1. Pressure tolerance of Mytilus edulis early life stages

3. Major Question How were deep sea environments colonized? High pressure environment Noxious environment Temperature extremes

4. Le Chatelier’s Principle Background  “If a chemical system at equilibrium experiences a change in concentration, temperature, volume or total pressure, the equilibrium will shift in order to counteract the imposed change” (Mestre et al. 2009)

5. Pressure  Effects biochemical reactions and membrane functionality Keq = [C][D]/[A][B],  G = -RTlnK eq and v = k[s] P sensitivity of reactions: K p = K 1 e (-P  V/RT) and k p = k 1 e -P  V‡/RT P therefore affects both K eq and k (Kinsey 2009) Increased pressure will move the equilibrium to the side with lowest volume  Limits depth range of marine organisms  Pressure tolerances are different for different life stages Some life stages are more suited for deep-sea colonization

6.  Along with pressure also plays an important role in biochemical reactions Can speed up or slow down metabolism  Response also varies with life stage  Can counteract pressure effects If membrane is more compressed because of pressure can be somewhat decompressed by higher temperature Temperature

7. What would limit organisms to a depth and temperature range in early ontogeny?

8. Biochemical reactions during fertilization

9. Program for post- fertilization changes in egg of sea urchins Epel 1975

10. Ca-Wave After Fertilization

11. Biochemical pathways are sensitive pathways  Could organisms, like mussels, with sensitive biochemical pathways colonize deep-sea habitat?

12. How were deep sea environments colonized by mussels? Mestre et al. chose a shallow- water relative of a deep-sea inhabitant

13. Phylogenetic Relationship subfamilys Mytilinae and Bathymodiolinae Shallow water species Mytilus edulis found here The rest are associated with one of the following: Hydrothermal vent Cold-water seep Wood/bone (Kyuno et al. 2009)

14. Free-spawning marine invertebrates Worldwide distribution Found in intertidal zones and estuaries Endure a wide range of temperatures and physical challenges Mytilus edulis

15. Origin of deep-sea mussels  2 Hypotheses: Deep sea species evolved from shallow-sea species in step-wise fashion via wood/bone habitat Direct colonization via larval transport from shallow-sea to deep-sea habitats.

16. Origin of deep-sea mussels  2 Hypotheses: Deep sea species evolved from shallow-sea species in step-wise fashion via wood/bone habitat Direct colonization via larval migration from shallow-sea to deep-sea habitats ○ Determine larval functional tolerance of pressure and temperature

17. Methods  3 Experiments Temperature effect on embryonic larvae and development Pressure effect on embryonic larvae and development with fertilization under pressure Pressure effect on embryonic larvae and development with fertilization at atmospheric pressure

18. Staging criteria for larvae:

19. Embryonic stages D-larvae Fertilized egg Sixteen-cell stage Multi-cell stage Early blastula Two-cell stage Four-cell stage

20. Temperature effect on embryonic larvae and development  5 temperature treatments 5, 10, 15, 20, and 25  C, at atmospheric pressure  Incubated until all treatments had reached D-larvae stage.

21. Pressure Vessels a) Plastic vial filled with the egg suspension and the microcentrifuge tube hald- filled with sperm suspension b) Pressure vessel showing the plastic vials inside Figure 1 from Mestre et al. 2009 Pressure Experiments

22. Pressure effect on embryonic and larvae development with fertilization under pressure  Placed sperm in separate vial which ruptured at pressure  Resulting embryos were incubated at different temperature/pressure treatments  Pressure/Temperature treatments Temperature treatments ○ 10, 15, and 20  C Pressure treatments ○ 1, 100, 200, and 300 atm ○ + 400 and 500atm for 4 hour treatments ○ Incubated for 4 and 24 hours

23. Pressure effect on embryonic and larval development with fertilization at atmospheric pressure  Fertilization at atmospheric pressure, at 15  C  Resulting embryos were incubated at 4 different pressures and 5 different temperatures Temperature treatments ○ 5, 10, 15, 20, and 25  C Pressure treatments ○ 1, 100, 200, and 300 atm  Incubated for 50 hours

24. Results  Divided results from 3 methods into 2 categories: Temperature effects on embryonic and larval development Pressure effects on embryonic and larval development

25. Temperature effects on embryonic and larval development  Mytilus edulis embryos develop faster at higher temperatures  Effect in the proportion of abnormally developing embryos

26. Pressure effects on embryonic and larval development with fertilization at atmospheric pressure after 50hrs

27. Pressure effects with fertilization under pressure at 4 hours

28. Pressure effects with fertilization under pressure at 24 hours

29. Krustal-Wallis analysis of variance

30. Conclusions  Temperature tolerance window is from approximately 10-20  C  Embryo development possible up to 500atm (~5000m) Hypothesized pressure presents no barrier to fertilization  Slower development with increasing pressure  Increase in abnormal cells with increasing pressure due to membrane rupture

31. Was hypothesis correct?

32. References  Kyuno, Akiko; Shintaku, Mifue; Fujita, Yuko; Matsumoto, Hiroto; Utsumi, Motoo; Watanabe, Hiromi; Fujiwara, Yoshihiro; Miyazaki, Jun-Ichi. 2009. Dispersal and Differentiation of Deep-Sea Mussels of the Genus Bathymodiolus (Mytilidae, Bathymodiolinae). Journal of Marine Biology. Vol. 2009. pp. 15.  Epel, David. 1975. The Program of and Mechanisms of Fertilization in the Echinoderm Egg. American Zoologist. 15, 3. pp. 507-522.  Mestre, Nelia C.; Thatje, Sven; Tyler, Paul A. 2009. The ocean is not deep enough: pressure tolerances during early ontogeny of the blue mussel Mytilus edulis. Proc. R. Soc. B 276, pp. 717-726.

33. Discussion Questions  What other effects of pressure could cause developmental problems?  Do their results support direct colonization? Last section of discussion implies it does