Rice Fields as Important Habitats for Three Anuran Species-Significance and Implications for Conservation.

Agriculture can have different effects on wildlife depending on land-use type and extensive/intensive practices. The aim of this study was to establish the significance of rice fields in Central Bulgaria as anuran habitats. We used Audiomoth acoustic loggers to record calling activity at three sites near the city of Plovdiv-one shallow pond and two rice fields-from June to September in 2022 and 2023. We registered the three most widespread species in the region-Bufotes viridis, Hyla orientalis and Pelophylax ridibundus-and created pattern-matching models for them using the free web interface Arbimon, which enabled us to perform presence/absence counts and abundance estimates. At the beginning and the end of the rice vegetation period, water samples were collected and analysed for 62 substances. Substance concentrations were compared between ponds and with LC50 data from the available literature. We registered 19 and 21 substances in 2022 and 2023, respectively, with concentrations within the accepted limits, and although some metals were near risk levels, this was not reflected in the presence counts or the abundance estimates. The results indicated that frog activity was not related to any of the registered substances, but that it was positively correlated with daily rainfall and was higher in the rice fields.

Analyses of Skin Secretions of Vipera ammodytes (Linnaeus, 1758) (Reptilia: Serpentes), with Focus on the Complex Compounds and Their Possible Role in the Chemical Communication.

Snakes rely heavily on chemical cues when foraging, searching for mates, etc. Snakes' sex attractiveness pheromones comprise mainly heavy, semi-volatile compounds such as ketones. Here we investigated the composition of skin secretions of adult Vipera ammodytes (Linnaeus, 1758) individuals. The samples were analyzed by gas chromatography/mass spectrometry and the identification of the compounds was performed using commercial mass spectral libraries and retention times. The relative concentrations of all detected compounds were tested for significant differences between (1) male vs. female live individuals, (2) shed skin vs. live individuals, and (3) pre-reproductive vs. reproductive live individuals. We detected fifty-nine compounds of which six were ketones. Two ketones (2-pentacosanone and 2-heptacosanone) were present in many of the samples and thus may have an important role in the V. ammodytes chemical communication. We did not find significant differences between the relative concentrations of the compounds between male and female individuals (only three compounds are exceptions). Significant differences were found between extracts from shed skins and live individuals and between live pre-reproductive individuals and live reproductive individuals. The results of the study suggest that chemical communication in V. ammodytes involves less compounds in comparison to the known literature data for other species.

Changes of erythrocyte-metric parameters in Pelophylax ridibundus (Amphibia: Anura: Ranidae) inhabiting water bodies with different types of anthropogenic pollution in Southern Bulgaria.

The article presents the basic erythrocyte-metric parameters: cell length (EL) and width (EW), EL/EW, erythrocyte size (ES), nucleus length (NL) and width (NW), NL/NW, nucleus size (NS) and nucleocytoplasmic ratio (NS/ES) in the wild populations of marsh frogs Pelophylax ridibundus from five water bodies in Southern Bulgaria (two rivers and three reservoirs) with different degrees and types of anthropogenic pollution (less disrupted water basins, domestic sewage pollution and heavy metal pollution). The changes in erythrocyte-metric parameters depend on concentrations and types of toxicant and, to a lesser extent, on the type of water basin. We found that when P. ridibundus populations live in conditions of domestic sewage pollution, EL, EW and ES increase in comparison with the control samples, with regard to an elongated elliptical cell shape. Simultaneously, NL, NW and NS did not undergo any significant changes when compared with the control samples. The nuclei had elliptical shape. In the populations from the water basins with heavy metal pollution, EL, EW, ES, NL, NW and NS decreased. The cells and nuclei had a circular shape. NS/ES decreased when compared with the control sample, regardless of the type of toxicants.

Seasonal changes of basic erythrocyte-metric parameters in Pelophylaxridibundus (Amphibia: Ranidae) from anthropogenically polluted biotopes in Southern Bulgaria and their role as bioindicators.

The purpose of this research work is to present data that show the seasonal changes (spring-summer-autumn) of basic erythrocyte-metric parameters (ЕL: Erythrocyte length, ЕW: Erythrocyte width, ЕL/ЕW, ES: Erythrocyte size; NL: Nucleus length, NW: Nucleus width, NL/NW; NS: Nucleus size, NS/ES: Nucleus-cytoplasmic ratio) in Pelophylax ridibundus populations from three biotopes located on two rivers in Southern Bulgaria (less disrupted biotope, with domestic sewage pollution and heavy metal pollution). Differences of high statistical significance were found among the different populations. Within the population living in conditions of domestic sewage pollution, for the entire period of the investigation the erythrocytes and their nuclei had an elliptical shape (a slight elongation of ellipses in autumn) and the biggest sizes (EL, EW, ES, NL and NS were constantly higher than the less disrupted biotope), NS/ES, became significantly smaller in autumn. Throughout the period of investigation, the values of all nine cellular and nuclear parameters were statistically-significantly the lowest in the population from the biotope with heavy metal pollution. The parameters: EL, ЕW, NL, NW and ES became significantly lower, progressively and statistically, during seasonal transitions. Cells and nuclei grew ovular in shape in comparison to the populations from the other two biotopes (this process was most pronounced in autumn) and NS/ES numbers were significantly decreased in summer and autumn.