Brno loanvirus (BRNV) in bats inhabiting the urban area of Brno, Czech Republic.

Bats are known reservoirs of various emerging pathogens, and have recently been found to host a novel hantavirus, named Brno loanvirus (BRNV), from the Mammantavirinae subfamily (family Hantaviridae, order Bunyavirales). Here we report BRNV detection in bats from the urban area of Brno, Czech Republic in March 2022. Specifically, we uncovered a high prevalence of BRNV (8.8%, 5/57) among hibernating bats (Nyctalus noctula) in urban area, which poses a risk of human exposure. The positive bats included adult females (3/9 positive), a juvenile female (1/32 positive), and an adult male (1/6 positive). All 10 juvenile males were negative. We used RT-qPCR to quantify the BRNV RNA levels in various bat organs, which yielded positive results for viral RNA in organs, including the kidneys, heart, spleen, brain, liver, lung, and gut, and in body cavity fluid. Among all tested organs, the liver showed the highest levels of viral RNA in 4 out of 5 animals examined (average Ct value of 20.8 ± 7.4).

Changes in body size and fertility due to artificial and natural feeding of laboratory common bed bugs (Heteroptera: Cimicidae).

Rearing common bed bugs (Cimex lectularius L.) and other hematophagous insects is essential for basic, medical, and pest-control research. Logistically, acquiring fresh blood can be a challenge, while biologically, the eventual effects of different rearing and blood preparation protocols on bed bug genotype and phenotype pose a risk of biased research results. Using bed bug populations that are either bat- (BL) or human-related (HL), we tested the short- and long-term effects of rearing bugs on live bats or human volunteers, or artificially on CPDA (citrate phosphate dextrose, adenine)-treated blood, measuring meal size, body size, and fertility. We found that artificial feeding did not affect meal size compared with feeding on natural hosts. Long-term rearing across many generations of HL on CPDA-preserved blood led to reduced body size and fertility compared with populations reared on human volunteers. Blood preservatives increased the proportion of sterile eggs even after a single feed. Finally, our results indicated that laboratory reared bed bugs were smaller, regardless of the blood source, than wild bugs. Similar effects of artificial feeding or laboratory rearing alone should be considered in future studies using bed bug cultures to choose an appropriate rearing protocol. With regard to switching between bat and human hosts, HL took smaller meals and BL had lower fertility when fed on bats than when fed on humans. We attribute these results to methodological constrains, specifically the inconsistency of bat feeding, rather than to host specialization. Nevertheless, BL can be easily reared using human blood and artificial feeding systems.

Despite genetic isolation in sympatry, post-copulatory reproductive barriers have not evolved between bat- and human-associated common bedbugs (Cimex lectularius L.).

BACKGROUND: The common bedbug Cimex lectularius is a widespread ectoparasite on humans and bats. Two genetically isolated lineages, parasitizing either human (HL) or bat (BL) hosts, have been suggested to differentiate because of their distinct ecology. The distribution range of BL is within that of HL and bedbugs live mostly on synanthropic bat hosts. This sympatric co-occurrence predicts strong reproductive isolation at the post-copulatory level. RESULTS: We tested the post-copulatory barrier in three BL and three HL populations in reciprocal crosses, using a common-garden blood diet that was novel to both lineages. We excluded pre-copulation isolation mechanisms and studied egg-laying rates after a single mating until the depletion of sperm, and the fitness of the resulting offspring. We found a higher sperm storage capability in BL, likely reflecting the different seasonal availability of HL and BL hosts. We also observed a notable variation in sperm function at the population level within lineages and significant differences in fecundity and offspring fitness between lineages. However, no difference in egg numbers or offspring fitness was observed between within- and between-lineage crosses. CONCLUSIONS: Differences in sperm storage or egg-laying rates between HL and BL that we found did not affect reproductive isolation. Neither did the population-specific variation in sperm function. Overall, our results show no post-copulatory reproductive isolation between the lineages. How genetic differentiation in sympatry is maintained in the absence of a post-copulatory barrier between BL and HL remains to be investigated.

Age-related mating rates among ecologically distinct lineages of bedbugs, Cimex lectularius.

Understanding how many mates an animal has in its lifetime is a critical factor in sexual selection. At the same time, differences in an organism's ecology, such as the quantity and quality of food, could be reflected in different mating rates. Mating rate had a significant effect on female net fitness (i.e., lifetime offspring production), however, laboratory measurements cannot well mirror the situation in wild. The common bedbug (Cimex lectularius) is a well-established model for studying traumatic insemination and sexual conflict. The species comprises two host lineages that feed on bats (BL) or humans (HL). HL can constantly feed on human hosts throughout the year, while BLs feed only during summer months when their bat hosts occupy the roosts. Because mating in female bedbugs is closely linked to foraging, this system provides a valuable model to study mating variation in the field. We established a new method for estimating age-dependent mating rates of females in the wild by relating the fluorescent pigment accumulation in the eyes of females to the number of mating scars that manifest as melanized spots caused by the injection of sperm through the wall of the female abdomen by the male into the spermalege. In addition, using laboratory bedbugs we found that three and a half observed matings on average lead to one observed melanized mating scar. Although young BL and HL females (with low pteridine concentrations) did not differ in the number of matings, the mating rate increased with age only in HL but not in BL females. We sampled on average older BL than HL females. The lack of access to food (bat blood) during winter could explain the lack of increase in the number of scars with age in BL. In species where mating leaves visible marks, using fluorescent pigments to determine female age (applicable to most arthropods) could be an important tool to study sexual selection and mating rate in the wild. The method can help formulate sustainable and biologically lucid approaches for their control.

Detection of Leptospira species in bat cadavers, Czech and Slovak Republics.

Kidney samples from 300 bat cadavers from the Czech and Slovak Republics were tested for Leptospira DNA using PCR and sequencing of three genes (lipL32, flab, and 16S ribosomal RNA). Overall detection rate was 4.7% and two bat species (Myotis myotis and Nyctalus noctula) were PCR-positive for at least one gene. Detected Leptospira sequences were similar to L. interrogans and L. borgpetersenii, and included a potentially novel species related to L. weilii.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Ecological values of intermittent rivers for terrestrial vertebrate fauna.

Rivers are generally considered critical habitats for biodiversity; however, this often ignores the fact that many rivers may run dry and support terrestrial as well as aquatic fauna. Here, we investigated the ecological value of intermittent rivers for terrestrial vertebrates by installing camera traps along rivers subject to varying dry periods in two contrasting European climatic zones. We then analysed i) species presence and behaviours (as a proxy of ecological functions) on perennial and intermittent streams; ii) environmental (hydrological and geomorphological) and anthropogenic factors affecting the frequency of occurrence and number of species recorded; and iii) the importance of hydrological factors as regards ecological functioning. In both study areas, we recorded a higher number of species and individuals along intermittent streams than perennial streams, with highest values in intermittent reaches exhibiting shorter dry periods. Both abundance and species richness were strongly affected by hydrological factors in both study areas, including not only the occurrence but also the duration of the dry period. Dry channels played a key role as migration corridors and as a source of food, being used more frequently than riparian habitats when the river ran dry. Our findings indicate that terrestrial vertebrate fauna benefit from dry phases in rivers. Intermittent rivers, supporting a high abundance and diversity of fauna, should be considered as target ecosystems for wildlife conservation. Not doing so will jeopardise urgently needed conservation strategies in the face of accelerating global climate change.

Cold arousal - A mechanism used by hibernating bats to reduce the energetic costs of disturbance.

During the season of hibernation, temperate bats alternate between prolonged bouts of torpor with reduced body temperature and short arousals with a return to normothermy. Hibernating bats are sensitive to non-tactile stimuli and arouse following changes in microclimatic conditions or disturbance from other bats, potential predators, or humans. Here, we used temperature data loggers to register the skin temperature of 38 Myotis myotis bats over two winters (between January and March), during which regular visits were made to the hibernaculum. Two kinds of arousal were observed, normothermic (Tsk > 25 °C) and cold (Tsk < 15 °C). Although bats responded to the presence of a researcher by arousals of both kinds, cold arousals were more frequent (63.8%). We found that mass loss was not affected by the number of disturbances, however it was in positive relationship with the mass at the beginning of the observation and differed between sex and age categories. Furthermore normothermic bats crawling among cluster-mates initiated arousal cascades, which mainly consisted of cold arousals. We failed to detect any effect of age or sex on the number of arousals initiated by normothermic individuals. Warming by only a few degrees requires less energy than a normothermic arousal and we propose it is sufficient to activate the sensory system in order to assess the relevance of external stimuli. Our results indicate that cold arousals reflect a physiological and behavioural adaptation aimed at avoiding the energetic costs of disturbance that can lead to depletion of fat reserves.

Bat aggregational response to pest caterpillar emergence.

Moths (Lepidoptera) are major agricultural and forest pests in many parts of the world, including Europe, with many causing great economic damage to crops, horticultural plants, stored items, and wool products. Here, we focus on two ecologically similar inchworms, Operophtera brumata and Erannis defoliaria, known for their high foliage consumption during the spring emergence of caterpillars. We hypothesise that bats could play a role in reducing pests such as caterpillars by switching to this abundant emerging prey. At two infested and one control forest sites, caterpillars were sampled during spring to determine levels of infestation. At the same time, bat flight activity was monitored during the peak in caterpillar abundance. During the spring caterpillar outbreak, we collected faecal samples of forest-dwelling bats capable of using gleaning. The majority of samples were positive for our focus species, being 51.85% for O. brumata and 29.63% for E. defoliaria faecal samples. The foraging activity of two gleaning bats, Myotis nattereri and Myotis bechsteinii, increased at both infested sites, but not at the control site, during caterpillar emergence, as did foraging of Plecotus auritus/austriacus, which used both gleaning and aerial hawking. We conclude that both specialists and occasional gleaners, which prefer different prey but are able to switch their foraging strategies, aggregate at sites during pest emergence and, as such, our results confirm the high potential of bats to reduce numbers of pest species such as caterpillars.

Male diet affects female fitness and sperm competition in human- and bat-associated lineages of the common bedbug, Cimex lectularius.

Sperm performance can vary in ecologically divergent populations, but it is often not clear whether the environment per se or genomic differences arising from divergent selection cause the difference. One powerful and easily manipulated environmental effect is diet. Populations of bedbugs (Cimex lectularius) naturally feed either on bat or human blood. These are diverging genetically into a bat-associated and a human-associated lineage. To measure how male diet affects sperm performance, we kept males of two HL and BL populations each on either their own or the foreign diet. Then we investigated male reproductive success in a single mating and sperm competition context. We found that male diet affected female fecundity and changed the outcome of sperm competition, at least in the human lineage. However, this influence of diet on sperm performance was moulded by an interaction. Bat blood generally had a beneficial effect on sperm competitiveness and seemed to be a better food source in both lineages. Few studies have examined the effects of male diet on sperm performance generally, and sperm competition specifically. Our results reinforce the importance to consider the environment in which sperm are produced. In the absence of gene flow, such differences may increase reproductive isolation. In the presence of gene flow, however, the generally better sperm performance after consuming bat blood suggests that the diet is likely to homogenise rather than isolate populations.

Urinary shedding of leptospires in palearctic bats.

Leptospirosis is a bacterial zoonotic infection of worldwide occurrence. Bats, like other mammalian reservoirs, may be long-term carriers that maintain endemicity of infection and shed viable leptospires in urine. Direct and/or indirect contact with these Leptospira shedders is the main risk factor as regards public health concern. However, knowledge about bat leptospirosis in the Palearctic Region, and in Europe in particular, is poor. We collected urine from 176 specimens of 11 bat species in the Czech Republic, Poland, Republic of Armenia and the Altai Region of Russia between 2014 and 2019. We extracted DNA from the urine samples to detect Leptospira spp. shedders using PCR amplification of the 16S rRNA and LipL32 genes. Four bat species (Barbastella barbastellus n = 1, Myotis bechsteinii n = 1, Myotis myotis n = 24 and Myotis nattereri n = 1) tested positive for Leptospira spp., with detected amplicons showing 100% genetic identity with pathogenic Leptospira interrogans. The site- and species-specific prevalence range was 0%-24.1% and 0%-20%, respectively. All bats sampled in the Republic of Armenia and Russia were negative. Given the circulation of pathogenic leptospires in strictly protected Palearctic bat species and their populations, non-invasive and non-lethal sampling of urine for molecular Leptospira spp. detection is recommended as a suitable surveillance and monitoring strategy. Moreover, our results should raise awareness of this potential disease risk among health professionals, veterinarians, chiropterologists and wildlife rescue workers handling bats, as well as speleologists and persons cleaning premises following bat infestation.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Reproductive compatibility among populations and host-associated lineages of the common bed bug (Cimex lectularius L.).

As populations differentiate across geographic or host-association barriers, interpopulation fertility is often a measure of the extent of incipient speciation. The bed bug, Cimex lectularius L., was recently found to form two host-associated lineages within Europe: one found with humans (human-associated, HA) and the other found with bats (bat-associated, BA). No unequivocal evidence of contemporary gene flow between these lineages has been found; however, it is unclear whether this is due to an inability to produce viable "hybrid" offspring. To address this question and determine the extent of compatibility between host-associated lineages, we set up mating crosses among populations of bed bugs based on both their host association (human-HA vs. bat-BA) and geographic origin (North America vs. Europe). Within-population fecundity was significantly higher for all HA populations (>1.7 eggs/day) than for BA populations (<1 egg/day). However, all within-population crosses, regardless of host association, had >92% egg hatch rates. Contrary to previous reports, in all interlineage crosses, successful matings occurred, fertile eggs were oviposited, and the F1 "hybrid" generation was found to be reproductively viable. In addition, we evaluated interpopulation genetic variation in Wolbachia among host-associated lineages. We did not find any clear patterns related to host association, nor did we observe a homogenization of Wolbachia lineages across populations that might explain a breakdown of reproductive incompatibility. These results indicate that while the HA and BA populations of C. lectularius represent genetically differentiated host-associated lineages, possibly undergoing sympatric speciation, this is in its incipient stage as they remain reproductively compatible. Other behavioral, physiological, and/or ecological factors likely maintain host-associated differentiation.

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.

Low seasonal variation in greater mouse-eared bat (Myotis myotis) blood parameters.

The greater mouse-eared bat (Myotis myotis) is a flagship species for the protection of hibernation and summer maternity roosts in the Western Palearctic region. A range of pathogenic agents is known to put pressure on populations, including the white-nose syndrome fungus, for which the species shows the highest prevalence and infection intensity of all European bat species. Here, we perform analysis of blood parameters characteristic for the species during its natural annual life cycle in order to establish reference values. Despite sexual dimorphism and some univariate differences, the overall multivariate pattern suggests low seasonal variation with homeostatic mechanisms effectively regulating haematology and blood biochemistry ranges. Overall, the species displayed a high haematocrit and haemoglobin content and high concentration of urea, while blood glucose levels in swarming and hibernating bats ranged from hypo- to normoglycaemic. Unlike blood pH, concentrations of electrolytes were wide ranging. To conclude, baseline data for blood physiology are a useful tool for providing suitable medical care in rescue centres, for studying population health in bats adapting to environmental change, and for understanding bat responses to stressors of conservation and/or zoonotic importance.

Identification and age-dependence of pteridines in bed bugs (Cimex lectularius) and bat bugs (C. pipistrelli) using liquid chromatography-tandem mass spectrometry.

Determining the age of free-living insects, particularly of blood-sucking species, is important for human health because such knowledge critically influences the estimates of biting frequency and vectoring ability. Genetic age determination is currently not available. Pteridines gradually accumulate in the eyes of insects and their concentrations is the prevailing method. Despite of their stability, published extractions differ considerably, including for standards, for mixtures of pteridines and even for light conditions. This methodological inconsistency among studies is likely to influence age estimates severely and to hamper their comparability. Therefore we reviewed methodological steps across 106 studies to identify methodological denominators and results across studies. Second, we experimentally test how different pteridines vary in their age calibration curves in, common bed (Cimex lectularius) and bat bugs (C. pipistrelli). Here we show that the accumulation of particular pteridines varied between a) different populations and b) rearing temperatures but not c) with the impact of light conditions during extraction or d) the type of blood consumed by the bugs. To optimize the extraction of pteridines and measuring concentrations, we recommend the simultaneous measurement of more than one standard and subsequently to select those that show consistent changes over time to differentiate among age cohorts.

Spatial networks differ when food supply changes: Foraging strategy of Egyptian fruit bats.

Animals are faced with a range of ecological constraints that shape their behavioural decisions. Habitat features that affect resource abundance will also have an impact, especially as regards spatial distribution, which will in turn affect associations between the animals. Here we utilised a network approach, using spatial and genetic data, to describe patterns in use of space (foraging sites) by free-ranging Egyptian fruit bats (Rousettus aegyptiacus) at the Dakhla Oasis in Egypt. We observed a decrease in home range size during spring, when food availability was lowest, which was reflected by differences in space sharing networks. Our data showed that when food was abundant, space sharing networks were less connected and more related individuals shared more foraging sites. In comparison, when food was scarce the bats had few possibilities to decide where and with whom to forage. Overall, both networks had high mean degree, suggesting communal knowledge of predictable food distribution.

Resistance is futile: RNA-sequencing reveals differing responses to bat fungal pathogen in Nearctic Myotis lucifugus and Palearctic Myotis myotis.

Resistance and tolerance allow organisms to cope with potentially life-threatening pathogens. Recently introduced pathogens initially induce resistance responses, but natural selection favors the development of tolerance, allowing for a commensal relationship to evolve. Mycosis by Pseudogymnoascus destructans, causing white-nose syndrome (WNS) in Nearctic hibernating bats, has resulted in population declines since 2006. The pathogen, which spread from Europe, has infected species of Palearctic Myotis for a longer period. We compared ecologically relevant responses to the fungal infection in the susceptible Nearctic M. lucifugus and less susceptible Palearctic M. myotis, to uncover factors contributing to survival differences in the two species. Samples were collected from euthermic bats during arousal from hibernation, a naturally occurring phenomenon, during which transcriptional responses are activated. We compared the whole-transcriptome responses in wild bats infected with P. destructans hibernating in their natural habitat. Our results show dramatically different local transcriptional responses to the pathogen between uninfected and infected samples from the two species. Whereas we found 1526 significantly upregulated or downregulated transcripts in infected M. lucifugus, only one transcript was downregulated in M. myotis. The upregulated response pathways in M. lucifugus include immune cell activation and migration, and inflammatory pathways, indicative of an unsuccessful attempt to resist the infection. In contrast, M. myotis appears to tolerate P. destructans infection by not activating a transcriptional response. These host-microbe interactions determine pathology, contributing to WNS susceptibility, or commensalism, promoting tolerance to fungal colonization during hibernation that favors survival.

Numerous cold arousals and rare arousal cascades as a hibernation strategy in European Myotis bats.

Hibernating bats optimise the duration of torpor bouts and arousals in relation to hibernaculum microclimatic conditions and fat reserves. Clustering has significant physiological and ecological benefits, promoting successful hibernation of individuals. Such aggregations may help maintain optimal temperatures, allowing better energy utilisation than in solitarily bats. However, aroused bats in a cluster could conceivably disturb those still hibernating, starting an energy-demanding arousal process. Our study was conducted over two winters in two different hibernacula (cave and mine) in the Czech Republic, where Greater mouse-eared bats (Myotis myotis) have previously been diagnosed with white-nose syndrome. In 118 arousal episodes we recorded 193 individual arousals in which a warming phase was observed, 135 (69.9%) being cold arousals, where bats ceased increasing their body temperatures at ≤ 10 °C. The remaining arousals were standard normothermic arousals, where body (fur) surface temperatures reached > 20 °C. Cold arousals occurred during the mid- and late hibernation periods, suggesting they were a response to disturbance by a neighbour in the same cluster. Arousal cascades, where bats aroused in series, were rare (12.7%) and reached a maximum in mid-January. Our data suggest that Myotis bats prolong their torpor bouts using numerous cold arousals but few arousal cascades. Upon arrival of a bat, the clustered bats show tolerance to disturbing by conspecifics.

Alterations in the health of hibernating bats under pathogen pressure.

In underground hibernacula temperate northern hemisphere bats are exposed to Pseudogymnoascus destructans, the fungal agent of white-nose syndrome. While pathological and epidemiological data suggest that Palearctic bats tolerate this infection, we lack knowledge about bat health under pathogen pressure. Here we report blood profiles, along with body mass index (BMI), infection intensity and hibernation temperature, in greater mouse-eared bats (Myotis myotis). We sampled three European hibernacula that differ in geomorphology and microclimatic conditions. Skin lesion counts differed between contralateral wings of a bat, suggesting variable exposure to the fungus. Analysis of blood parameters suggests a threshold of ca. 300 skin lesions on both wings, combined with poor hibernation conditions, may distinguish healthy bats from those with homeostatic disruption. Physiological effects manifested as mild metabolic acidosis, decreased glucose and peripheral blood eosinophilia which were strongly locality-dependent. Hibernating bats displaying blood homeostasis disruption had 2 °C lower body surface temperatures. A shallow BMI loss slope with increasing pathogen load suggested a high degree of infection tolerance. European greater mouse-eared bats generally survive P. destructans invasion, despite some health deterioration at higher infection intensities (dependant on hibernation conditions). Conservation measures should minimise additional stressors to conserve constrained body reserves of bats during hibernation.