Host–Microbiome Interactions as Modulators of Embryogenesis and Developmental Toxicity in Teleost Fish: Implications for Evolved Chemical Resistance

Abstract

Animals have evolved in environments abundant with microorganisms, resulting in deeply integrated host-microbe interactions that profoundly shape development, physiology, and survival. The microbiome is now recognized as essential for maintaining normal vertebrate physiology, including immune function, energy metabolism, nutrient processing, behavior, xenobiotic metabolism, and overall physiological homeostasis. Yet, this understanding is largely derived from post-embryonic, free-living life stages, when organisms are in direct contact with microbes. Because oviparous embryos develop within a protective chorion and viviparous organisms are shielded by the placental environment, embryonic development has long been assumed to be insulated from microbial influence until these barriers are breached. While emerging evidence challenges this paradigm, suggesting that microbial cues may exert lasting effects during early embryonic stages, the specific developmental processes affected and the mechanisms involved remain poorly characterized.This gap is particularly consequential given that embryonic development represents one of the most sensitive windows of vertebrate development, during which environmental perturbations can have lasting impacts on developmental trajectories. Accordingly, microbial cues during this window may influence not only development itself, but how organisms respond to environmental stressors, such as chemical pollutants. Consistent with this idea, emerging research highlights a key role for host-associated microbiomes in facilitating host responses to chemical stressors and enhancing resilience to changing ecological conditions. Yet, whether microbial-driven modulation of early developmental trajectories contribute to long-term environmental resilience or adaptation, particularly in oviparous species that develop surrounded by diverse microbial communities, remains unclear.Therefore, this dissertation investigates the mechanisms by which microbial communities influence early vertebrate development and shape organismal responses to chemical stressors, providing insight into the broader role of the microbiome in host resilience and adaptation. Specifically, this work defines how environmental microbial cues regulate embryonic development, elucidates the sub-cellular metabolic pathways through which microbes influence developmental resilience, and evaluates how host-microbe interactions contribute to evolved resistance in polluted environments. This research was guided by two central hypotheses: 1) microbial cues from the external environment shape developmental and xenobiotic response pathways during embryogenesis and 2) microbiota-mediated modulation during early life contributes to adapted resilience to chemical exposure across generations. Developing a mechanistic understanding of these interactions is essential for predicting and evaluating organismal resilience to rapid environmental change. In Chapter 2, I demonstrate for the first time, that environmental microbial communities signal to developing embryos across the chorion and alter host developmental trajectories. Using zebrafish (Danio rerio) embryos reared germ-free or conventionalized with a microbial community at 6 or 24 hours post-fertilization, I assessed transcriptomic, proteomic, and metabolomic responses during embryogenesis. Our results reveal the surrounding microbial environment altered pathways related to energy metabolism, neurodevelopment, and xenobiotic responses, including cytochrome P4501A (cyp1a) activation, in a time-dependent manner. To determine whether these molecular changes influenced chemical stress responses, embryos were exposed to benzo(a)pyrene, a CYP1A activator, and embryonic mitochondrial function and larval behavior were evaluated. This revealed that early microbial colonization was critical for mitochondrial and behavioral resilience to chemical exposure, demonstrating that microbial-driven molecular programming during embryogenesis manifests as functional physiological outcomes. These findings falsify the long-held assumption that embryonic development is insulated from microbial influence and reveal that host-microbe interactions begin earlier than previously recognized. Building on these results, Chapter 3 investigates the mechanisms by which microbial cues regulate energy metabolism during embryogenesis, when metabolic programming is first established. Since energy metabolism is critical for supporting cellular and physiological processes, especially during embryonic development, identifying the role of host-microbe interactions is essential for understanding how embryos sustain energetic demands and how early disruptions may influence later life outcomes. Again, using zebrafish embryos raised germ-free or conventionalized at 6 or 24 hours post-fertilization, with and without benzo(a)pyrene exposure, I quantified mitochondrial efficiency, energy production, glycolysis, β-oxidation, and redox homeostasis. Our results revealed that in the absence of a microbial community, embryos sustained mitochondrial respiration, but with reduced efficiency likely due to impaired proton motive force, alongside decreased glycolytic activity, collectively reducing overall energy production. Under germ-free conditions, embryos also upregulate β-oxidation gene expression yet rely less on β-oxidation-derived electron substrates, indicating disrupted lipid homeostasis and a compensatory increase in mitochondrial respiration to offset reduced efficiency. Although benzo(a)pyrene exposure did not substantially alter microbial effects on embryonic energy production, it impaired redox homeostasis in germ-free embryos. Together, these findings establish microbial cues as essential regulators of energetic efficiency and redox stability during embryogenesis, emphasizing a central role for the microbiome in mitochondrial resilience and metabolic programming. In Chapter 4, I extended these mechanistic insights from earlier chapters to test whether early-life host-microbe interactions shape evolutionarily encoded chemical resilience. Using wild-caught Atlantic killifish (Fundulus heteroclitus) populations from the Elizabeth River, VA, including a PAH-tolerant Republic (REP) population and a PAH-sensitive King’s Creek (KC) population, I examined whether population divergence in gut microbial communities contributes to adaptive phenotypes in contaminated environments. Germ-free killifish embryos from both populations were cross-colonized with REP or KC gut microbiota, exposed to benzo(a)pyrene, and assessed for transcriptomic and physiological responses related to cytochrome P4501A activity, mitochondrial function, and larval behavior. Our results reveal that the REP microbiota produced AhR-activating metabolites that induced CYP1A activity in KC killifish, while REP embryos were selectively resistant to these effects. Furthermore, disrupting native host-microbiome pairings in the REP population destabilized evolved aryl hydrocarbon receptor recalcitrance, heightening xenobiotic sensitivity to benzo(a)pyrene. Under benzo(a)pyrene exposure, the REP microbiota also promoted distinct transcriptional programs, enhanced mitochondrial resilience, and improved behavioral tolerance in REP, but not KC, embryos. Collectively, these results demonstrate that host-microbe interactions can contribute mechanistically to pollution resistance, highlighting microbial divergence as a partial driver of rapid adaptation to anthropogenic chemical stressors. Overall, this work challenges the paradigm of embryonic sterility by demonstrating that host-microbe interactions actively regulate developmental, metabolic, and xenobiotic response pathways during embryogenesis. By directly linking early-life microbial cues to the expression and stabilization of evolutionarily encoded adaptive traits, this research advances understanding of molecular adaptation in microbial environments. Together, these findings address critical knowledge gaps regarding microbial drivers of early vertebrate development, xenobiotic responses, and evolutionary adaptation. By doing so, they provide a mechanistic framework for understanding how microbiota shape the developmental origins of host resilience and vulnerability in microbially-dynamic natural habitats.