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