Lab 2 - Brine Shrimp Experiment Final (2)

Lab 2: Brine Shrimp ( Artemia ) Experiment Purpose: The purpose of this lab is for you to practice experimental design while learning about the effects of the external environment on brine shrimp. This lab will help you practice the following skills: 1. Performing serial dilutions to make solutions of different concentrations to test the response of brine shrimp to different environmental conditions 2. Using pipettes to make accurate and precise measurements of liquids to create solutions with specific concentrations 3. Using a dissecting microscope to make observations of brine shrimp 4. Writing a testable hypothesis and prediction for an experiment based on your observations 5. Designing an experiment to test your hypothesis and prediction This assignment will help you gain the following knowledge : 1. Familiarity with the appearance and behavior of Artemia 2. Predicting the effects of different environments on brine shrimp 3. Being able to explain the role of osmosis and tonicity on an aquatic organism Introduction: Artemia Biology and Ecology Cells have a narrow range of conditions that will allow them to function and remain living. Some organisms have evolved adaptations that will help them maintain the correct balance for pH, temperature, and/or water. For example, the single celled Paramecium has evolved a compartment to collect excess water and expel it. The organism used in this lab, Artemia or brine shrimp, lives in saline waters around the world and serves as a food source for a diverse multitude of organisms, such as birds, insects, and fish. As an important component of multiple food webs, Artemia is frequently studied to find adverse conditions for the organism. Artemia is found in isolated habitats, namely natural salt lakes and man-made salterns, in temperate to tropical regions. The occurrence of Artemia is constrained to sites where conditions allow the animals to survive throughout the year, and to regions where the seasonality of the environment is stable (Lenz, 1987; Amat et al., 1995). The common feature of all Artemia habitats is high salinity. Brine shrimp do not possess any anatomical defense mechanism against predation, but they have developed unique physiological adaptations to high salinity which provides an ecological defense against predation as they can thrive in salinities that are lethal to many common aquatic predators. These adaptations consist of:  a highly efficient osmoregulatory system  the capacity to synthesize highly efficient respiratory pigments to cope with the low O2 levels at high salinities  the ability of females to produce and release dormant cysts instead of normal eggs when environmental conditions endanger the survival of the species 1

Artemia are incapable of active dispersion, therefore wind and waterfowl (especially flamingos) are the most important natural dispersion vectors. Brine shrimp eggs float and adhere to the feet and feathers of birds, who will incidentally transport the eggs to new locations. If the eggs are ingested by birds, they remain intact for at least a couple of days within the digestive tract. Artemia Life Cycle Under favorable conditions, fertilized eggs develop into free-swimming nauplii (ovoviviparous reproduction), which are released by the mother. In extreme conditions (e.g., too high or too low salinity, low oxygen levels) the embryos only develop up to the gastrula stage. At this point they are surrounded by a thick shell, enter a state of metabolic dormancy (diapause), and are then released by the female as cysts (oviparous reproduction). In principle, both oviparity and ovoviviparity are found in all Artemia strains, and females can switch reproductive modes from one ovulation to the next. The cysts usually float in the high salinity waters and are blown ashore where they accumulate and dry. As a result of this dehydration process the diapause mechanism is generally inactivated; cysts are now in a state of quiescence and can resume further embryonic development when hydrated in optimal hatching conditions. Under optimal conditions brine shrimp can live for several months, grow from nauplius to adult in only 8 days and reproduce at a rate of up to 300 nauplii or cysts every 4 days. (Information adapted from: CABI, 2019. Artemia sp. In: Invasive Species Compendium. Wallingford, UK: CAB International. www.cabi.org/isc. ) Figure 1: Artemia life cycle Water Balance An important and extensively investigated biological phenomenon is the process of cellular transport. This process allows regulation of the composition of the cytoplasm and response to whatever conditions exist in the internal and external environments. The cell membrane structure is a phospholipid bilayer in which a number of proteins and other organic molecules 2

Experimental Organisms Biologists often need to study cellular processes, such as osmosis, because they constantly occur within living organisms. To do this, they conduct experiments using living organisms as a model in a controlled environment (specific diet, living conditions, genes, etc.). Since closely controlled experiments involving humans would often be unethical, biologists frequently use results from experiments on smaller, simpler animals to predict what would happen in more complex organisms, like humans. The most common experimental organisms are easy to obtain and work with, and they also grow and reproduce quickly. Artemia fit all these criteria and can be used to understand how environmental conditions affect aquatic organisms and other animals. Examples of other commonly used experimental organisms in biology include yeast, fruit flies, nematode worms, and mice. Concentration and Molarity In this lab, salt concentration is expressed as molarity (M). Molarity is the number of moles of a specified substance dissolved in one liter of liquid. Molarity describes solutions in which components are specified in proportion to the number of molecules of each component, rather than by weight or volume. One mole is equal to 6.02 x 10 23 molecules, so a 1M solution contains that number of molecules dissolved in 1 Liter of water. One mole of any substance is equal to the gram molecular weight of that substance. The gram molecular weight of NaCl is 58.44 g, so 1M of NaCl contains 58.44 g of NaCl dissolved into 1 Liter of water. Let’s say you only need to make 10 ml of a 1M NaCl solution. a. (0.25 pts) To determine the amount of NaCl that would be used to prepare any volume under or over one liter, 58.44 g (one mole of NaCl) is multiplied by the desired volume (in ml) and divided by 1000 ml. b. (0.25 pts) How much dry NaCl do you need to mix with 10 ml of distilled water (the solvent) to make a 1M NaCl solution? 0.5844 c. (0.25 pts) Show your calculations to the above question: 58.44(10)/1000 4

WEEK 1 – Brine Shrimp Behavior in Response to Changes in Salinity Part A – Serial dilution of a 4M NaCl solution To test the effects of different salinity environments on brine shrimp behavior, we will need a variety of NaCl solutions at different concentrations. A serial dilution involves a series of steps where each concentration is diluted (usually) with water to become half of the previous concentration. For example, a 4M NaCl solution will be diluted to become a 2M NaCl. Then, the 2M NaCl solution will be diluted to become a 1M NaCl solution. Then, the 1M NaCl solution will be diluted to become a 0.5M NaCl solution. And so on … Figure 2: Diagram of the serial dilution method Follow the steps below to create the different NaCl concentrations that we will use later. 1. Obtain two tubes: one containing 15 ml of 4M NaCl solution and one containing 15 ml of DI water. 2. Obtain four empty screw cap centrifuge tubes and label them as “2”, “1”, “0.5”, and “0”, respectively. 3. Use the serial dilution method to make NaCl solutions that are progressively halved in concentration. a. Transfer 2.5ml of the 4M NaCl solution to the tube labeled “2”, then add 2.5ml of DI water to the same tube. Screw on the tube’s cap and shake the tube to mix the solution. The tube now contains a 2M NaCl solution. b. Transfer 2.5ml of the 2M NaCl solution to the tube labeled “1”, then add 2.5ml of DI water to the same tube. Screw on the tube’s cap and shake the tube to mix the solution. The tube now contains a 1M NaCl solution. 5

7 Part C – Using the dissection microscope and the collection of preliminary data Prior to performing the formal experiment of testing brine shrimp in different NaCl concentrations, we will conduct a few simple steps to help us create a hypothesis and prediction in a later step. Since a hypothesis is an explanation based on observations and assumptions, preliminary testing is helpful. Use the results from this section and the information in this worksheet to write a hypothesis and prediction in Part D. To use the microscope, plug it in and turn on the light. After correctly positioning the oculars to fit your eyes, practice using the scope by viewing Artemia . Manipulate the focus knob to bring the organisms into focus. The light source for your microscope may be a separate light or built into the microscope. Light can be transmitted through a specimen on the microscope stage from below. Light can also be reflected from the surface of the specimen by positioning the light source above the specimen so that it shines downward onto the microscope stage. Always turn off the light source when the microscope is not in use to help prolong the life of the bulb. 1. Place 2.5 mL of the Artemia solution into a new petri dish. Look at them closely with low power (10-30x) magnification. These are Artemia under ideal conditions. (0.25 pts) Which illumination methods is your microscope capable of? Incident illumination (0.25 pts) If your microscope can provide both types of illumination, which works better (i.e., allows you to see the most detail of your specimen)? The incident illumination works better because it allows us to see the most detail of the brine shrimp. (0.25 pts) What parts of the brine shrimp can you identify? We can see the secondary antennas and eye of the brine shrimp, along with its overall shape in general. We cannot see many of its mandibles though. Also, some of the shrimps are thicker than the others. (0.25 pts) What are their swimming habits? They move quite jaggedly, almost as if they’re glitching. Their swimming habits aren’t smooth, but rather a bit harsh.

8 (0.25 pts) How do they distribute themselves in the solution? They distribute themselves equally, for the most part. They’re just spread out; no shrimp going to different sides, like they’re cliques or something of the sorts. (0.25 pts) What color are they? The brine shrimp are orange. (0.25 pts) What are the dark circular objects? The dark circular objects are dormant cysts. Cells that are in solutions of a different tonicity can undergo changes in the amount of water they contain. Either too much or too little water in the cell can be a fatal condition. In the introduction to this lab, you have information on the possible consequences of changes in water balance in a cell. You will now make observations of Artemia exposed to hypertonic and hypotonic solutions with a dissecting microscope. 2. Place a drop of the 4M NaCl on one edge of the dish and observe how the brine shrimp respond. (0.25 pts) Describe the change in brine shrimp behavior observed. Before, the brine shrimp were hitting the eggs, and only that, but when one of them dies, the others begin to eat them. They seem to be very cannibalistic. The stronger shrimp were beating up the weaker ones. They also kept hitting their heads on the walls. There are more broken eggs. (0.25 pts) Include sketches of the shrimp. n/a

10 Part D – Developing a hypothesis and prediction 1. Based on your observations of brine shrimp, develop a hypothesis for the experiment you set up in Part A. A hypothesis is an explanation based on observations and assumptions that leads to a testable prediction. (0.25 pts) Hypothesis: The different salinity levels of NaCl will affect the movement, pattern, and speed of the swimming and overall movement of the shrimp in comparison to the environments they’re used to. 2. From your hypothesis, write a prediction. Predictions are related to your hypothesis but are specific to your experiment. Think of what you would do in your experiment to test your hypothesis. These can be written as “If… then…” statements. (0.25 pts) Prediction: If the brine shrimp are placed into NaCl solutions that are different from their usual environments, a change in their behavior will be documented. 3. What led you to form the hypothesis and prediction you chose? This should reference your observations. (0.25 pts) Reasons for choosing: It’s only natural to assume that an animal that is removed from an environment it’s already adapted to will react noticeably differently when placed into a new environment with added factors. 4. We will be testing the effects of salinity on brine shrimp behavior. (0.25 pts) What specific behaviors will you look for changes in? Choose at least three different behaviors. Think of what you observed in Part C. These will be your dependent variable(s). Your independent variable is the condition that you are testing the effects of. We will look for changes in the brine shrimp’s swim movement, how fast or slow they move, and the constant patterns of which they respond to the change of salinity. (0.25 pts) Independent variable: The salinity concentrations (0.25 pts) Dependent variable(s): The brine shrimp’s swim movement, pattern, and speed

11 (0.25 pts) Define the control group: The control group is the brine shrimp that didn’t have any salt added, just water. (0.25 pts) Define the experiment group: The experiment group is the brine shrimp that has salinity added. Data falls into two categories: qualitative data (descriptive data; for example, observations of brine shrimp swimming and behavior), and quantitative data (measured data; for example, the number of brine shrimp that died in a certain solution). These data are often organized in tables and/or presented in graphs. Choose at least one of the dependent variables to be recorded as quantitative data. For example, it would be possible to observe and record an estimated mortality percentage. Others may choose to create a number system to compare swimming speeds. The control group could be set to 0. Groups that swam faster could be designated by a +1. Slower, by a -1. (0.25 pts) Write the dependent value(s) that will be recorded as quantitative data here: The brine shrimp that have more salinity placed inside of their groups die 20% more than the previous groups that have less salinity. Part E - Performing the brine shrimp salinity experiment 1. Obtain a tube containing 15ml of brine shrimp in a 0.5M NaCl solution (salt water). 2. Obtain five petri dishes and label them as “4”, “2”, “1”, “0.5”, and “0”, respectively. 3. Use a serological pipette to transfer 2.5 ml of the brine shrimp solution to each of the five petri dishes. Be sure to invert the tube containing the brine shrimp before each withdrawal to distribute them evenly throughout the solution, thereby assuring each petri dish will have an equal amount of brine shrimp. 4. Transfer 2.5ml from each of the NaCl solutions into the corresponding petri dishes with brine shrimp. 5. Let these dishes sit for 30 minutes. Be careful not to shake the dishes and influence the behavior of the shrimp. 6. Continue to Part F while waiting for the brine shrimp to incubate in their different solutions.

13 4. Reflect on the observations by answering the following questions. a. (0.25 pts) Compared to the control group, are they more or less active? The brine shrimp are less active. They get slower and slower each trial. b. (0.25 pts) Compared to the control group, did any of them die? Yes, the more salinity that was added, the more the brine shrimp died. Poor things! c. (0.25 pts) If so, how many? Lots and lots, depending on the salinity. If we grab the 2m of salinity, then there is about 70% that died compared to the control, which was about 2%. d. (0.25 pts) Could you use this to determine which treatment is closest to the natural conditions for the brine shrimp? Yes! e. (0.25 pts) How could you tell? Well, depending on the salinity added, we can see that the more added, the more they change from their original state. f. (0.25 pts) What does this tell you about the normal conditions of salinity for brine shrimp? (These are your conclusions about the normal salinity for brine shrimp.) The normal conditions of salinity allow the brine shrimp to thrive as it is closest to their natural environment. Cleanup : dump the petri dish solutions into the waste container under the fume hood and dump any excess salt solutions in the centrifuge tubes down the sink. Rinse the petri dishes and centrifuge tubes and return them to your lab bench for reuse. Do not dump the stock solutions provided to you . Wipe down all the equipment (dissecting microscope, pipette holders, etc.) that you touched and your lab bench. Then, continue to the section G.

14 Part G – Experimental design The experiment you performed today was designed for you; the experiment you perform next week will be one that you design. Go back and review the design of the experiment you just performed and consider the importance of technique in performing the experiment. (0.25 pts) Why was it important to handle all tubes and petri dishes the same way? The tubes and petri dishes must be handled in the same correct manner because it increases consistency and decreases error. (0.25 pts) If you hadn’t, how would that have affected the data you collected? The data would have been flawed or inconsistent if the tubes and petri dishes weren’t handled in the correct manner. There could also be more room for error, which isn’t exactly ideal. You will have similar materials available to use for your experiment next week, namely: a. Buffers at three pH levels: 2, 6, 7, 8, and 12 b. Water baths at several temperatures: 0, 23, 30, and 50 o C c. Solutions with different concentrations of alcohol in salt water: 100%, 50%, 25%, and 0%. Note: you will use the serial dilution method to make the lower concentrations . d. Solutions with different concentrations of caffeine in salt water: 4mg/ml, 2mg/ml, 1mg/ml, and 0mg/ml. Note: you will use the serial dilution method to make the lower concentrations. (0.25 pts) Which of these will you examine? We will examine how the brine shrimp reacts in different levels of pH. 1. (0.25 pts) What will the independent variable be? The pH levels in the water. 2. (0.25 pts) What will the dependent variable(s) be? The behavior of the brine shrimp 3. (0.25 pts) What things must you keep the same between all treatments? We must keep the amount of brine shrimp at 2.5 ml for all treatments.

16 6. (0.5 pts) Create a table below that you will fill in with the data you collect from your experiment. Ph Speed Pattern # Dead Shrimp 2 No speed No pattern 100% 6 Moderate Jagged 1% 7 Slow Twitching 0% 8 Moderate Jagged 0% 12 Slow Circles 0% 6. (0.25) How will you display your quantitative data? (Bar graph, line graph, pie chart) 1. We will display our quantitative data with a bar graph. Obtain a tube containing 15ml of brine shrimp in a 0.5M NaCl solution (salt water). 2. Obtain five petri dishes and label them as “4”, “2”, “1”, “0.5”, and “0”, respectively. 3. Use a serological pipette to transfer 2.5 ml of the brine shrimp solution to each of the five petri dishes. Be sure to invert the tube containing the brine shrimp before each withdrawal to distribute them evenly throughout the solution, thereby assuring each petri dish will have an equal amount of brine shrimp. 4. Transfer 2.5ml from each of the NaCl solutions into the corresponding petri dishes with brine shrimp. 5. Let these dishes sit for 30 minutes. Be careful not to shake the dishes and influence the behavior of the shrimp. 6. Continue to Part F while waiting for the brine shrimp to incubate in their different solutions.

17 Bring these pages to lab next week along with extra paper to record your experimental findings.