SUPPLEMENTAL INFO AND FIGURES:

Care/Handling: 

• Warning:  Live fiddler crabs will be used during this procedure.

• Safety Note: Wear lab attire throughout the experiment: lab coat and nitrile gloves. 

• Safety Note: Ensure that you wash your hands before and after experimenting to lessen the potential for bacteria transfer to occur. It is possible that bacteria that you are exposed to could disrupt the crab’s environment, and it’s important that this transfer is controlled to the greatest extent possible. Also, crabs produce their own bacteria that has the potential to be infectious. It is important to minimize this contact as well. 

• Care Note: When placing the terrarium on your workstation, ensure that it is outside of direct sunlight. Exposure to direct sunlight could cause premature death in the fiddler crabs. 

• Care Note: Be sure to transport the fiddler crabs with care in between experiments by using the 400 mL beakers and your hands to gently move the crabs between each of their containers. 

• Care Note: The crabs habitat temperature should be occasionally heated to ensure crab health as a habitat temperature that is too low will cause crabs survival rate to decrease. Ensure that you use a power trip with a mechanical timer as provided in the materials list above, so heating does not occur for an extended period of time. Extended heating can cause crab death or harm. If needed, please reference the video guide on how to set up the mechanical timer linked here. The heat lamp from the materials list will need to be used for roughly 35 minutes per day to ensure that the crabs receive the correct amount of heat per day. Additionally, the steps for heat lamp setup are contained in the positive control section of the experiment. 

• Care Note: Initial Setup steps should be completed within the vicinity of a functional electrical outlet. 

• Care Note: Ensure that you change out the water in the terrarium once per week using the brackish solution created in steps 1-3 of the Initial Setup. Ensure that the 9 L bucket of water is sealed using Aluminum Foil as described in step 25 of the Initial Setup. Replace the saran wrap using this method every time the water is changed.

• Care Note: Ensure that crabs are fed on a regular basis by providing them with 6 pellets of crab food every 3 days. The crab food is provided in each fiddler crab kit. To ensure that each crab receives at least two pellets of food, use your hands to distribute the food evenly to each crab in the tank. 

Initial Setup

Note: The anticipated time needed to complete this section of the experiment is 2 hours and 30 minutes.

1. Place the 9 L measuring pail under a sink faucet. 

2. Turn on the faucet to fill the 9 L measuring pail with 2 L of tap water using the measurement markings on the sides of the measuring pail.

   1. Note: Use the cold setting of the faucet.

3. Move the 9 L measuring pail to a new location, such as the top of your work station, or the corner of the room. Leave the pail in this location overnight in order to dechlorinate the water. 

   1. Note: The measuring pail should be placed in a location where it will not be disturbed, nor will it cause a disturbance to others. 

   2. Note: Naturally, some water will evaporate overnight. However, this will be a negligible amount, and you will still have more than enough dechlorinated water for the experiment to continue the next day.

   3. Note: This is a necessary stopping point. Resume the next day.

4. Open the box containing the first fiddler crab kit.

5. Gather 4 new 400 mL beakers, the sharpie, and the masking tape. Tear off an approximately 5-7 cm long strip of masking tape and secure it to one side of each beaker. Use the sharpie to label two beakers “M” and two “F” 

   1. Note: These will be used as gender-specific holding tanks for the fiddler crabs. 

   2. Note: The crabs must be divided by gender because if they are kept together in a confined space with members of the opposite sex, then they will engage in combative behavior. 

6. Gather a pair of scissors and the shipping bag containing the crabs from the kit. Place the scissors on the side of your workstation in a place where they are easily accessible. Place the shipping bag above a sink with a drain stopper. 

7. Open the shipping bag containing the crabs by using the pair of scissors to remove the zip tie from the top of the bag.

8. Discard the shipping water in the bag by grabbing the stem of the bag with one hand and the bottom of the bag with the other. Then, tilt the bag towards the sink to release the shipping water.

   1. Note: Make sure the angle that you tilt the bag is not too steep so that the crabs don’t fall into the sink.

9. Identify the male and female crabs in the bag by observing the claw size of each specimen.

   1. Note: Male crabs have one small claw and one major claw, and female crabs have two small claws. See reference image below for further detail. 

Female (A) and Male (B)

(Thaler, 2011)

10. Pick up one crab by scooping it out of the bag with one hand. Maintain the crab in your hand while holding your palm horizontally and slowly transfer the crab to the appropriate 400 mL beaker based on its gender. 

    1. Note: This step should be completed over a workstation. 

    2. Note: Maintain the crab at a safe height, which should be 10-20 cm above the surface of the workstation. 

    3. Note: Crabs will naturally cling to your hand with their 8 legs, which enable them to grip to foreign objects. This means that as long as you maintain a slow, steady pace of transfer and a horizontal hold, the crab will transfer easily.

11. Repeat step 10 five additional times until all crabs have been placed in an appropriate 400 mL beaker based on their gender.  

    1. Note: You will receive an equal number of crabs of each gender.

    2. Note: You should receive 6 crabs per kit, as per the instructions given by Carolina Biological. However, if this is not the case and you receive more, add any extra crabs to the appropriate beaker based on their gender. 

12. Gather the provided terrarium from the fiddler crab kit. 

13. Use scissors to open the 3 lb. packet of sand provided in the fiddler crab kit. 

14. Pour the sand inside of the provided terrarium such that it covers the bottom of the habitat. Spread the sand evenly across the floor of the terrarium using your hands. 

    1. Note: You do not need to measure this quantity of sand because it was premeasured for you and provided with the terrarium. 

15. Use your hands to dig a small valley in the sand in the terrarium. This should be done next to one of the terrarium’s glass walls. 

    1. Note: Excavate the valley to be approximately as deep as the plastic storage box provided in the first fiddler crab kit. 

    2. Note: A video example is provided here if needed. 

16. Gather the small plastic storage box and place it in the valley in the sand. 

    1. Note: The top rim of the box should be flush with the surface of the sand.

    2. Note: If necessary, dig a deeper valley to ensure that the top rim of the small plastic storage box is flush with the surface of the sand. 

17. Gather the 9 L measuring pail of dechlorinated tap water that was created in Steps 1-3.

18. Gather a 500 mL beaker and use it to retrieve 400 mL of water by scooping it out of the bucket with dechlorinated water. 

    1. Note: This is a good stopping point.

19. Get a change of gloves.

    1. Note: This is to prevent possible contamination of the dechlorinated water.

20. Place your finger in the water contained within the 500 mL beaker to roughly determine its temperature.

    1. Note: This temperature measurement does not need to be exact. The purpose is to ensure that the temperature is lukewarm.

    2. Note: If the water feels cold, follow steps 21-23. If the water feels lukewarm, go to step 24. 

1. Note: Crabs do not fare well in cold water temperatures so it is crucial that the water is lukewarm.

21. To warm the water, gather the 500 mL beaker containing the dechlorinated water and transfer it to near the sink faucet. 

22. Turn on the sink faucet and place your hand underneath the water to ensure that it's warm.

    1. Note: It may be necessary to use water to heat the inside of the beaker. This will help warm the water quicker if the water is very cold.

23. Place the outside of the 500 mL beaker under the warm water for 3 minutes to heat the dechlorinated water inside the beaker.

    1. Note: Once completed go back to back to step 20. 

    2. Note: Ensure water doesn't spill. 

       1. If water spills, go back to step 18.

24. Slowly pour the warmed water into the small plastic storage box to fill it with exactly 400 mL of dechlorinated water.

    1. Note: This amount of water will fill the small plastic storage box ¾ of the way full. 

25. Gather the aluminum foil and the wooden metric ruler. Use the ruler to measure 31 cm of aluminum foil. Then, use the perforated edge of the aluminum foil to remove this length from the roll. Carefully place this over the top of the 9 L bucket such that it completely covers the opening. 

    1. Note: This step is necessary as the dechlorinated water is used throughout multiple parts of the procedure which will be conducted across multiple days. The aluminum foil will act as a seal for the water so that it doesn't evaporate in between each use.

26. Gather the 1/16 tsp measuring spoon and the salt packet provided with the terrarium. Fill the teaspoon up to the edge to deliver 1/16 tsp of salt into the plastic storage box. 

27. Gather a second 500 mL beaker. Use your hands to fill the 500 mL beaker with 100 mL of sand gathered from the quantity you have poured inside of the terrarium in Step 14.

28. Pour the sand from the 500 mL beaker into the small plastic storage container. Ensure that the sand is leveled across the bottom of the container. If necessary, use your hands to smooth the surface of the sand.

    1. Note: This layer of sand will make it easier for the crabs to move in and out of the small plastic storage box.

29. Gather the petri dish provided with the first fiddler crab kit. Place it in the center of the remaining sand in the terrarium. This will be the location where all of the crab’s food is placed. See the image below for further clarification:

    1. Note: See reference image below

30. Gather all four 400 mL beakers containing the fiddler crabs. 

31. Tilt the 400 mL beakers one at a time over the sand in the terrarium to encourage the crabs to enter the terrarium. The crabs will naturally fall out of the beakers due to the container’s slippery nature. 

    1. Note: Ensure the angle at which you tilt the 400 mL beaker is not too steep so that the crabs don’t free fall directly into the terrarium.

32. Gather a black sharpie, the masking tape. Tear off an approximately 5-7 cm long strip of masking tape and secure it to one side of the terrarium. Use the black sharpie to label the terrarium that comes with the fiddler crab kits “Tera 1” on the masking tape that is on the side of the terrarium.

    1. Note: It doesn’t matter which side of the container that you choose, as long as this name is clearly marked. 

    2. Note: This is a good stopping point

33. Gather one 400 mL beaker, the stopwatch, the acrylic paint pens, and the terrarium labeled Tera 1. Place these on your workstation. 

34. Use one hand to pick up 1 crab from the terrarium in the same method described in step 10. Use the stopwatch to set a 15-minute timer. The crabs must stay in the beaker for the entire duration of this period to dry off. This will help ensure that the ink from the acrylic pen stays on in subsequent steps.

35. Gather the waterproof acrylic paint pen in the color “red." Remove the cap from the red acrylic paint pen. 

    1. Note: Use the chart on the back of the acrylic paint pen package to choose the correct color. 

36. Using the acrylic paint pen, gently blot the tip of the pen onto the top of the crab’s shell to create a red colored marking. 

    1. Note: This crab with the red marking will henceforth be referred to as “Crab 1.”

37. Use the stopwatch to set a timer for 30 minutes. Allow Crab 1 to remain in the 400 mL beaker during this time period.

    1. Note: This enables the ink from the acrylic paint pen to completely dry and ensure that the crabs are clearly labeled for the duration of the experiment.

    2. Note: This is a good stopping point. 

38. Use one hand to pick up 1 crab from the 400 mL beaker in the same method described in step 10 to transfer the crab back into Tera 1 once the stopwatch has gone off. 

39. Repeat steps 34-38 five additional times to paint each unique crab.

    1. Note: Use the chart on the back of the acrylic paint pen package to choose the correct color. 

    2. Note: Paint the second crab the color “green.” This crab will henceforth be referred to as “Crab 2."

    3. Note: Paint the third crab the color “orange.” This crab will henceforth be referred to as “Crab 3." 

    4. Note: Paint the fourth crab the color “blue.” This crab will henceforth be referred to as “Crab 4." 

    5. Note: Paint the fifth crab the color “pink.” This crab will henceforth be referred to as “Crab 5." 

    6. Note: Paint the sixth crab the color “purple.” This crab will henceforth be referred to as “Crab 6." 

    7. Note: Paint the seventh crab the color “white.” This crab will henceforth be referred to as “Crab 7."

40. Gather the second Fiddler Crab Kit.

41. Repeat Steps 4-39 using the second fiddler crab kit.

    1. Note: When repeating step 32, use a black sharpie to label the second terrarium that comes with the fiddler crab kit “Tera 2.”

    2. Note: When repeating steps 35-38, paint the crabs using the chart on the back of the acrylic paint pen package to choose the correct shade. 

    3. Note: Paint the seventh crab the color “white.” This crab will henceforth be referred to as “Crab 7” from this point on. 

    4. Note: Paint the eighth crab the color “black.” This crab will henceforth be referred to as “Crab 8” from this point on. 

    5. Note: Paint the ninth crab the color “sky blue.” This crab will henceforth be referred to as “Crab 9” from this point on. 

    6. Note: Paint the tenth crab the color “leaf buds.” This crab will henceforth be referred to as “Crab 10” from this point on. 

    7. Note: Paint the eleventh crab the color “light purple.” This crab henceforth will be referred to as “Crab 11” from this point on. 

    8. Note: Paint the twelfth crab the color “yellow.” This crab will henceforth be referred to as “Crab 12” from this point on. 

42. Use the qualitative data collection section in the Google Sheet linked here to record daily observations of Fiddler crab behavior. The day that you receive the crabs should be noted as “Day 0.”  

    1. Note: You should check for frequency of watering and feeding behaviors: How often are the crabs eating, and how much? Also note how often the brackish water solution in the small plastic storage box needs to be changed. You should also be observing crab behavior.  

    2. Note: When the crabs are behaving normally, they are minimally active in that they move around approximately every 3-5 minutes. Sometimes, you may notice that they are interacting with other crabs; this is normal. However, when crabs are stressed, they are in an almost constant state of motion and interact more often with their environment and their neighbors. You may notice that they are moving much more quickly and that they are restless. Ensure you are noting these observations in the qualitative data collection section.

43. Clean all equipment used by washing the beakers over the sink with soap and warm water, and return all other materials to their respective storage spaces. 

44. Safely remove all PPE and dispose of the nitrile gloves in the trash.

    1. Note: This is a good stopping point.

Care/Handling: 

• Safety note:  Wear lab attire throughout this section of the experiment: lab coat, safety goggles, nitrile gloves.

• Care Note:  The substrate created in Steps 12-15 of this section will need to be hydrated every 2 days to keep the surface moist. Use a 50 mL spray bottle to deliver 50 mL of water evenly across the surface of the substrate every 2 days.  

Tank Set Up 

Note: The total time for this experiment is 2 hours, but the steps can be broken up to fit the time provided to you. See notes for appropriate breaks. 

1. Gather the unused terrarium from RUBBERMAID , a black sharpie, and masking tape. Tear off an approximately 5-7 cm long strip of masking tape and secure it to one side of the terrarium. Use the black sharpie to label the terrarium that comes with the fiddler crab kits “Tera 3” on the masking tape that is on the side of the terrarium.

   1. Note: It doesn’t matter which side of the container that you choose, as long as this name is clearly marked. 

2. Open the RUBBERMAID container and carefully lay all contents on the workstation.

3. Gather a pair of scissors and the 20 lb bag of Marine sand. Use the scissors to open the 20 lb bag of sand.

4. Gather a new 9 L measuring pail. Pour the entire 20 lb bag of sand into a new 9 L measuring pail by tipping the opening over the measuring pail. Use your hands to encourage reluctant amounts of sand to enter the measuring pail. 

5. Gather the package containing silt sand mud and a new 9 L measuring pail. Use scissors to open the box.

6. Remove the bag of mud from the cooler inside of the package. 

7. Place the bag of mud inside of the new 9 L measuring pail.

   1. Note: Ensure that you hold from the bottom of the bag so that the plastic does not break.

8. Use your hands to remove the rubber bands fastened to the top of the bag of mud. 

9. Use the scissors to open the first layer of the bucket wrapping.

10. Lift the bag of mud and slowly turn it upside down to pour the mud into the 9 L measuring pail. 

    1. Note:  You may have to gently shake the bag during this process to encourage all of the mud in the package to release into the measuring pail.

    2. Note: You will receive 6 L of mud, you can use the gradations inside of the bucket to measure this quantity. 

11. Gather a 1000 mL beaker. Use the beaker to scoop 1000 mL of sand into the beaker from the 9 L measuring pail used in Step 5. 

12. Pour the sand gathered in the 1000 mL beaker into the 9 L measuring pail of mud that was created in Step 11. 

13. Gather a stopwatch and set a timer for 10 minutes. Use one hand, does not matter which one, to mix the mud and sand together for 10 minutes in the 9 L measuring pail. Use your hand to repeatedly scoop the substrate from the bottom of the measuring pail to the top. As you are mixing you will know that the sand and the mud are thoroughly combined when the color of the substrate is a medium brown throughout the entire mixture. The texture of the substrate should also be smooth, and devoid of any individual clumps.

    1. Note: It may also be necessary to mix for longer so if further time needs to be added for mixing ensure that the time is added. 

    2. Note:  It should take approximately 8-10 minutes of consistent mixing to achieve this result.

    3. Note: For further clarification on mixing techniques, texture, and color, see the video linked here.

14. Repeat steps 12-14 two additional times to create the final substrate mixture.

    1. Note: Remove nitrile gloves used in Steps 1-15. Put on a fresh pair of gloves after completing this step. 

15. Gather a new 1000 mL beaker and the terrarium labeled Tera 3.

16. Use the beaker to scoop 1000 mL of the substrate from the mixture created in steps 12-15.

17. Pour 1000 mL of the substrate into Tera 3.

18. Repeat steps 17-18 until all of the substrate has been added into Tera 3.

    1. Note: If there is not enough substrate to fill the storage container lay foam to ensure an even layer as shown here

19. Use your hand to flatten the surface of the substrate in Tera 3. Use a packing motion to create an even surface. 

    1. Note: See reference image below for a visual of what the substrate in Tera 3 should look like when this step is complete. 

20. Use a wooden metric ruler to measure the depth of the sand by placing the end of the ruler labeled “0” vertically into the sand. Look at the ruler and observe the height of the sand based on its measurement against the ruler.

21. Record the height of substrate in cm and the date when recording in the Google sheet titled “Qualitative data of substrate height” inside of the quantitative data table linked here. 

    1. Note: The height of the sand should be 7 cm based on the amount of substrate that was added to the terrarium. 

22. Remove the ruler from the sand.

    1. Note: This is a good stopping point

23. Gather the terrarium thermometer and the included Duracell lithium coin batteries. 

24. Place one lithium battery in the terrarium thermometer and turn on the device by pressing the “on” button. 

25. Mount the terrarium thermometer inside of Tera 3 by securing the suction cup onto the glass. The device should be placed approximately 10 cm above the sand so that it is not an obstacle for the fiddler crab. Use the metric ruler to confirm that the device is placed at this height. 

    1. Note: It does not matter which side of the terrarium you place the thermometer as long as you can read the measured temperature. 

26. Gather a stopwatch and set a timer for 5 minutes.

    1. Note: This is to let the thermometer acclimate to room temperature. 

27. Record the measured temperature of the terrarium when the stopwatch goes off. Do so in the table titled “Time Spent Interacting With Sand at a Room Temperature” that was created in Step 8 of the data collection setup section. Use the column titled “Temperature Per Session” to record this value.   

    1. Note: This section of the experiment may be completed over the span of several unique sessions, so the temperature of the environment may vary. Record the temperature of the terrarium at the start of each new session in the “Temperature per Session” column of the data table titled “Time Spent Interacting With Sand at a Room Temperature.” This will be found in the sheet titled “Negative Control” created in step 6 of the data collection setup section. 

       1. Note: A session is defined as a unique span of time spent completing an arm of the experiment. For example, a timespan spent setting up, working on the experiment, and putting away all materials represents one session. The act of resetting materials, completing additional work, and cleaning up materials represents a new session, and would require a new temperature to be taken.

28. Gather the tripod and set it up beside the terrarium such that the iPhone can be positioned to have a complete aerial view of the terrarium.

Care/Handling: 

Safety note:  Wear lab attire throughout this section of the experiment: lab coat, safety goggles, nitrile gloves.

Negative Control

Note: The total time for this experiment is 2 hours, but the steps can be broken up to fit the time provided to you. See notes for appropriate breaks. 

1. If the tripod positioning has been adjusted since completing Tank Set Up, repeat step 48 of the Tank Set Up. If not, go straight to step 2.

2. Gather the iPhone and place it inside the tripod. Secure it tightly by turning the knobs on the side of the tripod clockwise.

3. Place the tripod with the iPhone across from the container wall where the meter stick is attached. 

4. Enter the Camera app on the iPhone and select the Video recording feature. Your video frame should capture the full length of the meter stick and the entire length of the container. 

   1. Note: You may have to adjust how close the tripod is to the container in order to capture the entire testing arena.

   2. Note: The tripod's height should be adjusted so that the entire testing arena is viewable without obstruction from the container walls parallel to the wall with the meter stick. Additionally, you should be able to clearly read the gradations on the meter stick.

   3. See reference image below.

5. If the burrow created in the Tank Set Up has been altered or needs to be re-made, use your pointer finger to establish a burrow at the 75 cm mark on the meter stick.

   1. Note: The burrow should be created so that it is at an angle rather than completely vertical. The burrow should also be as deep as the length of your entire finger.

6. Gather the 500 g weight, the spool of string, and a pair of scissors.

7. Cut a small piece of string and attach it to the top of the weight.

8. Gather an iPad. Open the polarized bird image shown below using the link here, and set aside the iPad with the image open as it is imperative that you have quick access to it for testing. 

9. Then gather a heat lamp and secure it to a stable object at an empty workstation. For example, you can attach this to the hinge of an overhead cabinet.

   1. Note: If there isn't an available object to which you can attach the lamp, a lamp stand can also be used to secure the heat lamp.

   2. Note: Ensure that the lamp is roughly 0.5 meters above the surface of your workstation as it will be used to warm the crabs prior to trials. If the heat lamp is too close to the fiddler crabs, this can cause their internal body temperatures to become too high which can lead to decreased physiologic response and even death.

10. Obtain a 1 Tbsp measuring spoon, a new 400 mL beaker, Tera 1, and Tera 2. Use the spoon to gently encourage one of the fiddler crabs out of either Tera 1 or Tera 2 depending on which crab is being used for experimentation, and move the crab into the 400 mL beaker.

11. Carefully transport the 400 mL beaker with the crab to the empty workstation with the heat lamp, and place the beaker under the heat lamp.

12. Gather a stopwatch. 

13. Turn on the heat lamp and set a 5 minute timer using the stopwatch.

    1. Note: Depending on your environment, the environmental temperatures of Tera 1 and Tera 2 may be below fiddler crabs' standard habitat temperature causing a lower body temperature which can lead to decreased physiologic responses. Placing the crab under the heat lamp prior to testing will help  ensure that the crab's internal body temperature is warm enough to allow for normal physiologic responses. 

14. Once the 5 minutes have passed, turn off the heat lamp.

15. Gather the 400 mL beaker and carefully transport it to the testing arena area.

16. Hit the record button on the iPhone.

17. Tilt the 400 mL beaker over the substrate Tera 3 to transfer a crab to the end of the storage container opposite the burrow you created in step 5.

    1. Note: Make sure the angle that you tilt the beaker is not too steep, so that the crabs don’t free fall into the terrarium.

    2. Note: If a crab unintentionally falls out, using your hands, gently encourage the crab to the starting area of the testing arena. 

18. Gather the stopwatch and the 500g weight with the string attached.

19. Ensure that you line up the weight so that it does not hit or injure any crabs, and hold it approximately 30 cm above the sand.  

20. Release the string to drop the weight in the testing arena ensuring it's behind the crab, and start the stopwatch.

    1. Note: The weight should scare the crab and encourage it to run towards the burrow.  

    2. Safety note: Ensure you do not drop the weight on the crab as this can cause harm and possibly result in death. 

21. Quickly after dropping the weight, pick up the iPad displaying the polarized image from step 3 and place it horizontally against the external wall of Tera 3 closest to the crab.

    1. Note: The polarized image and weight should scare the crab towards the burrow at the end of the container.

22. Once 2 minutes have passed on the stopwatch, stop the recording.

23. Using your hands, gently encourage the crab back to the starting area of the testing arena.

24. Repeat steps 20-25 two additional times using the same biological replicates each time. 

    1. Note: You will generate three unique data points from each biological replicate. 

25. Gather the spoon and use it to transfer the crab from Tera 3 into a 400 mL beaker. 

26. Return the crab to its respective tank by tilting the 400 mL beaker over the sand at the bottom of the terrarium.

    1. Note: Make sure the angle that you tilt the beaker is not too steep, so that the crabs don’t free fall into the terrarium.

    2. Note: If need be, use the spoon to gently encourage the crab to move out of the 400 mL beaker.

27. Remove the iPhone from the tripod.

28. Enter the Photo app on your device. 

29. Select the three videos taken in steps 17-27.

30. Press the share button in the bottom left-hand corner of the screen.

31. Scroll to find the Google Drive app icon and select.

32. Tap on the name of the first video to rename. Rename the video based on the trial arm, crab number, and trial number, recorded in the data collection table, (e.g. Negative Control, Crab 6, Trial 1).

    1. Note: The numbers within the names of the videos correspond to the order in which they were taken. As such, the first video will be Trial 1, the second Trial 2, and the third Trial 3. 

    2. Note: Choose the destination for the video using the option titled “Folder.”

33. Repeat step 36 for the other two videos renaming them based on their respective trial numbers.

34. Select “MyDrive” and create a folder named “SRD Study” Select “Save Here.”

35. Repeat steps 11-38, 8 additional times using a new biological replicate each time. 

    1. Note: To ensure that you are using a unique biological replicates for each trial, use the painted colors on the crab’s shells and the videos of previous trials recorded in steps 17-27 to identify which crabs have not been tested. 

    2. Note: You will have 9 biological replicates and will create 3 unique data points per replicate.

    3. Note: It is appropriate to take breaks as needed in between conducting each trial.

36. Clean all equipment used in this section by washing the beakers, spoon, and graduated cylinder over the sink with soap and warm water, and return all other materials to their respective storage spaces. 

    1. Note: This could be a good time to stop if there is limited time left in the research period.

37. Safely remove all PPE and dispose of the nitrile gloves in the trash.

38. Advance to step 8 of the Data Processing section of the procedure for further instruction on data processing for this arm.

    1. Note: The decision to not separate the crabs by gender until the data collection phase is a practical approach to streamline the experiment. It reduces unnecessary handling and allows for a more focused analysis later on.

Care/Handling: 

Safety note:  Wear lab attire throughout this section of the experiment: lab coat, safety goggles, nitrile gloves.

Positive Control

Note: The total time for this experiment is 2 hours, but the steps can be broken up to fit the time provided to you. See notes for appropriate breaks. 

1. If the tripod positioning has been adjusted since completing Tank Set Up, repeat step 48 of the Tank Set Up. If not, go straight to step 2.

   1. Gather the iPhone and place it inside the tripod. Secure it tightly by turning the knobs on the side of the tripod clockwise.

2. Place the tripod with the iPhone on across from the meter-stick wall of the container. Your video frame should capture the meter stick and the entire length of the container. 

1. Note: You may have to adjust how close the tripod is to the container in order to capture the entire testing arena.

2. Note: The tripod's height should be adjusted so that the entire testing arena is viewable without obstruction from the container walls while still being able to read the gradations on the meter stick.

3. See reference image below.

2. Gather 3 heat lamps. 

   1. Note: Place 2 of the 3 heat lamps at opposite ends of the container wall with the meter stick, and place the remaining heat lamp on the container wall between the other two.

3. Turn on all three heat lamps. Monitor the thermometer in the testing arena. Once the thermometer reads 31℃ ± 3℃, turn off all of the heat lamps.

4. Gather the 500 g weight, the spool of string, and a pair of scissors.

5. Cut a small piece of string and attach it to the top of the weight.

6. Gather an iPad. Open the polarized bird image shown below using the link here, and set aside the iPad with the image open as it is imperative that you have quick access to it for testing. 

7. Then gather a heat lamp and secure it to a stable object at an empty workstation. For example, you can attach this to the hinge of an overhead cabinet.

   1. Note: If there isn't an available object on which you can attach the lamp, a lamp stand can also be used to secure the heat lamp.

   2. Note: Ensure that the lamp is roughly 0.5 meters above the surface of your workstation as it will be used to warm the crabs prior to trials. If the heat lamp is too close to the fiddler crabs, this can cause their internal body temperatures to become too high which can lead to decreased physiologic response and even death.

8. Obtain a 1 Tbsp measuring spoon, a new 400 mL beaker, Tera 1, and Tera 2. Use the spoon to gently encourage one of the fiddler crabs out of Tera 1 to move into the 400 mL beaker.

9. Carefully transport the 400 mL beaker with the crab to the empty workstation with the heat lamp, and place the beaker under the heat lamp.

10. Turn the heat lamp on and set a 5 minute timer.

    1. Note: Depending on your environment, the environmental temperatures of Tera 1 and Tera 2 may be below fiddler crabs' standard habitat temperature causing a lower body temperature which can lead to decreased physiologic responses. Placing the crab under the heat lamp prior to testing will help  ensure that the crab's internal body temperature is warm enough to allow for normal physiologic responses. 

11. Once the 5 minutes have passed, hit the record button on the iPhone.

    1. Ensure that the testing arena is the proper temperature utilizing the tank VIVOSUN thermometer that can determine sand temperature. Finally, once you are sure of the temperature 

12. Gather the 400 mL beaker and tilt it over the substrate Tera 3 to transfer a crab to the end of the storage container opposite the burrow you created in the experimental set-up. 

    1. Note: Make sure the angle that you tilt the beaker is not too steep, so that the crabs don’t free fall into the terrarium.

    2. Note: If a crab unintentionally falls out, pick up the crab using the method used in step 10 of the initial set up, and place it in the testing arena .

13. Gather the 500g weight with a short string attached.

14. Release the string to drop the weight in the testing arena ensuring it's behind the crab, and start the stopwatch.

    1. Note: The weight should scare the crab and encourage it to run towards the burrow.  

    2. Safety note: Ensure you do not drop the weight on the crab as this can cause harm and possibly result in death. 

    3. image?

15. Quickly after dropping the weight, pick up the iPad displaying the polarized image from step 3 and place it horizontally against the external wall of Tera 3 closest to the crabs.

    1. Note: The polarized image should then scare the crabs to the other end of the lane. 

    2. Note: See reference image below for set up.

16. Once 2 minutes have passed from the time the weight was dropped, stop the recording.

17. Using your hands, gently encourage the crab back to the starting area of the testing arena.

18. Repeat steps 10-22 two additional times using the same biological replicate each time. 

    1. Note: You will generate three unique data points from each biological replicate. 

19. Gather the spoon and use it to transfer the crabs from Tera 3 into a 400 mL beaker. 

20. Return the crab to its respective tank by tilting the 400 mL beaker over the sand at the bottom of this terrarium.

    1. Note: Make sure the angle that you tilt the beaker is not too steep, so that the crabs don’t free fall into the terrarium.

    2. Note: If need be, use the spoon to gently encourage the crab to move out of the 400 mL beaker.

21. Remove the iPhone from the tripod.

22. Enter the Photo app on your device. 

23. Select the three videos taken in steps 5-10.

24. Tap on the name of the first video to rename. Rename the video based on the session number, crab number, and trial number, recorded in the data collection table, (e.g. Positive Control 1, Crab 6, Trial 1).

    1. Note: The numbers within the names of the videos correspond to the order in which they were taken. As such, the first video will be Trial 1, the second Trial 2, and the third Trial 3.Choose the destination for the video using the option titled “Folder.”

25. Press the share button in the bottom left-hand corner of the screen.

26. Scroll to find the Google Drive app icon and select.

27. Tap on the name of the first video to rename. Rename the video based on the session number, crab number, and trial number, recorded in the data collection table, (e.g. Negative Control 1, Crab 6, Trial 1).

    1. Note: The numbers within the names of the videos correspond to the order in which they were taken. As such, the first video will be Trial 1, the second Trial 2, and the third Trial 3.

28. Choose the destination for the video using the option titled “Folder.”

29. Select “MyDrive” and scroll until the Google Drive folder labeled “SRD Study” is found. This was created in step 2 of the Data Collection Setup section. 

30. Scroll to find the folder labeled “Positive Control” within the folder “SRD Study,” that was created in step 3 of the Data Collection setup section. Select “Save Here.”

31. Repeat steps 32–35 two additional times to ensure that each of the three trials are properly saved.

32. Repeat steps 2-28, 8 additional times using a new biological replicates each time. 

    1. Note: To ensure that you are using unique biological replicates for each trial, use the painted colors on the crab’s shells and the videos of previous trials recorded in steps 5-24 to identify which crabs have not been tested. 

    2. Note: You will have 9 biological replicates and will create 3 unique data points per replicate.

    3. Note: It is appropriate to take breaks as needed in between conducting each trial.

33. Clean all equipment used in this section by washing the beakers, spoon, and graduated cylinder over the sink with soap and warm water, and return all other materials to their respective storage spaces. 

    1. Note: This could be a good time to stop if there is limited time left in the research period.

34. Safely remove all PPE and dispose of the nitrile gloves in the trash.

35. Advance to step 8 of the data collection section for further instruction on data processing for this arm.

    1. Note: The decision to not separate the crabs by gender until the data collection phase is a practical approach to streamline the experiment. It reduces unnecessary handling and allows for a more focused analysis later on.

36. Repeat this procedure again from the start this time using the temperature range of 27-29℃

Data Processing

Procedure:

Note: The total time for this experiment is approximately 14 hours, due to the volume of trial videos needed to be watched. However, this section can be broken up to fit the time provided to you.

1. Gather the computer being used for data processing.

2. Open Google Drive and navigate to the folder titled “SRD Study” which you created in step 36 of the Negative Control procedure. 

3. Open the Google Sheet titled “Copy of Google sheet for crab data" Navigate to the sheet labeled "Negative Control."

4. Gather an iPhone, a pen, paper, and a stopwatch.

5. Navigate to the Google Drive app on your iPhone. Open the folder titled “SRD Study.” 

6. Open the video titled "Negative Control, Crab 1, Trial 1” Watch the video and determine fiddler crab displacement from the start position 5 seconds after the weight has been dropped. Use the Google Sheet to record this information under "Displacement for 5 second reaction time cm"

   1. Note: The 5 seconds for measuring displacement should begin after the weight is dropped. 

7. Next go to the "Burrowing Y,N" column. 

   1. Note: A crab positioned below the sand indicates burrow retreat. Burrow retreat is defined as the amount of time the fiddler crab spends under the sand in their previously established burrow. If burrowing occurs, put a "Y" indicating the crab burrowed. In the column ,  If burrowing did not occur, put an "N" indicating the crab did not burrow. 

8. Then record "displacement after 2 minutes cm." Watch the video and determine fiddler crab displacement from the start position 2 minutes after the weight has been dropped. Use the Google Sheet to record this information under "Displacement for 2 minute reaction time cm"

   1. Note: The 2 minutes for measuring displacement should begin after the weight is dropped. 

9. Record the temperature in degrees℃

10. Record whether the crab is male or female.

    1. Note: A crab with a large claw is male while a crab with a small claw is female. 

11. Once this is complete repeat steps 6-10 for all test subjects and trials. This should be 27 videos in total – three videos for each of the 9 biological replicates..

    1. Note: Breaks are appropriate as needed both during and in between reviews of each unique video. 

12. Navigate to the Google Drive app on your iPhone. Open the folder titled “SRD Study." Then, open the sheet “Positive Control.” 

13. Repeat steps 6-12 using the 27 videos collected in the positive control section. These will be labeled "Positive Control, Crab #, Trial #." 

14. Convert the answers recorded in inches to cm. Then change the titles from inches to cm. 

15. Repeat steps 6-11 using the 27 videos collected in the experimental arm section. These will be labeled "Experimental Arm, Crab #, Trial #."