Zoological Science

Although primary olfactory systems in various animals display spontaneous oscillatory activity, its functional significance in olfactory processing has not been elucidated. The tentacular ganglion, the primary olfactory system of the terrestrial slug Limax marginatus, also displays spontaneous oscillatory activity at 1–2 Hz. In the present study, we examined the relationship between odor-evoked spike activity and spontaneous field potential oscillations in the tentacular nerve, representing the pathway from the primary olfactory system to the olfactory center. Neural activity was recorded from the tentacular nerve before, during and after application of various odors (garlic, carrot, and rat chow) to the sensory epithelium and the changes in firing rate and spontaneous oscillations were analyzed. We detected the baseline amplitude of the oscillations and baseline spike activity before stimulation. Odor stimulations for 20 s or 60 s evoked a transient increase in the firing rate followed by a decrease in the amplitude of spontaneous oscillations. The decrease in the amplitude was larger in the first 8 s of stimulation and subsequently showed recovery during stimulation. The amplitude of the recovered oscillations often fluctuated. Odor-evoked spikes appeared when the amplitude of the recovered oscillations was transiently small. These results suggest that the large oscillations could inhibit spike activity whereas the first transient increase in spike activity was followed by the decrease in the oscillation amplitude. Our results indicate that there is a significant negative correlation between spontaneous oscillations and odor-evoked spike activity, suggesting that the spontaneous oscillations contribute to the olfactory processing in slugs.

The tentacular ganglion, the primary olfactory system of terrestrial slugs, exhibits spontaneous oscillations with a spatial coherence. The digit-like extensions (digits) of the tentacular ganglion presumably house the cell bodies of the neurons underlying the oscillations. The present study was designed to identify the anatomical and physiological determinants of these oscillations with a special focus on whether the neurons located in the digits contribute to the coherent oscillations. We recorded field potentials from the spatially separated sites in the digits in the terrestrial slug Limax marginatus. We also simultaneously recorded tentacular nerve to monitor the coherent oscillations. The spatially separated regions in the digits oscillated at the same frequency as the tentacular nerve, indicating a single coherent activity. To study the neural networks underlying the coherent oscillations, we examined the distributions of acetylcholinesterase (AChE)-containing and γ-aminobutyric acid immunoreactive (GABA-ir) neurons. AChE-containing and GABA-ir fibers were found to connect the neurons in a branch of the digits with those in other branches. We also used a vital staining technique with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate to examine the projections of neurons in the digits. Large stained cells were detected in many branches of the digits after placing the dye on one of the cell masses located in right and left sides of the tentacular ganglion. They were detected in the cell masses and in many branches of the digits after placing the dye on a branch of the digits. Our results showed that the slug primary olfactory system has highly interconnected neural networks.

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH₂, homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.

Odors play important roles in the communication of house mice. They release behaviors and prime changes of the physiological conditions of other individuals. In our previous study, we showed that sperm motility was lowered in the subordinate mice comparing with dominant mice. Our hypothesis is that the lowered sperm motility was due to some primer effects by odor substances derived from dominant mice. To test the hypothesis, we destroyed the vomeronasal organ (VNO) of male mice (VNX male) at 5 weeks of age and paired them with intact male mice (Experimental Group). As control group males, intact male mice were kept in pairs (Control Group). At 15 weeks of age, the sperm motility and weights of reproductive organs, and social dominance was analyzed. The subordinate VNX males were found to have high sperm motility comparable to the dominant males. It was suggested that there is male-to-male primer effects, mediated by VNO, that suppress sperm motility of the subordinate mice.

Organizational relationships between endothelial stress fibers and fibronectin fibrils in the developing chick abdominal aorta, from 5th day embryos to 3rd day young chicks, were studied with immunofluorescence and electron microscopy. Stress fibers, axially aligned parallel to the longitudinal cell axis, were expressed in the largely elongated endothelial cells, in embryos older than 8th day of incubation. Fibronectin fibrils in the aortic basal lamina, changed its organizational pattern from the network-like form to the straight bundles arranged parallel to the vessel's longitudinal axis after 9th day of incubation. Such axial alignment was dominant in the matrix beneath the elongated cells containing stress fibers, suggesting the existence of stress fibers may possibly modify the fibronectin's organizational pattern. The vinculin-containing dense plaque, which shaped like as the adhesion plaque in the cultured cells, was located at the ends of or lateral associating sites of stress fibers in embryos older than 8th day stage. The expression of stress fibers, as well as the formation of stress fiber's end plaques, may closely relate to the alignment between the stress fiber and fibronectin fibrils in the extracellular matrix.

Axopodial contraction of the centrohelid heliozoon Raphidiophrys contractilis was induced by mechanical or electrical stimulation. For inducing contraction, extracellular Ca²⁺ was required. The threshold level of extracellular Ca²⁺ was between 10⁻⁶−10⁻⁷ M. The speed of axopodial contraction was faster than 3.0 mm/sec. Re-elongation of axopodia started just after contraction, and its initial velocity was ∼0.30 μm/sec. Electron microscopic observations were carried out using an improved fixative that contained 1 mg/ml ruthenium red and 15 μM Taxol. This fixative prevented artificial retraction of axopodia and resulted in better fixation. A bundle of hexagonally-arranged microtubules was observed in each axopodium, but no other filamentous structures were detected, suggesting that the contractile machinery of axopodia in R. contractilis may be different from that in actinophryid heliozoons in which Ca²⁺-dependent contractile filaments are employed for contraction.

It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 × g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 × g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 × g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.

In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.

The expression of mouse two-cell stage specific genes was studied using the modified DDRT-PCR method, which overcame the paucity of the experimental materials of preimplantation embryos. Embryo tissues equivalent to that of four blastomeres are sufficient for amplification of target genes as visualized using polyacrylamide gel. Sequence analyses and reverse Northern blots indicate that the genes of ATPase 6 and Ywhaz are expressed specifically in two-cell embryos. ATPase 6 is essential for one-cell to two-cell transition and plays an important role in establishment of oxidative phosphorylation, while Ywhaz is related to initiating cellular communication system.

Two DM-domain genes, DMY (sex-determining gene) and DMRT1, have been reported to be expressed in the testis of medaka. In this study, a specific RT-PCR assay was used to determine the expression patterns of DMY and DMRT1 in various tissues of medaka during different stages of development. The results show that the transcripts of both DMY and DMRT1 are present not only in testes but also in several other tissues from fry and adults of medaka.

The honeycomb grouper shows protogynous hermaphroditism. The endocrine mechanisms involved in gonadal restructuring throughout protogynous sex change are largely unknown. In the present study, we investigated changes in the gonadal structures and levels of serum sex steroid hormones during female to male sex change in the honeycomb grouper. On the basis of histological changes, entire process of sex change was assigned into four developmental phases: female, early transition (ET), late transition (LT), and male phase. At the female phase, the oocytes of several developmental stages were observed including gonial germ cells in the periphery of ovigerous lamellae. At the beginning of ET phase, perinucleolar and previtellogenic oocytes began degenerating, followed by proliferation of spermatogonia toward the center of lamella. The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cells throughout the gonad. At the male phase, no ovarian cells were observed and testis had germ cells undergoing active spermatogenesis. Serum levels of estradiol-17β (E2) were high in females in the breeding season, but low in the non-breeding female, transitional and male phase, and those of 11-ketotestosterone (11-KT) and testosterone (T) were low in females and gradually increased in the transitional and male phase. The present results suggest that low serum E2 levels and degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of spermatogenic germ cells are probably the events mediating protogynous sex change in the honeycomb grouper.

Effects of macromolecule synthesis inhibitors on the light-induced phase shift of the circa-dian clock in the photoreceptive pineal organ of a teleost, ayu (Plecoglosus altivelis) were investigated using melatonin release as an indicator. A single light pulse during the early- and late-subjective night delayed and advanced the phase of the circadian rhythm in melatonin release, respectively. During the late subjective-night, protein synthesis inhibitor cycloheximide (CHX) delayed the rhythm while RNA synthesis inhibitor 5,6-dichlorobenzimidazole riboside (DRB) had little effect. Light-induced phase advance was diminished by the treatment of CHX but not by DRB. During the early subjective-night, DRB, CHX, light and combination of these (DRB+light, CHX+light) all phase-delayed the rhythm. There were no additive effects of light and DRB or CHX. These results indicate that macromolecule synthesis is somehow involved in generation of circadian oscillation, and that de novo protein synthesis is required for light-induced phase shift of the circadian clock in the ayu pineal organ.

The entire length of the beard worm, Oligobrachia mashikoi (Pogonophora), including the posterior end, the “opisthosoma” was collected successfully. This species is exclusive to Tsukumo Bay in Ishikawa Prefecture, Japan. Although the portion preceding the opisthosoma was similar to a fine filament, it abruptly assumed a shape similar to a shovel and appeared to be composed of many segmental structures. The number of segments exceeded 50. The dorsal side of the opisthosoma differed from that of the ventral side in morphology. The opisthosoma was equipped with 4 lines of setae arranged longitudinally and a sucker on the tip. When considering the fact that the Family Oligobrachiidae is the most primitive group of pogonophores, the external morphology of the opisthosoma is interesting as it may be reminiscent of the ancestral condition. This is the first report of the opisthosoma in Oligobrachiidae.

The origin and genetic relationships of the Baikal seal, Phoca sibirica, were studied by restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA). Using 17 different six-base recognition restriction endonucleases, we examined 98 Baikal seals, and two other related species, the ringed seal, P. hispida, (n=87), and the Caspian seal, P. caspica, (n=94). Analysis revealed the existence of 87 mtDNA haplotypes in the total of 279 specimens. The haplotypes of each species were divided into different clusters on a dendrogram obtained by UPGMA based on haplotype frequency and mtDNA base substitution. No common haplotypes were found among the species examined. The Baikal seal is much more closely related to the ringed seal than the Caspian seal. The amount of divergence suggested that an ancestor of the Baikal seal came down to the lake approximately 0.4 million years ago as was previously indicated by paleontological studies. The seals examined here showed lower variabilities.

Phylogenetic relationships between two sibling species of Japanese tideland snails, namely, Batillaria multiformis from the Japanese Islands and B. flectosiphonata from the Ryukyu Islands, were analyzed on the basis of the nucleotide sequences of the mitochondrial gene for cytochrome oxidase I. Populations of B. multiformis were genetically distinct from those of B. flectosiphonata with the exception of a population from Amami-Oshima Island, which corresponded to the boundary between the distributions of these two species. Individuals with the mitochondrial gene of B. multiformis and those with the mitochondrial gene of B. flectosiphonata were collected from the same tidal flat on Amami-Oshima Island. All the snails with the mitochondrial gene of B. multiformis could be divided into two genetically distinct groups but there was no geographical structure to the distribution of these two groups. Individual populations of B. flectosiphonata in the Amami, Okinawa, Miyako and Yaeyama insular groups each consisted exclusively of a unique set of haplotypes, with the exception of a population at a northern site on Okinawajima Island, which included a few individuals with sequences related to those of individuals in the Amami insular group. All individuals from South Ryukyu formed a well-supported monophyletic group, while the monophyly of individuals from Central Ryukyu was not supported. The monophyly of B. multiformis was clearly demonstrated but there was no evidence to support that of B. flectosiphonata. Batillaria multiformis might have been derived from immigrants from the Ryukyu Islands, which became isolated and diverged genetically on the Japanese Islands.