the area and timing of the Deepwater Horizon oil spill highlight the need to study oil and hypoxia exposure in early life stage fishes. Though critical to health, little research has targeted the effect of oil and hypoxia exposure on developing immune systems. to this end, we exposed sheepshead minnows (Cyprinodon variegatus) at three early life stages: embryonic; post-hatch; and post-larval, to a high energy water accommodated fraction (HEWAF) of oil, hypoxia, or both for 48 hours. We performed RnAseq to understand how exposures alter expression of immune transcripts and pathways. Under control conditions, the embryonic to post-hatch comparison (first transition) had a greater number of significantly regulated immune pathways than the second transition (post-hatch to post-larval). The addition of oil had little effect in the first transition, however, hypoxia elicited changes in cellular and humoral immune responses. In the second transition, oil exposure significantly altered many immune pathways (43), and while hypoxia altered few pathways, it did induce a unique signature of generally suppressing immune pathways. these data suggest that timing of exposure to oil and/or hypoxia matters, and underscores the need to further investigate the impacts of multiple stressors on immune system development in early life stage fishes. The functionality of the immune system is key to organismal health and survival. Alteration of the immune system can have serious consequences, including immunosuppression or autoimmunity. The immune system is so critical to life that it starts developing very early on: for example, the innate immune system in zebrafish (Danio rerio) is detectable at day 1 of embryogenesis (via the appearance of macrophages) 1,2 and embryonic zebrafish exposed to Salmonella typhimurium respond with induction of two matrix metalloproteinase genes (mmp9 and mmp13) 3. In addition, the zebrafish immune system is both morphologically and functionally mature 4-6 weeks after fertilization 4. However, since fish are a highly diverse taxa, this period of development can widely vary depending upon species and environmental conditions. The area of oiling from the 2010 Deepwater Horizon oil spill impacted sites where embryos and other developing fishes reside 5. Given the fact that the immune system develops during early life stages, it is important to examine the immunological consequences of oil exposure on developing fish. In addition to contending with oil exposure, the Deepwater Horizon oil spill took place in areas that are prone to experiencing hypoxic conditions 6,7. Literature has clearly established that exposure to polycyclic aromatic hydrocarbons (PAHs) (for example, via oil spills) results in reduced immune function in multiple fish species 8 , but there is also evidence of immunological effects of hypoxia exposure in fishes. For example, fish experiencing hypoxic conditions can have decreased complement activity 9 , reduced production of reactive oxygen species 10 , depressed respiratory burst activity
