Sperm structure and locations in the hermaphroditic freshwater snail Planorbella trivolvis

ABSTRACT

The freshwater snail Planorbella trivolvis provides a model system for investigating hermaphrodite reproduction. Self-fertilization is rare, and individuals may mate as male, female, or reciprocally; after mating, sperm is stored with recipients laying eggs for 16–18 weeks after a single mating. Some key unanswered questions in this and other related species involve sperm. Very little is known about sperm structure, the pathway of sperm post-copulation or the location of sperm in the hours just after mating or in the long term. We carried out experiments to describe and determine the location of sperm produced by snails (autosperm) as well as that of sperm received after copulation (allosperm) using staining and fluorescence microscopy. We searched for and described sperm using phase contrast microscopy and used the DNA stain DAPI to visualize their nuclei with fluorescence microscopy. Sperm was found primarily in the seminal vesicles but also in the ovotestes and hermaphroditic duct; these cells have elongate cone-shaped heads with a helical keel and long helically twisted tails. We then performed mating experiments to track sperm location post-copulation. We incubated snails in a treated tap water with the less toxic stain Hoechst 33342 to label sperm in donor snails, mated them to unstained individuals, then tracked the location of sperm in recipients either several days after mating or weeks later. Just after mating, sperm was primarily located in the sperm receptacle sac, while long-term storage appeared to occur in or near the seminal vesicles and hermaphroditic duct. Further analysis will be necessary to determine how sperm are allocated for fertilization, particularly how autosperm are distinguished from allosperm.

INTRODUCTION

Understanding and predicting reproductive strategies requires knowledge of mating systems as well as gamete production, transfer and storage. Hermaphrodites are unique in that mating systems may involve self- or cross-fertilization; during outcrossing individuals may act as sperm donor, sperm recipient or both (Charnov, 1982; Jarne & Auld, 2006). Freshwater pulmonate snails serve as a useful system for investigating reproductive strategies in hermaphrodites, as they are easy to maintain in the laboratory and many aspects of their reproductive physiology and behaviour have been studied (Jarne et al., 2010; Nakadera & Koene, 2013; Koene, 2021). However, less is known about the pathway of sperm post-copulation or the location of sperm in the hours just after mating or in the long term. Sperm has been observed and described in several pulmonate snails (Soldatenko & Shatrov, 2016) as well as in many other molluscs (Anderson & Personne, 1976) and may be stored for weeks post-copulation (Nakadera et al., 2014), but the location of sperm storage remains elusive in many species (Jarne et al., 2010; Koene, 2021). Since it is not possible to visually distinguish between sperm produced by the individual (autosperm) and that transferred from a partner (allosperm) (Koene et al., 2009), appropriate methods of labelling sperm are required.

Since 2000, our laboratory has used the freshwater snail Planorbella trivolvis Say 1817 (Hygrophila: Planorbidae) (previously Helisoma; Johnson et al., 2013), as a model system to understand reproductive strategies of hermaphrodites. Planorbella trivolis almost never self-fertilizes (Norton & Newman, 2016) and simultaneous reciprocity seems to be the norm, especially when snails have not previously mated (Norton, 2024). Snails typically mate in the dark with bodies closely aligned, and physical coupling is not a reliable indicator of sperm transfer (Norton, 2024). Anatomical studies in P. trivolvis (Abdel-Malek, 1954) and Biomphalaria glabrata (Paraense, 1976) and information from other related species (Jarne et al., 2010) suggest the following process for gamete production and transfer: eggs are produced in the ovaries of the ovotestis and are presumed to be fertilized in the carrefour area where the hermaphroditic duct bifurcates into the oviduct and spermiduct (Tome & Ribeiro, 1998). Fertilized eggs then move through the female reproductive tract where they receive secretions from the albumin glands and are packed into capsules containing multiple eggs. Autosperm is produced in the seminal vesicles of the ovotestis, moves through the hermaphroditic duct, is mixed with seminal fluid proteins produced in the prostate gland and leaves the body through the vas deferens and penis. Allosperm enters the female gonopore, may be held in the short term in the sperm receptacle sac (also referred to as the bursa copulatrix or spermatheca), where it is ultimately digested, and some portion of the allosperm travels up through the female reproductive tract to the hermaphroditic duct or other proximal reproductive structures where presumably fertilization occurs.

Once mated, snails begin to lay eggs approximately 1–2 days post-copulation (C.G. Norton, unpubl.) and produce numerous egg masses containing c. 20–25 eggs; embryos are easily observed during early development and hatch about 7–10 days after laying. Snails store sperm for c. 16 weeks post-mating (Norton & Newman, 2016), yet very little is known about the pathway of sperm post-copulation or the location of sperm storage in P. trivolvis (Abdel-Malek, 1954). In B. glabrata, Paraense (1976) traced the fate of exogenous sperm (allosperm) using radioactive iron and found that 24 h post-copulation, sperm was found primarily in the ovotestis (where it may fertilize eggs) and spermatheca, where it is eventually digested. Montiero & Kawano (2000) tracked sperm in Biomphalaria tenagophila using tritiated thymidine and similarly found sperm in multiple locations along the reproductive tract, more proximally when a longer time had elapsed post-pairing. In Lymnaea stagnalis, another pulmonate, sperm was found in the distal parts of the female reproductive tract soon after mating, but by 24 or 48 h was found primarily in proximal areas of the system, that is the ovotestis, seminal vesicles and hermaphroditic duct (Koene et al., 2009). Here, sperm were labelled only after sectioning, so allosperm were not distinguished from autosperm. In order for allosperm to fertilize eggs, it must be stored near the seminal vesicles or the part of the hermaphroditic duct (termed the fertilization sac by Abdel-Malek, 1954) near the oviduct (Jarne et al., 2010; Koene, 2021) or transferred to this region before fertilization.

The aim of this exploratory study was to determine whether we could distinguish between allosperm and autosperm using fluorescent labelling, identify the locations of sperm immediately after copulation and identify potential long-term storage sites post-copulation. While doing so, we also described sperm structure in this species. To localize and describe sperm, we dissected snails to observe concentrated sperm, which we then examined microscopically using fluorescent DNA staining. To determine allosperm locations and potential sites of storage, we labelled sperm in potential partners, mated them to unlabelled snails and then dissected recipients to determine the location of labelled sperm at various times post-mating.

MATERIAL AND METHODS

Sperm identification and description

To detect the presence and examine the structure of sperm, we collected snails from communal tanks, dissected them and sampled sperm for observation with fluorescence microscopy. Snails originated from two 115 l tanks with populations maintained in the laboratory for over 10 years. Tanks were filled with dechlorinated tap water and filtered with water changes approximately monthly. Snails were fed with boiled romaine lettuce ad libitum every 3–5 days.

We collected 77 snails (two trials with 39 and 38 snails each) from the above aquarium cultures and maintained them in two smaller communal tanks (c. 1 l) for 1 week. We then measured the shell diameter of each snail as an estimate of body size and prepared them for dissection under a Leica MZ75 digital stereomicroscope. We anesthetized snails with 50 mM MgCl₂ x 6H₂O using a 1 mm syringe inserted between the foot and mantle, removed their shell and dissected the snails to visualize their reproductive structures. We removed the shell by pinching off small portions of shell with small forceps, beginning on the right side. Once most of the shell has been removed, the snails can be carefully removed from the remaining shell. We pinned the head and foot to a silicone filled petri dish using minutien pins with the right side up (opposite the genital openings). Holding the body with microforceps, we used spring scissors to cut down towards the head and then up towards the rest of the body, gently teasing the body wall apart. Additional pins were used to secure the spire. We were then able to carefully tease away the digestive and other non-reproductive structures to reveal the reproductive system. In the second trial, we measured the size of the sperm receptacle sac (Abdel-Malek, 1954) from tip to vagina using Image-Pro Express v. 7.0 software.

We noted and removed material with microforceps or a small dissecting needle from any area that had presumptive sperm as indicated by concentrations of opaque white fluid since ejaculate is typically white. Samples were placed on microscope slides and stained with 0.10 μg/ml DAPI (4′,6-diamidino-2-phenylindole), which binds to DNA and fluoresces at 461 nm (Thermo Fisher Scientific). Images were observed with oil immersion under a Leica DM6B microscope with phase contrast, epifluorescence and a LAS-X image acquisition system to confirm the stained cells were in fact sperm. Samples from 15 snails were examined further to determine sperm structure and to measure the length of both heads and tails using ImageJ. We measured one sperm from each sample that was complete and tails were extended enough to measure.

Identification and location of allosperm

To identify allosperm, as distinct from autosperm, we soaked donor individuals in a less toxic DNA stain, then allowed them to mate with unlabelled recipient snails. Any labelled sperm would therefore have come from the mate and not the recipient. We then dissected the recipients to search for labelled sperm at various times post-mating in the sperm receptacle sac and reproductive structures proposed to be sperm storage sites.

Snails were all collected from laboratory tanks. We incubated 30 snails in individual 295 ml plastic cups with 25 μM Hoechst 33,342 (Invitrogen Thermo Fisher Scientific) for 24 h. This stain also binds to DNA and fluoresces at a similar wavelength to DAPI (454 nm) but is less toxic and better able to permeate living cells. We then replaced the solution with dechlorinated tap water every 45–60 min for 3 h to wash away any unbound stain. Snails were kept in this final wash for 3 days and fed boiled romaine lettuce ad libitum. Donor snails were marked with small identification numbers glued to their shells (Queen Bee markers from The Bee Works, USA) and paired with an untreated partner for 48 h . This time frame was chosen to ensure that at least some portion of the snails would mate. Because they originated from larger tanks with available mates and therefore could have stored allosperm, we did not expect every snail to mate during these pairings, nor could we assume that donor snails mated in the male role and recipients in the female role.

After this mating opportunity, donors were discarded and recipients were placed in one of three groups. Snails in the first group were dissected 24 h later, the second after 1 week and the third 2 weeks after isolation. These times were selected to identify where sperm were located immediately after mating and then to locate potential longer-term storage sites. Upon dissection and visualization of the reproductive structures, samples were taken from the sperm receptacle sac, near the seminal vesicles, and in any other area where there were concentrations of white substance. Note that we did not quantify the amount of sperm in any of these locations. All samples were examined under the fluorescence microscope for the presence of labelled sperm. We then replicated the entire experiment several weeks later. Two snails died in the first replicate, so were omitted from analyses.

RESULTS

Sperm identification and description

Even at this broad level of observation large amounts of sperm were found in many locations as an opaque white substance. These structures included the seminal vesicles (where sperm were often in large quantities), the hermaphroditic duct and sperm receptacle duct and sac. In a few snails, there was a large collection of sperm between the ovotestis and seminal vesicles. Snails averaged 13.63 ± 0.22 mm in diameter and sperm receptacle sacs averaged 1.64 ± 0.07 mm in length. There was a significant positive relationship between body size and size of the receptacle sac (linear regression under the JASP platform; R² = 0.18; F_(1,37) = 8.137, P = 0.007; Fig. 1).

Prior to staining, sperm were visible, but did not autofluoresce. Sperm stained well with DAPI and were prevalent in the samples (Fig. 2). Heads fluoresced bright blue and were consistent with the location of heads in the phase contrast images. Sperm had elongate cone-shaped heads with a helical keel and long helically twisted tails (Fig. 3). Heads averaged 5.76 ± 0.21 μm and tails were on average 715.8 ± 27.8 μm for an average total size of 721.5 ± 27.8 μm.

Identification and location of allosperm

Fluorescently labelled sperm were found in 49 of the 58 recipient snails in two locations, the sperm receptacle sac and the area in or near the seminal vesicles (Table 1). No concentrations of sperm were found in other regions. Initially, within 24 h of mating, sperm were found primarily in the sperm receptacle sac (15/20 individuals), although 6 of these individuals also had labelled sperm in the seminal vesicles. One week post-mating, about half the snails had allosperm in the sperm receptacle (10/19) and about 80% in the more proximal reproductive tract 8/19); 2 snails had sperm in both places. By 2 weeks post-mating, most allosperm were found in the area near the seminal vesicles (13/19 individuals), while only 6 of 19 snails had visible sperm in the sperm receptacle. At 2 weeks, two snails had sperm in both locations.

DISCUSSION

Sperm identification and description

Reproductive anatomy was consistent with that described by Abdel-Malek (1954). The average length of the sperm receptacle sac (1.6 mm) was also similar to that found in the closely related species Helisoma duryi (1.8 mm) by Clelland et al. (2001). Clelland et al. observed differences not only in the structure of this organ in mated vs virgin individuals, but also in size. They suggest that the bursa copulatrix increases in size to accommodate received sperm and that size might reflect the amount of sperm received. If this is the case, the positive relationship we document between this organ and body size may help to explain the complex relationship between body size and egg production. We previously reported that 19% of the variation in overall egg production and 24% of variation in the number of eggs per mass can be explained by body size of the egg donor in Planorbella trivolvis (Norton & Bronson, 2005). Although this relationship is typically explained in terms of energetics, it may also be that larger snails have larger receptacles so can initially receive more sperm from their partner.

As expected, when mature snails were allowed to freely mate, sperm was found in several areas of the hermaphroditic reproductive tract, primarily in the sperm receptacle sac, seminal vesicles and hermaphroditic duct, often in large numbers as evidenced by prominent white globules, which likely also contain seminal fluid. When removed from the snails and stained with DAPI, sperm were easily identifiable by their long tails and heads which corresponded to areas of fluorescence. However, as others have noted (Jarne et al., 2010; Koene, 2021), there was no way to distinguish whether the sperm had been produced by the individual in which they were found or by a mating partner.

Sperm structure and size was consistent with that of other Planorbidae (Soldatenko & Shatrov, 2016). Heads were elongate and cone shaped with a helical keel and tails (indistinguishable from a midpiece at this magnification) were long relative to the size of the head. Pulmonate sperm described by Soldatenko & Shatrov (2016) ranged from 420 to 810 μm in total length, with heads ranging 4–5 μm, similar to our measurements. As our main purpose was to identify and locate sperm, we did not pursue structural studies at the level of electron microscopy.

Identification and location of allosperm

When donor snails were stained with Hoechst and then allowed to interact with untreated recipients, labelled sperm was found in the majority of recipients, indicating that mating had occurred and sperm had been transferred during the 48 h period when they were together. Although sperm was not found in every individual, this was not unexpected since snails may have previously mated and might not have been receptive to the current mate. Twenty-four hours post-mating, approximately 75% of the recipients with labelled sperm had sperm located in the sperm receptacle sac. One week later, only half of them had sperm in the sac, and 2 weeks later, even fewer. This pattern was expected, as sperm remaining in this structure (often called the bursa copulatrix or sperm digesting organ) are usually digested or move on to other locations (Jarne et al., 2010).

Soon after mating, very few recipient individuals had labelled sperm in the more proximal regions of the reproductive tract, but after 1 week almost half of the individuals in which we found labelled sperm had sperm in or near the seminal vesicles. This is consistent with our observations that snails do not usually begin laying eggs until 24–48 h after mating occurs. By 2 weeks, post-mating labelled sperm was found in this region in almost 70% of the individuals. This pattern indicates that allosperm moves out of the sperm receptacle sac (or is digested) and up the reproductive tract to an area near the seminal vesicles. This long-term storage location makes sense with respect to location of fertilization as sperm and egg must meet upstream of the carrefour/fertilization pouch area. This is the region in which Paraense (1976) and Montiero & Kawano (2000) found labelled allosperm after mating in Biomphalaria species and proposed as a likely long-term storage site. Koene et al. (2009) also proposed seminal vesicles as a storage site but were unable to distinguish between allo- and autosperm. The labelling technique described here provides the first demonstration to our knowledge that allosperm is concentrated in a location proximal to the site of fertilization several weeks after mating. More data concerning the quantities of sperm in each location, specific anatomical location, and an extended time frame of study would further our understanding of allosperm storage. Studies with snails isolated before maturity, which have had no previous mating experience would also be likely to result in higher proportions of successful matings.

The observed pattern of sperm location is also consistent with what is known about sperm precedence in this species. Planorbella trivolvis exhibits strong first sperm precedence; when individuals are mated to one partner and then another 48 h later, more than 80% of progeny result from fertilization by sperm from the first mate (Norton & Wright, 2019). We suggested this pattern results because the sperm receptacle sac is full so that additional allosperm from an immediate second mating is either unable to be transferred, or that snails that recently received sperm would resist mating in this role again, preferring to donate sperm to a partner. In snails recently mated in the female role, the long-term storage area would also be taken up by allosperm. If sperm from first mating fills the receptacle then moves out to another location for long-term storage, we would predict not only that fewer sperm would be found in the receptacle sac after a second mating, but that as time between matings increases, more space would become available for sperm from the second mate and thus more eggs would be fertilized by that partner. Preliminary work has shown this to be the case (C.G. Norton, unpubl.). As the amount of time between matings increases, the proportion of eggs fertilized by the second mate also increases.

One additional important question remains: how is allosperm distinguished from autosperm by the recipient snail? Planorbella trivolvis almost exclusively uses allosperm to fertilize eggs (Norton & Newman, 2016) and most other hermaphroditic freshwater gastropods predominantly outcross (Jarne et al., 1993), which means that allosperm is almost exclusively used for fertilizing the recipient’s eggs, whereas autosperm passes through the hermaphroditic duct to the male reproductive structures to fertilize the eggs of other individuals. While some stylomatophorans have a separate spermatheca in the fertilization pouch for allosperm (Duncan, 1975; Baur & Baur, 2021), this does not seem to be the case with bassomatophorans (Koene et al., 2009; Koene, 2021). It may be that auto- and allosperm are differentially activated such that only allosperm are able to fertilize eggs (as proposed by Koene et al., 2009), but the mechanism by which snails control paternity remains elusive. Differential staining techniques to distinguish between auto- and allosperm along with more detailed structural and biochemical analysis may provide a way to answer this question.

ACKNOWLEDGEMENTS

We thank Kay Tweeten for sharing the staining method she developed to track sperm in Lumbriculus and her and Andrea Kalis for help with the fluorescence microscopes.

FUNDING

This project was funded by a 3M Student-Faculty Collaborative Grant from St. Catherine University. A National Science Foundation Major Research Instrumentation Grant (MRI-1,827,514) supported the microscopy work.Data available on request.

CONFLICT OF INTEREST

The authors have no conflict of interest that might bias our work and have not used data from other sources.

DATA AVAILABILITY

Data available on request.

References