General anatomy and organisation of the ventral nervous system and position of trunk ganglia in tardigrades. (A) Light micrograph of a specimen of Hypsibius dujardini in dorsal view. (B–D) Confocal micrographs of Macrobiotus cf. harmsworthi specimens (ventral view, anterior is up). Note the anteriorly shifted position of the four trunk ganglia with respect to each leg pair. (B) DNA labelling of cell nuclei (SYBR® Green). (C) Combined anti-tyrosinated and anti-acetylated α-tubulin immunolabelling to reveal nerve tracts. (D) Combined anti-tyrosinated and anti-acetylated α-tubulin immunolabelling (green) and phalloidin-rhodamine staining (red) to reveal the musculature (same specimen as in C). Abbreviations: cn, somata-free connectives; ga1–ga4, trunk ganglia 1 to 4; le1–le4, walking legs 1 to 4. Scale bars: 25 μm (A–D).