No intromission is involved in the mating of Eptesicus serotinus, a novel copulatory pattern in mammals.

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Nicolas Fasel, Jan Jeucken, Kseniia Kravchenko, Marcus Fritze, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2813754/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Copulatory behaviours stand as cornerstones of sexual selection, yet they remain mysterious in many species, especially in bats. An extremely large penile erection with a heart-shape terminal swelling has been observed in the serotine bat ( Eptesicus serotinus ). The engorgement of the erectile tissues could take place after penetration to generate a copulatory lock. Alternatively, the erected penis, long and mobile, could be used to pass by the protective tail membrane to reach the vulva. In the latter scenario, the penis may however not be able to penetrate the vagina. In order to find out which of the two scenarios occurs, we investigated the morphology of female and male genitalia of E. serotinus . We found that the vagina is more than seven times shorter than the erected penis, supporting that the penis is used as a copulatory arm rather than an intromittent organ. We could confirm our hypothesis with direct observations of copulations. During mating, the male grasps the female in a dorsoventral position. The male probes the female’s ventral part with its erected penis until the penis is tightly pushed against the vulva. Afterward, the pair stops moving and can hold the position for several hours. In addition, the timing of copulations, which exhibits a peak in October, implies that visits to swarming sites throughout the year may serve other functions besides mating. This research reveals a novel copulatory behaviour that has not been previously documented in mammals and sheds light on the poorly understood area of bat reproduction. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Copulations and the transfer of gametes occupy a derisory fraction of any animal’s lifetime but are crucial events in terms of evolutionary fitness. Copulatory behaviours may be subject to post-copulatory sexual selection. Indeed, they can influence fertilisation success (Dixson, 2021a ; Edvardsson and Göran, 2000 ; Gallup et al., 2003 ) and, in some species, induce ovulation (e.g. domestic cat; (Shille et al., 1979 )). Also, they may evolve in response to ecological factors, as a result of natural selection (Birkhead et al., 1987 ; Dixson, 2021b ). Finally, copulatory behaviours may influence the access to fertile females by strengthening social bonds (Birkhead et al., 1987 ), and could consequently be considered as pre-copulatory traits. While standing as cornerstones of sexual selection, copulatory behaviours and their fitness consequences remain largely mysterious in most species. The available information on the act of mating in mammals has been recently synthesised (Dixson, 2021c ). Copulatory patterns have been described to classify these behaviours based on the presence or absence of a copulatory lock, pelvic thrusting movements performed by males, multiple intromissions before ejaculation and finally prolonged intromissions (Dewsbury, 1972 ; Dixson, 2021b ). Because of their nocturnal and hidden lifestyle, the copulatory behaviours of bats remain mostly overlooked. Hitherto, behaviours related to mating activities have been investigated in just a handful of bat species (McCracken and Wilkinson, 2000 ), whereas for most of them, knowledge about the act of copulation itself remains anecdotal. Nevertheless, penis morphology of Vespertilionidae is diverse and can represent an important criterion for species identification (Dietz and von Helversen, 2004 ; Fasel et al., 2020 ). The variation in penis morphology may accompany diversity in copulatory patterns. In the serotine bat ( Eptesicus serotinus ), a disproportionately large penile erection with a heart-shaped terminal swelling has been observed both during anaesthesia and during naturally conducted intercourse (see methods and results sections). As intromission of the erected penis may not be expected due to its size, the engorgement of the erectile tissues may take place after intromission. Such copulatory pattern is observed in some mammal species, as canids, and leads to a so-called “copulatory lock” (Dixson, 2021b ). It has also been suggested to occur in some bat species (Wilson, 1971 ), where the penis is erected before intromission, but could be retracted rapidly following disturbance (Wimsatt, 1945 ). Observations of captive E. serotinus pairs in copula for several hours (Racey and Kleiman, 1970 ) hint towards the occurrence of a copulatory lock in the species. Alternatively, the long motile and stable erected penis may be used to pass by the protective tail membrane (i.e. uropatagium) of the female in order to reach the vulva. Under such conditions however, the penis cannot be expected to penetrate the vagina, leaving as only option a contact mating. This copulatory pattern (i.e. cloacal contact, e.g. (Drachmann et al., 1997 )) is common in birds but remains unknown in mammals. In this study, we provide an anatomical description of the penis and female genital tract and describe mating events that we observed in order to define the copulatory behaviour of E. serotinus . Specifically, we want to establish whether the penis enables a copulatory lock during the mating process or functions as a copulatory arm without penetrating the vulva, which would represent a copulatory pattern still unknown in mammals. Additionally, to draw a full picture about the mating behaviours of this species, we present the phenology of copulations and compare it with seasonal activity patterns near underground sites. To the best of our knowledge, this is the first comprehensive compilation of knowledge on reproduction of a bat species so far. Methods Model species E. serotinus is a palearctic species whose distribution spans from Spain to China (Artyushin et al., 2018 ; Juste et al., 2013 ). At the global scale, it is listed as “Least Concern” on the IUCN Red List of Threatened Species (Godlevska et al., 2021 ), but locally has been considered as a vulnerable species (e.g. (Bohnenstengel et al., 2014 )). Yearling males can reach sexual maturity, produce enough sperm to fill up the epididymides and copulate during their first year of life (Racey and Kleiman, 1970 ). Sexual maturity in females also may be reached already during their first year of life (Pelikan, Gaisler and Rödl (1979) cited by (Haensel, 1994 )). Females of E. serotinus , like most of Vespertilionidae species, can store sperm until ovulation, which occurs in spring (Crichton, 2000 ), Personal observations of the authors). Ovulation in bats remains poorly investigated. Evidence indicates that it is spontaneous in Carollia perspicillata (Rasweiler IV et al., 2011 ), as well as in sperm storing species ((Oxberry, 1979 ), but see also (deCatanzaro et al., 2014 )). The flaccid penis of E. serotinus is pendulous and widens towards the tip. A weak medial ridge can be observed on the upper side (Dietz and von Helversen, 2004 ). The baculum measures are approximately 1.10mm, which is relatively short in comparison to other Vespertilionidae species (Hill and Harrison, 1987 ; Hosken et al., 2001 ). Erected penis and female genital tract anatomy In the framework of different research projects in Germany and Poland, ten male bats were captured and anaesthetised using Isoflurane (5 vol. % for induction, 1–2 vol. % for maintenance; flow rate 1.5 l/min ambient air). This procedure is known to provoke erection in Vespertilionidae (Fasel et al., 2020 , 2018 ). Penis length was measured while fully erected as the ventral length between the base of the penis, where the skin becomes bright red, and the tip of the penis (Fig. 1 B). Terminal swelling was measured at the tip of the penis, where the width is maximal (Fig. 1 C). Measurements were reported only when a complete erection was verified (i.e., tight skin, bright sharp red colour and rigid structure). All measures were performed by a single observer (NJF). Selected measurements were replicated, and repeatability was estimated using the function “rpt” from package “rptR” (Stoffel et al., 2017 ). We performed necropsy of eleven frozen-thawed females and seven males that deceased at the bat rehabilitation centre at Zitadelle Spandau, Germany. Of these, we performed histology of the genital tracts of four females (three in longitudinal orientation and one in cross-section) as well as of two males. Organ samples collected during necropsy were fixed in 4% neutral buffered formalin and embedded in paraffin wax for histology. Sections (3 µm) were stained with haematoxylin and eosin (HE). Histological images were recorded using a digital photo-microscope (Keyence VHX-1000, Osaka, Japan); organ size was measured post-hoc on scaled digital images. Behavioural observation Mating events were recorded opportunistically at two different sites: St Matthias Church in Castenray (Netherlands) and the Ukrainian Bat Rehabilitation Centre (UBRC) located in Kharkiv (Ukraine). St Matthias Church in Castenray (Netherlands) has been hosting a colony of around 150 females E. serotinus in the attic (JJ personal observation). A maximum of eighteen cameras have been installed since 2016 in order to document behaviours of this species. The date and duration of each recorded copulation event have been reported between October 25th 2016 and March 22nd 2022. Copulation events of captive E. serotinus in short- and long-term rehabilitation were documented by the staff of the UBRC. Bats lived in cages with custom-made wood and paper roosts, but only outside copulations were recorded. Copulations were recorded between August 2018 and December 2021. For each copulatory event, the date, time and duration of copulation were recorded and the bat`s behaviour was described. Videos focused on the genitalia were carefully reviewed to describe the type of copulatory behaviours and the female and male anatomical interactions. Activity at underground site Eptesicus serotinus were captured at the entrance of abandoned mines (Baulmes, Switzerland: 46°47’N, 6°31’W). Captures were part of a survey of the mines running annually from 2001 to 2022. The mine characteristics have been previously described by (Giavi et al., 2020 ). Results Flaccid and erected penis and female genital tract anatomy The flaccid penis was measured on digital scaled microscopic images of formalin-fixed material post-mortem in 7 individuals. Penis shaft length averaged 6.07 ± 1.87 mm and width 3.57 ± 0.98 mm. The histology of the vascular penis of E. serotinus follows the general vespertilionid anatomy (Fig. 2 ). It contains three distinct erectile tissues, (i) the corpora cavernosa (CC), (ii) the corpus spongiosum (CS) surrounding the urethra (u), both enveloped by their respective tunicae albugineae (TC), and (iii) the accessory cavernous body formed by accessory cavernous tissue (AT). An extended, folded and vascularized prepuce surrounds the glans penis, which is formed by accessory cavernous tissue . The baculum is located dorsal to the urethra , located partly in the penile body and partly in the glans penis . The erected penis was measured in 10 anaesthetised males, between one and three times (average 2.18 times). The repeatability of these measurements was 0.70 ± 0.17, P = 0.004 for length of the erected penis and 0.70 ± 0.17, P = 0.007 for the width of the terminal swelling respectively. The length of the penis was 16.40 ± 0.88 mm, and the maximum width of the terminal swelling was 7.46 ± 0.61 mm (Fig. 3 ). The terminal swelling is composed of two large erectile tissues, distinct from the corpora cavernosa (Figs. 1 and 2 ). The female reproductive tract length as measured on digital scaled microscopic images of formalin-fixed material post-mortem in 7 individuals is 13.89 ± 1.35 mm, cervical diameter 1.29 ± 0.41 mm, and ovarian diameter 1.94 ± 0.46 mm x 1.58 x 0.27 mm. The vagina is approximately 2.3 ± 0.2 mm long, the cervix 8.6 ± 1.7 mm, and the uterine horns 3.7 ± 0.2 mm (Fig. 4 ). Copulatory behaviours We recorded 93 copulations in St. Matthias church and four in the UBRC via video monitoring. Recorded copulatory behaviours are described as follows: The male grasped the female in a dorsoventral position, mostly on vertical surfaces, both animals with their head upside down (Fig. 5 ). The male bit the skin on the nape. Lateral movements of the male hindquarters were then accompanied by rapid probing movements of the fully erected penis (supplementary information). Social calls were emitted, probably by the female. The pair stopped moving when the penis was firmly pushed against the vulva. Once the pair remained immobile, no more vocalisation was heard. The fur on the female abdomen appeared wet. In St. Matthias the church, the first copulation after the weaning of the young can be observed in September, peaking in October with 49.5% of all recorded copulation events (Fig. 6 ). Then the frequency of copulatory decreased with the latest event recorded on the 31st of December. Infrequent copulations were also recorded in March and beginning of April. Half of the copulations lasted for less than 53 min and the longest one for 12.7 hours (Fig. 7 ). Activity at underground site At the abandoned mines (Baulmes, Switzerland), the number of surveys per year varied from 0 to 16 (mean: 3.15). No captures took place between November and February. In total, 233 E. serotinus were captured and sexed. Individuals were captured from March to October, with a peak in July and August (Fig. 8 ). The sex ratio was male biased (0.82). Discussion The mating events described in this study validate that copulation in E. serotinus does not comprise vaginal penetration of the shaft of the penis. Indeed, the erectile tissues of the penis are enlarged before the contact with the vulva and form a shape unsuitable for intromission. Furthermore, the erected penis length exceeds the vaginal length by approximately four times. Our observations support that the penis remains in contact with the vulva, but without vaginal intromission. The relatively small size of the baculum, as well as its shape, additionally indicate an absence of any specific role of the penis bone in the intromission. The baculum of E. serotinus may serve to protect the urethra from compression by the erectile tissues during erection as suggested for E. furinalis (Comelis et al., 2015 ). To our knowledge, this study is the first to report a mammalian copulatory pattern without intromission (Dixson, 2021b ). The stiffened penis of E. serotinus may be important as a means to pass by the uropatagium (i.e. tail membrane) and reach the vulva. To some extent, the tail membrane could be used by the female to avoid copulation. The long penis of E. serotinus may therefore serve as a ‘copulatory arm’, as observed in some cetaceans e.g. (Keener et al., 2018 ), in order to overpass the uropatagium of the female. Once the penis is firmly positioned on the top of the vulva, the movements of the hindquarters and of the penis stopped. While half of the observed copulations did not exceed 30 min, the longest one recorded lasted 12.7 hours, confirming previous observations (Racey and Kleiman, 1970 ). The hollow structure on the dorsal side of the erected penis (Fig. 1 C) is formed by two accessory cavernous tissues, according to the terminology of (Wimsatt and Kallen, 1952 ). This structure might act as a suction cup and support the maintenance of the copulatory contact. Thus, the large size and characteristic shape of the penis as well as the prolonged embrace might further support the transport of sperm cells to the vagina and to the cervix. From our observations, males appear to chase females, without any courtship behaviour. Furthermore, a minority of observed copulations took place during the hibernation period when females were torpids. The absence of courtship and winter copulations support a reduced pre-copulatory female choice. Male selection by the female could supposedly take place after the copulation through selective sperm transport through the cervix or active degradation by immune cells or maintenance within the uterus (Orr and Zuk, 2013 ). Such mechanisms remain to be investigated. As this mating pattern remained unobserved so far, it may concern only a few species and probably only bats. In Myotis lucifugus , a similar subterminal swelling of the penis has been described, produced by the engorgement of the accessory cavernous tissues (Wimsatt and Kallen, 1952 ). In this latest species, the intromission of the penis within the vagina was however suggested, despite its large size (Wimsatt, 1945 ). Males of E. serotinus are known to visit breeding roosting sites as early as the end of July (Haensel, 1994 ). This later article does, however, not mention the observation of copulations. In the St Matthias Church, copulations were observed from the beginning of August to the end of April. This result suggests that the mating season starts when the males terminate spermatogenesis and females wean the young, and continues until the ovulation in spring, confirming earlier findings (Eisentraut, 1936 ). Our observations also demonstrate that both males and females can store sperm for months in the epididymides and in the genital tract, respectively. Our study clearly supports that males visit female roosting sites. It remains unknown however whether males also attract sexual partners in other locations. E. serotinus are known to visit swarming sites in Germany (Pfeiffer and Mayer, 2013 ) and in the Netherlands (Van Schaik et al., 2015 ). In Switzerland, our results show that individuals are visiting the mine all year round. After the weaning of the young, the activity at the mines is higher in July and August, similar to what was observed in the Netherlands (Van Schaik et al., 2015 ). Similar phenology of activity near underground roosts was shown for E. fuscus (Mumford and Whitaker, 1974 ). However, as summer activity occurs earlier than the peak of copulations, it might have other functions than mating, as for example transferring information to yearling individuals about potential hibernacula and/or mating sites (i.e. colony member guidance hypothesis (Piksa, 2008 ; Stumpf et al., 2017 ; Van Schaik et al., 2015 ; Veith et al., 2004 )). It should also be considered that E. serotinus visit the entrance of underground sites to forage (suggested by (Mumford and Whitaker, 1974 ) for E. fuscus ) or that individuals are attracted there by the activity of other species. In conclusion, our results suggest that the erected penis of E. serotinus serves to pass by the tail membrane of the female and is used to reach the vulva without vaginal intromission. This copulatory pattern is unknown in other mammals. Additional studies of the copulatory behaviours of bats may reveal other species using copulation without sexual penetration. We also demonstrate that males visit breeding colonies and copulate there with females. The time lag between summer activity at swarming sites and the peak of copulation supports that the visits of E. serotinus at underground sites might serve functions other than mating. This study contributes to a better understanding of animal mating and further fertilisation-involved processes. Such knowledge can improve conservation of endangered species, and open doors for further research in physiology, ethology and ecology. Declarations Acknowledgements We are thankful to Jörg Harder and Robert Henning from the Zitadelle Spandau, Germany for providing female carcasses, to Bernd Ohlendorf and the administration of the Karst Landscape South Harz Biosphere Reserve for supporting our field work and providing field lab space. We thank Alona Prylutska and Maryna Yerofeieva from Ukrainian Bat Rehabilitation Center, Ukraine, for coordination and assistance during data collecting in Ukraine. We thank Gudrun Wibbelt for interpretation of the histology, Doris Krumnow and Monique Schmückert for preparation of histology slides. We are grateful to all who have got involved in the survey of the Swiss mines. We especially thank Taisiia Kravchenko for the professional drawings: [email protected] , Instagram: taisiya.kravchenko Compliance with Ethical Standards All captures and manipulations were conducted under permission of local conservation and animal ethics authorities (Germany: conservation authority of Mansfeld-Südharz county (authorization no. NB30A_18001_Fie) and Veterinary Affairs Office of Sachsen-Anhalt (LVwA, auth. no. 42502-2-1525 LIZW); Poland: Regional Director for Environmental Protection in Białystok (auth. no. WPN.6401.57.2015.WL) and Ethical Commissions in Białystok and Olsztyn (auth. no. 43/2015; 152/2015; 153/2015; 16/2016). The Bat Rehabilitation Center of Feldman Ecopark (Ukraine) works under the general permission of the Kharkiv Oblast Authority of Ecology and Natural Resources in 2013-2022. Authorizations of capture in Switzerland were delivered by the Direction Générale de l’Environnement, canton de Vaud. Funding This project was founded by the National Science Centre, Poland, on the basis of decision number DEC-2013/10/E/NZ8/00725, the Swiss National Science Foundation (grant number: P2BEP3_168709 to NJF). The Bat Rehabilitation Center of Feldman Ecopark worked (in 2013-2022) under financial support of International Charity Foundation “Oleksandr Feldman Foundation” (Kharkiv, Ukraine). References Artyushin, I. V., Kruskop, S. V., Lebedev, V.S., Bannikova, A.A., 2018. Molecular Phylogeny of Serotines (Mammalia, Chiroptera, Eptesicus ): Evolutionary and Taxonomical Aspects of the E. serotinus Species Group. Biol. 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Ecology of Myotis nigricans (Mammalia: Chiroptera) on Barro Colorado Island, Panama Canal Zone. J. Zool. 163, 1–13. https://doi.org/10.1111/j.1469-7998.1971.tb04521.x Wimsatt, W.A., 1945. Notes on Breeding Behavior, Pregnancy, and Parturition in Some Vespertilionid Bats of the Eastern United States. J. Mammal. 26, 23. https://doi.org/10.2307/1375029 Wimsatt, W.A., Kallen, F.C., 1952. Anatomy and histophysiology of the penis of a vespertilionid bat, Myotis lucifugus lucifugus , with particular reference to its vascular organization. J. Morphol. 90, 415–465. https://doi.org/10.1002/jmor.1050900303 Supplementary Files Epsero.mp4 Cite Share Download PDF Status: Posted Version 1 posted 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. 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Also discoverable on Platform About In Review Editorial Policies 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-2813754","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":192222521,"identity":"ce4e299f-fda7-4005-b09e-f0ae5bdc1618","order_by":0,"name":"Nicolas Fasel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYBAC9gYg8YCBgYeBgRnIrADymEEMPIDnAJBIAGthBKo8A9LCSJwWBrAWxjYoA68W9uMPHyQw3JHhb29s/PhzXm00fztQy4+Kbbi18OQYGyQwPOOROHOwWZp32/HcGYcZGxh7ztzGqcWeIYdNIoHhMI+BRGKDNOO2Y7kNQC3MjG24tfDwP3/+A6ql+efPOcdy5xPUIpFgxgDV0ibB21CTu4GwljfGEgkGh0F+abPmOXYgdyNQy0F8fuHhT3/44UPFYXv+9ubDN3/U1OXOO3/44IMfFbi1QIABnHUYTB4goB4F1JGieBSMglEwCkYIAAAUeloXHz2eWAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2600-7652","institution":"Universite de Lausanne","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nicolas","middleName":"","lastName":"Fasel","suffix":""},{"id":192222523,"identity":"d0e5b8c5-ca44-4ffa-8beb-743510cb4dab","order_by":1,"name":"Jan Jeucken","email":"","orcid":"","institution":"Stichting De Laatvlieger","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jan","middleName":"","lastName":"Jeucken","suffix":""},{"id":192222524,"identity":"10dcaefd-0f1d-4e20-8d5a-5df454c84635","order_by":2,"name":"Kseniia Kravchenko","email":"","orcid":"","institution":"UNIL: Universite de Lausanne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kseniia","middleName":"","lastName":"Kravchenko","suffix":""},{"id":192222527,"identity":"48b4aee9-a3b8-4369-b65e-3c036e31516e","order_by":3,"name":"Marcus Fritze","email":"","orcid":"","institution":"German bat observatory","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marcus","middleName":"","lastName":"Fritze","suffix":""},{"id":192222530,"identity":"1a3aacbf-079c-4435-926a-dd2c16c46c61","order_by":4,"name":"Ireneusz Ruczynski","email":"","orcid":"","institution":"Polish Academy of Sciences Mammal Research Institute: Polska Akademia Nauk Instytut Biologii Ssakow","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ireneusz","middleName":"","lastName":"Ruczynski","suffix":""},{"id":192222533,"identity":"16b6f00c-b9f7-4a78-a6dd-710e8ba71d25","order_by":5,"name":"Ewa Komar","email":"","orcid":"","institution":"Polish Academy of Sciences Mammal Research Institute: Polska Akademia Nauk Instytut Biologii Ssakow","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Komar","suffix":""},{"id":192222536,"identity":"1a5f33e5-68df-4ecf-ab42-136a4b6980d7","order_by":6,"name":"Marharyta Moiseienko","email":"","orcid":"","institution":"Jagiellonian University: Uniwersytet Jagiellonski w Krakowie","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marharyta","middleName":"","lastName":"Moiseienko","suffix":""},{"id":192222539,"identity":"4f533e08-7697-4679-9f3f-f223a4438ec9","order_by":7,"name":"Alona Shulenko","email":"","orcid":"","institution":"Ukrainian bat rehabilitation center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alona","middleName":"","lastName":"Shulenko","suffix":""},{"id":192222543,"identity":"e636b398-3b15-4a42-a682-d7df6622b8ce","order_by":8,"name":"Anton Vlaschenko","email":"","orcid":"","institution":"Ukrainian bat rehabilitation center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Vlaschenko","suffix":""},{"id":192222547,"identity":"d5f2ac5f-e92c-4a56-b81e-073a41fafb15","order_by":9,"name":"Philippe Christe","email":"","orcid":"","institution":"UNIL: Universite de Lausanne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Philippe","middleName":"","lastName":"Christe","suffix":""},{"id":192222549,"identity":"f85fbc91-e1fa-4377-9956-6ab13dd4b7f3","order_by":10,"name":"Olivier Glaizot","email":"","orcid":"","institution":"UNIL: Universite de Lausanne","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Glaizot","suffix":""},{"id":192222552,"identity":"f7c49831-1327-4150-8e80-0223b936706f","order_by":11,"name":"Susanne Holtze","email":"","orcid":"","institution":"IZW: Leibniz-Institut fur Zoo- und Wildtierforschung (IZW) im Forschungsverbund Berlin eV","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Holtze","suffix":""}],"badges":[],"createdAt":"2023-04-13 15:57:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2813754/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2813754/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":35944684,"identity":"7d726b43-8a68-4581-8ae1-42e7e0eb8ee6","added_by":"auto","created_at":"2023-04-18 14:48:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":792212,"visible":true,"origin":"","legend":"\u003cp\u003eLateral (A), ventral (B) and dorsal (C) views of the erected penis (drawing T. Kravchenko).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/89b828f731b1963a3cb06ba0.png"},{"id":35941678,"identity":"392d1be0-3bef-4762-8d63-fe072b1a29bc","added_by":"auto","created_at":"2023-04-18 14:32:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":904655,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic photograph (A), and histological images of the penis of \u003cem\u003eE. serotinus\u003c/em\u003e in coronal (B), and mid-sagittal (C) planes stained with hematoxylin-eosin (B-C). b \u003cem\u003ebaculum\u003c/em\u003e, AT \u003cem\u003eaccessory cavernous tissue\u003c/em\u003e, CC \u003cem\u003ecorpora cavernosa\u003c/em\u003e, CS \u003cem\u003ecorpus spongiosum\u003c/em\u003e, N nerve, TC \u003cem\u003etunica albuginea\u003c/em\u003e, p prepuce, s secretion containing sperm cells, u \u003cem\u003eurethra\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/42bd95ed2577e7348dc70234.png"},{"id":35941679,"identity":"e1f00dd9-f575-437e-a38b-61005d1248fc","added_by":"auto","created_at":"2023-04-18 14:32:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":109006,"visible":true,"origin":"","legend":"\u003cp\u003eLength and width in mm of the erected penis and terminal swelling respectively.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/3dcdd0ea0bf94360119d04d2.jpg"},{"id":35941682,"identity":"ef823eea-9367-407a-b8dd-0948019b775f","added_by":"auto","created_at":"2023-04-18 14:32:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":655671,"visible":true,"origin":"","legend":"\u003cp\u003eScaled digital microscopic photographs of \u003cem\u003eE. serotinus\u003c/em\u003efemale genital tract: (A) a formalin-fixed and (B) a HE-stained histology slide of a female genital tract. c = cervix, ov = ovary, r = rectum, ub = urinary bladder, ut = uterine horn, v = vagina. The female died on the 14th of May 2015 and was not pregnant. The scale in the lower right corner accounts for both images. [1] Length of the erected penis shaft (16.4 mm). [2] Width of the erected penis terminal swelling (7.5 mm). [3] Outer diameter of the cervix (0.7 mm) and [4] vagina (1.1 mm).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/4bc1e4886539983b30f698b8.png"},{"id":35943594,"identity":"79a48c93-ee2a-4fc3-be5f-d74ff2f5cb93","added_by":"auto","created_at":"2023-04-18 14:40:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1647696,"visible":true,"origin":"","legend":"\u003cp\u003eCopulation of an \u003cem\u003eEptesicus serotinus\u003c/em\u003e pair. The male, above on the drawing, uses its erected penis to pass by the uropatagium of the female, below on the drawing. The terminal swelling of the erected penis is firmly pressed against the vulva, without vaginal intromission (drawing T. Kravchenko).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/dce6028ed7d3a1ed52967426.png"},{"id":35944685,"identity":"ff385d89-2c94-4a3a-84b5-ea889d438796","added_by":"auto","created_at":"2023-04-18 14:48:22","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128065,"visible":true,"origin":"","legend":"\u003cp\u003eNumbers of copulations merged by month in St. Matthias church between the 25th of October 2016 and the 22nd of March 2022.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/ad86100747a998e3888d9781.jpg"},{"id":35943596,"identity":"490d01aa-63a2-4063-a43f-c815bbb60dc4","added_by":"auto","created_at":"2023-04-18 14:40:22","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":95594,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of copulation duration of \u003cem\u003eE. serotinus\u003c/em\u003e in hours in St. Matthias church between the 25th of October 2016 and the 22nd of March 2022.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/f555c509449b8d55d9b098bb.jpg"},{"id":35941685,"identity":"491b2555-9b21-468a-b77b-3694561dda65","added_by":"auto","created_at":"2023-04-18 14:32:22","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":187012,"visible":true,"origin":"","legend":"\u003cp\u003eAverage number of bats captured at underground site per night. The numbers of capture events between 2001 and 2022 occurring during the respective months are indicated in brackets.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/d7bcbcf6a90b8d8698aed272.jpg"},{"id":40209712,"identity":"5ebf5d07-4089-40bc-bb36-718342c84939","added_by":"auto","created_at":"2023-07-18 17:05:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1836657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/ec09722a-5e74-40f2-abca-f788f41555f7.pdf"},{"id":35941686,"identity":"afb4861a-83e5-4431-87c5-b81418f3ba62","added_by":"auto","created_at":"2023-04-18 14:32:22","extension":"mp4","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":18156867,"visible":true,"origin":"","legend":"","description":"","filename":"Epsero.mp4","url":"https://assets-eu.researchsquare.com/files/rs-2813754/v1/ab6d09ef3f5aa97d08c43447.mp4"}],"financialInterests":"","formattedTitle":"No intromission is involved in the mating of Eptesicus serotinus, a novel copulatory pattern in mammals.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCopulations and the transfer of gametes occupy a derisory fraction of any animal\u0026rsquo;s lifetime but are crucial events in terms of evolutionary fitness. Copulatory behaviours may be subject to post-copulatory sexual selection. Indeed, they can influence fertilisation success (Dixson, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e; Edvardsson and G\u0026ouml;ran, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Gallup et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and, in some species, induce ovulation (e.g. domestic cat; (Shille et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1979\u003c/span\u003e)). Also, they may evolve in response to ecological factors, as a result of natural selection (Birkhead et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Dixson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Finally, copulatory behaviours may influence the access to fertile females by strengthening social bonds (Birkhead et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), and could consequently be considered as pre-copulatory traits. While standing as cornerstones of sexual selection, copulatory behaviours and their fitness consequences remain largely mysterious in most species.\u003c/p\u003e \u003cp\u003eThe available information on the act of mating in mammals has been recently synthesised (Dixson, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021c\u003c/span\u003e). Copulatory patterns have been described to classify these behaviours based on the presence or absence of a copulatory lock, pelvic thrusting movements performed by males, multiple intromissions before ejaculation and finally prolonged intromissions (Dewsbury, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Dixson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Because of their nocturnal and hidden lifestyle, the copulatory behaviours of bats remain mostly overlooked. Hitherto, behaviours related to mating activities have been investigated in just a handful of bat species (McCracken and Wilkinson, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), whereas for most of them, knowledge about the act of copulation itself remains anecdotal. Nevertheless, penis morphology of Vespertilionidae is diverse and can represent an important criterion for species identification (Dietz and von Helversen, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Fasel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The variation in penis morphology may accompany diversity in copulatory patterns.\u003c/p\u003e \u003cp\u003eIn the serotine bat (\u003cem\u003eEptesicus serotinus\u003c/em\u003e), a disproportionately large penile erection with a heart-shaped terminal swelling has been observed both during anaesthesia and during naturally conducted intercourse (see methods and \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003eresults\u003c/span\u003e sections). As intromission of the erected penis may not be expected due to its size, the engorgement of the erectile tissues may take place after intromission. Such copulatory pattern is observed in some mammal species, as canids, and leads to a so-called \u0026ldquo;copulatory lock\u0026rdquo; (Dixson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). It has also been suggested to occur in some bat species (Wilson, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), where the penis is erected before intromission, but could be retracted rapidly following disturbance (Wimsatt, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1945\u003c/span\u003e). Observations of captive \u003cem\u003eE. serotinus\u003c/em\u003e pairs \u003cem\u003ein copula\u003c/em\u003e for several hours (Racey and Kleiman, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1970\u003c/span\u003e) hint towards the occurrence of a copulatory lock in the species.\u003c/p\u003e \u003cp\u003eAlternatively, the long motile and stable erected penis may be used to pass by the protective tail membrane (i.e. uropatagium) of the female in order to reach the vulva. Under such conditions however, the penis cannot be expected to penetrate the vagina, leaving as only option a contact mating. This copulatory pattern (i.e. cloacal contact, e.g. (Drachmann et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1997\u003c/span\u003e)) is common in birds but remains unknown in mammals.\u003c/p\u003e \u003cp\u003eIn this study, we provide an anatomical description of the penis and female genital tract and describe mating events that we observed in order to define the copulatory behaviour of \u003cem\u003eE. serotinus\u003c/em\u003e. Specifically, we want to establish whether the penis enables a copulatory lock during the mating process or functions as a copulatory arm without penetrating the vulva, which would represent a copulatory pattern still unknown in mammals. Additionally, to draw a full picture about the mating behaviours of this species, we present the phenology of copulations and compare it with seasonal activity patterns near underground sites. To the best of our knowledge, this is the first comprehensive compilation of knowledge on reproduction of a bat species so far.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eModel species\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. serotinus\u003c/em\u003e is a palearctic species whose distribution spans from Spain to China (Artyushin et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Juste et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). At the global scale, it is listed as \u0026ldquo;Least Concern\u0026rdquo; on the IUCN Red List of Threatened Species (Godlevska et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but locally has been considered as a vulnerable species (e.g. (Bohnenstengel et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eYearling males can reach sexual maturity, produce enough sperm to fill up the epididymides and copulate during their first year of life (Racey and Kleiman, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). Sexual maturity in females also may be reached already during their first year of life (Pelikan, Gaisler and R\u0026ouml;dl (1979) cited by (Haensel, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1994\u003c/span\u003e)). Females of \u003cem\u003eE. serotinus\u003c/em\u003e, like most of Vespertilionidae species, can store sperm until ovulation, which occurs in spring (Crichton, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), Personal observations of the authors). Ovulation in bats remains poorly investigated. Evidence indicates that it is spontaneous in \u003cem\u003eCarollia perspicillata\u003c/em\u003e (Rasweiler IV et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), as well as in sperm storing species ((Oxberry, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), but see also (deCatanzaro et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eThe flaccid penis of \u003cem\u003eE. serotinus\u003c/em\u003e is pendulous and widens towards the tip. A weak medial ridge can be observed on the upper side (Dietz and von Helversen, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The baculum measures are approximately 1.10mm, which is relatively short in comparison to other Vespertilionidae species (Hill and Harrison, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Hosken et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eErected penis and female genital tract anatomy\u003c/p\u003e \u003cp\u003eIn the framework of different research projects in Germany and Poland, ten male bats were captured and anaesthetised using Isoflurane (5 vol. % for induction, 1\u0026ndash;2 vol. % for maintenance; flow rate 1.5 l/min ambient air). This procedure is known to provoke erection in Vespertilionidae (Fasel et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Penis length was measured while fully erected as the ventral length between the base of the penis, where the skin becomes bright red, and the tip of the penis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Terminal swelling was measured at the tip of the penis, where the width is maximal (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Measurements were reported only when a complete erection was verified (i.e., tight skin, bright sharp red colour and rigid structure). All measures were performed by a single observer (NJF). Selected measurements were replicated, and repeatability was estimated using the function \u0026ldquo;rpt\u0026rdquo; from package \u0026ldquo;rptR\u0026rdquo; (Stoffel et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe performed necropsy of eleven frozen-thawed females and seven males that deceased at the bat rehabilitation centre at Zitadelle Spandau, Germany. Of these, we performed histology of the genital tracts of four females (three in longitudinal orientation and one in cross-section) as well as of two males. Organ samples collected during necropsy were fixed in 4% neutral buffered formalin and embedded in paraffin wax for histology. Sections (3 \u0026micro;m) were stained with haematoxylin and eosin (HE). Histological images were recorded using a digital photo-microscope (Keyence VHX-1000, Osaka, Japan); organ size was measured post-hoc on scaled digital images.\u003c/p\u003e \u003cp\u003eBehavioural observation\u003c/p\u003e \u003cp\u003eMating events were recorded opportunistically at two different sites: St Matthias Church in Castenray (Netherlands) and the Ukrainian Bat Rehabilitation Centre (UBRC) located in Kharkiv (Ukraine).\u003c/p\u003e \u003cp\u003eSt Matthias Church in Castenray (Netherlands) has been hosting a colony of around 150 females \u003cem\u003eE. serotinus\u003c/em\u003e in the attic (JJ personal observation). A maximum of eighteen cameras have been installed since 2016 in order to document behaviours of this species. The date and duration of each recorded copulation event have been reported between October 25th 2016 and March 22nd 2022.\u003c/p\u003e \u003cp\u003eCopulation events of captive \u003cem\u003eE. serotinus\u003c/em\u003e in short- and long-term rehabilitation were documented by the staff of the UBRC. Bats lived in cages with custom-made wood and paper roosts, but only outside copulations were recorded. Copulations were recorded between August 2018 and December 2021.\u003c/p\u003e \u003cp\u003eFor each copulatory event, the date, time and duration of copulation were recorded and the bat`s behaviour was described. Videos focused on the genitalia were carefully reviewed to describe the type of copulatory behaviours and the female and male anatomical interactions.\u003c/p\u003e \u003cp\u003eActivity at underground site\u003c/p\u003e \u003cp\u003e \u003cem\u003eEptesicus serotinus\u003c/em\u003e were captured at the entrance of abandoned mines (Baulmes, Switzerland: 46\u0026deg;47\u0026rsquo;N, 6\u0026deg;31\u0026rsquo;W). Captures were part of a survey of the mines running annually from 2001 to 2022. The mine characteristics have been previously described by (Giavi et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFlaccid and erected penis and female genital tract anatomy\u003c/p\u003e \u003cp\u003eThe flaccid penis was measured on digital scaled microscopic images of formalin-fixed material post-mortem in 7 individuals. Penis shaft length averaged 6.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87 mm and width 3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 mm. The histology of the vascular penis of \u003cem\u003eE. serotinus\u003c/em\u003e follows the general vespertilionid anatomy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It contains three distinct erectile tissues, (i) the \u003cem\u003ecorpora cavernosa\u003c/em\u003e (CC), (ii) the \u003cem\u003ecorpus spongiosum\u003c/em\u003e (CS) surrounding the \u003cem\u003eurethra\u003c/em\u003e (u), both enveloped by their respective \u003cem\u003etunicae albugineae\u003c/em\u003e (TC), and (iii) the accessory cavernous body formed by accessory cavernous tissue (AT). An extended, folded and vascularized prepuce surrounds the glans penis, which is formed by \u003cem\u003eaccessory cavernous tissue\u003c/em\u003e. The \u003cem\u003ebaculum\u003c/em\u003e is located dorsal to the \u003cem\u003eurethra\u003c/em\u003e, located partly in the penile body and partly in the \u003cem\u003eglans penis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe erected penis was measured in 10 anaesthetised males, between one and three times (average 2.18 times). The repeatability of these measurements was 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, P\u0026thinsp;=\u0026thinsp;0.004 for length of the erected penis and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, P\u0026thinsp;=\u0026thinsp;0.007 for the width of the terminal swelling respectively. The length of the penis was 16.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 mm, and the maximum width of the terminal swelling was 7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The terminal swelling is composed of two large erectile tissues, distinct from the \u003cem\u003ecorpora cavernosa\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe female reproductive tract length as measured on digital scaled microscopic images of formalin-fixed material post-mortem in 7 individuals is 13.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35 mm, cervical diameter 1.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 mm, and ovarian diameter 1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 mm x 1.58 x 0.27 mm.\u003c/p\u003e \u003cp\u003eThe vagina is approximately 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mm long, the cervix 8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 mm, and the uterine horns 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCopulatory behaviours\u003c/p\u003e \u003cp\u003eWe recorded 93 copulations in St. Matthias church and four in the UBRC via video monitoring. Recorded copulatory behaviours are described as follows: The male grasped the female in a dorsoventral position, mostly on vertical surfaces, both animals with their head upside down (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The male bit the skin on the nape. Lateral movements of the male hindquarters were then accompanied by rapid probing movements of the fully erected penis (supplementary information). Social calls were emitted, probably by the female. The pair stopped moving when the penis was firmly pushed against the vulva. Once the pair remained immobile, no more vocalisation was heard. The fur on the female abdomen appeared wet.\u003c/p\u003e \u003cp\u003eIn St. Matthias the church, the first copulation after the weaning of the young can be observed in September, peaking in October with 49.5% of all recorded copulation events (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Then the frequency of copulatory decreased with the latest event recorded on the 31st of December. Infrequent copulations were also recorded in March and beginning of April. Half of the copulations lasted for less than 53 min and the longest one for 12.7 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eActivity at underground site\u003c/p\u003e \u003cp\u003eAt the abandoned mines (Baulmes, Switzerland), the number of surveys per year varied from 0 to 16 (mean: 3.15). No captures took place between November and February. In total, 233 \u003cem\u003eE. serotinus\u003c/em\u003e were captured and sexed. Individuals were captured from March to October, with a peak in July and August (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The sex ratio was male biased (0.82).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe mating events described in this study validate that copulation in \u003cem\u003eE. serotinus\u003c/em\u003e does not comprise vaginal penetration of the shaft of the penis. Indeed, the erectile tissues of the penis are enlarged before the contact with the vulva and form a shape unsuitable for intromission. Furthermore, the erected penis length exceeds the vaginal length by approximately four times. Our observations support that the penis remains in contact with the vulva, but without vaginal intromission. The relatively small size of the baculum, as well as its shape, additionally indicate an absence of any specific role of the penis bone in the intromission. The baculum of \u003cem\u003eE. serotinus\u003c/em\u003e may serve to protect the urethra from compression by the erectile tissues during erection as suggested for \u003cem\u003eE. furinalis\u003c/em\u003e (Comelis et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). To our knowledge, this study is the first to report a mammalian copulatory pattern without intromission (Dixson, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe stiffened penis of \u003cem\u003eE. serotinus\u003c/em\u003e may be important as a means to pass by the uropatagium (i.e. tail membrane) and reach the vulva. To some extent, the tail membrane could be used by the female to avoid copulation. The long penis of E. \u003cem\u003eserotinus\u003c/em\u003e may therefore serve as a \u0026lsquo;copulatory arm\u0026rsquo;, as observed in some cetaceans e.g. (Keener et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), in order to overpass the uropatagium of the female. Once the penis is firmly positioned on the top of the vulva, the movements of the hindquarters and of the penis stopped. While half of the observed copulations did not exceed 30 min, the longest one recorded lasted 12.7 hours, confirming previous observations (Racey and Kleiman, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). The hollow structure on the dorsal side of the erected penis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) is formed by two accessory cavernous tissues, according to the terminology of (Wimsatt and Kallen, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1952\u003c/span\u003e). This structure might act as a suction cup and support the maintenance of the copulatory contact. Thus, the large size and characteristic shape of the penis as well as the prolonged embrace might further support the transport of sperm cells to the vagina and to the cervix.\u003c/p\u003e \u003cp\u003eFrom our observations, males appear to chase females, without any courtship behaviour. Furthermore, a minority of observed copulations took place during the hibernation period when females were torpids. The absence of courtship and winter copulations support a reduced pre-copulatory female choice. Male selection by the female could supposedly take place after the copulation through selective sperm transport through the cervix or active degradation by immune cells or maintenance within the uterus (Orr and Zuk, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Such mechanisms remain to be investigated.\u003c/p\u003e \u003cp\u003eAs this mating pattern remained unobserved so far, it may concern only a few species and probably only bats. In \u003cem\u003eMyotis lucifugus\u003c/em\u003e, a similar subterminal swelling of the penis has been described, produced by the engorgement of the accessory cavernous tissues (Wimsatt and Kallen, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1952\u003c/span\u003e). In this latest species, the intromission of the penis within the vagina was however suggested, despite its large size (Wimsatt, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1945\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMales of \u003cem\u003eE. serotinus\u003c/em\u003e are known to visit breeding roosting sites as early as the end of July (Haensel, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This later article does, however, not mention the observation of copulations. In the St Matthias Church, copulations were observed from the beginning of August to the end of April. This result suggests that the mating season starts when the males terminate spermatogenesis and females wean the young, and continues until the ovulation in spring, confirming earlier findings (Eisentraut, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1936\u003c/span\u003e). Our observations also demonstrate that both males and females can store sperm for months in the epididymides and in the genital tract, respectively.\u003c/p\u003e \u003cp\u003eOur study clearly supports that males visit female roosting sites. It remains unknown however whether males also attract sexual partners in other locations. \u003cem\u003eE. serotinus\u003c/em\u003e are known to visit swarming sites in Germany (Pfeiffer and Mayer, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and in the Netherlands (Van Schaik et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In Switzerland, our results show that individuals are visiting the mine all year round. After the weaning of the young, the activity at the mines is higher in July and August, similar to what was observed in the Netherlands (Van Schaik et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similar phenology of activity near underground roosts was shown for \u003cem\u003eE. fuscus\u003c/em\u003e (Mumford and Whitaker, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). However, as summer activity occurs earlier than the peak of copulations, it might have other functions than mating, as for example transferring information to yearling individuals about potential hibernacula and/or mating sites (i.e. colony member guidance hypothesis (Piksa, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Stumpf et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Van Schaik et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Veith et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e)). It should also be considered that \u003cem\u003eE. serotinus\u003c/em\u003e visit the entrance of underground sites to forage (suggested by (Mumford and Whitaker, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1974\u003c/span\u003e) for \u003cem\u003eE. fuscus\u003c/em\u003e) or that individuals are attracted there by the activity of other species.\u003c/p\u003e \u003cp\u003eIn conclusion, our results suggest that the erected penis of \u003cem\u003eE. serotinus\u003c/em\u003e serves to pass by the tail membrane of the female and is used to reach the vulva without vaginal intromission. This copulatory pattern is unknown in other mammals. Additional studies of the copulatory behaviours of bats may reveal other species using copulation without sexual penetration. We also demonstrate that males visit breeding colonies and copulate there with females. The time lag between summer activity at swarming sites and the peak of copulation supports that the visits of \u003cem\u003eE. serotinus\u003c/em\u003e at underground sites might serve functions other than mating. This study contributes to a better understanding of animal mating and further fertilisation-involved processes. Such knowledge can improve conservation of endangered species, and open doors for further research in physiology, ethology and ecology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe are thankful to Jörg Harder and Robert Henning from the Zitadelle Spandau, Germany for providing female carcasses, to Bernd Ohlendorf and the administration of the Karst Landscape South Harz Biosphere Reserve for supporting our field work and providing field lab space. We thank Alona Prylutska and Maryna Yerofeieva from Ukrainian Bat Rehabilitation Center, Ukraine, for coordination and assistance during data collecting in Ukraine. We thank Gudrun Wibbelt for interpretation of the histology, Doris Krumnow and Monique Schmückert for preparation of histology slides. We are grateful to all who have got involved in the survey of the Swiss mines. We especially thank Taisiia Kravchenko for the professional drawings: [email protected], Instagram: taisiya.kravchenko\u003c/p\u003e\n\u003cp\u003eCompliance with Ethical Standards\u003c/p\u003e\n\u003cp\u003eAll captures and manipulations were conducted under permission of local conservation and animal ethics authorities (Germany: conservation authority of Mansfeld-Südharz county (authorization no. NB30A_18001_Fie) and Veterinary Affairs Office of Sachsen-Anhalt (LVwA, auth. no. 42502-2-1525 LIZW); Poland: Regional Director for Environmental Protection in Białystok (auth. no. WPN.6401.57.2015.WL) and Ethical Commissions in Białystok and Olsztyn (auth. no. 43/2015; 152/2015; 153/2015; 16/2016). The Bat Rehabilitation Center of Feldman Ecopark (Ukraine) works under the general permission of the Kharkiv Oblast Authority of Ecology and Natural Resources in 2013-2022. Authorizations of capture in Switzerland were delivered by the Direction Générale de l’Environnement, canton de Vaud.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis project was founded by the National Science Centre, Poland, on the basis of decision number DEC-2013/10/E/NZ8/00725, the Swiss National Science Foundation (grant number: P2BEP3_168709 to NJF). The Bat Rehabilitation Center of Feldman Ecopark worked (in 2013-2022) under financial support of International Charity Foundation “Oleksandr Feldman Foundation” (Kharkiv, Ukraine).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArtyushin, I. V., Kruskop, S. V., Lebedev, V.S., Bannikova, A.A., 2018. Molecular Phylogeny of Serotines (Mammalia, Chiroptera, \u003cem\u003eEptesicus\u003c/em\u003e): Evolutionary and Taxonomical Aspects of the \u003cem\u003eE. serotinus\u0026nbsp;\u003c/em\u003eSpecies Group. Biol. Bull. 45, 469\u0026ndash;477. https://doi.org/10.1134/S1062359018050035\u003c/li\u003e\n \u003cli\u003eBirkhead, T.R., Atkin, L., M\u0026oslash;ller, A.P., 1987. Copulation Behaviour of Birds. Behaviour 101, 101\u0026ndash;138. https://doi.org/10.1163/156853987X00396\u003c/li\u003e\n \u003cli\u003eBohnenstengel, T., Krättli, H., Obrist, M., Bontadina, F., Jaberg, C., Ruedi, M., Moeschler, P., 2014.\u0026nbsp;Liste rouge Chauves-souris. Esp\u0026egrave;ces menac\u0026eacute;es en Suisse, \u0026eacute;tat 2011. L\u0026rsquo;environnement Prat. 1412, 95.\u003c/li\u003e\n \u003cli\u003eComelis, M.T., Bueno, L.M., G\u0026oacute;es, R.M., Morielle-Versute, E., 2015.\u0026nbsp;Penile histomorphology of the neotropical bat \u003cem\u003eEptesicus furinalis\u003c/em\u003e (Chiroptera: Vespertilionidae).\u0026nbsp;Zool. Anz. 258, 92\u0026ndash;98. https://doi.org/10.1016/j.jcz.2015.08.001\u003c/li\u003e\n \u003cli\u003eCrichton, E.G., 2000.\u0026nbsp;Sperm Storage and Fertilization, in: Reproductive Biology of Bats. pp. 295\u0026ndash;320. https://doi.org/10.1016/B978-012195670-7/50008-4\u003c/li\u003e\n \u003cli\u003edeCatanzaro, D., Pollock, T., Greville, L.J., Faure, P.A., 2014. Estradiol transfer from male big brown bats (\u003cem\u003eEptesicus fuscus\u003c/em\u003e) to the reproductive and brain tissues of cohabiting females, and its action as a pheromone. Gen. Comp. Endocrinol. 208, 126\u0026ndash;133. https://doi.org/10.1016/j.ygcen.2014.09.011\u003c/li\u003e\n \u003cli\u003eDewsbury, D.A., 1972. Patterns of Copulatory Behavior in Male Mammals.\u0026nbsp;Q. Rev. Biol. 47, 1\u0026ndash;33. https://doi.org/10.1086/407097\u003c/li\u003e\n \u003cli\u003eDietz, C., von Helversen, O., 2004.\u0026nbsp;Illustrated identification key to the bats of Europe. https://doi.org/10.1007/s00436-016-4936-2\u003c/li\u003e\n \u003cli\u003eDixson, A.F., 2021a. Copulatory Interactions and Sexual Selection., in: Mammalian Sexuality: The Act of Mating and the Evolution of Reproduction. Cambridge University Press.\u003c/li\u003e\n \u003cli\u003eDixson, A.F., 2021b. Copulatory patterns: Phylogeny and modes of life, in: Mammalian Sexuality: The Act of Mating and the Evolution of Reproduction. Cambridge University Press.\u003c/li\u003e\n \u003cli\u003eDixson, A.F., 2021c. Mammalian Sexuality: The Act of Mating and the Evolution of Reproduction. 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Zool. 1\u0026ndash;7. https://doi.org/10.1093/cz/zoy094\u003c/li\u003e\n \u003cli\u003eFasel, N.J., Mamba, M.L., Monadjem, A., 2020. Penis morphology facilitates identification of cryptic African bat species. J. Mammal. 101, 1392\u0026ndash;1399. https://doi.org/10.1093/jmammal/gyaa073\u003c/li\u003e\n \u003cli\u003eGallup, G.G., Burch, R.L., Zappieri, M.L., Parvez, R.A., Stockwell, M.L., Davis, J.A., 2003. The human penis as a semen displacement device. Evol. Hum. Behav. 24, 277\u0026ndash;289. https://doi.org/10.1016/S1090-5138(03)00016-3\u003c/li\u003e\n \u003cli\u003eGiavi, S., Glaizot, O., Christe, P., 2020. Sex and Age Variation in the Phenology of a Common Pipistrelle Bat (\u003cem\u003ePipistrellus pipistrellus\u003c/em\u003e) Population in Front of a Hibernaculum. Acta Chiropterologica 22, 113. https://doi.org/10.3161/15081109acc2020.22.1.010\u003c/li\u003e\n \u003cli\u003eGodlevska, L., Kruskop, S.V., Gazaryan, S., 2021. \u003cem\u003eEptesicus serotinus\u003c/em\u003e. IUCN Red List Threat. Species. https://doi.org/10.2305/IUCN.UK.2021-1.RLTS.T85199559A195834153.en\u003c/li\u003e\n \u003cli\u003eHaensel, V.J., 1994.\u0026nbsp;Zum Eintritt der Geschlechtsreife bei der Breitfl\u0026uuml;gelfledermaus (\u003cem\u003eEptesicus serotinus\u003c/em\u003e) und zum Aufenthalt adulter M\u0026auml;nnchen in ihren Wochenstubengesellschaften.\u0026nbsp;Nyctalus 5, 181\u0026ndash;184.\u003c/li\u003e\n \u003cli\u003eHill, J.E., Harrison, D.L., 1987. The baculum in the Vespertilioninae (Chiroptera: Vespertilionidae) with a systematic review, a synopsis of \u003cem\u003ePipistrellus\u003c/em\u003e and \u003cem\u003eEptesicus\u003c/em\u003e, and the descriptions of a new genus and subgenus. Bull. Br. Museum (Natural Hist. 52, 225\u0026ndash;305.\u003c/li\u003e\n \u003cli\u003eHosken, D.J., Jones, K.E., Chipperfield, K., Dixson, A.F., 2001. Is the bat os penis sexually selected? Behav. Ecol. 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Bat Mating Systems, in: Reproductive Biology of Bats. pp. 321\u0026ndash;362. https://doi.org/10.1016/B978-012195670-7/50009-6\u003c/li\u003e\n \u003cli\u003eMumford, R.E., Whitaker, J.O., 1974. Seasonal activity of bats at an Indiana Cave. Proc. Indiana Acad. Sci. 84, 500\u0026ndash;507.\u003c/li\u003e\n \u003cli\u003eOrr, T.J., Zuk, M., 2013. Does delayed fertilization facilitate sperm competition in bats? Behav. Ecol. Sociobiol. 67, 1903\u0026ndash;1913. https://doi.org/10.1007/s00265-013-1598-2\u003c/li\u003e\n \u003cli\u003eOxberry, B.A., 1979. Female reproductive patterns in hibernating bats. Reproduction 56, 359\u0026ndash;367. https://doi.org/10.1530/jrf.0.0560359\u003c/li\u003e\n \u003cli\u003ePfeiffer, B., Mayer, F., 2013. Spermatogenesis, sperm storage and reproductive timing in bats. J. Zool. 289, 77\u0026ndash;85. https://doi.org/10.1111/j.1469-7998.2012.00970.x\u003c/li\u003e\n \u003cli\u003ePiksa, K., 2008. 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Ecology of \u003cem\u003eMyotis nigricans\u003c/em\u003e (Mammalia: Chiroptera) on Barro Colorado Island, Panama Canal Zone. J. Zool. 163, 1\u0026ndash;13. https://doi.org/10.1111/j.1469-7998.1971.tb04521.x\u003c/li\u003e\n \u003cli\u003eWimsatt, W.A., 1945. Notes on Breeding Behavior, Pregnancy, and Parturition in Some Vespertilionid Bats of the Eastern United States.\u0026nbsp;J. Mammal. 26, 23. https://doi.org/10.2307/1375029\u003c/li\u003e\n \u003cli\u003eWimsatt, W.A., Kallen, F.C., 1952. Anatomy and histophysiology of the penis of a vespertilionid bat, \u003cem\u003eMyotis lucifugus lucifugus\u003c/em\u003e, with particular reference to its vascular organization. J. Morphol. 90, 415\u0026ndash;465. https://doi.org/10.1002/jmor.1050900303\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2813754/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2813754/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCopulatory behaviours stand as cornerstones of sexual selection, yet they remain mysterious in many species, especially in bats. An extremely large penile erection with a heart-shape terminal swelling has been observed in the serotine bat (\u003cem\u003eEptesicus serotinus\u003c/em\u003e). The engorgement of the erectile tissues could take place after penetration to generate a copulatory lock. Alternatively, the erected penis, long and mobile, could be used to pass by the protective tail membrane to reach the vulva. In the latter scenario, the penis may however not be able to penetrate the vagina. In order to find out which of the two scenarios occurs, we investigated the morphology of female and male genitalia of \u003cem\u003eE. serotinus\u003c/em\u003e. We found that the vagina is more than seven times shorter than the erected penis, supporting that the penis is used as a copulatory arm rather than an intromittent organ.\u003c/p\u003e \u003cp\u003eWe could confirm our hypothesis with direct observations of copulations. During mating, the male grasps the female in a dorsoventral position. The male probes the female\u0026rsquo;s ventral part with its erected penis until the penis is tightly pushed against the vulva. Afterward, the pair stops moving and can hold the position for several hours.\u003c/p\u003e \u003cp\u003eIn addition, the timing of copulations, which exhibits a peak in October, implies that visits to swarming sites throughout the year may serve other functions besides mating.\u003c/p\u003e \u003cp\u003eThis research reveals a novel copulatory behaviour that has not been previously documented in mammals and sheds light on the poorly understood area of bat reproduction.\u003c/p\u003e","manuscriptTitle":"No intromission is involved in the mating of Eptesicus serotinus, a novel copulatory pattern in mammals.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-18 14:32:16","doi":"10.21203/rs.3.rs-2813754/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"189686f6-33b6-49a2-8149-73d1c71bfc1c","owner":[],"postedDate":"April 18th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-07-18T17:05:10+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-18 14:32:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2813754","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2813754","identity":"rs-2813754","version":["v1"]},"buildId":"pf3fE39SIOqb-0xH_OWvX","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}