We thank M.R. Helmus for the voice-over, C.M. Popelier, S. Ypenburg and C. Levesque for technical assistance. This research is financially supported by the Netherlands Organization for Scientific Research (NWO), the Royal Netherlands Academy of Arts and Sciences (KNAW) and the Japan Student Services Organization (JASSO). This publication was supported by an Open Access grant of NWO to JMK.

1. Breeding Facility
<ol>
	<li>Obtain adult specimens of Lymnaea stagnalis (L.) from a laboratory culture like the one at VU University.</li>
	<li>Maintain the low-copper water at 20 &#176;C in both the breeding facility (molluscarium) and experimental tanks, which have continuous water exchange.</li>
	<li>Set the light/dark cycle at 12 hr:12 hr.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Dissection
<ol>
	<li>Isolate the snails for 1&#160;week, and feed them ad libitum, to ensure that they have full sperm and seminal fluid stores.</li>
	<li>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.</li>
	<li>Euthanize a snail by injecting MgCl2 solution (generally 3 ml or more for adult snails with a shell length of 31.6 &#177; 2.1 mm and wet weight of 2.89 &#177; 0.55 g). Penetrate the foot with the injection needle at a 45&#176;&#160;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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Localize the female gonopore, which will later on be a reference point for dissection.</li>
	<li>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&#8217; 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.</li>
	<li>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&#160;CaCl2&#183;2H2O, 3.76 mmol/L&#160;MgCl2&#183;6H2O, 42.69 mmol/L&#160;NaCl, 37.53 mmol/L&#160;KCl) as a carrier medium.</li>
	<li>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.</li>
</ol>
3. Intravaginal Injection
<ol>
	<li>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&#160;MgCl2 solution fitted with a sharp injection needle.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Fit a blunted injection needle on each syringe.</li>
	<li>For injection use a 1 mm diameter silicon tube with a minimum length of 10 cm.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Lay the snail in the shell-mold to keep it in a stable position.</li>
	<li>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.</li>
	<li>Use a forceps to hold the end of the silicon tube and gently insert approximately 2-4 mm&#160;of the silicon tube into the female gonopore.</li>
	<li>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.</li>
	<li>Return the treated snail to its isolation container, where it can be monitored, and allow it to recover from sedation in the experimental tank.</li>
</ol>
4. Bioassay
<ol>
	<li>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.</li>
	<li>Keep each snail isolated in a 460&#160;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#160;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.</li>
	<li>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.</li>
	<li>Place each egg mass in its own labelled 20 ml glass vial for further development and hatching.</li>
	<li>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).</li>
</ol>
5. Measuring and Counting Eggs
<ol>
	<li>Install the freely-available image analysis software ImageJ (NIH, USA) and the cell counter plugin.</li>
	<li>Open ImageJ as well as a spreadsheet program&#160;to transfer the data to. Then open a scanned picture of egg masses in ImageJ.</li>
	<li>Set the scale by using the zoom tool to navigate to the millimeter&#160;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.</li>
	<li>Set the preferred measurements (length, width, circumference) by navigating to Analyze, and Set Measurements by selecting Area and Feret&#8217;s Diameter (length and width). Click OK to confirm and continue with the image analyses.</li>
	<li>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.</li>
	<li>Measure as many eggs as necessary and then save the measures as .xls worksheet. Optionally, copy the data directly into a worksheet.</li>
	<li>Subsequently count the eggs within an egg mass, by using the counter window and put the counts manually into the worksheet.</li>
	<li>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 &#39;keep original&#39; box and press &#39;initialize&#39;. 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.</li>
	<li>Count multiple egg masses within one counting event by adding counter types, if necessary.</li>
</ol>

<ol>
	<li>Mann, T., Lutwak-Mann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Male reproductive function and semen. , (1981).</li><li>Arnqvist, G., Rowe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sexual Conflict. , (2005).</li><li>Birkhead, T. R., Hosken, D. J., Pitnick, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Sperm Biology, an Evolutionary Approach. , (2008).</li><li>Koene, J. M., Ter Maat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allohormones":+A+class+of+bioactive+substances+favoured+by+sexual+selection.">Allohormones": A class of bioactive substances favoured by sexual selection.</a> Journal of Comparative Physiology A. 187 (5), 323-326 (2001).</li><li>Parker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+competition+and+its+evolutionary+consequences+in+the+insects.">Sperm competition and its evolutionary consequences in the insects.</a> Biological Reviews. 45 (4), 525-567 (1970).</li><li>Perry, J. C., Sirot, L., Wigby, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+seminal+symphony:+how+to+compose+an+ejaculate.">The seminal symphony: how to compose an ejaculate.</a> Trends in Ecology &amp; Evolution. 28 (7), 414-422 (2013).</li><li>Baldini, F., Gabrieli, P., Rogers, D. W., Catteruccia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+and+composition+of+male+accessory+gland+secretions+in+Anopheles+gambiae:+a+comparison+with+other+insect+vectors+of+infectious+diseases.">Function and composition of male accessory gland secretions in Anopheles gambiae: a comparison with other insect vectors of infectious diseases.</a> Pathogens and Global Health. 106 (2), 82-93 (2012).</li><li>Xu, J., Baulding, J., Palli, S. 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M., Chase, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+reproductive+system+of+the+snail+Helix+aspersa+caused+by+mucus+from+the+love+dart.">Changes in the reproductive system of the snail Helix aspersa caused by mucus from the love dart.</a> Journal of Experimental Biology. 201 (15), 2313-2319 (1998).</li><li>Van Duivenboden, Y. A., Pieneman, A. W., Ter Maat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+mating+suppresses+fecundity+in+the+hermaphrodite+freshwater+snail+Lymnaea+stagnalis:+a+laboratory+study.">Multiple mating suppresses fecundity in the hermaphrodite freshwater snail Lymnaea stagnalis: a laboratory study.</a> Animal Behaviour. 33, 1184-1191 (1985).</li><li>Koene, J. M., Brouwer, A., Hoffer, J. N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+egg+laying+caused+by+a+male+accessory+gland+product+opens+the+possibility+for+sexual+conflict+in+a+simultaneous+hermaphrodite.">Reduced egg laying caused by a male accessory gland product opens the possibility for sexual conflict in a simultaneous hermaphrodite.</a> Animal Biology. 59 (1), 435-448 (2009).</li><li>Hoffer, J. N. A., Schwegler, D., Ellers, J., Koene, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+rate+influences+female+reproductive+investment+in+a+simultaneous+hermaphrodite,+Lymnaea+stagnalis.">Mating rate influences female reproductive investment in a simultaneous hermaphrodite, Lymnaea stagnalis.</a> Animal Behaviour. 84 (3), 523-529 (2012).</li><li>Koene, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+accessory+gland+protein+reduces+egg+laying+in+a+simultaneous+hermaphrodite.">Male accessory gland protein reduces egg laying in a simultaneous hermaphrodite.</a> PLoS ONE. (5), (2010).</li><li>Nakadera, Y., Swart, E. M., Hoffer, J. N. A., Den Boon, O., Ellers, J., Koene, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receipt+of+seminal+fluid+proteins+causes+reduction+of+male+investment+in+a+simultaneous+hermaphrodite.">Receipt of seminal fluid proteins causes reduction of male investment in a simultaneous hermaphrodite.</a> Current Biology. 24, 1-4 (2014).</li></ol>

The authors have nothing to disclose.

The presented protocol shows how to collect seminal fluid and how to test this in a biologically meaningful way. Although this procedure here is shown using the prostate gland extract, the effect of a single purified seminal fluid protein can also be tested following the same procedure. Obviously, in such tests it is always crucial to include proper controls (sham treatments) to ensure that the procedure and/or the carrier medium do not affect the parameters in questions14,16. Using this method, it has already been shown that there is a single seminal fluid protein, namely Ovipostatin, that mediates a reduction in egg laying14,16. This protein, produced in the prostate gland and transferred during mating along with the spermatozoa, represents the first fully characterized seminal fluid component in a simultaneous hermaphrodite and does, so far, not show any resemblance to known proteins.
The finding that Ovipostatin reduces egg mass production in the recipient corroborates earlier work indicating that substances in the semen may influence egg laying13-15. By transferring biologically relevant amounts (see 3.216), one can convincingly demonstrate that it is a seminal fluid protein, and not the presence of spermatozoa themselves, that mediates the observed reduction in egg laying (Figure 1). Given that repeated receipt of seminal fluid via natural matings increases the investment per egg15, it now becomes highly relevant to quantify other parameters of egg laying. The presented protocol now provides the necessary methods to do so. Although no effect was found of Ovipostatin on hatching success of the eggs laid by the recipient16, there may be other seminal fluid proteins that act in concert with this protein. Hence, parameters that need to be quantified in this context - next to laying frequency and egg numbers - include egg size, hatching time, hatchling development and hatchling size. In addition, it is possible to look at effects on other female processes than egg laying, such as sperm storage and usage, as well as the allocation of reproductive resources towards the female and male function14,17. Clearly, the presented method is instrumental in addressing these followup issues, both in Lymnaea stagnalis and in other fresh water snails.
In sum, experiments using this protocol show that transferred seminal fluid proteins affect the female reproductive performance of the recipient in these simultaneous hermaphrodites. Given that few studies have addressed this, it is likely to be much more common in internally fertilizing hermaphrodites than is currently reflected in the scientific literature. Especially when one realizes that pre-copulatory mate choice and sperm competition have been shown to play an important role in these hermaphrodites14, which highlights the potential of such male accessory gland proteins to be sexually selected in this system. Finally, this research helps to support the developing notion that seminal fluid proteins are of equal importance for simultaneous hermaphrodites as they are for separate-sexed species.

Upon transfer, spermatozoa are usually accompanied by seminal fluid produced by male accessory glands. While some ejaculate components play roles in nourishing and activating sperm1, others influence female physiology in order to increase fertilization success of the sperm donor2-4. Such effects are especially selected for, via sexual selection, when a species is highly promiscuous and has efficient sperm storage and sperm digestion. Under such conditions, sperm donors compete heavily for the fertilization of eggs5. While there is abundant correlational evidence for the importance of seminal fluid substances for male fertilization success, direct evidence is more rare and few studies have delved into the details of the physiological change and/or substances involved6.
In species with separate sexes (i.e., males and females; gonochorists) there are a few well-investigated model species when it comes to seminal fluid composition and their effects. Examples include the fruit fly,&#160;Drosophila melanogaster6, the malaria mosquito,&#160;Anopheles gambiae7, and the red flour beetle,&#160;Tribolium castaneum8. Such seminal fluid studies are much rarer when it comes to species that are simultaneously hermaphroditic (i.e., individuals are functionally male and female at the same time), even though this mode of reproduction is common throughout the animal kingdom9. The most notable hermaphroditic example of the transfer of an accessory gland substance is probably the shooting of so-called love-darts in land snails2,10, where the substance is transferred by the sperm donor via hypodermic injection11,12, i.e., not along with the spermatozoa. But there are many simultaneous hermaphrodites that transfer male accessory gland products along with their sperm, such as the model species used here, the great pond snail Lymnaea stagnalis. Previous research has already demonstrated that the receipt of seminal fluid influences the female reproductive output of this species13-15, and several of the involved seminal fluid proteins have been identified16,17.
The overall goal of this protocol, which is an extension of the methods reported in Koene et al16, is to test effects of seminal fluid proteins on various parameters of egg laying in gastropod molluscs. This is accomplished by dissection of the prostate gland (i.e., the male accessory gland producing the seminal fluid) of sexually isolated donor snails from a standardized laboratory culture. Second, the donor&#8217;s sperm is collected from the seminal vesicles and different treatment solutions are made. Subsequently, test snails are anaesthetized and intravaginally injected with a test solution (for example, prostate gland extract alone, prostate gland extract with sperm, sperm alone, carrier medium alone). These test snails are then monitored for the following days to weeks, under standard conditions, to record egg laying. Egg masses are collected, digitally scanned for counting and measuring various parameters, and then put in individual vials for hatching. After about two weeks, the hatchlings are counted and digitally scanned. All the scans, of both egg masses and hatchlings, are then analyzed using a freely-available image analysis software package.
For this protocol the laboratory strain of the great pond snail, Lymnaea stagnalis, is used, which is bred at VU University in Amsterdam. This species serves as a model system for investigating questions about sexual selection and reproduction in hermaphrodites. The animal&#8217;s relatively large size makes it experimentally very accessible. In addition, because it is used as a model species in many biological disciplines we already know a lot about the basic biology of these animals. This method will help to study questions about processes of sexual selection that are mediated via seminal fluid proteins in general. In addition, because these are simultaneous hermaphrodites, it is possible to address whether such proteins act in similar and/or different ways as they do in species where the sexes are separated16,17.

<table><tbody><tr><td>Fish food flakes</td><td>TetraPhyll</td><td></td><td></td></tr><tr><td>Stereo microscope</td><td></td><td></td><td></td></tr><tr><td>Small surgical scissors</td><td>WPI</td><td></td><td></td></tr><tr><td>Coarse and fine forceps</td><td>WPI</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td></td><td></td><td></td></tr><tr><td>Injection needles (0.3 mm x 13 mm)</td><td></td><td></td><td></td></tr><tr><td>50 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>5.83 mmol/L CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>3.76 mmol/L MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>42.69 mmol/L NaCl</td><td></td><td></td><td></td></tr><tr><td>37.53 mmol/L KCl</td><td></td><td></td><td></td></tr><tr><td>Two component Silicon</td><td>Sylgard</td><td></td><td></td></tr><tr><td>1 mm syringes</td><td></td><td></td><td></td></tr><tr><td>Silicon tubing (diameter 1 mm)</td><td></td><td></td><td></td></tr><tr><td>Blunted injection needles (0.3 mm x 13 mm)</td><td></td><td></td><td></td></tr><tr><td>Tubes</td><td>Eppendorff</td><td></td><td></td></tr><tr><td>460 ml perforated containers</td><td></td><td></td><td></td></tr><tr><td>Metal leaf puncher (19.6 cm&lt;sup&gt;2&lt;/sup&gt;)</td><td></td><td></td><td></td></tr><tr><td>Spatula/spoon</td><td></td><td></td><td></td></tr><tr><td>(Laptop) computer</td><td></td><td></td><td></td></tr><tr><td>Flatbed scanner</td><td></td><td></td><td></td></tr><tr><td>Millimeter scale</td><td></td><td></td><td></td></tr><tr><td>Transferring plates</td><td></td><td></td><td></td></tr><tr><td>Glass tiles</td><td></td><td></td><td></td></tr><tr><td>20 ml glass vials</td><td></td><td></td><td></td></tr></tbody></table>

effect of male accessory gland products on egg laying in gastropod molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

Figure 1 shows the percentage of animals laying eggs after intravaginal injection of either the control substance (saline), Ovipostatin, or Ovipostatin accompanied with sperm. N = 15 for each treatment; see Koene et al.16 for details and statistics (Source: Figure 3 from Koene et al. 2010, PLoS ONE).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51698/51698fig1highres.jpg" width="500" /> 
Figure 1. Effect of Ovipostatin on egg mass production of Lymnaea stagnalis. The graph shows the percentage of animals laying eggs after intravaginal injection of either the control substance (saline), Ovipostatin, or Ovipostatin accompanied with sperm. N=15 for each treatment; see Koene et al. 2010 details and statistics. Reprint with permission from Koene et al. 2010 PLoS ONE 5: e10117.

Watch this Scientific Journal Video about Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs at JoVE.com

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

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

effect-male-accessory-gland-products-on-egg-laying-gastropod

Research

JoVE Journal

Biology

10.6K Views.  VU University. This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

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

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Environment

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of individual cell populations often makes it difficult to obtain enough material for molecular studies. For example, the accessory gland of the Drosophila male reproductive system contains two distinct secretory cell types. The main cells make up 96% of the secretory cells of the gland, while the secondary cells (SC) make up the remaining 4% of cells (about 80 cells per male). Although both cell types produce important components of the seminal fluid, only a few genes are known to be specific to the SCs. The rarity of SCs has, thus far, hindered transcriptomic analysis study of this important cell type. Here, a method is presented that allows for the purification of SCs for RNA extraction and sequencing. The protocol consists in first dissecting glands from flies expressing a SC-specific GFP reporter and then subjecting these glands to protease digestion and mechanical dissociation to obtain individual cells. Following these steps, individual, living, GFP-marked cells are sorted using a fluorescent activated cell sorter (FACS) for RNA purification. This procedure yields SC-specific RNAs from ~40 males per condition for downstream RT-qPCR and/or RNA sequencing in the course of one day. The rapidity and simplicity of the procedure allows for the transcriptomes of many different flies, from different genotypes or environmental conditions, to be determined in a short period of time.

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Here, we present a protocol to dissociate and sort a specific cell population from the Drosophila male accessory glands (secondary cells) for RNA sequencing and RT-qPCR. Cell isolation is accomplished through FACS purification of GFP-expressing secondary cells after a multistep-dissociation process requiring dissection, proteases digestion and mechanical dispersion.

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Genetics

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

Biochemistry

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Developmental Biology

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the induction of ovarian maturation is important to meet the consumer demand for mature mud crabs and hasten seed production. Eyestalk ablation is an effective tool to enhance ovarian maturation in mud crabs. However, there is no standard protocol for the eyestalk ablation of mud crabs. In this study, two eyestalk ablation techniques are described: cauterization (the use of hot metal to ablate the eyestalk of an anesthetized crab) and surgery (the removal of the eyestalk using surgical scissors). Before eyestalk ablation, sexually mature females (CW &#62; 86 mm) were anesthetized using an ice bag (&#8722;20 &#176;C) with seawater. When the water temperature reached 4 &#176;C, the ice bag was removed from the water. Flowing seawater (ambient temperature: 28 &#176;C) was used for recovery from the anesthesia immediately after eyestalk ablation. Mortality did not occur during or after the process of eyestalk ablation. The eyestalk ablation protocol presented here accelerated the ovarian maturation of the mud crabs.

Two eyestalk ablation protocols (i.e., cauterization and surgery&#160;approaches) were performed on anesthetized female crabs. The eyestalk ablation of mud crabs hastened the ripening of ovaries without decreasing the survival rate.

Mud crabs (Scylla spp.) are commercially important crustacean species that can be found throughout the Indo-West Pacific region. During culture, the ...

Culturing Larvae of Crepidula fornicata in Laboratory and Field Conditions

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded veliger larvae of this species were collected by siphoning onto a sieve after natural release by adults, distributed into the culture at a density of 200/L, and fed with Isochrysis galbana (strain T-ISO) at 1 x 105 cells/mL. Shell growth and acquisition of competence for metamorphosis were documented for sibling larvae reared in ventilated 800 mL cultures designed for equilibration to ambient air or to defined atmospheric gas mixtures. Contrasting with these laboratory culture conditions; growth and competence data were also collected for larvae reared in a 15 L flow-through ambient seawater mesocosm located in a field population of reproductive adults. Growth rates and timing of metamorphic competence in the laboratory cultures were similar to those reported in previously published studies. Larvae reared in the field mesocosm grew much faster and metamorphosed sooner than reported for any laboratory studies. Together, these methods are suited for exploring larval development under predetermined controlled conditions in the laboratory as well as under naturally occurring conditions in the field.

Author Spotlight: Bridging the Gap Between Field Observations and Lab Manipulations in Larval Ecology Research

The paper presents methods for larval culture of the gastropod Crepidula fornicata in a small-volume laboratory-scale system and in an ambient-seawater mesocosm system that can be deployed in the field.

The calyptraeid gastropod mollusk, Crepidula fornicata, has been widely used for studies of larval developmental biology, physiology, and ecology. Brooded ...

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans

Source: Lee, S. Y. and Kang, K. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2017).
C. elegans egg-laying behavior can provide important information in developmental, reproductive, and anatomical studies.&#160; This video introduces the important steps of an egg laying assay and shows a sample experiment in which a drug is tested for reproductive toxicity.

Source: Lee, S. Y. and Kang, K. Measuring the Effect of Chemicals on the Growth and Reproduction of&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. 
...

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

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Chapters in this video

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Two Types of Assays for Detecting Frog Sperm Chemoattraction

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs

Laboratory and Field Culture of Larvae of The Slipper Limpet, Crepidula fornicata

Egg Laying Assay: A Method to Quantify the Egg-Laying Behavior of C. elegans