Discussion

We investigated the role of captive housing, benzyl-butyl-phthalate (BBP) exposure, and chemical vehicle choice on F. heteroclitus gut flora diversity. Analysis of mucosal and luminal gut flora produced a Rarefaction curve (Figure 1A) indicating a reasonable number of samples were assessed: it is unlikely that additional sampling would yield more species. Faith’s Phylogenetic Diversity (Figure 1B), an alpha-diversity metric where higher values indicate greater diversity, demonstrates changes in gut flora diversity associated with captivity and chemical exposure. Unweighted PCoA (beta diversity metric more sensitive to rare species), identifies significant differences among treatment groups (Figure 2). While ethanol exposure, specifically, appears to support the Anna Karenina Principle of how hosts in the same environment respond uniquely to stressors6, core microbes (taxa found among all treatment groups), also suggest there are essential microbial taxa that persist regardless of stress (Table 1). These taxa may be linked to gut stability and/or essential gut flora associated physiological functions.

Chemical vehicle choice appears to influence BBP actions on gut flora (Table 2), particularly Actinobacteriota, Campliobacteriota, Fusobacteriota, and the anaerobic Desulfobacteriota. Unlike BBPE, BBPA significantly reduced the abundance of core family (Figure 3) Vibrionaceae and Lachnospiraceae while enhancing Mycoplasmataceae. It also selected for Mycoplasma mobile, a pathogenic species that binds to fish gills and causes necrosis. Acetone selected for Clostridium colinum, a pathogen responsible for causing ulcerative enteritis, which aligns with symptomology1 observed among our fish. Although a reduction in pathogenic Firmicutes among control fish, coupled with increased selection by BBP, suggests BBP is a prime selective force for Firmicutes (Table 2), BBPE did exhibit a greater change in distribution than BBPA further supporting chemical vehicle modification of BBP toxicity.

Metagenomic functional profiling of metabolic enzymes (Figure 4, Table 3), including several stress-related enzymes (SREs), indicate 76% greater enzymatic capabilities among field fish isolates as compared to captive controls. Control fish isolates, however, had greater metabolic capabilities when compared to ethanol or acetone (65% and 82%, respectively) indicating that chemical vehicles alone greatly alter gut microbiota functionality. BBP appears to further alter metabolic enzyme capabilities, specifically increasing the percent of SREs favoring the BBP groups over their respective controls (Table 3).

Fish guts were randomly pooled (N=6) to produce the replicates (n=2) for each treatment group with the expectation that this would reduce outliers and give a clearer picture of population rather than individual F. heteroclitus gut flora diversity. While this was an effective strategy, ethanol replicates were outliers in multiple metrics. The Anna Karenina Principle may explain why the ethanol replicates differ, but further studies are needed to determine if the observed differences are reproducible.

Overall, our results identified chemically-induced changes in F. heteroclitus gut flora taxonomic distribution and functionality which differed from those induced by captivity alone. Thus, our findings underscore the profound impact of plastic-associated plasticizers on the gut flora of F. heteroclitus by highlighting significant shifts in microbial diversity, functionality, and prevalence of pathogenic taxa—effects that may cascade to broader ecological and physiological consequences in estuarine environments. Ultimately, this approach provides critical insight into how anthropogenic pollutants impact gut microbiota and animal health.

Citations

1. Bano, L., Ilenia Drigo, Cosetta Bacchin, Marcon, B., Cocchi, M., Bonci, M., Vascellari, M., & Fabrizio Agnoletti. (2009). Application of a PCR method for the diagnosis of ulcerative enteritis: preliminary results. Italian Journal of Animal Science, 8(4), 757–760.

2. Ma, L., Hahn, M.E., Karchner, S.I., Nacci, D., Clark, B.W., Apprill, A. (2025). Environmental and population influences on mummichog (Fundulus heteroclitus) gut microbiomes. Microbiology Spectrium. 13(3).

3. Guamer F. (2007). Papel de la flora intestinal en la salud y en la enfermedad [Role of intestinal flora in health and disease]. Nutricion hospitalaria, 22 Suppl 2, 14–19.Jun Adan-Kubo, Yoshii, S., Kono, H., & Miyata, M. (2012). Molecular Structure of Isolated MvspI, a Variable Surface Protein of the Fish Pathogen Mycoplasma mobile. Journal of Bacteriology, 194(12), 3050–3057.

4. Kaplan, L. A. E., Nabel, M., Van Cleef-Toedt, K., Proffitt, A. R. & Pylypiw Jr., H. M. (2013). Impact of benzyl butyl phthalate on shoaling behavior in Fundulus heteroclitus (mummichog) populations. Marine Environmental Research 86, 70-75.

5. Zaneveld, J., McMinds, R. & Vega Thurber, R. Stress and stability: applying the Anna Karenina principle to animal microbiomes. Nat Microbiol 2, 17121 (2017).

Professional Application 

"This project has been instrumental in preparing me for a career in medicine by strengthening my skills in organization, critical thinking and scientific communication. Working with in this lab, I learned to manage feeding and dissection schedules while helping coordinating timelines to ensure samples were sent out at the correct stages. Once we received the results, I, along with other members of the group, engaged in in-depth data analysis to interpret the impact of chemical exposure on gut flora—examining distribution, pathogenicity and functionality. The process was tedious and time-intensive, but incredibly rewarding, as it deepened my understanding of how environmental factors can influence biological systems. Presenting our findings at scientific symposiums, both this year and last, boosted my confidence and refined my ability to clearly communicate complex ideas. This project reinforced the value of patience, precision and persistence, all of which are critical in medical research and clinical practice." - Elisa Torres-Yeckley '25

For Further Discussion

This serves as an overview of the project and does not include the complete work. To further discuss this project, please email Elisa Torres-Yeckley.

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