Investigating Respiration of Maggots

The aim of my investigation is to see how the rate of respiration of some maggots differs between room temperature and other temperatures, in terms of volume of oxygen produced.

I predict that as I raise the temperature of the maggots, the rate of respiration will increase. I think this because respiration is an enzyme-dependant process of reactions. Increasing the temperature will give the enzymes and substrates more kinetic energy and therefore will increase the rate of respiration. This is explained in further detail later on.

Method

1. Using a syringe filled with manometer fluid, I will half fill a manometer.
2. I will then attach a 3-way tap to one of the delivery tubes on the bung and to this tap I will attach a syringe.
3. Next I will put 2g of soda lime in the bottom of each of the two boiling tubes, and will then place a gauze into each, which will rest slightly above the soda lime.
4. I shall then weigh some maggots using some measuring scales, and then put these into one of the boiling tubes, ensuring that none come into contact with the soda lime. After doing this I will place another gauze on top of the maggots to prevent them climbing up the tube.
5. I will then connect this boiling tube up with the manometer, as shown in the diagram.
6. I will then putt some glass beads into the other boiling tube and also connect this to the manometer. After doing this I will attach a screw-clip to the boiling tube delivery tube.
7. I will then, if necessary, use a clamp to hold the boiling tubes in place. During this time I will leave the three-way tap and screw-clip open to allow the maggots to acclimatise to their new environment for approximately 10 minutes.
8. After several minutes I will close both the three-way tap and the screw-clip and make a note of the position of the manometer fluid. I will then begin the stopwatch and record how far the fluid has travelled after 1-minute time intervals.
9. Once I have carried out the experiment I will open the screw-clip and the three-way tap to allow the fluid to return back to normal. I will then repeat the experiment twice to allow for any anomalous results.
10. After completing the experiment for one temperature, I will carry out the experiment for other temperatures.

Fair Test

To ensure the experiment is fair there are many things I can do. Firstly I must ensure that no maggots come into contact with the soda lime, as this is harmful to them and may affect how they respire. As well as this I must ensure that when I put the maggots in a water bath, I will ensure that the maggots are enclosed in the water and not above it. This will ensure the maggots are all at the same temperature. I will also make sure I read the position of the manometer fluid at eye level, to prevent inaccurately reading the position of the fluid.

There are many apparatus that I have decided to use over others, and this is because the accuracy of the equipment is much greater. Below are written several reasons why I have decided to use some apparatus over others as well as outlining why I am using some of the apparatus that I have listed above.

I have decided to use a water bath as oppose to a Bunsen burner, as there is a smaller fluctuation of temperature. Using a Bunsen burner to maintain a temperature is very difficult and also inaccurate. However, a water bath can maintain the temperature to a much higher level of accuracy. This will ensure the experiment is fair throughout, in terms of the temperature of the maggots. A fluctuation in temperature would mean the enzymes and substrates have varying levels of kinetic energy, which would make the experiment inaccurate.

In my experiment I am using measuring scales that measure to 100th of a gram. This is because the rate of respiration is dependent on the mass of the maggots, so therefore I must know the exact mass of the maggots. I will then be able to calculate the volume of oxygen produced per minute per gram.

The way to reduce percentage error could be done by making a larger reading- i.e. instead of using 1g of maggots, use 10g. This would therefore give you a smaller percentage error. Alternatively, to reduce the percentage error, I must use a piece of apparatus with a smaller error margin. Using a measuring scale, which measures to 2 decimal places of a gram would be adequate, but if a more accurate measuring scale could be used, this would make my experiment even more accurate.

This is necessary so that I can calculate the rate of oxygen uptake. Simply recording the volume of oxygen produced would only allow me to see that the maggots are respiring, which is not very useful.

I am using a 1cm3 syringe as this is needed to put the manometer fluid into the manometer. The reason for using a 1cm3 syringe as oppose to a 10cm3 syringe is because it has a smaller error margin, and therefore makes it more accurate to use.

I am using a thermometer to ensure that the temperature of the water bath remains constant throughout. If it fluctuates then I can account for any changes in rate of respiration.

Maggots are cold blooded and so can not regulate their body temperature like mammals can. If they are put into a water bath at 40 degrees Celsius then they will stay at this temperature. For this reason maggots are being used to determine how the rate of respiration is affected by temperature.

There are various set-ups for measuring the oxygen uptake of any organism. Many of these set ups are boiling tubes or a test tube connected to a scale. Air is then drawn in from the surroundings and the bubble moves towards the invertebrates in the boiling tube. This set up is shown below.

However the problem arises when this set up is used when the invertebrates are put into a water bath. The air inside the boiling tube will heat up and therefore will expand. The air being drawn in from the outside to push the bubble along is at a lower pressure. As a result the air inside the boiling tube will exert a force on the bubble and push it outwards and away from the invertebrates. This will therefore not give an accurate indication of the volume of oxygen produced, as the bubble will not move as far as it should. This can be seen in the diagram below.

Using a closed system device of measuring oxygen consumption can eliminate this problem. This is the case for the manometer, which is connected to a control tube. When the invertebrates are placed in a water bath both the tubes are at the same temperature, and therefore the same pressure. Therefore the manometer fluid will not move away from the invertebrates, as there is an equal force to oppose this increase in pressure. As a result the true oxygen consumption can be measured. For this reason I have decided to use a manometer to measure the rate of oxygen consumption.

The reason for using soda lime is so that any carbon dioxide is absorbed. When the maggots respire they produce carbon dioxide. If soda lime was not present, the manometer fluid in the manometer would not move, as the volume of gas is not changing. When soda lime is placed in the tubes, the carbon dioxide given off by the maggots from respiration will be absorbed. As oxygen is being used up, the volume in the tube will decrease and this will push the manometer fluid towards the tube containing the invertebrates.

There are many variables that affect the rate or respiration, so I must take these into account when I am doing my experiments. These variables are detailed below.

•  Temperature – Respiration involves enzymes. Glucose is quite a stable molecule, so has a fairly high activation energy. This must be overcome before any of the glucose can be oxidised, so enzymes are used to lower this energy level. Therefore the maggots must remain in a known temperature so the rate of respiration is steady. As temperature is a variable in my experiment, I must make sure the temperature of the maggots remains constant throughout the experiments. I will vary the temperature from 10 degrees Celsius to 60 degrees Celsius in 10*c intervals.
•  Mass of Maggots- Clearly more maggots means more respiration, and therefore more oxygen is consumed. I must therefore keep the mass of the maggots constant.
•  Forms of Respiration- There are two forms of respiration- aerobic and anaerobic. In my experiments I am relying on the fact that the maggots will consume air- therefore respiring aerobically, and this will cause the bubble to be pushed towards the maggots. If at times they respire without oxygen- i.e. anaerobic respiration- the rate of oxygen uptake will be affected and will give inaccurate results. I must therefore keep this in mind when carrying out my experiments.
•  Concentration of Enzymes/Substrates- Respiration is an enzyme-dependant process and therefore the concentration of enzymes and substrates must be kept constant. This is not something I will be able to control in my experiment, except by keeping the mass of the maggots constant. I must therefore bear this in mind when carrying out my investigation.
•  Life Cycle- Just like any other living organism the maggots are part of a life cycle. This cycle is shown below.

As the maggot gets older and progresses through the life cycle it becomes a more specialised organism. A fly is a much more complex stage of the organism than the maggot, and so will respire faster. Although it is easy to see the differences between a fly and maggot, I must take into account the age of the maggot increases during the 2 weeks of my investigation. I must therefore also take this into consideration when carrying out my investigation.

There are several measurements I must take when carrying out the experiment.

I will need to record the initial position of the manometer fluid and the distance it has travelled after various time periods. I can then work out the cumulative oxygen consumption.

•  The temperature around the maggots
•  Mass of soda lime in each boiling tube
•  Mass of maggots in the boiling tube

To ensure the experiment is fair I will allow the maggots time to acclimatise to the new environment. I will then take readings for several minutes and then repeat each temperature twice. This will enable me to account for any anomalous results I may acquire during the experiments.

During my investigation there are various safety issues that I must abide by to ensure my experiment is safe. These are detailed below:

1.  I will be using a syringe to insert the manometer fluid into the manometer, which can easily cut you and injure someone. Therefore I will make sure that I cover the needle back up when I have finished using it. This will provide a safe working environment.
2.  Soda Lime is corrosive so I must ensure it does not come into contact with my skin. Therefore I will use a spatula when I need to remove some from the bottle. If I do come into contact with any I will immediately wash my hands thoroughly.
3.  There is a lot of glassware in my experiment so I will make sure I am careful when using them. If I do drop something I will clear the glass up using a dustpan and brush, whilst making sure I don’t touch any of the shattered pieces of glass.
4.  Finally I will be using a water bath at temperature up to 60 degrees Celsius. I will therefore have to work carefully and if I do burn myself I will immediately rinse my hand under cold water.

Theory

Respiration is a process in which organic molecules are broken down in several stages to release chemical potential energy. This is then used to synthesise adenosine triphosphate (ATP). Usually the organic molecule is glucose, but fatty acids and amino acids can also be used if glucose is not present.

The 4 main stages of respiration are glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation. Glycolysis is the stage in which glucose is broken up, and this occurs in the cytoplasm of a cell. Initially 2 ATP units are needed to break down the glucose (6-carbon molecule) into 2 molecules of pyruvate (3-carbon molecule). However, during the steps between the splitting of glucose and formation of pyruvate, energy is released and is then used to make 4 ATP units. The diagram below shows the glycolytic pathway.

From the diagram above you can see the steps in between the break down of glucose and the formation of pyruvate. Firstly the glucose, which consists of 6 carbons, is phosphorylated, which is a process requiring energy. As it is phosphorylated twice, to form hexose bisphosphate, 2 units of ATP are used. Glucose is very rich in energy, but is quite unreactive due to having a stable structure. It is therefore broken down to give 2 molecules of triose phosphate. 2 hydrogen atoms are then removed from this molecule by 2 nicotinamide adenine dinucleotide (NAD) molecules to form 2 molecules of pyruvate. The diagram below shows the structure NAD.

Therefore in summary glycolysis results in the net increase of two ATP molecules. However, pyruvate still contains a great deal of chemical potential energy, which is released in the next stages of respiration. The next stage of the process is the Link reaction, which involves many enzymes. These work to lower the activation energy of the reactions and their mechanisms will be discussed later on.

During the Link reaction, pyruvate is decarboxylated- i.e. carbon dioxide is removed. This is very significant in my investigation, as this carbon dioxide is what will be absorbed by the soda lime, and will therefore allow me to calculate the rate of oxygen uptake.

Pyruvate is then dehydrogenated and combined with coenzyme A (CoA) to form acetyl CoA, which is a 2-carbon molecule. Once again NAD is the carrier which removes the hydrogen atoms and forms reduced NAD.

The third stage of the respiration process is known as the Krebs cycle, which is a closed pathway of enzyme-controlled reactions. The product of the Link reaction (Acetyl-CoA) combines with oxaloacetate, a 4-carbon compound, to form citrate, a 6-carbon compound. Like pyruvate, this is then decarboxylated and dehydrogenated in several steps, and eventually oxaloacetate is regenerated to allow the cycle to start again.

Each turn of the cycle results in 2 carbon dioxide molecules forming, one FAD molecule and 3 NAD molecules are reduced and one ATP molecule is generated. The diagram below shows the Link reaction and the Krebs cycle.

It is in the final stage of respiration that the bulk of ATP is produced, and it is this stage which requires oxygen. It is in this stage that the hydrogen atoms being carried by the reduced NAD and reduced FAD are put to use, and the stage is known as Oxidative Phosphorylation.

During glycolysis, the Link reaction and the Krebs Cycle only a few molecules of ATP have been produced. It is during oxidative phosphorylation that most of the energy locked in the original glucose molecule will be released. The electron transport chain is a network of electron-carrying proteins located in the inner membrane of the mitochondrion. Reduced NAD and reduced FAD pass to the electron transport chain, and here hydrogen atoms are released from the two carriers. In doing so one ATP molecule can be synthesised. The hydrogen atoms then break down into hydrogen ions (H+) and electrons.

The hydrogen ions remain in the mitochondrial matrix, whilst the electrons are transferred to the first electron carrier. As it passes between the three electron carriers, the energy level of the carriers, in relation to oxygen, decrease. This releases energy, which is used to synthesise ATP. In this way two more molecules of ATP are produced. Finally the electron reaches the final electron acceptor, which is also located in the mitochondrial matrix, and is oxygen. 2 hydrogen ions will also be drawn up and the oxygen is reduced to water.

This is why in my investigation the manometer fluid in the manometer will move towards the maggots. The maggots require oxygen for this last stage of respiration, which is what I am measuring to calculate the rate of respiration.

However this only accounts for 3 of the 28 molecules of ATP formed during oxidative phosphorylation. The other ATP molecules are synthesised due to the process of chemiosmosis. The energy released by the electron transport chain is also used to pump hydrogen ions into the mitochondrial intermembrane space. Due to this the concentration of hydrogen ions in the intermembrane space increases. This sets up a concentration gradient, and the hydrogen ions then pass back through the membrane into the mitochondrial matrix via protein channels.

In each channel is ATP synthase, which acts as an enzyme and uses the potential energy of 3 hydrogen ions to convert ADP and Pi (inorganic phosphates) to ATP. The diagrams below show the process of oxidative phosphorylation and also how ATP synthase works. ATP synthase is an enzyme, which shows that respiration is enzyme-dependant and so I must also consider how enzymes work to make this process possible.

Now that we have seen how respiration is able to release energy from glucose, we can consider the role enzymes play in the process. The enthalpy diagram below shows the overall change of glucose into carbon dioxide and water.

Although there are many compounds formed between glucose and the end product, essentially they can all be regarded as one. This is because for each individual step to occur the activation energy must be overcome. The enzymes work by lowering this energy, which allows the glucose to be converted to pyruvate during glycolysis, the pyruvate to be converted to Acetyl-CoA during the Link reaction etc. I will now go into more detail about how the many enzymes involved in respiration are able to do this.

An enzyme can be defined as a biological catalyst and is affected by the environment it is in. The enzyme has a specific 3-dimensional shape and this means a certain enzyme can break down only substrates with a certain shape.

E.g. For this reason amylase can only break down starch, due to the substrates fitting into the active site. It cannot however break down lipids due the lipid substrates having a structure that does not allow it to fit in the active site of the amylase enzyme.

As stated earlier enzymes are complex 3-dimensional globular proteins. The active site, which is usually a cleft in structure, contains some amino acids that carry out the breakdown of a substance.

The enzyme is in the tertiary structure of a protein. It is held together by several bonds, which are hydrogen bonds, ionic bonds, disulphide bonds and hydrophobic interactions between non- polar side chains. The long amino acid chains coiling up on themselves bring about this structure. Hydrogen bonds then form between the -CO groups of one amino acid and the -NH group of another, which hold this shape in place. This is called an ?- helix and is the secondary structure. This structure may coil up into a precise three- dimensional shape, which is the tertiary structure. The diagram below shows how hydrogen bonding can form.

The R groups determine the shape of the active site in an enzyme. The large variety of different R groups means different shape active sites can exist, explaining why the enzymes are specific to one substrate type.

The above diagram relates to how the enzymes in respiration work. In the left diagram we can see that the enzyme and substrate are in a mixture. The substrate moves into the active site of the enzyme. The two then bind and form an enzyme-substrate complex. It is held in place using temporary bonds that form between the R groups of the enzyme’s amino acid and the substrate. These bonds are weak and thus are not covalent.

The lock and key diagram can be used to understand the specific shape of the enzyme. The substrate above fits the shape of the active site, so can bind with it. Any other shape will not fit this active site. A lock and a key theory can resemble this in that if the key, i.e. the substrate, is not the right shape it will not fit in the lock, which is the enzyme.

Finally the interactions between the substrate and active site of the enzyme cause the substrate to break down. The temporary bonds, which form during this process, cause a higher tendency for the breakdown of a substance, which in turn reduce the activation energy. This will be explained in more detail in the next section.

Now that we know what an enzyme is we can see how altering the temperature would cause a change in the rate of the reaction of respiration. For a reaction to occur the particles must collide with a certain minimum kinetic energy. The size of this kinetic energy needed varies between reactions due to different bond enthalpies. An enzyme works by reducing the activation energy as shown in the diagram below.