Azadirachtin-induced oxidative stress in early life stages of the endangered horseshoe crab Tachypleus tridentatus: Implications for coastal conservation.

The horseshoe crab Tachypleus tridentatus, listed as "Endangered" by the IUCN, is a keystone species in Asian coastal ecosystems and is critical for biomedical applications. This species currently faces multiple threats, including an emerging but overlooked hazard: botanical pesticide contamination in nursery habitats. Azadirachtin, a novel biopesticide, can trigger increased accumulation of reactive oxygen species. However, regulation of gene expression under environmental stress and their synergistic relationship with developmental processes in T. tridentatus remain unclear. We investigated the conservation implications of azadirachtin exposure in the most vulnerable life stages critical for population recruitment. Using embryonic (Stages IV-V) and first-instar larvae, we documented dose- and time-dependent mortality with heightened sensitivity in later developmental stages-periods crucial for successful recruitment. Azadirachtin induced oxidative stress through disruption of antioxidant defense systems, with rapid SOD activation (peaking at 12 h), elevated MDA levels indicating cellular damage, and biphasic T-AOC responses. Molecular analysis results revealed stage-specific expression patterns of key stress-responsive genes, including antioxidant enzymes (Mn-SOD, CAT), a Phase II detoxification enzyme (GST), and a neurotoxicity biomarker (AchE), suggesting limited adaptive capacity in early life stages. These findings challenge the assumption that plant-derived pesticides are environmentally benign and reveal a significant threat to horseshoe crab conservation. High sensitivity during embryonic development, when animals are confined to beach sediments, is particularly concerning given the overlap between agricultural runoff zones and spawning habitats. We recommend establishing pesticide-free buffer zones (minimum 500 m) around known spawning sites, implementing azadirachtin monitoring in horseshoe crab protection areas, and revising coastal pesticide regulations to account for endangered species vulnerability. Our results underscore the need to integrate emerging contaminant assessments into conservation strategies for this ecologically irreplaceable species.