Supplementary Material for: The role of temperature in shaping the nervous system phenotype in the epaulette shark (Hemiscyllium ocellatum)

Introduction Over the last century, sea surface temperatures have increased by more than 0.5˚C, with predictions suggesting an increase of 1-4˚C by 2100. Oceanic warming poses significant challenges to marine species, particularly those with physiological and developmental processes that are tightly linked to environmental conditions. In cartilaginous fishes, including sharks, the brain grows continually throughout life, supported by the capacity for lifelong neurogenesis. This feature suggests that the nervous system – both peripheral (sensory) and central (brain) – of sharks may be highly plastic and able to adapt dynamically to a changing environment. Methods We investigated the effects of elevated rearing temperature on brain development in the epaulette shark (Hemiscyllium ocellatum), a species known for its tolerance of environmental fluctuations in intertidal habitats. Eggs (n = 12) were sourced from a breeding stock at the New England Aquarium and reared in either ambient (27°C) or elevated (31°C, 4°C above ambient) seawater temperatures until two months post-hatch. Using histological analyses, we compared the relative volume of the nose (olfactory rosette), total brain, and major brain regions between treatment groups. Results Despite this species’ natural exposure to temperature variability, Generalized Linear Models revealed that elevated temperature significantly altered the volume of the olfactory sensory epithelium, olfactory bulbs, and medulla oblongata after accounting for overall brain size. Analyses of proportional brain region volumes also showed that elevated temperature was associated with reduced olfactory bulb size and increased subpallial volume relative to total brain size. These changes may suggest potential changes in cognitive capacity related to olfactory processing as well as sensory and / or motor functions at elevated temperatures. Conclusions While short-term studies, such as this one, cannot capture long-term adaptive potential, understanding the impacts of elevated temperature on brain phenotypes provides critical insights into how elasmobranchs may cope with changing ocean conditions. Such knowledge will be vital for predicting the resilience of these ecologically important species to future environmental stressors.