Methods

Auxin administration

Auxin treatment was performed by transferring animals to bacteria-seeded NGM plates containing Auxin. A 400 mM stock solution of Auxin indole-3- acetic acid (IAA) (Thermo Fisher, Alfa Aesar™ #A1055614) was created by dissolving Auxin in ethanol. The Auxin stock was then diluted into separate flasks of molten NGM agar to final concentrations of 0.025mM and 1mM (molten NGM was cooled to ~50°C before the addition of Auxin), and were poured into Petri plates. Auxin plates were allowed 72 hrs to dry in the dark, and were then seeded with 50 µl of OP50 liquid culture 48 hrs before use. Animals were continuously exposed to Auxin as they were age synchronized (as described below) onto Auxin plates and tested at 72 hrs post-lay.

Behavioral assays

Animals were age synchronized onto NGM and Auxin Petri plates which were seeded with 50 µl of OP50 liquid culture and dried in the dark for 72 hrs. Four to 6 plates were synchronized for each condition by picking five gravid adults onto each plate and allowing the animals to lay eggs for 4 hrs before removal (resulting in 50-100 worms per plate) (Figure 1B). Animals were then maintained in the dark in a 20°C incubator for 72 hrs.

The behavioral paradigm (Figure 1C) consisted of a 5-minute (min) acclimation period followed by a 5-min period to assess morphological and baseline locomotion phenotypes. Thirty mechanosensory stimuli were then administered to the side of the Petri plate by the MWT’s automated piston at a 10 second (sec) interval. In response to the mechanosensory stimuli, animals respond by briefly crawling backwards then resuming forward locomotion (i.e. a reversal response) (Rankin et al. 1990), allowing multiple features of response sensitivity and habituation to be assessed (Swierczek et al. 2011; McDiarmid et al. 2020). Following the 30th stimulus, animals were allowed to recover for 5 mins. A final stimulus was then administered to test spontaneous recovery from short-term habituation (short-term memory retention).

Multi-Worm Tracker behavioral analysis and statistics

We used the MWT software (version 1.2.0.2) for stimulus delivery and image acquisition (Swierczek et al. 2011), and Choreography software (version 1.3.0_r103552) for phenotypic quantification. The filters, “–shadowless”, “–minimum-move-body 2”, and “–minimum-time 20” were used to identify animals that moved ≥ 2 body lengths and were tracked for ≥ 20 secs. Morphology and baseline locomotion features were identified using standard choreography output commands (Swierczek et al. 2011). Reversals that occurred within 1 sec after the administration of a mechanosensory stimulus were identified using the MeasureReversal plugin (Swierczek et al. 2011). Comparisons of “final response” comprised the average of the final three stimuli. For a complete description of the 26 phenotypic features, see the MWT user guide (https://sourceforge.net/projects/mwt/). All data is freely available upon request. Custom R scripts organized and summarized Choreography output files which are freely available at https://github.com/troymcdiarmid/MWT_Wildtype_Auxin/releases/tag/v1.0. (An archived version of these data and scripts are available on Caltech Data https://doi.org/10.22002/d1.1639). Condition blinding was not necessary as the MWT scores behavior objectively (Swierczek et al. 2011). Phenotypic features were pooled across the 4-6 plate replicates (50-100 animals per plate) for each strain and means of each condition were compared with an unpaired t-test and Benjamini-Hochberg control of the false discovery rate at 0.01. Final figures were generated using the ggplot2 package in R (Wickham 2016). Images taken by the MWT were used to confirm lethality of CA1210 (dhc-1::AID::GFP) animals after 72 hrs of Auxin exposure.