Contributions of water-borne corticosterone as one non-invasive biomarker in assessing nitrate pollution stress in tadpoles of Rana temporaria.

Among a multitude of stressors to which wildlife is exposed, environmental pollution is a pervasive one that poses a serious threat. The permeable skin of amphibians is likely to increase direct contact of the body with pollutants, making them a group worth studying to access environmental quality. Consequently, finding reliable and complementary biomarkers that will present detectable and predictable changes in response to pollutants is essential to identify pollution sublethal effects on amphibians and to investigate whether these are in part responsible for population declines. The glucocorticoid hormone corticosterone (CORT), involved in many metabolic functions, is often used to measure the physiological stress response to environmental stressors in amphibians. In this study, we evaluated whether water-borne CORT can serve as a non-invasive biomarker for nitrate pollution stress in the European common frog (Rana temporaria) by comparing the effect of nitrate exposure on hormone release rates and on other physiological downstream biomarkers, i.e., ultimate physiological effects of the stressor. Specifically, we investigated the effect of different nitrate concentrations (0, 10, 50, and 100 mg/L) on water-borne CORT release rates, age, size, and body condition. Exposure to nitrate pollution significantly increased age at metamorphosis and water-borne CORT release rates, and led to reduced mass and body condition, but only at higher nitrate concentrations (i.e., 50 and 100 mg/L). Considering this similar sensitivity to other acknowledged biomarkers, water-borne CORT was a reliable biomarker of physiological stress in R. temporaria exposed to nitrate pollution stress in a controlled single-stressor laboratory approach. Thus, water-borne CORT is a promising method to be included in more holistic approaches. We recommend that such approaches keep testing multiple biomarker combinations, as species are exposed to several stressors likely to interact and produce varied outcomes in different biomarkers in their natural habitats.

Differentiation of Central Slovenian and Moscow populations of Rana temporaria frogs using peptide biomarkers of temporins family.

Skin secretion represents the only means of defense for the majority of frog species. That phenomenon is based on the fact that the main components of the secretion are peptides demonstrating greatly varying types of bioactivity. They fulfill regulatory functions, fight microorganisms and may be even helpful against predators. These peptides are considered to be rather promising pharmaceuticals of future generation as according to the present knowledge microorganisms are unlikely to develop resistance to them. Mass spectrometry sequencing of these peptides is the most efficient first step of their study providing reliably their primary structures, i.e., amino acids sequence and S-S bond motif. Besides discovering new bioactive peptides, mass spectrometry appears to be an efficient tool of taxonomy studies, allowing for distinguishing not only between closely related species, but also between populations of the same species. Application of several tandem mass spectrometry tools (CID, HCD, ETD, EThcD) available with Orbitrap mass analyzer allowed us to obtain full sequence of about 60 peptides in the secretion of Slovenian population of brown ranid frog Rana temporaria. The problem of sequence inside C-terminal cycle formed by two Cys and differentiation of isomeric Leu and Ile residues was done in top-down mode without any derivatization steps. Besides general biomarkers of Rana temporaria species, Central Slovenian population of Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be treated as biomarkers of that population.

Post-metamorphic carry-over effects of altered thyroid hormone level and developmental temperature: physiological plasticity and body condition at two life stages in Rana temporaria.

Environmental stress induced by natural and anthropogenic processes including climate change may threaten the productivity of species and persistence of populations. Ectotherms can potentially cope with stressful conditions such as extremes in temperature by exhibiting physiological plasticity. Amphibian larvae experiencing stressful environments display altered thyroid hormone (TH) status with potential implications for physiological traits and acclimation capacity. We investigated how developmental temperature (Tdev) and altered TH levels (simulating proximate effects of environmental stress) influence the standard metabolic rate (SMR), body condition (BC), and thermal tolerance in metamorphic and post-metamorphic anuran larvae of the common frog (Rana temporaria) reared at five constant temperatures (14-28 °C). At metamorphosis, larvae that developed at higher temperatures had higher maximum thermal limits but narrower ranges in thermal tolerance. Mean CTmax was 37.63 °C ± 0.14 (low TH), 36.49 °C ± 0.31 (control), and 36.43 °C ± 0.68 (high TH) in larvae acclimated to different temperatures. Larvae were able to acclimate to higher Tdev by adjusting their thermal tolerance, but not their SMR, and this effect was not impaired by altered TH levels. BC was reduced by 80% (metamorphic) and by 85% (post-metamorphic) at highest Tdev. The effect of stressful larval conditions (i.e., different developmental temperatures and, to some extent, altered TH levels) on SMR and particularly on BC at the onset of metamorphosis was carried over to froglets at the end of metamorphic climax. This has far reaching consequences, since body condition at metamorphosis is known to determine metamorphic success and, thus, is indirectly linked to individual fitness in later life stages.

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti-diabetic agents.

Temporin A (FLPLIGRVLSGIL-NH2 ), temporin F (FLPLIGKVLSGIL-NH2 ), and temporin G (FFPVIGRILNGIL-NH2 ), first identified in skin secretions of the frog Rana temporaria, produced concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 µM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca2+ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration-dependent stimulation of insulin release from 1.1B4 human-derived pancreatic ß-cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 µM), but not temporin G, protected BRIN-BD11 cells against cytokine-induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon-like peptide-1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.

Subtle effects of environmental stress observed in the early life stages of the Common frog, Rana temporaria.

Worldwide amphibian populations are declining due to habitat loss, disease and pollution. Vulnerability to environmental contaminants such as pesticides will be dependent on the species, the sensitivity of the ontogenic life stage and hence the timing of exposure and the exposure pathway. Herein we investigated the biochemical tissue 'fingerprint' in spawn and early-stage tadpoles of the Common frog, Rana temporaria, using attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectroscopy with the objective of observing differences in the biochemical constituents of the respective amphibian tissues due to varying water quality in urban and agricultural ponds. Our results demonstrate that levels of stress (marked by biochemical constituents such as glycogen that are involved in compensatory metabolic mechanisms) can be observed in tadpoles present in the pond most impacted by pollution (nutrients and pesticides), but large annual variability masked any inter-site differences in the frog spawn. ATR-FTIR spectroscopy is capable of detecting differences in tadpoles that are present in selected ponds with different levels of environmental perturbation and thus serves as a rapid and cost effective tool in assessing stress-related effects of pollution in a vulnerable class of organism.

Cool tadpoles from Arctic environments waste fewer nutrients - high gross growth efficiencies lead to low consumer-mediated nutrient recycling in the North.

Endothermic organisms can adapt to short growing seasons, low temperatures and nutrient limitation by developing high growth rates and high gross growth efficiencies (GGEs). Animals with high GGEs are better at assimilating limiting nutrients and thus should recycle (or lose) fewer nutrients. Longer guts in relation to body mass may facilitate higher GGE under resource limitation. Within the context of ecological stoichiometry theory, this study combines ecology with evolution by relating latitudinal life-history adaptations in GGE, mediated by gut length, to its ecosystem consequences, such as consumer-mediated nutrient recycling. In common garden experiments, we raised Rana temporaria tadpoles from two regions (Arctic/Boreal) under two temperature regimes (18/23 °C) crossed with two food quality treatments (high/low-nitrogen content). We measured tadpole GGEs, total nutrient loss (excretion + egestion) rates and gut length during ontogeny. In order to maintain their elemental balance, tadpoles fed low-nitrogen (N) food had lower N excretion rates and higher total phosphorous (P) loss rates than tadpoles fed high-quality food. In accordance with expectations, Arctic tadpoles had higher GGEs and lower N loss rates than their low-latitude conspecifics, especially when fed low-N food, but only in ambient temperature treatments. Arctic tadpoles also had relatively longer guts than Boreal tadpoles during early development. That temperature and food quality interacted with tadpole region of origin in affecting tadpole GGEs, nutrient loss rates and relative gut length, suggests evolved adaptation to temperature and resource differences. With future climate change, mean annual temperatures will increase. Additionally, species and genotypes will migrate north. This will change the functioning of Boreal and Arctic ecosystems by affecting consumer-mediated nutrient recycling and thus affect nutrient dynamics in general. Our study shows that evolved latitudinal adaption can change key ecosystem functions.

Absorption capacity of renal proximal tubular cells studied by combined injections of YFP and GFP in Rana temporaria L.

The capacity for protein reabsorption in the renal proximal tubule (PT) was studied in Rana temporaria frogs by separate, simultaneous and sequential introduction of yellow fluorescent protein (YFP) and green fluorescent protein (GFP). The uptake patterns of YFP and GFP in PT epithelial cells were investigated 15-120min after their bolus intravenous and intraperitoneal injection. As shown by confocal microscopy, the tubular uptake of YFP and GFP was time- and dose-dependent. These proteins are absorbed in similar way and can be accumulated in the same endocytic vesicles after their combined injections. When GFP was injected 30 and 90min before YFP, and vice versa, the number of vesicles with pre-injected protein increased and the percentage of vesicles with colocalized GFP and YFP reduced. At the same time, the uptake rate of a protein injected later progressively and significantly decreased. Subcellular localization of endocytic receptors, megalin and cubilin, in renal PT cells after intravenous YFP introduction were revealed by immunofluorescent microscopy. Colocalization of internalized YFP with megalin or cubilin in the endocytic vesicles was demonstrated. The data suggest the possibility of protein uptake by receptor-mediated endocytosis and the existence of a mechanism limiting the protein absorption rate in wintering frogs.

Composition and antimicrobial activity of the skin peptidome of Russian brown frog Rana temporaria.

A nano-HPLC-ESI-OrbiTrap study involving HCD and ETD spectra has been carried out to clarify the composition of the skin peptidome of brown Russian frogs Rana temporaria. This approach allowed determinantion of 76 individual peptides, increasing 3-fold the identified portion of the peptidome in comparison to that obtained earlier with FTICR MS. A search for the new bradykinin related peptides (BRPs) was carried out by reconstructing mass chromatograms based on the ion current of characteristic b- and y-ions. Several peptides were reported in the secretion of R. temporaria for the first time. The overall antibacterial activity of the skin secretion in general and of one individual peptide (Brevinin 1Tb) was determined using PMEU Spectrion (Portable Microbe Enrichment Unit) technology. The inhibitory effects of these peptides on Staphylococcus aureus and Salmonella enterica Serovar typhimutium were equal in scale to that reported for some antibiotics.

Geographic variation in corticosterone response to chronic predator stress in tadpoles.

Chronic stress often affects growth and development negatively, and these effects are often mediated via glucocorticoid hormones, which elevate during stress. We investigated latitudinal variation in corticosterone (CORT) response to chronic predator stress in Rana temporaria tadpoles along a 1500-km latitudinal cline in Sweden tadpoles, in a laboratory experiment. We hypothesized that more time-constrained high-latitude populations have evolved a lower CORT response to chronic stress to maintain higher growth under stressful conditions. Southern tadpoles had higher CORT content in response to predators after 1 day of exposure, whereas there was no increase in CORT in the northern populations. Two weeks later, there were no predator-induced CORT elevations. Artificially elevated CORT levels strongly decreased growth, development and survival in both northern and southern tadpoles. We suggest that the lower CORT response in high-latitude populations can be connected with avoidance of CORT-mediated reduction in growth and development, but also discuss other possible explanations.

Alteration of mitochondrial efficiency affects oxidative balance, development and growth in frog (Rana temporaria) tadpoles.

Mitochondria are known to play a central role in life history processes, being the main source of reactive oxygen species (ROS), which promote oxidative constraint. Surprisingly, although the main role of the mitochondria is to produce ATP, the plasticity of mitochondrial ATP generation has received little attention in life history studies. Yet, mitochondrial energy transduction represents the physiological link between environmental resources and energy allocated to animal performance. Studying both facets of mitochondrial functioning (ATP and ROS production) would allow better understanding of the proximate mechanisms underlying life history. We have experimentally modulated the mitochondrial capacity to generate ROS and ATP during larval development of Rana temporaria tadpoles, via chronic exposure (34 days) to a mitochondrial uncoupler (2,4-dinitrophenol, dNP). The aim was to better understand the impact of mitochondrial uncoupling on both responses in terms of oxidative balance, energy input (oxygen and feeding consumption) and energy output (growth and development of the tadpole). Exposure to 2,4-dNP reduced mitochondrial ROS generation, total antioxidant defences and oxidative damage in treated tadpoles compared with controls. Despite the beneficial effect of dNP on oxidative status, development and growth rates of treated tadpoles were lower than those in the control group. Treatment of tadpoles with 2,4-dNP promoted a mild mitochondrial uncoupling and enhanced metabolic rate. These tadpoles did not increase their food consumption, and thus failed to compensate for the energy loss elicited by the decrease in the efficiency of ATP production. These data suggest that the cost of ATP production, rather than the oxidative balance, is the parameter that constrains growth/development of tadpoles, highlighting the central role of energy transduction in larval performance.

Effects of acetylcholinesterase inhibitor paraoxon denote the possibility of non-quantal acetylcholine release in myocardium of different vertebrates.

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5 × 10(-6)-5 × 10(-5) M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10(-5) M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10(-4) M) and inhibitor of vesicular acetylcholine transporters vesamicol (10(-5) M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.

David and goliath: a mitochondrial coupling problem?

An organism's size, known to affect biological structures and processes from cellular metabolism to population dynamics, depends upon the duration and rate of growth. However, it is still poorly understood how mitochondrial function affects the energetic basis of growth, especially in ectotherms, which represent a huge majority of animal biodiversity. Here, we present an intraspecies comparison of neighboring populations of frogs (Rana temporaria) that have large differences in body mass even at the same age. By investigating liver mitochondrial bioenergetics, we find that frogs with high growth rates and large body sizes exhibit higher ATP synthesis rates and more efficient oxidative phosphorylation compared to the smaller frogs with low growth rates. This higher energy transduction efficiency is not associated with significant increased oxidative capacity or membrane potential values, but instead may rely on a higher mitochondrial phosphorylation system activity in combination with a lower inner membrane proton leakage. Overall, the present study introduces the mitochondrial energy transduction system as an important mechanism for balancing physiological and ecological trade-offs associated with body size. Whether phenotype differences in mitochondrial function result from local ecological constraints or reflect a natural genetic variability within wild populations of common frogs remains an open question. However, our findings highlight the need for closer consideration of all aspects of mitochondrial metabolism for a better understanding of the physiological basis of the link between size, metabolism, and energy production in wild-dwelling organisms.

Ammonia as a pheromone in anuran tadpoles.

In laboratory experiments, conspecific excretions and ammonia solutions evoked avoidance reactions in tadpoles of three anuran species, Bufo bufo, Rana temporaria, and Rana arvalis. A differential sensitivity of ammonia chemoreception was determined for two anuran species. For Bufo bufo tadpoles, these characteristics at ammonia background concentration of 0.2 mg/l lied in the range 150 % < dI/I < 500 %, and for background of 0.4 mg/l the value lied in the range 400 % < dI/I < 500 %. For Rana temporaria tadpoles, differential threshold against ammonia background concentration of 0.15 mg/l was close to 200 % and against background ammonia concentration of 1.1 mg/l was close to 100 %. These results suggest that such sensitivity of both anurans is sufficient for using ammonia in intra- and interspecies communication.

[Comparative sensitivity of liver monoamine oxidases of frog and whitefish to some tricyclic compounds].

There is performed a comparative analysis of action of four acridine derivatives and of one xanthene derivative (pyronine G) on activity of liver monoamine oxidase (MAO) of two species of poikilothermal freshwater animals: a representative of amphibians--the common frog Rana temnporaria and a representative of the order Salmonidae--the European whitefish Coregonus lavaretus. The studied synthetic hexamerous tricyclic compounds show the irreversible character of inhibition of intermediate potency towards the enzyme from both biological sources. There are obtained qualitative and quantitative differences in the reactional ability and selectivity of action of the studied inhibitors for liver MAO of frog and whitefish. The obtained data of the inhibitory analysis with use of specific substrates are an indirect proof for the existence in liver of the studies frog species of two molecular forms, whereas in the whitefish liver--single molecular MAO form.

[Mechanisms of interactions between glycine- and GABA-mediated responses in spinal cord neurons].

The interactions of GABA- and glycine-mediated responses have been analyzed, the whole cell patch-clamp method being used. The response induced by co-application of glycine and GABA was a lesser one than the sum of responses induced by applying two transmitters separately. The molecular mechanisms underlying this effect have been determined. Due to applications of high concentrations of neurotransmitters it was revealed that GABA could activate glycine receptors in frog spinal motoneurons with relatively high efficiency (EC50 = 1.2 mM). The sequential application of neurotransmitters showed that even a single application of glycine could significantly boost the "run-down" of the GABA-mediated current, suggesting that there was a strong phosphorylation-dependent mechanism of GABAa-receptors inhibition. These mechanisms are likely to take place in frog spinal motoneurons when GABA and glycine are co-released from the same synaptic terminal.

[Mast cells of lymph hearts during ontogenesis of frogs Rana temporaria].

Electron microscopic observations of the lymph hearts of tadpoles and yearling frogs of Rana temporaria showed that mast cells (MCs) were present not only between muscle fibers (population of resident MCs), but in the cavities of lymph heart (population of circulating MCs), too. There were some differences in the ultrastructure of the resident MCs at each studied stage of larval development. The first recognizable MCs were revealed in the lymph hearts at premetamorphosis (stages 39-41). MCs presented as mononuclear relatively small and slightly elongated cells with a few immature secretory granules and numerous free ribosomes, polysomes and short cisternae of rough endoplasmic reticulum (RER) in the cytoplasm. Chromatin of their nuclei was poorly condensed; the Golgi apparatus was moderately developed. At pro-metamorphosis (stages 44-45), we revealed MCs at different levels of their differentiation. Some MCs demonstrated an active process of granulogenesis in their cytoplasm. Among densely packed cytoplasmic organelles, immature secretory granules were closely associated with cisternae of RER and free ribosomes. Other MCs appeared as more differentiated cells. They were characterized by a predominantly heterochromatic nuclei and cytoplasm filled with polymorphic and heterogeneous granules. MCs also showed a reduction in the number of free ribosomes and cisternae of RER in the cytoplasm. On the contrary, the Golgi apparatus was well developed. Stacks of Golgi cisternae, detaching vacuoles, and progranules occupied the perinuclear region. The majority of the outlines above ultrastructural features of differentiated MCs were typical for MCs of yearling frogs. At metamorphic climax (stages 52-53), MCs often tightly contacted with macrophages. We did not reveal apoptotic MCs. However, some MCs exhibited morphological features typical for programmed necrosis-like death, which was characterized by mitochondria swelling, dilatation of cisternae of RER and nuclear envelope, plasma membrane rupture and subsequent loss of intracellular contents. Electron microscopical immunocytochemistry revealed the localization of atrial natriuretic peptide (ANP), substance S (SP) and heat shock protein (Hsp70) in the secretory granules of the resident and circulating MCs at different stages of tadpole development and in yearling frogs.

[Gaba- and glycine-immunoreactive synapses in the spinal cord of the frog Rana temporaria].

Postembedding immunogold method was used to examine the distribution of gamma-aminobutyric acid- and glycine-immunoreactives synapses on the motoneurons and primary afferent axons in frog spinal cord. Analysis of all labeled boutons on dendrites and somata of motoneurons showed that 7% were labeled for GABA, 23% only for glycine and approximately 70% were immunoreactive for both GABA and glycine. These results confirm the predominant role of glycine in postsynaptic inhibition of motoneuronal activity. Three populations of synaptic boutons were found on primary afferent axons: GABA-immunoreactive (25%), glycine-immunoreactive (5%) and the majority of the immunoreactive synapses exhibited colocalization of two inhibitory transmitters. Greater proportion of axo-axonal synases was organized in synaptic triads. The possible roles of glycine in the axo-axonal synapses on the primary afferent fibers are discussed.

Role of glycogen in processes of cerebellar glial cells under conditions of its damage with sodium nitrite.

Ultrastructure of processes of glial cell, astrocytes of the molecular layer of cerebellar cortex in Rana temporaria frog, under conditions of damage to the cerebellum caused by NO-generating compound sodium nitrite was studied under an electron microscope. It was found that astrocytes have at least two types of processes: the first (fibrillar) primarily contained numerous fibrils and few glycogen granules and the second (granular) primarily containing glycogen granules. In the presence of NO-generating compound in toxic doses, fibrillar processes are damaged or completely degrade more rapidly than granular ones. The processes containing glycogen can protect both damaged synapses and individual synaptic buttons by forming a compact structure, wrapping, around them. We analyzed the possible role of glycogen of cerebellar glial cell processes in neuroglial interactions in the presence of sodium nitrite.