Apoptosis and the selective survival

of host animals following thermal

bleaching in zooxanthellate corals

Noelia I. Aponte SilvaGraduate 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.

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 bythe production of ROS plays a key role inzooxanthellate coral bleaching and death

▫ Regulation of the apoptotic cascade can preventzooxanthellate coral death

Methodology

• Zooxanthellate corals

• Relationship▫ Irradiance▫ Caspase activity▫ Increased temperature (thermal stress)

• Conditions▫ Aquaria - highly controlled▫ Field - natural environmental

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-regulationTchernov, 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

▫ Species-specific processes

• Apoptosis▫ Induces host mortality

• Caspases▫ Involved in apoptotic

response

• Caspase cascade▫ Not activated

▫ Down-regulated

▫ Interrupted

▫ Inhibitor of caspases

▫ Avoid apoptosis and bleaching

Discussion

• Personal opinion

• If sea surface temperatures continue to increase:

▫ What will be the future of corals?

▫ Would global climate change favor a particularsymbiotic phenotype?

Discussion

• If sea surface temperatures continue to increase:

▫ A strong selection of corals with algal symbiontsthat generate less ROS or hosts that do notactivate a caspase cascade in response to theproduction of ROS is to be expected.

▫ High temperature resistant symbiotic phenotypeshave persisted in some locations and arepotentially poised to become increasinglydominant.