Mating without intromission in a bat.

Copulatory behaviours stand as cornerstones of sexual selection, yet they remain mysterious in many species. Because of their nocturnal and elusive lifestyle, the copulatory behaviours of bats have been mostly overlooked1. Several aspects of bat reproduction differ from other mammals (e.g. prolonged sperm storage2, delayed development3). Here, we show that in serotine bats (Eptesicus serotinus) the penis is used as a 'copulatory arm' rather than an intromittent organ, revealing a novel copulatory behaviour in mammals.

Assigning metabolic rate measurements to torpor and euthermy in heterothermic endotherms: 'torpor', a new package for R.

Torpor is a state of controlled reduction of metabolic rate (M) in endotherms. Assigning measurements of M to torpor or euthermy can be challenging, especially when the difference between euthermic M and torpid M is small, in species defending a high minimal body temperature in torpor, in thermolabile species, and slightly below the thermoneutral zone (TNZ). Here, we propose a novel method for distinguishing torpor from euthermy. We use the variation in M measured during euthermic rest and torpor at varying ambient temperatures (Ta) to objectively estimate the lower critical temperature (Tlc) of the TNZ and to assign measurements to torpor, euthermic rest or rest within TNZ. In addition, this method allows the prediction of M during euthermic rest and torpor at varying Ta, including resting M within the TNZ. The present method has shown highly satisfactory results using 28 published sets of metabolic data obtained by respirometry on 26 species of mammals. Ultimately, this novel method aims to facilitate analysis of respirometry data in heterothermic endotherms. Finally, the development of the associated R-package (torpor) will enable widespread use of the method amongst biologists.

Energy allocation shifts from sperm production to self-maintenance at low temperatures in male bats.

The ability of animals to produce endogenous heat provides a buffer against environmental changes but also incurs high energetic costs. Especially small endothermic mammals have high energy demands. Some temperate-zone species (heterotherms) regularly use torpor, which slows down their entire metabolism but also potentially delays reproduction, to compensate for this. We used a unique experimental approach to test the consequences of extended low and high ambient temperatures on the trade-off in energy allocation to body mass maintenance, thermoregulation effort and seasonal sexual maturation in temperate zone male bats. We showed that long exposure to low ambient temperature shifts energy allocation away from sexual maturation to self-maintenance and results in a delay of sperm maturation by as much as an entire month. This effect was partially buffered by higher body mass. Heavier bats were able to afford more intensive thermoregulation and consequently speed up maturation. Interestingly, bats at constant high temperatures avoided deep torpor and matured faster than those at low temperatures, but sperm production was also slower than under natural conditions. Our results show that not only low, but also constant high ambient temperatures are detrimental during seasonal sexual maturation and the trade-off between investing into self-maintenance and fitness is a finely tuned compromise.

Malignant Peripheral Nerve Sheath Tumour in a Seba's Short-Tailed Bat (Carollia perspicillata).

There are few reports of tumours in bats, despite the fact that members of the Chiroptera order include 22.4% of all currently known mammal species. We now describe a case of malignant peripheral nerve sheath tumour (MPNST) in an adult female Seba's short-tailed bat (Carollia perspicillata) from a private zoo in Switzerland. The macroscopical and histological findings indicated that the MPNST originated from a dorsal spinal nerve root sheath in the lumbar region and metastasized to the spleen. Immunohistochemical labelling for glial fibrillary acid protein, S100 protein, neuron specific enolase and synaptophysin was negative. The cytoplasm of a few neoplastic cells labelled for smooth muscle actin and desmin. To our knowledge, this is the first report of a PNST in a member of the Chiroptera order.

DNA methylation predicts age and provides insight into exceptional longevity of bats.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Food restriction delays seasonal sexual maturation but does not increase torpor use in male bats.

Balancing energy budgets can be challenging, especially in periods of food shortage, adverse weather conditions and increased energy demand due to reproduction. Bats have particularly high energy demands compared to other mammals and regularly use torpor to save energy. However, while torpor limits energy expenditure, it can also downregulate important processes, such as sperm production. This constraint could result in a trade-off between energy saving and future reproductive capacity. We mimicked harsh conditions by restricting food and tested the effect on changes in body mass, torpor use and seasonal sexual maturation in male parti-coloured bats (Vespertilio murinus). Food-restricted individuals managed to maintain their initial body mass, while in well-fed males, mass increased. Interestingly, despite large differences in food availability, there were only small differences in torpor patterns. However, well-fed males reached sexual maturity up to half a month earlier. Our results thus reveal a complex trade-off in resource allocation; independent of resource availability, males maintain a similar thermoregulation strategy and favour fast sexual maturation, but limited resources and low body mass moderate this latter process.

Experimental manipulation of reproductive tactics in Seba's short-tailed bats: consequences on sperm quality and oxidative status.

To reproduce, males have to fertilize the female's eggs, sometimes in competition with ejaculates of other males. In species where males display alternative reproductive tactics, whereby territorial males secure mating and non-territorial males have to sneak copulations, the latter might be expected to invest relatively more resources towards sperm quality compared with the territorial males. Sperm cells are especially vulnerable to oxidative stress, which reduces male fertility. Therefore, antioxidant resources are expected to modulate sperm quality, and might be allocated differently between reproductive tactics. To test the link between reproductive tactics, redox profile and sperm quality, we experimentally induced changes in the reproductive tactics of 39 captive males Seba's short-tailed bats Carollia perspicillata. We monitored the blood and ejaculate oxidative balance, and the sperm quality before, 7 days and 21 days after the manipulation of reproductive tactic. Although ejaculates' oxidative damage was negatively related to sperm velocity, males exhibited similar blood and ejaculates redox profiles and similar sperm quality, regardless of their reproductive tactic. Possibly, these results arise as a consequence of some constraints having been lifted during the experiment. Our results also suggest that, in Seba's short-tailed bats, the expression of alternative reproductive tactics is not subjected to strong oxidative constraints. Furthermore, our results could reflect an absence of trade-off between pre- and post-copulatory traits in harem males, as they could be selected to invest both in female attraction and sperm quality, as a consequence of their inability to fully monopolize females.

First cryopreservation of phyllostomid bat sperm.

Bats, the second-largest mammalian order, are widely distributed and provide crucial ecosystem services. Their reproductive biology comprises noteworthy characteristics, such as long-term sperm storage, delayed implantation or even delayed fetal development. The understanding of these mechanisms remains limited. Research in reproductive biology may become crucial for the conservation of endangered bat species. Indeed, the IUCN Red List of Threatened Species currently lists 76 bat species as endangered or critically endangered, for which conservation measures should be taken. The development of assisted reproductive technologies (ART) in bats would thus open new perspectives. In this context we here describe the first successful cryopreservation of bat sperm in the Seba's short-tailed bat (Carollia perspicillata). Epididymal sperm of 30 males was pooled in 6 sample sets and diluted using four different cryo-diluents: Canifreeze, modified Canifreeze, BotuCrio® and Test egg yolk extender. Glycerol and a mixture of glycerol and methylformamide were used as cryoprotectants. Best post-thaw results were achieved when sperm was diluted in Test egg yolk extender containing a mixture of glycerol and methylformamide as cryoprotectant. Post-thaw sperm motility, progressive motility and acrosome integrity of 42.0 ±â€¯3.4%, 28.3 ±â€¯6.3% and 87.7 ±â€¯1.7%, respectively were superior to all other variants tested. It remains to be determined if the post-thaw quality of cryopreserved bat spermatozoa achieved in this study is sufficient for the use in artificial inseminations and produces acceptable pregnancy rates. Successful cryopreservation of spermatozoa in C. perspicillata may serve as the first model to establish gamete rescue programs and develop ART in other, critically endangered bat species.

Valuable carcasses: postmortem preservation of fatty acid composition in heart tissue.

In order to effectively conserve species, we must understand the structure and function of integral mechanisms at all levels of organismal organisation, from intracellular biochemistry to whole animal ecophysiology. The accuracy of biochemical analyses depend on the quality and integrity of the samples analysed. It is believed that tissue samples collected immediately postmortem provide the most reliable depiction of the living animal. Yet, euthanasia of threatened or protected species for the collection of tissue presents a number of ethical complications. Polyunsaturated fatty acids (PUFA) are essential to the cardiovascular system of all animals and the structure of PUFA can be degraded by peroxidation, potentially modifying the fatty acid composition of the tissue over postmortem time. Here, we assessed the composition of PUFA in cardiac tissue of bats (Carollia perspicillata) over the course of 12-h postmortem. We show that PUFA are resistant to naturally occurring postmortem degradation in heart tissue, with no difference in the overall composition of fatty acids across all time classes (0, 3, 6 or 12-h postmortem). Our results suggest that carcasses that would otherwise be discarded may actually be viable for the assessment of fatty acid composition in a number of tissues. We hope to spur further investigations into the viability of carcasses for other biochemical analyses as they may be an untapped resource available to biologists.

Modification of sperm fatty acid composition during epididymal maturation in bats.

Biochemical properties of polyunsaturated fatty acids (PUFAs) are fundamental to sperm movements. Amongst all adjustments operated during epididymal maturation, sperm membrane lipid composition is remodelled. Specifically, the proportion of PUFAs usually increases from the caput towards the cauda epididymidis. In mammals, PUFAs are predominantly acquired through the diet, which can consequently impact male fertility. We aimed at analysing to what extent n-6 and n-3 PUFAs are incorporated into sperm in the Seba's short-tailed bat (Carollia perspicillata), and at demonstrating the effect of the sperm fatty acid composition on sperm mobility. We therefore provided food varying in fatty acid composition to males of C. perspicillata and measured the fatty acid composition and mobility traits in spermatozoa collected from the caput and cauda epididymides. We found that n-6 and n-3 PUFAs and saturated fatty acids were significantly related to sperm velocity but not to the proportion of progressive sperm (i.e. motility). Concomitant to an increase in sperm velocity, the level of fatty acid saturation increased from the caput to the cauda epididymidis, while the proportion of PUFAs remained similar along the epididymis. A reduction in n-6 PUFAs counterbalanced an increase in n-3 PUFAs. The food treatments did not affect the sperm fatty acid composition. Our results suggest that a precise endogenous control rather than dietary effects determines sperm fatty acid composition in C. perspicillata.

Protection against cutaneous leishmaniasis in outbred vervet monkeys using a recombinant histone H1 antigen.

Infection with Leishmania major parasites results in the development of cutaneous ulcerative lesions on the skin. We investigated the protective potential of a single, recombinant histone H1 antigen against cutaneous leishmaniasis in an outbred population of vervet monkeys, using Montanide adjuvant. Protection was assessed by challenging the animals with a mixture of vector sand fly salivary-gland lysate and a low dose of in vitro-derived parasites, thus more closely mimicking natural infection induced by L. major. The course of infection in immunized monkeys was compared with that of animals that had healed from a primary infection and were immune. The monkeys immunized with recombinant histone H1 showed a reduced development of lesion size, compared with controls. Our study therefore illustrates the potential use of histone H1 as a vaccine candidate against cutaneous leishmaniasis in humans.