Artemia Lab PDF

MARI 3603, BIO5603 – Practical Aquaculture Artemia Lab Student Learning Outcomes 1. 2. 3. 4. 5. Identify different morphological and anatomical components in several life cycle stages of Artemia Identify supplier-reported characteristics and quality indices of Artemia cysts Practice hydration, dec...

MARI 3603, BIO5603 – Practical Aquaculture Artemia Lab Student Learning Outcomes 1. 2. 3. 4. 5. Identify different morphological and anatomical components in several life cycle stages of Artemia Identify supplier-reported characteristics and quality indices of Artemia cysts Practice hydration, decapsulation and hatching of cysts Estimate, from hatched samples, several indices of cyst quality and performance Compare the quality and performance indices of cysts hatched under different hatching conditions Materials • • • • • • • • • 1 can of Artemia cysts (with label) 1 culture of Artemia nauplii (hatched 1 day prior to lab) 1 culture of adult Artemia (hatched 2 weeks prior to lab) Dissecting microscope Scale 125 µm mesh sieve Pipette (250 µl) Small petri dishes Large petri dishes • • • • • • • • • • Thermometer Spatula Squirt bottle Plastic container for ice bath Glass hydration beaker (100 ml) Glass decapsulation container (100 ml) Decapsulation solution (Bleach) Deactivation solution (0.1% Na2S2O3) Lugol solution (dye/fixative for microscopy) ice General Instructions • • • • • Work in teams. All lab-related work is to be done in teams Lab reports are individual. Each individual students must submit her/his own report Prepare and submit a digital report (.docx) containing the questions below with their respective answers. Make sure to include your Name, B00 number and Lab name Reports have to be submitted via Brightspace Submission deadline is shown in the syllabus MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 1 of 18 Lab Overview The lab consists of (1) several descriptive sections and (2) a hatching experiment that includes the collection and analysis of a few samples, and then plotting and analysis of a “synthetic” dataset produced by a computer model. Descriptive sections: In these sections you are required to (1) extract supplier-provided information from a can of cysts (or supplier’s website), and (2) to take photos and identify body parts from different life cycle stages of Artemia. Hatching experiment: Here we are going to simulate an experiment that involves hatching several batches of Artemia cysts at different temperatures and with or without decapsulation. Many hatching indices need to be calculated throughout the hatching process and at the end of the experiment. The two objectives of the experiment are to evaluate (1) the effect of temperature and (2) the effect of cyst decapsulation, on the estimated hatching indices. In “real life” this experiment would require sampling every hour over several days. We don’t have this timeframe in this course, so we will instead work with synthetic data (i.e. simulated in a computer). However, we will take and analyse a few “real” samples as an example, to get a feel on what entails to do sample processing without having to do a multi-day experiment. Cyst hydration Later in the Lab you will need “hydrated cysts”. The hydration step takes about 1 hour. Therefore you want to start the hydration process first, then continue with the next sections while the cysts get hydrated. Hydration instructions (from FAO 1996, Worksheets 4.2.4 and 4.2.7): - Weigh 0.6 g of cysts - Place cysts in the glass hydration beaker (100 ml) - Gently stir the cysts every 10 minutes or so - After 1 hour, the cysts should be hydrated and ready for decapsulation 1. Answer the following questions: a. At what time did you immerse the cysts? __________ b. At what time do they have to come out of immersion? __________ c. What is the weight of dry cysts that you are hydrating? _________________ d. What is the water temperature? _____________________ Hint: make sure you include the appropriate units 5 Points: 1 point each (wrong or missing units = -0.5 per question) MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 2 of 18 Descriptive Sections Supplier-reported characteristics and quality indices The “quality” of Artemia cysts is reported by the suppliers using several indices. It is important to be aware of these indices, and to understand what they mean; particularly when comparing products before purchasing. Later in this lab we will estimate some of these indices, and compare them against the reported by the supplier. But for now, let’s start by simply extracting information from the label. For simplicity, use the labels below: 2. From the label on the can, get the information below (write “Not Specified” if information is missing): a. Hatch rate: _____________________ b. Guaranteed minimum hatch rate: ______ c. Hatching efficiency: _________________ d. Cysts per gram: _____________________ e. Nauplii per gram: ___________________ f. Moisture content: _________________ g. HUFA content: _____________________ h. Net weight (i.e. weight of cysts within a new can): ____________ 1 point each MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 3 of 18 Morphology and Anatomy As you will see in the questions below, you will have to place cysts or hatched Artemia under the microscope and take photos using your phone or camera (through the microscope’s ocular). Then you will need to include the photos in your report with appropriate figure captions. In some questions you will be asked to ad arrows and labels to your photos to identify different body parts. Cysts 3. Take a photo through the microscope’s scope of a dry cyst. Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption 4. Take a photo through the microscope’s scope of a hydrated cyst. Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption Note: For the next few questions, you will need to take a sample from the beaker with Artemia nauplii hatched 1 day prior to lab. 5. Take a photo through the microscope’s scope of a hatched cyst (i.e. empty shell). Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption Umbrella and Nauplius Below are some photos and diagrams to help you identify life stages and body parts in your own photos. Figure 1. Cyst in breaking stage. (1) Nauplius eye (source: FAO, 1996) MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 4 of 18 Figure 2. Embryo in “umbrella” stage (left) and instar I nauplius (right). (1) Nauplius eye; (2) antennula; (3) antenna; (4) mandible. (source: FAO, 1996) Figure 3. Diagram of Artemia nauplius (source: Fox 2006) 6. Take a photo through the microscope’s scope of an Artemia umbrella. Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 5 of 18 7. Take a photo through the microscope’s scope of a recently hatched nauplius. Include the photo in your report (with figure caption). Add arrows and labels identifying the parts below (if needed you can use more than one photo) a. Naupliar eye b. Antenna c. Mandible d. Swimming setae e. Gut 5 points: 1 point for each correct identification of body part + 1 point for correct figure caption Note: For the next few questions, you will need to take a sample from the beaker with adult Artemia hatched 1 week prior to lab . Adult Artemia Below are some photos and diagrams to help you sex and identify body parts in adult Artemia. Figure 4. Adult male Artemia (source: FAO, 1996) Figure 5. Adult female Artemia (source: FAO, 1996) MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 6 of 18 Figure 6. Head of an adult male. (1) antenna; (2) antennula; (3) lateral complex eye; (4) mandible (source: FAO, 1996) Figure 7. Diagram of adult Artemia (source: Fox, 2006) 8. Take a photo through the microscope’s scope of a male Artemia. Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption 9. Take a photo through the microscope’s scope of a female Artemia. Include the photo in your report with an appropriate figure caption. 2 points: 1 point for correct photo and 1 point for correct figure caption MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 7 of 18 10. Take one (or several) photo(s) through the microscope’s scope of an adult artemia. Include the photo in your report (with figure caption). Add arrows and labels identifying the parts below (if needed you can use more than one photo) a. Head b. Thorax c. Abdomen d. Naupliar eye e. Compound eyes f. Thoracopods g. Telson h. Gonad 9 points: 1 point for each correct identification of body part + 1 point for correct figure caption Nauplii response to light 11. Shine the light from your cellphone to hatched nauplii. Are the nauiplii attracted to light (i.e. positive phototaxis), repelled by light (i.e. negative phototaxis), or indifferent (i.e. no phototaxis)? Note that you may have to do this test in a dark environment. 1 point Hatching Experiment As mentioned earlier in the Lab overview, we are going to simulate an experiment that involves hatching several batches of Artemia cysts at different temperatures and with or without decapsulation. The two objectives of the experiment are to evaluate (1) the effect of temperature and (2) the effect of cyst decapsulation, on the estimated hatching indices. We won’t be running the full experiment in this lab, because it would take too long to collect samples during the full artemia hatching process (24 to 48 hrs). However, this lab includes explanations on how you will need to set up the experiment if you were running it for real. Additionally, in this lab we will (1) practice the process of cysts decapsulation, (2) collect and analyze a few “real” samples, (3) calculate the Instantaneous Hatching Properties of the “real” sample we collected, and (4) analyse a synthetic data set that represents the data that we would have collected if we have done the real experiment for 24 to 48 hours. A graphical representation of the simulated setup is shown in Figure 1. MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 8 of 18 Figure 8. Graphical representation of the Artemia hatching experiment. Note that there 4 conical hatching tanks. Two tanks are kept at room temperature, one at 24°C and one at 28°C. One of the two tanks are room temperature hold decapsulated artemia cysts. The other three tanks hold non-decapsulated (i.e. full-chorion) cysts. Cyst Decapsulation Decapsulation is the process of dissolving the chorion layer of the cyst. Decapsulation makes it easier for nauplii to hatch, thus hatched nauplii have a higher energy content than nauplii hatched from non-decapsulated cysts. Decapsulation also sterilizes cysts thus reducing the risk of introducing pathogens. While we don’t have the time to wait for the decapsulated cysts to hatch (hence doing a synthetic experiment), we will practice “in real life” how to decapsulate cysts. 12. Take photo(s) of cysts BEFORE decapsulation through the microscope’s ocular. Include the photo(s) in your report. Make sure to describe their color in the figure caption. 2 points: 1 point for photo + 1 point for correct figure caption 13. Take a photo of the decapsulation beaker with cysts within BEFORE decapsulation. Include the photo in your report. Make sure to describe their color in the figure caption. 2 points: 1 point for photo + 1 point for correct figure caption Decapsulation instructions (from FAO, Worksheet 4.2.4 and 4.2.7): - Locate your hydrated cysts (you placed them earlier in a decapsulation beaker) - Collect hydrated cysts on a 125 µm mesh sieve and rinse them. Use a squirt bottle with seawater to help you take the cysts out of the hatching bottle - Transfer the cysts from the sieve to a decapsulation container (small glass beaker). Again, use a squirt bottle with seawater to assist you, but try to use as little water as you can - Place the decapsulation container in a bath filled with ice water MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 9 of 18 - - - - - Decapsulation step: Record how much seawater + cysts you have in your beaker. You need to add the recorded amount of “decapsulation solution” to the beaker with cysts, effectively doubling the contents of the beaker (example: if you have 20 ml of seawater + cysts, you need to add 20 ml of decapsulation solution to the beaker). Start a timer. Decapsulation should finish in 3 to 15 minutes Gently stir the cysts using a spatula. You can squirt a bit of seawater to wash down cysts from the sides of the beaker Check evolution of decapsulation process regularly under the dissecting microscope (250 μl samples). Note that you need to take a sample, assess it, and then return the sample to the decapsulation beaker. When microscopic examination shows almost complete dissolution of the cyst shell (after approx. 3 to 15 minutes), stop the decapsulation process by collecting the decapsulated cysts on a 125 µm mesh sieve, and then wash/deactivate them as explained below. It is crucial not to leave the embryos in the decapsulation solution longer than strictly necessary, since this will affect their viability Washing step: Rinse decapsulated cysts with fresh water until no chlorine smell is detected anymore Deactivation step: Deactivate all traces of decapsulation solution by dipping the cysts (< 1 min.) in deactivation solution, then rinse again with water. It is easiest to do this by placing the sieve with cysts in a large petri dish, then poor some deactivation solution in the sieve (be careful not to add too much solution because it may overflow from the petri dish). You may want to answer some of the questions below before you continue Place rinsed cysts in a hatching bottle with exactly 300 ml of seawater and place bottle in Tank A (here it helps to fill a squirt bottle with exactly 300 ml of seawater. Use the water from the squirt bottle to rinse the sieve, then empty the remaining seawater from the squirt bottle to the hatching bottle) Use a permanent ink marker to mark the water level in the hatching bottle (i.e. create a 300 ml mark) 14. Take photo(s) cysts AFTER decapsulation through the microscope’s ocular. Include the photo(s) in your report. Make sure to describe their color in the figure caption. 2 points: 1 point for photo + 1 point for correct figure caption 15. Take a photo of the decapsulation beaker with cysts within AFTER decapsulation. Include the photo in your report. Make sure to describe their color in the figure caption. 2 points: 1 point for photo + 1 point for correct figure caption Setup of non-decapsulated (full chorion) batches We will NOT setup any of the hatching tanks. However, for reference, below are the steps that you would need to follow when setting up a hatching tank. Read the instructions below, but note that you do not have to do anything: - - Using a weighing-tray, weigh exactly 0.6 g of cysts and place them in your hatching tank (you may have to use a squirt bottle pre-filled with exactly 300 ml to rinse tray contents into the hatching bottle SEE BELOW) Fill in the hatching tank with exactly 300 ml of seawater (as mentioned above, here it helps to pre-fill a squirt bottle with exactly 300 ml of seawater. Use the water from the squirt bottle to rinse the weighing-tray, then empty the remaining seawater from the squirt bottle to the hatching bottle) MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 10 of 18 - Add an aeration stone into the hatching tank. Aeration is to keep all cysts in suspension, however make sure aeration is not too strong (i.e. it is important to prevent foaming) You may have to use a 10 ml pipette to collect water from inside the hatching bottle and wash down cysts stuck in the sides of the bottle above the water line. Repeat this step several times as needed. Use a permanent ink marker to mark the water level in the hatching bottle (i.e. create a 300 ml mark) After 8 to 40 hours (depending on temperature), the cysts should begin to hatch Sampling In the real-life experiment, you would need to collect (and process) samples every hour until all cysts hatched (about 1 to 2 days later), using the protocol shown below. In this lab we provide you with synthetic data so you don’t have to spend 2 days on a sample collection/processing marathon. However, in this lab you will take and process a few samples (2 to 3) to get a felling of what entails in sample collection and processing. Note: For the next few questions, you will need to take sample from the beaker with Artemia nauplii hatched 1 day prior to lab. Sampling protocol: - You don’t have to do this step, but it is here to remind you of the importance of maintaining a constant cyst concentration during the experiment. “Before you sample, make sure the water level inside your hatching tank is at the 300 ml mark. If some water evaporated, add distilled water until the water level is back at the 300 ml mark. Make sure there are no cysts stuck on the sides of the bottle. You can use the 10 ml pipette to collect water from inside the hatching bottle and wash down the cysts from the sides of the bottle” - Using a pipette, take two 250 µl sub-samples out of the bottle to be sampled (i.e. beaker with Artemia nauplii hatched 1 day prior to lab). - Place each sub-sample into a separate small Petri dish - Sub-sample 1: o Here you need to decapsulate unhatched cysts and dissolve empty cyst shells by adding 5 drops of domestic bleach solution (5.25% NaOCl) to the counting plate with your sample (note that this step is not required for the decapsulated cysts). o Leave this sample to decapsulate (3 to 15 minutes). While you wait, you can deal with the Subsample 2 in the other Petri dish o After you finished with Sub-sample 2, come back to Sub-sample 1 and count the number of embryos (Et), which can be identified by their orange color - Sub-sample 2: o Inspect the sample under the dissection microscope to assess the motility of the hatched nauplii o Add few drops of lugol solution to fixate the nauplii (wait until they stop moving) o Count the number of nauplii (Nt) and the number of umbrellas (Ut) Ideally you would enter the observed numbers (i.e. Nt, Ut and Et) in a spread sheet like the one shown in the example below. MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 11 of 18 Sample # Date Time Student Name Bottle # Temperature Nt °C Counts Measured Calculated Ut Et t Ht Counts Counts hours from start % HEt No. Nauplii 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 9. Example work sheet. Estimation of Instantaneous Hatching Properties Several properties can be calculated from the counts of nauplii, umbrellas and embryos measured over time (i.e. Nt, Ut and Et, respectively). For now we’ll focus on some properties that can be estimated throughout the experiment. Later we will introduce other properties that you can only estimate after the experiment has finished and you collected all the data. The following properties can be estimated on each sample, by entering their respective formulas (below) in your spread-sheet (see example in Figure 9). You should estimate these properties as soon as you finish processing a sample, so you can monitor the progress of the experiment and do adjustments if you detect any anomaly. Instantaneous hatching percentage (Ht, units: %) refers to the percentage of cysts that have hatched into nauplii up to time t (units: hours from start of experiment), and is calculated using the following formula: 𝐻𝑡 = 𝑁𝑡 × 100 𝑁𝑡 + 𝑈𝑡 + 𝐸𝑡 Instantaneous hatching efficiency (HEt, units: number of nauplii per g of cysts) refers to the efficiency at which nauplii are produced (from time zero to time t), and is calculated using the following formula: 𝐻𝐸𝑡 = 𝑁𝑡 × 4 × 300 = 𝑁𝑡 × 2000 0.6 where Nt, (units: number of nauplii) is the number of nauplii at time t. Synthetic data An individual-based model was used to run in-silico the experiment from this lab to produce a Synthetic Dataset (see Artemia Synthetic Data.xlsx), which represents the data that would have been collected if the experiment MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 12 of 18 worked perfectly. The Synthetic Dataset includes natural variability (i.e. not two cysts are the same), but it is free of human errors that may had happened during data collection. Initial plotting and visualization of your data As mentioned above, it is a good practise to plot your raw data as well as the instantaneous hatching properties, to detect any anomalies and correct your practices if needed. However, since we supplied the synthetic data, you don’t have to wait a couple of days to see the entire hatching evolution of your “virtual cysts”. Below you will be asked to plot the synthetic data. Your plots should look somewhat like the ones in Figure 10. Note that, as the embryos start to hatch, their number (Et) starts to decrease; at the same time the number of umbrella (Ut) and nauplii (Nt) start to increase. At the end of the experiment, there are almost no embryos nor umbrellas, and the number of nauplii becomes constant. All viable cysts are now hatched! Figure 10. Left panel: Hatching evolution showing the change in numbers over time of nauplii (Nt), umbrellas (Ut) and embryos (Et). Right panel: Evolution of hatching percentage (Ht) 16. Using the synthetic data, make four graphs of the hatching evolution of each of the four treatments (similar to the graph shown in Figure 10 left panel). In each graph you have to show the change in numbers over time of nauplii (Nt), umbrellas (Ut) and embryos (Et). 16 points: 4 points per figure with correct axes labels, legends, units and figure caption 10 points 17. Using the synthetic data, make one graph combining the four curves each showing the evolution of hatching percent (Ht) of a different treatment of your experiment. 4 points: 1 points per correct axes labels, legend, units and figure caption MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 13 of 18 Estimation of other Hatching Properties The following properties are estimated at the end of the experiment. They are calculated from the entire ensemble of samples collected from a single bottle. Maximum hatching percentage (Hmax, units: %) refers to the percentage of cysts that have hatched into nauplii by the end of the experiment. It essentially is the maximum Ht, and can be estimated from the spread-sheet as the last number in the Ht column. Manufacturers often refer to this property simply as “hatching percentage” or “hatch rate” or “hatching rate” or “hatch-out”. This is one of the most important properties used to determine the “grade” or quality of the product. 18. What are the calculated Hmax for each of the experimental conditions modelled in the synthetic data: a. Hmax of decapsulated cysts hatched at room temperature _________ b. Hmax of full-chorion cysts hatched at room temperature _________ c. Hmax of full-chorion cysts hatched at 24°C _________ d. Hmax of full-chorion cysts hatched at 28°C _________ 4 points Maximum hatching efficiency (HEmax, units: number of nauplii per g of cysts) refers to the maximum efficiency at which nauplii are produced, and is simply the largest HEt of the experiment (likely at the end of the experiment). Note that HEmax is often referred simply as “Hatching Efficiency” or “Nauplii per gram (NPG)” by Artemia suppliers. 19. What data: a. b. c. d. are the calculated HEmax for each of the experimental conditions modelled in the synthetic HEmax of decapsulated cysts hatched at room temperature _________ HEmax of full-chorion cysts hatched at room temperature _________ HEmax of full-chorion cysts hatched at 24°C _________ HEmax of full-chorion cysts hatched at 28°C _________ 4 points Adjust your data to focus on the section linear dynamics: Your data contains measurements at the beginning and at the end of the time-series that will need to be excluded from the following analyses. Explanation: To ensure that we capture the entire hatching process, it is important to start sampling way before the hatching process starts and continue sampling well after the process ended. This means that we have many samples taken at the beginning when nothing was happening (start tail), and also many samples taken at the end when the process had ended and thus also nothing was happening (end tail). These data will skew the estimated hatching properties and thus have to be removed before you do your calculations. In the example below, we estimate the Hatching Rate ( HR, see formula below) using all the data and only using the data when hatching was occurring (i.e. after removing the start and end tails). Note that the calculated HR using all the data (HR = 2157) is four times smaller than the HR calculated after excluding the start and end tails (i.e. HR = 8720). MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 14 of 18 Figure 11. Hatching Efficiency vs time of all data (left) and data excluding the start and end tails (right). Note that the data on the right is contained within the yellow box in the panel on the left. The linear regressions (red dotted lines) as well as their respective slopes, intercepts and R 2 (see equations in red) are drastically different. To adjust your data: 1) Make a copy of your spread-sheet (save it with a different name) 2) Delete all rows of raw data before the hatching process started 3) Delete all rows of raw data after the hatching process ended Hatching rate (HR: nauplii per gram of cyst per hour) refers to the average rate at which nauplii hatch from cysts. It can be loosely interpreted as a “hatching speed”. To calculate hatching rate, first you have to plot “time” (t, unit: hours) on the x axis, against the “Instantaneous Hatching Efficiency” (i.e. HEt) on the y axis. Using Excel’s Data Analysis (within “Data” tab), use a Regression to estimate the parameters HR (i.e. slope) and b (i.e. intercept) shown in the equation below: 𝐻𝐸𝑡 = (𝐻𝑅 × 𝑡) + 𝑏 20. What are the calculated HR for each of the experimental conditions modelled in the synthetic data: a. HR of decapsulated cysts hatched at room temperature _________ b. HR of full-chorion cysts hatched at room temperature _________ c. HR of full-chorion cysts hatched at 24°C _________ d. HR of full-chorion cysts hatched at 28°C _________ 4 points MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 15 of 18 Hatching synchrony (HS, units: hours) refers to the number of hours that it takes for 80% of the cysts to hatch. To calculate hatching synchrony, first you have to plot “time” (t, unit: hours) on the x axis, against “hatching percentage” (Ht, units: %) on the y axis. Using Excel’s Data Analysis (within “Data” tab), use a Regression to estimate the parameters m (i.e. slope) and n (i.e. intercept) shown in the equation below: 𝐻𝑡 = (𝑚 × 𝑡) + 𝑛 Then use the estimated parameters m and n, and the formula below, to calculate t10 (units: hours), which is the time that it takes for 10% of cysts to hatch. 𝑡10 = 10 − 𝑛 𝑚 Once again, use the estimated parameters m and n, and the formula below, to calculate t90 (units: hours), which is the time that it takes for 90% of cysts to hatch. 𝑡90 = 90 − 𝑛 𝑚 Finally, the hatching synchrony (HS) is estimated from t10 and t90 using the formula below: 𝐻𝑆 = 𝑡90 − 𝑡10 21. What are the calculated HS for each of the experimental conditions modelled in the synthetic data: a. HS of decapsulated cysts hatched at room temperature _________ b. HS of full-chorion cysts hatched at room temperature _________ c. HS of full-chorion cysts hatched at 24°C _________ d. HS of full-chorion cysts hatched at 28°C _________ 4 points Effect of decapsulation on Hatching Properties 22. Using the synthetic data, compare all the estimated hatching indices (i.e. Hmax, HR, HE, t10, t90, HS) between the full-chorion cysts and the decapsulated cysts (both hatched at room temperature). If there are any differences, please comment on the reasons why do you think those differences occurred. 5 points MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 16 of 18 Effect of temperature on Hatching Properties 23. Using the synthetic data, compare all the estimated hatching indices (i.e. Hmax, HR, HE, t10, t90, HS) among the full-chorion cysts hatched at room temperature, 24°C and 28°C. If there are any differences, please comment on the reasons why do you think those differences occurred. 5 points Predicting t90 using temperature 24. Use the synthetic data from the full-chorion cysts hatched at room temperature, 24°C and 28°C, to create a scatter-plot with t90 (x axis) against temperature (y axis). Include the scatter-plot in your report with labels, figure caption, and a trend line with Equation and R-squared (see hint) Hint: To add a trend line (Excel). Hover mouse over a point in your scatter-plot. Right click. Select “Add Trendline”. Check boxes of “display equation on chart” and “Display R-squared value on chart” 5 points 25. Scenario: Nauplii are most nutritious immediately after hatching. You may want to manipulate hatching temperature to synchronize the hatching of your nauplii with the next feeding time of your farmed fish larvae. Question: Using the synthetic data’s scatter-plot (temperature vs t90) and Equation from the question above, deduce what temperature you need to incubate your cysts to ensure that 90% of your nauplii are hatched 28 hours after initiating cyst hatching. Show your reasoning and/or calculations. 5 points 26. Considering the synthetic data, how do your estimated hatching indices compare against the supplier-provided hatching indices @ ideal conditions (see label and/or answers of question 2)? Hint: If there are any discrepancies, please comment on the reasons why do you think those discrepancies occurred. 5 points 27. Using the synthetic data, do you think the cyst used in this experiment reflect the properties advertised in the label? …or do you think we got scammed? 1 points MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 17 of 18 References FAO. 1996. Manual on the Production and Use of Live Food for Aquaculture procedures. Eds. Lavens and Sorgeloos. FAO FISHERIES TECHNICAL PAPER 361. http://www.fao.org/docrep/003/W3732E/w3732e00.htm#Contents Fox, R. 2006. Invertebrate Anatomy Online. Lander University. http://lanwebs.lander.edu/faculty/rsfox/invertebrates/artemia.html MARI 3603 Practical Aquaculture - Dalhousie University | Instructor: [email protected] Page 18 of 18