Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas).

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.

Possible conservation units of the sun bear (Helarctos malayanus) in Sarawak based on variation of mtDNA control region.

The mitochondrial DNA control region of the sun bear (Helarctos malayanus) was sequenced using 21 DNA samples collected from confiscated sun bears to identify conservation units, such as evolutionarily significant units and management units, in Sarawak, Borneo Island. A total of 10 haplotypes were observed, indicating the presence of at least two lineages in the sun bear population in Sarawak. Presumably, these two lineages could represent evolutionarily significant units. However, the geographical distributions of the two lineages remained unknown due to the lack of information regarding the exact capture locations of the confiscated sun bears. It is essential to elucidate the geographical distributions of these lineages in order to create a proper conservation plan for the sun bears in Sarawak. Therefore, further studies examining the haplotype distributions using DNA samples from known localities are essential.

Seasonal changes in serum leptin of the feral raccoon (Procyon lotor) determined by canine-leptin-specific ELISA.

Several reports have been published on blood leptin concentrations in feral animals, including members of the Carnivora, using a commercially available multi-species radioimmunoassay (RIA) kit with anti-human leptin antibody. However, we observed weak immunoreactivity between recombinant canine leptin and anti-human leptin antibody, suggesting a limitation in the applicability of the RIA kit for leptin assays in Carnivora species. We tested the applicability of RIA and sandwich enzyme-linked immunosorbent assay (ELISA) with anti-canine leptin antibody to assay blood leptin in the dog (Canis familiaris) and the raccoon (Procyon lotor). When RIA was used for recombinant canine leptin and dog sera, values were much lower than those determined by ELISA at higher concentrations (>10 ng/ml), while rather higher at lower concentrations (<2 ng/ml). A similar discrepancy between the two methods was found for serum leptin concentrations in raccoons. Clear seasonal variations were observed by ELISA, but not by RIA, with high values in autumn (3.46+/-0.45 ng/ml) and low values in spring and summer (0.71+/-0.07 ng/ml). Serum leptin concentrations in raccoons correlated positively with their body weight (r=0.753) and body mass index (r=0.755), corroborating our previous findings of a strong positive correlation between serum leptin concentrations and body fat content in dogs. Thus, the canine leptin ELISA is useful for assays of dog and raccoon leptin, and blood leptin is a good marker of nutritional condition in the species of Carnivora assayed in this study.

Short report: Echinococcus multilocularis confirmed on Kunashiri Island, 15 kilometers from the eastern part of Hokkaido, Japan.

There are two foci of alveolar echinococcosis (AE) caused by Echinococcus multilocularis in Japan. The first focus is on Rebun Island where AE patients were found from 1937, and the second is in eastern Hokkaido where patients have been found since the 1960s. The origin of the second focus is unknown. To further investigate AE in eastern Hokkaido, wild rodents (Muridae) were captured and examined for infection on Kunashiri Island, which is located 15 km off the northeastern coast of Hokkaido. Metacestodes of E. multilocularis were isolated from two of 31 voles, all of which were identified to be Clethrionomys rufocanus. Mitochondrial DNA sequencing data of recovered cestodes showed total identity with the cestode reported from Hokkaido. These results suggest that E. multilocularis may have been introduced to Hokkaido from Kunashiri Island during or after 1965.

Dental incremental lines in Sika deer (Cervus nippon); polarized light and fluorescence microscopy of ground sections.

Periodic growth incremental lines are found universally in dental hard tissues. This periodicity theoretically allows for estimation of age, even in days, which would be useful in studies of wild animals. In the present study, enamel and dentin increments of the sika deer (Cervus nippon) were observed in ground sections with a polarized light microscope, and their periodicity was examined by the use of a chronological labeling method with fluorochromes. Enamel increments occurred at a mean interval of 10.6 (SD=1.5) microm, and mean spacing of dentin increments was 17.3 (SD=1.8) microm. Fluorochromic marking revealed that incremental lines form each day in enamel and almost every second day in dentin. The fluorescence-labeled lines suggest that enamel formation of the first molar is complete by the age of 5 months. Due to its longer interval of incremental lines and longer term of formation, we conclude that dentin is more suitable than enamel for day-age estimation in sika deer. Experimental confirmation of incremental growth periodicity in various species can improve the reliability of use of tooth increments for age estimation and life history reconstruction.

Histological variations in myoepithelial cells and arrectores pilorum muscles among caudal, metatarsal and preorbital glands in Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884).

The morphological characteristics of myoepithelial cells and arrectores pilorum muscles were investigated in caudal, metatarsal and preorbital glands of Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884) using immunohistochemistry for alpha-smooth muscle actin. In the metatarsal, preorbital and general skin glands, myoepithelial cell layers continuously embraced the secretory epithelium, while in the caudal gland, discontinuous myoepithelial cell rows surrounded the apocrine tubules. There was a trend that the widths of the myoepithelial cells of the caudal and preorbital glands appeared to be thinner than those of the metatarsal and general skin glands. In the metatarsal gland, the arrectores pilorum muscles were highly developed and considerably larger than those in other skin glands.

Mitochondrial DNA variation in the Japanese marten Martes melampus and Japanese sable, Martes zibellina.

Genetic relationship among Japanese sables, Martes zibellina and the introduced Japanese martens, Martes melampus in northern Japan was revealed by analyzing a 521-524bp DNA sequence from the cytochrome b (112bp)/transfer RNA-threonine (67bp)/tRNA-proline (65bp) and control region (277-280bp) of the mitochondrial genome. Intraspecific differences in sequences of M. zibellina and M. melampus (3.8-15.0% and 1.9-16.4%, respectively) were similar to interspecific differences between these two species (5.8-16.6%). Comparison of sequence data exhibited five haplotypes of M. melampus and four haplotypes of M. zibellina, which clustered into two groups (clusters-A and-B). Cluster-A included two haplotypes of M. melampus and two haplotypes of M. zibellina, whereas cluster-B included three haplotypes of M. melampus and two haplotypes of M. zibellina. Results of this study lead three possible explanations. Firstly, past hybridization between M. zibellina and M. melampus might have occurred. Secondary, these two species might have similar heteroplasmy of mtDNA. Thirdly, these haplotypes might have come from nuclear genome. Although further intensive studies are needed to make a conclusion, detection of hybridization with the Japanese marten are occurred or not is quite important to conserve the Japanese sable.

Identification and morphological characteristics of dental neonatal line in sika deer (Cervus nippon).

The dental neonatal line of the sika deer (Cervus nippon) was identified experimentally using chronological labeling methods. In the enamel, prominent dark lines were observed under transmitted light, and the number of increments between the dark line and labeling line was almost consistent with the day-age at the time of labeling injection. Therefore, we identified the dark line as the enamel neonatal line. In the dentin, the bright line was observed under polarized light. Since the bright line corresponded to the enamel neonatal line, we recognized the bright line as the dentin neonatal line. Neonatal lines intersected with the enamel-dentin junction at approximately one-third cervical in the first molar. Using these features, it would make possible to distinguish the neonatal line in wild sika deer.

Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season.

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.

Evaluation of fetal growth and estimation of fetal age based on skeletal growth in Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884).

We investigated fetal development and the estimation of fetal age of 127 Hokkaido sika deer fetuses, categorizing them into three groups according to the nutritional condition of populations. The order and time of the appearance of ossification centers were clarified, and fetal age was determined based on bone length and the appearance of ossification centers. Then we observed the differences in fetal growth among three populations, and discussed the effect of poor nutrition on the fetal growth. The results suggest that fetal diaphysial length of the femur was affected very little by nutritional conditions, whereas conception dates were delayed and fetal weight was restricted as the nutritional condition became poorer. Although it is impossible to know the exact accurate fetal age in wild populations, it was possible to create a standard to estimate fetal age more precisely by the method described in this study. Both the bone length and the appearance of ossification centers are reliable indices to estimate fetal age precisely in measurements available from fetuses of unknown age, and can be applied to estimate the fetal age of other populations of sika deer, whereas estimation of fetal age based on weight is prone to great errors.

Population genetic structure of the spotted seal Phoca largha along the Coast of Hokkaido, based on mitochondrial DNA sequences.

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 (Phi(st )= -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.

Age-related change and allometry of skull and canine of sea otters, Enhydra lutris.

Skulls and canines of 460 sea otters from Lopatka Cape, Kamchatka, were examined to assess development patterns, individual variation and sexual differences. An allometric formula was applied to morphometrical data, and the relative growth of each character to total length of skull was analyzed. In both sexes, most morphometrical characters ceased growth at about 2 years of age. Canine root length increased rapidly during the first year of life, while crown length decreased due to remarkable wear. There was large individual variation in the feeding and breathing/sniffing apparatus, while there was little variation in braincase size. There were sexual differences in most characteristics, although males and females showed similar growth patterns. The coronoid process of the mandible showed positive allometry in both sexes, and we attributed this finding to feeding habits. The fact that only male mastoids showed positive allometry may be due to the need for male otters to maintain a passing territory.

Reproductive characteristics of the feral raccoon (Procyon lotor) in Hokkaido, Japan.

Reproduction of feral raccoons (Procyon lotor) in Hokkaido, Japan, was examined during a 2-year period by analysis of placental scars or fetuses in the uterus. Of 242 collected females, 69 (29%) were juveniles, 71 (29%) yearlings, and 102 (42%) adults. The pregnancy rate averaged 66% in yearlings and was significantly lower than the 96% average observed in adults (p<0.01). Litter size ranged from 1 to 7 offspring per female, and averaged 3.6 in yearlings and 3.9 in adults. There was no significant difference in mean litter size between yearlings and adults. In Hokkaido, the raccoon mating season peaked in February and the majority of litters were born between March and May, similar to patterns described in North America, but some females mated in summer. The reproductive potential of feral raccoons in Hokkaido was similar to that reported in North America. The recent increase in raccoon numbers can be explained by their high productivity. Harvest data suggest that hunting pressure on juveniles is lower than that for older age classes when using box traps in summer. In order to reduce the feral raccoon population, alternative hunting methods that increase juvenile mortality rates are needed.

Growth pattern and seasonal weight changes of the feral raccoon (Procyon lotor) in Hokkaido, Japan.

The growth pattern and seasonal weight fluctuations of feral raccoons in Hokkaido were evaluated between 1999 and 2001. The growth rates inbody length and body weight were described for juveniles (young of the year) and yearlings (animals born in the previous season) using the Gompertz growth model. The asymptotic body sizes for males were greater than those for females. Young raccoons born during spring in the study area could potentially grow up to their asymptotic size at the beginning of their first winter, but they would not reach their full adult sizes until at least their second fall. Adult raccoons (animals > or = 2 -year-old) had seasonal weight fluctuations with annual weight loss of 25% to 28% of mean maximum weights in west-central Hokkaido, but these result would be an underestimate of the degree of annual weight fluctuations. Juvenile raccoons can be distinguished from the older animals by measuring body length or body weight during fall in Hokkaido.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for mtDNA typing in hokkaido brown bear (Ursus arctos yesoensis).

To develop an easy method of typing of mitochondrial DNA (mtDNA) in Hokkaido brown bears (Ursus arctos yesoensis), the PCR-RFLP technique was improved using four restriction enzymes: Mbo 1, Cfr 13 I, TspE 1, and Fok 1. This approach identified seven groups of mtDNA haplotypes, HB1/2/5-7, HB 3, HB4, HB8/9, HB10/11, HB12 and HB13 from 102 brown bears of northern, central and eastern Hokkaido.

Plastic casts and confocal laser scanning microscopy applied to the observation of enamel tubules in the red Kangaroo (Macropus rufus).

Scanning electron microscopy for plastic casts and confocal laser scanning microscopy for Villanueva bone-stained ground sections were used together to observe enamel tubules in red kangaroo molars. Although the tubular structures such as terminals, bends, expansions, splits, divergences and rejoinings in this species were within the variations of marsupial species, their morphological characteristics were demonstrated with extremely clear and persuasive images. Thus, the combined observations of plastic casts by scanning electron microscopy and Villanueva bone-stain sections by confocal laser scanning microscopy were found to be of value for the investigation of enamel tubules and tubular structures in other hard tissues.

Daily incremental lines in sika deer (Cervus nippon) dentine.

This work was designed to observe the dentine incremental lines of the sika deer (Cervus nippon) fawns and to investigate their periodicity using the chronological labeling method with fluorochromes. The incremental lines were observed in decalcified specimens stained by Bodian's silver technique, and the fluorescence-labeled lines were observed in undecalcified and ground specimens. In the silver stained specimens, there were two types of lines, deeply stained thick lines and faintly stained minute regular incremental lines. The intervals and staining intensities of the deeply stained thick lines were very similar to those of the fluorescence-labeled lines in the ground specimens obtained from the same tooth, and hence, it appeared that the both lines were identical. The number of minute incremental lines between the deeply stained thick lines was the same as that of days between the time when each fluorescent labeling injection was made. Therefore, it seemed that each minute incremental line was formed each day. The possibility of age estimation in days using diurnal dentine increments was discussed.

Molecular phylogeography of the red deer (Cervus elaphus) populations in Xinjiang of China: comparison with other Asian, European, and North American populations.

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.

Annual changes in fecal estradiol-17beta concentrations of the sun bear (Helarctos malayanus) in Sarawak, Malaysia.

Fecal estradiol concentrations were measured in three captive unmated female sun bears (Helarctos malayanus) from August 1998 to July 1999 in Sarawak, Malaysia and vaginal smears from one of the females was observed in August 1998 and March 1999. A single peak in fecal estradiol concentration was obvious for each bear in August or September 1998, and there was a much higher percentage of superficial vaginal anuclear cells in August 1998 than in March 1999. These results suggest that sun bears in Sarawak are likely to be a seasonal breeder associated with a peak of estrogen production in August or September.

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.