(A). The time for the first fly to climb 17.5 cm. dfmr1 and FS (dfmr1 with a wild-type dfmr1 transgene that contains a frameshift mutation in the dfmr1 open reading frame) do not express functional dFMRP, and show a progressive change in climbing behavior over the course of 35 days. The abnormal climbing is rescued by a transgene containing the genomic wild-type dfmr1 locus (control). Data presented are the Mean +/- SEM (8 trials, total flies n = 80 for each genotype tested at each time point). (B). Total percentage of flies that successfully reach the 17.5 cm mark within 3 min. For all data, *p<0.05, **p<0.01, and ***p<0.001.

(A). At 5 days old, dfmr1 and FS flies groom significantly more than control flies. Grooming increases with age in mutant flies; control flies show consistent levels of grooming at all ages tested. (B). The average duration of grooming bouts in mutant flies follows a similar trend to the total time spent grooming (A). In contrast, control flies show little change in the duration of grooming bouts from 5 to 35 days of age. Data are represented as the mean percentage of time single flies spend grooming during a 5 min period (Mean +/- SEM; n = 10–15 flies for each genotype at each time point). For all data, *p<0.05, **p<0.01, and ***p<0.001.

(A). Treatment of dfmr1 mutant male flies with MPEP improves courtship of naïve females. Flies were grown as larvae and maintained as adults on either control food or food containing 86 µM MPEP. Data are presented as the mean courtship index (CI, +/- SEM) with sample sizes shown above each bar. dfmr1 and FS male flies treated with MPEP engage in courtship activity with wild-type virgin females significantly more than when treated with no drug. Control flies court less when treated with MPEP, but this difference is not significant. (B). 15 day-old dfmr1 and FS flies treated with 86 µM MPEP groom significantly more than when treated with no drug. MPEP does not affect grooming activity in control flies. Data are presented as the mean percentage of time single flies spend grooming during a 5 min period (Mean +/- SEM); sample sizes are displayed above each bar. For all data, *p<0.05 and ***p<0.001 (Two-tailed students t-test).

Grooming activity of 15 day-old flies grown as larvae and maintained as adults, or only maintained as adults on food containing 5 mM LiCl. Data are presented as the mean percentage of time single flies spend grooming during a 5 min period (Mean +/- SEM); sample sizes are displayed above each bar. For all genotypes, LiCl causes no significant change in grooming activity (Kruskal-Wallis one-way ANOVA with Dunn's post-hoc comparison).

(A). Grooming activity of 15 day-old flies grown as larvae and maintained as adults on food containing no drug, 10, 30, or 50 µM reserpine (15 and 20 µM are omitted from figure). Reserpine suppresses grooming in dfmr1 and FS flies, but only at 50 µM; control flies show significantly reduced grooming at the lowest concentration, 10 µM. (B). 15 day-old flies treated with reserpine only as adults (i.e. larvae were grown on control food), show a similar response. Suppressed grooming in mutant flies is only significant at 50 µM. In contrast, reserpine significantly decreases grooming activity in control flies at 15 µM. Data are presented as the mean percentage of time single flies spent grooming during a 5 min period (Mean +/- SEM); sample sizes are displayed above each bar. For all data, *p<0.05 and ***p<0.001 (Kruskal-Wallis one-way ANOVA with Dunn's post-hoc comparison).

Quantitative real-time PCR experiments indicate that dVMAT mRNA levels are increased in 5 day-old dfmr1 mutant flies (A) and in 25 day-old dfmr1 mutant flies (B) relative to control flies. Western blot analyses indicate that dfmr1 mutant flies also have increased levels of dVMAT protein at both (C) 5 days and (D) 25 days of age. (E) Representative blot showing increased dVMAT protein levels in dfmr1 and FS flies compared to control flies. Real-time PCR data are presented as the average of three biological replicates. Western blot data are shown as the average of three independent trials. *p<0.05 (One-way ANOVA with Dunnett's post-hoc comparison).