1. C HANGE IN THE G ENETIC E XPRESSION OF A POPTOTIC AND A UTOPHAGIC P ROTEINS AFTER T HERMAL S TRESS IN S YMBIOTIC A IPTASIA PALLIDA By: Jessica Flesher Mentors: Dr. Virginia Weis Dr. Camille Paxton Zoology Department HHMI Summer Program 2011 Aiptasia pallida

2. C ORALS Reef-building corals Biogenic habitat Trap nutrients and provide shelter Coral polyps contain symbiotic dinoflagellate algae, zooxanthellae Coral Reef

3. T HE S YMBIOSIS Corals Waste removal and photosynthetic products Zooxanthellae Acquire shelter and nutrients for photosynthesis Zooxanthellae in coral polyp

4. T HE S YMBIOSIS NOAA Ocean Service Education 2008

5. C ORAL B LEACHING 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

6. A M ODEL S YSTEM Corals are hard to grow Aiptasia pallida-Symbiodinium sp. Same symbiosis Replicate pedal lacerations Symbiodinium in tentacles of a coral polyp Aiptasia pallida

7. M ECHANISMS FOR C ORAL B LEACHING Weis, Journal of Experimental Biology, 2008

8. A POPTOSIS Programmed cell death Caspases Partially characterized mechanism Apoptosis proteins acasp Caspase 8 Cheung, Clinical Cancer Research, 2006 Vertebrate Apoptosis Model

9. A UTOPHAGY 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

10. H YPOTHESIS Under stress caused by increased temperature, the expression will change of the identified caspase and ATG sequences Bleached coral, Acropora sp.

11. P REDICTION 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.

12. S EQUENCES 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

13. E XPERIMENTAL S ETUP 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

14. A FTER I NCUBATION 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

15. PCR actinacaspcaspase 8ATG 12ATG 8 ATG 7 200bp 75bp 200bp 75bp 24 hr 168 hr 25 ° 27° 30° 33° 200bp 75bp 200bp 75bp

16. F URTHER A PPLICATIONS Continue RNA extractions and cDNA synthesis Perform quantitative PCR Determine time points for ATG8 western blot analysis and localization assays

17. S UMMARY 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

18. A CKNOWLEDGEMENTS 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