Zoological Science

Bone morphogenetic protein-4 (BMP-4) has been shown as an essential factor in differentiation of the primitive blood cells in Xenopus laevis embryo. Organizer factors, in contrast, function as a negative regulator for the blood cell differentiation. Differentiation of both blood cells and muscle tissue are negatively regulated by the organizer activity. However, blood cells but not muscle tissue can differentiate in the organizer-depleted embryos produced by removal of vegetal cortex cytoplasm at the one-cell stage. Thus the blood cells are totally independent cells from the organizer activity. The down-stream target molecules of the BMP-4 signaling, such as vent-1/2 and msx-1/2 are the positive regulators for muscle tissue differentiation, whereas these factors do not promote blood cell formation. It has not yet been elucidated how the BMP-4 signaling promotes the differentiation of blood cells, but it is likely that transcription factors such as GATA-2, SCL, LMO-2, biklf and GATA-1 are at least involved in the initial blood program. Experiments using combined germ layer explants suggest that interaction between ectoderm and mesoderm at the gastrula stage is important for the blood cell formation in mesoderm. BMP-4 produced in the ectodermal cells is essential for this interaction. For understanding the whole program in the embryonic blood cell differentiation, it is important to elucidate the molecular mechanisms underlying the tissue-tissue interaction, in addition to the analysis of the regulatory cascade that takes place in the mesodermal cells.

During the last five years, enormous progress has been made in understanding the molecular basis of circadian systems, mainly by molecular genetic studies using the mouse and fly. Extensive evidence has revealed that the core clock machinery involves “clock genes” and “clock proteins” functioning as molecular cogs. These participate in transcriptional/translational feedback loops and many homologous clock-components in the fruit fly Drosophila are also expressed in mammalian clock tissues with circadian rhythms. Thus, the mechanisms of the central clock seem to be conserved across animal kingdom. However, some recent studies imply that the present widely accepted molecular models of circadian clocks may not always be supported by the experimental evidence.

Diapause hormone (DH) originally identified to be a factor originating from neurosecretory cells in the suboesophageal ganglion acts on developing ovaries to produce diapause eggs in a female silkmoth, Bombyx mori. A male silkmoth has homologous neurosecretory cells, but little is known of the physiological nature of the cells and actions of their products. We examined the long-term firing activity of putative DH-producing neurosecretory cells and hormonal activity of their products in male pupae that had been experienced different environmental regimens for diapause induction. Firing activity patterns of male labial cells strongly depended on diapause types of pupae: cells in a diapause-type male were active throughout the pupal period, whereas the same cells in a non-diapause-type male were usually inactive during the early two-thirds of the pupal period. A male pupa with electrically active labial cells could induce diapause eggs in a female pupa connected parabiotically to that male. The firing activity of male neurosecretory cells and hormonal action of their products are qualitatively the same as in the female previously examined. We suggest that there is no evident sexual dimorphism in the physiological and biochemical nature of neurosecretory cells producing DH and the amidated peptide DH has different functions in a male.

Influences of photoperiod, temperature and melatonin were examined on development and color patterns of nymphs, and sternum color and reproductive fate of adults in Halyomorpha halys (brevis). Short-day accelerated nymphal development, whereas long-day accelerated reproductive maturation. The two types of photoperiodic responses at different stages may help maintain the univoltinism of this species in the field, assuring the right timing for diapause and reproduction. The pronotum of fifth instar nymphs reared under LD 11:13 shows a brown-marbled color pattern darker, with less creamy-yellowish speckles, than that of nymphs reared under LD 16:8. Short-day-reared fifth instar nymphs, which are destined to diapause in adults, had shorter white stripes on the pronotum, smaller body size, less frequent feeding and more lipid accumulation than the long-day-reared. The longer the exposure to LD 13:11 at 20°C during the nymphal stage, the greater the expression of short-day associated characteristics observed in the fifth nymphal instar and adult stage. Melatonin orally administered (500 μg/ml) to insects under LD 16:8 produced slightly reduced body size, suppressed feeding, extended nymphal period and increased accumulation of lipid compared to the untreated bugs, but did not affect the incidence of diapause in females. In males, melatonin retarded gonadal development under LD16:8. These results may suggest a possible role of melatonin for the control of seasonal polyphenism and development.

There are three known clusters of neurosecretory cells expressing a gene encoding dia-pause hormone (DH) and four related peptides in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings were made from the axonal tract (NCC-3) of a pair of cells localized in the labial (posterior) neuromere of SOG during pupal-adult development. There was a significant difference in firing activity patterns of the labial neurosecretory cells between diapause-egg and non-diapause-egg producers: labial cells in the former were active throughout pupal-adult development, whereas the same cells in the latter usually maintained an inactive state until the last quarter of pupal-adult development, a time at which a secretion of DH seems to be too late to act on the developing ovary for the induction of dia-pausing eggs. This observation strongly supports the notion that labial cells release DH and are responsible for determination of embryonic diapause in the silkmoth.

A gene encoding a precursor polyprotein of diapause hormone (DH) and four related peptides is expressed by three groups of neurosecretory cells in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings of firing activities were made from a common axonal tract (the maxillary nerve) of two groups of cells localized in the mandibular and maxillary neuromeres of SOG during pupal-adult development. Mandibular and maxillary cells usually produced a cluster of action potentials at an interval of 30–60 min during pupal-adult development and there was no significant difference in the firing activity profile between diapause-egg and non-diapause-egg producers. We suggest that rather than DH secretion, pupal mandibular and maxillary cells are involved in the secretion of DH-related neuropep-tides. DH secretion seems to be assigned to the third group of cells (labial cells); hence, there may be different posttranslational processing of the precursor polyprotein in different neurosecretory cell groups.

The starfish Asterina pectinifera of the family Asterinidae is endemic Japanese species and commonly found in Japanese waters. In order to examine the degree of genetic variation and the main-tenace mechanism of polymorphism within population, we studied the allozyme variation in five northern Japanese local populations of the starfish by electrophoresis. The species showed much higher genetic variability than many other shallow water echinoderms. Based on other allozyme studies and the ecological data, it was suggested that the high genetic variation of the starfish was closely related to the population size. Additionally, the relation between the degree of enzyme variation and the quaternary structure of enzymes was also examined, and the results suggested the close relation between the enzyme variability and functional constraints.

Ayu (Plecoglossus altivelis) is a teleost whose gonadal development is stimulated by shortened daylength and is a useful model to study the mechanism of photoperiodism. However, localization and characteristics of the photoreceptor that mediates photoperiodism in gonadal development remain to be determined. To identify the photoreceptive molecule that regulates photoperiodic responses, in the present study, we have cloned and characterized the cDNA encoding an opsin gene expressed in the ayu brain, a putative site of the photoreceptor for photoperiodism. The identified opsin was rhodopsin that is identical to the rhodopsin expressed in the retina. Phylogenetic analysis demonstrated that this rhodopsin belongs to the retina-type but not to the pineal-specific rhodopsin group. Genomic polymerase chain reaction (PCR) demonstrated that the ayu rhodopsin gene is intron-less. Southern and Northern blots and reverse-transcription PCR analyses indicate that the same rhodopsin gene is expressed in the retina and the brain but not in the pineal organ of ayu. These results indicate that the rhodospin gene is expressed in the retina and brain and mediates not only visual but also nonvisual functions such as photoperiodism and entrainment of the circadian clock.

In order to determine the titer of molt-inhibiting hormone (Prc-MIH) in the hemolymph of the American crayfish Procambarus clarkii, a time-resolved fluoroimmunoassay (TR-FIA) was established using specific antibodies against N-terminal and C-terminal segments of Prc-MIH. The lowest limit of detection of Prc-MIH in TR-FIA was 10 amol/assay. The Prc-MIH titers in the hemolymph were 6.53 fmol/ml at the intermolt stage and 1.25 fmol/ml at the early premolt stage. This result is consistent with the long-known hypothesis that the Y-organ is inhibited by MIH during the intermolt stage, whereas the Y-organ is activated by being freed from the inhibitory regulation of MIH.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuro-modulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunore-active PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.

Daily variations in plasma melatonin levels in the rainbow trout Oncorhynchus mykiss were studied under various light and temperature conditions. Plasma melatonin levels were higher at mid-dark than those at mid-light under light-dark (LD) cycles. An acute exposure to darkness (2 hr) during the light phase significantly elevated the plasma melatonin to the level that is comparable with those at mid-dark, while an acute exposure to a light pulse (2 hr) during the dark phase significantly suppressed melatonin to the level that is comparable with those at mid-light. Plasma melatonin kept constantly high and low levels under constant darkness and constant light, respectively. No circadian rhythm was seen under both conditions. When the fish were subjected to simulative seasonal conditions (simulative (S)-spring: under LD 13.1:10.9 at 13°C; S-summer: under LD 14.3:9.7 at 16.5°C; S-autumn: under LD 11.3:12.7 at 13°C; S-winter: under LD 10.1:13.9 at 9°C), melatonin levels during the dark phase were significantly higher than those during the light phase irrespective of simulative seasons. The peak melatonin level in each simulative season significantly correlated with temperature but not with the length of the dark phase employed. In addition, the peak melatonin level in S-autumn was significantly higher than those in S-spring although water temperature was the same under these conditions. These results indicate that the melatonin rhythm in the trout plasma is not regulated by an endogenous circadian clock but by combination of photoperiod and water temperature.

Patterns of roost use by Formosan leaf-nosed bats (Hipposideros armiger terasensis) were studied from November 1998 to April 2000. Structural characteristics, microclimates, and disturbance levels of 17 roosts used by H. a. terasensis and 15 roosts either used by other bat species (2) or not occupied by any bat species were compared. Roosts used by these bats were significantly larger in size and had greater areas covered by water compared to unused roosts. Entrances of active roosts were more likely to be east-west oriented. Hibernacula had lower entrances and ceilings than did roosts used only in summer.

Higher temperatures were recorded in non-breeding roosts than in breeding roosts, but temperature gradients in these two types of roosts did not differ. In winter, hibernacula were warmer, and the temperature fluctuated less than in non-hibernacula. The relative humidities in summer roosts and hibernacula were nearly 100%. Disturbance levels were significantly higher in non-breeding roosts than in breeding roosts, and in non-hibernacula than in hibernacula.

These results suggest that the Formosan leaf-nosed bats are selective of their roosts, but the pattern of their roost selection differs from those reported for bats of temperate regions. The reasons for such differences may be related to differences in body size, behavior, and reproductive strategy of the Formosan leaf-nosed bats living in a subtropical climate in Taiwan.

The endostyle is a pharyngeal organ of Urochordata, Cephalochordata and larval Cyclostomata. This organ secretes mucus-proteins for internal filter feeding, a feeding system that must have developed in the common ancestor of these subphyla. Therefore, the endostyle is a key structure to understanding the origin and evolution of chordates. A previous study of the overall gene expression in Ciona intestinalis young adults yielded several candidates for ascidian endostyle-specific genes. In the present study, we determined in detail the expression profiles of six novel endostyle-specific genes. Ci-VWFL1 and CiVWFL2 encode related proteins similar to vertebrate von Willebrand factor, and were continuously expressed in zones 4 and 2 of the developing endostyle, respectively. The expression of Ci-Ends8 was observed in the entire region of zone 6 in young adults; however, the expression of this gene was restricted to the dorsal- and ventral-regions of zone 6 in the adult endostyle. The expression of Ci-Ends9 and Ci-Ends10 was observed in zones 6 and 4 in young adults, respectively, and was downregulated in the adult endostyle. Ci-Ends11 showed an expression pattern similar to that of Ciona TTF-1, which encodes a thyroid-related transcription factor. The predicted amino acid sequence of Ci-Ends10 showed similarity to Trip230, and that of Ci-Ends11 resembled Ptp4E. These molecules might be useful for further analysis of the development, function and evolution of the endostyle.

The scleractinian coral Goniastrea aspera (Verrill) undergoes both broadcast spawning and planulae brooding in the Ryukyu Archipelago of southern Japan. Genetic variation and gene flow in G. aspera were studied using allozyme electrophoresis. We tested the hypothesis that gene flow is determined by the competency period of the planulae. We also assessed the relative contributions of sexual and asexual reproduction to recruitment. For the five staining systems surveyed, G. aspera encoded five polymorphic loci and one monomorphic locus. The genotype frequencies in each population significantly differed from the expected Hardy-Weinberg equilibrium (HWE), indicating that the local populations of G. aspera are not fully panmictic. The high ratio of the observed number of genotypes to the number of individuals (0.90±0.07, mean N_G :N±SD) and the observed to expected genotypic diversity (0.84±0.11, mean G_O :G_E ±SD) suggested that each population is likely maintained by sexual reproduction. The genetic differentiation (F _ST) and value of average number of migrants per generation (N _e m) among and within regions ranged from 0.025 to 0.104 and 2.2 to 9.6, respectively. Comparisons with other species demonstrated that larva survival rates also influence gene flow. In addition, gene flow on distant reefs by planulae originating from spawning might prevent divergence by planulae originating from brooding for short-distant dispersal among and within populations of G. aspera in the Ryukyu Archipelago.