Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

Histochemical and biochemical plasticity of muscle fibers in the little brown bat (Myotis lucifugus)

Summary Fiber composition, and glycolytic and oxidative capacities of the pectoralis, gastrocnemius, and cardiac muscles from active and hibernating little brown bats (Myotis lucifugus) was studied. The data were used to test two hypotheses: First, since hibernating bats maintain the capability of flight and make use of leg muscles to maintain a roosting position all winter, the fiber composition of the pectoralis and gastrocnemius muscles should not change with season. Second, we tested the hypothesis of Ianuzzo et al. (in press), who propose that the oxidative potential of mammalian cardiac muscle should increase with increasing heart rate while glycolytic potential should not. Our results indicate that the fiber composition of the pectoralis muscle was uniformly fast-twitch oxidative (FO)_ regardless of the time of year, as predicted. However, the gastrocnemius muscle exhibited a change in FO composition from 83% in active to 61% in hibernating animals. Contrary to the variable change in histochemical properties with metabolic state, a trend of reduced maximal oxidative (CS) and glycolytic (PFK) potential during hibernation in both flight and leg muscles was apparent. The oxidative potential of flight and leg muscles decreased by 15.2% and 56.5%, respectively, while the glycolytic potential of the same muscles decreased by 23.5% and 60.5%, respectively. As predicted, the glycolytic potential of cardiac muscle remained constant between active and hibernating bats, although there was a significant decrease (22.0%) in oxidative potential during hibernation.Abbreviations FO fast-twitch oxidative - FG fast-twitch glycolytic - SO slow-twitch oxidative - Vmax maximal enzyme activity - PFK phosphofructokinase - CS citrate synthase     

Evolution of opsin genes reveals a functional role of vision in the echolocating little brown bat (Myotis lucifugus)

Echolocating bats are able to orientate, navigate and forage without visual cues. To probe the role of vision in bats, we studied the visual opsin genes from the echolocating little brown bat (Myotis lucifugus). Short-wavelength sensitive (SWS1) opsin, middle/long-wavelength sensitive (M/LWS) opsin and rhodopsin cDNA sequences were identified from the Ensembl database and validated by the sequencing of genomic DNA. We retrieved the published orthologous genes from eleven additional representative species of mammals from GenBank and conducted an evolutionary analysis. We found that the M/LWS opsin and rhodopsin genes were both under strong purifying selection, whereas the SWS1 opsin gene has undergone positive selection at two amino acid sites and one lineage, though the main evolutionary force is still purifying selection. Two-ratio model of the SWS1 opsin gene revealed that the ω ratio for the little brown bat lineage was nearly three times lower than the background ratio, suggesting a much stronger functional constraint. Our relative rate tests show the little brown bat has a lower nonsynonymous substitution rate than those in other mammals (on average 32% lower) for the SWS1 opsin gene. However, no such significant differences were detected for the M/LWS opsin and rhodopsin genes. The results of the relative ratio tests are consistent with that of tests for selection, showing a history of purifying selection on the little brown bat opsin genes. These findings suggest a functional role of vision in the little brown bat despite being nocturnal and using echolocation. We speculate that this echolocating bat may be able to use visual cues to orientate, navigate and forage at night, to discriminate color under moonlight and starlight conditions, or to avoid predation by diurnal raptors.     

Enzymes of the normothermic and hibernating bat,Myotis lucifugus: Metabolites as modulators of pyruvate kinase

Summary The previous paper (Borgmann, A., and Moon, 1976) suggested that temperature differentially affected the binding of the substrates phosphoenol pyruvate (PEP) and ADP to bat tissue pyruvate kinases (PK) under hibernating and normothermic conditions. Since the regulatory properties of most mammalian type-L PKs are temperature dependent, a study of these properties for the bat enzymes was initiated.M. pectoralis PK ofM. lucifugus is modulator insensitive, although ATP does increase theK _m(PEP) slightly (Table 1). This effect is most pronounced at low temperatures for HM-PK (Fig. 1) and may be of some regulatory significance.Modulators affect bat liver PK in a manner analogous to other mammalian type-L enzymes, and marked quantitative differences exist between the NL-and HL-enzymes. TheK _m(PEP) is increased only slightly by alanine and ATP (Table 3), althoughV _max decreases markedly for NL-PK; fructose-1,6-diphosphate (FDP) decreases theK _m(PEP) and overrides the inhibitory action of ATP and alanine (Table 3). As temperature decreases, the proportional change inK _m(PEP) with or without effectors is unchanged (Table 3).HL-PK is markedly affected by these modulators. The extent of this interaction, as indicated byn _H-values (Table 3) andK _i orK _a values (Table 2; Figs. 1, 2, 3), between the effectors and the binding of PEP to HL-PK is relatively greater than for the NL-enzyme. However, the temperature sensitivity of these interactions is reduced (Table 3).Therefore, the strategies associated with enzymes of hibernating and normothermic bats are similar to those previously reported by Hochachka and Somero (1973) for certain enzymes of poikilotherms, and argues for the existence of distinct enzyme forms in the two physiological states.Abbreviations ala alanine - FDP fructose-1,6-diphosphate,HL, HM, hibernator liver, and muscle, respectively - LDH lactate dehydrogenase - NL, NM normothermic liver, and muscle, respectively - PEP phosphoenol pyruvate - PK pyruvate kinase     

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.     

Behavioural flexibility: the little brown bat, Myotis lucifugus, and the northern long-eared bat,M. septentrionalis , both glean and hawk prey

We present behavioural data demonstrating that the little brown bat, Myotis lucifugus, and the northern long-eared bat, M. septentrionalis, can glean prey from surfaces and take prey on the wing. Our data were collected in a large outdoor flight room mimicking a cluttered environment. We compared and analysed flight behaviours and echolocation calls used by each species of bat when aerial hawking and gleaning. Our results challenge the traditional labelling ofM. lucifugus as an obligate aerial-hawking species and show that M. septentrionalis, which is often cited as a gleaning species, can capture airborne prey. As has been shown in previous studies, prey-generated acoustic cues were necessary and sufficient for the detection and localization of perched prey. We argue that the broadband, high-frequency, downward-sweeping, frequency-modulated calls used by some bats when gleaning prey from complex surfaces resolve targets from background. First, because calls of lower frequency and narrower bandwidth are sufficient for assessing a surface before landing, and second, because there are few, if any, simple surfaces in nature from which substrate-gleaning behaviours in wild bats would be expected. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.     

Seasonal biochemical plasticity of a flight muscle in a bat, Murina leucogaster

Cellular and biochemical responses of the pectoral muscle to variation in seasonal activity were studied in the bat, Murina leucogaster ognevi. We collected bats in mid-hibernation (February), end-hibernation (April), and mid-summer (August) to track major activity periods in their annual cycle. Our findings indicated that myofiber cross-sectional area decreased to 68% between mid- and end-hibernation, but returned to the winter level in mid-summer. Total soluble protein and total RNA concentrations were not altered over these sampling periods. Oxidative potential gauged by citrate synthase activity increased 1.47-fold from mid- to end-hibernation and then remained at the similar level in mid-summer. Glycolytic potential gauged by lactate dehydrogenase activity changed little between mid- and end-hibernation but increased 1.42-fold in summer, compared with the winter level. Thus, the myofibers underwent disuse atrophy during hibernation, while enzymatic catalytic function recovered towards the level of mid-summer.     

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.     

Histochemical and immunohistochemical properties of skeletal muscle fibres fromRana andXenopus

Summary Modern histochemical and immunohistochemical techniques have been used to type skeletal muscle fibres from threeRana species andXenopus laevis.Differing myosin properties and metabolic capacities (representing various contractile properties) define a minimum of four fibre types inRana and five inXenopus. TheRana andXenopus types are sufficiently similar so that a single nomencclature can be applied to them. This nomenclature uses an initial letter indicating the probable contractile performance (F=fast-twitch, S=slow-twitch and T=tonic), and a number indicating rank order of presumed shortening velocity.The largest, fastest fibres-F1-have low oxidative and, at best, moderate glycolytic capacities. Commonly adjacent to them are smaller, F2 fibres with variable but at least moderate metabolic capacities. F3 fibres are rarer and have on average the highest oxidative capacity, and at least moderate glycolytic capacity. They usually occur in the reddest parts of the muscle and, inRana, only in the vicinity of tonic fibres.Metabolically weak, classical amphibian tonic fibres (T5) occur in bothXenopus andRana, but onlyXenopus also has an S4 fibre type. This has moderate metabolic capacity and myosin properties suggesting it is probably capable of slow shortening as well as tonic hold. Immunohistochemically, S4 fibres are most similar to avian slow-twitch fibres.     

Histochemical properties of skeletal muscle fibers in streptozotocin-diabetic rats

Summary The response of rat gastrocnemius muscle fibers to chronic streptozotocin-diabetes was studied. Transverse sections of this muscle from normal and diabetic rats were histochemically assayed for reduced diphosphopyridine nucleotide-diaphorase, myofibrillar adenosine triphosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and alkaline phosphatase activities. Cross-sectional areas of the fiber types were measured, and fiber capillarization and populations estimated. Chemically-induced diabetes appeared to have little effect on the metabolic or morphological properties of slow-twitch fibers. However, a general dedifferentiation occurred in the 2 fast-twitch fiber populations. There was a loss of oxidative potential in the fast-twitch-oxidative-glycolytic fibers, and a significant decrease in size in the fast-twitch-glycolytic fibers. No change in the proportions of slow- and fast-twitch fibers in the muscles of diabetic rats occurred. It is concluded that hypoinsulinism has differential effects on the 3 fiber types in heterogeneous rat skeletal muscle, and that slow-twitch fibers are least affected by the diabetic condition.     

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.     

Histochemical, enzymatic, and contractile properties of skeletal muscle fibers in the lizard Dipsosaurus dorsalis

Lizard skeletal muscle fiber types were investigated in the iliofibularis (IF) muscle of the desert iguana (Dipsosaurus dorsalis). Three fiber types were identified based on histochemical staining for myosin ATPase (mATPase), succinic dehydrogenase (SDH), and alphaglycerophosphate dehydrogenase (alphaGPDH) activity. The pale region of the IF contains exclusively fast-twitch-glycolytic (FG) fibers, which stain dark for mATPase and alphaGPDH, light SDH. The red region of the IF contains fast-twitch-oxidative-glycolytic (FOG) fibers, which stain dark for all three enzymes, and tonic fibers, which stain light for mATPase, dark for SDH, and moderate for alphaGPDH. Enzymatic activities of myofibrillar ATPase, citrate synthase, and alphaGPDH confirm these histochemical interpretations. Lizard FG and FOG fibers possess twitch contraction times and resistance to fatigue comparable to analogous fibers in mammals, but are one-half as oxidative and several times as glycolytic as analogous fibers in rats. Lizard tonic fibers demonstrate the acetylcholine sensitivity common to other vertebrate tonic fibers.     

Uniform myosin isoforms in the flight muscles for little brown bats, Myotis lucifugus

The present study used muscle histochemistry and polyacrylamide gel electrophoresis of native myosin and myosin heavy chains to establish a correlation, if any, between chiropteran histochemical fiber types and myosin isoform composition. Histochemical analysis of the primary flight muscle, the pectoralis profundus, documented the presence of a single histochemical fiber type, here termed Type II. Electrophoresis of native myosin isolated from pectoralis muscle yielded a single isoform that comigrated with the FM-3 isoform of rat diaphragm. Heavy chain analysis of the Myotis pectoralis demonstrated a single heavy chain with comparable electrophoretic mobility to rat IIa myosin heavy chain. These data demonstrate unique histochemical and biochemical homogeneity in the myosin composition of the pectoralis muscle of Myotis lucifugus. Thus this muscle is extremely specialized for flight at histochemical, morphologic, and molecular levels. These data contrast with the mixed myosin and histochemical fiber types found in other mammals, as well as in other muscles of Myotis lucifugus.