Effect of sex and reproductive status on the immunity of the temperate bat Myotis daubentonii.

Studies of immunity in bat species are rare. However, it is important to determine immunological variations to identify factors influencing the health status of these endangered mammals from an evolutionary, ecological, conservation, and public health point of view. Immunity is highly variable and can be influenced by both internal (e.g. hormone levels, energy demand) and external factors (e.g. pathogens, climate). As bats have some peculiar ecological, energetic, and putative immunological characteristics, they are outstanding study organisms for ecoimmunological studies. We tested if (i) female bats have a higher immunity than males similar to most other mammalian species and (ii) individuals differ according to their energy demand (e.g. reproductive status). To study these questions, we sampled female and male Myotis daubentonii with different reproductive states and estimated their bacterial killing activity, hemolysis/hemagglutination titer, immunoglobulin G (IgG) concentration, and total and differential white blood cell counts. These methods characterize the cellular and humoral branches of both the adaptive and the innate immune responses of these individuals. Reproductively active males had lower cellular immunity compared to non-reproductive individuals. Pregnant females had increased IgG concentrations while hemolysis was enhanced during lactation. No clear trade-off between immunity and reproduction was found; instead immunity of males and female bats seems to be modulated differently due to varying hormonal and energetic states. Our data suggest that both adaptive and innate immunity as well as individual differences (i.e. sex and reproductive state) need to be considered to get a comprehensive overall picture of immunity in wild mammals.

Similar but not the same: metal concentrations in hair of three ecologically similar, forest-dwelling bat species (Myotis bechsteinii, Myotis nattereri, and Plecotus auritus).

Recently, a number of studies demonstrated the suitability of hair analysis to assess metal exposure of bats. As many bat species are endangered, such a non-destructive method is particularly suited for this taxon. The present study analyzed the levels of two non-essential (cadmium and lead) and four essential metals (copper, manganese, molybdenum, and zinc) in hairs of three ecologically similar, sympatric bat species, Bechstein's bat (Myotis bechsteinii), Natterer's bat (Myotis nattereri), and Brown long-eared bat (Plecotus auritus) from an area in Central Hesse (Germany), as well as metal concentrations in soil samples from the bats' foraging habitats. Applying a previously established protocol, the analyses were performed using microwave-assisted extraction followed by inductively coupled plasma optical emission spectrometry. Cadmium and lead concentrations in hair did not differ significantly among the three studied species, whereas the following significant differences existed for levels of essential metals in hair. Manganese concentrations in hair were higher in M. bechsteinii and P. auritus than in M. nattereri and Cu concentrations were higher in M. nattereri than in P. auritus. Myotis bechsteinii showed higher Zn concentrations compared to P. auritus and lower Mo concentrations compared to M. nattereri. Reasons for the observed differences among the three studied species could be differential exposure to these metal elements in their foraging areas, related to variation in the species composition of their arthropod diet in combination with different metal levels in the respective prey species, and/or species-specific requirements for essential metals and related variation in physiological regulation of these elements in the bats.

Reduction of metal exposure of Daubenton's bats (Myotis daubentonii) following remediation of pond sediment as evidenced by metal concentrations in hair.

Transfer of contaminants from freshwater sediments via aquatic insects to terrestrial predators is well documented in spiders and birds. Here, we analyzed the metal exposure of Myotis daubentonii using an urban pond as their preferred foraging area before and after a remediation measure (sediment dredging) at this pond. Six metal elements (Zn, Cu, Cr, Cd, Pb and Ni) were measured in the sediment of the pond, in EDTA extracts of the sediment and in hair samples of M. daubentonii foraging at the pond. Samples were taken before remediation in 2011 and after remediation in 2013. Metal concentrations were quantified by ICP-OES after miniaturized microwave assisted extraction. In 2011, the pond sediment exhibited a high contamination with nickel, a moderate contamination with copper and chromium and low contents of zinc, cadmium and lead. While sediment metal contents declined only weakly after remediation, a much more pronounced reduction in the concentrations of zinc, copper, chromium and lead concentrations was observed in bat hair. Our results suggest a marked decline in metal exposure of the bats foraging at the pond as a consequence of the remediation measure. It is concluded that Daubenton's bats are suitable bioindicators of metal contamination in aquatic environments, integrating metal exposure via prey insects over their entire foraging area. We further suggest that bat hair is a useful monitoring unit, allowing a non-destructive and non-invasive assessment of metal exposure in bats.

Shedding of Infectious Borna Disease Virus-1 in Living Bicolored White-Toothed Shrews.

BACKGROUND: Many RNA viruses arise from animal reservoirs, namely bats, rodents and insectivores but mechanisms of virus maintenance and transmission still need to be addressed. The bicolored white-toothed shrew (Crocidura leucodon) has recently been identified as reservoir of the neurotropic Borna disease virus 1 (BoDV-1). PRINCIPAL FINDINGS: Six out of eleven wild living bicoloured white-toothed shrews were trapped and revealed to be naturally infected with BoDV-1. All shrews were monitored in captivity in a long-term study over a time period up to 600 days that differed between the individual shrews. Interestingly, all six animals showed an asymptomatic course of infection despite virus shedding via various routes indicating a highly adapted host-pathogen interaction. Infectious virus and viral RNA were demonstrated in saliva, urine, skin swabs, lacrimal fluid and faeces, both during the first 8 weeks of the investigation period and for long time shedding after more than 250 days in captivity. CONCLUSIONS: The various ways of shedding ensure successful virus maintenance in the reservoir population but also transmission to accidental hosts such as horses and sheep. Naturally BoDV-1-infected living shrews serve as excellent tool to unravel host and pathogen factors responsible for persistent viral co-existence in reservoir species while maintaining their physiological integrity despite high viral load in many organ systems.

Trace metal concentrations in hairs of three bat species from an urbanized area in Germany.

Metal-contaminated soils and sediments are widespread in urbanized areas due to atmospheric deposition close to emission sources. These metals are bio-available for organisms, e.g., insects, and accumulate in food chains of insectivorous mammals. Especially bats, which live in urban regions and ingest large amounts of food relative to their body mass, are at risk of being poisoned due to the accumulation of trace metals. To determine species-specific trace metal contents in bats from urban environments, hair samples were analyzed by ICP-OES. Observed trace metal concentrations were related to species-specific foraging habitat, prey spectrum and degree of synanthropy. The species studied were Pipistrellus pipistrellus, Myotis daubentonii and Nyctalus noctula. P. pipistrellus showed the highest concentrations of lead and zinc and slightly higher concentrations of cadmium than the other two species, which was related to its high degree of synanthropy with foraging habitat mostly located in cities. In contrast, N. noctula displayed the highest contents of manganese and copper. The reason might be found in its prey spectrum, as N. noctula feeds mainly on beetles that are caught in cultured areas. Trace metal concentrations determined in hair samples of M. daubentonii ranged between the values of P. pipistrellus and N. noctula, probably reflecting an intermediate level of synanthropy. Positive correlations were observed between the concentrations of cadmium and lead and those of manganese and copper. Hair samples from bats are suitable monitoring tools to study trace metal exposure and can be used to determine differences in trace metal levels between species.

Silvicolous on a small scale: possibilities and limitations of habitat suitability models for small, elusive mammals in conservation management and landscape planning.

Species distribution and endangerment can be assessed by habitat-suitability modelling. This study addresses methodical aspects of habitat suitability modelling and includes an application example in actual species conservation and landscape planning. Models using species presence-absence data are preferable to presence-only models. In contrast to species presence data, absences are rarely recorded. Therefore, many studies generate pseudo-absence data for modelling. However, in this study model quality was higher with null samples collected in the field. Next to species data the choice of landscape data is crucial for suitability modelling. Landscape data with high resolution and ecological relevance for the study species improve model reliability and quality for small elusive mammals like Muscardinus avellanarius. For large scale assessment of species distribution, models with low-detailed data are sufficient. For regional site-specific conservation issues like a conflict-free site for new wind turbines, high-detailed regional models are needed. Even though the overlap with optimally suitable habitat for M. avellanarius was low, the installation of wind plants can pose a threat due to habitat loss and fragmentation. To conclude, modellers should clearly state the purpose of their models and choose the according level of detail for species and environmental data.

Stage of pregnancy dictates heterothermy in temperate forest-dwelling bats.

Bats face high energetic requirements, as powered flight is costly and they have a disadvantageous surface-to-volume-ratio. To deal with those requirements energy saving mechanisms, such as heterothermy (torpor), have evolved. Torpor during pregnancy, however, reduces rates of foetal development and consequently prolongs pregnancy. Therefore, heterothermy has a great effect on reproduction, as an unhindered parturition can only be assured by high body temperatures. Regardless of these adverse affects of torpor the energetic requirements of bats during reproduction urge for energy savings and bats are known to enter torpor during pregnancy. The species in the current study differ in their torpor patterns and thus their heterothermic strategy. However, we hypothesized, that species-specific heterothermic behaviour should be revoked at the end of pregnancy. We analyzed skin temperatures of Myotis bechsteinii, Myotis nattereri and Plecotus auritus during pregnancy and found no differences in torpor depth between species during the last phase of pregnancy. Furthermore, we could show that individuals entered torpor frequently during pregnancy and only minimized torpor during the last stage of pregnancy. This suggests that close to the end of pregnancy, heterothermy is restricted but not species-specific and the required energy is allocated otherwise.

No short-term effect of handling and capture stress on immune responses of bats assessed by bacterial killing assay.

Ecoimmunology of wild animals becomes increasingly important. However, there are methodical limitations, especially when working on small mammals, e.g. small sample volume and acute stress associated with capture, handling and sampling that can influence immune parameters. The plasma bacterial killing assay measures innate humoral immune responses, mainly complement activity. It is a powerful tool with many methodical advantages. To avoid investigation of artefacts in future ecoimmunological studies the influence of acute stress on the bacterial killing activity was assessed. Bats (Nyctalus noctula, n = 9) were repeatedly sampled in three time intervals up to 97 min after capture. Bacterial killing activity against Escherichia coli was measured using a microplate absorbance reader. Bacterial killing activity was not influenced by capture, handling and sampling. Hence, released stress hormones did not affect circulating complement activity. To conclude, the plasma bacterial killing assay is reliable and efficient ecoimmunological tool in wildlife studies even of small mammals.

Insectivorous bats digest chitin in the stomach using acidic mammalian chitinase.

The gastrointestinal tract of animals is adapted to their primary source of food to optimize resource use and energy intake. Temperate bat species mainly feed on arthropods. These contain the energy-rich carbohydrate chitin, which is indigestible for the endogenous enzymes of a typical mammalian gastrointestinal tract. However, the gastrointestinal tract of bat species should be adapted to their diet and be able to digest chitin. We hypothesized that (i) European vespertilionid bat species have the digestive enzyme chitinase and that (ii) the chitinolytic activity is located in the intestine, as has been found for North American bat species. The gastrointestinal tracts of seven bat species (Pipistrellus pipistrellus, Plecotus auritus, Myotis bechsteinii, Myotis nattereri, Myotis daubentonii, Myotis myotis, and Nyctalus leisleri) were tested for chitinolytic activity by diffusion assay. Gastrointestinal tracts of P. pipistrellus, P. auritus, M. nattereri, M. myotis, and N. leisleri were examined for acidic mammalian chitinase by western blot analysis. Tissue sections of the gastrointestinal tract of P. pipistrellus were immunohistochemically analyzed to locate the acidic mammalian chitinase. Chitinolytic activity was detected in the stomachs of all bat species. Western blot analysis confirmed the acidic mammalian chitinase in stomach samples. Immunohistochemistry of the P. pipistrellus gastrointestinal tract indicated that acidic mammalian chitinase is located in the stomach chief cells at the base of the gastric glands. In conclusion, European vespertilionid bat species have acidic mammalian chitinase that is produced in the gastric glands of the stomach. Therefore, the gastrointestinal tracts of insectivorous bat species evolved an enzymatic adaptation to their diet.

Balancing the energy budget in free-ranging male Myotis daubentonii bats.

Mammals use five main, mutually nonexclusive mechanisms to balance energy budgets: torpor, metabolic compensation, change in activity patterns, change in ingested energy, and/or variability in digestive efficiency. Bats, as small and actively flying mammals, have a high mass-specific energy demand; therefore, balancing mechanisms should be pronounced in this group. We found that male Myotis daubentonii exhibited marked variation in the relative importance of these different mechanisms during their period of seasonal activity in response to extrinsic (ambient temperature, insect abundance) and intrinsic (reproduction, body condition) factors. Cold ambient temperatures in spring facilitated long and frequent daily torpor bouts, whereas in early summer, increased energy intake was the dominant factor in energy balancing. Intake was further increased in late summer, when insect abundance was highest, and daily torpor bouts were shorter and less frequent than in early summer. In autumn, males used metabolic compensation to reduce their resting metabolic rate in addition to daily torpor. Metabolic compensation might be one of the mechanisms that allow males to maintain high body temperature during the day while decreasing the need for foraging time at night, thus maximizing their opportunities to mate.

Landscape features and reservoir occurrence affecting the risk for equine infection with Borna disease virus.

Borna disease (BD) is a severe endemic and fatal disorder caused by the neurotropic Borna disease virus (BDV) which mainly occurs in horses and sheep. Borna disease virus belongs to the order Mononegavirales, which includes many reservoir-bound viruses with high zoonotic and pathogenic properties including the filoviruses and lyssaviruses. Clinically manifest BD occurs in endemic areas of Germany, Switzerland, Liechtenstein, and Austria. A seasonal accumulation of cases in spring and summer, incidences that vary from year to year, and the recent detection of BDV in bicolored shrews (Crocidura leucodon) in Swiss endemic areas argue for a natural reservoir. We established a geographic information system analysis of the distribution of 485 equine BD cases in Bavarian (Germany) endemic areas and of the occurrence of 285 records of C. leucodon captured in Bavaria. Boosted regression trees were used to identify driving factors of habitat choice and virus prevalence. The distribution model of C. leucodon and the prevalence model for BDV had very good accuracy. Mean annual precipitation <900 mm, mean annual temperatures of 8 C, elevation <350 m, low forest cover, and a high percentage of urban fabric and arable land describe the optimal habitat for C. leucodon. Occurrence probability of C. leucodon was significantly higher in Bavarian BDV-endemic areas than in random areas in Bavaria. The prevalence of BD was higher in urban areas with annual mean precipitation of 800-900 mm, annual mean temperature of 8 C, and elevation >500 m. Our results indicate that the distribution model can accurately predict BD occurrence. Based on these results, practical safety precautions could be derived. The BDV model represents a suitable system for reservoir-bound, neurotropic Mononegavirales because it allows analyzing ecologic and biologic aspects that determine virus abundance, maintenance in reservoir species, and transmission to end host species.

The effects of reproductive state on digestive efficiency in three sympatric bat species of the same guild.

The functional link between food as an energy source and metabolizable energy is the digestive tract. The digestive organs may change in size, structure, or retention time in response to energetic demands of the animal. Very efficient digestive tracts may be better at processing food but require higher energetic investments for maintenance even when post-absorptive. These costs influence the resting metabolic rate (RMR) that is defined as the energy necessary to fuel vital metabolic functions in a resting animal. In bats a trade-off between the necessity for a highly efficient digestive tract and moderate energetic maintenance costs may be particularly important. We hypothesized that low RMR coincides with low digestive efficiency (defined as apparent metabolizable energy coefficient (MEC)) and that phases of increased energetic demand are compensated for by increased digestive efficiency. We measured RMR and apparent MEC in the bats species Myotis nattereri, M. bechsteinii, and Plecotus auritus. In support of our hypothesis, M. nattereri has the lowest mass-specific RMR of the three species and the lowest apparent MEC. However, apparent MEC did not change during phases with differing energetic demands in any of the bat species, probably because bats operate at the limit of their sustainable energy demand.