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
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
Partially bleached coral head, Montastrea cavernosa

Interest in the cellular and molecular pathways
A MODEL SYSTEM
Corals are hard to grow
 Aiptasia pallida-Symbiodinium sp.

Same symbiosis
 Replicate pedal lacerations

Aiptasia pallida
Symbiodinium in tentacles of a coral
polyp
MECHANISMS FOR CORAL BLEACHING
Weis, Journal of Experimental Biology,
2008
APOPTOSIS
Programmed cell death
 Caspases
Vertebrate Apoptosis Model
 Partially characterized mechanism
 Apoptosis proteins



acasp
Caspase 8
Cheung, Clinical Cancer Research, 2006
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

Electrophoresis Gel
With many sequence errors
Primers used to isolate, clone, and sequence
genes
 Accurate sequences used to develop qPCR
primers

Aiptasia pallida
EXPERIMENTAL SETUP

Four temperatures:


Length of incubation (hours):


25°C (control) , 27°C, 30°C, 33°C
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

Symbiotic and bleached Aiptasia
pallida
cDNA library for use in analysis
 PCR as initial test


24 and 168 hour time points
PCR
actin
acasp
caspase 8
ATG
ATG
ATG 12
257° 27° 30° 33° 258° 27° 30° 33° 25 ° 27° 30° 33°
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
24 hr
200bp
75bp
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
200bp
75bp
25 ° 27° 30° 33° 25 ° 27° 30° 33° 25 ° 27° 30° 33°
168 hr
200bp
75bp
200bp
75bp
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