fected worms have been harvested and mechanically disrupted applying 1 mm diameter Zirconia beads (BioSpec). Resulting lysate was filtered through five m filters (Millipore Sigma) to remove nematode debris. Spore preparations had been tested for contamination and those free of charge of contaminating bacteria have been stored at -80 .N. parisii infection assays and several pressure adaptation D4 Receptor manufacturer assaysP0 populations of 2500 animals had been mixed with 1 ml of 10saturated E. coli OP50-1 or P. vranovensis in addition to a low dose of N. parisii spores (see Table three) and plated on a ten cm plate. This low dose restricted the detrimental effects on animal fertility that happen to be observed with larger doses, though ensuring most animals had been nevertheless infected. F1 populations of 1000 animals have been mixed with 400 l of 10saturated E. coli OP50-1 in addition to a high dose of N. parisii spores (see Table three) and plated on a 6 cm plate. Table 3. Details of N. parisii doses employed. To test for inherited immunity to N. parisii Plate Millions of spores applied in C. elegans, C. briggsae, C. tropicalis, and C. N. parisii concentration kamaaina, synchronized animals have been infected dose (spores/cm2) 6 cm plate 10 cm plate from the L1 larval stage using a low dose of N. Low 32,000 two.five parisii. C. elegans and C. briggsae had been grown for Higher 88,000 2.five 72 hr at 21 ; C. tropicalis and C. kamaaina wereBurton et al. eLife 2021;ten:e73425. DOI: doi.org/10.7554/eLife.17 ofResearch articleEvolutionary Biology | Genetics and Genomicsgrown for 96 hr at 21 . Ten percent of total P0 animals had been fixed in acetone for DY96 staining, as described beneath. Embryos in the remaining animals had been collected by bleaching and synchronized by BD1 Biological Activity hatching overnight in M9. Resulting F1 animals have been infected in the L1 larval stage with a higher dose of N. parisii. C. elegans and C. briggsae were fixed at 72 hr postinfection (hpi) at 21 ; C. tropicalis and C. kamaaina were fixed at 96 hpi at 21 . For many anxiety adaptation assays applying N. parisii and osmotic pressure, animals had been grown on NGM agar plates seeded with 10saturated E. coli OP50-1 till the L4 stage. Next, animals have been collected and mixed with 1 ml of either E. coli OP50-1 alone or supplemented using a low dose of N. parisii spores and plated on either 50 mM NaCl or 250 mM NaCl plates. Animals had been grown for 24 hr at 21 . Embryos from these animals had been collected by bleaching. To test adaptation to osmotic stress, 2000 F1 embryos were transferred to 420 mM NaCl plates seeded with E. coli OP50-1. Percentage of animals hatched was scored right after 48 hr at 21 , as previously described in Burton et al., 2017 and Burton et al., 2020. To test adaptation to N. parisii, the remaining embryos have been synchronized by hatching overnight in M9. Resulting F1 animals were either not infected as controls, or infected in the L1 larval stage having a higher dose of N. parisii. Animals have been fixed immediately after 72 hr at 21 for DY96 staining and evaluation. For a number of strain adaptation assays applying N. parisii and P. vranovensis, animals were grown on NGM agar plates seeded with E. coli OP50-1 till the L4/young adult stage. Next, animals were collected and mixed with 1 ml of either E. coli OP50-1 alone or E. coli OP50-1 supplemented with a low dose of N. parisii spores, or 1 ml of P. vranovensis BIGb0446 alone or P. vranovensis BIGb0446 supplemented having a low dose of N. parisii spores. Animals have been plated on NGM and grown for 24 hr at 21 . Embryos from these animals have been collected by bleaching. To test adaptation to P. vra
