1. Changes in hemolymph osmolality and gill Na+ K+ -ATPase activity in the land hermit crab Coenobita clypeatus in response to salinity variations Joseph B. Oliver (’06) Thesis Adviser: Dr. Holliday

2. Osmoregulation Osmoregulation is a form of homeostasis The methods of adjusting to the environment – Avoid – Conform – Compensate The classification of osmoregulation – Stenohaline – Euryhaline

3. Na +, K + -ATPase Mechanism

4. Crustacean Gills – Osmoregulatory Organs: Model 1

5. Crustacean Gills – Osmoregulatory Organs: Model 2

6. Coenobita clypeatus Develops in marine habitat and lives adult life on land Inhabits snail shell – vital for survival Found throughout Western Atlantic coastal areas http://foreverpurple.com/gallery/displ ayimage.php?album=24&pos=17

7. Experimental Procedures Animal preparation – treatment conditions – Open air – Submerged Hemolymph sampling Gill Na+, K+ -ATPase assay

8. Animal Care and Treatment Open Air: plastic shoe boxes (10.2 cm x 19.1 cm x 33 cm) and provided 2 water dishes and food, gravel on bottom Fully Submerged: cylindrical container (10.2 cm diameter, 7.6 cm height) with water replaced every 1-2 days, small air stone, and periodic feeding

9. Hemolymph Sampling Wescor 5100C vapor pressure osmometer 8  l of hemolymph assayed Measured in millimole solute / kg water - milliosmolality (mOsm x kg -1 )

10. Na+, K+ -ATPase assay 4 parts to assay: – Preparation of homogenates – Homogenate ATPase assay – Homogenate protein assay – Calculations Enzyme Specific Activity (ESA) results: micromoles of phosphate liberated x mg -1 of protein x hour -1 by the sodium pump

11. Hemolymph Osmolality in Open Air Conditions with Commercial Hermit Crab Food

12. Gill Enzyme Specific Activity in Open Air Conditions with Commercial Hermit Crab Food

13. Comparison of Hemolymph Osmolality of Open Air Crabs Fed Pellets and Fed Vegetation

14. Comparison of Gill ESA of Open Air Crabs Fed Pellets and Fed Vegetation

15. Hemolymph Osmolality of the Fully Submerged Hermit Crabs

16. Gill ESA of Fully Submerged Hermit Crabs

17. Range

19. Comments on Pattern Very high gill ESA when not osmotically stressed, little fluctuation under osmotic stress Seen also in beach hopper Megalorchestia californiana Advantage: Respond quickly to changes in available water by modulation of enzyme

20. Interesting Tidbits Control animals closely resembled freshwater animals Seawater conditions are less osmotically stressful Not all salt comes from ion pumping by gills Diet may play an important role

21. Conclusions on C. clypeatus Euryhaline crab species when submerged Strong hyperregulators in dilute conditions Well adapted to terrestrial lifestyle Osmoconformers in seawater and concentrated conditions

22. Future Considerations Are changes in gill salt pumping due to modulation of existing enzyme or creation of new enzyme? What is the complete range of salinities C. clypeatus can survive in, and how long can they survive in these stressed conditions?

23. THANK YOU FOR YOUR TIME AND ATTENTION At this time, I am able to field any questions