EFFECTS OF AIR EXPOSURE IN CRABS
Description:1500 words on the oxygen intake levels under water and outside water and during stress+ the guidelines of the report should be followed as it is . Kindly find the uploaded practical. Please note that all the results are recorded and questions answered but not in the version uploaded .
1 Integrative Zoology BSX2030 Practical 1 Schedule 2019-20 Nia Whiteley 2 General Introduction The purpose of the two practicals in BSX2030 is to introduce you to two lab based experiments related to physiology and endocrinology. The first practical aims to introduce you to the measurements of oxygen uptake rates as part of the respiration lectures given by Dr Nia Whiteley. The second practical aims to introduce you to a hormone related response, which can be easily observed in crabs, in association with the endocrinology lectures delivered by Prof Simon Webster. Both practicals will focus on the intertidal shore crab, Carcinus maenas, because it is readily available from the Menai Strait and is particularly tolerant of air exposure when exposed by the tides. This is remarkable because water-breathers, such as the shore crab, with well-developed gills, are not well suited to breathe in air. Many intertidal organisms, face twice daily emersion during ebbing tides, which potentially expose them to several environmental stressors. For example: dehydration, rapid changes in temperature and salinity, reduction in oxygen availability, predation risk, to name but a few. Within physiological limits, intertidal animals adapt, and show clear responses to these environmental stressors, to maintain internal homeostasis. Even though oxygen levels are abundant in air relative to water, air-breathing may not be possible as the gills collapse. In Practical 1 you will investigate the effect of air exposure and forced exercise (simulated predator attack) on rates of oxygen uptake in shore crabs. In Practical 2 you will investigate the hormonal control of chromatophore dispersal and concentration in juvenile shore crabs. Timetable Practical 1 runs 30/01/20 & 31/01/20: Group A Thurs 30th Jan 10.00-13.00 Group B Thurs 30th Jan 14.00-17.00 Group C Fri 31st Jan 10.00-13.00 Group D Fri 31st Jan 14.00-17.00 Practical 2 runs 06/03/20 & 10/03/20: Group A Fri 6 th March 10.00-13.00 Group B Fri 6 th March 14.00-17.00 Group C Tues 10 th March 10.00-13.00 Group D Tues 10 th March 14.00-17.00 Deadlines for practical assessments: Practical 1 Monday 17th Feb Practical 2 Wed 25th March 3 PRACTICAL 1 OXYGEN UPTAKE RATES IN A TYPICAL GILL BREATHER: EFFECTS OF AIR EXPOSURE Aim: To investigate the ability of the shore crab, Carcinus maenas, to consume oxygen from both water and air. Introduction In this practical you will determine rates of oxygen uptake in a typical water breather, the shore crab, Carcinus maenas. Crabs typically have 8 pairs of gills arranged in two branchial chambers on either side of the body. These structures are highly specialised for gas and ion exchange, having large surface areas and thin diffusion distances between the external seawater and the blood (haemolymph) supply. When crabs are removed from water, the gills are no longer supported and they tend to collapse and clump together. This disrupts gas exchange, especially oxygen uptake, as it increases diffusion distances. C. maenas, however, has strengthened gills, which enable it to survive in the intertidal zone for short periods when exposed by the tide. You will use this practical to investigate whether the shore crab can consume oxygen from air as well as it can from water by determining rates of oxygen uptake of crabs held in both respiratory media. You will then exercise the crabs in both respiratory media to see whether there is scope to increase rates of oxygen uptake. You will need to organise yourselves into groups of 4 students to do this practical. Materials and Methods Each group will be provided with 4 crabs in individual white buckets. There are 4 experimental scenarios: 1) Undisturbed crabs in seawater (Water); and 2) Forced exercise to simulate predator attack in crabs in seawater (Water & Exercise) 3) Undisturbed crabs exposed to air (Air) 4) Forced exercise to simulate predator attack in crabs exposed to air (Air & Exercise). 4 Check that you have 4 white buckets, labelled one to 4, and that you have a crab in each one. Each crab at this stage should be in seawater. Try not to disturb the crabs i.e. do not lean over the buckets etc. We are aiming for resting crabs at the start of the experiment. You should also have 3 plastic boxes labelled one to 3. These are respirometers as they will be used to measure rates of oxygen uptake. Method To measure rates of oxygen uptake: 1. Take Crab 1 and place individually and very carefully into a labelled respirometer containing 2 marbles. Place the box into the larger orange bucket, half-full with seawater. Slide the lid across the surface of the water to eliminate any air bubbles and seal the container. Make a note of the time on Table 1 on page 6. N.B. Air bubbles are a problem, so take some care at this step to get rid of them. You will also have to handle the crabs. This can be done perfectly safely by picking the crabs up firmly from behind as demonstrated by the staff running the practical. In theory, the crabs cannot pinch you with their claws if you use this approach. Try and keep the time out of water to a minimum. 2. Once sealed within its respirometer, remove from the larger bucket and mop away any access seawater. Take the labelled respirometer immediately to the water bath and place onto the platform to maintain the temperature at 15°C. DO NOT fully submerge the respirometer but follow instructions given by staff. 3. Take an oxygen reading as quickly as possible using the fibre optic pen. We will show you how to do this. This will involve reading the bar code that relates to each respirometer. Make a note of the time on Table 1. 4. Repeat steps 1 to 3 with the remainder of the crabs. Each group should now have 3 respirometers in the water bath. For the last crab, you will have to wait until the measurements have been completed for Crabs 1 to 3. 5. Leave for 15 mins then take another oxygen reading with the fibre optic pen. Before you do this, gently move the respirometer backwards and forwards so that the marbles roll from one side of the respirometer to the other. This will mix the water and prevent gradients of O2 partial pressure (PO2) from forming. Note the time at which you take the oxygen reading and enter onto Table 1. 5 6. Remove the respirometer from the water bath and take back to a splash tray. Remove the lid and pour the water from the respirometer into a measuring cylinder via a funnel. Record the volume of the seawater in Table 1. Then return the crab to its original bucket. 7. Repeat with the remaining crabs, and return the crabs to their respective buckets when you have finished. As you only have 3 respirometers per group, and you need to take measurements from 4 crabs, you will have to stagger the measurements. Further information will be provided in the practical. Scenarios and treatments 8. Once the first set of oxygen uptake measurements have been taken, leave Crabs 1 and 2 in their buckets with aerated seawater, but drain the water carefully from Buckets 3 and 4. Start timing from the moment both crabs are out of water. 9. Leave all 4 crabs for 60 min. Place the lids loosely on the buckets and try not to disturb the crabs. Remember that Crabs 1 and 2 will be in seawater. The other 2 crabs will be in air, so leave the crabs in air to take the oxygen uptake measurements. Our measurement system will also measure oxygen levels in air as well as in seawater. 10. After 60 min, repeat steps 1 through to 7 to get a second set of readings for rates of oxygen uptake. Enter you data onto Table 2 on page 6. You may have to leave the crabs in air for longer in order to record a drop in PO2. 11. Once the crabs have been returned to their buckets, exercise Crabs 2 and 4. This can be achieved by making them run after pushing them gently with a pencil. Try to make sure the crabs are always running. You may find that the crabs become increasingly reluctant to run after about 7-8 minutes exercise, and may not be able to right themselves if turned upside down. This is normal, and is a consequence of increased lactate levels due to profound hypoxia. Start timing from the moment you start exercising the crabs. Exercise for 10 min and repeat the determination of oxygen uptake rates for all crabs (steps 1 through to 7). Remember that Crab 2 will be exercised in seawater and Crab 4 will be exercised when exposed to air. Enter your data onto Table 3 on page 7. 12. Once the rates of oxygen uptake have been determined for the third and final time then weigh all crabs before returning them to seawater for recovery. 13.N.B. It is important that you weigh the crabs and enter the data into Table 3 on page 7. Record the weight in kg. You will need this information to calculate rates of oxygen uptake. 6 Tables for entering data Table 1. Summary of the values required for calculating rates of oxygen uptake in all 4 crabs 1, 2 and 3 at Time = 0 min. Crab no First PO2 reading (Torr) Time of 1st reading 2 nd O2 reading (Torr) Time of 2nd reading Total time in resp. (min) Volume of seawater (ml) Crab 1 Crab 2 Crab 3 Crab 4 PO2 = partial pressure of oxygen; Torr = mmHg While you are waiting to take the 60 min readings, have a go at completing the first part of the calculations required to determine rates of oxygen uptake from your data by trying to complete Table 4. Table 2. Summary of the values required for calculating rates of oxygen uptake in all 4 crabs at Time = 60 min. Crab no First O2 reading (Torr) Time of 1st reading 2 nd O2 reading (Torr) Time of 2nd reading Total time in resp (min) Volume of seawater/air (ml). Same as Table 1 Crab 1 Crab 2 Crab 3 Crab 4 N.B. Remember to leave Crabs 3 and 4 out of water. 7 Table 3. Summary of the values required for calculating rates of oxygen uptake in all 4 crabs at Time = 70 min with and without exercise. Crab no First O2 reading (Torr) Time of 1st reading 2 nd O2 reading (Torr) Time of 2nd reading Total time in resp (min) Volume of seawater/air (ml). Same as Table 1 Weight of crab (kg) Crab 1 Crab 2 Crab 3 Crab 4 Table 4. First steps towards the calculation of oxygen uptake rates Crab no. Treatment First PO2 Reading (Torr) 2 nd PO2 Reading (Torr) Difference (Δ) (Torr) Time interval (min) 1 Water 0 min 2 Water 0 min 3 Water 0 min 4 Water 0 min 1 Water 60 min 2 Water 60 min 3 Air 60 min 4 Air 60 min 1 Water 70 min 2 Water & Ex 70 min 3 Air 70 min 4 Air & Ex 70 min Ex = exercise 8 Calculation of Oxygen Uptake Rates After completing the experiments calculate the oxygen uptake rate for the crabs under the various conditions (Water, Water & Exercise, Air, Air & Exercise). Calculations differ depending on the respiratory medium. Rates of oxygen uptake: crabs in water Before you can do this you will need to know:- 1. The volume of seawater in the respirometer. This is the volume of water minus the volume taken up by the crab. You should have this data in Table 1. Make sure that the volume is recorded in ml. 2. You will also have the weight of the crabs recorded in Table 3. 3. Calculate the drop in PO2 over a given unit of time. e.g. 8 to 10 Torr over 15 min. This is shown as ∆ PO2 in Equation 1. You have calculated this value in Table 4. 4. Convert ∆ PO2 into oxygen content (ml O2 ml-1 of seawater) using the solubility coefficient for O2 in seawater (salinity and temperature have a marked effect on the solubility of O2 in water). Use the table below to select the most appropriate coefficient. You will need to know the temperature at which the measurements were taken and the salinity. You will find both values on the white board in the lab. Table 5. Solubility coefficients for O2 in seawater at various temperatures Temperature (°C) Salinity Solubility coefficient (ml O2 litre-1 155 Torr-1 ) Solubility coefficient* (ml O2 ml-1 Torr-1 ) 14 32 6.11 3.94 x10-5 15 32 6.00 3.87×10-5 16 32 5.89 3.80×10-5 *= use this value 9 5. Calculate O2 uptake rates per hour for each whole crab (ml O2 h -1 ) using the following equation: ∆ PO2 x solubility coefficient x Vol of water in respirometer (ml) x (60 min/15 min) = Equation 1 6. In order to compare crabs of different body mass, calculate the weight-specific O2 uptake rate (ml O2 kg -1 h -1 ). Take the value calculated in Equation 1 and divide by the body mass of the crab in kg (see Table 3). 7. Convert the values you have obtained from ml O2 kg-1 h -1 into mmol O2 kg-1 h -1 . This is necessary to compare values between crabs breathing in water and in air. One mole of O2 occupies 22.414 litres (ml O2 kg-1 h -1 (litres)/22.414) x 1000 = mmol O2 kg-1 h -1 Equation 2 Rates of oxygen uptake: crabs in air 1. You need to know the volume of air in the respirometer minus the crabs. The only way to estimate this is to use the volume of seawater previously measured for the same crab at the start of the experiment (Table 1). This is equivalent to the volume of air within the respirometer. 2. The weight or body mass of the crabs will also be the same. 3. Now, all you need to know is the amount of O2 taken up by the crab over the measurement period. This will involve converting partial pressures into moles using the Ideal Gas Law: PV = nRT Equation 3 Where ∆ PO2 = change in partial pressure between the start and the end of the measurement period, V= vol of air in respirometer (litres), n= number of moles, R= universal gas constant (62.36 Torr L-1 mol-1 K-1 ), T = temperature in degrees Kelvin (at 15°C = 288.15 K) This equation can be re-arranged to calculate n: (P/RT) x V = n Equation 4 (∆ PO2/(62.36 x 288.15)) x V = n (mmol O2) 10 4. Then calculate the weight-specific O2 uptake rate (mmol O2 kg -1 h -1 ). Take the value calculated in Equation 4 and divide by the body mass of the crab in kg and account for the amount of time that it has been in the respirometer to get the value per hour. 5. You should now have the relevant data to complete the final Tables, Tables 7 and 8. Notice that there are two columns for rates of oxygen uptake when the crabs are waster breathing. Table 7. Rates of oxygen uptake in crabs submerged in seawater at the beginning of the experiment. Crab number Treatment Rates of oxygen Uptake (ml O2 kg-1 h -1 ) Rates of oxygen Uptake (mmol O2 kg -1 h -1 ) 1 Water 2 Water 3 Water 4 Water Table 8. Rates of oxygen uptake in crabs left in seawater and exposed to air for 60 min. Crab number Treatment Rates of oxygen uptake (ml O2 kg-1 h -1 ) Rates of oxygen uptake (mmol O2 kg -1 h -1 ) 1 Water 60 min 2 Water 60 min 3 Air 60 min _____________ 4 Air 60 min _____________ 11 Table 9. Rates of oxygen uptake in crabs left in seawater and exposed to air for 70 min, either with or without a period of exercise. Crab number Treatment Rates of oxygen uptake (ml O2 kg-1 h -1 ) Rates of oxygen uptake (mmol O2 kg -1 h -1 ) 1 Water 2 Water & Exercise 3 Air ______________ 4 Air & Exercise ______________ Instructions for the write-up For Practical 1, the write-up is limited to a Results section and a Discussion. For Results include one table and 4 figures. The class data will be collected to enable you to calculate mean values for rates of oxygen uptake in all 4 crabs at time 0 and after 60 min. It will also enable you to calculate mean values for crabs in water after 70 min with and without exercise. Display the mean values in a table. Give mean values ± standard errors. You should have 8 mean values to enable you to compare: Crabs 1 & 2 in water at time = 0 to the same crabs after 60 min in water (Fig. 1). Crabs 3 & 4 in water at time = 0 to the same crabs after 60 min in air (Fig. 2). Crabs 1 & 2 in water after 70 min to the same crabs after exercise in water (Fig. 3). Crabs 3 & 4 in air after 70 min to the same crabs after exercise in air (Fig. 4). Use this data to plot 4 figures either using box plots or a bar graph. In order to determine whether there has been a significant increase in rates of oxygen uptake in each crab in each respiratory medium after 60 min, use SPSS to compare the mean values in Figures 1 & 2. Use the same process to investigate whether exercise has an effect on rates of oxygen uptake in both water and in air in Figures 3 & 4, respectively. 12 First, you will need to test for normality (i.e. is the data parametric or nonparametric?), and either use a paired students t-test, or a Wilcoxon signed ranks test. N. B. There are several ways of testing for normality in SPSS. Here is one way. Place data for each group into a separate column. Select ‘Analyze’ then ‘Non-parametric’. Then ‘Legacy Dialoges’ and finally ‘1-sample Kolmogorov-Smirnov Test’. Move the groups of data across into the Test Variables box. The important bit is the outcome: Asymp. Sig. If this value is <0.05 then the distribution of the data is significantly different from a normal distribution. If this applies, even to one of the 2 groups, then use a Wilcoxon signed ranks test. Describe the resulting trends. Discussion – explain your results in the light of previous studies. Ask yourself whether shore crabs are capable of oxygen uptake when they are out of water. Discuss the effects of activity on rates of oxygen uptake when the crabs are in and out of water. Information relating to the analyses, calculations and statistical tests will be covered in the workshop that follows this Practical on Friday 7th Feb 9.00-12.00 in A1 Brambell. Reference Simonik E & Henry RP 2014. Physiological responses to emersion in the intertidal green crab, Carcinus maenas (L.). Marine and Freshwater Behaviour and Physiology 47, 101-115.
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OXYGEN UPTAKE RATES IN A TYPICAL GILL BREATHER:
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