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Figure 1. Lifelong Auxin exposure does not affect a suite of phenotypes scored by the Multi-Worm Tracker in N2 and PD1074 wild-type C. elegans strains: The Auxin-Inducible Degradation (AID) system relies on genetically tagging a protein of interest (POI) with an AID degron peptide tag and transgenic expression of the inducible E3 ubiquitin ligase TIR1 (A). The presence of Auxin triggers TIR1 to polyubiquitinate the POI, causing subsequent protein degradation (A). N2 and PD1074 animals were age synchronized onto standard Nematode Growth Media (NGM) plates or 0.025mM or 1mM Auxin plates (B). Animals were subjected to a short-term habituation behavioral paradigm and (C) the Multi-Worm Tracker (MWT) was used to quantify the effects of Auxin on morphological and behavioral phenotypes. For N2 and PD1074 animals, Auxin exposure at either 0.025mM or 1mM did not affect initial response sensitivity or habituation of response probability, speed, or duration (D and E), or 5 commonly studied morphological (animal length and width) and locomotion (movement speed, directional bias, and degree of kink in body posture (related to curvature of body posture)) phenotypes (F and G). A detailed description of all phenotypes can be found in the MWT user guide (Swierczek et al. 2011). Description 10/8/2020 Open Access The Auxin-Inducible Degradation (AID) system is a powerful technique in the C. elegans toolkit that enables conditional and reversible protein depletion with high temporal and spatial specificity (Zhang et al. 2015; Martinez et al. 2020; Ashley et al. 2020; Martinez and Matus 2020). This system relies on tagging a gene of interest with a short AID degron sequence and transgenic expression of TIR1, an inducible E3 ubiquitin ligase normally found only in plants (Nishimura et al. 2009; Zhang et al. 2015). Upon exposure to the plant-derived hormone Auxin, TIR1 is activated and targets AIDtagged proteins for proteasomal degradation (Nishimura et al. 2009; Zhang et al. 2015) (Figure 1A). While there are qualitative reports that Auxin does not overtly affect the morphology or behavior of wild-type C. elegans (Zhang et al. 2015), this has not been quantitatively assessed. Determining whether Auxin significantly affects C. elegans morphology and behavior, even in subtle ways, is important given the C. elegans community’s rapid uptake of the AID system (Kasimatis et al. 2018; Nance and Frøkjær-Jensen 2019; Ashley et al. 2020; McDiarmid et al. 2020). Here, we use our high-throughput machine vision tracking system, the Multi-Worm Tracker (MWT) (Swierczek et al. 2011), to investigate whether exposure to Auxin affects a suite of morphological, locomotor, mechanosensory, and short-term habituation learning phenotypes in our lab’s derivative of Bristol N2 wild-type worms and the CGC wild-type reference strain, PD1074 (Yoshimura et al. 2019) (Figure 1B). We report that lifelong exposure to 0.025mM or 1mM Auxin does not significantly affect any of the objectively quantified phenotypic features in either N2 or PD1074 wild-type strains (Figure 1D-G). We tested 1mM Auxin as it is commonly used to quickly and fully degrade AID tagged proteins, whereas, in our experience, 0.025mM Auxin still enables protein degradation but is dilute enough to allow AID tagged proteins to be re-expressed within 24 hours (hrs) of the animals being off Auxin. The Auxin plates used were indeed effective, as plates from the same batch were able to induce lethality in the CA1210 (dhc-1::AID::GFP) positive control strain (this is expected due to ubiquitous degradation of the essential dynein heavy chain DHC-1) (Zhang et al. 2015). An additional concern has been that Auxin is dissolved in ethanol (Martinez et al. 2020), and, as many of us know first-hand, ethanol can indeed alter animal behavior (Mitchell et al. 2007; Spear 2018). Importantly, we find that the ethanol used to dissolve Auxin does not affect these phenotypes when tested on agar plates, likely because the ethanol evaporates during the 72 hrs plates are allowed to dry. NOTE: The ethanol that Auxin is dissolved in will likely affect these phenotypes if using liquid culture (not tested here). We hope this article will serve as a resource to show that with proper preparation, Auxin can be used with the AID system for effective protein depletion in C. elegans with minimal effects on a suite of morphological and behavioral phenotypes studied by the community. However, we still recommend the inclusion of a wild-type on Auxin negative control for behavioral experiments to ensure there is nothing wrong with the Auxin solution that may affect behavior (e.g. contamination). Methods Request a detailed protocol Auxin administration Auxin treatment was performed by transferring animals to bacteria-seeded NGM plates containing Auxin. A 400 mM stock solution of Auxin indole-3acetic acid (IAA) (Thermo Fisher, Alfa AesarTM #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.

Auxin does not affect a suite of morphological or behavioral phenotypes in two wild-type C. elegans strains.