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Hunting, habitat loss and fragmentation have caused a rapid decline in the distribution and abundance of the clouded leopard Neofelis nebulosa across its range, and in several areas, including Taiwan, the species is now extirpated. Taiwan, a former stronghold for the species, is a candidate for its reintroduction, based on increasing prey abundance and high forest coverage. Such future reintroduction efforts, however, are hampered by a lack of analysis of potential clouded leopard habitat on the island. To address this, we explore habitat suitability for the species in Taiwan. We used a multi-scale, multivariate habitat selection model based on clouded leopard presence–absence data from extensive camera-trap surveys across its current range to predict suitable habitat in Taiwan. Our findings indicate that 38% of Taiwanese territory is potentially suitable habitat for the clouded leopard, of which 46% is under protection. This demonstrates the high potential of Taiwan's habitat for clouded leopard reintroduction.

Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia. Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy. Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4 × 10(-5)). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice. Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.

Context Elucidating how demography and contemporary landscape features regulate functional connectivity is crucial to implementing effective conservation strategies. We assessed the impacts of landscape features on the genetic variation of a locally endangered carnivore, the leopard cat ( Prionailurus bengalensis ) in Taiwan. Objectives We aim to evaluate the association between genetic structure and landscape features. We further predicted the changes in genetic diversity and suitable habitats in the future. Methods We genotyped 184 leopard cats in western Taiwan using 12 nuclear microsatellites and a mitochondrial marker. We applied a landscape optimization procedure with two genetic distances to identify major genetic barriers and employed ecological niche modeling to predict the future distribution of the leopard cat. Results Bayesian demographic inferences revealed a dramatic population decline for all leopard cat populations in Taiwan. Genetic clustering and resistance surface modeling supported that the population connectivity was influenced by highways and high elevation. Niche modeling indicated low temperature was one of the primary factors limiting the occurrence of leopard cats that may inhibit their movement in high elevations. We predicted the suitable habitats of leopard cats would shrink northward and towards higher altitudes with rugged topography in response to global warming. Conclusions Our study provided genetic evidence that leopard cats in Taiwan had undergone a dramatic population decline that may be associated with anthropogenic impacts. We also inferred the anthropogenic linear feature compromised the connectivity and persistence of leopard cats in human-mediated landscapes. Our finding serves as a model for landscape genetic studies of island carnivores in subtropical regions.

Among the five species of the Mustelidae found in Taiwan, four terrestrial species are potentially sympatric. These species include yellow‐throated marten Martes flavigula, Siberian weasel Mustela sibirica, Taiwan least weasel Mustela nivalis formosana, and Chinese ferret‐badger Melogale moschata. Scat from the first three mustelids are similar in morphology. Therefore, differentiating the scats of these three species is challenging, and without accurate species identification, scatology‐related techniques such as dietary analysis are problematic. Here, we developed a novel set of primers capable of effectively identifying scat samples of these three species through a simple PCR amplification process. This allows for collection of more accurate field data on these species, enabling a better understanding of their ecological niche and basic biological information for future conservation strategies.

Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR). A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.

During 1997–2012 we conducted a nationwide camera-trapping survey and assessed the availability of prey and habitat for the clouded leopard Neofelis nebulosa in Taiwan. We surveyed 1,249 camera-trap sites over 113,636 camera-trap days, from the seashore to an altitude of 3,796 m and covering various types of vegetation. No clouded leopards were photographed during 128,394 camera-trap days, including at 209 sites in other studies, confirming the presumed extinction of clouded leopards in Taiwan. Assessment of the prey base revealed altitudinal distribution patterns of prey species and prey biomass. Areas at lower altitudes and with less human encroachment and hunting supported a higher prey biomass and more of the typical prey species of clouded leopards. Habitat analysis revealed 8,523 km2 of suitable habitat but this was reduced to 6,734 km2 when adjacent areas of human encroachment were subtracted. In the absence of hunting and large mammalian carnivores the major prey of clouded leopards in Taiwan, such as Formosan macaques Macaca cyclopis, Reeves's muntjacs Muntiacus reevesi, Formosan serow Capricornis swinhoei and sambar Rusa unicolor, could become over-abundant. Thus, it is important to address the cascading effect of the disappearance of top-down predator control. Our assessment indicated that, with proper regulation of hunting, habitat restoration and corridor improvement, it may be possible to reintroduce the clouded leopard.

Sound management of the Mandarin Duck (Aix galericulata) requires an understanding of its survival and cause-specific mortality, yet these parameters have not been studied in Taiwan. From January 1999 to December 2002, a study was conducted along the Tachia River, in central Taiwan, where the largest population of Mandarin ducks resides. The sex ratio was male-biased for adults (average 2.10:1) but not for juveniles (average 0.92:1). Ducks (34 males and 42 females) were radio-tagged and monitored two to three times per month for 1–47 months. Annual survivorship was 0.80 and 0.44 for male and female ducks, respectively; lower female survival may lead to a male-biased sex ratio. Forty-three ducks died, the greatest proportion from unknown causes, followed by avian predators, mammalian predators and anthropogenic activity. Mortality occurred year-round, but female mortality was greatest from spring to fall, 9–10 carcasses, while male mortality (seven) was greatest in the winter.

Little is known about clouded leopards (Neofelis nebulosa), who have a vulnerable population that extends across southern Asia. We reviewed the literature and synthesized what is known about their ecology and behavior. Much of the published literature either note detections within and on the edges of their range, or are anecdotal observations, many of which are decades if not over a century old. Clouded leopards are a medium-sized felid, with distinctive cloud-shape markings, and notably long canines relative to skull size. Estimates for population densities range from 0.58 to 6.53 individuals per 100 km2. Only 7 clouded leopards have been tracked via radio-collars, and home range estimates range from 33.6-39.7 km2 for females and 35.5-43.5 km2 for males. Most accounts describe clouded leopards as nocturnal, but radio telemetry studies showed that clouded leopards have arrhythmic activity patterns, with highest activity in the morning followed by evening crepuscular hours. There has never been a targeted study of clouded leopard diet, but observations show that they consume a variety of animals, including ungulates, primates, and rodents. We encourage future study of their population density and range to inform conservation efforts, and ecological studies in order to understand the species and its ecological niche.

The EGF signaling system plays a major role in the embryo-uterine dialogue during implantation and heparin-binding EGF-like growth factor (HBEGF) serves a role in coordinating embryonic and endometrial development at the site of blastocyst attachment. Implantation in mutant mice lacking HBEGF is delayed, but partially rescued by compensatory upregulation of amphiregulin. As implantation proceeds, HBEGF stimulates trophoblast differentiation and invasive activity essential for placentation. Therefore, we investigated the effects of embryonic HBEGF deficiency on placental development by breeding mice heterozygous for the HBEGF null mutation. Placentas and fetuses were removed on gestation day 14 and weighed. Fetuses were assayed to establish the genotype of corresponding placentas. Placental discs were halved, fixed and embedded in paraffin for sectioning. Central cross sections were deparaffinized for hematoxylin and eosin staining, immunohistochemistry or cell death (TUNEL) assay. Image analysis was used for semiquantification of immunohistochemical staining intensity. Weights of wild type (77.4 ± 3.5 mg; N=12) and heterozygous (76.5 ± 19.3 mg; N=32) placentas were similar, but homozygous null placentas had reduced average weights (59.8 ± 16.0 mg; N=21, p<0.01). Average fetal weight was not significantly affected by genotype. HBEGF immunostaining was gene dose-dependent and differed significantly among the three groups, with heterozygotes having an intermediate level. Other members of the EGF family, amphigreglin, TGF-alpha and EGF were expressed equally in wild type and homozygous null placentas. Histological examination revealed no apparent differences in trophoblast giant cells, but the spongiotrophoblast region was reduced compared to labyrinth in homozygous null placentas. Morphometric assessment of relative area demonstrated that spongiotrophoblast comprised a smaller (p<0.05) proportion of the cross sectional area in null placentas than in the wild type; however, heterozyotes were intermediate and did not differ significantly from the other two genotypes. There were no differences in apoptosis, but proliferation, assessed by nuclear Ki67 staining, was elevated (p<0.05) in the labyrinth and decreased (p<0.05) in the spongiotrophoblast of HBEGF null placentas compared to the wild type. Labyrinth morphology appeared disrupted in HBEGF null placentas stained with laminin to highlight capillary basement membrane. Indeed, the density of laminin-labeled capillaries was decreased (p<0.05) in HBEGF null placentas compared to the wild type, based on morphometric analysis. Immunohistochemical staining revealed reduced vascular endothelial growth factor (VEGF) in both spongiotrophoblast (p=0.06) and labyrinth (p<0.01) regions of HBEGF null placentas, suggesting that poor angiogenesis was due to reduced VEGF. These findings support an active role for HBEGF in placentation. Production of HBEGF by adjacent maternal tissues could have limited the detrimental effects of embryonic HBEGF deficiency. While there was no clear evidence of aberrant giant cell invasion in placentas lacking HBEGF, roles for HBEGF in spongiotrophoblast formation and fetal angiogenesis within the labyrinth were supported. HBEGF could promote fetal capillary formation by inducing VEGF or other angiogenic factors in the developing placenta. Supported by NIH grant HD12304 and the intramural research program of NICHD.

During 2000-2004 I studied the population status of the Formosan clouded leopard (Neofelis nebulosa brachyurus) and the ecology of its prey and other sympatric carnivores in the largest remaining lowland primary forest in southern Taiwan. My research team and I set up 232 hair snare stations and 377 camera trap sites at altitudes of 150-3,092m in the study area. No clouded leopards were photographed in total 13,354 camera trap days. Hair snares did not trap clouded leopard hairs, either. Assessment of the prey base and available habitat indicated that prey depletion and habitat loss, plus historical pelt trade, were likely the major causes of extinction of clouded leopards in Taiwan. Using zero-inflated count models to analyze distribution and occurrence patterns of Formosan macaques (Macaca cyclopis) and 4 ungulates, we found habitat segregation among these 5 herbivore species. Formosan macaques, Reeve's muntjacs (Muntiacus reevesi micrurus), and Formosan serows (Nemorhaedus swinhoei) likely were the most important prey species of Formosan clouded leopards given their body size and high occurrence rates in lower altitudes. In contrast, sambar deer (Cervus unicolor swinhoii) tended to occur more frequently as altitude increased. Formosan macaques exhibited seasonal differences in occurrence rates and were absent at altitudes > 2,500m in winter. Only Formosan serows showed preference for cliffs and rugged terrain, while the other 4 species, except wild boars (Sus scrofa taivanus), avoided these areas. Habitat segregation in forest understory and structure were more pronounced among the 4 ungulates. Forest structure rarely affected occurrence rates of Formosan macaques on the ground. Niche relationships of the other sympatric carnivores were studied through habitat, diet, and temporal dimensions. Resource partitioning by carnivores was observed. Altitude was the strongest factor explaining the composition of the carnivore community in the local study-area scale and in the landscape scale across Taiwan. Carnivores could be divided into 2 groups: low-mid altitude consisting of Formosan ferret badgers (Melogale moschata subaurantiaca ), gem-faced palm civets (Paguma larvata taivana), lesser oriental civets (Viverricula indica taivana), crab-eating mongooses (Herpestes urva formosanus), leopard cats ( Prionailurus bengalensis chinensis), and feral cats (Felis catus), and the mid-high altitude group consisting of yellow-throated martens (Martes flavigula chrysospila), Siberian weasels (Mustela sibirica taivana), and Asiatic black bears ( Ursus thibetanus formosanus). Carnivore richness was higher at mid altitudes where these 2 groups overlapped (i.e. mid-domain effect). The low-mid altitude carnivores were more nocturnal and tolerant of human activity and forest alteration except crab-eating mongooses, which were diurnal and avoided human encroachment. Similar to crab-eating mongooses, the mid-high altitude carnivores also avoided human encroachment and were diurnal except for Siberian weasels, which were more nocturnal. Diet summary based on their major food items for all sympatric carnivores revealed 3 groups of foragers which foraged on: invertebrates, small mammals, and plant fruits. Felidae, yellow-throated martens, and Siberian weasels preyed on small mammals. Asiatic black bears and gem-faced palm civets ate mostly plant fruits. The other 3 carnivores were mainly invertebrate foragers. These 9 carnivores partitioned resource uses in the 3 niche dimensions except for some overlap in resource use by leopard cats and feral cats. Prey base for Formosan clouded leopards and the carnivore richness in Taiwan were found to be lower in areas with higher levels of human activity. On the other hand, Formosan macaques and ungulates could become over-abundant without human hunting and top carnivore predation. Mesopredator release may occur because of vanishing top carnivores, causing reduction of the lower trophic level prey species. It is important to assess the cascading impacts of the loss of the Formosan clouded leopards and Eurasian otters ( Lutra lutra chinensis) and the declining Asiatic black bears and to consider reintroduction of Formosan clouded leopards, as well as active management of the other larger mammals. These results provided baseline information for reintroduction of clouded leopards and management of their prey and generated new hypotheses regarding the ecology of these large mammals for future investigation.