Manabu Onuma

Molecular Phylogeography of the Red Deer (Cervus elaphus) Populations in Xinjiang of China: Comparison with other Asian, European, and North American Populations

Abstract To illustrate phylogeography of red deer (Cervus elaphus) populations of Xinjiang, we determined their mitochondrial DNA (mtDNA) control region sequences, and then investigated geographic variations and phylogenetic relationships between Xinjiang populations and other populations from Asia, Europe, and North America. The C. elaphus mtDNA control region shared different copy numbers of tandem repeats of 38 to 43-bp motifs which clearly distinguished the Western lineage from the Eastern lineage of this species in Eurasia. The western lineage comprised the Tarim populations from southern Xinjiang and the European populations, all of which had four copies of the motifs. By contrast, the Eastern lineage consisted of populations from northern Xinjiang (Tianshan and Altai Mountains), other Asian areas (Alashan, Gansu, Tibet, Mongolia, and northeastern China), and North America, all of which shared six copies of the motifs. MtDNA phylogenetic trees showed that there are two major clusters of haplotypes which referred to the Western and Eastern lineages, and that subgroupings of haplotypes in each cluster were congruent with their geographic distributions. The present study revealed that a boundary separating the Western lineage from the Eastern lineage occurs between Tarim Basin and Tianshan Mountains in Xinjiang. Meanwhile, North American populations were genetically closer to those of northern Xinjiang, northeastern China, and Mongolia, supporting that C. elaphus immigrated from northeastern Eurasia to North America through the glacier-induced land-bridge (Beringia) which had formed between the two continents after Late Pleistocene.

Characterization of Highly Pathogenic Avian Influenza Virus A(H5N6), Japan, November 2016.

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Population Genetic Structure of the Spotted Seal Phoca largha along the Coast of Hokkaido, Based on Mitochondrial DNA Sequences

Abstract Population genetic structure of the spotted seal, Phoca largha, along coastal regions of Hokkaido was investigated, using mitochondrial DNA (mtDNA) sequences. A 571-bp fragment of the mtDNA control region and adjacent threonine and proline transfer RNA genes was sequenced from 66 seals. We categorized all individuals into three groups considering sampling area and season: twenty-four seals from the Sea of Okhotsk in winter, 11 seals from the Sea of Okhotsk coast in fall, and 31 seals from the Sea of Japan coast in winter. From the 66 animals, 57 haplotypes were identified. Compared with the harbor seal sequence, all spotted seals examined shared two deletions in the control region, which distinguished between the two species. Forty-nine haplotypes were represented by a single individual, and haplotypes shared by more than two animals were generally restricted to same sampling-groups. Phylogenetic trees did not indicate clear geographic differences among the three groups. An Analysis of molecular variances (AMOVA) did not showed any significant population genetic structure in Hokkaido spotted seals (Φst = −0.003). Our results showed a high level of diversity but no genetic structure, and did not deny the possibility that seals in the Okhotsk breeding concentration mainly stayed in the fall Okhotsk and also inhabited in the winter Sea of Japan.

Seasonal fluctuations of body condition in northern sika deer on Hokkaido Island, Japan

We analyzed seasonal and sexual fluctuations in kidney mass (KM) and kidney fat mass (KFM) as indices of condition in Hokkaido sika deerCervus nippon yesoensis Heude, 1884. For 76 male and 132 female sika deer, seasonal fluctuations in KM and KFM were given by fitted sine wave growth curves. Although the kidney fat index (KFI) is used frequently to evaluate animal condition, we reject it because it is based on the assumption that kidney mass is proportional to body mass in all seasons. Our data did not support this assumption. KFM is a better indicator of Hokkaido sika deer condition than KFI. Although sex-based differences in cervid KFM are said to reflect differences in reproductive cycles, the seasonal similarities in sika deer KFM levels may represent adaptations to the long severe Hokkaido winter. Because in our study deer populations were at low densities and had high pregnancy rates, our sine wave growth models can be regarded as reference for fat level fluctuations in Hokkaido sika deer.

Sperm Preservation by Freeze-Drying for the Conservation of Wild Animals

Sperm preservation is a useful technique for the maintenance of biological resources in experimental and domestic animals, and in wild animals. A new preservation method has been developed that enables sperm to be stored for a long time in a refrigerator at 4°C. Sperm are freeze-dried in a solution containing 10 mM Tris and 1 mM EDTA. Using this method, liquid nitrogen is not required for the storage and transportation of sperm. We demonstrate that chimpanzee, giraffe, jaguar, weasel and the long-haired rat sperm remain viable after freeze-drying. In all species, pronuclei were formed after the injection of freeze-dried sperm into the mouse oocytes. Although preliminary, these results may be useful for the future establishment of “freeze-drying zoo” to conserve wild animals.

Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata)

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

STEATITIS IN EGRETS AND HERONS FROM JAPAN

More than 70 egrets and herons were found sick or dead at an agricultural water reservoir in Kanagawa Prefecture, Japan between September and October 2008. The birds showed weakness, lethargy, and inability to fly before death. Postmortem findings included large amounts of firm subcutaneous and cavitary fat comprised of necrotic adipose tissues with infiltrates of heterophils and macrophages. The birds were diagnosed with steatitis on the basis of the gross lesions and histopathology. Egrets with steatitis had low blood levels of vitamin E. High counts of cyanobacteria (Microcystis aeruginosa) were found in the reservoir concurrent with the outbreak of steatitis. No microcystin was detected in the reservoir water or the livers from the egrets. This is the first report of steatitis in wild birds in Japan.

Significant relationships between the serum leptin concentration and the conventional fat reserve indices in a wildlife species, Hokkaido sika deer (Cervus nippon yesoensis)

We assayed the serum leptin concentration and determined its correlation with the kidney fat mass (KFM), kidney fat index (KFI), and femur marrow fat (FMF) indices in 12 Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884) collected in April, June, and October 2000. The KFM and KFI in April and June were both significantly lower than those in October. The leptin concentrations in April and June and in October were 1.222–1.521 ng mL–1 HE (Human Equivalent) units and 1.676–2.63 ng mL–1 HE, respectively, without overlapping. The relationships between the leptin concentration and the KFM, KFI, and FMF were all significant. Therefore, we conclude that the serum leptin concentration can be used as a new index for fat reserves in Hokkaido sika deer. However, reliable results could not be obtained from four samples collected in April and June when the deer were in a lower nutritional status. This may be due to the limited sensitivity of the antisera in the commercial multi-species leptin RIA kit used. For practical use in wildlife biology, species-specific leptin assays should be developed to eliminate this problem.

Effects of environmental radiation on testes and spermatogenesis in wild large Japanese field mice (Apodemus speciosus) from Fukushima.

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident that occurred after the Great East Japan Earthquake in March 2011 released large quantities of radionuclides to the environment. The long-term effects of radioactive cesium (Cs) on biota are of particular concern. We investigated the accumulation of radioactive Cs derived from the FDNPP accident, and chronic effects of environmental radionuclides on male reproduction, in the large Japanese field mouse (Apodemus speciosus). In 2013 and 2014, wild mice were captured at 2 sites in Fukushima Prefecture and at 2 control sites that were distant from Fukushima. Although the median concentrations of 134Cs and 137Cs in the mice from Fukushima exceeded 4,000 Bq/kg, there were no significant differences in the apoptotic cell frequencies or the frequencies of morphologically abnormal sperm among the capture sites. Thus, we conclude that radiation did not cause substantial male subfertility in Fukushima during 2013 and 2014, and radionuclide pollution levels in the study sites would not be detrimental to spermatogenesis of the wild mice in Fukushima.

Acid-Base Status and Blood Gas Arterial Values in Free-Ranging Sika Deer Hinds Immobilized with Medetomidine and Ketamine

Ten free-ranging female sika deer (Cervus nippon) were captured to obtain the reference values for acid-base status and blood gas when immobilized with the combination of medetomidine and ketamine. The mean ± SE of PaCO2, PaO2, and HCO3− were 58.1 ± 6.1 mmHg, 58.8 ± 6.4 mmHg, and 36.0 ± 4.4 mmol/l, respectively. Although acidosis and alkalosis occurred in three and two animals, respectively, no serious conditions were observed. The blood values, however, suggest that some degree of hypoxemia and respiratory acidosis with metabolic alkalosis are developed. The trapped deer showed a significantly higher than normal rectal temperature reflective of exertion.