Biochemical parameters in skin and muscle of Pelophylax kl. esculentus frogs: Influence of a cyanobacterial bloom in situ.

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludas (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required.

Low Temperature and Polyploidy Result in Larger Cell and Body Size in an Ectothermic Vertebrate.

Previous studies reported that low temperatures result in increases in both cell size and body size in ectotherms that may explain patterns of geographic variation of their body size across latitudinal ranges. Also, polyploidy showed the same effect on body size in invertebrates. In vertebrates, despite their having larger cells, no clear effect of polyploidy on body size has been found. This article presents the relationship between temperature, cell size, growth rate, and body size in diploid and polyploid hybridogenetic frog Pelophylax esculentus reared as tadpoles at 19° and 24°C. The size of cells was larger in both diploid and triploid tadpoles at 19°C, and triploids had larger cells at both temperatures. In diploid and triploid froglets, the temperature in which they developed as tadpoles did not affect the size of their cells, but triploids still had larger cells. Triploid tadpoles grew faster than diploids at 19°C and had larger body mass; there was no clear difference between ploidies in growth rate at 24°C. This indicates better adaptation of triploid tadpoles to cold environment. This is the first report on the increase of body mass of a polyploid vertebrate caused by low temperature, and we showed relationship between increase in cell size and increased body mass. The large body mass of triploids may provide a selective advantage, especially in colder environments, and this may explain the prevalence of triploids in the northern parts of the geographic range of P. esculentus.

Post-zygotic selection against parental genotypes during larval development maintains all-hybrid populations of the frog Pelophylax esculentus.

BACKGROUND: Hybridization between two species usually leads to inviable or infertile offspring, due to endogenous or exogenous selection pressures. Yet, hybrid taxa are found in several plant and animal genera, and some of these hybrid taxa are ecologically and evolutionarily very successful. One example of such a successful hybrid is the water frog, Pelophylax esculentus which originated from matings between the two species P. ridibundus (genotype RR) and P. lessonae (LL). At the northern border of the distribution all-hybrid populations consisting of diploid (LR) and one or two triploid (LLR, LRR) frog types have been established. Here, the hybrid has achieved reproductive independence from its sexual ancestors and forms a self-sustaining evolutionary unit. Based on the gamete production of these hybrids, certain mating combinations should lead to LL and RR offspring, but these parental forms are absent among the adults. RESULTS: In order to investigate the mechanisms that maintain such an all-hybrid system, we performed a field study and a crossing experiment. In the field we sampled several ponds for water frog larvae at different developmental stages. Genotype compositions were then analysed and life-history differences between the genotypes examined. In the experiment we crossed diploid and triploid males and females from different ponds and determined fertilization success as well as development speed and survival rates of the offspring under high, medium and low food availability. In both parts of the study, we found numerous LL and RR offspring during the egg and early larval stages; but the frequency of these parental genotypes decreased drastically during later stages. In natural ponds almost all of them had disappeared already before metamorphosis; under the more benign experimental conditions the last ones died as juveniles during the following year. CONCLUSIONS: From the combined results we conclude that the absence of parental genotypes in all-hybrid populations is due to post-zygotic selection against them, rather than to pre-zygotic mechanisms that might prevent their formation in the first place. For this post-zygotic selection, genetic mechanisms resulting from low genetic diversity and fixation of deleterious mutations seem to be a more likely explanation than ecological factors.

[Ploidy and genetic structure of hybrid populations of water frogs Pelophylax esculentus (L., 1758) complex (Amphibia, Ranidae) of Ukraine].

The present study of green frog hybrid populations of Ukraine, including analysis of allozyme variability and planimetric analysis oferythrocytes size has confirmed that the unique region in this area is the Severski Donets basin The allopolyploid individuals there are met very frequently (5.7% of all investigated frogs). In other areas of Ukraine only two polyploid hybrids have been recorded. Beside that, one frog was defined as triploid Rana ridibundus. According to our investigations, all triploid hybrids from the Severski Donets basin are identified as P. esculentu (=lessonae)--2 ridibundus males.

Modeling species occurrence dynamics with multiple states and imperfect detection.

Recent extensions of occupancy modeling have focused not only on the distribution of species over space, but also on additional state variables (e.g., reproducing or not, with or without disease organisms, relative abundance categories) that provide extra information about occupied sites. These biologist-driven extensions are characterized by ambiguity in both species presence and correct state classification, caused by imperfect detection. We first show the relationships between independently published approaches to the modeling of multistate occupancy. We then extend the pattern-based modeling to the case of sampling over multiple seasons or years in order to estimate state transition probabilities associated with system dynamics. The methodology and its potential for addressing relevant ecological questions are demonstrated using both maximum likelihood (occupancy and successful reproduction dynamics of California Spotted Owl) and Markov chain Monte Carlo estimation approaches (changes in relative abundance of green frogs in Maryland). Just as multistate capture-recapture modeling has revolutionized the study of individual marked animals, we believe that multistate occupancy modeling will dramatically increase our ability to address interesting questions about ecological processes underlying population-level dynamics.

Examining the single and interactive effects of three insecticides on amphibian metamorphosis.

Although aquatic communities frequently are exposed to a number of pesticides, the effects of chemical mixtures are not well understood. In two separate studies, I examined how insecticide mixtures influenced the likelihood of unpredictable, nonadditive effects on American toad (Bufo americanus) and green frog (Rana clamitans) tadpoles reared in outdoor aquatic communities. I exposed tadpoles to single or multiple insecticides at approximately half the reported median lethal concentrations using insecticides that were either acetylcholinesterase inhibitors (carbaryl or malathion) or a sodium-channel disruptor (permethrin). I found that combinations of insecticides with the same mode of action were more likely to have nonadditive effects on amphibian metamorphosis compared with those having different modes of action. Additionally, in one study, a commercial formulation of permethrin led to near-complete elimination of American toads, suggesting that this formulation could have adverse effects on aquatic communities. Many community studies exploring the ecological effects of expected environmental concentrations of pesticides have suggested that indirect effects in the food web, rather than direct effects on individual physiology, have the largest effect on amphibians. The present study indicates that direct effects of pesticides may become particularly important when insecticides with the same mode of action are present in the environment.

Food supply modifies the trade-off between past and future reproduction in a sexual parasite-host system (Rana esculenta, Rana lessonae).

Life history theory is concerned with the costs of survival, growth and reproduction under different ecological conditions and the allocation of resources to meet these costs. Typical approaches used to address these topics include manipulation of food resources, followed by measures of subsequent reproductive traits, and measures of the relationship between current and future reproductive investment. Rarely, however, do studies test for the interaction of past investment, present resource availability and future investment simultaneously. Here, we investigate this interaction in females of a sexual parasite-host system consisting of the hybridogenetic frog Rana esculenta (E) and one of its parental species Rana lessonae (L). We kept females from each of two groups (with or without previous reproduction) under two food treatments (low or high) and regularly recorded their growth as well as their body condition and hormone titres as measures of future reproductive condition. After keeping them in hibernation until the following spring, we exposed the females to males, recorded whether they spawned or not and related this response to their condition in the previous autumn. Past reproduction negatively affected growth during summer and condition during autumn which, in turn, reduced the following year's reproductive output. These costs of previous reproduction were less pronounced under the high than under the low food treatment and lower in R. lessonae than in R. esculenta. Increasing food supply improved reproductive condition more in L than in E females. These species differences in reproductive costs and food requirements provide a mechanistic explanation for why E females skip annual reproduction almost twice as often as L females. Since R. esculenta is a sexual parasite that depends on R. lessonae for successful reproduction, these species-specific life history patterns not only affect individual fitness but also the spatial structure and temporal dynamics of mixed LE populations.

Evaluation of Rana snk esculenta blood cell response to chemical stressors in the environment during the larval and adult phases.

The assessment of the biological effects on aquatic vertebrate species is frequently employed to monitor water pollution, as it provides significant information on bioavailability and actual concentration levels. In anamniote vertebrates (fish and amphibians), significant correlations have been observed between exposure to contaminants - both natural and experimental - and blood modification. We investigated the changes in some circulating blood cell parameters of green frog (Rana snk esculenta) tadpoles and adults collected at two sample rice fields, one heavily polluted and the other relatively unpolluted. The frequency of eosinophilic leucocytes, mitotic, anucleated and micronucleated erythrocytes was evaluated also regarding the haemopoietic/haemocatheretic and NOS expression of the liver. Haematological indicators in polluted samples were found to be significantly different from controls as regards both larval and adult exposure, and provided information on long-term background pollution of the habitats under investigation. The population of the polluted area showed evident effects of chronic exposure to contaminants, to a degree which could lead to sub-lethal alterations of their health status. The general nature of responses to this kind of stress emphasizes the role of amphibian peripheral blood as a sensitive indicator regarding contamination in aquatic environments.

[Quality evaluation on eighteen samples of forest frog's oviduct from different habitat of northeast China].

OBJECTIVE: To compare eighteen samples of Forest frog's oviduct from different regions of northeast China, in order to fomulate the quality evaluation standard. METHODS: According to the documents, comparing the target constituent of Forest frog's oviduct, including the mositure, ash, protein, lipid and expansibility were analysed. RESULTS: It was similar to the chemical constituent in Forest frog's oviduct from different habitiat of northeast China. CONCLUSION: The germplasm of this species is stable.

Proliferative activity in the frog brain: a PCNA-immunohistochemistry analysis.

By means proliferating cell nuclear antigen (PCNA) immunohistochemistry, we have provided a detailed neuroanatomical mapping of proliferative activity during development and adulthood in the frog (Rana esculenta) brain. Western blot analysis confirmed the presence of this protein in brain extracts from adults and tadpoles. Proliferative activity was observed in the ventricular and subventricular zones throughout the brain. The present study provides details as to which of the morphologically distinguishable brain region(s) has a long-lasting proliferative activity and in which region this activity undergoes a progressive decrease during development. In the subventricular zones of the third ventricle, PCNA-labeled cells were particularly abundant in the magnocellular preoptic nucleus and the ventromedial thalamic nucleus. It was observed that proliferation zones are present practically in all major subdivisions of the forebrain, midbrain and hindbrain, including the cerebellum in which PCNA-labeled cells were located in the outer granular layer and the inner molecular layer. The habenulae, epiphysis and isthmic nuclei never showed the presence of PCNA-immunoreactive nuclei. The widespread proliferative activity implies that the frog brain has a great potential for neurogenesis/gliogenesis not only during larval development but also in the adulthood.

Ontogeny of the somatostatin variant [Pro2,Met13]somatostatin-14 in the brain, pituitary, and sensory organs of the frog Rana esculenta.

Two forms of somatostatin are expressed in the frog brain, i.e., somatostatin-14 (SS1) and the [Pro(2), Met(13)]somatostatin-14 variant (SS2). We have previously described the ontogeny of SS1-immunoreactive cells in the brain of Rana esculenta. In the present study, we have investigated the distribution of prepro-SS2 (PSS2)-expressing neurons in the brain of the same species during development by using antibodies directed against the N-flanking region of SS2 (PSS2(54-66)). Immunoreactive perikarya first appeared in the ventral hypothalamus at stages IV-VII. Subsequently, positive neurons were seen in the nucleus of the diagonal band of Broca, the anterior preoptic area, the posterior tuberculum (stages VIII-XII), as well as the dorsal (stages XIII-XV) and medial (stages XIX-XX) periventricular preoptic nucleus. At metamorphic climax and in newly metamorphosed frogs, positive perikarya were found in the striatum and in the interpeduncular nucleus. PSS2(54-66)-immunoreactive fibers were already widely distributed during the first stages of development, indicating that SS2 may act as a neuromodulator and/or neurotransmitter during ontogeny. The presence of PSS2(54-66)-positive nerve fibers in olfactory structures suggests that, in tadpoles, SS2 may be involved in the processing of olfactory information. The occurrence of PSS2(54-66)-like immunoreactivity in taste buds, and in the olfactory and vomeronasal organs indicates that SS2 may mediate the unconditioned and reinforcing properties of natural chemicals. Finally, the intenseexpression of PSS2(54-66)-like immunoreactivity in melanotrope cells of the pituitary suggests that SS2 may diffuse toward the pars distalis to regulate the activity of adenohypophysial cells during tadpole development.

Cardiac morpho-dynamics in Rana esculenta: influence of sex and season.

Morpho-dynamic aspects of the frog (Rana esculenta) heart were correlated to seasonal and sexual traits. The statistical analysis of the data (696 frog specimens: 448 males and 248 females), collected during 8 years of routine research on frog cardiac physiology, indicated that cardiac biology is characterised by sexual dimorphism. The relative cardiac weight of males was higher than that of females of similar size. With respect to the males of similar size, female frogs revealed an increased relative ventricular weight and ventricular contractile capacity. The morphometric analysis showed that the ventricular growth was achieved through significant myocardial enlargement accompanied by a reduction of the lacunary spaces. This is more pronounced in females during the breeding period. The correlation between sexual maturity and ventricular morpho-functional changes revealed that in male frogs sexual maturity correlates with a remarkable increment of the ventricular weight associated with higher values of pulse pressure. This data indicate that sex and season-related factors influence cardiac morpho-dynamics in R. esculenta.

Connexin 43 expression in the testis of the frog Rana esculenta.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

Multiple sublethal chemicals negatively affect tadpoles of the green frog, Rana clamitans.

Many habitats may be exposed to multiple chemical contaminants, particularly in agricultural areas where fertilizer and pesticide use are common; however, the singular and interactive effects of contaminants are not well understood. The objective of our study was to examine how realistic, sublethal environmental levels of ammonium nitrate fertilizer (0, 10, 20 mg/L and ammonium chloride control) and the common insecticide carbaryl (0 or 2.5 mg/L) individually and interactively affect the development, size, and survival of green frog (Rana clamitans) tadpoles. We reared tadpoles for 95 d in outdoor 1,000-L polyethylene ponds. We found that the combination of carbaryl and nitrate had a negative effect on development and mass of tadpoles compared to the positive effect that either contaminant had alone. Presence of carbaryl was generally associated with short-term increases in algal resources, including ponds exposed to both carbaryl and nitrate. However, with exposure to nitrate and carbaryl, tadpole mass and development were not positively affected as with one chemical stressor alone. The combination of these sublethal contaminants may reduce the ability of amphibians to benefit from food-rich environments or have metabolic costs. Our study demonstrates the importance of considering multiple stressors when evaluating population-level responses.

Ontogeny of 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase in the frog brain.

The distribution of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 5alpha-reductase (5alpha-R) has been studied in the frog brain during development. Soon after hatching, 3beta-HSD- and 5alpha-R-immunoreactive (ir) cells appeared first in the olfactory bulb and in the rhombencephalon. Subsequently, 3beta-HSD-ir cells were seen in the hypothalamus and cerebellum, whereas 5alpha-R-ir cells were visualized in the pallium, preoptic nucleus, posterocentral nucleus, cerebellum, and pituitary gland. At stages XIII-XVIII, additional 3beta-HSD- and 5alpha-R-ir cells appeared in several regions of the telencephalon, diencephalon, and mesencephalon. At stages XIX-XXI, the number of 5alpha-R-ir cells increased in the preoptic nucleus. These observations indicate that biosynthesis of biologically active steroids occurs in the brain of tadpoles, suggesting that neurosteroids may play a role in brain development.

The epithalamus of the developing and adult frog: calretinin expression and habenular asymmetry in Rana esculenta.

Expression of the calcium binding protein (CaBP) calretinin (CR) was studied with immunohistochemistry in the pineal complex and habenular nuclei (HN) of the developing and adult frog Rana esculenta. The frog pineal complex is a medial structure formed by two interconnected components, the frontal organ and the pineal organ or epiphysis; the habenular nuclei are bilateral and are asymmetric due to subdivision of the left dorsal nucleus into medial and lateral components. In the pineal complex, calretinin immunostaining of cells and fibers was consistently observed in developing and adult frogs. In the habenulae, calretinin immunoreactivity exhibited instead marked variations during development, and was expressed only in cells of the medial subnucleus of the left dorsal habenula. In particular, calretinin was detected at larval stages, peaked during metamorphosis, was markedly downregulated at the end of metamorphosis, and was evident again in adulthood. This sequence of calretinin expression was confirmed by quantitative analysis of immunoreactive cells in the left habenula. In tadpoles, calretinin-positive cells exhibited a dorsoventral gradient of density, while in adulthood, they were distributed throughout the dorsoventral extent of the medial subnucleus. The study demonstrates a peculiar developmental pattern, with transient downregulation, of asymmetric calretinin expression in the frog epithalamus. The findings indicate that calcium and calcium buffering systems may play critical roles in neurogenetic and neuronal migration processes implicated in the formation of the asymmetric habenular portion in amphibians. In addition, the reappearance of calretinin expression in the adult frog supports a distinct functional role of the asymmetric habenular component in amphibians.

First evidence of prothymosin alpha in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta.

A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha (fProt-alpha) predicted a 109 amino acid protein with a high homology to the mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid residues and presents the typical basic KKQK amino acid sequence in the close carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is highly expressed in the testis. A different expression of fProt-alpha transcript was found during the frog reproductive cycle with a peak in September/October in concomitance with germ cell maturation, strongly suggesting a role for this protein in the testicular activity. In situ hybridization evidenced that the only germ cells expressing fProt-alpha are the primary and secondary spermatocytes; in addition, the hybridization signal was stronger in the October testis. Taken together, our findings indicate that fProt-alpha might contribute to the efficiency of frog spermatogenesis with a role during the meiosis. This study is the first report on the isolation and characterization of a Prot-alpha in a non-mammalian vertebrate. In addition, our results indicate that the testis of the frog R. esculenta may be a useful model to increase the knowledge concerning the physiological role of Prot-alpha in vertebrates.

Fixation of deleterious mutations in clonal lineages: evidence from hybridogenetic frogs.

The hemiclonal waterfrog Rana esculenta (RL genotype), a bisexual hybrid between R. ridibunda (RR) and R. lessonae (LL), eliminates the L genome from its germline and clonally transmits the R genome (hybridogenesis). Matings between hybrids produce R. ridibunda offspring, but they generally die at an early larval stage. Mortality may be due to fixed recessive deleterious mutations in the clonally inherited R genomes that were either acquired through the advance of Muller's ratchet or else frozen in these genomes at hemiclone formation. From this hypothesis results a straightforward prediction: Matings between different hemiclones, that is, between R. esculenta possessing different R genomes of independent origin, should produce viable R. ridibunda offspring because it is unlikely that different clonal lineages have become fixed for the same mutations. I tested this prediction by comparing survival and larval performance of tadpoles from within- and between-population crossings using R. esculenta from Seseglio (Se) in southern, Alpnach (Al) in central, and Elliker Auen (El) in northern Switzerland, respectively. Se is isolated from the other populations by the Alps. Enzyme electrophoresis revealed that parents from Se belonged to a single hemiclone that was different from all hemiclones found north of the Alps. Parents from Al also belonged to one hemiclone, but parents from El belonged to three hemiclones, one of which was indistinguishable from the one in Al. Rana esculenta from Se produced inviable tadpoles when crossed with other hybrids of their own population, but when crossed with R. esculenta from Al and El, tadpoles successfully completed metamorphosis, supporting the hypothesis I tested. Within-population crosses from Al were also inviable, but some within-population crosses from El, where three hemiclones were present, produced viable offspring. Only part of the crosses between Al and El were viable, but there was no consistent relationship between hemiclone combination and tadpole survival. When backcrossed with the parental species R. ridibunda, hybrids from all source populations produced viable offspring. Performance of these tadpoles with a sexual and a clonal genome was comparable to that of normal, sexually produced R. ridibunda tadpoles. Thus, in the heterozygous state, the deleterious mutations on the clonal R genomes did not appear to reduce tadpole fitness.

[Evaluation of developmental stability and cytogenetic homeostasis in populations of European green frogs (Rana esculenta-complex) under natural and anthropogenic conditions]. / Otsenka stabil'nosti razvitiia i tsitogeneticheskogo gomeostaza v populiatsiiakh evropeiskikh zelenykh liagushek (kompleks Rana esculenta) v estestvennykh i antropogennykh usloviiakh.

The status of amphibians in populations subjected to anthropogenic influence of various natures has been evaluated. We studied the effect of complex anthropogenic influences as well as chemical and radiation pollution. The status of the specimens in populations was evaluated by morphological (developmental stability as the level of fluctuating asymmetry) and cytogenetic (micronucleus test) methods. Disturbed developmental stability and cytogenetic homeostasis have been observed in populations affected by anthropogenic factors, which indicates the changed status of the organism.

Ontogenetic profile of FSH and LH in Rana esculenta.

Circulating levels and pituitary content of FSH and LH were determined by specific radioimmunoassays in Rana esculenta starting a few days after hatching until the completion of metamorphosis. Both gonadotropins were found in the pituitary as well as in the blood plasma at all stages of development examined here. The plasma concentrations of FSH and LH were more or less uniform during pre- and prometamorphosis, but increased significantly at the onset of metamorphic climax. The plasma levels of FSH and LH remained high at the completion of metamorphosis. The pituitary content of FSH and LH was low in early premetamorphosis. It increased slightly through prometamorphosis and metamorphic climax, following which a highly significant increase occurred. Whereas plasma concentrations of FSH and LH were essentially similar within a single stage of development, the pituitary FSH content was severalfold higher than pituitary LH. The significance of these results is discussed in relation to the functional maturation of the brain-pituitary-gonadal axis in the frog.