Eco‐Metabolomic Signatures of Frog Skin Secretions Reveal Environmental Stress Biomarkers Across Pond Disturbance Gradients

Frogs are highly sensitive bioindicators whose skin secretions reflect physiological responses to environmental change. Yet, no study has directly linked amphibian skin metabolomes to quantified habitat parameters across a disturbance gradient. We applied an eco-metabolomics approach to Hylarana erythraea from eight pond sites in northern Peninsular Malaysia, spanning natural to heavily modified habitats. Thirty ecological parameters defined two habitat categories: minimally disturbed and moderately-to-heavily disturbed. Untargeted QTOF LC-MS/MS revealed distinct metabolomic profiles between categories (PERMANOVA, F = 11.78, R² = 0.67, p = 0.001). Frogs from minimally disturbed habitats had higher lysophosphatidylcholines (LPC 18:0, LPC 18:1), consistent with membrane integrity and physiological homeostasis, whereas disturbed sites showed elevated xenobiotic-related compounds (e.g., Triton X-45, Lauramine oxide) and histamine, indicating anthropogenic stress. Generalized linear models identified water clarity, aquatic vegetation, and pond size as the strongest predictors of metabolomic variation (ΔAIC ≤ 2). Our findings provide the first molecular-level evidence linking frog skin metabolites to quantified ecological drivers, demonstrating their value as early-warning biomarkers within amphibian conservation and One Health monitoring frameworks.