Methods and Materials
For this experiment the blue crab (Callinectes sapidus) was used. They were kept in tanks in room temperature, artificial seawater with a ‘normal’ osmolarity until the experiment was started. Great caution was taken keep hands out of the way of the crab’s claws because they are very aggressive and can pinch and crush fingers. To ensure safe handling, the crabs were picked up by the posterior portion of the carapace near the swimmerets and placed on their ventral surfaces. This is to keep them calm, and it also allows easier access to obtain a 50 μL hemolymph sample from their fleshy joints on a walking leg (which is unprotected compared to most of the leg where there is a hard exoskeleton) using a 28 gauge (or similar) needle with a 1 ml syringe.
Four different experimental tanks were filled with artificial seawater with five different osmolarities: 1200, 1100, 1000, 600, and 400 mosmols. To be accurate in the composition of the water in all five tanks, it all came from one common source -- a large bin of the artificial seawater that was made to be 1250 mosmol and diluted to the specified amount. It was tested using an osmometer to get an exact osmolarity of the salt water.
The crabs were taken from their resting place and a sample of hemolymph was extracted using a syringe and put into the osmometer to get an initial reading which was recorded. Next, the crabs were each placed in one of the five tanks of different salinities and left to sit for a half hour. After a half hour the crabs were taken out of the water and the hemolymph extraction was repeated and recorded. The crabs were then allowed to sit for 24 hours to allow acclimation time. After 24 hours the crabs were taken out and again, the hemolymph salt concentration was obtained. After the crab’s hemolymph was measured, it was moved into a different, randomly pre-determined tank with a different salinity than the one it was previously in....