Grass Frog Research Articles

The hidden threat: Exploring the parasite burden and feeding habits of invasive raccoon dogs (Nyctereutes procyonoides) in central Europe

Originally from Asia, the raccoon dog Nyctereutes procyonoides is an invasive alien species in Europe, listed since 2019 on the List of invasive alien species of Union concern. The raccoon dog is considered to have negative impact on native biodiversity, as well as a crucial role in hosting and transmitting diverse parasites and pathogens of human and veterinary importance. In the present study, stomach content analyses and parasitological examinations were performed on 73 raccoon dogs from Germany. In addition, fecal samples were analyzed. The results of the study confirm the assumption that the examined raccoon dogs were infested with a various ecto- and endoparasite fauna. A total of 9 ecto- and 11 endoparasites were detected, with 6 of the endoparasites having human pathogenic potential. Trichodectes canis (P = 53.42%), Toxocara canis (P = 50.68%) and Uncinaria stenocephala (P = 68.49%) were the most abundant parasite species. The stomach contents consisted of approximately one-third vegetable and two-thirds animal components, composed of various species of amphibians, fish, insects, mammals and birds. Among them were specially protected or endangered species such as the grass frog Rana temporaria. The study shows that the raccoon dog exerts predation pressure on native species due to its omnivorous diet and, as a carrier of various parasites, poses a potential risk of infection to wild, domestic and farm animals and humans.

Crossing the Rift valley: using complete mitogenomes to infer the diversification and biogeographic history of ethiopian highlands Ptychadena (anura: Ptychadenidae).

The Ethiopian Highlands are considered a biodiversity hotspot, harboring a high number of endemic species. Some of the endemic species probably diversified in situ; this is, for example, the case of a monophyletic clade containing 12 known species of grass frogs of the genus Ptychadena. The different species occur at elevations ranging from 1,500 to above 3,400m and constitute excellent models to study the process of diversification in the highlands as well as adaptations to high elevations. In this study, we sampled 294 specimens across the distribution of this clade and used complete mitogenomes and genome-wide SNP data to better understand how landscape features influenced the population structure and dispersal of these grass frogs across time and space. Using phylogenetic inference, population structure analyses, and biogeographic reconstructions, we found that the species complex probably first diversified on the south-east side of the Great Rift Valley. Later on, species dispersed to the north-west side, where more recent diversification occurred. We further demonstrate that Ptychadena species have dispersed across the Great Rift Valley at different times. Our analyses allowed for a more complete understanding of the contribution of geological events, biogeographic barriers and climatic changes as drivers of species diversification and adaptation in this important biogeographic region.

Renal protein reabsorption impairment related to a myxosporean infection in the grass frog (Rana temporaria L.).

A morphophysiological study of tubular reabsorption and mechanisms of protein endocytosis in the kidney of frogs (Rana temporaria L.) during parasitic infection was carried out. Pseudoplasmodia and spores of myxosporidia, beforehand assigned to the genus Sphaerospora, were detected in Bowman's capsules and in the lumen of individual renal tubules by light and electron microscopy. Remarkable morphological alteration and any signs of pathology in kidney tissue related to this myxosporean infection have not been noted. At the same time, significant changes in protein reabsorption and distribution of molecular markers of endocytosis in the proximal tubule (PT) cells in infected animals were detected by immunofluorescence confocal microscopy. In lysozyme injection experiments, the endocytosed protein and megalin expression in the infected PTs were not revealed. Tubular expression of cubilin and clathrin decreased, but endosomal recycling marker Rab11 increased or remained unchanged. Thus, myxosporean infection resulted in the alterations in lysozyme uptake and expression of the main molecular determinants of endocytosis. The inhibition of receptor-mediated clathrin-dependent protein endocytosis in amphibian kidneys due to myxosporidiosis was shown for the first time. Established impairment of the endocytic process is a clear marker of tubular cell dysfunction that can be used to assess the functioning of amphibian kidneys during adaptation to adverse environmental factors.

Neuro-immunohistochemical and molecular gene expression variations during hibernation and activity phases between Rana mascareniensis and Rana ridibunda

Low temperatures and the lack of food during the winter lead the marsh frog Rana ridibunda and the grass frog Rana mascareniensis to hibernate in order to survive. The present study aimed to investigate the cytoarchitecture of brain sub-regions affected by the thermal cycle's fluctuations during the hibernation and activity period, besides the regional distribution quantitative expression of Na(+)/K(+)-ATPase and Pax6 transcriptional factor, the molecular gene expressions of some heat shock proteins, uncoupling protein, and metallothionein. The two frog species were isolated from the field during summer and hibernation time in winter. During hibernation it was notable the destitution of degenerated, pyknotic and vasogenic neurons in different brain areas with high rate nearby the pallium. The immunohistochemical expression of Na+/ K+-ATPase and Pax 6 is decreased during hibernation in different brain sub-regions in the two species suggesting their tendency for energy conservation strategy during hibernation. Additionally, RT-qPCR recorded the up regulation of a number of heat shock protein genes during hibernation with sharing increase between two species for hsp90 besides and the non-significant expression in summer and hibernation periods for hsp47 for both species. Moreover, uncoupling protein (ucp1and ucp2) and metallothionein genes in olfactory bulb were with significant up regulation during the hibernation suggesting that these proteins possibly have a protective effect against reactive oxygen species ROS. So, brain adaptations to low temperature play a crucial role in coordinating stress responses. The present study shed light on the importance of the olfactory bulb in the thermoregulation and sensation of temperature elevations during the hibernation period and defended by the expression of heat shock proteins and uncoupling proteins preventing the cellular damage and proteins misfolding. Neuronal energy production and regeneration activities among amphibians are markedly reduced with decreasing body temperature.

Comparative spermatozoa ultrastructure of neotropical grass frogs (genus Leptodactylus) with comments on anuran reproductive modes and phylogeny

The sperm cell ultrastructure provides important insights into fertilization mechanism and phylogenetic relationships. However, it has rarely been included in phylogenetic analysis of anurans. This work aims to characterize the ultrastructure of the mature spermatozoa in the genus Leptodactylus, to relate the observed variations with the different reproductive strategies and define characters to optimize in a previously published cladogram of this genus. We studied 24 species distributed into four groups: L. fuscus, L. latrans, L. melanonotus and L. pentadactylus where morphological characters were coded and optimized using the parsimony method. The analyzed spermatozoa have three conspicuous regions: head, middle zone and tail. Regarding the head, a short and thin acrosomal vesicle is observed in species of the groups L. fuscus and L. pentadactylus, but it is long and thick in most of the studied species of the groups L. latrans and L. melanonotus. Furthermore, nuclear lacunae are observed in most species. In relation to the middle zone, an organelle-free cytoplasmic region was observed in species of the groups L. fuscus, L. latrans and L. melanonotus, but it was absent in species of the L. pentadactylus group. Most of the studied species showed a mitochondrial collar and cytoplasmic canal. Finally, in the tail all of the species have an axoneme, an undulating membrane, and an axial fiber, but only some have a juxtaxonemal fiber. Results show the thick acrosomal vesicle as a synapomorphy of the clade L. latrans + L. melanonotus. The presence of nuclear lacunae and the absence of a sub-acrosomal cone optimizes as a putative synapomorphy of Leptodactylidae. In conclusion, we observed a relationship between reproductive strategies and spermatozoa morphology, which also supports the phylogenetic relationships in this genus.

Karyosphere Capsule in Oocytes of the Grass Frog: To Be or Not to Be? A Critical View

Karyosphere Capsule in Oocytes of the Grass Frog: To Be or Not to Be? A Critical View

Genomic Analyses Reveal Association of ASIP with a Recurrently evolving Adaptive Color Pattern in Frogs.

Traits shared among distantly related lineages are indicators of common evolutionary constraints, at the ecological, physiological, or molecular level. Here, we show that the vertebral stripe, a cryptic color pattern, has evolved hundreds of times in the evolutionary history of anurans (frogs and toads) and is favored in terrestrial habitats. Using a genome-wide association study, we demonstrate that variation near the Agouti signaling protein gene (ASIP) is responsible for the different vertebral stripe phenotypes in the African grass frog Ptychadena robeensis. RNAseq and real-time quantitative PCR revealed that differential expression of the gene and an adjacent long non-coding RNA is linked to patterning in this species. Surprisingly, and although the stripe phenotypes are shared with closely related species, we found that the P. robeensis alleles are private to the species and unlikely to evolve under long-term balancing selection, thus indicating that the vertebral stripe phenotypes result from parallel evolution within the group. Our findings demonstrate that this cryptic color pattern evolved rapidly and recurrently in terrestrial anurans, and therefore constitutes an ideal system to study repeated evolution.

Mitogenomics of historical type specimens clarifies the taxonomy of Ethiopian Ptychadena Boulenger, 1917 (Anura, Ptychadenidae).

The taxonomy of the Ptychadenaneumanni species complex, a radiation of grass frogs inhabiting the Ethiopian highlands, has puzzled scientists for decades because of the morphological resemblance among its members. Whilst molecular phylogenetic methods allowed the discovery of several species in recent years, assigning pre-existing and new names to clades was challenged by the unavailability of molecular data for century-old type specimens. We used Illumina short reads to sequence the mitochondrial DNA of type specimens in this group, as well as ddRAD-seq analyses to resolve taxonomic uncertainties surrounding the P.neumanni species complex. The phylogenetic reconstruction revealed recurrent confusion between Ptychadenaerlangeri (Ahl, 1924) and P.neumanni (Ahl, 1924) in the literature. The phylogeny also established that P.largeni Perret, 1994 represents a junior synonym of P.erlangeri (Ahl, 1924) and distinguished between two small species, P.nana Perret, 1994, restricted to the Arussi Plateau, and P.robeensis Goutte, Reyes-Velasco, Freilich, Kassie & Boissinot, 2021, which inhabits the Bale Mountains. The phylogenetic analyses of mitochondrial DNA from type specimens also corroborate the validity of seven recently described species within the group. Our study shows how modern molecular tools applied to historical type specimens can help resolve long-standing taxonomic issues in cryptic species complexes.

Amphibian and fish eye lens used as biomarker of remote and chronic environmental contamination

The lens of vertebrates has a general structural plan and reacts similarly to various factors of anthropogenic impact. Rainbow trout (Oncorhynchus mykiss) and grass frog (Rana temporaria L.) are the most well-studied objects of aquatic toxicology in Russia, which react to the level of exposure to toxicants in the urbanized environment and retain the consequences of this exposure. The study aims to determine the possibility of using the lens of fish and amphibians as a biomarker of chronic and long-term background environmental contamination. The fundamental importance of the work lies in the fact that for the first time the mechanisms of the response of the epithelial cells of the eye lens and lens fibers to the spectrum of various toxicants entering the environment with industrial effluents are considered. In chronic experiments, the sensitivity of the lens of Oncorhynchus mykiss fingerlings to three toxicants (dibutyl sebacate-DBS, polyterpenes-PTP, and 3-aminopropyltriethoxysilane-AGM-9) in concentrations close to the normalized valuesin the environment was studied. On adult individuals of Rana temporaria, the appearance of abnormities in the lens was studied over a long period, depending on the total environmental contamination. To assess remote environmental contamination, studies were carried out on the constantly growing lens of the grass frog, which can preserve the resulting fiber abnormities throughout its life in the form of local opacities from the harmful environmental factors impacts. On the epithelium of the lens of fish, it was found that under the influence of certain pollutants, the mitotic index (MI) in the germinal area can change over a wide range. With the combined effect of toxicants, inhibition of mitosis is also observed. Using biomicroscopy of the eye, it was possible to show that frogs from the urbanized zone of Moscow have 1.5 times more genotypic and modification changes in the lens than their relatives taken from an ecologically clean zone of the Moscow region, Mozhaisky district in the water area of the Ozerninskoye reservoir. For the first time, the sensitivity of the lens of aquatic organisms to chronic effects under experimental and background contamination of the terrestrial and aquatic environment has been shown. Therefore, taking into account the uniformity of the structure of the eye lens of fish and amphibians, as well as in other vertebrates, the lens can be used as a biomarker for assessing chronic and remote environmental contamination. The research results can be applied for a comprehensive assessment of the action of anthropogenic factors.

Molecular determinants of protein reabsorption in the amphibian kidneys

Participation of molecular determinants of endocytosis in the processes of glomerular filtration and tubular reabsorption of albumin and lysozyme in the mesonephros of grass frogs (Rana temporaria L.), lake frogs (Rana ridibunda P.), and newts (Triturus vulgaris L.) is investigated. In all studied species, the constitutive expression of endocytic receptors in proximal tubule (PT) cells is established using immunofluorescence microscopy and immunoblotting. The certain stages of lysozyme and albumin endocytosis involving megalin/LRP2, cubilin, clathrin and protein Rab11 are detailed, and the central role of ligand-induced megalin/LRP2 activity in this process is shown. Increased ligand-induced expression for clathrin and Rab11was also found. In grass frogs, the different patterns of endocytic receptors and both absorbed proteins in the initial parts of proximal tubules suggest the proximo-distal specialization of absorptive processes along these tubule segments, similar to this in more complex mammalian nephrons. This data, as well as the revealed peculiarities of ligand-receptor interactions during intracellular trafficking of proteins prove that megalin is mainly involved in the absorption of lysozyme. At the same time, albumin absorption is mediated by both receptors, or cubilin contributes the most. The detection of endocytic receptor in glomerular structural elements in frogs and newts suggests the participation of filtration barrier components in endocytosis of filterable proteins. The results represent a new contribution to the study of the fundamental mechanisms of renal protein uptake in the amphibian mesonephros as a more primitive kidney compared to mammalian metanephros.

A new species of Hannemania Oudemans, 1911 (Trombidiformes: Leeuwenhoekiidae) from Brazil

ABSTRACT Hannemania has 27 described species parasitizing amphibians in the Americas. To date, five species have been recorded from Brazil. Here, we describe a new species for the country, collected parasitising the South American white-lipped grass frog, Miranda’s white-lipped frog, basin white-lipped, Bahia dwarf frog and granular toad all from Ceará state, Brazil. http://www.zoobank.org/urn:lsid:zoobank.org:pub:CD88F94F-ED46-4BA9-8E8E-B4AA1D2815B1

Taxonomic revision of grass frogs (Ptychadenidae, Ptychadena) endemic to the Ethiopian highlands.

Frogs of the genus Ptychadena that inhabit the Ethiopian highlands serve as a model system to understand biogeography, diversification, and adaptations to high elevations. Despite recent studies focusing on the systematics of this group, the taxonomy of the Ptychadena neumanni species complex remains only partially resolved, owing largely to the morphological resemblance of its members. Here, the taxonomy of this historically problematic group of frogs is revised by integrating morphological and molecular analyses on both century-old type specimens and more recently collected material. Based on these multiple lines of evidence, the P. neumanni species complex is shown to be more speciose than previously thought and four new species are described. With the aim of clarifying and stabilizing the taxonomy of the group, six species are also re-described and morphological and acoustic identification keys are provided. This study also establishes species distribution maps and reveals important differences in range size between the members of the P. neumanni complex, calling for adapted conservation measures across the Ethiopian highlands.

Reasons of time shifting of animals’ seasonal events in the Central Forest Biosphere Reserve

The paper considers dynamics of phenological phenomena of animals in the south taiga Central Forest biosphere reserve. As initial data we used materials of meteorological and phenological observations made under the program of maintaining the nature chronicle on the basis of the meteorological post Forest reserve in the Roshydromet network. Of the 46 meteorological series only ten (22%) show significant long-term changes over the period 19902019: an increase in the average annual air temperature, temperature in May, August, September and November; a decrease of total precipitation in September and an increase in December; a positive displacement of occurrence dates of maximum temperature less than 0C and the final formation of ice on the pond, a negative shift of the phenological summer beginning dates. The authors prove that there has been a shift of time when winter comes or an increase in the duration of the frost-free period on the reserve territory over a 30-year period. For the spring phenodates of the considered animal species, there is a negative long-term linear trend in the variability of phenological phenomena towards an earlier start of events. Such signs as the first meeting of the small tortoiseshell, the first bites of the biting mosquito, the first appearance of the grass frog and the viviparous lizard are recommended to be used as indicators of the climate change. The shift of insect and amphibian phenodates is associated with a shortening of the cold season in the reserve climate system. A significant change of lizards phenological phenomenon time is caused by abiotic as well as by biotic (forage) factors.

ЗМЕНШЕННЯ ЧИСЕЛЬНОСТІ ТРАВ’ЯНОЇ ЖАБИ (RANA TEMPORARIA) ЯК ПОКАЗНИК НЕСТАБІЛЬНОСТІ ЛОКАЛЬНОЇ ЕКОСИСТЕМИ

In the 21st century, the existence of many species of amphibians on our planet has become quite problematic due to a number of adverse environmental factors of both natural and anthropogenic origin. Amphibian fauna in Ukraine is not an exception. By conducting a series of field studies and observations, the author has established changes that occurred in the population of the grass frog (Rana temporaria) in the territory of the Lypnyky Forestry of the State Enterprise "Lvivlis". From 2005 to 2019 a number of field studies were conducted and the fact of a sharp decrease in the number of this species was established. During 2005-2009, the number of frogs reached 16 individuals per 1,000 m route. In the last few years, the number is 0.1 individuals per 1000 m of route. The drying up of reservoirs in which amphibians lay eggs spawned a reason for the low success of young frogs after the metamorphosis. First of all, we are talking about large, previously stable reservoirs and, to a lesser extent, ephemeral forest puddles. Over the last 5-7 years, the number of inhabitants has increased significantly in the study area. We are talking about the villages of Solonka, Lypnyky, Kovyri and others located near the forest. Thus, due to the rise of population, the volume of sewage drains also increased. This is a major cause of pollution and degradation of wintering frogs. Due to the low level of oxygen in local ponds, it has become virtually impossible for amphibians to winter. In 2019, due to the drought, we determined the death of caviar laying in different places of the Lypnyky forest. In one of the locations where thousands of frogs metamorphosis successfully completed ten years ago, spawning of single individuals occurred with complete loss of eggs. Therefore, we have obtained and summarized valuable data for a long period of research, formed a prediction for the population of herb frogs for the future. We believe that once numerous species in the Lypnyky forest, in the next few years will be on the verge of extinction, in some forest quarters completely cease to occur. The instability of the local ecosystem prevents the successful existence of amphibians, which requires two environments in the life cycle - terrestrial for spring and autumn habitat and aquatic for reproduction and wintering.

Phylogenomic characterization of ranaviruses isolated from cultured fish and amphibians in Thailand

Ranaviruses are emerging pathogens associated with worldwide epizootics in farmed and wild ectothermic vertebrates. In this study, we determined the full genomes of eight ranaviruses isolated from marbled sleeper goby ( Oxyeleotris marmorata), goldfish ( Carassius auratus), guppy ( Poecilia reticulata), tiger frog ( Hoplobatrachus tigerinus), Asian grass frog ( Fejervarya limnocharis), and East Asian bullfrog ( H. rugulosus) cultured or imported into Thailand. These ranaviral isolates induced the same cytopathic effects (i.e., progression of coalescing round plaques) in epithelioma papulosum cyprini (EPC) cell cultures. Transmission electron microscopy of infected EPC cells revealed cytoplasmic viral particles with ultrastructural features typical for ranaviruses. Pairwise genetic comparisons of the complete major capsid protein coding sequences from the Thai ranaviruses displayed the highest identity (99.8%–100%) to a ranavirus (tiger frog virus; TFV) isolated from diseased tiger frogs cultured in China, a slightly lower identity (99.3%–99.4%) to a ranavirus (Wamena virus; WV) isolated from diseased green tree pythons ( Morelia viridis) illegally exported from Papua New Guinea, and a lower identity to 35 other ranaviruses (93.7%–98.6%). Phylogenomic analyses supported the eight Thai ranaviruses, Chinese TFV, and WV as a subclade within a larger frog virus 3 clade. Our findings confirm the spread of TFV among cultured fish and amphibians in Asia and likely in reptiles in Oceania. Biosecurity measures are needed to ensure TFV does not continue to spread throughout Southeast Asia and to other parts of the world via international trade.

ELECTROMAGNETIC FIELD INFLUENCE ON PECULIARITIES OF RANA TEMPORARIA LINNAEUS, 1758 (ANURA, RANIDAE) ONTOGENY

This work is dedicated to study of electromagnetic irradiation influence on peculiarities of its impact on linear features and mortality of European grass frog tadpoles. Among the effects observed the intensification of growth and mortality were detected according to the increasing of number of irradiation sessions. Also the presence of morphological abnormalities in post-metamorphic individuals was assessed. The experiment showed that electromagnetic irradiation did’t cause the mass occurrence of morphological anomalies. The preliminary results of the work show that this field of experiment is promising and further investigations should be conducted. The results of the experiment can be used in herpetoculture while creating of stable breeding stock of amphibians, development of nature conservation management-plans for objects of nature reserve fund as well as in studying of impact of electromagnetic field on living organisms in general.

Actin depolymerization disrupts karyosphere capsule integrity but not residual transcription in late oocytes of the grass frog Rana temporaria.

Late diplotene oocytes are characterized by an essential decrease in transcriptional activity. At this time, chromosomes condense and form a compact structure named a karyosphere. The karyosphere of grass frogs Rana temporaria is surrounded by a fibrillar karyosphere capsule (KC). One of the main protein constituents of R. temporaria KC is actin. In this study, we used antibodies against different actin epitopes to trace different forms of actin in the KC. We also investigated the effect of F-actin depolymerization on the oocyte nuclear structures and transcription of chromatin DNA and rDNA in the amplified nucleoli. It was determined that disruption of actin filaments leads to chromosome shrinkage, nucleoli fusion, and distortion of the KC structure, but does not inhibit residual transcription in both the karyosphere and the nucleoli.

On Skeletochronology of Asian grass frog Fejervarya limnocharis (Gravenhorst, 1829) from Java to support management conservation

Asian grass frog Fejervarya limnocharis is being utilized as pets, for laboratory experiments, for a mixture of traditional medicine and for cuisine. The harvest of F. limnocharis in high volume can threat its population. Biological data such as the age when the specimens are harvested is valuable information to manage the harvesting system in sustainable way. We conducted the skeletochronology technique using paraffin methods and hematoxylin staining from 69 samples (46 males, 21 females, 2 juveniles). The results showed that the age harvested male ranged from 1 to 3 years old, while the female ranged from 2 to 3 years old. The snout-vent length (SVL) of harvested specimens ranges between 39.84−52.37 mm for both sexes. We propose an intervention in the harvesting system by limitation of the size for harvested specimens to at least 46 mm. In this minimum size, individuals of F. limnocharis have reproduced several times and have contributed to the population in the wild.

The karyosphere capsule in Rana temporaria oocytes contains structural and DNA-binding proteins

ABSTRACTDuring the last stages of oogenesis, oocyte chromosomes condense and come close together, forming the so-called karyosphere. Karyosphere formation is accompanied by an essential decrease in transcriptional activity. In the grass frog Rana temporaria, the karyosphere is surrounded by an extrachromosomal capsule that separates the chromosomes from the rest of the nucleoplasm. The karyosphere capsule (KC) of R. temporaria has been investigated in detail at the ultrastructural level, but its protein composition remained largely unknown. We demonstrate here that nuclear actin, especially F-actin, as well as lamins A/C and B are the most abundant proteins of the KC. Key proteins of nuclear pore complexes, such as Nup93 and Nup35, are also detectable in the KC. New antibodies recognizing the telomere-binding protein TRF2 allowed us to localize TRF2 in nuclear speckles. We also found that the R. temporaria KC contains some proteins involved in chromatin remodeling, including topoisomerase II and ATRX. Thus, we believe that KC isolates the chromosomes from the rest of the nucleoplasm during the final period of oocyte growth (late diplotene) and represents a specialized oocyte nuclear compartment to store a variety of factors involved in nuclear metabolism that can be used in future early development.Abbreviations: BrUTP: 5-bromouridine 5’-triphosphate; CytD: cytochalasin D; IGCs: interchromatin granule clasters; IgG: immunoglobulin G; KC: karyosphere capsule; Mw: molecular weight; NE: nuclear envelope; PBS: phosphate buffered saline; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; Topo II: topoisomerase II

A new grass frog with rupicolous tadpoles from northern Zambia (Anura: Ptychadenidae).

We describe a new species of Ptychadena from Mutinondo Wilderness in northern Zambia. It has rupicolous tadpoles that develop in a film of water. The species is distinguished on morphology, advertisement call and DNA sequences from other grass frogs and was found to be most closely related to P. broadleyi. It has no contrasting longitudinal bands on the posterior thigh, the foot is longer than half SVL, it has no light triangle on the snout, three phalanges of the fourth toe free of web and the snout has no skin folds. The species should be considered Near Threatened in terms of the IUCN Red List criteria given its limited extent of occurrence and population size.