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Mammals exhibit several types of diel activity pattern, including nocturnal, diurnal, crepuscular, and cathemeral. These patterns vary inter- and intra-specifically and are affected by environmental factors, individual status, and interactions with other individuals or species. Determining the factors that shape diel activity patterns is challenging but essential for understanding the behavioral ecology of animal species, and for wildlife conservation and management. Using camera-trap surveys, we investigated the species distributions and activity patterns of terrestrial mammals on the Shiretoko Peninsula, Hokkaido, Japan, with particular focus on brown bears and sika deer. From June to October 2019, a total of 7,530 observations were recorded by 65 camera-traps for eight species, including two alien species. The diel activity pattern of brown bears was diurnal/crepuscular, similar to that of bears in North America, but different from European populations. Bear observations were more frequent during the autumnal hyperphagia period, and adult females and sub-adults were more diurnal than adult males. In addition, bears inside the protected area were more diurnal than those outside it. These findings suggest that appetite motivation, competitive interactions between conspecifics, and human activities potentially affect bear activity patterns. Similar to other sika deer populations and other deer species, the diel activity patterns of sika deer were crepuscular. Deer showed less variation in activity patterns among months and sex-age classes, while adult males were observed more frequently during the autumn copulation period, suggesting that reproductive motivation affects their activity patterns.

In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5-15 V) or low (3-6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37 degrees C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25 degrees C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.

Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia -positive and Borrelia -negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis , Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella -like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.

Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.

In this study, Babesia screening was conducted in 55 rodents and 160 tick samples collected from primary forests and an oil palm plantation in Sarawak, Malaysian Borneo. PCR targeting the 18S ribosomal DNA revealed the presence of Babesia spp. DNA detected in two questing male Haemaphysalis shimoga ticks collected from the oil palm plantation. Sequence analysis revealed that both sequences were identical and had 98.6% identity to a Babesia macropus sequence obtained from Eastern grey kangaroos (Macropus giganteus) in Australia. Phylogenetic tree revealed clustering with marsupial-associated Babesia spp. in the Babesia sensu stricto clade. Whether or not H. shimoga is the competent vector and the importance of the Babesia sp. detected in this study warrants more investigation.

Robust estimates of demographic parameters are critical for effective wildlife conservation and management, but are difficult to obtain for elusive species. We estimated the breeding and adult population sizes, as well as the minimum population size, in a high-density brown bear population on the Shiretoko Peninsula, in Hokkaido, Japan, using DNA-based pedigree reconstruction. A total of 1,288 individuals, collected in and around the Shiretoko Peninsula between 1998 and 2020, were genotyped at 21 microsatellite loci. Among them, 499 individuals were identified by intensive genetic sampling conducted in two consecutive years (2019 and 2020) mainly by noninvasive methods (e.g., hair and fecal DNA). Among them, both parents were assigned for 330 bears, and either maternity or paternity was assigned to 47 and 76 individuals, respectively. The subsequent pedigree reconstruction indicated a range of breeding and adult (≥4 years old) population sizes: 128–173 for female breeders and 66–91 male breeders, and 155–200 for female adults and 84–109 male adults. The minimum population size was estimated to be 449 (252 females and 197 males) in 2019. Long-term continuous genetic sampling prior to a short-term intensive survey would enable parentage to be identified in a population with a high probability, thus enabling reliable estimates of breeding population size for elusive species.

Body condition in mammals fluctuates depending on energy intake and expenditure. For brown bears (Ursus arctos), high‐protein foods facilitate efficient mass gain, while lipids and carbohydrates play important roles in adjusting dietary protein content to optimal levels to maximize energy intake. On the Shiretoko Peninsula, Hokkaido, Japan, brown bears have seasonal access to high‐lipid pine nuts and high‐protein salmon. To assess seasonal and annual fluctuation in the body condition of adult female brown bears in relation to diet and reproductive status, we conducted a longitudinal study in a special wildlife protection area on the Shiretoko Peninsula during 2012–2018. First, analyses of 2,079 bear scats revealed that pine nuts accounted for 39.8% of energy intake in August and salmon accounted for 46.1% in September and that their consumption by bears varied annually. Second, we calculated the ratio of torso height to torso length as an index of body condition from 1,226 photographs of 12 adult females. Results indicated that body condition continued to decline until late August and started to increase in September when salmon consumption increased. In addition, body condition began to recover earlier in years when consumption of both pine nuts and salmon was high. Furthermore, females with offspring had poorer body condition than solitary females, in particular in late August in years with low salmon consumption. Our findings suggest that coastal and subalpine foods, which are unique to the Shiretoko Peninsula, determine the summer body condition of female brown bears, as well as their survival and reproductive success., To clarify seasonal and annual fluctuation in the body condition of adult female brown bears in relation to food habits and reproductive status, we conducted a 7‐year longitudinal study that included scat sampling and direct observation of bears in a special wildlife protection area on the Shiretoko Peninsula. Using photographic evaluation to assess the body condition of free‐ranging brown bears, we noninvasively monitored the body condition of identifiable bears throughout the study period. Our findings suggest that coastal and subalpine foods, which are unique to the Shiretoko Peninsula, determine the summer body condition of female brown bears as well as their survival and reproductive success.

Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.

Human habituation of large carnivores is becoming a serious problem that generates human–wildlife conflict, which often results in the removal of animals as nuisances. Although never tested, human habituation potentially reduces the fitness of adult females by reducing their offspring’s survival as well as their own, due to an increased likelihood of human-caused mortality. Here, we tested this hypothesis in brown bears inhabiting Shiretoko National Park, Japan. We estimated the frequency of human-caused mortality of independent young (aged 1–4 years) born to mothers living in areas with different maternal levels of human habituation and different proximities to areas of human activity. The overall mortality rate was higher in males than in females, and in females living near a town than those in a remote area of park. Surprisingly, more than 70% of males born to highly habituated mothers living around a remote wildlife protection area were killed by humans; this proportion is greater than that for males born to less-habituated mothers living in almost the same area. The current study clarified that interactions among maternal human habituation, birthplace (proximity to town), age, and sex determine the likelihood of human-caused mortality of brown bears at an early stage of life.

Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi, Babesia sp. UR2-like group, Cytauxzoon sp. UR1, Babesia sp. UR1, and Babesia microti) were detected from bear blood DNA samples. B. microti was detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens, H. ursi, Babesia sp. UR2-like pathogens, and Cytauxzoon sp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence of H. ursi were significantly higher in Japanese black bear (0% vs 96.4%) while that of Babesia sp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence of Babesia sp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.

Male reproductive behavior has not been thoroughly investigated in large, non-social mammals. We studied male reproductive input using field observations and microsatellite DNA data for the brown bear (Ursus arctos yesoensis Lydekker, 1897) population in a special wildlife protection area on the Shiretoko Peninsula in Hokkaido, Japan. We identified the father of 82 offspring born between 2006 and 2018 from 54 litters produced by 20 females and revealed that they were sired by a total of 19 males. Overall, paternity was distributed relatively evenly among different males, although litter production tended to be skewed to a few bears in some years. Male breeding tenure varied among individuals, but most tenures were within the range of 1 to 6 years. Human-caused mortality, mostly occurring near human residential areas, induced male turnover, indicating that the special wildlife protection area located in the inner part of the Shiretoko Peninsula is not free from human impact. The frequency of successful reproduction was highest in 10- to 14-year-old bears, which is consistent with the period when males reach physical maturity. These results contribute to an understanding of breeding systems in large solitary mammals and to appropriate conservation and management strategies for brown bear populations.

Mammals around the world face serious threats and experience massive decline in their populations and geographic ranges. The sloth bear has become extinct from two of five range countries and the existing population face serious conservation threats. Despite being endangered in Nepal, decades of conservation obliviousness have hindered their conservation and management. We assessed the distribution and habitat use by sloth bears in relation to landscape, environmental, and anthropogenic factors in Chitwan National Park, Nepal. We conducted a sign survey from March-June 2020 in 4*4 km2 grids (n=45) in a checkerboard approach. An observation was spatially replicated four times by recording information in four continuous search trails of 1 km each. We recorded presence/absence of sloth bears, termites, fruits and disturbance in the field and extracted information on tree cover, terrain ruggedness and vegetation index from remotely sensed image of the study area. The data was analyzed using single species-single season occupancy method in PRESENCE software. By using repeated observations these models account for imperfect detectability of species to provide robust estimates of species occupancy. Averaged habitat occupancy estimate obtained from the best models was Ѱ = 0.53 ± 0.04SE with detection probability p = 0.44 ± 0.1SE. Selection of variable based on summed AIC weight from the top models (AIC

This report describes the genome sequences of two Mycobacterium tuberculosis isolates, S1 and S3, recovered from Asian elephants in Nepal. These genome sequences will enhance our understanding of the genomic epidemiology of Mycobacterium tuberculosis in Asian elephants.

Natal dispersal likely plays an important role in avoiding inbreeding among large carnivores. We tested the hypothesis that male-biased dispersal reduces close inbreeding by limiting the spatial overlap of opposite-sex pairs of close relatives in brown bears (Ursus arctos) in the Shiretoko Peninsula, Hokkaido, Japan. We genotyped 837 individuals collected in 1998–2017 at 21 microsatellite loci and performed parentage analysis. To calculate natal dispersal distance, we considered the site where the mother was identified as the birthplace of her offspring, and the site where the offspring were identified as their dispersed place. As predicted, we found that dispersal distances were significantly greater for males (12.4 km ± 1.0) than for females (7.7 km ± 0.9), and those for males increased from 3 years old, indicating that males begin to disperse around the time sexual maturation begins. Relatedness decreased with distance among pairs of females, and the mean relatedness was significantly higher between pairs of females than between pairs of males or between female–male pairs within 3 km. Closely related female–male pairs rarely (5–6%) resided in close proximity (< 3 km), compared with pairs of closely related females. Our study revealed that the potential for close inbreeding was low in Hokkaido brown bears because males are effective dispersers.

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear

Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and β-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The β-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the β-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

In Japan, there are concerns that invasive alien raccoons prey on rare native species during their spawning season from late winter to early summer. We investigated raccoon predation impact by examining the predation presence using DNA metabarcoding and extent of predation on rare native species using stable isotope analysis. We captured raccoons in Hokkaido, Japan from April to August in 2018 and 2019. We analysed raccoon faeces and gastric contents by DNA metabarcoding to detect the rare native Hokkaido salamander and Japanese crayfish. Hokkaido salamanders were detected from gastric contents, but Japanese crayfish were not detected in any samples. Stable isotope analysis of raccoon muscle samples and the Bayesian mixing model were used to estimate each food resource’s contribution to the raccoon diet. Animal food resources accounted for 70% of total consumed food. The foraging ratios of amphibians and crustaceans were about 9% and 5%, respectively. Raccoons have been found to use amphibians at a higher rate than previously reported, including a rare endangered species, the Hokkaido salamander. Hokkaido salamander and Japanese crayfish spawn in the spring, and increased predation pressure by raccoons may directly impact populations of these rare native species.

Piroplasms, which include

Hair biomarkers have been widely used to study wildlife physiology and ecology. To apply this technique to practical studies, detailed information on hair growth is needed for each study species. This study measured hair growth patterns, including growth rate, periods of growth and shedding, and hair bulb structural changes in the brown bear. Hair growth was examined monthly in captive bears, and gross observations of fur were performed in wild bears. Guard hairs grew from late April to early October at an approximate rate of 0.5 mm/day for captive bears, and from late May to late September at an approximate rate of 0.7 mm/day for wild bears. Underfur began growing in August and continued into winter. The growth periods for intermediate hairs began at different times and lasted 3–5 months, depending on individuals. Most shedding was complete by August, and all shedding was complete by the end of September. Hair bulbs were classified into three types: white spheres (WS-type), black hooks (BH-type), and white hooks (WH-type). This classification can be used to discriminate between previous year's hair (WS-type) and current year's hair (BH-type or WH-type). The current findings will enhance the understanding of brown bear ecology in the wild.

Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp.

Bears do not suffer from osteoporosis during hibernation, which is associated with long-term inactivity, lack of food intake, and cold exposure. However, the mechanisms involved in bone loss prevention have scarcely been elucidated in bears. We investigated the effect of serum from hibernating Japanese black bears (Ursus thibetanus japonicus) on differentiation of peripheral blood mononuclear cells (PBMCs) to osteoclasts (OCs). PBMCs collected from 3 bears were separately cultured with 10% serum of 4 active and 4 hibernating bears (each individual serum type was assessed separately by a bear PBMCs), and differentiation were induced by treatment with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). PBMCs that were cultured with the active bear serum containing medium (ABSM) differentiated to multi-nucleated OCs, and were positive for TRAP stain. However, cells supplemented with hibernating bear serum containing medium (HBSM) failed to form OCs, and showed significantly lower TRAP stain (p< 0.001). On the other hand, HBSM induced proliferation of adipose derived mesenchymal stem cells (ADSCs) similarly to ABSM (p> 0.05), indicating no difference on cell growth. It was revealed that osteoclastogenesis of PBMCs is hindered by HBSM, implying an underlying mechanism for the suppressed bone resorption during hibernation in bears. In addition, this study for the first time showed the formation of bears' OCsin-vitro.

Hibernating bears survive up to 6 months without feeding while yet maintaining metabolic homeostasis. We previously reported expression changes in energy metabolism-related genes in the liver of hibernating Japanese black bears. The present study examined the role of microRNAs in the regulation of hepatic gene expression during hibernation. The quantitative analyses revealed significant increases in the expression of 4 microRNAs (miR-221-3p, miR-222-3p, miR-455-3p, and miR-195a-5p) and decreases of 2 microRNAs (miR-122-5p and miR-7a-1-5p) during hibernation. RNA sequencing and in silico target prediction regarding 3 upregulated microRNAs (miR-221-3p, miR-222-3p and miR-455-3p) found 13 target mRNAs with significantly decreased expression during hibernation. The transfection of microRNA mimics into cells showed that miR-222 and miR-455 reduced solute carrier family 16 member 4 (SLC16A4) and fatty acid synthase (FASN) mRNA expression, respectively. Our results suggest that the increased levels of hepatic miRNA during hibernation (miR-222-3p and miR-455-3p) negatively regulate the expression of targeted genes predicted to be involved in the transport of energy source and de novo fatty acid synthesis, is consistent with a regulatory role of these miRNAs in energy metabolism in hibernating black bears.

Understanding the breeding ecology of a species is essential for the appropriate conservation and management of wildlife. In brown bears, females occasionally copulate with multiple males in one breeding season, which may lead to multiple paternity in a single litter. In contrast, inbreeding, a potential factor in the reduction of genetic diversity, may occur, particularly in threatened populations. However, few studies have reported the frequency of these phenomena in brown bear populations. Here, we investigated the incidence of multiple paternity and inbreeding in a high-density brown bear population on the Shiretoko Peninsula in Hokkaido, Japan. A total of 837 individuals collected from 1998 to 2017 were genotyped at 21 microsatellite loci, and parentage analysis was performed. Out of 70-82 litters with ≥2 offspring, 14.6-17.1% of litters were sired by multiple males. This was comparable to the rate reported in a Scandinavian population, although population density and litter size, factors that potentially affect the incidence of multiple paternity, differed between the 2 populations. Out of 222 mother-father mating pairs, 6 litters (2.7%) resulted from matings between fathers and daughters. Additionally, 1 (0.5%) and 4 (1.8%) cases of mating between maternal half-siblings and between paternal half-siblings, respectively, were observed; however, no cases of mating between mothers and sons or between full siblings were observed. Our results suggest that male-biased natal dispersal effectively limits mating between closely related individuals (aside from fathers and daughters) in brown bears.

In the testes of the Sunda porcupine (Hystrix javanica), the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes, such as cytochrome P450 side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom), was immunohistochemically examined to clarify the location of steroidogenesis. In this study, complete spermatogenesis (spermiogenesis) was observed in the testes of the examined Sunda porcupine, and spermatozoa of the Sunda porcupine had a spatulate sperm head unlike that of rats and mice which has an apical hook. On immunostaining of StAR, P450scc, 3β-HSD, P450c17 and P450arom, immunoreactivity for all proteins was only detected in the Leydig cells and not observed within the seminiferous tubules, suggesting that the Leydig cells can synthesize both androgen and estrogen from cholesterol in the Sunda porcupine testes.

Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.

Phylogeographic studies can resolve relationships between genetic population structure of organisms and geographical distributions. Raccoons have become feral in Japan, and in Hokkaido island, they have been rapidly increasing in number and spreading since the 1970s. We analyzed mitochondrial (mtDNA) and microsatellite DNA to understand the current phylogenetic distribution and invasive founder events. Overall, Hokkaido raccoons maintained high genetic diversity (i.e., the level of heterozygosity was comparable to the original habitat, North America). Based on mtDNA distribution and microsatellite diversity, Hokkaido raccoons were divided into six management units. However, mtDNA haplotype distributions and genetic structures based on microsatellites did not always correspond to each other (e.g., two geographically and genetically separated populations showed similar mtDNA distributions). In addition, a high degree of genetic admixture was observed in every unit, and the degree of genetic differentiation was low even between regions separated by long distances. Compared with other countries in Europe where genetic distribution of introduced raccoons is more clearly structured, the current results represent a unique and complex phenomenon of pet escape/abandonment in Hokkaido: i.e., genetically related colonies were introduced into multiple regions as founder events, resulting in the current state in which raccoons are not clearly genetically differentiated even 40 years after introduction.

In the testes of the Sunda porcupine (Hystrix javanica), the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes, such as cytochrome P450 side chain cleavage (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 17α-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom), was immunohistochemically examined to clarify the location of steroidogenesis. In this study, complete spermatogenesis (spermiogenesis) was observed in the testes of the examined Sunda porcupine, and spermatozoa of the Sunda porcupine had a spatulate sperm head unlike that of rats and mice which has an apical hook. On immunostaining of StAR, P450scc, 3β-HSD, P450c17 and P450arom, immunoreactivity for all proteins was only detected in the Leydig cells and not observed within the seminiferous tubules, suggesting that the Leydig cells can synthesize both androgen and estrogen from cholesterol in the Sunda porcupine testes.

The reproductive tracts of 130 adult female brown bears (Ursus arctos) in Hokkaido, Japan, euthanized during 2010–2013 for conflict management, were examined for the presence and number of corpora lutea (CL) in the ovary and placental scars (PS) in the uterine horns. We scored PS on a 0–5 scale based on morphological features including their rim and color. They were characterized as representing either current-year parturition, as determined by accompanying cubs, or previous-year parturition, as determined by accompanying yearlings. In total, 128 females were divided into 3 reproductive categories: 44 females with current-year parturition, 39 with previous-year parturition, and 45 solitary females that had not given birth for >2 years. The estimated ovulation rate was 2.21 ± 0.55 (mean ± SD), the implantation rate was 1.91 ± 0.74, and the litter size was 1.54 ± 0.51. Implantation rates were lower than ovulation rates and changed annually during 2010–2013. Systematic observation of PS and CL allow...

Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period.