Zoological Science

In anuran amphibians transitions of hemoglobins (Hbs) and red blood cells (RBCs) from the larval to the adult type have been reported to occur at metamorphosis, depending on certain influence of thyroid hormones (THs). Contrary to this, transition of RBCs/Hbs from the larval to the adult type during the metamorphosis in a urodele, Hynobius retardatus occurs almost independently of thyroid activity, but dependent on certain pituitary factor(s). All findings reported so far support the idea that the Hb switching in H. retardatus occurs in a single RBC population (“Hb switching” model), rather than the concept that larval RBCs are replaced by new, adult RBCs (“RBC replacement” model) as is known to occur in many anurans. Erythropoiesis in vertebrates occurs with two distinct phases, termed primitive and definitive. Primitive erythropoiesis generally provides embryonic/larval erythroids, and definitive hematopoiesis contributes to adult RBCs. Primitive erythropoiesis in Xenopus laevis occurs in the ventral blood island (VBI), and the dorsolateral plate (DLP) cells remain undifferentiated until later for definitive hematopoiesis. H. retardatus embryos also have two distinct hematopoietic sites, the VBI and DLP. The DLP cells of H. retardatus, however, differentiate in situ to RBCs containing larval globin, suggesting that both the VBI and DLP contribute to “primitive” erythropoiesis. Some DLP cells may be set aside in an undifferentiated state during embryogenesis for future “definitive” erythropoiesis coming to express only adult globin during metamorphosis. A tentative model was proposed to explain similarities and dissimilarities in erythropoiesis and conversion of RBCs/Hbs between anurans and urodeles.

The position of oxygen equilibrium curve (OEC) for human adult Hb at rest is optimized with respect to the effectiveness of the Bohr shift which is measured by the change in O₂ saturation at a venous O₂ pressure of 40 torr per unit change in partial pressure of O₂ at half saturation (dS₍₄₀₎/dP₅₀). The effectiveness of the Bohr shift at the physiological P₅₀ of 27 torr depends on the cooperativity of O₂ binding calculated by the Hill coefficient n, being maximized at n = 4.

The effectiveness of the Bohr shift during exercise, which was expressed as dS(PvO2)/dP₅₀, was the highest at the PvO₂ (venous O₂ pressure) of 28 torr. The effectiveness of the Bohr shift at PvO₂ of 28 torr increased with increases n value (n value ranged from 2.65 to 3.27), while below PvO₂ of 15 torr the opposite was true.

As a whole, the position of the OEC of human adult Hb at rest is optimized with respect to the effectiveness of the Bohr shift while the efficiency of O₂ delivery is moderately maintained. On the other hand, during exercise the position of the OEC is adjusted to make the efficiency of O₂ delivery at high levels while the effectiveness of the Bohr shift is maintained at the same level as that at rest.

The egg-jelly of C. pyrrhogaster is composed of six layers: the J0, J1, J2, J3, J4 and sticky layers (from the innermost to outermost). In this study, we investigated the localizations of carbohydrate moieties in the egg-jelly using the FITC-conjugated N-acetylgalactosamine/galactose, glucose/mannose, Nacetylglucosamine and fucose group lectins. Eight of nine kinds of tested lectins belonging to every group were bound to the J1 and J3 layers, while Dolichos biforus agglutinine (DBA) were bound only to the J0 and the J2 layers. Though Triticum vulgaris agglutinine was bound to all the four layers, the binding was inhibited only in the J0 and J2 layers by N-acetylglucosamine. Five kinds of lectins belonging to every group except for the fucose group uniformly bound to the J4 layer, while DBA and Arachis hypogaea agglutinine did not. Ulex europaeus agglutinine and Glycine max agglutinine bound only to the inner portion of the layer. These results suggest that the inner four layers are composed of two types of layers and that the J4 layer has a unique carbohydrate composition. In the oviduct, most lectins bound to the regions where the egg-jelly layers bound by the lectins were added. However, DBA bound to the anteriormost portion of the regions where the layers next to those bound by DBA was added. These results suggest that carbohydrate components of egg-jelly layers are fundamentally added in the corresponding regions of the oviduct while some molecules may be added after the matrices of the layer have already been accumulated.

Motility of trout sperm is suppressed by extracellular K⁺ at rest and initiated by the decrease in the extracellular K⁺ concentration, and Ca²⁺ and cyclic AMP participate in the cell signaling for the initiation of sperm motility. We showed here that tetraethyl ammonium (TEA) and some blockers used as the voltage-dependent K⁺ channel blockers in the neuron inhibited the motility in the sperm of the rainbow trout and the steelhead trout, Oncorhynchus mykiss. Other types of K⁺ channel blockers, e.g., apamin, a blocker of small conductance Ca²⁺-dependent K⁺ channels, did not inhibit sperm motility. L-type Ca²⁺ channel blockers such as nifedipine, nimodipine, etc. also inhibited the motility, but other types of Ca²⁺ channel blockers did not. On the other hand, the gradual increase in external K⁺ concentration caused both gradual decreases in the amplitude of hyperpolarization of the sperm plasma membrane and cyclic AMP synthesis. TEA and nifedipin suppressed both hyperpolarization and cyclic AMP synthesis, and these suppressions were relieved by addition of the K⁺ ionophore, valinomycin. The calmodulin inhibitors W-7, trifluoroperazine and calmidazol-Cl inhibited the sperm motility, membrane hyperpolarization and cyclic AMP synthesis, although only at rather high concentrations. These results suggest that K⁺ efflux through K⁺ channels and Ca²⁺ influx through Ca²⁺ channels that are sensitive to specific channel blockers cause the changes of membrane potential and lead to a synthesis of cyclic AMP in the cell signaling for the initiation of trout sperm motility.

Complementary DNAs (cDNAs) encoding follicle-stimulating hormone (FSH) β, luteinizing hormone (LH) β and glycoprotein hormone (GPH) α subunits were isolated and characterized from ayu pituitary using a PCR technique. The FSH β, LH β and GPH α subunit cDNAs were found to be 556, 588 and 621 base pair (bp) long, encoding 384, 432 and 357bp long open reading frames, respectively. The deduced amino acid sequences of the putative mature forms of ayu GPH α and LH β subunits were on average 63% homologous with those of other teleosts, whereas ayu FSH β subunit was on average 42% homologous. These results show that ayu has two different types of gonadotropins (FSH and LH), as in other teleosts. FSH β subunit mRNA was mainly detected during early vitellogenesis and spermatogenesis. In contrast, LH β subunit mRNA was detected during the late phase of gonadal development, suggesting that the primary function of FSH may be to initiate gametogenesis, while LH may have a role in the development of gametes.

The bambooleaf wrasse, Pseudolabrus sieboldi, is a diandric protogynous fish with a diurnal rhythm of ovarian development, and females spawn daily during the spawning season. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in vitellogenic ovarian follicles of the bambooleaf wrasse. We incubated follicles in vitro with radioactively labeled steroid precursors, and measured serum steroid levels using microtiter plate enzyme-linked immunosorbent assays (ELISAs). ELISAs for estrone (E1) and testosterone (T) were developed. The experiments showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, dehydroepiandrosterone, androstenedione, and E1. T was not produced from any radiolabeled precursors, and exogenous T was not converted to E2. During the spawning season, serum levels of E2 and E1 showed similar patterns, with a diurnal rhythm of high levels at 03:00 hr associated with active ovarian vitellogenic follicles. In contrast, serum T levels were constant and relatively low compared to levels of E2 and E1. These results indicate that E2 is synthesized via E1, rather than T, in the ovarian follicles, and suggest that T detected in the blood is likely derived from extra-follicular tissues.

Response to stress during the spawning period of sockeye salmon (Oncorhynchus nerka) was investigated by measuring cortisol, selected steroids and glucose levels in the plasma of cultured fish under acute confinement conditions. Fish were individually placed in nets and sacrificed to obtain blood samples after each confinement period: 0, 3, 5,15 and 30 min, respectively. In males, acute stress increased cortisol levels in the plasma at 15 min and testosterone, 11-ketotestosterone and glucose levels in the plasma within 3 min. In females, the cortisol level was higher than that in males and did not significantly change at 15 and 30 min. Testosterone levels in females decreased at 15 min in confinement. Progesterone, 17-hydroxyprogesterone and 17,20 β-dihydroxy-4-pregnen-3-one levels did not change with confinement time in females. To know the reproductive stage of the fish used in the stress experiment we collected blood and measured sex steroid hormones in different fish from the same pond one week and one day before the experiment. In these fish, the sex steroids in males and females changed with the pattern of breeding. Cortisol levels were higher in females than in males for one week. This study demonstrates that the cortisol level in females is higher than in males during their spawning period, and that females might be less sensitive to changes in the level of cortisol caused by acute stress than are the males.

The growth-retarded (grt) mouse, originally isolated from a closed colony of Snell's dwarf mouse (DW/J strain), shows growth retardation that is inherited in a recessive manner. We have already reported that this strain exhibits severe primary hypothyroidism with significantly reduced plasma levels of thyroxine (T₄), dramatically elevated plasma titers of thyroid-stimulating hormone (TSH) and an increase in the number of immunoreactive TSH cells in the pituitary gland. The thyroid gland of the grt mouse exhibits characteristically numerous smaller follicles with poor colloid accumulation. In order to elucidate the possible site of the defect in the grt mouse, and in particular to clarify the discrepancy between elevated plasma TSH titers and reduced T₄ levels, we examined the bioactivity of TSH recovered from the plasma sample of the grt animal and the responses of the grt thyroid gland to exogenous TSH and other thyroid hormone secretagogues. Plasma samples from the grt mice invariably exhibited significant levels of TSH bioactivity following injection into normal test mice. Thus, the reduced responsiveness to TSH exhibited by the grt mice is not due to the reduced bioactivity of TSH. Administration of exogenous TSH to the grt mice failed to elevate the plasma T₄ and triiodothyronine (T₃) levels in vivo or to stimulate free T₄ and free T₃ releases from the grt thyroid gland in vitro. The thyroid gland of the grt mouse exhibited a markedly diminished response of adenylate cyclase to exogenous TSH as compared to the gland of euthyroid littermates. Production of cAMP in the grt mouse was significantly increased following stimulation of the thyroid glands with forskolin, cholera toxin, prostaglandin (PG) E₁ and isoproterenol. These results strongly suggest a defect in TSH responsiveness, particularly in TSH-TSH receptor-Gs protein-adenylate cyclase signalling system including the expression and the function of TSH receptor and the TSH receptor-Gs protein coupling, in the thyroid gland of the grt mice.

Ambient temperature exerts strong influences on physiological processes in ectothermic animals. Thus, we have examined the effects of changes in water temperature on drinking and arterial blood pressure in seawater-adapted eels, Anguilla japonica. Temperature dependence was also examined with respect to the effects of atrial natriuretic peptide (ANP) on drinking and arterial pressure. When water temperature was altered abruptly from 18°C to 11°C or to 25°C, drinking rate consistently decreased or increased, respectively, in all fish examined. The temperature-response relationship was highly linear (p<0.001) between 11°C and 25°C with a Q₁₀ value of 2.45. Arterial pressure was significantly lower at 11°C compared with the level at 18°C, but no difference was observed between 18°C and 25°C. The change in hematocrit did not parallel that of temperature. Plasma Na⁺ concentration did not change at different temperatures. Bolus injections of eel ANP at 10, 100, and 1000 pmol/kg decreased drinking rate and arterial pressure dose-dependently. The effects of ANP did not differ in terms of potency and efficacy at different temperatures except that the effects continued longer at lower temperatures. These results show that the ambient temperature causes profound effects on drinking rate in seawater eels even though Na⁺ balance and blood volume were well maintained. The sensitivity to ANP, a physiological regulator of drinking in eels, was not altered by the temperature, suggesting an involvement of other factors in the thermogenic drinking that is demonstrated in this study.

Respiration rates of six free-living nematode species dominant in the subtidal coarse-sand habitat of Otsuchi Bay, Northeastern Honshu, Japan, were measured discriminating sex and adult or juvenile. To measure the respiration rate, each nematode was put in a temperature-controlled microchamber of 45 μl in volume, and the dissolved oxygen concentration of the seawater was monitored for more than 3 hrs. Measurements were done at 5, 10, 15, and 20°C, covering the yearly temperature range of the habitat. Metabolic intensities (M.I.) of these species at 20°C were within the range of values previously reported for other nematode species. Juveniles of Mesacanthion sp. had the highest M.I. among the species measured, reflecting the carnivorous nature of the species. For Polygastrophora sp.., Mesacanthion sp., Metachromadora sp.. and Monoposthia sp.., M.I. decreased at lower temperature, and the Q₁₀ value was close to 2. On the other hand, M.I. values of Symplocostoma sp.. and Theristus sp. were independent of the change of temperature and the Q₁₀ value was close to 1. For Symplocostoma sp.., Polygastrophora sp.. and Mesacanthion sp., M.I. of male was higher than that of female. Males of all these species were rare and may need to be more active than females, to copulate with many females.

The physiological cost of parental care and the necessity of parental care for larval growth were examined in the burying beetle, Nicrophorus quadripunctatus, by removing adult pairs during the first reproduction and allowing them to reproduce again. When the reproduction was interrupted after hatching of the first clutch, the number and mass of the second clutch did not decrease as the interruption was performed later. These results demonstrated that the physiological cost of parental care was relatively small. The parental care for one day or less after hatching was necessary for the growth of progeny larvae. Parental care later than one day after hatching influenced neither survival nor growth of progeny larvae. Moreover, progeny larvae grew normally with either a male or female parent. Nevertheless, all parental females and most males stayed with their progeny until the larvae finished feeding. The evolution of parental care in N. quadripunctatus will be discussed along with both its cost and benefit.

A unique scale worm Hesperonoe hwanghaienssi Uschakov and Wu, 1959 (Polychaeta: Polynoidae) was collected from tidal flats in Japan and is described here taxonomically as the second record of this species since its original description from Chinese specimens. Some morphological features of our specimens differed slightly from the original description of this species. Marked differences in growth pattern, morphology of elytra and microhabitat were demonstrated between juveniles (1.8 mm or less in body width) and adults (1.9 mm or more).

We estimated the outline of the life history of the scale worm by sampling it in various seasons. The scale worm was commensal with the burrowing shrimp Upogebia major. Juveniles of the scale worm were commonly attached to the ventral or lateral surface of the thorax or abdomen of the host throughout May to September in Isahaya Bay in the Ariake Sea, Kyushu. In July 1998, 89% of the host shrimps were infested by the scale worm. Several juveniles (maximum: 7) of the scale worm often aggregated on a host body, with no adults found on the hosts. The adults were collected from sediment samples. These results suggest that the juveniles mainly live on the surface of the host body and that they later detach themselves from the host body and live freely on the inner surface of the burrow of the host.

The taxonomy of the leucosiid crab genus Merocryptoides Sakai, 1963 is revised, and two new species, M. ohtsukai and M. peteri are described from Japan. These new species resemble the type species of the genus, M. frontalis Sakai, 1963 from Japan, but can be distinguished from it by the form of the cardiac tubercle, male abdomen, male telson and first male pleopods. Merocryptoides ohtsukai is also distinguished from M. peteri by the broadly V-shaped ridge on the gastric tubercle, the lateral margin of the hepatic region not ridged and the form of the median tooth of the epibranchial margin.

A detailed analysis was done on the karyotypes of four species of mole in the genera Euroscaptor and Mogera using a G-banding technique. All four species examined had a chromosome number of 2n = 36, as reported previously. Euroscaptor mizura and M. wogura from Aichi Prefecture had almost the same chromosome constitution and G-banding patterns. These common karyotypes were considered to be equivalent to the hypothetical ancestral karyotype of Mogera, since two distinct genera derived from an ancestor share G-banding homologies. According to this hypothesis, the karyotype of M. imaizumii might have been derived from the ancestral karyotype through pericentric inversion in one pair of acrocentrics, chromosome 11. Two geographically isolated populations of M. tokudae had different karyotypes: the Echigo population had the same karyotype as M. imaizumii, whereas the Sado population had a derivative karyotype that was able to be explained by pericentric inversions in three pairs of acrocentrics, remaining chromosome 11 as subtelocentric. Mogera wogura from South Korea differed considerably in terms of chromosome constitution from the Japanese population of M. wogura. However, we deduced that pericentric and paracentric inversions in four pairs of acrocentrics would explain the karyotype differences between the Japanese and Korean populations. Furthermore, from the morphology of chromosome 11, we can conclude that the Korean mole was derived from primitive M. wogura through four inversions, quite separately from the lineage of M. imaizumii and M. tokudae. Thus, inversion rearrangements appear to have played a major role in the chromosomal evolution of Japanese talpine moles.

Early ontogeny of the Japanese Common Squid Todarodes pacificus was described for artificially inseminated and collected specimens to present new criteria for developmental stages in relation to its ecological adaptation. For the purpose, details for formation of the following organs and tissues were observed with special attention: cilia on the integument, mouth part, shell sac and stellate ganglia, visceral mass, funnel-collar complex, statocysts, eye parts, and ventral photosensitive vesicles. At the embryonic stage (i.e., pre-hatching), various types of epidermal cilia that seem to work as the embryonic rotation were detected. At the early postembryonic stage (i.e., post-hatching), the epidermal lines were characteristically arranged at the scattered condition on arms, tentacles, head, and funnel. Novel strong muscle fibers were distinct in the base of tentacles and funnel retractor muscles at the early postembryonic stage, which is clearly related to the head withdrawal behavior of the paralarvae. The lip cilia and toothed beak developed at the early postembryonic stage, but disappeared later; these apparatus were considered to be related with a change of unique feeding mode in the paralarval life. Based on such morphological features, four distinct stages, namely, paralarval stage 1, 2, 3, and juvenile stage are proposed. The present observations are discussed in relation to survival strategy at early life of T. pacificus and they are compared with those in other cephalopods.

The Japanese rose bitterling, Rhodeus ocellatus kurumeus, is an endangered cyprinid species. The main reason of drastic diminution of R. o. kurumeus has been suggested to be due to hybridization between this subspecies and R. o. ocellatus introduced from China. Both RFLP analysis of mitochondrial DNA and RAPD-PCR analysis of genomic DNA were performed to elucidate the genetic introgression of R. o. ocellatus into R. o. kurumeus. The two subspecies were distinguished in the D-loop region of mtDNA by six restriction endonucleases. Rhodeus o. kurumeus had eleven subspecies-specific RAPD markers and R. o. ocellatus had two. Except some R. o. kurumeus populations, most of the populations of R. ocellatus in Japan were hybrids, equipped with subspecies-specific RAPD markers for the two subspecies, respectively. The genetic constitution of these markers in hybrids, however, greatly differed among populations. The R. o. ocellatus mtDNA was predominantly observed in hybrid populations, except two populations with mtDNAs of the two subspecies. Judging from the genetic dominance of morphological and physiological characters of R. o. ocellatus against R. o. kurumeus, hybrids probably have the same ecological dominance as R. o. ocellatus against R. o. kurumeus. Therefore, it is considered that R. o. kurumeus not only has its genetic property spoiled by hybridization with R. o. ocellatus, but also is expelled by R. o. ocellatus and hybrids. The replacement of mtDNA and genomes of R. o. kurumeus with those of R. o. ocellatus in hybridization might be accelerated by the backcross between hybrids and R. o. ocellatus.