Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Through transplantation experiments with Xenopus laevis tadpoles, we found a new morphogenetic phenomenon consisting of limb bud formation at the boundary between transplanted whole limb buds and the tail surface. This phenomenon occurs without limb—limb stump interaction and has a number of unique features: (1) Only one extra limb bud was formed per transplant and the new limb and the transplanted limb were bilaterally symmetrical, forming a pair of limb girdles. (2) Extra new limb bud formation occurred not only at the tail but also at other non-limb regions, including abdominal and head surfaces. (3) Successful limb formation required the presence of basal 1/4 region (presumptive limb girdle) of a limb bud explant. (4) New limb formation was host-stage-dependent: before metamorphosis, limb bud formation ratio was high (> 90%), but as the host tadpole entered metamorphosis, this potential declined and morphological abnormalities of new limbs increased. (5) Cell lineage analysis showed that epidermis of the new limb bud always contained many (about 60%) host-derived cells, while new limb cartilage cells were completely graft-derived. These results suggest that heterotopic new limb formation occurs through interaction between graft mesenchyme and host epidermis. Thus, the present study has clarified the two important aspects of limb ontogeny: the importance of presumptive limb girdle for the limb bud initiation and the relationship between limb bud formation potential and metamorphic tissue remodeling. The present experimental system may help to improve our understanding of epithelial-mesenchymal interaction during limb bud initiation and subsequent limb cell differentiation during metamorphosis.

The nymphalid groundplan has been proposed to explain diverse butterfly wing color patterns. In this model, each symmetry system is composed of a core element and a pair of paracore elements. The development of this elemental configuration has been explained by the induction model for positional information. However, the diversity of color patterns in other butterfly families in relation to the nymphalid groundplan has not been thoroughly examined. Here, we examined aberrant color pattern phenotypes of a lycaenid butterfly, Zizeeria maha, from mutagenesis and plasticity studies as well as from field surveys. In several mutants, the third and fourth spot arrays were coordinately positioned much closer to the discal spot in comparison to the normal phenotype. In temperature-shock types, the third and fourth array spots were elongated inwardly or outwardly from their normal positions. In field-caught spontaneous mutants, small black spots were located adjacent to normal black spots. Analysis of these aberrant phenotypes indicated that the spots belonging to the third and fourth arrays are synchronously changeable in position and shape around the discal spot. Thus, these arrays constitute paracore elements of the central symmetry system of the lycaenid butterflies, and the discal spot comprises the core element. These aberrant phenotypes can be explained by the black-inducing signals that propagate from the prospective discal spot, as predicted by the induction model. These results suggest the existence of long-range developmental signals that cover a large area of a wing not only in nymphalid butterflies, but also in lycaenid butterflies.

To investigate geographic genetic structures and taxonomic relationships among isolated populations of Buergeria japonica, occurring very widely in various habitats of the Ryukyu Archipelago and Taiwan, we conducted phylogenetic and demographic analyses among individuals from various localities, representing their entire distributional ranges. Buergeria japonica is genetically greatly differentiated and comprises three major clades (the Southern Taiwan [ST] clade, the Northern Taiwan Southern Ryukyu [NT/SR] clade, and the Central Northern Ryukyu [CR/NR] clade), each of which seems to represent independent species. The first divergence in the species is estimated to have occurred in the middle to late Miocene in areas of current Taiwan, then eastern periphery of the Asian continent. Split of the ST and the remaining clades, and subsequent divergence between the NT/SR and the CR/NR clades in the latter, indicate consecutive south to north vicariant diversifications. However, these vicariances are not always associated with formation of significant barriers such as deep straits. Less but still prominently diverged subclades (the Amami Tokara [AM/TK] and the Okinawa [ON] subclades) in the CR/NR clade were recognized in spite of the absence of an intervening deep strait. Contrariwise, individuals from Amami and Tokara Groups formed the AM/TK subclade in spite of the presence of the intervening Tokara Gap (a long-standing deep tectonic strait). Furthermore, in the AM/TK subclade, low but definite genetic divergence was found between the Northern Amami Tokara (NAM/TK) lineage and the Southern Amami (SAM) lineage. Estimated divergence time and gene flow rate within the NAM/TK lineage indicate that this species reached northern Tokara from the south by overseas dispersal over the Tokara Gap long after its formation, but not by more recent artificial transportation. This overseas dispersal would have been facilitated by its more frequent occurrence around coastal habitats than other frogs.

Chironomid midges (Diptera; Chironomidae) are found in various environments from the high Arctic to the Antarctic, including temperate and tropical regions. In many freshwater habitats, members of this family are among the most abundant invertebrates. In the present study, the genome sizes of 25 chironomid species were determined by flow cytometry and the resulting C-values ranged from 0.07 to 0.20 pg DNA (i.e. from about 68 to 195 Mbp). These genome sizes were uniformly very small and included, to our knowledge, the smallest genome sizes recorded to date among insects. Small proportion of transposable elements and short intron sizes were suggested to contribute to the reduction of genome sizes in chironomids. We discuss about the possible developmental and physiological advantages of having a small genome size and about putative implications for the ecological success of the family Chironomidae.

This study demonstrates the potential of the Japanese marten (Martes melampus) to serve as a directed seed disperser of Actinidia arguta, a representative forest edge liane. Fecal compositions of the Japanese marten in a western part of Tokyo, Japan were analyzed by the point-frame method. It fed on fruits in autumn (73.1%) and winter (63.0%), and the seeds of A. arguta were most frequently eaten (47.4%). Although the vegetation in the study area was dominated by forest (95.5%), seeds found in the marten feces were dominated by those of forest edge plants (92.1%), suggesting a strong selective bias, both habitat and food, toward these species. The density of marten feces was also higher at forest edges than forest interiors. A. arguta plants were more abundant at forest edges than within the forest at Afan Wood, Nagano Prefecture. These results suggest that the Japanese marten selectively uses forest edges as a location for feeding and defecation and thus functions as a directed seed disperser of A. arguta.

Although populations of the coconut crab, Birgus latro, have declined in the tropical Indo-Pacific region, insufficient knowledge exists for the management of this species. We investigated the growth of the northernmost coconut crab population, located at Ocean Expo Park, Okinawa, southern Japan, using a mark—recapture method based on the identification of individual carapace grooving patterns. Of the 485 crabs photographed (264 males, 221 females; 14.3–68.8 mm thoracic length [ThL]), 64 males and 62 females were recaptured (recapture rate 26%). The liberty period ranged from two to 2384 days. The annual data indicated that most crabs molted during winter, except for juveniles and crabs near the maximum size. Using the GROTAG program, the asymptotic ThL (L_∞) was estimated as 80.72 and 49.89 mm for males and females, respectively. The Brody growth coefficient (K) was 0.063 for both sexes. The growth curves from these parameters showed that males grew larger than females because of a difference in growth speed. Longevity was estimated at approximately 50 years for both sexes. The growth data obtained in the present study, which are the most precise gathered for the coconut crab to date, can be compared with the results of studies performed in other regions.

Day length is one of the most important factors that organisms use to predict seasonal changes in their environment. Several amphibians regulate their growth and development in response to photoperiod. However, many studies have not focused on the ecological effects of the photoperiodic response on growth and development because they use tropical animals, animals from a commercial source or from unknown localities, or extreme light regimens for experiments. In the present study, we examined the effects of photoperiod on growth and development in the clouded salamander (Hynobius nebulosus) by raising larvae under different photoperiods and at different temperatures in the laboratory. The average larval period under a long-day photoperiod of L16:D8 was longer than that under L12:D12 at 15°C or 20°C, although the difference between the photoperiods was only significant for 15°C. Juveniles weighed more at metamorphosis under L16:D8 than those under L12:D12, irrespective of temperature, suggesting that a longer developmental period results in a heavier body weight. The head width of juveniles did not differ for different photoperiods at either temperature. However, the growth rate of the head width under L12:D12 was faster than that under L16:D8 at 15°C. Long day length appears to produce larger H. nebulosus juveniles in a relatively stable aquatic environment with a low population density. Thus, development may be accelerated when the day length becomes shorter as winter approaches, and larvae may have increased the growth rate of their head widths to compensate for the shorter growing period under shorter day lengths.

Mammary cancer is a disease that affects many women. Extensive research has been conducted to elucidate which variables are involved in the development of this cancer. Studies have highlighted thyroid function as a modulator of tumor growth and development. Thyroxine and 3,3′,5-triiodothyronine are responsible for regulating the development, differentiation, homeostasis, and metabolism of cells in the body including mammary tissue. Thyroid hormones also have estrogen-like effects on mammary cancer cell growth by regulating the estrogen receptor. The present study was designed to determine whether medically induced hyperthyroidism increases the multiplicity, prevalence, and mammary tumor burden in rats; and to elucidate whether surgically induced hypothyroidism conversely attenuates the rate of mammary cancer cell growth. Female Sprague-Dawley rats were randomly divided into three groups (euthyroid-control, hyperthyroid, and hypothyroid). Hyperthyroidism was induced via oral administration of levothyroxine; whereas, hypothyroidism was induced by thyroidectomy. Mammary carcinogenesis was induced with a single intraperitoneal injection of N-methyl-N-nitrosurea (MNU). Rats were sacrificed at 38 weeks, and the mammary tumors were excised, fixed for histology and analyzed. Analysis included evaluation of malignancy and immunohistochemistry for ER. MNU-induced mammary carcinogenesis among the groups resulted in a significant difference in tumor burden. The hyperthyroid group had a statistically higher tumor burden than did the euthyroid group, and the hypothyroid group had no tumors of mammary tissue origin at 38 weeks. All excised mammary tumors were ER alpha negative. These data support the hypothesis that thyroid function is one of potentially many factors that contribute to modulation of MNU-induced mammary tumor growth.

The present study compares the morphology of the head, appendages, and cervical region of three species of the butterflies Archaeoprepona demophon demophon (Linnaeus, 1758), Archaeoprepona licomedes licomedes (Cramer, 1777), and Prepona pylene pylene Hewitson, [1854], through descriptions, illustrations, and scanning electron micrographs. The results are compared with Prepona claudina annetta (Gray, 1832), Memphis moruus stheno Hübner, [1819], and Zaretis itys itylus (Westwood, 1850), showing unique characteristics for each species and/or genus, or characteristics shared among the species analyzed. The detailed morphology of these three species was previouslyunknown.

The present report, the second part of a study of the external morphology of Preponini, compares the thorax and thoracic appendages of Archaeoprepona demophon demophon (Linnaeus, 1758), Archaeoprepona licomedes licomedes (Cramer, 1777) and Prepona pylene pylene Hewitson, [1854], through descriptions and illustrations. The results are compared with three other species, Prepona claudina annetta (Gray, 1832), Memphis moruus stheno Hübner, [1819] and Zaretis itys itylus (Westwood, 1850), revealing previously unrecognized similarities among species of Charaxinae.

The present paper is the final part of a study of the external morphology of Preponini, which compares the abdomen and genitalia of Archaeoprepona demophon demophon (Linnaeus, 1758), Archaeoprepona licomedes licomedes (Cramer, 1777) and Prepona pylene pylene Hewitson, [1854], through descriptions and illustrations. The results are compared with three other species, Prepona claudina annetta (Gray, 1832), Memphis moruus stheno Hübner, [1819] and Zaretis itys itylus (Westwood, 1850). The abdomen is commonly the most informative tagma for butterflies. In Charaxinae, this tagma supports diagnoses of both genera and species, besides providing a solid morphological base for recent molecular findings for Preponini.

In order to study the freeze-tolerance mechanism in the Japanese tree frog, Hyla japonica, we cloned a cDNA encoding aquaporin (AQP) 9 from its liver. The predicted amino acid sequence of H. japonica AQP9 (AQP-h9) contained six putative transmembrane domains and two conserved Asn-Pro-Ala motifs, which are characteristic of AQPs. A swelling assay using Xenopus laevis oocytes injected with AQP-h9 cRNA showed that AQP-h9 facilitated water and glycerol permeation, confirming its property as an aquaglyceroporin. Subsequently, glycerol concentrations in serum and tissue extracts were compared among tree frogs that were hibernating, frozen, or thawed after freezing. Serum glycerol concentration of thawed frogs was significantly higher than that of hibernating frogs. Glycerol content in the liver did not change in the freezing experiment, whereas that in the skeletal muscle was elevated in thawed frogs as compared with hibernating or frozen frogs. Histological examination of the liver showed that erythrocytes aggregated in the sinusoids during hibernation and freezing, and immunoreactive AQP-h9 protein was detected over the erythrocytes. The AQP-h9 labeling was more intense in frozen frogs than in hibernating frogs, but nearly undetectable in thawed frogs. For the skeletal muscle, weak labels for AQP-h9 were observed in the cytoplasm of myocytes of hibernating frogs. AQP-h9 labeling was markedly enhanced by freezing and was decreased by thawing. These results indicate that glycerol may act as a cryoprotectant in H. japonica and that during hibernation, particularly during freezing, AQP-h9 may be involved in glycerol uptake in erythrocytes in the liver and in intracellular glycerol transport in the skeletal muscle cells.

Seven nuclear and mitochondrial DNA markers were examined in 12 specimens of Ptychodera flava, a model acorn worm used in molecular biology, collected in Japan from three local populations with different modes of living. A comparison of intraspecific results did not show genetically isolated populations despite the species' enclave habitats and asexual reproduction. Moreover, both the nuclear 18S ribosomal RNA gene and mitochondrial 16S ribosomal RNA gene sequences were identical to those from Moorea in French Polynesia, nearly 10,000 kilometers away from Japan. I also provide the first definitive information regarding polymorphisms in 18S ribosomal RNA gene, the external transcribed spacer (ETS), internal transcribed spacers (ITS), and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) sequence in hemichordates using newly designed primer sets, and I show both high larval vagility and certain criteria for the molecular identification of this species.

Mature galls induced by Daphnephila truncicola, D. taiwanensis, D. sueyenae, D. stenocalia, and D. ornithocephala on Machilus thunbergii in northern Taiwan were examined to verify the dictum that the morphology of galls is an expression of the extended phenotype of the respective gall-inducing insect. Based on their length-width ratio, the materials were grouped into either fleshy (those induced by D. taiwanensis and D. sueyenae) or slim galls (those induced by D. truncicola, D. stenocalia, and D. ornithocephala). Stem galls induced by D. truncicola showed an energy level of 0.0178 kJ/g. Among leaf galls, the greatest energy level was in the one induced by D. stenocalia (0.0193 kJ/g), followed by D. sueyenae (0.0192 kJ/g), D. taiwanensis (0.0189 kJ/g), and D. ornithocephala (0.0160 kJ/g). The numbers of reserve and nutritive cell layers in galls were greater in the stem galls induced by D. truncicola, similar to those in the fleshy leaf galls, than in the slim leaf galls. Based on the fungal taxa isolated from the larval chambers and considering the similarities and divergences among gall characteristics, the galls induced by D. truncicola and D. taiwanensis clustered into one, whereas those of D. sueyenae aligned with the ‘D. stenocalia—D. ornithocephala’ cluster. The present study verified that shapes, structure, nutritive tissues, energy levels, and multiple coexisting fungal taxa within galls reinforce that they are extended phenotypes of the respective gall-inducing Daphnephila species and they represent adaptive evolution of Daphnephila on M. thunbergii.