Hematological Reference Intervals and Diagnostic Biomarkers for High-Temperature Stress in Starry Flounder (Platichthys stellatus)

Species-specific hematological reference intervals and reliable blood biomarkers are essential for physiological health assessment and disease diagnosis in aquaculture, yet such information remains unavailable for cultured starry flounder (Platichthys stellatus). This study aimed to establish hematological and plasma biochemical reference intervals for cultured starry flounder under optimal culture conditions and to identify diagnostic biomarkers associated with high-temperature stress. A total of 124 clinically healthy sub-adult starry flounder (mean total length, 28.9 ± 0.3 cm; mean body weight, 402.2 ± 13.7 g) were investigated under low—(4.1–5.7 °C, n = 30), optimal—(16–19 °C, n = 62), and high-temperature (26–28 °C, n = 32) conditions. Thirteen hematological and plasma biochemical variables, including osmolality, calcium, magnesium, cortisol, heat shock protein 70 (HSP70), glucose, total cholesterol, total protein, immunoglobulin M (IgM), aspartate aminotransferase (AST), alkaline phosphatase (ALP), hemoglobin, and hematocrit, were analyzed. Reference intervals were established for fish maintained under optimal temperature following ASVCP recommendations. Significant differences were observed among temperature groups in most blood variables (p < 0.05). Plasma cortisol exhibited the highest diagnostic performance for discriminating high-temperature fish (AUC = 0.870), followed by osmolality (AUC = 0.812). Principal component analysis demonstrated distinct physiological responses among temperature groups, while Random Forest analysis identified cortisol and osmolality as the most influential variables, with ALP, total protein, and AST also contributing to discrimination between optimal- and high-temperature fish. HSP70 concentrations tended to increase under high-temperature conditions, although the difference among temperature groups was not statistically significant. These findings provide the first hematological reference intervals for cultured starry flounder and identify plasma cortisol as the biomarker with the highest diagnostic performance for detecting high-temperature stress under the conditions examined. The proposed reference intervals and diagnostic biomarkers provide a practical framework for physiological health assessment and early thermal stress monitoring in starry flounder aquaculture.