The mouse pink-eyed dilution (p) locus is known to control the melanin content in melanocytes. However, it was not known whether the p gene is involved in regulating the proliferation and differentation of melanocytes during development, especially the biogenesis of melanosomes and other organelles. Epidermal cell suspensions of neonatal dorsal skin derived from mice wild type for the p locus (black, C57BL/10JHir-P/P) and their congenic mutant phenotype (pink-eyed dilution, C57BL/10JHir-p/p) were cultured in serum-free melanocyte-proliferation medium (MDMD). The supplement of additional L-tyrosine (Tyr) into the MDMD stimulated the differentiation of p/p melanoblasts into melanocytes. Electron microscopy revealed that in p/p melanoblasts and melanocytes treated with L-Tyr, the number of stage II and III melanosomes dramatically increased. Moreover, p/p melanoblasts possessed smaller but more numerous mitochondria than P/P melanocytes. The treatment of p/p melanoblasts and melanocytes with L-Tyr decreased the number of mitochondria. The supplement of 2, 4-dinitrophenol (DNP), an inhibitor of mitochondrial function, into the MDMD stimulated both the proliferation and differentiation of p/p melanoblasts. Simultaneous treatment of DNP and L-Tyr dramatically stimulated the differetiation of p/p melanocytes. These results suggest that L-Tyr and some unknown factors related to mitochondrial function may influence the differentiation of melanoblasts in the epidermis of p/p mice.

Homothermal animals need to keep their body temperature within a narrow range. Only a few degrees Celsius change in temperature has a dynamic influence on many physiological processes. To investigate the effect of the body temperature on muscle cell differentiation, we cultured the mouse myoblast cell lines C2C12 and Sol8 at lower temperatures than mouse body temperature. At 38°C, the cells fused into multinucleated myotubes within 4 days after the induction of differentiation. However, myotube formation was blocked at 30°C, whereas it was delayed but relatively normal at 35°C. The myoblasts expressed MyoD, but not myogenin, at 30°C. Id3, which acts as a negative regulator of myogenic regulatory factors (MRFs), was expressed at a higher level at 30°C than at 38°C, whereas the expression level of E2A, which acts as a positive regulator of MRF expression, exhibited no difference between these temperatures. We also found that the expression of muscle-enriched microRNAs decreased at 30°C. In addition, we investigated the expressions of MyoD and myogenin during mouse satellite-cell activation in single-fiber culture as an in-vivo model, and found that the expression of myogenin, but not of MyoD, was inhibited. These results suggest that skeletal muscle formation can be regulated by temperature, and that the physiological body temperature plays a crucial role in the myogenesis of homothermal animals.

Crinoids, including feather stars, are the most basal group among extant echinoderm classes and share a basic body plan. In spite of their importance for evolutionary developmental study, information on the development of crinoids has been limited, because there are not many species whose spawning season is known, and artificial spawning is impossible. Therefore, it is not easy to obtain fertilized eggs of crinoids. We have observed the spawning and development of the feather star Oxycomanthus japonicus for 7 years. We have established a cultivation system that has enabled us to culture large numbers of O. japonicus from eggs through to sexually mature adults. In the present study, we show that (1) individuals take 2 years to reach sexual maturity; (2) the skeleton of the theca of a stalked juvenile consists of five orals, five basals, five radials, five infrabasals, and an anal plate; and (3) the onset of spawning has shifted by about two weeks since 60 years ago. Our cultivation system can provide enough embryos, larvae, juveniles, and adults for further experiments, extending the possibilities for crinoid research.

In the present study, morphological examinations, crossing experiments and molecular analyses were performed to elucidate the degree of genetic divergence and phylogenetic relationships within the genus Fejervarya from Bangladesh and other Asian countries. Morphological characteristics revealed that Fejervarya species from Bangladesh were divided into four distinct groups: large, medium, small, and mangrove types. Crossing experiments indicated the involvement of three reproductive isolating mechanisms: gametic isolation between the large type and mangrove type, hybrid inviability between the large type and two other types, and hybrid sterility between the medium and small types. Experimental results also indicated that these four types of frogs merit the status of individual species of Fejervarya. Molecular analyses based on mtDNA gene sequences showed that the Bangladesh Fejervarya species were largely divided into three groups: the mangrove type, large type, and others, with the last further subdivided into the medium and small types. Comparison with other Asian Fejervarya species revealed that the Bangladesh mangrove type (which resembled F. cancrivora in morphology) was closely related to F. cancrivora from India, Thailand, and the Philippines; the large type belonged to the F. iskandari group and closely resembled F. orissaensis; the small type was included in the South Asian or Indian group, and was closest to F. syhadrensis from India and Sri Lanka, whereas the medium type was most closely related to F. limnocharis from Myanmar among all described species of this genus.

The British and Japanese subspecies of the cabbage butterfly, Pieris rapae, differ in terms of the UV reflectance of their wings (Obara and Majerus, 2000). We studied the biogeographical distribution of the female cabbage butterfly having wings with UV reflectance around the Eurasian continent, and between Britain and Japan. For the study, we collected specimens from various locations. A gradient in the UV reflectance of the wings appears to exist along the west-east axis; reflectance was higher toward the east and reached a peak in butterflies in Japan. The UV-reflecting Japanese subspecies Pieris rapae crucivora was found exclusively along the east coast of the Eurasian continent. This suggests that the Japanese subspecies has evolved from a continental ancestor, with females having UV-absorbing wings. We discuss the results of our study with regard to the evolution and adaptive significance of UV coloration in the Japanese subspecies.

There are two karyotypes among Japanese populations of the leaf beetle Chrysolina aurichalcea. In “Type 31” beetles, the diploid number of chromosomes is 2n=32 in females and 31 in males, while in “Type 41” individuals, the diploid number of chromosomes is 2n=42 in females and 41 in males. Both karyotypes occur allopatrically or parapatrically in the wild, depending on location; generally, Type 41 beetles inhabit mountainous areas and Type 31 beetles occur on lowland plains. To assess genetic differentiation among C. aurichalcea populations and between the two karyotypes, we collected 606 individuals throughout Japan, determined the karyotypes of 203 individuals, and examined sequence variation in the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene for all 606 individuals. Karyotypic analysis indicated that hybrids between the two karyotypes are rare. A haplotype phylogeny shows that neither karyotype is monophyletic. These results may be explained by ancestral polymorphism and/or interkaryotype hybridization. Population genetic analysis showed that genetic divergence between the two karyotypes is relatively low, and similar to variation within karyotypes. Our data suggest that chromosomal changes occurred rapidly and recently. We also discuss the phylogeography of this species and the association between speciation and chromosomal rearrangements.

The brown anole (Anolis sagrei) occurs naturally in various localities in Central America, and an exotic invasive population was first reported in Sheishan District, Chiayi County, Taiwan, in 2000. Previous studies showed that following the invasion of A. sagrei, the diversity and abundance of local terrestrial arthropods, such as orb spiders and arboreal insects, were severely affected. In this study, we assessed the impact of A. sagrei on arthropod diversity in Taiwan by comparing spider and insect diversities among betelnut palm plantations, in which this lizard species was either present or absent, and a secondary forest. In addition, enclosures were established in which the density of A. sagrei was manipulated to investigate the effect of this predator on spiders. The results of a lizard stomach content analysis showed that spiders comprised 7% and insects 90% of the prey consumed. Among the insects consumed by A. sagrei, more than 50% were ants. The abundances of the major arthropod prey of A. sagrei, such as jumping spiders and hymenopterans, in the lizard-present sites were much lower than in the lizard-removed sites. The enclosure experiments also showed that predation by the lizards significantly reduced the abundance of jumping spiders. All these results indicated that the introduced lizard greatly affected the diversity and abundance of terrestrial arthropods in agricultural areas in southern Taiwan.

When hemocytes of two different individuals of the solitary ascidian Halocynthia roretzi come into contact (allogeneic recognition), they devacuolate in several seconds following contact, release phenoloxidase (PO) into the supernatant, and form coagulates. These coagulates show brown pigmentation. This reaction is referred to as the contact reaction (CR). In this study, the CR-inhibitory monoclonal antibody ku-4-96, which inhibits devacuolation, increase in PO activity, coagulation, and pigmentation, was constructed. This antibody is thought to exert its inhibitory action at an early stage in the CR. A differential display analysis was conducted by using ku-4-96 to search exhaustively for differentially expressed genes involved in the CR. One of the genes cloned was downregulated in the presence of ku-4-96 and upregulated during the CR. This gene showed very high similarity to the Cl^– channel gene ClC-2 and was named HrClC-2. We examined the effects of Cl^– channel inhibitors on the CR to examine whether the Cl^– channel was involved in the CR signal cascade. Devacuolation, coagulation, and pigmentation were not affected by different concentrations of these inhibitors, which inhibited PO activity. This suggests that the PO activity is independent of these other phenomena occurring during the CR.

This study combined nearly complete 28S and 18S rRNA gene sequences (>4100 nt long) to investigate the phylogenetic relationships of basal hexapods (Protura, Collembola, and Diplura). It sequenced more 28S genes, to expand on a previous study from this lab that used 18S plus only a tiny part of the 28S gene. Sixteen species of basal hexapods, five insects, six crustaceans, two myriapods, and two chelicerates were included in the analyses. Trees were constructed with maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded consistent results: (1) Hexapoda was monophyletic and nested in a paraphyletic Crustacea, and Hexapoda was divided into Entognatha [Collembola Nonoculata (Protura plus Diplura)] and Insecta (=Ectognatha), but the Nonoculata clade must be accepted with caution because of its strong nonstationarity of nucleotide composition. (2) Within Diplura, the monophyly of Campodeoidea and of Japygoidea were supported respectively, and all methods united Projapygoidea with Japygoidea. (3) Within Protura, Sinentomidae was the sister group to Acerentomata. (4) Within Collembola, the modern taxonomical hierarchy of Collembola (Poduromorpha, Entomobryomorpha, Symphypleona and Neelipleona) was confirmed.

The fruit fly, Drosophila melanogaster, shows a bimodal circadian activity rhythm with peaks around light-on and before light-off. This rhythm is driven by seven groups of so-called clock neurons in the brain. To dissect the multioscillatory nature of the Drosophila clock system, the process of reentrainment to a reversed light cycle was examined by using wild-type flies and cry^b mutant flies that carry a strong loss-of-function mutation in cryptochrome (cry) gene. The wild-type flies showed that the morning peak dissociated into two components, while a substantial fraction of cry^b flies exhibited dissociation of the evening peak into two components that shifted in different directions. When the temperature cycle was given in constant darkness in such a manner that the thermophase corresponded to the previous night phase, the morning peak also split into two components in wild-type flies. These results suggest that both morning and evening peaks are driven by two separate oscillators that have different entrainability to light and temperature cycles. Examination of the process of reentrainment to a reversed LD in mutant flies that lack some of the four known circadian photoreceptors (compound eyes, ocelli, CRYPTOCHROME [CRY], and Hofbauer-Buchner [H-B] eyelets) revealed that these four photoreceptors play different roles in photic entrainment of the four putative oscillators.

The state of the art of turrid (=Turridae s. l.) systematics is that shells — when they include the protoconch — are reliable species-level identifiers, but inadequate proxies for allocation to genera or subfamilies. Generally, the radula is used for allocation to a (sub)family, but the hypothesis that the radula is a more adequate proxy than the shell for relationships has not yet been tested by molecular data. Species of Xenuroturris may have drastically different radulae, with either “semi-enrolled” or “duplex” marginal teeth, although their shells are very similar or even almost indistinguishable. Molecular data confirm that specimens with different types of radulae constitute different species, but two species of a pair with respectively semi-enrolled and duplex teeth end up being not closely related. However, it is still unresolved whether species with semi-enrolled (=Iotyrris) and duplex teeth (=Xenuroturris) form two supported monophyletic clades. Iotyrris devoizei n.sp. and I. musivum n.sp. are described from Vanuatu, where they occur sympatrically with I. cingulifera and Xenuroturris legitima.

A new solitary entoproct, Loxosomella plakorticola sp. nov., was found on a sponge, Plakortis sp., on a coral reef slope on the western coast of Okinawa Island, Ryukyu Archipelago, Japan. This species has a medium-sized body (up to about 1.2 mm), slender proportion (the stalk is 0.83–1.76 times longer than the calyx), a slug-like foot with a foot gland and foot groove, and 14 to 18 tentacles. Small black pigment granules are visible only in the living stage in the calyx, stalk, and buds. This is the first report of a commensal loxosomatid from the Ryukyu Archipelago and the second species inhabiting sponges reported from Japan. Detailed morphological observations indicate that this species attaches to sponges by narrowing the foot groove; the sponge surface is pinched in the deepest part of the groove, which is free of the cuticle layer but covered by microvilli of epidermal cells. The accessory cells lining the foot groove have long been believed to be gland cells, but they are not gland cells in ultrastructure, at least in this species.