Slipper limpet males avoid copulating with parasitic castrated females

Slipper limpet males avoid copulating with parasitic castrated females | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Slipper limpet males avoid copulating with parasitic castrated females Emiliano Hernán Ocampo, Carmen Gilardoni, Jesús D. Nuñez, Florencia Cremonte, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5654364/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Marine Biology → Version 1 posted 4 You are reading this latest preprint version Abstract Discrimination mechanisms that allow males to select female partners are expected to evolve when a significant portion of the female population is sterilized. In this study, we investigated whether males of the slipper limpet species Bostrycapulus odites discriminate between parasitically castrated by a Microphallidae trematode and non-castrated females. We hypothesized that males would prefer healthy females, spend shorter periods on parasitized females, displace longer distances when encountering parasitized females, and abandon females if castrated. Field data revealed that only 7% of the males observed in copulatory positions were on parasitized females. Laboratory experiments confirmed that males primarily select non-parasitized females and exclusively copulate with healthy individuals. The duration of time spent by males on non-parasitized females was almost twice as long as that spent on parasitized females. These results indicate that males bias their mating efforts toward healthy females, and in the few cases where they interact with parasitized females, copulation does not occur. Instead, males spend brief periods on castrated females. Although uncommon in nature, this discriminative ability would be expected to benefit males by enhancing their reproductive success through mating with healthy females. Mating choice calyptraeid limpet digenean parasite intertidal rocky shore Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Parasitism can induce changes in the host behavior (Rosenkranz et al. 2018; Shaw and Wilson 2018), resulting in direct benefits to the parasites, such as an increased transmission rate to other hosts (Helluy and Helmus 1990). Conversely, hosts may evolve mechanisms whereby individuals of one sex can detect the presence of parasites and avoid copulating with infected individuals (e.g., Hamilton and Zuk 1982). The evolution of such mechanisms in hosts largely depends on the magnitude of the parasite effect (physiological and/or anatomic) (Ashby and Boots 2015). If the impact of parasitism on host fitness is substantial, host responses are expected to emerge. Castration is a parasitic strategy characterized by extreme effects on fitness (Sorensen and Minchella 2001) since sterilized individuals cannot reproduce (Lafferty and Kuris 2009). Therefore, sexual selection would favour individuals featuring pre-copulatory discrimination mechanisms permitting them to avoid mating with those castrated partners. Nevertheless, in very few host species the reproductive behavior preventing mating with castrated individuals has been identified (Rosenqvist and Johansson 1995). In the present study, we focus on revealing whether males of a slipper limpet species discriminate among parasitically castrated and non-castrated limpet females. Slipper limpets (family Calyptraeidae) are filter-feeding gastropods that typically inhabit intertidal and subtidal marine environments (Chaparro et al. 2002). They are protandric hermaphrodites, with a mobile male phase preceding the sessile female stage (Collin 2006). During copulation, the male climbs over the female and assumes a “copulatory position” in which it introduces its penis into the female’s vagina to transfer sperm (Beninger et al. 2016). Females may store spermatozoa from one or multiple males (Brante et al. 2011) for at least one year (Hoagland 1978). The infection by sporocyst (asexual larvae) of microphallid trematodes has been reported in some calyptraeids (Gilardoni et al. 2018; Quinn et al. 2022). These parasites exhibit a complex life cycle: free-living cercariae are released from mollusks (first intermediate host) and infect crustaceans (second intermediate host) to then be trophically transmitted to birds or mammals (the definitive host) (Diaz and Cremonte 2010). The trematode asexual larvae were reported to castrate the female mollusk by eliminating their ovarian tissues (Sorensen and Minchella 2001). In populations with a high prevalence of castrating parasites, the sexual maturation of limpet females occurs early, compensating for the future loss of reproduction (Gilardoni et al. 2012). The slipper limpet Bostrycapulus odites is distributed from São Paulo, Brazil, to Puerto Madryn, Argentina, with its native range also extending to the south coast of South Africa (Collin 2005). This species was also reported in the Spanish Mediterranean (Izquierdo et al. 2007), where it may be considered a potentially invasive species (Cledón et al. 2015). This limpet exhibits a direct development pattern with fully formed juveniles emerging from maternal capsules (Collin 2005). Adult females of B. odites are castrated by a pea crab, which does not remove its ovary but occupies the space necessary for oviposition (Ocampo et al. 2014). An unidentified trematode of the family Microphallidae also castrates B. odites ; however, in this case, the sporocysts of this trematode destroy the female gonadal tissue (unpubl data). A preliminary population survey suggested a low frequency of male limpets in copulatory positions with trematode-infected females, providing an excellent opportunity to investigate potential pre-copulatory discrimination mechanisms. In the present study, we experimentally assessed whether limpet males discriminate between non-parasitized females and castrated individuals. We hypothesized that males would prefer (herein preference and choice are used as synonyms, see Rosenthal 2017) healthy females over trematode-castrated individuals. We also experimentally analyzed whether the time that males spend over healthy females is greater than over castrated and if the abandon rate (males that leave females) varied between parasitized and non-parasitized females. In this case, we hypothesized that if males select parasitized females, these males would abandon parasitized individuals soon after they chose them. Lastly, we analyzed the distances traversed by males on different categories: those selecting only non-parasitized females versus those selecting parasitized females at least once. We speculated that males choosing parasitized females would face a cost in terms of transverse longer distances while finding the right mating partner. 2. MATERIAL AND METHODS 2.1. Sampling site Samples were collected during February 2020 at the low intertidal zone of San Antonio Oeste (S 40°43′, W 64°55′); a bay with a vast intertidal area (143 km 2 ) that opens to the San Matías Gulf, North Patagonia, Argentina. The limpet Bostrycapulus odites occurs in the intertidal and subtidal zones mainly attached to small-sized rocks. Multiple limpets often share the same rock, in most cases, they are solitary though stacks of up to three individuals are also observed. As a semidiurnal macro-tidal regime dominates this area, limpets are exposed to air during low tide and submerged up to 9 m depth when high tide floods the area. Specimens were sampled by hand during low tides in January 2020 (austral summer). Limpets attached to their substrata were maintained in plastic containers with aerated seawater and transported to the laboratory (Zoology of Invertebrates, University of Mar del Plata, Argentina). In the laboratory, limpets were maintained and acclimatized in containers of 20 L with aerated seawater at 20–22°C and 34 PSU for two weeks before the start of the experiment. During this period, limpets were fed daily with ad libitum aliquots of unicellular algae Nannochloropsis oculata and Isochrysis galbana . Some limpets attached to rocks were transported to the Laboratory of Parasitology (CENPAT, Puerto Madryn, Argentina) and analyzed soon after sampling to identify and estimate the prevalence of a trematode species (infected limpets/total limpets) by host sex and size. For this purpose, these limpets were coldly anaesthetized, dissected, and tissues were observed under steromicroscope to verify the presence of sporocysts. Due to the diagnostic characters in the cercaria, some specimens were stained with Nile Blue or Neutre Red, observed under a light microscope, and identified at the family level using specialized bibliography (Yamaguti 1975; Gilardoni et al. 2011). The sex of each limpet host was determined by observing the presence of the penis or genital pore (= papilla). When both sexual structures were present, the individual was classified as intersex (i.e., transitioning from male to female). Small individuals without penis were categorized as sexually immature juveniles. 2.2. Laboratory experiment on male behavior We evaluated whether ( 1 ) male limpets chose non-parasitized females over infected ones; ( 2 ) the rate at which males abandon parasitized females is greater than that of males leaving non-parasitized females; ( 3 ) the time that males spend over parasitized females is shorter than that spent over non-parasitized individuals; we evaluated ( 4 ) the differences in the displacement of males that chose non-parasitized females versus those that chose parasitized females. Additionally, we compared the displacement of males that abandoned a non-parasitized female to find another non-parasitized female, against those that abandoned a parasitized female to find a non-parasitized female. For these purposes, we conducted an experiment in which 22 females naturally attached to small-sized rocks were cultured surrounding a group of 15 males (Fig. S1). We used four independent aquaria of 10 L each, thus a total of 88 females and 60 males were employed. We set the experiment positioning females in a circle separated from the centre by 3 cm. Males were positioned on a rock in the center of the aquarium and from this place they dispersed looking for females (Fig. S1). Males used in the experiment were randomly extracted from rocks or females other than those used for the experiment. Thus, these experimental males were independent of females used in the experiment. Males were cultured in small containers (2 L) at least 1 week before the experiment. The sex of experimental males was confirmed by observing the penis. The shell length (SL) of each limpet was measured with a digital calliper to the nearest 0.1 mm. Each male and female individual was identified by painting unique numbers on shells. Females used were larger than 17.5 mm SL which is the mean body size of females in San Matias Gulf (Cledón et al. 2015). Selection of this size ensured that these individuals were not intersexual. Female sex and the presence of parasites were verified after the experiment finished. The number of limpets per rock varied from 1 to 4 depending on the size of the rock. No females were forming stackings. The rock containing the experimental males was included in the center of the container when the experiment started. In the field, we noticed that limpets without males over them exhibited a prevalence of sporocysts trematode of ~ 44%. Thus, we used in the experiment females which did not carry males when sampled to achieve an equivalent number of infected and non-infected individuals. Two times per day (early in the morning and late in the afternoon) we observed the experiment and computed the position of males. Photos were taken from a standardized height and position when we visually verified changes in the positions of males. A scale was included inside the experimental container to then measure the distance covered by males. In the few cases in which a male was not visually found, we carefully flipped each rock to determine if they were attached to the opposite side. In these cases, as pictures did not capture the actual place, the position of these males was computed as the center of the rock in which they were found. The total displacement of each male was calculated using basic functions of the Rstudio, after creating an x-y matrix of positions with the ImageJ software (Schneider et al. 2012). The displacement was used as a proxy for the movements made by males; however, it is important to note that we are not measuring the paths of those displacements. We considered males to be roaming if they were recorded on rocks and their position had changed from the previous record (i.e., 12 hours earlier). We considered males to be staying fixed in one place if they did not change relative to the previous record. Lastly, we considered that they were on the female’s shell (indicating they had chosen a female) when they climbed onto the female and positioned themselves over the anterior and right area of the female's shell (i.e., the copulatory position). The experiment was interrupted after 10 days when at least 70% of the males selected females. Two weeks before the start of the experiments, biological filters were confectioned on each experimental container and covered with substrata collected from the sampling site (fine sediment and little rocks). Limpets were fed daily as explained before. Aerated seawater was partially replaced every 4 days. After the experiment finished, each limpet was detached, cold anesthetized, sacrificed, and then prospected on a stereomicroscope to verify the presence of sporocysts trematode in the organs. There are several experimental designs commonly used to study mate choice (Dougherty 2020). However, our study system imposes certain limitations on the use of the no-choice design (a method in which a male decides whether to accept a given female or reject it and wait for a potentially better option). This design is typically used to measure absolute preference values. First, it is not possible to determine whether females are parasitized or non-parasitized prior to the experiment, as this requires sacrificing and dissecting the limpet to confirm its condition. Second, females of this limpet species begin to move once detached from their original substrate to be cultured individually, whereas under natural conditions they are sessile. These two particularities prevent the use of single females per trial, as required in no-choice experiments, because it involves removing them from their substrate. Given these constraints, we opted for an experimental design in which both parasitized and non-parasitized females are simultaneously presented to the male. The preference measured in such experiments reflects a relative preference, allowing us to assess whether males discriminate between parasitized and non-parasitized females. We used a two-way contingency table to evaluate differences in the proportion of males over parasitized versus non-parasitized females. The infected/uninfected females and the presence/absence of males on females were used as categorical variables. Similarly, we used a two-way contingency table to evaluate differences in the proportion of males that abandoned parasitized versus non-parasitized females. The infected/uninfected females and the abandonment/permanency of males were used as categorical variables. In both tables, the observed frequencies (either of female choice or male abandonment) were compared with the expected frequencies calculated under the null hypothesis of independence between the infection and the presence of males. In both tables, we did not use data from second or third female choices because selection of one female can influence following choice decisions. Significant differences between the observed and expected frequencies were examined using a Chi-square test of independence or Fisher’s exact test. The difference in the duration (in hours) that males spent over parasitized and non-parasitized females was evaluated using a Generalized Linear Mixed Model (GLMM). We considered only the time spent over the first selected female by each male; time spent on second or third females was excluded. The treatment (parasitized or non-parasitized female) was included as a fixed factor, and the time males spent on females as the response variable. Similarly, a second GLMM was used to assess differences in the displacements of males that selected parasitized versus non-parasitized females. In this case, the response variable was the total distance covered by each male from the start of the trial until the first female was selected. A third GLMM was fitted to compare the distances covered by males that initially selected a parasitized female and then moved to a non-parasitized one, versus males that selected a non-parasitized female and then moved to another non-parasitized female. For all models, a Gaussian distribution was specified using the lme4 package (Bates et al. 2015), following the recommendations of Zuur et al. (2009). All models initially included both the random effect of replicate (i.e., experimental aquarium) and its interaction with female categories (parasitized and non-parasitized females), to account for potential non-independence in the data structure. In cases there were unbalance number of observations per treatment, we used a type III Wald chi-square test to evaluate the effect of treatment using the car package (Fox and Weisberg 2019 ). Residual diagnostics were performed using the DHARMa package (Hartig 2022) to verify assumptions of homoscedasticity and normality. All statistical analyses described above and in the next section were conducted using R version 4.4.0. (R Core Team 2024). 2.3. Direct observation of copulation Field data and experiments (previous section) indicate a relative preference of males for non-parasitized females. To verify whether females with males over them are copulating or not, we conduct a series of direct observations. In the laboratory, 36 limpets that did not carry males over their shells when sampled were detached from their substrata and reattached to the glass sides of three different aquaria (12 females in each aquarium) of 22 L. Sex of these individuals was confirmed by observing the absence of penis or presence of the papilla. In each aquarium, 12 males were also included. Three times per day during 15 days the periphery of each aquarium was inspected to look for mating pairs. Once copulation was detected (by the observation of the penis introduced into the female papilla) the pairs were removed, the female was sacrificed, and trematode infection was evaluated as detailed before. One week before the experiments, biological filters were set up in those aquaria. Aerated seawater was partially replaced every 4 days and limpets were daily fed as detailed before. To determine whether copulation in limpets occurs independently of parasitic infection, we constructed a two-way contingency table. The infected/uninfected females and the occurrence of copulation were used as categorical variables. The observed frequencies of copulation were compared with expected frequencies calculated under the null hypothesis of independence between the infection and the copulation. Significant differences between the observed and expected frequencies were examined using Fisher's exact test (Sokal and Rohlf 1981). 3. Results 3.1. Prevalence of parasitism in the field A total of 251 limpets were sampled and then analyzed to evaluate the prevalence by host size and sex. 110 of them were males, 23 were intersex individuals, 109 were females, and 7 were small non-sexually differentiated juveniles (Fig. 1 ). The infection by the sporocysts of the trematode species of the family Microphallidae was identified in intersexual individuals and females while neither juveniles nor males showed sporocysts after carefully inspecting their organs (Fig. 1 ). The total prevalence of infected limpets was 19% but this value scaled up to 35.1% when we considered only intersex and female individuals. The smallest parasitized limpet was an intersexual individual of 13 mm of shell length. The prevalence increases from the smallest parasitized sizes up to ~ 15 mm of SL and over this size the prevalence remains between 30–40% approximately (Fig. 1 ). A total of 59 female and intersex individuals carried males over the shell. 93% of these male-carrying individuals were non-parasitized while 7% (4 individuals) of them were parasitized. By contrast, the prevalence of parasites among intersex and females which did not bear males was 44%. We consider these observations an indication that males prefer climbing over non-parasitized limpets over those parasitized. 3.2. Male behavior: female selection, female abandonment rate, duration over females and displacement. From the total of 88 female limpets used in the experiment, 55 individuals were non-parasitized (average ± SD of shell length: 18.5 ± 1.5) and 33 parasitized (18.9 ± 1.8) by the trematode. From the total of 60 males used (10.8 ± 1.7), 58 individuals were active while two of them remained in the same position where they were attached at the beginning of the experiment. These two males were discarded from the analysis. Males selected up to four females, including non-parasitized and parasitized individuals (Fig. 2 a). More males chose 1 female than 0, 2, 3 or 4 females (Fig. 2 a). A gradual increment in the selection of uninfected females was observed from the beginning of the experiment until the seventh day (Fig. 2 b). At the same time, the number of males displaying roaming declined during this period (Fig. 2 b). The number of males selecting parasitized females remained relatively constant throughout the experiment. Beyond this pattern, most males that chose females were observed to choose females immediately and remain over these limpets the time the experiment lasted. Comparatively fewer males were observed to choose females and then abandon them to roam around other females or select a new one; others roamed among rocks during the days, selecting females later, at the middle/end of the experiment, or even not selecting any female at all (see displacements by males in Fig. S2). For the few males that abandoned their first choice, we avoided including subsequent choices into the analyses to prevent non-independent data. A total of 37 males chose, firstly, non-parasitized females during the experimental period (Fig. 3 a). A total of eight males were observed to choice, firstly, parasitized females. The frequency of males choosing parasitized females is significantly lower than expected if mate selection were independent of parasitism, indicating that males discriminate between infected and non-infected females (Chi-square: χ² = 13.61, P < 0.0001; Fig. 3 a). The rate at which males abandon parasitized females after climbing and positioning themself over the shell was 62.5% (5 out of 8 individuals) while this rate was 24.3% (9 out of 37) in the case of uninfected females (Fig. 3 b). No differences, however, were detected in our analysis (Fisher's Exact Test: odds ratio = 4.96, 95% CI = 0.79 to 38.6, P = 0.085) indicating the rate of abandonment of males is independent of the parasitic infection of the female. On average, the 37 males that selected uninfected females spent 132.3 ± 76.2 hours (mean ± SD), while the 8 males that selected infected females spent 68.5 ± 56.7 hours over them (Fig. 4 a). We initially fitted a model including both the random effect of replicate and its interaction with female categories (infected vs. uninfected). The model showed singularity due to zero variance in the replicate term. Therefore, the final model retained only the replicate-by-female category interaction (accounting for approximately 6.6% of the total variance) as a random effect. A significant effect of female categories on the time males spent over females was detected (Type III Wald chi-square test, χ² ( 1 ) = 4.16, P = 0.041), indicating that males spent significantly more time over uninfected females than over infected ones. The average (± SD) displacement of males that chose a parasitized versus those that chose non-parasitized females was 11.68 ± 6.82 and 10.53 ± 3.21 centimeters, respectively (Fig. 4 b and 2 S). The initial model, which included replicate and its interaction with female categories as random effects, showed singularity due to zero variance components. We therefore simplified the model by excluding the random terms. No significant differences on the displacement of males (Type III Wald chi-square test, χ² ( 1 ) = 0.22, P = 0.644). For males that switched from one female to another, the fitted model included replicate and its interaction with female categories as random effects. The replicate term explained part of the variance in distance (30.47%), while its interaction had zero variance and was excluded to avoid singularity. The analysis showed a marginal effect of treatment on the displacement of males (estimate = 19.35 ± 7.00, t(4.05) = 2.76, P = 0.0501), suggesting that males switching from parasitized to non-parasitized females may have displaced greater distances than those switching between non-parasitized females (Fig. 4 c). However, given the small sample size in this test, this result should be interpreted with caution, and no strong conclusions can be drawn at this stage. 3.3. Direct observation of copulation A total of 12 out of the 36 limpets (average ± SD of shell length: 18.6 ± 2.6) exhibited trematode sporocysts in the ovary and other tissues while the other 24 (18.3 ± 2.5) individuals were not parasitized. Males used in this experiment exhibited an average (± SD) shell length of 11.6 ± 2.6 mm. The parasitized females did not copulate during the entire experimental period of 15 days (Fig. 5 a) which represents a lower frequency than expected under the null hypothesis of independence (Fisher's Exact Test: odds ratio = 0, 95% CI = 0 to 0.69, P = 0.009). In contrast, half of the non-parasitized limpets (i.e., 12 out of 24 individuals) were observed copulating during this period (Fig. 5 a). Copulation in these individuals was observed through the transparent glass; the male extends its large penis from the right size of the male’s head, and the penis passes under the female shell and it penetrates the female’s genital pore (Fig. 5 b). Then, our results showed the infection by the trematode avoided the copulation. 4. DISCUSSION Females and intersex individuals of the slipper limpet Bostrycapulus odites are parasitized by the asexual larvae of a trematode species of the family Microphallidae. This parasite reaches a considerably high prevalence in the analyzed population. The female gonadal acini of limpets are occupied by sporocysts (data not shown), similar to that observed in several other limpets parasitized by trematodes (Pechenik et al. 2012). Considering that these females are castrated, it would be expected that limpet males featured pre-copulatory discriminatory behavior to avoid copulating with parasitized females. Parasitic castrators may constitute a force that strongly shapes host life history traits (Lafferty and Kuris 2009); thus, sexual selection would favor individuals exhibiting mating choice mechanisms. Consistently with that expectation, in this study we find males of B. odites discriminated between parasitized and non-parasitized females. Our visual observations confirmed that the pairs engaged in copulation consisted of males and non-parasitized females. Our experimental results also revealed that the mean period of males over non-parasitized is more than twice that of males over parasitized individuals. Overall, field observations and experimental data indicate that males of B. odites bias their sexual efforts toward healthy females. In the few cases in which males climb over parasitized individuals, copulation does not occur; rather these males spend short periods over castrated females. In our experimental approach we excludes individuals that do not select females from the analysis and that exclusion may introduce some bias into the results (Dougherty and Shuker 2015). That is, by discarding non-choosing individuals, the proportion of selected males could be inflated and the preference overestimated. Nevertheless, the observed pattern is so pronounced (with only 7% of males choosing exclusively parasitized females in the laboratory, a result consistent with field observations) that it becomes challenging to explain the findings without considering parasitism as a key factor influencing male behavior. This strong discrimination towards non-parasitized females suggests that parasitism is indeed a primary determinant in mate choice. Our data showed that both males paired with castrated and non-parasitized females may abandon their chosen partners. The abandonment rate was higher for castrated females, although the difference was not statistically significant compared to healthy females. Regardless of parasitism, limpet males appear capable of shifting between females. In other slipper limpet species, males shift partner and copulate with multiple males (Brante et al. 2011). One possible explanation for the observed change in mating partner, independent of parasitism, could be that males of this species also mate multiple times. This possibility should be explored in future studies. The pre-copulatory discrimination behavior associated with the avoidance of parasitized individuals is common (e.g., Córdoba-Aguilar et al. 2003; Dass et al. 2011), particularly in hosts infected by sexually transmitted parasites (Ashby and Boots 2015). In contrast, pre-copulatory discrimination against parasitic castrated hosts has been observed in a few isolated studies. For example, females of a pipefish species exhibit a drastic reduction of fecundity when infected by a trematode (Rosenqvist and Johansson 1995). When given a choice, male pipefish discriminate between parasitized and healthy females (Rosenqvist and Johansson 1995). In the case of castrated mollusk species, the few studies conducted to date reported that hosts lack such pre-copulatory mechanisms. Males in the mud snails Potamopyrgus antipodarum persist in copulating with females infested by castrating trematodes (Neiman and Lively 2005). Similarly, males of the marine snail Littorina littorea engage in copulatory pairs despite females being infected by the castrator trematode Cryptocotyle lingua (Zimmerman 2007), although copulation with castrated females may be shorter (Saur 1990). The present study appears to be the first report of discrimination of castrated hosts in mollusks. It is not surprising that a species from the family Calyptraeidae exhibits this key of behaviors, as members of this group are recognized for displaying plastic behaviors associated with reproduction (Brante et al. 2016). For instance, slipper limpets exhibit multiple paternity (Dupont et al. 2006), and the degree of this phenomenon varies according to population context (Brante et al. 2011). Similarly, the timing of sex change can vary within a species depending on the social context (Mérot and Collin 2012). Furthermore, sex change has been observed as a plastic trait that may fluctuate with the intensity of parasitic infection (Gilardoni et al. 2012). Sexual selection appears to operate strongly within Calyptraeidae limpets. We do not know, based on our results, why the ability to discriminate among females evolved in this species. However, one possibility is that it relates to a network of direct benefits for males. By avoiding copulation with castrated females, males would increase their chances of reproducing successfully and prevent wasting costly sperm. This type of mechanism would be especially relevant in systems where trematode parasites that castrate their hosts reach high prevalence. While many trematodes show low prevalence in mollusks (< 5%) (Bagnato et al. 2015; Faltýnková et al. 2016), some groups, such as microphallids, can exceed 10% (Galaktionov and Bustnes 1999; Gilardoni et al. 2011, 2018). The high prevalence observed in our study could therefore represent a selective pressure favoring the evolution of pre-copulatory discrimination. In addition to potential benefits, males may also incur costs associated with mate searching. While we found no difference in the distances traveled by males that encountered parasitized versus non-parasitized females, some variation (though based on small sample sizes) was observed among males that abandoned a female and continued searching. Specifically, males that first encountered a parasitized female and later found a non-parasitized one traveled longer distances than those that abandoned non-parasitized females. This suggests that encountering parasitized females during mate searching may involve higher costs for certain males. Future studies should further investigate male movement patterns during mate searching and the potential energetic costs associated with these behaviors. In this study, we report that males of B. odites avoid castrated females. It is unknown, however, the mechanism by which male-female attraction fails. It may involve waterborne cues where males either perceive the infection or do not perceive a pheromone that is lost in castrated females but remains in healthy females. Alternatively, physical contact may mediate the avoidance mechanism. Contact-based chemical cues were reported to modulate the timing of the sex transition on a slipper limpet species (Carrillo-Baltodano and Collin 2015). Distinguishing between these possibilities will require further experiments. It is also unknown whether the trematode species promotes premature changes in the sex transition of B. odites . In the vicinities of the location we sampled B. odites another limpet species ( Crepipatella dilatata ) is castrated by another species of Microphallidae (Gilardoni et al. 2012). Sex transition from male to female occurs earlier in this population which may compensate for the loss of future reproduction (Gilardoni et al. 2012). In the locality we studied B. odites sizes at the female transition are smaller than in a potentially non-parasitized subtidal population (Cledón et al. 2015). This may be an indication that B. odites also responds to trematodes by transitioning soon from males to females, as occurs in C. dilatata . However, further information is required to unveil this possibility. Declarations Data availability Data will be made available on request to authors. Funding This work was partially supported by the followed grants: ANPCyT, PICT 2017-0373; ANPCyT, PICT 2019-2385; UNMdP, 2020 EXA953/20; CONICET, PIP 11220200102466CO. Competing Interests The authors declare no financial or non-financial conflict of interest. Supplementary Information The online version contains supplementary material available at XXX. Ethical standards No ethical approval was required. All applicable international, national, and institutional guidelines for sampling and experimental use of organisms have been followed. Author contributions All authors contributed to the study conception and sampling of individuals. Experimental design, data analysis, and interpretation were performed by Emiliano Ocampo, Tomás Luppi, and Jesús Nuñez. Florencia Cremonte and Carmen Gilardoni identified and analyzed parasites from limpets. Emiliano Ocampo wrote the first draft of the manuscript, and all authors commented on and edited previous versions. All authors read and approved the final manuscript. Acknowledgments We would like to thank Dr. Barbara Gorriti for providing us with algae species to feed the limpets during the experimental setup. We also thank the three anonymous reviewers and the associate editor for their invaluable contributions during the revision of this manuscript References Ashby B, Boots M (2015) Coevolution of parasite virulence and host mating strategies. Proc Natl Acad Sci USA 112(43):13290–13295. 10.1073/pnas.1508397112 Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67(1):1–48. 10.18637/jss.v067.i01 Beninger PG, Valdizan A, Le Pennec G (2016) The seminal receptacle and implications for reproductive processes in the invasive gastropod Crepidula fornicate . Zoology 119:4–10. https://doi.org/10.1016/j.zool.2015.09.001 Bagnato E, Gilardoni C, Di Giorgio G, Cremonte F (2015) A checklist of marine larval trematodes (Digenea) in molluscs from Argentina, Southwestern Atlantic coast. Check List 11:1706–1706. https://doi.org/10.15560/11.4.1706 Brante A, Fernández M, Viard F (2011) Microsatellite evidence for sperm storage and multiple paternity in the marine gastropod Crepidula coquimbensis . J Exp Mar Biol Ecol 396:83–88. https://doi.org/10.1016/j.jembe.2010.10.001 Brante A, Quiñones A, Silva F (2016) The relationship between sex change and reproductive success in a protandric marine gastropod. Sci Rep 6:29439. https://doi.org/10.1038/srep29439 Carrillo-Baltodano A, Collin R (2015) Crepidula slipper limpets alter sex change in response to physical contact with conspecifics. Biol Bull 229:232–242. 10.1086/BBLv229n3p232 Chaparro OR, Thompson RJ, Pereda SV (2002) Feeding mechanisms in the gastropod Crepidula fecunda . Mar Ecol Prog Ser 234:171–181. 10.3354/meps234171 Cledón M, Nuñez JD, Ocampo E, Sigwart JD (2015) Sexual traits plasticity of the potentially invasive limpet Bostrycapulus odites (Gastropoda: Calyptraeidae) within its natural distribution in South America. Mar Ecol 37:433–441. http://dx.doi.org/10.1111/maec.12329 Collin R (2005) Development, phylogeny, and taxonomy of Bostrycapulus (Caenogastropoda: Calyptraeidae), an ancient cryptic radiation. Zool J Linn Soc 144(1):75–101. https://doi.org/10.1111/j.1096-3642.2005.00162.x Collin R (2006) Sex ratio, life history invariants, and patterns of sex change in a family of protandrous gastropods. Evolution 60:735–745. https://doi.org/10.1111/j.0014-3820.2006.tb01152.x Córdoba-Aguilar A, Salamanca-Ocaña JC, Lopezaraiza M (2003) Female reproductive decisions and parasite burden in a calopterygid damselfly (Insecta: Odonata). Anim Behav 66:81–87. 10.1006/anbe.2003.2198 Diaz JI, Cremonte F (2010) Development from metacercaria to adult of a new species of Maritrema (Digenea: Microphallidae) parasitic in the kelp gull, Larus dominicanus , from the Patagonian coast. Argentina J Parasitol 96(4):740–745. http://dx.doi.org/10.1645/GE-2343.1 Dass SA, Vasudevan A, Dutta D, Soh LJ, Sapolsky RM, Vyas A (2011) Protozoan parasite Toxoplasma gondii manipulates mate choice in rats by enhancing attractiveness of males. PLoS ONE 6(11):e27229. 10.1371/journal.pone.0027229 Dougherty LR (2020) Designing mate choice experiments. Biol Rev 95:759–781. https://doi.org/10.1111/brv.12586 Dougherty LR, Shuker DM (2015) The effect of experimental design on the measurement of mate choice: a meta-analysis. Behav Ecol 26:311–319 Dupont L, Richard J, Paulet YM, Thouzeau G, Viard F (2006) Gregariousness and protandry promote reproductive insurance in the invasive gastropod Crepidula fornicata : evidence from assignment of larval paternity. Mol Ecol 15:3009–3021. 10.1111/j.1365-294X.2006.02988.x Faltýnková A, Sures B, Kostadinova A (2016) Biodiversity of trematodes in their intermediate mollusc and fish hosts in the freshwater ecosystems of Europe. Syst Parasitol 93:283–293. https://doi.org/10.1007/s11230-016-9627-y Fox J, Weisberg S (2019) An R Companion to Applied Regression, Third edition. Sage, Thousand Oaks CA. https://www.john-fox.ca/Companion/ Galaktionov KV, Bustnes JO (1999) Distribution patterns of marine bird digenean larvae in periwinkles along the southern coast of the Barents Sea. Dis Aquat Org 37:221–230. 10.3354/dao037221 Gilardoni C, Etchegoin J, Diaz JI, Ituarte C, Cremonte F (2011) A survey of larval digeneans in the commonest intertidal snails from Northern Patagonian coast, Argentina. Acta Parasitol 56:163–179. 10.2478/s11686-011-0021-2 Gilardoni C, Ituarte C, Cremonte F (2012) Castrating effects of trematode larvae on the reproductive success of a highly parasitized population of Crepipatella dilatata (Caenogastropoda) in Argentina. Mar Biol 159:2259–2267. 10.1007/s00227-012-2011-9 Gilardoni C, Di Giorgio G, Bagnato E, Cremonte F (2018) Survey of trematodes in intertidal snails from Patagonia, Argentina: new larval forms and diversity assessment. J Helminthol 1–10. https://doi.org/10.1017/S0022149X18000329 Hamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites? Science 218:384–387. 10.1126/science.7123238 Hartig F (2022) DHARMa: Residual Diagnostics for Hierarchical (Multi-Level/Mixed) Regression Models. R package version 0.4.6. 10.32614/CRAN.package.DHARMa Helluy S, Holmes JC (1990) Serotonin, octopamine, and the clinging behavior induced by the parasite Polymorphus paradoxus (Acanthocephala) in Gammarus lacustris (Crustacea). Can J Zool 68:1214–1220. https://doi.org/10.1139/z90-181 Hoagland KE (1978) Protandry and the evolution of environmentally-mediated sex change: a study of the Mollusca. Malacologia 17:365–391 Izquierdo A, Loya A, Díaz-Valdés M, Ramos-Espla AA (2007) Non-indigenous species at the Alicante Harbor (SE Spain): Oculina patagonica de Angelis, 1908 and Bostrycapulus aculeatus (Gmelin, 1791). Rapport Comm Int Mer Mediterranee 38:50–506 Lafferty KD, Kuris AM (2009) Parasitic castration: the evolution and ecology of body snatchers. Trends Parasitol 25:564–572. https://doi.org/10.1016/j.pt.2009.09.003 Mérot C, Collin R (2012) Effects of stress on sex change in Crepidula cf. marginalis (Gastropoda: Calyptraeidae). J Exp Mar Biol Ecol 416–417:68–71. https://doi.org/10.1016/j.jembe.2012.02.012 Neiman M, Lively CM (2005) Male New Zealand mud snails ( Potamopyrgus antipodarum ) persist in copulating with asexual and parasitically castrated females. Am Midl Nat 154:88–96. 10.1674/0003-0031 (2005)154[0088:MNZMSP]2.0.CO;2 Ocampo EH, Nuñez JD, Cledón M, Baeza JA (2014) Parasitic castration in slipper limpets infested by the symbiotic crab Calyptraeotheres garthi . Mar Biol 161:2107–2120. https://doi.org/10.1007/s00227-014-2490-y Pechenik JA, Fried B, Bolstridge J (2012) The marine Gastropods Crepidula plana and Crepidula convexa do not serve as first intermediate hosts for larval Trematode. Dev Comp Parasitol 79:5–8. https://doi.org/10.1654/4520.1 Poulin R, Mouritsen KN (2003) Large-scale determinants of trematode infections in intertidal gastropods. Mar Ecol Prog Ser 254:187–198. 10.3354/meps254187 Quinn EA, Thomas JE, Malkin SH, Eley M-J, Coates CJ, Rowley AF (2022) Invasive slipper limpets Crepidula fornicata are hosts for sterilizing digenean parasites. Parasitology 149:811–819. 10.1017/S0031182022000257 RStudio. Integrated Development Environment for R. RStudio Team, RStudio, Boston MA (2024) Disponible en: http://www.rstudio.com Rosenkranz M, Poulin R, Selbach C (2018) Behavioural impacts of trematodes on their snail host: Species-specific effects or generalised response? Ethology 124:790–1795. 10.1111/eth.12808 Rosenthal GG (2017) Mate Choice. Princeton University Press, Princeton Rosenqvist G, Johansson K (1995) Male avoidance of parasitized females explained by direct benefits in a pipefish. Anim Behav 49:1039–1045. https://doi.org/10.1006/anbe.1995.0133 Saur M (1990) Mate discrimination in Littorina littorea (L.) and L. saxatilis (Olivi) (Mollusca: Prosobranchia). Hydrobiologia 193:261–270. https://doi.org/10.1007/BF00028082 Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. 10.1038/nmeth.2089 Shaw JC, Wilson K (2018) Reproductive behavior and parasites: invertebrates. Academic, London Sokal RR, Rohlf FJ (1981) Biometry. Freeman, San Francisco Sorensen RE, Minchella DJ (2001) Snail–trematode life history interactions: past trends and future directions. Parasitology 123:S3–S18. 10.1017/s0031182001007843 Yamaguti S (1975) A synoptical review of life histories of digenetic trematodes of vertebrates with special reference to the morphology of their larval forms. Keigaku Publishing Co. Tokyo Zimmerman AF (2007) Shooting blanks: Mate choice and determination in a parasitically castrated snail, Littorina littorea . Honors Thesis. Cornell University Zuur AF, Ieno EN, Walker N, Saveliev AA, Smith GM (2009) GLMM and GAMM. Mixed Effects Models and Extensions in Ecology with R, pp. 323–341. 10.18637/jss.v032.b01 Supplementary Files Fig1S.jpg Fig. S1 Position of limpets at the onset (a) and end (b) of the experiment. At the beginning, the males were positioned on a central rock, which is outlined in yellow in the picture. Rocks containing females are outlined with light blue. All female and male individuals are outlined with black to facilitate visualization Fig2S.jpg Fig. S2 Trajectories of males that roamed around rocks without selecting any female (light red), males that selected one or more non-parasitized females (green), and males that selected at least one parasitized female (blue) during the experiments. White dots show the start points of males and colored dots indicate the final points. The images correspond to the four experimental replicates at the end of the experiment. The heat map shows the density of dots of the male trajectories Cite Share Download PDF Status: Published Journal Publication published 30 Sep, 2025 Read the published version in Marine Biology → Version 1 posted Reviewers agreed at journal 22 Apr, 2025 Reviewers invited by journal 22 Apr, 2025 Editor assigned by journal 21 Apr, 2025 First submitted to journal 19 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5654364","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446589206,"identity":"d182f726-83d4-4388-8131-80ed633b756f","order_by":0,"name":"Emiliano Hernán Ocampo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBADOQaGBCDFRoIWY9K1JDYQrYWfvfnhp5s7bNLXtuceYPhQdpiBf/YB/Foke44ZS+eeScvdduZdAuOMc4cZJM4l4NdicCOHQTq37XDuths5Bsy8bYcZGM4QcJj9jRzm30At6WYgLX+BWuQJaTGQyGED2ZIA1sII1GJASIvEmWNm1kC/GIL8crDnXDqPISEt/O3Nj2/n7rCRNzuee/DBjzJrOTlCWsCAsQFE8jAcAJNEAZiWUTAKRsEoGAVYAQDBTUWYuwynwgAAAABJRU5ErkJggg==","orcid":"","institution":"IIMYC: Instituto de Investigaciones Marinas y Costeras","correspondingAuthor":true,"prefix":"","firstName":"Emiliano","middleName":"Hernán","lastName":"Ocampo","suffix":""},{"id":446589207,"identity":"67c6cd4a-c477-4c4a-ac8b-ce4c694585c6","order_by":1,"name":"Carmen Gilardoni","email":"","orcid":"","institution":"Patagonian National Centre: CONICET CENPAT","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Gilardoni","suffix":""},{"id":446589208,"identity":"9585b80c-7e58-408a-b1ff-b569e95ca836","order_by":2,"name":"Jesús D. Nuñez","email":"","orcid":"","institution":"IIMYC: Instituto de Investigaciones Marinas y Costeras","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"D.","lastName":"Nuñez","suffix":""},{"id":446589209,"identity":"cb080d79-f7cc-4ae7-8e2f-3795ffd63b98","order_by":3,"name":"Florencia Cremonte","email":"","orcid":"","institution":"Centro Nacional Patagónico: CONICET CENPAT","correspondingAuthor":false,"prefix":"","firstName":"Florencia","middleName":"","lastName":"Cremonte","suffix":""},{"id":446589210,"identity":"70e38534-1682-4c8a-8022-107b242f5400","order_by":4,"name":"Tomás A. Luppi","email":"","orcid":"","institution":"IIMYC: Instituto de Investigaciones Marinas y Costeras","correspondingAuthor":false,"prefix":"","firstName":"Tomás","middleName":"A.","lastName":"Luppi","suffix":""}],"badges":[],"createdAt":"2024-12-16 13:44:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5654364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5654364/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00227-025-04715-3","type":"published","date":"2025-09-30T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81512534,"identity":"f31aa45d-6099-4016-a58b-64159b16a0eb","added_by":"auto","created_at":"2025-04-28 06:27:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51061,"visible":true,"origin":"","legend":"\u003cp\u003eTop, prevalence (percentage of occurrence) of the microphallid trematode in limpets of \u003cem\u003eBostrycapulus odites \u003c/em\u003efrom different size classes. At left, an \u003cem\u003ein vivo\u003c/em\u003e light microscope photograph of the microphallid cercaria extracted from a sporocyst. Bottom, The distribution of body sizes in juveniles, intersexes, males, and females of the limpet.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/7bf57e7c24b0c076b2c8803c.jpg"},{"id":81512529,"identity":"acb2fc13-5eae-40c6-b25f-68be170ec875","added_by":"auto","created_at":"2025-04-28 06:27:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71075,"visible":true,"origin":"","legend":"\u003cp\u003ea. Distribution of the number of female limpets of \u003cem\u003eBostrycapulus odites \u003c/em\u003eselected by males in the experiment. b. Proportion of the behaviors displayed by limpet males during the experimental period\u003c/p\u003e","description":"","filename":"figura2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/11b81062e8f17cd127aa7ec3.jpg"},{"id":81512558,"identity":"b0e6612f-2b08-41cd-8826-0b0d8bf5c6ff","added_by":"auto","created_at":"2025-04-28 06:27:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59550,"visible":true,"origin":"","legend":"\u003cp\u003ea. Expected and observed number of male limpets of \u003cem\u003eBostrycapulus odites \u003c/em\u003eover the shell of parasitized and non-parasitized females in the experiment. b. Expected and observed number of males that abandoned parasitized and non-parasitized females in the experiment\u003c/p\u003e","description":"","filename":"figura3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/09208cdcf27e3d287e7a1bd5.jpg"},{"id":81512525,"identity":"6ad4f92a-86ea-4d40-8690-8d3c7cf619c5","added_by":"auto","created_at":"2025-04-28 06:27:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52588,"visible":true,"origin":"","legend":"\u003cp\u003ea. Boxplot showing the time (in hours) that Bostrycapulus odites males spent on parasitized versus non-parasitized females. b. Boxplot of the displacement of males that chose non-parasitized versus parasitized females. c. Boxplot of the displacement of males that selected a non-parasitized female after abandoning either a non-parasitized or a parasitized female.\u003c/p\u003e","description":"","filename":"figura4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/0d0a9b15ad11212cd699a210.jpg"},{"id":81512533,"identity":"c1307092-babf-43be-be8e-c8ec265bff18","added_by":"auto","created_at":"2025-04-28 06:27:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":156080,"visible":true,"origin":"","legend":"\u003cp\u003ea. Number of copulations observed and expected between parasitized and non-parasitized female limpets of \u003cem\u003eBostrycapulus odites \u003c/em\u003eduring the experimental period. b. A ventral view of a female limpet detailing the insertion of the male´s penis (surrounded by an oval) partially covered by an anterior fold of the female’s foot\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/2cff1b8050f9a16e416c06cd.png"},{"id":92883990,"identity":"6d503f62-0cf0-4bd5-8cf6-adae5e9bcaff","added_by":"auto","created_at":"2025-10-06 16:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":975732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/d950a795-f2cc-4ad4-9a42-4f39c4dfc6dc.pdf"},{"id":81512550,"identity":"0ba84783-57fe-4995-9e84-d2d4e967fc3c","added_by":"auto","created_at":"2025-04-28 06:27:09","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":149858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e Position of limpets at the onset (a) and end (b) of the experiment. At the beginning, the males were positioned on a central rock, which is outlined in yellow in the picture. Rocks containing females are outlined with light blue. All female and male individuals are outlined with black to facilitate visualization\u003c/p\u003e","description":"","filename":"Fig1S.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/863559bc7c4ef46b36e3877d.jpg"},{"id":81512527,"identity":"7e020f17-7ca0-42ea-afa7-d130c8be3e5a","added_by":"auto","created_at":"2025-04-28 06:27:06","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":122439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2\u003c/strong\u003e Trajectories of males that roamed around rocks without selecting any female (light red), males that selected one or more non-parasitized females (green), and males that selected at least one parasitized female (blue) during the experiments. White dots show the start points of males and colored dots indicate the final points. The images correspond to the four experimental replicates at the end of the experiment. The heat map shows the density of dots of the male trajectories\u003c/p\u003e","description":"","filename":"Fig2S.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654364/v1/2fad5478efd1ddfe8eb880bc.jpg"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSlipper limpet males avoid copulating with parasitic castrated females\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eParasitism can induce changes in the host behavior (Rosenkranz et al. 2018; Shaw and Wilson 2018), resulting in direct benefits to the parasites, such as an increased transmission rate to other hosts (Helluy and Helmus 1990). Conversely, hosts may evolve mechanisms whereby individuals of one sex can detect the presence of parasites and avoid copulating with infected individuals (e.g., Hamilton and Zuk 1982). The evolution of such mechanisms in hosts largely depends on the magnitude of the parasite effect (physiological and/or anatomic) (Ashby and Boots 2015). If the impact of parasitism on host fitness is substantial, host responses are expected to emerge. Castration is a parasitic strategy characterized by extreme effects on fitness (Sorensen and Minchella 2001) since sterilized individuals cannot reproduce (Lafferty and Kuris 2009). Therefore, sexual selection would favour individuals featuring pre-copulatory discrimination mechanisms permitting them to avoid mating with those castrated partners. Nevertheless, in very few host species the reproductive behavior preventing mating with castrated individuals has been identified (Rosenqvist and Johansson 1995). In the present study, we focus on revealing whether males of a slipper limpet species discriminate among parasitically castrated and non-castrated limpet females.\u003c/p\u003e \u003cp\u003eSlipper limpets (family Calyptraeidae) are filter-feeding gastropods that typically inhabit intertidal and subtidal marine environments (Chaparro et al. 2002). They are protandric hermaphrodites, with a mobile male phase preceding the sessile female stage (Collin 2006). During copulation, the male climbs over the female and assumes a \u0026ldquo;copulatory position\u0026rdquo; in which it introduces its penis into the female\u0026rsquo;s vagina to transfer sperm (Beninger et al. 2016). Females may store spermatozoa from one or multiple males (Brante et al. 2011) for at least one year (Hoagland 1978). The infection by sporocyst (asexual larvae) of microphallid trematodes has been reported in some calyptraeids (Gilardoni et al. 2018; Quinn et al. 2022). These parasites exhibit a complex life cycle: free-living cercariae are released from mollusks (first intermediate host) and infect crustaceans (second intermediate host) to then be trophically transmitted to birds or mammals (the definitive host) (Diaz and Cremonte 2010). The trematode asexual larvae were reported to castrate the female mollusk by eliminating their ovarian tissues (Sorensen and Minchella 2001). In populations with a high prevalence of castrating parasites, the sexual maturation of limpet females occurs early, compensating for the future loss of reproduction (Gilardoni et al. 2012).\u003c/p\u003e \u003cp\u003eThe slipper limpet \u003cem\u003eBostrycapulus odites\u003c/em\u003e is distributed from S\u0026atilde;o Paulo, Brazil, to Puerto Madryn, Argentina, with its native range also extending to the south coast of South Africa (Collin 2005). This species was also reported in the Spanish Mediterranean (Izquierdo et al. 2007), where it may be considered a potentially invasive species (Cled\u0026oacute;n et al. 2015). This limpet exhibits a direct development pattern with fully formed juveniles emerging from maternal capsules (Collin 2005). Adult females of \u003cem\u003eB. odites\u003c/em\u003e are castrated by a pea crab, which does not remove its ovary but occupies the space necessary for oviposition (Ocampo et al. 2014). An unidentified trematode of the family Microphallidae also castrates \u003cem\u003eB. odites\u003c/em\u003e; however, in this case, the sporocysts of this trematode destroy the female gonadal tissue (unpubl data). A preliminary population survey suggested a low frequency of male limpets in copulatory positions with trematode-infected females, providing an excellent opportunity to investigate potential pre-copulatory discrimination mechanisms.\u003c/p\u003e \u003cp\u003eIn the present study, we experimentally assessed whether limpet males discriminate between non-parasitized females and castrated individuals. We hypothesized that males would prefer (herein preference and choice are used as synonyms, see Rosenthal 2017) healthy females over trematode-castrated individuals. We also experimentally analyzed whether the time that males spend over healthy females is greater than over castrated and if the abandon rate (males that leave females) varied between parasitized and non-parasitized females. In this case, we hypothesized that if males select parasitized females, these males would abandon parasitized individuals soon after they chose them. Lastly, we analyzed the distances traversed by males on different categories: those selecting only non-parasitized females versus those selecting parasitized females at least once. We speculated that males choosing parasitized females would face a cost in terms of transverse longer distances while finding the right mating partner.\u003c/p\u003e"},{"header":"2. MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sampling site\u003c/h2\u003e \u003cp\u003eSamples were collected during February 2020 at the low intertidal zone of San Antonio Oeste (S 40\u0026deg;43\u0026prime;, W 64\u0026deg;55\u0026prime;); a bay with a vast intertidal area (143 km\u003csup\u003e2\u003c/sup\u003e) that opens to the San Mat\u0026iacute;as Gulf, North Patagonia, Argentina. The limpet \u003cem\u003eBostrycapulus odites\u003c/em\u003e occurs in the intertidal and subtidal zones mainly attached to small-sized rocks. Multiple limpets often share the same rock, in most cases, they are solitary though stacks of up to three individuals are also observed. As a semidiurnal macro-tidal regime dominates this area, limpets are exposed to air during low tide and submerged up to 9 m depth when high tide floods the area. Specimens were sampled by hand during low tides in January 2020 (austral summer). Limpets attached to their substrata were maintained in plastic containers with aerated seawater and transported to the laboratory (Zoology of Invertebrates, University of Mar del Plata, Argentina). In the laboratory, limpets were maintained and acclimatized in containers of 20 L with aerated seawater at 20\u0026ndash;22\u0026deg;C and 34 PSU for two weeks before the start of the experiment. During this period, limpets were fed daily with \u003cem\u003ead libitum\u003c/em\u003e aliquots of unicellular algae \u003cem\u003eNannochloropsis oculata\u003c/em\u003e and \u003cem\u003eIsochrysis galbana\u003c/em\u003e. Some limpets attached to rocks were transported to the Laboratory of Parasitology (CENPAT, Puerto Madryn, Argentina) and analyzed soon after sampling to identify and estimate the prevalence of a trematode species (infected limpets/total limpets) by host sex and size. For this purpose, these limpets were coldly anaesthetized, dissected, and tissues were observed under steromicroscope to verify the presence of sporocysts. Due to the diagnostic characters in the cercaria, some specimens were stained with Nile Blue or Neutre Red, observed under a light microscope, and identified at the family level using specialized bibliography (Yamaguti 1975; Gilardoni et al. 2011). The sex of each limpet host was determined by observing the presence of the penis or genital pore (=\u0026thinsp;papilla). When both sexual structures were present, the individual was classified as intersex (i.e., transitioning from male to female). Small individuals without penis were categorized as sexually immature juveniles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Laboratory experiment on male behavior\u003c/h2\u003e \u003cp\u003eWe evaluated whether (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) male limpets chose non-parasitized females over infected ones; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) the rate at which males abandon parasitized females is greater than that of males leaving non-parasitized females; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) the time that males spend over parasitized females is shorter than that spent over non-parasitized individuals; we evaluated (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) the differences in the displacement of males that chose non-parasitized females versus those that chose parasitized females. Additionally, we compared the displacement of males that abandoned a non-parasitized female to find another non-parasitized female, against those that abandoned a parasitized female to find a non-parasitized female. For these purposes, we conducted an experiment in which 22 females naturally attached to small-sized rocks were cultured surrounding a group of 15 males (Fig. S1). We used four independent aquaria of 10 L each, thus a total of 88 females and 60 males were employed. We set the experiment positioning females in a circle separated from the centre by 3 cm. Males were positioned on a rock in the center of the aquarium and from this place they dispersed looking for females (Fig. S1). Males used in the experiment were randomly extracted from rocks or females other than those used for the experiment. Thus, these experimental males were independent of females used in the experiment. Males were cultured in small containers (2 L) at least 1 week before the experiment. The sex of experimental males was confirmed by observing the penis. The shell length (SL) of each limpet was measured with a digital calliper to the nearest 0.1 mm. Each male and female individual was identified by painting unique numbers on shells. Females used were larger than 17.5 mm SL which is the mean body size of females in San Matias Gulf (Cled\u0026oacute;n et al. 2015). Selection of this size ensured that these individuals were not intersexual. Female sex and the presence of parasites were verified after the experiment finished. The number of limpets per rock varied from 1 to 4 depending on the size of the rock. No females were forming stackings. The rock containing the experimental males was included in the center of the container when the experiment started. In the field, we noticed that limpets without males over them exhibited a prevalence of sporocysts trematode of ~\u0026thinsp;44%. Thus, we used in the experiment females which did not carry males when sampled to achieve an equivalent number of infected and non-infected individuals. Two times per day (early in the morning and late in the afternoon) we observed the experiment and computed the position of males. Photos were taken from a standardized height and position when we visually verified changes in the positions of males. A scale was included inside the experimental container to then measure the distance covered by males. In the few cases in which a male was not visually found, we carefully flipped each rock to determine if they were attached to the opposite side. In these cases, as pictures did not capture the actual place, the position of these males was computed as the center of the rock in which they were found. The total displacement of each male was calculated using basic functions of the Rstudio, after creating an x-y matrix of positions with the ImageJ software (Schneider et al. 2012). The displacement was used as a proxy for the movements made by males; however, it is important to note that we are not measuring the paths of those displacements. We considered males to be roaming if they were recorded on rocks and their position had changed from the previous record (i.e., 12 hours earlier). We considered males to be staying fixed in one place if they did not change relative to the previous record. Lastly, we considered that they were on the female\u0026rsquo;s shell (indicating they had chosen a female) when they climbed onto the female and positioned themselves over the anterior and right area of the female's shell (i.e., the copulatory position). The experiment was interrupted after 10 days when at least 70% of the males selected females. Two weeks before the start of the experiments, biological filters were confectioned on each experimental container and covered with substrata collected from the sampling site (fine sediment and little rocks). Limpets were fed daily as explained before. Aerated seawater was partially replaced every 4 days. After the experiment finished, each limpet was detached, cold anesthetized, sacrificed, and then prospected on a stereomicroscope to verify the presence of sporocysts trematode in the organs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThere are several experimental designs commonly used to study mate choice (Dougherty 2020). However, our study system imposes certain limitations on the use of the no-choice design (a method in which a male decides whether to accept a given female or reject it and wait for a potentially better option). This design is typically used to measure absolute preference values. First, it is not possible to determine whether females are parasitized or non-parasitized prior to the experiment, as this requires sacrificing and dissecting the limpet to confirm its condition. Second, females of this limpet species begin to move once detached from their original substrate to be cultured individually, whereas under natural conditions they are sessile. These two particularities prevent the use of single females per trial, as required in no-choice experiments, because it involves removing them from their substrate. Given these constraints, we opted for an experimental design in which both parasitized and non-parasitized females are simultaneously presented to the male. The preference measured in such experiments reflects a relative preference, allowing us to assess whether males discriminate between parasitized and non-parasitized females.\u003c/p\u003e \u003cp\u003eWe used a two-way contingency table to evaluate differences in the proportion of males over parasitized versus non-parasitized females. The infected/uninfected females and the presence/absence of males on females were used as categorical variables. Similarly, we used a two-way contingency table to evaluate differences in the proportion of males that abandoned parasitized versus non-parasitized females. The infected/uninfected females and the abandonment/permanency of males were used as categorical variables. In both tables, the observed frequencies (either of female choice or male abandonment) were compared with the expected frequencies calculated under the null hypothesis of independence between the infection and the presence of males. In both tables, we did not use data from second or third female choices because selection of one female can influence following choice decisions. Significant differences between the observed and expected frequencies were examined using a Chi-square test of independence or Fisher\u0026rsquo;s exact test.\u003c/p\u003e \u003cp\u003eThe difference in the duration (in hours) that males spent over parasitized and non-parasitized females was evaluated using a Generalized Linear Mixed Model (GLMM). We considered only the time spent over the first selected female by each male; time spent on second or third females was excluded. The treatment (parasitized or non-parasitized female) was included as a fixed factor, and the time males spent on females as the response variable. Similarly, a second GLMM was used to assess differences in the displacements of males that selected parasitized versus non-parasitized females. In this case, the response variable was the total distance covered by each male from the start of the trial until the first female was selected. A third GLMM was fitted to compare the distances covered by males that initially selected a parasitized female and then moved to a non-parasitized one, versus males that selected a non-parasitized female and then moved to another non-parasitized female. For all models, a Gaussian distribution was specified using the lme4 package (Bates et al. 2015), following the recommendations of Zuur et al. (2009). All models initially included both the random effect of replicate (i.e., experimental aquarium) and its interaction with female categories (parasitized and non-parasitized females), to account for potential non-independence in the data structure. In cases there were unbalance number of observations per treatment, we used a type III Wald chi-square test to evaluate the effect of treatment using the car package (Fox and Weisberg 2019\u003cb\u003e).\u003c/b\u003e Residual diagnostics were performed using the DHARMa package (Hartig 2022) to verify assumptions of homoscedasticity and normality. All statistical analyses described above and in the next section were conducted using R version 4.4.0. (R Core Team 2024).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Direct observation of copulation\u003c/h2\u003e \u003cp\u003eField data and experiments (previous section) indicate a relative preference of males for non-parasitized females. To verify whether females with males over them are copulating or not, we conduct a series of direct observations. In the laboratory, 36 limpets that did not carry males over their shells when sampled were detached from their substrata and reattached to the glass sides of three different aquaria (12 females in each aquarium) of 22 L. Sex of these individuals was confirmed by observing the absence of penis or presence of the papilla. In each aquarium, 12 males were also included. Three times per day during 15 days the periphery of each aquarium was inspected to look for mating pairs. Once copulation was detected (by the observation of the penis introduced into the female papilla) the pairs were removed, the female was sacrificed, and trematode infection was evaluated as detailed before. One week before the experiments, biological filters were set up in those aquaria. Aerated seawater was partially replaced every 4 days and limpets were daily fed as detailed before.\u003c/p\u003e \u003cp\u003eTo determine whether copulation in limpets occurs independently of parasitic infection, we constructed a two-way contingency table. The infected/uninfected females and the occurrence of copulation were used as categorical variables. The observed frequencies of copulation were compared with expected frequencies calculated under the null hypothesis of independence between the infection and the copulation. Significant differences between the observed and expected frequencies were examined using Fisher's exact test (Sokal and Rohlf 1981).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Prevalence of parasitism in the field\u003c/h2\u003e \u003cp\u003eA total of 251 limpets were sampled and then analyzed to evaluate the prevalence by host size and sex. 110 of them were males, 23 were intersex individuals, 109 were females, and 7 were small non-sexually differentiated juveniles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The infection by the sporocysts of the trematode species of the family Microphallidae was identified in intersexual individuals and females while neither juveniles nor males showed sporocysts after carefully inspecting their organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The total prevalence of infected limpets was 19% but this value scaled up to 35.1% when we considered only intersex and female individuals. The smallest parasitized limpet was an intersexual individual of 13 mm of shell length. The prevalence increases from the smallest parasitized sizes up to ~\u0026thinsp;15 mm of SL and over this size the prevalence remains between 30\u0026ndash;40% approximately (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A total of 59 female and intersex individuals carried males over the shell. 93% of these male-carrying individuals were non-parasitized while 7% (4 individuals) of them were parasitized. By contrast, the prevalence of parasites among intersex and females which did not bear males was 44%. We consider these observations an indication that males prefer climbing over non-parasitized limpets over those parasitized.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Male behavior: female selection, female abandonment rate, duration over females and displacement.\u003c/h2\u003e \u003cp\u003eFrom the total of 88 female limpets used in the experiment, 55 individuals were non-parasitized (average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of shell length: 18.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5) and 33 parasitized (18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8) by the trematode. From the total of 60 males used (10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7), 58 individuals were active while two of them remained in the same position where they were attached at the beginning of the experiment. These two males were discarded from the analysis. Males selected up to four females, including non-parasitized and parasitized individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). More males chose 1 female than 0, 2, 3 or 4 females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). A gradual increment in the selection of uninfected females was observed from the beginning of the experiment until the seventh day (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). At the same time, the number of males displaying roaming declined during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The number of males selecting parasitized females remained relatively constant throughout the experiment. Beyond this pattern, most males that chose females were observed to choose females immediately and remain over these limpets the time the experiment lasted. Comparatively fewer males were observed to choose females and then abandon them to roam around other females or select a new one; others roamed among rocks during the days, selecting females later, at the middle/end of the experiment, or even not selecting any female at all (see displacements by males in Fig. S2). For the few males that abandoned their first choice, we avoided including subsequent choices into the analyses to prevent non-independent data. A total of 37 males chose, firstly, non-parasitized females during the experimental period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). A total of eight males were observed to choice, firstly, parasitized females. The frequency of males choosing parasitized females is significantly lower than expected if mate selection were independent of parasitism, indicating that males discriminate between infected and non-infected females (Chi-square: χ\u0026sup2; = 13.61, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The rate at which males abandon parasitized females after climbing and positioning themself over the shell was 62.5% (5 out of 8 individuals) while this rate was 24.3% (9 out of 37) in the case of uninfected females (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). No differences, however, were detected in our analysis (Fisher's Exact Test: odds ratio\u0026thinsp;=\u0026thinsp;4.96, 95% \u003cem\u003eCI\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.79 to 38.6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.085) indicating the rate of abandonment of males is independent of the parasitic infection of the female. On average, the 37 males that selected uninfected females spent 132.3\u0026thinsp;\u0026plusmn;\u0026thinsp;76.2 hours (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), while the 8 males that selected infected females spent 68.5\u0026thinsp;\u0026plusmn;\u0026thinsp;56.7 hours over them (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). We initially fitted a model including both the random effect of replicate and its interaction with female categories (infected vs. uninfected). The model showed singularity due to zero variance in the replicate term. Therefore, the final model retained only the replicate-by-female category interaction (accounting for approximately 6.6% of the total variance) as a random effect. A significant effect of female categories on the time males spent over females was detected (Type III Wald chi-square test, \u003cem\u003eχ\u0026sup2;\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.16, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041), indicating that males spent significantly more time over uninfected females than over infected ones. The average (\u0026plusmn;\u0026thinsp;SD) displacement of males that chose a parasitized versus those that chose non-parasitized females was 11.68\u0026thinsp;\u0026plusmn;\u0026thinsp;6.82 and 10.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21 centimeters, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eS). The initial model, which included replicate and its interaction with female categories as random effects, showed singularity due to zero variance components. We therefore simplified the model by excluding the random terms. No significant differences on the displacement of males (Type III Wald chi-square test, \u003cem\u003eχ\u0026sup2;\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.644). For males that switched from one female to another, the fitted model included replicate and its interaction with female categories as random effects. The replicate term explained part of the variance in distance (30.47%), while its interaction had zero variance and was excluded to avoid singularity. The analysis showed a marginal effect of treatment on the displacement of males (estimate\u0026thinsp;=\u0026thinsp;19.35\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00, \u003cem\u003et(4.05)\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.76, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0501), suggesting that males switching from parasitized to non-parasitized females may have displaced greater distances than those switching between non-parasitized females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). However, given the small sample size in this test, this result should be interpreted with caution, and no strong conclusions can be drawn at this stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Direct observation of copulation\u003c/h2\u003e \u003cp\u003eA total of 12 out of the 36 limpets (average\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of shell length: 18.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6) exhibited trematode sporocysts in the ovary and other tissues while the other 24 (18.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5) individuals were not parasitized. Males used in this experiment exhibited an average (\u0026plusmn;\u0026thinsp;SD) shell length of 11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 mm. The parasitized females did not copulate during the entire experimental period of 15 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) which represents a lower frequency than expected under the null hypothesis of independence (Fisher's Exact Test: odds ratio\u0026thinsp;=\u0026thinsp;0, 95% \u003cem\u003eCI\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0 to 0.69, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009). In contrast, half of the non-parasitized limpets (i.e., 12 out of 24 individuals) were observed copulating during this period (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Copulation in these individuals was observed through the transparent glass; the male extends its large penis from the right size of the male\u0026rsquo;s head, and the penis passes under the female shell and it penetrates the female\u0026rsquo;s genital pore (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Then, our results showed the infection by the trematode avoided the copulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eFemales and intersex individuals of the slipper limpet \u003cem\u003eBostrycapulus odites\u003c/em\u003e are parasitized by the asexual larvae of a trematode species of the family Microphallidae. This parasite reaches a considerably high prevalence in the analyzed population. The female gonadal acini of limpets are occupied by sporocysts (data not shown), similar to that observed in several other limpets parasitized by trematodes (Pechenik et al. 2012). Considering that these females are castrated, it would be expected that limpet males featured pre-copulatory discriminatory behavior to avoid copulating with parasitized females. Parasitic castrators may constitute a force that strongly shapes host life history traits (Lafferty and Kuris 2009); thus, sexual selection would favor individuals exhibiting mating choice mechanisms. Consistently with that expectation, in this study we find males of \u003cem\u003eB. odites\u003c/em\u003e discriminated between parasitized and non-parasitized females. Our visual observations confirmed that the pairs engaged in copulation consisted of males and non-parasitized females. Our experimental results also revealed that the mean period of males over non-parasitized is more than twice that of males over parasitized individuals. Overall, field observations and experimental data indicate that males of \u003cem\u003eB. odites\u003c/em\u003e bias their sexual efforts toward healthy females. In the few cases in which males climb over parasitized individuals, copulation does not occur; rather these males spend short periods over castrated females.\u003c/p\u003e \u003cp\u003eIn our experimental approach we excludes individuals that do not select females from the analysis and that exclusion may introduce some bias into the results (Dougherty and Shuker 2015). That is, by discarding non-choosing individuals, the proportion of selected males could be inflated and the preference overestimated. Nevertheless, the observed pattern is so pronounced (with only 7% of males choosing exclusively parasitized females in the laboratory, a result consistent with field observations) that it becomes challenging to explain the findings without considering parasitism as a key factor influencing male behavior. This strong discrimination towards non-parasitized females suggests that parasitism is indeed a primary determinant in mate choice.\u003c/p\u003e \u003cp\u003eOur data showed that both males paired with castrated and non-parasitized females may abandon their chosen partners. The abandonment rate was higher for castrated females, although the difference was not statistically significant compared to healthy females. Regardless of parasitism, limpet males appear capable of shifting between females. In other slipper limpet species, males shift partner and copulate with multiple males (Brante et al. 2011). One possible explanation for the observed change in mating partner, independent of parasitism, could be that males of this species also mate multiple times. This possibility should be explored in future studies.\u003c/p\u003e \u003cp\u003eThe pre-copulatory discrimination behavior associated with the avoidance of parasitized individuals is common (e.g., C\u0026oacute;rdoba-Aguilar et al. 2003; Dass et al. 2011), particularly in hosts infected by sexually transmitted parasites (Ashby and Boots 2015). In contrast, pre-copulatory discrimination against parasitic castrated hosts has been observed in a few isolated studies. For example, females of a pipefish species exhibit a drastic reduction of fecundity when infected by a trematode (Rosenqvist and Johansson 1995). When given a choice, male pipefish discriminate between parasitized and healthy females (Rosenqvist and Johansson 1995). In the case of castrated mollusk species, the few studies conducted to date reported that hosts lack such pre-copulatory mechanisms. Males in the mud snails \u003cem\u003ePotamopyrgus antipodarum\u003c/em\u003e persist in copulating with females infested by castrating trematodes (Neiman and Lively 2005). Similarly, males of the marine snail \u003cem\u003eLittorina littorea\u003c/em\u003e engage in copulatory pairs despite females being infected by the castrator trematode \u003cem\u003eCryptocotyle lingua\u003c/em\u003e (Zimmerman 2007), although copulation with castrated females may be shorter (Saur 1990). The present study appears to be the first report of discrimination of castrated hosts in mollusks. It is not surprising that a species from the family Calyptraeidae exhibits this key of behaviors, as members of this group are recognized for displaying plastic behaviors associated with reproduction (Brante et al. 2016). For instance, slipper limpets exhibit multiple paternity (Dupont et al. 2006), and the degree of this phenomenon varies according to population context (Brante et al. 2011). Similarly, the timing of sex change can vary within a species depending on the social context (M\u0026eacute;rot and Collin 2012). Furthermore, sex change has been observed as a plastic trait that may fluctuate with the intensity of parasitic infection (Gilardoni et al. 2012). Sexual selection appears to operate strongly within Calyptraeidae limpets.\u003c/p\u003e \u003cp\u003eWe do not know, based on our results, why the ability to discriminate among females evolved in this species. However, one possibility is that it relates to a network of direct benefits for males. By avoiding copulation with castrated females, males would increase their chances of reproducing successfully and prevent wasting costly sperm. This type of mechanism would be especially relevant in systems where trematode parasites that castrate their hosts reach high prevalence. While many trematodes show low prevalence in mollusks (\u0026lt;\u0026thinsp;5%) (Bagnato et al. 2015; Falt\u0026yacute;nkov\u0026aacute; et al. 2016), some groups, such as microphallids, can exceed 10% (Galaktionov and Bustnes 1999; Gilardoni et al. 2011, 2018). The high prevalence observed in our study could therefore represent a selective pressure favoring the evolution of pre-copulatory discrimination. In addition to potential benefits, males may also incur costs associated with mate searching. While we found no difference in the distances traveled by males that encountered parasitized versus non-parasitized females, some variation (though based on small sample sizes) was observed among males that abandoned a female and continued searching. Specifically, males that first encountered a parasitized female and later found a non-parasitized one traveled longer distances than those that abandoned non-parasitized females. This suggests that encountering parasitized females during mate searching may involve higher costs for certain males. Future studies should further investigate male movement patterns during mate searching and the potential energetic costs associated with these behaviors.\u003c/p\u003e \u003cp\u003eIn this study, we report that males of \u003cem\u003eB. odites\u003c/em\u003e avoid castrated females. It is unknown, however, the mechanism by which male-female attraction fails. It may involve waterborne cues where males either perceive the infection or do not perceive a pheromone that is lost in castrated females but remains in healthy females. Alternatively, physical contact may mediate the avoidance mechanism. Contact-based chemical cues were reported to modulate the timing of the sex transition on a slipper limpet species (Carrillo-Baltodano and Collin 2015). Distinguishing between these possibilities will require further experiments. It is also unknown whether the trematode species promotes premature changes in the sex transition of \u003cem\u003eB. odites\u003c/em\u003e. In the vicinities of the location we sampled \u003cem\u003eB. odites\u003c/em\u003e another limpet species (\u003cem\u003eCrepipatella dilatata\u003c/em\u003e) is castrated by another species of Microphallidae (Gilardoni et al. 2012). Sex transition from male to female occurs earlier in this population which may compensate for the loss of future reproduction (Gilardoni et al. 2012). In the locality we studied \u003cem\u003eB. odites\u003c/em\u003e sizes at the female transition are smaller than in a potentially non-parasitized subtidal population (Cled\u0026oacute;n et al. 2015). This may be an indication that \u003cem\u003eB. odites\u003c/em\u003e also responds to trematodes by transitioning soon from males to females, as occurs in \u003cem\u003eC. dilatata\u003c/em\u003e. However, further information is required to unveil this possibility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request to authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by the followed grants: ANPCyT, PICT 2017-0373; ANPCyT, PICT 2019-2385; UNMdP, 2020 EXA953/20; CONICET, PIP 11220200102466CO.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no financial or non-financial conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at XXX.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eEthical standards\u003c/h3\u003e\n\u003cp\u003eNo ethical approval was required. All applicable international, national, and institutional guidelines for sampling and experimental use of organisms have been followed.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and sampling of individuals. Experimental design, data analysis, and interpretation were performed by Emiliano Ocampo, Tom\u0026aacute;s Luppi, and Jes\u0026uacute;s Nu\u0026ntilde;ez. Florencia Cremonte and Carmen Gilardoni identified and analyzed parasites from limpets. Emiliano Ocampo wrote the first draft of the manuscript, and all authors commented on and edited previous versions. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Barbara Gorriti for providing us with algae species to feed the limpets during the experimental setup. We also thank the three anonymous reviewers and the associate editor for their invaluable contributions during the revision of this manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAshby B, Boots M (2015) Coevolution of parasite virulence and host mating strategies. Proc Natl Acad Sci USA 112(43):13290\u0026ndash;13295. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1508397112\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1508397112\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBates D, M\u0026auml;chler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67(1):1\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18637/jss.v067.i01\u003c/span\u003e\u003cspan address=\"10.18637/jss.v067.i01\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeninger PG, Valdizan A, Le Pennec G (2016) The seminal receptacle and implications for reproductive processes in the invasive gastropod \u003cem\u003eCrepidula fornicate\u003c/em\u003e. Zoology 119:4\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.zool.2015.09.001\u003c/span\u003e\u003cspan address=\"10.1016/j.zool.2015.09.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBagnato E, Gilardoni C, Di Giorgio G, Cremonte F (2015) A checklist of marine larval trematodes (Digenea) in molluscs from Argentina, Southwestern Atlantic coast. Check List 11:1706\u0026ndash;1706. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15560/11.4.1706\u003c/span\u003e\u003cspan address=\"10.15560/11.4.1706\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrante A, Fern\u0026aacute;ndez M, Viard F (2011) Microsatellite evidence for sperm storage and multiple paternity in the marine gastropod \u003cem\u003eCrepidula coquimbensis\u003c/em\u003e. J Exp Mar Biol Ecol 396:83\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jembe.2010.10.001\u003c/span\u003e\u003cspan address=\"10.1016/j.jembe.2010.10.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrante A, Qui\u0026ntilde;ones A, Silva F (2016) The relationship between sex change and reproductive success in a protandric marine gastropod. Sci Rep 6:29439. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep29439\u003c/span\u003e\u003cspan address=\"10.1038/srep29439\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrillo-Baltodano A, Collin R (2015) \u003cem\u003eCrepidula\u003c/em\u003e slipper limpets alter sex change in response to physical contact with conspecifics. Biol Bull 229:232\u0026ndash;242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1086/BBLv229n3p232\u003c/span\u003e\u003cspan address=\"10.1086/BBLv229n3p232\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaparro OR, Thompson RJ, Pereda SV (2002) Feeding mechanisms in the gastropod \u003cem\u003eCrepidula fecunda\u003c/em\u003e. Mar Ecol Prog Ser 234:171\u0026ndash;181. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3354/meps234171\u003c/span\u003e\u003cspan address=\"10.3354/meps234171\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCled\u0026oacute;n M, Nu\u0026ntilde;ez JD, Ocampo E, Sigwart JD (2015) Sexual traits plasticity of the potentially invasive limpet \u003cem\u003eBostrycapulus odites\u003c/em\u003e (Gastropoda: Calyptraeidae) within its natural distribution in South America. Mar Ecol 37:433\u0026ndash;441. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1111/maec.12329\u003c/span\u003e\u003cspan address=\"10.1111/maec.12329\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollin R (2005) Development, phylogeny, and taxonomy of \u003cem\u003eBostrycapulus\u003c/em\u003e (Caenogastropoda: Calyptraeidae), an ancient cryptic radiation. Zool J Linn Soc 144(1):75\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1096-3642.2005.00162.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1096-3642.2005.00162.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCollin R (2006) Sex ratio, life history invariants, and patterns of sex change in a family of protandrous gastropods. Evolution 60:735\u0026ndash;745. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.0014-3820.2006.tb01152.x\u003c/span\u003e\u003cspan address=\"10.1111/j.0014-3820.2006.tb01152.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC\u0026oacute;rdoba-Aguilar A, Salamanca-Oca\u0026ntilde;a JC, Lopezaraiza M (2003) Female reproductive decisions and parasite burden in a calopterygid damselfly (Insecta: Odonata). Anim Behav 66:81\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1006/anbe.2003.2198\u003c/span\u003e\u003cspan address=\"10.1006/anbe.2003.2198\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiaz JI, Cremonte F (2010) Development from metacercaria to adult of a new species of \u003cem\u003eMaritrema\u003c/em\u003e (Digenea: Microphallidae) parasitic in the kelp gull, \u003cem\u003eLarus dominicanus\u003c/em\u003e, from the Patagonian coast. Argentina J Parasitol 96(4):740\u0026ndash;745. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1645/GE-2343.1\u003c/span\u003e\u003cspan address=\"10.1645/GE-2343.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDass SA, Vasudevan A, Dutta D, Soh LJ, Sapolsky RM, Vyas A (2011) Protozoan parasite \u003cem\u003eToxoplasma gondii\u003c/em\u003e manipulates mate choice in rats by enhancing attractiveness of males. PLoS ONE 6(11):e27229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0027229\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0027229\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDougherty LR (2020) Designing mate choice experiments. Biol Rev 95:759\u0026ndash;781. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/brv.12586\u003c/span\u003e\u003cspan address=\"10.1111/brv.12586\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDougherty LR, Shuker DM (2015) The effect of experimental design on the measurement of mate choice: a meta-analysis. Behav Ecol 26:311\u0026ndash;319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDupont L, Richard J, Paulet YM, Thouzeau G, Viard F (2006) Gregariousness and protandry promote reproductive insurance in the invasive gastropod \u003cem\u003eCrepidula fornicata\u003c/em\u003e: evidence from assignment of larval paternity. Mol Ecol 15:3009\u0026ndash;3021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1365-294X.2006.02988.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-294X.2006.02988.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalt\u0026yacute;nkov\u0026aacute; A, Sures B, Kostadinova A (2016) Biodiversity of trematodes in their intermediate mollusc and fish hosts in the freshwater ecosystems of Europe. Syst Parasitol 93:283\u0026ndash;293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11230-016-9627-y\u003c/span\u003e\u003cspan address=\"10.1007/s11230-016-9627-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFox J, Weisberg S (2019) An R Companion to Applied Regression, Third edition. Sage, Thousand Oaks CA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.john-fox.ca/Companion/\u003c/span\u003e\u003cspan address=\"https://www.john-fox.ca/Companion/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalaktionov KV, Bustnes JO (1999) Distribution patterns of marine bird digenean larvae in periwinkles along the southern coast of the Barents Sea. Dis Aquat Org 37:221\u0026ndash;230. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3354/dao037221\u003c/span\u003e\u003cspan address=\"10.3354/dao037221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilardoni C, Etchegoin J, Diaz JI, Ituarte C, Cremonte F (2011) A survey of larval digeneans in the commonest intertidal snails from Northern Patagonian coast, Argentina. Acta Parasitol 56:163\u0026ndash;179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2478/s11686-011-0021-2\u003c/span\u003e\u003cspan address=\"10.2478/s11686-011-0021-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilardoni C, Ituarte C, Cremonte F (2012) Castrating effects of trematode larvae on the reproductive success of a highly parasitized population of \u003cem\u003eCrepipatella dilatata\u003c/em\u003e (Caenogastropoda) in Argentina. Mar Biol 159:2259\u0026ndash;2267. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00227-012-2011-9\u003c/span\u003e\u003cspan address=\"10.1007/s00227-012-2011-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGilardoni C, Di Giorgio G, Bagnato E, Cremonte F (2018) Survey of trematodes in intertidal snails from Patagonia, Argentina: new larval forms and diversity assessment. J Helminthol 1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0022149X18000329\u003c/span\u003e\u003cspan address=\"10.1017/S0022149X18000329\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamilton WD, Zuk M (1982) Heritable true fitness and bright birds: a role for parasites? Science 218:384\u0026ndash;387. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.7123238\u003c/span\u003e\u003cspan address=\"10.1126/science.7123238\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartig F (2022) DHARMa: Residual Diagnostics for Hierarchical (Multi-Level/Mixed) Regression Models. R package version 0.4.6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.32614/CRAN.package.DHARMa\u003c/span\u003e\u003cspan address=\"10.32614/CRAN.package.DHARMa\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelluy S, Holmes JC (1990) Serotonin, octopamine, and the clinging behavior induced by the parasite \u003cem\u003ePolymorphus paradoxus\u003c/em\u003e (Acanthocephala) in \u003cem\u003eGammarus lacustris\u003c/em\u003e (Crustacea). Can J Zool 68:1214\u0026ndash;1220. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1139/z90-181\u003c/span\u003e\u003cspan address=\"10.1139/z90-181\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoagland KE (1978) Protandry and the evolution of environmentally-mediated sex change: a study of the Mollusca. Malacologia 17:365\u0026ndash;391\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIzquierdo A, Loya A, D\u0026iacute;az-Vald\u0026eacute;s M, Ramos-Espla AA (2007) Non-indigenous species at the Alicante Harbor (SE Spain): \u003cem\u003eOculina patagonica\u003c/em\u003e de Angelis, 1908 and \u003cem\u003eBostrycapulus aculeatus\u003c/em\u003e (Gmelin, 1791). Rapport Comm Int Mer Mediterranee 38:50\u0026ndash;506\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLafferty KD, Kuris AM (2009) Parasitic castration: the evolution and ecology of body snatchers. Trends Parasitol 25:564\u0026ndash;572. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pt.2009.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.pt.2009.09.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026eacute;rot C, Collin R (2012) Effects of stress on sex change in \u003cem\u003eCrepidula cf. marginalis\u003c/em\u003e (Gastropoda: Calyptraeidae). J Exp Mar Biol Ecol 416\u0026ndash;417:68\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jembe.2012.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.jembe.2012.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeiman M, Lively CM (2005) Male New Zealand mud snails (\u003cem\u003ePotamopyrgus antipodarum\u003c/em\u003e) persist in copulating with asexual and parasitically castrated females. Am Midl Nat 154:88\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1674/0003-0031\u003c/span\u003e\u003cspan address=\"10.1674/0003-0031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e(2005)154[0088:MNZMSP]2.0.CO;2\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOcampo EH, Nu\u0026ntilde;ez JD, Cled\u0026oacute;n M, Baeza JA (2014) Parasitic castration in slipper limpets infested by the symbiotic crab \u003cem\u003eCalyptraeotheres garthi\u003c/em\u003e. Mar Biol 161:2107\u0026ndash;2120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00227-014-2490-y\u003c/span\u003e\u003cspan address=\"10.1007/s00227-014-2490-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechenik JA, Fried B, Bolstridge J (2012) The marine Gastropods \u003cem\u003eCrepidula plana\u003c/em\u003e and \u003cem\u003eCrepidula convexa\u003c/em\u003e do not serve as first intermediate hosts for larval Trematode. Dev Comp Parasitol 79:5\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1654/4520.1\u003c/span\u003e\u003cspan address=\"10.1654/4520.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoulin R, Mouritsen KN (2003) Large-scale determinants of trematode infections in intertidal gastropods. Mar Ecol Prog Ser 254:187\u0026ndash;198. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3354/meps254187\u003c/span\u003e\u003cspan address=\"10.3354/meps254187\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuinn EA, Thomas JE, Malkin SH, Eley M-J, Coates CJ, Rowley AF (2022) Invasive slipper limpets \u003cem\u003eCrepidula fornicata\u003c/em\u003e are hosts for sterilizing digenean parasites. Parasitology 149:811\u0026ndash;819. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/S0031182022000257\u003c/span\u003e\u003cspan address=\"10.1017/S0031182022000257\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRStudio. Integrated Development Environment for R. RStudio Team, RStudio, Boston MA (2024) Disponible en: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rstudio.com\u003c/span\u003e\u003cspan address=\"http://www.rstudio.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenkranz M, Poulin R, Selbach C (2018) Behavioural impacts of trematodes on their snail host: Species-specific effects or generalised response? Ethology 124:790\u0026ndash;1795. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/eth.12808\u003c/span\u003e\u003cspan address=\"10.1111/eth.12808\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenthal GG (2017) Mate Choice. Princeton University Press, Princeton\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenqvist G, Johansson K (1995) Male avoidance of parasitized females explained by direct benefits in a pipefish. Anim Behav 49:1039\u0026ndash;1045. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/anbe.1995.0133\u003c/span\u003e\u003cspan address=\"10.1006/anbe.1995.0133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaur M (1990) Mate discrimination in \u003cem\u003eLittorina littorea\u003c/em\u003e (L.) and \u003cem\u003eL. saxatilis\u003c/em\u003e (Olivi) (Mollusca: Prosobranchia). Hydrobiologia 193:261\u0026ndash;270. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00028082\u003c/span\u003e\u003cspan address=\"10.1007/BF00028082\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671\u0026ndash;675. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nmeth.2089\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2089\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaw JC, Wilson K (2018) Reproductive behavior and parasites: invertebrates. Academic, London\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSokal RR, Rohlf FJ (1981) Biometry. Freeman, San Francisco\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorensen RE, Minchella DJ (2001) Snail\u0026ndash;trematode life history interactions: past trends and future directions. Parasitology 123:S3\u0026ndash;S18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/s0031182001007843\u003c/span\u003e\u003cspan address=\"10.1017/s0031182001007843\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamaguti S (1975) A synoptical review of life histories of digenetic trematodes of vertebrates with special reference to the morphology of their larval forms. Keigaku Publishing Co. Tokyo\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZimmerman AF (2007) Shooting blanks: Mate choice and determination in a parasitically castrated snail, \u003cem\u003eLittorina littorea\u003c/em\u003e. Honors Thesis. Cornell University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuur AF, Ieno EN, Walker N, Saveliev AA, Smith GM (2009) GLMM and GAMM. Mixed Effects Models and Extensions in Ecology with R, pp. 323\u0026ndash;341. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18637/jss.v032.b01\u003c/span\u003e\u003cspan address=\"10.18637/jss.v032.b01\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Mating choice, calyptraeid limpet, digenean parasite, intertidal rocky shore","lastPublishedDoi":"10.21203/rs.3.rs-5654364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5654364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiscrimination mechanisms that allow males to select female partners are expected to evolve when a significant portion of the female population is sterilized. In this study, we investigated whether males of the slipper limpet species \u003cem\u003eBostrycapulus odites\u003c/em\u003e discriminate between parasitically castrated by a Microphallidae trematode and non-castrated females. We hypothesized that males would prefer healthy females, spend shorter periods on parasitized females, displace longer distances when encountering parasitized females, and abandon females if castrated. Field data revealed that only 7% of the males observed in copulatory positions were on parasitized females. Laboratory experiments confirmed that males primarily select non-parasitized females and exclusively copulate with healthy individuals. The duration of time spent by males on non-parasitized females was almost twice as long as that spent on parasitized females. These results indicate that males bias their mating efforts toward healthy females, and in the few cases where they interact with parasitized females, copulation does not occur. Instead, males spend brief periods on castrated females. Although uncommon in nature, this discriminative ability would be expected to benefit males by enhancing their reproductive success through mating with healthy females.\u003c/p\u003e","manuscriptTitle":"Slipper limpet males avoid copulating with parasitic castrated females","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 06:26:56","doi":"10.21203/rs.3.rs-5654364/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-23T00:20:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-22T23:37:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-21T13:49:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Marine Biology","date":"2025-04-19T12:03:20+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"marine-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mabi","sideBox":"Learn more about [Marine Biology](https://www.springer.com/journal/227)","snPcode":"227","submissionUrl":"https://submission.nature.com/new-submission/227/3","title":"Marine Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6f78adc3-4cf6-4e91-9345-7b8fb5a2b094","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:07:02+00:00","versionOfRecord":{"articleIdentity":"rs-5654364","link":"https://doi.org/10.1007/s00227-025-04715-3","journal":{"identity":"marine-biology","isVorOnly":false,"title":"Marine Biology"},"publishedOn":"2025-09-30 15:57:57","publishedOnDateReadable":"September 30th, 2025"},"versionCreatedAt":"2025-04-28 06:26:56","video":"","vorDoi":"10.1007/s00227-025-04715-3","vorDoiUrl":"https://doi.org/10.1007/s00227-025-04715-3","workflowStages":[]},"version":"v1","identity":"rs-5654364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5654364","identity":"rs-5654364","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}