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.

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.

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.

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.

[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.

Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths.

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.

[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.

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.

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.

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.

Ontogeny, phylogeny, and morphology in anuran larvae: morphometric analysis of cranial development and evolution in Rana tadpoles (Anura: Ranidae).

Comparative studies of chondrocranial morphology in larval anurans are typically qualitative in nature, focusing primarily on discrete variation or gross differences in the size or shape of individual structures. Detailed data on chondrocranial allometry are currently limited to only two species, Rana sylvatica and Bufo americanus. This study uses geometric morphometric and multivariate statistical analyses to examine interspecific variation in both larval chondrocranial shape and patterns of ontogenetic allometry among six species of Rana. Variation is interpreted within the context of hypothesized phylogenetic relationships among these species. Canonical variates analyses of geometric morphometric datasets indicate that species can be clearly discriminated based on chondrocranial shape, even when whole ontogenies are included in the analysis. Ordinations and cluster analyses based on chondrocranial shape data indicate the presence of three primary groupings (R. sylvatica; R. catesbeiana + R. clamitans; and R. palustris + R. pipiens + R. sphenocephala), and patterns of similarity closely reflect phylogenetic relationships. Analysis of chondrocranial allometry reveals that some patterns are conserved across all species (e.g., most measurements scale with negative allometry, those associated with the posterior palatoquadrate tend to scale with isometry or positive allometry). Ontogenetic scaling along similar allometric trajectories, lateral transpositions of individual trajectories, and variable allometric relationships all contribute to shape differences among species. Overall patterns of similarity among ontogenetic trajectories also strongly reflect phylogenetic relationships. Thus, this study demonstrates a tight link between ontogeny, phylogeny, and morphology, and highlights the importance of including both ontogenetic and phylogenetic data in studies of chondrocranial evolution in larval anurans.

[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.

Population differentiation in G matrix structure due to natural selection in Rana temporaria.

The additive genetic variance-covariance matrix (G) is a concept central to discussions about evolutionary change over time in a suite of traits. However, at the moment we do not know how fast G itself changes as a consequence of selection or how sensitive it is to environmental influences. We investigated possible evolutionary divergence and environmental influences on G using data from a factorial common-garden experiment where common frog (Rana temporaria) tadpoles from two divergent populations were exposed to three different environmental treatments. G-matrices were estimated using an animal model approach applied to data from a NCII breeding design. Matrix comparisons using both Flury and multivariate analysis of variance methods revealed significant differences in G matrices both between populations and between treatments within populations, the former being generally larger than the latter. Comparison of levels of population differentiation in trait means using Q(ST) indices with that observed in microsatellite markers (F(ST)) revealed that the former values generally exceeded the neutral expectation set by F(ST). Hence, the results suggest that intraspecific divergence in G matrix structure has occurred mainly due to natural selection.

On the structural and phyletic origin of the aminergic nerves of the hypophysis of frog tadpoles (Rana temporaria) with special reference to pars distalis.

With the aid of lesion experiments and the Falck-Hillarp fluorescence technique, it is shown that the aminergic nerves of the hypophysis, including pars distalis, originate in the caudal hypothalamus and reach the gland via a fibre tract in the floor of tuber cinereum. However, some contribution to the eminentia mediana from the preoptic area could not be excluded. A vasomotor function of the pars distalis nerves is unlikely. Based on a comparative survey the hypothesis is put forward, that the pars distalis nerves, which disappear during the metamorphic climax, are remnants of an aminergic innervation existing together with a hypophyseal portal system as a functional link between brain and pituitary in the fish-like ancestor of amphibians. Aspects are given on the problem of why a direct pars distalis innervation has neither been retained nor evolved during evolution of tetrapods.

Evidence for an orderly arrangement of optic axons within the optic nerves of the major nonmammalian vertebrate classes.

The pathways of selected optic axons were traced in representative urodele, anuran, teleost, reptile, and avian species by filling the fibers with HRP or by tracing, at the light and electron microscopic (EM) level, the degeneration caused by focal retinal or optic nerve lesions. In all species it was shown that fibers retain retinotopic neighborhood relationships throughout their transit of the optic nerve. Additionally, in anurans, it was found that a subset of large diameter, myelinated fibers take up a random arrangement in the nerve. It is argued that retinotopic fiber organisation is a reflection of contact guidance of axons during fiber outgrowth in the embryo and that this organisation could account for the arrival of fibers in orderly arrays at central nuclei during normal embryonic development.

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog.

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.

Descending serotonergic spinal projections and modulation of locomotor rhythmicity in Rana temporaria embryos.

The neuroanatomy of descending spinal projections from serotonergic raphe interneurons in embryos of the amphibian, Rana temporaria, has been examined around the time of hatching by using immunocytochemical techniques. The results illustrate that at this early stage in development the ventrolateral spinal cord is richly innervated by 5HT immunoreactive (5HTi) raphe spinal axons and associated growth cones. Other regions are devoid of processes. In conjunction, the effects of bath applied 5-hydroxytryptamine (5HT, serotonin) and its metabolic precursor, 5-hydroxytryptophan (5HTP) on locomotor activity, was also investigated by monitoring ventral root activity during fictive swimming in immobilized animals. Fictive swimming activity is similarly modulated by both exogenously applied 5HT and enhanced endogenous release of 5HT (using 5HTP). These agents increase the duration and intensity of ventral root burst, decrease cycle frequency, lengthen rostrocaudal phase delays and reduce swimming episode duration. We conclude that by the time of hatching in Rana temporaria a functional endogenous serotonergic system is established in the spinal cord which modulates the output of the central pattern generator for swimming. We compare and contrast these results with homologous descending pathways in other vertebrates, especially in a related amphibian Xenopus laevis at equivalent stages in development.

Season-specific thymic architecture in the frog, Rana temporaria: SEM studies.

It is already known that the thymus of the adult common frog, Rana temporaria, undergoes conspicuous annual cyclic changes. Light microscopic (LM) observations are at present confirmed by three-dimensional scanning electron microscopy (SEM) pictures showing the presence of a characteristic cortico-medullary division of summer thymuses and cystic appearance of winter organs. Summer thymuses are larger, much heavier and populated by a significantly higher number of cells than winter organs. The season-specific thymic size, cell content and distribution are reflected by its angioarchitecture. The microcorrosion casts of vascular system of winter thymuses are much smaller and more compact than those of the summer organs in which vessels are arranged at larger spatial intervals. It seems that thymic enlargement following the winter atrophy results from vernal repopulation of the thymic cortex by thymocytes which in consequence induces a reshaping of the existing capillary network.