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.
Apoptosis and the selective survival of host animals
Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate coralsNoelia I. Aponte SilvaGraduate Student – Environmental ScienceUniversity of Puerto Rico – Rio Piedras CampusTchernov, 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.
Authors• Israel ▫ Dan Tchernov ▫ Hagit Kvitt ▫ Hanna Rosenfeld• Rutgers, NJ ▫ Liti Haramaty ▫ Thomas S. Bibby ▫ Maxim Y. Gorbunov ▫ Paul G. Falkowski
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
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
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
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
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)
Transmission electron micrographs• Transmission electron microscope• Samples ▫ Thin sections ▫ Preserve ▫ Chemically fixed ▫ Dehydrated ▫ Embedded in a polymer resin
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)
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
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)
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.
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.
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
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
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
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.
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
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?
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.