Tsukasa Okano

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.

Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in the Ryukyu Islands Tree Rat (Diplothrix legata)

Abstract Angiostrongylus cantonensis is a zoonotic nematode with rodents serving as natural definitive hosts. We report A. cantonensis in the Ryukyu Islands tree rat (Diplothrix legata, Thomas, 1906), a native endangered species in Japan. Adult and larvae of A. cantonensis were macroscopically, histologically, and genetically detected in three tree rats collected between August 2011 and January 2012 in the Yambaru area of Okinawa Prefecture, Japan. Pathologic observations of the lungs of rats showed that infection may be lethal. We also conducted a retrospective genetic survey of helminths parasitic in lung in cryopreserved lung samples of Ryukyu Islands tree rats collected between 2007 and 2011 in the Yambaru area and found A. cantonensis DNA in one of 29 samples, which was collected in December 2010.

Coexistence of two different genotypes of Sarcoptes scabiei derived from companion dogs and wild raccoon dogs in Gifu, Japan: The genetic evidence for transmission between domestic and wild canids

Sarcoptes scabiei is the causal agent of sarcoptic mange in domestic/companion dogs (Canis lupus familiaris) and raccoon dogs (Nyctereutes procyonoides). Although there have been successful cases of experimental transmission of S. scabiei from mangy wild Canidae hosts to healthy dogs, and suspected cases of transmission between raccoon dogs and companion dogs, no clear-cut evidence has been obtained. In the present study, the genetic relationships between Sarcoptes mites from raccoon dogs and companion dogs living in the same region were elucidated.One hundred and thirty Sarcoptes mites from 22 raccoon dogs and 5 companion dogs were collected from the Gifu area in Japan. Using 9 microsatellite markers, the genotypes were compared, and the genetic structure of these mites was analyzed. In 6 pairs of companion dog- and raccoon dog-derived mites, 17 out of the 18 alleles analyzed were identical. Using a Bayesian approach, these 130 mites were separated into at least two groups, and companion dog- and raccoon dog-derived mites were segregated into both groups. In addition, comparatively large numbers of alleles at these loci were revealed by comparison with data from past studies. These results demonstrated that the host specificity at the 9 microsatellite-level could not be confirmed, strongly suggesting the transmission of Sarcoptes mites between raccoon dogs and companion dogs. This is the first report to provide a genetic evidence of Sarcoptes transmission between domestic and wild mammals in the natural environment. The possibility of a prior introduction of mites with novel genotypes (e.g., spillover of sarcoptic mange from domestic/companion dogs to raccoon dogs) could not be eliminated when considering the cause of the large number of alleles, and the coexistence of 2 mite groups in sympatric raccoon dogs and companion dogs in this local area.

Classification of the spermatogenic cycle, seasonal changes of seminiferous tubule morphology and estimation of the breeding season of the large Japanese field mouse (Apodemus speciosus) in Toyama and Aomori prefectures, Japan

The large Japanese field mouse, Apodemus speciosus, is a potential indicator of environmental stress, but this function has not been confirmed by histological studies. Since environmental stress affects the reproductive function of mice, we determined the reproductive characteristics of this species at two locations: Toyama (36°35ʹN, 137°24ʹE) and Aomori (40°35ʹN, 140°57ʹE). Mice were captured during May–November (n=119) and July–November (n=146) at these locations, respectively. We classified the breeding season from the numbers of pregnant females and young, in addition to the spermatogenic cycle and seasonal changes in seminiferous tubule morphology of males. Testicular weight was measured, and seminiferous tubule morphology was examined histologically. Fourteen stages were found in the seminiferous epithelium cycle based on acrosome formation and spermatid head morphology. At both locations, the breeding season peaked from late summer to early autumn and possibly in spring. Spermatogenic activity was classified into 4 periods from June to November: resting around June and October–November; resumptive around July; active around August; and degenerative around September. During the resting period, the seminiferous tubules consisted of Sertoli cells, spermatogonia and spermatocytes. Spermatogenesis began during the resumptive period, and spermatids were observed. During the active period, active spermatogenesis and a broad lumen were observed. During the degenerative period, spermatogenesis ended, and Sertoli cells, spermatogonia, spermatocytes and degenerating exfoliated round spermatids were observed. This study provides scientific information about the testicular histopathological evaluations of the large Japanese field mouse for its use as an index species of environmental pollution.

Reproductive traits of the Ryukyu long-furred rat (Diplothrix legata) on Okinawa-jima Island.

The Ryukyu long-furred rat, Diplothrix legata, is a large rodent distributed only on Amami-ohshima Island, Tokuno-shima Island and Okinawa-jima Island, Japan. This animal is endangered as a result of deforestation, predation by introduced carnivores and mortality caused by vehicles. We performed theriogenological examinations of 32 male and 25 female Ryukyu long-furred rats carcasses collected from wild populations on northern Okinawa-jima Island from December 2005 to September 2013. Adult males had remarkably large preputial glands. Seminiferous diameter of adult was significantly small (136 ± 28 µm, n=8) from April to August. Numerous spermatozoa were observed from September through February, and seminiferous diameter was significantly large (216 ± 27 µm, n=12) during this time in adults; testes length changed in a similar pattern. These findings indicate that the mating season may occur from September through February. Size (body length) at sexual maturity was estimated to be >560 mm in both sexes. From observation of corpora lutea and placental scars, litter size was estimated to range from 2 to 12 (average=6, n=4). These results provide fundamental knowledge that will be beneficial for in situ and ex situ conservation of this rare species.

Dietary habits of the endangered Okinawa Rail

Abstract Many species of flightless rail are endemic to small islands and endangered, or have become extinct during historical times. However, our understanding of their basic ecological knowledge remains limited. The Okinawa Rail Hypotaenidia okinawae is a flightless species of rail endemic to Okinawajima Island in the Ryukyu Archipelago, Japan. In this study we have quantified the dietary components of the Okinawa Rail using gizzard contents classified by the frequency of occurrence and wet weight. The Okinawa Rail feeds on a wide variety of animals and plants throughout the year, but the frequency of occurrence and wet weight of animal matter significantly exceeds those of plant matter. The number of items present in the gizzard did not differ among seasons, ages and between sexes. Grit was present in almost all individuals and the wet weight did not differ between sexes or among age groups. Although the diet was observed to be diverse, the primary dietary component of both sexes, all ages, and throughout the year appears to be land snails, indicating their great significance as a source of food.

Characterizing the temporal patterns of avian influenza virus introduction into Japan by migratory birds

The objectives of the present study were to observe the temporal pattern of avian influenza virus (AIV) introduction into Japan and to determine which migratory birds play an important role in introducing AIV. In total, 19,407 fecal samples from migratory birds were collected at 52 sites between October 2008 and May 2015. Total nucleic acids extracted from the fecal samples were subjected to reverse transcription loop–mediated isothermal amplification to detect viral RNA. Species identification of host migratory birds was conducted by DNA barcoding for positive fecal samples. The total number of positive samples was 352 (prevalence, 1.8%). The highest prevalence was observed in autumn migration, and a decrease in prevalence was observed. During autumn migration, central to southern Japan showed a prevalence higher than the overall prevalence. Thus, the main AIV entry routes may involve crossing the Sea of Japan and entry through the Korean Peninsula. Species identification was successful in 221 of the 352 positive samples. Two major species sequences were identified: the Mallard/Eastern Spot-billed duck group (115 samples; 52.0%) and the Northern pintail (61 samples; 27.6%). To gain a better understanding of the ecology of AIV in Japan and the introduction pattern of highly pathogenic avian influenza viruses, information regarding AIV prevalence by species, the prevalence of hatch-year migratory birds, migration patterns and viral subtypes in fecal samples using egg inoculation and molecular-based methods in combination is required.

Isolation and characterization of 25 polymorphic microsatellites of the large Japanese wood mouse (Apodemus speciosus)

The large Japanese wood mouse (Apodemus speciosus) is common, but endemic to Japan, and its population structure was affected by habitat fragmentation because of urbanization. It suggested that the species might be one of the important models for the conservation of ecosystems and biodiversity affected by humans, including the effect of radioactive discharge caused by nuclear power plant accidents at Fukushima. We developed and characterized 25 novel polymorphic microsatellite markers from the next-generation sequencing data in an effort to provide an effective tool for genetic studies on this species. In 8 individuals from Aomori, Japan, the number of alleles and expected heterozygosities ranged from 5 to 13 and from 0.795 to 0.991, respectively, suggesting the availability of these markers for genetic studies in this species.