Chronic Thermal Stress During Early Zebrafish (Danio rerio) Development Induces Morphological, Molecular, and Liver Histopathological Changes

Abstract Temperature is a critical abiotic factor mediating the physiological fitness of fish. While the impact of acute high-temperature exposure is well documented in teleost fishes, the effects of chronic thermal stress, especially during early stages, remain poorly understood. This study examined the effects of prolonged exposure to elevated temperatures (34 ºC) on zebrafish ( Danio rerio ) development, survival, molecular responses and liver histology during pre-independent (24–120 h post fertilisation hpf) and independently feeding (240–480 hpf) stages. While survival was not affected by elevated temperature, normal development was significantly impaired in both stages. Compared to control conditions (28 °C), heat exposure (34 ºC) increased the incidence of deformities, including spinal and yolk sac abnormalities during the pre-independent feeding stage, and spinal and growth-related deformities during independent feeding. Heat-induced changes in gene expression were most evident during independent feeding, with the upregulation of heat shock proteins (HSP90AA1, SERPINH1A, SERPINH1B) and downregulation of growth (GH, GHRA, IGF-1) genes. By 480 hpf, pronounced liver changes were also observed in heat-exposed fish, characterised by marked cellular vacuolation and hepatic glycogen accumulation. These results highlight the complex, age-specific responses to chronic thermal stress, reflected in altered heat shock response, development, and hepatocyte morphology. These findings contribute to the assessment of stage-specific responses relevant to biomarker development for prolonged heat exposure in developing finfish.