Fig. 3.

Behavioral characterization of SNXs deletion mutants. a Locomotion behavior of young adult worms (day 4, post-hatching) was analyzed for the indicated genotypes. Δsnx-3 mutant displays increased motor impairments at standard growth conditions. Approximately 50% of the Δsnx-3 mutant worms were unable to exit the 1 cm circle during the duration of the assay (60 s). Three independent experiments were performed with N > 100 per assay. One-way ANOVA analysis was performed and data represented as mean ± SEM; b average locomotion velocities were assessed in the presence/absence of a food source, such as OP50 or in the presence/absence of an external stimulus, such as tapping. Independent samples Mann–Whitney U test was performed. Disruption of SNX-3 gene significantly affects worm’s locomotive behavior, and reduces its crawling speed. c, d Chemotaxis index scores for SNXs mutants. Represented are the chemotaxis results of wild-type and SNXs mutant animals towards two volatile attractants (10% isoamyl alcohol, or 1% diacetyl). Chemotaxis index was calculated as CI (adults at the attractant minus the number of worms at the solvent, divided by the total number of worms in the plate [46]). Three independent experiments were performed with N > 100 per assay. One-way ANOVA analysis was performed and data represented as mean ± SD; e thermotaxis behavior of wild-type and Δsnx-3 mutant in a temperature gradient. Δsnx-3 mutant (second row) displays a cryophilic behavior, and is not able to perform isothermal tracking. f Locomotion behavior of young adult worms (day 4, post-hatching) was analyzed for the indicated genotypes. Δsnx-3 mutant displays motor impairments at standard growth conditions, such as the stronger Wls (mig-14) mutant (ga62), the Wnt ligand mutant and the general retromer mutant. However, the weaker Wls (mig-14) mutant (mu71) presented no notorious locomotive defects, comparing to the wild-type strain. Approximately 46% of the snx-3 mutant worms, 59% of mig-14 mutant worms (stronger mutant), 47% of the egl-20 mutant and 49% of the vps-35 mutant worms were unable to exit the 1 cm circle during the duration of the assay (60 s). Three independent experiments were performed with N > 100 per assay. One-way ANOVA analysis was performed and data represented as mean ± SD. g Chemotaxis index scores for Wnt and retromer mutants. Represented are the chemotaxis results of wild-type and Wnt/retromer mutant animals towards a volatile attractants (10% isoamyl alcohol). Chemotaxis index was calculated as CI (adults at the attractant minus the number of worms at the solvent, divided by the total number of worms in the plate [46]). Clearly, Wnt signaling, Wls and retromer mutants display no deficits in its chemotaxis behavior towards IA, when compared to the wild-type strain (N2) or to osm6 (perturbed chemotaxis to IA) and osm9 (standard chemotaxis to IA) mutant controls. Data are the mean ± SD. ***p < 0.001. h qRT-PCR expression analysis of distinct snx-s in all the tested snx-s, retromer, and Wnt-related mutants. Snx-s are expressed in all the backgrounds, and snx-5 is the most expressed Snx. It’s noteworthy that snx-3 expression is similar in wild-type and mutant strains, particularly in the retromer and Wnt-related mutants [F(11,12) = 2.10 p = 0.109]. Independent experiments were performed with n > 200 per mutant background. One-way ANOVA was performed and data represented are mean ± SD