DNA methylation as a biomarker of salinity and contaminant stress in mangrove crab Ucides occidentalis.

Epigenetic modifications provide underlying mechanisms of organism responses to environmental stressors, yet their role in estuarine species that are exposed to combined natural and anthropogenic pressures remains poorly understood. Here, we quantified global 5-methylcytosine (5-mC) representing epigenetic alteration in the red mangrove crab Ucides occidentalis across 14 sites with varying salinity levels and contamination profiles in the Guayas Estuary (Ecuador). To obtain comprehensive view of crabs' environmental stressors, we measured concentrations of 10 metals and three common pesticides in both sediments and crab tissues using multi-compartment analysis. The metal panel (Zn, Cu, Fe, Cr, As, Cd, Ni, Al, Pb, Hg) and pesticides (diuron, azoxystrobin, cadusafos) were selected to reflect the documented multi-source contamination profile of the estuary, urban-industrial metal inputs and agricultural pesticide runoff, and their known or suspected relevance to epigenetic stress responses. Global DNA methylation strongly increased with salinity, with crabs from the high-salinity Salado sub-estuary showing up to 10-fold higher methylation levels than those from the low-salinity Churute sub-estuary. Among contaminants, cadmium and arsenic were consistently and positively associated with methylation across environmental compartments, whereas other metals and pesticides showed compartment-specific relationships. Random forest models identified salinity and tissue metals (Cu, As, Cd) as primary methylation predictors, while their sediment concentrations were of minor importance. These findings demonstrate that global 5-mC reflected the combined effects of natural salinity level and anthropogenic-driven bioaccumulated contaminants. The strong associations between DNA methylation and environmental conditions imply its potential as a biomarker in multi-stressor assessment.