www.nature.com/scientificreports OPEN Estrogen induces shift in abundances of specifc groups of the coral microbiome Caren L. S. Vilela1, Helena D. M. Villela1, Gustavo A. S. Duarte1, Erika P. Santoro1, Caio T. C. C. Rachid1 & Raquel S. Peixoto1,2* Synthetic estrogens such as ethinylestradiol (EE2) are persistent micropollutants that are not efectively removed from wastewater by conventional treatments. These contaminants are released into waterbodies, where they disrupt endocrine systems of organisms and cause harmful efects such as feminization, infertility, reproduction problems and genital malformations. The consequences of this pollution for key marine ecosystems such as coral reefs and their associated microbiomes are underexplored. We evaluated the efects of EE2 concentrations of 100 ng L−1 and 100 µg L−1 on the coral metaorganism Mussismilia harttii. The results indicated no efects on visible bleaching or Fv/Fm ratios in the corals during a 17-day microcosm experiment. However, next-generation sequencing of 16S rDNA revealed a statistically signifcant efect of high EE2 concentrations on OTU richness, and shifts in specifc microbial groups after treatments with or without EE2. These groups might be bioindicators of early shifts in the metaorganism composition caused by EE2 contamination. Estrogenic hormones are part of a group of endocrine-disrupting compounds (EDCs) that can alter the endo- crine system of animals 1–3. 17α-ethinylestradiol (EE2) is a synthetic hormone used in birth-control pills and in hormone-replacement drugs in the menopausal period, or to treat other hormonal defciencies4. EE2 is derived from the main endogenous human estrogen, estradiol (E2), but is 10 to 50 times more powerful 5,6. Its longer half-life makes EE2 more available for bioaccumulation by other animals 7–10. Tis compound is released into the environment through human excreta, and is persistent 4,11. Studies have shown that EE2 can cause estrogenic efects in very low concentrations (1 to 5 ng L −1), with a predicted no-efect concentration of 0.35 ng L−112. However, EE2 concentrations of 0.001 to 0.042 µg L−113 or higher, of 0.831 μg L −114 have been reported in some wastewater-treatment plants and their receiving water bodies, and may come into direct contact with wildlife2,7,15–17. Te presence of estrogens in water bodies is responsible for female infertility, feminization of male fsh, and sexual dysfunction of many aquatic species 2,3,18–20. Tese compounds also afect the microbiome associated with diferent organisms, such as the insect Megaselia scalaris, which had its development impacted by a hormone diet (including EE2), with a signifcant efect on microbial groups compared with control samples and the appearance of Mycobacterium, Sphingobacterium, Nocardioides, Acinetobacter and other genera 21. Mosquitoes exposed to hormone treatment in environmentally important concentrations showed a signifcant diference in their microbiome compared to untreated samples 22,23. In one study, Microbacterium laevaniformans was the most abundant species in hormone-treated samples, whereas Wol- bachia pipientis was most abundant in the control23. Continuous use of human oral contraceptive pills was found to alter the normal vaginal microbiome and increase yeast colonization 24. Tese examples from humans and insects are used as references due to the lack of information regarding the efects of EE2 on the coral-associated microbiome. Te investigation of the impacts of EE2 on other animal models and their associated microbiomes will add to the current body of information regarding the importance of this pollutant in potentially symbiotic microbial populations. Te efects of estrogens on corals have been examined in a few studies focusing on their metabolism, repro- duction and physiology 25–29. Sex steroids are biologically active in invertebrates, including corals, although their mechanisms of action remain unclear 26,30. It is suggested that estrogens stimulate the process of gamete release and coral spawning, and that their eggs contain estrogenic compounds that will aid in their fnal maturation 27,31. It is also speculated that diferences in estrogen concentration will regulate the reproductive processes of corals32. 1Department of General Microbiology, Paulo de Goes Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil. 2Red Sea Research Center (RSRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuval, Saudi Arabia. *email: [email protected] Scientifc Reports | (2021) 11:2767 | https://doi.org/10.1038/s41598-021-82387-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ Tarrant et al.33 found that corals have the capacity to take up estrogens from the water and accumulate these compounds in their tissue, although the consequences of this bioaccumulation are unknown. Corals are metaorganisms, i.e., a biological unit that includes the host and its associated microbiome, whether stable or transitory34. Coral-associated microbes are diverse, complex, dynamic, and essential for the function- ing and balance of the coral reef ecosystem35–38. Tese microorganisms have several functions that aid in coral ftness36,37,39,40, such as nitrogen fxation41, ammonia metabolism42, removal of nitrogenous waste42, production of antibiotics, physical occupation of coral space, and competition with other microorganisms, in order to protect the host from invasion of opportunistic pathogens 37,43,44. Te coral-associated microbial community is sensitive to environmental changes and can quickly respond and adapt to new environmental conditions, which is important in maintaining the homeostasis of the metaorganism37,42,44. Diferent environmental stressors, such as thermal stress 38,45,46, oil47,48 the presence of pathogens40,49,50 or ocean acidifcation51,52 can cause shifs in the coral microbiome, which may change their diversity, abundance and functionality for long periods of time53. For example, exposure of the coral Acropora muricata to relatively high levels of nickel and copper resulted in coral bleaching. Copper pollution also modi- fed the coral microbiome (eukaryotes and prokaryotes)54. One study showed that the bacterial core associated with corals can respond to a gradient of anthropogenic pollution55. Diferent stressors, such as climate change, pollution and overfshing are correlated with an increase in the richness and diversity of specifc coral-associated microbes, including the commonly found association of higher abundances of members of Vibrionaceae and Altermonadaceae with stressed and diseased corals56–58. Further, coral micro-organisms are able to degrade or neutralize toxic substances that threaten coral health, suggesting that the microbiome can protect coral health and mitigate the efects of pollution47,59. However, no reports about the efects of EDCs on the health of corals and their microbiome are currently available. Here, we evaluated the efects of EE2 on the coral Mussismilia harttii in a microcosm system, and, for the frst time, the impacts of an EDC on a coral-associated microbiome. Results Visual health status and Fv/Fm. No signifcant variations in water-quality parameters (temperature, salin- ity and pH) (Supplementary Table S1) or visual changes (Fig. S1) were observed during the experiment. Tese Scientifc Reports | (2021) 11:2767 | https://doi.org/10.1038/s41598-021-82387-x 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Non-metric multidimensional scaling (NMDS) ordination using Bray–Curtis dissimilarity, based on abundances of OTUs in samples from Mussismilia harttii treatments (n = 4). NMDS ordination of the OTU results demonstrated that, despite a slight trend toward treatment-based clus- tering at the last sampling time, the total coral-associated microbiome was not signifcantly afected by EE2 con- tamination, and was clustered only by time (days 0, 3, 9 and 17, i.e. T0, T3, T9 and T17 respectively) (p < 0.0001, pseudo F = 1.7321), regardless of the treatment (Fig. 2). Taxonomic investigation showed that Proteobacteria was the most abundant phylum in the coral samples, with or without estrogen. Tis group comprised about 60% of the bacterial community in all samples, followed by unclassifed Bacteria, Firmicutes and Bacteroidetes. Additionally, Acidobacteria showed signifcant diferences between times T0 and T17, as did Deinococcus-Termus, which also showed interaction among factors, according to ANOVA followed by a Tukey test (p < 0.05) (Fig. S4). At the class level, Alphaproteobacteria predominated, comprising about 40% of the total microbiome. Betaproteobacteria showed a signifcant reduction in relative abundance compared to the control with 100 μg of EE2 (Two-Way ANOVA, p < 0.05) and Gammaproteobacteria and Acidobacteria groups GP4 and GP10 showed signifcant difer- ences as a function of the incubation time (Two-Way ANOVA, p < 0.05) (Fig. 3A). At the genus level, taxonomic diversity and relative abundance were similar (with few diferences) among all samples. Rhizobiales_unclassifed, Verrucomicrobiaceae_unclassifed, Sphingomonadaceae_unclassifed, Erythrobacter, and Blastopirellula difered signifcantly among the diferent collection times; the last genus showed interaction among the factors (Fig. 3B). Most genera were unclassifed, related to Rhodobacteriaceae, Bacteria, Gammaproteobacteria, Alphaproteobac- teria, Ruegeria, Clostridiales and Pseudobacteriovorax. An Indicator Species