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Multiple mating, sperm storage, and mating preference in Aplysia californica.


Darwin (1877) was the first to discuss sexual selection and its relationship to the evolution of mating systems. Experimentally, "Bateman's Principles" attempt to explain the common observation of "coy" females and "eager" males on the basis of the relationship between fecundity and mating success (Bateman, 1948; Leonard, 1991; Arnold, 1994). Using Drosophilia, Bateman demonstrated that reproductive fitness in males tends to be limited by access to cafemales, and in females by the energetic costs of egg production (Bateman, 1948; Charnov, 1979; Leonard and Lukowiak, 1985; Leonard, 1991; Arnold, 1994; Anthes et at, 2006). Thus, in order to increase individual reproductive fitness, males should attempt to mate with as many females as possible and females should be selective with their limited valuable resources. Therefore, a conflict of interest exists between males and females, leading to sexual selection and the evolution of a wide variety of mating systems.

Charnov (1979, 1996), using resource allocation models, showed that these same principles can be applied to simultaneous hermaphrodites. In simultaneous hermaphrodites, reproduction in the male role should be preferred, assuming the costs associated with sperm donation are less than those with receipt (Charnov, 1979, 1996). Since each individual is reproductively both male and female, a conflict should arise during each mating encounter (Angeloni et al., 2002). Therefore, hermaphrodite mating systems should evolve to impose costs on reproduction via the male role (Charnov, 1979, 1996; Eberhard, 1996). The complex internal structure of the reproductive systems of hermaphrodites, including sperm storage and digestion organs, may have evolved as a means of controlling the access of sperm to eggs (Angeloni et al., 2003). In fact, multiple mating and allosperm digestion can increase the costs of reproduction in the male role to the point that available resources, rather than access to eggs, become limiting (Greff and Michiels, 1999).

Control of fertilization (the Gamete-Trading Hypothesis), rather than allocation of resources, may explain sexual conflict in simultaneous hermaphrodites (Leonard and Lukowiak, 1984, 1985, 1991). Given that not all copulations result in zygotes, an important difference between the sexes could be the extent to which control is exerted over fertilization. In simultaneous hermaphrodites, the sexual role that controls fertilization affords the greatest certainty that their investment in gametes will result in zygotes (Leonard and Lukowiak, 1984, 1985, 1991; Michiels, 1998). As a result, an "arms race" for the control of fertilization may have led to the evolution of the diverse array of mating systems and bizarre accessory glands and organs found in simultaneous hermaphrodites (Michiels, 1998).

In spite of this diversity, multiple mating involving reciprocation, long-term storage of allosperm, and the ability to digest allosperm are common among many hermaphrodite species (Baur, 1998; Michiels, 1998). This suite of common reproductive features sets the stage for sperm competition or cryptic female choice to occur in these simultaneous hermaphrodites (Michiels, 1998). Sperm competition arises when the sperm of two or more males competes for the ability to fertilize a female's eggs (Parker, 1998). Cryptic, or post-copulatory, female choice involves biases on male reproductive success through female-controlled processes or structures (Eberhard, 1996, 1998). Unfortunately, it may be particularly difficult to distinguish between the two since the female's morphology ultimately determines the arena for sperm competition (Eberhard, 1998; Jennions and Petrie, 2000).

Aplysia californica is an opisthobranch mollusc commonly used by researchers as a model animal for the study of neurophysiology (Kandel. 1979). Aplysia californica's natural history is well known, and research organisms are bred and reared in captivity at the NIH National Center for Research Resources' National Resource for Aplysia at the University of Miami. Like other opisthobranchs, A. californica, a simultaneous hermaphrodite, possess two evaginations of the common genital gonoduct: the seminal receptacle and the copulatory bursa (Hadfield and Switzer-DunSap, 1984). The seminal receptacle is known to be the storage site for exogenous sperm received during mating (allosperm) (Beeman, 1970: Hadfield and Switzer-Dunlap, 1984). Recently, it has been shown that A. californica can use stored sperm for up to 41 days to fertilize eggs after a single copulation (Ludwig and Walsh, 2004). In aplysiids, the copulatory bursa is believed to be a site for the digestion and absorption of sperm (Beeman, 1970; Hadfield and Switzer-Dunlap, 1984). In nature and in the laboratory, Aplysia individuals will frequently mate with several partners and participate in chain copulations (MacGinitie, 1934; Hadfield and Switzer-Dunlap, 1984; Carefoot, 1987; Zaferes et al., 1988; Pennings, 1991; Yusa. 1996; Angeloni et al., 2003). As a consequence of this behavior and Aplysia's reproductive anatomy, the opportunity for sperm competition and post-copulatory female choice exists (Charnov, 1979, 1996; Maynard Smith, 1991; Yusa, 1994; Baur, 1998; Eberhard, 1998; Michiels. 1998; Moller. 1998). In this context, the paternity of the offspring from an individual of A. californica that has mated with multiple partners should remain in question. Ultimately, the paternity of the offspring could depend upon mixing or displacement of sperm within the seminal receptacle, competition between sperm for eggs, the recipient's ability to discriminate between sperm from different mates, or a combination of the three factors. Additionally, A. californica mates unilaterally, with one animal acting as the sperm donor and the other as the sperm recipient (Kandel 1979; Carefoot, 1987). During copulation, mating animals assume a customary orientation with the donor on top of the recipient (Kandel. 1979; Carefoot, 1987), making it relatively simple to determine the mating role of each individual. In this study, we used controlled matings and behavioral observations to determine whether mating preferences exist during multiple mating in Aplysia californica.

Materials and Methods

Isolation and identification of broodstock

Specimens of Aplysia californica Cooper, 1863. at an average age of 135 days and average mass of 58.3 g, were selected from the hatchery-reared population at the National Resource for Aplysia. Grown under hatchery conditions and at this age, sea hares of this species have yet to reach sexual maturity (Capo et al., 2002). Following Mg[Cl.sup.2] anesthesia, broodstock individuals were weighed to the nearest 0.1 g and tagged, for later identification, by attaching color-coded beads to the animal's parapodia with a small suture of 000 silk. To ensure that broodstock animals did not receive sperm prior to maturation, it was necessary to grow potential broodstock in isolation (presumptive "virgins'"). Tagged juvenile broodstock were quarantined in separate 16-1 flow-through seawater tanks maintained at 19-23[degrees]C and fed ad libitum on the macroalga Gracillaria ferox. Reared under these conditions, juveniles reached sexual maturity in 2-3 weeks (Ludwig and Walsh, 2004). When reared in isolation in the laboratory, this species commonly lays unfertilized egg masses at the onset of sexual maturity (Ludwig and Walsh, 2004). Therefore, the presence of an unfertilized egg mass was used as an indicator that isolated broodstock animals had reached sexual maturity.

Multiple mating I

Prior to pairing with a potential mate, sexually mature isolated broodstock of Aplysia californica were weighed to the nearest 0.1 g. Animals were paired with potential mates of similar mass to control for sexual preference choices that could be based on size differences between mating pairs. Additionally, care was taken to ensure that each pair of potential mates came from a different outbred family to avoid any effects associated with inbreeding. For the initial mating attempts, pairs were placed in separate 16-1 flow-through seawater cages maintained at 19-23[degrees]C in the absence of food. Behavioral observations were recorded every 5 mm to determine the onset of mating and the sexual role of each animal. Evidence suggests that members of the genus Aplysia are not sperm-limited after mating encounters lasting less than 1 h (Yusa, 1996; Ludwig and Walsh, 2004). Therefore, in an attempt to limit the number of sperm transferred and maintain a willingness to accept more sperm, mating was only allowed to proceed for 30-40 min. After this time, mating animals were manually separated, and penis intromission was used as an indicator of successful mating. The sperm donor (functional male) and sperm recipient (functional female) were removed and placed into separate cages containing a second potential mate of similar mass. Behavioral observations were recorded every 5 min to determine the onset of mating and the sexual role of each animal during the second mating encounter. As previously indicated, these matings were also manually interrupted after 30-40 min of copulation. All of the experimental animals were returned to separate cages and fed ad libitum.

Multiple mating II

A second group of mating experiments was designed to determine whether the preference of singly mated individuals of Aplysia californica to act as the sperm donor, when presented immediately with a potential mate (see Results section), is related to the use or depletion of sperm to fertilize eggs. For these controlled matings, broodstock animals were treated as previously described in Isolation and identification of broodstock, and initial experimental procedures were the same as in Multiple mating I. After mating had proceeded for 30-40 min, the experimental animals were manually separated, returned to separate cages, and fed ad libitum. The sperm recipient (functional female) was checked daily for the presence of a newly spawned egg mass. Egg masses were collected, subsampled, and cultured, as previously described by Ludwig and Walsh (2004), to determine the percent fertilization of each clutch of eggs. As shown in the 2004 study, there was no apparent sampling bias due to clustering of fertilized or unfertilized eggs. Shortly after laying an egg mass, sperm recipients were moved to a clean cage and paired with a sexually mature virgin animal as a new potential mate. Observations and procedures were as previously described. After the experimental animals were returned to separate cages, the initial sperm recipient was checked daily for eggs. This process of egg collection and re-mating of the initial sperm recipient was continued until the animal accepted sperm from a second donor. Egg mass deposition and fertilization data were then compared to those of the initial sperm recipient to determine whether use or depletion of stored allosperm is related to sexual behavior. To determine whether previously mated individuals exhibited a preference for a particular mating role during their second mating encounter, observed behavior was compared to expectations that there would be no preference for mating role. The chi square test for goodness of fit with continuity correction for small sample size (Zar, 1984) was used for this comparison.


Multiple mating I

Over the course of several months (May 2003 - October 2003), 21 pairs of Aplysia califonica were successfully mated as initial partners. Immediately after manual separation, each individual of the initial pairing was given the opportunity to re-mate with an isolated "virgin" partner. All 42 previously mated animals were willing to mate during the second mating opportunity. However, an interesting pattern was detected. Animals who acted as sperm donors in their initial mating encounter showed no preference for second mating role when paired with a virgin partner ([x.sup.2] = 0.429, P = 0.5127, df = l, n = 21) (Fig. 1). Yet animals who acted as sperm recipients in their initial mating encounter showed a highly significant preference for acting as the sperm donor in subsequent matings, when paired with a virgin partner [x.sup.2] = 10.714, P = 0.0011, df = 1, n = 21) (Fig. 1). In fact, it was quite difficult to obtain an experimental animal who accepted sperm in two sequential matings. Of the 21 animals who initially accepted sperm for their first mating encounter, only 3 acted as sperm recipients during their second mating encounter.

Multiple mating 11

In the first mating experiment {Multiple mating I), animals who acted as sperm recipient in the initial mating bout showed a significant preference for acting as sperm donor when presented, immediately, with a second potential mate (Fig. 1). However, animals who acted as sperm recipient in the initial mating bout did not demonstrate a significant preference for either mating role when allowed to lay fertilized eggs prior to their second mating bout ([x.sup.2] = 1.24, P = 0.265, df = 1, n = 13) (Fig. 2). On average, singly mated animals laid 2.85 [+ or -] 0.39 (mean [+ or -] SE, n = 13) egg masses before accepting sperm from a second donor. The average fertilization rate of the final egg mass prior to accepting a second sperm donation was 84.0 [+ or -] 7.5 percent (n = 13), indicating that some of these animals may have begun to deplete stored allosperm.
Figure 1 Frequency of multiple mating behavior in Aplysia californica
from multiple mating experiment I. Firsts and second mating role are
abbreviated as "R" for receipient and "D" for donor. Excepted results
were determined for no preferene as recipient or donor during second
mating encounter.

     observed  expected

R/R      3       10.5
R/D     18       10.5
R/R      4        6.5
R/D      9        6.5

Note: Table made from bar graph.

Figure 2. Frequency of multiple mating behavior in Aplysia california
from multiple mating experiment II. Animals were offered a second
opportunity to mate only after laying fertilized eggs. First and second
mating roles are abbreviated as "R" for recipient and "D" for donor.
Excepected results were determined for no preference as recipient or
donor during second mating encounter.

     observed  expected

1st Mating Role/2nd Mating Role

R/R      4       6.5
R/D      9       6.5

Note: Table made from bar graph.

Three singly mated animals did accept a second sperm donation immediately after laying their first egg mass (Fig. 3). Two of these exhibited greater than 95% fertilization of their initial egg mass. The third sea hare fertilized only 33.3% [+ or -] 2.4% (n = 5 replicate samples of egg mass) of the eggs spawned, suggesting this animal did not receive ample sperm from its initial mating. Most of the experimental animals did not accept a second sperm donation until they had spawned their fourth egg mass (Fig. 3). These five animals, who accepted a second sperm donation after their fourth spawning, fertilized an average of 77.6% [+ or -] 15.1% (n = 5 egg masses) of the eggs spawned. Additionally, when presented with a potential mate after each spawning event, these same animals preferred to donate sperm to each partner, until they ultimately accepted a second sperm donation after spawning their fourth egg mass. The general trend for individuals of A. californica who acted as a sperm recipient during their initial mating encounter was to spawn a fertilized egg mass, then either refuse to mate or mate as a sperm donor when presented with a virgin partner. This process was repeated several times until sperm was finally


Expected results were determined for no preference as recipient or donor during second mating encounter. accepted from a second donor. As an example, specimen #23 laid four fully fertilized egg masses and mated as a sperm donor after each spawning. Finally, after depositing a fifth egg mass (92.2% [+ or -] 0.9% fertilized, n = 5), this specimen acted as a sperm recipient for a second time.


Multiple mating has been reported as common behavior in Aplysia californica (MacGinitie, 1934; Hadfield and Switzer-Dunlap, 1984; Carefoot, 1987; Zaferes et al., 1988; Pennings, 1991; Yusa, 1996; Angeloni et at, 2003). In the current study, A. californica individuals readily mated with a second partner immediately after an initial mating encounter of 30-40 min. However, prior mating behavior did influence behavior with a second partner. Sea hares that acted as the initial sperm donors were just as likely to donate or accept sperm when presented immediately with a second virgin partner (Fig. 1). Yet animals who acted as the initial sperm recipient were 6 times more likely to immediately donate sperm to a second isolated partner (Fig. 1). This behavior demonstrates a significant preference for donating sperm immediately after the initial act of receiving sperm. Proponents of conditional reciprocal mating as a resolution for sexual conflict in simultaneous hermaphrodites may view this as a behavioral constraint for switching of mating roles between partners--behavior known as "sperm-trading" (Fischer, 1980, 1987; Leonard and Lukowiak, 1984. 1985, 1991; Leonard, 1991; Sella and Ramella, 1999; Michiels and Bakovski, 2000; Michiels et al 2003; Angeloni, 2003; Anthes and Michiels, 2004). If this were the case, a similar but opposite preference should have been observed in the initial sperm donors when immediately presented with a second mate. Yet our initial sperm donors showed no preference for either sexual role during their second mating encounter. Additionally, because individuals of A. californica commonly lose contact after the completion of mating, conditional reciprocity is considered unlikely in the wild in this species (Pennings, 1991).

Other researchers have suggested that isolation of hermaphroditic flatworms and opisthobranchs prior to mating may enhance their attractiveness as a female (Michiels and Bakovski, 2000; Michiels et al 2003). These "sexy mothers" are believed to be more attractive as sperm recipients because the opportunity for sperm competition is low and egg fertilization is more likely in isolated animals (Michiels and Bakovski, 2000; Michiels et al 2003). We do not believe our results are an artifact of this phenomenon of mating experienced animals with isolated animals. First, initial sperm donors showed no preference to act as sperm donors when presented with isolated animals for their second mating opportunity. Second, after spawning a fertilized egg mass, initial sperm recipients lost their preference to mate as sperm donors when presented with isolated animals for their second mating opportunity (Fig. 2). Thus, we speculate that in A. californica a sperm recipient's preference to mate as a male in subsequent matings is related to the use and depletion of stored allosperm rather than to the attractiveness of a potential mate.

It has been previously shown that singly mated A. californica individuals are not sperm-limited after matings of 30-40 min (Ludwig and Walsh, 2004): the sea hares fertilized over 16 million eggs over a period of 22 days from the sperm received from a single 30-40-min mating. Given high rates of mate encounter and an abundant food supply, A. californica individuals who act initially as a sperm recipient may not obtain substantial benefits from acting as a multiple sperm recipient until stored allosperm becomes depleted. Once a sperm recipient's seminal receptacle is full, fitness gains may be maximized by alternating between using stored allosperm to spawn fertilized eggs and donating sperm to potential mates until depletion of stored allosperm begins to limit the production of fertilized eggs. At such a time, the animal may then accept sperm from an additional donor as fertility assurance for future clutches. In support of this notion, we found that previously mated individuals' preference as a sperm donor decreased when they were allowed to spawn prior to additional mating attempts (Figs. 1 and 2). Furthermore, most of the animals did not re-mate as a sperm recipient until after they had spawned at least four fertilized egg masses with an average fertilization rate of about 78% (Fig. 3). In a previous study, we observed an initial period of greater than 90% fertilization for the first four to six egg masses, followed by an 18% drop in fertilization success for each subsequent egg mass in singly mated individuals of A. californica (Ludwig and Walsh, 2004). In concert, these results support the notion that depletion of stored allosperm plays an important role in the resolution of sexual conflict and the mating decisions of A, californica. Similar results were observed in the simultaneous hermaphrodite snail Physa heterostropha: snails recently inseminated with allosperm exhibited "rejective behavior" when mounted by a second potential sperm donor (Wethington and Dillon, 1996).

If individuals of A. californica are depleting their first mate's sperm prior to accepting a second sperm donation, then we would expect to observe a high frequency of mixed paternity with second-mate sperm precedence in the wild and in the laboratory. Angeloni et al. (2003), using micro-satellites, observed mixed paternity in 76% of egg masses spawned by A. californica specimens collected from the wild. In 75% of the clutches they sampled, the last known sperm donor fathered more than half of the offspring (Angeloni et al., 2003). In unpublished results, we also detected second-mate sperm precedence, using single-copy nuclear DNA- restriction fragment length polymorphism (scnDNA-RFLP) markers, in two egg masses from one of our multiple sperm recipients. On the basis of the results of our behavioral studies, we believe second-mate sperm precedence to be the result of differences in sperm volume rather than cryptic-female choice or sperm competition in the strictest sense. Since most of our animals did not accept a second sperm donation until after their fourth spawning, it follows that the second sperm type would constitute most of the stored allosperm after a second mating. This pattern may also help to explain the high incidence of multiple paternity of egg masses observed in wild populations (Angeloni etal., 2003).

Our study of multiple mating in Aplysia californica did not detect any form of the reciprocal or conditional mating often associated with the resolution of sexual conflict in simultaneous hermaphrodites (Leonard and Lukowiak, 1984, 1985, 1991; Fischer, 1987; Baur, 1998; Michiels, 1998; Michiels and Bakovski, 2000; Michiels et al 2003; Koene and ter Maat, 2005). We speculate that, at least for A. californica, individual mating role decisions are more appropriately determined case by case rather than by an insistence upon reciprocal behavior for all encounters as the means for resolving mating conflicts between simultaneous hermaphrodites. Each individual must consider its own condition during each potential mating encounter. Sperm-storing simultaneous hermaphrodites must balance the available resources for the production of fertilized eggs and the donation of sperm with the current state of stored allosperm against the benefits of accepting more sperm and the chance of encountering a second mate. Given the dynamic nature of each of these factors, flexibility in sexual behavior rather than insistence upon reciprocation should be the rule. This flexibility should be especially valuable in a simultaneous hermaphrodite with an annual life cycle such as A. californica. To maximize reproductive fitness over a single breeding season, A. californica could employ a mixture of strategies or change gender preferences depending on the unique set of circumstances associated with each mating encounter.

Anthes et ml. (2006) proposed a new "gender ratio" hypothesis in which mating role derives from the potential fitness gain for each mating event. By assessing one's own reproductive state and cues from potential mates and the environment, sex role preferences and various forms of reciprocation may be favored (Anthes et al., 2006). However, hermaphrodite mating systems should exhibit an inherent flexibility to shift with fluctuating environmental conditions, as evidenced by the success of hermaphroditism in general (Anthes et at, 2006). Using hatchery-reared animals, future studies of A. californica should focus on changes in mating behavior and sex role preferences under different conditions; such experiments, in conjunction with controlled breeding studies, should better quantify evidence of second-donor sperm precedence.


The authors wish to acknowledge T. Capo, A. Bardales, A. Boyd, and D. Stommes for their assistance in raising sea hares, and Drs. C. Hughes, L. Fieber, and M. Schmale for advice on experimental design. The research was supported by NIH grant RR10294 and an NSERC Discovery grant to PJW.

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Division of Marine Biology and Fisheries, NIH NCRR National Resource for Aplysia, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, Miami, Florida 33149

Received 1 April 2008; accepted 4 August 2008.

* To whom correspondence should be addressed, at Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON KIN 6N5, Canada. E-mail: pwalsh@uottawa.
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Author:Ludwig, Adam N.; Walsh, Patrick J.
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1USA
Date:Dec 1, 2008
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