Multi-biomarker assessment of Apis mellifera as a sentinel of environmental stress: A two-year field study across diverse land-use gradients in Tuscany (Italy)

Honey bees (Apis mellifera) are essential pollinators currently facing global declines driven by the interaction of multiple stressors, including habitat fragmentation, climate change, diseases, invasive species acting as predators, and exposure to anthropogenic contaminants. While laboratory studies have extensively explored these factors, field-based assessments of cumulative sublethal effects remain limited. Biomarkers represent sensitive methodologies for identifying early-warning signals of physiological impairment before population-level declines occur. In this study, we evaluated the ecotoxicological health status of honey bee adults across ten locations in Tuscany (Italy) with varying degrees of anthropogenic impact, including high-naturality sites (wood and wildflower field), agricultural areas (orchard, vineyards, olive grove, berries field, wheat crop and clover field), and urban sites. Sampling was conducted over two consecutive years (2020–2021) to account for inter-annual climatic variations. A multi-biomarker approach was applied to assess neurotoxicity (acethylcolinesterase and carboxylesterase activities), detoxification and metabolism (glutathione S-transferase and alkaline phosphatase), immune response (lysozyme activity and haemocyte counts), and genotoxicity (Nuclear Abnormalities assay). The results demonstrated that biomarker responses are significantly modulated by land use (p < 0.05). Clearer signs of physiological disturbance consistently emerged in anthropized sites, whereas the areas with a high degree of naturality exhibited comparatively lower biomarker values. These findings highlight that climatic conditions influenced the sublethal responses observed in organisms. However, multiple stressors, besides climate-related ones, should be taken into account for a more complete evaluation of honey bees’ ecotoxicological status in a field scenario.