Cynops Pyrrhogaster Research Articles

A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Ecological niche modeling shed light on new insights of the speciation processes and historical distribution of Japanese fire-bellied newt Cynops pyrrhogaster (Amphibia: Urodela)

Environmental factors play vital roles in determining the range of organisms and application of selective pressures that drive their evolution. Investigation of how these ecological factors influence the diversification of species has long been a point of interest among ecologists. In this study, we used climate-based ecological niche models to project the suitable distributions of four main clades of Japanese fire-bellied newt Cynops pyrrhogaster at the Last Glacial Maximum (LGM), Mid-Holocene (MH), and the present. Then we tested hypotheses regarding ecological niche diversification and the processes of diversification among these clades. The potential distribution of the Northern clade encompassed the Tohoku and Kanto districts, along with the northeastern Chubu district. The Central clade had a potential distribution across the Chubu and Kinki districts, and northeastern Chugoku district. The range of the Western clade was projected to encompass Kyushu, Shikoku, the southern Kinki region, and western Chugoku. Finally, the projected distribution of the Southern clade was limited to the southern Kyushu district. The lack of equivalent niches among the tested pairs indicated the absence of ecological interchangeability among the clades. The results of a similarity test supported significant parapatric divergence of the Northern and Central clades from the remaining clades. On the other hand, the Western and Southern clades probably underwent divergence through an allopatric process. Our paleomodels suggest that the distribution of C. pyrrhogaster may have been reduced and fragmented along the coastal regions during the LGM. The distribution models for the MH estimated a suitable area larger than for the LGM, but smaller than predicted by the current distribution. Our findings serve as a warning that the potential distributions of species could be significantly influenced by future climate change.

Coronary artery established through amniote evolution.

Coronary arteries are a critical part of the vascular system and provide nourishment to the heart. In humans, even minor defects in coronary arteries can be lethal, emphasizing their importance for survival. However, some teleosts survive without coronary arteries, suggesting that there may have been some evolutionary changes in the morphology and function of coronary arteries in the tetrapod lineage. Here, we propose that the true ventricular coronary arteries were newly established during amniote evolution through remodeling of the ancestral coronary vasculature. In mouse (Mus musculus) and Japanese quail (Coturnix japonica) embryos, the coronary arteries unique to amniotes are established by the reconstitution of transient vascular plexuses: aortic subepicardial vessels (ASVs) in the outflow tract and the primitive coronary plexus on the ventricle. In contrast, amphibians (Hyla japonica, Lithobates catesbeianus, Xenopus laevis, and Cynops pyrrhogaster) retain the ASV-like vasculature as truncal coronary arteries throughout their lives and have no primitive coronary plexus. The anatomy and development of zebrafish (Danio rerio) and chondrichthyans suggest that their hypobranchial arteries are ASV-like structures serving as the root of the coronary vasculature throughout their lives. Thus, the ventricular coronary artery of adult amniotes is a novel structure that has acquired a new remodeling process, while the ASVs, which occur transiently during embryonic development, are remnants of the ancestral coronary vessels. This evolutionary change may be related to the modification of branchial arteries, indicating considerable morphological changes underlying the physiological transition during amniote evolution.

Hormonal and pheromonal studies on amphibians with special reference to metamorphosis and reproductive behavior.

In this article, we have reviewed studies which have been conducted to investigate the hormonal influence on metamorphosis in the bullfrog (Rana catesbeiana) and Japanese toad (Bufo japonicus) larvae, in addition to studies conducted on the hormonal and pheromonal control of reproductive behavior in red-bellied newts (Cynops pyrrhogaster). Metamorphosis was studied with an emphasis on the roles of prolactin (PRL) and thyrotropin (TSH). The release of PRL was shown to be regulated by thyrotropin-releasing hormone (TRH) and that of TSH was evidenced to be regulated by corticotropin-releasing factor. The significance of the fact that the neuropeptide that control the secretion of TSH is different from those encountered in mammals was discussed in consideration of the observation that the release of TRH, which stimulates the release of PRL, is enhanced when the animals are subjected to a cold temperature. Findings that were made by using melanin-rich cells of Bufo embryos and larvae, such as the determination of the origin of the adenohypophyseal primordium, identification of the pancreatic chitinase, and involvement of the rostral preoptic recess organ as the hypothalamic inhibitory center of α-melanocyte-stimulating hormone (α-MSH) secretion have been mentioned in this article. In addition, the involvement of hormones in eliciting the courtship behavior in the male red-bellied newts and discovery of the peptide sex pheromones and hormonal control of their secretion have also been included in the present article. This article is protected by copyright. All rights reserved.

Structural and functional analysis of the newt lymphatic system

Regeneration competent vertebrates such as newts and salamanders possess a weakened adaptive immune system characterized by multiple connections between the lymphatic system and the blood vascular system called lymphatic hearts. The role of lymphatic vasculature and these lymphaticovenous connections in regeneration is unknown. We used in-vivo near-infrared lymphangiography, ultra-high frequency ultrasonography, micro-CT lymphangiography, and histological serial section 3-dimentional computer reconstruction to evaluate the lymphatic territories of Cynops pyrrhogaster. We used our model and supermicrosurgery to show that lymphatic hearts are not essential for lymphatic circulation and limb regeneration. Instead, newts possess a novel intraosseous network of lymphatics inside the bone expressing VEGFR-3, LYVE-1 and CD-31. However, we were unable to show Prox-1 expression by these vessels. We demonstrate that adult newt bone marrow functions as both a lymphatic drainage organ and fat reservoir. This study reveals the fundamental anatomical differences between the immune system of urodeles and mammals and provides a model for investigating lymphatics and regeneration.

Longevity in salamandrid newts – a rule, not an exception? Verified cases of Japanese Fire-bellied Newts (Cynops pyrrhogaster) reaching a lifespan of more than 40 years

We report on verified cases of longevity in the Japanese Fire-bellied Newt Cynops pyrrhogaster. Two individuals obtained as adults in 1978 died in July 2008 and November 2017, resulting in an individual minimum lifespan of 33 and 42 years, respectively, possibly even longer. Another four individuals of C. pyrrhogaster were received in 1975 and living now for more than 47 years, with two individuals received as adults likely having reached a lifespan of 50 years already. Another adult male C. pyrrhogaster was obtained in 1991 and died in 2022 at an accordingly estimated age of >34 years. Previously reported lifespans for C. pyrrhogaster in human care were 20-25 years, with only a single report of 40 years. However, other species of Cynops and Hypselotriton are also apparently long-lived, such as H. cyanurus chuxiongensis (25 years), H. orientalis (32 years), and C. ensicauda (43 years). With these documented cases, the lifespan of Fire-bellied Newts is exceeded only by few other species of Caudata, such as Andrias japonicus (70 years), Cryptobranchus alleganiensis, Salamandra salamandra (>50 years), and Proteus anguis (100 years). Our report thus indicates that decades-long lifespans in salamandrid newts, viz. substantial longevity, might be rather common and could be considered a rule, not an exception.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.

Issue Information

Upper left: Some of the red blood cells (RBCs) in the liver of metamorphic Xenopus laevis express both larval globin (magenta) and the proliferation marker PCNA (green), indicating that larval type cells as well as adult ones (not shown) proliferate during metamorphosis in this species (see Yamaguchi et al. issue 8, pp. 420‐432).Upper middle: in vitro reconstruction of nuclei using Xenopus laevis sperm chromatin and X. laevis (left) or X. tropicalis (right) egg extract. Note that cytoplasmic factors influence the size of reconstructed nuclei consisting of chromatin DNA (magenta) and membrane (green) (see Heijo et al. pp. 501‐507).Upper right: Sox2‐ (green) and radial glia‐like cell marker GFAP‐ (magenta) immunoreactive cells around the lateral ventricle in the adult telencephalon of the newt Cynops pyrrhogaster. This study showed the proliferative and neurogenic zones as well as the fate of proliferated cells in the adult brain, providing a clue to clarify the significance of neurogenesis in adulthood (see Iwasa et al. pp. 474‐485).Middle left: Glucose is considered to act as one of the cryoprotectants in the Japanese tree frog (Hyla japonica) as indicated by a marked elevation of the type 2 glucose transporter (red) expression in the liver of the frozen frog (see Okada et al. pp. 486‐493).Lower left: Dynamic post‐translational modifications of histone H3 during early Xenopus development (see Zhou et al. pp. 508‐516).Lower right: Neurospheres generated from spinal cord cells of the newt Pleurodeles waltl (upper left) expressed the radial glia‐like cell marker GFAP (red) and the neural stem cell marker Nestin (green; weakly) (upper right); or the neural stem cell marker Vimentin (green) and the proliferation marker phospho‐histone H3 (red) (lower left). Cells in the neurosphere differentiated into beta‐III tubulin‐positive neurons (green) and GFAP‐positive astrocytes (magenta) (lower right) (see Seki‐Omura et al. pp. 494‐500). image

Cell proliferation and neurogenesis in the adult telencephalon of the newt Cynops pyrrhogaster.

Urodele amphibians have the ability to regenerate several organs, including the brain. For this reason, the research on neurogenesis in these species after ablation of some parts of the brain has markedly progressed. However, detailed information on the characteristics and fate of proliferated cells as well as the function of newly generated neurons under normal conditions is still limited. In this study, we focused on investigating the proliferative and neurogenic zones as well as the fate of proliferated cells in the adult brain of the Japanese red-bellied newt to clarify the significance of neurogenesis in adulthood. We found that the proximal region of the lateral ventricles in the telencephalon and the preoptic area in the diencephalon were the main sites for continuous cell proliferation in the adult brain. Furthermore, we characterized proliferative cells and analyzed neurogenesis through a combination of 5-ethynyl-2'-deoxyuridine (EdU) labeling and immunohistochemistry using antibodies against the stem cell marker Sox2 and neuronal marker NeuN. Twenty-four hoursafter EdU injection, most of the EdU-positive cells were Sox2-immunopositive, whereas, EdU-positive signals and NeuN-immunoreactivities were not colocalized. Two months after EdU injection, the colocalization ratio of EdU-positive signals with Sox2-immunoreactivities decreased to approximately 10%, whereas the ratio of colocalization of EdU-positive signals with NeuN-immunoreactivities increased to approximately 60%. Furthermore, a portion of the EdU-incorporated cells developed into γ-aminobutyric acid-producing cells, which are assumed to function as interneurons. On the basis of these results, the significance of newly generated neurons was discussed with special reference to their reproductive behavior.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, Cynops pyrrhogaster.

In adult newts, when a limb is amputated, a mesenchymal cell mass called the blastema is formed on the stump, where blood vessels filled with premature erythrocytes, named polychromatic normoblasts (PcNobs), elongate. We previously demonstrated that PcNobs in the blastema express an orphan gene, Newtic1, and that they secrete growth factors such as BMP2 and TGFβ1 into the surrounding tissues. However, the relationship between Newtic1 expression and growth factor secretion was not clear since Newtic1 was thought to encode a membrane protein. In this study, we addressed this issue using morphological techniques and found that the Newtic1 protein is a component of globular structures that accumulate at the marginal band in the cytoplasm along the equator of PcNobs. Newtic1-positive (Newtic1(+)) globular structures along the equator were found only in PcNobs with a well-developed marginal band in the blastema. Newtic1(+) globular structures were associated with microtubules and potentially incorporated TGFβ1. Based on these observations, we propose a hypothesis that the Newtic1 protein localizes to the membrane of secretory vesicles that primarily carry TGFβ1 and binds to microtubules, thereby tethering secretory vesicles to microtubules and transporting them to the cell periphery as the marginal band develops.

Isolation and Characterization of Peritoneal Macrophages from Salamanders.

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders.

The emergence of a cryptic lineage and cytonuclear discordance through past hybridization in the Japanese fire-bellied newt,Cynops pyrrhogaster(Amphibia: Urodela)

AbstractDiscrepancies in geographic variation patterns between nuclear DNA and mitochondrial DNA (mtDNA) are the result of the complicated differentiation processes in organisms and are the key to understanding their true evolutionary processes. The genetic differentiation of the northern and Southern-Izu lineages of the Japanese newt, Cynops pyrrhogaster, was investigated through their single nucleotide polymorphism variations obtained via multiplexed ISSR genotyping by sequencing (MIG-seq). We found three genetic groups (Tohoku, N-Kanto and S-Kanto), that were not detected by mtDNA variations, in the northern lineage. N-Kanto has intermediate genetic characteristics between Tohoku and S-Kanto. The genetic groups are now moderately isolated from each other and have unique genetic characteristics. An estimation of the evolutionary history using the approximate Bayesian computation (ABC) approach suggested that Tohoku diverged from the common ancestor of S-Kanto and S-Izu. Then, S-Kanto and S-Izu split, and the recent hybridization between Tohoku and S-Kanto gave rise to N-Kanto. The origin of N-Kanto through the hybridization is relatively young and seems to be related to changes in the distributions of Tohoku and S-Kanto as a result of climatic oscillation in the Pleistocene. We conclude that the mitochondrial genome of S-Kanto was captured in Tohoku and that the original mitochondrial genome of Tohoku was entirely removed through hybridization.

Motilin is a regulator of gastric contraction in Japanese fire belly newts (Cynops pyrrhogaster), in vitro studies using isolated gastrointestinal strips of newts, rabbits, and chickens

The effects of newt motilin on the contractility of the isolated gastrointestinal (GI) tract from Japanese fire belly newts (newt) were examined to clarify whether motilin regulates GI motility in urodele amphibians. In addition, contractile responsiveness to motilins from seven species of vertebrates (human, chicken, turtle, alligator, axolotol, newt and zebrafish) were compared in GI preparations from three different animals (rabbit duodenum, chicken ileum and newt stomach) to determine the species-specific action of motilin. Newt motilin (10-10 M – 10−6 M) caused a contraction of cognate gastric strips, while the upper, middle, and lower intestinal strips were insensitive. The rank order of motilins for contractile activity in newt gastric strips was newt > alligator > axolotol > chicken > turtle > human ≫ zebrafish. On the other hand, newt motilin caused a weak contraction in the rabbit duodenum (human > alligator = chicken > turtle > newt ≧ axolotol > zebrafish), and it was ineffective in the chicken ileum (chicken > turtle > alligator > human ≫ newt, axolotol and zebrafish). This study demonstrates that motilin induces contraction in the GI tract of a urodele amphibian, the newt, in a region (stomach)-specific manner and further indicates that a ligand-receptor interaction of the motilin system is a species-specific manner probably due to differences in the amino acid sequence of motilin.

The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth

Newts can regenerate their limbs throughout their life-span. Focusing on muscle, certain species of newts such as Cynops pyrrhogaster dedifferentiate muscle fibers in the limb stump and mobilize them for muscle creation in the regenerating limb, as they grow beyond metamorphosis. However, which developmental process is essential for muscle dedifferentiation, metamorphosis or body growth, is unknown. To address this issue, we tracked muscle fibers during limb regeneration under conditions in which metamorphosis and body growth were experimentally shifted along the axis of development. Our results indicate that a combination of metamorphosis and body growth is necessary for muscle dedifferentiation. On the other hand, ex vivo tracking of larval muscle fibers revealed that newt muscle fibers have the ability to dedifferentiate independently of metamorphosis and body growth. These results suggest that newt muscle fibers have an intrinsic ability to dedifferentiate, but that metamorphosis and body growth are necessary for them to exhibit this hidden ability. Presumably, changes in the extracellular environment (niche) during developmental processes allow muscle fibers to contribute to limb regeneration through dedifferentiation. This study can stimulate research on niches as well as gene regulation for dedifferentiation, contributing to a further understanding of regeneration and future medical applications.

Identification of motilin in Japanese fire bellied newt

Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.

Phylogenetic Origins of a Newly Found Japanese Red-Bellied Newt Population in Yokohama City and Other Populations in Kanagawa Prefecture

We uncovered the phylogenetic origins of the Japanese red-bellied newt (Cynops pyrrhogaster) population newly found in Yokohama city and 11 neighboring populations in Kanagawa Prefecture based on mitochondrial NADH6-tRNAGlu-cytochrome b DNA sequences. The Yokohama city population was found to be an alien population introduced from Western Japan. On the other hand, the other populations we investigated belong to either of two genetic lineages of the newt (CENTRAL and NORTHERN), and they are distributed parapatrically around the foot of the Tanzawa Mountains.

Dermocystid infection in Japanese fire-bellied newt, <i>Cynops pyrrhogaster</i>

Here, we report details of a new infectious disease in wild-caught Japanese fire-bellied newts (Cynops pyrrhogaster), a Near Threatened species. Skin lesions consisting of numerous masses were found in the animals near Lake Biwa, Shiga Prefecture, Japan. The gross appearance of the skin lesions showed blister-, cyst-, and/or tumor-like morphology. Various sizes of skin lesions were observed on their entire body surface. Histologically, spherical basophilic cysts, including numerous spores, were observed in the dermis layer. Ultrastructural analysis indicated the presence of main bodies of flagellated zoospores within the spores. While 18s rRNA gene sequencing indicated that the skin lesions were due to dermocystid infection. To our knowledge, this is the first report of dermocystid infection in this amphibian in Japan. Further studies are needed to prevent epidemics and to establish diagnostic and treatment methods.

A record of the Japanese Fire-bellied Newt (Cynops pyrrhogaster) from Aoba, Yokohama City, Kanagawa Prefecture

A record of the Japanese Fire-bellied Newt (Cynops pyrrhogaster) from Aoba, Yokohama City, Kanagawa Prefecture

Skin Wound Healing of the Adult Newt, Cynops pyrrhogaster: A Unique Re-Epithelialization and Scarless Model.

In surgical and cosmetic studies, scarless regeneration is an ideal method to heal skin wounds. To study the technologies that enable scarless skin wound healing in medicine, animal models are useful. However, four-limbed vertebrates, including humans, generally lose their competency of scarless regeneration as they transit to their terrestrial life-stages through metamorphosis, hatching or birth. Therefore, animals that serve as a model for postnatal humans must be an exception to this rule, such as the newt. Here, we evaluated the adult newt in detail for the first time. Using a Japanese fire-bellied newt, Cynops pyrrhogaster, we excised the full-thickness skin at various locations on the body, and surveyed their re-epithelialization, granulation or dermal fibrosis, and recovery of texture and appendages as well as color (hue, tone and pattern) for more than two years. We found that the skin of adult newts eventually regenerated exceptionally well through unique processes of re-epithelialization and the absence of fibrotic scar formation, except for the dorsal-lateral to ventral skin whose unique color patterns never recovered. Color pattern is species-specific. Consequently, the adult C. pyrrhogaster provides an ideal model system for studies aimed at perfect skin wound healing and regeneration in postnatal humans.

Antipredator behavior of newts (Cynops pyrrhogaster) against snakes.

Newts and salamanders show remarkable diversity in antipredator behavior, developed to enhance their chemical defenses and/or aposematism. The present study reports on the antipredator behavior of newts (Cynops pyrrhogaster) in response to snakes. Newts displayed a significant amount of tail-wagging and tail-undulation in response to a contact stimulus from the snake’s tongue, which is a snake-specific predator stimulus, as compared to a control stimulus (behavioral scores: tongue, 1.05 ± 0.41; control, 0.15 ± 0.15). Newts that were kept in warm temperature conditions, 20°C (at which snakes are active in nature), performed tail displays more frequently than newts kept in low-temperature conditions, 4°C (at which snakes are inactive in nature). Our results suggest that the tail displays of C. pyrrhogaster could function as an antipredator defense; they direct a snake’s attention to its tail to prevent the snake from attacking more vulnerable body parts. We also discussed the reason for inter-populational variation in the tendency of newts to perform tail displays.