Outbreeding depression in the common frog, <Emphasis Type="Italic">Rana temporaria</Emphasis>

Theory suggests that parental relatedness is a continuous variable with a fitness optimum that we heretoforth will refer to as optimal outbreeding. In the present paper, we test this proposition from a conservation (translocation) perspective. Amphibians are facing a global decline and many amphibian populations are today small and threatened by extinction. Because genetic differentiation is often high between amphibian populations, they could be particularly sensitive to outbreeding depression, e.g. due to breakdown of locally adapted gene complexes. We tested if outbreeding would reduce fitness in common frogs, Rana temporaria, crossed from a large and an isolated, small population, separated by 130km, using artificial fertilization. For females from the large population, tadpoles were significantly smaller and more malformed in crosses with males from the small population, than with males from the large population. For the small population, however, no significant paternal genetic effects could be found. The difference in response to outbreeding between populations was accompanied with significant differences in the importance of maternal effects. We conclude that care should be taken when translocating frogs between distantly related populations to avoid outbreeding depression.     

Photopreferendum of migratory and nonmigratory larvae of European river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

In the light gradient installation, the larvae of European river lamprey Lampetra fluviatilis of 0+ age, migrating downstream (downstream migrants), are distributed predominantly over the zone of dusk illumination (150–1 lx) and less so over the daylight zone (2500–150 lx) and night illumination zone (<1 lx). In 0+ larvae that passed over to the sedentary lifestyle, the photopreferendum changes: they avoid the dusk and daytime illumination and prefer nighttime illumination. Such photopreferendum is retained in later larvae of lamprey (1+ and older).     

Peculiarities of functioning of liver mitochondria of river lamprey Lampetra fluviatilis and common frog Rana temporaria at periods of suppression and activation of energy metabolism

The work dealt with study of mitochondria in reversible metabolic suppression of hepatocytes of the river lamprey Lampetra fluviatilis in the course of prespawning starvation and of liver mitochondria of the common frog Rana temporaria during hibernation and activity. In winter the metabolic depression of lamprey hepatocytes, unlike that of frog hepatocytes, has been found to be due to deactivation of complex I of the electron transport mitochondrial chain, a low rate of NAD-dependent substrate oxidation, a low content of adenine nucleotide content, and a high degree of mitochondrial membrane permeability to H+ and other monovalent ions (KCl-, K+). The mitochondrial membrane permeability decreases in the presence of ethyleneglycoldiamineethyltetraacetic acid (EGTA), cyclosporine A (CsA), adenosine-5'-diphosphate (ADP), and Mg+. These facts indicate the presence in these mitochondria of the Ca2+ -dependent unspecific pore in the low-conductance state. Histological studies showed the lamprey and the frog to have principal differences in use of energy substrates at the period of metabolic depression. Lampreys utilize predominantly lipids, whereas frogs--glycogen. The clearly pronounces activation of lipid consumption is observed at the spring period before spawning and death of lamprey. Possible causes of metabolic depression are discussed as well as similarity and difference in behavior of mitochondria of cyclostomes and amphibians throughout metabolic depression and activity.     

Kinetic properties of sodium transport pathways in the river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> erythrocytes

To activate Na⁺/H⁺ exchange, intracellular pH (pH_i) of erythrocytes of the river lamprey Lampetra fluviatilis were changed from 6 and 8 using nigericin. The Na⁺/H⁺ exchanger activity was estimated from the values of amiloride-sensitive components of Na⁺ (²²Na) inflow or of H⁺ outflow from erythrocytes. Kinetic parameters of the carrier functioning were determined by using Hill equation. Dependence of Na⁺ and H⁺ transport on pH_i value is described by hyperbolic function with the Hill coefficient value (n) close to 1. Maximal rate of ion transport was within the limits of 9–10 mmol/l cells/min, and the H⁺ concentration producing the exchanger 50% activation amounted to 0.6–1.0 μM. Stimulation of H⁺ outcome from acidified erythrocytes (pH_i 5.9) with increase of H⁺ concentration in the incubation medium is described by Hill equation with n value of 1.6. Concentration Na⁺ for the semimaximal stimulation of H⁺ outcome amounted to 10 mM. The obtained results indicate the presence in lamprey erythrocytes of only binding site for H⁺ from the cytoplasm side and the presence of positive cooperativity in Na⁺-binding from the extracellular side of the Na⁺/H⁺ exchanger. Na⁺ efflux from cells in the Na⁺-free medium did not change at a 10-fold increase of H⁺ concentration in the incubation medium. The presented data indicate differences of kinetic properties of the lamprey erythrocyte Na⁺/H⁺ exchanger and of this carrier isoforms in mammalian cells. In intact erythrocytes the dependence of the amiloride-sensitive Na⁺ inflow on its concentration in the medium is described by Hill equitation with n 1.6. The Na⁺ concentration producing the 50% transport activation amounted to 39 mM and was essentially higher as compared with that in acidified erythrocytes. These data confirm conception of the presence of two amiloride-sensitive pathways of Na+ transport in lamprey erythrocytes.     

Temporal peculiarities of responses of auditory system neurons of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> to tone bursts with different modulation frequencies

Activity of medullar and midbrain auditory neurons at action of amplitude-modulated tone burst was recorded in immobilized common frogs Rana temporaria. Depth of modulation amounted to 10% or 80%, frequency of modulation varied from 5 to 150 Hz, and carrier intensity was in the range of 20–30 dB. Phasic neurons in medulla clearly reproduced the modulation frequency, but only at the 80% modulation depth. However, during presentation of signal with the 10% modulation depth, these neurons practically did not respond. Tonic neurons were able to reproduce the modulation frequency up to 10–150 Hz, but at the 10% modulation depth, the degree of reproduction of envelope was rather low, although for several first modulation periods it rose statistically significantly. In midbrain, the highest frequency of the reproduced modulation sharply fell. At greater modulation frequencies, the response of these neurons qualitatively reminds that of medullar neurons. At the low modulation frequencies, there is identified a group of midbrain neurons with a prominent increase of the signal modulation. This occurs in the frequency diapason up to 60 Hz; at an increase of the modulation frequency the time of achievement of maximal synchronization decreases. The optimal modulation frequency in many neurons of semicircular torus corresponds to parameters of the male nuptial call.     

Polymorphism of spawners of the resident form of European river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> (Petromyzontidae)

Data on polymorphism (plastic and meristic characters) are obtained for spawners of resident nonparasitic form of European river lamprey Lampetra fluviatilis from the Serebristaya River. The new data verify the diagnosis of this species and contribute to the formulation of a hypothesis on the presence of several phenotypic groups: dwarf, small-sized, and common. Analysis of plastic characters and of the number of trunk myomeres demonstrated that the values of these characters reliably depend on position of lampreys in a certain discerned group. Some aspects of biology of this species are discussed: spawning and interaction between resident nonparasitic river and migrating parasitic lampreys.     

The influence of anthropogenic factors on reproduction of <Emphasis Type="Italic">Rana temporaria</Emphasis> and <Emphasis Type="Italic">Rana arvalis</Emphasis>

Studies of spawning water bodies in the city of Moscow have shown that the urban populations of common (Rana temporaria) and moor (R. arvalis) frogs are small compared with suburban populations, and their individuals lead a hidden mode of life. The recorded increase in the fecundity of females from several populations of the city of Moscow may be accompanied by an increase or the preservation of the diameter of eggs as compared with the same indicator for suburban populations. The populations in which the females produced many small eggs died out during the study period. The most prosperous populations are urban populations of brown frogs whose females spawned eggs of various sizes. We consider the formation of these clutches as a manifestation of the bet-hedging strategy compensating for mortality in adverse and unstable environmental conditions.     

GABA- and glycine-immunoreactive synapses in spinal cord of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Using the method of the double immune label combined with two antibodies, i.e., monoclonal antibodies to gamma-aminobutyric acid (GABA) and polyclonal antibodies to glycine, the distribution of gamma-aminobutyric acid- and glycine-immunoreactive synapses on motoneurons and primary afferent axons was studied in the frog Rana temporaria spinal cord. An analysis of all labeled boutons on the dendrites and soma of motoneurons showed the existence of three categories of immunoreactive synapses as follows: 7% were labeled for GABA, 23% were labeled for glycine, and approximately 70% were immunoreactive to both GABA and glycine. These results confirm the predominant role of glycine in the postsynaptic inhibition of motoneuronal activity. Three similar populations of synaptic boutons were also founded on primary afferent axons, including one GABA-immunoreactive (25%) and one glycine-immunoreactive (5%); the majority of the immunoreactive synapses had the colocalization of two inhibitory transmitters. As a rule, the higher proportion of axo-axonal synapses was organized in synaptic triads. The possible simultaneous roles of glycine as a transmitter of postsynaptic inhibition and as a transmitter that mediates the process of the autoreception of glutamate in the axo-axonal synapses on the primary afferent fibers are discussed.     

Molecular Cloning of Synucleins in River Lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Synaptic proteins synucleins are found in pathologic aggregates in human brain during neurodegenerative diseases and in some tumors. Normal functions of these proteins in synapses are still unclear. In the present study, we used cDNA cloning to determine amino acid sequences of synucleins in the central nervous system of river lamprey (Lampetra fluviatilis), which is used as a model organism to study molecular mechanisms of synaptic transmission. Three genes are identified. High similarity in amino acid sequences as compared to other vertebrate species is revealed. The bioinformatic analysis predicts that the river lamprey synucleins relate to the group of gamma-synucleins. High homology with human alpha-synuclein is reported. The hydrophobic region required for the formation of alpha-synuclein amyloid fibers is also present in the river lamprey synucleins. The latter suggests that this region appeared at early stages of evolution. The obtained amino acid sequences of synucleins in the river lamprey brain will allow generating novel molecular tools for dissecting physiological functions of these proteins.     

Temporal characteristics of downstream migration of smolts of the European river lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> in the Chernaya River

Data on the seasonal and diurnal dynamics of downstream migration of smolts of the European river lamprey Lampetra fluviatilis are obtained. On the basis of published data and surveys in nature, it is shown that in the largest part of the area the downstream migration of smolts occurs mainly in spring, in the period of high water in rivers caused by snow melting. The smolts undertake downstream migration at night, under minimal illumination. It has been determined experimentally that the smolts are characterized by negative photopreferendum (>90% of smolts preferred illumination <0.4 lx).     

Transport of monovalent thallium across the membrane of oocyte of the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis>

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using ²⁰⁴Tl. The Tl⁺ transport in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by mechanisms of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl₂, 5 glucose, 10 Tris-HCl-in the presence of ouabain, the coefficient of the ²⁰⁴Tl stationary distribution (cell/medium) was within the range of 2.3–2.5, while the time necessary to reach its 50% value amounted to 40–45 min at 20°C. In potassium-free media, transport of ²⁰⁴Tl via Na/K-pump was described by simple kinetics with saturation and was characterized by the value V _max = 520 pmol/(cell h) and K _M = 0.3 mM. In the presence of 4 mM K⁺ and 0.1 mM/1 Tl⁺, the ouabain-sensitive Tl⁺ flux decreased to 75 pmol/(cell h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na⁺ (in Na-NMDG media of different composition) the total influx of Tl⁺ reached 193 ± 20 pmol/(cell h), while the bumetanide-sensitive component—119 ± 12 pmol/(cell h) with K _M for Na⁺ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl⁻ by NO₃) the total Tl⁺ flux reached 220 ± 21, while via the mechanisms of Na,K,Cl-cotransport—87 ± 8 pmol/(cell h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl⁺ influx. The potassium channels that usually are permeable also to monovalent thallium ions were not revealed.     

Comparative study of inhibitory specificity of liver monoamine oxidase in frogs <Emphasis Type="Italic">Rana ridibunda</Emphasis> and <Emphasis Type="Italic">Rana temporaria</Emphasis>

A comparative enzymological investigation of inhibitory specificity of the liver monoamine oxidases (MAO) from the two frog species, lake frog Rana ridibunda and grass frog Rana temporaria, revealed certain interspecies similarities and distinctions of this enzyme. The anti-monoamine oxidase effect of five derivatives of acridine, three derivatives of phenothiazine and one derivative of xanthene (pyronine G) was comparatively analyzed. The tested six-membered tricyclic compounds were shown to exert an irreversible inhibitory effect on the enzyme from both biological sources, displaying the same substrate deamination specificity. Thus, the rate of interaction of acridine and phenothiazine derivatives with the MAO active center in both frog species was considerably higher when activity was determined using noradrenaline versus N-methylhistamine, while that of pyronine G—when activity was determined using N-methylhistamine versus noradrenaline. Interspecies quantitative differences were found in the inhibitory efficacy and degree of selectivity of the tested tricyclic compounds, indicative of the differences in catalytic properties of liver MAO at the interspecies level in the representatives of the genus Rana, family Ranidae. The data of substratespecific inhibitory analysis provide indirect evidence of the existence of two molecular MAO forms in the liver of the studied frog species.     

Developmental responses to UV-B radiation in common frog <Emphasis Type="Italic">Rana temporaria</Emphasis> embryos from along an altitudinal gradient

Solar ultraviolet radiation (UV) is harmful for developing amphibians. As UV increases with altitude and latitude, it is suggested that high altitude and latitude populations have evolved tolerance to high levels of UV. Using laboratory experiments, we tested the hypothesis that Rana temporaria populations from several altitudes (438–2,450 m above sea level) were adapted to UV by assessing the effects of artificial UV on embryos mortality rate, malformations, and body length at hatching. We also tested the protective role of the jelly surrounding the embryos. Without artificial UV exposure, hatching success decreased with altitude of the population. Malformation rates were low for all populations (mean 1.36%), and hatching size increased with altitude. The artificial UV (UV-B, UV-A, and visible) used was similar to the solar spectrum received at high altitude. Exposed embryos had performance similar to that of embryos without exposure: a decreased hatching success with altitude and a low malformation rate (mean 0.85%). However, hatching size did not vary with altitude, and UV-exposed embryos tended to be smaller at hatching than non-exposed embryos. Removal of the protective jelly envelope greatly decreased the performance of UV-exposed embryos: hatching success strongly decreased with altitude and embryos of the highest population (2,450 m asl) did not develop. Malformation reached 4.98%, without population differences, and hatching size of embryos without jelly was smaller than hatching size of non-manipulated embryos with no population effect. This study demonstrates that lowland jellyless embryos were less sensitive (i.e., exhibited a higher survival rate) than highland embryos and, on the other hand, that the jelly envelope was a more efficient protection for embryos in highland than in lowland populations. A trade-off hypothesis is presented to explain this difference in UV tolerance and protection among populations: in a harsh, highland environment (i.e., with a curtailed activity period), embryos have to invest in development and growth and, thus, embryo protection should be a female investment (via the jelly). In a lowland environment, embryonic growth and development are less constrained and embryos are able to invest and use protection or repair mechanisms inherited from their parents.     

Absorption of arginine-vasopressin and arginine-vasotocin in small intestine of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Experiments in vitro demonstrated a partial absorption of arginine-vasopressin (AVP) in the frog small intestine. Dynamics and efficiency of the nonapeptide absorption are studied with use of hydroosmotic method of recording of the osmotic permeability of the frog urinary bladder epithelium and immunoenzyme analysis. In the process of absorption there were preserved intactness of the hormone cyclic structure and its physiological activity, like in the case of the arginine-vasotocine (AVT) absorption. The AVP absorption increased at its administration into the gut with inhibitor of proteases. By methods of immunoelectron and immunofluorescent microscopy with use of polyclonal antibody to AVP, location of the label to the hormone was shown in the enterocyte cytoplasm. Thus, there was obtained a morphological evidence for the AVP absorption and transepithelial transfer in the frog small intestine. These data enlarge the concept of the poorly studied properties of the absorbing epithelium of the vertebrate intestine with respect to absorption of intact molecules of polypeptides.     

Synaptic and electotonic contacts on primary afferent axons in the lamprey <Emphasis Type="Italic">Lampetra fluviatilis</Emphasis> spinal cord

Distribution of GABA and glycine immunoreactivity was studied in synapses on primary afferent axons of the lamprey Lampetra fluviatilis spinal cord using a double labelling technique. Approximately 25% of synapses exhibit GABA immunoreactivity, while more than 70% are immunoreactive to both neurotransmitters. As in other vertebrates, axo-axonal contacts represent three-component synaptic complexes, the so-called triads, where the immunoreactive terminal make synaptic contact simultaneously with the afferent axon and the dendrite contacting this afferent. Contact zones with gap junction-like cell membrane specializations were found between adjacent afferents suggesting the presence of electrotonic interaction between them. This interaction appears to serve for the synchronization of the afferent flow and represents a structural correlate of the mechanism of rapid interneuronal communication between functionally uniform neurons, which is an important element in the organization of coordinated locomotor acts. Besides, our studies provide evidence that afferent–afferent interaction may be mediated not only electrotonically but also with the aid of chemical synapses. This finding suggests that glutamate-induced depolarization of primary afferents results not only from autoreception but also from the direct effect of glutamate on the afferent’s cell membrane.     

Genetic diversity of Mexican brook lamprey <Emphasis Type="Italic">Lampetra (Tetrapleurodon) geminis</Emphasis> (Alvarez del Villar, 1966)

Lampreys are the only surviving representatives of the oldest known vertebrates. The Mexican lamprey L. geminis (nonparasitic), is particularly interesting, because it is an endemic, biogeographical relict, and a threatened species. RAPD markers were used to describe genetic diversity in L. geminis A total of 77 specimens were collected from five populations, three in the R'o Grande de Morelia-Cuitzeo basin and two in the R'o Duero-Lerma-Chapala basin, Mexico. Eighty-eight RAPD markers were obtained from eight primers. Genetic diversity within each population was estimated using Shannon's index (S), heterozygosity (H) and gene diversity (h). These estimates revealed significant variation within populations, although a variance homogeneity test (HOMOVA) showed no significant differences among populations or between basins. Nei genetic distance values indicate a low genetic differentiation among populations. Analysis of molecular variance (AMOVA) indicates that most of the genetic diversity occurs within populations (91.4%), but that a statistically significant amount is found among populations (P0.001). Principal coordinates and cluster analyses of RAPD phenotypes show that specimens are not grouped by geographical origin. The genetic diversity found within L. geminispopulations may be explained by its breeding system and an overlapping of generations. The scarce genetic differentiation among populations is likely to the low rate of DNA change that characterizes the lamprey group.     

Features of the spatial distribution and size structure of the Pacific lamprey <Emphasis Type="Italic">Lampetra tridentata</Emphasis> in the North Pacific

Results of long-term research on the spatial and vertical distribution of the Pacific lamprey Lampetra tridentata (Richardson, 1836) (the family Petromyzontidae) in the North Pacific, and data on its size structure are submitted. It was shown, that L. tridentate reached its greatest number in the Bering Sea. The maximum concentration of the Pacific lamprey was observed all year round about Navarin Cape, in the Koryak shelf area, at the East Aleutian Islands and at the west coast of the USA, which, apparently, spoke of the increased number of its prey there. On the bottom, the Pacific lamprey was the most numerous at depths of less than 500 m, and in the pelagic, in the upper 100-meter layer. The length of individuals varied in catches from 12 up to 85 cm. The existence of several size groups may indicate that the Pacific lamprey spend several years in the sea. No correlation was revealed between the length of body and fishing depth. The correlations between body length and body weight, body length and condition factor were analyzed. Seasonal dynamics of these parameters were considered.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Spawning ecology of the American brook lamprey, <Emphasis Type="Italic">Lampetra appendix</Emphasis>

Synopsis We observed spawning American brook lamprey, Lampetra appendix, in coldwater streams in Minnesota to assess various aspects of their spawning behavior and spawning habitat requirements. Spawning occurred during April and May, at water temperatures ranging from 8.7 to 15.5°C. Average adult lamprey length and mass differed significantly among streams, but there were no significant differences in length or mass between males and females. Overall sex ratio was 1:1, although one stream had significantly more males than females and one stream held significantly more females. Lampreys spawned in groups of 2–14 individuals, averaging 4.2 adults per nest across all streams. Nests were constructed in gravel and cobble substrate just upstream of riffles, spaced at an average density of three nests m⁻². The typical nest was 16 cm in diameter in water 31 cm deep with a bottom current velocity of 14 cm s⁻¹, and was excavated to a depth of 4 cm below the stream bottom; however, some nest characteristics varied significantly in a few streams. Nests were larger in streams with larger spawning groups, deeper water, and slower current velocities. American brook lamprey exhibited spawning behaviors and spawning habitat requirements similar to those of other species of lamprey in North America.