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.

Why do green rods of frog and toad retinas look green?

Amphibian "green" rods express a blue-sensitive cone visual pigment, and should look yellow. However,when observing them axially under microscope one sees them as green. We used single-cell microspectrophotometry (MSP) to reveal the basis of the perceived color of these photoreceptors. Conventional side-on MSP recording of the proximal cell segments reveals no selective longwave absorbing pigment explaining the green color. End-on MSP recording shows, in addition to the green rod visual pigment, an extra 2- to 4-fold attenuation being almost flat throughout the visible spectrum. This attenuation is absent in red (rhodopsin) rods, and vanishes in green rods when the retina is bathed in high-refractive media, and at wide illumination aperture. The same treatments change the color from green to yellow. It seems that the non-visual pigment attenuation is a result of slender green rod myoids operating as non-selective light guides. We hypothesize that narrow myoids, combined with photomechanical movements of melanin granules, allow a wide range of sensitivity regulation supporting the operation of green rods as blue receptors at mesopic-to low-photopic illumination levels.End-on transmittance spectrum of green rods looks similar to the reflectance spectrum of khaki military uniforms. So their greenness is the combined result of optics and human color vision.

Giants, dwarfs and the environment - metamorphic trait plasticity in the common frog.

In order to understand adaptation processes and population dynamics, it is central to know how environmental parameters influence performance of organisms within populations, including their phenotypes. The impact of single or few particular parameters in concert was often assessed in laboratory and mesocosm experiments. However, under natural conditions, with many biotic and abiotic factors potentially interacting, outcomes on phenotypic changes may be different. To study the potential environmental impact on realized phenotypic plasticity within a natural population, we assessed metamorphic traits (developmental time, size and body mass) in an amphibian species, the European common frog Rana temporaria, since a) larval amphibians are known to exhibit high levels of phenotypic plasticity of these traits in response to habitat parameters and, b) the traits' features may strongly influence individuals' future performance and fitness. In 2007 we studied these metamorphic traits in 18 ponds spread over an area of 28 km2. A subset of six ponds was reinvestigated in 2009 and 2010. This study revealed locally high variances in metamorphic traits in this presumed generalist species. We detected profound differences between metamorphing froglets (up to factor ten); both between and within ponds, on a very small geographic scale. Parameters such as predation and competition as well as many other pond characteristics, generally expected to have high impact on development, could not be related to the trait differences. We observed high divergence of patterns of mass at metamorphosis between ponds, but no detectable pattern when metamorphic traits were compared between ponds and years. Our results indicate that environment alone, i.e. as experienced by tadpoles sharing the same breeding pond, can only partly explain the variability of metamorphic traits observed. This emphasizes the importance to assess variability of reaction norms on the individual level to explain within-population variability.

A stockpile of ova in the grass frog Rana temporaria is established once for the life span. Do ovaries in amphibians and in mammals follow the same evolutionary strategy?

Most anuran amphibians produce high numbers of eggs during several consecutive breeding seasons. The question is still open whether oocytes are formed anew as a result of oogonial proliferation after each spawning or the definitive pool of oocytes is established during the juvenile period and is sufficient for the whole reproductive life span of a female. Our quantitative studies show that primary oogonia in adult female frogs can proliferate, but they fail to differentiate further and do not enter meiosis, and thereby there is no supplementation of new generations of oocytes after each spawning. Ovaries of one-year-old grass frogs contain (median) 53,447 diplotene oocytes, in two-years-old frogs this number decreased to 33,583 and eventually reached 25,679 in virgin mature females. More than 50% decrease in the total oocyte number was accompanied by massive degeneration (atresia) of oocytes. The final number of oocytes in a female forms a stock for 11-12 breeding seasons and exceeds the number of eggs produced during the potential reproductive life span of this species. The phylogenetic context of oocyte recruitment modes in the major clades of vertebrates is discussed in respect to their ability to replenish the stock (a renewable stock in ovaries named "open" vs. a non-renewable stock in ovaries named "closed").

Discordant patterns of nuclear and mitochondrial introgression in Iberian populations of the European common frog (Rana temporaria).

Amphibians often show complex histories of intraspecific and interspecific genetic introgression, which might differ in mitochondrial and nuclear genes. In our study of the genetic differentiation of the European common frog, Rana temporaria (159 specimens from 23 populations were analyzed for 24 presumptive allozyme loci; 82 specimens were sequenced for a 540-bp fragment of the mitochondrial 16S rRNA gene), multilocus correspondence analysis (CA) and Bayesian assignment tests of the nuclear data were concordant in identifying 2 population groups corresponding to 1) the Pyrenees in the east and 2) the Galicia and Asturias regions in the west, the latter corresponding to the subspecies R. temporaria parvipalmata. Geographically intermediate populations were genetically intermediate in the allozyme CA and, less clearly in the Bayesian assignment, with mitochondrial haplotypes exclusively belonging to the parvipalmata group. This indicates different degrees of introgression in the mitochondrial and nuclear genomes. Although Pyrenean high-altitude populations are morphologically distinct from low-altitude populations, these 2 groups were not separate clusters in any analysis. This suggests that the morphological differences may be due to fast adaptation to elevational gradients, likely under maintenance of gene flow, and that the underlying genetic changes are not detectable by the analyzed markers. We argue that a parsimonious explanation for the observed pattern along the east-west axis in northern Spain may be competition between invading and resident populations, with no need to invoke selection. However, in order to conclusively rule out selective processes, additional and finer scale data are required to test for asymmetric mating preference/behaviour, sex-biased gene flow, or sex-biased survival of potential hybrids.

Brain plasticity over the metamorphic boundary: carry-over effect of larval environment on froglet brain development.

Brain development shows high plasticity in response to environmental heterogeneity. However, it is unknown how environmental variation during development may affect brain architecture across life history switch points in species with complex life cycles. Previously, we showed that predation and competition affect brain development in common frog (Rana temporaria) tadpoles. Here, we studied whether larval environment had carry-over effects in brains of metamorphs. Tadpoles grown at high density had large optic tecta at metamorphosis, whereas tadpoles grown under predation risk had small diencephala. We found that larval density had a carry-over effect on froglet optic tectum size, whereas the effect of larval predation risk had vanished by metamorphosis. We discuss the possibility that the observed changes may be adaptive, reflecting the needs of an organism in given environmental and developmental contexts.

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.

Allen's rule revisited: quantitative genetics of extremity length in the common frog along a latitudinal gradient.

Ecogeographical rules linking climate to morphology have gained renewed interest because of climate change. Yet few studies have evaluated to what extent geographical trends ascribed to these rules have a genetic, rather than environmentally determined, basis. This applies especially to Allen's rule, which states that the relative extremity length decreases with increasing latitude. We studied leg length in the common frog (Rana temporaria) along a 1500 km latitudinal gradient utilizing wild and common garden data. In the wild, the body size-corrected femur and tibia lengths did not conform to Allen's rule but peaked at mid-latitudes. However, the ratio of femur to tibia length increased in the north, and the common garden data revealed a genetic cline consistent with Allen's rule in some trait and treatment combinations. While selection may have shortened the leg length in the north, the genetic trend seems to be partially masked by environmental effects.

[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.

Trait performance correlations across life stages under environmental stress conditions in the common frog, Rana temporaria.

If an organism's juvenile and adult life stages inhabit different environments, certain traits may need to be independently adapted to each environment. In many organisms, a move to a different environment during ontogeny is accompanied by metamorphosis. In such organisms phenotypic induction early in ontogeny can affect later phenotypes. In laboratory experiments we first investigated correlations between body morphology and the locomotor performance traits expressed in different life stages of the common frog, Rana temporaria: swimming speed and acceleration in tadpoles; and jump-distance in froglets. We then tested for correlations between these performances across life stages. We also subjected tadpoles to unchanging or decreasing water levels to explore whether decreasing water levels might induce any carry-over effects. Body morphology and performance were correlated in tadpoles; morphology and performance were correlated in froglets: hence body shape and morphology affect performance within each life stage. However, performance was decoupled across life stages, as there was no correlation between performance in tadpoles and performance in froglets. While size did not influence tadpole performance, it was correlated with performance of the metamorphosed froglets. Experiencing decreasing water levels accelerated development time, which resulted in smaller tadpoles and froglets, i.e., a carry-over effect. Interestingly, decreasing water levels positively affected the performance of tadpoles, but negatively affected froglet performance. Our results suggest that performance does not necessarily have to be correlated between life stages. However, froglet performance is size dependent and carried over from the tadpole stage, suggesting that some important size-dependent characters cannot be decoupled via metamorphosis.

[Resident and circulating mast cells in propulsative organs of the frog Rana temporaria].

Mast cells (MCs) of the "blood" and lymph hearts of the adult frog Rana temporaria were investigated at histochemical and ultrastructural levels. Two populations of MCs were revealed in these propulsative organs: population of resident MCs and population of circulating MCs. It has been shown that the resident cardiac MCs have an oval or elongated form and are located between atrial or ventricular myocytes and under endocardial endothelium. The resident cardiac MCs are situated in connective tissue of epicardium, too. Avascular myocardium of the frog ventricle consists of a spongy network of muscle trabeculae. We revealed circulating MCs in intertrabecular spaces and clefts of the spongy myocardium and in the blood of the main central cavity. Circulating MCs are round in shape and contain a large central nucleus enriched with condensed chromatin. They resemble the lymphocytes, but show cytoplasm filled with granules. These granules ultrastructure is much like that of the granules of the cardiac resident MCs. In the lymph heart, oval and somewhat elongated resident MCs are located in the interstitial space among cross-striated muscle fibers and among smooth muscle cells of tubular (afferent and efferent) valves. Sometimes lymphocyte-like circulating MCs are revealed in the cavity of lymph heart. Circulating MCs are also present in the lymphatics located adjacent to the lymph hearts. In certain parts of the lymphatic walls MCs are in close adhesion to the mesothelial cells lining the lymphatic cavity. Our histochemical investigation revealed that both the resident and circulating MCs of the propulsative organs give a strongly positive reaction with alcian blue, but weakly red with safranin and weakly metachromatic with toluidine blue. The presence of population of circulating MCs in the frog suggests that there are differences in biology of MCs between lower and higher vertebrates.

An eye-tectum preparation allowing routine whole-cell recordings of neuronal responses to visual stimuli in frog.

We propose an in vitro eye-tectum preparation enabling whole-cell recordings of tectal neurons combined with visual stimulation. The recordings were made from the tectum, which was cut in order to facilitate access to the cell bodies located in the inner tectal layers. The preparation remains viable for up to 5h while routine prolonged whole-cell recordings could be obtained from tectal neurons. Cutting of the tectum did not disrupt endogenous synaptic circuits and sensory inputs allowing examination of functional neuronal responses evoked with visual stimuli. Recordings from layer 6 tectal neurons indicated that neuronal responses were shaped by a mixture of excitation and inhibition generated by sensory input and local neuronal network. Visually evoked synaptic responses could also activate fast dendritic currents. Thus, the preparation brings about the benefits of in vivo recordings without the effects of anesthetics that could influence processing of sensory inputs. Using the proposed preparation, the network circuit function, which operates during central processing of a visual input, can be studied as well as the role of intrinsic properties of neurons in detection and processing of visual information.

Antipredator defenses along a latitudinal gradient in Rana temporaria.

Antipredator defenses are expected to decrease toward higher latitudes because predation rates are predicted to decrease with latitude. However, latitudinal variation in predator avoidance and defense mechanisms has seldom been studied. We studied tadpole antipredator defenses in seven Rana temporaria populations collected along a 1500-km latitudinal gradient across Sweden, along which previous studies have found increasing tadpole growth and development rates. In a laboratory common garden experiment, we measured behavioral and morphological defenses by raising tadpoles in the presence and absence of a predator (Aeshna dragonfly larva) in two temperature treatments. We also estimated tadpole survival in the presence of free-ranging predators and compared predator densities between R. temporaria breeding ponds situated at low and high latitudes. Activity and foraging were generally positively correlated with latitude in the common garden experiment. While all populations responded to predator presence by decreasing activity and foraging, high-latitude populations maintained higher activity levels in the presence of the predator. All populations exhibited defensive morphology in body and tail shape. However, whereas tail depth tended to increase with latitude in the presence of predator, it did not change with latitude in the absence of the predator. Predator presence generally increased larval period and decreased growth rate. In the southern populations, predator presence tended to have a negative effect on metamorphic size, whereas in the northern populations predators had little or a positive effect on size. Latitude of origin had a strong effect on survival in the presence of a free-ranging predator, with high-latitude tadpoles experiencing higher mortality than those from the low latitudes. In the wild, predator densities were significantly lower in high-latitude than in mid-latitude breeding ponds. Although the higher activity level in the northern populations seems to confer a significant survival disadvantage under predation risk, it is probably needed to maintain the high growth and development rates. However, the occurrence of R. temporaria at high latitudes may be facilitated by the lower predator densities in the north.

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.

Effect of NO-generating compound NaNO2 on ultrastructure of synaptic vesicles of glutamatergic synapses.

Ultrastructure of synaptic vesicles in axon terminals of granule cells from isolated cerebellum of Rana temporaria frogs under the influence of NO-generating compound NaNO2 in various concentrations and electrical stimulation was evaluated by the method of electron microscopy. NO-generating compound in low concentration induced translocation of synaptic vesicles and formation of small clusters. The size and structure of synaptic vesicles remained unchanged under these conditions. Increasing the concentration of NaNO2 led to swelling of synaptic vesicles, formation of arranged heaps from individual vesicles or fusion of their content. Electrical stimulation of the cerebellum in the presence of NaNO2 increased damage to synaptic vesicles. These experimental data model some stages observed in stroke. The formation of clusters from synaptic vesicles is a compensatory and adaptive response maintaining the structure of synaptic vesicles and protecting neurons from high concentrations of glutamate. Glutamate produces a toxic effect on nerve cells and glial cells of the cerebellum under pathological conditions, which is accompanied by impairment of signal transduction from presynaptic to postsynaptic neurons.

Non-equivalence of growth arrest induced by predation risk or food limitation: context-dependent compensatory growth in anuran tadpoles.

1. To gain insight into the evolution of compensatory growth, we studied the growth patterns of anuran (Rana temporaria) larvae following either a period of exogenous growth depression (food restriction) or a period of endogenous depression (exposure to predators). We also investigated the potential deferred costs that larval compensatory growth could impose on post-metamorphic individuals. 2. Food-deprived larvae exhibited full compensatory growth in response to reduced growth rates caused by food limitation, and the growth trajectories of low- and high-rations tadpoles converged before the onset of metamorphosis. 3. According to our predictions, individuals exposed to larval predators did not show growth compensation following predator removal despite undergoing a significant reduction in growth rate associated with low activity levels. 4. Jumping ability of individuals exposed to predators during only 20 days from the commencement of the larval phase was equivalent to that of non-exposed animals, and greater than the jumping capacity of those maintained with predators until the time of metamorphosis. This pattern was consistent with the pattern observed for variation in relative leg length. 5. These results support the suggestion that submaximum and compensatory growth could have evolved to minimize the overall growth/mortality costs in environments with high spatiotemporal variation in predation intensity.

Flexible defense strategies: competition modifies investment in behavioral vs. morphological defenses.

Competition is predicted to affect the expression of inducible defenses, but because costs of behavioral and morphological antipredator defenses differ along resource gradients, its effects on defenses may depend on the traits considered. We tested the predictions from different defense models in tadpoles of the common frog Rana temporaria, which exhibit both types of defenses. In an outdoor experiment, we exposed the tadpoles to nonlethal predators (Aeshna dragonfly larvae) and to a gradient of intraspecific competition. Morphological responses did not follow any of the expected patterns, since investment in defense was not affected by resource level. Instead, tail depth decreased in the absence of predators. Behavioral defenses followed a state-dependent model. Overall, the defense strategy of the tadpoles revealed a shift from morphological and behavioral defenses at low tadpole density to morphological defense only at high density. This difference probably reflects the different efficiency of the defenses. Hiding is an effective means of defense, but it is unsustainable when resources are scarce. Morphological responses become more important with increasing density to compensate for the increase in behavioral risk-taking. Our results indicate that competition can strongly affect reaction norms of inducible defenses and highlight the importance of integrating ecological parameters that affect the cost-benefit balance of phenotypic plasticity.

Genetic variability predicts common frog (Rana temporaria) size at metamorphosis in the wild.

We investigated associations between genetic variability and two fitness-related traits--size and age at metamorphosis--in two subartic populations of the common frog, Rana temporaria. We found that metamorphic size was positively correlated with individual heterozygosity (as estimated using eight microsatellite loci) and that maternal heterozygosity also explained a significant amount of variation in this trait. In contrast, age at metamorphosis was only explained by environmental factors. Since size at metamorphosis is positively correlated with fitness in amphibians, these results suggest that genetic variability may be an important component of individual fitness in common frogs. The environmental variation underlying timing of metamorphosis may indicate that strong selection pressure on this trait in the Nordic environment is likely to override genetic effects.

Spikelet currents in frog tectal neurons with different firing patterns in vitro.

Neuronal potential-dependent membrane currents are important in shaping the integration of synaptic inputs. Our recordings in voltage-clamp mode indicate that the small fast inward currents (spikelet currents), which were several times smaller than action potential (AP) currents, are a distinguished feature of 33% of neurons from 8 to 6 layers of the frog tectum. Out of all neuronal types described previously, only phasic cells and neurons with 'sag' in response to hyperpolarizing step current injection did not show spikelet currents. These small fast inward currents were sensitive to the intracellular administration of the sodium channel blocker QX-314, but not to the extracellular application of a glutamate receptor antagonist kynurenic acid. This suggests that spikelet currents are mediated by fast voltage-dependent Na(+) channels. Since spikelet currents could also be elicited with synaptic stimulation it is possible that spikelets are generated in dendrites and, thus, are important for fast integration of visual signals in tectal neurons.