Incomplete versus Sustained Innate-Immune Responses in the Medicinal Leech following Chronic, Environmentally Relevant Per- and Polyfluoroalkyl Substance Exposure

Per- and polyfluoroalkyl substances (PFAS) persist in aquatic systems, yet hazard evaluations still rely largely on shortterm tests. We chronically exposed the medicinal leech Hirudo verbana for two months to four PFAS congeners at 1 nmol L−1 aligned with the EU Drinking Water Directive thresholds (PFAS total = 0.50 μg L−1): a legacy long-chain acid (PFOA), two shortchain ether replacements (HFPO-DA and PFMoBa), and an intermediate methoxy analogue (PFMOPrA). Histology, enzymatic histochemical staining, immunofluorescence, and quantitative polymerase chain reaction tracked the canonical inflammatory sequence, represented by CD11b+ granulocyte influx, CD31+ neovessel formation, and recruitment of CD45+/ACP+ macrophage-like cells, together with transcriptional responses of oxidative stress genes (SOD and GST) and two immune mediators (HvRNASET2 and HmAIF-1), chosen for their known role in leech innate immunity. PFOA and PFMOPrA sustained the full CD11b/CD45/CD31 cascade, maintained high ACP activity, and strongly upregulated HvRNASET2, HmAIF-1, SOD and GST, signaling stability, and tissue-remodeling inflammation. By contrast, HFPO-DA and PFMoBa induced a granulocyte influx but failed to promote macrophage recruitment or neovascularization; HvRNASET2 transcripts rose, whereas HmAIF-1 expression, ACP activity, and CD31 signal remained at the baseline. These outcomes suggest that while all PFAS trigger the same innate-immune program, only PFOA and PFMOPrA sustained its full development. Taken together, these observations suggest that multiple structural and bioactivity determinants, potentially including but not limited to the chain length, shape the sustained response at the chronic level in leeches. This supports the need for multitemporal, policy-relevant PFAS hazard assessment.