Cal fluor 610 fluorophore

Orsay viral preps were prepared as previously described (7 (link)). Synchronized L1 animals were exposed to a mixture of OP50-1 bacteria and Orsay virus for 18 h at 20 °C (Fig 6A). For infection at the L4 stage (Fig 6B), synchronized animals were grown on rde-1 RNAi plates (1,000 animals per plate, 2 plates per strain) for 72 h (pals-17(*) mutants) or 48 h (all other strains) at 20 °C. rde-1 RNAi increases susceptibility to Orsay virus infection (78 (link), 79 (link)). L4 animals were top-plated with a mixture of Orsay virus, OP50-1 and M9, and incubated at 20 °C for 24 h. Following incubation, animals were collected and washed in M9, and subsequently fixed in 4% paraformaldehyde for 30 min. Fixed worms were washed and then stained at 46 °C overnight using FISH probes conjugated to the red Cal Fluor 610 fluorophore (Biosearch Technologies), targeting Orsay virus RNA1 and RNA2 (80 (link), 81 ). Analyses were performed visually using a Zeiss AxioImager M1 compound microscope; at least 300 animals were scored for the presence of the FISH fluorescent signal per strain per replicate. Due to relatively high variation in infection levels between replicates, the percentage of infected wild-type animals was normalized to one. Data analysis was performed using GraphPad Prism 9 software. Normalized data are shown in S1 Table.

N. parisii spores were prepared as previously described [82 ]. Spores (1 million per plate) were mixed with food and L1 (Fig 6C) or L4 (Fig 6D) synchronized animals. To reach the L4 stage, worms were grown on NGM plates for 72 h (pals-17(*) mutants) or 48 h (all other strains) at 20 °C. During infection, animals were incubated at 25 °C for 30 h. Animals were collected and fixed in 4% paraformaldehyde for 30 min. Fixed worms were stained at 46 °C overnight using MicroB FISH probe conjugated to the red Cal Fluor 610 fluorophore (Biosearch Technologies), targeting microsporidia ribosomal RNA [81 (link),82 ]. N. parisii pathogen load was measured with the COPAS Biosort machine (Union Biometrica). Data analysis was performed using GraphPad Prism 9. Raw data are shown in S1 Table.

Orsay virus filtrates were prepared as previously described [29 (link)]. A 1:5 dilution of Orsay virus filtrate was mixed with OP50-1, M9, and 1,000 synchronized L1 animals, and the mix was top-plated onto 6-cm NGM plates; at least two technical replicates (two plates) per genotype were set up per infection assay. Four independent infection assays were performed. Animals were infected at 20°C for 18 h before fixation in 4% paraformaldehyde. Fixed worms were incubated at 46°C overnight with two FISH probes, mixed equally, that were both conjugated to the red Cal Fluor 610 fluorophore and hybridize to either Orsay RNA1 or RNA2 genome segments (Biosearch Technologies). Samples were analyzed for Orsay virus infection using a AxioImager M1 compound microscope (Zeiss). 100 animals per genotype, per infection assay, were scored and the percentage of infected animals in the population was quantified.