Zoological Science

We examined quantitative changes in biogenic amines in relation to effects of population density on growth and behavior in the cricket Gryllus bimaculatus, which were reared in two different densities, completely isolated solitary and 40 crowded insects. In the crowded animals, the rate of increase in body weight was slow, and also the day of imaginal moult was late, when compared to the isolated ones. Development and growth were thus suppressed in the crowded group. The body color of isolated ones was darker than that of crowded ones. Behavioral tests indicated that the isolated crickets were significantly more aggressive than the crowded ones. The levels of biogenic amines, their precursors and metabolites were examined in the brain, corpora allata, corpora cardiaca and frontal ganglion of isolated and crowded crickets using high-performance liquid chromatography with electrochemical detection. The brains of crowded crickets contained significantly higher amounts of octopamine, dopamine, and 5-hydroxytryptamine (5-HT) than those in the isolated ones. On the contrary, the level of N-acetyldopamine in the isolated crickets was significantly higher than that in the crowded ones. In crowded animals the corpora allata contained significantly higher levels of 5-HT, and the corpora cardiaca contained more octopamine. These results indicate that high population density affects aminergic systems which in turn modulate various biological events, such as development, growth and behavior.

Immunocytochemistry of diapause hormone (DH) or pheromone biosynthesis activating neuropeptide (PBAN) revealed three clusters of neurosecretory cells present on the ventral midline of the suboesophageal ganglion (SG) of the silkworm: there were 4 cells in the anterior, 6 cells in the medial, and 2 cells in the posterior cluster. Intracellular injection of Lucifer Yellow into an anterior or a medial cell revealed that the cell has a dendritic arborization at the anterior region of the SG and that it projects an axon to the corpus cardiacum (CC) via a branch of the maxillary nerve and an associated nerve of the CC (NCC-V). A dye-filled posterior cell in the larva projects bilaterally-symmetric dendritic branches to the anterior half of the SG that is to expand laterally to fuse the brain after pupation. The axon of the cell, passing through the brain, enters the NCC 3 to spread varicose terminal branches in the CC and associated nerves of the CC.

The distribution of RFamide-like immunoreactivity was examined in the terminal abdominal ganglion (TAG)-related peripheral nervous system, and in the visceral tissues of the female cricket, Gryllus bimaculatus. In the TAG, most of the RFamide-like immunoreactive (RFaLI) neurons were bilaterally paired, and were found in the neuromeres of the 7th to the 10/11th segment. The remaining neurons were found in the midline region. These included anterior unpaired neurons, a cluster of neurons in the posterior end, and ventral paired neurons in the 9th segment. Immunocytochemistry combined with Lucifer Yellow back-filling indicated that the immunoreactive paired medial neurons innervated the rectum. In the visceral tissues innervated by the TAG, RFaLI fibers were seen in the oviducts and rectum, while the spermathecal duct and spermatheca did not exhibit any immunoreactivity. In the peripheral nervous system, RFaLI peripheral neurosecretory cells (PNCs) were found on the fifth and seventh segmental nerve roots of the TAG in both sexes. The perikaryon of the PNCs contained several immunoreactive large electron-dense granular vesicles. Intracellular dye injection showed that the PNCs probably form neurohaemal-release sites for RFamide-like peptide. The PNCs did not project any processes into the TAG.

We have identified and characterized the hygroreceptive sensilla on the antenna of the adult male firefly, Luciola cruciata, by coupling extracellular electrophysiological recordings from single sensilla with observation of morphology of the sensilla using a field emission scanning electron microscope (FE-SEM). Seven morphologically different types of sensilla were present on the antenna: pored chaetic, poreless chaetic, basiconic, trichoid, capitular and campaniform sensilla and a new type of sensillum. To determine which is the hygroreceptive sensillum, responses to humidity changes were electrophysiologically recorded from each type of these sensilla. Impulses from moist and dry receptor cells could be obtained from only the capitular sensillum along with impulses from a cold receptor cell. The results clearly showed that the capitular sensillum is hygro- and thermoreceptive. The capitular sensillum had a cuticular apparatus that extends about 8 μm above the antennal surface and is externally composed of three parts: an inner stem, an outer wall and a basal dome. This cuticular apparatus was not perforated in any region. About 30 capitular sensilla per antenna were distributed only on the lateral surface of flagellar segments. A new type of sensillum, ‘sensillum gemmiformium’ was found. The cuticular apparatus of the gemmiform sensillum consists of a main spheroidal body and one to five protrusions. We discuss the common external features of the hygro- and thermoreceptive sensilla, the role of hygroreception in the behavior of fireflies and possible functions of the other types of sensilla.

Ascidians have several types of blood cells which have been reported to be involved in various functions. However, it is still unclear what type of blood cell participates in each of various functions and in which tissue each type of blood cell localize in ascidians. As a necessary step to make clear the important and intractable problems, the localization of four different types of blood cells was examined using two kinds of cell marker, monoclonal antibodies and autonomous fluorescence. Consequently, it was revealed that signet ring cells were the only blood cells that were localized in the connective tissues around the alimentary canal and the amoebocytes were mainly distributed in the transverse vessels of the branchial basket. The morula cells and compartment cells were distributed under the epidermis of the mantle around the visceral region.

Retinal cells dissociated from adult newt eyes were seeded onto plastic dishes which were coated with various cell adhesion substances. They were cultured in Leibovitz's L-15 medium with no serum. Concanavalin A (ConA) and poly-L-lysine (PLL) caused relatively good cell attachment, regardless of retinal cell types, in comparison with uncoated dishes. On the other hand, extracellular matrix (ECM) components such as collagen, fibronectin and laminin tended to inhibit the cell attachment.

In both ConA- and PLL-coated dishes, ganglion cells survived for longer periods than other retinal cells and extended neurites with time in culture. The outgrowth of neurites by ganglion cells was found to be promoted to a far greater degree by ConA than PLL. Branching of neurites also appeared to occur more frequently on ConA. From these observations, ConA may be an useful tool for investigating retinal regeneration in vitro.

The frequency and distribution of mitomycin C (MMC)-induced sister chromatid exchanges (SCEs) were investigated in the fibroblast chromosomes of three mammalian species, Microtus montebelli, Apodemus argenteus and Chimarrogale himalayica, by the fluorescence-plus-Giemsa (FPG) and C-band staining methods, paying special attention to the large C-band area (C-block)-carrying and/or nucleolus organizer region (NOR)-carrying chromosomes. The junctions of heterochromatin and euchromatin (HE-junctions) and NORs were found to be “hot spots” of SCEs in all the species examined: their SCE frequencies were 35.3% and 24.2% in the HE-junction of the X chromosomes of M. montebelli and A. argenteus, and 16.7% and 17.8% in the NORs of the No. 1 chromosomes of M. montebelli and C. himalayica, respectively. In M. montebelli and A. argenteus the SCE frequency was apparently lower in the C-block region than in the euchromatic one, when compared with each other based on equal length, while in C. himalayica no such marked difference in the SCE frequency was found between these two regions of the chromosome. These findings may indicate that occurrence of SCEs is significantly suppressed in the C-block region of M. montebelli and A. argenteus, but not in that of C. himalayica. In addition, the C-blocks of the No. 1 homologue of C. himalayica showed a highly varied individual-to-individual heteromorphism in length. The biological implication of SCEs was discussed in connection with the generation of heteromorphism.

The role of morula cells (MC) in the formation of necrotic regions characterizing the rejection reaction between incompatible (i.e. nonfusible) colonies was investigated in the colonial ascidian Botryllus schlosseri. These blood cells share several chemical and histochemical properties with the pigment of the necrotic masses: both of them show strong reducing activities and contain high quantities of sulphur and iron. Sulphur is present mainly as sulphates and thiols in MC and as sulphates and disulphides in necrotic regions; iron is in the form of ferrous iron in MC and as ferric iron in the necrotic regions. Inside MC vacuoles phenoloxidase (PO) activity and polyphenol substrata are present: the latter are oxidized by PO to quinones, which then polymerize to form the melanin-like substances of dark-brown colour of the necrotic regions. When hemocytes are incubated with heterologous incompatible blood plasma (BP), MC change their morphology, and a significant increase in PO activity is found in the recovered medium as compared with the activity of untreated plasma. No increase in PO activity is observed after incubation with autologous or heterologous compatible blood plasma. These results are consistent with the hypothesis of a humoral factor diffusing from incompatible colonies which is recognized by MC and leads to their degranulation, with the consequent release of the content of their vacuoles, mainly oxidative enzymes, responsible for the localized cell death in necrotic regions.

Myosin was purified to a homogeneity from sea sponge, Halichondria okadai. The myosin consisted of 220 kDa heavy chain, 18 kDa calcium binding light chain and 21 kDa phosphorylatable light chain. Rotary shadowed images showed the two headed myosin (myosin II) with a 160 nm tail. The myosin was less soluble in a KCl solution as compared to rabbit skeletal myosin.

The K⁺-stimulated and Ca²⁺-stimulated ATPase activities of sea sponge myosin were 0.46 and 0.07 μmol Pi min⁻¹ mg⁻¹, respectively. The Mg²⁺-activated myosin ATPase activity showed no significant enhancement by the addition of rabbit skeletal muscle actin despite that the light chain was phosphorylated by myosin light chain kinase from chicken gizzard. Sea sponge myosin 18 kDa light chain bound to Ca²⁺ ion but was not phosphorylated like Physarum plasmodia myosin light chains.

An acid thiol protease that was activated at an early stage of embryogenesis was purified from Xenopus embryos. The N-terminal amino acid sequence(16 residues) of the heavy chain of the enzyme was similar to that of cathepsin L. The proteolytic activity of the protease was enhanced by ATP. Other nucleoside triphosphates, AMP and nucleic acids also enhanced the proteolytic activity. The possible mechanism and biological significance of the activation of the protease in Xenopus embryos are discussed.

The rate of DNA synthesis of wing discs and the effects of X ray irradiation and mitomycin C injection were observed during last larval and early pupal stage of Bombyx mori. The rate of DNA synthesis in wing discs of Bombyx mori was estimated by means of the incorporation of ³H-thymidine during the fifth larval stage. Mitomycin C injection and X ray irradiation were performed during the fifth larval instar and pupal stage to investigate the effects of inhibition of DNA synthesis. The rates of ³H-thymidine incorporation were high on days 0, 1, 2, 4, 5, 7, 8 and 10 of the fifth larval instar. Wings of eclosed adults were affected by injections of mitomycin C between the 7th day of the fifth larval instar and the second day of the pupal stage. Exposing the insects to X rays during the fifth larval instar affected wing and scale formation, and at earlier stages, it had a more deleterious effect. Irradiation at 24–36 hours after pupation inhibited wing expansion and scale formation most among pupal stages. The results suggested that the peaks of ³H-thymidine incorporation during the fifth larval instar and the pupal stage have specific significance for wing development.

To elucidate the induction mechanism of abnormal apyrene sperm by high temperature (32°C, 72 hr) treatment during the wandering stage of the silkmoth, Bombyx mori, ecdysteroid titres in the haemolymph and in the testes were determined. The fluctuation of ecdysteroids in the haemolymph was not disturbed by the high temperature treatment. The amount of ecdysteroids in the testes of control animals was low during the two-thirds of the last larval stage and an abrupt increase was observed on day 8. On the other hand, the elevation of ecdysteroid titre was suppressed in the testes of the treated animals during the period of treatment. After pupation, the fluctuation pattern of the ecdysteroid titre was almost the same both in control and treated animals. These results indicate that the deficiency of ecdysteroids in the testes could be the cause of the abnormal differentiation of the apyrene spermatozoa brought about by the high temperature treatment during the wandering stage of the silkmoth, Bombyx mori.

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of female Japanese eel, Anguilla japonica. A single class of high affinity binding sites was found, with a Kd=0.97 nM for the cytosolic estrogen receptor (cER) and Kd=0.85 nM for the nuclear estrogen receptor (nER). Binding of both the cER and the nER was specific for estrogens (diethylstilbestrol: DES>estradiol-17β: E₂>>estriol: E₃>estrone: E₁). These binding characteristics of ERs were quite different from those of the serum estrogen-binding component; [³H]E₂ binding to serum was not saturable, and was displaced by testosterone but not by DES, E₁ or E₃. The relationships between the levels of hepatic ERs, circulating E₂ and vitellogenin (VTG) during artificial maturation of cultivated female eels were examined, using eels injected weekly with chum salmon pituitary homogenate at a dose of 20 μg/g-body weight. Serum E₂ levels were constantly low during pre- to midvitellogenesis, and dramatically increased in the migratory nucleus stage. However, VTG levels gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly increased in early vitellogenesis, and then increased significantly from midvitellogenesis to the migratory nucleus stage. In contrast, nER levels did not change significantly, although nER levels in the migratory nucleus stage were higher than those at other stages. The changes in cER levels represent increased hepatic responsiveness to estrogenic stimuli during artificial maturation. Lack of change in nER levels may be a feature of artificial maturation compared to sexual maturation in nature.

When pea aphids, Acyrthosiphon pisum, were raised on an artificial diet which mimics the amino acid composition of the total tissue of aphids, their intracellular symbionts in the bacteriocyte gradually decreased in number as the host generation proceeded, accompanied with a decrease of the host's fecundity. It is possible that host sacrificed its symbionts to supplement the artificial diet that is nutritionally deficient. This inevitably incurs aposymbiosis, which will, in turn, lead to the host's sterility, as observed repeatedly.

In Bengalese finches, adult male distance calls are narrow-banded in the spectral domain and continuous in the time domain while female calls are wide-banded and pulsed. To examine the degree of syringeal control on this sexually dimorphic behavior, the nerve bundle from the hypoglossal nucleus to the syrinx was cut unilaterally in adult birds under deep Nembutal anesthesia. The sexual dimorphism in the time-amplitude profile of the distance calls did not disappear by cutting neither the left nor the right nerve. On the other hand, the sex difference in the spectral domain of the distance call became more ambiguous by cutting the left nerve but only minor effect was observed by cutting the right nerve. Results suggest that only spectral information is conveyed by the tracheosyringeal nerves and calls of the both sexes are actively controlled by the left tracheosyringeal nerve. Some of these results are in contrast with the zebra finch whose right tracheosyringeal nerve is dominant over the left and the female call does not change by the nerve cut operation.

The green paramecium, Paramecium bursaria, has endosymbiotic algae in the cytoplasm. Here, we report that all endosymbiotic algae are destroyed, producing symbiotic algae-free strains of paramecia, when green paramecia are cultivated in the presence of a herbicide paraquat.

Dendritic chromatophores that contained blue pigmentary organelles were found in the bluish parts of the skin of two callionymid species, the mandarin fish, Synchiropus splendidus, and the psychedelic fish, S. picturatus. We named these novel cells “cyanophores” and the organelles “cyanosomes”. In response to various stimulatory cues, the cyanophores responded by the aggregation or dispersion of cyanosomes. In addition to their role in the revelation of bluish hues, these cyanophores may participate in the changes in shades of blue of these gorgeous fish.

In the early stage of courtship, the sexually developed male newt (Cynops pyrrhogaster) vibrates its tail in front of the female. Administration of androgen and prolactin (PRL) to sexually inert male newts is known to elicit this behavior. The effects of ovariectomy and hormonal treatment on the induction of male-like tail vibration behavior were studied in female newts, which never show such behavior in an intact state. Ovariectomy elicited the behavior with a low incidence and frequency. Estradiol supplementation almost nullified the effect of ovariectomy. Treatment of ovariectomized females with a combination of testosterone propionate and PRL markedly increased the frequency of the behavior.