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Joseph B. Oliver (’06) Thesis Adviser: Dr. Holliday

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. Osmoregulation. Osmoregulation is a form of homeostasis

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Joseph B. Oliver (’06) Thesis Adviser: Dr. Holliday

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  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/displayimage.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 Range Range

  18. 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

  19. 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

  20. 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

  21. 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?

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

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