Cynops Ensicauda Research Articles

Lack of variations in the salamander chytrid fungus, Batrachochytrium salamandrivorans, at its alleged origin: Updating its Japanese distribution with new evidence.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

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.

Differences of Extracellular Cues and Ca2+ Permeable Channels in the Signaling Pathways for Inducing Amphibian Sperm Motility.

Low osmolality of freshwater and/or sperm motility-initiating substance (SMIS) induce amphibian sperm motility through increases in intracellular Ca2+. In the internally fertilizing newt Cynops pyrrhogaster, the sperm motility-initiating substance engages T type voltage-dependent Ca2 + channels and N-methyl D-aspartate-type glutamate receptors to initiate sperm motility and L type voltage-dependent Ca2+ channels to enhance motility. In the present study, differences in the usages of SMIS and Ca2+ permeable channels for sperm motility regulation were examined in amphibians that undergo different reproductive modes. Proteins of 14-17 kDa were detected by antibody against the active site peptide of SMIS in the oviduct secretion of internal fertilizers (C. pyrrhogaster, Cynops ensicauda, and Ambystoma mexicanum) and arboreal fertilizers (Rhacophorus arboreus and Rhacophorus schlegelii), but not in Buergeria japonica, an external fertilizer in freshwater. In the pharmacological study, a blocker of some transient receptor potential channels (RN1734) additionally suppressed enhancement of sperm motility in C. pyrrhogaster. In R. schlegelii, blockers of four types of channels differently suppressed sperm motility induced by low osmolality with or without the active site peptide of SMIS. Notably, blockers of L type voltage-dependent Ca2+ channels (nifedipine) and N-methyl D-aspartate-type glutamate receptors (MK801) suppressed sperm motility in the presence and the absence of the peptide, respectively. Low osmolality-induced sperm motility was suppressed by RN1734 and MK801 in B. japonica, but not in Xenopus laevis. These results reveal complex differences in the signaling pathways for inducing sperm motility that may be partly related to reproductive modes in amphibians.

Diet of the American Bullfrog Lithobates catesbeianus Naturalized on Okinawajima, Ryukyu Archipelago, Japan

Abstract: The diet of the nonnative American Bullfrog Lithobates catesbeianus occurring in Ogimi Village of Okinawajima, Ryukyu Archipelago, is investigated. Seventy two of 89 frogs (nine adults, five subadults, and 58 juveniles) captured had food items in its stomach. We identified a total of 64 taxa from 253 food items. Our analyses show that (1) the diet consists mainly of terrestrial prey, (2) mollusks and vertebrates are the essential prey groups for adults and subadults, and (3) odonates, mollusks, and arachnids are the main prey for juveniles. Such dietary habits are quite different from those of several conspecific nonnative populations in mainland Japan and other regions. Part of this is most likely related to the absence of the frog's favorite prey, nonnative American crayfish Procambarus clarkii, in the study area. We also confirm the predation of a poisonous newt Cynops ensicauda popei (Salamandridae) and several aquatic insects by this frog and present some implications for these results.

Isolation and Biological Activity of 8- Epitetrodotoxin and the Structure of a Possible Biosynthetic Shunt Product of Tetrodotoxin, Cep-226A, from the Newt Cynops ensicauda popei.

Tetrodotoxin (TTX, 1), a potent neurotoxin, has been found in various animal species in both marine and terrestrial environments. In this study, a new TTX analogue, 8- epiTTX (2), and a possible biosynthetic shunt compound of TTX, Cep-226A (3), were isolated from the newt Cynops ensicauda popei. The voltage-gated sodium ion channel (Nav) blocking activity of 2 and 6- epiTTX (4), a known analogue, were investigated by a colorimetric cell-based assay and compared with that of 1. The EC50 values for 2 and 4 were determined to be 110 ± 40 and 33 ± 11 nM, respectively, which were larger than that of 1 (1.9 ± 0.7 nM). The results indicated that the equatorial hydroxy group at C-8 in TTX significantly contributes to its Nav blocking activity, whereas the 6-epimer of TTX retains substantial activity, consistent with its previously reported toxicity in mice and binding affinity to rat brain membrane preparations. The presence of these epimers of TTX (2 and 4) and Cep-226A (3) in newts supports our hypothesis that TTX is derived from a monoterpene in terrestrial environments.

Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan

Leptospires, which cause the zoonotic disease leptospirosis, persist in soil and aqueous environments. Several factors, including rainfall, the presence of reservoir animals, and various abiotic and biotic components interact to influence leptospiral survival, persistence, and pathogenicity in the environment. However, how these factors modulate the risk of infection is poorly understood. Here we developed an approach using environmental DNA (eDNA) metabarcoding for detecting the microbiome, vertebrates, and pathogenic Leptospira in aquatic samples. Specifically, we combined 4 sets of primers to generate PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. Using our method to analyze the eDNA of leptospirosis-endemic areas in northern Okinawa, Japan, we found that the microbiota in each river shifted over time. Operating taxonomic units corresponding to pathogenic L. alstonii, L. kmetyi, and L. interrogans were detected in association with 12 nonpathogenic bacterial species. In addition, the frequencies of 11 of these species correlated with the amount of rainfall. Furthermore, 10 vertebrate species, including Sus scrofa, Pteropus dasymallus, and Cynops ensicauda, showed high correlation with leptospiral eDNA detection. Our eDNA metabarcoding method is a powerful tool for understanding the environmental phase of Leptospira and predicting human infection risk.

Glycoconjugate expression in the olfactory bulb of the premetamorphic larva of the Japanese sword-tailed newt (Cynops ensicauda).

We examined the organization of the olfactory organ and assessed the lectin histochemistry to investigate the glycoconjugate distribution of the olfactory bulb in premetamorphic larvae of Cynops ensicauda. The nasal cavity was an oval chamber that contained olfactory epithelium and a primitive vomeronasal organ. Secretory products were found in the supporting cells of the two sensory epithelia and in the respiratory cells. Ten lectins bound to the olfactory and vomeronasal nerve fibers as well as to the glomeruli in the olfactory bulb. The binding intensity in larvae was weaker than that reported previously in mature animals. This difference suggests a functional correlation between the expression change of glycoconjugates and the developmental refinement of the olfactory system during metamorphosis.

Heterogeneous expression of glycoconjugates in the primary olfactory centre of the Japanese sword-tailed newt (Cynops ensicauda).

Histochemical organization of the Caudata olfactory system remains largely unknown, despite this amphibian order showing phylogenetic diversity in the development of the vomeronasal organ and its primary centre, the accessory olfactory bulb. Here, we investigated the glycoconjugate distribution in the olfactory bulb of a semi-aquatic salamander, the Japanese sword-tailed newt (Cynops ensicauda), by histochemical analysis of the lectins that were present. Eleven lectins showed a specific binding to the olfactory and vomeronasal nerves as well as to the olfactory glomeruli. Among them, succinylated wheat germ agglutinin (s-WGA), soya bean agglutinin (SBA), Bandeiraea simplicifolia lectin-I (BSL-I) and peanut agglutinin showed significantly different bindings to glomeruli between the main and accessory olfactory bulbs. We also found that s-WGA, SBA, BSL-I and Pisum sativum agglutinin preferentially bound to a rostral cluster of glomeruli in the main olfactory bulb. This finding suggests the presence of a functional subset of primary projections to the main olfactory system. Our results therefore demonstrated a region-specific glycoconjugate expression in the olfactory bulb of C. ensicauda, which would be related to a functional segregation of the olfactory system.

Sperm motility initiating substance may be insufficient to induce forward motility of Cynops ensicauda sperm.

Sperm motility-initiating substance (SMIS) is a key protein for internal fertilization of the newt, Cynops pyrrhogaster, and commonly enhances forward sperm motility in some amphibian species, including external fertilizers. SMIS action varies among different species in correlation with a species-specific reproductive environment. In the present study, we identified the gene of C. ensicauda SMIS (CeSMIS) and examined the mechanism of SMIS action with reference to that of the closely related Cynops species. The CeSMIS was identified by a 176-amino acid sequence including seven amino acids critical for the initiation of sperm motility. The amino acid sequence showed 91% homology to the whole sequence of C. pyrrhogaster SMIS (CpSMIS). By immunostaining with an anti-CpSMIS antibody, CeSMIS was shown to be localized in the outer layer of the egg jelly. A peptide presenting the active site of SMIS was observed to bind to the axial rod of the midpiece in C. ensicauda sperm. The localization and binding patterns of CeSMIS were fundamentally similar to those of CpSMIS. However, the SMIS peptide did not induce forward motility of C. ensicauda sperm, although it induced a fast wave of the undulating membrane. Forward sperm motility was induced in the egg jelly extract containing CeSMIS. These results suggest that the mechanism of initiation of sperm motility is differentiated between C. ensicauda and C. pyrrhogaster.

Development of Nine Microsatellite Markers forParamesotriton hongkongensis(Hong Kong Newt) Through Shotgun Sequencing and Cross-species Amplification in Other Asian Salamandrids

The Hong Kong newt (Paramesotriton hongkongensis) is a tropical stream-dwelling salamandrid native to Hong Kong and coastal areas of Guangdong Province, China, inhabiting small rocky hill streams and surrounding forests. It is categorized as “near threatened” on the IUCN Red List because of range-wide habitat degradation and vulnerability to over-exploitation for the pet trade. Nine novel polymorphic microsatellite markers were developed and characterized using Illumina paired-end shotgun sequencing. Number of alleles per locus ranged from 10 to 19 (median=14). Cross-species amplification was attempted in four species of Asian newt: Cynops ensicauda, Pachytriton granolosus, Paramesotriton chinensis, and Tylototriton shanjing, which included two species of conservation concern, with varying degrees of success. The nine novel markers can be used to quantify genetic variability among isolated populations and have potential forensic applications in a number of Asian newt species that are highly threatened by over-exploitation throughout their ranges.

Molecular structures of two tetrodotoxin analogs containing a monooxa-hydrocarbon cage: A computational study

Using quantum chemical calculations we investigate the molecular structures of two tetrodotoxin (TTX) analogs recently isolated from the Japanese toxic newt Cynops ensicauda popei. These novel analogs are characterized by a monooxa-hydrocarbon cage with a direct C5–C10 bond that replaces one of the ether bridges in the canonical dioxa-adamantane cage of TTX. The computed change in the 13C NMR chemical shifts is in good agreement with the change in the corresponding experimental values that results from the above chemical modification. This confirms the chemical structure assigned to the TTX analogs. A topological analysis of the theoretical electronic charge density indicates that the removal of the oxygen bridge in TTX increases the magnitude of the charge density at the cage critical point. A database search indicates that the monooxa-hydrocarbon cage is also present in other natural products such as cinnzeylanine and platensimycin whose molecular structures have been characterized by single-crystal X-ray diffraction analyses.

C5-C10 directly bonded tetrodotoxin analogues: possible biosynthetic precursors of tetrodotoxin from newts.

The identification of novel tetrodotoxin (TTX, 1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5-C10 directly bonded TTX analogues, 4,9-anhydro-10-hemiketal-5-deoxyTTX (2) and 4,9-anhydro-8-epi-10-hemiketal-5,6,11-trideoxyTTX (3), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC-LC-MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (rs =0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9-anhydroTTX (4) by Baeyer-Villiger-like oxidation or via 4,9-anhydro-5-deoxyTTX formed by cleavage of the C5-C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX.

C5–C10 Directly Bonded Tetrodotoxin Analogues: Possible Biosynthetic Precursors of Tetrodotoxin From Newts

AbstractThe identification of novel tetrodotoxin (TTX, 1) analogues would significantly contribute to the elucidation of its biosynthetic pathway. In this study, the first C5–C10 directly bonded TTX analogues, 4,9‐anhydro‐10‐hemiketal‐5‐deoxyTTX (2) and 4,9‐anhydro‐8‐epi‐10‐hemiketal‐5,6,11‐trideoxyTTX (3), were found in the newt Cynops ensicauda popei by using a screening method involving HILIC‐LC–MS/MS focused on the fragment ions of TTX analogues, and their structures were elucidated by spectroscopic methods. Compound 2 was detected in a wide range of newt species, and the 2 and TTX contents of 22 newt specimens were correlated (rs=0.88). Based on these results and its structural features, 2 was predicted to serve as a precursor of TTX that would be directly converted into 4,9‐anhydroTTX (4) by Baeyer–Villiger‐like oxidation or via 4,9‐anhydro‐5‐deoxyTTX formed by cleavage of the C5–C10 bond. The bicyclic carbon skeletons of 2 and 3 suggested a possible monoterpene origin for TTX.

Stem stiffness plays a role in determining the foraging success of predators

The presence of macrophytes in the littoral zone provide prey animals with protection from predators. Two macrophyte characters, stem density and branching, are known to hinder predator foraging in macrophyte beds. Stem stiffness is a character that allows the macrophyte to withstand current power in the intertidal zone, but its effect on predator movements in macrophyte beds has not been studied to date. In this study I examined whether the foraging success of predators is constrained by stem stiffness, as well as stem density and the presence of branches. Artificial macrophytes were constructed using two types of rubber that differed in stiffness. The newt Cynops ensicauda popei and larvae of the damselfly Paracercion melanotum were used as predator and prey, respectively, in this model system. The results revealed that all three plant characters studied influenced the survival rate of prey. Stiff stems consistently increased the survival rate compared with flexible stems. Stem density had the highest positive influence on survival rate. The direct effect of branches was negative and minute, but it altered the dependency on stem density. Although stiffness did have an effect on the survival rate of prey, its magnitude was relatively low. The effect of stiffness in other settings should be examined in future studies.

Locomotion pattern and trunk musculoskeletal architecture among Urodela.

We comparatively examined the trunk musculature and prezygapophyseal angle of mid-trunk vertebra in eight urodele species with different locomotive modes (aquatic Siren intermedia, Amphiuma tridactylum, Necturus maculosus and Andrias japonicus; semi-aquatic Cynops pyrrhogaster, Cynops ensicauda; and terrestrial Hynobius nigrescens, Hynobius lichenatus and Ambystoma tigrinum). We found that the more terrestrial species were characterized by larger dorsal and abdominal muscle weight ratios compared with those of the more aquatic species, whereas muscle ratios of the lateral hypaxial musculature were larger in the more aquatic species. The lateral hypaxial muscles were thicker in the more aquatic species, whereas the M. rectus abdominis was more differentiated in the more terrestrial species. Our results suggest that larger lateral hypaxial muscles function for lateral bending during underwater locomotion in aquatic species. Larger dorsalis and abdominal muscles facilitate resistance against sagittal extension of the trunk, stabilization and support of the ventral contour line against gravity in terrestrial species. The more aquatic species possessed a more horizontal prezygapophyseal angle for more flexible lateral locomotion. In contrast, the more terrestrial species have an increasingly vertical prezygapophyseal angle to provide stronger column support against gravity. Thus, we conclude trunk structure in urodeles differs clearly according to their locomotive modes.

Differential survival rates of damselfly larvae in the presence of newt and dragonfly predators

The damselfly species Paracercion melanotum has been found to be the most abundant species in damselfly larval communities on Okinawa-zima Island in southwest Japan. To clarify differential susceptibility to predation, a possible factor affecting relative population densities in larval communities, between Paracercion melanotum and a less common damselfly species, Ischnura senegalensis, laboratory experiments were conducted using three abundant predator species: the sword-tailed newt (Cynops ensicauda popei), anisopteran larvae (Crocothemis servilia servilia), and a planktivorous fish (Poecilia reticulata). Paracercion melanotum survived predation by the newt and the dragonfly well compared to I. senegalensis. Fishes consumed approximately equal numbers of the two damselfly species. From these results, the newt and the dragonfly were suggested as the most probable predators regulating damselfly larval communities on Okinawa-zima Island. Predators could be a crucial factor determining relative abundance in...

Development of Microsatellite Markers forCynops ensicauda(Amphibia: Caudata) by Using Next-Generation Sequencing Technology

We isolated and characterized seven microsatellite markers including di-, tri-, and tetra-nucleotide repeats for Cynops ensicauda, which is a near-threatened species and endemic to the Central Ryukyus, Japan, using next-generation sequencing. Six out of the seven loci that showed polymorphism in Cynops ensicauda popei on Okinawajima Island were also amplified in C. e. ensicauda on Amamioshima Island. Expected heterozygosities of these markers were 0.00–0.738 (mean, 0.429) and 0.00–0.791 (mean, 0.459) in C. e. popei and C. e. ensicauda, respectively. These markers will be useful for genetic studies, such as evaluating the genetic diversity within and between populations of this species.

Isolation and Structural Determination of the First 8-epi-type Tetrodotoxin Analogs from the Newt, Cynops ensicauda popei, and Comparison of Tetrodotoxin Analogs Profiles of This Newt and the Puffer Fish, Fugu poecilonotus

Identification of new tetrodotoxin (TTX) analogs from TTX-possessing animals might provide insight into its biosynthesis and metabolism. In this study, four new analogs, 8-epi-5,6,11-trideoxyTTX, 4,9-anhydro-8-epi-5,6,11-trideoxyTTX, 1-hydroxy-8-epi-5,6,11-trideoxyTTX, and 1-hydroxy-4,4a-anhydro-8-epi-5,6,11-trideoxyTTX, were isolated from the newt, Cynops ensicauda popei, and their structures were determined using spectroscopic methods. These are the first 8-epi-type analogs of TTX that have been found in a natural source. Furthermore, we examined the composition of the TTX analogs in this newt and in the ovary of the puffer fish, Fugu poecilonotus, using LC/MS. The results indicate that TTX and 11-deoxyTTX were present in both sources. However, 6-epiTTX and 8-epi-type analogs were detected only in the newt, while 5,6,11-trideoxyTTX was a specific and major analog in the puffer fish. Such considerable differences among analog compositions might reflect differences in the biosynthesis or metabolism of TTX between these animals.

P-33 沖縄産シリケンイモリ(Cynops ensicauda popei)の新規テトロドトキシン類縁体の単離と構造決定(ポスター発表の部)

P-33 沖縄産シリケンイモリ(Cynops ensicauda popei)の新規テトロドトキシン類縁体の単離と構造決定(ポスター発表の部)

Sexual Interference in the Sword-tailed Newt, Cynops ensicauda popei (Amphibia: Salamandridae)

This paper describes competitive interaction between males of the salamandrid Cynops ensicauda popei. The sexual behaviour of the male is influenced by the presence of a second, rival, male. Competition for access to the female and other male interactions continuously interrupt ongoing courtships. Spermatophore pick-up success is lower than in encounters involving single pairs. During triad encounters, fewer spermatophores were deposited than in dyad encounters, due to interference during the creep stage of courtship. Two basic forms of male sexual interference were distinguished: ‘lure away’ during the creeping stage, from a position that is different from the direction of creep; and ‘female mimicry’, during which the male presses his snout alternately against the courting male's tail and the female's snout. In both forms, a rival male may shove one of the courting individuals away. The repertoire of sexual behaviour patterns of Cynops ensicauda popei appears to be less complex and varied than that of most Triturus species. The courtship of Cynops places less emphasis on display behaviour with tail and body, and a stronger emphasis on the creeping stage, where the male carefully leads the responsive female over a series of spermatophores, but during which rival males may interfere in a variety of ways.