changes in hemolymph osmolality and gill na+ k+ -atpase activity in the land hermit crab coenobita...
TRANSCRIPT
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 The methods of adjusting to the environment
– Avoid– Conform– Compensate
The classification of osmoregulation– Stenohaline– Euryhaline
Na+, K+ -ATPase Mechanism
Crustacean Gills – Osmoregulatory Organs:Model 1
Crustacean Gills – Osmoregulatory Organs: Model 2
Coenobita clypeatus
Develops in marine habitat and lives adult life on land
Inhabits snail shell – vital for survival
Found throughout Western Atlantic coastal areas
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Experimental Procedures
Animal preparation – treatment conditions– Open air– Submerged
Hemolymph sampling Gill Na+, K+ -ATPase assay
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
Hemolymph Sampling
Wescor 5100C vapor pressure osmometer 8l of hemolymph assayed Measured in millimole solute / kg water -
milliosmolality (mOsm x kg-1)
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
Hemolymph Osmolality in Open Air Conditions with Commercial Hermit Crab Food
0
200
400
600
800
1000
1200
1400
1600
1800
Control Freshwater only Saltwater only Hypersaline water only
External Medium
Hemolymph Osmolality (mOsm x kg-1)
*
*
n=8n=9
n=9
n=9
0
5
10
15
20
25
30
35
Control Freshwater Only Saltwater Only Hypersaline Only
External Medium
ESA (umol x mg-1 x hr-1)
**
*
n=8
n=9
n=9
n=9
Gill Enzyme Specific Activity in Open Air Conditions with Commercial Hermit Crab Food
Comparison of Hemolymph Osmolality of Open Air Crabs Fed Pellets and Fed Vegetation
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Control Freshwater Only Saltwater Only Hypersaline Only
External Medium
Hermit Crab Pellets Vegetation
*
*
*
*
Hemolymph Osmolality
(mOsm x kg-1)
n=8 n=9
n=9
n=9
n=7n=7
n=8
n=8
Comparison of Gill ESA of Open Air Crabs Fed Pellets and Fed Vegetation
0
5
10
15
20
25
30
35
Control Freshwater Only Saltwater Only Hypersaline Only
External Medium
Hermit Crab Pellets Vegetation
ESA (umol x mg-1 x hr-1)
*
*
*
n=8
n=9
n=9
n=9
n=7n=7
n=8
n=8
Hemolymph Osmolality of the Fully Submerged Hermit Crabs
0
200
400
600
800
1000
1200
1400
400 mOsm 800 mOsm 1200 mOsm
External Medium
Hemolymph Osmolality (mOsm x kg-1)
*
*
n=8n=8
n=6
Gill ESA of Fully Submerged Hermit Crabs
0
5
10
15
20
25
30
35
400 mOsm 800 mOsm 1200 mOsm
External Medium
ESA (umol x mg
-1 x hr
-n=8
n=8
n=6
Osmotically Unstressed Gill ESA of Various Species
Artenia salinaCarcinus maenasCoenobita clypeatus
Idotea wosnesenskii
IsopodsCallinectes sapidus
0
5
10
15
20
25
30
35
Species
ESA (umol x mg
-1 x hr
-
Ran
ge
Ran
ge
Ran
ge
Increase in Gill ESA in response to Osmotic Stress
Cyprinodon variegates hu...Oreochromis mossambicus
Callinectes smilis
Idotea wosesenskii
Coenobita clypeatus
Carcinus maenas
0
1
2
3
4
5
6
Species
Relative Increase
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
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
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
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?
THANK YOU FOR YOUR TIME AND ATTENTION
At this time, I am able to field any questions