The document summarizes a study that experimentally demonstrates caspase-induced apoptosis plays a key role in coral bleaching and death from thermal stress. The study shows increased temperature leads to higher reactive oxygen species production by symbiotic algae, which triggers caspase activity in the coral host and apoptosis. Inhibiting caspase activity prevented bleaching and apoptosis. The findings help explain how selective survival of coral hosts occurs following thermal bleaching and provides insight into impacts of climate change on coral reefs.
MULTIDISCIPLINRY NATURE OF THE ENVIRONMENTAL STUDIES.pptx
Apoptosis and the selective survival of host animals
1. Apoptosis and the selective survival
of host animals following thermal
bleaching in zooxanthellate corals
Noelia I. Aponte Silva
Graduate Student – Environmental Science
University of Puerto Rico – Rio Piedras Campus
Tchernov, D., Kvitt, H., Haramaty, L., Bibby, T. S., Gorbunov, M. Y., Rosenfeld, H., & Falkowski, P. G. (2011).
Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.
PNAS, 108(24), 9905-9909.
2. Authors
• Israel
▫ Dan Tchernov
▫ Hagit Kvitt
▫ Hanna Rosenfeld
• Rutgers, NJ
▫ Liti Haramaty
▫ Thomas S. Bibby
▫ Maxim Y. Gorbunov
▫ Paul G. Falkowski
3. Coral bleaching
• Environmental triggers
▫ Increase sea surface temperature
• Expulsion, digestion, or loss of pigmentation
▫ Photosynthetic dinoflagellate symbiotic algae
▫ Selective survival of host animals
▫ Zooxanthellate corals
4. Apoptosis
• Genetically directed cell self-destruction
▫ Programmed cell death
▫ Presence or removal of a stimulus
▫ Eliminate damaged or unwanted DNA
• Cell morphological changes
▫ Cytoplasmic shrinkage
▫ Chromatin condensation
▫ Plasma membrane blebbing
▫ DNA degradation or fragmentation
5. Apoptotic cascade
• Environmental trigger
▫ Coral stress (thermal stress)
• Reactive oxygen species (ROS)
▫ Coral endodermal cells
▫ Photosynthetic activity of the symbiotic algae
▫ Aerobic respiration activity in the host mitochondria
• Cysteine proteases (caspases)
▫ Caspase activity regulated by ROS
6. Objective
• Experimentally demonstrate
▫ Caspase-induced apoptotic pathway initiated by
the production of ROS plays a key role in
zooxanthellate coral bleaching and death
▫ Regulation of the apoptotic cascade can prevent
zooxanthellate coral death
8. Collection and maintenance of corals
• Seriatopora hystrix and Stylophora pistillata
▫ Grown at 26°C for 6 mo
▫ Transferred to 32°C for 2 mo
▫ Osborn Laboratories, New York Aquarium
• Montipora capitata and Pocillopora damicornis
▫ Kaneohe Bay, Oahu, Hawaii
▫ Full natural sunlight and low light
▫ Ambient temperature 26°C
▫ Increased temperature 32°C
▫ Induced bleaching: 9 mo darkness stress
▫ Caspase inhibitor Z-VAD-FMK (VAD)
9. Transmission electron micrographs
• Transmission electron microscope
• Samples
▫ Thin sections
▫ Preserve
▫ Chemically fixed
▫ Dehydrated
▫ Embedded in a polymer resin
10. Fenton reaction and ROS assay
• Fenton reaction
▫ Reaction mixture
▫ Production of hydroxyl radicals anions (OH-)
• ROS assay
▫ Dihydrorhodamine (DHR 123) fluorescence
▫ Oxidation
▫ Rhodamine 123 (fluoresces at 515nm)
11. Caspase activity determination
• Incubation of cell lysate
▫ Fluorogenic substrate for caspases
Fluorescence measured every 5 min
▫ Irreversible caspase inhibitor VAD and
Fluorogenic substrate for caspases
• Statistical significance
▫ Control vs. treatment
▫ ANOVA and t-test
12. Western blots
• Immunochemical analysis
▫ Equal protein basis
▫ Separated
Gel electrophoresis (12% polyacrylamide gels)
▫ Transferred to membrane
PVDF
▫ Probed
Polyclonal antibodies for recombinant human caspase 3
▫ Chemiluminescence detection
Horse-radish peroxidase enzyme (HRP)
13. Thermal stress
• Grown at 26°C and transferred to 32°C
• Seriatopora hystrix
▫ Bleached
▫ Morphological integrity
▫ Decreased caspase activity
• Stylophora pistillata
▫ Bleached
▫ Shift in chromatin
▫ Membrane blebbing
▫ Lost all recognizable tissue
▫ Increased caspase activity
Tchernov, D., Kvitt, H., Haramaty, L., Bibby, T. S., Gorbunov, M. Y., Rosenfeld, H., & Falkowski, P. G. (2011). Apoptosis and the
selective survival of host animals following thermal bleaching in zooxanthellate corals. PNAS, 108(24), 9905-9909.
14. Cascade triggers
• Irradiance and Temperature (4)
▫ Increased temperature (32°C)
Increased caspase activity (full sunlight)
• Montipora capitata
• Pocillopora damicornis
▫ Fragmentation of DNA
▫ Lost all recognizable tissue
• Caspase inhibitor VAD
▫ Caspase activity reduced
▫ Bleaching and apoptosis prevented
Tchernov, D., Kvitt, H., Haramaty, L., Bibby, T. S., Gorbunov, M. Y., Rosenfeld, H., & Falkowski, P. G. (2011). Apoptosis and the
selective survival of host animals following thermal bleaching in zooxanthellate corals. PNAS, 108(24), 9905-9909.
15. Role and source of ROS
• Measured steady-state pool of ROS
• Montipora capitata
▫ Zooxanthellae colonies
▫ Dark-bleached colonies
▫ Thermal stress
• Increased temperature (32°C)
▫ Higher ROS production in zooxanthellae colonies
▫ Higher caspase activity in zooxanthellae colonies
16. Caspase activity induced by ROS
• Add exogenous ROS
▫ Hydroxyl anion radicals
• Montipora capitata colonies grown at 26°C
▫ Higher caspase activity in dark-bleached colonies
▫ Host animal death
• Montipora capitata colonies grown at 32°C
▫ Higher caspase activity in zooxanthellae colonies
17. Phenotypic combinatorial matrix
• Two components
▫ Factors that trigger the apoptotic response
Zooxanthellae thermally sensitive
Lipid composition of thylakoid membranes
High temperature and irradiance
Production of ROS
▫ Initiation of the caspase cascade
Host cell
ROS production threshold
Bleach and apoptosis
18. Physiological model
• Relates mortality and bleaching
• Two factors
▫ Algal symbiont thermal sensitivity
▫ Activation of the caspase cascade by host
• Two responses *
▫ Bleach / Death
Sensitive symbiont
Activation
▫ Bleach / Recovery
Sensitive symbiont
No activation / down-regulation
Tchernov, D., Kvitt, H., Haramaty, L., Bibby, T. S., Gorbunov, M. Y., Rosenfeld, H., & Falkowski, P. G. (2011). Apoptosis and the
selective survival of host animals following thermal bleaching in zooxanthellate corals. PNAS, 108(24), 9905-9909.
19. Conclusions
• Bleaching and host death
▫ Independent • Caspase cascade
▫ Species-specific processes ▫ Not activated
▫ Down-regulated
• Apoptosis ▫ Interrupted
▫ Induces host mortality ▫ Inhibitor of caspases
▫ Avoid apoptosis and
• Caspases bleaching
▫ Involved in apoptotic
response
20. Discussion
• Personal opinion
• If sea surface temperatures continue to increase:
▫ What will be the future of corals?
▫ Would global climate change favor a particular
symbiotic phenotype?
21. Discussion
• If sea surface temperatures continue to increase:
▫ A strong selection of corals with algal symbionts
that generate less ROS or hosts that do not
activate a caspase cascade in response to the
production of ROS is to be expected.
▫ High temperature resistant symbiotic phenotypes
have persisted in some locations and are
potentially poised to become increasingly
dominant.
Hinweis der Redaktion
Dr. Dan TchernovDeputy director of the Leon H.Charney School of Marine Sciences in the University of HaifaHead of the Marine Biology DepartmentBA in Biology - Hebrew University in 1994PhD in Oceanography -Hebrew University at 2003Postdoctoral studies -IMCS in Rutgers University, NJ
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are highly reactive due to the presence of unpaired valence shell electrons.Proteases are enzymes that degrade polypeptides.
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image.Biological specimens typically require to be chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers and similar materials may require special `staining' with heavy atom labels in order to achieve the required image contrast.
Oxidation is the loss of electrons or an increase in oxidation state by a molecule, atom, or ion.
Lysate: the cellular debris and fluid produced by lysis.
The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.Enhanced chemiluminescence is a common technique for a variety of detection assays in biology. A horseradish peroxidase enzyme (HRP) is tethered to the molecule of interest (usually through labeling an immunoglobulin that specifically recognizes the molecule).
ROS are produced primarily by the algal symbionts.
The phenotypic expression of this symbiont/host relationship places a selective pressure on the symbiotic association. Predicts the survival of the host animals in which the caspase-mediated apoptotic cascade is down-regulated.
We need to give more credit to nature’s ability to adapt to environmental changes.