Zoological Science

Annelids are segmented animals that display a high degree of metamerism in their body plan. This review describes the segmentation of clitellate annelids (i.e., oligochaetes and leeches) and polychaetes with special reference to cellular and molecular mechanisms elaborating the metameric body plan. In clitellate embryos, segments arise from five bilateral pairs of longitudinal coherent columns (bandlets) of primary blast cells that are generated by five bilateral pairs of embryonic stem cells called teloblasts (M, N, O, P and Q). Recent cell-ablation experiments have suggested that ectodermal segmentation in clitellates consists of two stages, autonomous morphogenesis of each bandlet leading to generation of distinct cell clumps (i.e., segmental elements; SEs) and the ensuing mesoderm-dependent alignment of separated SEs. In the N and Q lineages, SEs are each comprised of clones of two consecutive primary blast cells. In contrast, in the O and P lineages, individual blast cell clones are distributed across SE boundaries; each SE is a mixture of a part of a more-anterior clone and a part of the next more-posterior clone. In contrast, the metameric segmentation in the mesoderm (M lineage) is a one-step process in that it arises from an initially simple organization (i.e., a linear series) of primary blast cells, which individually serve as a founder cell of each segment; the boundary between mesodermal segments is determined autonomously. Cell-autonomous properties of primary blast cells have also been suggested in two fundamental aspects of segmentation, viz., specification of segment polarity and determination of segmental identities. Recent cell-ablation and -transplantation studies have suggested that segmental identities in primary blast cells derived from M teloblasts are determined according to the genealogical position in the M lineage and that the M teloblast possesses a developmental program through which the sequence of blast cell identities is determined. It is unlikely that either a segment polarity gene engrailed or Hox genes are involved in specifying polarity or identities of segments of clitellates, since these genes (in leeches) are reportedly expressed long after the establishment of these segmental properties. As in clitellates, segments in polychaetes arise from descendants of teloblasts located in a posterior growth zone, but it is only during trochophore larval stages that overt segmental organization becomes recognizable. At present, it is not known how the posterior growth zone generates trunk segments either during larval stages or after metamorphosis. However, the recent finding that Hox genes are expressed in the growth zone (probably in teloblasts) suggests that segmentation mechanisms in polychaetes are distinct from those in clitellates.

We identified a Cl⁻ channel, two K⁺ channels and a cAMP-gated channel which were isolated from bullfrog fungiform papilla cell membranes and incorporated into phospholipid bilayers using the tip-dip method. The 156 pS channels were inhibited by 100 μM 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and displayed the reversal potential identical to the equilibrium potential of Cl⁻, it was identified as a Cl⁻ channel. Two types of K⁺ channel had unitary conductances of 79 and 43 pS, which may correspond to those of Ca²⁺-activated and cAMP-blockable K⁺ channels observed in isolated intact frog taste cell membranes, respectively. These results suggest that the tip-dip method is useful for stable investigation of the properties of ion channels already identified in the taste cell. Furthermore, the 23 pS channels were newly found and were activated directly by internal cAMP as cyclic nucleotide-gated (CNG) nonselective cation channels established in olfactory receptor cells. Thus, our results suggest the possibility that besides Cl⁻ and K⁺ channels, the cAMP-gated channels contribute to taste transduction.

Phallusia nigra, a solitary ascidian, has no epibionts on the surface of tunic. Moreover, the tunic contains sulfuric acid. The pH inside the tunic was about 1 as measured with a needle-tip pH electrode, and the pH at the tunic surface was 4–5 as measured with a flat-surfaced pH electrode. The surface pH decreased to 2 when the tunic surface was gently wiped with tissue paper. Thus, the tunic seems to release acid in response to mechanical stimuli. The time course of the pH recording showed that the acidity is stable at pH 1–2 for more than five minutes after the needle-tip electrode is inserted, so neutralization of tunic acid with seawater would not occur rapidly. The acid is contained in highly vacuolated cells (tunic bladder cells), and most of these cells are located beneath the tunic surface (Stoecker, 1978; Hirose, 1999). The major anion in the tunic acid was sulfuric acid, and the SO₄ ²⁻/Cl⁻ ratio was 4.63. In histological sections, the vacuolar lumen appear to occupy 25% of the total tunic and 75% of the tissue lying just beneath the surface of the tunic. The relationship between the properties of tunic acid and anti-predation, anti-fouling, and anti-infection is discussed.

Changes in gill vacuolar-type H⁺-ATPase B subunit, and Na⁺,K⁺-ATPase α and β subunit mRNA expression were examined during the course of smoltification in Salmo salar. We cloned and sequenced cDNA fragments of S. salar gill i) vacuolar-type H⁺-ATPase (V-H⁺-ATPase) B subunit, ii) Na⁺,K⁺ATPase α₁ subunit, and iii) Na⁺,K⁺-ATPase β₁ subunit, and used these as Northern blotting probes. During smoltification, the salmon showed a typical increase in gill Na⁺,K⁺-ATPase activity and improved hypo-osmo-regulatory ability as judged by their ability to regulate plasma [Cl⁻] in a 24-hr seawater challenge test (35 ppt). Gill Na⁺,K⁺-ATPase α₁ and β₁ subunit mRNA levels were regulated at a constant ratio during smoltification. Both transcripts were elevated during the build-up of gill Na⁺,K⁺-ATPase activity, underlining the importance of increased mRNA levels for increased enzyme activity. Gill V-H⁺-ATPase B subunit mRNA levels were high during the early phase of smoltification. These results support our hypothesis that gill V-H⁺-ATPase expression may be elevated during the early stages of smoltification in order to counter the effects of increased ionic efflux when in FW. The peak smolt stage was, however, characterized by simultaneously elevated gill Na⁺,K⁺ATPase expression and low V-H⁺-ATPase expression, and possibly ensures the complete transformation of the gill into a hypo-osmoregulatory organ and hence the development of optimal SW-tolerance of the smolt.

Extraretinal photoreceptor cells have been found in the pineal complex and deep brain of a variety of non-mammalian vertebrates. Light signals received by these photoreceptor cells seem to be a potent regulator of diverse physiological responses. Here, the pineal complex and deep brain of the Japanese grass lizard, Takydromus tachydromoides, were immunohistochemically analyzed to localize the photoreceptive molecule (opsin) and the light signal-transducing G-protein (transducin). In addition to the pineal organ and parietal eye constituting the pineal complex, we unexpectedly found a parapineal organ, which is located just below the parietal eye and is morphologically similar to the pineal organ. Both organs had photoreceptor-like cells with outer segments immunostained by anti-rhodopsin and anti-pinopsin antibodies. Neither opsin- nor transducin-like immunoreactivities were detected in the parietal eye with all the antibodies tested in this study, although its morphology resembles that of the lateral eyes. In the deep brain region, rhodopsin-like immunoreactivities were observed in the posterior pallial commissure and median eminence. The cerebrospinal fluid-contacting neurons in the paraventricular organ were immunoreactive to an antibody against α-subunit of cone transducin. In lizards, this is the first report showing (1) rhodopsin- and pinopsin-like immunoreactivities in the parapineal organ, (2) rhodopsin-like immunoreactivity in the deep brain, and (3) putative photoreceptive areas in the hypothalamus.

Cortactin was initially identified as a substrate for Src tyrosine kinase. It interacts with the filamentous actin in the cell cortex through the tandem repeats of 37-amino acid. In this report, we describe the identification of a Xenopus homolog of cortactin. The deduced amino acid sequence shares over 70% identity with human, mouse, and chicken cortactin. Northern and Western blot analyses revealed that Xenopus cortactin is widely expressed in Xenopus tissues. Analysis of the transcripts using polymerase chain reaction revealed two isoforms being different in the number of the tandem repeats. The major isoform has 6.5 tandem repeats but the minor one has 5.5 tandem repeats. As the sixth repeat, which is missed in the minor isoform, is encoded by a single exon flanked by introns on both sides, these two isoforms are likely to be generated by alternative splicing. We propose that cortactin regulates the construction of actin cytoskeleton by altering the number of its tandem repeats.

Cytoplasmic polyadenylation regulates translational activation of dormant cyclin B1 mRNA stored in immature oocytes, a process required for the initiation of oocyte maturation in goldfish and Xenopus. As a first step towards understanding the molecular mechanisms of translational activation of cyclin B1 during oocyte maturation, we have isolated cDNA clones encoding proteins involved in cytoplasmic polyadenylation and produced specific antibodies against recombinant proteins. These include poly(A) polymerase (PAP), poly(A)-binding protein (PABP) and cytoplasmic polyadenylation element-binding protein (CPEB). Monoclonal antibodies raised against goldfish PAP recognized several forms of PAP in goldfish and Xenopus oocytes. Besides ordinary PAPs with high molecular weight (ca. 100 kDa), the antibodies also detected those with low molecular weight (ca. 40 kDa) that are present specifically in the cytoplasm, raising new players that might be responsible for cytoplasmic polyadenylation. An antibody against goldfish PABP showed for the first time in Xenopus oocytes the protein expression of PABPII, another PABP distinct from the well-characterized PABPI. Monoclonal antibodies raised against Xenopus CPEB recognized both unphosphorylated 62-kDa and phosphorylated 64-kDa forms but did not cross-react with goldfish CPEB, which was specifically detected by anti-goldfish CPEB monoclonal antibodies produced previously. The cDNAs, recombinant proteins and antibodies produced in this study are expected to provide useful tools for investigating the regulatory mechanisms of cyclin B1 translation during oocyte maturation in goldfish and Xenopus.

Disulfiram causes undulation of the notochord and bending of pharyngeal cartilages in fish embryos. Using flounder embryos, this study aimed to elucidate which process of pharyngeal arch development was affected by the drug. Since disulfiram is known to block the synthesis of retinoic acid (RA) in vivo, we first examined whether the drug suppresses the expression of Hoxd-4 and shh, RA responsive genes related with the pharyngeal arch development. Disulfiram at a concentration which induces undulation of the notochord and bending of cartilage elements did not affect the expression of these genes. On the other hand, similar phenotypes of anomalies were found to be reproduced both in the notochord and pharyngeal cartilages by α, α′-dipyridyl which reduces the mechanical stability of collagen. Thus, we suppose that disulfiram causes anomalies by decreasing the mechanical stability of collagen and not by suppressing the expression of RA-responsive genes. Since disulfiram blocks ascorbate dehydrogenase, it is our hypothesis that this drug inhibits the maturation of collagen by affecting ascorbic acid metabolism.

Peronella japonica is a direct-type developing sea urchin that metamorphoses in 3 days without feeding, providing a good system to analyze developmental mechanisms especially at later larval stages to metamorphosis. We surveyed the Hox genes of P. japonica by PCR and RT-PCR, and isolated 13 Hox-type sequences. Phylogenetic analysis and database searches resulted in identification of all the P. japonica orthologs of the Hox genes that have been reported in other sea urchins, plus 3 Hox-type genes: the Evx-type, the Xlox-type genes, and an additional posterior Hox gene. This is the first report to show the Evx in echinoderms. The present results revealed the putative Hox gene cluster of P. japonica, which contains three anterior, four medial, and four posterior genes plus the Evx. The organization is essentially similar to the vertebrate Hox cluster except for only a single gene for Hox4-5 types. The C-terminal amino acid sequence outside the homeodomain of Hox4/5 suggested that loss of Hox4 might have occurred in the echinoderm lineage.

Cell lineage analysis of the ascidian Halocynthia roretzi demonstrated that a pair of cells situated on the right and left sides of 64-cell stage embryos, termed the A7.6 cells, give rise to trunk lateral cells (TLCs) of the tadpole larva and that after metamorphosis TLCs give rise to various mesodermal tissues of the adult, in particular all of the blood cells or coelomic cells. Here we report the isolation and characterization of cDNA clones for two TLC-specific genes HrTLC1 and HrTLC2. HrTLC1 encodes for a novel protein while HrTLC2 encodes for a polypeptide with RNA recognition motifs. Zygotic expression of HrTLC1 and HrTLC2 begins at the neural plate stage and transcripts of both these genes are restricted to TLCs.

Polyclonal antisera to Manduca sexta allatotropin and allatostatin were utilized to localize allatotropin- and allatostatin-immunoreactivities in the central nervous system of larvae, pupae and adults from the silk moth Bombyx mori. In larva the first allatotropin-immunoreactivity appeared in the brain and terminal abdominal ganglion of first instar larva. In the third, fourth and fifth instar larvae, there was allatotropinimmunoreactivity in the suboesophageal ganglion, three thoracic ganglia, and eight abdominal ganglia with immunoreactivity in some axons of N 1 and N 2. Allatostatin-immunoreactivity, which could be not demonstrated in the first and second instar larvae, appeared first in the brain and suboesophageal ganglion of the third instar larva. Allatostatin-immunoreactive cells increased to seven pairs in brain of the fifth instar larva, in which immunreactivity also appeared in eight abdominal ganglia. Allatotropin- and allatostatinimmunoreactive cell bodies in the brain projected their axons into corpora allata without terminations in the corpora cardiaca. During pupal and adult stages, brains had no allatotropin-immunoreactivity in the brains, but most ventral ganglia contained allatotropin-immunoreactive cells. There was allatostatin-immunoreactivity in the brains of the 5- and 7-day-old pupae and adult and suboesophageal ganglion of the 7-day-old pupa.

Opalina sp. were cultivated under an anaerobic condition in the complex medium containing DME/F-12 HAM mixture (Sigma) as a basic medium, and fetal bovine serum and autoclaved rumen fluid as additional components. In the medium, the opalinid cells reproduced by binary fission, and in the longest case they were maintained for more than one month, following successive transfers. When cells collected from the host in breeding season were cultured in the same medium, encystation occurred after rapid cell divisions. In this case, the population obtained from the frog had contained a sub-population of cells with small number of nuclei and small cell size. The sub-population might have been in the phase of palintomy that is a rapid sequence of binary fissions without cell growth and finally produces small cysts. Our successful maintenance of the opalinid cells can offer the first step for the establishment of the in-vitro culture method.

Effects of the coastal oceanographic conditions on sexual maturation, salinity tolerance, and expression of vasotocin (VT) gene in homing chum salmon, Oncorhynchus keta, were studied by four years of fieldwork and transfer experiments. In fieldwork in 1992 and 1993, fish were sampled at three sites along their migratory pathway on the Sanriku coast, Japan. In transfer experiments in 1994 and 1995, fish captured in the seawater (SW) environment were transferred to SW or freshwater (FW) aquaria and sampled 1 to 4 days later. The distribution of cold and warm currents, which governs the oceanographic conditions of the Sanriku coast, were deduced from the mean sea surface temperature. Maturity of homing fish was estimated by gonadal states. Salinity tolerance was estimated by plasma Na⁺ levels and mortality in SW environment. Changes in VT gene expression were assessed by a quantitative dot blot analysis of the hypothalamic levels of VT mRNA. Homing fish were fully mature, and showed high plasma Na⁺ levels and high mortality in the SW environment in 1992 and 1994 when the warm current dominated. In the 1994 transfer experiment, VT mRNA levels markedly increased in the SW-retained males, whereas the levels were decreased by FW-transfer in both sexes. Homing fish were not fully matured in 1993 and 1995 when the branch of cold current reached the Sanriku coast. In the 1995 transfer experiment, VT mRNA levels did not change in both SW-retained and FW-transferred fish. In conclusion, the warmer oceanographic conditions affected the maturity and salinity tolerance in homing salmon, which in turn altered the osmotically-induced expression pattern of VT gene.

The formative processes of the brooding organs of two botryllid ascidians, Botryllus delicatus and Botryllus sexiens, were observed. The structure of these organs was also studied in detail histologically. The brooding organs of these two species were formed from the branchial sac, in contrast to the brooding organs of other botryllids, which are formed from the peribranchial wall. The structures of the brooding organs of B. delicatus and B. sexiens were different, but the processes of their formation were almost the same. As the maturation of the oocytes occurred, a part of the epithelium of the branchial sac extended toward the peribranchial epithelium covering the ovary. The extension of the branchial epithelium looked like a bowl and made a surrounding space with the peribranchial epithelium in a peribranchial cavity. The oocyte was ovulated into this space and fertilized there. After ovulation, the extension of the branchial epithelium formed a sac-like brooding organ in combination with the peribranchial wall and held the embryo tightly, like the incubatory pouch of the genus Botrylloides. The unusual characteristics of the brooding organs in these two species lead to reconsideration of phylogenetic relationships and taxonomic criteria in botryllids.

Two new species of lysianassoid amphipods, Anonyx omorii sp. nov. and A. abei sp. nov. were described from shallow waters of Onagawa Bay, northeastern Japan. The genus Anonyx has been divided into five informal groups by the morphological characters of the uropod 2 (Steele, 1979). According to the morphological features of uropod 2, A. omorii sp. nov. is assigned to A. compactus group with the inner ramus of uropod 2 completely constricted beyond the insertion point of a long distal spine, and A. abei sp. nov. to A. nugax group with the inner ramus of uropod 2 constricted at the insertion point of the long distal spine.

In the present study, we have cloned and determined the nucleotide sequence of telomeric DNA of a freshwater gastropod Biwamelania habei. The sequence was comprised of (TTAGGG)n tandem repeat, which is identical with the sequence of vertebrate telomere. The characteristic sequences, together with the chromosomal localization revealed by FISH analysis and the Bal 31 exonuclease sensitivity of the hybridization signal, support the sequences to be located at chromosomal ends. This is the first report for identification of molluscan telomere DNA and is also the first report of invertebrate telomere with (TTAGGG)n sequence. Although the telomere sequence of B. habei is different from that of any other invertebrates reported to date, the sequence universally occurs among various eukaryotes including some metazoans. It is thus evident that B. habei retains ancestral sequence for invertebrate telomeres. Interstitial telomeric signals were observed in some chromosomes. Some of such interstitial telomeric sites coincide with the presumed fusion point of Robertsonian rearrangement.

Caligus lalandei Barnard, 1948 (Caligidae, Siphonostomatoida) is redescribed based on the materials occurring in the waters of East Asia. This parasitic copepod is so far known only from the amber-jacks (Seriola spp.) outside of the western North Pacific. However, in 1989 it was discovered on “mojako” (juveniles) of Seriola quinqueradiata collected off Azuma-cho and Yamakawa-cho in Kogoshima Prefecture, Japan. In 1998 it appeared on Seriola lalandi caught in a set net installed off the coast near Kangreung, Korea and in the following year it was found parasitic on S. lalandi cultured in Tsukumi of Oita Prefecture, Japan. The possible origin of the occurrence of C. lalandei in Japan and Korea is discussed; but, due to several unknown factors, it is inconclusive at this point of time whether the occurrence of C. lalandei in Japan and Korea is due to the “natural” or “artificial” process. However, the parasite has well established in its new habitat - the northern region of the western North Pacific.

Karyotypes of the greater Japanese shrew-mole, Urotrichus talpoides (Talpidae), collected from 57 localities in Japan were studied by conventional, G- and C-band staining techniques to elucidate geographical chromosomal variations, the mechanism of changes in chromosomes, and the distribution patterns of karyotypic races. Shrew-mole samples examined could be divided geographically into two slightly divergent chromosomal forms designated as the western and eastern races. These two karyotypic races had the same diploid number (34) and fundamental number (64), but they had autosomes with different makeups. A comparison of conventional karyotypes showed a distinct intraspecific variation in shape of autosomal pair no. 14 which was classified as subtelocentric in the western race and as metacentric in the eastern race. G- and C-banding analyses revealed that karyotypic variation found in no. 14 pair was involved in pericentric inversion and quantitative changes in constitutive heterochromatin. Intraspecific and geographical variation in chromosomes caused by such karyological events is rare and unique among members of the family Talpidae examined so far. Furthermore, our results demonstrated that the clear boundary between the two parapatric karyotypic races was actually located along the Kurobe-Fuji line in the central part of Honshu, but not along the Owari-Tsuruga isthmian line previously postulated by Tsuchiya (1987, 1988). Zoogeographical implications of the boundary of parapatric distribution in U. talpoides are also discussed.