Experience and relatedness influence mating interactions in a simultaneously hermaphroditic snail, Physa gyrina

The means by which animals assess potential mates is an important issue in studies of reproductive systems. I tested whether an individual’s previous experiences and the relatedness of mates affected mating behavior in a simultaneous hermaphrodite snail, Physa gyrina. Previous work with this species showed reduced reproductive success resulting from both strong outbreeding and inbreeding. Thus, I predicted that individuals should prefer partners of intermediate relatedness. During activity trials, snails moved longer distances when exposed to chemical cues from conspecifics of lesser relatedness. Furthermore, during mating interactions, behavioral responses to relatedness varied with gender-role: male-role behaviors did not vary across relatedness treatments, while snails paired with either closely related or highly dissimilar partners increased their female-role resistance behaviors as interactions escalated. Experiences with their current partner also affected behavioral dynamics. Familiar pairs had fewer matings and longer latency times until a mating occurred than unfamiliar pairs. Snails acting in the female role also exhibited higher resistance rates in familiar pairs than in unfamiliar pairs. Previous, brief exposure to chemical cues in a non-mating context also influenced behavior during a subsequent mating interaction. Snails that were previously exposed to chemical cues from unfamiliar individuals tended to be more likely to occupy the male role following an encounter, and had significantly lower copulation frequencies and higher female-role resistance rates (i.e. were choosier) than those previously exposed to cues from familiar individuals. Overall, the results show that: 1) relatedness, past exposure to conspecific chemical cues, and experience with a current partner all influence mating behaviors in these snails; and 2) in these simultaneous hermaphrodites, an individual’s responses depend on whether it is occupying the male or female role.


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A major focus in behavioral ecology is the study of mate choice. Considerable effort has 39 been dedicated to studying kin recognition, its mechanisms and its implications [see [1][2][3]. Many 40 studies have also examined the fitness effects of inbreeding and outbreeding [4][5][6]. Most animals 41 prefer some potential mates to others and do not mate indiscriminately [7][8][9]. Consequently, 42 mating behaviors should reflect fitness patterns [10][11][12]. One way to minimize the deleterious 43 fitness effects of inbreeding and outbreeding is to be able to recognize kin [10] and alter mating 44 behaviors accordingly. However, individuals' mating behaviors and mate-choice decisions may 45 often be influenced, sometimes in disparate ways, by their previous experiences (e.g. exposure to 46 phenotypes during development, or previous social and mating interactions as adults) [13]. 47 Genetic similarity of mates may dramatically affect fitness through changes in the 48 number, viability or quality of offspring [11,14,15]. Optimal outbreeding can be viewed as a  Since inbreeding and outbreeding affect fitness, it is logical to ask what processes 58 mitigate these consequences in natural populations [20]. In the context of mating and genetic 59 similarity, behavioral studies have focused on kin recognition and species recognition. While [1] and individual familiarity [10,13,18,23]. Not surprisingly, researchers examining these 66 processes typically use vertebrate species that live in defined social groups or have considerable 67 parental care. However, hermaphroditic invertebrate systems that lack social organization and 68 parental care afford an exciting opportunity to address questions of how mating patterns are 69 influenced by both innate abilities and individual experience. 70 Physa is a genus of aquatic snails common throughout North America [24]. Physids are 71 considered to be good dispersers and colonizers [25,26], and some species are globally invasive 72 (e.g. Physa acuta) [27][28][29]. Physa live in diverse aquatic habitats ranging from large lakes and 73 rivers to small streams, ditches, and temporary ponds [30]. Within these habitat types, population 74 densities vary from hundreds of snails per m 2 to fewer than one snail per m 2 [31]. Given the high 75 reproductive rates of physid snails, individuals may encounter relatives as potential mates, 76 especially in smaller populations living in restricted habitats. In contrast, individuals may also 77 encounter conspecifics from foreign populations as a result of transport via other organisms (e.g.  including itself, is a potential mate. Physa produce many offspring and mate readily, with easily 92 observed mating behaviors [39,43]. For a successful copulation to occur, snails must physically 93 contact each other, and then one snail assumes the male gender role by crawling onto the shell of 94 the other snail, which becomes the prospective female by default. The snail acting as a male will 95 position itself along the margin of the shell aperture of the prospective female by crawling in 96 small circles on the female's shell. Once in the proper alignment, the attempting male everts the 97 penile complex (preputium); copulation occurs when the male's preputium contacts the female's 98 gonopore and sperm is transferred. However, presumptive females can resist males through a 99 variety of behaviors [39,43,44], including 'biting' behaviors where the female turns its head and 100 the mouth region contacts the male's preputium, presumably scraping it with the radula.

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Pheromones likely play important role in attracting mates in many gastropods [45] that previous experiences should also influence mating behavior with a current partner: previous 119 exposure to preferred snails (in terms of relatedness or familiarity) should reduce motivation to 120 mate with a current partner, whereas, previous exposure to non-preferred snails should increase 121 mating with a current partner.  iii. inner cup; iv. mesh window; v. waterline). Activity levels (II: Experiment 1) of individual 144 snails were then recorded in Petri dishes (vi.) while exposed to either familiar (F C ) or unfamiliar 145 (U C ) chemical cues. Next, partners were allowed to physically interact for 72 h (III). Last,  Paired snails were maintained together for two weeks so that individuals were exposed to 150 chemical cues from their partner without being able to directly interact with each other. I placed 8 151 each pair into an apparatus consisting of a smaller 250 ml plastic cup with two mesh windows 152 resting inside a larger 900 ml plastic cup (Fig 1: I.B). One snail was placed in the small, inner 153 cup while its partner was placed into the large, outer cup. Thus, partners were not able to  This stage of the experiment tested whether the locomotion rates of mature, virgin P. 164 gyrina were influenced by either chemical familiarity or the degree of relatedness between 165 individuals. Since mating propensity is associated with high locomotion rates in some gastropods 166 [61], I predicted that crawling rates could serve as a proxy representing mate-search effort and 167 would increase in response to chemical cues from unfamiliar snails and from snails of 168 intermediate relatedness. To test these hypotheses, I exposed focal snails to chemical cues from 169 conspecifics that varied in relatedness and prior chemical exposure (i.e. novel or familiar cues). I 170 compared mean travel distances between treatments to determine whether snails differentially 171 respond to conspecifics without direct interaction.

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Snails from each of five relatedness categories were randomly assigned to a chemical-173 exposure treatment (Fig 1: II.). Snails in the chemically-familiar treatments were exposed to 9 174 chemical cues from their own holding apparatus: snails from the inner cup received water drawn 175 from the outer cup and vice versa. It should be noted that as a result of the mesh windows in the 176 small cups, each snail was exposed to chemical cues from two potential sperm donors: 177 themselves (because physids are simultaneous hermaphrodites) and the other snail in the 178 apparatus. Snails assigned to the chemically-unfamiliar treatments were exposed to chemical 179 cues generated by another pair of snails maintained in a different apparatus. Again, this exposed 180 each focal snail to cues from two potential mates. In order to reduce confounding effects, each 181 snail in the chemically-unfamiliar treatment was exposed to cues from an apparatus containing a 182 pair that was related to the focal snail to the same degree as was its apparatus-partner. For 183 example, a focal CN snail was exposed to cues from snails in an unfamiliar CN pair that were 184 also cousins to the focal snail. Thus, the degree of relatedness remained constant while the 185 familiarity changed. 186 I exposed each snail to the chemical cues by placing it in a Petri dish containing 50 ml of 187 the appropriate treatment water. Snails' paths were traced during a 3-minute observation period.

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Tracings were photographed, the images were digitized using ImageJ [62], and the travel 189 distances were calculated for each snail. I compared crawl-distances between treatments, with 190 shell length as a covariate (ANCOVAs). I also used orthogonal contrasts to specifically examine 191 how genetic similarity influenced crawl-distances: a treatment group was compared to all more-    proportional 'size differences' between paired individuals were initially used as covariates but 233 were dropped from the analyses since they did not significantly contribute to the models.

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Female-role resistance behaviors ('bites') were examined using linear regression analyses.    The degree of relatedness between snails did not influence the interaction outcome (Table   262 1), the latency of copulation (failure-time analysis:  2 = 3.90, df = 4, p = 0.42), the escalation of 263 male-role behaviors (        Table 1; mean number of copulations per pair  SE: familiar 312 snails = 0.31  0.08, unfamiliar snails = 0.68  0.12). Unfamiliar snails also mated sooner than 313 familiar partners (Fig 3; failure-time analysis:  2 = 4.60, df = 1, p = 0.03). These differences  (Table 4B).  The chemical-cue familiarity treatments that snails experienced in the first experiment 330 also significantly affected the mating interactions observed in the second experiment. Individuals 331 that were exposed to chemical cues from unfamiliar snails in the first experiment were more 20 332 likely to mount their partners ('Cue-trt' in Table 2A; mean mounting frequencies ± SE: exposed 333 to familiar cues = 0.47 ± 0.04, exposed to unfamiliar cues = 0.62 ± 0.05), but also tended to 334 reject the female more often during an interaction ('Cue-trt' in Table 3B -marginally   335 nonsignificant; mean 'male' rejection frequencies ± SE: exposed to familiar cues = 0.58 ± 0.06, 336 exposed to unfamiliar cues = 0.70 ± 0.04). Positioning frequencies were affected by a significant 337 interaction between the familiarity treatments of the two experiments (Table 2B; Fig 4).

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Bonferroni post hoc tests indicated that snails exposed to familiar cues during the first  (Table 2D; mean copulation frequencies ± SE: 343 exposed to familiar cues = 0.65 ± 0.09, exposed to unfamiliar cues = 0.50 ± 0.08). Snails 344 occupying the female role that had been exposed to familiar chemical cues during the first 345 experiment resisted highly motivated males during interactions, but this was not the case for 346 snails that had been exposed to unfamiliar cues in the first experiment (Table 4C).  Neither a snail's size nor its crawl-distance (first experiment) was related to the 355 individual's conditional frequencies of behaviors during mating interactions (Table 5). 356 Moreover, there was no trend for size-based gender occupation during successful copulations 357 (mean size (mm) ± SE: females = 7.25 ± 0.04, males = 7.23 ± 0.05; paired t-test: t 33 = 0.14, p = 358 0.89).  This study indicates that the degree of relatedness between individuals and the contexts in  activity in response to close relatives, or they increased search efforts for less related mates.

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While snails acting as males exhibited few preferences for mates based on relatedness, those 404 acting in the female role significantly increased resistance to highly motivated SS and IP males.

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Thus, one interpretation for the lower crawling rates in response to cues from close relatives, 406 given the gender-role specific strategies, is that reduced activity is a mechanism to avoid fitness 407 costs should that individual become 'female' during an interaction. Consequently, the snails' 408 gender-specific behavioral strategies (no discrimination when acting as a male, but alter 409 resistance levels to mates when acting as a female) should maximize individual fitness.