Influence of Nest Microbiota on Hatching Success of Caretta Caretta on Lampedusa Island

Egg hatching success in sea turtle nests can be influenced by multiple abiotic and biotic factors. Although interest in nest microbiota as a determinant of embryo development and viability is increasing, its role has not yet been fully elucidated. In this study, we profiled the bacterial communities of four Caretta caretta nests on Lampedusa Island: Cala Pisana (P1 and P2) and Spiaggia dei Conigli (C1 and C2), which showed different hatching success rates (P1 = 85.2%, P2 = 1.1%, C1 = 1.1%, C2 = 0.0%). Using 16S rRNA gene (V3-V4) sequencing, we analyzed different sample types, including sand from inside and outside the nest chamber, eggshells, and inner membranes. Alpha diversity was highest in sand and lower in eggshells and inner membranes. β-diversity clearly separated the only successful nest (P1) from the others (P2, C1, and C2) (PERMANOVA p < 0.001). Across all nests, the dominant phyla were Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota; notably, the Firmicutes/Bacteroidota (F/B) ratio was lowest in P1 compared with P2, C1, and C2. Nests with low hatching success were Firmicutes-dominated, enriched in Bacillus and Pseudomonas, and harbored hydrocarbon-degrading genera (Pseudoxanthomonas and Devosia), suggesting environmental influences. Opportunistic pathogens (Ochrobactrum and Simkaniaceae), likely associated with vertical transmission, were detected exclusively in C1 and C2 nests. Overall, our findings highlight the potentially critical role of nest microbiota in reproductive success. Both vertical (maternal) and horizontal (environmental and anthropogenic) transmission appear to shape microbial composition, potentially affecting hatchling viability and offering useful insights for conservation monitoring.