Vegerina et al. (2013) Determination of the ratio of diploids and triploids among metamorphs of green frogs

A. O. Vegerina, E. V. Meleshko, I. S. Pirina, V. A. Sapozhnikova, O. V. Biryuk. Determination of the ratio of diploids and triploids among metamorphs of green frogs in the North-Donetsk center of diversity of the Pelophylax esculentus complex // Bulletin of V. N. Karazin Kharkiv National University. Series: Biology. – 2013. – Issue 18, No. 1079. – P. 107–113. UDC: 597.851 Determination of the ratio of diploids and triploids among metamorphs of green frogs in the North-Donetsk center of diversity of the Pelophylax esculentus complex A. O. Vegerina, E. V. Meleshko, I. S. Pirina, V. A. Sapozhnikova, O. V. Biryuk V. N. Karazin Kharkiv National University (Kharkiv, Ukraine) mykhailova.o.v@gmail.com The hypothesis that the proportion of triploids among metamorphs of representatives of the Pelophylax esculentus complex in the North-Donetsk center of green frog diversity is higher than among sexually mature individuals was tested. The ploidy of 30 metamorphs collected in July 2013 was determined. It was found that the average erythrocyte length in metamorphs is lower than in sexually mature frogs. Ploidy was determined by counting the number of chromosomes in metaphase plates obtained from intestinal endothelium. To determine ploidy in the absence of dividing cells, Ag-staining was used, which allows the detection of nucleoli in interphase nuclei. All studied individuals turned out to be diploid. Considering the sample studied in 2012, one triploid was registered in the combined sample of 47 young individuals; the hypothesis tested was not confirmed. The reasons for the low proportion of triploids in the studied sample are suggested. Keywords: Pelophylax esculentus, triploids, erythrocytes, Ag-staining, ploidy.

The determination of the ratio of diploid and triploid green frogs underyearlings from the Pelophylax esculentus complex Seversky Donets diversity center
A. O. Vegerina, O. V. Meleshko, I. S. Pyrina, V. O. Sapoznikova, O. V. Biryuk
The hypothesis has been tested that the percentage of triploids among the metamorphs of Pelophylax esculentus complex representatives in the Seversky Donets green frogs diversity center is higher than that among the mature individuals. The ploidy of 30 metamorphs collected in July, 2013 was defined. It was found that the metamorphs erythrocytes mean length is lower than that in the mature frogs. Ploidy was defined by counting chromosomes in metaphase plates derived from intestine endothelium. To determine the ploidy in the absence of dividing cells the Ag‑staining was used, which reveals the nucleoli in interphase nuclei. All the studied specimens were diploid. Given the sample studied in 2012, one triploid was registered in the combined sample of 47 young individuals; the tested hypothesis was not confirmed. The assumptions about the causes of the low percentage of triploids in the studied sample were made.
Key words: Pelophylax esculentus, triploids, red blood cells, Ag‑staining, ploidy.

Introduction The hybrid complex of green frogs (Pelophylax esculentus complex) includes two parental species: the pool frog Pelophylax lessonae (Camerano, 1882) and the lake frog Pelophylax ridibundus (Pallas, 1771), from the crossing of which interspecific hybrids are formed. To denote these hybrids (with some convention), a name analogous to the species name is used – the edible frog Pelophylax esculentus (Linnaeus, 1758). Different forms of hybrids differ in ploidy and the nature of their gametogenesis; in the typical case, they hemizonally (without recombination) transmit one of the parental genomes to gametes (Plötner, 2005). Individuals of the parental species live and reproduce together with hybrids, forming hemizygous population systems. The region located in the Seversky Donets river basin, characterized by high diversity of hybrid green frogs, including the distribution of triploid hybrids, was named the North-Donetsk center of diversity of the P. esculentus complex (Shabanov et al., 2009).

In the first work reporting the detection of triploid green frog hybrids in this area, the registered proportion of triploids among hybrids was relatively high, at 30% (Borkin et al., 2004). The sample of green frogs studied in this work included a significant number of juveniles. In several other studies of sexually mature individuals, a lower proportion of triploids in the total number of hybrids was recorded: 9% (Mezhzherin et al., 2010) or 11% (Kozak et al., 2012).

The described difference in results allows us to hypothesize that the proportion of triploids among P. esculentus in the North-Donetsk center of green frog diversity decreases with age. This hypothesis is supported by the fact that triploid hybrids are not a separate taxon and likely do not have an independent genealogy separate from diploids. The genomes of such frogs are the result of the evolution of diploid representatives of the parental species, and their triploidy can be considered a cytogenetic anomaly. Based on this, it can be assumed that selection reduces the proportion of triploids in older age classes compared to younger ones.

In 2012, some of the authors of this work studied a sample of 17 juveniles and immature individuals collected near the village of Gaidary, Zmiiv district, Kharkiv region. The ploidy of frogs in this work was determined using the pressed preparation method (Mikhailova et al., 2011). The proportion of triploids registered in this work turned out to be unexpectedly low: out of 17 individuals, 1 was triploid and 16 were diploid.

In this work, we tested the same assumption using a larger sample size and combining an indirect method of ploidy determination with two direct methods. The indirect method of ploidy determination involves measuring the average erythrocyte size of each individual. As is known, the nuclei of triploid hybrid cells contain one and a half times more chromatin (Plötner, 2005); this leads to an increase in the size of nuclei and cells in general. The boundary between the sizes of diploid and triploid erythrocytes in different regions of triploid P. esculentus distribution may vary slightly. Previously, during studies of erythrocyte sizes in semi-adult and sexually mature green frogs from the North-Donetsk center of their diversity, it was established that the boundary erythrocyte length separating diploids and triploids is 26–28 μm (Bondareva et al., 2012). Furthermore, it was shown that erythrocyte size in immature frogs can depend on body length (Iskenderova et al., 2012), and erythrocyte length in immature triploids may not reach the boundary size established for larger individuals (Temnikov et al., 2012). Direct methods of ploidy determination included karyoanalysis and the study of nucleolar organizers in interphase nuclei (see below).

Materials and Methods To test the proposed hypotheses, we studied a sample of 40 metamorphs (juveniles that recently completed metamorphosis) of green frogs, caught in the Seversky Donets riverbed near the village of Gaidary, Zmiiv district, Kharkiv region, in late July 2013. The sample consisted of individuals with body lengths ranging from 18.5 to 33.3 mm. A blood smear was taken from the caught metamorphs. 8 individuals were not studied due to technical reasons. For the remaining frogs, intestinal fragments were taken for karyological studies, but for 2 individuals, the material turned out to be unsuitable for analysis, so they were also excluded.

Obtaining and studying blood smears was carried out according to a published methodology (Bondareva et al., 2012).

For karyoanalysis, the studied metamorphs were injected with a 0.04% colchicine solution intraperitoneally (0.04–0.08 ml per individual depending on weight). After 24 hours, the animals were anesthetized with ethyl chloride. During dissection, an intestinal fragment was removed. The obtained material was incubated for 20 minutes in a hypotonic solution (0.07 M KCl), and then transferred to Carnoy's fixative (3 parts methanol and 1 part glacial acetic acid). Preparations were made by the scraping method on a heating table at 60 °C. To do this, the tissue fragment was immersed in a 70% acetic acid solution, causing the tissue to become looser, and the studied fragment disintegrated into individual cells, forming a suspension. Then, drops up to 1 cm in diameter were applied to heated glass slides using a Pasteur pipette. The applied drops were immediately removed with a pipette, leaving a small number of individual cells on the slide. The preparations were dried and kept in an incubator for 3 weeks at 37 °C.

Ploidy was determined by counting the number of chromosomes (26 for diploids and 39 for triploids) in at least seven metaphase plates for each individual. On the prepared slides, Ag-staining (silver staining) was applied – a method that allows the detection and localization of the nucleolar organizer region (NOR, sites of 18S + 28S rDNA) in the chromosome (Birshtein, 1984) (Fig. 4). In somatic cell nuclei in interphase, nucleoli are detected in diploid individuals due to Ag-staining, appearing as two clearly stained regions (Fig. 3) (Schmid, 1982). In triploid individuals, three such regions are detected, making this method suitable for determining ploidy. For individuals for whom no metaphase plates suitable for analysis were found on the karyological preparations, ploidy was determined by counting the number of nucleolar organizers in at least 20 nuclei.

We used the silver staining method (Birshtein, 1984) with modifications. For staining, a 30% aqueous solution of AgNO₃ was prepared, which was filtered through a Simplepure NY syringe filter (0.22 μm pores) before use. Then, 200 mg of gelatin was dissolved in 10 ml of warm water. After dissolution, 100 μl of formic acid was added to the gelatin. 75 μl of gelatin solution was applied to the slides, followed by 150 μl of AgNO₃ solution. The slides were covered with coverslips and placed in a humid, light-proof chamber, which was left in an incubator at 60 °C for 3 minutes, after which they were removed, washed under running water, and dried. The slides were then stained with a 2% Giemsa stain solution. After drying, the slides were microscopically examined, and the number of nucleolar organizers in interphase nuclei was counted.

Results and Discussion Based on the average erythrocyte length of each metamorph, it was assumed that all studied individuals were diploid. The average erythrocyte sizes of metamorphs ranged from 15.38 to 22.56 μm, which is significantly lower than the established boundary between the sizes of di- and triploid erythrocytes of 26–28 μm (Bondareva et al., 2012). For comparison, the average erythrocyte sizes of adult diploid individuals normally range from 21–26 μm. It is worth noting that one immature triploid, previously studied by us in a sample of 17 individuals collected in the same area, had an average erythrocyte length of 23.5 μm at a body length of 48 mm, which exceeds the average size of erythrocytes of all frogs considered in this work, but is significantly less than the boundary established for sexually mature individuals (Temnikov et al., 2012).

In the studied sample, the relationship between frog body size and the average length of their erythrocytes (Fig. 1) was not significant (p = 0.36).

In blood smears of metamorphs, both small oval erythrocytes and larger round cells were observed. This pattern may be due to the fact that the individuals considered had just completed metamorphosis and possibly still had blood cells characteristic of larvae (Hollyfield, 1966) (Fig. 2).

Therefore, determining the ploidy of immature individuals by average erythrocyte length proves unreliable. The method requires further research and determination of the boundary between erythrocyte sizes of different ploidies in individuals of the same size class or developmental stage. At the same time, Ag-staining of karyological preparations of somatic tissues allows for ploidy determination even when metaphase plates are scarce or absent.

According to the results of karyological analysis, all metamorphs studied in this work also turned out to be diploid. Specifically, metaphase plates containing 26 chromosomes, corresponding to a diploid set, were found in preparations from 13 individuals. For the remaining 17 metamorphs, the conclusion about ploidy was made based on the presence of two clearly stained nucleolar organizers in interphase nuclei (Fig. 3). Fig. 1. The plot of the frogs’ average red blood cells length dependence on their body length Fig. 2. The red blood cells of different shapes and sizes in the blood smear of one individual When discussing the reasons for the absence of triploids in the studied sample, it should be noted that, unlike the works cited above (Borkin et al., 2004; Mezhzherin et al., 2010; Kozak et al., 2012), this study's sample consisted not only of hybrids but also, likely, of juveniles of P. ridibundus. Determining the species affiliation of metamorphs by morphological features is extremely unreliable due to the insufficient distinctness of their diagnostic characteristics. Among juveniles of the entire green frog hybrid complex, triploids should occur less frequently than in a sample of sexually mature hybrids.

In the area where metamorphs were collected, the ratio of P. ridibundus to P. esculentus individuals was close to 1:1 (D. A. Shabanov, personal communication; Moskalev et al., 2012). Combining the samples studied in the 2012 paper (Temnikov et al., 2012) and in this study, we can establish that in the combined group of 47 juveniles, one was triploid, and 46 were diploid. Fig. 3. Small intestine epithelial cells with silver nitrate stained nucleoli Fig. 3. Small intestine epithelial cells with silver nitrate stained nucleoli Fig. 4. Metaphase plate (26 chromosomes) with silver nitrate stained NORs Fig. 4. Metaphase plate (26 chromosomes) with silver nitrate stained NORs

The obtained result allows us to reject the hypothesis tested in this work at this stage of the study. To reconcile the low proportion of triploids registered in our study with the results of publications indicating a significantly higher proportion of triploids among P. esculentus, it is necessary to assume the action of some factors not accounted for by us. The population of metamorphs we studied either differs significantly in the proportion of triploids it contains, or differs significantly in the ratio of P. ridibundus to P. esculentus from previously studied samples of older individuals.

Two hypotheses can be put forward to explain the obtained result. 1. It is possible that among metamorphs and juveniles, the proportion of triploids may not be greater, but, on the contrary, smaller than in older age classes. It is known that when crossing hybrids that clonally transmit gametes of the same parental species, individuals of this parental species are separated. This phenomenon has been called hybridolysis. However, diploid individuals arising from hybridolysis are found to be poorly viable and die before reaching sexual maturity (Plötner, 2005). If this process occurs frequently enough in the studied population system, it can lead to an increase in the proportion of triploids in older age classes due to the selective death of a certain part of diploid individuals (representatives of parental species separated as a result of hybrid crossing). Since a significant proportion of gametes produced by P. esculentus in the studied population system belong to P. ridibundus (Borkin et al., 2005), a substantial proportion of hybridolytic individuals belonging to this species may die after metamorphosis. 2. The obtained result may be a consequence of the uneven distribution of juveniles belonging to the parental species or triploid hybrids along the riverbed. It was previously shown that the distribution of sexually mature frogs throughout the riverbed is not random and does not depend on their genotype. Individuals of different forms and genotypes concentrate in different microhabitats and exhibit different behaviors (Korshunov, 2010; Moskalev et al., 2012).

The proposed hypotheses can be further tested on a broader material using the methods applied in this work.

Acknowledgments The authors express their sincere gratitude to Associate Professor D. A. Shabanov, as the author of the idea and scientific supervisor of this work.

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