The concentration of solutes in the bodily fluids of most marine invertebrates is roughly isosmotic to their environment (Raven, 2008). Because there is no osmotic gradient there is no tendency for the net diffusion of water away from the animal’s cells to occur. When a change in salinity occurs some organisms have the ability to maintain a constant internal homeostasis despite these external changes and are known as osmoregulators (Oxford, 2008). Other animals lack this ability and as such are called osmoconformers; their internal osmolarity matches that of their environment although their ionic composition may be different (Oxford, 2008).
In this experiment the osmoregulatory capability of two marine invertebrates was investigated, Carcinus maenas (shore crab) and Arenicola marina (lugworm). The lugworm’s capability to regulate cell volume over a period of 90 minutes was observed by measuring the change in weight of an entire worm exposed to different seawater concentrations in 15 minute intervals. The shore crab’s haemolymph ionic composition was analysed in response to a longer term exposure to hypo-osmotic conditions over 3 days. The haemolymph was analysed for osmolarity, Sodium ion concentration and chloride ion concentration.
Methodology Instructions were followed for both organisms as supplied by the lab schedule. Carcinus maenus Haemolymph was extracted from a crab that had been stored in a seawater concentration of 75% for 3 days prior to the experiment. 200µl was then pipetted into an Eppendorf tube, avoiding transferring any coagulated proteins into the Eppendorf tube. The osmolarity of the sample was measured by pipetting 100µl of the sample into another Eppendorf tube and placing this inside a Camlab Freezing Point Osmometer, ensuring the probe is clean and dry.
Sodium levels were measured using a flame photometer, placed in a fume cupboard and left to run with distilled water for 15 minutes prior to running a sample. The lab schedule stated to dilute the sample with deionized water at a ratio of 1:800. 7µl of haemolymph was pipetted into a beaker and 5. 6ml of water was pipetted in 1000µl at a time to reach the required dilution. The flame photometer in the lab was set to a 1:200 dilution and as such all results have been multiplied by 4 for the correct sodium concentrations in the haemolymph.
Chloride levels were measured by pipetting 14µl of undiluted haemolymph into a beaker filled with electrolyte solution and measured using a Corning Chloride Meter Mark II conditioned and calibrated by filling a beaker with standard electrolyte solution, pipetting 100µl of 0. 1M NaCl into the beaker and pressing “condition”. 14µl of the 0. 1M NaCl solution is then pipetted in and “titrate” is pressed, noting the reading, which should be 100±5. This is repeated to ensure the readings are consistent. Arenicola marina A single live animal was chosen at random from a selection stored in full strength seawater.
A 75% seawater solution was selected. The lugworm was blotted carefully, by placing one sheet of tissue under and one sheet over the worm and carefully rolling until dry. The initial weight was noted to 2 decimal places and then the worm was placed into a container of the 75% seawater solution. The worm was removed at 15 minute intervals, dried using the above method and weighed again over a 90 minute period. The percentage weight change relative to the original weight was calculated using the following formula: % change=Tx-T0T0? 100 T0=Initial Weight Tx=Weight at time x Results Carcinus maenus
Figure 1 shows a significant difference (p<0. 001) between the 4 haemolymph treatments, with the trend line in closely matching the hatched equivalence line, indicating the change in osmolarity of the haemolymph when seawater concentration was changed. Figure 1. Haemolymph osmolarity as a function of seawater osmolarity at varying concentrations, 100%, 75%, 50% and 25%. Values are mean ± SEM. N=7 for all groups. As seen in Figure 2 the haemolymph at each concentration closely follows the equivalence line and each reading at the different seawater concentrations is significantly different from every other data point (p<0.
001). Figure 2. Haemolymph chloride levels as a function of seawater chloride at 4 concentrations; 100%, 75%, 50% and 25%. Values are mean ± SEM. N=7 for all groups. After 3 days in the diluted seawater the crab’s haemolymph Sodium levels were significantly lower (p<0. 001) than the crabs stored in full strength seawater as seen in figure 3. There was no significant difference between the mean haemolymph Sodium values of the three dilutions at 75%, 50% and 25%. Figure 3. Haemolymph Sodium levels as a function of seawater Sodium at 4 concentrations; 100%, 75%, 50% and 25%. Values are mean ± SEM. N=7 for all groups.