Estimation of breeding population size using DNA-based pedigree reconstruction in brown bears.

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 1288 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.

Diel and monthly activity pattern of brown bears and sika deer in the Shiretoko Peninsula, Hokkaido, Japan.

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.

Dining from the coast to the summit: Salmon and pine nuts determine the summer body condition of female brown bears on the Shiretoko Peninsula.

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.

Development of a noninvasive photograph-based method for the evaluation of body condition in free-ranging brown bears.

Body condition is an important determinant of health, and its evaluation has practical applications for the conservation and management of mammals. We developed a noninvasive method that uses photographs to assess the body condition of free-ranging brown bears (Ursus arctos) in the Shiretoko Peninsula, Hokkaido, Japan. First, we weighed and measured 476 bears captured during 1998-2017 and calculated their body condition index (BCI) based on residuals from the regression of body mass against body length. BCI showed seasonal changes and was lower in spring and summer than in autumn. The torso height:body length ratio was strongly correlated with BCI, which suggests that it can be used as an indicator of body condition. Second, we examined the precision of photograph-based measurements using an identifiable bear in the Rusha area, a special wildlife protection area on the peninsula. A total of 220 lateral photographs of this bear were taken September 24-26, 2017, and classified according to bear posture. The torso height:body/torso length ratio was calculated with four measurement methods and compared among bear postures in the photographs. The results showed torso height:horizontal torso length (TH:HTL) to be the indicator that could be applied to photographs of the most diverse postures, and its coefficient of variation for measurements was <5%. In addition, when analyzing photographs of this bear taken from June to October during 2016-2018, TH:HTL was significantly higher in autumn than in spring/summer, which indicates that this ratio reflects seasonal changes in body condition in wild bears. Third, we calculated BCI from actual measurements of seven females captured in the Rusha area and TH:HTL from photographs of the same individuals. We found a significant positive relationship between TH:HTL and BCI, which suggests that the body condition of brown bears can be estimated with high accuracy based on photographs. Our simple and accurate method is useful for monitoring bear body condition repeatedly over the years and contributes to further investigation of the relationships among body condition, food habits, and reproductive success.

Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus).

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.

Maternal human habituation enhances sons' risk of human-caused mortality in a large carnivore, brown bears.

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.

PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Incidence of Multiple Paternity and Inbreeding in High-Density Brown Bear Populations on the Shiretoko Peninsula, Hokkaido, Japan.

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.

Reproductive parameters and cub survival of brown bears in the Rusha area of the Shiretoko Peninsula, Hokkaido, Japan.

Knowing the reproductive characteristics of a species is essential for the appropriate conservation and management of wildlife. In this study, we investigated the demographic parameters, including age of primiparity, litter size, inter-birth interval, reproductive rate, and cub survival rate, of Hokkaido brown bears (Ursus arctos yesoensis) in the Rusha area on the Shiretoko Peninsula, Hokkaido, Japan, based on a long-term, individual-based monitoring survey. A total of 15 philopatric females were observed nearly every year from 2006 to 2016, and these observations were used to estimate reproductive parameters. The mean age of primiparity was 5.3 ± 0.2 (SE) years (n = 7, 95% CI = 5.0-5.6). We observed 81 cubs in 46 litters from 15 bears. Litter size ranged from one to three cubs, and averaged 1.76 ± 0.08 (SE) cubs/litter (95% CI = 1.61-1.91). Inter-birth intervals ranged from 1 to 4 years, and the mean value was estimated as 2.43 (95% CI = 2.16-2.76) and 2.53 (95% CI = 2.26-2.85) years in all litters and in litters that survived at least their first year, respectively. The reproductive rate was estimated from 0.70 to 0.76 young born/year/reproductive adult female, depending on the method of calculation. The cub survival rate between 0.5 and 1.5 years ranged from 60 to 73%. Most cub disappearances occurred in July and August, suggesting that cub mortality is mainly due to poor nutrition in the summer. All reproductive parameters observed in the Rusha area on the Shiretoko Peninsula fell within the range reported in Europe and North America, and were among the lowest or shortest age of primiparity, litter size, and inter-birth intervals, and ranked at a high level for reproductive rate.

Serologic evidence of Brucella infection in pinnipeds along the coast of Hokkaido, the northernmost main island of Japan.

Brucella infection in Hokkaido was serologically surveyed in four species of pinnipeds inhabiting Cape Erimo during 2008-2013 and the Shiretoko Peninsula in 1999 by ELISA using Brucella abortus and B. canis as antigens. Anti-Brucella positive sera showed higher absorbance to B. abortus than B. canis in almost all samples. Anti-B. abortus antibodies were detected in serum samples from 24% (n = 55) of Western Pacific harbor seals (Phoca vitulina stejnegeri) in Cape Erimo and from 66% (n = 41) of spotted seals (P. largha), 15% (n = 20) of ribbon seals (Histriophoca fasciata) and 18% (n = 17) of Western Steller's sea lions (Eumetopias jubatus jubatus) in the Shiretoko Peninsula. Anti-Brucella antibodies were detected at higher absorbance in 1- to 4-year-old harbor seals than in the pups and mature animals, suggesting either that Brucella infection mainly occurs after weaning or that it is maternally transmitted to pups with premature or suppressed immunity. Anti-Brucella antibodies were detected in both immature and mature spotted seals and ribbon seals, with higher absorbance in the former. The antibodies were detected only in mature Western Steller's sea lions. Western blot analysis of the serum samples showed some differences in band appearances, namely discrete versus smeary, and in the number of bands, indicating that multiple different Brucella may be prevalent in pinnipeds in Hokkaido. Alternatively, the Brucella of pinnipeds may have some intra-species diversity.

Maternal-to-fetal transfer and concentration profiles of PCB congeners for Steller sea lions (Eumetopias jubatus) from Hokkaido, Japan.

The concentrations of PCB congeners in the blubber and liver of mother Steller sea lions (Eumetopias jubatus; SSLs) and their fetuses from the coast of Hokkaido, Japan in 2008, 2010 and 2012 were analyzed by HRGC-HRMS, in order to elucidate PCB congener profiles and maternal-to-fetal transfer of PCBs in SSLs. ΣPCBs in the fetuses were 1400 ± 660 (the mean ± SD) ng/g-fat in the blubber and 570 ± 320 ng/g-fat in the liver, respectively. There was a concern that SSLs had been contaminated by PCBs during the fetal period. The concentrations of the major congeners in the blubber and liver were a correlation between the fetus and mother (blubber: r=0.9934, liver: r=0.9160; P ≦ 0.05). The results indicated that PCBs in the fetuses came from the mothers. #177 and #199 showed no correlations between the fetus and the mother in the blubber and liver. This indicated a selective capture by some natural protector such as the placenta.

Sex determination of sika deer (Cervus nippon yesoensis) using nested PCR from feces collected in the field.

We describe a method for determining the sex of sika deer (Cervus nippon yesoensis) from feces collected in the field. Using a nested polymerase chain reaction (nested PCR), partial sequences of the sex determination region of the Y chromosome (SRY) gene and X zinc finger protein (ZFX) gene were amplified. In 19 individuals with sex information, the correct sex was successfully detected and sequences of target amplicons were completely matched between muscle and feces from the rectum. Among 75 fecal samples collected noninvasively in the field, 68-71 samples (90.7-94.7%) yielded successful sex determinations. Using this technique, feces collected in the field would enhance the utility of genetic analysis. As a result, instead of biomaterials, these samples can serve as new or alternative materials. Finally, it can be expected that this technique will contribute to reveal in advanced detail of the population dynamics and genetic diversity that needed to carry out effective population control and to reduce the extinction risk of sika deer.

Flow cytometric analysis of canine umbilical cord blood lymphocytes.

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.

Optimum dose of LH-RH analogue Fertirelin Acetate for the induction of superovulation in mice.

The optimum dose for establishing superovulation in mice of Fertirelin Acetate (FA), an LH-RH analogue, was examined. Mice were subcutaneously injected with 5 IU of hCG at 17:00 (Day 0), and with various doses of FA (0.001 to 1.0 microg) five times at 4 h intervals on and after 22:00 on Day 0. To induce ovulation, 5 IU of hCG was again injected subcutaneously at 17:00 on Day 2. In the groups administered with doses ranging from 0.01 to 0.5 microg of FA, the number of ovulated eggs was significantly (p<0.05) larger than in the control group (12.9 +/- 5.9). The greatest number of ovulated eggs (22.6 +/- 7.3) was obtained in the group administered with 0.025 microg of FA. The results indicate that the effective dose of LH-RH analogue, FA, is between 0.1 and 0.5 microg for superovulation induction in mice.

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy.

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.