Zoological Science

Medaka (Oryzias latipes) has been recognized as a potential model vertebrate to which modern molecular genetics can be applied. Recent progress in transgenic techniques emphasizes the importance of the use of medaka in a variety of biological fields, particularly in view of the development of transgenic vectors. The first transgenic medaka was produced by microinjection of DNA into the pronucleus of unfertilized oocytes. Several new methods have since been developed for producing transgenic medaka, and an appropriate method can be selected according to the aims of experiments and application. Transgenic medaka have been used in many fields, including aquaculture, toxicology, developmental biology (phenotypic rescue experiments), and for successful characterization of transcriptional regulatory regions. Here, we describe examples of the application of transgenics in these fields. We also summarize the recent progress in transgenic techniques and transgenic vectors, focusing on the elements and the marker genes that enable identification and/or induction of the expression of exogenous genes in transgenics.

Fish undergo a brief growth spurt known as “compensatory growth” when they resume adequate feeding after a restricted food intake. In the present study, changes in formation and resorption of acellular bone during the compensatory growth period were quantified using bone histomorphometry. Juvenile tilapia (Oreochromis niloticus) were starved for 15 days and subsequently refed for 15 days. In the starvation period, osteoblastic activity decreased linearly with time and almost reached zero on day 15, whereas the significant reduction in osteoclastic activity first became evident on day 15. This resulted in an increased percentage of eroded bone surface, which is the bone surface affected by osteoclasts. In the refeeding period, osteoblastic activity recovered to the levels of fed fish on day 3, briefly increased above the levels of fed fish on day 7, then returned to those of fed fish on day 15. Osteoclastic activity gradually increased and returned to the levels of fed fish from 7 days onwards. The eroded surface rapidly decreased to the levels of fed fish on day 3, became significantly lower than those of fed fish on day 7, and then returned to those of fed fish on day 15. It is concluded that starvation leads to a decreased turnover rate in tilapia acellular bone, which can be reversed by subsequent refeeding. A brief increase in bone-forming activity above the levels of fed fish, “the compensatory bone-formation”, is a characteristic feature of the recovery period.

We explored whether the calcium imaging can be applicable to detection of spike activity of Aplysia wide-spread neurons. Well-used procedures, the membrane permeable acetoxymethyl (AM) types and the retrograde labeling could not be used for loading the calcium sensitive dye into many neurons, however, impalement of each cell body with a microelectrode containing high concentration of the dye solution in turn could load the dye iontophoretically into many large neurons in a relatively short time and into several small neurons in a very short time. Spike activity just induced a fluorescent increase in the cell body while depolarization without spikes induced it in the axon hillock. In the cell body the slope of the fluorescent increase was almost relative to the spike frequency while the period of it almost corresponded to the firing duration. Therefore, the phase relationship of the rhythmic firing responses in plural neurons could be precisely detected. Moreover stable responses could be repeatedly obtained for a long time from small neurons unsuitable for a long time recording with microelectrodes. Removal of the extracellular Ca²⁺ completely suppressed the spike-induced increase in fluorescence in the cell body and application of nifedipine partly reduced it, suggesting contribution of L-type channels distributing in Aplysia wide-spread neurons.

Neurosecretory cells releasing the pheromone biosynthesis-activating neuropeptide (PBAN) in Bombyx mori exhibit diurnal firing activity. Diel changes in the firing activity of the PBAN producing cells persisted in both constant dark and constant dim light (0.1 lx) at mean periods of 23.0± 1.6 hr and 22.6± 0.8 hr, respectively, thereby suggesting that the neurosecretory cell system is under the control of a circadian pacemaker. The circadian firing rhythm was greatly modified by background illumination: (1) the period of free-running activity rhythm was significantly short (18.5± 1.6 hr) under continuous illumination of a moderate intensity (100 lx) and (2) the duration of a firing period of cells elongated by 2.1± 0.7 hr, when light intensity during a photophase was lowered to 0.01 lx. The suppressive effect of light on the firing activity may induce a nocturnal component of a daily activity pattern by releasing PBAN cells from suppression after termination of illumination.

We examined morphologically innervation of the heart of the ostracod crustacean Vargula hilgendorfii. The heart is single chambered and composed of a single layer of myocardial cells characterized by localization of myofibrils at the epicardial side. A nerve net in the heart was determined by vital staining with methylene blue. Electron microscopy revealed that a single neuron situated on the outer surface of the dorsal heart wall sends an axon into the heart wall. The axon of the dorsal neuron is branched widely and forms many neuromuscular junctions on the myocardial cells. A pair of extrinsic nerves, each of which contains several axons, enters the heart bilaterally and forms numerous nerve terminals on the dorsal neuron and myocardial cells, while no synaptic structures were found in the nerve terminals. The results suggest that the heart of V. hilgendorfii is neurogenic, with a single cardiac neuron having both pacemaker and motor functions.

To understand the functional role of the insect brain in mating behavior, copulatory actions in response to a model stimulus were compared between intact and decerebrated male crickets. Decerebrated males were then tested to examine whether their copulatory actions were modifed by biogenic amines or electrical stimulation. The main difference in copulatory actions between intact and decerebrated males was in the body thrusts consisting of the protraction and retraction of the abdomen for hooking. These movements became slower after the removal of the brain, as measured by the average interval between responses, called the cycle length. The cycle length increased to twice the length in intact males within about 20 min. Intraperitoneal injection of octopamine in decerebrated males shortened the cycle length dose-dependently (10⁻⁶–10⁻²M) and restored it nearly to the level in intact males at 10⁻⁴–10⁻²M. Octopamine-mimicking agents forskolin, IBMX and synephrine, and cyclic AMP analogue DB-cyclic AMP had effects similar to that of octopamine, while serotonin, dopamine, noradrenaline and adrenaline did not. Electrical stimulation of the neck connectives mimicked the effect of octopamine, which was blocked by octopamine antagonist phentolamine. Perfusion of the hemocoel with Ringer's solution to eliminate the octopamine previously injected abolished the effect of extrinsic octopamine, whereas it did not abolish the effect of electrical stimulation. These results suggest that the brain in the male cricket is involved in facilitating the activity of the pattern generator for mating behavior via intraganglionic octopamine.

Melanophores of the isolated tail fin of the Xenopus tadpole respond to light, resulting in melanin aggregation in the melanophores.

Western blot analysis showed that a protein in the Xenopus tail fins, in which photosensitive melanophores exist, had reacted with the antibody against bovine rhodopsin.

RT-PCR and nested-PCR using rhodopsin-specific primers showed the expression of rhodopsin mRNA in the tail fins. The amino acid sequences deduced from the PCR products were completely identical to those of rhodopsin.

We also detected the mRNA of melanopsin in the tail fin, another opsin originally described in cultured melanophores of Xenopus.

These results indicate that these two types of opsin molecules exist in Xenopus tail fin and may take part in the photo-response in melanophores of the Xenopus tadpole.

We cloned cDNAs encoding two peroxins, PEX-1 and PEX-6, of the nematode Caenorhabditis elegans. Peroxins are proteins that play essential roles in peroxisome biogenesis and are encoded by pex genes. Among the peroxins, PEX-1 and PEX-6 constitute the subfamily 2 of AAA (ATPases associated with diverse cellular activities) proteins. Each cDNA agreed well with the respective mRNA in size (3.4 kb for pex-1 and 2.3 kb for pex-6) and did not carry any spliced leader sequence. The pex-1 cDNA was composed of 24 exons, which were encoded by a genomic region containing three open reading frames (ORFs), c11h1.4, c11h1.5, and c11h1.6; the predicted ORF c11h1.5 was encompassed in the 15th intron. Although many exon-intron borders in pex-1 were inconsistent with those predicted for c11h1.4 and c11h1.6, those in pex-6 coincided with those for the ORF f39g3.7. The pex-1 and pex-6 genes encoded proteins with 996 and 720 amino acid residues, respectively. Both pex-1 mRNA and pex-6 mRNA were detectable mainly in intestinal cells throughout the life cycle of C. elegans.

We characterized the chromosomes of the lancelet Branchiostoma belcheri Gray. Testes were removed from adult males, cut up, treated with colchicine (25–50 μg/ml) for 3–6 hr, and dissociated into single cells by pippeting. The dissociated cells were swollen with 5.0–6.5% sodium citrate for 10 min and fixed with methanol-acetic acid (3:1). The fixed cells were dropped onto a glass slide and air-dried. The haploid chromosome number was 18 and diploid number was 36.

We obtained the full-length cDNA clone (DsPTGC04) encoding a membrane guanylyl cyclase expressed in the testis of the sea urchin Diadema setosum, the egg jelly of which contains sperm-activating peptide IV. The cDNA was 4305 bp long and an open reading frame predicted a protein of 1127 amino acids including an apparent signal peptide of 24 residues. The mature protein of 1103 amino acids is composed of a single transmembrane domain of 25 amino acids that divides the mature protein (Mw 123818) into an amino-terminal, extracellular domain of 484 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids, with the latter consisting of two clearly defined subdomains, a protein kinase-like and a cyclase catalytic. Four potential N-linked glycosylation sites are present in the extracellular domain and 4 presumed phosphorylatable serine residues are conserved in the cyclase catalytic domain. Northern blot analysis demonstrated that the 4.5 kb mRNA for DsPTGC04 is expressed only in the testis. Antibodies raised against two synthetic peptides, ⁸⁰⁰WVENPDERPN⁸⁰⁹ and ¹⁰⁸⁰KPPPQKLSAEVMEAAANREIPEDL¹¹⁰³, corresponding to two carboxyl-terminal portions of DsPTGC04, reacted with a protein of about 120 kDa in D. setosum spermatozoa and testis, but not with any protein in the ovary, eggs, or intestine. Immunohistochemistry showed that both antibodies react with the mature spermatozoa in the testis.

The ter (teratoma, chromosome 18) mutation causes a deficiency in primordial germ cells (PGCs) of ter/ter embryos from ter congenic mouse strains at around 8.0 days post coitum (dpc). Our previous studies indicated that in vivo, apoptosis of PGCs was caused by ter/ter gonadal somatic cells. To examine survival and proliferation in ter/ter PGCs and the deficiency caused by ter/ter gonadal somatic cells in vitro, we performed “exchange-co-culture” of PGCs and gonadal somatic cells by combining different tergenotypes, on Sl/Sl4-m220 feeder cells. The number of PGCs after 3 days culture of 9.5 dpc ter/ter PGCs with +/+ 12.5 dpc gonadal somatic cells increased similar to that of +/ter or +/+ PGCs. The numbers of PGCs after 12 hr culture of +/+ and ter/ter 11.5 dpc PGCs with 11.5 dpc ter/ter gonadal somatic cells decreased significantly when compared to those cultured with +/+ somatic cells. PGCs preferred the WT1-positive gonadal somatic cells, Sertoli cells, to the feeder cells. Both TUNEL and BrdU assays showed that ter/ter somatic cells induced apoptosis but were independent of DNA synthesis in PGCs “exchange-co-cultured”. Through these results, we demonstrated for the first time that in vitro ter/ter PGCs showed survival and proliferation activities in response to the gonadal somatic cells and that ter/ter gonadal somatic cells caused apoptosis in PGCs through cell-cell contact.

We describe the normal embryonic development of the Chinese soft-shelled turtle, Pelodiscus sinensis. We identified 23 stages between the late neurula and hatching, based on chronology and the external morphology of embryos. The anlage of the sensory organs appeared in early stages of the pharyngula, followed by limb buds. The lateral ridge of the carapace, or the carapacial ridge, could be recognized by two weeks of incubation, and the basal structure of the shell was complete by the mid stage (24 days) of development. The hatchling was well pigmented, and the yolk had been entirely absorbed. The developmental stages of P. sinensis were comparable with those of other turtle species. The developmental pattern of P. sinensis resembled basal taxa rather than more derived groups. Some peculiarities were recognized in the development of P. sinensis, including the appearance of cutaneous papillae on the carapace instead of horny scutes. Those features are shared by Carettochelys, possibly the species taxonomically closest to the Trionychids.

Thriving chemosynthetic communities were located for the first time in the Indian Ocean between 2420 and 2450 m, on a volcanic knoll at the eastern crest of an axial valley, approximately 22 km north of the Rodriguez Triple Junction. The communities were distributed in a 40m by 80m field around the knoll. At least seven active vent sites, including black smoker complexes that were emitting superheated water at 360°C, were observed at the field. The faunal composition of the Indian Ocean hydrothermal vent communities had links to both Pacific and Atlantic vent assemblages. This discovery supports the hypothesis that there is significant communication between vent faunas in the Pacific and Atlantic Oceans via active ridges in the Indian Ocean.

Tunic is a unique integument that contains cellulosic components and various types of free cells (tunic cells), and this tissue is exclusively found in the subphylum Urochordata (= Tunicata). In order to discuss the ascidian phylogeny, the presence or absence of the two characteristic types of tunic cells (tunic bladder cells and the tunic net cells) are examined in 65 ascidian species covering 11 families out of the 15 recognized ones. The tunic bladder cells are exclusively distributed in the species of Didemnidae, Holozoinae, Diazoninae, and Ascidiidae. The species of Corellidae have hemocytes, giant cells, that are very similar to the bladder cells, but these cells are not distributed in the tunic. The tunic pH of these species is usually acidic, because the tunic bladder cells contain strong acid in the vacuoles. The tunic net cells are found in Polyclinidae, Polycitorinae, and Didemnidae. The tunic rounding assay suggests that the tunic net cells may be involved in the contraction of the tunic at least in the species of Polyclinidae and Polycitorinae. If these types of tunic cells can be considered as synapomorphic characters, the character-state distribution suggests that the suborders Phlebobranchia and Aplousobranchia are not monophyletic groups.

The taxonomic status of the Senkaku mole,Nesoscaptor uchidai Abe, Shiraishi et Arai, 1991 (Mammalia: Insectivora: Talpidae), described from Uotsurijima in the Senkaku Group, Ryukyu Archipelago, was re-evaluated. Morphological analyses suggest that N. uchidai is most similar to Mogera insularis from Taiwan, although several morphological characters, such as the number of premolars and the shapes of the anterior portion of the palate, zygomatic arch, auditory bulla, and coronoid process, differentiate N. uchidai from M. insularis. Therefore, we synonymize the monotypic genus Nesoscaptor Abe, Shiraishi et Arai, 1991 with the genus Mogera Pomel, 1848, and define Mogera uchidai (Abe, Shiraishi et Arai, 1991) as a valid species endemic to Uotsurijima. In addition, we analyzed morphological variation within M. insularis, such as variation in the second upper premolar pair, overall cranial size, and the breadth of the rostrum and palate. Some of this variation may be associated with topographical and environmental factors in its habitat.

Ancient DNA was analyzed from skull remains of 12 brown bears (Ursus arctos) excavated from the archeological site of the Okhotsk Culture on Rebun Island of Hokkaido, where no natural populations of brown bears currently occur, in order to trace their original habitats. The Okhotsk Culture developed around southern coastal regions of the Okhotsk Sea (southern Sakhalin, Rebun and Rishiri Islands, northern and eastern Hokkaido, and southern Kuril Islands) during 6–11th centuries, A.D. The ancient people of those days are considered to have involved brown bears for traditional ceremonies and rituals. From the skull remains, partial fragments (approximately 250–360 base pairs) of the mitochondrial DNA (mtDNA) control region were successfully sequenced. Compared with sequence data of modern brown bears of the Hokkaido main land, ancient mtDNAs of Rebun Island were phylogenetically classified into either of two lineages of modern mtDNA: the north-central Hokkaido lineage and southern Hokkaido lineage. The southern Hokkaido lineage was identified from three juvenile (less than one year old) ancient bears, while the north-central Hokkaido lineage was mainly from adults (more than three years old). Our findings demonstrated that juvenile ancient bears of Rebun Island were originated from southern Hokkaido, which was an outside area of the Okhotsk Culture and belonged to the Epi-Jomon Culture with a close relation to a northern part of the Tohoku district. The molecular phylogeographic study on ancient and modern brown bears provides an insight to further understanding zooarcheology and ancient people's cultures around Hokkaido.