Rana Lessonae Research Articles

Discovery of alien water frogs (gen. Pelophylax) in Umbria, with first report of P. shqipericus for Italy

Allochthonous water frogs (gen. Pelophylax ) have been repeatedly introduced in several European coun- tries, causing dramatic consequences for the conservation of indigenous taxa. In Italy, invasive populations are known for northern regions, where they were introduced mainly for edible and scientific purposes. Here, we report the first detection of an alien population of water frogs in Central Italy, along the Resina valley (Umbria). Genetic analysis of the mitochondrial ND3 gene polymorphism assigned some specimens to two different Pelophylax ridibundus clad - es widespread in Central and Eastern Europe. By contrast, two samples matched the mitochondrial DNA (mtDNA) sequence of Pelophylax lessonae bergeri, an autochthonous taxon widespread in Central Italy, suggesting possible hybridization between alien and indigenous frogs. Finally, the specific haplotype of Pelophylax shqipericus, the Alba - nian Pool Frog, was also identified according to mtDNA polymorphism. This record, firstly reported for Italy, poses concerns for the conservation of this cryptic taxon, suggesting that international water frog trade may involve also particularly endangered species.

Batrachochytrium dendrobatidis in Germany: distribution, prevalences, and prediction of high risk areas

In Germany, the pathogenic fungus Batrachochytrium dendrobatidis (Bd) was detected in 11 indigenous frog species, 4 newt species, and 1 salamander species in 64 out of the 181 locations (35%) investigated. Among the 3450 samples collected between 2003 and 2011, 284 (8.2%) were positive for Bd infections. The highest prevalences were observed in Alytes obstetricans (17.8% of individuals, 20% of populations), followed by Ichthyosaura alpestris (14.7%, 22.2%), Bombina variegata (13.9%, 38.5%), and water frogs comprising 2 species, Pelophylax lessonae and P. ridibundus, and their hybrid form P. esculentus (13.5%, 29.0%). Bd is widespread; areas of higher prevalence were detected in eastern, southeastern, western, and southwestern Germany. Our data indicate that drift fencing of amphibians is not a risk factor for the anthropogenic spread of Bd. Although chytridiomycosis outbreaks have never been observed in Germany, it cannot be excluded that Bd infections affect the dynamics of local amphibian populations. Among the questions still to be answered is whether juveniles are more susceptible to Bd infections than adults. Further work, especially long-term observations including capture-mark-recapture studies, is required to clarify the impact Bd has on amphibians in Germany and Central Europe.

Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentuscomplex) from the Eastern Ukraine

BackgroundHybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid – P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes.ResultsIn order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)n-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes.ConclusionsThe constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.

Genetic and Cytogenetic Characteristics of Pentaploidy in Water Frogs

We describe a pentaploid froglet (LLLRR; three Pelophylax lessonae and two Pelophylax ridibundus genomes) that has never been reported before within the Water Frog (Pelophylax esculentus) hybrid complexes. The pentaploid specimen was found among almost all triploid siblings obtained from a diploid female P. esculentus (LR) crossed with a diploid male P. lessonae (LL). We confirmed ploidy levels of the parents and the offspring by karyotyping, microsatellite analysis (18 loci), and measurements of DNA content and erythrocyte size. Microsatellite analysis indicated that the pentaploid originated from a tetraploid ovum (LLRR) fertilized by a haploid sperm (L). Surprisingly, the erythrocytes of the pentaploid were not proportionally larger than in triploids, despite a higher DNA content. Only 6.7% of the erythrocytes were distinctly large, whereas the others varied strongly in shape and size; besides typical ovoid mature erythrocytes there were small, tear-shaped, or enucleated ones. We discuss the possibility of loss of some cytoplasm by large erythrocytes as a result of mechanical damages during circulation through the narrow vessels; when the erythrocytes achieve a relatively higher surface-to-volume ratio, they may function more effectively in a proper gas exchange.

Gonadal Sex Differentiation in Frogs: How Testes Become Shorter than Ovaries

Testis differentiation in anuran amphibians is the result of two opposing processes: degeneration of the distal part, and development of the proximal part, which becomes a functional male gonad. Undifferentiated gonad differentiates directly into a testis without a transition phase. We described the morphology of developing testes in Rana temporaria and Hyla arborea, and made careful histology and ultrastructure in Pelophylax lessonae. The developing testis was divided into 10 stages (I-III, undifferentiated gonad, IV-X, testis). The earliest morphological symptoms of testis differentiation were observed in 4- to 5-week-old tadpoles at Gosner stage 27-28. At that time an undifferentiated gonad, composed of 6-9 metameres, differentiates into a testis. The proximal metameres (2-3 in the right gonad and 3-4 in the left one) differentiate into a functional testis, while the distal ones degenerate. The difference between left and right gonad size is maintained until adulthood (stage X). Degeneration of the distal part progresses along the posterior-anterior gradient and starts at stage IV. It affects first the germ cells with accompanying precursor Sertoli cells, and then the mesenchymal cells and fibroblasts. Finally the external epithelium forms a "sleeve" around the almost empty distal part. The total length of the testes stays the same until stage VIII at Gosner stage 41 (age 74-148 days). Active spermatogenesis starts at stage IX (juveniles after their first hibernation), during which the distal part eventually disappears and the proximal part starts growing considerably due to progressing spermatogenesis.

Genome dosage effect and colouration features in hybridogenetic water frogs of the Pelophylax esculentus complex

There are three taxons of central European water frogs of the Pelophylax esculentus complex: two morphologically distinct species, Pelophylax lessonae (LL) and Pelophylax ridibundus (RR), and hybrids Pelophylax esculentus, which can be either diploid (RL) or triploid (LLR or RRL). The morphology of hybrids is supposed to follow genome dosage effect. We describe colouration of water frogs with genome composition verified by chromosome analysis. Typical colouration features in LL were: spots on the ventral side, brown limbs, “weak” femur spotting pattern, brown dorsal folds and yellow-green colour in “waist”. Typical RR had dark-green or olive-green limbs, “full” femur spotting pattern, no hip spot and no yellow colour in “waist”. For all hybrids the most typical features were strong spots on the ventral side and a mosaic of green and brown colour on limbs. Typical LLR had brown-greenish dorsal folds underlined by a partial black line, “weak” femur spotting pattern and yellow-greenish colour in “waist” and on femur. Typical RL had greenish hind limbs, green dorsal folds, no yellow colour in “waist”, and no hip spot. Typical RRL was similar to RL, but had a continuous black line under dorsal folds. There were, however, numerous exceptions to these trends, with the most prominent being much higher than expected variability of colouration of Pelophylax lessonae. Therefore caution must be advised when trying to estimate genome composition of water frogs solely on the basis of colouration.

Genomic effects on advertisement call structure in diploid and triploid hybrid waterfrogs (Anura, Pelophylax esculentus).

BackgroundIn anurans, differences in male mating calls have intensively been studied with respect to taxonomic classification, phylogeographic comparisons among different populations and sexual selection. Although overall successful, there is often much unexplained variation in these studies. Potential causes for such variation include differences among genotypes and breeding systems, as well as differences between populations. We investigated how these three factors affect call properties in male water frogs of Pelophylax lessonae (genotype LL), P. ridibundus (RR) and their interspecific hybrid P. esculentus which comes in diploid (LR) and triploid types (LLR, LRR).ResultsWe investigated five call parameters that all showed a genomic dosage effect, i.e. they either decreased or increased with the L/R ratio in the order LL-LLR-LR-LRR-RR. Not all parameters differentiated equally well between the five genotypes, but combined they provided a good separation. Two of the five call parameters were also affected by the breeding system. Calls of diploid LR males varied, depending on whether these males mated with one or both of the parental species (diploid systems) or triploid hybrids (mixed ploidy systems). With the exception of the northernmost mixed-ploidy population, call differences were not related to the geographic location of the population and they were not correlated with genetic distances in the R and L genomes.ConclusionsWe found an influence of all three tested factors on call parameters, with the effect size decreasing from genotype through breeding system to geographic location of the population. Overall, results were in line with predictions from a dosage effect in L/R ratios, but in three call parameters all three hybrid types were more similar to one or the other parental species. Also calls of diploid hybrids varied between breeding systems in agreement with the sexual host required for successful reproduction. The lack of hybrid call differences in a mixed-ploidy population at the northern edge of the water frog distribution is likely to be associated with genetic particularities, including a) low genetic variability and/or b) a local loss of genes coding for genotype-dependent call differentiation under conditions where female discrimination between diploid and triploid males is not beneficial.

Hybrid advantage in skin peptide immune defenses of water frogs (Pelophylax esculentus) at risk from emerging pathogens

Heterogeneity in immune defense effectors can benefit hosts encountering a variety of parasites and pathogens. Antimicrobial peptides (AMPs) are a diverse set of immune defense effectors in many amphibians, and are secreted from dermal granular glands to protect the skin from infection. Over 50 different skin peptides have been reported from the European water frog hybridogenic complex (Pelophylax esculentus complex), consisting of the hybrid P. esculentus, and the parent species Pelophylax lessonae and Pelophylax ridibundus. In central Europe the hybrid is sympatric with only P. lessonae, while in other areas all three species can co-occur. Amphibian immune defenses are likely under selective pressure from emerging pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd). To assess if hybridization affects immune defenses against Bd, we compared skin peptides of the three species in terms of (i) quantity, (ii) activity against Bd, (iii) repertoire, and (iv) stability. Hybrids secreted AMPs at higher quantities and with greater fungicidal activity compared to cohabiting P. lessonae. Compared to P. ridibundus, AMPs from hybrids were of similar quantity but slightly greater antifungal activity. Mass spectrometric analyses (MALDI-TOF) revealed that of all three species P. esculentus has the greatest peptide diversity, a repertoire inclusive of peptides occurring in either one or the other parent species. Measurements of degradation dynamics indicate that peptides remain relatively stable on the skin of all species for over an hour after induction of skin gland secretions. Our data demonstrate that the hybrid has more effective peptide defenses against Bd and a richer peptide repertoire than either parent species. Hybrid advantage in environments hosting virulent pathogens may contribute to disassortative mating preferences, and we suggest that AMP diversity may be analogous to major histocompatibility complex (MHC) heterozygosity by benefiting hosts encountering multiple parasites.

Rhabdias esculentarum n. sp. (Nematoda: Rhabdiasidae) from green frogs of the Rana esculenta species complex in Italy: molecular evidence, morphological description and genetic differentiation from its congeners in frogs and toads

A new taxon, Rhabdias esculentarum n. sp., is described based on DNA sequence analysis at multiple loci (i.e. mtDNA cox-1, 12S rRNA, ITS-1 and partial ITS-2 regions of the nuclear rDNA) and morphometric analysis carried out on specimens collected from the green frogs of the Rana esculenta species complex in Italy (i.e. R. lessonae Camerano and R. esculenta Linnaeus, identified genetically by diagnostic allozyme loci). Rhabdias esculentarum n. sp. was differentiated genetically, at both mitochondrial and nuclear levels, from Rh. bufonis (Schrank, 1788) (sensu Hartwich, 1972) and Rh. sphaerocephala Goodey, 1924 recovered from the toad Bufo bufo Linnaeus collected sympatrically with the specimens of Rana lessonae and R. esculenta examined in the present study. Moreover, the new taxon proved to be different from the other species of Rhabdias from anurans, which had previously been sequenced using the same genes and deposited in GeneBank. Phylogenetic analyses (MP and ML) inferred from mitochondrial (mtDNA cox-1 and 12S ribosomal RNA) and nuclear (ITS-1 and ITS-2 of the rDNA regions) sequences datasets were congruent in depicting Rh. esculentarum n. sp. as forming a highly supported clade distinct from the sympatric species Rh. bufonis, as well as from Rh. sphaerocephala, characterised on the basis of the same loci. Morphometric analysis and the differential diagnosis of genetically characterised specimens of the new species have revealed differences in several features in comparison with the type-species, Rh. bufonis. Material of the latter species included voucher specimens from Germany deposited by Hartwich (1972) and other specimens collected from B. bufo in Italy. Among the diagnostic characters, the particular cup-shaped buccal capsule characterising Rh. esculentarum is clearly different from the tear-shaped buccal capsule observed in material of R. bufonis obtained from Berlin Museum and collected in the same geographical area as the green frogs under study. Rh. esculentarum was also found to differ in some measurements and allometric characters from Rh. bufonis (sensu Moravec et al., 1997). The data so far collected appear to indicate a host-preference of Rh. esculentarum for Rana lessonae and R. esculenta, which belong to the R. esculenta hybridogenetic species complex in Italy.

LC–MS/MS with 2D mass mapping of skin secretions’ peptides as a reliable tool for interspecies identification inside Rana esculenta complex

Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC–MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.

Investigation of skin secretory peptidome of Rana lessonae frog by mass spectrometry

Amphibian skin secretion represents a cerain scientific interest as a source of biologically active natural peptides. In the present research skin peptidome of wide-spread European frog Rana lessonae (Camerano, 1882) was studied for the first time ever. Peptide sequencing was accomplished with Fourier transform ion cyclotron resonance mass spectrometer in collision-induced and electron capture dissociation modes. A portion of amphibian peptides contains intramolecular C-terminal disulfide cycle which obstructs mass spectrometric sequencing. Two methods were utilized to overcome this difficulty: reduction with dithiotreithol followed by thiol group alkylation and oxidation into sulfonic acid groups with performic acid. Integrated approach employed in the present study allowed the identification of 49 peptides (of 6 to 37 amino acid residues), including 19 novel species.

Mitochondrial genome organization and divergence in hybridizing central European waterfrogs of the Pelophylax esculentus complex (Anura, Ranidae)

Natural transfer of mitochondrial DNA has occurred between three western Palaearctic waterfrog taxa: Pelophylax lessonae, Pelophylax ridibundus and their hybridogenetic hybrid, Pelophylax kl. esculentus. The transfer is asymmetric with most P. kl. esculentus and approximately one third of all central European P. ridibundus having mtDNA derived from P. lessonae (L-mtDNA). We obtained complete nucleotide sequences of multiple mitochondrial genomes (15,376–78 bp without control regions) from all 3 taxa, including a P. ridibundus frog with introgressed L-mtDNA. The gene content and organization of the mitogenomes correspond to those typical of neobatrachians. Divergence between the mtDNAs of P. lessonae and P. ridibundus is high with an uncorrected p-distance of 11.9% across the entire mitogenome. However, the rate of nucleotide substitution depends on the degree of functional constraint with up to 30-fold differences in levels of divergence. In general, mitochondrial genes encoding the translational machinery evolve very slowly, whereas genes encoding polypeptides of the electron transport system, especially the ND genes, evolve rapidly. Only 25 of 211–213 observed amino acid replacements could be classified as radical and are therefore more likely to be exposed to selection. A disproportionately high number of amino acid substitutions has occurred in the ND4, ND4L and cytb genes of the P. lessonae lineage (including 36% of all radical changes). In contrast to the interspecific divergence, nucleotide polymorphism within L- and R-mtDNA is very low: L-mtDNA haplotypes differed on average by only 19 nucleotides, while there was no variation within two mtDNAs derived from P. ridibundus. This is an expected finding considering that we have sampled a post-glacial expansion area. Moreover, the introgressed L-mtDNA on a P. ridibundus background differed from other L-mtDNAs by only a few substitutions, indicative of a very recent introgression event. We discuss our findings in the context of natural selection acting on L-mtDNA and its potential significance in cytonuclear epistasis.

Endoparasitic helminths of water frog complex in Poland: do differences exist between the parental species Pelophylax ridibundus and Pelophylax lessonae, and their natural hybrid Pelophylax esculentus?

Abstract Parasitic fauna of water frogs was mainly studied in the second half of the 20th century. However, these studies were done without differentiation into species and hybrids and pooled the 3 taxa as “water frogs” or “green frogs”. The aim of this study was to make an inventory of helminth species as well as their prevalence and intensity of infection in the two parental species (Pelophylax ridibundus and P. lessonae) and the hybrid (P. esculentus) of water frogs from 3 big populations composed of hundreds or thousands of individuals inhabited natural and seminatural landscapes in Poland. Eight helminth species were found: Polystoma integerrimum, Diplodiscus subclavatus, Opisthoglyphe ranae, Gorgodera cygnoides, Haematoloechus variegatus, Oswaldocruzia filiformis, Cosmocerca ornata and Acanthocephalus ranae. The results were compared with data from other, polish and European studies. Additionally we compared the level of infection among water frog taxa.

RrS1-like Sequences of Water Frogs from Central Europe and Around the Aegean Sea: Chromosomal Organization, Evolution, Possible Function

RrS1-like sequences of water frogs (genus Pelophylax) display varied genomic organization, whereas the centromeric hybridization pattern reveals species-specific differences. Using fluorescent in situ hybridization, Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, and Pelophylax ridibundus showed a hybridization signal at centromeres of chromosomes 1-5, but in P. kurtmuelleri the medium-small chromosome labeled was 10 rather than 8. Pelophylax cretensis had almost 16 of 26 centromeres labeled, as did Pelophylax lessonae from Poland when its chromosomes are hybridized with a homologous probe. When StuI-digested genomic DNA was hybridized with RrS1 probe, hybridization ladders for P. ridibundus from Poland have evenly spaced steps (about 100bp) of uniform intensity from about 200bp upward. Steps in hybridization ladders from circum-Aegean taxa vary in intensity: larger, odd-numbered steps are often fainter. A strong double band (800/900bp) in Anatolian P. cf. bedriagae, emphasized by a weak 700bp band, distinguishes them from P. kurtmuelleri from the Peloponnisos, in which the 900bp band is almost absent. The ladder in P. cretensis lacks odd-numbered steps. A and B repeats, observed originally within the RrS1 satellite of P. ridibundus, occur also in the circum-Aegean frogs and in P. lessonae, Pelophylax epeiroticus, Pelophylax saharicus, and Pelophylax shqipericus. It is plausible that AB dimers or ABB trimers rather than A or B monomers correspond to functional/evolutionary units. The presence of regions similar to yeast CDEs and mammalian CENP-B boxes suggests a role for RrS1 sequences in centromere organization.

Genome Dosage Effect and Hybrid Morphology — The Case of the Hybridogenetic Water Frogs of thePelophylax esculentusComplex

Western Palearctic water frogs offer a unique possibility to study the genome dosage effect (GDE). There are two morphologically distinct species, Pelophylax ridibundus RR and Pelophylax lessonae LL, and their hybridogenetic hybrid Pelophylax esculentus (RL, LLR or RRL). It is supposed that RL have intermediate morphological features, LLR are more similar to P. lessonae, and RRL more similar to P. ridibundus. We tested if the morphology of the water frogs reflects the GDE, and whether it can be used in the field for determination of the genome composition. Mean values of the indices DP/CI, T/CI and F/T followed the order LL-LLR-RL-RRL-RR. After applying discriminant and canonical analyses 89% RR, 95% LL, 91% RL, 84% LLR and 52% RRL were correctly classified. Surprisingly, the L haplotype had bigger influence on morphology than the R haplotype — all hybrid genotypes were morphologically closer to P. lessonae than to P. ridibundus.

Influence of genotype of froglets belonging to the Rana esculenta hybridogenetic complex in relation to learning capacity to eat pellets

Amphibians generally require moving preys to elicit feeding behaviour. In addition, since feeding capacity is linked to learning and memory capacities in many animals, the idea has long prevailed that amphibians were somewhat impoverished in this regard. As a result, there is currently no frog farming in Europe. However, in the 1990s, we succeeded in getting Rana ridibunda to accept pellets. R. ridibunda is a European frog belonging to the esculenta complex, which is composed of two species: Rana lessonae (LL) and R. ridibunda (RR) and their natural hybridogenetic hybrid Rana esculenta (RL). This latter taxon can be either diploid or triploid (RLL or RRL). For rearing purposes, we need to compare the learning and memory capacities of the components of this complex in relation to artificial feeding. While froglets of all taxa from the esculenta complex seem able to eat pellets, various results are obtained according to the genotype and origin of the parents. Generally, froglets from RR pairs have high survival rates (0.59–0.79), but the presence of only one L genome in the hybrid male is sufficient to reduce the survival rate (0.08–0.54). This reduction in survival is also observed in all pairs made up of an LL female and a male from various genotypes. The RL hybrid females produce smaller outputs (0–0.49) irrespective of the male genotype. The froglets from wild RR females paired with the common triploid RLL show a good survival rate (0.54), but this drops sharply with the rare RRL (0.08). The RR females from intensive rearing yield good results with RR males (0.66–0.78), somewhat lower with triploids (0.26 with RLL or RRL). LL females from one site paired with local diploid males give rise to progenies with a good survival rate (0.45–0.54), but this becomes worse (0.17) with RLL triploid. On the other hand, LL females from another site yield lower rates (0.07–0.11), which even fall to zero for corresponding RL females, whatever the genotype of the males. While pellet odour can be a feeding stimulus learnt during the tadpole stage, it is more associated with a particular stimulation of froglets during the beginning of their sub-aerial life, which varies according to the population history. Social facilitation seems to act as a feeding stimulus by direct and indirect effects (free-moving among floating pellets). Frogs, as primitive animals, are thus able to carry out cognitive tasks under the extreme conditions of artificial rearing.

A new PCR‐RFLP‐based method for an easier systematic affiliation of European water frogs

We describe a non-invasive, PCR-RFLP-based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum-likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P.lessonae and P.ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR-RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression.

Degeneration of germ line cells in amphibian ovary

AbstractOgielska, M., Rozenblut, B., Augustyńska, R., Kotusz, A. 2010. Degeneration of germ line cells in amphibian ovary. —Acta Zoologica (Stockholm) 91: 319–327We studied the morphology of degenerating ovarian follicles in juvenile and adult frogs Rana temporaria, Rana lessonae and Rana ridibunda. Degeneration of primordial germ cells was never observed and was extremely rare in oogonia and early oocytes in a cyst phase in juveniles. Previtellogenic oocytes were rarely affected. Three main types of atresia were identified. In type I (subdivided into stages A–D), vitellogenic oocytes are digested by proliferating follicle cells that hypertrophy and become phagocytic. A – germinal vesicle shrinks, nucleoli fuse, oocyte envelope interrupts, and follicular cells hypertrophy; B – follicular cells multiply and invade the oocyte; C – entire vesicle is filled by phagocytic cells; D – degenerating phagocytes accumulate black pigment. Type II is rare and resembles breakdown of follicles and release of ooplasm. In type III, observed in previtellogenic and early vitellogenic oocytes, ooplasm and germinal vesicle shrink, follicle cells do not invade the vesicle, and condensed ooplasm becomes fragmented. The residual oogonia in adult ovaries (germ patches) multiply, but soon degenerate.

Molecular characterization and expression of a heat-shock cognate 70 (Hsc70) and a heat-shock protein 70 (Hsp70) cDNAs in Rana (Pelophylax) lessonae embryos

Heat-Shock Protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. In this work, two cDNAs encoding a constitutive Hsc70 and an inducible Hsp70 from the water frog Rana (Pelophylax) lessonae, belonging to the Rana (P.) esculenta complex of central Italy, have been isolated and characterized. The two cDNA clones, named Rl-Hsc70 and Rl-Hsp70, encode 646 and 640 amino acid proteins respectively, which present extremely conserved functional domains characteristic of cytosolic members of the HSP70 family. Comparative studies of the amino acid sequences showed that Rl-Hsc70 and Rl-Hsp70 had the highest homology with constitutive and inducible HSP70 members of other amphibian species. The phylogenetic analysis also demonstrated a separate clustering of the Rl-Hsc70 and Rl-Hsp70 with constitutive and inducible members from other vertebrate species. Heat-inducibility assays performed during embryogenesis showed that the two isolated mRNAs displayed different expression profiles. Rl-Hsp70 was induced only in heat shock-treated embryos, whereas Rl-Hsc70 transcript levels, which were constitutively modulated in non-stressed embryos, did not increase following the heat treatment. In situ hybridization experiments demonstrated that both transcripts showed a tissue-specific enrichment in the central nervous system and in the somites.

Paternity control for externally fertilised eggs: behavioural mechanisms in the waterfrog species complex

Male competition for mates and female mate choice are key mechanisms involved in sexual selection. Surprisingly, these mechanisms have often been investigated separately although they appear to interact in many species. Male–male competition for territories located at the best places or to establish dominance relationships often explain mating patterns. Such male behaviours may affect and sometimes even hinder female mate choice, as in the case of sexual coercion. While in many species females are able to exert cryptic control over paternity (i.e. a process allowing females to bias offspring production toward certain males after intromission), in other species external fertilisation prevents females from doing so. This is the case in the waterfrog hybridisation complex where the hybrid Pelophylax esculentus can only produce viable offspring by pairing with the parental species Pelophylax lessonae (hybridogenetic reproduction). We examined two potential processes that could enhance such mating combinations. Firstly, by monitoring male spatial distribution within six choruses, we showed that the proportion of P. lessonae males located at the edge (in the best position to grasp females arriving at the chorus) cannot explain the frequency of mating combinations observed. Secondly, an experimental approach emphasised a new way for anuran females to favour paternity of a particular male in a sexual coercion context. When females are forcefully paired with an incompatible male, they cannot remove the male grasped on their back by themselves. Nevertheless, by controlling the movement of the pair within the chorus, these females often change mates by enhancing male competition instead of laying eggs. In many species with externally fertilised eggs, it may be thus necessary to take into account this new possibility for females to control offspring paternity.