The present study used an immunohistochemical technique to examine the expression of cadherins in the regenerating retina of adult newts. After surgical removal of the neural retina, retinal pigment epithelial (RPE) cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, retinal cells originating from RPE cells are undifferentiated precursor cells. Both E-cadherin and R-cadherin are expressed at the surface of these precursor cells. As regeneration proceeds, precursor cells differentiate into retinal neurons. R-cadherin is expressed at the surface of the differentiated neurons, but E-cadherin is lost to the differentiated neurons. The difference in expression pattern suggests that each cadherin plays distinctive roles in retinal regeneration. And our finding that Ecadherin is expressed transiently by undifferentiated precursor cells implies the importance of cell-cell adhesions by E-cadherin for differentiation.

There are four pairs of neurosecretory cells producing bombyxin, an insulin-related peptide, in the brain of Bombyx mori. In the present study, electrical signals of the bombyxin-producing cells were identified and short- and long-term activity patterns of the cells throughout the pupal period were analyzed. Activity pattern of the cells was characterized by periodic discharges of a volley of action potentials. Periodicity of the electrical activity depended on the temperature and mean±SD of periods was 34.7±4.8 min at 26°C. Four cells in a brain hemisphere were functionally heterogeneous: individual cells had specific activity amplitudes and rhythms, thereby suggesting that an ultradian activity rhythm of a cell originates from its intrinsic pacemaker activity. There was no significant diel (circadian) oscillation in the electrical activity. Daily activity patterns had a maximum at an early stage (day-2 or 3) of pupal period. Periodic secretion of bombyxin, like insulin, seems to be an efficient temporal pattern for hormonal actions.

It has been reported that amphibians can smell not only airborne odorants but also amino acids. It is not clear, however, whether the signal transduction pathway of the amino acid responses is same as that of volatile odorant responses. In this study, we use patch-clamp recordings of newt olfactory receptor neurons to show that amino acid (200 μM glutamic acid, acidic; 200 μM arginine, basic; 200 μM alanine or cysteine, neutral) responses are accompanied by inducing depolarizing currents. Moreover, responses to both amino acids and forskolin, a stimulator of adenylate cyclase, were observed in the same cells, which indicates that the cells responding to amino acids possess the cAMP-system. In addition, our EOG (electro-olfactogram) studies show that forskolin attenuates not only responses to volatile odorants, but also those to amino acids. These data provide evidence that the cyclic AMP system might underlie the signal transduction pathway of amino acid responses in addition to volatile odorant responses.

The general background hue of the skin of the domino damsel (Dascyllus trimaculatus; Pomacentridae) is velvet-black, but three white spots, one on the median parietal region and the other two around the base of the dorsal fin, form a characteristic, conspicuous pattern for this species. In the dark background region, dermal melanophores form a dense monolayer, while in the white spots, a thick accumulation of non-dendritic iridophores is present in the dermis, which is lined with a layer of melanophores. The melanophores in the velvet-black area extend their processes parallel to the plane of the skin, and respond to various stimuli by aggregating or dispersing their melanosomes, which result in the hue changes there. Iridophores in the white spots contain many piles of thin light-reflecting platelets which are responsible for the whiteness of the spots through the multilayered thin-film interference phenomenon. In response to certain stimuli, the spectral reflectance peak shifts to some extent, and such responses may be due to simultaneous changes in the distance of platelets in those piles. The melanophores underlying the white spots extend their cellular processes through spaces among iridophores in order to cover them. When melanosomes migrate peripherally into the tips of the dendrites, they cover the underlying iridophores and the spot darkens; the whiteness reappears when the melanosomes move back down the dendrites into the perikarya of the melanophores.

Butterflies of both sexes have two pairs of extraocular photoreceptors on the genitalia. Here we demonstrate in female Papilio xuthus that a pair of the genital photoreceptors, P1s, is crucial for oviposition. Mated females of Papilio lay eggs on citrus leaves. When a female finds a food plant of the larvae, the female lands on its leaf and curls the abdomen often pushing out the ovipositor. As soon as the ovipositor touches the leaf surface, the female deposits an egg and glues it on to the leaf. We observed the oviposition behavior of individuals whose P1s or the mechanoreceptors on the ovipositor were ablated. Females treated in either way could no longer lay eggs, although they actively curled the abdomen and pushed the leaf, often quite strongly, with exposed ovipositor. This indicates that the females first confirm whether the ovipositor is sufficiently pushed out by using the P1 response as the measure, and then they deposit an egg in response to the mechanical stimulation of the ovipositor.

In Drosophila ananassae, female remating with respect to productivity and sperm displacement was studied by employing different mutant strains and a wild type strain. In all the experiments, the continuous confinement technique was used. The comparison of productivity between once-mated (control) and remated females reveals that the productivity of remated females is significantly higher than that of once-mated ones in all the crosses. The P2′ values (the proportion of second male progeny produced after remating) were calculated to test sperm displacement in each cross of remated females. In all the crosses, high P2′ values (0.91–0.94) were found which indicate sperm precedence of second male to mate suggesting the existence of sperm displacement in D. ananassae. Furthermore, female productivity is increased after remating in D. ananassae.

Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes.

The blood cells of a solitary ascidian, Halocynthia roretzi exhibit the allogeneic cellular reaction in vitro denoted as the “contact reaction”(Fuke, 1980). Nine cell types have been recognized in the blood and body fluid of H. roretzi (Fuke and Fukumoto, 1993). In the present study, the precise role of each cell type in allogeneic reactions is investigated in vitro. The vacuolated cells show devacuolation after contact with almost all other cell types from different reactive animals. These cells include hyaline amoebocytes, granular amoebocytes, macrogranular cells, small amoebocytes and giant cells (large basophilic cells), as well as vacuolated cells. The hyaline amoebocytes and small amoebocytes, which belong to the phagocyte series exhibit contact reactions with the cells of phagocyte series from other specimens of H. roretzi. They also show a contact reaction when in contact with granular amoebocytes. Therefore, almost all cell types have the materials responsible for individuality on their cell surface and can directly show the allogeneic cellular reactivity when they contact each other. To determine whether the contact reaction is involved in cell death, the loss of plasma membrane integrity was examined using fluorescent dyes. The number of cells showing uptake of ethidium bromide increased immediately after mixing of allogeneic cells. Almost the same cell types as those showing allogeneic behavior by light microscopy, as described above, exhibited loss of cell membrane integrity.

These results are discussed in the context of immune systems in invertebrates and vertebrates.

Five enzymes involved in the generation and scavenging of reactive oxygen species, i.e., NADH/NADPH oxidase, xanthine oxidase (XOD), superoxide dismutase (SOD), ascorbate peroxidase, and catalase were assayed in various tissues of the Japanese monkey. Their activities were largely different between tissues. Generally, small intestine, kidney, and cerebellum contained larger amounts of these enzymes than other tissues. Multiplicities of these enzymes were analyzed by staining of their enzymatic activities after electrophoresis. The number of isozymes was 2 in the case of NADPH oxidase and catalase, and 3 in the case of XOD, SOD, and ascorbate peroxidase. The expression of these isozymes differed between tissues, suggesting the occurrence of tissue-specific systems to generate and scavenge reactive oxygen species in the Japanese monkey.

In this study, primordial germ cells (PGCs) in zebrafish were described histologically using eosinophilic granules as a marker. PGC-like cells (PL-cells) with eosinophilic granules were identified initially at the sphere stage (4-hr postfertilization), and were observed until the bud stage, the earliest stage to which PGCs with proper morphology could be traced. The morphology and distribution of eosinophilic granules in PL-cells changed during epiboly. Mitoses of PL-cells were observed only from the shield to bud stage, after eosinophilic granules aggregated to the perinuclear region in PGCs. These shifts of eosinophilic granules corresponded histologically to those of germ plasm described in Xenopus. These results suggest that eosinophilic granules represent germ plasm in fish and that PL-cells with these granules correspond to the PGCs or presumptive PGCs (pPGCs).

Yolk-free follicle cells during final meiotic maturation were incubated with a radioactively labeled steroid precursor, and the maturation-inducing activity of the steroid metabolites produced was tested in an in vitro assay based on GVBD. Of the metabolites obtained by TLC, whose purity and final characterization were confirmed by recrystallization to constant specific activity, two steroids, 17,20β-P and 20β-S, exhibited maturation-inducing activity in vitro. However, 20β-S was overwhelmingly more effective and faster at inducing GVBD in vitro than 17,20β-P. During final oocyte maturation in the tiger puffer, a shift in steroidogenic enzymes, a decrease in C17,20-lyase activity, and an increase in 20β-HSD activity, were found, as previously reported in salmonids and medaka. In addition to this shift in steroidogenic enzyme activity, however, high and increasing 21-hydroxylase activity throughout all the oocyte developmental stages was a distinctive feature in tiger puffer follicles. This 21-hydroxylase activity likely enables ovarian follicles to accumulate enough 11-deoxycortisol during vitellogenesis for 20β-S production on a massive scale during GVBD. Thus, this study provides not only evidence on the physiological role of 20β-S as a MIH in the tiger puffer, but also fact on enzymatic kinetics in 20β-S biosynthesis.

Application of 20-hydroxyecdysone (20E) or juvenile hormone analogue (JHA) to the last (fourth) larval instar of a recessive trimolter Bombyx strain induced extra larval ecdysis. The ecdysteroid titer, the PTTH-secreting activity of the brain, and the ecdysteroid-secreting activity from the PG were compared among the untreated and 20E- and JHA-treated recessive trimolter. The ecdysteroid titer in the hemolymph and ecdysteroid secretory activity of the PG decreased in the mid-fourth larval instar in untreated control larvae, while those in 20E- and JHA-treated larvae did not decrease during the same stage. An application of JHA enhanced the PTTH-secreting activity of the brain. Results indicate that the recessive trimolter strain of Bombyx mori lost the responsiveness of the PG to PTTH in the early stage of the fourth larval instar, resulting in precocious metamorphosis. Results also indicate that the application of 20E induced extra larval ecdysis by maintaining substantial levels of ecdysteroid in the hemolymph. However, JHA is suggested to induce larval ecdysis through activation of the brain and maintenance of the responsiveness of the PG to PTTH.

The organization of the gonadotropin-releasing hormone (GnRH) system originating from the terminal nerve (TN) neuron in the olfactory bulb was examined immunohistochemically by tracing the changes in the distribution of salmon type (sGnRH) and chicken-II type (cGnRH-II) GnRH fibers after olfactory tract section (OTX) in the female goldfish. Following OTX, which blocks the axonal transport of GnRH from the TN to other brain regions, the density of sGnRH-ir fibers in various brain regions decreased from 7 days to 28 days, with the exception of rather restricted areas surrounding cell bodies in the ventral telencephalon, preoptic area, and hypothalamus. The density of cGnRH-II-ir fibers decreased only in the telencephalon from 7 days to 14 days. In spite of the decrease of GnRH-ir fibers in several brain areas, neither type of GnRH fibers showed marked changes in the pituitary gland during the experiment. These results indicate that the TN GnRH system project fibers to wide brain areas and that most of sGnRH and some of the cGnRH-II-ir fibers in the brain of goldfish are TN origin. Furthermore, the GnRH system that project fibers to the pituitary does not primarily originate from the TN-GnRH system.

The Japanese spined loach, Cobitis takatsuensis, has some unique morphological and ecological features among Japanese Cobitis species. Mitochondrial DNA analyses were conducted to investigate the magnitude of intraspecific differentiation and phylogenetic relationships among Japanese congeners of C. takatsuensis. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis of the ND1 region with 16 restriction enzymes was carried out for thirteen C. takatsuensis populations throughout the species' distributional range. Populations in each river system possessed their own haplotypes, with marked genetic differentiation between the populations from Chugoku and Kyushu (ChugokuKyushu group) and those from Shikoku (Shikoku group). The two allopatric groups also showed different color pattern. Subsequently, sequencing analysis of part (725 bp) of the cytochrome b gene was carried out for C. takatsuensis and six other closely-related Japanese species. The phylogenetic tree indicated the presence of two major mtDNA lineages within Japanese Cobitis. It was noteworthy that the Chugoku-Kyushu and Shikoku groups of C. takatsuensis were included in separate mtDNA major lineages, and each group was closely related to other species. It is inferred that the distinct mtDNA relationship between the two allopatric C. takatsuensis groups is a result of the parallel evolution or mtDNA introgression, rather than divergence by geographic isolations.

An investigation was made of the morphology and life history of an unidentified botryllid ascidian that was first collected from the stony shore of Shikine Island of the Izu Islands in Japan. This species has a very soft and transparent tunic, which is different from that of other botryllids. The arrangement of ovary and testis in this species is the same as those of other species of Botryllus. The embryo develops in the sac-like brooding organ formed in the peribranchial cavity of a zooid. Some colonial parts of significant size that include only the vascular system (i.e., without zooids) often extend from the colony margins, and in these parts vascular budding often occurs. Then, the processes and features of the allorecognition reaction of this ascidian were observed. Allorejection occurred after fusion of the vascular systems between two incompatible colonies. This feature is also observed in Botryllus scalaris, but it takes this ascidian much longer to initiate the allorejection reaction than B. scalaris after fusion of blood vessels between two incompatible colonies. It was concluded, on the basis of this study, that this ascidian should be designated as a new species belonging to the genus Botryllus.

The presence of monomer and dimer subunits was revealed, by means of native polyacryl-amide gel electrophoresis (PAGE), in examined arachnid hemocyanins. We determined the N-terminal amino acid sequences of nine monomer subunits prepared from hemolymph of a whipscorpion, Typopeltis crucifer, and a primitive spider, Heptathela kimurai, and two constituent monomers of a dimer subunit from the whipscorpion. Based on a comparison of the sequences, we confirmed that the orthologous hemocyanin subunits are shared between the whipscorpion and the scorpion, Liocheles australasiae, between the primitive spider and the scorpion, and among the whipscorpion, the scorpion, and mygalomorph spiders. This study is the first to demonstrate the presence of orthologus hemocyanin subunits in different orders. Furthermore, it is evident that one of the constituent monomers of the hemocyanin dimer from the whipscorpion is orthologous to the constituent monomers (the group G subunits) of the hemocyanin dimers in mygalomorph spiders and to the subunit LA8 (a constituent monomer of a dimer subunit) of the scorpion, suggesting that these constituent monomers of arachnid hemocyanin dimers originated from a common ancestral gene which existed in a common ancestor of these arachnids.

Eleven adult females of the viviparous scorpion, Liocheles australasiae, parthenogenetically repeated pregnancies and parturitions up to three times under the separate rearing. Only two of these females experienced the fourth parturition after the fourth pregnancy; one was dissected before the fifth pregnancy and the other early in the fifth pregnancy. In the ovaries of all these females, there were large empty ovarian diverticula, which had lost their embryos by the last parturition, and small ones as remnants of the previous parturitions. The number of all the empty ovarian diverticula in each female was roughly equal to the total number of her neonates. Even in the female in the fifth pregnancy, there were young ovarian diverticula containing oocytes other than the large ones containing embryos. The number of the young ovarian diver-ticula in the female seemed enough for one or two additional pregnancies, but further pregnancies and parturitions should become difficult because of deterioration of conditions for embryonic development under the long rearing and/or by the maternal ageing.