Abstract
This study investigated the uric acid and ammonia excretion rates in both crickets (terrestrial insect) and cricket organs (hindgut, midgut, and malphigian tubules) as well as the excretion rates in mosquito larva (aquatic insect). After all 5 (hindgut, midgut, malphigian tubules, cricket, and mosquito larva) were suspended in water, they underwent an ammonia and uric acid assay. The results were such that midgut only excreted uric acid, the malphigian tubules ole excreted uric acid, the hindgut excreted 78% uric acid, and 22% ammonia, the cricket only excreted uric acid, and the mosquito larva excreted only ammonia. This supports the idea that terrestrial animals excrete mostly uric acid where as aquatic animal excrete mostly ammonia.
Introduction
Discovered in 1772 by Daniel Rutherford Nitrogen is crucial to the health of humans (Galloway ,2013). It is an important element found in amino acids and proteins which is vital to human survival. These amino acids make up muscles, hair, skin and a variety of other tissues. The majority of nitrogen is acquired through dietary means, however it can also be absorbed through the epidermis . Proteins and amino acids are regularly undergoing catabolic metabolism creating an excess of nitrogen to build up in the human body. Nitrogen excretion consists almost always in three forms: ammonia (NH3), urea (CH4N2O), and uric acid (C5H4N4O3) (Weiner, 2014). Ammonia is the most toxic because it raises the pH of bodily fluids putting health in a critical state . Aquatic species are typically able to handle a more elevated blood-ammonia level thus excreting a by-product of ammonia. Highly soluble in water, ammonia is able to easily penetrate cell membranes and readily defuses across providing water is available. For every gram of ammonia, an animal must dilute with approximately 400 mls of water, to maintain a stable blood-ammonia level and avoid toxicity. During the transition from water to land natural selection favoured less toxic by-products such as urea and uric acid, because these species were no longer surrounded by water thus no longer having a favourable osmotic gradient. Both have the capability of being concentrated in a higher number in bodily fluids than ammonia with no toxicity effects (Wright, 1995). Terrestrial animals cannot excrete nitrogen in the form of ammonia because it require a large quantity of water and would suffer of dehydration. Compared to ammonia urea – a soluble molecule – requires approximately 10 times less water. Uric acid – an insoluble molecule and the least toxic – requires approximately 50 times less water than ammonia (Wright, 1995). The ability to excrete all three waste products are found within most organisms, however the form which is excreted depends on environmental conditions (Weiner, 2014). Crickets, mosquito larva, cricket hindgut, cricket midgut, and cricket malphigian tubules will be placed in water and undergo an ammonia and uric acid assay to determine the excretion rate of ammonia and the amount of uric acid in the samples. It is hypothesized that the rate of ammonia excretion will increase starting from: cricket, mlaphigian tubules, midgut, hindgut, and mosquito larva. It is also believed that uric acid concentrations will increase starting from: mosquito larva, midgut, malphigian tubules, hindgut and the cricket.
Methods and materials
The experiment was preformed as described in the Biol3030 laboratory protocol, however, the ammonia assay tubes were placed in the dark for 45 minutes as oppose to the 90 minutes stated in the laboratory manual.
Results Figures
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Percent of Ammonia and Uric Acid found in Cricket Organs
Uric Acid Ammonia
Midgut
Hindgut
Malphigian Tubules
Figure 1. displays the relationship between the percentage of uric acid (C5H4N4O3) and ammonia (NH3) found in cricket midgut, cricket hindgut, and cricket malphigian tubules. Organs were harvested from live crickets during the laboratory experiment ensuring accurate data. All cricket midgut, hindgut, and malphigian tubules were suspended in 4 millilitres of water and shaken to lyse the organs establishing that any potential uric acid and ammonia was in suspension. Percents were calculated based upon excretions rates which were calculated from concentrations of the sample, the amount of water the organs were lysed in (4 mls), and the weight. The masses of the midgut, hindgut, and malphigian tubules were 0.000044 kilograms, 0.000014 kilograms and 0.00001kg respectively. Note: This data was collected under standard room temperature settings.
Percent (%)
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Uric Acid Ammonia
Percent of Ammonia and Uric Acid found in Crickets and Mosquito Larva
Cricket
Mosquito Larva
Figure 2. displays the relationship between the percentage of uric acid (C5H4N4O3) and ammonia (NH3) found in crickets and mosquito larva. Percents were calculated based upon excretions rates which were calculated from concentrations of the sample, the amount of water the cricket and mosquito larva were excreting in, the weight and the length of time they were excreting in the water for. The cricket was excreting in 20 millilitres of water for 1.866 hours and had a mass of 0.296 grams where as 10 mosquito larva were excreting in 5 millilitres of water for 7.5 hours and had an individual mass of 2.7mg (27 mg cumulatively). Note: This data was collected under standard room temperature settings.
Percent (%)
Results Text
The midgut was determined to have excreted 100% uric acid, resulting in no ammonia excretion (figure 1). When suspended in 4 millilitres of water and prepared for a uric acid assay, the midgut had an optical density of 0.063 from which it was determined to have a uric acid sample concentration of 1.012847595 umol/L. When prepared for the ammonia assay, the midgut had a optical density reading of 0.528, and was the determined from the standard ammonia curve to have a negative concentration of ammonia thus being null. With a mass of 44 mg the midgut had an uric acid excretion rate of 92.07705364 umol/N/Kg. Likewise from figure 1 the malphigian tubules were determined to have excreted 100% uric acid resulting in no ammonia being excreted. When suspended in 4 millilitres of water and prepared for a uric acid assay, the malphigian tubules had an optical density of 0.152. It was then determined to have a uric acid sample concentration of 2.443695789 umol/L. With a mass of 10 mg the midgut had a uric acid excretion rate of 977.4783136 umol/N/Kg. From the ammonia assay the malphigian tubules had an optical density of 0.195, however when compared on the ammonia standard curve, the concentration of ammonia was negative this being null. From figure 1 it was established that the hindgut had 78% uric acid excretion and 22% ammonia excretion. When suspended in 4 millilitres of water and prepared for a uric acid assay, the hindgut had an optical density of 0.207 from which it was determined to have a uric acid sample concentration of 3.327927811 umol/L. Likewise when prepared for the ammonia assay it was determined to have a optical density of 0.757 and a ammonia sample concentration of 0.9333024548 umol/L. With a mass of 14 mg the midgut had an uric acid excretion rate of 950.8365174 umol/N/Kg, and an ammonia excretion rate of 266.6578442 umol/N/Kg. From figure 2 it was determined that the cricket had excreted 100% uric acid, resulting in no ammonia excretion. When suspended in 20 millilitres of water and prepared for a uric acid assay, the cricket had an optical density of 0.075 from which it was determined to have a uric acid sample concentration of 1.205770946 umol/L. When prepared for the ammonia assay, the cricket had a optical density reading of 0.153, and was the determined from the standard ammonia curve to have a negative concentration of ammonia thus being null. With a mass of 0.296 grams the cricket had a uric acid excretion rate of 43.64518901 umol/N/Kg/Hr. Similarly from figure 2 it was determined that the mosquito larva had excreted 100% ammonia, resulting in no uric acid excretion. When suspended in 5 millilitres of water and prepared for a uric acid assay, the mosquito larva had an optical density of 0 indicating there was no uric acid excreted. When prepared for the ammonia assay, the mosquito larva had an optical density reading of 1.772, and was the determined from the standard ammonia curve to have an ammonia concentration of 5.634553034. With a total mass of 27 mg the mosquito larva had a ammonia excretion rate of 139.1247663 umol/N/Kg/Hr.
Discussion
This study investigated the uric acid and ammonia excretion rates in both crickets (terrestrial insect) and cricket organs (hindgut, midgut, and malphigian tubules) as well as the excretion rates in mosquito larva (aquatic insect). It was hypothesized that the rate of ammonia excretion will increase starting from: cricket, mlaphigian tubules, midgut, hindgut, and mosquito larva. It is also believed that uric acid concentrations will increase starting from: mosquito larva, midgut, malphigian tubules, hindgut and the cricket. Supported by the data show in figure 1 and figure 2 the hypothesis was show to be accurate. Figure 1 depicts that midgut and malphigian tubules contain solely uric acid and that the hindgut contains both uric acid and ammonia with and approximate 5:1 ratio respectively. Figure 2 depicts that the cricket solely excreted uric acid where as the mosquito larva only excreted ammonia. Literature indicates that there is a proportional effect between type of excretion (ammonia, uric acid, and urea) and environment (terrestrial and aquatic). Terrestrial animals have a higher uric acid excretion as oppose to ammonia, and that aquatic animals have a higher ammonia excretion as oppose to uric acid (Wright, 1995).
The data collected in this lab is similar to that found in literature such that it revealed that the common house cricket (acheta domestics) and other common insects such as Oncopeltus faciatus, Periplaneta americana, Tenebrio molitor and Galleria mellonella all have a major nitrogen excretion product of uric acid (Nation, 2003).
Similarly, literature supports the finding for the mosquito larva. Although there is no literature specifically for mosquito larva, there is some for a common similar insect: the alderfly (Sialis lutaria). With this insect when they are in the larval form in aqueous solution this species excretes ammonia, however once they turn into the adult alderfly they then begin to excrete uric acid (Staddon, 1954). Supporting the belief that nitrogen excretion methods depends on environmental factors. This evidence supports the fact that terrestrial animals cannot excrete nitrogen in the form of ammonia because it require a large quantity of water and would suffer of dehydration compared to uric acid requires approximately 50 times less water than ammonia
Although the results are fairly accurate to the literature, data could have effected due to the fact that the cricket was diseased when the laboratory experiment began, therefore there is no guarantee of long the cricket was excreting in the water, possibly affecting the rate of excretion of both ammonia and uric acid.
Conclusion
To conclude the hypothesis was supported such that rate of ammonia excretion will increase starting from: cricket, mlaphigian tubules, midgut, hindgut, and mosquito larva. It is also believed that uric acid concentrations will increase starting from: mosquito larva, midgut, malphigian tubules, hindgut and the cricket. This was based upon the knowledge that aquatic animals have a higher tendency to excrete nitrogen in the form of ammonia, and that terrestrial animals usually excrete nitrogen in the form of uric acid.
Appendix
2
R2 = 0.9456
y = 0.2159x + 0.5555
Ammonia Standard Curve
1.5
1
0.5
0
25 50 75 100 125 150
Calculations were hand written.
Concentration of Ammonia (umol/L)
Optical Density (OD)
References
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- Nation, J.L., Patton, R.L. (2003). A study of nitrogen excretion in insects. Science Direct, 6(4), 99-308.
- Staddon, B.W. (1954). E EXCRETION AND STORAGE OF AMMONIA BY THE AQUATIC LARVA OF SIALIS LUTARIA (NEUROPTERA). The Journal of Experimental Biology, 84-94.
- Weiner, I. D., Mitch, W. E., & Sands, J. M. (2014). Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion. Clinical journal of the American Society of Nephrology : CJASN, 10(8), 1444-58.
- Wright, P. A. (1995). NITROGEN EXCRETION: THREE END PRODUCTS, MANY PHYSIOLOGICAL ROLES. The Journal of Experimental Biology 198, 273– 281