Metallothionein gene methylation reflects epigenetic changes in white stork (Ciconia ciconia L., 1758) nestlings

The white stork (Ciconia cico nia), a migratory bird from the Ciconiidae family, thrives in open habitats such as flood plains and wet meadows. In recent years, they have in creasingly turned to foraging at landfills. Given that, white storks are apex predators and environmental pollutants accu mulate in their bodies through the food web. Such exposure to pollutants can result in envi ronmental stress, leading to epigenetic modifications that can influence gene expression. In this study, the percentage of the methylation index of the regulatory region of the metallothionein gene was analysed using methylation-specific real-time PCR to investigate the potential epigenetic effects of environmental pollution on white storks. The results suggest that both hypermethylation and hypomethylation of the MT gene may be associated with exposure to specific metals, depending on the pollutant profile and the nestling's detoxification capacity. Variations in the meth ylation index point to potential epigenetic responses in white stork nest ling populations, which could serve as sensitive indicators of environ mental stress. These findings represent the first successful application of DNA methylation analysis in the white stork nestlings in Croatia. Further detailed studies are needed to elucidate the causes of these methylation differences and to explore their relationship with metallothionein gene expression and broader biological outcomes. These results provide a foundation for future research aimed at understanding the influence of environmental pollutants on wildlife health through epigenetic pathways, and highlight the potential of epigenetic tools for wildlife biomonitoring and conservation strategies.