Visualisation of microplastics in the gut of arthropods: An experimental approach on millipedes (Diplopoda, Julida)

Microplastics (MPs) are a growing threat to terrestrial ecosystems, yet the localisation of ingested MPs within the digestive tract of soil arthropods remains poorly understood. Here, we developed and validated a reliable fluorescence microscopy approach to visualise MP particles in different gut regions of millipedes (Diplopoda). MPs (LDPE and PLA, 3–5 mm) were pre-stained with Nile red and millipedes were exposed for five days. After gut isolation, cryosections were counterstained with Sytox Green for nuclei and examined under UV excitation, which provided optimal discrimination between blue‑white MP fluorescence and bright green cell nuclei. Whole‑body sectioning proved unsuitable due to fragmentation and autofluorescence of the cuticle, leading to false‑positive signals. In contrast, isolated gut preparations yielded clear images. We observed a region‑specific MP distribution: particles adhered closely to the cuticle‑lined wall of the foregut and hindgut but remained mostly in the lumen of the midgut, likely separated by the peritrophic membrane. This differential localisation may have functional implications for MP residence time, translocation risk and interaction with compartment‑specific gut microbiomes. Our protocol offers a practical and reproducible tool for future studies on MP ingestion, fragmentation and biodegradation by arthropods.