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CHANGE IN THE GENETIC EXPRESSION OF APOPTOTIC AND AUTOPHAGIC PROTEINS AFTER THERMAL STRESS IN
SYMBIOTIC AIPTASIA PALLIDA
By: Jessica Flesher
Mentors: Dr. Virginia Weis
Dr. Camille Paxton
Zoology Department
HHMI Summer Program 2011 Aiptasia pallida
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CORALS
Reef-building corals Biogenic habitat
Trap nutrients and provide shelter Coral polyps contain symbiotic dinoflagellate
algae, zooxanthellae
Coral Reef
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THE SYMBIOSIS
Corals Waste removal and photosynthetic products
Zooxanthellae Acquire shelter and nutrients for photosynthesis
Zooxanthellae in coral polyp
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THE SYMBIOSIS
NOAA Ocean Service Education 2008
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CORAL BLEACHING
Loss of zooxanthellae from the host Increase with ocean temperature
Worldwide in 2008 19% lost 15% critical 20% threatened
Interest in the cellular and molecular pathways
Partially bleached coral head, Montastrea cavernosa
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A MODEL SYSTEM
Corals are hard to grow Aiptasia pallida-Symbiodinium sp.
Same symbiosis Replicate pedal lacerations
Symbiodinium in tentacles of a coral polyp
Aiptasia pallida
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MECHANISMS FOR CORAL BLEACHING
Weis, Journal of Experimental Biology, 2008
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APOPTOSIS
Programmed cell death Caspases Partially characterized mechanism Apoptosis proteins
acasp Caspase 8
Cheung, Clinical Cancer Research, 2006
Vertebrate Apoptosis Model
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AUTOPHAGY
Autophagy – the degradation of the symbiont by the host cell
Highly conserved pathway – yeast to mammals
Unknown mechanism Autophagy related (ATG) proteins
ATG 7 ATG 8 ATG12
Mizushima, Genes and Development, 2007
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HYPOTHESIS
Under stress caused by increased temperature, the expression will change of the identified caspase and ATG sequences
Bleached coral, Acropora sp.
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PREDICTION
If there is an increase in thermal stress, than there will be an increase in the genetic expression of the caspase and ATG sequences
Bleached coral, Acropora sp.
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SEQUENCES Acasp previously identified (Dunn, et al. 2006)
Caspase 8, ATG7, ATG8, ATG12 unknown No full genomic sequence for Aiptasia Initial primers using Transcriptome (Pringle Lab)
Partial genomic database With many sequence errors
Primers used to isolate, clone, and sequence genes
Accurate sequences used to develop qPCR primers
Aiptasia pallida
Electrophoresis Gel
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EXPERIMENTAL SETUP
Four temperatures: 25°C (control) , 27°C, 30°C, 33°C
Length of incubation (hours): 12, 24, 48, 96, 168
Six-well plates Anemones were starved Artificial seawater, changed every other day 12 hour light/dark cycle
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AFTER INCUBATION
Freeze anemones Extract RNA Purify RNA DNase RNA Convert RNA to cDNA
cDNA library for use in analysis PCR as initial test
24 and 168 hour time points
Symbiotic and bleached Aiptasia pallida
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PCR
actin acasp
caspase 8
ATG 12ATG 8
ATG 7
200bp75bp
200bp75bp
24 hr
168 hr
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
200bp75bp
200bp75bp
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FURTHER APPLICATIONS
Continue RNA extractions and cDNA synthesis
Perform quantitative PCR
Determine time points for ATG8 western blot analysis and localization assays
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SUMMARY
Coral bleaching occurs when zooxanthellae are lost from the host
My focus is on apoptosis and autophagy as mechanisms for coral bleaching
The sequences of Caspase 8, ATG7, ATG8, and ATG12 were identified
There are visual differences in the expression of the genes at different time points and temperatures
Future work will begin with qPCR
Aiptasia pallida
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ACKNOWLEDGEMENTS
Dr. Virginia Weis Dr. Camille Paxton The rest of the Weis Lab
Angela Poole, Sheila Kitchen, Jamie Jo McGraw, and Ben Haslam
Bayne, Chappell, Pringle and Taylor Lab CGRB
HHMI Program Dr. Kevin Ahern
Aiptasia pallida