Early-Life Microbial Responses to Environmentally Relevant 17α-Ethinylestradiol Exposure in Danio rerio and Kryptolebias marmoratus

Steroid hormones released into aquatic environments are increasingly recognized as contaminants of emerging concern due to their biological activity at very low concentrations and their potential to interfere with endocrine and developmental processes in aquatic organisms. While the effects of estrogenic compounds on fish physiology and development have been extensively investigated, considerably less is known about their influence on embryo-associated microbial communities during early ontogeny. This study investigates the response of microbial communities associated with embryos of two teleost species, Danio rerio and Kryptolebias marmoratus, to environmentally relevant exposure to 17α-ethinylestradiol (EE2).

Embryos were exposed to 1 ng/L EE2 during species-specific pre-hatching developmental periods. Exposure lasted 4 days in D. rerio and 14 days in K. marmoratus and was terminated before hatching in both species. Baseline samples collected before exposure (day 0) were included to characterize microbial community composition at the beginning of the experimental period and to distinguish developmental changes from those associated with EE2 exposure. For each species, independent control and EE2-exposed groups consisted of eight biological replicates. Owing to differences in embryo size and biomass, individual K. marmoratus embryos were analysed separately, whereas D. rerio samples consisted of pools of 4–5 embryos per biological replicate. DNA was extracted following exposure and microbial communities were characterized by long-read sequencing on an Oxford Nanopore Technologies PromethION P2 Solo platform using the Native Barcoding Kit V14. Alpha-diversity indices and Bray–Curtis-based beta-diversity analyses, including PCoA, NMDS, and pairwise PERMANOVA, were used to compare microbial community profiles.

Preliminary analyses revealed significant differences among experimental groups in observed richness (p = 0.0028), Chao1 richness (p = 0.0029), Shannon diversity (p = 0.0373), and Simpson diversity (p = 0.0415). At the exposure endpoint, D. rerio control and EE2-exposed communities were significantly separated in Bray–Curtis space (pairwise PERMANOVA: F = 18.44, p = 0.001). In contrast, no significant separation was observed between the corresponding K. marmoratus control and EE2-exposed groups (F = 0.53, p = 0.882). These preliminary findings suggest contrasting microbiome responses to low-dose EE2 exposure between the two fish species during embryonic development.