Methods

Strain, Growth, and Media: The PD1074 strain of C. elegans was grown and maintained on 10 cm NGM agar plates seeded with an LB-cultured, OP50 strain of Escherichia coli at 22° C. The worms were chunked onto new 10 cm seeded plates once a week. Additionally, a 10 cm stock plate of PD1074 C. elegans was stored at 15° C. Every three to four weeks, a new set of 10 cm plates were chunked from the stock plate in order to maintain a homogeneous experimental population and prevent significant genetic drift.

Experimental Plate Preparation: A stock concentration of 1-HP was diluted using DMSO in order to produce the following five experimental concentrations: 100 μM, 1 mM, 5 mM, 10 mM, and 20 mM. The concentrations at plate surface are the effective concentrations and were corrected by the ratio of the volume of the agar to the volume of DMSO (90 x). 100 μL of each toxin concentration was distributed onto the total surface area of 6 cm NGM plates using a cell spreader and left to dry for 30 min. Then, each toxin plate was seeded with 25 μL of OP50 bacteria and spread to create a small lawn at the center of the plate. After an additional 30 minutes of drying, the plates were placed at 22°C in an insulated container overnight.

Bleach Synchronization: A plate of C. elegans was washed with 3 mL of M9 and pipetted into a 15 mL falcon tube. A second wash of the plate was performed with an additional 3 mL of M9 and left to sit for 5 min to ensure removal of any remaining worms. After 5 min, the contents were pipetted into the falcon tube. The worm/M9 mixture was centrifuged for 1 min at 2000 rpm. A worm pellet was formed at the bottom of the tube and isolated through aspiration. The pellet was then mixed with 2.5 mL of the bleach solution (Porta-de-la-Riva et al., 2012). The tube was shaken gently to disperse the pellet throughout the solution. Every 30 sec, 5 μL of the worm solution was aliquoted onto a glass slide and observed under a microscope to check the state of the worms. Once eggs could be seen breaking out of the worm bodies, the bleaching process was stopped by adding 12.5 mL of M9 and quickly inverting the tube to ensure dilution. The solution was centrifuged for 1 min at 2000 rpm and the supernatant was aspirated. The pellet underwent 2 additional washes and centrifugations at 1200 rpm for 3 min. The egg pellet was resuspended in 5 mL of M9 and then transferred to a 25 mL Erlenmeyer flask and covered with foil. The flask was left to shake at 300 rpm in a 20°C shaker for 24 hours until the worms reached L1 arrest life-stage.

Experimental Worm Plating: After the worms reached the L1 stage, the worms were transferred to a 15 mL falcon tube. The liquid culture was centrifuged for 3 min at 1200 rpm and the supernatant was aspirated to leave a volume of about 300 μL in the tube. The tube was gently shaken to disperse the worm pellet and the 300 μL mixture was pipetted on the outer edge of a seeded 10 cm plate. The plate of L1 worms were incubated at 22° C for about 35-42 hours until they reached the L4 life-stage. Then the worms were washed with 3 mL M9 and pipetted into a 15 mL falcon tube, twice. An average of 15 L4 worms were aliquoted onto each experimental toxin plate. This was done by aliquoting 10 μL of the worm and M9 mixture onto a glass slide to determine the average amount of worms present per μL of M9. Based on this the volume of M9 to be aliquoted onto the experimental plate was determined. After the M9 dried, the experimental plates were placed in an incubator set at 22° C for an exposure time of 7 hours.

Fluorescent Bead Assay and Data Collection: A fluorescent bead assay was used to distinguish between living and dead worms following toxin exposure. The solution was prepared with Fluoresbrite® Polychromatic Red Microspheres 0.5µm, according to a preexisting protocol (Kiyama et al., 2012). After the 7-hour exposure period, the bead solution was dropped in 2 μL aliquots onto each worm on the experimental plates. The worms were left to feed on the bead solution for 5 minutes, and then observed under a fluorescent microscope with a Texas Red Filter. If there was visible fluorescence in the pharynx or anus, the worms were scored as alive. If there was no visible fluorescence, then the worms were scored as dead. Each plate was scored twice, by separate researchers, and the counts were averaged to determine the final count.

Statistical Analysis: The statistical test performed was a Tukey-Kramer HSD test. This test has three assumptions: observations being tested are independent within and among the groups, the groups associated with each mean in the test are normally distributed, and there is homogeneity of variance (Montgomery, 2013). Our data met these three assumptions thus we chose to carry out this test. The Tukey HSD test was performed using a publicly available software (Pro, 2015).