1. Enzymatic responses against anthracene are compromised in yellow band disease tissues of the reef-building species Orbicella faveolata Luis Miguel Montilla luismmontilla@usb.ve Laboratorio de Ecología Experimental Ruth RamosElia García Aldo Cróquer Universidad Simón Bolívar

2. 2 Enzymatic defense ROS (Reactive Oxygen Species) Xenobiotics ( alellochemicals )

3. 3 Antioxidant activity Biotransformation paths Scavenge free radicals Phase I & II Enzymatic defense Xenobiotic Substrate GST Excretable conjugate OO H H Hydrogen peroxide GPx O O H H Water H H

4. 4 PAH’s Biocides Hg Cu Pb Cd Heavy metals

5. 5 There is a lack of controlled experiments that combine both stressful conditions Activity or expression of immune response Response to xenobiotics Healthy corals Diseased corals

6. 6 Why are we doing this bioassay? In Venezuela we have three things

7. 7 How diseased corals are responding to the presence of xenobiotics in comparison with healthy ones? Our question

8. 8 The presence of pollution represents an additional challenge for corals that are defending against a disease Response Anti Xenobiotic Immune Presence Xenobiotic Disease Our model ? ?

9. 9 A diseased coral will have a lower enzymatic response when exposed to a xenobiotic in comparison with a healthy one Response Anti Xenobiotic Immune Presence Xenobiotic Disease Our hypothesis ? ?

10. Orbicella faveolata Important reef builder Affected by several diseases Listed as Endangered by the IUCN (2008) 9

11. 11 Caribbean Yellow band disease A. Cróquer Highly prevalent Alters microbial assemblages Deteriorates zooxanthellae integrity Compromises reproductive capacity

12. 12 Anthracene One of the EPA priority PAHs Toxicity increased by exposure to light An important PAH largely produced and used as chemical reagent

13. Orthogonal design: 13 Concentration 4 levels 0 ppb 10 ppb 40 ppb 100 ppb Condition: 2 levels Healthy YBD Two factors

14. Our variables: Enzyme Involved in… CatalaseFree radical reduction Glutathione-s transferase Xenobiotic biotransformation Glutathione peroxidase Free radical reduction Indicator of lipid peroxidation

15. 15 Specific activity of CATdfSSMSPseudo-FP(perm)Unique perms Condition11.99E-4 10.060.0029816 Concentration36.25E-52.08E-51.030.379958 Cond x Conc33.16E-41.05E-45.310.0029963 Residuals1212.40E-31.99E-5 Total1282.97E-3 What did we find? Univariate Permanova There is a simultaneus effect of both Condition and the concentration

16. Catalase 16 What did we find? 75%

17. 17 Specific activity of GSTdfSSMSPseudo-FP(perm)Unique perms Condition14.18E-7 0.160.689813 Concentration38.94E-62.98E-61.140.349958 Cond x Conc33.30E-51.10E-54.210.0089949 Residuals1303.39E-42.61E-6 Total1373.81E-4 What did we find? Univariate Permanova So far, GST is consistent with the behaviour of CAT

18. Glutathione S-tranferase 18 What did we find? 65%

19. 19 Specific activity of GPxdfSSMSPseudo-FP(perm)Unique perms Condition13.81E-02 0.570.469830 Concentration32.31E-017.71E-0211.460.339958 Condition x Concentration32.57E-018.57E-0212.740.299950 Residuals734.91E+006.73E-02 Total805.48E+00 What did we find? CAT Pseudo-FP(perm) 5.310.002 GST Pseudo-FP(perm) 4.210.008 Power: 1-β= 99% Effect size GPx71% Cat~73% GST~ 64%

20. Glutathione peroxidase 20 What did we find?

21. 21 What does this tell us? Catalase and Glutathione-S transferase in YBD-tissues failed to respond to anthracene as healthy corals Highly variable More replicates for a better size effect

22. 22 Our hypothesis was fulfilled Response Anti Xenobiotic Immune Presence Xenobiotic Disease Our hypothesis ? The antixenobiotic response of O. faveolata to anthracene is compromised when is infected with YBD

23. 23 Although we can't tell what’s the process underlying our findings… PAH may induce bleaching YBD affects zooxanthellae integrity Zooxanthellae contributes to xenobiotic response YBD alters bacterial assemblage Bacterial assemblages contributes to xenobiotic response Bleaching compromises immune response Biomarker expresión depends on concentration thresholds We need a bigger picture! Next steps

24. 24 Use additional biomarkers Homeostatic condition, Immune response, Anti-xenobiotic response Would there be an effect on the microbial assemblage? We need a bigger picture! Pollutants aren’t present alone… Heavy metals, Mixes of different PAHs How the xenobiotics affect the dynamic of the disease?

25. 25 Thanks for your attention!