Zoological Science

The submandibular glands (SMGs) were removed in male genetically obese yellow A^y mice at 24 weeks of age. The mice lost weight postoperatively unlike the sham-operated controls. Furthermore, the coat color changed after sialoadenectmy from yellow to black on part of the back. The pattern and tone of the black regions varied from mouse to mouse. The black color was conspicuous 4 to 8 weeks postoperatively, then gradually faded, but was still noticeable for at least 5 months. In A^y mice, the agouti gene is overexpressed, and the pleiotropic effects of excess agouti protein are believed responsible for their obesity and yellow coat color; agouti antagonizes both the hypothalamic melanocortin-4 receptor responsible for obesity and the skin melanocortin-1 receptor responsible for yellow pigmentation. Our observations suggest that a factor or factors in the SMG are essential for the expression of two separable phenotypes. The SMG presumably affects the expression level or action or both of agouti to alter the phenotypes.

Ascidians have the unusual physiological ability to accumulate high levels of vanadium and reduce it to the +3 oxidation state (V^III) in vanadocytes, the vanadium-containing blood cells. We are characterizing several polypeptides specific to vanadocytes that may participate in this. This study revealed that a 100-kDa antigen, recognized by a newly prepared monoclonal antibody, S8E4, is exclusively localized in vanadocytes, and identified the antigen as glycogen phosphorylase (EC 2.4.1.1) by sequencing the encoded cDNA. Since two enzymes, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), both in the pentose phosphate pathway, have already been identified in vanadocytes, at least three enzymes involved in carbohydrate metabolism are localized in vanadocytes in huge amounts.

Vacuolar-type H⁺-ATPases (V-ATPases), which are composed of at least ten different subunits, can generate a proton-motive force by hydrolyzing ATP and acidify the contents of various intracellular organelles. Subunits A and B of V-ATPase have been detected immunologically in ascidian blood cells, predominantly in signet ring cells (vanadocytes), which accumulate vanadium in their vacuoles. The action of V-ATPase in ascidian blood cells has been demonstrated by the fact that bafilomycin A₁, a specific inhibitor of V-ATPases, inhibits the acidification of the vacuoles of vanadocytes. As the next step in studying the function of V-ATPase in vanadocytes, we isolated cDNAs encoding subunits A and B of V-ATPase from the blood cells of an ascidian, Ascidia sydneiensis samea. The nucleotide sequences of the cDNAs for subunits A and B encoded proteins of 619 and 509 amino acids, respectively, both of which were highly conserved among organisms.

The termination of the escape behavior in the cockroach Periplaneta americana was investigated. Escape behavior was effectively terminated when cockroaches were allowed to select a dark shelter and hide beneath it. This shade-induced pause in escape running (a shade response) was observed even in very low-light levels (less than 0.01 lux). Contributions of the ocelli and the compound eyes to the shade response were examined. Removal of both compound eyes resulted in complete disappearance of the shade response. Animals with just the ocelli removed were less likely to shelter in the shadowed area, especially under a low-light condition. Input from compound eyes seems to be essential to the shade response. The ocellus may enhance the function of compound eye, and its modulatory function is effective in low-light conditions.

Young songbirds use auditory feedback to match their own vocalizations with a memorized tutor's song in order to develop their songs normally. After learning, songs of adult birds are stereotyped and remain stable. Recent studies showed that auditory feedback is necessary for maintaining the stability of adult bird song in some species including Bengalese finches. We deprived adult male Bengalese finches of auditory input by removing their cochleae and acoustically analyzed the songs of the deafened birds. After the operation, song patterns altered within a week and remained unstable for approximately a month. The post-operative songs were finally stabilized within 60 days after the operation, although their patterns were different from those of the pre-operative songs. Compared with the pre-operative songs, syllables with higher fundamental frequencies decreased and those with lower fundamental frequencies increased in the stabilized post-operative songs of all the birds examined. These results suggest that adult male Bengalese finches need auditory feedback in order to maintain their normal songs and that this feedback is involved in retaining the syllables of higher fundamental frequencies. They also suggest that the stabilized post-operative songs consisting of syllables with lower fundamental frequencies may be maintained by other sensory feedback systems.

The developmental pathways of neuter castes in the processional nasute termite Hospitalitermes medioflavus, which has monomorphic soldiers and trimorphic workers, was examined. Hospitalitermes is one of the genera of Nasutitermitinae which make processional foraging columns. The castes engaged in foraging are soldiers, and minor, medium and major workers. Sex determination by dissection and histological staining showed that soldiers and minor workers were males, while medium and major workers were females. In larval head width, three well-defined size peaks in the frequency distribution were found. The smallest peak consisted of both males and females of the first instar. The intermediate and largest peaks consisted respectively of males only and females only, of the second instar. Molting individuals and observations of mandibles confirmed the developmental relationships among castes. We concluded that the developmental pathway of the neuter castes in H. medioflavus is parallel to that found in Nasutitermes. Males change tasks from gnawing food (as minor workers) to defense (as soldiers), whilst females specialize in foraging (gnawing and carrying food). Division of labor during foraging depends primarily on sex and the developmental stage of neuter castes. This species thus displays both temporal and sexual polyethism.

During autogamy or conjugation in Paramecium, the micronucleus undergoes prezygotic and postzygotic nuclear divisions to result in new micro- and macronuclei, while the old macronucleus breaks down into fragments. The fragments appear to remain functional for some time, as evidenced by their ability to generate an intact macronucleus, via the process called macronuclear regeneration. On the other hand, macronuclear DNA degradation must take place, as suggested by a transfer of isotope-labeled material from the fragments to the developing macronuclear anlagen. We monitored electrophoretic profiles of DNA in autogamous and exautogamous cells of P. tetraurelia at 1-day intervals for 5 days, and in conjugating and exconjugant cells of P. multimicronucleatum treated with conjugation-inducing chemicals at 1–2 hr intervals for 2 days. We failed to detect DNA degradation in P. tetraurelia, but found a prominent DNA electrophoretic smear in starving exconjugant cells of P. multimicronucleatum 33–40 hr after the initiation of conjugation. This DNA degradation was remarkable in that it occurred 10–20 hr after the appearance of macronuclear anlagen, in that it occurred suddenly and transiently, showing no trace one hour before and one hour after the smear was detected, and in that most of the macronuclear fragments persisted after the smear was detected. These results show that, during the starvation period in P. multimicronucleatum exconjugants, partial autolysis of macronuclear fragments occurs at a specific stage. We propose that this stage might be the critical point after which macronuclear fragments could not be regenerated to a functional macronucleus.

Contraction of molluscan adductors has been classified into three states; 1) resting state, 2) contracted state, 3) prolonged “catch” state. Among these, the “catch” state is considered a peculiar state, which requires little expenditure of energy, but in which contraction can be maintained for long periods. It is not yet known whether “catch” muscle contraction is regulated by Ca, or where Ca translocates during resting state through “catch” state, if the muscle contraction is regulated by Ca. We attempted to observe Ca translocation in muscle cells during contraction by the K-pyroantimonate method. We fixed “catch” muscle cells for electron microscopy with fixative including K-pyroantimonate, and observed where electron-dense precipitates, in which Ca is concentrated, were located in the muscle cells in the three states of contraction. At the resting state, precipitate was located at cell peripheries, in positions such as at the inner surface of cell membranes and in sarcoplasmic reticular systems (SRs). In the contracted state, they were located within the cytoplasm. At the “catch” state, they were found in both the cytoplasm and at peripheries, although the number of precipitates in peripheral areas was small. Thus, we show that calcium translocates in the cells during resting-contraction-catch cycles of “catch” muscle contraction.

Ultrastructural and physiological studies have shown that planarian muscles have some characteristics of smooth and some characteristics of striated muscles. To characterize planarian muscles, we isolated two myosin heavy chain genes (DjMHC-A and DjMHC-B) from a planarian, Dugesia japonica, by immunological screening, and analyzed their structures and spatial expression patterns. Structural analysis indicated that both MHC genes are striated-muscle-type myosin genes, although planarian muscles do not have any striation. In situ RNA hybridization showed that expression of the two myosin genes is spatially strictly segregated. DjMHC-A was expressed in pharynx muscles, pharynx cavity muscles, muscles surrounding the intestinal ducts, a subpopulation of body-wall muscles and several muscle cells in the mesenchymal region around the base of the pharynx. DjMHC-B was expressed in body-wall muscles (including circular, diagonal and longitudinal muscles), vertical muscles and horizontally oriented muscles. Double staining with DjMHC-A and -B probes clearly demonstrated that expression of the DjMHC-A and -B genes do not occur in the same cell. During regeneration, the number of cells positive for expression of each gene increased in the blastema region, suggesting that both types of muscle may be involved in blastema formation. DjMHC-B-positive cells disappeared from the body-wall muscle layer in the pharynx-cavity-forming region, whereas DjMHC-A-positive cells were markedly accumulated there, suggesting that the two types of muscle in the body wall layer may have distinct functions. These results indicate that planarians have at least two types of muscle that express striated-muscle-type MHC genes, but do not form striation.

BMP (Bone Morphogenetic Protein) acts as a morphogen for dorso-ventral patterning and organogenesis in both vertebrate and invertebrate development. A cDNA encoding BMP (named Djbmp) has been cloned and sequenced from the planarian Dugesia japonica. The mature form of DjBMP which was deduced from the cDNA sequence was composed of 114 amino acid residues. The position of seven cysteine residues of the mature DjBMP was highly conserved among the TGF-β superfamily. DjBMP had high similarity to human BMP-2A (50% amino acid identity), BMP-4 (49%) and Drosophila decapentaplegic protein (48%), indicating that DjBMP belongs to DVR (decapentaplegic-Vg1-related) group. The expression pattern in intact and regenerating planarians revealed by whole mount in situ hybridization suggested that the DjBMP plays a role not only in dorso-ventral but also in mid-lateral body patterning.

We have demonstrated that the tissue differentiation patterns along the dorsoventral and anteroposterior axes can be controlled by a combination of activin A, concanavalin A (Con A), and retinoic acid. Xenopus blastula animal caps, normally fated to form epidermal tissues, differentiated into ventral mesoderm tissues such as coelomic epithelium and blood-like cells following treatment with activin A (0.5 ng/ml). Dorsal mesoderm tissues like muscle and notochord, were induced by graded addition of Con A. Conversely, Con A (1 mg/ml) induced anterior neural tissues, forebrain accompanied by eyes and cement glands, in the animal caps. Posterior neural tissues, hindbrain with ear vesicles and spinal cord, were induced by graded addition of activin A. Retinoic acid was also capable of shifting the Con A-induced anterior neural tissues to more posterior tissue phenotypes, however, its caudalizing activity was slightly different from that of activin A. These results suggest that the concentration gradients of these three factors can regulate the differentiation patterns along the embryonic axes. The present study provides a suitable test system for analyzing the establishment of the fundamental body plan in early vertebrate development.

The differentiation patterns of animal cap explants from the Japanese salamanders Hynobius lichenatus and Hynobius nigrescens were examined after exposure to various concentrations of activin A. A wide range of concentrations of activin A (0.5-100 ng/ml) induced various mesodermal tissues such as ventral mesoderm, somitic muscle, and notochord. At concentrations higher than 50 ng/ml, yolk-rich endodermal tissue was induced in many of the explants. Activin A is also known to have mesoderm-and/or endoderm-inducing activity on the animal caps of Xenopus laevis and Cynops pyrrhogaster, but their response patterns are slightly different. The mode of differentiation of activin-treated Hynobius animal caps was compared with that of Xenopus and Cynops in relation to the structure of the animal caps.

The adult ovary was examined in a freshwater crayfish, Procambarus clarkii, to clarify the ovarian structure and the mode of oogenesis. A Y-shaped ovary consisting of a pair of anterior ovarian sacs and a single posterior ovarian sac was located in the cephalothorax, on the dorsal side of the stomach. An oviduct connected each of the posterior ends of the paired anterior ovarian sacs with the genital pore on the coxa of the 6th appendage. The wall of the ovarian sacs, consisting of a layer of the ovarian epithelium, folded inwards to form a number of oogenetic pouches of various sizes. Each oogenetic pouch contained one egg or large oocyte, vitellogenic or previtellogenic, sometimes followed by a few early previtellogenic oocytes in the oogenetic pouch lumen. Germaria containing oogonia, very early previtellogenic oocytes and somatic interstitial cells were located in the ovarian epithelium near the bases of the oogenetic pouches. In a cross-section of the ovarian sac, the germaria were concentrated in the center of the ovarian sac as a central germarial cluster. An early previtellogenic oocyte beginning to grow left its germarium and raised the ovarian epithelium to form a new oogenetic pouch, in which it remained until mature. Mature eggs were ovulated from the oogenetic pouches into the central ovarian lumen, transferred into the oviducts, and oviposited through the genital pores. The female reproductive system was surrounded wholly and tightly by a thin muscular sheath, which has often been mistaken as the ovarian epithelium in some decapod crustaceans.

The process of neural crest cell migration differs between the lamprey and swordtail [Hirata et al., Zoological Science 14: 305-312 (1997)]. In swordtail embryos, neural crest cells in the ventral pathway are distributed ventrally beyond the notochord at all axial levels of the trunk. In contrast, no neural crest cells are seen ventrally beyond the notochord in post-branchial regions of lamprey embryos, although in the branchial regions some cells migrate ventrally beyond the notochord. Since sclerotomal development is essential in the formation of ventral pathways in avian and mammalian embryos, sclerotomal development in lamprey and swordtail embryos was examined to understand the mechanism underlying the differences in neural crest cell migration between these two animals. Sclerotomal development and neural crest cell migration progressed concurrently in the swordtail trunk. In the lamprey, the formation of the sclerotome was regionally different along the antero-posterior axis. In the branchial region, where neural crest cells are observed ventrally beyond the notochord, sclerotomal development was closely correlated with neural crest cell migration as in the swordtail. The overt sclerotome was not observed in the lamprey trunk posterior to the branchial region during the period of neural crest cell migration. These observations suggest that differences in the timing of neural crest cell migration and sclerotomal development generate the differences in migration of lamprey and swordtail neural crest cells along the ventral pathways. Heterochronic differences in sclerotomal development correlated with neural crest cell migration may have some significance in the evolution of the trunk body organization from agnathans to gnathostomes.

In early Xenopus embryos, continuous exposure of embryos to aphidicolin and inactivation of the nucleus by ultraviolet-irradiation induce prolongation of the cell division interval. The extent of prolongation of the cell division interval appears to depend on how heavily DNA replication is suppressed by the treatments. Embryos showing significantly prolonged cell division intervals tend to fail normal cell divisions, often forming abortive furrows. There appears to be a critical point for the extent of prolongation of the cell division interval at 30% that divides the success and failure of normal cell division. This percentage, 30%, coincides with that for the prolongation of the interval of oscillatory activities seen in enucleated eggs. The presence of an intact nucleus that can undergo DNA replication rescues normal cell divisions with normal intervals and a normal furrow. Histologically, the nucleus in embryos showing more than 30% prolongation of cell division intervals appears to fail DNA replication and remain unduplicated with a round morphology like the interphase nucleus. A number of unduplicated asters lacking chromosomes are found in these embryos. We conclude from these results that early Xenopus embryos, like late embryos, have a cell cycle control system that is affected by DNA replication and is involved in establishing the cell division interval.

A sensitive quantitation system using reverse transcription-polymerase chain reaction (RTPCR) was developed to measure the low estrogen receptor (ER) mRNA levels in Japanese eel (Anguilla japonica). Two types of cytoskeletal actins, β- and γ-actins, were distinguished in Japanese eel and used as the internal control for exact quantitation. Actin and ER primers of this study annealed to different exons, allowing for the skipping of DNase treatment. Accordingly, ER and actin RT-PCR products showed a single band and were amplified with the same efficiency during PCR. The ER mRNA amount was calculated as a relative value, normalized over actin (β and γ) mRNA. The results thus obtained by RT-PCR agreed with the results from Northern blot analysis of liver from pre-vitellogenic and hormone-treated early vitellogenic eel. Using this system, the ER mRNA levels were further measured in coelomic epithelium, pituitary, brain and ovary. In the liver, ER mRNA levels of the early vitellogenic eel increased about by 2.7 folds compared to those of the immature eel. In contrast, changes in levels of ER transcripts were not observed in other tissues. This system can be used to detect relative ER mRNA levels around 100-fold lower than those of actin mRNA in all tissues in which it has been difficult to measure ER mRNA by Northern blot analysis.

We have purified and characterized two molecular forms of insulin from the Brockmann bodies of barfin flounder, Verasper moseri: a normal type of insulin (insulin-I), which consisted of 21 amino acid residues for the A-chain and 30 residues for the B-chain, and a novel type of insulin (insulin-II), which had an extension of two amino acid residues at the N-terminus of the B-chain. The additional two residues at the N-terminus of B-chain of insulin-II were Pyr-Ala which had not yet been reported in vertebrate insulins. Except for these two residues, the amino acid sequence of insulin-II was completely identical with that of insulin-I. Each Brockmann body extract from five individuals contained both insulins, indicating that insulin-I and -II were the products of non-allelic expression. By polymerase chain reaction, only one nucleotide sequence of preproinsulin gene encoding insulin-I and -II was obtained, and the amino acid sequence of A-and B-chains deduced from the nucleotide sequence was identical with that of insulin-I and also insulin-II established by Edman degradation. Furthermore, genomic Southern blot analysis using a part of nucleotide sequence of the preproinsulin gene as a probe showed a single positive band in all cases of genomic DNA digested with each of three restriction endonucleases. These results indicate that insulin-I and -II of barfin flounder arise from a single preproinsulin by proteolytic cleavage at different sites of the signal peptide region.

Insulin-I and -II were purified from stone flounder (Kareius bicoloratus), and their primary structures were determined. The amino acid sequences of insulin-I and -II from stone flounder were identical with those of barfin flounder (Verasper moseri) except for position 2 of both B-chains. Insulin-II of stone flounder had an extension of the two amino acid residues (Pyr-Ala), at the N-terminus of the B-chain. These structural characteristics of insulins from stone flounder support the idea (Andoh and Nagasawa, Zool. Sci. 15: 901, 1998) that insulin-I and -II of flounders arise from a single preproinsulin in each species by proteolytic cleavage at different sites of the signal peptide region, and suggest that this generation system of two molecular forms of insulin is not specific for barfin flounder. In the course of the purification of insulins, somatostatin-14 and two glucagons (glucagon-I and -II) were also purified from the extract of the Brockmann body. The amino acid sequence of somatostatin-14 of stone flounder was identical with those of mammals. Five amino acids were different between glucagon-I and -II of stone flounder. The amino acid sequences of both glucagons were highly conserved among several acanthopterygian and paracanthopterygian fish. These results suggest that their common ancestral species had both glucagons.

The distribution of mRNA encoding thyrotropin-releasing hormone (TRH) precursor in the brain of sockeye salmon was studied by in situ hybridization histochemistry using digoxigenin-labeled riboprobes as a basis to investigate its physiological functions in the salmon brain. Since seasonal variation in TRH gene expression was expected in relation to smolting or maturation, fish were sampled in February and October. In both groups, TRH mRNA was widely distributed in discrete brain regions including the internal cellular layer (ICL) of the olfactory bulb, postcommissural nucleus of area ventralis telencephali (Vp), nucleus preopticus parvocellularis anterioris (PPa), nucleus preopticus magnocellularis, dorsal zone of periventricular hypothalamus (Hd), torus semicircularis, and also the motor nucleus of vagus nerve in the medulla oblongata. TRH neurons in ICL and Hd are round and small with diameters of 5–10 μm. In contrast, TRH neurons in the ventral telencephalon and the preoptic area are medium-sized (10–20 μm), and appear to have multiple processes. Most of these cells are restrictively localized along the lateral margin of the preoptic nuclei. The number of TRH neurons in Vp and PPa was smaller in February than in October, suggesting a seasonal change of TRH neurons in the preoptic area. In the medulla oblongata, a cluster of large oval-shaped cells (20–30 μm) showed signals for TRH mRNA. The present results suggest that TRH may function as a neurotransmitter or neuromodulator involved in olfactory activity and also autonomic motor integration, in addition to neurohormonal control of secretion of pituitary hormones.

In both seminal vesicle (SV) and testis of the catfish Clarias batrachus, a large number of interstitial cells are distributed, singly or in groups close to blood capillaries in the interstices of the secretory lobules and seminiferous tubules, respectively. They showed strong reactions for 3β-HSD, G-6-PD, NADH diaphorase and UDPGD activities. Both the germinal epithelium and SV epithelium showed moderate to strong reaction for UDPGD activity. These results strongly indicate that the SV is steroidogenic and capable of steroid glucuronidation, like the testis. It is suggested that the interstitial cells of the testis and SV are homologous having a common embryonic origin from the genital ridge.

The present paper presents an application of the Triple Mark Recapture technique on two Italian populations of the subterranean termite (Reticulitermes lucifugus). Two large infested areas in the historical center of the town Bagnacavallo (Ravenna) were chosen. Two dye markers were utilized, Nile Blue A or Neutral Red respectively for the two sites. The first population was estimated at 1,085,000 ± 92,000 insects, foraging on a surface of 1500 m²; the second, smaller, population has been estimated to be 640,000 ± 33,000 insects, foraging on an area of 675 m². Maximum linear distance observed in both sites was 45-50 m. Population dynamics look different in the two sites: in the first site the spread distribution with a few insects at each feeding point probably indicates an old, well established, population which grows slowly in all directions during the hot season. In the second site the expansion was much more rapid and it is possible to hypothesise that the termite colony settled in this area more recently and that it is a relatively young, growing colony. The gathered data appear of great utility in extending the knowledge about biology, ecology and behaviour of the Italian subterranean termite.

Historically, the taxonomy and nomenclature of Japanese salmon have been in a state of confusion. Masu, amago and biwa salmon have been variously classified as distinct species, subspecies, or often conflicting or overlapping combinations of the two. In particular, the taxonomy of masu and amago salmon is obscured by their similarity in ecological and morphological traits. Here, DNA sequence analyses of nuclear and mitochondrial DNA were applied to clarify the genetic relationship between masu and amago salmon. No fixed differences were detected in the mitochondrial ND3 gene and control (D-loop) region, or in the nuclear growth hormone type-2 gene (GH2). However, the frequency of single nucleotide substitution alleles within GHZ intron C and size variants at a microsatellite locus nested within intron D differed markedly, providing genetic evidence to support a taxonomic distinction between the two types. The genetic data were related to previous mitochondrial DNA sequence analyses and alternative classification schemes for masu and amago salmon. The best-supported scheme arranges the two types as subspecies; masu as Oncorhynchus masou masou Brevoort and amago as Oncorhynchus masou ishikawae Jordan and McGregor.

We report for the first time the chromosome numbers, karyotypes and C-banding patterns of Hynobius chinensis and Hynobius amjiensis from the middle-eastern region of mainland China. Both species had a diploid set of 56 chromosomes, with similar karyotypes. Among Hynobius species, the C-banding patterns of H. chinensis and H. amjiensis were the most similar to those of H. leechii from the northern region of South Korea. The chromosome number of H. chinensis and H. amjiensis was identical to that of 11 Hynobius species distributed throughout Korea and Japan. This identity indicates close phylogenetic relationships among the 13 species.

Intraspecific differentiation of the Japanese brown frog Rana japonica was investigated by analyzing nucleotide sequences of the mitochondrial cytochrome b (cyt b) gene in order to clarify phylogenetic relationships among three population groups known to exist in this species. The nucleotide sequences of 447 base pair (bp) segments were determined by the PCR-direct sequencing method on 31 individuals from 14 populations of R. japonica from Honshu, and phylogenetic trees were constructed by the UPGMA and NJ methods using Rana catesbeiana as an outgroup. A sequence alignment provided 92 variable sites (15 corresponded to the first codon position, three to the second, and 74 to the third), and 19 haplotypes were identified from 31 frogs. The sequence divergences were 0.22∼2.50% (x̄ = 0.65%) within populations, 0.22∼12.02% (x̄ = 7.34%) between populations, and 23.59∼27.89% (x̄ = 26.19%) between R. japonica and R. catesbeiana. Although many nucleotide substitutions were silent mutations, 12 amino acid replacements were found to occur within R. japonica. A high frequency of transitions relative to transversions was observed within R. japonica. The present nucleotide sequence data showed that the eastern and western groups of R. japonica was considerably differentiated to each other, and that the Akita population of the northwestern group was evidently derived from the eastern group, but the Nakajo and Izumozaki populations of the northwestern group diverged considerably from each of the eastern and the western groups.