Immunocytochemical localization of neuropeptides in the hypothalamus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary To elucidate the role of hypothalamic neuropeptides in regulation of reproductive phenomena of seasonally breeding feral mammals, we used Japanese long-fingered bats, Miniopterus schreibersii fuliginosus, for immunocytochemical study of distribution of the following neuropeptides in the hypothalamus: arginin vasopressin, oxytocin, luteinizing hormone-releasing hormone, somatostatin, corticotropin-releasing factor, and growth hormone-releasing factor. The size, shape and location of supraoptic, paraventricular, suprachiasmatic, and arcuate nuclei of the bat were determined. Arginin vasopressin-and oxytocin-immunoreactive magnocellular neurons were found in the supraoptic and paraventricular nuclei, where they exhibited separate distribution into two distinct groups. Parvocellular arginin vasopressin neurons occurred only in the suprachiasmatic nucleus. The hibernating bats exhibited slightly increased numbers of vasopressin and oxytocin neurons in the supraoptic and paraventricular nuclei. The pregnant bat displayed further increased numbers of vasopressin and oxytocin neurons in both nuclei. Somatostatin-immunoreactive neurons in the paraventricular nucleus were also immunopositive to anti-oxytocin serum, while those in the ventromedial and arcuate nuclei reacted solely to anti-somatostatin serum. They projected to the anterior median eminence and infundibular stalk. Luteinizing hormone-releasing hormone-immunoreactive perikarya were scattered throughout the basal hypothalamus, being particularly abundant in the arcuate nucleus. They were larger in size in hibernating bats than those in normal (non-pregnant) and pregnant females. They projected fibers mainly to the internal layer of the median eminence and infundibular stalk. A few luteinizing hormone-releasing hormone-reactive fibers were also observed in the organum vasculosum laminae terminalis, lateral habenular nuclei, pineal stalk, retroflexus fasciculus, and olfactory tubercle. Corticotropin releasing factor-immunoreactive perikarya were distributed in the paraventricular nucleus and medial preoptic area and projected into the external layer of the anterior median eminence, while growth hormone-releasing factor-immunoreactive perikarya occurred only in the arcuate nucleus and projected into the posterior part of the median eminence.     

Development of the main and accessory placentae in the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus

The placentae of the Japanese long-fingered bat were characterized by their morphological and functional transition from the main placenta to the accessory placentae. The main placenta transformed from an endotheliodichorial to a haemodichorial (one layer of syncytiotrophoblast and one layer of cytotrophoblast cells) condition. Degeneration of the main placenta was accompanied by development of two accessory placentae. These developed on both sides (fetal side) of the main placenta, and subsequently converted from a haemodichorial (two layers of cytotrophoblast cells) to a haemomonochorial condition.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Noradrenergic and cholinergic nerves in the uterus of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus,change with reproductive cycle

The pattern of uterine innervation by noradrenergic (NA) and acetylcholinesterase-positive (AChE) nerves in different reproductive stages of the adult Japanese long-fingered bats were investigated histochemically and immunohistochemically. In the non-pregnant bat, the uterine horn was supplied with abundant NA and AChE nerves. These two types of nerves were closely associated with the uterine arteries and myometrial smooth muscles. In the pregnant bat, NA and AChE nerves supplying the uterus did not degenerate much during hibernating period, but reduced markedly after arousal. In the postpartum bat, the density of nerves recovered progressively. The significant change in the innervation pattern of uterine NA and AChE nerves in the pregnant bats under and after hibernation, and in the postpartum bat must be considered in relation to the adrenergic and cholinergic controlling mechanisms on the uterine function that is matched for the unique reproductive cycle of this bat.     

Ultrastructural development of chorioallantoic placenta in the Indian Miniopterus bat, Miniopterus schreibersii fuliginosus (Hodgson).

The chorioallantoic placental interhemal membrane of Miniopterus schreibersii fuliginosus has been described electron-microscopically. Morphologically there are three main types of placentae which develop in chronological sequence. They are (1) primary placenta, (2) secondary placenta and (3) tertiary placenta. In neural groove and limb-bud embryos the primary placenta consists of the following elements which separate the maternal and fetal circulations: (1) a continuous ectoplasmic layer, (2) intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The primary placenta degenerates until term when it consists of a thin syncytiotrophoblastic layer resting on basal lamina. Mesenchyme does not show the presence of fetal capillaries. The secondary placenta is formed in early limb-bud embryos. The electron microscope has revealed that the placenta is of the endotheliomonochorial type and (1) consists of a well-developed maternal endothelium, (2) the trophoblast surrounding the maternal blood tubule is cellular, not syncytial as previously thought and the apical plasma membrane of these trophoblastic cells is in direct contact with the discontinuous interstitial membrane, (3) basal lamina, (4) mesenchyme and (5) fetal endothelium. Tertiary placenta at full term stage is of the hemodichorial type having the following elements: (1) thin ectoplasmic layer, (2) a thick intrasyncytial lamina, (3) syncytiotrophoblast, (4) cytotrophoblast, (5) basal lamina, (6) mesenchyme and (7) fetal endothelium. The definitive chorioallantoic placental barrier in this bat thus differs from the organization earlier proposed by Chari and Gopalakrishna [Proc. Indian Acad. Sci. 93: 463-483, 1984] on the basis of light-microscopic observations: (1) the absence of maternal endothelium in the primary placenta from the neural groove and early limb-bud embryos, (2) the existence of only cellular trophoblast in the secondary placenta throughout the gestation and (3) the presence of well-developed hemodichorial tertiary placenta is the unique feature of the interhemal membrane in higher Chiroptera.     

Isolation and characterization of microsatellite loci in the long-fingered bat Miniopterus fuliginosus

We isolated and characterized 10 microsatellite loci in the long-fingered bat Miniopterus fuliginosus. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.     

Plasma progesterone concentrations in the female Natal clinging bat (Miniopterus schreibersii natalensis)

Plasma progesterone concentrations measured by radioimmunoassay in the Natal clinging bat remained below 2.01 ng/ml during lactational anoestrus but increased significantly during the period of delayed implantation. Values peaked at implantation but were followed by a significant decrease thereafter. Concentrations remained low (less than 6.0 ng/ml) during the initial period of fetal development (153-180 days post coitum) and attained peak values (85.6-181.3 ng/ml) 216-222 days after fertilization. The marked post-implantation increase in progesterone concentrations coincided with a significant increase in placental weight.     

Reproductive synchrony and annual variation in foetal growth rate in the long-fingered bat (Miniopterus schreibersii)

Mating in the long-fingered bat (Miniopterus schreibersii) from the East Cape region of South Africa occurred during a four-week period in April and May, and implantation was delayed until August. Analyses of foetal age and estimated dates of conception indicate that after implantation, the progression of pregnancy within the population was synchronous both within and between years. It is suggested that implantation, probably initiated by increasing day length, occurred at about the same time throughout the population and thus synchronized reproduction. In the final month of pregnancy foetal growth rate and, consequently, the timing of parturition in the three years of the study varied. These variations were correlated with variations in rainfall and it is suggested that the failure of the November rains in 1990 would have caused a reduction in insect abundance and that this was responsible for the decrease in foetal growth rate and the delay in parturition.     

Patterns of Leydig cell and LH gonadotroph activity, and plasma testosterone concentrations in the seasonally reproducing Schreibers' long-fingered bat (Miniopterus schreibersii)

Spermatogenesis in Schreibers' long-fingered bat from approximately 33 degrees S in South Africa was seasonal, and occurred in the 3 months (February-April) preceding ovulation. The ultrastructure of the Leydig cells indicated a period of increased steroidogenesis at this time, and plasma testosterone concentrations were elevated from March to May (10.3 ng/ml). The reproductive accessory glands were secretorily active between March and May, and copulation occurred at the end of this period of activity. Changes in LH-beta immunoreactivity suggest that the LH gonadotrophs were secretorily active 1 month before the onset of spermatogenesis and that peak activity coincided with peak plasma testosterone concentrations, spermiogenesis and spermiation. During winter (May-August) there was no reproductive activity and the bats remained active, only entering prolonged periods of torpor during particularly cold spells. A secondary elevation in plasma testosterone concentration, during reproductive inactivity (October; 9.3 ng/ml), was not accompanied by any change in Leydig cell ultrastructure, and the biological significance of this peak is unknown. Such synchronous activity of the pituitary, Leydig cells, seminiferous epithelium and accessory glands is associated with the typical reproductive cycle of long-fingered bats in which copulation and fertilization are restricted to a brief period at the end of summer, and in which neither sperm storage nor a prolonged period of copulation occur.     

普通长翼蝠食性结构及其回声定位与体型特征

在普通长翼蝠(Miniopterus fuliginosus)的捕食区内用灯诱法和网捕法调查潜在食物(昆虫)种类; 用粪便分析法鉴定普通长翼蝠的食物组成,发现其主要捕食体型较大的鳞翅目和鞘翅目昆虫,体积百分比分别为55%和38%.普通长翼蝠具有相对狭长的翼,翼展比为6.94 ± 0.13;翼载为(9.85 ± 0.83)N/m2,相对较大.飞行状态下普通长翼蝠的回声定位叫声为调频下扫型,声脉冲时程为(1.45 ± 0.06)ms,脉冲间隔为(63.08 ± 21.55)ms,主频较低,为(44.50 ± 2.26)kHz.研究表明,普通长翼蝠的形态特征和回声定位特征与其捕食行为有着密切的联系.     

Plasma progesterone and luteinizing hormone concentrations and the role of the corpus luteum and LH gonadotrophs in the control of delayed implantation in Schreibers' long-fingered bat (Miniopterus schreibersii)

In Schreibers' long-fingered bat from South Africa (approximately 33 degrees S) copulation, ovulation and fertilization occurred in April and May, implantation was delayed until August, and parturition occurred in December. Delayed implantation coincided with winter, during which the bats remained active, only entering prolonged periods of torpor during particularly cold spells. Plasma progesterone concentration was low during non-pregnancy (1.54 ng/ml) and during delayed implantation (1.67 ng/ml), and thereafter increased to reach a peak mean of 64.82 ng/ml in late pregnancy. Changes in size and ultrastructure of the luteal cells indicated periods of steroidogenesis just after formation of the corpus luteum, and for about 2 months after implantation; reduced steroidogenic activity during delayed implantation; and luteolysis in the last 2 months of pregnancy. Plasma luteinizing hormone (LH) concentration and pituitary LH-beta immunoreactivity were highest during follicular development and peaked just before ovulation. During early delayed implantation, plasma LH concentration was low, and both plasma LH and pituitary LH-beta immunoreactivity increased from July, reaching peaks in late pregnancy. LH may be required to activate the corpus luteum and terminate delayed implantation, or, as in some small carnivores, it may be required for luteal maintenance.     

Isolation and characterization of microsatellite loci in the western long-fingered bat, Miniopterus magnater

We isolated and characterized 10 microsatellite loci in the western long-fingered bat, Miniopterus magnater. These loci were tested on 48 individuals from Anhui Province of China, and all loci were highly polymorphic. The mean number of observed alleles per locus was 13.6 (range from six to 27). Observed and expected heterozygosity values ranged from 0.364 to 0.957, and from 0.676 to 0.951, respectively. After Bonferroni correction, four loci deviated significantly from Hardy-Weinberg equilibrium. No pairs of loci were in linkage disequilibrium. These polymorphic markers will be used to examine population structure and genetic diversity in this species.     

The effect of cave microclimate on winter roosting behaviour in the bat,Miniopterus schreibersii blepotis

The microclimate at Thermocline Cave (lat, 30° 45′S, long, 149° 43′E) was investigated by measuring air temperature and relative humidity at five stations on 18 occasions from September 1971 to December, 1973. The activity, body weight and roosting sites of the bat Miniopterus schreibersii blepotis in the cave were recorded on each visit. Relative humidity in the cave was generally high and paralleled temperature. The cave exhibited a range of temperatures from 9 to 19.5°C but bats selected roosting sites only in a part of this range. During the autumn when the bats arrived and were feeding, their body weights were low, and they roosted in a domed area at the rear of the cave with a temperature of 19.5°C. As they became less active and body weight increased they moved to cooler parts (9.5-11°C) towards the front of the cave and underwent periods of torpor, in one case lasting for at least 12 days. From July to September body weight decreased. The bats became more active in September and most had left the cave by October. It appears that M.s. blepotis can detect temperature differences of 1°C. They used this ability to select cold areas with stable high humidity in Thermocline Cave to under go periods of winter torpor.     

Progesterone concentrations and contents in the plasma, ovary, adrenal gland and placenta of the pregnant Natal clinging bat Miniopterus schreibersii natalensis

Reproduction in the Natal clinging bat in South Africa at 25°S is characterized by fertilization in April, implantation during July and parturition in November. In the present study plasma progesterone concentrations peaked two months before implantation, this coinciding with significant peaks in ovarian progesterone content and concentration. Plasma progesterone concentrations remained relatively low during and after implantation, but increased significantly during the limb bud stage to peak levels one month before parturition. Adrenal progesterone content and concentration did not follow a specific trend; post-implantation ovarian progesterone content and concentration remained at baseline values. Placental progesterone content increased significantly with foetal weight (age). The present study showed that pregnancy in these bats is characterized by two major sources of progesterone, with the ovary as the primary source before implantation and the placenta the primary source during embryonic and foetal development.     

Reproductive biology of the male bent-winged bat, Miniopterus schreibersii (Vespertilionidae) in southeast South Australia

The seasonal chronology of the events of the reproductive cycle, and changes in the structure and function of the primary and accessory organs of the male bent-winged bat, Miniopterus schreibersii, were studied at latitude 37 degrees S in temperate southeastern Australia. The testicular cycle commenced in late spring (November), and sperm appeared in the seminiferous tubules and epididymides in early fall (March). The cycle of the accessory sex gland complex generally paralleled the testicular cycle, reaching maximum hypertrophy at the time of insemination in late fall (April/May). Thereafter, the primary and secondary sex glands (except the ampullary gland) involuted as the animals entered winter torpor. However, a cauda epididymal store of sperm persisted until late spring, and sperm were often observed, as well, in the ampullary gland duct and alveoli throughout winter. This study has confirmed that male Miniopterus differs from other vespertilionids in that accessory gland activity declines following the fall breeding in keeping with the fact that, unlike in other vespertilionids, insemination, ovulation and conception are concurrent events in the fall in this species. The reduced secretory status of the Leydig cells and exceptionally low levels of circulating androgens throughout the year, in combination with the presence of viable epididymidal sperm for most of gestation, are all interesting features of this reproductive cycle.     

Genetic diversity of coronaviruses in Miniopterus fuliginosus bats

Coronaviruses, such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, pose significant public health threats. Bats have been suggested to act as natural reservoirs for both these viruses, and periodic monitoring of coronaviruses in bats may thus provide important clues about emergent infectious viruses. The Eastern bent-wing bat Miniopterus fuliginosus is distributed extensively throughout China. We therefore analyzed the genetic diversity of coronaviruses in samples of M. fuliginosus collected from nine Chinese provinces during 2011–2013. The only coronavirus genus found was Alphacoronavirus. We established six complete and five partial genomic sequences of alphacoronaviruses, which revealed that they could be divided into two distinct lineages, with close relationships to coronaviruses in Miniopterus magnater and Miniopterus pusillus. Recombination was confirmed by detecting putative breakpoints of Lineage 1 coronaviruses in M. fuliginosus and M. pusillus(Wu et al., 2015), which supported the results of topological and phylogenetic analyses. The established alphacoronavirus genome sequences showed high similarity to other alphacoronaviruses found in other Miniopterus species, suggesting that their transmission in different Miniopterus species may provide opportunities for recombination with different alphacoronaviruses. The genetic information for these novel alphacoronaviruses will improve our understanding of the evolution and genetic diversity of coronaviruses, with potentially important implications for the transmission of human diseases.     

Molecular systematics and biogeography of the bent-wing bat complex Miniopterus schreibersii (Kuhl, 1817) (Chiroptera: Vespertilionidae)

The complete mitochondrial ND2 gene (1037 bp) was sequenced to examine relationships within the bent-wing bat complex, Miniopterus schreibersii (Family Vespertilionidae). It was found that M. schreibersii is a paraphyletic assemblage comprising several species. Two major lineages were identified, one of which was restricted to the Palearctic-Ethiopian regions and the other to the Oriental-Australasian regions. This pattern of differentiation was mirrored by the genus as a whole. Speciation and differentiation within the genus Miniopterus appears to have a hierarchical geographical pattern. The earliest divergence corresponds to the Ethiopian-Palearctic and the Oriental-Australasian biogeographical zones. This early divergence is then followed by radiations within each of the Ethiopian, Oriental and Australasian regions. The study also revealed that the number of species currently recognized (11 or 13) is a gross underestimate of the number of actual species. The emerging picture is one of a relatively speciose genus with most species having relatively restricted distributions; few, if any, occur in more than one biogeographical region.     

Erythrocyte metabolism in two species of bats: common bent-wing bat (Miniopterus schreibersii) and red fruit bat (Pteropus scapulatus).

1. Metabolic intermediates, substrate utilization and enzyme activities were determined in the red blood cells of the common bent-wing bat and the red fruit bat. Standard haematological parameters and oxy-haemoglobin dissociation curves were also determined in both species. 2. The glycolytic rate as measured by lactate production was much higher for all substrates in the bent-wing bats. The activities of the glycolytic enzymes were also much higher in this species. 3. The standard haematological parameters were similar for the two species. The levels of 2,3-diphosphoglycerate (DPG) in the red cells of the fruit bat were nearly twice as high as those in the bent-wing bats. 4. The oxy-haemoglobin dissociation curve for the red fruit bat was located to the right of that for the bent-wing bat and both these curves were located to the right of that normally seen for human blood. 5. Both species of bat show blood characteristics well adapted to carrying the increased oxygen demands of flight.     

Diet and prey selection in the trawling long-fingered bat

The diet of the long-fingered bat Myotis capaccinii is poorly known, and there is no previously recorded information on this species' prey preferences. To investigate these subjects, we captured 51 individuals at a nursery cave in the Iberian Peninsula, from pre-breeding to post-lactation seasons. Each bat's diet composition was assessed by faecal content analysis and its foraging places (rivers, pools and channels) identified by radio-telemetry. To estimate prey availability, we sampled arthropods in the individual bats' identified foraging places and also emulated the bats' hunting technique. The bats' diet comprised of arthropods, dominated by small insects with aquatic larvae and flying adult phases. The most consumed taxon was Nematocera (mainly Chironomidae), including adults and pupae, which were also found to be the most abundant prey over water. Other frequently consumed prey were brachycerans, lepidopterans, arachnids, trichopterans and neuropterans. Diet proportions were compared with prey availability to infer a rank of preferences. The preferred prey were lepidopterans and arachnids, both having a terrestrial life cycle and a bigger size than any other taxa consumed. Without discarding the possible underestimation of prey's aerial availability, the observed preference pattern seems to be a consequence of selection for size more than for specific taxa. Apparently M. capaccinii efficiently exploit water-related prey according to availability when the bats hunt low over the water's surface, and are also able to take more profitable prey found higher in the air.     

On the occurence of a short period of delayed implantation in Schreibers'long-fingered bat (Miniopterus schreibersii) from a tropical latitude in Zimbabwe

Mating in Schrcibers'long-fingered bat ( Miniopterus schreibersii ) from central Zimbabwe (18°S) occurred between mid-April and mid-May, after a five-month period of spermatogenesis. Implantation was delayed until early July and parturition occurred between late October and mid-November, about four months after implantation and six to seven months after fertilization. The timing and length of gestation at 18°S are similar to that described for M. schreibersii from Zaire (11σS) and when these date are compared with those for the same species from higher latitudes in Africa, it is apparent that there is a small, but distinct, increase in the total length of pregnancy and the length of delayed implantation with increasing latitude. The reproductive bilogy of Miniopterus schreibersii from Africa and France differs fundamentally from that of the same species from Australia and Japan, and it is suggested that studies of the relationship between latitude and duration of delayed implantation should be based on comparisons within single species from different latitudes on the same continent, and that comparisons between species and between continents should be avoided.