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Apoptosis and the selective survival of host animals

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Apoptosis and the selective survival of host animals

  1. 1. 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.
  2. 2. Authors• Israel ▫ Dan Tchernov ▫ Hagit Kvitt ▫ Hanna Rosenfeld• Rutgers, NJ ▫ Liti Haramaty ▫ Thomas S. Bibby ▫ Maxim Y. Gorbunov ▫ Paul G. Falkowski
  3. 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. 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. 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. 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
  7. 7. Methodology• Zooxanthellate corals• Relationship ▫ Irradiance ▫ Caspase activity ▫ Increased temperature (thermal stress)• Conditions ▫ Aquaria - highly controlled ▫ Field - natural environmental
  8. 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. 9. Transmission electron micrographs• Transmission electron microscope• Samples ▫ Thin sections ▫ Preserve ▫ Chemically fixed ▫ Dehydrated ▫ Embedded in a polymer resin
  10. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.

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