NITROGENOUS EXCRETION IN THE CRAYFISH IN RELATION TO DIETARY PROTEIN

Introduction

Dietary protein is broken down in the gut to its constituent amino acids which are then absorbed into the blood. Here excess amino acids, not needed for synthesis of other compounds, are metabolised in the citric acid cycle after removal of the a-amino groups as ammonia. The ammonia is then excreted, sometimes after conversion to other compounds; there are marked differences in the nitrogen excretion products of aquatic and terrestrial animals.

In this lab class you will investigate ammonia production by an aquatic animal, the crayfish (Crustacea: Decapoda), in relation to diet. There is one native species of crayfish found in UK waterways (the White-clawed Crayfish Austropotamobius pallipes), which is now endangered by competition and diseases introduced with non-native crayfish species. This practical uses farmed American Signal Crayfish (Pacifastacus leniusculus), which have been fed either high-protein (bloodworm; Tubifex) or low-protein (potato) diets for the preceding week. The prediction is that crayfish fed on tubifex worms will have more excess amino acids, and thus higher excretion of ammonia. For this practical you will work in pairs, but will use the data from your entire laboratory class to investigate the effect of diet on ammonia excretion by crayfish.

http://news.bbc.co.uk/1/hi/uk/england/1562118.stm (Crayfish make the news)

http://www.wbrc.org.uk/worcRecd/Vol1Iss2/crayFish.htm (Status of native and introduced crayfish)

http://www.sac.ac.uk/management/External/diversification/Fishfarm/crayfish.asp (Crayfish farming)


Procedure

Ammonium Excretion

  1. Label the two 2 l beakers at your lab bench "potato" and "worm". Using a graduated cylinder, add 400 ml of the treated water (from the "stock" tank) to each of the beakers.

  2. Pipette 10 ml of water from the water "stock" and place in a 25 ml test tube. Mark this tube "pre-exposure" and seal with parafilm.

  3. Select two crayfish of approximately equal size from the two holding tanks, for potato- and worm-fed animals. Remove superficial water with tissue paper and transfer the crayfish to your beakers, noting the time exactly. Ensure that your crayfish cannot escape.

  4. At the end of 30 min gently swirl the beaker to mix the water, draw 10 ml of water, place this in an appropriately labelled 25 ml test tube and seal the top with parafilm. Take two samples for each crayfish. Label each tube "post-exposure" and with the diet. Record the total exposure time.

  5. As soon as you have collected all four 10 ml "post-exposure" water samples, begin the ammonia analysis of these samples, plus the "pre-exposure" sample, a blank and standards as outlined under the section on Determination of Ammonium Excretion (below).

  6. Once your ammonia assay has been started, remember to weigh your crayfish.

Determination of Ammonium Excretion (nitroprusside method)

Calculation of the rate of ammonium excretion (μmol g-1 h-1)


Writing your report

Your report will use your own data, plus a statistical analysis of the class results which will be sent to you by email. All data must be added to the class sheet before you leave, including the mass of each crayfish, the diet and the excretion rate. What is the effect of diet on the rate of excretion of ammonia?

Read the appropriate sections of Chapter 58 in your text book "Raven & Johnson" but especially page 1191. Then write a paragraph (ie about half a page) to accompany your results, explaining why aquatic organisms excrete ammonia and why terrestrial animals largely do not. What do they excrete to rid themselves of excess and waste nitrogen and why these substances?

If the rate of nitrogenous excretion depends on catabolism of proteins, why were one group of crayfish fed potatoes (a low protein, high carbohydrate diet) rather than simply fasted (starved)?