Refrigerated storage of European common frog Rana temporaria oocytes.

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1-5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1-5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.

European common frog Rana temporaria (Anura: Ranidae) larvae show subcellular responses under field-relevant Bacillus thuringiensis var. israelensis (Bti) exposure levels.

Bacillus thuringiensis var. israelensis (Bti) is presumed to be an environmental friendly agent for the use in either health-related mosquito control or the reduction of nuisance associated with mosquitoes from seasonal wetlands. Amphibians inhabiting these valuable wetlands may be exposed to Bti products several times during their breeding season. Up until now, information regarding effects on the non-targeted group of amphibians has to be considered rather inconsistent. On this account, we evaluated how three repeated exposures to frequently used Bti formulations (VectoBac®12AS, VectoBac®WG) in field-relevant rates affect European common frog (Rana temporaria) larvae. In a laboratory approach, we assessed potential effects with regard to enzymatic biomarkers (glutathione-S-transferase (GST), glutathione reductase (GR), acetylcholine esterase (AChE)), development, body condition and survival until the end of metamorphosis. Although survival and time to metamorphosis were not significantly affected, larval development tended to be shortened in the Bti treated water phase. Furthermore, exposure to Bti induced significant increases of GST (37-550%), GR (5-140%) and AChE (38-137%) irrespectively of the applied formulation, indicating detoxification, antioxidant responses as well as an alteration of neuronal activity. GST activity increased twice as much after two repeatedly executed Bti applications within a time period of 6 days. The examination of several biochemical markers is needed to fully evaluate the ecotoxicological risk of Bti for amphibian populations, especially in the context of worldwide amphibian declines. Nevertheless, following the precautionary principle, it may be advisable to implement certain thresholds for application numbers and intervals in order to ensure environmentally friendly mosquito control programs, especially in areas designated for nature conservation.

Four-dimensional analysis of early pelvic girdle development in Rana temporaria.

A key event in vertebrate evolution is the linkage of the appendicular to the axial skeleton. The present study investigates the developmental dynamics of pelvic girdle morphogenesis in Rana temporaria up until metamorphosis, with respect to its functional and spatio-temporal organization. The main questions to be addressed are: initial location and the number of elements contributing to pelvic girdle formation, mechanism of bridging between the pelvic anlage and the sacrum and arthrogenesis. Serial histological sections of specimens from Gosner Stages 30 to 41 were bright-field microscopically examined and 3D-reconstructed. 3D-models were merged to 4D-animations illustrating the complex developmental dynamics through time. The results reveal the initial formation of a single mesenchymal condensation located close to the appendicular skeleton, but far from the axial skeleton. In addition, our analysis detects a thin connective tissue strand in R. temporaria guiding the elongation of the ilium towards the sacrum. The 4D-visualization allows novel insight into the ilio-sacral bridging process and the reorientation of the pelvis.

Effects of single and combined embryonic exposures to herbicide and conspecific chemical alarm cues on hatching and larval traits in the common frog (Rana temporaria).

Recent studies indicate that amphibian embryos can exhibit hatching plasticity in response to chemical cues indicative of a predation risk. However, data are lacking concerning the possible impacts of waterborne contaminants on such a process. To investigate this impact, we raised eggs of the common frog (Rana temporaria) until hatching in water contaminated with sublethal concentrations (0.01, 0.1, and 1 mg/L) of amitrole, a widely used triazole herbicide, either with or without the presence of chemical alarm cues from crushed conspecific tadpoles. Embryonic exposure to conspecific alarm cues resulted in a delay in hatching, reduced growth, and decreased larval activity, regardless of the amitrole concentration present during the incubation. Conspecific cues also induced morphological changes, but only in individuals incubated in water contaminated with the highest amitrole concentration. The herbicide impacts on hatching time were restricted to embryos incubated in the presence of conspecific cues, with individuals exposed to 0.1 and 1 mg/L showing an extended embryonic period compared to controls in uncontaminated water. Whether tested alone or in combination with conspecific cues, amitrole also induced slight morphological changes but did not affect larval growth or behavioral activity. Thus, depending on the trait considered, both chemical stressors exhibited either single or interactive effects. Furthermore, our data indicate that a stressing factor without apparent impact when tested alone could exert effects when combined with another stressor. Such results highlight the importance of considering multiple environmental factors and biological traits when examining stress-induced phenotypic variability.

Variation in UV sensitivity among common frog Rana temporaria populations along an altitudinal gradient.

Solar ultraviolet-B (UV-B) radiation can be harmful for developing amphibians. As the UV-B dose increases with altitude, it has been suggested that high-altitude populations may have an increased tolerance to high levels of UV-B radiation as compared to lowland populations. We tested this hypothesis with the common frog (Rana temporaria) by comparing populations from nine altitudes (from 333 to 2450m above sea level). Eggs collected in the field were used for laboratory experiments, i.e., exposed to high levels of artificial UV-B radiation. Eggs were reared at 14+/-2 degrees C and exposed to UV treatments until hatching. Embryonic developmental rates increased strongly and linearly with increasing altitude, suggesting a genetic capacity for faster development in highland than lowland eggs. Body length at hatching varied significantly with UV-B treatments, being lower when eggs developed under direct UV-B exposure. Body length at hatching also increased as the altitude of populations increased, but UV-B exposure times were shorter as altitude of population increased. However, the body length difference between exposed and non-exposed individuals in each population decreased as altitude of populations increased, suggesting a costly effect of UV exposure on growth. Type of UV exposure did not influence the mean rates of embryonic mortality and deformity, but both mortality and deformity rates increased as the altitude of populations increased (while UV-B exposure duration decreased). The effect of UV-B on body length at hatching, mortality, and deformities suggests that the sensitivity to UV-B varied among populations along the altitudinal gradient. These results are discussed in evolutionary terms, specifically the potential of R. temporaria high-altitude populations to develop local genetic adaptation to high levels of UV-B.

[Mechanisms of adaptive regulation of early development of Anuran amphibians dwelling the anthropogenically polluted water bodies].

Comparison of the gastrulation in three Anuran species has shown the possibility of evolution of earlier morphogenesis directed to adaption to such new environmental factor as anthropogenic pollution of water bodies. Changing proportion of variability and correlation of the developing embryonic structures causes changes in the character of morphogenetic processes to the point of discoordinated development and formation of heterochronies and/or increased death rate. In the studied amphibian species, the increase of the concordent development in different populations resulted either from increased general correlation of morphogenesis or from the emergence of a group of traits with more interdependent development. Mechanisms of early development regulation comply with a single scheme, which indicates the nonspecificity of early embryogenesis reaction based on epigenetic regulation mechanisms.

[Survival of amphibian embryos after continuous ultrasound treatment].

The influence of continuous ultrasound on the embryonic development of grass frog Rana temporaria has been investigated. Intact embryos at the blastula stage were treated by ultrasound of different frequency (0.88 and 2.64 MHz), intensity (0.05-1.0 W/cm2), and duration (1-15 min). The treatment with ultrasound of frequency 0.88 MHz and intensity 0.05 W/cm2 for 1-5 min tended to increase the proportion of normally developing embryos up to hatch (10-25% of control). Increasing the intensity of ultrasound (0.88 MHz) to 0.7-1.0 W/cm2 and the duration of its action to 5-15 min induced the death of almost all of treated embryos. No significant differences were found between the development of control embryos and embryos treated with ultrasound of middle intensity (0.2-0.7 W/cm2) for 1-5 min. The exposure of amphibian embryos to ultrasound of frequency 2.64 MHz and intensity 0.05-0.7 W/cm2 for 1-5 min did not change their survival. Increasing the intensity of ultrasound (2.64 MHz) to 1.0 W/cm2 and the duration of its action to 5 min decreased the number of normal developing embryos (by 35%).

Stereological analysis of mitochondria in embryos of Rana temporaria and Bufo bufo during cleavage.

Total numbers of mitochondria and their morphology have been quantitatively determined in mature oocytes and in cleaving embryos of two anuran species Rana temporaria and Bufo bufo using stereological methods. Surface densities of inner mitochondrial membranes for both studied species during cleavage ranged from 5.43 m2/cm3 to 7.53 m2/cm3, whereas volume densities of mitochondria did not exceed 1.65%. Since values of these parameters were low, thus embryos during cleavage may be considered as metabolically "silent". Transition of ultrastructural morphology of mitochondria towards that characterising actively respiring organelles occurs at stage 9 for R. temporaria and at stage 8 for B. bufo, correlated with blastula-gastrula and mid-blastula transition, respectively. The total numbers of mitochondria N(c) in mature oocytes are as high as 114.8 and 107.2 millions for R. temporaria and B. bufo, respectively, and during cleavage at late blastula stages they increase to 300 millions for both species under study. We suggest that an undefined mechanism might eliminate during cleavage those amphibian embryos which contain small number of mitochondria and low levels of nutrient substances.

[Comparison of variability of Rana temporaria (Amphibia, Anura) gastrula from different populations developing under the conditions of antropogenic pollution].

The gastrulation of Rana temporaria embryos was studied at the natural spawning sites of Moscow city and Moscow district by means of morphometric analysis. We demonstrated that anthropogenic pollution plays an important role in morphogenesis of Anura. The measurements of embryos from the Moscow ponds made it possible to show an increased variability of gastrulation. Moreover, the level of gastrulation variability depends on the degree of pollution of a spawning pond and is coupled with increased correlations between the morphological features. This increase of correlations could be due to: (1) an increased number of correlating features (appearance of new correlations) and (2) strengthening of the existing correlations. The first way makes the gastrulation process more coordinated, and the second one causes the appearance of morphological structures, which are normally formed only at the next developmental stages.

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos.

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT(1,5A,7) receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT(1,7) receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT(1A) receptors, as in Xenopus, but also 5-HT(7) receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT(1A) and/or 5-HT(7) receptor antagonism, implicating 5-HT(1B/1D) receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT(1A,7) and 5-HT(1B/1D) receptors act as gain-setters of burst durations, whilst 5-HT(5A) receptors are involved in the effects of bath-applied 5-HT on locomotion.

[Distribution of the Xvent-2 mRNA between informosomes and polysomes in early frog development]. / Raspredelenie mRNK Xvent-2 mezhdu informosomami i polisomami v rannem razvitii liagushek.

Translational expression regulation was studied for Xvent-2 coding for a transcription factor involved in dorsoventral differentiation of amphibian embryos. The Xvent-2 mRNA was detected predominantly in informosomes at all developmental stages examined, and only its minor fraction was associated with polysomes. Expression of Xvent-2 was assumed to be regulated at the translational level, with Xvent-2 produced immediately before polypotent cells enter a certain differentiation pathway.

Development of the basement membrane and formation of collagen fibrils below the placodes in the head of anuran larvae.

The development of the basement membrane and collagen fibrils below placodes, including the corneal region of the ectoderm, lens epithelium, nasal plate, and auditory vesicle in anuran larvae was observed by transmission electron microscopy and compared with that in nonplacodal regions such as the epidermis, neural tube, and optic vesicle. In the corneal region the lamina densa becomes thick concomitantly with the development of the connecting apparatuses such as hemidesmosomes and anchoring fibrils. The collagen fibrils increase in number and form a multilayered structure, showing similar morphology to the connective tissues below the epidermis. These two areas, i.e., the corneal region and epidermis, possess much collagenous connective tissue below them. On the other hand, the neural tube and ophthalmic vesicle that originated from the neural tube each have a thin lamina densa and a small number of underlying collagen fibrils. The lamina densa does not thicken and the number of collagen fibrils do not significantly increase during development. These two areas possess little extracellular matrix. The nasal plate and auditory vesicle show intermediate characteristics between the epidermis-type and the neural tube-type areas. In these areas, the lamina densa becomes thick and hemidesmosomes and anchoring fibrils develop. The number of collagen fibrils increases during development, but does not show an orderly arrangement; rather, they are randomly distributed. It is thought that the difference in the arrangement of collagen fibrils in different tissues is due to differences in the extracellular matrix around the collagen fibrils. Placodal epithelia have the same origin as epidermis, but during development their morphological characteristics differ and they are not associated with the pattern of extracellular matrix with characteristics of epidermal and corneal multilayered collagen fibril areas.

Latitudinal and temperature-dependent variation in embryonic development and growth in Rana temporaria.

Variation in seasonal time constraints and temperature along latitudinal gradients are expected to select for life history trait differentiation, but information about the relative importance of these factors in shaping patterns of divergence in embryonic traits remains sparse. We studied embryonic survival, growth and development rates in the common frog (Rana temporaria) along a 1,400-km latitudinal gradient across Sweden by raising embryos from four populations in the laboratory at seven temperatures (9 degrees C, 12 degrees C, 15 degrees C, 18 degrees C, 21 degrees C, 24 degrees C, 27 degrees C). We found significant differences in mean values of all traits between the populations and temperature treatments, but this variation was not latitudinally ordered. In general, embryonic survival decreased at the two highest temperatures in all populations, but less so in the southernmost as compared to the other populations. The northernmost population developed slowest at the lowest temperature, while the two mid-latitude populations were slowest at the other temperatures. Hatchling size increased with increasing temperature especially in the two northern populations, whereas the two southern populations showed peak hatchling size at 15 degrees C. Analyses of within-population genetic variation with a half-sib design revealed that there was significant additive genetic variation in all traits, and egg size-related maternal effects were important in the case of hatchling size. Overall, our results indicate that unlike larval growth and development, variation in embryonic development and growth in R. temporaria cannot be explained in terms of a latitudinal gradient in season length. While adaptation to a latitudinal variation in temperature might have contributed to the observed differentiation in embryonic performance, the effects of other, perhaps more local environmental factors, seem to have overridden them in importance.

[Fluorescent analysis of cryopreserved totipotent cells of amphibian embryos]. / Fluorestsentnyi analiz kriokonservirovannykh totipotentnykh kletok zarodyshei amfibii.

The cryotolerance of totipotent cells from dissociated embryos of amphibian (grass frog Rana temporaria and grey toad Bufo bufo) was studied. Cell integrity and preservation of the cell barrier function were evaluated by fluorescent analysis. It was shown that the best cryopreservation of the cells was achieved by using the cryoprotective agent 10% dimethyl sulfoxide and 10% saccharose. These cells were successfully used for the homotransplantation of nuclei into enucleated eggs. The development of reconstructed eggs to the blastula stage was noted.

Utilization of yolk platelets during early embryonic development of Rana temporaria and Bufo bufo.

Utilization of yolk platelets in cleaving embryos of Rana temporaria and Bufo bufo was studied by different methods. Morphological observations of yolk platelets of R. temporaria embryos at tail bud stage by transmission electron microscopy indicated four initial phases of platelet degradation. The pattern of these events is similar to that found in embryos of B. bufo. The morphological observations were confirmed by energy-dispersive X-ray microanalysis of the elemental content of platelets and by selected-area electron diffraction of platelet cores. Covalently bound sulphur content decreased during cleavage and the content of different inorganic ions changed, whereas the structure of crystalline core remained constant. Morphological changes found in the amorphous cortex of yolk platelets were due to their utilization. Stereological measurements indicated that utilization during cleavage increased, but only the initial phases of yolk platelet degradation were seen. The volume of the cortex did not decrease and the crystalline core did not fragment.

Carry-over effects of ultraviolet-B radiation on larval fitness in Rana temporaria.

A number of studies have failed to find evidence for negative effects of ultraviolet-B radiation (UVBR) on amphibian early-embryonic performance, leading to the conclusions, first, that the embryonic stages of many species are tolerant to UVBR, and second, that the increased amount of UVBR reaching the Earth's surface is not likely to have any direct negative effects on many amphibian populations. However, possible carry-over effects of exposure to UVBR in the embryonic stages to the larval stages have received less attention. We studied the effects of UVBR experienced during the embryonic stages (age less than 11 days) on the later performance (age 11-75 days) of common frog, Rana temporaria, larvae. In a factorial laboratory experiment, newly fertilized embryos were divided into three different UVBR treatments (no UVBR (control), 1.25 kJm(-2) (normal) and 1.58 kJm(-2) (26% enhanced)), after which the individual larvae were raised until metamorphosis in the absence of UVBR. No effects of UVBR on embryonic survival rates, frequency of developmental anomalies or hatchling size were found, corroborating the earlier results indicating that R. temporaria embryos are tolerant to UVBR. However, analyses of larval performance revealed that larvae exposed to enhanced levels of UVBR as embryos suffered from an increased frequency of developmental anomalies and metamorphosed later and at a smaller size than larvae that had been protected from UVBR as embryos. These results suggest, in contrast to the earlier studies, that UVBR has direct negative effects on R. temporaria embryos, but these effects are expressed mostly or only during the later life stages. To this end, our results support the contention that carry-over effects from one life stage to another may be an important source of phenotypic variation in fitness.

Induction of a non-rhythmic motor pattern by nitric oxide in hatchling Rana temporaria embryos.

Nitric oxide (NO) is a ubiquitous neuromodulator with a diverse array of functions in a variety of brain regions, but a role for NO in the generation of locomotor activity has yet to be demonstrated. The possibility that NO is involved in the generation of motor activity in embryos of the frog Rana temporaria was investigated using the NO donors S-nitroso-n-acetylpenicillamine (SNAP; 100--500 micromol l(-1)) and diethylamine nitric oxide complex sodium (DEANO; 25--100 micromol l(-1)). Immobilised Rana temporaria embryos generate a non-rhythmic 'lashing' motor pattern either spontaneously or in response to dimming of the experimental bath illumination. Bath-applied NO donors triggered a qualitatively similar motor pattern in which non-rhythmic motor bursts were generated contra- and ipsilaterally down the length of the body. The inactive precursor of SNAP, n-acetyl-penicillamine (NAP), at equivalent concentrations did not trigger motor activity. NO donors failed to initiate swimming and had no measurable effects on the parameters of swimming induced by electrical stimulation. Intracellular recordings with potassium-acetate-filled electrodes revealed that the bursts of ventral root discharge induced by NO donors were accompanied by phasic depolarisations in motor neurons. During the inter-burst intervals, periods of substantial membrane hyperpolarization below the normal resting potential were observed, presumably coincident with contralateral ventral root activity. With KCl-filled electrodes, inhibitory potentials were strongly depolarising, suggesting that inhibition was Cl(-)-dependent. The synaptic drive seen in motor neurons after dimming of the illumination was very similar to that induced by the NO donors. NADPH-diaphorase histochemistry identified putative endogenous sources of NO in the central nervous system and the skin. Three populations of bilaterally symmetrical neurons were identified within the brainstem. Some of these neurons had contralateral projections and many had axonal processes that projected to and entered the marginal zones of the spinal cord, suggesting that they were reticulospinal.

Studies in developmental cytomechanic.

One of the most promising trends in modern developmental and cell biology, recently defined as <>, or <>, is directed towards revealing the role of mechanical stresses, chemomechanical transduction and active stress responses of cells antissues of developing embryos. We review here the results obtained in this field by our research group and compare them with those from other labs. Our studies relate to the buds of hydroid polypes and to amphibian embryos. We describe the space-temporal patterns of mechanical stresses in these species, analyze their morphogenetical role and the tissue responses to the experimental modulations of stress patterns. In hydroid polypes we explore also the molecular events involved in mechanochemical coupling. A model, linking the passive mechanical stresses with the active stress-responses of embryonic tissues is suggested. We consider these investigations as a first approach to a developing embryo as to an <>.