Acidified freshwater disrupts skeletal mineralization and bone remodeling gene expression in zebrafish (Danio rerio)...

Freshwater acidification is a growing environmental threat to fish physiology; however, its specific impact on bone mineralization during critical early developmental stages is poorly understood. This study used zebrafish larvae (Danio rerio) to investigate the effects of acidified freshwater (pH 7, 6, and 5) on survival, growth, craniofacial cartilage, and skeletal ossification. Although survival rates were unaffected, exposure to pH 5 substantially reduced body length and impaired bone mineralization in both the vertebrae and skull at 8-d post-fertilization (dpf). The craniofacial cartilage morphology and cartilage-related gene expression remained resilient, indicating that mineralized bone was more vulnerable to pH stress than cartilage. Molecular analysis indicated that exposure to pH 5 shifted the bone remodeling balance by suppressing osteoblast-related gene expression and substantial upregulating osteoclast-related gene expression. In zebrafish larvae, exposure to acidified freshwater stimulated the efflux of calcium and phosphorus. Elevated bone resorption serves as a compensatory response to maintain bodily fluid calcium and phosphorus levels. However, this response compromises skeletal integrity, which may ultimately impair swimming capacity. The upregulation of genes encoding parathyroid hormone, epithelial calcium channel, and sodium-phosphate cotransporter IIa indicate a coordinated homeostatic effort. This response not only counteracts the accelerated whole-body mineral loss but also represents a physiological attempt to balance the impaired bone mineralization observed under pH 5 conditions. These findings provide critical insights into the molecular basis of environmental acidification-impaired bone mineralization in fish larvae.