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
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
Osmoregulation is a form of homeostasis
The methods of adjusting to the environment
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–
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
Avoid
Conform
Compensate
The classification of osmoregulation
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–
Stenohaline
Euryhaline
Na+, K+ -ATPase Mechanism
Crustacean Gills – Osmoregulatory Organs:
Model 1
Crustacean Gills – Osmoregulatory Organs:
Model 2
Coenobita clypeatus
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http://foreverpurple.com/gallery/displ
ayimage.php?album=24&pos=17
Develops in marine
habitat and lives adult
life on land
Inhabits snail shell –
vital for survival
Found throughout
Western Atlantic coastal
areas
Experimental Procedures
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Animal preparation – treatment conditions
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–
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Open air
Submerged
Hemolymph sampling
Gill Na+, K+ -ATPase assay
Animal Care and Treatment
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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
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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
Hemolymph Sampling
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Wescor 5100C vapor pressure osmometer
8l of hemolymph assayed
Measured in millimole solute / kg water milliosmolality (mOsm x kg-1)
Na+, K+ -ATPase assay
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4 parts to assay:
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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
Hemolymph Osmolality in Open Air Conditions with
Commercial Hermit Crab Food
Hemolymph Osmolality (mOsm x kg-1)
1800
n=9
*
1600
1400
1200
n=9
*
1000
n=8
n=9
800
600
400
200
0
Control
Freshwater only
Saltwater only
External Medium
Hypersaline water only
Gill Enzyme Specific Activity in Open Air Conditions with
Commercial Hermit Crab Food
35
ESA (umol x mg-1 x hr-1)
30
n=9
n=8
n=9
25
**
n=9
*
20
15
10
5
0
Control
Freshwater Only
Saltwater Only
External Medium
Hypersaline Only
Comparison of Hemolymph Osmolality of Open Air
Crabs Fed Pellets and Fed Vegetation
2000
1800
n=8
*
Hermit Crab Pellets
Vegetation
n=9
Hemolymph Osmolality
(mOsm x kg-1)
1600
1400
1200
n=7
1000
n=9
n=7
*
n=8
n=8
*
n=9
*
800
600
400
200
0
Control
Fres hwater Only
Saltwater Only
External Medium
Hypers aline Only
Comparison of Gill ESA of Open Air Crabs Fed Pellets and
Fed Vegetation
n=9
35
n=8
ESA (umol x mg-1 x hr-1)
30
n=7
*
Hermit Crab Pellets
n=7
*
n=9
25
Vegetation
n=8
n=9
n=8
20
*
15
10
5
0
Control
Freshw ater Only
Saltw ater Only
External Medium
Hypersaline Only
Hemolymph Osmolality of the Fully Submerged Hermit
Crabs
Hemolymph Osmolality (mOsm x kg-1)
1400
n=6
*
1200
n=8
1000
n=8
800
*
600
400
200
0
400 mOsm
800 mOsm
External Medium
1200 mOsm
Gill ESA of Fully Submerged Hermit Crabs
35
n=8
-1
ESA (umol x mg x hr
-
30
n=8
25
n=6
20
15
10
5
0
400 mOsm
800 mOsm
External Medium
1200 mOsm
Range
Range
Range
Comments on Pattern
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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
Interesting Tidbits
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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
Conclusions on C. clypeatus
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Euryhaline crab species when submerged
Strong hyperregulators in dilute conditions
Well adapted to terrestrial lifestyle
Osmoconformers in seawater and
concentrated conditions
Future Considerations
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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?
THANK YOU FOR YOUR TIME
AND ATTENTION
At this time, I am able to
field any questions