Zoological Science

On the occasion of the hundredth anniversary of the term “synapse”, gradual progress of the knowledges conducting to the notion of chemical transmission was related. It was particularly stressed how the synapse was understood by the major contributors in this field. The soundness of the choice of the term synapse, instead of syndesm that was proposed at first, was underlined by a comparison of the original meanings of the two words and confirmed by their use in a treatise of Aristotle. It appeared that the notion of humoral immunity influenced the birth of humoral theory of chemical transmission, which was illustrated in the textbook of M. Verworn.

Major contributions to the research on the cholinergic synapse in the last five decades was described. The original notion of active zone, composed of a presynaptic dense projection and associated synaptic vesicles, was analyzed in order to underline its functional meaning in synaptic transmission. An overview was done on the findings made on acetylcholine release (ultrastructural aspect, cytochemistry, non-vesicular hypothesis and mediatophore), acetylcholinesterase (ultrastructural localization, discovery of molecular polymorphism) and acetylcholine receptor (characterization, purification). The structural base of the smallest functional unit in a synapse was attributed to a single synaptic vesicle of active zone and corresponding area of synaptic cleft and postsynaptic membrane interacting with the neurotransmitter released from the vesicle.

An improved photoelectric method for recording motile responses of individual leucophores was developed. Leucophores from scales of the medaka, Oryzias latipes, were used as a model. The intensity of light scattering from the peripheral dendritic zone (S _p) and that from the cell body (S _c) of each leucophore were separately transduced to the current changes, which were then converted to voltages, and appropriately amplified. Output voltages of both channels were represented as “V _p = A₁ × S _p”, and “V _c = A₂ × S _c”, where A₁ and A₂ were the overall amplification coefficients for these channels, respectively. When leucosomes dispersed from the cell body into the dendrites, the V _p increased and the V _c decreased, while the reverse changes occurred when the leucosomes aggregated. Using a simple electronic circuit, the remainder of the two outputs, namely, V _p − V _c, could easily be obtained and recorded. As alternative expressions of the response, the ratio of the outputs (V _p/V _c), its square root ( ), or could also be recorded using integrated circuits developed for such purposes (v _p, v _c: voltage biases applied to V _p and V _c). The relative merits of these types of analysis are discussed. Similar analysis should also be applicable to the motile activities of iridophores of the dendritic type, such as those of some gobiid fish.

Mammalian endothelins (ETs: ET-1, -2 and -3) effectively disperse the light-scattering organelles (leucosomes) in leucophores of the medaka, Oryzias latipes, in a dose-dependent manner. They were almost equally effective, their minimal effective concentrations being less than 100 pM, with EC₅₀ value about 8.3–8.4 nM. Endothelins act directly on the leucophores, since denervated cells responded to the peptides quite similarly. Phentolamine, an α-adrenergic blocker, propranolol, a β-adrenergic blocker, and BQ-123, an inhibitor of mammalian ET_A receptor, did not interfere with the action of ETs. By contrast, BQ-788, an inhibitor of the mammalian ET_B receptor, potently blocked the action of ETs. Sarafotoxin S6c and IRL 1620, both mammalian ET_B receptor-selective agonists, were also found to disperse leucosomes effectively, mimicking the effect of ETs. Thus, ETs may act through the mediation of specific receptors existing in the leucophores. Along with their recently disclosed actions on light-absorbing chromatophores, ETs may play an important role in the delicate and exquisite control of integumentary hues and patterns.

In the isopod Bathynomus doederleini, the cardioarterial valves of all five pairs of lateral arteries and the pair of anterior lateral arteries are innervated by inhibitory (dilator) nerves which consist of one or two axons arising from the central nervous system. Stimulation of the valve dilator nerves produced inhibitory junctional potentials (IJPs) in valve muscle cells which arose one-to-one in response to stimulus pulses. Acetylcholine (ACh) hyperpolarized muscle cells of the valves. Both the IJPs and ACh-induced hyperpolarization brought about an increase of haemolymph pressure in the arteries, through relaxation of valve muscles. The muscarinic agonists, muscarine, carbamylcholine and arecoline, mimicked ACh-induced hyperpolarizing responses of the valve muscle cells. Atropine and methylxylocholine antagonized both the IJPs and ACh-induced hyperpolarizing potentials, while d-tubocurarine did not antagonize IJPs. These results indicate that ACh may be the transmitter for the valve dilator nerves. IJPs did not invert in Cl⁻-free saline. Amplitude of IJPs increased in low K⁺ salines, and decreased in high K⁺ salines. It is likely that IJPs are mediated predominantly by K⁺ ions. This could be the first case of cholinergic inhibitory transmission at neuromuscular junctions in crustaceans.

Planthopper harbors eukaryotic endosymbionts that are essential for normal development and growth of the host. Our previous studies suggested the possibility that the symbionts play an important role in nitrogen metabolism of the host through utilization of uric acid, a nitrogenous waste product. To examine the precise role of the symbiont, we prepared synthetic diets with various concentrations of amino acids, and measured uric acid contents stored in the insects reared on these diets. The results showed that planthopper synthesizes uric acid not only as a waste product, but also as a storage product when it ingests an excess amount of amino acids. We also investigated effects of the uric acid storage on growth of the normal and symbiont-depleted host. It turned out that in nitrogen deficiency the stored uric acid is consumed by the symbiont in order to sustain the growth of the host. In addition, we noted that the uric acid content of the host egg is highest at oviposition, and decreases significantly with its development, suggesting that the egg is supplied with uric acid by its parent prior to oviposition. These results are reminiscent of the nitrogen recycling reported for cockroaches.

The behavioral responses to a wind stimulus were studied in the freely moving cricket, Gryllus bimaculatus. The observed responses included walking, running, jumping, turning, withdrawing, abdominal lifting, hind-leg lifting, kicking, cercal cleaning, antennal swinging, and “no action” and were classified into five behaviors as functional categories: escape, evasion, offense, surveillance and “no action”. The elicitation of each type of behavior by an identical stimulus was variable and unpredictable. However, the stochastic approach showed that the probabilities of the behaviors converged in a series of responses obtained from one cricket and in mass response data collected from 48 crickets. These probabilities, the values calculated from the different populations, were the same. The statistical analysis, using the probabilities, revealed that the probabilities of these wind-evoked behaviors were affected by the intensity of the wind stimulus. The escape and “no action” were dominant, together constituting more than 60% of the total responses. The stimulus method and the animal conditions determining a high probability of escape are also discussed.

C6 glioma cells were arborized by cytochalasin D (2 μM), while the processes induced by the drug were destroyed by colcemid (2 μM). Distribution of actin and tubulin during the arborization was investigated by fluorescence microscopy to identify core structures involved in arborization. There were two types of actin filaments in C6 cells: one was oriented radially and the other was concentric. Cytochalasin D destroyed the actin filaments in sequence and transiently left a radial structure in the early stage (the first 3 min) of arborization. Microtubules were also organized similarly. At a later stage (15 min), few radial actin filaments remained, whereas microtubule bundling proceeded and seemed to support a starfish-like cell morphology. In the last stage (2 hr), most actin-staining was found around the plasma membrane undercoat area, whereas the microtubules were bundled in the core part of the processes. Radial actin filaments, which showed higher stability, seemed to be important collectively in the first step of arborization as a transient organizer of the cytoskeleton. Distribution of α-actinin and vinculin on actin filaments was investigated as potential factors stabilizing the radial filaments because of their affinity for the barbed ends of actin filaments for which cytochalasin D also had affinity. Results showed that α-actinin and vinculin were preferentially located on the radial filaments rather than on the concentric filaments.

Melatonin binding sites in the goldfish retina were characterized by radioreceptor assay using 2-[¹²⁵|]iodomelatonin as the radioligand. The specific binding to goldfish retinal membranes is rapid, stable, saturable and reversible. Saturation studies demonstrated that 2-[¹²⁵|]iodomelatonin binds to a single class of sites with an affinity constant (K _d) of 61.9 ± 5.7 pM, a total binding capacity (B _max) of 6.52 ± 0.79 fmol/mg protein and Hill coefficients (n _H) of 1.07 ± 0.03 (mean ± SEM, n = 6). Competition experiments with various indoles and neurotransmitters revealed the following order of affinities: 2-iodomelatonin > melatonin > 6-hydroxymelatonin > 5-methoxytryptamine = N-acetylserotonin > 5-methoxytryptophol. The other indoles and neurotransmitters tested were much less effective. The order resembles with those reported for the goldfish brain and the ML-1 subtype melatonin receptors in vertebrates. Co-incubation of retinal membranes with a non-hydrolyzable GTP analog, guanosine 5′-O-(3-thiotriphosphate), significantly reduced the specific binding. These results suggest that in the goldfish, ocular melatonin plays neuromodulatory roles in the retina via G protein-coupled melatonin receptors with picomolar affinity.

We cloned the cDNA for Japanese monkey Testis-Specific Protein Y (TSPY). The cDNA contained an open reading frame of 246 amino acids. This coding fragment shared 89% nucleotide sequence identity and 81% amino acid sequence identity with the homologous fragment of previously isolated human TSPY cDNA. Monkey TSPY was assumed to be a molecular mass of 28 kDa and an isoelectric point of pH 5.35. This protein was hydrophilic and contained an Arg and Lys-rich region which was a potential DNA binding site. Expression of the TSPY gene examined by reverse transcription PCR showed that the transcript was detectable only in testis, suggesting that TSPY plays an important role in spermatogenesis of primates.

Bombyxin F1 gene, a new bombyxin family gene, has been identified. The F1 gene forms a pair with bombyxin B10 gene with an opposite transcriptional orientation and the gene pair F1/B10 is located between bombyxin gene pairs B9/C1 and A7/B7 in a bombyxin gene cluster. The nucleotide sequence of the F1 gene and its deduced amino acid sequence deviate moderately from those characterized previously for the family-A, family-b, family-C, family-D, and family-E bombyxin genes; the bombyxin F1 gene and preprobombyxin F1 share no more than 62% and 53% sequence identities with other bombyxin members, respectively. Harr-plot analysis indicated that the spacer of the F1/B10 gene pair has low sequence similarity with that of other bombyxin gene pairs characterized. The bombyxin F1 mRNA in Bombyx mori brain was shown to locate in four pairs of medial neurosecretory cells, which also produce other bombyxin family mRNAs. Genomic Southern hybridization indicated that the Bombyx haploid genome contains a single copy of the family-F bombyxin gene.

An experimental study on sperm entry into the micropyle of the medaka egg was carried out in order to analyze the effect of micropylar size on fertilization. The eggs and micropyles of Oryzias latipes were larger than those of Oryzias melastigma. When eggs of O. latipes and O. melastigma were simultaneously inseminated by spermatozoa of O. latipes, O. melastigma or Aplocheilus panchax, O. melastigma spermatozoa rapidly entered the conspecies eggs as well as the large micropyles of O. latipes eggs, while most of the O. latipes and A. panchax spermatozoa failed to rapidly enter the small O. melastigma micropyles. Moreover, when O. latipes eggs with a small or a large vestibule were simultaneously inseminated by O. melastigma spermatozoa, the size of the micropylar vestibules (range of 17-23 μm in diameter) also affected rapid sperm entry into the egg. However, this effect was not recognized when the eggs were inseminated by conspecies spermatozoa. On the other hand, when O. latipes eggs were inseminated by a mixture of O. latipes and O. melastigma spermtozoa or O. latipes and A. panchax spermatozoa, the faster swimming spermatozoa fertilized significantly more frequently than the slower O. latipes spermatozoa. These results suggest that rapid sperm entry into the micropyle is not only affected by differences in the morphology and size of the micropyles, but also might be accelerated by the linear swimming velocity of the spermatozoa.

Light irradiation, at a light fluence rate sufficient for the strong photo-reactivation of the CO-inhibited cytochrome c oxidase in mitochondria isolated from the sperm of fish, oyster and sea urchin, weakly activated the CO-inhibited respiration only in the sea urchin sperm, with peaks of photo-reactivation corresponding to those in the absorption spectrum of CO-bound cytochrome aa ₃. NADH cytochrome c reductase was inhibited by CO, weakly in mitochondria from sea urchin sperm and completely in those from fish and oyster sperm. The CO-induced complete inhibition of cytochrome c reduction in fish and oyster sperm probably does not allow the photo-reactivation of CO-inhibited cytochrome c oxidase to augment CO-blocked respiration. At a light fluence rate higher than that mentioned above, photo-activation of NADH cytochrome c reductase, found in the sperm of oyster and sea urchin, occurred even in the presence of CO in mitochondria isolated from sea urchin sperm and strongly activated CO-inhibited respiration in sea urchin sperm, with peaks corresponding to those in the absorption spectrum of reduced cytochrome b. The acceleration of cytochrome c reduction due to the photo-activation of this complex enzyme in sea urchin sperm probably induces another activation of CO-inhibited respiration at the high light fluence rate.

A simple method for determination of the early stages of nymphs of the cricket, Gryllus bimaculatus, was presented. The width of the head-capsules was found to be a useful value to determine the nymphal stages. On this evidence, the testes from 3rd- to 6th-instar nymphs were investigated and histologically analyzed. The number of spermatogonia rapidly increased between the 3rd- to 5th-instars. Primary spermatocytes entered into meiotic prophase at the latest 2 days after ecdysis into the 5th-instar.

In order to establish a reliable and reproducible method for cryopreservation of medaka spermatozoa, we tested several procedures. The vapor phase of liquid nitrogen (LN), the liquid phase of LN, and dry ice were used for freezing, and dimethyl sulfoxide and N, N-dimethylformamide (DMF) were used as cryoprotectants. The best results were obtained using the following method. Medaka spermatozoa were collected in a plastic tube containing 50 μl of fetal bovine serum supplemented with 10% DMF by squeezing an isolated testis. The sperm suspension was frozen by holding the tube for 10 or 20 min in the vapor phase of LN at a depth of 9 or 10 cm from the top of a container. The frozen sample was immersed and stored in LN. After more than one week of storage, the sample in the tube was rapidly thawed by being incubated in a waterbath for 0.5–1 min at 30°C, and then immediately diluted with 2 volumes of Iwamatsu's solution. Fertilization tests using fresh unfertilized eggs showed that the stored spermatozoa could fertilize 96–100% of the eggs. The hatchability of the fertilized eggs was 84-100%. Thus, this study provides, for the first time, a practical method for preserving medaka spermatozoa.

Pituitary adenylate cyclase activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal polypeptide family of peptides, consists of a 38-residue (PACAP 38) and a truncated 27-residue (PACAP 27) form that play several roles in mammals. Recently, we isolated a PACAP-like peptide with a sequence highly homologous with that of tetrapod PACAP from the brain of a teleost, the blue spotted-stargazer (Gnathagnus elongatus). In this study, a PACAP-like peptide obtained from the brain of the stargazer, Uranoscopus japonicus, was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis using an anti-PACAP 27 serum. The stargazer PACAP-like peptide and synthetic human PACAPs were reacted with this antiserum. The distribution of PACAP-like immunoreactivity in the stargazer brain was also studied immunohistochemically using the peroxidase-antiperoxidase method. PACAP-like immunoreactive (LI) cells were found in the nucleus preopticus, pars parvicellularis of the hypothalamus. PACAP-LI nerve fibers and terminals were observed in the anterior neurohypophysis and in the rostral pars distalis, and many PACAP-LI nerve fibers were seen in the medulla oblongata. These results suggest that PACAP-like peptide may be involved in pituitary regulation and/or other neural functions in the stargazer brain.

Hormonally induced changes in synaptic inputs to Mauthner cells (M cells) were examined in adult male red-bellied newts, Cynops pyrrhogaster. Hypophysectomized, castrated newts were treated with saline, prolactin, testosterone propionate or both prolactin and testosterone propionate every other day for three weeks. The medulla oblongata containing M cells was examined at ultrastructural level. In the hypophysectomized, castrated newts treated with prolactin, testosterone propionate or both prolactin and testosterone propionate, the mean number of synapses onto somata of M cells were significantly larger than that in the hypophysectomized, castrated animals treated with saline. There were no differences in the parameter among the prolactin-, testosterone propionate- and both prolactin and testosterone propionate-treated animals. These findings suggest that prolactin and androgen are critical for maintaining reorganization of synaptic inputs to somata of M cells in the adult male newts.

The relationship of the development of uterine adenomyosis with the changes in the cell arrangement and cell membrane permeability, and incidence of DNA fragmentation was examined in mice. In uterine areas showing the invasion of endometrial tissues into musculature, rhodamine-phalloidin staining for actin fibers revealed that the stromal cells ran parallel to the direction of the infiltration, and the muscle cells lost their regular arrangement, unlike those in the normal uteri of control mice. Inner myometrium showed positive fluorescence with Evans blue, which is known to stain only cells with increased membrane permeability. Outer myometrium also became fluorescence-positive, when the disease was advanced to severe state bearing invasion of endometrial tissues into the outer myometrium. No fluorescence with Evans blue was observed in the normal uterine tissues. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL method)-positive nuclei were exclusively found in some smooth muscle cells near both the blood vessels and tip of the invading endometrial tissue. These findings indicate that in adenomyotic uteri, apoptotic cell death developed in certain cells in myometrium, though disrupted cell arrangement and increased cell membrane permeability occurred in almost all of the inner myometrial cells. The increased membrane permeability in the myometrial cells might participate in the local occurrence of cell death near the blood vessels. Therefore, endometrial tissue would invade the myometrium through the space produced by the cell death along the blood vessels.

Using a double immunostaining technique, we examined the relationships between neuropeptide Y (NPY)- and urotensin I (UI)- and/or urotensin II (UII)-positive structures in the caudal spinal cord of various species of fishes, particularly in the teleost Oncorhynchus masou and in elasmobranchs Scyliorhinus torazame, Triakis scyllium, and Raja kenojei. Primitive actinopterygians, i.e., Acipenser transmontanus, Polypterus senegalus, and Lepisosteus productus, and cyclostomes Lethenteron japonica and Eptatretus atami were also studied from the viewpoint of comparative endocrinology. In the teleost and elasmobranchs, NPY-positive fibers were demonstrated to be often in contact with UI- and/or UII-positive cells and fibers, suggesting a control of the caudal neurosecretory neurons by NPY or a related substance. Similar association was seen in the primitive actinopterygians. As to the cyclostomes, immunoreactivities for NPY and UII were found in the lamprey, but none of the immunoreactivity for these peptides was seen in the hagfish. In the lamprey, NPY-positive varicose fibers abutting on the UII-positive cells were also demonstrated.

Radfordia (Microtimyobia) mites infesting arvicoline voles in Japan comprise three new subspecies of Radfordia (M.) lemnina (Koch, 1841). These new subspecies are defined on the basis of the form of the male genitalia, the shape of the anterior gnathosomal setae, and the chaetotaxy of the female tritonymph. Radfordia (M.) lemnina hata ssp. n. is a parasite of Microtus montebelli (Milne-Edwards, 1872). Radfordia (M.) lemnina mikado ssp. n. infests only Clethrionomys rutilus mikado Thomas, 1905 in Hokkaido, and it differs from Radfordia (M.) lemnina rutila Fain and Lukoshus, 1977 parasitizing Clethrionomys rutilus (Pallas, 1979) in Europe. Radfordia (M.) lemnina japonica ssp. n. is found widely on the other Clethrionomys and Eothenomys voles in Japan. Among these host voles, Clethrionomys rufocanus bedfordiae Thomas, 1905 is designated as the type host of this new subspecies. Immature stages of the three new subspecies are described and illustrated. Radfordia mites parasitic on Eothenomys voles in Taiwan and Japan are much more different from each other than would be expected on the basis of the current systematics of the Arvicolinae proposed by Japanese mammalogists.

Fine structure of the ascidian tunic cuticle was surveyed for minute protrusions in 30 species of the suborder Aplousobranchia, 10 species of Phlebobranchia and 23 species of Stolidobranchia (Ascidiacea, Chordata). The present results, combined with previously published data, describe the fine structure of the tunic cuticle in 116 species of ascidians covering all the families and subfamilies of the class Ascidiacea except for the phlebobranch families Octacnemidae and Plurellidae. These data confirm the general stability of the character-state distribution (presence or absence) of the tunic cuticular protrusions within families or subfamilies. Cuticular protrusions occur only in Polyclininae and Clavelininae of the order Enterogona (Aplousobranchia and Phlebobranchia) and in all families of the order Pleurogona (Stolidobranchia and Aspiraculata). A few exceptions were the occurrence of cuticular protrusions in Polycitor proliferus (Polycitorinae), Distaplia dubia (Holozoinae), Pterygascidia longa (Cionidae) and Adagnesia vesiculiphora (Agneziidae), and the absence in some styelids and pyurids that have hard tunics. Possible character-state changes in the ascidian phytogenies are discussed briefly.

Balaenophilus umigamecolus n. sp., the second species known at present in the aberrant harpacticoid family Balaenophilidae, has been collected from the neck skin of a juvenile loggerhead sea turtle Caretta caretta in the aquarium of the Kushimoto Marine Park Center, Wakayama Prefecture, Japan. This is the first record of copepods occurring on a sea turtle. This new species differs from Balaenophilus unisetus, which has been recorded only as an associated species on baleen plates of baleen whales, in its small size, the number of apical claws of leg 1, the setal arrangement of leg 4, and the length of the caudal rami. Gut contents indicate that the copepod scrapes the skin of the host sea turtle directly for food.

Wolbachia, a member of rickettsia found in the cells of many arthropod species, are cytoplasmically inherited bacteria which interfere with host's sexuality and reproduction. Wolbachia strains have been phylogenetically divided into A and B groups based on the nucleotide sequences of their ftsZ genes. In an attempt to further define the phylogenetical relationship among these endosymbionts, we cloned and sequenced the entire length of the groE operon of a Wolbachia harbored by a cricket. The operon encoded two heat shock proteins, which represented the third and fourth proteins of any Wolbachia ever characterized. Also, 800 bp stretches of the groE operons of several other Wolbachia were sequenced, and a phylogenetic tree was constructed based on the results. The groE tree defined the relationship among A group Wolbachia strains that had not been successfully resolved by the ftsZ tree, and suggested unexpected horizontal transmission of these bacteria.