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

Interest in the cellular and molecular pathways

Partially 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