Histological and physiological analysis of skin discoloration in Scophthalmus maximus induced by low salinity stresss

Turbot (Scophthalmus maximus) is a commercially important aquaculture species in China. As farming expands into coastal mudflats and inland regions, the breeding of varieties with enhanced low-salinity tolerance has become critical. However, the lack of efficient and quantifiable phenotypic markers for stress resistance remains a major bottleneck. Previous studies observed that juvenile S. maximus exposed to extreme low-salinity stress (0) exhibit a consistent and reversible pattern of abnormal coloration: transitioning from the appearance of black stripes (termed the "qingsi" period) to a regionalized mosaic pattern of darkening and blanching (termed the "shikong" period). Notably, coloration reverts to normal upon return to seawater, even in near-moribund individuals. This phenomenon suggests that the chromatic response is intrinsically linked to physiological tolerance and may serve as an intuitive phenotypic marker for low-salinity resilience. To investigate the underlying mechanisms, this study subjected fish to 0 (low salinity) and 30 (control) environments. The low-salinity group was further stratified into "color-changing" and "color-maintaining" subgroups based on their phenotypic response. During a one-week stress-recovery experiment, body color dynamics and melanophore morphology were systematically monitored through histological observation. Concurrently, serum levels of key hormones—including norepinephrine (NE), epinephrine (E), α-melanocyte-stimulating hormone (α-MSH), melanin-concentrating hormone (MCH), acetylcholine (ACh), and thyroid hormones (T₃, T₄, fT₃, fT₄)—were quantified using ELISA analysis. Results showed that control fish maintained normal coloration and melanophore morphology (partially dispersed state) throughout the experiment. Under low-salinity stress, the color-maintaining group showed no significant differences in coloration or melanophore morphology compared to controls. Conversely, the color-changing group exhibited dynamic transitions: from normal coloration to black stripes, followed by a mosaic pattern, and finally reverting towards normal upon seawater restoration. Melanophore dynamics mirrored these changes: during the "qingsi"period, melanophores were fully dispersed in black stripes but partially dispersed in normal areas; in the "shikong"period, melanophores were fully dispersed in darkened regions and completely aggregated in blanched areas. Hormone analysis revealed that NE, E, and α-MSH levels in the color-changing group exhibited a dynamic pattern: rising gradually during the stress period, peaking at the “shikong” period, and subsequently declining to near-baseline levels during the recovery period. specifically, concentrations of these three hormones were significantly elevated in the color-changing group compared to both the control and color-maintaining groups during the “shikong” period (P < 0.05). At the “qingsi” period, NE and α-MSH levels remained significantly higher in the color-changing group (P < 0.05). Levels in the color-maintaining group were generally intermediate between those of the color-changing and control groups. In contrast, other endocrine indicators (MCH, T₃, T₄, fT₃, fT₄, and ACh) showed minimal fluctuations under low-salinity stress, with no significant differences or clear trends observed across groups. These findings suggest that low-salinity stress induces abnormal coloration through melanophore alterations driven by the interplay of NE, E, and α-MSH. This study provides new insights into stress physiology and highlights these color-associated hormones as key potential indicators for cultivating new aquaculture varieties with enhanced low-salinity tolerance.