Zoological Science

Phototactic behaviors of the garden spider Araneus ventricosus and the jumping spiders Menemerus confusus and Hasarius adansoni were examined as they walked on a Y-maze globe. On both dark- and light-backgrounds, Araneus, a nocturnal web spider, tended to turn at Y-arms away from a test light given to the eyes (negative phototaxis), but Menemerus and Hasarius, diurnal hunting spiders, tended to turn toward the light (positive phototaxis). Araneus tended to turn transiently toward the light after the cessation of background illumination.

Ascidians are known to accumulate high levels of vanadium. Vanadium-accumulating blood cells (vanadocytes) have one large and highly acidic vacuole. Recently, it was found unexpectedly that the number of vanadocytes increased rapidly and significantly when ascidians were immersed in 10 mM or 20 mM NH₄Cl solution for 20 hr to neutralize vacuole content. Suspecting that changes in intra-organellar pH and in levels of ATP caused by the treatment might be involved, we examined whether or not several reagents that perturb either acidic pH or ATP synthesis affected the increase in the number of vanadocytes. SF6847 (a proton conductor), nigericin, monensin, valinomycin (ionophores), 2,4-dinitrophenol (an uncoupler), bafilomycin A₁ (a V-ATPase inhibitor), oligomycin and NaN₃ (F-ATPase inhibitors) all increased the number of vanadocytes by about three- to five-fold over that of control. However, treatment with NaCl, KCl, LiCl, CaCl₂, TJ24373, sporeamycin (macrolide antibiotics), ouabain and Na₃VO₄ (P-ATPase inhibitors) had no effect on the increase. These results suggest that neutralization of intra-organellar pH triggers an increase in the number of vanadocytes. Vanadocytes that increased in number in the coelomic fluid after treatment were revealed by immunohistochemical study, to have originated in the connective tissues around the alimentary canal.

Measurements of pH in minced cuticle from the alloscutum of non-copulated and copulated female Haemaphysalis longicornis revealed a descent of cuticular pH after copulation. Sample cuticular loops were cut from the alloscutum of non-copulated females and immersed in buffers adjusted to pH 4, 5, 6, 7 or 8. Extensions of the cuticular loops subjected to a constant applied load were measured using a creep test. The cuticular loops at pH 7 and 8 showed small extensibilities. However, as the acidity of the cuticle increased, the sample loops displayed larger extensibilities. Extensibilities of sample loops from copulated females were significantly larger than those from non-copulated females. Based upon this difference of extensibility, the descent of cuticular pH after copulation is presumed to be about 0.5 pH units. These data suggest that the cuticular plasticization is caused by the cuticular acidification in the female tick. Moreover, novel acidophilic epidermal cells were situated around dermal glands. Their cytoplasm have an affinity for eosin, because of numerous mitochondria. A smooth endoplasmic reticulum like tubulovesicular system appeared in the apical cytoplasm after copulation. Such morphological features suggest that the acidophilic epidermal cells actively transport ions and provoke the pH descent of the cuticle after copulation.

Japanese quail chicks have various patterns of calling. The chicks of both sexes emit distress call at a high rate when they are socially isolated. Chronical subcutaneous implantation of testosterone (T) induced the chick crowing which has similar in acoustical structural characteristics to male adult crowing. The chick crowing which was induced by T appeared in a mixed manner with the distress call at high rate when isolated. After T implantation, the frequencies of the chick crowing gradually increased and the distress call disappeared. The chick crowing was induced as the result of androgenic action of T, and the chick crowing disappeared after removal of T. Implantation of a small amount of T directly into mesencephalon, also induced chick crowing after a short latency. The same dose of subcutaneous T implantation failed to induce the chick crowing. This study suggested that T acts on quail chick brain to transform the distress call into the chick crowing.

Japanese quail chicks produce the distress calls at a high rate when socially isolated from other individuals. Under the same conditions, chicks which were given chronical subcutaneous implantation of testosterone (T) produced the chick crowing which has similar acoustical structural characteristics to both the chick distress call and the adult crow. Electrical stimulation of dorso-medial areas of chick mesencephalon through a chronically implanted electrode elicited calls in freely moving chicks. The acoustical structure of the elicited call resembled that of the distress call with the harmonic structures and constant frequency before T implantation. At 6 days after T implantation, the chicks produced the chick crowing similar to the adult crow with a trill structure by electrical stimulation. The area in brainstem of chicks that exhibit two different kinds of vocal behavior upon electrical stimulation lies in the medial intercollicular nucleus (ICo). This suggests that the medial ICo of chicks has two different function, production of the distress call and the chick crowing with T implantation. We concluded that continual exposure of the neural substrate in the medial ICo of chick to T induced the functional change from emitting the distress call to producing the chick crowing.

Adults of Aulacophora nigripennis terminated diapause when transferred from outdoor conditions to a long photoperiod before February but remained in diapause when transferred to a short photoperiod. Both mating and vitellogenesis occurred at either photoperiod in adults examined in February or later. The supercooling point (SCP) of adults was about −6°C in summer and −10 to −11°C in late autumn and winter. Summer adults rapidly decreased the SCP to about −16°C after deprivation of food for 4 days at 25°C, suggesting that the gut contents play an important role in lowering the SCP. Among the cryoprotectants known in insects, only myo-inositol was found accumulated in overwintering adults of A. nigripennis. Female adults accumulated more myo-inositol than males in February. An exposure of overwintering beetles to 25°C caused a rapid decrease in myo-inositol level even at a short photoperiod. This indicated that the reduction in inositol may be independent of the termination of diapause.

Distribution of opine dehydrogenases and lactate dehydrogenase (LDH) among different body parts of a sipunculid are reported for the first time. Alanopine dehydrogenase (ADH), octopine dehydrogenase (ODH) and strombine dehydrogenase (SDH) were present in the body wall and the introvert of Phascolosoma arcuatum. However, no activity of ADH or SDH was detected in the internal organs of this sipunculid. LDH activities were detected in the three body parts studied. In the body wall and the introvert, the activities of LDH represented only a small fraction of the total pyruvate oxidoreductase activities. In the internal organs, however, the activity of LDH was approximately half that of ODH. The lactate oxidizing:pyruvate reducing ratio of the LDH from the internal organs was greater than those from the body wall and the introvert. The electrophoretic pattern of LDH isozymes from the internal organs was also different from those of the latter body parts. The distribution patterns of pyruvate oxidoreductases in these body parts of P. arcuatum were discussed in relation to the physiology of this sipunculid.

Ocular melatonin contents in a cyprinid teleost, oikawa Zacco platypus were determined by newly-developed radioimmunoassay. Under light-dark (LD) cycles of 12:12 hr, melatonin levels in the oikawa eye exhibited daily rhythms with higher levels during the dark phase than those during the light phase. However, the rhythms disappeared under either constant light (LL) or constant darkness (DD). Ocular melatonin contents under LL remained at low levels while those under DD kept high titers. These results indicate ocular melatonin rhythms in oikawa are driven not by a circadian clock but by LD cycles.

Two experiments are described in which the interaction of prolactin, cortisol and growth hormone on hypoosmoregulation in the anadromous brown trout was studied. In experiment 1, fish at the postsmolt stage in freshwater (FW) were given four injections on alternate days of 5 μg cortisol (F)/g in combination with 0, 0.2, 1 or 2 μg ovine prolactin (oPRL)/g. Additional groups received 2 μg oPRL/g or saline as control. In experiment 2, FW parr were given four injections on alternate days with 5 μg F/g and 2 μg ovine growth hormone (oGH)/g in combination with 0, 0.2, 1 or 2 μg oPRL/g. Additional groups received 2 μg oPRL/g, 2 μg oGH/g, 5 μg F/g or saline as control. In both experiments, the fish were subjected to a 48 hr seawater (SW) challenge test 24 hr after the last injection. Muscle water content, plasma osmolality and ion levels, kidney and gill Na⁺, K⁺-ATPase activity were measured. In experiment 1, F-treated fish had better hypoosmoregulatory capacity than control fish as judged by a higher level of muscle water content and lower plasma osmolality after 48 hr in SW. All three doses of oPRL completely abolished this action of F. Gill Na⁺,K⁺-ATPase activity was stimulated by F and unaffected by oPRL at any dose. In experiment 2, oPRL impaired, whereas F and oGH (injected individually or together) improved performance in the 48 hr SW test relative to control fish, judged by plasma osmolality and muscle water content. Ovine PRL inhibited the combined action of F and oGH in a dose-related manner but could not completely counteract the combined effect of these hormones. F and oGH had additive stimulatory effects on gill Na⁺,K⁺-ATPase activity. This activation was counteracted by coinjection of oPRL in a dose-related manner but not below the level found in the F-treated fish. We conclude that exogenous oPRL inhibits the hypoosmoregulatory action of F. However, oPRL does not antagonize the F-dependent stimulation of gill Na⁺,K⁺-ATPase activity. The observed antagonism between PRL and F in SW-acclimation may therefore occur on a target different from gill Na⁺,K⁺-ATPase.

Larval life histories of Kyoto and Tokyo populations of the stream breeding salamander Hynobius kimurae are compared in the field. The Kyoto population has smaller adults and ovarian egg numbers, but larger egg sizes. Minimum water temperature is lower, and the stream partly dries up between late October and late November in Kyoto, while the amount of water is always stable in Tokyo. In Kyoto, metamorphosis occurs between late July and late September and no larva is found in the water in and after October. In Tokyo, only 21% of larvae metamorphose in the first year and most overwinter and metamorphose between late May and mid June of the second year. Size of hatched larvae does not differ between Kyoto and Tokyo, but metamorphs from Kyoto are smaller than first year metamorphs from Tokyo, which in turn are smaller than the second year metamorphs. Differences seen in larval life histories between the two populations may represent adaptations for differential stability of water level and water temperature, and resultant differences in the size of metamorphs possibly induce differential adult body size, and hence fecundity.

Normal serum from the hagfish Eptatretus burgeri strongly enhanced the phagocytic activity of homologous peritoneal macrophages against rabbit erythrocytes (RRBC). Such phagocytosis was effectively but incompletely inhibited by rabbit antibodies against hagfish C3 (HC3). However, when RRBC were pretreated with purified HC3 or normal hagfish serum in the presence of EDTA, little enhancement of ingestion was observed. The results indicate that HC3 binds to RRBC, in collaboration with other serum factor(s), and that bound HC3 functions as an opsonin. Additionally, we obtained evidence for the possible presence of another opsonin (HOP) that was resistant to EDTA in C3-depleted hagfish serum. HOP prepared from plasma had a molecular mass of approximately 1,000 kDa, consisting of various small polypeptides that were able to form aggregates via disulfide bonds and/or noncovalent interactions. The functions of HC3 and HOP might be essential to immunity in hagfish because no clear evidence for the production of antibodies in cyclostomes has been reported to date.

The plasma of a solitaly ascidian, Halocynthia roretzi, stimulates the proliferation of mouse thymocytes in the presence of a mitogen, phytohemagglutinin P (PHA-P). Eight factors capable of stimulating the proliferation of mouse thymocytes were isolated from H. roretzi plasma by procedures including hydrophobic chromatography, gel filtration, and anion-exchange chromatography. Four active fractions with molecular weights of 100–110 K, 75–90 K, 10–30 K, and 5–6 K were first separated by gel filtration. Each of these fractions was then separated into two fractions with different ionic properties by anion-exchange chromatography. The eight factors thus obtained have different heat stabilities, heparin-binding abilities, and PHA-P sensitivities.

In order to elucidate the nucleotide sequence divergence of mitochondrial DNAs between two Japanese pond frog species Rana nigromaculata and R. brevipoda, and the mode of inheritance of cytoplasmic genomes in the female hybrid lineages of the two species, the cleavage patterns of mtDNAs digested with 10 restriction endonucleases were examined by agarose gel electrophoresis using a total of 52 frogs including Rana nigromaculata and R. brevipoda, their reciprocal hybrids and the backcross offspring (B₁ and B₂) derived from female hybrids by crossing with the paternal species. The cleavage patterns for mtDNA of Rana nigromaculata were different from those of R. brevipoda digested with all the restriction endonucleases used except EcoRV. The nucleotide sequence divergence of mtDNAs between these two species was roughly estimated to be 8.5%. The cleavage patterns for mtDNAs of the reciprocal hybrids and the B₁ and B₂ offspring were clearly similar to those of the maternal species, and paternal mtDNAs could not be detected. On the other hand, the proportions of original maternal nuclear genes at the 22 allozyme loci were 50% in the reciprocal hybrids, 21.9% or 25.6% in the B₁ offspring, and 7.5% in the B₂ offspring. These results demonstrate that nuclear genomes decrease the original maternal constitution in the female hybrid lineages generationally, whereas the mtDNAs are inherited maternally during repeated backcrossing.

We describe a culture method in which larval muscle cells of the starfish Asterias amurensis develop from epithelial cells, probably deriving from the coelomic pouches. The nature of the muscle which appears in the culture is described morphologically, physiologically and ultrastructurally. Cells were dissociated from the late gastrula stage, treated with 0.6 M of glycine in half-strength sea water free of Ca²⁺ and Mg²⁺ for 12 hr, and cultured for various periods. Elongated cells appeared after about a week from small aggregates of epithelial cells which were found among the mesenchymal network on the 1st day of culture. The characteristics of the elongated cells are as follows: (1) they possess two or more arms; (2) they adhere to the cultural substratum, mesenchyme cells, and themselves at all parts of the cell body; (3) they contract in response to acetylcholine; (4) they contain an abundance of fibrous actin and myosin throughout their cytoplasm; (5) the cytoplasm contains bundles of thick (12–19 nm) and thin (5–8 nm) filaments without any dense material; and (6) no proliferative activity was observed while the cells were kept in culture for up to 14 days. These features were compared with those of the larval muscle cells in vivo.

Molgula tectiformis is a new ascidian species recently described by Nishikawa (1991). In Otsuchi Bay, Iwate, Japan, they are easily obtainable from cages for culturing scallops. We report here that M. tectiformis is another example of a direct developer: their embryonic development is lacking the tadpole larva. The fertilized egg is orange and about 150 μm in diameter. At 18°C, the egg cleaves at about 20 min intervals and gastrulation occurs about 5 hr after fertilization. In contrast to conventionally-developing ascidians, M. tectiformis does not form a tadpole larva. Immediately before hatching, three stolons or ampullae begin to extend from the tailless embryo. After hatching the stolons mediate the attachment of the juvenile body to the substratum. Histochemistry for tissue-specific enzyme activity did not detect muscle-specific acetylcholinesterase, endoderm-specific alkaline phosphatase, and pigment cell-specific tyrosinase. In addition, in situ hybridization could not prove the presence of muscle actin gene transcripts in the embryo. These results suggest that these larval tissues do not differentiate in M. tectiformis embryos. Because M. tectiformis is common and gravid year-around in Otsuchi Bay, this direct developer provides the opportunity for further analysis of molecular changes during evolution that cause an alternative mode of development.

Migration and colonization patterns of neural crest cells were analyzed histochemically in embryos of the brook lamprey, Lampetra reissneri, and the swordtail, Xiphophorus helleri, using HNK-1 monoclonal antibody, which recognizes migratory neural crest cells and crest derivatives in many groups of vertebrates including teleosts. We demonstrated that HNK-1 recognizes a subpopulation of naural crest cells and crest derivatives in the lamprey as well as in the swordtail. In the trunk of lamprey and swordtail embryos, HNK-1-positive cells were observed in the major migratory pathways of neural crest cells, that is ventral and dorso-lateral pathways. In the swordtail embryos, neural crest cells in the ventral pathway were ubiquitously observed along the rostro-caudal axis at the neural tube level, but at the notochord level their migration was restricted to the middle region of the somite. In the lamprey, by contrast, no HNK-1-immunoreactive neural crest cells migrated ventrally beyond the notochord level in the ventral pathway at axial levels behind the gill pouch region. This migration pattern of neural crest cells in the lamprey trunk might be closely related to the absence of anatomically distinct, sympathetic chains in the lamprey, which is one of characteristic features of the lamprey's body organization. Furthermore, we found dorsal pathways, which extended to the embryonic dorsal fin, of neural crest cells in both swordtail and lamprey embryos. In the lamprey, dorsal cells (Rohon-Beard cells) and cells that resemble chromaffin cells immunoreacted with HNK-1.

We developed a serum-free culture technique for chick epiblast explants in low density. In the condition free of unknown serum factors, we examined the invasiveness of chick epiblast cells by dissociating the cells into single cells and seeding them onto basement membrane analogues. At stage X–XI of Eyal-Giladi and Kochav's table (1976), 5–11% of the epiblast cells invaded laminin gel or the reconstituted basement membrane (EHS gel). The proportion of invasive cells increased quickly up to 50% at stage XII. Single prestreak epiblast cells on laminin gel still showed invasive behavior even in the simple medium without any growth factors or vitamins. They were also invasive on fibronectin gel, but not on type I collagen gel or a mixture of EHS gel and type I collagen gel. As for regionality, invasive epiblast cells were more densely distributed in the posterior region of area pellucida than in the anterior region. The early commitment of invasiveness in individual epiblast cells precedent to gastrulation was proved in our serum-free culture. In addition, we confirmed that prestreak epiblast cells are already heterogeneous in the invasive potency; they can be classified into two groups, invasive cells and non-invasive ones.

Abnormal development in Bh quail embryos was histologically examined. In addition to the abnormal plumage pigmentation in Bh-heterozygotes and homozygotes, and subcutaneous hemorrhage and liver degeneration in homozygotes as previously reported, the lungs in all 10-day homozygotes examined showed hemorrhage, suggesting that the Bh gene may also be expressed in this organ. Other organs, including the esophagus, the gizzard, the small intestine, the large intestine, the pancreas, the metanephros, the heart, the gonads and the hemopoietic organs in heterozygotes and homozygotes were histologically normal on days 7 and 10, though the development of homozygotes was slightly delayed. Primordial germ cells and hemopoietic cells were normally developed in gonads and hemopoietic organs such as the bursa of Fabricius and the spleen of the homozygote, respectively. These results suggest that Bh is a mutation inducing pleiotropic effects such as plumage pigmentation changes in both heterozygotes and homozygotes, and abnormal development of the blood vessels in the skin, the feather germs and the lung in homozygotes.

We investigated the effect of activin A on the In Vitro differentiation of primary myogenic cells isolated from chick embryonic breast muscle. As judged by the morphology of the cells and accumulation of creatine kinase, activin A inhibited myogenesis in a dose-dependent manner. The inhibitory activity was reversibly suppressed by follistatin. Activin A also inhibited the expression of MyoD1. Our data also suggest that the inhibitory activity of activin A is exerted neither on proliferation of myoblasts or the growth of myotubes, but rather an early phase of differentiation following the withdrawal from cell cycle. A comparison of actions of activin A and TGF-β suggests that points of action is similar.

Proliferation and differentiation of ependymal cells in the injured carp spinal cord were studied by immunohistochemistry using proliferating cell nuclear antigen (PCNA) and ³H-thymidine (³H-TdR) autoradiography. A surge of proliferation of ependymal cells occurred in the lesion with a peak around 6th day after surgical operation (complete transection) of the spinal cord. The proportion of PCNA-positive ependymal cells at 6th post-operative day (6 POD) was over 14 times that in the normal state. Electron microscopic autoradiography revealed that most of the ependymal cells incorporating ³H-TdR at 5 POD, especially those located in the caudal side of the transection site, contained numerous free ribosomes in their apical portion, but the other organellae, such as rough endoplasmic reticulum and Golgi apparatus, were poorly developed in this portion. In three weeks thereafter, the ependymal cell layer was reconstructed through the lesion, and the apical part of the ³H-TdR-labeled ependymal cells became elongated and morphologically differentiated: that is, it had free ribosomes decreased and glial filaments increased in number. Many bundles of regenerating axons were observed to course within the reconstructed ependymal cell layer. These results may suggest that proliferation, differentiation and reconstruction of the ependymal cell layer following injury of the carp spinal cord are requisite to make the permissive milieu for elongation of the regenerating axons.

The prowing, T (9; 10)/9; 10 Pw, is a homeotic mutant of the German cockroach, Blattella germanica. Adults are characterized by winglike extensions from the pronotum. This trait is associated with a reciprocal translocation of chromosomes 9 and 10. Translocation heterozygotes express the trait and homozygotes are lethal. The expression of Pw is variable in adults. Individuals with excellent expression show that the pronotal extension is undoubtedly a primitive wing, judging from its venation. We crossed adults with different degrees of expression and found that crosses between parents with excellent expression tended to produced F₁ with excellent expression and those with poor expression produced F₁ with poor the expression. This suggests that more than one factor is involved in the expression of the trait, and that the different expressions cannot be due to different “expressivity”. We also found that the expression Pw was greatly reduced when prowings were crossed with the Nara wild strain, although this did not happen when they were crossed with individuals with wild-type expression from the Pw strain. This suggests that at least one more factor participates in the expression of Pw. We observed meiosis clearly even in the adult stage. We examined many male adults and confirmed that the reciprocal translocation cause Pw expression without an exception. We also observed the frequencies and stages of embryonic death of F₁ from both Pw and Pw × wild type parents. The results agreed well with the ratio between alternate and adjacent disjunction at metaphase I. Pw individuals tended to be delay in nymphal development compared with the wild type.

Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, inhibited the activity of 26S proteasome in goldfish oocytes and arrested the oocytes at two distinct stages of maturation. One was prior to the migration of germinal vesicle (GV) toward the animal pole, and the other was between the attachment of GV to the oocyte plasma membrane and GV breakdown (GVBD). The first DFP-sensitive period corresponded to the period during which the activity of proteasomes increased, but maturation-promoting factor (MPF) was still inactive. During the second DFP-sensitive period, MPF was abruptly activated, although the proteasome activity detectable in the oocyte cytosol extracted by ultracentrifugation reached the lowest level during this period. These results suggest the requirement of 26S proteasome for at least two stages of oocyte maturation, the early stage before GV migration and the later stage, including MPF activation immediately before GVBD.

We studied the effect of temperature and photoperiod on prolactin (PRL) gene expression in the pituitaries of summer-aclimatized carp (C. carpio). To assess the level of PRL gene transcription, we used a 24mer synthetic oligonucleotide probe derived from the known carp PRL gene sequence. Employing in situ hybridization assays, we observed high expression of PRL mRNA in the rostral pars distalis of summer-acclimatized carp in contrast to the almost negligible PRL transcription which occurs in the winter-acclimatized fish. When experimental combinations of long and short photoperiods with 10°C and 20°C environmental temperatures were studied, only a short photoperiod (8L-16D) in summer-acclimatized carp acclimated to a winter temperature (10°C) markedly depressed PRL gene expression. Our observations indicate that photoperiod constitutes a particularly relevant modulator in the neuroendocrine cascade that activates PRL transcription in the carp.

The effects of somatostatin (SRIF) on in vitro avian adrenal steroid secretion have been investigated in the domestic turkey (Meleagris gallopavo) and fowl (Gallus gallus domesticus). SRIF did not affect either basal or ACTH-stimulated aldosterone and corticosterone production by dispersed turkey adrenocortical cells, but it concentration-dependently inhibited the secretory response to angiotensin II (ANG II). It is concluded that in the turkey, like in mammals, SRIF specifically interferes with the intracellular mechanisms transducing the ANG II secretagogue signal. Fowl adrenocortical cells did not display any secretory response to ANG II, and accordingly SRIF did not alter their basal and agonist-stimulated secretory activity.

Ontogeny and cellular localization of the lutropin receptor (LH-R) mRNA were studied in fowl gonads between day 5 of incubation and day 1 of posthatch. In situ hybridization using an antisense cRNA probe demonstrated that LH-R gene expression began at embryonic day 7 in females and day 14 in males. In female embryos, the specific hybridization was detected in not only developing left ovaries but also regressing right ovaries. The LH-R mRNA expression in the right ovary was detectable until hatch. LH-R mRNA was localized in the medullary cords in ovaries and interstitial tissues in testes. These results are consistent with previous reports on ontogenic analysis of LH-dependent steroidogenesis in chicken embryo gonads.

The masseter muscles of the Japanese field vole Microtus montebelli were studied histochemically and electron microscopically. The masseter muscles were composed of only fast-twitch oxidative (FO) fibers. As to ultrastructural features of the fibers, mitochondria, sarcoplasmic reticula and transverse tubules were well developed. It is concluded that the masseter muscles in M. montebelli have the structural characteristics that they can contract fast and enduringly. Thus, they appear to be highly adapted to herbivorous food habit.