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. 1B). Terminal swelling was measured at the tip of the penis, where the width is maximal (Fig. 1C). 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).