Student Works after the Second-Year Field Practice - 2017

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Gorensky G. G., Andreev D. A., Onyshchenko K. S., Pustovalova E. S. Study of the Population System Structure of Green Frogs (Pelophylax esculentus complex) of the Iskov Pond in the Zmiiv District of Kharkiv Region // "Biology: From Molecule to Biosphere." Materials of the XII International Conference of Young Scientists (November 26 – December 1, 2017, Kharkiv, Ukraine). – Kharkiv: FOP Shapovalova T. M., 2017. – pp. 132-133. Lukan R. M., Pustovalova E. S., Biryuk O. V. Method for Intravital Determination of Ploidy in Green Frog Tadpoles // "Biology: From Molecule to Biosphere." Materials of the XII International Conference of Young Scientists (November 26 – December 1, 2017, Kharkiv, Ukraine). – Kharkiv: FOP Shapovalova T. M., 2017. – pp. 141-142. Fomicheva A. Yu., Loboyko D. I. Study of the Composition of the Hemi-Clonal Population System of Green Frogs of Koryakov Yar (Zmiiv District, Kharkiv Region) // "Biology: From Molecule to Biosphere." Materials of the XII International Conference of Young Scientists (November 26 – December 1, 2017, Kharkiv, Ukraine). – Kharkiv: FOP Shapovalova T. M., 2017. – pp. 149-150. Fomenko K. S., Trokhymchuk R. R., Lukan R. M., Makaryan R. N., Pustovalova E. S. The study of hemiclonal population system Pelophylax esculentus complex from the Lower Dobritskiy pond (the territory of the National Park "Gomolshanski lisy") // "Biology: From Molecule to Biosphere." Materials of the XII International Conference of Young Scientists (November 26 – December 1, 2017, Kharkiv, Ukraine). – Kharkiv: FOP Shapovalova T. M., 2017. – pp. 152-153. Stepanenko K. R. Hemiclonal population systems features’ influence on spermatozoid size in Pelophylax esculentus complex // "Biology: From Molecule to Biosphere." Materials of the XII International Conference of Young Scientists (November 26 – December 1, 2017, Kharkiv, Ukraine). – Kharkiv: FOP Shapovalova T. M., 2017. – p. 153. Study of the Population System Structure of Green Frogs (Pelophylax esculentus complex) of the Iskov Pond in the Zmiiv District of Kharkiv Region Gorensky G. G., Andreev D. A., Onyshchenko K. S., Pustovalova E. S. V. N. Karazin Kharkiv National University, Faculty of Biology, Svobody Sq., 4, Kharkiv, Ukraine, 61022 e-mail: gorenskiyg@outlook.com The group of water frogs, Pelophylax esculentus complex, consisting of two parental species: P. lessonae (Camerano, 1882) and P. ridibundus (Pallas, 1771), as well as their natural hybrids P. esculentus (Linnaeus, 1758), forms hemiclonal population systems, HPS (Shabanov et al., 2009). We studied the HPS of green frogs of the Iskov pond (outskirts of the village of Haidary, Zmiiv District, Kharkiv Region). Observations of this HPS have been conducted since 1995, when a large population of P. esculentus, consisting of diploid individuals with effective reproduction, was discovered in the pond (Lada, 1996). The composition of the HPS has changed over the course of the study. In 2000, the HPS entered a critical state due to the pond drying up; in 2005, during spawning, only males were observed in the pond; in 2011, successful reproduction of this HPS was recorded. In this regard, the study of its structure is of significant interest. The aim of our research is to study the sex and species composition of the HPS of the Iskov pond for further monitoring of its status. Species and sex were determined by a complex of external characteristics (Shabanov, 2015). Ploidy of individuals was determined by measuring the average erythrocyte length from microphotographs of blood smears (Bondareva et al., 2012) using the PDF-XChangeViewer program. Statistical processing of the obtained data was carried out using the Statistica 8.0 program. A total of 137 individuals were studied. The proportion of males in the sample was 95.6%; females – 2.9%; immature individuals – 1.5%. According to literature data, typical erythrocyte sizes for diploids range from 21 to 26 µm, while triploids are characterized by erythrocytes larger than 26 µm (Bondareva et al., 2012). In the sample we studied, the proportion of diploid individuals of P. esculentus was 94.2% (129 individuals), triploid – 1.5% (2 individuals), and 4.4% were attributed to the species P. ridibundus. The average erythrocyte size in diploids was 23.3 µm, and in triploids – 29.6 µm. Compared to the data from 2015 (Cherepashuk et al., 2015), no significant changes in the proportion of triploids were recorded (p = 0.35). No dependence between sex and erythrocyte size was found. Thus, it is confirmed that a population system of the R-E-Ep type, close to the pure E type, with a significant predominance of diploids, exists in the Iskov pond. Summary. Group of water frogs in hemiclonal population system from Iskov pond was studied. We tried to find out the difference in size of erythrocyte between diploids and triploids of Pelophylax esculentus complex. The method we used to spot the ploidy was measuring the average length of red blood cells. The total number of individuals was 87; the percentage of triploids was 1.5 %. The average size of diploid erythrocytes was 23.3 μm and triploid red blood cells were estimated at 29,6 μm. Thus, we confirmed, that population system of Iskov pound belongs to R-E-Ep-type. The authors thank Professor D. A. Shabanov, Doctor of Biological Sciences, Head of the Department of Zoology and Animal Ecology at KNU, for the idea and scientific guidance, as well as all participants of the catch. Method for Intravital Determination of Ploidy in Green Frog Tadpoles Lukan R. M., Pustovalova E. S., Biryuk O. V. V. N. Karazin Kharkiv National University, Faculty of Biology, Department of Zoology and Animal Ecology, Svobody Sq., 4, Kharkiv, Ukraine, 61022 e-mail: minihobbit29@gmail.com Hemi-clonal inheritance is an unusual, rare mode of reproduction among vertebrates, associated with an interesting area of research. This mode of inheritance accelerates evolution (Shabanov, Litvynchuk, 2010), and its study may lead to new methods in animal breeding, biotechnology, and genetic medicine in the future. Complex hemiclonal population systems (HPS) of the hybridogenic complex of green frogs (Pelophylax esculentus complex), such as those inhabiting water bodies of the Kharkiv region, are objects of monitoring their composition (Meleshko et al., 2014). Within these HPS, diploid and triploid interspecies hybrids P. esculentus (Linnaeus, 1758) of both sexes, as well as individuals of the parental species P. ridibundus (Pallas, 1771), have been registered (Shabanov, 2015). To date, it is not precisely known how the composition of HPS is regulated. It is hypothesized that selective mortality of certain forms plays an important role in regulating their composition (Biryuk et al., 2016). The development of a method for intravital determination of tadpole ploidy is a relevant task, given that in tadpoles, unlike adult individuals, ploidy cannot be determined from blood smears (Bondareva et al., 2012). Such a method would expand the possibilities for studying hemiclonal inheritance in green frogs, allowing tadpoles to be raised for further study and assessment of selective mortality among tadpoles of different ploidies. The method for intravital determination of tadpole ploidy presented in this work involves the intravital extraction of tissue samples by amputating part of the tail fin after determining the developmental stage (Gösner, 1960) and size of the tadpoles (McDiarmid and Altig, 1999). For this purpose, we collected a sample of 15 tadpoles of the genus Pelophylax from the Lower Dobritsky pond (Zmiiv district, Kharkiv region). Using this material, we tested the possibility of incubating the amputated tissue fragment in a colchicine solution as a means of obtaining enough metaphase plates for karyoanalysis. Preliminary studies showed that an isotonic colchicine solution needs to be prepared (Sherstyuk et al., 2016). We tried several dilution options: in distilled water, hypotonic solution, and Versene solution. Next, the samples were incubated for 20 minutes in a pure hypotonic solution of 0.07 M KCl, and then immersed in Carnoy's fixative. The suspension of cells was dropped and stained with Romanowsky-Giemsa stain according to a previously described method (Veherina et al., 2013). The best preparations were obtained from samples incubated in solutions made from 0.07 M KCl – a large number of normal cells and isolated metaphase plates were observed. This may be due to the low proliferative activity of tail tissues in tadpoles at late developmental stages (tadpoles at stages 26-37 according to Gosner, 1960 were studied). It is recommended to make several slides with dropped cells. Among the 15 tadpoles examined, all turned out to be diploid. Subsequently, their ploidy was confirmed by the number of nucleoli using Ag-staining (Biryuk et al., 2015). Thus, we found that tail fin tissues can be used for intravital determination of green frog tadpole ploidy. For this, it is necessary to incubate them in a 0.2% colchicine solution in 0.07 M KCl for 4-5 hours, followed by immersion in a hypotonic solution (0.07 M KCl) for 20 minutes. After fixation, such tissues are ready for obtaining chromosome preparations. Summary. The study was performed to determine the proportion of triploids among tadpoles of Pelophylax genus and to improve an intravital method of ploidy determination. 15 tadpoles from the population of Pelophylax esculentus complex inhabited the Lower Dobritsky pond (Zmiyiv district of the Kharkiv region, the floodplain of the Gomolsha River at the National Natural Park "Gomilshanski lisy") were studied. No triploids have been found. The authors express their gratitude to Doctor of Biological Sciences, Professor D. A. Shabanov of the Department of Zoology and Animal Ecology for the idea, scientific guidance, and comprehensive support in processing the results. Study of the Composition of the Hemi-Clonal Population System of Green Frogs of Koryakov Yar (Zmiiv District, Kharkiv Region) Fomicheva A. Yu., Loboyko D. I. V. N. Karazin Kharkiv National University, Faculty of Biology, Department of Zoology and Animal Ecology, Svobody Sq., 4, Kharkiv, Ukraine, 61022 e-mail: anastasiafomichova1904@gmail.com The two parental species of green frogs, Pelophylax ridibundus (Pallas, 1771) and Pelophylax lessonae (Camerano, 1882), when crossed, produce a hybrid – Pelophylax esculentus (Linnaeus, 1758). Such hybrids are capable of hemiclonal inheritance: transmission of parental genomes from generation to generation clonally, i.e., without recombination. The joint reproduction of P. esculentus and, as a rule, representatives of the parental species, leads to the formation of hemiclonal population systems (HPS), in which both clonal and recombinant genomes are transmitted. HPS of green frogs inhabiting Koryakov Yar (outskirts of the V. N. Karazin Kharkiv National University Biological Station, village of Haidary, Zmiiv District, Kharkiv Region) have been studied for over 20 years. In 1995-1996, a population system consisting only of diploid P. esculentus was discovered here (Lada, 1998). In the 2000s, there were almost no frogs in Koryakov Yar. In 2015, according to unpublished data (Yu. Artemova, T. Beshentseva, E. Meleshko), 83 individuals were registered. The aim of this work is to study the composition of the HPS of green frogs in Koryakov Yar in 2017 and to compare the obtained data with the results of the 2016 study. Animals were caught by hand at night using a flashlight. Species and sex were determined by a complex of morphological features (Shabanov, 2015). Body length was measured with calipers. Frogs were marked by cutting off the longest toe on the hind right limb, and blood smear preparations were processed using a generally accepted method (Bondareva et al., 2012). Frogs with erythrocytes longer than 28 µm were diagnosed as triploids. We studied 230 individuals, among whom were 3 ♀♀ and 5 ♂♂ P. ridibundus, 10 ♀♀ and 136 ♂♂ P. esculentus (2n), 1 ♀ P. esculentus (3n) and 9 ♂♂ P. esculentus (3n), as well as 66 juvenile individuals. The proportion of triploid individuals was 6% of the total sample. The difference in the composition of the 2017 and 2016 samples was statistically insignificant (p = 0.53). No marks from 2016 were found in the 2017 sample. Thus, Koryakov Yar is inhabited by an R-E-Ep-HPS (i.e., an HPS that includes P. ridibundus, as well as diploid and triploid P. esculentus) with a significant predominance of male diploid P. esculentus and a low proportion of triploids (6%). Compared to 2016 (Stakh V. O. et al., 2016), the HPS of green frogs in Koryakov Yar has not changed significantly. Summary. The water frogs, living in Koryakov ravine (near the village Gaydary in Zmievsky district of the Kharkov region), form hemiclonal population systems. Diploid males of P. esculentus are prevailing. The share of triploids is 6 %. In comparison with the 2016 HPS Pelophylax esculentus complex structure has not changed significantly (p = 0.53). Scientific supervisor: D. A. Shabanov, Doctor of Biological Sciences, Professor of the Department of Zoology and Animal Ecology.

**Study of Hemiclonal Population Systems in the Pelophylax esculentus Complex from Lower Dobritskiy Pond** This research focuses on the hemiclonal population system (HPS) of the *Pelophylax esculentus* complex, which includes the pool frog (*Pelophylax lessonae*), marsh frog (*Pelophylax ridibundus*), and their hybrid, the edible frog (*Pelophylax esculentus*). These species reproduce together, forming HPS. The study specifically examined the Lower Dobritskiy pond within the Gomolshanski lisy National Park, noting it as a thriving HPS with a significant proportion (around 30%) of triploid adult frogs. In 2016, a sample of frogs from this pond consisted of 36 diploids (*P. esculentus* and *P. ridibundus*) and 14 triploid *P. esculentus*. The objective of this study was to determine the ratio of diploid to triploid tadpoles in this HPS. A random sample of 27 tadpoles collected on June 29, 2017, was analyzed for ploidy using karyoanalysis of intestinal cells. All tadpoles in this sample were found to be diploid. This finding aligns with previous research by R. R. Lukan and colleagues, who also found only diploid tadpoles among 15 tadpoles from the same collection using a different ploidy determination method. In total, across both studies of tadpoles from this pond, all 42 individuals examined were diploid. The proportion of triploid tadpoles was significantly lower (p = 0.0003) than the proportion of triploid adult frogs. The researchers hypothesize that the stability of the Lower Dobritskiy pond HPS might be due to the high mortality rate of diploid tadpoles. They illustrate this with an example: if only one out of 50 tadpoles is triploid, but 47 out of 49 diploid tadpoles die, it's possible to find no triploid tadpoles while the adult frog population shows a 2:1 ratio of diploids to triploids. This could potentially be linked to the death of individuals from the parent species that originated from hybrid broods carrying specific clonal genomes. Alternative explanations, such as a drastic change in the HPS composition or spatial separation of diploid and triploid tadpoles, were considered but not supported by the current field data. **Influence of Hemiclonal Population System Features on Spermatozoid Size in the *Pelophylax esculentus* Complex** This research investigates factors affecting spermatozoid size within the *Pelophylax esculentus* complex, which comprises *P. ridibundus*, *P. lessonae*, and their hybrid *P. esculentus*. Hemiclonality, characterized by the inheritance of non-recombinant genomes (R from *P. ridibundus* and L from *P. lessonae*) in *P. esculentus*, and the presence of stable triploid populations can lead to anomalies in spermatogenesis, including meiosis violations and sterility. The study aimed to identify factors influencing spermatozoid size. Sperm samples were collected, and the heads of the spermatozoa were measured to create size distribution profiles. Comparisons were made between *P. ridibundus*, *P. lessonae*, and diploid and triploid *P. esculentus* from various types of hemiclonal systems in the Volyn' and Kharkiv regions of Ukraine. These systems could include parental species (R- or L-), diploid hybrids (E-), and triploid hybrids (Ep-). Males from R-E-, R-E-Ep, and L-E-R hemiclonal population systems were examined. The researchers initially hypothesized that spermatozoid size would be primarily determined by DNA content, expecting additional peaks in the size distribution for aneuploid and diploid sperm. However, these additional peaks were indistinct, preventing confirmation or refutation of diploid sperm presence. Statistical analysis (Kruskal-Wallis test and median test) revealed significant differences (p < 0.05) between some comparison groups, suggesting a complex relationship between spermatozoid size, frog species, ploidy, and the type of hemiclonal system. Notably, the most significant differences in spermatozoid size distribution were observed between *P. esculentus* from different Ukrainian regions, unlike *P. ridibundus* from the same localities, which showed similar distributions. The distribution for *P. ridibundus* and *P. lessonae* from L-E-R HPS was the most uniform, indicating high reproductive stability within this system. The authors conclude that features of the HPS, such as the presence of diploid or triploid hybrids, both parental species, and geographical factors, influence individual reproduction and the stability of gametogenesis, ultimately affecting spermatozoid size.