Frog Research Articles

Ouro proteins are not essential to tail regression during Xenopus tropicalis metamorphosis.

Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated ouro-knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1- and ouro2-knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8(+) T cells, which play a major role in cytotoxic reaction. Furthermore, T-cell-dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.

The Australasian frog family Ceratobatrachidae in China, Myanmar and Thailand: discovery of a new Himalayan forest frog clade.

In an effort to study the systematic affinities and specieslevel phylogenetic relationships of the enigmatic anurans variably assigned to the genera Ingerana or Limnonectes (family Dicroglossidae), we collected new molecular sequence data for five species including four Himalayan taxa, Limnonectes xizangensis, Lim. medogensis, Lim. alpina, [corrected] Ingerana borealis and one southeast Asian species, I. tasanae, and analyzed these together with data from previous studies involving other ostensibly related taxa. Our surprising results demonstrate unequivocally that Lim. xizangensis, Lim. medogensis and Lim. alpina [corrected] form a strongly supported clade, the sister-group of the family Australasian forest frog family Ceratobatrachidae. This discovery requires an expansion of the definition of Ceratobatrachidae and represents the first record of this family in China. These three species are distinguished from the species of Ingerana and Limnonectes by the: (1) absence of interdigital webbing of the foot, (2) absence of terminal discs on fingers and toes, (3) absence of circumarginal grooves on the fingers and toes, and (4) absence of tarsal folds. Given their phylogenetic and morphological distinctiveness, we assign them to the oldest available generic name for this clade, Liurana Dubois 1987, and transfer Liurana from Dicroglossidae to the family Ceratobatrachidae. In contrast, Ingerana tasanae was found to be clustered with strong support with the recently described genus Alcalus (Ceratobatrachidae), a small clade of otherwise Sundaic species; this constitutes a new record of the family Ceratobatrachidae for Myanmar and Thailand. Finally, Ingerana borealis clustered with the "true" Ingerana (family Dicroglossidae), for which the type species is I. tenasserimensis.

A New Species of the Frog Genus Pseudocallulops from the Foja Mountains in Northwestern New Guinea (Amphibia, Microhylidae)

We describe a new species in the recently erected microhylid frog genus Pseudocallulops , increasing the number of species in this genus from two to three. The new species differs from congeners in body proportions, color and advertisement calls which consist of a series of 4 – 5 soft, distinctly pulsed «creaking» notes. It is presently known only from the Foja Mountains, an isolated mountain range in northern Papua Province of Indonesia.

Synuclein expression in the african clawed frog Xenopus laevis

The synuclein (syn) family comprises three proteins (α-, β- and γ- syns) encoded by different genes (snca, sncb and sncg). In mammals, α- and β- syn are primarily expressed in the brain where they are localized in pre-synaptic terminals while γ-syn is mainly expressed in the peripheral nervous system. In humans, synucleins are involved in neurodegenerative diseases such as Parkinson’s disease and in tumors. However, the normal cellular functions of the three syns have not yet been fully clarified. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce. Our research focused on the evolution of syns with the aim of analyzing their molecular and cellular expression in the CNS of representative vertebrates such as the carp Cyprinus carpio for teleost fish (1,2) and the green lizard Anolis carolinensis for reptiles (3). Current model of our comparative analysis for amphibians is the african clawed frog Xenopus laevis. The only information available on syn expression in this species relate to embryonic stages but data on syn expression in the adult are still lacking. At larval stages, amphibian snca is expressed in the brain, branchial arches and somites, and sncb signals were detected in the entire brain and spinal cord whereas sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion (4). Preliminary data are here reported on syn expression in adult specimen of X. laevis, obtained by RT-qPCR, Western blot and IHC. The results demonstrated that syns are expressed both in neuronal and non-neuronal tissues suggesting differences in the expression pattern between developmental and adult stages.

Geographic variation in the characteristics of Rana temporaria metamorphs

Geographical differences and the structure of intrapopulation phenotypic variation in metamorphs’ life-history traits from 6 spatially distant populations of Rana temporaria were studied. In frogs from northern populations, heritability of larval period duration, growth rate, and size at metamorphosis was relatively low. Growth rate and size at metamorphosis in laboratory frogs were significantly higher than in those collected in nature, whereas the larval period, in turn, was lower. The revealed differences within and between populations represent a counter-gradient variation. The genetic and phenotypic correlations by pairwise comparisons these three traits were similar (both in magnitude and sign) in all populations, with the exception of one population from Kamchatka. This population was successfully introduced 10 years ago from the Moscow region to the habitat with the minimal duration of activity season. The genetic correlation between body size at metamorphosis and larval period was significantly negative. Thus the response to shortening of activity season was not only the growth rate increase but decrease in larval period, partially at the expense of metamorph size. Refs 6. Tables 2.

Assessing the threat status of amphibians in China

In order to clarify the threat status of Chinese amphibians and the conditions threatening these species, we compiled a red list of amphibians in China based on the IUCN Red List Categories and Criteria (Version 3.1), and Guidelines for Application of IUCN Red List Criteria at Regional and National Levels (Version 4.0). This red list, which includes details on population and habitat status, rates of population decline, and projected population trends, will facilitate development of protective management agreements for Chinese administrative departments, the Chinese public, and international organizations. We evaluated 408 amphibian species and discovered that 43.1% of the evaluated species, i.e., 176 species, were threatened, which exceeds the net percentage of threatened amphibian species throughout the entire world. One species was classified as “Extinct” and another species was classified as “Regionally Extinct”. There are 272 amphibian species endemic to China, and 48.9% of them were threatened. The tailed amphibian order (Urodela) possessed the highest ratios of threatened species, followed by the tailless amphibian order (Anura). The 第 5 期 江建平等: 中国两栖动物受威胁现状评估 589 families with the highest percentages of threatened species were Cryptobranchidae (100% threatened), Hynobiidae (86.7% threatened), and Dicroglossidae (78.1% threatened). In eleven provinces, more than 30% of the local amphibian species were classified as threatened species, with the provinces Sichuan (40.8%), Guangxi (39.2%), and Yunnan (37.0%) having the highest percentage of threatened amphibians. Most of China’s amphibians are distributed in southwestern and southern China and below 2,000 m altitude. Habitat degeneration and loss, human capture, and pollution were the three leading threats to amphibians in China. In order to restore endangered amphibian populations and conserve amphibian diversity in China, more population surveys and monitoring projects as well as scientific research on Chinese amphibians are necessary.

Sequencing and analysis of the complete mitochondrial genome of Hyla ussuriensis (Anura: Hylidae)

In this study, the complete mitogenome sequence of Hyla ussuriensis (Anura: Hylidae) is first determined using long PCR. It is a circular molecule of 18 023 bp in length (GenBank accession no. KT964710). Similar to the typical mtDNA of amphibians, the complete mtDNA sequence of Hyla ussuriensis contained two rRNA genes (12S rRNA and 16S rRNA), 22 tRNA genes, 13 protein-coding genes (PCGs), and a control region (D-loop). The nucleotide composition was 29.9% A, 25.4% C, 14.5% G, and 30.2% T. Mitochondrial genomes analyses based on NJ method yield phylogenetic trees, indicating 13 reported Anura frogs belonging to five families (Hylidae, Bufonidae, Microhylidae, Ranidae, and Rhacophoridae). These molecular data presented here provide a useful tool for systematic analyses of genus Hyla and family Hylidae.

Competitive Interactions between Cricket Frogs (Acris blanchardi) and Other Anurans

Abstract: In recent decades, created ponds have become one of the dominant aquatic habitats in anthropogenic landscapes. Understanding how competition between colonizing species influences community assembly in these new habitats is important for predicting species distributions across the landscape. The objective of this study was to examine competition between larval Cricket Frogs (Acris blanchardi), which are showing declines in parts of their range, and other anurans including Bullfrogs (Lithobates catesbeianus), Green Frogs (Lithobates clamitans), and Cope’s Gray Treefrogs (Hyla chrysoscelis). Using two separate experiments, we examined competitive interactions between larvae of (1) Cricket Frogs and different size classes of Bullfrogs; and (2) Cricket Frogs and Green Frogs, Cricket Frogs and Cope’s Gray Treefrogs, or all three species together. We found no effect of either recently hatched or overwintered Bullfrog larvae on Cricket Frog metamorphosis. However, we found that Green Frogs reduced Cric...

THE DIVERSITY OF REPTILES ON SEVERAL HABITAT TYPES IN CAMPUS AREA OF SRIWIJAYA UNIVERSITY INDRALAYA, OGAN ILIR

The Diversity of Reptiles on Several Habitat Types in Campus Area of Sriwijaya University Indralaya, Ogan Ilir was aimed to determine the diversity of amphibian species present in this area. The research has been done in March until May 2015, in the campus of the Sriwijaya University, Indralaya. The method in this study is use the transect method by placing seventh transect line on line with the direction of cutting contour lines. Data collection techniques using the Visual Encounter Survey (VES) and Pit-Fall Trap method in combination with the Straight Line Method and Fence (Drift Fence). Results from this study recorded 11 species of amphibians from five families of the Anura Order. Average diversity index at Sriwijaya University Indralaya region is 1.70 which fall into the medium category. Keywords : Sriwijaya university, amphibians, transect,diversity

Correlation between development and increase of number of labial tooth rows in ghost frog tadpoles (Anura: Heleophrynidae)

The family Heleophrynidae is restricted to Southern Africa and comprises two genera with seven species. Tadpoles are well adapted, with huge sucker mouths, to live in the fast flowing headwaters of mountain streams. The unique sucker-like mouth has numerous transverse rows of labial teeth, which are used to attach themselves to the rock surface and to scrape algae from the rocks. In this paper data on the ontogenetic increase of labial tooth rows in tadpoles of four species of ghost frog are presented. Notes on the development of tadpoles and mouthparts are also presented.

Comparison of thyroid hormone-dependent gene responses in vivo and in organ culture of the American bullfrog (Rana (Lithobates) catesbeiana) lung

Postembryonic frog development requires a thyroid hormone (TH)-dependent metamorphic transition from an aquatic larva to a terrestrial frog. Such change in environment involves lung maturation in preparation for breathing air. However, little is known regarding the underlying molecular events and the role of THs in this process. Using quantitative real-time polymerase chain reaction, we evaluated Rana (Lithobates) catesbeiana lung mRNA transcripts representing key elements of TH and oxidative stress signaling pathways during natural and TH-induced precocious metamorphosis. TH induction was evaluated in two ways: 1) in vivo through interperitoneal injection of 10pmol/g body weight of 3,3′, 5-triiodothyronine (T3) into premetamorphic tadpoles and analysis after 48h, and 2) in serum-free organ culture in the presence of 10nM T3 after 48h. Abundance of transcripts encoding the transcriptional regulators TH receptors α and β, TH-induced bZip protein, and CCAAT/enhancer binding protein 1 was increased during postembryonic development and following administration of exogenous THs to premetamorphic tadpoles in vivo and culture. In contrast, mRNA representing Krüppel-like factor 9 and cold-inducible RNA binding protein revealed differential effects between natural and precocious metamorphosis. Elevated levels of catalase and Cu/Zn superoxide dismutase mRNA were observed at the end of metamorphosis with transcript levels displaying minimal TH-dependency. No change in stress-responsive heat shock protein 30 mRNA abundance was noted. The results support a role for TH-dependent reprogramming of the lung transcriptome during frog development and reveal a requirement for increased antioxidant capacity following anuran metamorphosis.

Body size, reproduction and feeding ecology of Pleurodema diplolister (Amphibia: Anura: Leiuperidae) from Caatinga, Pernambuco state, Northeastern Brazil

We present data about body size, sexual dimorphism, reproductive traits and diet ecology of Pleurodoma diplolister. This species is sexually dimorphic with females larger than males, corroborating others Leiuperidae species. The number of eggs varied from 62 to 1241 and we found a positive relationship between SVL of females and number of mature ovarian eggs but there is no relationship between SVL and volume of eggs. The diet of P. diplolister was composed by 11 categories of which Formicidae, Coleoptera and Orthopterans were the most important items and showed generalist and oportunistic predator habits. Data presented here should be considered in the development of future conservation strategies of anurans from Caatinga biome and other semiarid/arid environments.

Species Richness of Montane Herpetofauna of Southern Eastern Ghats, India: A Historical Resume and a Descriptive Checklist

We examined the amphibian and squamate reptilian species richness of Southern Eastern Ghats based on a long term-field survey with nearly two years of field days. We surveyed high elevation slopes (>900 m a.s.l.) of four select hill ranges namely Jawadi, Shevaroys, Kolli, and Sirumalai hills which comprehensively represented full geographical and spatial coverage. We present a descriptive species-account with basic morphological data supported by voucher photographs. We summarize the history of herpetological explorations in this landscape and also comment on some of the major previous works in the region. Our study revealed the presence of 62 species in the montane zones, including 32 (51%) new records involving all the three target taxa (frogs, lizards and snakes) and all the four hill ranges that testify the poor knowledge on the region’s herpetofauna till date. Lastly, we remark on the unresolved taxonomic status of some species recorded in the present study. We recommend specimen-based revisionary works in the nearby Western Ghats, where such taxa are much more diverse, to enable taxonomic studies in this region.

Evolution of a reproductive character in 26 Neotropica anuran species

Evolution of a reproductive character in 26 Neotropica anuran species

Regulation of thyroid hormone-induced development in vivo by thyroid hormone transporters and cytosolic binding proteins

Differential tissue sensitivity/responsivity to hormones can explain developmental asynchrony among hormone-dependent events despite equivalent exposure of each tissue to circulating hormone levels. A dramatic vertebrate example is during frog metamorphosis, where transformation of the hind limb, brain, intestine, liver, and tail are completely dependent on thyroid hormone (TH) but occurs asynchronously during development. TH transporters (THTs) and cytosolic TH binding proteins (CTHBPs) have been proposed to affect the timing of tissue transformation based on expression profiles and in vitro studies, but they have not been previously tested in vivo. We used a combination of expression pattern, relative expression level, and in vivo functional analysis to evaluate the potential for THTs (LAT1, OATP1c1, and MCT8) and CTHBPs (PKM2, CRYM, and ALDH1) to control the timing of TH-dependent development. Quantitative PCR analysis revealed complex expression profiles of THTs and CTHBPs with respect to developmental stage, tissue, and TH receptor β (TRβ) expression. For some tissues, the timing of tissue transformation was associated with a peak in the expression of some THTs or CTHBPs. An in vivo overexpression assay by tail muscle injection showed LAT1, PKM2, and CRYM increased TH-dependent tail muscle cell disappearance. Co-overexpression of MCT8 and CRYM had a synergistic effect on cell disappearance. Our data show that each tissue examined has a unique developmental expression profile of THTs and CTHBPs and provide direct in vivo evidence that the ones tested are capable of affecting the timing of developmental responses to TH.

The use of animal models in homeopathic research – a review of 2010–2014 PubMed indexed papers

In the 1990s, a study was performed on the effects of highly diluted thyroxine on frog metamorphosis. This model represented one of the most discussed examples of the biological effects of high dilutions over the next two decades. In 2010, another critical conceptual review of the use of animal models in homeopathy and high-dilution research was published. The main contribution of these studies was the elucidation of the biological features and phenomenology of the effects of high dilutions on living systems, representing an important step forward in our understanding of the mechanisms of action of homeopathic medicines. We performed a further review of this line of investigation using the same methods. Fifty-three articles that were indexed in the PubMed database and used 12 different animal species were systematically evaluated. Only a fraction of the studies (29/53) reported herein were performed with "ultra high" dilutions. The other studies were performed with dilutions in ranges below 10(-23) (14/53 articles) or commercial complexes (10/53 articles). Only two articles reported negative results; both used in vivo protocols to test commercial complexes, one in fish and one in bees. The quality of the employed techniques improved in 2010-2014 compared with the studies that were reviewed previously in 2010, with the inclusion of more ethically refined protocols, including in vitro primary cell cultures and ex vivo studies (10/53 articles), often with three or more replicates and analyses of epigenetic mechanisms that were previously unknown in 2010. In our updated review of the past 5 years, we found further demonstrations of the biological effects of homeopathy using more refined animal models and in vitro techniques.

Checklist of nematode parasites of amphibians from Argentina.

This review includes information about 47 taxa of nematode parasites reported from 34 species of Argentinean amphibians, all belonging to order Anura (33 native species and 1 introduced species). Thirty four nematode species have been reported as adults and 13 species were reported as larvae (10 taxa) or juveniles (3 taxa). Two species, Cosmocerca parva and C. podicipinus (Cosmocercidae), collected as adults, are the most commonly occurring adult nematodes in Argentinean amphibians; each of them parasitize 14 amphibian species. The bufonid Rhinella schneideri and the leptodactylid Leptodactylus bufonius present the highest species richness of parasitic nematodes (9 species); followed by Rhinella fernandezae, R. arenarum and Leptodactylus chaquensis, each of which is parasitized by 8 nematode species. Mean species richenss was highest for the family Bufonidae (4.5±3.4; range: 1-9); followed by the Leptodactylidae (3.5±2.8; range: 1-9). Data on hosts, geographical distribution, site of infection, location of deposited materials, and information about life cycles are provided. This is the first compilation of information on nematode parasites of amphibians in Argentina.

Re-description of the external morphology of Phyllomedusa iheringii Boulenger, 1885 larvae (Anura: Hylidae), with comments on the external morphology of tadpoles of the P. burmeisteri group

Phyllomedusa iheringii is a leaf frog endemic to the Uruguayan Savanna, which reproduces in water bodies in open areas. Here, based on the lack of some informative characteristics observed on the first description of this species, we re-describe the larval external morphology of P. iheringii from localities in Brazil and Uruguay, and compared them with other species from the P. burmeisteri group. The tadpoles of P. iheringii belong to the suspension-rasper guild. The body length corresponds to approximately one-third of the total length. The body is pyriform in dorsal view and laterally triangular. The snout is rounded in a dorsolateral view. The spiracle is single and almost ventral, not forming a free tube, and sinistral. The opening of vent tube is dextral. The oral disc is anteroventral and anteriorly directed, with single ventral emargination. Marginal papillae uniseriate, interrupted in a wide dorsal gap, and with pointed tips. The labial tooth row formula is 2(2)/3(1). The third lower row is four times shorter than the others. Furthermore, the tadpoles of P. iheringii showed morphological differences in relation to other species of the P. burmeisteri group, this species seems to be smaller in average and have fewer marginal papillae in the oral discs.

Reproductive ecology of Sichuan digging frogs (Microhylidae: Kaloula rugifera )

Abstract. We investigated the reproductive ecology of Sichuan digging frogs (Microhylidae: Kaloula rugifera ) in Mianyang, China during the wet season of 2013. Male Sichuan digging frogs first appear at temporary ponds following the first heavy rain of the wet season and initiate calling. Females arrive at ponds shortly after males. Male frogs chorus extensively throughout the wet season during the evenings and nights following rainstorms. Female frogs leave the pond after laying eggs, and likely only lay one clutch annually. Amplexus lasted up to three hours. Females were larger than males in terms of body size, but we found no evidence of size-assortative mating. Clutch size varied from 920 to 2200 eggs, with egg diameter ranging from 1.33 to 1.93 mm. Larger female frogs laid more eggs, and there was no correlation between egg number and egg size. Tadpoles hatched from eggs within 18-20 hours of oviposition, and grew for 30-40 days before complete metamorphosis occurred. The initial body length of tadpoles ranged from 3-5 mm snout-vent length. Growth was fastest immediately after hatching, and declined asymptotically with increasing tadpole body size. Overall, Sichuan digging frogs have a breeding biology characterized by strong male-male competition with prolonged breeding coinciding with the annual wet season. Key w ords . Breeding ecology; Kaloula rugifera ; life history; mating system

Direct Activation of Amidohydrolase Domain-Containing 1 Gene by Thyroid Hormone Implicates a Role in the Formation of Adult Intestinal Stem Cells During Xenopus Metamorphosis.

The T3-dependent anuran metamorphosis resembles postembryonic development in mammals, the period around birth when plasma T3 levels peak. In particular, the remodeling of the intestine during metamorphosis mimics neonatal intestinal maturation in mammals when the adult intestinal epithelial self-renewing system is established. We have been using intestinal metamorphosis to investigate how the organ-specific adult stem cells are formed during vertebrate development. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells. A tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis has identified a number of candidate stem cell genes. Here we have carried out detailed analyses of one such gene, amidohydrolase domain containing 1 (AMDHD1) gene, which encodes an enzyme in the histidine catabolic pathway. We show that AMDHD1 is exclusively expressed in the proliferating adult epithelial stem cells during metamorphosis with little expression in other intestinal tissues. We further provide evidence that T3 activates AMDHD1 gene expression directly at the transcription level through T3 receptor binding to the AMDHD1 gene in the intestine. In addition, we have reported earlier that histidine ammonia-lyase gene, another gene in histidine catabolic pathway, is similarly regulated by T3 in the intestine. These results together suggest that histidine catabolism plays a critical role in the formation and/or proliferation of adult intestinal stem cells during metamorphosis.