Expression of glycogenic genes in the oviduct of Chinese brown frog (Rana dybowskii) during pre-brumation.

The oviduct of Chinese brown frog (Rana dybowskii) displays seasonal morphological and functional changes, which expands specifically during pre-brumation. To uncover the molecular mechanism underlying this phenomenon, we firstly confirmed the increased weight and enlarged diameter of the oviduct in pre-brumation by morphological observation. Interestingly, the glycogen content in the oviduct increased significantly during pre-brumation, indicating Rana dybowskii stores energy in the oviduct before brumation. Transcriptome analysis further identified the differentially expressed genes in the synthesis and metabolism pathways of carbohydrates in the oviduct during pre-brumation. Based on that evidence, we focused on the mRNA and protein expression of glycogenic genes in the oviduct of Rana dybowskii. qPCR confirmed that the expression of glycolysis and glycogenesis-related genes were up-regulated while gluconeogenesis-related genes were down-regulated during pre-brumation. Western blot data showed that glucose transporter GLUT1 and glycogen synthesis-regulation proteins including GYS, and p-GSK-3ß were highly expressed in the oviduct during pre-brumation. Moreover, immunohistochemical data showed that GLUT1, GYS, p-GYS, GSK-3ß and p-GSK-3ß were expressed regionally in the oviduct of Rana dybowskii. The data suggests that glycogen synthesis may be involved in the oviductal expansion of Rana dybowskii during the pre-brumation.

Histology and structure of the testicles in three species of Atelopus frogs (Anura: Bufonidae) endemic to the Sierra Nevada de Santa Marta, Colombia / Histología y estructura de los testículos en tres especies de ranas Atelopus (Anura: Bufonidae) endémicas de la Sierra Nevada de Santa Marta, Colombia

Abstract Introduction: Testicular histology constitutes one of the least explored aspects in frogs of the genus Atelopus. This taxonomic group shows an alarming population decline; therefore, its reproductive biology is one of the greatest topics of interest for its conservation. Objective: To describe the testicular morphology and the spermatogenetic lineage cells in adult males of Atelopus laetissimus, Atelopus nahumae, and Atelopus carrikeri in the Sierra Nevada de Santa Marta, Colombia. Methods: During June - July 2017 and 2018, sampling was conducted in the localities of San Lorenzo and Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), to collect 15 adult males, 5 per species. Testes samples were fixed in Bouin to be processed by the standard paraffin-embedding technique. Histological sections (3 μm) were stained with Hematoxylin-eosin and Mallory-Heidenhain-Azan-Gomori's. For the description and photographic register of the germ cells, the photonic microscopy technique was used with the differential interference contrast system. Results: The testes are oval organs, compact, light yellow color, and with little vascularization. Externally, they are surrounded by a thin albuginea tunic constituted by regular dense connective tissue. Inside this layer, they are composed of numerous seminiferous tubules of hexagonal contour, in which germ cell cysts are distinguished at different stages of spermatogenesis (spermatogonia I and II, spermatocyte I and II, and early and late spermatids) and spermiogenesis (spermatozoa in fascicles and free spermatozoa). Separating the seminiferous structures is the interstitial tissue in which Leydig cells and blood vessels stand out. Additionally, in the cranial part of the testis, the Bidder's organ was found, formed by two distinguishable regions, the cortex and the medulla. In the cortex, there are previtellogénic oocytes of different sizes surrounded by a monolayer of flat follicular cells. For its part, the medullary region is the connective tissue that nourishes the oocytes and is constituted by blood capillaries. Conclusions: The gonads of the three species analyzed present a cystic cellular organization similar to other anurans, where all stages of spermatogenesis and spermiogenesis were identified, possibly indicating a continuous reproductive activity. Likewise, the Bidder's organ is reported for the first time in the three Atelopus species, which allows suggesting a possible sexual reversion in case of a population decrease of females as a reproductive strategy.

Resumen Introducción: La histología testicular constituye uno de los aspectos menos explorados en las ranas del género Atelopus. Este grupo taxonómico ostenta un declive poblacional alarmarte, es por ello, que su biología reproductiva resulta uno de los temas de mayor interés para su conservación. Objetivo: Describir la morfología testicular y las células del linaje espermatogénico en machos adultos de Atelopus laetissimus, Atelopus nahumae y Atelopus carrikeri en la Sierra Nevada de Santa Marta, Colombia. Métodos: Durante Junio - Julio de 2017 y 2018 se realizaron muestreos en las localidades de San Lorenzo y Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), para recolectar 15 machos adultos, 5 por especie. Las muestras de testículo se fijaron en Bouin para ser procesadas mediante la técnica estándar de inclusión en parafina. Las secciones histológicas (3 μm) se tiñeron con Hematoxilina-eosina y Mallory-Heidenhain-Azan-Gomori's. Para la descripción y registro fotográfico de las células germinales, se utilizó la técnica de microscopía fotónica con el sistema de contraste diferencial de interferencia. Resultados: Los testículos son órganos ovalados, compactos, de color amarillo claro y con poca vascularización. Externamente, están rodeados por una delgada túnica albugínea constituida por tejido conectivo denso regular. Al interior de esta capa se componen por numerosos túbulos seminíferos de contorno hexagonal, en los que se distinguen quistes de células germinativas en diferentes etapas de la espermatogénesis (espermatogonia I y II, espermatocito I y II y espermátidas tempranas y tardías) y espermiogénesis (espermatozoides en fascículos y espermatozoides libres). Separando las estructuras seminíferas se halla el tejido intersticial en el que se destacan las células de Leydig y los vasos sanguíneos. Adicionalmente, en la parte craneal del testículo se encontró el órgano de bidder formado por dos regiones diferenciables, la corteza y la medula. En la corteza se aprecian ovocitos previtelogénicos en diferente tamaño rodeados por una monocapa de células foliculares planas. Por su parte, la región medular es el tejido conectivo que nutre los ovocitos y está constituido por capilares sanguíneos. Conclusiones: Las gónadas de las tres especies analizadas presentan una organización celular quística de manera similar con otros anuros, donde se identificó todos los estadios de la espermatogénesis y espermiogénesis indicando posiblemente una actividad reproductiva continua. Así mismo, se reporta por primera vez el órgano de bidder en las tres especies de Atelopus, lo cual permite sugerir una posible reversión sexual en caso de una disminución poblacional de las hembras como una estrategia reproductiva.

Congolius, a new genus of African reed frog endemic to the central Congo: A potential case of convergent evolution.

The reed frog genus Hyperolius (Afrobatrachia, Hyperoliidae) is a speciose genus containing over 140 species of mostly small to medium-sized frogs distributed in sub-Saharan Africa. Its high level of colour polymorphism, together with in anurans relatively rare sexual dichromatism, make systematic studies more difficult. As a result, the knowledge of the diversity and taxonomy of this genus is still limited. Hyperolius robustus known only from a handful of localities in rain forests of the central Congo Basin is one of the least known species. Here, we have used molecular methods for the first time to study the phylogenetic position of this taxon, accompanied by an analysis of phenotype based on external (morphometric) and internal (osteological) morphological characters. Our phylogenetic results undoubtedly placed H. robustus out of Hyperolius into a common clade with sympatric Cryptothylax and West African Morerella. To prevent the uncovered paraphyly, we place H. robustus into a new genus, Congolius. The review of all available data suggests that the new genus is endemic to the central Congolian lowland rain forests. The analysis of phenotype underlined morphological similarity of the new genus to some Hyperolius species. This uniformity of body shape (including cranial shape) indicates that the two genera have either retained ancestral morphology or evolved through convergent evolution under similar ecological pressures in the African rain forests.

Active escape of prey from predator vent via the digestive tract.

Predation pressures can lead to the evolution of escape behavior in prey animals [1,2]. Most previous studies investigated how prey can escape from predators before contact [1,2], whereas recent studies have focused on the post-contact escape of prey [2]. Predators can damage prey by biting or chewing, and the predator's digestive system ultimately kills almost all prey after swallowing. However, several species can survive passage through a predator's gut and are ultimately excreted with feces [3-5]. Such escapes from the predator's vent (cloaca or anus) are considered passive [3-5]. Survival in the extreme pH and anaerobic conditions of the predator digestive tract depends on the speed of passage and activity of the prey. This lethal environment may impose selective pressures on the evolution of quick and active escape behavior in swallowed prey species. Here, I report active escape of the aquatic beetle Regimbartia attenuata (Coleoptera: Hydrophilidae) from the vents of five frog species via the digestive tract. Although adult beetles were easily eaten by frogs, 90% of swallowed beetles were excreted within 6 h (0.1-6.0 h) after being eaten and, surprisingly, were still alive. When beetle legs were experimentally fixed with wax, all of the treated beetles were killed in the frogs' digestive system and finally excreted >24 h (38.3-150.3 h) after consumption. Therefore, swallowed beetles likely used their legs to move through the digestive tract toward the frog vent, hastening their escape. This study is the first to document active prey escape from the vent of a predator and to show that prey may promote predator defecation to hasten escape from inside the predator's body.

A Phylogenetically Distinct Group of Glandirana rugosa Found in Kyushu, Japan.

The Japanese wrinkled frog Glandirana rugosa is separated into five genetically different groups. One group in western Japan is further divided into three subgroups, found in Kyushu, Shikoku, and western Honshu. We collected G. rugosa frogs at 39 sites in Kyushu and determined nucleotide sequences of the mitochondrial 12S and 16S rRNA genes for phylogenetic analysis. Unexpectedly, we found a group of frogs in southeastern Kyushu that did not cluster with any of the pre-existing five groups of G. rugosa on the phylogenetic trees. The frogs in the new group and G. rugosa in Kyushu were externally similar, but there were a few significant differences in morphological features between the two populations. In addition, we observed significant differences in the frogs' calls . Thus, the group of the frogs in southeastern Kyushu may represent a new candidate species in the genus Glandirana. We discuss the possibility of a new species.

A new species of the genus Amolops (Anura: Ranidae) from Yunnan, China.

A new species of the genus Amolops, Amolops tuanjieensis sp. nov., is described from Yunnan, China. The new species can be distinguished by the following characters: dorsolateral folds present; dorsal and ventral surfaces smooth; top of head and dorsum brown-red with irregular gray and dark spots; flank green; side of head black, from tip of snout, diffusing posteriorly to axilla, continuing as black streak below edge of dorsolateral fold; SVL 39.5-40.4 mm in males, 56.8-60.7 mm in females; tympanum distinct; supratympanic fold indistinct; vomerine teeth in two oblique rows between choanae, closer to each other than choanae; vocal sacs present; nuptial pads present; outer metatarsal tubercle absent, supernumerary tubercles absent; all fingertips expanded into discs; limbs dorsally brown with dark brown bars and irregular dark brown blotches.

Patterns of fluctuating asymmetry in the limbs of anurans.

It has been hypothesized that fluctuating asymmetry (FA) may provide an indication of the functional importance of structures within an organism, with structures that more strongly impact fitness being more symmetric. Based on this idea, we predicted that for tetrapods in which the forelimbs and hindlimbs play an unequal role in locomotion, the less functionally important limb set should display higher levels of FA. We conducted a multispecies test of this hypothesis in anurans (frogs and toads), whose saltatory locomotor mode is powered by the hindlimbs. We also tested whether FA in the forelimbs, which play a more important role during landing, differed between families that differ in the degree of forelimb use in locomotion (Bufonidae vs. Ranidae). We calculated FA from the lengths of humeri and femora measured from disarticulated skeletal specimens of four anuran taxa (Bufonidae: Anaxyrus americanus, Rhinella marina; Ranidae: Lithobates catesbeianus, Lithobates clamitans). Our findings were consistent with the hypothesis that natural selection for increased locomotor performance may influence patterns of FA seen in vertebrate limbs, with all species displaying lower mean FA in the hindlimbs. More subtle functional roles between the forelimbs of bufonids and ranids, however, did not elicit different levels of FA.

Ecological Character Displacement in Non-Congeneric Frogs.

Character displacement is phenotypic divergence driven by competition (ecological character displacement) or reproductive interference (reproductive character displacement). Although previous studies have examined these phenomena separately, recent evidence suggests that reproductive interference can drive both reproductive and ecological character displacement, in that certain traits are related to both competition and reproduction. Thus, to evaluate the effect of competition, the effect of reproductive interference must be excluded. Here, we analysed ecological character displacement between non-congeneric frogs, which show little reproductive interference. Odorrana amamiensis inhabits the Amami and Tokunoshima Islands, Japan, whereas its non-congeneric competitor Babina subaspera inhabits the Amami Island. We tested three of the Schluter (2000) criteria for ecological character displacement: phenotypic changes in O. amamiensis between the two islands, phenotypic change related to prey preference, prey availability between the two islands. We demonstrated that the three criteria in Schluter (2000) were likely to be satisfied, indicating the occurrence of ecological character displacement in non-congeners without reproductive interference. Thus, we conclude that competition is potentially the main driver of this phenotypic divergence, and that non-congeners may be a suitable model for evaluating ecological character displacement in a variety of organisms, as the influence of reproductive interference can be excluded.

A new species of the genus Amolops (Anura: Ranidae) from high-altitude Sichuan, southwestern China, with a discussion on the taxonomic status of Amolops kangtingensis.

A new species of the genus Amolops Cope, 1865 is described from Xinduqiao, Kangding, Sichuan. It was previously identified as Amolops kangtingensis, which is synonymized to Amolops mantzorum in this study. The new species, Amolops xinduqiao sp. nov., is distinguished from all other congeners by the following combination of characters: (1) medium body size, adult males SVL 41.2-47.5 mm ( n=15, average 43.9 mm), adult females SVL 48.5-56.6 mm ( n=15, average 52.5 mm); (2) head length equal to width or slightly wider than long; (3) tympanum small, but distinct; (4) vomerine teeth in two tiny rows, separated by a space about one vomerine teeth row; (5) bony projections on lower jaw absent; (6) dorsolateral folds usually absent; (7) tarsal folds or glands on tarsus absent; (8) circummarginal groove on disc of finger I absent; (9) tibiotarsal articulation reaching nostril or beyond; (10) webs of toe IV reaching to distal articulation, other toes fully webbed to disc; and (11) vocal sac absent in males.

Prespermatogenesis and early spermatogenesis in frogs.

Spermatogenesis in frogs was for the first time divided into two phases: prespermatogenesis, when gonocytes proliferate in developing tadpole testes, and active spermatogenesis when spermatogonial stem cells (i.e. descendants of gonocytes), either self-renew or enter into meiotic cycles within cysts formed by Sertoli cells. We argue that amphibian larval gonocytes are homologues to mammalian gonocytes, whereas spermatogonial stem cells (SSCs) in adult frogs are homologous to mammalian single spermatogonia (As). Gonocytes constitute sex cords, i.e. the precursors of seminiferous tubules; they are bigger than SSCs and differ in morphology and ultrastructure. The nuclear envelope in gonocytes formed deep finger-like invaginations absent in SSCs. All stages of male germ cells contained lipid droplets, which were surrounded by glycogen in SSCs, but not in gonocytes. Mitochondria in gonocytes had enlarged edges of cristae, and in SSCs also lamellar mitochondria appeared. Minimal duration of prespermatogenesis was 46days after gonadal sex differentiation, but usually it lasted longer. SSCs give rise to secondary spermatogonia (equal to mammalian A, In, and B). Their lowest number inside a cyst was eight and this indicated the minimal number of cell cycles (three) of secondary spermatogonia necessary to enter meiosis. We sorted them according to the number of cell cycles (from 8 to 256 cells). This number is similar to that recorded for mammals as the result of a single As proliferation. The number of secondary spermatogonia correlates with the volume of a cyst. The general conclusion is that spermatogenesis in amphibians and mammals follows basically the same scheme.

Expression of leptin receptor in the oviduct of Chinese brown frog (Rana dybowskii).

The oviduct of Chinese brown frog (Rana dybowskii) expands specifically during prehibernation instead of in the breeding period. In this study, we investigated the expression of leptin receptor (Ob-Rb) in Rana dybowskii oviduct during the breeding period and prehibernation. Histologically, the oviduct of Rana dybowskii consists of glandular cells, tubule lumen, and epithelial cells. The oviductal weight and pipe diameter also revealed significant differences, which were higher in prehibernation than that of the breeding period. Ob-Rb was observed in stromal cells of oviductal tissue in both the breeding period and prehibernation. The mean protein and mRNA levels of the Ob-Rb were significantly higher in prehibernation as compared with the breeding period. In addition, oviductal content of leptin was also higher in prehibernation than that of the breeding period. These results suggested that oviduct of Rana dybowskii might be a target organ of leptin, and leptin may play an autocrine/paracrine role mediated by Ob-Rb in regulating the oviductal hypertrophy during prehibernation.

Contacts with large, active individuals intensify the predation risk of small conspecifics.

Size variation within a population can influence the structure of ecosystem interactions, because ecological performance differs between individuals of different sizes. Although the impact of size variation in a predator species on the structure of interactions is well understood, our knowledge about how size variation in a prey species might modify the interactions between predators and prey is very limited. Here, by examining the interactions between predatory Hynobius retardatus salamander larvae and their prey, Rana pirica frog tadpoles, we investigated how large prey individuals affect the predation mortality of small prey conspecifics. First, in an experiment conducted in a field pond in which we manipulated the presence of salamanders and large tadpoles (i.e., large enough to protect them against salamander predation) with small tadpoles, we showed that in the presence of large tadpoles the mortality of small tadpoles from salamander predation was increased. On the basis of our observations of the activity of individuals, we hypothesized that active large tadpoles caused physical disturbances, which in turn caused the small tadpoles to move, and thus increased their encounter frequency with the predatory salamanders. To test this hypothesis, we conducted a laboratory experiment in small tanks with three players (i.e., one salamander as predator, one small tadpole as focal prey, and either a small or a large tadpole as the prospective movement inducer). In each tank, we manipulated the presence or absence of a movement inducer, and, when present, its size (large or small) and access (caged or uncaged) to the focal prey. In the presence of a large, uncaged movement inducer, the focal prey was more active and suffered from higher predation mortality compared with the other treatments, because the large movement inducer (unlike a small movement inducer) moved actively and, when uncaged, could stimulate movement of the focal prey through direct contact. The results indicated that high activity of large prey individuals and the resulting behavioral interactions with small conspecifics via direct contact indirectly increased the mortality of the small prey.

ICE on the road to auditory sensitivity reduction and sound localization in the frog.

Frogs and toads are capable of producing calls at potentially damaging levels that exceed 110 dB SPL at 50 cm. Most frog species have internally coupled ears (ICE) in which the tympanic membranes (TyMs) communicate directly via the large, permanently open Eustachian tubes, resulting in an inherently directional asymmetrical pressure-difference receiver. One active mechanism for auditory sensitivity reduction involves the pressure increase during vocalization that distends the TyM, reducing its low-frequency airborne sound sensitivity. Moreover, if sounds generated by the vocal folds arrive at both surfaces of the TyM with nearly equal amplitudes and phases, the net motion of the eardrum would be greatly attenuated. Both of these processes appear to reduce the motion of the frog's TyM during vocalizations. The implications of ICE in amphibians with respect to sound localizations are discussed, and the particularly interesting case of frogs that use ultrasound for communication yet exhibit exquisitely small localization jump errors is brought to light.

A new species of Amolops (Anura: Ranidae) from southern China.

A new species, Amolops albispinus sp. nov. is described based on a series of specimens collected from Mt. Wutong, Shenzhen, Guangdong Province, China. The new species can be distinguished from other known congeners by molecular divergence in the mitochondrial COI and 16S rRNA gene and morphological characters including presence of white conical spines on the lips, loreal and temporal regions, excluding the tympanum; small body, SVL 36.7-42.4 mm in adult males and 43.1-51.9 mm in adult females; very rough dorsal skin of body with numerous raised large warts; olive-brown dorsum with dark brown blotches; strongly developed vomerine teeth; absence of vocal sacs; absence of tarsal glands; absence of dorsolateral folds; presence of circummarginals groove on the disk of first finger; and absence of outer metatarsal tubercles. At present, the genus Amolops contains 51 species, of which 23 occur in China.

A new species of Odorrana (Amphibia: Anura: Ranidae) from Vietnam.

A new species of Odorrana is described from the karst forests in northeastern Vietnam based on morphological differences and molecular divergence. Morphologically, the new species is distinguishable from its congeners on the basis of a combination of the following diagnostic characters: (1) size large (SVL 85.9-91.6 mm in males, 108.7-110.1 mm in females); (2) head longer than wide; (3) vomerine teeth present; (4) external vocal sacs absent; (5) snout short (SL/SVL 0.16-0.17); (6) tympanum large (TD/ED 0.70 in males, 0.68 in females); (7) dorsal surface of head and anterior part of body smooth, posterior part of body and flanks with small tubercles; (8) supratympanic fold present; (9) dorsolateral fold absent; (10) webbing formula I0-0II0-0III0-1/2IV1/2-0V; (11) in life, dorsum green with dark brown spots; (12) flanks greyish brown with dark brown spots; (13) throat and chest grey, underside of limbs with large dark brown spots, edged in white, forming a network. In the phylogenetic analyses, the new species is unambiguously nested within the O. andersonii group, and placed as the sister taxon to O. wuchuanensis.

Nanoscale friction and adhesion of tree frog toe pads.

Tree frogs have become an object of interest in biomimetics due to their ability to cling to wet and slippery surfaces. In this study, we have investigated the adhesion and friction behavior of toe pads of White's tree frog (Litoria caerulea) using atomic force microscopy (AFM) in an aqueous medium. Facilitating special types of AFM probes with radii of ∼400 nm and ∼13 µm, we were able to sense the frictional response without damaging the delicate nanopillar structures of the epithelial cells. While we observed no significant adhesion between both types of probes and toe pads in wet conditions, frictional forces under such conditions were very pronounced and friction coefficients amounted between 0.3 and 1.1 for the sliding friction between probes and the epithelial cell surfaces.