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The placenta of the Korean water deer was anatomically examined to accumulate basic information regarding its reproductive system. The convex placentomes with five to nine well-developed pedicles were observed in the whole uterine horns, and therefore, the placenta was classified as oligocotyledonary. The evidence indicating the migration of binucleate cells (BNCs) from trophectoderm to the uterine epithelium led to the histological classification of the placenta as synepitheliochorial. The number of fetuses was markedly higher than that in other ruminant species. However, the number of placentomes was found to be similar to the other Cervidae species. Therefore, these results suggest that the Korean water deer may possess special mechanisms or structures at the fetus attachment site to maintain this unusally high number of fetuses.

Anatomy of bat genital organs has been conventionally studied by gross and microscopic observations to date. Here, we employ both histological observation and diceCT (diffusible iodine-based contrast-enhanced computed tomography) to study the detailed three-dimensional morphological structure of the male genital organs in bats, using the greater horseshoe bat, Rhinolophus ferrumequinum. This is the first study to three-dimensionally describe the whole reproductive organs of bats in detail. Our highly resolved three-dimensional reconstruction reveals that the male organs of R. ferrumequinum consist of paired testes, epididymides, deferent ducts, and five accessory genital glands. The boundary between the ampullary and vesicular glands has been difficult to identify in previous observations, but our diceCT imaging allowed us to clearly differentiate the two. We found that the ampullary gland is located at the terminal part of the deferent ducts, and the vesicular gland lies distal to the ampullary glands. This species possesses a single and carrot-shaped urethral gland, which is not found in most chiropteran families. The presence of the urethral gland in this species and its secretions suggest that after copulation this species is capable of forming a vaginal plug, which can seal the female’s vaginal orifice to block the entrance of spermatozoa from other males. The presence of the urethral gland and elongated epididymal tail and the fact that some individuals can terminate their hibernation and reactivate imply forced copulation on hibernating females can occur in R. ferrumequinum.

To better understand the genetic relatedness of the eastern bent-wing bat Miniopterus fuliginosus across East Asia, mitochondrial D-loop sequences were analyzed from 39 M. fuliginosus individuals living on Jeju Island, South Korea. Seven different haplotypes were identified. Neighbor-joining analysis was performed and divergence time was calculated in combination with available online data on D-loop and ND2 sequences of M. fuliginosus in East Asia, respectively. Results suggest that M. fuliginosus populations living on Jeju Island and in Japan originated from China in the past 20,000 years.

Haeckel's recapitulation theory has been a controversial topic in evolutionary biology. However, we have seen some recent cases applying Haeckel's view to interpret the interspecific variation of prenatal ontogeny. To revisit the validity of Haeckel's recapitulation theory, we take bats that have undergone drastic morphological changes and possess a characteristic ecology as a case study. All members of Rhinolophoidea and Yangochiroptera can generate an ultrasonic pulse from the larynx to interpret surrounding objects (laryngeal echolocation) whereas Pteropodidae lacks such ability. It is known that the petrosal bone is particularly derived in shape and expanded in laryngeal echolocators. If Haeckel's recapitulation theory holds, the formation of this derived trait should occur later than those of other bones. Therefore, we compared the prenatal ossification timing of the petrosal in 15 bat species and five outgroup species. We found that the ossification of the petrosal is accelerated in laryngeal echolocators while it is the last bone to ossify in non-laryngeal echolocating bats and non-volant mammals, which runs counter to the prediction generated by Haeckel's recapitulation theory. We point out the evolutionarily labile nature of trait developmental timing and emphasize that Haeckel's recapitulation theory does not hold in many cases. We caution that generating predictions on ancestral conditions and evolutionary history leading from Haeckel's recapitulation theory is not well supported.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

Highlights • EMCV genome was widely found in fecal guanos in Taiwanese, Korean, and Japanese caves. • Miniopterus fuliginosus is the main source of the fecal guano. • It is possible that Miniopterus fuliginosus is one of the natural hosts of EMCV in East Asia., Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

In order to gather basic reproductive information of the water deer and Reeves' muntjac, the immunolocalization of the cytoskeleton proteins in the testes and epididymides of these two species was investigated. The distribution pattern of cytoskeletal proteins in these two species was similar. The desmin was detected in the peritubular myoid cells of the testes and the sub-epithelial cells of the epididymal ducts. Vimentin was observed in the myoid cells, Leydig cells and perinuclear region of the Sertoli cells. Intense immunoreactions for α-smooth muscle actin were restricted to the smooth vascular muscle cells and the peritubular myoid cells in the testes. From the present results, it appears that these distribution patterns of cytoskeletal proteins may be common in the cervids.

Most of the Cyclodextrin glucanotransferase (Gtases) which have been produced fromB. subtilis were found to be excreted from the cells during cultivation. Immobilized whole cell CGTase fromB. subtilis was prepared by encapsulating the broth solution which had been concentrated ten times with a rotary vacuum evaporator. Cyclization activity of CGTase was reduced by about 10% during the concentrating process, however, its transglycosylation activity, to convert xylitol to glucosyl-xylitol, using dextrin as glucosyl donor, increased by a factor of 3 or 5.