Zoological Science

We have previously demonstrated that hemocytes of the solitary ascidian Halocynthia roretzi respond to several stimuli, such as calcium ionophore, lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA), and to release metalloproteases. Here, we show that H. roretzi hemocytes contained two substances, named protease-releasing factors A and B, which induced the release of metalloproteases from the hemocytes. Factor A was isolated from the acid-ethanol extract of hemocytes by gel filtration, while factor B was isolated from the hypotonic extract of hemocytes by cation exchange chromatography. The former factor was a heat-labile, large molecule and the latter was a heat-stable, small molecule. We found that these factors existed only in some types of hemocytes.

Accumulative inactivation of a cloned Aplysia K⁺ channel (AKv1.1a) was examined in Xenopus oocyte expression system by the patch clamp technique. AKv1.1a inactivates by both N-type and C-type mechanisms. The amino-terminal domain of the channel is indispensable for N-type inactivation, whereas other parts of the channel is involved in C-type inactivation. The accumulative inactivation induced by repetitive pulses (0.2–0.5 Hz) was relatively insensitive to the pulse duration (10–900 msec). The accumulative inactivation was inhibited when the external K⁺ concentration ([K⁺]_out) was increased, or when tetraethylammonium (TEA) was added in the external solution. The accumulative inactivation of the amino-terminal deletion mutant (ΔN) which lacks N-type inactivation was dependent on the pulse duration such that it was less pronounced for short repetitive pulses (<100 msec). The accumulative inactivation of ΔN was also inhibited by high [K⁺]_out and external TEA. By contrast, the accumulative inactivation induced by pair-pulse protocol was not perturbed by external TEA, and was not observed in ΔN. The accumulative inactivation of AKv1.1a was enhanced when the membrane patch was excised out of the cell. Paradoxically, the macroscopic inactivation of AKv1.1a became slower in the excised patch. The accumulative inactivation of ΔN was less sensitive to the patch excision. Some synthetic peptides which were designed based on the amino-terminal sequences of K⁺ channels induced a use-dependent block of ΔN which was apparently similar to the inactivation of AKv1.1a. Our results suggest that either N-type or C-type inactivation can induce the accumulative inactivation of K⁺ channels, and that C-type inactivation coupled to N-type inactivation plays substantial roles in the frequency dependent accumulative inactivation of AKv1.1a.

Ascidians are known to accumulate high levels of vanadium in their blood cells. Recently, we found a vanadium-associated protein (VAP) in blood cells of a vanadium-rich ascidian, Ascidia sydneiensis samea. In this paper, we raised a monoclonal antibody against VAP, designated F8DH. Immunoblot analysis showed that F8DH recognized 2 related peptides of 15 kDa and 16 kDa of VAP. Using F8DH, VAP was shown to be in the cytoplasm of vanadocytes and compartment cells, both of which were reported to contain vanadium. F8DH also stained the vanadocytes distributed in the connective tissues around the alimentary canal, suggesting that vanadocytes in the connective tissue contained VAP. Furthermore, blood cells of 3 different species of ascidian having high levels of vanadium, A. sydneiensis samea, A. ahodori, and Ciona intestinalis, showed reactivity of F8DH but little reactivity was observed in 2 species having less vanadium, Halocynthia roretzi and Pyura michaelseni, suggesting that VAP recognized by F8DH is a common protein in vanadium-rich ascidians.

The biochemical-genetic analysis of 19 enzyme systems coded by 33 structural gene loci in the diploid Leuciscus cephalus have revealed the existence of two genetic loci never described in other cyprinid fishes. The electromorphs of mitochondrial aspartate aminotransferase (mAAT) and fumarate hydratase (FH) in larvae and in different adult tissues of Leuciscus cephalus suggest that these enzymes are coded by two loci each, at variance of available literature data on cyprinids, in which a single structural locus is known to code the mAAT and FH enzymes. These observations are confirmed for the adults of 8 additional Italian cyprinids.

A soluble alkaline trehalase was purified from embryos and larvae of the brine shrimp, Artemia, by acetone treatment, chromatography on columns of DEAE-Sepharose Fast Flow, Con A-Sepharose and TSKgel AF-Chelate TOYOPEARL 650M, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme subjected to SDS-PAGE showed a single protein band, suggesting a molecular mass of 70,000 Da. The enzyme exhibited an apparent molecular mass of 58,000 Da on gel filtration. Endoglycosidase H digestion of the enzyme did not affect the activity of the trehalase, and resulted in a molecular mass of 66,000 Da on SDS-PAGE. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7∼4.8. The catalytic activity showed a maximum at pH 8.0, and a specific activity of 140 μmoles glucose liberated from α,α-trehalose min⁻¹ × mg⁻¹ was observed at 30°C. The Km value for α,α-trehalose was estimated to be 8.4 mM. Among the eleven oligosaccharides and two α-glucoside derivatives studied, the enzyme hydrolyzed only α,α-trehalose. The enzyme was maximally active at 55°C and had an activation energy of 55.8 kJ × mol⁻¹. The enzymatic reaction was completely inhibited by 0.1 mM HgCl₂. The activity of the purified enzyme was inhibited by 1 mM EDTA in the presence of 50 mM phosphate buffer, and the additions of appropriate amounts of MnCl₂, MgCl₂ and CaCl₂ to the reaction mixture each protected the activity.

It has long been known that only L-lactic acid is found in animals and that D-lactic acid is produced in microbial organisms. During the course of study of D-lactate formation from methylglyoxal (the methylglyoxal bypass) in animals, we found that D-form is mainly present in Octopus vulgaris and very little L-lactate was found in octopus muscle. There is an inverse relationship between octopus and normal animals for concentrations of D-and L-lactate. The activities of D-lactate dehydrogenase (D-LDH) was predominantly found in octopus muscle, while L-LDH activity was scarcely detected. Methylglyoxal was the best substrate for D-lactate formation in octopus foot homogenate and pyruvate was the second best substrate. It was also found that D-lactate is present in much higher amounts than the L-form in some animals or plants.

In the eri-silkworm, Samia cynthia ricini, the adult antennal flagellum is segmented into many annuli. Although the number of annuli is an important parameter in the morphogenesis of adult antenna, it is not clear when and how the number of annuli is determined. In the present study the fifth instar larva of the eri-silkworm was studied histologically to clarify when the antennal imaginal disk began morphogenesis and when the number of annuli of adult antennal flagellum was determined. In addition we studied whether the length of the embryonic and larval period might have any influence on the number of annuli in the erisilkworm, and the influence of other factors such as body size and sex was also examined. Serial histological study of the imaginal disk during the larval period suggested that the number of annuli was determined by the second day after gut-purging, since the segmentation of the pupal antenna was almost finished by this time and the number of segments of pupal antenna was nearly equal to the number of annuli of adult antenna. The embryonic and larval period was closely related with the number of annuli. When the insects were reared at 25°C, the number of annuli was almost equal to the number of the days in which the insects passed from oviposition to gut-purging. In addition, the number of annuli tended to increase one by one from 27 to 34 as the embryonic and larval period was extended day by day from 28 to 35 days. When the insects were reared at 18°C, the larval period was doubled, whereas the number of annuli remained in the same range (28–34) as that reared at 25°C. The body size did not correlate with the number of annuli. Although the number of annuli was significantly larger in female than in male, this difference seemed to be due to the difference in the length of the embryonic and larval period in both sexes.

The highly diverse Perciformes are the most numerous and most advanced group of the teleosts, but experimental embryology has almost completely neglected them. The ice goby (shiro-uo), Leucopsarion petersii, deserves attention as a useful species for this purpose. It is readily available commercially from fishermen, and their mature adults can be easily kept and bred in the laboratory. Its totally transparent eggs and embryos develop swiftly and easily under simple culture conditions. Cleavage is finished in 17 hr at 20°C and most organs are established in 3 days. Main stages of embryonic development and methods of handling are described.

The time of appearance and location of three distinct collagen gene transcripts termed 1α, 2α, and 3α, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1α and 2α transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3α transcripts of the basement membrane collagen appeared earlier than 1α and 2α, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3α at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3α mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.

The distribution of glycoconjugate in testicular germ cells of the cricket, Gryllus bimaculatus, was cytochemically investigated using a panel of lectins and several kinds of glycosidases. Observations were focused on the early process of spermatogenesis. The binding pattern with N-acetylgalactosamine-binding lectins, especially with Dolichos biflorus agglutinin, and their susceptibility to peptide-N-glycosidase F (PNGase F) suggested the stage-specific expression of N-linked glycoproteins with terminal α-N-acetylgalactosamine in the early meiotic prophase. In the primary spermatocyte, the transitory expression of PNGase F-resistant glycoproteins was detected by several mannose-binding and fucose-binding lectins such as Lens culinaris agglutinin and Anguilla anguilla agglutinin. Hyaluronidase-sensitive glycoconjugates also distributed widely in cricket testes, exemplified by perinuclear granular structures recognized by Helix pomatia agglutinin and nuclear staining with Phaseolus lunatus agglutinin. These lectin-binding affinities in meiotic prophase were discussed in reference to many informations about carbohydrate-lectin binding specificities.

We have characterized the nucleotide sequence of Na⁺/K⁺-ATPase alpha subunit (NKA) cDNA in embryos of the sea urchin, Hemicentrotus pulcherrimus. The primer extension experiments showed that the sea urchin NKA gene generated multiple lengths of transcript. To obtain the 5′-ends of the transcripts, we isolated cDNA clones by the rapid amplification of cDNA ends (RACE). These clones were classified into 2 types on the basis of their 5′ leader sequences. The sequences of the clones were identical except their 5′ leaders. By Northern blot analysis, 1 of the 2 types of transcripts was always detectable in sea urchin embryos during early development, and another was not detected before the morula stage. Genomic PCR demonstrated that the two 5′ leaders were coded by different exons separated by an intron in a single gene. These results show that the transcripts coding 2 isoforms were expressed from a single gene.

To clarify when the free neuromasts of fish become functional, the neuromast formation in the embryonic development of the cod fish Gadus macrocephalus Tilesius was investigated. The initial appearance of placodes and an increase in the number and distribution of free neuromasts were followed using light microscopy and scanning electron microscopy. Subsequently, the appearance of afferent and efferent nerve endings of the free neuromasts was investigated by transmission electron microscopy. Sections of embryos 72 hr before hatching revealed a pair of placodes on the head and two pairs on the trunk. The placodes increased in number, and some of them differentiated into free neuromasts during the embryonic stage. There were three pairs of free neuromasts on the head and four pairs on the trunk in newly hatched larvae. The afferent nerve endings were recognized first in the free neuromasts on the trunk at about 36 hr before hatching, and the efferent nerve endings were recognized on the trunk at about 24 hr before hatching. On the head, afferent nerve endings were seen at about 12 hr before hatching; the efferent nerve endings were not yet seen in newly hatched larvae.

The anterior pituitary gland has recently been shown to contain a number of bioactive peptides other than inherent pituitary hormones. One of these peptides is vasoactive intestinal peptide (VIP). The localization of VIP has not known and so the present study was undertaken to determine which type of pituitary cell contains this peptide. The adult rat anterior pituitary was immunohistochemically examined with two VIP antisera. One of these antisera successfully stained some pituitary cells. Both the double immunostaining technique and the flip-flop section method revealed VIP-like immunoreactivity, mainly in gonadotropes. This immunostaining was lost when the anti-VIP was preabsorbed with synthetic VIP or with pituitary adenyl cyclase activating peptide (PACAP), 68% of the 1–28 portion of which is homologous to VIP. When tested with anti-PACAP, however, no immunoreactivity was observed in anterior pituitary tissue. These results indicate that VIP is localized in gonadotropes in the anterior pituitary. The physiological significance of such localization of VIP is discussed in relation to the regulation of PRL secretion.

The early stage of cell migration from the olfactory placode to the forebrain was studied immunohistochemically in chick embryos to investigate the nature of early migrated cells and the role of these cells in the sebsequent migration of luteinizing hormone releasing hormone (LHRH) neurons. The initial cells migrating from the olfactory placode stained strongly positive for the highly polysialylated neural cell adhesion molecule (NCAM-H), but were negative for LHRH. These migrating NCAM-H immunoreactive cells were observed on embryonic day (ED) 2.5 as a bulge with a few cells from the base of the placode. By ED 3, they formed a wide cellular strand and developed into a cellular bridge between the olfactory placode and the ventro-rostral surface of the forebrain. These migrating cells are neurons because they stained positive for growth associated protein-43 (GAP-43), microtubule associated protein (MAP) 2, and MAP 5. Some of these cells seemed to migrate caudally along the ventro-lateral surface of the forebrain. LHRH-immunoreactive cells were not detected in the olfactory epithelium until after ED 3.5, which was one day after formation of the cellular bridge. Then, LHRH-immunoreactive cells appeared and began to cross the cellular bridge. The outgrowth of the olfactory nerve axon bundles from the olfactory epithelium was detected at around the same time when LHRH-immunoreactive cells first appeared. These olfactory nerve axons expressed NCAM-H, GAP-43, and MAP 5, as assessed by immunochemistry. After bundle formation, the olfactory nerve appeared to provide the migratory routes for LHRH neurons. These results suggest that the cellular bridge formed by the NCAM-H-immunoreactive neurons plays an important supportive role for LHRH neurons at the initial stage of their migration in chick embryos.

The male reproductive cycle of the toad, Bufo melanostictus, was studied with emphasis on spermatogenic activity, plasma androgen, and changes in the weights of testes, liver, and fat bodies. A total of 98 toads were collected between March 1990 and March 1991 in central Taiwan. Histological evidence indicated that the spermatogenic cycle of this toad is of a fluctuating continuous type. Although cell nests of all spermatogenic types were present every month of the year, the greatest intensity of spermatogenic activity, as expressed by the presence of sperm bundles, occurred in March. Both testicular weight and plasma androgen levels peaked in March. The weights of fat bodies peaked in July, which was not coincident with the beginning of breeding. Combined data from spermatogenic activity, plasma androgen levels, and changes in the weights of testes, fat bodies, and livers revealed that B. melanostictus is a continuous breeder. However, its annual reproductive cycle could be divided into 4 periods: 1) breeding period (February–April); 2) post-breeding period (May–June); 3) reproductive energy preservation period (July–September); and 4) torpid period (October–January).

The relationship between body size and the results of territorial conflicts was studied in males of the dragonfly, Orthetrum japonicum japonicum. Territorial residents were larger than intruders in body width, but not in hind wing length. Winners of territorial conflicts were larger than losers in body width, but not in hind wing length. This difference was attributed to the fact that residents were larger than intruders. The results of territorial conflicts were more strongly affected by the role of the opponents (resident or intruder) than by the difference in their body sizes. Territorial males arrived at the territorial sites earlier than non-territorial ones on a given day. The body size of males arriving at the study area earlier in a day was not larger than that of males arriving later.

Embryonic temperature sensitivity of the solitary ascidian, Ciona savignyi, was examined with special reference to acclimatization to seasonal change in seawater temperature. This ascidian spawns throughout the year; its life span is completed within six months and depends on the cumulative environmental temperature. The optimal temperature range for development from early cleavage stage to metamorphosis in embryos produced by individuals raised in warmer seasons differed significantly from that of individuals raised in colder seasons. Within the common optimal temperature range, developmental times at any given temperature were the same for both groups of embryos. The thermal acclimation of ascidian embryos is discussed and compared with that of echinoid embryos.

Analysis of mitochondrial DNA (mtDNA) restriction fragment length polymorphism in Japanese wild populations of the medaka, Oryzias latipes revealed a large number of mtDNA haplotypes that form three distinct clusters (clusters A, B and C). The average nucleotide diversities among these three clusters are 8.9% (A versus B), 8.4% (A versus C), and 7.3% (B versus C). Cluster A consists of seven haplotypes and was subdivided into two subclusters. The nucleotide diversity in cluster A is low, ranging from 0.3% to 1.4% (mean 0.8%). Cluster B has 55 haplotypes and was subdivided into 11 subclusters. The nucleotide diversity in cluster B is high, ranging from 0.1 to 4.8% (mean 1.5%). Cluster C consists of only one haplotype, found in two sites of the Kanto district. The geographic distributions of mtDNA haplotypes in clusters A and B appear fully concordant with the previously described ranges of the Northern Population and the Southern Population defined by allozymes. Moreover, the distributions of mtDNA haplotypes in the subclusters show strong geographical associations. The distribution patterns of mtDNA haplotypes suggest some migration events of the medaka.

Phylogenetic relationships among 31 operational taxonomic units of shrews (Soricidae, Mammalia), mainly from eastern Eurasia, were inferred from partial nucleotide sequences of the mitochondrial cytochrome b gene by maximum likelihood (ML) and neighbor joining (NJ) methods. Eleven monophyletic groups were recognized among the soricine shrews examined in the ML tree. However, branching orders of the groups were obscure judging from the local bootstrap values, and two out of the 11 groups were not monophyletic in the NJ tree. The phylogenetic relationships among Sorex caecutiens, S. shinto, and S. sadonis in the Japanese and Sakhalin islands, whose taxonomy was controversial, were clarified. S. shinto in the Honshu and Shikoku Islands is genetically differentiated enough to be considered a separate species from S. caecutiens, while S. sadonis could be treated as a subspecies of S. shinto. Some other taxonomic problems are also discussed.