Fig 4. Treatment with a TRPV agonist dysregulates chemotaxis of B. pahangi infective larvae.

(A) L3 parasites were extracted from mosquitoes and subjected to chemotaxis assays with or without 250 μM NAM treatment. Chemotaxis assays were performed by adding L3s to the middle of a 0.8% agarose plate (M), with either test cue (T) or water (C) added to the opposite sides of the plate. The plate was placed at 37°C for 30 minutes, scored after incubation, and the CI was calculated. (B) NAM dysregulates attraction of freshly extracted L3s to serum and NaCl but has no effect on aversion to 3-methyl-1-butanol. (C) NAM has no effect on translational movement of freshly extracted L3s. (D) Bpa-osm-9 expression is unchanged by in vitro culture at physiological or room temperature 4 HPE. (E) L3s cultured for 1 DPE do not show chemotaxis toward serum and have reduced motility on the chemotaxis plate when compared with untreated freshly extracted parasites (p = 0.028, t test). Data for (A–C) represent the combined results of three independent biological replicates, except for the experiments with 3-methyl-1-butanol, which included two replicates (cohorts of mosquito infections). Data for (E) represent the results of two biological replicates. Each point represents a single chemotaxis plate with 8–10 L3s. Red diamonds and bars indicate the mean and standard error of the mean. Comparisons of means were performed using t tests (**p ≤ 0.01). Raw data for (B) through (E) can be found at https://github.com/zamanianlab/BrugiaChemo-ms. CI, chemotaxis index; DPE, days postextraction; FBS, fetal bovine serum; HPE, hours postextraction; L3, third stage larvae; NAM, nicotinamide; ns, not significant; TRP, transient receptor potential.