Ontogeny of flight in the little brown bat,Myotis lucifugus: behavior,morphology, and muscle histochemistry

Summary Postnatal changes in wing morphology, flight ability, muscle morphology, and histochemistry were investigated in the little brown bat, Myotis lucifugus. The pectoralis major, acromiodeltoideus, and quadriceps femoris muscles were examined using stains for myofibrillar ATPase, succinate dehydrogenase (SDH), and mitochondrial -glycerophosphate dehydrogenase (-GPDH) enzyme reactions. Bats first exhibited spontaneous, drop-evoked flapping behavior at 10 days, short horizontal flight at 17 days, and sustained flight at 24 days of age. Wing loading decreased and aspect ratio increased during postnatal development, each reaching adult range before the onset of sustained flight. Histochemically, fibers from the three muscles were undifferentiated at birth and had lower oxidative and glycolytic capacities compared to other age groups. Cross-sectional areas of fibers from the pectoralis and acromiodeltoideus muscles increased significantly at an age when dropevoked flapping behavior was first observed, suggesting that the neuromuscular mechanism controlling flapping did not develop until this time. Throughout the postnatal growth period, pectoralis and acromiodeltoideus muscle mass and fiber cross-sectional area increased significantly. By day 17 the pectoralis muscle had become differentiated in glycolytic capacity, as indicated by the mosaic staining pattern for -GPDH. By contrast, the quadriceps fibers were relatively large at birth and slowly increased in size during the postnatal period. Fiber differentiation was evident at the time young bats began to fly, as indicated by a mosaic pattern of staining for myosin ATPase. These results indicate that flight muscles (pectoralis and acromiodeltoideus) are less well developed at birth and undergo rapid development just before the onset of flight. By contrast the quadriceps femoris muscle, which is required for postural control, is more developed at birth than the flight muscles and grows more slowly during subsequent development.     

Ontogeny of the Pectoralis muscle in the little brown bat,Myotis lucifugus

The ontogeny of a primary flight muscle, the pectoralis, in the little brown bat (Myotis lucifugus: Vespertilionidae) was studied using histochemical, immunocytochemical, and electrophoretic techniques. In fetal and early neonatal (postnatal age 1–6 days) Myotis, histochemical techniques for myofibrillar ATPase (mATPase) and antibodies for slow and fast myosins demonstrated the presence of two fiber types, here called types I and IIa. These data correlated with multiple transitional myosin heavy chain isoforms and native myosin isoforms demonstrated with SDS-PAGE and 4% pyrophosphate PAGE. There was a decrease in the distribution and number of type I fibers with increasing postnatal age. At postnatal age 8–9 days, the adult phenotype was observed with regard to muscle fiber type (100% type IIa fibers) and myosin isoform profile (single adult MHC and native myosin isoforms). This “adult” fiber type profile and myosin isoform composition preceeded adult function by about 2 weeks. For example, little brown bats were incapable of sustained flight until approximately postnatal day 24, and myofiber size did not achieve adult size until approximately postnatal day 25. Although Myotis pectoralis is unique in being composed of 100% type IIa fibers, transitional fiber types and isoforms were present. These transitional forms had been observed previously in other mammals bearing mixed adult muscle fibers and which undergo transitional stages in muscle ontogeny. However, in Myotis pectoralis, this transition transpires relatively early in development. © 1994 Wiley-Liss, Inc.     

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.     

Effects of preservation on wing morphometry of the little brown bat (Myotis lucifugus)

The effects of formalin fixation and subsequent alcohol preservation on various morphometric variables and their derivatives (lifting surface area, wingspan, mass, aspect ratio, wing loading and minimum power speed) and on the results of procedures that estimate lifting surface area of the little brown bat ( Myotis lucifugus ) are identified and quantified. Statisitical analysis demonstrates that the values of all of the examined morphometric variables depends upon the specimen type from which they are obtained (live animal; freshly killed specimen; immediately following formalin fixation; or after 36 weeks in alcohol). Over the short term, the choice of preservation fluid is ot important with respect to determination of the six variables studied. The fixation positin of the wing is an important factor in the determination of all variables except mass. Although originally suggested for study skins and not fluid-preserved specimens, 'intermediate' and 'extended' wing positions are demonstrably better than the conventional 'compressed' position. The estimation procedures of both Pirlot (1977) and Blood & McFarlane (1988) significantly underestimate analogous lifting surface areas determinedby tracing live bats. Smith & Starrett's (1979) procedure was found to yield accurate estimates occasionally: on live animals and preserved specimens with wings fixed in the extended position. Aldridge's (1988) method also yields accurate estimates of lifting surface area, but is limited to those museum specimens where the live or freshly-killed mass is known. Such conclusions permit recommendation of procedures that minimize changes arising through the fixation and preservation process in fluid-preserved museum specimens when compared to the live animal.     

Distribution of somatostatinlike immunoreactivity in the brain of the little brown bat (Myotis lucifugus)

The distribution of somatostatinlike immunoreactive (SLI) perikarya, axons, and terminals was mapped in subcortical areas of the brain of the little brown bat, Myotis lucifugus, using light microscopic immunocytochemistry. A preponderance of immunoreactivity was localized in reticular, limbic, and hypothalamic areas including: 1) in the forebrain: the bed nucleus of the stria terminalis; lateral preoptic, dorsal, anterior, lateral and posterior hypothalamic areas; amygdaloid, periventricular, arcuate, supraoptic, suprachiasmatic, ventromedial, dorsomedial, paraventricular, lateral and medial mammillary, and lateral septal nuclei; the nucleus of the diagonal band of Broca and nucleus accumbens septi; 2) in the midbrain: the periaqueductal gray, interpeduncular, dorsal and ventral tegmental, pretectal, and Edinger-Westphal nuclei; and 3) in the hindbrain: the superior central and parabrachial nuclei, nucleus incertus, locus coeruleus, and nucleus reticularis gigantocellularis. Other areas containing SLI included the striatum (caudate nucleus and putamen), zona incerta, infundibulum, supramammillary and premammillary nuclei, medial and dorsal lateral geniculate nuclei, entopeduncular nucleus, lateral habenular nucleus, central medial thalamic nucleus, central tegmental field, linear and dorsal raphe nuclei, nucleus of Darkschewitsch, superior and inferior colliculi, nucleus ruber, substantia nigra, mesencephalic nucleus of V, inferior olivary nucleus, inferior central nucleus, nucleus prepositus, and deep cerebellar nuclei. While these results were similar in some respects to those previously reported in rodents, they also provided interesting contrasts.     

Pectoralis muscle morphology in the little brown bat,Myotis lucifugus: A non-convergence with birds

Recent studies of muscle architecture demonstrate that many mammalian muscles are composed of short, interdigitating fibers. In addition, the avian pectoralis, a muscle capable of producing high frequency oscillations has been shown to possess a serially arranged pattern of muscle endplate in all sizes of birds studied. The pectoralis muscle of the little brown bat, Myotis lucifugus (Chiroptera: Vespertilionidae), is composed of fairly uniform fibers that span the length of the muscle and is characterized by a zone of motor endplates within the middle third of the muscle. The homogeneous fiber architecture of the bat pectoralis muscle is in contrast to the serial arrangement of endplates (and presumably muscle muscle fibers) in the avian pectoralis in species equivalent in size to Myotis. The short fiber organization and motor endplate pattern observed in most birds is thus not a requisite design for flying vertebrates. © 1994 Wiley-Liss, Inc.     

Functional innervation of the myometrium of Myotis lucifugus, the little brown bat

Myometria of pregnant and nonpregnant Myotis lucifugus were studied in vitro by using electrical field stimulation as well as autonomic agonists and antagonists to determine whether functional responses corresponded with structural evidence showing abundant adrenergic and sparse cholinergic innervation, which uniquely does not disappear during pregnancy. Field stimulation (70 V, 0.6 ms, 5.0-s pulse train, 2.5 - 60 Hz) of myometria from nonpregnant (hibernating) bats produced graded responses consisting of an initial alpha-adrenergic contraction and a subsequent beta-adrenergic relaxation phase. Responses were sensitive to both the nerve poison tetrodotoxin and the adrenergic antagonist guanethidine, demonstrating that they resulted from stimulation of intrinsic adrenergic nerves. Field stimulation responses were unaffected by atropine indicating that there was no functional cholinergic innervation, even though carbachol-induced contraction showed that muscarinic receptors were present. In contrast, functional innervation of cervical tissue was cholinergic and nonadrenergic-non-cholinergic, but not adrenergic. At the beginning of active gestation, some myometrial preparations exhibited little of no response to field stimulation. However, as uterine size increased, the biphasic response to field stimulation was enhanced, particularly the inhibitory (beta-adrenergic) phase. Moreover, the contractile phases, though reduced, was not abolished by alpha-adrenergic antagonists. The residual contractile response was also tetrodotoxin-resistant, suggesting that the myometrium was sensitive to direct electrical stimulation. Near the end of pregnancy, myometrial tissue became nonresponsive to both field stimulation and autonomic agonists, suggesting an absence of available receptor sites on muscle cells.     

A morphometric study of the cochlea of the little brown bat (Myotis lucifugus).

A detailed morphometric study of the basilar membrane was made from serial sections and graphic reconstructions of the cochlea of three little brown bats. Four distinct morphometric changes were observed within the basilar membrane. First, between 0-1.4 mm from the basal end of the cochlea, there is a rapid increase in width and cross-sectional area of the basilar membrane. Secondly, between 1.4-2.5 mm, there is little change in width of the basilar membrane (its cross-sectional area is at its greatest in this region). Thirdly, between 2.7-3.1 mm, there is a sudden decrease in cross-sectional area concomitant with an increase in the width of the basilar membrane. Finally, between 3.1 mm and the apex, there is a gradual decrease in cross-sectional area concomitant with an increase in the width of the basilar membrane. The magnitudes of the cross-sectional areas of the scalae media and vestibuli decrease from base to apex, but this is not true for the scala tympani. The cross-sectional area of the scala tympani appears to decrease from the base to 0.7 mm, then it increases up to 1.4 mm, and then it decreases to the apex. These morphometric changes in the basilar membrane of the little brown bat are compared to those in other echolocating and non-echolocating mammals. The significance of these changes is discussed in relation to the range of hearing in the little brown bat.     

Histochemical and biochemical properties of flight muscle fibers in the little brown bat,Myotis lucifugus

Summary The purpose of this investigation was (1) to determine the fiber composition of pectoralis muscle of the little brown bat,Myotis lucifugus; (2) to compare the fiber composition of this muscle with two of the animal's accessory flight muscles; and (3) to study the effect of hibernation on pectoralis muscle fiber composition. Bat skeletal muscle fibers were also compared with those of white laboratory rats (Rattus norvegicus). Bat pectoralis muscles possessed exceptionally high oxidative capacities as indicated by their succinate dehydrogenase activities, but relatively low glycolytic potentials (phosphofructokinase activities). Muscle histochemistry demonstrated that fiber composition of bat pectorlis muscle was homogeneous; all fibers possessed high aerobic and low glycolytic potentials, and high myofibrillar ATPase activities indicating fast contractile properties. In contrast, accessory flight muscles possessed three distinguishable fiber types. During hibernation there was a significant decline in oxidative potential, no change in glycolytic potential, and no alteration in basic fiber composition of bat pectoralis muscle. The findings of this study suggest that pectoralis muscles ofM. lucifugus may approach the ultimate adaptation of a mammalian locomotory muscle for aerobic generation of muscular power.Abbreviations FG fast-twich glycolytic - FOG fast-twitch-oxydative-glycolytic - -GPDH -glycerophosphate dehydrogenase - LDH lactate dehydrogenase - NADH-D reduced nicotinamide adenine dinucleotide diaphorase - PFK phosphofructokinase - SDH succinate dehydrogenase - SO slowtwich-oxidative     

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.     

Dynamic aspects of the LHRH system associated with ovulation in the little brown bat (Myotis lucifugus)

Adult female bats were collected from natural roosting sites in pre-ovulatory and post-ovulatory conditions. LHRH neurones of these animals were examined using light and electron microscopic immunocytochemistry, and LHRH tissue contents were measured by radioimmunoassay. Comparisons between the two groups of bats revealed that the number of LHRH perikarya detected immunocytochemically, as well as hypothalamic LHRH content, were significantly reduced in post-ovulatory animals. Distributions of immunoreactive perikarya were, however, strikingly similar in both groups. The reduction in immunoreactive cell number observed after ovulation was therefore not restricted to an anatomically defined subset of neurones, but was evident throughout the population. The projection of LHRH-immunoreactive fibres that extend into the pituitary neural lobe in this species also exhibited changes related to endocrine condition. Morphometric indices of fibre density in the neural lobe were significantly reduced in post-ovulatory bats, as was LHRH content of the lower infundibular stalk and neural lobe. Fine structural study of perikarya revealed complex anatomical interactions between LHRH-immunopositive elements, especially in post-ovulatory bats. These interactions included direct apposition of perikarya, as well as more elaborate networks involving various combinations of perikarya and large- and small-caliber processes. These changes in the LHRH system associated with ovulation suggest reduction of stored peptide within perikarya and depletion from terminals within the lower infundibular stem and neural lobe. Parallel reductions in hypothalamic and neural lobe LHRH content during the periovulatory period support the hypothesis that the neural lobe component of the system contributes to control of gonadotrophin secretion in this species. Finally, increased complexity of anatomical contact between components of the LHRH system may be related to activation of this cell population in spring.     

Genetic connectivity among swarming sites in the wide ranging and recently declining little brown bat (Myotis lucifugus)

Characterizing movement dynamics and spatial aspects of gene flow within a species permits inference on population structuring. As patterns of structuring are products of historical and current demographics and gene flow, assessment of structure through time can yield an understanding of evolutionary dynamics acting on populations that are necessary to inform management. Recent dramatic population declines in hibernating bats in eastern North America from white‐nose syndrome have prompted the need for information on movement dynamics for multiple bat species. We characterized population genetic structure of the little brown bat, Myotis lucifugus, at swarming sites in southeastern Canada using 9 nuclear microsatellites and a 292‐bp region of the mitochondrial genome. Analyses of F_ST, Φ_ST, and Bayesian clustering (STRUCTURE) found weak levels of genetic structure among swarming sites for the nuclear and mitochondrial genome (Global F_ST = 0.001, P < 0.05, Global Φ_ST = 0.045, P < 0.01, STRUCTURE K = 1) suggesting high contemporary gene flow. Hierarchical AMOVA also suggests little structuring at a regional (provincial) level. Metrics of nuclear genetic structure were not found to differ between males and females suggesting weak asymmetries in gene flow between the sexes. However, a greater degree of mitochondrial structuring does support male‐biased dispersal long term. Demographic analyses were consistent with past population growth and suggest a population expansion occurred from approximately 1250 to 12,500 BP, following Pleistocene deglaciation in the region. Our study suggests high gene flow and thus a high degree of connectivity among bats that visit swarming sites whereby mainland areas of the region may be best considered as one large gene pool for management and conservation.     

Fetal development, and placental and maternal plasma concentrations of progesterone in the little brown bat (Myotis lucifugus)

Progesterone concentrations measured in plasma samples from 280 bats captured during pregnancy or early lactation were related to fetal attributes indicative of stage of pregnancy. Fetal weight increased exponentially from 40 mg at crown-rump length of 6 mm to 2000 mg at 23 mm (term). Fetal weights at term accounted for up to 35% of the weight of intact pregnant animals. Progesterone concentrations increased from less than 5 ng/ml at 2 mm estimated crown-rump length to plateau values of approximately 65 ng/ml (geometric means) from 16 mm crown-rump length until the most advanced stages of pregnancy. Mean concentration in 8 post-partum bats, most of which were actively lactating, was 8.4 ng/ml; 11.6 ng/ml was measured in one animal that was carrying a wet neonate when sampled yet was still pregnant when captured 5 h earlier. Placental concentrations of progesterone ranged from 43 to 964 ng/g wet weight of tissue and mean values increased in a similar fashion though were about 4-fold greater than changes in plasma concentrations of the steroid. The concentrations in placental tissue were at least 15- to 20-fold higher than could be expected from blood contamination, indicating that placental steroidogenesis is likely to occur in this species.