Materials

Protocol

Preparing arenas and agar casts

1. Obtain 20 B. glabrata snails (medium to large size) and 25 mL of artificial pond water in a 250 mL beaker. Place this beaker in an incubator set to 28C. Incubate for 3 hours. Then, pipette the pond water out of the beaker - this will be the “snail conditioned water” (SCW) used in the assays.

2. Measure out two samples of 0.025g. agarose powder each using a precision scale and place them in 15 mL conical tubes. Label one “0.5% Agar - SCW” and the other “0.5% Agar - APW”.
3. Add 5 mL of artificial pond water to the APW tube and microwave for ~8 seconds. Mix will be inverting, and use a p1000 to pipette it into the “Control” agar molds. Repeat this process for the SCW tube, but use 5 mL of SCW (made in step 1), and use the “Snail” agar mold instead of the control mold.
4. Set both molds in a container with a wet paper towel. Place this container in the refrigerator for 12-15 minutes. The agar will solidify during this time, and the humid chamber will ensure it does not dry out.

5. Obtain arenas for use in the experiment and flush them with 1,000 µL artificial pond water.
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Pipette 1000µL artificial pond water into the arena. Tilt the arena around so the water passes through all of it, especially the corners (air bubbles tend to occur there if this is not done properly). Remove as much of the water as possible with the pipette (through the entry port or the cue cut-outs).
6. Wipe down the outside of each arena with 70% ethanol on a Kimwipe to remove as much dust as possible.

7. Fill the arena with artificial pond water, inserting it through the entry port of the arena.
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TIPS: - Pipette slowly, be patient. - Ensure there is not air in the tip of the pipette when inserting water into the arena - You may have to try a couple times - that’s normal. Remove the water and start over. - Fill until the water level is flat across the cue cutouts - not convex or concave! (This will lead to bubbling over when the agar cubes are inserted.)
8. Once the arenas are filled and the agar cubes are solidified, obtain a p10 pipette tip and a metal spatula. Use the p10 pipette tip to trace the outside of the agar cube in the mold (unsticking it from the edges - making it easier to remove). Line up the spatula (it’s rectangular side) with the edge of the agar cube you want to remove and use the p10 pipette tip to maneuver it out of the mold and onto the edge of the spatula.

9. Gently use the p10 pipette tip to push the agar cube off the spatula and into the cue cutout in the arena.
10. Once both agar cubes are placed in the arena, allow the arena to sit for 1 hour before beginning the assays. This creates a cue gradient within the arena.

Miracidia preparation

1. Harvest miracidia from infected mouse livers (Harvesting Protocol); through step 15, but do not extract miracidia from flask.
2. Record the time that the schistosome eggs are in the flask (once it is filled with APW and there is a light source added). Wait 1 hour from this time before extracting.
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This is considered the first “hatch” time point (”0 hours post hatch”). The first time miracidia hatch from their eggs. For these assays, miracidia will be used “1 hour post hatch”.
3. After 1 hr., extract 8 mL of miracidia with a sterile transfer pipette from the top of the flask and transfer them to a 15 mL conical tube.
4. Centrifuge this tube for 1 minute at 4°C at 500 rcf. Then, remove excess APW (as much as possible) and resuspend the miracidia to 1 mL with fresh APW. Transfer this to a clean 1.5 mL tube.
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On the centrifuge we have, this reads as 0.5 rcf (x1000).

5. Pipette 10 µL aliquots of the miracidia sample and 10 µL aliquots of 1:5 diluted Lugol’s iodine in order to perform counts using the Zeiss Stemi Microscope. Count how many miracidia there are in each aliquot (5+ replicates).
6. Calculate the amount of miracidia in the 1.5 mL tube based on the counts and perform necessary dilutions to have 1 miracidia/µL concentration.

7. This sample will be used in 20 µL aliquots for each assay that is performed.

Performing behavioral assays

1. Obtain titered miracidia sample (1 miracidia/µL) and the prepared arenas with the cues of interest (SCW and APW). Ensure InVision is set up and ready to record (protocol).
2. Remove the stage from inside of the InVision imaging system. Wipe it down with 70% ethanol, removing as much dust as possible.
3. Place the arena in the stage of InVision. Make sure the SCW agar cube is on the right side so that camera 24568744 is always recording the cue of interest and camera 24568709 is recording the control.
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If this orientation gets switched up by accident, be sure to make a note of it! There is no other way to know which side is SCW and which is APW.

4. Pipette 20 µL of the miracidia sample into the entry port of the arena(s) at a 90° angle.
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It’s important that the miracidia are not pipetted toward (or away) from the cue. Pipetting slowly and at a 90° angle will ensure the miracidia are not given a “head start” toward one cue or another.
5. After the miracidia are in the arena, efficiently but carefully place the stage back into the InVision imaging system, close the door, and press the blue “Record” button on the computer screen. Do your best to not disturb the miracidia or gradient.
6. Place the agar cubes into the next arena (next replicate) after this, ensuring it sits for ~1 hour before recording.
7. Allow InVision to record for 1 hour, then repeat the process for each replicate arena.