The bdelloid rotifer of the genus Adineta is a freshwater metazoan characterized by anhydrobiosis, a highly stable state of suspended animation induced by desiccation. This study investigated the influence of anhydrobiosis on the thermal habituation by use of an index, Activity Ratio (AR = the number of active rotifers at each experimental temperature/ number of active rotifers at the 25°C stage). In the first experiment, rotifers were divided into two groups: one group was cultivated at 25°C throughout experiment, and another group was transferred to 15°C for two days. AR was estimated during heating up to 40°C, or during cooling down to 5°C in each group. The largest difference in AR occurred at 35°C and 10°C, indicating that AR was changed depending on the pretreated medium temperature. In the next experiment, rotifers were maintained at 15°C, and were desiccated (anhydrobiosis). AR was estimated in the high temperature range (25°C to 40°C), using rotifers that had recovered from anhydrobiosis. AR was significantly different between the groups with and without desiccation, suggesting that thermal habituation at 15°C was completely cancelled by anhydrobiosis. Possible mechanisms on the influence of anhydrobiosis on the thermal habituation have been discussed in terms of neural changes and proteins.

Many molluscs perform sex reversal, and sex hormones may be involved in the process. In adult scallops, Patinopecten yessoensis, gonadotropin releasing hormone and 17β-estradiol (E₂) are involved in male sexual maturation, however, little is known about the effects of E₂ and testosterone (T) on the gonadal differentiation in young scallops. In the present study, scallop gonadal development was analyzed to determine the sex reversal stage in Funka bay, and effects of E₂ and T were examined. In Funka bay, almost all scallops were male at month 12. Scallops equipped with ambiguous gonads were 61.1% at month 16 and disappeared at month 18. Therefore, sex reversal in Funka bay occurs at around month 16. For establishment of organ culture systems for bivalves, Manila clam gonads were cultured in 15% L-15 medium diluted with HBSS containing 10% KSR on agarose gel at 10°C, and the gonads survived for 14 days. Scallop gonads were also able to be cultured in 30% L15 medium diluted with ASW containing 10% KSR on agarose gel for seven days. At mature stage, Foxl2 and Tesk were predominantly expressed in ovary and testis, respectively. When scallop gonads at sex reversal stage were organ-cultured, sex steroid treatment decreased Tesk expression in the majority of scallop gonads at sex reversal stage. However, no obvious change in Foxl2 and Tesk expression was detected in mature gonads in response to either E₂ or T in culture, suggesting sex steroid treatment might affect gonadal development at sex reversal stage.

Xenopus vasa-like gene 1 (XVLG1), a DEAD-Box Helicase 4 (DDX4) gene identified as a vertebrate vasa homologue, is required for the formation of primordial germ cells (PGCs). However, it remains to be clarified when and how XVLG1 functions in the formation of the germ cells. To gain a better understanding of the molecular mechanisms underlying XVLG1 during PGC development, we injected XVLG1 morpholino oligos into germ-plasm containing blastomeres of 32-cell stage of Xenopus embryos, and traced cell fates of the injected blastomere-derived PGCs. As a result of this procedure, migration of the PGCs was impaired and the number of PGCs derived from the blastomeres was significantly decreased. In addition, TUNEL staining in combination with in situ hybridization revealed that the loss of PGCs peaked at stage 27 was caused by apoptosis. This data strongly suggests an essential role for XVLG1 in migration and survival of the germ cells.

The African clawed frog Xenopus laevis has a female heterogametic ZZ/ZW-type sex-determining system. We previously discovered a W-linked female sex-determining gene dm-W that is involved in ovary formation, probably through the up-regulation of the estrogen synthesis genes cyp19a1 and foxl2. We also reported that a unique “mass-in-line structure”, which disappears from ZZ gonads during early testicular development, might serve as the basis for ovary differentiation in ZW gonads. However, the molecular mechanisms underlying early masculinization are poorly understood. To elucidate the development of bipotential gonads into testes after sex determination in this species, we focused on the orthologs of five mammalian sex-related genes: three nuclear factor genes, dax1, sf1 (also known as ad4bp), and sox9, and two genes encoding members of the tumor growth factor-β (TGF-β) family, anti-Müllerian hormone (amh) and inhibin βb (inhbb). Quantitative RT-PCR analysis revealed that the expression of dax1, sox9, amh, and inhbb or sf1 was greatly or slightly higher in ZZ than in ZW gonads during early sex development. In situ hybridization analysis revealed that amh and inhbb mRNAs were expressed in somatic cells on the inner and outer sides of cell masses in the mass-in-line structure, respectively, in the developing ZZ gonads. Interestingly, estrogen exposure prevented the disappearance of the mass-in-line structure in early developing ZZ tadpoles. These findings suggest that TGF-β signaling is involved in the destruction of the mass-in-line structure, which may be maintained by estrogen.

To study the effects of post-glacial isolation by islands on population genetic diversity and differentiation of the large Japanese field mouse, Apodemus speciosus, we examined partial nucleotide sequences of the mitochondrial Dloop region (ca. 300 bp) in 231 individuals collected from islands in the Seto Inland Sea and adjacent regions on Honshu and Shikoku Islands in the western part of the Japanese archipelago. Molecular phylogenetic and network analyses showed that haplotypes in each island tended to form monophyletic groups, while those in Honshu and Shikoku (the major Japanese islands) showed scattered relationships and were connected with island haplotypes. These observations suggest that a set of Honshu and Shikoku haplotypes became the ancestral lineages of the island population. No gene flow was detected among island populations, indicating that independent evolution occurred on each island, without the influence of human activities, since the establishment of the islands in the Holocene. Population genetic diversities on each island were lower than those on Honshu and Shikoku. Comparison between genetic diversity and island area size showed positive correlations and supported the suggestion that genetic drift is a major factor that shaped the current haplotype constitution of the islands in the Seto Inland Sea.

Using preserved specimens, we studied the basic life history of the topotypic population of the unique Asian plethodontid salamander, Karsenia koreana. Of 51 individuals examined, 11 males and 13 females were judged as mature from the development of gonads. The ovarian eggs were large (diameter 3.7–4.8 mm) and yellow to orange in color, and the clutch size was about 8–10. These values approximate those of actually spawned eggs recently reported. Skeletochronological analyses revealed the average age of males (5.3 years) to be lower than females (7.3 years). The age at maturity and maximum observed longevity were four and nine years in males and five and 10 years in females, respectively. In the growth curves estimated by a von Bertalanffy growth model, the growth coefficient and asymptotic SVL did not differ between the sexes, although males (40.6 mm) were smaller than females (45.3 mm) in the average snout-vent length. The time and place of courtship behavior, oval development, hatching, and especially, whether the species shows aquatic larval stage or direct development, are important topics to be resolved in future.

We investigated the life cycle of the tanaidid Zeuxo sp. 1 (Crustacea: Peracarida: Tanaidacea), which lives epiphytically in dwelling tubes on the algae Sargassum spp. and Neorhodomela aculeata in Oshoro Bay, Hokkaido, Japan. We obtained data on its population size distribution, age structure, and reproductive phenology through monthly sampling from April 2011 to June 2012. From these data, we detected an overwintering group (F₀) that arises from mancae that are released in autumn, overwinters, reproduces the following spring, and then disappears. The overwintering group produces mancae (F₁) that reach maturity and themselves reproduce within six weeks after release; the F₁, mancae give rise to an F₂ and possibly an F₃ generation within a single summer. We refer to individuals that reproduce in the same summer that they were released as the ‘annual group’. Reproduction overall was restricted to the period from May to October, when the seasurface temperature exceeded 10°C. During both years, overwintering females first became reproductive in May. Reproduction in the annual group began in June and continued through mid-October. Mancae were observed in samples from June through October. The two groups differed significantly in size at reproduction; compared to the annual group, females in the overwintering group reproduced at larger minimum and average body sizes, and males began to express enlarged chelae, a secondary sexual character, at a larger size. The difference in size at maturity may be related to the differences in water temperature during the main period of growth and maturation.

We compared the reliability of visual diagnostic criteria to DNA diagnostic techniques, including newly designed primers, to discriminate Japanese marten (Martes melampus) feces from those of other sympatric carnivore species. Visual criteria proved > 95% reliable for fresh, odoriferous scats in good condition. Based upon this verification, we then examined if and how Japanese marten diet differs among seasons at high elevation study site (1500–2026 m). We also considered how intra-specific competition with the Japanese red fox (Vulpes vulpes japonica) may shape marten feeding ecology. From 120 Japanese marten fecal samples, high elevation diet comprised (frequency of occurrence) 30.6–66.0% mammals, 41.0–72.2% insects and 10.6–46.2% fruits, subject to seasonal variation, with a Shannon-Weaver index value of 2.77. These findings contrast substantially to seasonal marten diet reported in adjacent lowland regions (700–900 m), particularly in terms of fruit consumption, showing the trophic adaptability of the Japanese marten. We also noted a substantial dietary overlap with the red fox (n = 26 scats) with a Shannon-Weaver index of 2.61, inferring little trophic niche mutual exclusion (trophic niche overlap: 0.95), although some specific seasonal prey selection differences were likely related to relative differences in body size between foxes and martens. This additional information on the feeding ecology of the Japanese marten enables a better assessment of the specific risks populations face in mountainous regions.

The thiazine dye toluidine blue (TB) is well known to stain mast cells and hyaline cartilage metachromatically, and thus is mostly often used for their identification. However, TB is not suitable for counterstaining in immunohistochemistry, because of its high-background staining in the cytoplasm of other cell species and in extracellular structures. To expand the knowledge about dyestuffs staining mast cells in consideration with their usage in immunohistochemistry, we determined the stainability of several thiazines and oxazines, which are structurally related compounds to TB, using sections of mast cell-containing tissues. We found that all azine dyes used metachromatically stained mast cells and cartilage. Among these dyes, an oxazines cresyl violet (CV) stained mast cells with lower background, suggesting that those are useful for detecting mast cells and for counterstaining in immunohistochemistry. To ascertain its utility, CV was used in immunostaining of bHSDs in sections from adult rat ovary. Immunopositive signals reflected by DAB development in brown were clearly detected even after CV staining. We conclude that, similar to thiazines, oxazines stain mast cells metachromatically, and that of these, CV is more useful as a counterstain in immunohistochemistry than TB.

The eclosion gate in insect development is controlled by the circadian clock and hormonal cascade. To study mechanisms underlying the eclosion gate, we examined eclosion-timing signals from the circadian clock, and the role of 20-hydroxyecdysone in the eclosion gate of the flesh fly, Sarcophaga crassipalpis. Phase responses of the eclosion rhythm were examined by applying a low-temperature pulse in the day prior to the first eclosion peak. A low-temperature pulse applied about 5.4 h before eclosion advanced an eclosion peak by 0.9 h. This indicates that an interval from the Zeitgeber (external environmental cues) input to the behavioral output by the circadian clock is 4.5 h. Signals released by the circadian clock in the last 4.5 h before eclosion could change eclosion time. In the prothoracic gland, daily changes in immunoreactivity against a circadian clock protein PERIOD were observed in the last two days before eclosion. Hemolymph titers of 20-hydroxyecdysone were very low in the last two days of the pupal period. 20-hydroxyecdysone injections caused a delay, not an advancement, in eclosion time in a time dependent manner: pharate adults were sensitive to 20-hydroxyecdysone about 20 and 16 h before eclosion, whereas no significant effects were observed about 12 and 8 h before eclosion. These results suggest that 20-hydroxyecdysone is not a timing signal submitted by the circadian clock but an indicator to suppress premature eclosion. The circadian clock in the prothoracic gland presumably sends a signal distinct from ecdysteroids from several hours before eclosion to time the onset of eclosion.

A quadrannulate species of Orobdella, O. kanaekoikeae sp. nov., from the mountainous region of Shikoku, Japan is described. This new species is small with a body length of mature individuals reaching only ca. 5 cm. Phylogenetic analyses using nuclear 18S, histone H3, mitochondrial cytochrome c oxidase subunit I, tRNA^Cys, tRNA^Met, 12S rRNA, tRNA^Val, 16S rRNA, tRNA^Leu, and NADH dehydrogenase subunit 1 markers showed that O. kanaekoikeae is a sister species of the quadrannulate small O. brachyepididymis. Phylogenetic relationships among populations of this new species were also estimated using mitochondrial DNA markers. Additionally, transmission electron micrographs of a capsule-like structure obtained from a post-copulatory individual revealed that the capsule contained spermatozoa, and was thus defined as the spermatophore. The gastroporal duct of Orobdella leeches is an accessory copulatory organ that receives a spermatophore during copulation.