High-pressure freezing and freeze substitution was conducted as described previously for morphometric analysis (Weimer, 2006 ) with modifications. In short, about 10 young adult hermaphrodites were loaded into the 100 μm deep well of the freezing chamber (Specimen Carriers Type A (100 μm) and B (0 μm), Bal-Tec AG, Liechtenstein, acquired by Leica Microsystems, Wetzlar, Germany) filled with a mixture of 10% BSA and pelleted OP50 bacteria (ratio: 20:100, v/v) and processed by an EM HPM100 (Leica Microsystems, Wetzlar, Germany) at a freezing speed > 20 000 K/s and a pressure > 2000 bar. High-pressure frozen worm/bacteria pellets were directly transferred in liquid nitrogen to an EM AFS2 freeze substitution system (Leica Microsystems, Wetzlar, Germany). Freeze chambers were opened to allow better penetration during freeze substitution. Samples were incubated in 0.1% tannic acid, 0.5% glutaraldehyde in anhydrous acetone at -90 °C for 96 h, washed 4 times for 1 h with anhydrous acetone at -90 °C and fixed in 2% OsO4 in anhydrous acetone at -90 °C for 28 h, then temperature was ramped for 14 h to -20 °C, incubated 16 h at -20 °C, ramped again to 4 °C in 4 h and immediately washed with anhydrous acetone at 4 °C 4 times at 0.5 h intervals. Then the temperature was gradually raised to 20 °C in 1h and samples were subsequently transferred for embedding in a series of freshly prepared araldite solutions at increasing concentrations (50% araldite in acetone for 3 h at room temperature, 90% araldite in acetone overnight at 4 °C followed by 2 times pure araldite at room temperature). Araldite infiltrated samples were polymerized for 48 h at 60 °C.