Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

1. Breeding Facility

1. Obtain adult specimens of Lymnaea stagnalis (L.) from a laboratory culture like the one at VU University.
2. Maintain the low-copper water at 20 °C in both the breeding facility (molluscarium) and experimental tanks, which have continuous water exchange.
3. Set the light/dark cycle at 12 hr:12 hr.
4. For breeding, feed the adult snails pesticide-free lettuce ad libitum, feed the young juveniles with TetraPhyll (fish food) flakes. Maintain snails in separate tanks according to their age, starting from egg masses which are laid within a 24 hr time frame.
5. Once snails are removed from the breeding tanks they are not returned, to avoid effects of having been kept under slightly different conditions as well as to prevent pseudoreplication.

2. Dissection

1. Isolate the snails for 1 week, and feed them ad libitum, to ensure that they have full sperm and seminal fluid stores.
2. Prepare a stereo microscope, a dissection plate with pins, small surgical scissors, coarse and fine forceps, a 10 ml syringe filled with a 50 mM MgCl₂ solution, injection needles (0.3 mm x 13 mm) and some paper towels.
3. Euthanize a snail by injecting MgCl₂ solution (generally 3 ml or more for adult snails with a shell length of 31.6 ± 2.1 mm and wet weight of 2.89 ± 0.55 g). Penetrate the foot with the injection needle at a 45° angle and inject by gently applying pressure continuously until the snail relaxes and remains extended (within seconds). Remove the injection needle and check whether the snail is euthanized (for example, by checking whether the tentacle withdrawal reflex is absent).
4. Carefully remove the shell following its curvature by using coarse forceps. Halfway, gently detach the columellar muscle from the shell by scraping with the forceps. Gently twist the snail out of its shell, following the winding direction of the shell.
5. Place the snail on the dissection plate and pin down the back end of the foot. Then, place a pin through the head and put some tension on the snail to facilitate dissection.
6. Localize the female gonopore, which will later on be a reference point for dissection.
7. Using the surgical scissors and fine forceps, make the first incision, just under the edge of the mantle and cut towards the female gonopore. When one of the scissors’ blades is pushed under the body wall, lift it up slightly to ensure that only the body wall, and none of the internal organs, is cut. Continue cutting above the gonopore towards the head and cut in a straight line.
8. Localize the prostate gland (posterior) and seminal vesicles (anterior). Dissect these out carefully and keep them on ice. Use these to make the test solutions containing seminal fluid and/or sperm. Use physiological Lymnaea saline (5.83 mmol/L CaCl₂·2H₂O, 3.76 mmol/L MgCl₂·6H₂O, 42.69 mmol/L NaCl, 37.53 mmol/L KCl) as a carrier medium.
9. Collect the seminal fluid that is present in the lumen of the prostate gland as well as the sperm from the seminal vesicles. Subsequently, suspend both in saline in separate vials by gently expressing their contents and removing the organ tissue with a forceps. Keep on ice and use the same day. Later, mix these suspensions to create the different treatment solutions.

3. Intravaginal Injection

1. Before starting the intravaginal injection procedure, prepare a silicon shell-mold (made to fit the tip of the shell), 1 mm syringes, 10-12 cm silicon tubing with a diameter of 1 mm, blunted injection needles (0.3 mm x 13 mm), coarse forceps and the same 10 ml syringe filled with a 50 mM MgCl₂ solution fitted with a sharp injection needle.
2. Collect the sperm, needed to add to the seminal fluid (or individual seminal fluid proteins) in some treatments; a biologically relevant dose equals 1/3 of a prostate gland and seminal vesicle per injection.
   NOTE: In the representative results section (Figure 1) the results of an experiment with three different treatments are shown: Control (carrier medium, i.e., saline), Ovipostatin (one of the identified seminal fluid proteins) alone and Ovipostatin accompanied with sperm. In addition, to estimate the biologically relevant dose in other species, determine the difference in weight of the prostate gland of individuals that have recently mated and individuals that remained sexually-isolated¹⁶.
3. Prepare 1 ml syringes for each of the prepared test solutions. Fill each syringe with one solution and ensure that there are no air bubbles inside.
4. Fit a blunted injection needle on each syringe.
5. For injection use a 1 mm diameter silicon tube with a minimum length of 10 cm.
6. Carefully slide the silicon tube over the injection needle, making sure not to pierce the tube, and remove the air from the tube by gently applying the pressure on the syringe.
7. Anaesthetize a snail by following the same protocol as in point 2.3. This time make sure not to inject more than 2-3 ml of 50 mM MgCl₂.
8. Lay the snail in the shell-mold to keep it in a stable position.
9. Locate the female gonopore, which is clearly visible as a white spot on the right side of the animal, anterior to the right tentacle and male gonopore (this location also applies to other species of freshwater snails). Make sure to have unobstructed access.
10. Use a forceps to hold the end of the silicon tube and gently insert approximately 2-4 mm of the silicon tube into the female gonopore.
11. Carefully apply pressure to the syringe and inject 0.03 ml of the test solution. Wait half a minute to allow the pressure in the tube to spread into the female tract before removing the tube.
12. Return the treated snail to its isolation container, where it can be monitored, and allow it to recover from sedation in the experimental tank.

4. Bioassay

1. Label each snail for identification and measure its shell length. The latter is a good indicator of the size, which is relevant when quantifying reproductive output.
2. Keep each snail isolated in a 460 ml container that is perforated to allow for water exchange within the experimental tank. It is important to place all containers in the right orientation, with the perforations in the direction of the water flow, to allow for efficient water exchange.
3. During the bioassay, feed snails regularly with a standardized amount of lettuce. Use a round, metal puncher to cut lettuce. In this protocol lettuce discs of 19.6 cm² are used, which approximates the amount they maximally can eat in one day.
4. Collect an egg mass using the flat side of a spatula and scrape the egg mass from the container wall. Use the spoon side to collect the egg mass.
   NOTE: Opaque egg masses that have just been laid cannot be used for scanning (they become transparent within a few hours after laying). For the model species in this protocol, Lymnaea stagnalis, check for egg masses every day, since they produce 2-3 egg masses per week. This can differ in other species.
5. Scan the collected egg masses digitally, using a (laptop) computer containing the scanner driver, a flat bed scanner, a standard millimeter scale, a spatula, transferring plates and glass tiles.
6. Place each egg mass on the transferring plates and repeat until the plates are full. Subsequently, dab the egg masses dry on paper towel and transfer them onto the glass tiles in the same order as on the transferring plates.
7. Turn each tile upside down (the egg masses will stick to the glass tile) and carefully place it on the flatbed scanner, just under the millimeter scale. Apply a bit of pressure to flatten the egg masses, which will distribute the eggs evenly so they are all visible within the same focus plane. Fit the tiles with a standardized amount of tape around two edges to assure that all eggs are flattened equally.
8. Adjust the scanner settings. It is important to adjust the resolution to its maximum, make a preview scan, crop the working area, and adjust the brightness and contrast. Scan the egg masses.
9. Place each egg mass in its own labelled 20 ml glass vial for further development and hatching.
10. Refresh the water in hatching vials every other day to keep it properly oxygenated. Do this according to hatchling numbers and size in the following order: decanting/pouring off (no hatchlings), overflowing (start of hatching), and extracting out the water with a pipette (many hatchlings).

5. Measuring and Counting Eggs

1. Install the freely-available image analysis software ImageJ (NIH, USA) and the cell counter plugin.
2. Open ImageJ as well as a spreadsheet program to transfer the data to. Then open a scanned picture of egg masses in ImageJ.
3. Set the scale by using the zoom tool to navigate to the millimeter scale. Zoom in and adjust the window for 1 mm to fill up the screen. Draw a line using the line tool between the two lines that indicate 1 mm. Navigate to Analyze, and Set scale. In the pop-up menu the measured length option becomes visible, adjust Known distance to 1 and unit of length to mm. Check the box Global, and close the window.
4. Set the preferred measurements (length, width, circumference) by navigating to Analyze, and Set Measurements by selecting Area and Feret’s Diameter (length and width). Click OK to confirm and continue with the image analyses.
5. To measure the eggs, zoom in on an egg of the first egg mass that needs to be measured. A zoom of 800% is recommended. Then, using the elliptical tool draw an oval exactly around the egg, press ctrl+D to draw the circumference on the image (to prevent measuring the same egg twice). Then press ctrl+M to record the measure of the properties of the oval (length, width, circumference); a pop-up screen opens displaying the measurements.
   NOTE: The use of the elliptical tool to measure eggs does not apply to all species. For species with non-uniformly shaped eggs, use the freehand selections instead.
6. Measure as many eggs as necessary and then save the measures as .xls worksheet. Optionally, copy the data directly into a worksheet.
7. Subsequently count the eggs within an egg mass, by using the counter window and put the counts manually into the worksheet.
8. Alternatively, count the individual eggs per egg mass with the help of the cell counter plug-in. For this, navigate to Plug-ins, then, Analyze and select Cell counter. The cell counter window will pop up, check the 'keep original' box and press 'initialize'. A new counter window appears, select the crosshair tool from the toolbar and select a counter type in the cell counter menu. Count the individual eggs by clicking on the individual eggs in the cell counter window. Observe the total count next to the counter type in the cell counter menu. Copy this number manually to a spreadsheet or obtain the results by pressing Results.
9. Count multiple egg masses within one counting event by adding counter types, if necessary.