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Procambarus clarkii (crayfish) are crustaceans often found in fresh water climates (Britannica). The crayfish are close relatives to those of lobster, shrimp and crab. Yet, crayfish are better subjects for this study because it is an easier organism to extract the hemolymph from, rather than that of a lobster. The crayfish stores the CHH around its eyes and when compared to humans, who store it in their pancreases and liver, it is easier to extract the hormone from. In addition, you can control the crayfish by eliminating the x-organ which controls the release of CHH into the blood stream.
The particular hormone that affects the glucose level in crayfish is CHH, crustacean hyperglycemic hormone. When the hormone is released, it causes the hepatopancreas to release glucose into the blood (hymolymph). (Sullivan 2009). The CHH hormone is created in the X-organ and is stored in the sinus gland before it is released into the blood (Chung Sook). The CHH hormone is extremely important in maintain the metabolism and emotions in crustaceans (Chung Sook). This hormone is particularly important for the crayfish and other crustaceans to survive.
This study is particularly important for a comparison on how a similar hormone like glucagon can affect the stress levels in humans. Glucagon is the hormone that is produced that is similar to glucose like that in crustaceans. In a study done by S.R Bloom and colleagues found that, when a human was stressed they had released a significant amount of glucagon. This is similar to what is said to happen when a crustacean is put into a stressful stimulus.
This study was conducted in order to become familiar with how stress levels can affect the glucose level in Procambarus clarkii (crayfish). The working hypothesis of this experiment was, P.clarkii (crayfish) with eyes are going to release higher levels of the CHH and cause the glucose concentration to rise because they still present the working x-organ. The null hypothesis states that the P.clarkii (crayfish) would not show a difference between the two groups under the stress of chloroform.
Methods and Materials
Prior to the lab two crayfish (Carolina Biological Supply Burlington, NC), had been obtained; one crayfish had its eyes removed along with its sinus cavity containing its X-organ the other had eyes and the X-organ. In addition to the beginning of the study, two insulin needles were prepared, one holding the ringers solution. The other will be what is used to extract the hemolymph. During the first segment of the study, 0.1mL hemolymph was extracted from the last leg of the crayfish. After doing so immediately replace the hemolymph with 0.1 mL Ringers Solution (Department of Biology and Environmental Science). Then allow the crayfish to rest for fifteen minutes to be able to produce more hemolymph. Next the crayfish has to be put under a stressor, chloroform (Department of Biology and Environmental Science), for twenty seconds. Then again, allow the crayfish to rest for fifteen minutes to allow the CHH to be released. Following the resting period extract an additional 0.1mL of hemolymph, and then again immediately replace with 0.1 mL of Ringer's solution. After the samples were taken, then obtain four small test tubes. One is made into a blank that contains ~0.1mL of Ringer's solution and an enzyme solution obtained from the Department of Biology and Environmental Science. And a glucose standard (~0.1 mL glucose, ~0.1 mL Ringer's solution, and ~1.5 mL enzyme solution) to test the hemolymph against. In each of the tubes, add the hemolymph obtained from the crayfish with into one tube and the without eyes to a second tube. To both tubes, add ~1.5 mL of enzyme solution. After the solution is added take all four test tubes and place them in a water bath of 37° C for 30 minutes. When the time is up take the test tubes out of the water bath and place them into separate cuvettes. After blanking the Spectronic 20 plus (Thermo Spectronic) with the Ringer's solution you then place the glucose standard in the Spectronic 20 plus and read the concentration. Following the glucose standard obtain the concentration of the test tub for the crayfish with and without eyes, being sure to sure to re-zero the Spectronic 20 plus with the Ringer's solution.
Below Table 1 shows the average change in hemolymph glucose concentration in Crayfish over the years of 2009 to 2012. The crayfish without eyes had shown a higher increase of hemolymph glucose concentration after stress. And the crayfish with eyes had shown a lower increase of hemolymph glucose concentration after stress in chloroform. The t-test shown is not statistically significant. This could be a result of there being more crayfish with eyes than crayfish without eyes. In order for the results to be statistically significant the T-test should be 0.05 and lower, this shows that it is not statically significant being at 0.29.
Table 1: Average change in Hemolymph Glucose Concentration in Procambarus clarkii over the
years of 2009-2012 with standard deviation, and a t-test of 0.580559.
Average Change Standard Deviation T-Test
Without eyes 0.011634271 0.033922 0.29028
With eyes 0.000684232 0.097702
Figure 1 shows the relationship between Procambarus clarkii (Crayfish) without eyes and without the x-organ and Procambarus clarkii (Crayfish) with the x-organ and with eyes. The figure below shows that crayfish without eyes had a higher change in glucose concentration than those with eyes (p=0.29028).
Figure 1.0: Hemolymph glucose concentration of Procambarus clarkii with and without eyestalks and x-organ had been sampled before and after exposure to chloroform. Average change in glucose concentration (mg/ml) over several experiments from 2009-2012. (p>0.05)
We tested Procamburas clarkii (crayfish) without eyes and no X-organ against those with eyes and an organ. We found that Procamburas clarkii (crayfish) without eyes had produced more change in glucose concentration than Procamburas clarkii (crayfish) with eyes. Based on the data, the working hypothesis is rejected because the Procamburas clarkii (crayfish) with the x-organs did not produce a higher hemolymph glucose concentration than the crayfish without the x-organs. This is rejected because Figure 1 shows that the crayfish without eyes had shown a higher glucose concentration level than those with eyes.
A study conducted by Ernest S. Chang tested the CHH level in stressed out lobsters. They tested lobsters on three different levels of stress: emersion, temperature elevation and salinity changes (Ernest S. Chang). The results that they found was that emersion had caused a significant increase of CHH after a rest of 15 minutes. This result is rather important because it helps prove that when we emerged the crayfish into chloroform we as well should have found a significant change in CHH levels after a rest of 15 minutes. Another finding was a study done by Junying Zheng and colleagues on blue crab and CHH levels. The results that they had obtained were that the CHH was dominate in the eyestalks of the blue crab. This result also helps prove that when a crayfish is lacking the eyes and x-organ of the crayfish is missing it will affect the CHH levels produced into the crustaceans.
Ways to improve this experiment and come out with results that are closer to other groups that have tested CHH in other crustacean is that we used younger crayfish. A lot of the crayfish had died off allowing for a miscalculation in numbers and not providing a number that is more statistically significant. With the use of more and younger crayfish, this would lower the risk of the percent error in the data.
Another way to improve is to handle the crayfish carefully. If the crayfish had been dropped or shaken, this could have caused them to release the CHH and would put more stress on them causing a difference in glucose concentration that was obtained. When the crayfish had obtained more stressful situations other than the chloroform it had allowed them to release more of the CHH hormone at various times causing the hemolymph level to be slanted.
The CHH ( crustacean hyperglycemic hormone) is critical for all crustaceans because it ensures that they are in a state of homeostasis of their body functions. Without the proper organs, their bodies would not have been able to respond to a stressful stimulus causing their bodies to produce higher levels of glucose into their hemolymph and causing their bodies to essentially go into shock or even death. This study cannot conclude or deny that crayfish without the x-organs are able to produce more hemolymph glucose concentration than those with the x-organs, or if the crayfish without the x-organ are able to withstand stressful situations.
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