Morphology of thyroid C-cells and parathyroid glands in summer-active little brown bats, Myotis lucifugus lucifugus, with particular reference to pregnancy and lactation

Morphological studies of thyroid C-cells and morphometric analyses of parathyroid glands in summer-active little brown bats indicated concomitant regulatory endocrine functions correlating with bone remodeling. C-cells apparently maintain maximal activity throughout the summer-active period in all bats. However, the hyperactivity of the parathyroid glands in summer-active female bats can be correlated with the maintenance of plasma calcium concentrations during lactation, when the female skeleton undergoes a period of bone demineralization. In summer-active male bats, which did not lose bone, the parathyroid glands did not show morphological evidence of hyperactivity; instead they were found to exhibit moderate parathyroid activity.     

Seasonal variations in pituitary LH-gonadotropes of the hibernating bat Myotis lucifugus lucifugus: an immunohistochemical study

Pituitary gonadotropes were identified throughout the year in the seasonally breeding, hibernating bat Myotis lucifugus lucifugus by means of light microscopic immunohistochemistry. In both male and female bats, these cells were immunoreactive with an antiserum directed to the beta subunit of luteinizing hormone. Some gonadotropes were aggregated near a portion of the infundibular stalk which crosses the anterior lobe, while most were scattered singly in a uniform manner throughout the rest of the pars distalis. This cell population exhibited seasonal variations in both sexes. In males, the proportional volume of the pars distalis occupied by immunoreactive gonadotropes (volume fraction) was significantly reduced in late July, when plasma testosterone levels were approaching their seasonal peak. In females, the volume fraction declined in April, following ovulation, and remained low during pregnancy and lactation. The size and shape of gonadotropes appeared relatively constant throughout the annual reproductive cycle in male bats; the immunoreactive cells were irregular in shape, with cytoplasmic extensions insinuating between and often "cupping" other secretory cell types. In females, the gonadotropes resembled those of males throughout most of the year, except during pregnancy, when these cells became enlarged and ovoid. No evidence of involution was observed in these anterior pituitary cells in either males or females during hibernation.     

Plasma progesterone levels during pregnancy in the little brown bat Myotis lucifugus (Vespertilionidae)

Plasma progesterone was measured by radioimmunoassay in individual female Myotis lucifugus throughout pregnancy and lactation. Progesterone levels, which averaged 6.7 +/- 0.7 ng/ml in late hibernation, rose to a mean of 18.9 +/- 6.7 ng/ml in unimplanted bats collected in the first two weeks after arrival at a maternity roost. Analysis of progesterone levels in bats in which the developmental stage of the embryo was known revealed two sharp, transient increases in plasma progesterone during the preimplantation period. The first, with values of 30-45 ng/ml, occurred at ovulation. The second, with values of 20-30 ng/ml, coincided with blastocyst formation. Progesterone levels increased exponentially from a mean of 7.4 +/- 1.0 ng/ml during early implantation to peak values of 100-200 ng/ml (means = 136.2 +/- 15.6) in the last two weeks of pregnancy, and showed no evidence of either a midpregnancy or prepartum decline. Despite involution of the corpus luteum at the end of pregnancy, progesterone levels averaged 9.0 +/- 1.0 ng/ml during lactation and did not decline until the end of lactation. In bats undergoing abortion, mean levels of plasma progesterone were already less than 6 ng/ml, equivalent to levels in nonbreeding females. The results indicate that the progesterone profile of pregnant M. lucifugus, though generally resembling those of other bats, exhibits several distinctive features. The sharp rise in plasma progesterone coinciding with blastocyst formation has not been reported in other mammals and suggests a possible role of progesterone in the cavitation process. In addition, peak values of plasma progesterone in late pregnancy were conspicuously higher than levels reported in other verpertilionid bats. The levels did not appear to fall before parturition, although such falls have been reported in other bats.     

Mammalian hibernation as a model of disuse osteoporosis: the effects of physical inactivity on bone metabolism, structure, and strength

Reduced skeletal loading typically leads to bone loss because bone formation and bone resorption become unbalanced. Hibernation is a natural model of musculoskeletal disuse because hibernating animals greatly reduce weight-bearing activity, and therefore, they would be expected to lose bone. Some evidence suggests that small mammals like ground squirrels, bats, and hamsters do lose bone during hibernation, but the mechanism of bone loss is unclear. In contrast, hibernating bears maintain balanced bone remodeling and preserve bone structure and strength. Differences in the skeletal responses of bears and smaller mammals to hibernation may be due to differences in their hibernation patterns; smaller mammals may excrete calcium liberated from bone during periodic arousals throughout hibernation, leading to progressive bone loss over time, whereas bears may have evolved more sophisticated physiological processes to recycle calcium, prevent hypercalcemia, and maintain bone integrity. Investigating the roles of neural and hormonal control of bear bone metabolism could give valuable insight into translating the mechanisms that prevent disuse-induced bone loss in bears into novel therapies for treating osteoporosis.     

Specific alterations in complement protein activity of little brown myotis (Myotis lucifugus) hibernating in white-nose syndrome affected sites

White-nose syndrome (WNS) is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans), depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus) based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI) during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist between energy conservation and immunological responses. Relationships between immune activity and torpor, including associated energy expenditure, are likely critical components in the development of WNS.     

Timing of ovulation and early embryonic development in Myotis lucifugus (Chiroptera: Vespertilionidae) from northern central Ontario

Female Myotis lucifugus lucifugus, obtained from a hibernaculum in northern central Ontario, were aroused during the week that normal departures from hibernation began, maintained in large cages at 28-30 degrees C, and fed mealworms daily. Some bats ovulated less than 24 hr after arousal, and with one exception, all bats with normal, mature follicles ovulated within 72 hr. Histological examination of reproductive tracts from ovulated bats indicated that oviductal transit was completed in 24-48 hr, and that blastocyst formation and implantation occurred 4 and 10 days, respectively, after fertilization. These time intervals were somewhat shorter than those reported in previous studies; however, the differences appeared to be due to the relative accuracy in determining the time of ovulation, not to differences in rates of development among different populations.     

A comparison of milk composition in Myotis lucifugus and Eptesicus fuscus (chiroptera: vespertilionidae)

Milk composition in two species of insectivorous bats (Myotis lucifugus and Eptesicus fuscus) was determined. No significant differences in fat, lactose, protein, or energy content were evident during the lactation period for M. lucifugus. The percentage of fat, lactose, and protein averaged 13.5, 3.3, 7.4, respectively, for M. lucifugus, and 16.4, 2.5, 6.2, respectively, for E. fuscus. Energy content of milk averaged 7.32 kJ/g for M. lucifugus and 8.37 kJ/g for E. fuscus. The percentage of lactose in milk from M. lucifugus was significantly greater than in E. fuscus, but there were no significant interspecific differences in percentages of fat or protein. The two species also differed significantly in content of five out of nine fatty acids.     

Changes in Leydig cell activity during the annual testicular cycle of the bat Myotis lucifugus lucifugus: histology and lipid histochemistry

Changes in Leydig cell histology and testicular sudanophilic lipids were examined in relation to spermatogenic activity in the bat Myotis lucifugus lucifugus (Chiroptera: Vespertilionidae) throughout the annual cycle in the northeastern United States. These changes were correlated with annual variations in plasma testosterone concentrations which have recently been described for this species. Gametogenic activity occurred during the months of May-August when bats were metabolically most active. During hibernation (October-April), when sperm are stored in the epididymides, and accessory glands are hypertrophic, the seminiferous tubules were at rest, and the germinal epithelium was reduced to reserve spermatogonia and Sertoli cells. Based on their structure and cyclic pattern of sudanophilic lipids, Leydig cells exhibited a pattern of activity that closely paralleled that of the seminiferous epithelium. On renewal of spermatogenesis in spring, Leydig cells became hypertrophied and accumulated lipid inclusions. These inclusions, seen as vacuoles in plastic sections and sudanophilic droplets in frozen sections, reached maximal accumulations in late June. In late July and during August, when peak testosterone levels occur in blood, lipid droplets were dramatically depleted, and Leydig cells were weakly sudanophilic. In September, when testosterone titers return to low baseline levels, Leydig cells had regressed but exhibited a marked increase in sudanophilic inclusions which appeared to be mostly lipofuscins. During the ensuing mating and hibernation periods, Leydig cells were involuted and filled with lipofuscins. During the periarousal period, however, Leydig cells became weakly Sudan-positive while many large, intensely sudanophilic cells were scattered throughout the interstitium. In electron micrographs these cells were identified as macrophages. They appear to play an important role in the annual testicular cycle by phagocytizing the residues of Leydig cell involution in preparation for a new steroidogenic cycle. Seasonal changes in lipid inclusions were also observed in the seminiferous tubules. In addition, the relationship of the Leydig cell cycle to androgen action and the accessory organs in this bat is discussed.     

Polar bears (Ursus maritimus), the most evolutionary advanced hibernators, avoid significant bone loss during hibernation

Some hibernating animals are known to reduce muscle and bone loss associated with mechanical unloading during prolonged immobilisation,compared to humans. However, here we show that wild pregnant polar bears (Ursus maritimus) are the first known animals to avoid significant bone loss altogether, despite six months of continuous hibernation. Using serum biochemical markers of bone turnover, we showed that concentrations for bone resorption are not significantly increased as a consequence of hibernation in wild polar bears. This is in sharp contrast to previous studies on other hibernating species, where for example, black bears (Ursus americanus), show a 3-4 fold increase in serum bone resorption concentrations posthibernation,and must compensate for this loss through rapid bone recovery on remobilisation, to avoid the risk of fracture. In further contrast to black bears, serum concentrations of bone formation markers were highly significantly increased in pregnant female polar bears compared to non-pregnant,thus non-hibernating females both prior to and after hibernation. However, bone formation concentrations in new mothers were significantly reduced compared to pre-hibernation concentrations. The de-coupling of bone turnover in favour of bone formation prior to hibernation, suggests that wild polar bears may posses a unique physiological mechanism for building bone in protective preparation against expected osteopenia associated with disuse,starvation, and hormonal drives to mobilise calcium for reproduction, during hibernation. Understanding this physiological mechanism could have profound implications for a natural solution for the prevention of osteoporosis in animals subjected to captivity with inadequate space for exercise,humans subjected to prolonged bed rest while recovering from illness, or astronauts exposed to antigravity during spaceflight.? 2008 Elsevier Inc. All rights reserved.     

Post-Pleistocene range expansion of the recently imperiled eastern little brown bat (Myotis lucifugus lucifugus) from a single southern refugium

Myotis lucifugus, once among the most widespread and common bats in North America, has been forecast to be extirpated east of the Rockies in as few as 16 years by the spread of white-nose syndrome. Recent genetic research has demonstrated that this species is paraphyletic and part of a broader species complex; however, only one lineage (Myotis lucifugus lucifugus [M. l. lucifugus]) is present in eastern North America. I used molecular tools and niche modeling to validate this and investigate the role that historical biogeography has played in the phylogenetic and population genetic structure of this species to determine if the eastern subspecies represents an evolutionarily distinct population.To establish the genetic structure within M. l. lucifugus, I densely sampled maternity colonies in Minnesota and sequenced 182 individuals for a portion of cytochrome b. Phylogenetic reconstruction and a haplotype network were used to infer the relationships among mitochondrial haplotypes. Population growth statistics were calculated to determine if there was evidence of significant expansion, and an environmental niche model (ENM) was constructed based on conditions during the last glacial maximum (LGM) to illustrate potential glacial refugia. All individuals derived from a single mitochondrial lineage. Genetic evidence points to population growth starting approximately 18 kya. ENM results show that there was likely a single large southern refugium extending across the southeastern United States and possibly several isolated refugia in western North America. Myotis lucifugus lucifugus likely maintained both a large range and a large population during the peaks of the glacial cycles, and its population appears to have expanded following the retreat of the Laurentide ice sheet. This imperiled lineage likely diverged in isolation from other members of the M. lucifugus/western long-eared Myotis during the Pleistocene.     

Enzymes of heterotherms: LDH of hibernating and normothermic little brown bats, Myotis lucifugus.

1. Lactate dehydrogenase (LDH) was isolated from pectoral or flight muscle, liver and heart of hibernating and normothermic bats, Myotis lucifugus. 2. Activities at high substrate concentrations and the interactions between substrate and enzyme were studied to evaluate possible adaptations associated with the extended period of depressed metabolism of this bat. 3. Activity levels in all tissues decreased with hibernation, although the magnitude was dependent upon both temperature and substrate concentration. In general, the extent of decrease was in the order pectoral muscle greater than liver much greater than heart. 4. No correlation was found between the temperature sensitivity of the tissue LDHs as estimated by Ea-values, and the physiological state. 5. However, affinity parameters as estimated by Km-values were less temperature sensitive in hibernator tissues in contrast to the marked perturbations exhibited at temperature extremes in the normotherm enzymes. 6. It is suggested that changes in subunit quantities are in part responsible for these kinetic differences, and not major qualitative isozyme changes. 7. The net result of these effects is a reduced carbon flux to lactate during hibernation, but the maintenance of a potential for LDH catalyses during arousal.     

The tradeoff between torpor use and reproduction in little brown bats (Myotis lucifugus)

In mammals, reproduction, especially for females is energetically demanding. Therefore, during the reproductive period females could potentially adjust patterns of thermoregulation and foraging in concert to minimise the energetic constraints associated with pregnancy and lactation. We assessed the influence of pregnancy, lactation, and post-lactation on torpor use and foraging behaviour by female little brown bats, Myotis lucifugus. We measured thermoregulation by recording skin temperature and foraging by tracking bats which carried temperature-sensitive radio-tags. We found that individuals, regardless of reproductive condition, used torpor, but the patterns of torpor use varied significantly between reproductive (pregnant and lactating) females and post-lactating females. As we predicted, reproductive females entered torpor for shorter bouts than post-lactating females. Although all females used torpor frequently, pregnant females spent less time in torpor, and maintained higher skin temperatures than either lactating or post-lactating females. This result suggests that delayed offspring development which has been associated with torpor use during pregnancy, may pose a higher risk to an individual’s reproductive success than reduced milk production during lactation. Conversely, foraging behaviour of radio-tagged bats did not vary with reproductive condition, suggesting that even short, shallow bouts of torpor produce substantial energy savings, likely obviating the need to spend more time foraging. Our data clearly show that torpor use and reproduction are not mutually exclusive and that torpor use (no matter how short or shallow) is an important means of balancing the costs of reproduction for M. lucifugus.     

Frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome

White-nose syndrome (WNS), an emerging infectious disease that has killed over 5.5 million hibernating bats, is named for the causative agent, a white fungus (Geomyces destructans (Gd)) that invades the skin of torpid bats. During hibernation, arousals to warm (euthermic) body temperatures are normal but deplete fat stores. Temperature-sensitive dataloggers were attached to the backs of 504 free-ranging little brown bats (Myotis lucifugus) in hibernacula located throughout the northeastern USA. Dataloggers were retrieved at the end of the hibernation season and complete profiles of skin temperature data were available from 83 bats, which were categorized as: (1) unaffected, (2) WNS-affected but alive at time of datalogger removal, or (3) WNS-affected but found dead at time of datalogger removal. Histological confirmation of WNS severity (as indexed by degree of fungal infection) as well as confirmation of presence/absence of DNA from Gd by PCR was determined for 26 animals. We demonstrated that WNS-affected bats aroused to euthermic body temperatures more frequently than unaffected bats, likely contributing to subsequent mortality. Within the subset of WNS-affected bats that were found dead at the time of datalogger removal, the number of arousal bouts since datalogger attachment significantly predicted date of death. Additionally, the severity of cutaneous Gd infection correlated with the number of arousal episodes from torpor during hibernation. Thus, increased frequency of arousal from torpor likely contributes to WNS-associated mortality, but the question of how Gd infection induces increased arousals remains unanswered.     

Bending properties, porosity, and ash fraction of black bear (Ursus americanus) cortical bone are not compromised with aging despite annual periods of disuse

In many species, including humans, disuse causes an imbalance in bone remodeling that leads to increased bone porosity as a result of increased bone resorption and decreased bone formation. However, black bears (Ursus americanus) may not develop disuse osteopenia, to the extent that other animals do, during long periods of disuse (i.e. hibernation) because they maintain osteoblastic bone formation during hibernation, even though bone resorption is increased during hibernation. Black bears may also have a mechanism to rapidly and completely recover the bone lost (by increased resorption during hibernation) during their remobilization period. Our findings suggest that cortical bone bending strength (211-328 MPa), bending modulus (16.0-29.5 MPa), fracture energy (0.0118-0.0205 J mm(-2)), porosity (2.3-7.1%), and ash fraction (0.638-0.672) are not compromised with age in black bears, despite annual periods of disuse. In fact, the ultimate strength (p=0.01), modulus (p=0.04), and ash fraction (p=0.03) of cortical bone were shown to significantly increase with age (2-14 yrs). Female bears give birth and nurse during hibernation; however, we found no significant (p>0.16) differences between male and female bone properties. Other animals require remobilization periods 2-3 times longer than the immobilization period to recover the bone lost during disuse. Our findings support the idea that black bears, which hibernate 5-7 months annually, have evolved a biological mechanism to mitigate the adverse effects of disuse on bone porosity and mechanical behavior.     

Recovery of little brown bats (Myotis lucifugus) from natural infection with Geomyces destructans, white-nose syndrome

Geomyces destructans produces the white fungal growth on the muzzle and the tacky white discoloration on wings and ears that characterize white-nose syndrome (WNS) in cave-hibernating bats. To test the hypothesis that postemergent WNS-infected bats recover from infection with G. destructans, 30 little brown bats (Myotis lucifugus) were collected in May 2009 from a WNS-affected hibernation site in New Jersey. All bats were confirmed to be infected with G. destructans using a noninvasive fungal tape method to identify the conidia of G. destructans and polymerase chain reaction (PCR). The bats were then held in captivity and given supportive care for 70 days. Of the 26 bats that survived and were humanely killed after 70 days, 25 showed significant improvement in the external appearance of wing membranes, had no microscopic evidence of infection by G. destructans, and had wing tissue samples that were negative for G. destructans by PCR. A subset of the bats was treated topically at the beginning of the rehabilitation study with a dilute vinegar solution, but treatment with vinegar provided no added advantage to recovery. Provision of supportive care to homeothermic bats was sufficient for full recovery from WNS. One bat at day 70 still had both gross pathology and microscopic evidence of WNS in wing membranes and was PCR-positive for G. destructans. Dense aggregates of neutrophils surrounded the hyphae that remained in the wing membrane of this bat.     

Cancellous and cortical bone mechanical properties and tissue dynamics during pregnancy, lactation, and postlactation in the rat

There are substantial changes in maternal skeletal dynamics during pregnancy, lactation, and after lactation. The purpose of this study was to correlate changes in cortical and cancellous bone mass, structure, and dynamics with mechanical properties during and after the first reproductive cycle in rats. Rats were mated and groups were taken at parturition, end of lactation and 8 wk after weaning, and were compared with age-matched, nulliparous controls. Measurements were taken on femoral cortical bone and lumbar vertebral body cancellous bone. At the end of pregnancy, there was an increase in cortical periosteal bone formation and an increase in cortical volume, but a suppression of turnover in cancellous bone with no change in cancellous or cortical mechanical properties. Lactation was associated with a decrease in cortical and cancellous bone strength with a decrease in bone volume, but an increase in turnover on cancellous and endocortical surfaces. After lactation, there was a partial or full restoration of mechanical properties. This study demonstrates substantial changes in bone mechanics that correlate with changes in bone structure and dynamics during the first reproductive cycle in rats. The greatest changes were observed during the lactation period with partial or full recovery in the postlactational period.