The loggerhead sea turtle (Caretta caretta) is the most widespread sea turtle species in the Mediterranean Sea and the only one regularly nesting along the Italian coasts. Understanding factors affecting hatching success is essential for conservation, particularly in nesting areas
increasingly exposed to environmental and human pressures. While abiotic drivers such as temperature and humidity have been extensively studied, the role of microbial communities remains poorly understood. Fungal colonization, in particular, has been recognized as a major cause of egg infection and embryonic mortality, yet its diversity and temporal dynamics during incubation are still largely unexplored.
This study investigated fungal and bacterial communities associated with three loggerhead nests monitored along the Ionian coast of Reggio Calabria, one of Italy’s most important nesting areas, during the 2025 nesting season. Environmental samples, including nest sand,
eggshell fragments, and egg surface swabs, were collected after egg deposition and during nest excavation after hatching. Environmental DNA (eDNA) was analysed using high-throughput metabarcoding targeting the fungal ITS1 region and the bacterial 16S rRNA gene. Diversity analyses, community comparisons, and functional predictions were performed to explore microbial dynamics and their relationship with hatching success. Hatching success varied markedly among nests: two showed high success rates (84.1% and 85.9%), whereas one experienced severe failure (13.3%) with uniform pre-embryonic arrest. Temperature (~31 °C) and moisture (1–3%) remained within suitable ranges, suggesting that abiotic conditions alone did not explain the failure. Fungal metabarcoding revealed highly diverse communities in nest sand, likely acting as a microbial reservoir, while eggshells hosted less diverse assemblages dominated by opportunistic fungi. Fusarium solani was the most recurrent and abundant species in unhatched eggs, consistent with its known pathogenic role in sea turtle nests. Other opportunistic taxa, including Aspergillus alabamensis and Purpureocillium lilacinum, were also frequently detected. Results suggest that early fungal colonization, particularly by F. solani, may impair embryonic development, while nest-specific environmental conditions influence microbial succession and community structure. Integrating molecular and environmental approaches
can support more effective conservation and nest management strategies.