Post by stuart alman on Feb 16, 2006 23:12:20 GMT
Measurements of crustacean glucose and lactate levels in the edible crab Cancer pagurus during emersion stress.
Abstract
The effects of emersion stress upon glucose levels and thus hyperglycaemic hormone levels (CHH) in the edible crab Cancer pagurus. This was tested using glucose and lactate assays to measure their levels. CHH levels increased rapidly after emersion of the treatment crabs while remaining fairly constant in the control crabs.
Introduction
This experiment was performed to attempt to better understand the stress levels of crabs by measuring glucose and lactate levels. The glucose levels will be measured because these are known to rise during periods of stress. Crabs respond differently to stress and so different crabs will give different responses. For example some crabs which live near busy roads will be stressed their entire lives due to the loud noise coming from traffic. The glucose levels are regulated by the release of CHH (hyperglycaemic hormone) from the eyestalks.
The glucose assay was used because the process oxidises chromogen which turns a vivid green colour and it is easy to observe high levels of glucose through this method. The lactate assay works on a similar process resulting in the oxidation of a different chromogen which turns purple.
As the treatment crabs stress levels rise the crab will go through a hyperglycaemic response which will result in the CHH hormone raising glucose levels significantly compared to the control crabs. Cancer pagurus was chosen for this practical because it is more sensitive to stress than other crabs.
During the experiment exterior factors should be taken into consideration such as temperature and the age of the crabs. These factors may also affect the stress levels of the crabs. There should be a mild rise in the stress levels of the control crabs as well due to the constant blood sampling over the four hour period.
Method
During this experiment 2 crabs were kept as control crabs and 2 were used as treatment crabs. The control crabs were kept submerged during the experiment and only removed from their tanks for blood sampling. The treatment crabs were left in air for the duration of the experiment. At the start of the experiment haemolymph samples were taken from the crabs as a base level. Due to the inexperience of handling crabs the first sample was probably higher than normal for the crabs. After this the crabs were put into their selected environments and left. Further samples were taken on the hour for the next four hours to sample the stress levels.
In between the sampling the glucose and lactate assays were prepared by taking samples of haemolymph, making sure there were no clots, and added to either water in the case of lactate assays or to 0.6M perchloric acid in the glucose assays. The glucose samples were then centrifuged and a sample was added to 0.2M phosphate buffer which neutralises the acid. Dilution series were also made during these intervals to compare the results.
At the end of the experiment all the samples were collected and the reagents were added to the samples to display the results. The samples were then placed in a microplate reader for more accurate results.
Results
Glucose T and P Values
Time T Value P Value
0 hours 0.6423713616 0.555
1 hour -3.3130722912 0.009
2 hours -2.3018809149 0.048
3 hours -2.9627029888 0.04
4 hours -2.6892884605 0.024
Lactate T and P Values
Time T Value P Value
0 hours 1.1831299885 0.26
1 hour -3.3118568721 0.009
2 hours -2.2850710712 0.04
3 hours -3.3880740822 0.008
4 hours -1.3666557241 0.204
Fig. 3 – The above two tables show the T and P values for the both the glucose and lactate levels. This was done using a paired T-test measuring the raw data of the Control crabs against the Treatment crabs.
Discussion
As Fig. 1 and 2 show both the glucose and lactate levels rose indicating stress in the treatment crabs. In Fig. 1 the control crabs glucose levels remain fairly constant throughout the experiment indicating limited stress from the experiment. However due to the lack of experience in handling crabs the control levels are probably higher than normal. In Fig.2 the control crabs seem more affected this may be due to their exposure to air during the sampling process. When removed from water the crab transfers immediately to anaerobic respiration and may even stop its heart beating, this will affect the crab for some time even when it’s placed back into water.
The experiment was done to a satisfactory standard as several good and significant results were collected. Although one crab from group ones sample didn’t show any signs of stress and thus was ignored in this practical the rest of the crabs gave a good example of stress induced glucose rises. If the experiment was to be repeated adding in a temperature gauge would probably aid the experiments results as this would remove temperature as an alternate factor affecting stress. Monitoring noise levels would also produce more accurate results.
In Fig. 3 the bold results in the P value column represent the results of significant interest which are represented in Fig. 1 and 2.
References
Campbell and Reece (2005) Biology sixth edition.
Warner, G.F. (1977) The Biology of crabs.
Webster, S.G. (1996) Measurement of crustacean hyperglycaemic hormone levels in the edible crab Cancer pagurus during emersion stress.
Wilcockson, David C (2000) The crustacean hyperglycaemic hormone precursor- related peptide (CPRP) of the crab Cancer pagurus.
Abstract
The effects of emersion stress upon glucose levels and thus hyperglycaemic hormone levels (CHH) in the edible crab Cancer pagurus. This was tested using glucose and lactate assays to measure their levels. CHH levels increased rapidly after emersion of the treatment crabs while remaining fairly constant in the control crabs.
Introduction
This experiment was performed to attempt to better understand the stress levels of crabs by measuring glucose and lactate levels. The glucose levels will be measured because these are known to rise during periods of stress. Crabs respond differently to stress and so different crabs will give different responses. For example some crabs which live near busy roads will be stressed their entire lives due to the loud noise coming from traffic. The glucose levels are regulated by the release of CHH (hyperglycaemic hormone) from the eyestalks.
The glucose assay was used because the process oxidises chromogen which turns a vivid green colour and it is easy to observe high levels of glucose through this method. The lactate assay works on a similar process resulting in the oxidation of a different chromogen which turns purple.
As the treatment crabs stress levels rise the crab will go through a hyperglycaemic response which will result in the CHH hormone raising glucose levels significantly compared to the control crabs. Cancer pagurus was chosen for this practical because it is more sensitive to stress than other crabs.
During the experiment exterior factors should be taken into consideration such as temperature and the age of the crabs. These factors may also affect the stress levels of the crabs. There should be a mild rise in the stress levels of the control crabs as well due to the constant blood sampling over the four hour period.
Method
During this experiment 2 crabs were kept as control crabs and 2 were used as treatment crabs. The control crabs were kept submerged during the experiment and only removed from their tanks for blood sampling. The treatment crabs were left in air for the duration of the experiment. At the start of the experiment haemolymph samples were taken from the crabs as a base level. Due to the inexperience of handling crabs the first sample was probably higher than normal for the crabs. After this the crabs were put into their selected environments and left. Further samples were taken on the hour for the next four hours to sample the stress levels.
In between the sampling the glucose and lactate assays were prepared by taking samples of haemolymph, making sure there were no clots, and added to either water in the case of lactate assays or to 0.6M perchloric acid in the glucose assays. The glucose samples were then centrifuged and a sample was added to 0.2M phosphate buffer which neutralises the acid. Dilution series were also made during these intervals to compare the results.
At the end of the experiment all the samples were collected and the reagents were added to the samples to display the results. The samples were then placed in a microplate reader for more accurate results.
Results
Glucose T and P Values
Time T Value P Value
0 hours 0.6423713616 0.555
1 hour -3.3130722912 0.009
2 hours -2.3018809149 0.048
3 hours -2.9627029888 0.04
4 hours -2.6892884605 0.024
Lactate T and P Values
Time T Value P Value
0 hours 1.1831299885 0.26
1 hour -3.3118568721 0.009
2 hours -2.2850710712 0.04
3 hours -3.3880740822 0.008
4 hours -1.3666557241 0.204
Fig. 3 – The above two tables show the T and P values for the both the glucose and lactate levels. This was done using a paired T-test measuring the raw data of the Control crabs against the Treatment crabs.
Discussion
As Fig. 1 and 2 show both the glucose and lactate levels rose indicating stress in the treatment crabs. In Fig. 1 the control crabs glucose levels remain fairly constant throughout the experiment indicating limited stress from the experiment. However due to the lack of experience in handling crabs the control levels are probably higher than normal. In Fig.2 the control crabs seem more affected this may be due to their exposure to air during the sampling process. When removed from water the crab transfers immediately to anaerobic respiration and may even stop its heart beating, this will affect the crab for some time even when it’s placed back into water.
The experiment was done to a satisfactory standard as several good and significant results were collected. Although one crab from group ones sample didn’t show any signs of stress and thus was ignored in this practical the rest of the crabs gave a good example of stress induced glucose rises. If the experiment was to be repeated adding in a temperature gauge would probably aid the experiments results as this would remove temperature as an alternate factor affecting stress. Monitoring noise levels would also produce more accurate results.
In Fig. 3 the bold results in the P value column represent the results of significant interest which are represented in Fig. 1 and 2.
References
Campbell and Reece (2005) Biology sixth edition.
Warner, G.F. (1977) The Biology of crabs.
Webster, S.G. (1996) Measurement of crustacean hyperglycaemic hormone levels in the edible crab Cancer pagurus during emersion stress.
Wilcockson, David C (2000) The crustacean hyperglycaemic hormone precursor- related peptide (CPRP) of the crab Cancer pagurus.