1. Metabolic Programming of Zebrafish, Danio rerio Physiological performance as explained by Dynamic Energy Budget Theory and life-cycle consequences of uranium induced perturbations April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Starrlight Augustine 1 Promoteren: Prof.Dr.Sebastiaan A.L.M. Kooijman, Prof.Dr. Christelle Adam-Guillermin Copromotor: Dr. Béatrice Gagnaire

2. Extinction Context : Environmental Risk Assessment Aquatic ecosystems are the recipient of byproducts of our industries: How much of each type of compound can be released into the environment without inducing deleterious side effects? U U U U 2 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

3. Extinction Context : Environmental Risk Assessment Aquatic ecosystems are the recipient of byproducts of our industries: How much of each type of compound can be released into the environment without inducing deleterious side effects? U U U U 2 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

4. Context: IRSN research programme ENVIRHOM-ECO Major cities Mine tailing storage sites still in use in 1989 Uranium mines and mine tailings storage in France Mine tailing storage closed in in 1989 3 Mission statement: Evaluate long term effects of chronic exposure to radionuclides on the ecosystem. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

5. From molecules to ecosystems … 4 Space April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

6. From molecules to ecosystems … 5 Space April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

7. From molecules to ecosystems … 5 Space Growth + reproduction April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

8. OECD test organism: zebrafish >90 jours 2-3 cm 5 jours 0.45 cm Embryo Juvenile Adult BIRTHPUBERTY >90 days 2-3 cm 5 days 0.45 cm 6 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit

9. Dynamic Energy Budget (DEB) Theory Internal concen- tration Enough Too much NEC Effects General unified theory in biology created over 30 years ago (Kooijman 1993, 2000, 2010) which specifies the acquisition and use of energy for all organisms. The theory respects mass and energy balancing. 7 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit NEC = No effect Internal Concentration 0 0 Too little

10. Phase I: characterizing the physiological performance of non-exposed zebrafish (the blank) Growth and Reproduction at different food levels + Literature data Zebrafish DEB model Ageing Temperature Starvation 8

11. Results: metabolic programming uncovered 9 Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

12. Results: metabolic programming uncovered 9 Feeding Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

13. Results: metabolic programming uncovered 9 Feeding Digestion Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

14. Results: metabolic programming uncovered 9 Feeding Digestion Storage Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

15. Results: metabolic programming uncovered 9 Feeding Digestion Storage Maintenance Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

16. Results: metabolic programming uncovered 9 Feeding Digestion Storage Maintenance Growth Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

17. Results: metabolic programming uncovered 9 Feeding Digestion Storage Maintenance Growth Development Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

18. Results: metabolic programming uncovered 9 Feeding Digestion Storage Maintenance Growth Development Processes specified by the DEB model April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Augustine et al 2011a Augustine et al 2011b Mueller et al 2012 Reproduction

19. 9 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance Results: metabolic programming uncovered Augustine et al 2011a Augustine et al 2011b Mueller et al 2012

20. Phase II: characterizing uranium induced deviations from the physiological performance of the blank + Literature data Zebrafish DEB model 0 nM U 84 nM U420 nM U Growth and Reproduction at three levels of uranium Uptake and elimination How effects depend on internal concentration 10

21. Results: effects of uranium 11 Uranium induces toxic effects almost as soon as it is internalized Effects on -growth (early life stages) -maintenance (adults) April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance

22. Results: effects of uranium 12 Dry mass (µg) Age, d Data: Stéphanie Bourrachot 2009 Lines: DEB model predictions 1054 nM 0 nM April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance

23. Results: effects of uranium 13 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 010203040 0 500 1000 1500 2000 0.2 0.4 0.6 0.8 1 1.2 Allocation to reproduction is a continuous process Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance

24. Results: effects of uranium 13 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 010203040 0 500 1000 1500 2000 0.2 0.4 0.6 0.8 1 1.2 Allocation to reproduction is a continuous process Spawning is discontinuous, but when the organism does not spawn the mass invested in reproduction remains inside the individual Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance

25. Results: effects of uranium 13 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 010203040 0 500 1000 1500 2000 0.2 0.4 0.6 0.8 1 1.2 Allocation to reproduction is a continuous process Spawning is discontinuous, but when the organism does not spawn the mass invested in reproduction remains inside the individual When energy assimilated from food no longer covers maintenance then missing energy is taken from that invested in reproduction Feeding Digestion Storage Growth Development Processes specified by the DEB model Reproduction Ageing Maintenance

26. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 14 010203040 0 500 1000 Experiment time, days Cumulated number of eggs Augustine et al 2012 Spawning

27. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 14 010203040 0 500 1000 Experiment time, days 866 eggs spawned in total is about 0.36 g (fresh weight) Cumulated number of eggs Augustine et al 2012

28. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 14 0.4 0.6 0.8 1 1.2 010203040 0 500 1000 Experiment time, days Wet mass, g Final observed mass DEB model prediction for the blank Cumulated number of eggs Augustine et al 2012

29. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 14 0.4 0.6 0.8 1 1.2 About half the final mass of the fish was spawned !! 010203040 0 500 1000 Experiment time, days Wet mass, g Cumulated number of eggs Final observed mass DEB model prediction for the blank Augustine et al 2012

30. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit 14 0.4 0.6 0.8 1 1.2 010203040 0 500 1000 Experiment time, days Cumulated number of eggs Wet mass, g Final observed mass DEB model prediction for the blank DEB model prediction for increasing maintenance costs Augustine et al 2012

31. 15 April 23, 2012 - Public Doctoral Defense – Vrije Universiteit How do these results link back to the initial problem ? Uranium turns out to be toxic and effects show up as soon as it is internalized. The zebrafish DEB model is a very sensitive tool for detecting effects The model is also useful for interpreting data and comparing studies Effects on populations and ecosystems follow from effects on individuals

32. From individual to ecosystems … 16 Space April 23, 2012 - Public Doctoral Defense – Vrije Universiteit From individual to molecules …

33. April 23, 2012 - Public Doctoral Defense – Vrije Universiteit Thank you for your attention Thanks are due to: Magalie Floriani (TEM) Virginie Cavalie (ICP-AES, TEM) Sylive Pierrisnard (ILC) Sandrine Pereira (immuno-histochemistry) Alban Carlat (data collection) « LRE » Laboratory and many more … 17