Japanese Newts Research Articles

Effects of the propylthiouracil on tooth development in newts (921.3)

Objective: Propylthiouracil (PTU) is originally a thiouracil‐derived drug used to treat hyperthyroidism by blocking the effect of thyroid hormone. Recently, PTU is using as a marker of pollutant as a potential thyroid hormone disruptor in aquatic environments. The purpose of this study was to evaluate the effects of exposure to PTU on the development of the teeth and the regeneration of alveolar bone following amputation.Method: Japanese newts (Cynops pyrrhogaster) were maintained in aqueous solutions with or without 100 ug/ml PTU for one month and tracked the sequential development of the tooth cap, the tooth bud, and ultimately the maturation of the sequential tooth germ. Fifty days after the amputation procedure of the right mandible alveolar bone, the newts were observed the effect of PTU by the expression levels of mRNA of thyroid hormone receptor detected in‐situ hybridization. A microscope and digital camera interfaced with Image Analysis & Measurement Software WinROOF (Mitani Corp., Japan) were used to measure the areas of regenerated alveolar bone.Result: The amputated mandible sites were observed microscopically. The regenerated area of alveolar bone in amputation with PTU treatment was significantly smaller than that in the amputation without PTU treatment (Student’s t‐test, p<0.001). There were no differences in the bone histology and tooth germ generation with or without PTU.Conclusion: These data indicate that PTU may affect the tooth germ development and the regeneration of alveolar bone in newts.

Limited Immune Diversity in Urodela: Chronic Transplantation Responses Occur Even with Family-disparate Xenografts

Urodele amphibians are thought to have poorer immune responses than evolutionary more ancestral vertebrate classes, such as bony fish. We investigated skin graft rejection and transplantation immunity in Urodele amphibians, Japanese newts, and Asiatic salamanders, and compared these findings to those from transplants in several species of frogs. The skin grafts used in this study were either allogeneic or xenogeneic. The mean survival time of the first set of allografts at 20°C was approximately 60 days for chronic responses in Urodela and 20 days for acute responses in Anura. As the graft survival times of urodeles were significantly longer than those of anurans, even when urodeles were repeatedly grafted from identical donors, there appear to be substantial differences in transplantation immunity between Urodela and Anura. These slow responses in Urodela may not be accompanied by the expansion of cytotoxic T cells, as observed in fish and anuran species, which are known to have functional major histocompatibility complex (MHC)-class I systems. In our study, approximately five histo-incompatible immunogenic components were found to be involved in chronic responses in newts. Similar chronic responses were also observed in xenograft rejection in newts. In contrast, xenografts were rejected in frogs due to an accelerated acute response, possibly involving natural killer cells. Our findings that some anti-allogeneic components appear to be shared with xenogeneic components indicate that the diversification of the acquired immune system is poorly developed in Urodela.

Hematological- and Neurological-Expressed Sequence 1 Gene Products in Progenitor Cells during Newt Retinal Development

Urodele amphibians such as Japanese common newts have a remarkable ability to regenerate their injured neural retina, even as adults. We found that hematological- and neurological-expressed sequence 1 (Hn1) gene was induced in depigmented retinal pigment epithelial (RPE) cells, and its expression was maintained at later stages of newt retinal regeneration. In this study, we investigated the distribution of the HN1 protein, the product of the Hn1 gene, in the developing retinas. Our immunohistochemical analyses suggested that the HN1 protein was highly expressed in an immature retina, and the subcellular localization changed during this retinogenesis as observed in newt retinal regeneration. We also found that the expression of Hn1 gene was not induced in mouse after retinal removal. Our results showed that Hn1 gene can be useful for detection of undifferentiated and dedifferentiated cells during both newt retinal development and regeneration.

Phase-II conjugation ability for PAH metabolism in amphibians: Characteristics and inter-species differences

The present study examines amphibian metabolic activity – particularly conjugation – by analysis of pyrene (a four ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry detector (MS) system and kinetic analysis of conjugation enzymes. Six amphibian species were exposed to pyrene (dissolved in water): African claw frog ( Xenopus laevis); Tago's brown frog ( Rana tagoi); Montane brown frog ( Rana ornativentris); Wrinkled frog ( Rana rugosa); Japanese newt ( Cynops pyrrhogaster); and Clouded salamander ( Hynobius nebulosus); plus one fish species, medaka ( Oryzias latipes); and a fresh water snail ( Clithon retropictus), and the resultant metabolites were collected. Identification of pyrene metabolites by HPLC and ion-trap MS system indicated that medaka mainly excreted pyrene-1-glucuronide (PYOG), while pyrene-1-sulfate (PYOS) was the main metabolite in all amphibian species. Pyrene metabolites in amphibians were different from those in invertebrate fresh water snails. Inter-species differences were also observed in pyrene metabolism among amphibians. Metabolite analysis showed that frogs relied more strongly on sulfate conjugation than did Japanese newts and clouded salamanders. Furthermore, urodelan amphibians, newts and salamanders, excreted glucose conjugates of pyrene that were not detected in the anuran amphibians. Kinetic analysis of conjugation by hepatic microsomes and cytosols indicated that differences in excreted metabolites reflected differences in enzymatic activities. Furthermore, pyrenediol (PYDOH) glucoside sulfate was detected in the Japanese newt sample. This novel metabolite has not been reported previously to this report, in which we have identified unique characteristics of amphibians in phase II pyrene metabolism.

Changes in the Olfactory Response to Amino Acids in Japanese Newts After Transfer from an Aquatic to a Terrestrial Habitat

Amphibians are capable of smelling both volatile and water-soluble (e.g., amino acids) odorants. Adult Japanese newts, Cynops pyrrhogaster, live mostly in water, except during hibernation, but sometimes on land. To examine olfactory responses of the newts to adaptation to a short-term stay on land (land adaptation), we measured the magnitude of the olfactory response at five different time points (land adaptation time: 0, 30, 54, 90, and 114 h after transfer from an aquatic to a terrestrial habitat by using electro-olfactogram (EOG) recordings. Statistical analysis by the weighted linear model (P < 0.05) indicated that the time to land adaptation had a significant effect on the magnitude of the EOG induced by 1 microM and 10 microM amino acid mixtures. Further, the slope estimates of the weighted linear model were significantly positive (P < 0.05). These results indicate that the magnitude of the EOG response to amino acid mixtures (arginine, alanine, proline, and glutamic acid) significantly increases with land adaptation time. On the other hand, we observed no significant relationship between the magnitude of the EOG response induced by an 0.05% volatile odorant mixture (isoamyl acetate, n-amyl acetate, cineole, and limonene) and land adaptation time. Our results indicate that olfactory sensitivity to amino acids significantly increases with land adaptation time in adult Japanese newts.

Daily and Circadian Variations of the Pineal and Ocular Melatonin Contents and their Contributions to the Circulating Melatonin in the Japanese Newt, Cynops pyrrhogaster

Daily and circadian variations of melatonin contents in the diencephalic region containing the pineal organ, the lateral eyes, and plasma were studied in a urodele amphibian, the Japanese newt (Cynops pyrrhogaster), to investigate the possible roles of melatonin in the circadian system. Melatonin levels in the pineal region and the lateral eyes exhibited daily variations with higher levels during the dark phase than during the light phase under a light-dark cycle of 12 h light and 12 h darkness (LD12:12). These rhythms persisted even under constant darkness but the phase of the rhythm was different from each other. Melatonin levels in the plasma also exhibited significant day-night changes with higher values at mid-dark than at mid-light under LD 12:12. The day-night changes in plasma melatonin levels were abolished in the pinealectomized (Px), ophthalmectomized (Ex), and Px+Ex newts but not in the sham-operated newts. These results indicate that in the Japanese newts, melatonin production in the pineal organ and the lateral eyes were regulated by both environmental light-dark cycles and endogenous circadian clocks, probably located in the pineal organ and the retina, respectively, and that both the pineal organ and the lateral eyes are required to maintain the daily variations of circulating melatonin levels.

An ultrastructural and carotenoid analysis of the red ventrum of the Japanese newt, Cynops pyrrhogaster.

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)-reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab-reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, beta-carotene and five other kinds of carotenoids were detected by thin-layer chromatography (TLC). In the ventral yellow skin of lab-reared newts, however, only beta-carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab-reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.

Hormonal control of urodele reproductive behavior

Hormonal control of expression of courtship behavior and of development of structures related to the reproductive behavior in two species of Japanese newts, Cynops pyrrhogaster and Cynops ensicauda, was described. Prolactin (PRL) and androgen were essential factors for eliciting courtship behavior. In addition, arginine vasotocin markedly enhanced the expression of courtship behavior. PRL induced migration to water, in which courtship and oviposition take place, and converted the integument from the terrestrial type to the aquatic one. PRL also stimulated the growth of the tail fin, which was blocked by estrogen. Cellular and nuclear size and number of synapses on the somata of Mauthner cells, which are involved in tail movement, were also increased by PRL and androgen. Synthesis of sodefrin, a female-attracting pheromone, in the abdominal gland as well as that of mucopolysaccharides constituting the sac of sperm in the lateral gland was enhanced by PRL and androgen. Structural development of oviducts was elicited by estrogen or PRL to a certain extent, and full oviducal development by the combination of these two hormones, PRL being indispensable for the oviducal jelly secretion.

Variation in age and size among breeding populations at different altitudes in the Japanese newts, Cynops pyrrhogaster

AbstractThe age distribution of Cynops pyrrhogaster was studied by skeletochronology on 12 breeding populations inhabiting altitudes ranging from 120 m to 1140 m on Shikoku Island, Japan. In populations inhabiting altitudes 500 m or less, the mean SVL were smaller than in those that lived at higher altitudes. In populations inhabiting altitudes less than 500 m, minimum age at maturation was three years. In populations inhabiting altitudes of 500 m or more, the minimum age at maturation was four to seven years. The number of testes lobes was influenced by age and body size and was variable among populations.

Behavioral rhythms of the Japanese newts, Cynops pyrrhogaster, under a semi-natural condition

Locomotor activity rhythms of the Japanese newt, Cynops pyrrhogaster, were recorded under a semi-natural condition using phototransistor systems. The daily activity rhythm showed a seasonal change: the locomotor activity was mainly diurnal (active during the daytime) from spring to early summer; mainly nocturnal (active during the night-time) from summer to autumn; and showed either a diurnal or nocturnal pattern, depending on the ambient temperature, in winter. To analyze the daily activity in detail, we observed the behavior of a group of newts (three males, three females) throughout 24 h. Four types of behavior (respiration, feeding, mating, and resting on the land) were observed. Each behavior had daily rhythms and showed a seasonal change. The behavior on land showed mainly a nocturnal or bimodal pattern (activity rhythms with two peaks) throughout the year and was more frequently observed in summer. Mating behavior also showed a seasonal change: high activity in spring, with peaks in the early morning and evening, but no activity in summer. Except in winter, feeding and respiratory behavior showed no seasonal changes in either activity period or frequency. Coupling between behavior and the clock seems to be weak in the Japanese newt because of indistinct daily rhythms and frequent phase changes of locomotor activity in water. Physical factors such as humidity and temperature seem to affect strongly the daily activity of the newts.

Ultrastructure of Rhabdias tokyoensis, a pulmonary nematode of the Japanese newt (Cynops pyrrhogaster)

Abstract The parasitic nematode, Rhabdias tokyoensis, Wilkie, 1930, which is found primarily in the saccular lungs of the Japanese newt, Cynops pyrrhogaster, Boie, 1826, is described from the clinical and ultrastructural perspectives. Newts are commonly infected with 1-3 nematodes per lung and often appear healthy, but may present in a wasted state and rarely, if the parasites have penetrated the lungs, will appear grossly bloated with air. This distention alters the newt diving behavior due to impaired buoyancy control. The nematodes induce ulcerative lesions on the inner, pulmonary surface and, if they escape from the lungs, may attach to abdominal organs such as the liver. Rhabdias tokyoensis has minor annulations in the head region, with an oral cavity devoid of crown or lips, and with narrow, sub-cuticular, bacillary band-like longitudinal ridges, as observed by scanning electron microscopy (SEM). Cuticular wrinkling due to dehydration is evident by SEM. Transmission electron microscopy revealed that the cuticle was distinctive, with a particularly thick inner cortex, two thin laminations comprising the basal, fibrous layer, and a convoluted, outer cortex which reflected the surface wrinkling observed by scanning electron microscopy. Cells lining the intestinal tract had apical surfaces lined by microvilli and both large (2.5 μm) and small (0.3 μm) electron dense secretory granules. Somatic muscles encircling the periphery of the nematode body were typical for this genus. The life cycle, prevalence in the natural environment, and possible intermediate hosts for R. tokyoensis remain unknown. Infection of Japanese newts by R. tokyoensis, which otherwise are in good health, may not significantly impair this primarily aquatic amphibian because it relies much on integumentary respiration and the nematode-damaged, primitive saccular lungs do not undergo significant hemorrhage.

Astronewts in Space: Observations on Pathology and Early Development of Japanese Newts, Cynops pyrrhogaster, On-board the Space Shuttle, Columbia

Astronewts in Space: Observations on Pathology and Early Development of Japanese Newts, Cynops pyrrhogaster, On-board the Space Shuttle, Columbia

Hormonal control of response to and secretion of sex attractants in Japanese newts

Hormonal control of sexual chemoattraction in the newt, Cynops pyrrhogaster, was investigated by a water preference test. Sexually inert newts injected with prolactin (PRL) and human chorionic gonadotropin (HCG) in combination preferred the water in which newts of the opposite sex had been kept, whereas saline-injected specimens did not. This indicates that PRL and HCG enhance the response of the newts to attractant(s) secreted by the opposite sex. The water in which PRL plus HCG-treated newts had been kept attracted the opposite sex more intensely than the water in which PRL-, HCG-, or saline-injected newts had been kept. In castrated males, PRL plus testosterone propionate (TP), and in ovariectomized females, PRL plus estradiol induced the secretion of substance(s) that attracted newts of the opposite sex. Removal of the abdominal gland of the cloaca in the male or the oviduct in the female attenuated the attracting activity of the water in which they were kept. Involvement of PRL and gonadal steroids in the secretion of sex attractants by the male abdominal gland and female oviduct was suggested.

Reduction of papilloma size by ultraviolet irradiation in the Japanese newt, Cynops pyrrhogaster

Experiments were conducted on 44 captive Japanese newts ( Cynops pyrrhogaster) to test the hypothesis that ultraviolet (UV) irradiation might inhibit growth or induce regression of spontaneous skin papillomas of probable viral origin. An empirically derived exposure to UV irradiation (42 μW per cm 2 per day) induced reduction of papilloma size which began approximately 4 weeks after irradiation was initiated. This regression included histological evidence of tissue vacuolization and cellular necrosis of papillomatous epithelium. These findings, taken together with previous data on seasonal, temperature and geographical influences on papilloma prevalence, lend support to the notion that solar UV radiation may modulate papilloma prevalence in the natural environment, possibly by direct effects on viral infection.

Gastric Leiomyomas in the Japanese Newt, Cynops pyrrhogaster; Ultrastructural observations

Nineteen of 700 outwardly healthy captive adult Japanese newts, Cynops pyrrhogaster, had nodular neoplasms protruding on the gastric serosal surface. These tumours were characterized as sharply circumscribed leiomyomas of the muscularis externa with occasional submucosal involvement. Ultrastructural analysis of tumour myocytes revealed irregular-shaped, well-differentiated cells with cytoplasmic processes, and reduced quantities of intracellular myofibrils. They were surrounded by enlarged intercellular spaces filled with disorganized and increased amounts of collagen. Vacuolation was seen both in tumour myocytes and in mucous epithelial cells. Virus-like particles were observed in nuclei of some tumour myocytes. Interstitial cells with laminated-type granules were seen amongst tumour cells. These relatively benign leiomyomas shared some morphologic features with human gastric leiomyomas. Their cause and prognosis remain unknown.

The structure of the kidney of Japanese newts, Triturus (Cynops) pyrrhogaster.

The construction of renal lobules in Triturus (Cynops) pyrrhogaster was studied by reconstruction from serial semithin sections, and the structure of nephrons, collecting ducts and ureters was investigated by means of light and electron microscopy. In T. pyrrhogaster the kidney was mesonephros in construction; renal lobules were arranged segmentally and each of them sent one ureter. Male ureters ran caudally and met together before joining the Wolffian duct. In renal lobules, long collecting ducts ran medio-laterally in the dorsal aspect of the kidney and sent several branches ventrally. Each branch duct or short collecting duct received one nephron. Each nephron had five segments; 1) renal corpuscle, 2) ciliated neck segment with or without a naphrostome, 3) proximal tubule, 4) ciliated intermediate segment and 5) distal tubule. Proximal and distal tubules were segregated spacially in renal lobules and occupied the peripheral and central zone respectively. The filtration barrier of the glomerulus consisted of both the basal lamina of podocytes and the subendothelial connective tissue, and was much thicker than the mammalian filtration barrier. Proximal tubule cells had a brush border, apical specialization for reabsorption of organic materials and well-developed smooth endoplasmic reticulum, but few baso-lateral interdigitations. In distal tubule cells, baso-lateral interdigitations and infoldings were well-developed. Collecting duct cells had a sparse cytoplasm. Ureter cells in males contained many secretory granules. On the basis of structural organization of the newt kidney as well as physiological data in literature, we suggest that in land vertebrates proximal tubules were primarily adapted to reabsorption of organic materials and distal tubules to reabsorption of electrolytes and water.

Karyotypes and banding patterns of Tylototriton andersoni boulenger, a newt endemic to the ryukyu islands.

The karyotypes of Tylototriton andersoni Boulenger were studied. This species is known to be an endemic salamander of the Ryukyu islands and may be the most primitive species of the Salamandridae. Somatic chromosomes (2n=24) prepared from intestinal tracts of adult males and females were examined with both conventional Giemsa stain and the BSG technique. Karyological characteristics of the primitive species was recognized in comparison with other Japanese newts Cynops pyrrhogaster and C. ensicauda. The C-band pattern also indicated the dissimilarity with other examined species of more advanced salamandrids. No heteromorphic pair of chromosomes was detected between male and female karyograms prepared with either conventional Giemsa or the C-banding method.

Studies on intramitochondrial inclusions in the pancreatic acinar cells of the Japanese newt, Triturus pyrrogaster: I. Occurrence of intramitochondrial inclusions with long-term starvation

Pancreatic acinar cells of starved Japanese newts, Triturus pyrrogaster, were examined using an electron microscope and paying particular attention to the intramitochondrial inclusions. Three types of intramitochondrial inclusions were detected only in the mitochondrial matrix every 2 to 4 weeks in starved animals. Type I inclusion is a localized enlargement of the mitochondrial matrix. Type II inclusion is a dense osmiophilic body. Type III inclusion is a crystal or crystalloid. This type showed the obvious internal ordered structures in suitable sections. Other unusual mitochondria have long parallel arrays apparently connected to their cristae.

Induced Ovulation in Amphibians by Injection of Antuitrin-S

Japanese newts, Triturus (Molge) pyrrhogaster, were given injections of Antuitrin-S, a sex-stimulating extract from the urine of pregnant women, twice daily for 5 days, with the result that eggs were laid on the sixth day. The total number of eggs laid was about 50 of which 27 were fertile and from which number 21 developed into swimming larvae. Both the males and the females were injected at the same time, 0.2 cc. twice a day, subcutaneously in the abdominal region.The same extract was used in experiments on the Mexican axolotl, generally considered a neotenous form of Amblystoma tigrinum, with the following results.Experiment I. 2 males and 2 females were injected during the same period, with 0.3 cc. of Antuitrin-S 3 times a day for 4 days. Several hundred eggs were laid on the fifth, sixth, and seventh days, These eggs were not fertile.Experiment II. 2 males only were injected with 0.3 cc. three times a day, after which they deposited 15-20 spermatophores on the fourth day. 2 females were then injected...