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Tardigrades are microscopic organisms with exceptional resilience to environmental extremes. Most protocols to visualize the internal anatomy of tardigrades rely on fixation, hampering our understanding of dynamic changes to organelles and other subcellular components. Here, we provide protocols for staining live tardigrade adults and other postembryonic stages, facilitating real-time visualization of structures including lipid droplets, mitochondria, lysosomes, and DNA. 

Abstract Pristionchus pacificusis a nematode model for the developmental genetics of morphological polyphenism, especially at the level of individual cells. Morphological polyphenism in this species includes an evolutionary novelty, moveable teeth, which have enabled predatory feeding in this species and others in its family (Diplogastridae). From transmission electron micrographs of serial thin sections through an adult hermaphrodite ofP. pacificus, we three‐dimensionally reconstructed all epithelial and myoepithelial cells and syncytia, corresponding to 74 nuclei, of its face, mouth, and pharynx. We found that the epithelia that produce the predatory morphology ofP. pacificusare identical toCaenorhabditis elegansin the number of cell classes and nuclei. However, differences in cell form, spatial relationships, and nucleus position correlate with gross morphological differences fromC. elegansand outgroups. Moreover, we identified fine‐structural features, especially in the anteriormost pharyngeal muscles, that underlie the conspicuous, left‐right asymmetry that characterizes theP. pacificusfeeding apparatus. Our reconstruction provides an anatomical map for studying the genetics of polyphenism, feeding behavior, and the development of novel form in a satellite model toC. elegans.