. As will be discussed in this workshop, the idea has
largely been shown not to hold true, especial y if the case is restricted to case of
"evolutionary switching" as opposed to "shuffling". In the former case, coral
bleaching has never been shown to result in a completely novel symbiotic
association, as would be necessary if an association were to "evolve" suitably new
thermal tolerances to deal with escalating sea temperatures due to climate change.
Shuffling, the case where changes occur in the relative proportions of different
Symbiodinium genotypes within multi-cladal hosts, does not lead to truly novel
symbioses and hence doesn't lead to required rapid changes in the thermal
tolerances needed to keep up with the current high pace of climate change.
The current debate requires resolution given its importance to how coral reefs may
fare under rapid climate change. From recent exchanges (e.g. Baker 2002 versus
Hoegh-Guldberg et al. 2002), it is clear that there is need to clarify terms that are
often used loosely yet the specific meaning of which is critical to any resolution of the
debate. It is also clear that time scales are important. In this regard, evidence of
evolutionary switching that occurs rarely over long time frames (at least 10,000's of
years if not 100,000s of years, Stat 2005) should not be used to support the idea that
corals will be able to change their thermal tolerances in the short intervals typified by
bleaching events and rapid climate change. That is, the observation that a coral has
a genotype of Symbiodinium (Clade E) which is normal y found in Foraminifera
(Rodriguez-Lanetty et al. 2000) is not proof that evolutionary switching occurs with
enough frequency to cause the required upward changes in thermal tolerances
required to allow a reef to remain robust under rapid climate change. One has only
to calculate the required change in the thermal tolerance for corals and their
Symbiodinium (between 0.21.0oC per decade, Donner et al 2005) to keep pace with
even mild rates of climate change to appreciate the challenge populations of corals
and their Symbiodinium face over this present century.
This workshop took up many of these issues and has provided a series of papers
that review the exciting new developments in the field. In doing this, the workshop
has indicated where we should be putting greater emphasis in future studies. Most
importantly, it has provided a consensus statement at the end of this section, which
summarises the current understanding of many key issues. The consensus that was
achieved in Mexico provides a very useful clarification and set of common terms by
which to discuss complex issues such as the flexibility of the symbiosis between
corals and Symbiodinium. This represents a great basis from which to start to
address the critically important issues of the adaptability of reef-building corals (or
not) to climate change.
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Microbiol., 48(3), 2001 pp. 368373
Pochon, X., LaJeunesse, T.C., Pawlowski, J. (2004) Marine Biology 146: 1727
Rodriguez-Lanetty, M., H. R. Cha., and J. I. Song (2000) Proc. 9th Int. Coral Reef
Symp. Vol. 1: 163-166.
Rowan R, Powers DA (1991) Science 251:13481351
Rowan, R., N. Knowlton, A. Baker, and J. Jara. (1997) Nature 388:265269.
Santos SR, Taylor DJ, Kinzie RA, Hidaka M, Sakai K, Coffroth MA (2002) Mol
Phylogenet Evol 23:97111
Santos, S.R, Shearer, T.L., Hannes, A.R. and Coffroth, M.A. (2004) Molecular
Ecology 13, 459-469
Schoenberg DA, Trench RK (1980a) Proc R Soc Lond B Biol Sci 207:405427
Schoenberg DA, Trench RK (1980b) Proc R Soc Lond B Biol Sci 207:429444
Schoenberg DA, Trench RK (1980c) Proc R Soc Lond B Biol Sci 207:445460
Stat. M. (2005) PhD, University of Sydney
Trench RK, Blank RJ (1987) J Phycol 23:469 481
van Oppen, M. J. H., F. P. Palstra, A. T. Piquet, and D. J. Miller. (2001). Proceedings
of the Royal Society, London B, 268, 1759-1767
42
Functional diversity of Symbiodinium: the
evidence and the history.
Roberto Iglesias-Prieto
Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún QR
77500, México.
Over the last 10 years we have witnessed a dramatic shift in the paradigms
addressed by scientists working on the biology of algal invertebrate symbioses. The
realization that not all "zooxanthellae" were created equal has had major implications
for our understanding of the biology, ecology and evolution of coral reefs. Contrasting
to the early realization that invertebrates harboring symbiotic phototrophic
dinoflagellates form a diverse assemblage, the assessment of the diversity within the
genus Symbiodinium has been a difficult task involving the establishment of axenic
mono-algal cultures, the taxonomic description of different Symbiodinium species
and the development of different genetic markers (Fig 1). As a result of progress in
these areas, we have supplanted the notion of a pandemic zooxanthellae, for our
current perception of the genus as a highly diverse and specific group. Although
progress in this field is the result of the work of many scientists, the pioneering work
of Bob Trench and his co-workers was instrumental for the development of our
current understanding.
Assessing phenotypic variability in Symbiodinium in cultures
In the absence of suitable genetic
markers, the assessment of
phenotypic variability within the
genus Symbiodinium was limited for
a number of years to the use of
axenic mono-algal cultures. The
successful establishment of these
cultures was crucial development.
Comparisons of different culture's
growth under identical conditions in
defined media permitted the
of the phenotypic
description
plasticity of symbionts isolated from
different sources. By the early part Fig 1. Major developments in the field.
of the 1980s the accumulation of
behavioral, physiological, biochemical and structural evidence lead to the suggestion
that symbiotic dinoflagellates isolated from different animal sources constitute dis-
continuous genetic entities (Schoenberg & Trench 1980). Based on detailed
ultrastructural analyses of cultured Symbiodinium, the first taxonomic descriptions of
species within the genus were published. Today only six species of Symbiodinium
have been formally described. Considering the importance of algal photosynthesis to
the well being of the intact associations, it is not surprising that significant efforts
were devoted to describe the variability of the photosynthetic responses of cultured
Symbiodinium isolated from different animal hosts (Iglesias-Prieto & Trench 1997).
Collectively, these studies show that different algal symbionts have significant
differences in their photoacclimatory abilities and that these differences probably
represent adaptation to different light climates. Although the physiological diversity of
43
the genus was firmly established by the end of the 1980s, the lack of suitable genetic
markers hindered the investigation of the ecological and evolutionary implications of
such diversity in the reef.
Linking genetic and physiological diversity in the field
This field of research experienced a dramatic change after the introduction of reliable
genetic markers. Restriction analyses of the ssuRNA gene permitted the
identification of several clades of uncertain taxonomic value (Rowan & Powers 1991)
which allowed us to initiate a global survey of the genetic diversity of the genus. One
of the most important limitations of the use of genetic analyses based on the ssuRNA
gene is its lack of resolution. This technique fails, for example, to distinguish between
two Symbiodinium types that have been identified by traditional techniques as
belonging to different species (Rowan & Powers 1991). Early attempts to correlate
clade designation and physiological performance were based the distribution of algal
types along environmental gradients. Unfortunately, the assignment of physiological
properties to each clade was premature and not based on empirical physiological
data. This approach resulted in unsubstantiated generalizations regarding the
functionality of the different clades. Different cultured Symbiodinium in clade "A"
exhibit a wide range of photoacclimatory responses comparable to those observed
among algae in different clades. The use of high resolution genetic markers such as
the ITS2 region has successfully identify all the described species in the genus, and
has the potential to describe genetic variation of the genus Symbiodinium in the field
at a scale close to the species level (LaJeunesse 2001). The combined use of high
resolution genetic markers and non-invasive techniques such as Pulse Amplitude
Modulated (PAM) flourometry al ow us to measure directly in the filed the
physiological performance of different algal genotypes (Iglesias-Prieto et al. 2004).
We showed that the vertical distribution of the two dominant scleractinians in the
Eastern Pacific along an irradiance gradient could be explained exclusively on the
basis of the photosynthetic performance of their respective specific symbionts. This
type of information suggests that for ecological and evolutionary purposes, the unit of
selection is the symbiotic phenotype (holosymbiont). It has been suggested that host
can respond to variations in the environment by changing the composition of their
symbionts (Buddemeier & Fautin 1993). Testing this possibility requires the
assessment of the physiological and ecological performance of the holosymbiont.
Conclusions and future directions
Currently, one of the most pressing questions that we face is how reef coral will
respond to the environmental chal enges imposed by climate change, in particular
what role will be played by Symbiodinium in determining the limits of acclimation and
adaptation of their hosts. In principle there is enough functional variability within the
genus to accommodate potential hosts to very different environments. In this context,
it is imperative that we determine how specificity controls host range in nature.
Addressing this question requires the simultaneous utilization of genetic and
physiological techniques.
References
Buddemeier RW, Fautin DG (1993) Coral bleaching as an adaptive mechanism.
BioScience 43:320-326
Iglesias-Prieto R, Beltrán VH, LaJuenesse TC, Reyes-Bonilla H, Thomé PE (2004)
The presence of different algal symbionts explains the vertical distribution
patterns of two dominant hermatypic corals. Proceedings of the Royal Society of
London B. 271:1757-1763
44
Iglesias-Prieto R, Trench RK (1997) Photoadaptation, photoacclimation and niche
diversification in invertebrate-dinoflagellate symbioses. In: Lessios HA, Macintyre
IA (eds) 8th International Coral Reefs Symposium, Panamá, p 1319-1324
LaJeunesse TC (2001) Investigating the biodiversity, ecology and phylogeny of
endosymbiotic dinoflagellates of the genus Symbiodinium using the internal
transcribed spacer region: in search of a "species" level marker. Journal of
Phycology 37:866-880
Rowan R, Powers DA (1991) A molecular genetic classification of zooxanthellae and
evolution of animal-algal symbioses. Science 251:1348-1351
Schoenberg DA, Trench RK (1980) Genetic variation in Symbiodinium
(=Gymnodinium) microadriaticum Freudenthal, and specificity in its symbiosis
with marine invertebrates. I. Isoenzyme and soluble protein patterns of axenic
cultures of Symbiodinium microadriaticum. Proceedings of the Royal Society of
London B 207:405-427
45
Diversity and specificity of Symbiodinium
William K. Fitt
This talk was designed to ask questions (that we don't know all the answers to), to
give partial answers that consist of real data, and then speculate wildly about the rest
of the answer.
What are the major patterns of diversity of algal symbionts?
There is an increased diversity of Symbiodinium in shal ow water vs. deep water, in
marine habitats closer to the equator, in hosts closer to shore, and in the Caribbean
vs. Indo-Pacific (e.g. LaJeunesse 2002, et al. 2003. 2004a,b). Symbionts that are
relatively rare at some reefs may be extremely common on other reefs.
We hypothesize that high-diversity Symbiodinium indicates reefs that are under more
stress than low-diversity reefs.
What are the steps in establishing specificity in the endosymbioses?
There are at least three studies documenting uptake of Symbiodinium from the water
column that suggests that there is little specificity for juveniles of broadcast spawners
they take up what is available and sort it out later (Coffroth et al. 2001, Thornhill et
al. 2006, Cabrera et al. 2006).
We hypothesize that there is no specificity in uptake (phagocytosis) of Symbiodinium,
and that all symbiotic cnidarians can take up all types of Symbiodinium throughout
their lives!
What is the evidence for, and significance of, free-living Symbiodinium?
Free-living Symbiodinium exist, and infect aposymbiotic hosts in nature and in the
laboratory. We don't know where they are coming from, nor what they are doing.
We hypothesize that most Symbiodinium are free-living, and that only a small subset
inhabits invertebrates.
What are the physiological correlates of having different types of
Symbiodinium?
Since Symbiodinium ranges in size from about 6 to 12 um, the volume has a 4x
range. Whether one looks at scyphistomae or giant clams, one can see a whole
range of potential differences in photosynthesis and respiration rates, calcification
rate, etc, as well as a way to competitively displace one symbiont by another.
We hypothesize that the host digestive cell acts as a "culture-tube" and plays a
passive role, but specificity resides in the host-cell properties.
46
What is the evidence that hosts are flexible in their associations with
Symbiodinium?
We hypothesize that ALL host species harbor several (dozens to hundreds?) of types
of Symbiodinium at the same time. Most hosts establish a relationship whereby one
type of Symbiodinium comprises over 95% of the cells.
What is the evidence that "stressed" hosts can "pick-up" or "change"
complements of Symbiodinium?
We hypothesize that during abnormally high temperature type D1a can become "life-
jackets" for the host. Under normal conditions D1a is not optimal and the host must
acquire an optimal zooxanthellae type to survive.
References
Cabrera MCG (2005) Some apects of the physiology and ecology of the Acropora
longicyathus multi-cladal symbiosis. U. Queensland, PhD thesis
Coffroth MA,, Santos R, Goulet TL (2001) Early ontogenetic expression of specificity
in cnidarian-algal symbiosis. Mar Ecol Prog Ser 222:85-96
LaJeunesse TC (2002) Diversity and community structure of symbiotic dinoflagelates
from Caribbean coral reefs. Marine Biology 141:387-400
LaJeunesse TC, Loh W, Van Woesik R, Hoegh-Guldberg O, Schmidt GW, Fitt WK
(2003) Low symbiont diversity in southern Great Barrier Reef corals relative to
those of the Caribbean. Limnol Oceanogr 48:2046-2054
LaJeunesse TC, Bhagooli R, Hidaka M, deVantier L, Done T, Schmidt GW, Fitt WK,
Hoegh-Guldberg O (2004) Closely related Symbiodinium spp. Differ in relative
dominance in coral reef host communities across environmental, latitudinal and
biogeographic gradients. Mar Ecol Prog Ser 284:147-161
LaJeuness TC, Thornhill DJ, Cox E, Stanton F, Fitt WK and Schmidt GW (2004
e
)
High diversity and host specificity observed among symbiotic dinoflagel ates in
reef coral communities from Hawaii. Coral Reefs 23:596-603
Thornhill DJ, LaJeunesse TC, Schmidt GW, Fitt WK (2006) Multi-year seasonal
geotypic surveys of coral-dinoflagellate symbioses reveal prevalent stability of
post-bleaching reversion. Marine Biology In press.
Thornhill D, Daniel M. LaJeunesse T, Schmidt G, Fitt W (2006) Patterns of infection
by free-living Symbiodinium populations in aposymbiotic scyphistomae of the
jellyfish Cassiopea xamachana. JEMBE In press.
47
Symbiodinium systematics: molecular markers
and the techniques appropriate for eco-
evolutionary investigations.
Todd C. LaJeunesse
Department of Biology, Florida International University, Oe 167,
n
U iversity Park Campus, Miami Fl 33199, USA
The analysis and characterization of Symbiodinium diversity is at a crossroads. A
total of eight divergent clades are recognized
taxonomy is largely in
and their
agreement. However, the classification of numerous genetic types/strains/sub-
clades within each of these main lineages is in disarray. Much of the confusion
resides in the use of different genes and/or certain techniques that do not allow for
cross-comparison between findings from different research laboratories. Progress in
the physiological, ecological and biogeographic research on the coral-algal
symbioses requires a consensus on sub-cladal taxonomy (and the methods used to
identify it). The taxonomical significance of within-clade diversity must first be
addressed. Viewed from one extreme, these "types" are merely sequence variants
within a single metapopulation (clade=species). At the other extreme, each variant
appears to represent an ecologically distinctive "species" on an independent
evolutionary trajectory. Interpretation of the true significance of this genetic diversity
appears to hinge on the techniques employed to measure it.
As for most microorganisms, molecular techniques are required in examining,
assessing, and identifying the diversity of coral endosymbionts. Commonly referred
to as zooxanthellae, dinoflagellates in the genus Symbiodinium were once
collectively assigned to a single panmictic species. Results from morphological,
biochemical, behavioral, and physiological studies by Trench and colleagues in the
1970's and 1980's began to dispel this established belief. However, it was the
publication of the first ribosomal DNA sequences by Rowan and Powers (1991) that
final y convinced many of, or made them aware of, the potential diversity that existed
within this symbiont group. Since then, numerous studies involving comparisons of
DNA sequence diversity have been published. Desire for increased genetic
resolution resulted in the analysis of more rapidly evolving genes. Starting with the
conservative nuclear ribosomal SSU 18S, analysis shifted to the LSU and then the
internal transcribed spacer regions (ITS). The recent application of comparing
microsatellite loci (and DNA fingerprinting) has initiated population level genetic
investigations of these microorganisms.
In efforts to increase the number of samples sequenced, speed the process, and
reduce costs, single strand conformational polymorphism (SSCP) and denaturing
gradient gel electrophoresis (DGGE) were employed. Both allow the resolution of
single base pair differences in the DNA region of interest.. Each method produces
repeatable fingerprint patterns that are consistently observed from sample to sample.
Both techniques, however have drawbacks. For SSCP, the quality of the fingerprint
image is relatively poor making the characterization and subsequent identification
among numerous unique fingerprints difficult. The direct excision of bands from
SSCP gels, followed by their re-amplification and direct sequencing, is not possible.
Instead, left over PCR products from the initial amplification must be shotgun cloned
and sequenced. Direct sequencing, especially of the ITS region, is often
problematical. This is most likely due to high levels of intragenomic variants that are
48
present throughout the ribosomal array of many Symbiodinium. The presence of a
mixed template in a sequencing reaction leads to nonsensical sequences. The
solution is to clone from these PCR products and then sequence. The major problem
with shotgun cloning is that a cloned gene copy may or may not be a representative
marker for the organism. Typical y, one or two variants are the most common in a
genome and are presu ably responsible for
m
diagnostic SSCP /DGGE fingerprints. If
they cannot be identified, numerous cloned copies must be sequenced in order to
properly characterize the extent of this intragenomic diversity. This method requires
too much time and money to be practical. In contrast, DGGE produces highly
repeatable and clear fingerprint profiles of which the most diagnostic bands are easily
excised, re-amplified, and directly sequenced. The main drawbacks of this technique
are; 1) the required investment of special electrophoresis equipment and 2) time
required to run each gel.
The application of different techniques targeting different DNA regions by so many
different research labs has led to confusion about the perceived genetic diversity of
Symbiodinium and its ecological significance. Work accomplished by one lab is not
readily assessable by others wishing to incorporate it into their own publications.
Uncertainty with evaluating and integrating previous work has been especially
problematical when describing Symbiodinium diversity within each clade. This lack
of consistency has stalled progress toward a fuller understanding of symbiont
biogeography, host-symbiont specificity, and differences in physiology. It would
seem that effort is needed in establishing a consistent technique that reliably
distinguishes between ecologically distinctive forms.
Standardizing a molecular taxonomy for Symb
yond the "clade" level
iodinium be
would dramatically improve the scientific progress of our field. Fundamental to any
endeavor in ecological or comparative physiological research is the accurate
identification of the organisms under study. A candidate approach using denaturing
gradient gel electrophoresis (DGGE) of the ITS rDNA does provide a consistent
identification of genetically similar yet ecologically and biogeographically distinct
Symbiodinium. Work is still required to "ground-truth" whether or not these 'types'
signify genetically isolated species. Because the ribosomal array contains a high
degree of intragenomic variability, careful analysis and interpretation of this variability
is necessary. The development of additional nuclear, mitochondrial, and chloroplast
markers will offer direct and indirect testing of the ecological and evolutionary
significance of these sub-cladal "types."
49
Coral bleaching as an exaptation that can
promote rapid and beneficial change in algal
symbiont communities
Andrew C. Baker1, 2
1. Wildlife Conservation Society, Marine Conservation Program, 2300 Southern
Boulevard, Bronx, New York 10460, USA. 2. Center for Environmental Research and
Conservation, Columbia University, MC 5557, 1200 Amsterdam Avenue, New York,
New York 10027, USA.
Semantic confusion
The Adaptive Bleaching Hypothesis (ABH, Buddemeier and Fautin 1993), now over a
dozen years old, has a name that is both powerful and dangerous. Powerful,
because most readers intuitively understand the general concept from the name
alone (resulting in the ready incorporation of the term into the research lexicon), but
dangerous because this intuitive understanding is often accompanied by secondary
meaning in the words used.
Much debate published or otherwise over the ABH has centered over the use of
the word "adaptive" (Buddemeier et al. 2004), which most biologists interpret as
implying a process acting over evolutionary time that favors certain heritable traits
over others, leading to a directional change in the relative abundance of genes in a
population over time. This perspective has led to criticism of the ABH on the grounds
that bleaching (and subsequent recovery, occurring over timescales of weeks to
months) provides insufficient time for evolution to act (e.g., Hoegh-Guldberg et al.
2002). However, these arguments have generally failed to address the real questions
of interest, which are less concerned with whether bleaching is "adaptive" (or not),
and more concerned with whether it promotes change in symbiont communities, and
what the effects of these changes might be.
Critics of the ABH maintain that bleaching evolved to remove damaged (and
therefore harmful) symbionts as part of the immune response. However, traits can be
adaptive for multiple reasons, not all of which need to contribute equally to the
selection pressure on a trait over the course of its evolution. Bleaching as an immune
response can still have added adaptive value if it has the side effect of promoting
rapid symbiont community change that benefits the coral host. Perhaps when viewed
in this way (as an exaptation), the ideas encapsulated by the ABH might be less
contentious.
Sublethal bleaching and the ABH
Most of the interest surrounding the ABH has little to do with evolution, and
everything to do with ecology. Does bleaching result in symbiont change within an
individual colony frequently enough to be ecologically meaningful? Or is bleaching
principally an agent of natural selection, causing differential mortality within a
population or community of corals determined to some degree by the symbiont
communities they contain? These two questions may be fundamentally linked if
periodic sublethal bleaching expels certain symbiont types over others, and
subsequent recovery involves the stochastic acquisition of novel symbionts from the
environment. If this is the case, mechanisms of symbiont regulation, including low-
50
level seasonal bleaching (Stimson 1997, Brown et al. 1999, Fagoonee et al. 1999,
Fitt et al. 2000), may introduce critical background variation into symbiont
communities that ultimately provide the backdrop against which shifts in symbiont
communities in response to severe bleaching and mortality events (Baker et al. 2004)
are able to occur (Fig. 1).
Acquisition of symbionts from environmental sources
Figure 1 emphasizes the importance of cryptic or "minor" symbionts in determining
the responses of corals to bleaching, and assumes that most (perhaps all) coral
species are able to acquire symbionts from the environment in the adult phase, as is
the case in certain non-scleractinian anthozoans. Probability theory supports this
assumption: if symbiont diversity is solely determined at the larval stage (as a subset
of maternal and/or environmental symbionts), and if adult colonies only possess
mechanisms for losi g
n this diversity (through bleaching), then symbiont diversity
would inevitably be purged from adult colonies over time. Consequently, large coral
colonies, as well as coral species that maternally transfer symbionts from generation
to generation, would become monotypic in their symbionts over time. Molecular
survey data indicate this is not the case, suggesting that adult colonies can and do
acquire symbionts from environmental sources.
A
B
C
D
E
Time
Fig. 1. Conceptual model illustrating how low-level bleaching and recovery of coral colonies
maintains critical background variation in algal symbiont communities. In this model, five
healthy conspecific coral colonies (A-E, larger circles) start with identical symbiont types
(smaller circles, with different symbionts represented in different colours). These colonies
experience different levels of sublethal bleaching over time (varying shades of grey). After
eight time steps, al colonies are dominated by the same symbiont type (yellow) but contain
different minor symbiont types. A severe bleaching event (red vertical line) leads to dramatic
loss of symbionts in all colonies, followed by recovery (increasing numbers of symbionts) or
mortality (X). In this model, orange symbionts are resistant to bleaching, leading to a shift in
colony B to favor these symbionts following severe bleaching. This change in dominance is a
result of variation in minor symbionts existing prior to the event.
51
Future questions and research directions
Current debate centers on the timescales over which symbiont change can occur, the
extent to which symbiont change involves endogenous vs. exogenous sources, and
the degree of bleaching and/or mortality of coral hosts that might be necessary for
changes to be detectable. Fortunately, all of these questions are tractable from a
research perspective. However, there has been surprisingly little research in these
areas over the last dozen years, with active research focusing instead on the
diversity and distribution of Symbiodinium in different host species. In order to make
real progress in this field, these questions should be moved to the top of the agenda.
References
Baker AC, Starger CJ, McClanahan TR, Glynn PW (2004) Corals' adaptive response
to climate change. Nature 430: 741
Brown BE, Dunne RP, Ambarsari I, Le Tissier MDA, Satapoomin U (1999) Seasonal
fluctuations in environmental factors and variations in symbiotic algae and
chlorophyll pigments in four Indo- Pacific coral species. Marine Ecology Progress
Series 191: 53-69
Buddemeier RW, Baker AC, Fautin DG, Jacobs JR (2004) The adaptive hypothesis
of bleaching. In: Rosenberg E, Loya Y (eds) Coral Health and Disease. Springer-
Verlag, Berlin, New York, pp 427-444
Buddemeier RW, Fautin DG (1993) Coral bleaching as an adaptive mechanism - a
testable hypothesis. Bioscience 43: 320-326
Fagoonee I, Wilson HB, Hassell MP, Turner JR (1999) The dynamics of
zooxanthellae populations: A long-term study in the field. Science 283: 843-845
Fitt WK, McFarland FK, Warner ME, Chilcoat GC (2000) Seasonal patterns of tissue
biomass and densities of symbiotic dinoflagellates in reef corals and relation to
coral bleaching. Limnol Oceanogr. 45: 677-685
Hoegh-Guldberg O, Jones RJ, Ward S, Loh WK (2002) Is coral bleaching really
adaptive? Nature 415: 601-602
Stimson J (1997) The annual cycle of density of zooxanthellae in the tissues of field
and laboratory-held Pocillopora damicornis (Linnaeus). Journal of Experimental
Marine Biology and Ecology 214: 35-48
52
Modeling the Adaptive Bleaching Hypothesis
John R. Ware
SeaServices, Inc., 19572 Club House Road, Montgomery Village, MD, 20886, USA.
The adaptive bleaching hypothesis asserts that coral bleaching (the loss of the
symbiotic algae that give corals their colour) may be an adaptive mechanism. By
providing an opportunity for recombining hosts with alternative algal types that might
be better adapted to altered circumstances, bleaching may result in improved
resistance to
stress. On
increasing
ce thought to be a single taxon, recent studies
have demonstrated that the symbiotic dinoflagellates which inhabit the coral body
cells have significant taxonomic diversity.
The ABH was first postulated by Buddemeier and Fautin in 1993. Shortly thereafter,
Ware, Fautin, and Buddemeier (1996) published a paper describing a simulation of
the ABH which provided some interesting results. More importantly, the 1996 paper
made explicit the assumptions of the ABH, some of which were implicit in the original
paper.
Since the publication of these two papers, the ABH has been the center of one of the
most intense controversies in coral reef science since Darwin's publication of his
coral reef hypothesis.
Clearly, if true, the ABH would imply that corals had some sort of `hedge' against
global warming. By shifting their symbionts to ones with higher temperature
resistance, corals might be less sensitive to long-term temperature increases than
previously thought. I summarized this thought with the following limerick:
An elderly coral was teaching
The younger corals 'bout bleaching:
A word to the wise:
As temperatures rise,
Change the zooxs that you all are hosting!
In addition to presenting simulation results related to the ABH from the original paper
that appeared in Ecological Modeling and from a presentation at the SICB in Boston
some years later, I discussed the process of developing mathematical models of
biological concepts and the advantages which accrue from such development.
From my viewpoint, the primary advantages of developing mathematical and
computer based models are that: (1) they focus and clarify what might have been
fairly vague thoughts during the formulation of a concept; (2) assumptions that may
have been implicit in the formulation phase, must now be made explicit; and (3)
despite the fact that nothing comes out of a computer model that was not put in,
some results may not have been anticipated.
One of the interesting aspects of the simulation approach is that the climate model
used to develop the results presented is not the more common, physics based
formulation. General Circulation Models (GCMs) attempt to model the physics of the
climate by dividing the atmosphere (and sometimes a portion of the oceans) into
blocks and follow the flow of energy into and out of each block. The resulting models
are extremely complex and, depending on the granularity with which the atmosphere
53
is modeled, require massive computer resources; thereby precluding multiple
simulations. In contrast, the basic temperature model that I have developed is based
on both long-term (centuries) and short-term (years) temperature observations. The
resulting model is extremely simple (by comparison) and permits hundreds to
thousands of
h
repeats wit differing initial conditions.
Several of the common fallacies and observations associated with computer-base
d
models are emphasized. These include:
- Complexity = Accuracy. The fact that GCMs are based on sound physical
principles does not equate with precise emulation of the Earth's temperature
evolution. For example, GCMs would not demonstrate the Little Ice Age, whereas
the simpler model does occasionally show LIA type behavior. In addition, GCMs
typically contain a large number of parameters whose true values are not accurately
known.
- Precision = Accuracy. Computer models may provide results to 6 decimal places.
This in no way implies that the results are accurate.
- Validation and verification of computer models are things that engineers do.
Science does not require V&V (that is, the more intelligent the programmer, the less
likely they are to check (verify) their computer programs).
Using temperature as a surrogate for all aspects of global climate change and
bleaching as a surrogate for all responses, I compare the response of corals under
the ABH and under a gradual acclimation model.
The simulation results show that, if the ABH is valid, reef corals may acclimate
rapidly and resist climate change for a substantial period, perhaps another 25 to 50
years. However, ultimately the rapid changes expected as a result of anthropogenic
perturbation of global climate, augmented by the hundreds of point sources of
pollution on or near reefs, may cause many coral reefs to change their community
structure precipitously.
The figure below, Figure 3G, al ows comparison of the probability of severe bleaching
events for corals which acclimate with a time lag of 50 years versus "ABH corals"
under a global warming scenario of 1.2 Co per century. As can be seen, the ABH
corals appear to be doing fairly well until approximately 2030 to 2050 at which time
probabilities of severe bleaching events increase precipitously leading to the ultimate
demise of the coral reef
ecosystem. If, on the other
hand, corals and/or their algal
symbionts can acclimate with
even a 50 year time lag, which
I consider highly unlikely, coral
reef survival is possible.
Ultimately, if sea surface
temperatures continue to
increase, acclimation potential
will be exceeded and the reef
system will collapse.
(Probabilities are estimated
from 1000 runs of a simplified
climate model.)
54
In summarizing, it is extremely important to note that current global climate change
should NOT be interpreted as being of benefit to coral reefs. The ABH suggests that
bleaching may allow corals to acquire symbionts that are more resistant to increased
temperatures than otherwise. This does not mean the holobiont (combination of
coral and symbiont) is better off than before the perturbation that caused the
bleaching. In fact, the new holobiont must be at some disadvantage in the absence
of perturbation or a shift to a new symbiont would have occurred without the
perturbation.
55
Dynamics of cnidarian-Symbiodinium
symbioses: Thoughts on flexibility, stability and
ontogeny of the symbiosis
Mary Alice Coffroth, A. R. Hannes, J. Holmberg, N. L. Kirk, C. L. Lewis, D.
M. Poland
Department of Biological Sciences, University at Buffalo, Buffalo NY 14260 USA
The endosymbiosis of the dinoflagellate Symbiodinium spp. with cnidarians is one of the
most striking and ecologically important relationships in the marine environment, having
profound effects on the hosts' physiology and ecology. Although the symbiosis had been
regarded as an almost invariant and unchanging feature, it is now recognized that
symbionts within a single host are both diverse and dynamic, changing ontogenetically
and in response to environmental conditions. Some Caribbean scleractinians harbor
multiple Symbiodinium taxa either simultaneously within a single host colony or across
reef habitats, while in other host taxa (i.e., octocoral) the symbiont complement is less
diverse and often does not vary with changes in environmental conditions. Thus,
cnidarian-Symbiodinium symbioses present a continuum in both Symbiodinium diversity
and in selectivity in the pairing of host and symbiont.
Given this potential for variability, it is important to determine how Symbiodinium diversity
in adult cnidarians is maintained and the factors that control the diversity and distribution
of the different taxa of Symbiodinium among their cnidarian hosts. To address the
effects of global warming on reefs and to understand phenomena such as bleaching, it is
necessary to determine if the established Symbiodinium complement is fixed or able to
undergo further change. Variation in small subunit ribosomal rDNA (ssrDNA), large
subunit ribosomal DNA of the chloroplast (cp-23S-DNA) and a series of microsatellite
loci were used to determine patterns of Symbiodinium diversity over spatial and temporal
scales and the flexibility (and stability) of the symbiosis under normal and stressful
environmental conditions.
Variation/diversity on a spatial scale
Symbiodinium diversity within the host species Porites divaricata, Gorgonia ventalina,
Briareum asbestinum and Pseudopterogorgia elisabethae was examined in the Florida
Keys and the Bahamas. Symbiodinium clades within the scleractinian P. divaricata
varied across the Florida Keys, with Symbiodinium clade A detected more frequently in
the middle keys than in the upper or lower keys. Within B. asbestinium, Symbiodinium
cp 23S rDNA type also varied with location with type B184 more common in the lower
keys. Symbiodinium diversity was also detected at the population level. For example,
within G. ventalina, variation in microsatellite allele frequencies was detected across the
Florida Keys. High levels of genetic structure was also found among symbiont
populations isolated from P. elisabethae host populations in the Bahamas (Santos et al
2003). On a single reef the majority of host colonies harbored the same symbiont
microsatellite genotype but that genotype differed between reefs. It is not known if this
distribution of zooxanthellae reflects local availability of the symbionts and if that
variability is generated by restricted dispersal or microhabitat differences and niche
partitioning among the symbionts.
Variation/ flexibility on a temporal scale
Symbiodinium diversity was also examined on a temporal scale by following the
zooxanthella complement in octocorals over time. Symbiont identity within individual
56
colonies of Plexaura kuna was followed for up to 10 years over multiple reefs using
multiloci DNA fingerprinting and ssrDNA. No change in symbiont complement was
observed (Goulet and Coffroth 1997, 2003ab). Symbiodinium abundance, chlorophyll
content and genotype within B. asbestinium colonies were monitored over a year. Cell
densities and chlorophyll a content varied, but no significant seasonal variation was
observed in the Symbiodinium cp-23S-rDNA genotype, suggesting that symbiont pairing
in this host species is stable.
Flexibility in algal taxon in early ontogeny
Flexibility in the symbiosis occurs across ontogeny in some host taxa. In primary polyps
of many octocorals, initial acquisition of symbionts is non-selective (Coffroth et al 2001).
Primary polyps placed over a range of habitats acquire multiple Symbiodinium clades
within the first 3 months and then over time these symbiotic taxa are winnowed to the
single clade found within the adult host. Although the mechanism that leads to the
establishment of the establishment of a single clade is not known, field and laboratory
experiments have demonstrated that the final adult complement is not dependent on
which taxon initially colonizes the host or on early survivorship of the polyps.
Flexibility/stability in response to stress
Among octocorals, the Symbiodinium complement within adult hosts does not appear to
vary under normal environmental conditions (time and space). However, what happens
when the coral is more severely stressed such as in a disease or bleaching event? The
symbiont complement within healthy G. ventalina and those infected with the fungal
pathogen, Aspergillus sydowii was monitored in the field and in the lab where the
disease was induced. In all cases there was no change in Symbiodinium cp-23S rDNA.
When colonies of B. asbestinium were induced to bleach experimentally, low levels of
zooxanthellae were detected and these often differed from the original dominant
symbiont type. This suggests that cryptic populations may be important in dealing with
stress. Bleached colonies of B. asbestinium were also exposed to cultures of an
isoclonal line of Symbiodinium that had a rare cp23S-rDNA allele. Subsequent analysis
of cp23S-rDNA of Symbiodinium from these B. asbestinium colonies recovered the rare
allele, confirming for the first time that adult corals retain the ability to acquire new
symbionts from the environment (Lewis and Coffroth 2004).
Conclusions and thoughts
These data suggest that within octocorals the typical adult zooxanthella population is
established early in ontogeny and does not change under moderate environmental
fluctuations (seasonally or disease). When the coral is more severely stressed such as
in a bleaching event, there appears to be a sequential loss of zooxanthellae. If the coral
survives, the repopulating algae can be from a residual population and/or from
exogenous sources. Surveys of zooxanthellae diversity across the Florida Keys and
Bahamas established Symbiodinium variation within a host species at the population
level. These data raise other questions such as what is the mechanism that leads to the
establishment of one Symbiodinium taxon in a host? Can a new symbiont colonize a
host without bleaching? Do patterns of host-alga selectivity reflect species/taxon specific
interactions between host and alga, or is Symbiodinium community dynamics a function
of the suitability of the host habi t to the al
ta
gal symbiont, or do the dynamics of the
zooxanthellae reflect stochastic processes of dispersal, colonization and population
expansion within hosts? What happens to these patterns if the established symbiosis is
perturbed, and final y, how do these processes vary among host taxa? Knowledge of
how Symbiodinium diversity within scleractinians and octocorals is established (via initial
infection, replacement and/or competition over time) and how flexible these symbioses
57
are in response to environmental perturbations is essential to understanding host and
symbiont distribution patterns and will aid in identifying the processes that are critical in
maintaining a viable symbiosis.
Coffroth, MA, TL Goulet, Santos, SR 2001 Early ontogenic expression of selectivity in
a cnidarian-algal symbiosis. Mar. Ecol. Prog.Ser. 222:85-96
Goulet, TL, Coffroth, MA 2003a Long term temporal and spatial stability of an
octocoral-algal symbiosis. Mar. Ecol. Prog. Ser. 250:117-124
Goulet, TL, Coffroth, MA 2003b Genetic composition of zooxanthellae between and
within colonies of the octocoral Plexaura kuna based on small subunit rDNA and
multilocus DNA fingerprinting Mar. Biol.142:233-239
Lewis, CL and Coffroth, MA 2004 The acquisition of exogenous algal symbionts by
an octocoral after bleaching. Science 304: 1490-92
Santos, SR, Gutierrez-Rodriguez, Lasker, HR, Coffroth, MA 2003 Patterns of
Symbiodinium associations in the Caribbean gorgonian Pseudopterogorgia
elisabethae: high levels of genetic variability and population structure in symbiotic
dinoflagellates of the Bahamas Mar Biol 143:111-120
58
Can the highly variable ITS2-region uncover
ecologically relevant patterns in the distribution
and persistence of Symbiodinium sp. in
Pocilloporid corals?
Eugenia Sampayo, Ove Hoegh-Guldberg and Sophie Do e
v
Centre for Marine Studies, University of Queensland, St Lucia, Qld 4072, Australia.
Coral reefs and stress
The persistence of coral reefs is determined by the finely regulated symbiosis
between the coral host and symbiotic algae (Symbiodinium sp), where each
component determines the success of the complex. Environmental disturbances,
such as global climate change, can disturb the finely regulated balance between
coral and algae, whereupon the symbionts are expelled from the host tissue
(becoming white or bleached) and an entire colony may die (Lesser, 1997; Fitt and
Warner, 1995). In addition to large scale bleaching, other localised sources of
damage to reefs such as pollution, nutrient run-off and over-fishing, have resulted
major damage to coral reefs around the world. Many believe that coral reefs now
face catastrophic decline as mass bleaching events and other stresses increase in
severity and frequency over the next 30 years (Hoegh-Guldberg, 1999).
The response of corals to bleaching has been found to vary among coral genera and
geographic location, indicating a possible role for both the host and symbiont in
determining the stress-response (Marshall and Baird, 2000; Brown, 1997). Symbionts
have been shown to have variable physiologies, and "same host-different symbiont"
combinations may therefore have alternate tolerance limits in accordance with
environmental parameters (Baker, 2003, Banaszak et al, 2000; Iglesias-Prieto and
Trench, 1997a,b; Buddemeier and Fautin, 1993). More importantly, bleaching
tolerant hosts are found in areas where the majority of the population is highly
affected (Edmunds, 1994). Unfortunately, very little information is available as to how
or why these individuals resist or cope with environmental stress better than those
that die.
Genetic variability
The use of molecular techniques has uncovered an enormous diversity of symbionts
(LaJeunesse 2001, 2002; Baker et al, 1997; Rowan and Powers, 1991) Here, the use
of the highly variable ITS2 DNA region to detect variability in host-symbiont
combinations on a local scale was tested to assess whether the huge variability has
a physiological function that can be related to the ecology of the symbionts within
their specific host species. Given the crucial role that symbiont availability and host-
specificity plays in determining what combinations of host and symbiont will be
successful, three major goals were to (a) determine if multiple host-symbiont
combinations are possible within a single host and whether these are determined by
local environmental gradients; and (b) whether these combinations can be adopted
by all individuals at any time, and finally (c) if these associations are flexible over time
and under altered conditions.
59
Flexibility
Three ubiquitous species of corals, viz. Stylophora pistillata, Pocillopora damicornis
and Seriatopora hystrix, were subjected to a broad sampling regime at multiple
depths on two locations around Heron Island (Great Barrier Reef, GBR). Pocil oporid
corals in this geographic location are generally reported to host Clade C symbionts,
and PCR and DGGE of the ITS2-region were selected to study the intra-cladal
variability of symbionts. Individual host colonies of both S. pistillata and P. damicornis
formed associations with multiple symbiont types, and within these species ITS2-
types exhibit a marked relation with depth. Each host species has its own community
of symbionts, in which each symbiont occupies a specific niche.
Additional y, to determine whether established host-symbiont associations were
flexible, a large-scale transplant experiment was established to monitor symbiont
populations over a period of two years. Preliminary results from the transplant
experiment also indicate that established host-symbiont combinations are not
necessarily fixed and may vary over time depending on the stress placed upon them.
This study forms part of my PhD research, and the transplants are still in the field for
continued collections.
Conclusions and future directions
Most studies to date have focused on biogeography and phylogeny of the genus
Symbiodinium using the ribosomal array (Rowan and Baker, Rodriguez-Lanetty and
Hoegh-Guldberg, 2003, Baker et al, 1997; Loh et al, 2001;
Lajeunesse et al, 2002,
2001). From these, some ecological relevance can be inferred about the function of
particular symbiont types, and predictions about the future of coral species are made
based on low replication within areas. Without a thorough understanding of the level
of flexibility in the coral symbiosis, we cannot accurately predict how corals will react
to certain stressors and levels thereof. Here, it has been shown that each host in the
family of Pocilloporid corals has multiple options in their symbiotic partnership and
that these are not only optimized to the environment that the colony is growing in but
is also flexible if the environment is changed. This suggests that corals may have the
potential to optimize their performance to a wider environmental range than
previously thought.
Even though the ITS regions have attracted some controversy as to whether they
can be used as an ecologically relevant marker due to the high rates of change, the
results of
m
this study see to validate the use of this region at this fine scale level and
suggest that small differences in the ITS-2 region may indeed confer functionality as
ITS2-types are regulated on a fine scale in relation to local environment. These
conclusions must remain limited to the species studied, and other species of corals
may not have the ability to associate with multiple symbionts, as each species does
not necessarily follow the same strategy. Some species may have a multitude of
symbionts suited to cover particular environments over the full range of their
distribution, whereas other species may associate with a single symbiont that has a
broad tolerance range over a particular environmental distribution. Of course there
are many possible combinations and this diversity indicates the importance of
studying these associations in depth so we can more accurately understand the
intricacies involved in the persistence of a successful symbiosis. More importantly, it
will yield information on the tolerance levels of each symbiont and the associations
as a whole. Moreover, th
is will help us predict how corals will respond to changes in
their environment and we can start to evaluate how damaged reef areas are most
likely to respond. If this information can be coupled with large scale monitoring efforts
and population genetics of the host, management programs can start to evaluate
60
which regions are most valuable in terms of sustaining reef health and providing
viable recruits to damaged areas.
References
Baker, AC (2003). Annu. Rev. Ecol. Evol. Syst. 34: 6618
Baker AC, Rowan R, Knowlton N (1997). Proc. Int. Coral Reef Symp., 8th, Panama,
2:1295300
Banaszak, AT, LaJeunesse, TC, and Trench, RK (2000). J. Exp. Mar. Biol. Ecol. 249:
219-233
Brown, BE (1997). Adv. Mar. Biol. 31:221-229
Buddemeier, RW and Fautin, DG (1993.) Bioscience 43: 320-326
Edmunds, PJ (1994). Mar. Biol. 121: 137-142
Fitt, WK and Warner, ME (1995). Biol. Bull. 189: 298-307
Hoegh-Guldberg, O (1999). Mar. Freshw. Res. 50: 839-866
Iglesias-Prieto, R and Trench, RK (1997a). Proc. 8th Int. Coral Reef Sym. 2: 1319-
1324
Iglesias-Prieto, R and Trench, RK (1997b). Mar. Biol. 130: 23-33
LaJeunesse, TC (2002). Mar. Biol. 141: 387-400
LaJeunesse, TC (2001). J. Phycol. 37: 866-880
Lesser, MP (1997). Coral reefs 16: 187-192
Loh WKW, Loi T, Carter D, Hoegh-Guldberg O (2001). Mar. Ecol. Prog. Ser. 222:97
107
Marshall, PA and Baird, AH (2000). Coral Reefs 19; 155-163
Rodriguez-Lanetty, M, Hoegh-Guldberg, O (2003). Mar. Biol. 143: 501509
Rowan, R and Powers, DA (1991). Mar.Ecol. Prog. Ser. 71: 65-73
61
Symbiodinium ITS-2 sequences: inter- or
intraspecific data? Implications for the detection
of ecological & biogeographic patterns
Adrienne M. Romanski1 and Andrew C. Baker 2,3
1. Department of Ecology, Evolution & Environmental Biology, Columbia University,
MC 5557, 1200 Amsterdam Avenue, New York, New York 10027, USA; 2. Wildlife
Conservation Society, Marine Conservation Program, 2300 Southern Boulevard,
Bronx, New York 10460, USA; 3. Center for Environmental Research and
Conservation, Columbia University, MC 5557, 1200 Amsterdam Avenue, New York,
New York 10027, USA.
Contemporary molecular systematic analyses generally employ multiple independent
markers to evaluate whether conclusions drawn from individual sequence datasets
are evolutionarily trivial or meaningful. The clade-level systematics of Symbiodinium
has largely fol owed this approach, using data from two independent markers
(cpDNA and mtDNA) to support phylogenies based on studies of nuclear rDNA (18S
and 24S) (. However, fine-scale (sub-clade) relationships within this genus are
unclear; the uniform application of multiple independent markers (to the same
samples) is required for their resolution.
The nuclear ITS-2 region has been extensively applied to investigating sub-clade
diversity within Symbiodinium, and sequence variants ("types") within this region
have been suggested as representing different species. However, this conclusion,
based on an analysis of 16 sequences (including 9 named taxa) in 2001, needs
validation in light of the rapidly increasing number (>100) of distinct sequences that
have been reported since.
Symbiodinium systematics and ecology: Limitations of the current framework
When interspecific algorithms are applied to Symbiodinium ITS-2 sequence data, the
resulting phylogenies contain many unresolved polytomies and lack bootstrap
support at many nodes (LaJeunesse 2002, LaJeunesse 2005). In addition, "living
ancestors" (C1 and C3) within clade C phylogenies violate assumptions of the
Phylogenetic Species Concept and the algorithms used to build bifurcating trees
(Figure 1 in LaJeunesse [2005]). Species distinctions within Symbiodinium based on
ITS-2 types are in some cases made on the existence of paralogs in the rDNA repeat
that differ by only a single base pair.
We suggest that systematic decisions based on these methods and criteria likely
obscure important patterns in the distribution of Symbiodinium. LaJeunesse (2005)
argues that Symbiodinium ITS-2 types occupy unique ecological niches (sometimes
based on symbionts' host distribution, endemicity and/or rarity) and may therefore be
considered distinct "species". However, explicit descriptions of these unique niches
are rarely reported for types, and seldom tested statistically. The community-level
sampling strategy commonly employed for Symbiodinium ITS-2 (in which a few
samples from each of many host species are identified) has been useful in exploring
the limits of Symbiodinium diversity but lacks the statistical power necessary to test
how diverse the symbionts of particular coral species really are. This is critical for
investigations of the Adaptive Bleaching Hypothesis.
62
Population-level theory explains some Symbiodinium ITS-2 variation
Observed patterns of Symbiodinium ITS-2 variation are more consistent with
interpretations based on population theory than with those based on interspecific
phylogenies. In populations, many ancestral haplotypes are expected to exist
alongside their descendants (Templeton et al.
se ance
1992). The
stral haplotypes
are relatively common and widely distributed (e.g., C1 and C3), while descendant
haplotypes are relatively rare and restricted in distribution (e.g., paralog types). The
older (and by extension, the more abundant) an ancestral haplotype, the more
descendant haplotypes are predicted to be associated with it (Watterson and Guess
1977). Therefore, rare haplotypes (e.g., C1a) are more likely to be related to
ancestral haplotypes (ex: C1) than to other paralogs (e.g., C1b) (Excoffier and
Langaney 1989). Single base pair differences between sequence pairs detected
using species-level markers are treated as intraspecific diversity (Avise et al. 1987,
Templeton et al. 1992) in both soft corals and dinoflagellates (Adachi et al. 1997,
McFadden and Hutchinson 2004, Shao et al. 2004). Intraspecific datasets are
expected to produce multifurcating trees (Templeton et al. 1992).
Traditional clade C phylogenies are reminiscent of Avise's "wall of death" (Avise
2004), predicted to occur when a species-level algorithm is applied to intraspecific
data. These algorithms can incorrectly depict extant ancestral haplotypes as "living
ancestors", occupying a branch of length zero at the basal node of a cluster (Posada
and Crandall 2001). In published bifurcating trees of Symbiodinium clade C, this is
the position occupied by sequences C1 and C3. Viewing C1 and C3 as the ancestral
haplotypes of two respective species explains the existence of their many paralogs
(C1a-k and C3a-m) and the lack of hierarchical organization within each lineage.
LaJeunesse (2005) correctly identifies the C1 and C3 sequences as ancestral, but
fails to arrive at the most parsimonious conclusion that they are haplotypes, not
species.
An alternative approach to Symbiodinium systematic
We suggest that much of the variation in Symbiodinium ITS-2 in fact represents
different populations diverging from extant (and often common) ancestral haplotypes.
This approach generally agrees with relationships assigned using interspecific tree-
building algorithms, but recognizes clusters of closely related sequences that
represent fewer, statistically better-supported "species". We propose that significant
fine-scale diversity arises through mutation during the asexual reproduction of
Symbiodinium living within hosts and is culled by selective purging events (such as
bleaching). Novel symbiont haplotypes arising in this manner are not necessarily
different species, even though they may achieve local abundance or patchy
geographic distributions (through host fragmentation and/or vertical transmission).
Our more conservative view of Symbiodinium taxonomy has important implications
for understanding which molecular patterns are ecologically meaningful, and impacts
our understanding of flexibility and specificity in coral-algal symbioses.
References
Adachi M, Sako Y, Ishida Y (1997) Analysis of Gymnodinium catenatum
Dinophyceae using sequences of the 5.8S rDNA-ITS regions and random
amplified polymorphic DNA. Fisheries Science 63: 701-707
Avise JC (2004) Speciation and Hybridization Molecular Markers, Natural History,
and Evolution. Sinauer Associates, Inc., Sunderla
321-399
nd, Massachusetts, pp
Avise JC, Arnold J, Ball RM, Bermingham E, Lamb T, Neigel JE, Reeb CA, Saunders
NC (1987) Intraspecific Phylogeography - The Mitochondrial-DNA Bridge
63
Between Population-Genetics And Systematics. Annual Review Of Ecology And
Systematics 18: 489-522
Excoffier L, Langaney A (1989) Origin and differentiation of human mitochondrial
DNA. American Journal of Human Genetics 44: 73-85
LaJeunesse TC (2002) Diversity and community structure of symbiotic dinoflagellates
from Caribbean coral reefs. Marine Biology 141: 387-400
LaJeunesse TC (2005) ``Species'' Radiations of Symbiotic Dinoflagellates in the
Atlantic and Indo-Pacific Since the Miocene-Pliocene Transition. Molecular
Biology and Evolution 22: 570-581
McFadden CS, Hutchinson MB (2004) Molecular evidence for the hybrid origin of
species in the soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia).
Molecular Ecology 13: 1495-1505
Posada D, Crandall KA (2001) Intraspecific gene genealogies: trees grafting into
networks. Trends in Ecology & Evolution 16: 37-45
Shao P, Chen Y-Q, Zhou H, Yuan J, Qu L-H, Zhao D, Lin Y-S (2004) Genetic
variability in Gymnodiniaceae ITS regions: implications for species identification
and phylogenetic analysis. Marine Biology 144: 215-244
Templeton AR, Crandal KA, Sing CF (1992) A Cladistic Analysis of Phenotypic
Associations With Haplotypes Inferred From Restriction Endonuclease Mapping
and DNA Sequence Data. III. Cladogram Estimation. Genetics 132: 619-633
Watterson GA, Guess HA (1977) Is the most frequent allele the oldest? Theoretical
Population Biology 11: 141-160
64
Symbiont shuffling represents a trade-off for
modern reef-builders
Madeleine van Oppen1, David Abrego1,2, Angela Little1,2, Jos Mieog1,3,
1
2
Ray Berkelmans and Bette Willis
1Australian Institute of Marine Science, Townsvil e, Qld 4810, Australia, School of
2
Marine Biology and Aquaculture, James Cook University, Townsville, Qld 4811,
Australia, 3Department of Marine Biology, Biological Centre, University of Groningen,
9750 AA Haren, Netherlands.
The recent discovery of the genetically diverse nature of the dinoflagellate genus
Symbiodinium (zooxanthellae) that forms symbiotic associations with stony corals
raises the possibility that physiological properties and tolerances of reef corals may
vary according to the association established. The genus Symbiodinium consists of
at least eight clades (A to H) based on sequence analysis of nuclear ribosomal DNA
(reviewed in Baker 2003, Pochon et al. 2004), as well as many genetic types within
each clade referred to as subclades or strains (e.g., C1, C2). In most broadcast
spawning corals, zooxanthellae are acquired from the environment in early ontogeny
and become established in the end dermal cells
o
of coral hosts as an endosymbiosis.
This creates an opportunity for the host to establish an association with a variety of
symbionts. Indeed, adults of some coral species form associations with more than
one Symbiodinium strain according to the local environment or microhabitats within a
coral (Rowan and Knowlton 1995, Rodriguez-Lanetty et al. 2001, van Oppen et al.
2001, Ulstrup and van Oppen 2003). Such polymorphic symbioses suggest that
corals within a species may not be physiologically uniform and that the taxonomic
identity of the Symbiodinium partner(s) may be as significant as that of the host in
determining the physiology of the holobiont (host-symbiont partnership).
Initial symbiont up-take is non-selective
The apparent specificity for strain C1 observed in adult populations of Acropora
tenuis is not present in the early stages of infection. Instead, new recruits take up a
mix of C and D strains and become dominated by Symbiodinium clade D after ~4
months (Little et al. 2004). The lack of specificity in initial uptake of zooxanthellae in
early ontogeny provides a mechanism for establishing associations with multiple
symbionts and, hence, may be adaptive, as different zooxanthella types can have
different physiological characteristics. The increase in Symbiodinium clade D is
unlikely to reflect greater mortality of C-juveniles, as accumulated mortality was only
slightly greater in C-juveniles three months after settlement but did not differ between
C- and D-juveniles seven months after settlement. The increase in juveniles
harbouring Symbiodinium clade D may therefore be caused either by competition
between algal types or by a host-mediated up-regulation of Symbiodinium clade D.
Role of symbiont type in holobiont physiology
Both A. tenuis as well as Acropora millepora juveniles grow 2-3 faster when
associating with Symbiodinium clade C compared to associations with clade D (Little
et al. 2004). Faster growth of holobionts infected with Symbiodinium C may reflect a
greater contribution of the symbiont to host nutrition through faster rates of population
growth inside the host (Fitt 1985). For A. tenuis, the faster growth rates of C1
juveniles may explain why C1 adults are the most common at Magnetic Island
(Ulstrup and van Oppen 2003). In contrast, the dominance of Symbiodinium D,
65
known to be associated with greater thermal tolerance (Baker et al. 2004, Fabricius
et al. 2004, Rowan 2004) in naturally infected, 6-month-old A. tenuis, is more difficult
to explain.
Conclusions and future directions
Shuffling from Symbiodinium clade C to D may increase thermal tolerance, but is
likely to result in impaired growth, competition and reproduction (trade-off).
Furthermore, it is currently not known how wide-spread symbiont shuffling is as a
mechanism to cope with environmental change, and symbiont changes are not
heritable in many species. Future work will therefore need to investigate the potential
for adaptation through selection on genetic variation and new mutations in both the
coral host as well as the algal endosymbionts.
References
Baker AC (2003) Flexibility and specificity in coral-algal symbioses: Diversity,
ecology, and Biogeography of Symbiodinium. Annual Reviews in Ecology and
Systematics, 34, 661-689
Baker AC, Starger CJ, McClanahan TR, Glynn PW (2004) Corals' adaptive response
to climate change. Nature, 430, 741
Fabricius KE, Mieog JC, Colin PL, Idip D, van Oppen MJH (2004) Identity and
diversity of coral endosymbionts (zooxanthellae) from three Palauan reefs with
contrasting bleaching, temperature and shading histories. Molecular Ecology, 13,
2445-2458
Fitt WK (1985) Effect of different strains of zooxanthella Symbiodinium
microadriaticum on growth and survival of their coelenterate and molluscan hosts
Proc. 5th Int. Coral Reef Symp. 6, 131
Little AF, van Oppen MJH, Willis BL (2004) Flexibility in algal endosymbioses shapes
growth in reef corals. Science, 304, 1492-1494
Pochon X, LaJeunesse TC, Pawlowski J (2004) Biogeographic partitioning and host
specialization among foraminiferan dinoflagel ate symbionts (Symbiodinium;
Dinophyta). Marine Biology, 146, 17-27
Rowan R (2004) Thermal adaptation in reef coral symbionts. Nature, 430, 742
Rowan R, Knowlton N (1995) Intraspecific diversity and ecological zonation in coral-
algal symbiosis. Proceedings of the National Academy of Science, 92, 2850-2853
Rodriguez-Lanetty M, Loh W, Carter D, Hoegh-Guldberg O (2001) Latitudinal
variability in symbiont specificity within the widespread scleractinian coral
Plesiastrea versipora. Marine Biology, 138, 1175-1181
Ulstrup KE, van Oppen MJH (2003) Geographic and habitat partitioning of genetically
distinct zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier
Reef Molecular Ecology, 12, 3477-3484
van Oppen MJH, Palstra FP, Piquet AT, Miller DJ (2001) Patterns of coral-
dinoflagellate associations in Acropora: significance of local availability and
physiology of Symbiodinium strains and host-symbiont selectivity. Proceedings of
the Royal Society, London B, 268, 1759-1767
66
"Checks and Balances" in the identification of
Symbiodinium diversity
Scott R. Santos.
Department of Biological Sciences & Cell and Molecular Biosciences Peak Program,
Auburn University, Auburn, AL 36849, USA.
The identification of genetic diversity in dinoflagellates belonging to the genus
Symbiodinium has been the subject of numerous studies over the last decade.
During this time, a variety of molecules have been utilized. Some of these loci offer a
"coarse" (i.e., 18S-rDNA) view while others provide a much "finer" (i.e., DNA
fingerprinting) portrait of diversity within Symbiodinium. The use of sequences from
the ribosomal internal transcribed spacer (ITS) region has become a standard in
categorizing genetic diversity of these dinoflagellates and has revealed a plethora of
ecological "types" within the genus. For some of these "types", data from
microsatel ite loci has uncovered additional, biologically relevant, diversity. However,
one cannot help to ask if this diversity is truly "real" or results from artifacts created
by molecular techniques. Here, I discuss ways in which Symbiodinium ITS and
microsatellite data can be "checked" for methodological and interpretation errors.
These processes not only lead to a "balance" in our quantification of Symbiodinium
diversity, but also provide novel insight into the biology of these unique organisms.
The ribosomal internal transcribed spacers (ITS)
Among eukaryotic organisms, the ribosomal DNA (rDNA) operon is organized in a
similar fashion. The operon consists of three ribosomal RNAs (5.8S, 18S and 28S),
each separated by external and internal transcribed spacer regions. The 5.8S rRNA
is separated from the small subunit (SSU or 18S) rRNA by the first of two internal
transcribed spacers (ITS1), while the large subunit (LSU or 28S) rRNA and 5.8S are
separated by the second internal transcribed spacer (ITS2). Typically, these three
rRNAs, as well as both the internal and external spacers, are transcribed by RNA
polymerase I into a single precursor molecule, the 35-45S pre-rRNA. This molecule
undergoes a series of processing steps that ultimately leads to mature and ful y
functional rRNAs. Mutations in the spacer regions flanking the rRNAs are known to
prevent formation of the mature molecules, suggesting that they contain essential
signals required for correct processing. In recent years, both ITS1 and ITS2 have
been recognized as vital components of the processing steps leading to rRNA
maturation; specifical y, it has been emphasized that a particular secondary structure
in these regions are required for correct processing (Coleman 2003). This being the
case, novel Symbiodinium ITS sequences could potentially be validated by
comparing them to established secondary structures for the genus as well as specific
Symbiodinium clades. For this reason, the ITS2 secondary structures from members
of the eight major Symbiodinium clades, A-H, were elucidated and compared.
The ITS2 secondary structure of Symbiodinium
In spite of large amounts of primary sequence divergence (>60%, in some cases), a
nearly common ITS2 secondary structure has been recovered from representatives
of all Symbiodinium clades (Fig. 1). This structure is consistent with the four-helix
model, which has been previously described from other organisms (Coleman 2003),
including some free-living dinoflagellates (Gottschling and Plotner 2004). However,
among Symbiodinium clades B, C, F and H, a subtle, but significant, difference in
67
secondary structure was apparent when
compared to the other Symbiodinium
clades, free-living dinoflagellates and
eukaryotes in general. This structural
difference was the presence of an additional
stem-loop (label ed IIIa in Fig. 1), which
results in a five-helix model for these
clades. By using the features inherent to
this ITS2 secondary structure, such as
Fig. 1. Consensus Symbiodinium
nucleotide bulges and conserved
ITS2 secondary structure
processing sites, as well as compensatory
base changes found among groups of closely-related taxa, the structural skeleton for
Symbiodinium ITS2 provided here will find use in the validation of novel sequences
and optimisation of alignments for phylogenetic reconstruction.
Microsatellites
Microsatellites are simple, tandemly repeated DNA sequences elements, distributed
abundantly in the genomes of virtually all organisms. For Symbiodinium, data from
microsatellite loci (e.g., either the presence or absence of an allele, size variability
between alleles or phylogenetically informative substitutions in the flanking regions
adjacent to the repeat array) have demonstrated the non-representative nature of
some zooxanthella cultures. When compared to the populations from which they
were established (Santos et al. 2001), this data confirmed that Symbiodinium spp.
are haploid in the vegetative life stage (Santos and Coffroth 2003), revealed striking
differentiation in Symbiodinium populations associated with the octocoral
Pseudopterogorgia elisabethae across the Bahamas (Santos et al. 2003) and
elucidated fine-scale diversity and specificity in the most prevalent lineage of
symbiotic dinoflagellates of the Caribbean, Symbiodinium "type" B1 (Santos et al.
2004). However, to date, the mutational behaviour of microsatellite loci in the
Symbiodinium genome has not been discussed. Data from two well-characterized
Symbiodinium microsatellites suggest that a range of evolutionary processes operate
on these loci.
Evolutionary patterns in Symbiodinium microsatellites
Substitutions, nucleotide insertion and deletions (indels), alterations to the repeat
array structure and non-stepwise changes in repeat number have been documented
from these two Symbiodinium microsatellites. Because the accurate estimation of
population structure and relationships using microsatellite data rests in the
assumption that alleles identical in state (i.e., size) have experienced a common
mutational history, this discovery may complicate future studies. However, although
microsatellite alleles homologous in size but resulting from different evolutionary
processes (e.g., size homoplasy) were identified, this phenomenon appears to pose
little problem for the interpretation of population structure if techniques capable of
detecting differences in the primary sequence of microsatellite alleles are employed.
Furthermore, mutations such as the ones de
ve are a rich source
scribed abo
of
information that complement and extend the population-level data inherent to these
markers. Analysing such mutational patterns will identify the forces shaping the
genome of these important organisms and provide new insight into Symbiodinium
biology.
68
References
Coleman, AW (2003). ITS2 is a double-edged tool for eukaryote evolutionary
comparisons. Trends in Genetics, 19: 370-375.
Gottschling, M & Plötner, J (2004). Secondary structure models of the nuclear
internal transcribed spacer regions and 5.8S rRNA in Calciodinelloideae
(Peridiniaceae) and other dinoflagellates. Nucleic Acid Research, 32: 307-315.
Santos, SR, & Coffroth, MA (2003). Molecular genetic evidence that dinoflagellates
belonging to the genus Symbiodinium Freudenthal are haploid. Biological.
Bulletin, 204: 10-20.
Santos, SR, Taylor, DJ, & Coffroth, MA (2001). Genetic comparisons of freshly
isolated vs. cultured symbiotic dinoflagellates: implications for extrapolating to the
intact symbiosis. Journal of. Phycology. 37: 900-912.
Santos, SR, Gutiérrez-Rodríguez, C, Lasker, HR, Coffroth, MA (2003). Symbiodinium
sp. associations in the gorgonian Pseudopterogorgia elisabethae in the
Bahamas: high levels of genetic variability and population structure in symbiotic
dinoflagellates. Marine Biology 143: 111-120.
Santos, SR, Shearer, TL, Hannes, AR, & Coffroth, MA (2004). Fine-scale diversity
and specificity in the most prevalent lineage of symbiotic dinoflagel ates
(Symbiodinium, Dinophyceae) of the Caribbean. Molecular Ecology 13: 459-469.
69
Transcriptome analysis of a cnidarian
dinoflagellate mutualism reveals complex
modulation of host gene expression
Mauricio Rodriguez-Lanetty, Wendy Phillips, and Virginia M. Weis
Department of Zoology, Oregon State University, Corvallis, OR 97331, USA.
Mutualistic symbioses are defined as the association between unrelated organisms
living together in a close, protracted relationship that benefits both partners.
Cnidarian dinoflagellate associations represent on of the most important
e
symbioses in the marine environment. These partnerships form the trophic and
structural foundation of coral reef ecosystems, and have been the driving force in the
radiation and biodiversity of cnidarian species. Despite the prevalence of these
marine symbioses and the overall interest in coral reef health, we still know very little
about the cellular and molecular basis of the intracellular cnidarian dinoflagellate
symbiosis. What are the key molecular modulators that initiate, regulate and
maintain the interaction between these two different biological entities?
Modulation of host gene expression as a function of symbiosis
The discovery and identification of host genes that modulate cnidarian
dinoflagellate symbioses is a topic that is ideally
suited to a comprehensive microarray
approach. We conducted a comparative host
anemone transcriptome analysis using a cDNA
microarray platform to identify genes involved in
cnidarian algal symbiosis. Following earlier
proteomic studies [1], we used the temperate
anemone Anthopleura elegantissima as a
model as it occurs naturally in both the
symbiotic and aposymbiotic state (Figure 1).
We discovered 91 unigenes (5.63% of the
genes originally spotted on the microarray) that
Fig.1 Symbiotic (brown) and
showed to be significantly different between aposymbiotic (white) anemones of
Anthopleura elegantissima
aposymbiotic and symbiotic anemones. From
the identified host unigenes only 32
genes showed significant BLAST
Down
Up
hits (E < 1.0 x 10-4) with homologue
14
known genes in the Genbank.
12
10
One important discovery is that our
8
data do not support the existence of
6
symbiosis-specific genes involved in
4
controlling and regulating the
2
symbiosis. Twelve of the 91
0
differential unigenes were very
7
6
5
4
3
2
5
1
1
5
2
3
4
5
6
7
8
9
10
15
20
25
30
35
40
45
50
55
8-
7-
6-
5-
4-
3-
1.
5-
5-
1.
3-
4-
5-
6-
7-
8-
9-
10-
2-
1.
1.
2-
15-
20-
25-
30-
35-
40-
45-
50-
55-
60-
highly expressed in symbiotic state
Ratio of gene expression
(Fig. 3). These were viewed with Fig. 2. Distribution of genes as a function of the ratio
suspicion as possible algal genes of expression. Arrows divide those down and up
regulated in symbiosis. Color bars mean as follow:
that were contaminating the host-
Black (contaminating algal symbiont unigenes) and
only cDNA library. Specific primers
Grey (host unigenes).
70
for these highly symbiotic unigenes were constructed and used in PCR reactions with
genomic host-only and algae-only symbiont template DNA. Successful DNA
amplification was only achieved in the algal DNA samples and no in the
t
host
genomic DNA (data not shown). These contaminating algal unigenes e
w re therefore
removed from further analyses. Al other up-regulated host unigenes in the symbiotic
state were also expressed in some extent in aposymbiotic hosts, as their fold change
expression was subtle; largely ranging between 1 and 2. Rather than finding genes
which expression change as a function of symbiosis in an ON/OFF manner, we
detected alterations of expression of genes regulating different functional processes
(Fig. 3).
10
s 8
ne
e
g 6
r
of
e 4
b
m
u
N 2
0
Cel growth and/or
Metabolism;
Protein
Regulation of
Cel signalling and
Transport
unclassified
maintenance
Energy pathways
metabolism
nucleic acid
communication
metabolism
Fig. 3. Distribution of the known host unigenes based on their functional classification. White and
grey bars are the genes up and down regulated in symbiosis, respectively.
Gene ontologies of the 32 differentially expressed genes exhibiting significant
homologies reveal the complexity of the interaction between the symbiotic state and
host gene expression. This suggests that symb
iosis is regulated and controlled, not
by pathways unique to the symbiotic state but
changes wit
rather by
hin existing
pathways used to control metabolism and growth of the animal as whole. Most of
these genes are putatively involved in metabolism and energy pathways, cell growth
and/or maintenance, and cell signaling.
Suppression of apoptosis and deregulation of cell cycle
We discovered that the gene expression of key biomolecules involved in cel cycl
e
progression and apoptosis are differential y modulated in symbiosis. For instance,
we detected a down regulation of the gene Sphingosine Phosphate Phosphatase
(SPPase) in symbiotic state, which may play a role in keeping the levels of the anti-
apoptotic sphingolipid, Shingosine-1-Phosphate (S1P) higher over the pro-apoptotic
sphingolipid, sphingosine. This increase of S1P would facilitate higher survival of
symbiont-harboring host cells, as it has been documented to occur in other animal
cells [2]. Moreover, high levels of S1P would also enhance cell proliferation by
expediting the G1/S transition in the cel cycle [3].
Our findings provide novel insight into the physiological roles of sphingolipids in
cnidarian algae symbiosis, and how the modulation of sphingolipid regulators, such
as SPPase, is emerging as putative mechanisms to regulate host cell apoptosis and
survival in host symbiont associations. We suggest that a suppression of apoptosis
together with a deregulation of the host cel cycle create a platform that might be
necessary to symbiont survival and/or symbiont-containing host cell survival. These
findings are very interesting as they adjust our perception of the interaction between
cnidarians and symbiotic dinoflagel ates from a cel ular perspective. We have always
71
visualized the cnidarian algae interaction as a cooperative since the ecological
outcome of the interaction is a mutualistic symbiosis; however, from a cellular level
the interaction between host and symbionts appear to have components of a
parasite/pathogenic interaction. Symbionts, like pathogens need to overcome the
host innate immunity to enter, reside and growth inside the host cell. But intriguingly,
the algae-induced changes in the host cell, which show some similarity to pathogen
host interactions, do not lead to the development of disease. Understanding the
nature of the molecular regulation of cnidarian algal symbiosis and by comparison
with parasite/pathogen associations, it will provide further insight into the evolution of
host symbiont/parasite associations.
References
1. Weis VM, Levine RP (1996) Differential protein profiles reflect the different
lifestyles of symbiotic and aposymbiotic Anthopleura elegantissima, a sea
anemone from temperate waters. J. Exp. Biol. 199: 883-892
2. Spiegel S, Milstien S (2002) Sphingosine 1-phosphate, a key cell signaling
molecule. J. Biol. Chem. 277: 25851-25854
3. Stunff H, Milstien S, Spiegel S (2004) Generation and metabolism of bioactive
sphingosine-1-phosphate. J. Cel. Biochem. 92: 882-899
72
Consensus statement on the current understanding
of the diversity, specificity and flexibility
of Symbiodinium symbioses.
Statement agreed to by participants at Puerto Morelos meeting (May 17 2005)
Dinoflagellates in the genus Symbiodinium are the principal symbionts of reef-
building corals as well as hosts from several other phyla. These single-cel ed
photosynthetic organisms general y occur intracellularly within host cells. Once
thought to represent a single species, Symbiodinium microadriaticum, they are now
considered to be phylogenetically diverse and include a number of described and
undescribed species.
Understanding the forces that have driven the distribution and evolution of
Symbiodinium may provide important insights into the response of corals to
environmental change. Distinguishing which processes operate at ecological as
opposed to evolutionary timescales is critical
h
to t is endeavour. Future efforts should
focus on relating genetic diversity to ecologically relevant physiological differences.
Corals and their dinoflagellate symbionts exhibit a range of specificities. Some coral
species transfer symbionts directly between generations, while others acquire
symbionts from the environment anew each generation. In the latter case, the events
that lead to the establishment of Symbiodinium symbioses are relatively specific
despite the fact that most genotypes can be taken up by host cells initially.
The initial types of Symbiodinium that enter newly settled corals appear to be a
subset of those available in the environment. This set of Symbiodinium types is
further narrowed down to the complement dominant within the adult host and its
environment, although some types may persist at background levels within the
tissues of the host coral.
The processes by which one or several symbionts become dominant within the host
have yet to be described, but probably involve host-symbiont recognition, specific
host factors and competition between Symbiodinium genotypes. Future studies need
to focus on understanding these mechanisms and their relative importance.
Adult corals may change their symbiotic complement in response to environmental
change. `Shuffling' and `switching' are two non-exclusive mechanisms by which this
may be accomplished. `Shuffling' is a quantitative (compositional) change in the
relative abundance of symbionts within a colony; `switching' is qualitative change
involving symbionts acquired from the environment. These exogenous symbionts
may represent types that are new to the colony but not the species, or may be truly
novel to the host species which is referred to as `evolutionary switching.'
While evolutionary switching is assumed to explain the phylogenetic patterns of
symbiont distribution within hosts, such events are thought to be very rare. Shuffling
and/or switching of existing symbionts are thought to be more common. However,
distinguishing between evolutionary (and truly novel) switches, and those that involve
existing symbionts, is a methodological challenge.
Some corals routinely shuffle symbionts as a consequence of seasonal regulation of
symbiont numbers with or without visual signs of bleaching. Corals can also shuffle
symbionts during and after bleaching. Switching is also likely to be promoted by
seasonal regulation and bleaching. Both shuffling and switching may be important
73
mechanisms that extend the ability of corals to acclimatize to changes in the
environment but requires further investigation to demonstrate true
l
physiologica
advantages of the changes involved.
Bleaching probably did not evolve directly as a mechanism for shuffling or switching
symbionts, and has clear pathological effects. A potential side-effect of bleaching is
an acceleration of symbiont change which has the potential to elevate the tolerance
threshold of coral reefs to environmental change. However, without evolutionary
switching, there will be no change in the tolerance threshold for any particular coral-
Symbiodinium symbiosis.
There continues to be widespread concern within the research community over the
future of coral reef c
e osystems. Increased sea temperatures as well as ocean
acidification and other anthropogenic challenges continue to pose grave threats to
the future of these diverse ecosystems and the people that depend on them. Unless
these threats are addressed as a priority soon, coral reefs will continue to degrade.
Scott Santos addresses the workshop on "Diversity, flexibility, stability, physiology of
Symbiodinium and the associated ecological ramifications".
74
Theme 3: Exploration of the Coral and Symbiodinium
genomes (May 19-21, 2005)
Co Chairs: Bill Leggat, Sophie Dove, David Yellowlees
Over the past five years, there has been a rapid growth of studies aimed using
molecular methods to explore the gene expression of corals and Symbiodinium using
molecular methods. This discussion evolved out of the perceived need to promote
better communication
research g
and synthesis between
roups in this area. The
major goal of this section of the workshop was to review the key research questions
and agenda for the future and where possible develop synergies and collaborations.
Participants
Andrew Baker; Merideth Meredith Bailey; Jeffry Deckenback; Sophie Dove;
Susana Enriquez; William Fitt; Ruth Gates; Ove Hoegh-Guldberg; Roberto
Iglesias Prieto; Michael Lesser; Todd LaJuenesseLaJeunesse; Mikail Matz;
David Miller; Mckenzie Manning; Maurico Rodiguez-Lanetty; Adrienne Romanski;
Hector Reyes; Baraka Ruguru.; Eugenia Sampayo; Jodie Swartz; Roee Segal;
Noa Shenkar; Madeleine van Oppen; Shakil Visram; Gidon Winters; Mark
Warner; David Yellowlees and Assaf Zenvoluni.
Theme coordinator Bill Leggat takes notes during discussions. Others in photo
Mark Warner (left) and Ross Hill (right).
75
Progress in coral genomics
David J Miller, L Grasso, D Hayward, P Maxwell, J
ld, S Rudd,
Maindona
U Technau, EE Bal l
Biochemistry & Molecular Biology, James Cook University, Townsville 4811 Australia
Anthozoan cnidarians such as corals are phylogenetically basal, and a substantial
body of morphological and molecular data supports the idea that they are the closest
extant relatives of Ur-eumetazoa the common ancestor of all higher animals. To
better define the basic metazoan gene complement, and also gain insights into the
genetic bases of coral-specific properties such as calcification, we are conducting
ESTs analyses on two anthozoans the coral Acropora millepora and the sea
anemone Nematostella vectensis. To date, we have examined 16,571 non-redundant
ESTs (12,547 predicted peptides) across the two species, and both projects are
ongoing. The resulting dataset is much more complex than might be assumed based
on morphology, and implies that much of the genetic complexity normally assumed to
have arisen much later in animal evolution is actually ancestral. For example, all of
the key developmentally-regulated cell-signaling pathways are represented, in most
of the types usually associated with vertebrate development. At least 5% of genes
have been independently duplicated in the anthoz
e, and emerging data fo
oan lineag
r
other cnidarians suggest complex patterns of duplication in some cases. The most
surprising implication of our analyses, however, is that anthozoans have retained a
substantial number of genes not previously known in the animal kingdom. These and
other anomalously distributed genes suggest unanticipated physiological properties,
and therefore that anthozoan stress responses may be complex. The redundant
Acropora EST collection, comprising almost 13,000 ESTs from three developmental
stages, has been printed to microarrays and we are using these to investigate many
aspects of development as well as stress responses. The arrays are available from
the Adelaide microarray facility at $US160 (or $A200). A third generation Acropora
microarray is in development this will add at least 4,000 ESTs from zooxanthellae-
free adult coral tissue to the previous release.
76
A microarray approach to understanding stress
responses and the functional biology of corals
Madeleine van Oppen1, Andrew Negri1, David J. Miller2
1
n
Australian I stitute of Marine Science, Townsville, Qld 4810, Australia
2Comparative Genomics Centre, Molecular Sciences Building 21, James Cook
University, Townsville, Qld 4811, Australia
Mass coral bleaching events have greatly increased in frequency and intensity over
the past 30 years, leading to widespread concern over the long-term survival of coral
reefs as we know them today. Anthropogenic toxicants from runoff and ship
groundings have also been reported to cause localised bleaching (Jones et al. 2003;
Smith et al. 2003). Coral bleaching has been studied at the level of the photosystems
of symbiotic algae (e.g. Warner et al. 1996, 1999; Jones et al. 1998, 2001; 2003; Hill
et al. 2004; Tchernov et al. 2004), however, the molecular responses of the coral
partner in relation to heat or toxicant stress are almost completely unknown. The
limited molecular studies conducted to date on stress responses in corals have
focussed on single candidate molecules, such as Hsp70, often using heterologous
probes or antibodies to assay changes in protein levels. For example, Brown et al.
(2002) examined a range of host and symbiont biomarkers in heat-stressed
Goniastrea aspera colonies and showed that the coral host is likely to play a
significant role in enhancing the thermal tolerance of certain tissues by increased
production of CuZnSOD, Hsp60 and Hsp70 at elevated temperature.
The availability of a microarray chip developed for the coral Acropora millepora by
James Cook University and the ARC Centre for the Molecular Genetics of
Development, as well as the availability of field coral samples from two non-bleaching
('00-'01 and '02-'03) and one bleaching ('01-'02) summers collected by the Australian
Institute of Marine Science, permits us to study the natural response of coral to
thermal stress using microarray technologies. In addition, we have exposed A.
millepora larvae to elevated temperatures and various concentrations of the heavy
metal copper, the antifoulant TBT and the herbicide diuron in replicated exposures
for 24 hours, allowing us to examine gene expression differences between these
stress treatments. . As a wide range of stressors are known to cause corals to
bleach, the specific cellular mechanism underlying the bleaching response is likely to
be different for each stressor. The approach proposed here aims at characterising
these differences and may lead to the development of diagnostic tools for use in the
field.
Materials & methods
The coral Acropora millepora was used for this study as this species is common on
the Great Barrier Reef and easily identifiable, it is relatively well-studied and coral
husbandry methods have been developed for this species. A pilot microarray
experiment was conducted, where RNA was isolated from 6 tagged coral colonies
sampled on 24 January 2000 (a non-bleaching summer) and the same 6 colonies on
24 January 2002 (a bleaching summer). Gene expression levels at ~3,000 ESTs
were compared between the two sampling time points.
77
Results and Discussion
Over a hundred genes consistently showed significant differential expression in the
bleaching and non-bleaching comparison. However, of the differential y expressed
genes, none showed a fold change higher than three. One of the most highly down-
regulated genes is clearly related to a human gene that has been implicated in
cancer metastasis, suggested the possibility of
coral gene in
involvement of the
expulsion of zooxanthellae. We are currently validating these results using real time
quantitative PCR. One of the challenges we are facing is the identification of
appropriate house-keeping genes for comparative analysis.
Conclusions and future directions
Real time PCR will aid in selection of the most informative time points during a
natural bleaching event to use in a future microarray experiment. We will also be
targeting some genes directly using real time PCR, such as some of the genes
encoding heat shock proteins and homologs of the bacterial universal stress proteins.
References
Brown et al. (2002) Mar. Ecol. Progr. Ser. 242:119-129
Hill et al. (2004) Mar. Biol. 144:633-640
Jones et al. (1998) Plant, Cell and Environm. 21:1219-1230
Jones and Hoegh-Guldberg (2001) Plant, Cell and Environm. 24:89-100
Jones et al. (2000) Mar. Freshw. Res. 51:63-71
Jones et al. (2003) Mar. Ecol. Progr. Ser. 251:153-167
Smith et al. (2003) Mar. Biol. 143:651657
Tchernov et al. (2004) Proc. Natl. Acad. Sci., USA 101:13531-13535
Warner et al. (1996) Plant, Cell and Environm. 19:291-299
Warner et al. (1999) Proc. Natl. Acad. Sci., USA 96:8007-8012
78
Coral Reef Genomics: A Genome-wide
Approach to the Study of Coral Symbiosis
Jodie Schwarz1, P. Brokstein1, C. Lewis2, C. Manohar3, D. Nelson3, A.
Szmant4, M.A. Coffroth2, M. Medina1
1Joint Genome Institute, 2SUNY Buffalo, 3Lawrence Livermore National Laboratory,
4UNC Wilmington
The symbiotic association between corals and Symbiodinium is one of the structuring
features of coral reef ecosystems. The dramatic decline in the health of coral reefs
worldwide has prompted great interest in understanding how the symbiosis breaks
down under conditions of environmental stress. Equally important is an
understanding of mechanisms that lead to the establishment and regulation of the
"normal" state of symbiosis. We are taking a genomics approach to identify genes
and gene networks in both partners that play a role in the establishment of the
symbiosis. To accomplish this, we are developing cDNA libraries and cDNA
microarrays to study the onset of symbiosis in two Caribbean corals, Montastraea
faveolata and Acropora palmata and several strains of
b
Sym iodinium. These corals
spawn egg/sperm bundles that lack zooxanthellae, providing an experimental system
for studying the onset of symbiosis. We rear
and then e
the larvae
xperimentally
establish the symbiosis, using cultures of Symbiodinium. We sample RNA from both
partners throughout the process to capture genes that are expressed at different
stages of symbiosis. We have created cDNA librari s
e from coral eggs, embryos,
larvae, and adults, and from coral larvae infected with Symbiodinium. We are
currently making cDNA libraries from the Symbiodinium strains with which the larvae
were infected. To date, we have generated over 10,000 ESTs and are developing
methods to annotate the ESTs to obtain as much information from the sequence data
as possible. Using the sequence information, we will select specific genes to include
in microarrays. We have conducted a pilot study using a 100 gene cDNA microarray
to examine relative gene expression levels in coral eggs vs. adult tissues. We are
now scaling up to examine gene expression levels in thousands of genes in various
stages of coral development, as well as in symbiotic vs. non-symbiotic larvae. To
examine the genomic context of genes that we identify as being related to symbiosis,
we have made BAC libraries from both coral species that can be probed and
examined for upstream regulatory sequence information.
79
Targeted Functional Genomics of Coral Stress
Theresa Seron, Karen Konzen and Mikhail Matz
Whitney Laboratory for Marine Bioscience,
r
University of Flo ida, 9505 Ocean Shore
Blvd, St Augustine, FL 32137, USA
The recently documented trend of gradual decline of coral reefs on a global scale is a
matter of high concern. Our awareness of the problems experienced by corals is
critically dependent on the ability to recognize stressed condition, as well as
determine the cause and magnitude of stress. The symptom of stress most
frequently quantified during field surveys is "bleaching" - loss of pigmentation
due to dramatically reduced number of algal endosymbionts ("zooxanthellae") within
coral's tissues (Jokiel and Coles, 1990). The bleached corals often die, sometimes
on the scale of the whole reef, although in many cases they are able to recover
(McClanahan, 2004). Although easy to observe in the field, the "bleaching" symptom
lacks the in discriminatory power: it signifies only the most severe stress and can be
caused by a variety of factors such as increased (Jokiel and Coles, 1990) or
decreased (Saxby, et alDennison and Hoegh-Guldberg, 2003) water temperature,
elevated visible light (Lesser and Farrell, 2004), low salinity (Kerswell and Jones,
2003), chemical insult (Brown, 2000), and bacterial infection (Ben-Haim et al., 1999).
Most importantly, without additional information it is not possible to predict whether
the bleached coral is going to die or recover.
Clearly, methods integrating several observable parameters that may change during
stress have a better chance to infer the details of the coral condition. We believe that
monitoring expression levels of 100-150 genes that respond to stress will provide
sufficient data to evaluate stress intensity as well as recognize the contribution of
particular stress factors. The first goal of our project is to develop such a technique.
We performed six subtractive hybridizations to obtain cDNA samples enriched by the
transcripts that are either up- or down-regulated in Porites lobata and/or Porites
compressa during any of following the three types of stress: elevated heat/light,
exposure to copper and mechanical injury. The array of 3456 randomly picked clones
from these samples has been printed. Its preliminary characterization indicates that
the subtractive cDNA libraries contain a high proportion of differentially expressed
genes: 285 clones have been identified thus far as possible candidates, some of
them common, some specific to particular stressors (Fig. 1). This array will be further
extended by adding similar subtractive analysis of three more stressors cold,
excessive light (but no temperature increase) and low salinity. Our aim is to discover
at least 150 stress-related genes, expression of which will be it quantified in corals
stressed under a variety controlled laboratory conditions. The results will be
subjected to multivariate statistical analysis to extract patterns of gene up- and down-
regulation characteristic for different stressors and stress intensities, using the same
algorithm that was recently developed for artificial olfaction (Carmel et al., 2003).
80
Fig. 1. Results of the primary array analysis of coral stress. A-C: Scatter plots of
the intensity ratio between two compared samples at each spot versus mean
spot intensity, in logarithmic coordinates. Spots significantly deviating from the
mean are highlighted: the ones above zero are up-regulated, the ones below -
down-regulated. D and E: numbers of common and unique differential spots for
the three types of treatment, following the results represented on panels A-C.
The plots and lists of differential spots were generated using the NIA Array Analysis
tool
The identification of the genes up- and down-regulated during stress will provide an
opportunity to investigate the molecular mechanism of stress response in corals,
which is the second goal of our project. We are going to sequence all the clones
showing expression changes in our experiments, obtain full-length cDNA sequences
for them to facilitate the analysis of their homology relationships, and compare the
resulting collection of genes to the known stress-related gene interaction networks
from other organisms. The information about the correlation between expression
levels of individual genes in our experiments, with reference to the organization of
characterized gene networks, will provide additional grounds for inference of the
stress-related gene regulation network in corals ( Herrgard, et al Covert and
Palsson, 2003). It can be expected that the newly identified network elements may
serve as key indicators of survival ability of corals under stress. For example, the
expression level of genes responsible for the non-specific stress response may
reflect the general susceptibility of the organism to stress. In the future, such
knowledge will provide the basis for assessing survival chance of different coral
species in different ecological zones and/or geographical regions in the face of
changing global conditions.
81
References
Ben-Haim, Y., E. Banim, A. Kushmaro, Y. Loya and E. Rosenberg. 1999. Inhibition of
photosynthesis and bleaching of zooxanthellae by the coral pathogen Vibrio
shiloi. Environmental Microbiology 1: 223-229.
Brown, B. E. 2000. The significance of pollution in eliciting the 'bleaching' response in
symbiotic cnidarians. International Journal of Environment and Pollution 13: 392-
415.
Carmel, L., N. Sever, D. Lancet and D. Harel. 2003. An eNose algorithm for
identifying chemicals and determining their concentration. Sensors and Actuators
B- Chemical 93: 77-83.
Herrgard, M. J., M. W. Covert and B. O. Palsson. 2003. Reconciling gene expression
data with known genome-scale regulatory network structures. Genome Research
13: 2423-2434.
Jokiel, P. L. and S. L. Coles. 1990. Response of Hawaiian and Other Indo-Pacific
Reef Corals to Elevated-Temperature. Coral Reefs 8: 155-162.
Kerswell, A. P. and R. J. Jones. 2003. Effects of hypo-osmosis on the coral
Stylophora pistillata: nature and cause of 'low-salinity bleaching'. Marine Ecology-
Progress Series 253: 145-154.
Lesser, M. P. and J. H. Farrell. 2004. Exposure to solar radiation increases damage
to both host tissues and algal symbionts of corals during thermal stress. Coral
Reefs 23: 367-377.
McClanahan, T. R. 2004. The relationship between bleaching and mortality of
common corals. Marine Biology 144: 1239-1245.
Saxby, T., W. C. Dennison and O. Hoegh-Guldberg. 2003. Photosynthetic response
s
of the coral Montipora digitata to cold temperature stress. Marine Ecology-
Progress Series 248: 85-97.
82
Using Molecular Markers A Cautionary Tale
Ruth D. Gates, Amy M. Apprill and Benjamin R. Wheeler
Hawaii Institute of Marine Biology, University of Hawaii, PO. Box 1346, Kaneohe,
Hawai 96744
The SSU (18S), ITS-1, ITS-2 and LSU (28S) rDNA are arguably the most common
markers used for exploring the taxonomy of dinoflagellate symbionts belonging to the
genus Symbiodinium (reviewed by Baker, 2003; Coffroth and Santos, 2005). These
four regions of the ribosomal array provide different levels of taxonomic resolution
based on their individual rates of evolution and thus provide insight into a variety of
fundamental aspects of coral biology that cross multiple temporal and spatial scales.
Col ectively work exploiting these markers has dramatical y improved our
understanding of the relationship of Symbiodinium with respect to other protist phyla,
revealed differences in host/symbiont specificity and niche preference in corals
across environmental gradients, allowed for the description of geographic patterns of
distribution, and resolved aspects of the temporal and spatial stability of given
symbionts with respect to their hosts and/or a particular geographic province.
An accurate and repeatable methodology is key to describing the diversity of
symbionts found in cnidarians and is an endeavour that is currently a component of
many studies that are being conducted on corals. The ITS-2 provides "species" level
resolution within the genus (Lajeunesse, 2001) and thus was an ideal choice for the
work ongoing in my own laboratory. To implement the day-to-day use of this marker
in defining symbiont diversity, we conducted a series of preliminary studies and
validation steps with this marker, and in doing so, encountered a number of problems
that we feel have relevance to the coral reef community.
The first issue unfolded as a consequence of our inexperience using the screening
methodology that has been developed for ITS-2, namely denaturing gradient gel
electrophoresis (DGGE). This technique allows for rapid screening of multiple
samples and promotes the identification of new sequences types based on new band
positions on a gel, which can then be validated using sequencing. In our preliminary
runs using this technique to explore symbiont diversity in ITS-2 amplified from
genomic DNAs isolated from Porites lobata and Porites evermanni, we obtained
complex banding patterns that were difficult to interpret. For speed, simplicity and
familiarity with the technique we chose to clone the PCR products as a means of
separating the individual ITS-2 types in the mixture for sequencing rather than using
DGGE. It is noteworthy that these two species had previously been analyzed from
the same geographic location and designated as containing ITS-2 type C15
symbionts using DGGE (LaJeunesse et al., 2004).
A survey of multiple clones originating from 6 colonies of P. lobata and 3 colonies of
P. evermanni revealed that they contained from 1 to 6 and 3 to 8 ITS-2 types
respectively, all of which belonged to clade C. A number of the newly characterized
sequences were shared by both species and multiple colonies within a species, and
the most commonly encountered ITS-2 type was a previously undescribed ITS-2
sequence type found in all six colonies of P. lobata and two of the three P. evermanni
colonies. Other novel sequences were only found in a single colony of one species.
Interestingly, C15 was only identified in only 4 of the 8 clone libraries and was not the
dominant type present in either coral species. Cloning has been criticized as a
downstream analytical tool in assessing symbiont diversity because of the potential
83
for introducing PCR and cloning error and generation of non-biologically relevant
variants. In reality, the generation of sequences using clone libraries is achieved
using fewer rounds of PCR than DGGE and cloning is prone to less error than PCR.
In an attempt to reconcile the large discrepancy between the ITS-2 diversity obtained
using a cloning approach and that reported as a result of DGGE, we examined how
our characterized cloned sequences behaved in DGGE. These analyses revealed
that very closely related sequences (13 bp difference) exhibited different migration
rates on the denaturing gradient gels, while sequences that exhibited higher level of
variation (14 bp change) migrated to a very similar position on the gel . The position
of bands on the DGGE is used as the criterion for selecting potential y new
sequences for further characterization. Those that occupy well-documented positions
are often bypassed and defined according to the initial sequence obtained for a band
at that specific location. Our results suggest that selecting new bands for further
analyses using this methodology may be skewed towards those bands that show the
least degree of sequence divergence and has the potential to completely overlook
novel sequences with the same migration characteristics as already defined bands.
The ITS-2 is considered to be a "species" level marker and is thus is widely utilized to
examine fundamental questions in coral biology. Assigning an individual membership
within a group based on ITS-2 sequence identity is grounded in the assumption that
each DNA sequence obtained is representative of an individual symbiont. Although it
is well known that ITS-2 is a multi-copy marker, for other systems it has been clearly
demonstrated that the multiple sequence copies within a genome are subject to
concerted evolution. This is a process by which differences in re-iterated copies of a
sequence within a genome become homogenized and thus in molecular analyses
appear as a single sequence type that is representative of the complement of copies
in the cell (e.g. Hillis et al., 1991). To validate this assumption for ITS-2 in
Symbiodinium, we developed an analytical protocol that al ows us to PCR amplify the
marker from individual symbiotic cel s. Our preliminary data show that individual cells
freshly isolated from the sea anemone Aiptasia pulchella possess up to seven
sequence copies of the ITS-2 and that these copies are not the same (Figure 1). In
fact, the sequence copies from a single symbiont cell represent ITS-2 identities
belonging to three different clades, B, C and E.
We have ruled out methodological error as a possible explanation for these finding
and our results have now been independently verified in a similar study exploring
ITS-1 copies in single Symbiodinium spp. cells isolated from Acropora millepora (van
Oppen et al., 2005). These data demonstrate that in both the ITS-1 and 2 regions of
the ribosomal array, the intragenomic ITS sequence copies are not uniform or
84
homogeneous. As such, the underlying assumption that the isolation of one or
multiple ITS sequence infers the presence of one or multiple individual, respectively,
is not met. Perhaps equally complicating from an interpretational standpoint is the
lack of a predictable pattern in the ITS-2 types belonging to different symbionts
individuals isolated from the same host. We have found that in some cases an
individual symbiont contains multiple ITS-2 types belonging to three different clades,
as shown in the example above, but other individuals possess multiple ITS-2 types
belonging to one only one clade.
In conclusion then, our data suggest that: 1) the diversity of ITS-2 sequence types
within individual coral colonies is greater than previously reported; 2) the diversity
ITS-2 sequence types varies among colonies of the same species; 3) the prominence
of a single band on a DGGE gel does not always infer dominance of a single
sequence type in a mixture; and 4) there is significant variation of ITS-2 sequence
types within individual symbiont cells (intragenomic variation) that confounds the
interpretation of the data generated using this marker. Collectively these data
highlight the importance of validating the molecular methodologies used to explore
biological diversity, contextualize a re-evaluation of the current literature based on
ITS-2 sequence data, and lastly, provide the rationale for the development of new
single copy markers for exploring diversity within the genus Symbiodinium.
References
Baker, AC (2003) Flexibility and specificity in coral-algal symbiosis: Diversity,
Ecology, and Biogeography of Symbiodinium. Annual Review of Ecology
Evolution and Systematics 34: 661-689
Coffroth, MA and Santos, SR (2005) Genetic Diversity of Symbiotic Dinoflagellates
inthe Genus Symbiodinium. Protist 156: 19-34
LaJeunesse, T. C. (2001). Investigating the biodiversity, ecology, and phylogeny of
endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS
region: In search of a "species" level marker. J. Phycol. 37, 866-880
Hillis DM, Noritz C, Porter CA and Baker RJ (1991). Evidence of biased gene
conversion in concerted evolution of ribosomal DNA. Science 251: 308-310
LaJeunesse TC (2001). Investigating the biodiversity, ecology, and phylogeny of
endosymbiotic dinoflagellates in the genus Symbiodinium using the ITS
region: in search of a ` species' level marker. Journal of Phycology 37: 866-
880
LaJeunesse TC, Bhagooli R. Hidaka M, Done T, deVantier L, Schmidt GW, Fitt WK,
Hoegh-Guldberg. (2004b) Closely-related Symbiodinium spp. differ in relative
dominance within coral reef host communities across environmental,
latitudinal, and biogeographic gradients. Mar. Ecol. Prog. Ser. 284: 147-161
van Oppen, M. J. H., Miego, J. C., Sanchez, C. A. & Fabricius, K.E. (2005). Diversity
of algal symbionts (zooxanthellae) in octocorals: the roles of geography and
host relationships Mol. Ecol. 14, 24032417
85
Distinct differences in a Symbiodinium EST
library compared to other dinoflagellates
William Leggat1, Ove Hoegh-Guldberg1, Sophie Dove1, David Yellowlees2
1 Centre for Marine Studies, University of Queensland, St Lucia 4067 Australia; 2
Biochemistry and Molecular Biology, James Cook University, Townsville, 4811,
Australia
Introduction
Coral reefs are dependent upon the symbiosis between the dinoflagellate
Symbiodinium sp. and their coral host. Coral are now severely threatened by a
variety of stressors, ranging from localised (point pollution) to broad-scale
anthropogenic stresses 2 (river run off, global warming). How the coral symbiosis will
respond to these stresses is dependent upon the genetic complement that the
individual partners, and therefore the symbiosis as a whole, contain. For example,
mass coral bleaching, where large portions of the symbiontic dinoflagellate
population symbionts areis expelled from the coral host, often resulting in death of
the host, have been correlated to increases in seawater temperatures only slightly
above long term summer averages, presumably driven by global warming 3. This
slight increase in temperature leads to a breakdown of the photosynthetic capacity of
4-6
the alga, the exact mechanism of which is not clearly understood , and subsequent
expulsion of the alga from the host. In this case how the symbiosis as a whole
responds to the thermal stress is dictated by the proteins which are expressed by
Symbiodinium. Therefore a greater knowledge of the repertoire of proteins that are
present in each of the symbiotic partners may provide clues to how they will respond
current and future stress events.
Until recently our knowledge of the genetic complement of Symbiodinium, and
dinoflagel ates as a whole, was extremely limited. In 2002 only 32 protein sequences
had been obtained for all dinoflagellates, some of which indicated that dinoflagellates
were significantly different from any other photoautotroph. The presence of a form II
Rubisco 7, previously found only in anaerobic purple non-sulfur bacteria, ; a
chloroplast genome replaced by plasmid-like minicircles, encoding an extremely
reduced number of genes 8,9, ; and a unique light harvesting protein (peridinin-
10
chlorophyll a binding protein, PCP) all indicated a unique genetic complement in
dinoflagellates. Since then there has been an explosion in the number of sequences
available for dinoflagellates, with four large scale expressed sequence tag (EST)
projects 1,11-13, totalling over 10000 sequences, being released. However all of these
studies used long term algal cultures grown under normal (non-stressful) conditions.
With this in mind we constructed and analysed a cDNA library for stressed
Symbiodinium (short term heat stress, long term heat stress, increased ammonia,
increased inorganic carbon) with the aim to better understand the genes that
dinoflagellates, in particular Symbiodinium, express under stressful conditions.
Analysis of the Symbiodinium EST library
A total of 2447 clones from a Symbiodinium cDNA library were randomly picked and
sequenced from the 5'end, with an average of 654bp of sequence obtained for each
clone. A total of 1698 ESTs were found more than once, and were grouped into 594
clusters/contigs giving a total of 1343 unique EST sequences. Given the large
number of dinoflagellate ESTs recently deposited in the GeneBank, the
86
Symbiodinium sequences were blasted against these ESTs. Of the 1343 ESTs
obtained for Symbiodinium, 102 were found to match sequences from other
dinoflagellates with a bit score greater
a.
than 100. Translated blast searches
against NCBI protein database identified
30
530 sequences that had bit scores unction 25
greater than 50, indicating a match. The
distribution of these EST was analysed
f
i
ed f 20
and sorted by organism "best hit" and by
function, here it cou
w
ld be assigned.
15
This distribution was then compared to
10
the distribution of genes from another
s
with identi
dinoflagellate EST project
EST 5
(Lingulodinium polyedrum1) (Figure 1).
The distribution of the Symbiodinium
% of 0
n
sequences was significantly different
from that seen in L. polyedrum.
unication
nknow
m
aintenance etabolism repliction
M
A
th/division Energy U
Metabolic genes (25%) were the largest
Stress/defense
N
ell m
D
row
Photosynthesis
C
category identified, followed by proteins
ell com
C
ell g
associated with cell communication
b.
Transcription/translation Transport
C
(13%) and transcription/translation
X Data
35
(13%) in Symbiodinium. In L.
30
polyedrum the largest categories were
metabolism (25%),
i
sm matches 25
transcription/translation (21%) and 20
unknown (17%). Perhaps the most
significant difference was associated
15
with stress and defence genes where
10
many more were identified in
Symbiodinium (7% vs 1%), reflecting the
5
stressful conditions used to generate % of ESTs with organ 0
this library. The organism group with the
te
t
s
ngi
ae
greatest number of protein matches
rate
aryote
Fu
l ate Alg
were vertebrates (32%), the majority of
nd Plan
Vertebrate
Aveola
Prok La
verteb
lage
In
which were metabolic (26%),
nof
Di
transcription/translation (18%) or cell Figure 1 Distribution of proteins identifie
communication (14%) proteins. The
d
through Blastx with a score greater than 50. .
a
alveolates as an entire group (including Distribution by function. b. Distribution by best
dinoflagellates) were next greatest hit to organisms. Black bars = Symbiodiniu
(alveolates 11%, dinoflagellates 8%)
m
(this study), white bars from the dinoflagel at
e
followed by the prokaryotes and land Lingulodinium polyedrum 1.
plant (13% each). Again this was
significantly different from L. polyedrum.
Conclusions
This study represents the first large scale exploration of the Symbiodinium genome
and the first attempt to examine the stress response genes of dinoflagellates. Initial
results demonstrate that the stress conditions used to generate this library has up-
regulated a set of genes which have not been previously characterised from
dinoflagellates. Analysis of these genes should enable use to determine how the
expression of these genes respond during stress events.
87
References
1. Tanikawa,N., Akimoto,H., Ogoh,K., Chun,W. & Ohmiya,Y. Expressed Sequence
Tag Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase.
Photochem. Photbiol. 80, 31-35 (2004).
2. Hughes,T.P. et al. Climate Change, Human Impacts, and the Resilience of Coral
Reefs. Science 301, 929-933 (2003).
3. Hoegh-Guldberg,O. Climate change, coral bleaching and the future of the world's
coral reefs. Mar. Fresh. Res. 50, 839-866 (1999).
4.
Smith,D.J., Suggett,D.J. & Baker,N.R. Is photoinhibition of zooxanthellae
photosynthesis the primary cause of thermal bleaching in corals? Global Change
Biology 11, 1-11 (2005).
5. Jones,R.J., Hoegh-Guldberg,O., Larkum,A.W.D. & Schreiber,U. Temperature-
induced bleaching of corals begins with impairment of the CO2 fixation
mechanism in zooxanthellae. Plant Cel Environ. 21, 1219-1230 (1998).
6. Warner,M.E., Fitt,W.K. & Schmidt,G.W. Damage to photosystem II in symbiotic
dinoflagellates: a determinant of coral bleaching. Proc. Natl. Acad. Sci. USA 96,
8007-8012 (1999).
7. Rowan,R., Whitney,S.M., Fowler,A. & Yel owlees,D. Rubisco in marine symbiotic
dinoflagellates: Form II enzyme in eukaryotic oxygenic phototrophs encoded by a
nuclear multigene family. Plant Cell 8, 539-553 (1996).
8. Zhang,Z., Green,B.R. & Cavalier-Smith,T. Single gene circles in dinoflagel ate
chloroplast genomes. Nature 400, 155-159 (1999).
9.
Barbrook,A.C., Symington,H., Nisbet,R.E.R., Larkum,A.W.D. & Howe,C.J.
Organisation and expression of the plastid genome of the dinoflagellate
Amphidinium operculatum. Molecular Genetics and Genomics 266, 632-638
(2001).
10. Weis,V.M., Verde,E.A. & Reynolds,W.S. Characterization of a short form
peridinin-chlorophyl -protein (PCP) cDNA and protein from the symbiotic
dinoflagellate Symbiodinium muscatinei (Dinophyceae) from the sea anemone
Anthopleura elegantissima (Cnidaria). J. Phycol. 38, 157-163 (2002).
11. Hackett,J.D. et al. Migration of the Plastid Genome to the Nucleus in a Peridinin
Dinoflagellate. Curr. Biol. 14, 213-218 (2004).
12. Bachvaroff,T.R., Concepcion,G.T., Rogers,C.R., Herman,E.M. & Delwiche,C.F.
Dinoflagellate Expressed Sequence Tag Data Indicate Massive Transfer of
Chloroplast Genes to the Nuclear Genome. Protist 155, 65-78 (2004).
13. Patron,N.J., Waller,R.F., Archibald,J.M. & Keeling,P.J. Complex Protein
Targeting to Dinoflagellate Plastids. J. Mol. Biol. 348, 1015-1024 (2005).
88
How does the Symbiodinium EST database add
to our knowledge of zooxanthellae and their
metabolism?
David Yellowlees1 and Bill William Leggat2.
1. Biochemistry and Molecular Biology, James Cook University, Townsville 4811
Australia; 2. Centre for Marine Studies, University of Queensland, St Lucia 4067
Australia.
Knowledge of the metabolism of corals and their symbionts is an essential aspect of
understanding how these unique organisms function and thrive in their natural
environment. A detailed appreciation of their metabolism and how it can change in
response to various factors can also inform environmental management decisions. In
the past metabolic studies have been addressed using standard biochemical
techniques of enzyme isolation and metabolite measurement. These have been
fraught with difficulties because of the small tissue volume available in corals.
Progress has therefore been slow a
nd our knowledge of carbon metabolism let alone
nitrogen and phosphate assimilation is poor.
The availability of molecular techniques has the potential to revolutionise our
understanding of coral metabolism and the availability of the coral EST library will be
instrumental in this. We have now established an EST database for zooxanthel ae
which, while not as extensive as that of coral, will be of major assistance in defining
some of the major aspects of metabolism, including photosynthesis, in Symbiodinium
and defining possible differences between the different clades. The library was
developed using corals (and zooxanthellae) that had been exposed to a number of
stress regimes including thermal, nutrient addition (ammonium), and the depletion
and addition of inorganic carbon. This was to maximise the expression of genes
crucial to the management of these conditions.
Analysis of the database has identified a number of avenues for future investigation
which wil significantly increase our understanding of the metabolism and other
processes in zooxanthellae. This includes the identification of Hsp proteins and the
ubiquitin protein targeting pathway initiated by thermal stress; the discovery of a
number of carbonic anhydrase isoforms not previously identified in zooxanthel ae; the
identification of a number of genes coding for carbohydrate metabolising enzymes
including those in photosynthesis; the identification of a number of genes coding for
proteins involved in nitrogen acquisition and assimilation.
Thermal Stress
With the relentless increase in ocean temperatures as a consequence of global
warming it is important to understand the responses of Symbiodinium. We have now
identified members of the Hsp100, Hsp90 (2 isoforms), Hsp70 protein families along
with DNAJ DnaJ (3 isoforms), a Hsp70 co-chaperone, and p23, a Hsp90 co-
chaperone (Leggat et al 2005). Previously only Hsp90 and Hsp70 have been
characterised from dinoflagellates. We have also identified a suite of proteins
essential for the ubiquitin mediated protein degradation cycle. This opens the way to
look at expression patterns under a variety of thermal conditions and the differences
between clades.
89
Nitrogen acquisition and assimilation
The relative roles of zooxanthellae and the host in the acquisition and assimilation of
nitrogen hashave been contentious. The database has identified a number of the
genes encoding crucial transport proteins and enzymes in this process. As a
consequence we can now look at the expression of nitrate and ammonium
transporters in zooxanthellae, the subsequent reduction of nitrate to ammonium and
its assimilation into glutamine by glutamine synthase. This represents a significant
advance in our knowledge and provides the capacity to determine how the
expression of these genes changes under different nitrogen regimes.
Carbon acquisition, fixation and assimilation
Efficient carbon acquisition and its subsequent fixation underpin the relationship
between host and zooxanthellae. An understanding of this process will assist in
predicting the consequences of an increase in seawater inorganic carbon
concentrations predicted to occur with global climate change. We have previously
demonstrated that zooxanthellae possess a carbon concentrating mechanisms
(CCM) to overcome the presence of a Form II Rubisco in Symbiodinium (Leggat et al
1999). However there is a lack of knowledge on how this CCM operates. The
database has revealed the presence of a number of genes encoding carbonic
anhydrase isoforms. These sequences will be useful in testing our model for CO2
acquisition and elucidating the localisation of these enzymes in zooxanthellae. The
downstream processes following carbon acquisition can also be investigated as EST
database contains a number of the genes encoding enzymes in the utilisation and
storage of the fixed carbon eg glyceraldehyde 3-phosphate dehydrogenase.
Conclusions and future directions
The availability of the database is going to be a
g
valuable tool in addressin a number
of issues that will contribute to our basic knowledge of this unicellular algal symbiont
but also as a powerful investigative tool in understanding how zooxanthellae and the
coral symbiosis in general respond to environmental changes. Given the current
focus on climate change and anthropogenic effects on coral reefs the bank of
information we have can now be applied to understanding how these organisms
respond to these challenges.
References
Leggat, W, Badger, MR, Yel owlees, D. (1999) Evidence for an inorganic carbon
concentrating mechanism in the symbiotic dinoflagellate Symbiodinium sp. Plant
Physiology, 121: 1247-1255.
Leggat, W, Hoegh-Guldberg, O, Dove, S, Yellowlees, D. (2005) Initial analysis of the
heat shock genes from an EST library from the dinoflagellate (Symbiodinium sp.)
symbiont of reef-building corals Submitted.
90
Theme 4: Targeted Research Working Group
joint field methods (May 22)
Co-Chairs: Rob van Woesik, Yossi Loya
Task: Our task was to assess the dynamics of corals and coral reef communities,
identify key process variables, and environmental change under different levels of
protection (e.g. MPAs)
Attending: Juan Carlos Oritiz, Ove Hoegh-Guldberg, Assaf Zenvoluni, Jeffry
Deckenback, Roberto Iglesias Prieto, Ron Johnstone, Mark Davy, Ayax Ruiz Diaz
Ruiz, Luisa Falconer, Noa Shenkar, Gidon Winters, Eugenia Sampayo, Paulina
Kaniewska, Jez Roff, Tracy Ainsworth, Jessica Gilner, Ranjeet Bhagooli, Peter Ralph,
Christian Wild, Baraka Ruguru, Glen Holmes.
Morning session:
1. Introduction to the Coral Reef Targeted Research Working Group joint field
methods (R. van Woesik).
2. What are the main ecological questions for the field based studies (generate
table).
3. What are the key hypotheses (e.g., Global climate change will alter coral
populations to become increasingly skewed toward larger colonies Bak &
Meesters, (1999). Am Zoo 39: 56-65) (generate table)
4. What are the key dependent state variables (e.g., coral cover), and process
variables (e.g., recruitment rates, mortality rates) that need to be measured
(generate table).
Outcomes:
Afternoon session:
Sampling design and statistical analyses
5. At what spatial scale do we observe the most variance (in for example coral
cover)? To what degree does that variance vary among regions and oceans -
results of pilot studies (including a short presentation by Juan Carlos Ortiz).
6. Compromises towards an optimal design.
7. Interpretation and applications (future design modifications).
91
Tracking coral populations through time
ya2
Rob van Woesik1 & Yossi Lo
1, Department of Biological Scienc
Institute of
es, Florida
Technology, 150 West
University Boulevard, Melbourne, Florida 32901-6988, USA, E-mail: rvw@fit.edu
2Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel, E-mail:
yosiloya@post.tau.ac.il
Critical concepts and long-term trends
Recent reports of wide-scale coral bleaching, or paling of corals through the loss of
their symbiotic algae (zooxanthellae) and/or their pigments because of stress at and
above the corals' acclimation capacity, from all of the world's tropical oceans are a
major concern among scientists and resource managers (Loya et al 2001). Clearly,
coral bleaching is a global phenomenon linked to global climate change and
increasing ocean temperatures (Glynn 1991; 1993; Brown 1997; Hoegh-Guldberg
1999). In the last 2 decades mass coral bleaching events have damaged reef in
many localities, yet whether these events cause long term changes to coral
communities is unknown.
In 1998, unprecedented worldwide coral bleaching coincided with some of the
warmest Sea Surface Temperatures (SST) on record. Coral bleaching was evident
from 25oN to 33oN in Japan since nearshore SST in Okinawa were 2.5oC above the
ten-year average (Loya et al. 2001). We witnessed another coral bleaching event in
2001 in southern Japan (Van Woesik et al 2004); the thermal anomaly was of similar
intensity and duration. In a 2001 publication we showed that some species are
tolerant to thermal stresses and wil become `the winners', while others are not so
tolerant, and are destined to become `the losers' (Loya et al 2001). In 2004, we
further show
me coral populations ar
ed that so
e adjusting to thermal stress events
(Van Woesik et al 2004).
Why coral-colony size matters
Many researchers are interested in why some coral species are physiologically more
tolerant than others, and why some species will adjust to global climate change. In
addition to this we are also interested in whether size frequency distributions (i.e.,
coral colony size) will be influenced by thermal stress events. Bak & Meesters (1999)
proposed t
alter cora
hat global climate change will
l populations to become
increasingly skewed toward large colonies. Size-frequency distributions, and relative
shifts toward large colonies may have little affect effect on fitness, unless
senescence is shown to be a common trait. However, if shifts occur toward small
corals, and we know that small juvenile coral colonies are not reproductive, by
definition, and neither are most coral colonies that regress back to juvenile size after
disturbances, then fitness will be compromised and adaptati
on unlikely.
We have shown that small coral colonies are more tolerant to anomalous SSTs than
large colonies; such small size facilitates mass transfer which in turn aids survival
(Loya et al 2001; Nakamura and Van Woesik 2001). This further suggests an
increasing trend toward smaller colonies if bleaching events become more frequent.
Smaller colonies may not be reproductively competent if they are remnants of once
larger colonies, and certainly not when they are new recruits and thus immature.
Since the foundation of adaptation is based upon the notion that differential-
92
reproductive rates are facilitated by environmental influences on different individuals
within populations, shifts in size-frequency distributions toward smal er corals maybe
a sign that adaptation is less likely to occur if bleaching events are frequent --
because of repeated setbacks toward immaturity.
Community shifts
Of the 1629 colonies examined in southern Japan (van Woesik et al 2004) we
recorded colony mortality and assessed the degree of resistance (where no
discernible visual change to the colonies were evident) and the resilience among
species, depths and colony sizes. While there were obvious differences in
accordance with species, there were also differential responses within genera. Coral
mortality was more immediate and higher on the shallow reef than on the deep reef,
producing a more marked structural shift. Small faviids showed a higher tolerance
than large faviids. In May 2002, after 2 thermal-stress events, 720 colonies were
recorded (i.e., 44% of those recorded in 1998), which included surviving colonies and
new recruits of all 12 species. While the environmental conditions were similarly
extreme in 1998 and in 2001, 42% of the coral populations showed an increase in
tolerance; more resistant populations included Pocillopora verrucosa, Porites
cylindrica, Pachyseris gemmae, Favia pallida, and Favia favus colonies at two depths
(3 & 10 m), which suggest that the populations had adjusted their constitution to the
thermal stresses, while
rians
Pavona va
appeared less resistant in 2001, and the
other 6 species, which included Porites lutea, responses remained relatively similar
over time. There are only two studies that have reported on coral community shifts
associated with 2 bleaching events (Glynn et al 2001; Van Woesik et al 2004).
Hence, current information on changes to coral communities fol owing coral
bleaching is very limited and there are unclear or controversial projections
concerning the long-term effects on the structure of coral communities.
Future directions
Projected climate change may drive temperature and seawater chemistry to levels
outside the envelope of modern reef experience. As a consequence, coral reef
communities wil change. Clearly, juvenile coral colonies are more resistant to a
combination of high SST and high irradiance compared with large corals (Loya et al
2001). These results suggest an increasing trend toward smaller colonies if bleaching
events become more frequent. Furthermore, we suggest that the relative shifts
toward the above-mentioned winners and losers are dynamic, and certainly not
static. Therefore, what may appear to be a winning strategy in the short term, through
survival of small colonies or the apparent short-term survival of a `winning' growth
form, may turn out to be detrimental in the long term, especially if thermal stress
events increase in frequency and the time period for colony growth is reduced. Yet,
the only means to understand these trends is through long-term assessments of
permanent sites.
References
Bak P.M & Meesters E.H. (1999) Population structure as a response of coral
communities to global change. Amer Zool 39: 56-65
Brown, B.E. (1997) Coral bleaching: causes and consequences. Coral Reefs 16,
129-138
Glynn, P.W. (1991) Coral reef bleaching in the 1980s and possible connections with
global warming. Trends Ecol. Evo, 6, 175-179
93
Glynn PW, Mate JL, Baker AC, Calderon MO (2001) Coral bleaching and mortality in
Panama and Ecuador during the 1997-1998 El Nino-Southern Oscillation event:
Spatial/temporal patterns and comparisons with the 1982-83 event. Bull Mar Sci
69" 79-109
Hoegh-Gulberg, O. (1999) Climate change, coral bleaching and the future of the
world's coral reefs. Mar. Freshwater Res., 50 (8), 839-866
Loya Y, Sakai K, Yamazato K, Nakano H, Sambali H, Van Woesik R (2001) Coral
bleaching: the winners and the losers. Ecology Letters 4:122-131
Nakamura T, Van Woesik R (2001) Water-flow rates and passive diffusion partially
explain differential survival of corals during 1998 bleaching event. Mar Ecol Prog
Ser 212: 301-304
Van Woesik R, A. Irikawa, Y Loya. (2004) Coral bleaching: signs of change in
southern Japan. In, Coral Health and Disease (eds. Eugene Rosenberg & Yossi
Loya), Springer, 119-141
94
Final design for the permanent monitoring program at Heron Island for the
GEF-UNESCO-World Bank "Global Coral Reef Targeted Research and Capacity
Building Project"
Juan Carlos Ortiz. Centre for marine studies, The University of Queensland,
Brisbane, Australia
Coral reefs are characterized as highly dynamic systems through space and time
(Veron 1995, Connel et al 1997). As a consequence of the rapid deterioration
processes affecting coral reefs in the world a better understanding of the way these
systems change and the factors affecting these changes is crucial for predicting the
future of coral reefs as well as its management (Done 1992, Hoegh-Guldberg 1999,
Hughes et al 2003).
Most of the ecological monitoring programs target coral cover as the main response
variable (Done 1992, Glynn 1994, Connel et al 1997). This is a very slow changing
variable (Hughes et al 2003, Palandro 2003) that should not be rely on its own as an
indicator of reef stress since by the time a significant change in coral cover is
observed little can be done in terms of management to protect the reef. In contrast,
relative abundance of coral species, evenness of coral taxa and specific population
dynamics may be much more dynamic variables that could provide managers with
earlier alerts about processes affecting the reef (Bak and Meesters 1999, Hughes
and Connell 1987, Tanner et al 1994, Loya et al 2001).
In this project a series of permanent sites will be set on the reef surrounding Heron
Island using different sampling techniques within the permanent sites. The main aim
of this project is to assess the dynamics of corals and coral reef organisms at the
population and community level, and identify key process variables under
environmental change
Hypotheses to be tested:
H1 Coral cover, Taxa evenness, relative abundance of coral species, coral
recruitment and coral population size frequency distributions will remain constant
over time.
H2 Coral taxa evenness responds to climate change at the same speed as coral
cover.
H3 Global climate change will alter coral populations to become increasingly skewed
toward larger colonies (Bak et al 1999).
H4 Population probability of quasi-extinction is constant across different coral
species. (Loya et all 2001)
H5 Coral population (x) probability of quasi-extinction is independent of surrounding
total coral cover.
H6 Coral population (x) probability of quasi-extinction is independent of surrounding
total coral taxa evenness
Methods:
The design explained below is the result of a comprehensive pilot study developed
between January and December 2005. The details of this pilot study will be
published separately.
Six to seven sites will be established around Heron Reef: 3 to the south, three to the
north and potentially (depending on the logistics) one to the east side of the reef
(Figure 1). Site is going to be considered as a random factor established
systematically. This will be the most important level of information for the analyses.
95
Coral
cascade
s
Second
point
Fourth
point
Coral
gardens
Potential
replacement of
Coral Gardens
Potential
7th site
Harry's
bommie
Figure 1: Selected sites around Heron Reef. In white: selected sites, in grey: potential
sites
Within each site two sampling areas are going to be fixed. One at 0 meters and one
at 5 meters at low tide (reef flat and upper reef slope). Each experimental area will be
defined as a 4m wide by 140m long band. Within each sampling area 3 different
sampling units will be used:
· 1m x 15m fixed photo transects (n=8). The transects will be fixed systematically in
relation to the long axis, and randomly in relation to the short axis. Since on the
short axis (4m) there are just 4 potential position to locate a 1m wide transect the
randomisation process will be done ensuring that each potential position is used
twice.
· 4x4m quadrates (n=3), the quadrates will be fixed systematically along the
sampling area (beginning, middle point and end of the sampling area).
· Tagged colonies of targeted species (the species will be chosen after the first set
of photo transects ha been taken)
s
. Between 5 and 10 colonies of each of 5
targeted species will be tagged. The colonies will be selected randomly from the
area that is not being photo-sampled within th
SE A/B
marker
e sampling area. The 5 targeted
species will not be the same at both depths due to spatial segregation (most of the
species found on he reef flat are different than the ones found on the upper reef
slope). The information obtained from the closest transect to each colony will be
extrapolated as the surrounded environment for that particular colony. These
colonies wil be sample each sampling time for physiological parameters. For a
schematic presentation of the sampling design see figure 2.
Each site will be sampled every 6 months (unless a big disturbance occurs, in which
case extra sampling times wil be included).
96
Information to be obtained from each sampling unit:
Photo transects:
Coral cover, coral community composition (relative abundance of the different
families of corals and other substrates). Percentage of bleaching, prevalence of
corals affected by diseases that can be detected from the photo, evenness, coral
mortality, number of new recruits, etc.
Population size distribution of 4-6 targeted species: These species will be selected
ensuring that their abundances, growth form and size range is appropriate to be
measured precisely from the photos.
Diagram of a site (6-7 around the island)
140m
0m
4m
depth
4 8 tagged
colonies per sp
(4 spp) randomly
8 Permanent 15x1 m transects
3 4x4m permanent quadrats
chosen from the
fixed systematically on the long
fixed systematically along the
studied area
axis and randomly on the short
140 m
(outside the
one
transects and
quadrats)
4m
4m
depth
140m
Figure 2: Diagram of an experimental site
Photo quadrates:
Coral cover, composition of coral community (relative abundance of the different
coral growth forms and other substrates), percentage of bleaching, prevalence of
corals affected by diseases that can be detected from the photo, evenness of
morphotypes, and coral mortality. This information will be used to validate remote
sensing images.
Tagged colonies:
From the fragments taken: Symbiont density, concentration of photosynthetic
pigments, protein concentration, and tissue thickness.
Statistical analysis
The data will be analysed using linear and non-linear regression models. The
number of factors and the way each factor will be treated will vary between variables.
"Bulk" variables: The design will allow to perform regression models relating the
dependent variables with the covariates as well as make comparisons between sites.
A finite population correction will be used to correct for the assumption of all classical
statistical analyses that the sampled area is a very small part of the statistical
universe, because the proportion of the site area that is being sampled is relatively
high (about 20% of the total area of the site).
Physiological variables: The design shown in table one will allow to perform
regression models relating the dependent variables with the covariates as well as
compare between sites.
97
Population size distribution: we propose to do an analysis in 3 stages where first we
use the mean size per site, then the square root of the variance of the mean size of
the site and finally the forth root of the skewness of the size distribution. All this
statistics are calculated per site generating a single number for the entire site. Then a
regression is done using the values of the six or seven sites trough time.
The only disadvantage with this approach is that in contrast with the analyses done in
the previous two sections, this analysis will not allow us to compare between the
sites at a specific time, although this comparison is not the main focus of this study.
Using this design we maximise the statistical power of detecting changes in the
different variables studied, considering the logistical limitation involved in the
sampling. Additional statistical analysis and predicting models could also be applied
to further explore more detailed information obtained simultaneously during the
sampling.
References:
Bak, R. P. M. and E. H. Meesters (1999). "Population structure as a response of
coral communities to global changes." American Zoologist 39: 56-65.
Connell, J. H., T. P. Hughes and C. C. Wal ace (1997). A 30-Year Study of Coral
Abundance, Recruitment, and Disturbance at Several Scales in Space and
Time. Ecological Monographs 67(4): 461-488.
Done, T. (1992). Constancy and Change in Some Great Barrier Reef Coral
Communities: 1980-1990. American Zoologist 32: 655-662.
Glynn, P. W. (1994). State of Coral-Reefs in the Galapagos Island. Natural Vs
Anthropogenic Impacts. Marine Pollution Bulletin 29(1-3): 131-140.
Hoegh-Guldberg, O. (1999). Climate Change, Coral Bleaching and the Future of the
World's Coral Reefs.
eshwater Research
Marine and Fr
50: 839-866.
Hughes, T. P., A. H. Baird, D. R. Bel wood, S. R. Connol y, C. Folke, R. Grosberg, O.
Hoegh-Guldberg, J. B. C. Jackson, J. Kleypas, J. M. Lough, P. Marshall, M.
Nystrom, S. R. Palumbi, J. M. Pandolfi, B. Rosen and J. Roughgarden (2003).
Causes of Coral Reef Degradation - Response. Science 302(5650): 1503-
1504.
Hughes, T. P. and J. H. Connell (1987). Population Dynamics Based on Size or Age?
A Reef-Coral Analysis. American Naturalist 129(6): 818-829
Loya, Y., K. Sakai, et al. (2001). "Coral bleaching: the winners and the losers."
Ecology Letters 4(2): 122-131.
Palandro, D., S. Andrefouet, F. E. Muller-Karger, P. Dustan, C. M. Hu and P. Hallock
(2003). Detection of Changes in Coral Reef Communities Using Landsat-5
Tm and Landsat-7 Etm+ Data. Canadian Journal of Remote Sensing 29(2):
201-209.
Tanner, J. E., T. P. Hughes and J. H. Connell (1994). Species Coexistence,
Keystone Species, and Succession: A Sensitivity Analysis. Ecology 75(8):
2204-2219
Veron, J. E. N. (1995). Corals in Space and Time: the biogeography and evolution of
the scleractinia. UNSW Press, 321p.
98
Field methods to detect change in the coral
communities of south eastern Mexico
Rob van Woesik1, Jessica Gilner1 and Eric Jordan-Dahlgren2
1 Department of Biological Sciences, Florida Institute of Technology, 150 West
University Boulevard, Melbourne, Florida 32901-6988, USA, E-mail: rvw@fit.edu
2 Unidad Academica Puerto Morelos, Instituto de Ciencias del Mar y Limnologia,
Universidad Nacional Autonoma de Mexico, Apartado Postal 1152, 77500 Cancun,
Mexico
Driving hypothesis
Coral bleaching is a global phenomenon linked to global climate change and
increasing ocean temperatures (Glynn 1991; Brown 1997; Hoegh-Guldberg 1999).
We are in general interested in which coral species will become `the winners', while
others that are not so tolerant, and are destined to become `the losers' (Loya et al
2001), and in particular whether size frequency distributions will be influenced by
thermal stress events. Our null hypothesis is that global climate change will alter
coral populations to become increasingly skewed toward large colonies (Bak &
Meesters 1999).
Reef monitoring
Rapid ecological assessments are less concerned about the statistical power of
detecting a change than long-term monitoring programs (Andrew and Mapstone
1987). The latter requires appropriate strategies to account for the spatial
arrangement of organisms that should yield accurate estimates of coral colony
abundance. Besides the spatial considerations associated with data collection,
monitoring programs must also decide on the degree of site permanency, and at
what level to randomize. A ful y randomized design provides characterizations that
account for the spatial arrangement of individuals in the communities. High statistical
power achieved through randomized designs means that any measured change is
indicative of a change in the community, yet sampling effort may need to be
tremendously intensive (Green and Smith 1997). It is often convenient to determine
geomorphological reef units a priori and sample within those units, instead of
identifying high variance among geomorphological units posteriori and then lack the
rigor to identify changes over time. A time series of permanent photo-quadrats
provides valuable insight into population dynamics, but lacks general information
regarding the spatial arrangement of organisms within the community as a whole.
Pilot study
Most pertinent to any long-term monitoring program is the over-riding question: do we
have enough information, or statistical power, to detect changes if changes occur?
We undertook a pilot study at Puerto Morelos, Mexico, in September 2002 to
understand the spatial variance in the distribution of coral communities as a
prerequisite for a longer-term monitoring program. Three evenly distributed sites
were set up in the back reef habitats in shallow water (~2-3 meters). Sampling was
conducted at 3 levels of resolution using replicated: (1) 50 m video transects, (2) 50
m x 2 m belt transects, where we identified each colony to species level and
99
measured each colony's maximum diameter, and (3) 1 m2 permanent quadrats,
where we photographed each quadrat as a set of 4 x 50 cm2 images using high
resolution digital cameras and assessed each quadrat for coral recruits. Still images
were randomly extracted from video tapes and used for analysis. To derive summary
statistics we recorded the taxa or substrate type under 10 random selected points per
image. Back calculations were made to determine the appropriate sample size
required at a 90% confidence level, and suggested that we need a h gh number of
i
transects to detect a change. Furthermore, we found that an assessment of size
frequency distributions of coral colonies using belt transects and permanent sites
may be one of the most useful sampling strategies for monitoring.
Proposed monitoring program
We propose to establish at least 10 Sites along the eastern Cancun Peninsula of
Mexico, which are being subjected to different levels of human disturbance (Fig.1).
There will be 2 permanent 50 m by 10 m Stations within each site. Three permanent
4 m x 4 m Quadrats will be permanently marked within each station. At least 6, 50
m x 1 m Belt Transects will be randomly placed in each station and each meter will
be photographed with a stereo set of digital cameras mounted on a 1 m2 frame, with
a white color bar attached. We also plan to tag a number of Colonies within each
Station to examine physiological attributes.
Fig. 1. Proposed study sites
along a suspected gradient of
human influence on the
Cancun Peninsula.
Proposed outcomes
Sites will be examined to assess whether a pollution gradient exists, and whether
different coral communities are reflected along that gradient. The advantage of long-
term monitoring of permanent quadrats allows the same colonies to be tracked
through time allowing elucidation of recruitment, death, and survival rates of the
organisms under question. There are some observations in the literature of partial
mortality pertaining to stress, but its quantification is limited. This study will elucidate
(i) to what degree fragmentation occurs under which conditions and track the fate of
the fragments, (i ), what proportion of colonies are new recruits, and (iii) what
proportion are survivors. In total, this study will quantify the dynamics of the coral
communities, examine size-frequency distributions over time, assess relative shifts in
species composition and determine differential colony growth rates, recruitment and
post-settlement success. Similar methods will be employed in other regions, for
example Heron Island and Zanzibar, which will standardize data and facilitate
comparisons.
100
References
Andrew, N. L., and B. D. Mapstone. 1987. Sampling and the description of spatial
pattern in marine ecology. Mar. Biol. Ann. Rev. 25:39-90.
Bak P.M & Meesters E.H. (1999) Population structure as a response of coral
communities to global change. Amer Zool 39: 56-65
Brown, B.E. (1997) Coral bleaching: causes and consequences. Coral Reefs 16,
129-138
Glynn, P.W. (1991) Coral reef bleaching in the 1980s and possible connections with
global warming. Trends Ecol. Evo, 6, 175-179
Glynn PW, Mate JL, Baker AC, Calderon MO (2001) Coral bleaching and mortality in
Panama and Ecuador during the 1997-1998 El Nino-Southern Oscillation event:
Spatial/temporal patterns and comparisons with the 1982-83 event. Bull Mar Sci
69" 79-109
Hoegh-Gulberg, O. (1999) Climate change, coral bleaching and the future of the
world's coral reefs. Mar. Freshwater Res., 50 (8), 839-866
Loya Y, Sakai K, Yamazato K, Nakano H, Sambali H, Van Woesik R (2001) Coral
bleaching: the winners and the losers. Ecology Letters 4:122-131
101
Theme 5: Integrated research on coral bleaching
and disease (May 24-26)
This workshop was held primarily to explore areas of overlap and synergy between
the GEF Targeted Research Groups on Coral Bleaching and Related Ecological
Factors (BTRG) and Coral Disease (DTRG).
Discussion conveners/coordinators
John Bythel (j.c.bythel @ncl.ac.uk, Newcastle University)
Drew Harvel (cdh5@cornell.edu, Cornell University)
Ove Hoegh-Guldberg (oveh@uq.edu.au; University of Queensland)
Goals of workshop
The goals of the workshop were to establish important areas of research overlap
between the two Targeted Research Groups and to
tial
explore areas of poten
synergy where integrating or coordinating research activities between the groups will
improve outputs. Specific areas considered were:
1. Survey and monitoring of coral populations, disease and bleaching
2. Biomarkers of stress and resistance (="health") in corals
3. Environmental and bacterial causes of coral bleaching.
The initial talks during this section of the workshop included details of planned survey
and monitoring of coral populations. The first talk was by Robert van Woesik, which
was followed by Ernesto Weil. Discussion then ensued as regard the fine tuning of
these field programs to accomplish the ambitions of the Disease Working Group.
Participants of the workshop on "Integrated research on coral bleaching and disease"
102
Bleaching and physical/chemical stress on coral reefs
v
O e Hoegh-Guldberg1, Michael P. Lesser2 and Roberto Iglesias Prieto3
1. Centre for Marine Studies, University of Queensland, St Lucia 4067 Australia; 2.
Department of Zoology and Center for Marine Biology University of New Hampshire,
Durham, NH 03824, USA; 3. Unidad Académica Puerto Morelos, Instituto de
Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apartado
Postal 1152, Cancún QR 77500, México
Corals loose colour (bleach) when they become stressed, which may arise for a
number of reasons including high irradiance or prolonged darkness, excess
ultraviolet radiation, low salinity, toxins such as Cu2+ or CN-, infection by pathogens
such as Vibrio and temperatures that are either too high or too low (Hoegh-Guldberg
1999). It may or may not result in the death of corals, an outcome which depends on
the extent and length of exposure of corals to a particular stress. Given the key role
that corals play in building coral reefs and the fact that recent global episodes of
mass coral bleaching have been tied directly to global warming, bleaching has
assumed a central importance in our understanding of stress on coral reefs. Despite
this importance, our understanding of bleaching is still evolving and during the past
several years, several discoveries have caused us to rethink models and
perspectives gained over the past 30 years of study. In this paper, we will discuss
some of these new ideas and perspectives.
The definition of "bleaching"
Bleaching has been defined by a number of authors as a paling of coral tissues due
to the loss of the cells and/or pigments of zooxanthel ae (Symbiodinium). While this
definition is true in the broadest sense, it fails to incorporate some interesting and
important details. Recently Enríquez et al (2005), have shown that the absorptance
(the percentage of incoming radiation absorbed) of coral surfaces have a non-linear
relation with pigment density. This effect is the result of multiple scattering of light on
the highly reflective coral skeleton. As a
result of multiple scattering, the optical
BL
Hidden variation
path length and thereby the probability of
absorption by the photosynthetic pigments
of the algae is larger in intact coral
100
structures than in isolated cells (see paper
by Enríquez, Mendez an Iglesias-Prieto,
80
)
this volume). The effect of the skeleton
e
(
%
60
on the optical path length is inversely
proportional to the density of pigment,
40
o
r
p
t
a
nc
which means that the probability of
Abs
absorption by a pigment decreases as the
20
pigment density of targets increases. This
0
property has an important effect on the
0
20
40
60
80
100
Symbiont density (%)
perceived colour of a coral undergoing Figure 1. The non-linear relationship
stress. Previous definitions of bleaching between absorptance and symbiont
have assumed that absorptance varies density in reef building corals. BL
linearly with the density of symbionts. But
indicates symbiont density below which
was shown in Figure 1, absorptance visible bleaching begins to occur.
(under the effect described above) will
103
vary asymptotically with the density of symbionts. That is, we will only see a visible
effect on the colour of corals (bleaching) in the last stages of any change in symbiont
e implicatio
density. Th
ns of this change are that we are missing much of the story if
we only record the colour of corals as perceived by our eyes. While colour is a useful
proxy under some circ m
u stances,
make caref
we need to
u
e
l measurements of th
actual density and pigment concentration of Symbiodinium in reef-building corals.
The cellular mechanisms of mass coral bleaching
Since 1979, the world's coral reefs have been undergoing an escalating frequency
and intensity of mass bleaching events. The latest two examples, the 2005
bleaching event in the Caribbean and the 2006 bleaching event in the Western
Pacific are among the most severe bleaching events ever. Mass bleaching events
are triggered by warmer than normal seas, which, under sunlit conditions result in
symbiotic dysfunction (Brown 1997, Hoegh-Guldberg 1999; Lesser 2004). These
changes result in a loss of pigment and/or symbionts, the last stages of which
manifest as stark paling of coral colonies. From here the prognosis may be recovery
or death of a coral colony, the difference being driven by the intensity of the thermal
stress and the length that the corals were exposed to it for. Roughly, corals exposed
to low amounts of thermal stress (1-8 degree heating weeks; anomaly size X
exposure time, Strong et al. 2000) tended to recover, while those exposed to greater
stress for longer (9-14 degree heating weeks) tend to die (Hoegh-Guldberg 2001).
Purely ecological studies of mass bleaching have provided a limited understanding of
mass bleaching events, largely due to the lack of an explicit mechanism to explain
mass bleaching. In this respect, studies that have focused on defining the underlying
mechanism of bleaching have added some very useful information (Hoegh-Guldberg
and Smith 1989; Glynn and D'Croz 1990; Iglesias-Prieto et al 1992; Lesser et al
1990, Lesser 2004; Fitt and Warner, 1995; Jones et al. 1998). In this respect,
consensus has focused on a model in which bleaching in response to elevated
temperature begins with decrease in
the photosynthetic activity of the
association, most probably associated
with a decrease in the ability of
Symbiodinium sp. to process captured
light. Whether this is associated with
a lesion close to or down stream from
Photosystem II (PSII) is still being
debated. However, most evidence
points to the dysfunction of PSII
leading to an accumulation of oxygen
radicals as the energy that normally
goes to photochemistry is donated to
oxygen via over-excited PSI and PSII
components. Under so-called normal
conditions, a range of components
such as superoxide dismutase (SOD)
and ascorbate peroxidase (APO)
quench and convert superoxide
molecules into less harmful
components. However, under high Figure 2. Photoinhibition model for
temperature and light, these systems thermal stress related bleaching
are over-run, with deleterious (adapted from Hoegh-Guldberg 19 9)
9
consequences for cellular
components.
104
What happens next is not as clearly defined. Work by Tchernov and others (See
Tchernov et al, this volume) has found clear signs that cellular integrity of the algae
may play an important role, and that some more resistant varieties of Symbiodinium
may have more thermally stable thylakoid membrane (TM) lipid compositions. In
other work, Tchernov and co-workers have shown that caspase inhibitors will slow
the process of bleaching, implying that apoptosis (programmed cell death) may be
triggered in coral host cells as they become stressed by active oxygen and other
stress related changes. This important insight follows on from the demonstration by
Dunn et al. (2002) that bleaching is associated with the activation of apoptosis in
Symbiodinium. These processes were hinted at in the early 1990s by Gates et al.
(1992), and while provocative, require further substantiation. One might summarise
the steps leading to bleaching as follows:
1. Environmental stress (physical, chemical or biological change)
2. Excitation processing by PSII is blocked
3. Oxidative stress increases
4. Membranes become dysfunctional
5. Apoptotic processes begin in response to general cellular dysfunction
6. Host cells with algae detach
7. Corals bleach in last stages of process.
Writing these steps down, immediately illuminates how little we do know about the
cellular mechanisms of bleaching. The linkage between oxidative stress and
membrane dysfunction is still unclear but the mechanism described by Tchernov and
coworkers, could be very important in explaining differences between thermally
tolerant and susceptible genotypes of Symbiodinium. It is also apparent that these
studies are heavily focused on the dysfunction of Symbiodinium. While
Symbiodinium is clearly in stress, it would be a mistake to downplay the role of the
host. By comparison to the multitude of studies focused on Symbiodinium, studies
on how thermal stress affects the host are relatively few. It is clear that we need to
focus some of the future efforts on the role and susceptibility of the coral host to
thermal stress.
One final twist: the role of "photic hell" in escalating stress.
One of the consequences of the extended optical path length within the coral skeletal
environment is the enhancement of light fields. As shown by Enriquez et al (2005),
irradiance, within the surfaces making up the coral suface, can be up to 4 fold higher
than the incidence of light. This, combined with the self-shading effect of pigment
targets, means that the light available to a Symbiodinium cell within the tissues of a
coral may vary many fold depending on the density of Symbiodinium within the
tissues of the coral host. At high densities of cells, the path length is short and light
is efficiently absorbed. At low densities of Symbiodinium like those seen in bleached
corals, light has a very much longer path length as it bounces around within the
highly reflective skeletal surfaces. Under these situations, irradiances can increase
many fold above that of the incoming irradiance, as shown experimentally by Kuhl
(1995). These light levels may be extremely damaging ("photic hell"), further
exacerbating the stress that had original y led to the decline of Symbiodinium within
the tissues. This, combined with the non-linearity of absorptance against
Symbiodinium density, may explain the rapidity with which mass coral bleaching
events seem to affect coral reefs. Not only are we not aware of the non-visible
changes that are occurring within the tissues of coral reefs, but falling densities of
Symbiodinium led to a dramatic escalation of stress as the efficiency of light
absorption is enhanced in the tissues of stressed corals. The resulting positive
105
feedback loop (less cells more stress) creates an environment that is extremely
hostile to Symbiodinium, which leads to the rapid removal of most of the remaining
cells. It is clear we need to explore this phenomenon further, especially the role it
probably plays in inhibiting recovery. In this regard, understanding this feedback
phenomenon may suggest ways that recovery may be enhanced following a thermal
event.
Implications for disease
The phenomenon of "photic hell" has implications for any situation in which the
density of Symbiodinium falls below a particular critical limit. This includes many
coral disease-like states in which white patches or areas develop in which
pigmentation has disappeared. While it is speculative, the rapid progression of these
syndromes may be driven by the extreme photic environments that develop within
tissues as pathogens and other influences reduce the population density of
Symbiodinium. A better understanding of how the consequence of these changes
affect the in-tissue light environments is necessary if we are to understand the
changes that occur as disease-like syndromes progress.
References
Brown, BE (1997) Coral Reefs, 16: 129-138.
Dunn, SR, Bythell, JC, Le Tissier, MDA, Burnett, WJ, Thomason, JC (2001) JEMBE:
272: 29 53
Enriquez, S., Mendez, E, Iglesias-Prieto, R (2005) Limnol. Oceanogr., 50(4), 2005,
10251032
Fitt WK, Warner ME (1995) Biol Bull 189 (3): 298-307
Gates, RD, Baghdasarian, G, Muscatine, L. (1992) Biol. Bull. 182: 324-332
Glynn, P. W., DCroz, L. (1990) Coral Reefs 8: 181-91.
Hoegh-Guldberg, O. (1999) Marine and Freshwater Research, 50:839-866.
Hoegh-Guldberg, O. (2001). in "Fingerprints" of Climate Change. Editor: Gian-Reto
Walther, KLUWER ACADEMIC/PLENUM PUBLISHERS, New York, U.S.A.
Hoegh-Guldberg, O. and G.J. Smith (1989) Exp. Mar. Biol. Ecol. 129:279-303.
Hoegh-Guldberg, O. (1999) "Coral bleaching, Climate Change and the future of the
world's Coral Reefs." Review, Marine and Freshwater Research Mar. Freshwater
Res. 50:839-866.
Iglesias-Prieto, R., Matta, J. L., Robins, W. A., Trench, R. K. (1992), Proceedings of
the National Academy of Science 89: 302-305.
Jones, R., Hoegh-Guldberg, O., Larkum, A. W. L., Schreiber, U. (1998) Plant Cell
and Environment 21: 1219-30.
Kühl, M., Cohen, Y., Dalsgaard, T., Jřrgensen, B.B., and Revsbech, N.P. (1995).
Marine Ecology Progress Series 117: 159-172.
Lesser, M. P., Stochaj, W. R., Tapley, D. W., Shick, J. M. (1990) Coral Reefs 8: 225-
232.
Lesser, M.P. (2004) Experimental Coral Reef Biology. Journal of Experimental
Marine Biology and Ecology, 300: 217-252.
106
Bacterial bleaching of corals
Eugene Rosenberg
Department of Molecular Microbiology & Biotchnology, George S. Wise Faculty of
Life Sciences, Tel Aviv University, Ramat Aviv, Israel 69978
Vibrio shiloi is the causative agent of bleaching in the coral Oculina patagonica in the
Mediterranean Sea. Bleaching of O. patagonica in the sea occurs only in the summer
when water temperatures exceed 26°C. High temperature also plays a key role in
bleaching by V. shiloi in laboratory aquaria experiments. At 29°C V. shiloi-induced
bleaching is rapid and complete; below 20°C no bleaching occurs, even with a very
high inoculum size of V. shiloi. The data indicate that several critical V. shiloi
virulence factors are produced only at the elevated summer water temperatures,
suggesting that primary effect of temperature is on the pathogen, not the host.
Adhesion and chemotaxis
The first step
ctious proce
in the infe
ss is the adhesion of V. shiloi to the coral surface.
V. shiloi is attracted to the mucus obtained from O. patagonica. Motility and
chemotactic behavior were present when the bacteria were grown either at 16°C or
25°C. The bacteria then adhere to a -galactoside-containing receptor on the coral
surface. The temperature of bacterial growth was critical for the adhesion of V. shiloi
to the coral. When the bacteria were grown at the low winter temperature (16-20°C),
there was no adhesion to the coral regardless of at what temperature the coral had
been maintained. However, bacteria grown at elevated summer temperatures (25-
30°C) adhered avidly to corals maintained either at low or high seawater
temperatures. The important ecological significance of these findings is that the
environmental stress condition (high temperature) is necessary for the coral
bleaching pathogen to initiate the infection and become virulent.
Penetration and intracellular multiplication.
Electron micrographs of thin sections of O. patagonica following infection with V.
shiloi demonstrated large numbers of bacteria in the epidermal layer of the coral.
Using monoclonal antibodies specific to V. shiloi, it was shown that the observed
intracellular bacteria were, in fact, V. shiloi. The gentamycin invasion assay was used
to measure the kinetics of V. shiloi penetration into the epidermal cells. The assay
relies on the fact that the antibiotic gentamicin does not penetrate into eukaryotic
cells. Thus, only V. shiloi cells which have penetrated into the coral cells escape the
killing action of gentamicin. After adhesion was complete (ca. 12 h), the bacteria
began to penetrate into the coral as determined by both total counts and colony
forming units. By 24 h, 40-50% of the inoculated V. shiloi had penetrated into corals
cel s. From 24-72 h, the intracellular bacteria multiplied (based on total counts),
aching 3 x 10 bacteria per 1 cm
8
coral frag
3
re
ment. When the infected corals were
maintained at the high summer temperatures, the bacteria remained at 108-109 cells
per cm3 for at least two weeks.
Differentiation into the viable-but-not-culturable (VBNC) state
At the same time that V. shiloi multiplies inside the coral tissue (24-48 h after
infection), the number of colony-forming units (cfu) decreases more than a thousand
fold. Entry of bacteria into a state described as VBNC has been reported repeatedly
107
with a large number of bacterial species. A bacterium in the VBNC state has been
defined as "a cel which can be demonstrated to be metabolical y active, while being
incapable of undergoing the sustained cellular division required for growth on a
medium normal y supporting growth of that cel ". Intracel ular V. shiloi cells fit that
definition, bu
VBNC that
t unlike most cases of
have been studied, this is not brought
about by starvation or low temperature. Rather, the entry of V. shiloi into the VBNC
state occurs inside the coral epidermis, where nutrients are abundant.
Toxin P production and mode of action
V. shiloi produces extracellular toxins that block photosynthesis, bleach and lyse
zooxanthellae. The toxin responsible for inhibition of photosynthesis, referred to as
Toxin P, is the following proline-rich peptide: PYPVYAPPPVVP. In the presence of
NH4Cl, the toxin causes a rapid decrease in the photosynthetic quantum yield of
zooxanthellae. The toxin binds irreversibly to algal membranes, forming a channel
that allows NH
+
3, but not NH4 , to rapidly pass, thereby destroying the pH gradient
across the thylakoid membrane and blocking photosynthesis. This mode of action of
Toxin P can help explain the mechanism of coral bleaching. Toxin P is produced at
more than ten-fold higher levels at 29°C compared to 16°C.
Role of superoxide dismutase
When corals are infected with V. shiloi at the permissive temperature of 28°C, the
bacteria adhere, penetrate and begin to multiply intracellularly. If the infected corals
are then shifted slowly (0.5°C per day) to lower temperatures, the bacteria die and
the infection is aborted. The failure of V. shiloi to survive inside coral tissue at
temperatures below 20°C is because it does not produce superoxide (SOD) at these
low temperatures. This hypothesis was supported by constructing an SOD minus
mutant. At 28°C, the mutant adhered to the coral, penetrated into the tissue and then
died. Death only occurred when the coral was exposed to light. The most reasonable
explanation for these data is that the high concentration of oxygen and resulting
oxygen radicals produced by the zooxanthel ae during photosynthesis is highly toxic
to bacteria and is one of the mechanisms by which corals resist infection. At high
temperatures, V. shiloi produces a potent SOD which helps it to survive in the coral
tissue.
Transmission of the disease
The observations that V. shiloi could not be found inside O. patagonica during the
winter and that the bacterium could not survive in the coral below 20°C indicate that
bleaching of O. patagonica requires a fresh infection each spring, rather than the
activation of dormant intracellular bacteria. Using fluorescence in situ hybridization
(FISH) with a V. shiloi-specific deoxyoligonucleotide probe, it was found that the
marine fireworm Hermodice carunculata is a winter reservoir for V. shiloi. Worms
taken directly from the sea during the winter contained 0.6-2.9 x 108 V. shiloi per
worm by FISH analysis. To test if worms carrying V. shiloi could serve as vectors for
transmitting the pathogen to O. patagonica, worms infected with V. shiloi were placed
in aquaria containing O. patagonica. Corals that came into contact with the infected
worms showed smal pa
bleached tiss
tches of
ue in 7-10 days and total bleaching in
17 days. Uninfected worms did not cause bleaching. Thus, H. carunculata is not only
a winter reservoir for V. shiloi, but also a potential vector for transmitting the
bleaching disease to O. patagonica.
108
Bleaching of Pocillopora damicornis by Vibrio coralliilyticus
Vibrio coralliilyticus is an etiological agent of bleaching of the coral Pocillopora
damicornis on coral reefs in the Indian Ocean and Red Sea. Strains of V.
coralliilyticus have been isolated from diseased corals on the Eilat coral reef, Red
Sea and in the Indian Ocean, near Zanzibar. All of these V. coralliilyticus strains
bleached P. damicornis in controlled aquaria experiments. The infection of P.
damicornis by V. coralliilyticus shows strong temperature dependence. Below 22°C
no infection occurred. At 24-26°C the infection resulted in bleaching, and at 27-29°C
the infection caused rapid tissue lysis and death of the coral. V. coraliilyticus
produces a potent metalloproteinase at temperatures above 26°C. This enzyme
shows high levels of amino acid sequence homology to a range of proteases found in
members of the family Vibrionaceae. Because the purified protease caused tissue
lysis of corals, it was suggested that at the elevated seawater temperature, where the
protease is produced, the bacterium attacks the coral tissues, whereas at the lower
temperatures the intracellular algae is the target and the outcome of the infection is
bleaching.
References
Ben-Haim, Y., M. Zicherman-Keren and E. Rosenberg. 2003. Temperature-regulated
bleaching and lysis of the coral Pocillopora damicornis by the novel pathogen
Vibrio coralliilyticus. Appl. Environ. Microbiol. 69: 4236-4242.
Banin, E., S. K. Khare, F. Naider and E. Rosenberg. 2001. A proline-rich peptide
from the coral pathogen Vibrio shiloi that inhibits photosynthesis of zooxanthellae.
Appl. Environ. Microbiol. 67: 1536-1541.
Rosenberg, E. L. Falkovitz. 2004. The vibrio shiloi / oculina patagonica model system
of coral bleaching. Ann. Rev. Microbiol. 58: 143-159.
Sussman, M., Y. Loya, M. Fine and E. Rosenberg. 2003. The marine fireworm
Hermodice carunculata is a winter reservoir and spring-summer vector for the
coral-bleaching pathogen Vibrio shiloi. Environ. Microbiol. 5: 250-255.
Undergraduate Jez Roff discusses coral disease with Professor Eugene Rosenberg's
fol owing his presentation.
109
Experimental analysis of bacterial ecology of
bleaching and disease
John Bythell1 and Olga Pantos2
1. Newcastle University UK and 2. San Diego State University
The potential interaction between GCC-mediated temperature increase and coral
disease prevalence and severity is an area that requires urgent attention. To date,
the processes of disease causation and the physiological effects of environmental
stress (e.g. bleaching) have been largely studied in isolation, despite some cases of
bleaching being directly attributable to pathogens. However, we clearly need a
holistic understanding of the sequence of events involved in environmental stress-
mediated alterations in disease susceptibility (Fig 1).
Health status
Zooxanthellae density, chl a,
tissue depth, C:N
stress
Microbial defences
Environmental change
Antimicrobial activity,
(e.g. temperature,
mucus reserves
irradiance, salinity)
Disease
Microbial ecology
Incidence and progression
Bacterial community
rates, histopathology
structure, population density
Figure 1. Environmental stress leads to a reduced health status which may be manifest
as visible bleaching. The extent to which this stress reduces the coral's antimicrobial
defences is unknown. Subtle effects of health status on the microbial community have
been shown, but the sequence of alterations in microbial communities versus appearance
of disease signs and specific pathogens has not yet been elucidated. Changes in
associated microbial community may also promote environmental stress via changes in
metabolite production, for example in surface mucus layer biomineralisation processes,
leading to a positive feedback loop and potential bacterial proliferation.
There are limitations to using solely traditional culture-based approach to assess
these processes. Most (>99%) marine microbes cannot be cultured using standard
plate techniques, we know that some coral diseases may be caused by multiple
agents (microbial consortia) and diseased and healthy corals are connected by
seawater, so potential pathogens are probably everywhere. These factors make it
difficult to prove that disease symptoms are unique or that identical symptoms are
produced in the laboratory during infectivity studies. Culture-independent methods,
including in situ localisation of bacteria and sequence analysis are powerful tools for
assessing these processes and this seminar outlines studies of known diseases and
experimental studies of stress experiments on Heron Island using these approaches.
We conclude that a significant area of overlap between the BTRG and DTRG is in
elucidating the processes of lesion generation, disease progression and mortality
post-bleaching.
110
Post-mortem Microbial Communities on Dead
Corals: Implications for Nutrient Cycling?
Ron Johnstone, Mark Davey an
d Glen Holmes.
Centre for Marine Studies, University of Queensland, St Lucia 4067 Australia.
Nutrient cycling (nitrogen, phosphorus, carbon) is an essential part of coral reef
ecosystems and is fundamental to their development and survival. While essential to
the ecosystem, nutrient loads and associated processes often vary both within and
between reefs. These variations also occur over both temporal and spatial scales.
Variations from "mean ambient" nutrient loads can lead to dramatic ecosystem wide
impacts as has been well documented in locations such as Kaneohe Bay, Hawaii and
Chapwani, Zanzibar.
Coral bleaching and mortality is becoming an increasingly common and regular event
and this increased mortality has the potential to significantly alter the nutrient
dynamics within reef ecosystems. Questions that are then raised include: How do
the microbial communities developing post-mortem on bleached corals influence
nutrient cycling?; What is the significance of changes in microbial community
structure?; and; What is the overall significance of such changes for ecosystem
functionality at different spatial and temporal scales?
Scale of Influence
Reports of areas of impact following a bleaching event are usually obtained via
remote sensing tools such as satellite or aerial imagery. These areas are typically
reported as a 2-dimensional area represented by coral colonies and other community
members. However, if the 3-dimensional surface area is considered, the actual area
of influence at microbial process scale is significantly larger. Hence, the functional
significance of processes based on these reported surfaces may be currently greatly
underestimated; particularly for reefs dominated by highly structured corals.
The challenge then becomes how to sensibly relate micro-scale processes and
community performance to the larger scale function and functionality of reefs both
spatial y and temporally. Aspects of this work include examining community
composition, critical conditions for processes (e.g. how much biomass is needed
before denitrification can occur
ever?), succession
if
and the
dynamics of these
communities/processes under
different conditions.
At the microbial scale we know
through DDGE and FISH
analysis (In prep) that there is
a distinct change in community
composition and abundance in
the days immediately following
coral mortality. While results
Day 0
y
Day
Day 4
Day
Day 8
y
Day 12
Day
Day
Day 1
Day 4
14
from these analyses are initial,
Figure 1: DGGE analysis indicating community
the findings indicate that within
structure change over time.
the first 10 days of mortality,
111
the bacterial and cyanobacterial community undergo a series of successions with an
increase in overall abundance for a range of species (as would be expected).
At the process level, experiments have thus far concentrated on nitrogen fixation,
with present research focussing upon denitrification, nitrification rates ongoing. The
initial results based on mortality of Acropora sp. show a dramatic increase
(approximately an order of magnitude) in the amount of nitrogen fixed pe
in
r unit area
the 12 days following coral mortality due to thermal stress. When considering the
actual surface area involved in mass mortality events (due to bleaching) compared to
that reported from remote sensing, the levels of new nitrogen entering the system are
therefore likely to be highly significant to the ecosystem as a whole.
Overall significance
It is generally accepted that there is a global increase in coral mortality (Wilkinson
2000,2002 & others). For example, in Fiji 40% of corals were lost during the 2000
and 2002 bleaching events. Fiji has approximately 10 000 km2 of coral reefs. If we
assume that 50% of this coverage is live coral, then the mortality following the 2000
and 2002 bleaching events led to approximately 2000 km2 of potential new surface of
nitrogen fixation. If we then consider that this 2000 km2 is based on a 2-dimensional
area, then it is not unreasonable to estimate that this "new substrate" is introducing
nitrogen into the ecosystem at rates several orders of magnitude above that which
would have otherwise been estimated based on background nitrogen fixation rates.
Fiji is not an isolated case. Other areas have reported far greater losses. For
example, 4,230 m2 (93%) of Acropora palmata and 1,760 m2 (98%) of Acropora
cervicornis lost at Looe Key, Florida between 1983 and 2000 (Miller et al, 2002).
Conclusions and future directions
The initial conclusions from this work are that the microbial processes occurring in
the immediate period following coral bleaching and mortality can have a significant
influence on the nutrient dynamics, and therefore function, of coral reef ecosystems.
Ongoing work includes:
· Closer examination of microbial functional groups nitrogen fixers, denitrifiers
and nitrifiers;
· The construction of oxygen, carbon, nitrogen, & phosphorous budgets
applicable across both temporal and spatial scales;
· An examination of critical states or conditions necessary to support different
processes;
· An examination of the influence of the physical complexity of coral surfaces
(different coral types) in terms of their microbial community development,
type, and process function
· An assessment of the po
ese change
tential for th
s in microbial community to
influence other key ecological processes such as the recruitment of primary
producing organisms and other functional groups onto newly exposed dead
coral surfaces.
Increasing our knowledge in this area will greatly aid coral reef managers in making
decisions about what, if any, action that should be taken in order to improve a reefs
ability to recover from this increasingly common event.
References
112
Miller MW, Bourque AS, Bohnsack JA (2002) An analysis of the loss of acroporid
corals at Looe Key, Florida, USA: 1993-2000. Coral Reefs 21: 179-182
Wilkinson CR (2000) State of the Coral Reefs of the World: 2000. Global Coral Reef
Monitoring Network (GCRMN). Australian Institute of Marine Science, Townsville
Wilkinson CR (2002) State of the Coral Reefs of the World: 2002. Global Coral Reef
Monitoring Network (GCRMN). Australian Institute of Marine Science, Townsville
Drew Harvell discusses aspects of John Bythell's presentation to workshop in
"Integrated research on coral bleaching and disease".
113
PAM fluorometry and Symbiodinium stress.
Roberto Iglesias-Prieto
Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología,
Universidad Nacional Autónoma de México, Apartado Postal 1152, Cancún QR
77500, México.
The development of non-invasive techniques based in the detection of chlorophyll a
(Chl a) fluorescence have revolutionize the way we investigate how primary
producers respond to different stressful conditions. In the particular case of
dinoflagellates in symbioses with invertebrates most of the studies have been
motivated by the pressing need to investigate the role played by temperature in coral
bleaching. During the last 10 years, the use of these techniques have been
instrumental for the study of the mechanisms used by these organisms to cope with
stressful levels of several other environmental variables such as light, nutrients and
salinity.
The origin of the variable fluorescence
Under physiological conditions, the
majority of the Chl a fluorescence
emitted by the cells is originated at
the antenna complexes associated
with photosystem II (PSII). PSII is a
supramolecular complex that
catalyze the light-mediated oxidation
of water and the reduction of the
plastoquinone pool (Fig. 1). Once
light is collected by the
photosynthetic pigments, the
excitation energy is transferred to
the reaction centers were primary
photochemistry takes place. Using Fig 1. Schematic representation of PSII.
this excitation energy, one of the two
Chl a molecules that form the core of the reaction center (P680) is oxidized, reducing
a pheophytin which in turn reduces the first stable electron acceptor QA.
Simultaneous to the formation of the first radial pair at the reaction center, P680- is
reduced by a tyrosine residue located near the core. Ultimately, electrons traveling
through PSII are supplied by the oxidation of water. Further down the PSII electron
transport chain, QA reduces QB, which once neutralized by two protons migrates
through the membrane to reduce the cytochromes. The intensity of the fluorescence
emitted by a sample would be dependent on the redox state of the internal electron
transport chain. When QA is oxidized, the fluorescence yield is minimal, as most of
the energy would be used for moving electrons through the chain. In contrast, when
QA is reduced, the excitons migrates back to the antenna, producing maximum
fluorescence yields. We can use these changes in the fluorescence yield as a
sensitive probe of the redox state of the electron transport chain.
Excitation pressure as a measure of stress
When the rate of primary photochemistry (delivery of electrons) is smaller or equal to
the rates of consumption of reducing power in the chloroplasts, the excitation
114
pressure in PSII is minimal. When the rate of photochemistry is larger than the
consumption of reducing power, excitation pressure raises. This condition can be
sensed by the photosynthetic apparatus as (i) an over-reduction of the platoquinone
pool and (ii) as an increase in the pH between the internal and external parts of the
tylakoid membrane. Co
result in increases in excitation pressure can be
nditions that
dependent on the light intensity (increases in the rate of light capture), and/or
reductions in the rates of reducing power consumption or sink limitations. As a
response to the increase in excitation pressure, photosynthetic organisms have
developed a series of mechanisms that provide protection against the formation of
free radicals that can destroy the membrane. The inductions of these mechanisms
can be detected using fluorescence techniques as an increase in non-photochemical
quenching (NPQ). These photoprotective mechanisms compete for the Chl a excited
states with the reaction centers, releasing harmlessly the excess excitation as heat.
Sink limitation as a source of light stress
When cultured Symbiodinium cells or intact corals are exposed to elevated
temperatures, one of the first detectable responses is an increment in the excitation
pressure relative to controls maintained at permissive temperatures. These
observations can be interpreted as indicative of a sink limitation, in particular it has
been suggested the inactivation of RUBISCO is responsible for the initiation of the
events that lead to coral bleaching (Jones et al. 1998). Similar results can be
obtained when corals are exposed to other stressors. Symbiodinium cells growing
actively consume approximately 1/3 of the reducing power generated by the
photosynthetic electron transport chain to reduce and assimilate nitrogen.
(Rodríguez-Román & Iglesias-Prieto 2005). Exposures to medium without nitrogen or
containing enough nitrogen as ammonium to support growth, result in significant
increases in the excitation pressure consistent with sink limitation. In general,
excitation pressure could be generated when one or more reactions consuming
reducing power are either inhibited, or have limitations in the supply of substrate. In
this context, exposure to any stressful condition would have synergic effects when it
is combined with reduced water flow. The short-term responses to increases in
excitation pressure include the induction of the photoprotective mechanisms, and the
induction of the PSII repair cycle. If the stressful conditions persist for longer periods
of time (days to weeks), a reduction in the optical cross section of the cells is
achieved by reducing the concentration of photosynthetic pigments. These
reductions effectively reduce the light capture rates in most primary producers. In the
particular case of corals, when the symbionts become optically thinner, the multiple
scattering of incident light by the highly reflective aragonite skeleton, result in
increases in the absorption rates (Enríquez et al. 2005). This effect could explain why
small increments in temperature can propagate into the complete collapse of the
symbiosis. It is important to notice that bleaching can be the result of exposures to
any environmental extreme, and that several signs of several coral diseases include
the loss of pigmentation.
Conclusions and future directions
Exposure to a variety of stressors results in increments in the excitation pressure
over PSII. From the perspective of the symbiont, any stress would be perceived as
an imbalance between the rates of light capture and the use of reducing power.
These results suggest a generalized unit of stress in Symbiodinium could be amount
of radiation absorbed but not use for photosynthesis. Although the use of PAM
fluorometers for the study of stress in Symbiodinium has been very fruitful, the use of
biochemical techniques to corroborate the results obtained from fluorescence
determinations in need.
115
References
Enríquez S, Méndez ER, Iglesias-Prieto R (2005) Multiple scattering on coral
skeletons enhances light absorption by symbiotic algae. Limnology and
Oceanography 50:1025-1032
Jones RJ, Hoegh-Guldberg O, Larkum AWD, Schreiber U (1998) Temperature-
induced bleaching of corals begins with impairement of the CO2 fixation
mechanism in zooxanthellae. Plant, Cell and Environment 21:1219-1230
Rodríguez-Román A, Iglesias-Prieto R (2005) Regulation of photochemical activity in
cultured symbiotic dinoflagellates under nitrate limitation and deprivation. Marine
Biology 146:1063-1073
Rob van Woesik, Eugene Rosenberg and Baraka Ruguru in discussion during
workshop.
116
Oxidative Stress, Bleaching, and Coral Disease
Michael P. Lesser
Department of Zoology and Center for Marine Biology University of New Hampshire,
Durham, NH 03824, USA
Coral reefs are experiencing unparalleled levels of anthropogenically-induced stress.
Current estimates on the rate of decline in the health of coral reefs, and the loss or
change in community structure of reefs are of worldwide concern (Wilkinson 2000). It
is estimated that a combination of physical, chemical and biological stresses will
cause the decline of between 40 to 60% of the world's coral reefs over the next 50
years unless appropriate steps are taken (Wilkinson 2000). Until recently, global
climate change was seen as just one of many factors (e.g., eutrophication, coastal
development, sedimentation, over-fishing) responsible for the decline in the health of
coral reefs (Wilkinson 1999) while the time scales of change due to global climate
effects was believed to be slow and other anthropogenic causes a higher priority for
study. In 1998, however, an estimated 16% of the world's living corals were
eliminated in a single warming event related to El Nińo (Wilkinson 2000). During this
event, sea temperatures warmed to 2-3°C above long-term average summer
temperatures and resulted in a catastrophic "bleaching" event that caused significant
mortality of several species of coral (e.g., both the expulsion of zooxanthallae and
host tissue death occurred). The impact of this thermal event on the percent cover of
shallow coral reefs worldwide and the projection of continued rising sea temperatures
under greenhouse warming (Hoegh-Guldberg 1999) has radically changed the focus
of a large proportion of the research community towards understanding the potential
impact of greenhouse driven climate change on the world's coral reefs. Bleaching as
a result of thermal stress is not the only threat from global climate change and coral
reef biologists from around the world have had to use new experimental tools at all
levels of biological organization in their efforts to understand how reefs work,
determine which corals will survive anthropogenically driven change, and predict
what reefs will look like at the end of the next century. In essence, who will be the
"winners" and the "losers" (Loya et al. 2001)?
Exposure to elevated temperatures alone, UVR alone, or in combination can result in
photoinhibition of photosynthesis in zooxanthellae. Photoinhibition occurs as a result
of the reduction in photosynthetic electron transport, combined with the continued
ption of
high absor
excitation energy. One consequence of reducing electron
transport is the production of reactive oxygen species (ROS) such as singlet oxygen
[1O
-
2 ] superoxide radicals [O2 ], hydrogen peroxide [H2O2], and hydroxyl radicals
[OH.]) for which there are many cellular targets including photosystem II and the
primary carboxylating enzyme, Rubisco in zooxanthellae (Lesser 2004). The
enzymes superoxide dismutase, catalase, and ascorbate peroxidase act in concert to
inactivate superoxide radicals and hydrogen peroxide, thereby preventing the
formation of the most reactive form of ROS, the hydroxyl radical, and subsequent
cellular damage. Enzymic defenses in the animal host occur in proportion to the
potential for photooxidative damage in symbiotic cnidarians. However, high fluxes of
ROS in the host or zooxanthellae can overwhelm the protective enzymatic response
and result in hydroxyl radical production via the Fenton reaction. Oxidative stress
has been proposed as a unifying mechanism for several environmental insults that
cause bleaching (Lesser 2004). Oxidative stress can lead to bleaching of
zooxanthellae via exocytosis from coral host cells or apoptosis. A cellular model of
bleaching in symbiotic cnidarians has been developed and includes oxidative stress,
117
PSII damage, membrane instability, DNA damage, and apoptosis as underlying
processes. This model is consistent with a variety of biomarker proteins expressed
in corals during thermal stress.
Damage to
er
photosystem II (PSII) reaction cent s in the zooxanthellae, specifically at
the D1 protein of PSII, following exposure to elevated temperatures and solar
radiation, is believed to be an important factor leading to the bleaching of corals and
caused by ROS (Lesser 2004). Damage or impairment of PSII function is easily
detected using non-destructive active chlorophyll fluorescence techniques.
Instruments have been developed that incorporate protocols to measure the multiple
photochemical turnover (pulse amplitude modulated [PAM]), and single
photochemical turnover of PSII (fast repetition rate [FRR]) in the laboratory and in the
field. These instruments measure, non-destructively, fluorescent transients that
provide information on the efficiency of PSII and can discern chronic photoinhibition
from dynamic photoinhibition, the former representing damage to PSII and the latter
a protective regulatory response of the photosynthetic apparatus. The underwater
FRR has been used to examine diel cycling and dynamic versus chronic
photoinhibition of corals in shallow and deep waters. One advantage of the FRR
versus the PAM instrument is that because of the protocol used to measure
fluorescent transients, a series of flashlets that saturate PSII in microseconds this
instrument can also measure the optical cross section of PSII which is a valuable
parameter for discerning dynamic versus chronic photoinhibition. An underwater
version of the PAM instrument is commercially available and has been widely used to
study diel changes in the quantum yield of PSII fluorescence and its relationship to
differences between photochemical and non-photochemical quenching, or dynamic
photoinhibition. Changes in PSII fluorescence have also been correlated with
changes in the concentration of D1 protein during exposure to thermal stress and/or
solar radiation. Other models of thermally induced bleaching have suggested that
the dark reactions of photosynthesis are affected initially, leading to sink limitation,
over reduction of photosynthetic electron transport, oxidative stress, and damage to
PSII. Lesser (2004) combined the PSII inhibition model with the dark reaction model
with oxidative stress as the common effector mechanism for the inhibition of
photosynthesis and subsequent bleaching.
One of the most significant changes on coral reefs has been the emergence of
diseases and the potential relationship to global climate change. While coral
bleaching is most commonly associated with thermal stress and its physiological
consequences, bleaching in at least one species of coral, Oculina patagonica, is
caused by the bacterium, Vibrio shiloi, subsequent to thermal stress. A virulence
factor associated with bleaching in this coral is the expression of superoxide
dismutase by the bacterium that allows it to survive the hyperoxic tissues of the coral.
From this observation one could reasonably ask does resistance and pathogenicity in
coral diseases related to the modulation of the ROS environment? Do corals depend
on hyperoxia and ROS production to defend themselves, and do pathogens have to
have, as one of several virulence factors, the ability to detoxify ROS? Is Oculina
patagonica a better model of coral disease than coral bleaching per se? What other
virulence factors are essential for successful attachment, penetration, multiplication,
and toxin production in a wide range of pathogens and how does the ROS
environment affect these steps in the infection process?
118
References
Hoegh-Guldberg, O. (1999) Coral bleaching, Climate Change and the future of the
world's Coral Reefs. Marine and Freshwater Research Mar. Freshwater Res.
50:839-866.
Lesser, M.P. (2004) Experimental Coral Reef Biology. Journal of Experimental
Marine Biology and Ecology, 300: 217-252.
Loya, Y., Sakai, K., Yamazato, K., Nakano, Y., Sembali, H., van Woesik, R. (2001).
Coral bleaching: the winners and the losers. Ecology Letters, 4: 122-131.
Wilkinson, C. (2000). Status of Coral Reefs of the World. Global Coral Reef
Monitoring Network, Australia.
Lunch time discussions with Michael Lesser. Left to right: Michael Kuhl, Mebrahtu
Ateweberhan and Michael Lesser.
119
Seafan epizootic and resistence to Aspergillosis.
Drew Harvell.
Department of Ecology and Evolutionary Biology, E- 321 Corson Hall, Cornell
University, Ithaca, NY 14853
This project is a test of the general hypothesis that compromised immunity drives the
Aspergillosis outbreak in seafan corals. This is a plausible hypothesis because
Aspergil us tends to be an o
that
pportunistic pathogen
colonizes immune
compromised hosts, for examples humans (Aspergillus fumigatus) and insects
(Aspergillus niger). We are examining the role of climate and environmental factors
as facilitators of Aspergillus infections. I present evidence for increased
temperatures, increasing fungal growth rate, and increased nutrients in the field,
increasing lesion severity. We are elucidating specific mechanisms of coral immunity
to disease. Our current focus is on peroxidases and chitinases, and I present
evidence of anti-fungal activity of both classes of proteins. The seafan- Aspergillus
pathosystem has been an unusually good experimental system and one future goal
is to investigate how general results from this patho-system can be extrapolated to
scleractinian-bacterial interactions.
Drew Harvell introduces workshop theme on "Integrated research on coral bleaching
and disease".
120
Ecology, Physiology and Cell Biology of `White
Syndrome' on the Great Barrier Reef
Roff J1, Ainsworth TD1,2, Kvennefors EC1, Henderson M1,2, Blackall LL 1,2,
Fine M 1,3, Hoegh-Guldberg O1
1Centre for Marine Studies, The University of Queensland, St. Lucia, QLD, 4072
Australia. 2Advanced Wastewater Management Centre, The University of
Queensland, St. Lucia, QLD, 4072 Australia. , 3 The Leon Recanati Institute for
Maritime Studies, University of Haifa, Mount Carmel, Haifa 31905, Israel.
During the past two decades there has
been a growing concern over the
worldwide degradation of coral reef
ecosystems (Hoegh-Guldberg 2004) with
recent reports estimating a global loss of
27% to date (Wilkinson 2002). Outbreaks
of disease-like syndromes have
dramatically increased in recent years,
with an exponential increase in the
number of coral diseases reported since
the initial observation of a disease
affecting scleractinian coral in 1965
Fig 1. WBD affecting Acropora palmata.
(Sutherland et al. 2004). As emphasised
Puerto Morelos, Mexico
by Richardson (1998), the incomplete
characterisation of such `new' disease-
like syndromes in the last decade has
caused confusion from not only differing terminology and symptoms for various
diseases, but also as to what constitutes a `disease' (e.g. Borger 2005).
There are at least seven different types of `white syndrome' that have been reported
from the Caribbean region; white plague (WP types I, II & III), white band (WBD type
I & II, Fig 1), white pox (WPD) and shut-down reaction (SDR - see Bythell et al. 2004
for review), four of which are known to affect Acroporid corals. Although collectively
white syndromes have played a significant contribution to a region-wide decline in
coral reefs (Aronson & Precht 2001), pathogens have only been identified within two
of these diseases. Whilst white syndromes vary in both rates of lesion progression
and patterns of tissue loss (Bythell et al. 2004), they are commonly characterised in
the field by distinct signs of tissue loss resulting from the sloughing of the coenosarc,
exposing the underlying white skeleton (Bythell et al. 2004), and are likely to
represent different etiologies.
The first qualitative descriptions of disease-like syndromes were recorded on the
Great Barrier Reef over a decade ago, and several recent studies have documented
epizootics in the northern sectors of the GBR (Dinsdale 2002, Jones et al 2004). The
results of a five year survey in conjunction with the Australian Institute of Marine
Science Long-Term Monitoring Program (Willis et al 2004) has provided a broad
overview and insight into novel syndromes and incidence across the GBR, describing
several novel syndromes (e.g. brown band, black necrosing syndrome), as well as
documenting previously reported diseases with global distributions (e.g. black-band
disease). The most notable increase in prevalence was for `white syndrome', a
collective term for symptoms analogous to Caribbean diseases (Fig 2). Distribution
121
and abundance of white syndrome has dramatically increased over the last 5 years,
with a 30 fold increase recorded in the Capricorn Bunker group (s u
o thern GBR)
between 2002/2003 (Willis et al 2004).
Our research to date has focused primarily
upon the ecology, physiology and cell
biology of white syndrome in tabular
Acropora spp. from the GBR and has
continual y emphasised a multi-disciplinary
approach to coral diseases. Although
significant mortalities have been
associated with disease, substantial
knowledge gaps exist regarding the
ecological processes and interactions
across spatial and temporal scales. Kinne
(1980) notes that "diseases affect basic
phenomena of life in oceans and coastal
Fig 2. White syndrome affecting tabular
waters... in short, diseases are a major
Acropora. Agincourt Reef, Northern GBR
denominator of population dynamics". To
gain an understanding of the epizootiology of white syndrome we conducted
intensive broad-scale surveys to determine the prevalence of disease at local and
regional scales, to describe the spatial distribution of disease-like syndromes and
effects of disease upon community structure. Concurrent monitoring of colonies
affected by white syndrome was conducted across sites at Heron Reef to determine
rates of lesion progression, examine the fate of affected colonies and to elucidate
potential interactions between progression of white syndrome and abiotic factors.
The concept of the cora
l as a holobiont (Rohwer et al 2002) is fundamental to
understanding the process of disease and determining the primary target of white
syndrome. Our research has investigated the effects of white syndrome on the
symbiotic zooxanthellae and phototrophic endolithic communities by investigating
both standard physiological parameters and chlorophyll fluorescence using novel
tools such as the Imaging-PAM, in collaboration with Karin Ulstrup and Dr. Peter
Ralph (University of Technology, Sydney). To further the understanding of the
microbial ecology of healthy corals and the shifts in microbial communities
associated with disease we have utilised the combination of DGGE as a potential
screening tool and sequencing of bacterial 16S rRNA genes in developing clone
libraries to investigate key functional groups and identify potential pathogens. At a
colony level, the understanding of the host response to white syndrome was
examined using 14C labelling to determine the intra-colonial translocation of
resources towards progressive white syndrome lesions.
Given previous difficulties associated with culture-dependant techniques in identifying
potential pathogens, fluorescent in-situ hybridisation (FISH) in association with
confocal laser scanning microscopy was conducted to visualise microbial
communities associated with white syndrome. Further investigations utilising
histological techniques including in-situ end labelling (ISEL) and haematoxylin and
eosin (H&E) were conducted to determine cell death mechanisms and investigate the
role of necrosis and apoptosis in white syndrome.
References
Aronson and Precht (2001) Paleobiology 23(3) 326-346.
Borger (2005) Coral Reefs 24 139-144
Bythell et al (2004) Coral health and disease 351-365
122
Dinsdale (2002) Proc. 9th Int. Coral Reef Symp. 2 1239-1243
Hoegh Guldberg (2004) Symbiosis 37 1-31
Jones et al (2004) Mar.Ecol. Prog. Ser. 281 63-77
Kinne (1980) Diseases of Marine Animals 466pp
Richardson (1998) T.R.E.E. 13(11) 438-443
Rohwer et al (2002) Mar. Ecol. Prog. Ser. 243 1-10
Sutherland et al. 2004 Mar. Ecol. Prog. Ser 266 273-302
Wilkinson (2002) Status of coral reefs of the world
Willis et al (2004) Coral health and disease pp 69-104
123
Towards an Understanding of Coral Disease
and Drivers on Indo-Pacific Reefs
Bette L Willis1, Cathie Page1, Elizabeth Dinsdale1, Meir Sussman1,2, Shelley
Anthony1, Holly Boyett1, Carole Lonergan , David Bourne
1
2
1School of Marine Biology & Aquaculture, James Cook University, Townsvil e, Q
4811
2Australian Institute of Marine Science, PMB No 3, Townsvil e MSO, Qld 4810.
Diseases of coral reef organisms have been escalating in the past few decades,
particularly in the Caribbean, but comparatively little is known about the prevalence
of coral disease on Indo-Pacific reefs or factors that affect their abundance and
severity. A targeted research project funded by the Global Environmental Facility
(GEF) seeks to understand the impacts of localized stress and compounding effects
of climate change on coral disease globally. As the Australian arm of the WB/GEF
Working Group on Coral Disease, we have commenced baseline surveys of coral
disease in the northern, central and southern sectors of the Great Barrier Reef (GBR)
Marine Park as a first step in understanding the epidemiology of coral disease in the
region.
We recognise seven disease states (tumors, skeletal eroding band (SEB), black
band disease (BBD), other cyanobacterial syndromes, white syndrome (WS), brown
band (BrB), and atrementous necrosis) that affect scleractinian corals and one that
affects gorgonians. White syndrome has been introduced as a general category for
disease states on Indo-Pacific reefs that manifest as an area of recently exposed
white skeleton adjacent to healthy or bleached tissue. There could be a variety of
causes or triggers for the apparently rapid tissue loss associated with the syndrome,
including a variety of pathogens associated with the Caribbean white diseases or
cellular mechanisms associated with stress events. Brown band is a new disease
first reported from the GBR (Willis et al. 2004). Six of these disease states (SEB, WS,
BBD, other cyanobacterial syndromes, BrB and tumors) were widely distributed,
being found in all sectors and associated with at least three of the more abundant
coral families (Acroporidae, Pocilloporidae, Faviidae). Thus, disease is a natural part
of the ecology of coral populations on GBR reefs.
Seasonal patterns in disease prevalence
Disease prevalence increased dramatically between winter and summer surveys on
reefs in the far northern sector, more than doubling in the acroporids and faviids (Fig
1). In particular, the number of cases of SEB, BBD and WS was greatest in the
austral summer (Fig 2), suggesting a link between higher temperatures and disease
incidence for these syndromes. In contrast, BrB prevalence did not differ significantly
between seasons over two years and experimental studies found no impact of
elevated temperatures on rates of progression across host colonies. We conclude
that coral pathogens vary in their response to temperature within seasonal ranges on
the Great Barrier Reef. However, given putative links between disease prevalence
and elevated temperatures for the majority of diseases surveyed, plus current trends
in global climate change and intensity of human-related activities that compound
stress in corals, studies such as ours on the Great Barrier Reef are critical for
124
establishing global baselines against which to judge whether background levels of
coral disease are increasing.
Fig 1. Sea
1. Se son
aso al Patterns in
nal Patterns Disea
in Disease
Fig
Fig 2. Sea
2. Se son
aso al Patterns in
nal Patterns Disea
in Disease
Preva
Pre lence
valen : Disea
ce: Dise se
ase Types
pes
Prevalence
len : Cora
ce: Co l
ral Families
Families
(ada
(a
pt
dapted f
ed from
om Wi
Willllis
is, Pag
,
e,
Page, Dins
Dinsdale 2004
2004)
(mod
odified f
ied from
om Wi
Willis
lis, Page,
, Page, Di
Dins
nsda
dale
le 2004
20
)
04)
) 18
Winter
%
6.0
e
( 16
Summer
)
E
e
14
% 5.0
S
i
s
eas
12
i
seas
D
nce (
nce ± 10
1.5
a
n
ale
e
8
ale
ean D
ev
M
ev 6
M
Pr 1.0
Pr 4
0.5
2
0
0.0
Acroporidae
SEB BBD/OT cyano BrB
WS
BNS
Other
Pocil oporidae
Poritidae
Favi dae
Other
Disease types
Prevalence increased in summer in 3 families
Prevalence increased in summer for 3 diseases
Coral disease outbreaks: the importance of diversifying approaches to
understand the impacts of disease on coral populations
A number of outbreaks of coral diseases have been detected on Indo-Pacific reefs in
the last 5 years, including an outbreak of white syndrome cases in 2002/03 on outer-
shelf reefs in the northern and southern sectors of the GBR (Fig 3), a white syndrome
outbreak on Marshall Island reefs in 2003/04 (D. Jacobsen, pers. comm.) and a white
syndrome outbreak on reefs in Palau in 2005 (GEF Coral Disease Working Group,
unpubl. data). In the latter 2 cases, surveys of disease prevalence did not detect the
outbreaks, highlighting the need for a research approach that combines quantitative
surveys of disease prevalence with qualitative reconnaissance surveys and
monitoring of tagged colonies in order to understand the impact of disease on coral
populations.
Cooktown/Lizard Is.
Inner
Mid
Outer
100
80
*
* *
Fig 3. WS Frequency
ta
Fig 3. WS Frequenc
60
Northern
40
20
Y Da
0
on th
t e GBR: 1998-03
Cairns
100
*
(modified
ied from
om Will
i is et
llis et al.
al. 2004
004)
80
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)
60
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Townsville
a
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e
s
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70
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80
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ree
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Central Whean # citsundays
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*
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S 60
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White Syndrome
/99
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/01 /02 /03
/00
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1998/99 1999
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/99 /00 1
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98 99 00/001/0202/0
125
References
Wil is BL, Page CA, Dinsdale EA (2004) in Rosenberg E & Loya Y (eds) Coral Health
and Disease, Springer-Verlag, Berlin, 69-104.
Participants during final days of Puerto Morelos workshop
126
Contact details of workshop participants
Participants
Ateweberhan, Mebrahtu
World Conservation Society, Kenya
tmcclanahan@wcs.org
Azam, Farooq
University of California, San Diego, USA
fazam@ucsd.edu
Bahgooli, Ranjeet
UAPM, ICML, UNAM, Mexico
coral@scientist.com
Bailey, Merideth
University of New Hampshire, USA
mab26@cisunix.unh.edu
Baker, Andrew
Columbia University, USA
abaker@wcs.org
Banaszak, Ania
UAPM, ICML, UNAM, Mexico
banaszak@mar.icmyl.unam.mx
Baraka Rugura
Interuniversity Institute for Marine Science, Tanzania
barakakuguru@hotmail.com]
Bythell, John
University of Newcastle, UK
J.C.Bythell@newcastle.ac.uk
Coffroth, Mary Alice
University of Buffalo, USA
coffroth@buffalo.edu
Dani Chernov
Tel Aviv University, Israel
yosiloya@post.tau.ac.il
Davy, Mark
University of Queensland, Australia
mdavey@marine.uq.edu.au
de Sampayo, Eugenia
University of Queensland, Australia
E.Sampayo@marine.uq.edu.au
Deckenback, Jeffry
University of Queensland, Australia
JeffryD@marine.uq.edu.au>
Díaz Ruíz, Ayax Rolando University of Queensland, Australia
A.Diaz-Ruiz@marine.uq.edu.au
Dove, Sophie
University of Queensland, Australia
sophie@uq.edu.au
Enriquez, Susana
UAPM, ICML, UNAM, Mexico
enriquez@icmyl.unam.mx
Falcon, Luisa
UNAM, Mexico
falcon@miranda.ecologia.unam.mx
Fitt, William K
University of Georgia, USA
fitt@sparrow.ecology.uga.edu
Gates, Ruth
University of Hawaii, USA
rgates@hawaii.edu
Gilner, Jessica
Florida Institute of Technology, USA
jgilner@fit.edu
Guppy, Reia
University of Newcastle, UK
reia.guppy@ncl.ac.uk
Harvell, Drew
Cornell University, USA
cdh5@cornell.edu
Hernandez Pech, Xavier
UAPM, ICML, UNAM, Mexico
iglesias@mar.icmyl.unam.mx
Hill, Ross
University of Technology, Sydney, Australia
Ross.Hill@uts.edu.au
o
H egh-Guldberg, Ove
University of Queensland, Australia
oveh@uq.edu.au
o
H lmes, Glenn
University of Queensland, Australia
rnje@uq.edu.au
Iglesias-Prieto, Roberto
UAPM, ICML, UNAM, Mexico
iglesias@mar.icmyl.unam.mx
Jatkar, Amita
University of Newcastle, UK
a.a.jatkar@ncl.ac.uk
Johnstone, Ron
University of Queensland, Australia
rnje@uq.edu.au
Jordan, Eric
UAPM, ICML, UNAM, Mexico
jordan@mar.icmyl.unam.mx
Kaniewska, Paulina
University of Queensland, Australia
p.kaniewska@marine.uq.edu.au
Kemp, Dusty
University of Georgia, USA
fitt@sparrow.ecology.uga.edu
Kinzie, Robert III
University of Hawaii, USA
kinzie@hawaii.edu
Kuhl, Michael
University of Copenhagen, Denmark
MKuhl@bi.ku.dk
Lanetty-Rodriquez, Maurici Oregon State University
rodrigm@science.oregonstate.edu
Leggat, Bill
University of Queensland, Australia
bleggat@marine.uq.edu.au
Lesser, Michael
University of New Hamshire, USA
mpl@cisunix.unh.edu
Loya, Yossi
Tel Aviv University, Israel
yosiloya@post.tau.ac.il
Manning, McKenzie
University of Hawaii, USA
rgates@hawaii.edu
Matz, Michael
University of Florida, USA
matz@whitney.ufl.edu
Méndez, Eugenio R.
CICESE, Ensanada, Mexico
emendez@cicese.mx
Miller, David
James Cook University, Australia
David.Miller@jcu.edu.au
Ortiz, Juan Carlos
University of Queensland, Australia
jortiz@marine.uq.edu.au
Padillo-Gamino, Jackie
University of Hawaii, USA
rgates@hawaii.edu
Pantos, Olga
San Diego State University, USA
opantos@sciences.sdsu.edu
Ralph, Peter
University of Technology, Sydney, Australia
Peter.Ralph@uts.edu.au
Reyes, Hector
Universidad Autónoma de Baja California Sur. Mexico
hreyes@uabcs.mx
Raymundo, Laura
University of Guam/Philippines
lauriejr@dgte.mozcom.com
o
R driguez Roman, Aime
UAPM, ICML, UNAM, Mexico
aime@mar.icmyl.unam.mx
Roff, Jez
University of Queensland, Australia
s4015960@student.uq.edu.au
Romanski, Adrienne
Columbia University, USA
amr2007@columbia.edu
Rosenberg, Eugene
Tel Aviv University, Israel
eros@post.tau.ac.il
Santos, Scott
University of Arizona
srsantos@email.arizona.edu
Segal, Roee
Tel Aviv University, Israel
yosiloya@post.tau.ac.il
Shenkar, Noa
Tel Aviv University, Israel
yosiloya@post.tau.ac.il
Schwarz, Jodi
DOE Joint Genome Institute, USA
JASchwarz@lbl.gov
127
Schwarz, Jodi
DOE Joint Genome Institute, USA
JASchwarz@lbl.gov
Smith, Garriet
University of South Carolina, USA
smithres@aiken.sc.edu
Todd LaJuenesse
Florida International University, USA
lajeunes@fiu.edu
l
U strup, Karen
University of Technology, Sydney, Australia
kulstrup@gmail.com
O
van ppen, Madeleine
James Cook University, Australia
m.vanoppen@aims.gov.au
van Woesik, Robert
Florida Institute of Technology, USA
rvw@fit.edu
Visram, Shakil
Bamburi, Mombasa, Kenya
shak@africaonline.co.ke
Ware, John
SeaServices, Gaithersburg, USA
jware@erols.com
Warner, Mark
University of Deleware, USA
mwarner@udel.edu
Wegley, Linda
San Diego State University, USA
opantos@sciences.sdsu.edu
Weil, Ernesto
University of Puerto Rico, PR
eweil@caribe.net
Willis, Bette
James Cook University, Australia
Bette.Wil is@jcu.edu.au
Winters, Gidon
Tel Aviv University, Israel
wintersgidon@hotmail.com
Yellowlees, David
James Cook University, Australia
david.yellowlees@jcu.edu.au
Zevuloni, Assaf
Tel Aviv University, Israel
zvuloni@post.tau.ac.il
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