Analysis of Crop Consumption Using Scatological Samples from the Red-Crowned Crane Grus japonensis in Eastern Hokkaido, Japan.

Total DNA extracts from the intestinal contents of 60 flying red-crowned cranes (juveniles, subadults and adults) found dead in 2006-2021, and the feces of 25 chicks collected in June and July of 2016-2018, were used for PCR reactions with primers specific for 16 crops, followed by high-throughput sequencing. The most predominant crop detected was corn in adult and subadult cranes (61.7%). Other grains (barley, wheat, soybean) (5.0-8.3%) and vegetables (tomatoes, Chinese cabbage, etc.) (1.7-6.7%) were also detected in flying cranes. Surprisingly, some of the detected crops were not grown in the Kushiro and Nemuro regions. There was no significant difference in crop intake status in winter and that in other seasons for most of the crops. Corn (28.0%), soybeans (8.0%), wheat and beet (4.0%) were detected in crane chicks in summer, though the detection rates were generally lower than those in flying cranes. Alfalfa, which is not grown in eastern Hokkaido but is used in some cattle feed, was detected in some cranes. Rice, buckwheat, adzuki beans, common beans, potatoes and carrots were not detected at any life stage, indicating the preferences of red-crowned cranes. The results suggest that red-crowned cranes in Hokkaido are dependent on dairy farmers for their feed supply.

Metabarcoding of feces and intestinal contents to determine carnivorous diets in red-crowned cranes in eastern Hokkaido, Japan.

The red-crowned crane Grus japonensis in Hokkaido, Japan forms a closed population as a residence that is independent of the mainland population. Based on observations of a limited number of individuals as well as cranes in captivity, red-crowned cranes are omnivores and eat fish, worms, insects and plants in their own territories except in winter, when they are fed with dent corn that is supplied in eastern Hokkaido. DNA metabarcoding based on high throughput sequencing was carried out using universal primer sets for cytochrome oxidase subunit I gene. Feces from 27 chicks collected in June and July in the period from 2016 to 2018 and intestinal contents from 33 adult and subadult cranes that were found dead almost throughout year in 2006-2013 in the field in eastern Hokkaido were used. Although compositions varied considerably in the cranes, both insects and fish were found in adults and subadults to the same extents, while insects were predominant in chicks. Both insects and fish were detected in all seasons for adults and subadults. Horse flies, scarab beetles and weevils accounted for the most of the insects regardless of the life stage. Dace, stickleback, flatfish and sculpin were the major fish species in adults, while chicks ate almost only stickleback. The results provide the first comprehensive data on carnivorous diets in wild red-crowned cranes in eastern Hokkaido as basis for conservation of red-crowned cranes, for which the life style and area continue to change.

Middle upper beak fracture in a Red-crowned crane that completely recovered with external skeletal fixation.

An adult Red-crowned crane in captivity that had a displaced compound fracture of the middle upper beak caused by an accident was treated by using Type I-a external skeletal fixator (ESF). The ESF that was equipped with a unilateral epoxy putty fixator and with five half-pins was fixed on the premaxilla bone. The crane with the ESF on the beak was able to feed smoothly by itself. The ESF was removed 49 days after the first surgery. Beak malocclusion, which was observed in the latter half period of fixing, spontaneously improved within one month after removal of the ESF. This is the first successful case of repair of an upper beak fracture in a Red-crowned crane.

Hepatic transcriptional profile and tissue distribution of cytochrome P450 1-3 genes in the red-crowned crane Grus japonensis.

The endangered red-crowned crane (Grus japonensis) is a protected species in eastern Hokkaido and injured specimens are treated with medication. The present study aimed at understanding the expression profiles of cytochrome P450 (CYP) 1-3 genes in red-crowned crane tissues. We used 14 individuals found dead in the wild in eastern Hokkaido or in Kushiro City Zoo. Nine CYP1-3 genes expressed in the liver of the red-crowned crane were identified by high-throughput sequencing, and phylogenetically classified as CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2U1, CYP2AC1, CYP3A37, and CYP3A80. Based on the quantitative real-time PCR of 13 samples, the rank order of their median expression levels was as follows: CYP3A37 > CYP2AC1 > CYP2C45 > CYP2D49 > CYP2G19 > CYP1A5 > CYP3A80 > CYP2C23. The tissue distribution of the CYP transcripts indicated that many of the CYP1-3 genes examined were mainly expressed in the tissues where drug metabolism occurs, such as the liver, kidneys, and lungs. We found that CYP3A37 was dominant at the transcript level in the liver, indicating it might play a crucial role in liver physiology and xenobiotic metabolism. Similarly, an "orphan" CYP2AC1 was expressed at relatively high levels in the kidneys and liver, suggesting a possible role in renal and liver physiology and xenobiotic metabolism. Our results establish a foundation for future studies on red-crowned cranes aiming to further understand drug sensitivity and develop medication protocols, but also contribute to national and local projects for the conservation of red-crowned crane.

Physical body parameters of red-crowned cranes Grus japonensis by sex and life stage in eastern Hokkaido, Japan.

Red-crowned (or Japanese) cranes Grus japonensis are native to eastern Hokkaido, Japan--the island population, and mainland Asia--the continental population that migrates from breeding grounds along the Amur River Basin to winter in east China and the Korean Peninsula. The island population was reduced to about 50-60 birds in early part of the 20th century. Since 1950s, the population has increased to more than 1,300 as a consequence of human-provided food in winter, resulted in change of their habitats and food resource. From the carcasses of 284 wild cranes from the island population, collected in Hokkaido since 1976 until 2010, we measured six physical parameters (body weight and lengths of body, wing, tarsus, tail and exposed culmen) and divided into groups by sex and three developmental stages (juvenile, yearling and adult). All parameters of males were larger than those of females at the same stage. Total body length of females tends to grow up earlier than males, in contrast to body weight. Obvious time trends were not observed in these all parameters during 34 years for these six categories measured, except total length of male juveniles, which showed a significant increasing trend. These results provide the first extensive data on body size and mass in the wild red-crowned cranes.

Intestinal undifferentiated carcinoma in a red-crowned crane (Grus japonensis).

A 33-year-old red-crowned crane (Grus japonensis) had a diffuse intestinal thickening from the duodenum to colon. Microscopically, neoplastic cells were arranged in sheets and occasionally nests or cords without gland or squamous differentiation. Metastatic tumor cells were found in the lungs, heart, kidneys and adrenal glands. Immunohistochemically, the neoplastic cells were strongly positive for pan-cytokeratin and cytokeratin 8 and 18 and only partly positive for E-cadherin antibodies. Immunostaining for CD3 was positive in normal lymphocytes, and NSE was also positive in normal nerve fibers. But, the neoplastic cells were not immunoreactive to CD3 and NSE. Based on the histological features and the epithelial characteristics in the immunohistochemical stain, the present case was diagnosed as undifferentiated carcinoma originating from the intestine. Interestingly, the neoplastic cells showed a unique growth pattern; they never invaded the submucosa or muscularis throughout the intestine, whereas they spread lymphogenously or hematogenously to other organs.

Mycobacterium ulcerans infection in an Indian flap-shelled turtle (Lissemys punctata punctata).

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.