CHANGE IN THE GENETIC EXPRESSION OF APOPTOTIC AND AUTOPHAGIC PROTEINS AFTER THERMAL STRESS IN
SYMBIOTIC AIPTASIA PALLIDA
By: Jessica FlesherMentors: Dr. Virginia Weis
Dr. Camille PaxtonZoology DepartmentHHMI Summer Program 2011 Aiptasia pallida
CORALS Reef-building corals Biogenic habitat
Trap nutrients and provide shelter Coral polyps contain symbiotic dinoflagellate
algae, zooxanthellae
Coral Reef
THE SYMBIOSIS Corals
Waste removal and photosynthetic products Zooxanthellae
Acquire shelter and nutrients for photosynthesis
Zooxanthellae in coral polyp
THE SYMBIOSIS
NOAA Ocean Service Education 2008
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 pathwaysPartially bleached coral head, Montastrea cavernosa
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
MECHANISMS FOR CORAL BLEACHING
Weis, Journal of Experimental Biology, 2008
APOPTOSIS Programmed cell death Caspases Partially characterized mechanism Apoptosis proteins
acasp Caspase 8
Cheung, Clinical Cancer Research, 2006
Vertebrate Apoptosis Model
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
HYPOTHESIS Under stress caused by increased
temperature, the expression will change of the identified caspase and ATG sequences
Bleached coral, Acropora sp.
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.
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
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
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
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
FURTHER APPLICATIONS Continue RNA
extractions and cDNA synthesis
Perform quantitative PCR
Determine time points for ATG8 western blot analysis and localization assays
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
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