Small Molecule Nematicides Screening Assay: A Medium Throughput Method to Screen Potential Nematicides Against Ditylenchus dipsaci

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board

1. In vitro small molecule screen

1. Prepare the assay plates following the steps below.
   1. Pour autoclaved distilled water into a sterile trough and, using a multichannel pipette, dispense 40 µL of distilled water from the trough into each well of a flat-bottom 96-well plate.
2. Prepare pinning tool and add the chemicals.
   1. Set up pinner trays (see Table of Materials) near a Bunsen burner flame on a lab bench. Add the following to successive pinner trays: 25 mL of pin cleaning solution, 35 mL of 50% DMSO (in water), 45 mL of distilled water, 55 mL of 70% EtOH, and 65 mL of 95% EtOH. Place one piece of blotting paper in front of each tray.
   2. Clean the pinning tool by emersing the pins in the cleaning solution and moving the pins up and down three times (3x) in the solution. In this protocol, '3x' and '10x' are defined as moving the pinner up and down in a solution three or ten times, respectively. Blot the pins on the blotting paper. Repeat this procedure once more.
      1. Next, rinse the pins 3x in distilled water, followed by blotting the pins. Repeat the procedure once more.
      2. Lastly, rinse the pins 3x in 95% EtOH, followed by blotting the pins. Repeat once more. Flame the pinner and allow the ethanol to evaporate.
   3. Add chemicals from the 96-well chemical stock plates to assay plates by pinning 3x into the chemical plate, then transferring the pins 10X into the assay plate. Blot onto paper in front of the cleaning solution.
   4. Clean pinning tool between plates by washing in the following order, blotting in between on blotting paper: 3x in 50% DMSO (once), 3x in distilled water (once), 3x in 75% EtOH (once), 3x in 95% EtOH (twice). Flame the pinner and allow ethanol to evaporate.
   5. Repeat step 1.2.2 when all pinning is completed.
      NOTE: The screening was performed at a final concentration of 60 µM.
3. Addition of worms
   1. Count the number of nematodes from the collection by first resuspending and then pipetting 5 µL using low retention tips onto a slide for observation. Count the number of nematodes in 5 µL using a dissection microscope.
      1. Adjust the concentration to 2 worms/µL using sterile distilled water. Using a multichannel pipette and a trough, add 10 µL (~20 worms) to each well of the 96-well plates.
         NOTE: Approximately 15,000 D. dipsaci nematodes will be collected per culture plate using the described culture and collection method. Twenty worms are used per well because the small number facilitates the clear visualization and accounting of mobile and immobile ones.
   2. Seal the plates in laboratory wrapping film and wrap with a damp paper towel. Place in box and affix on a sticky pad in 20°C shaking incubator set at 200 rpm. Ensure that plates are stabilized in the box by adding an extra damp paper towel to ensure minimal movement of plates.

2. Data collection and analysis

1. Observe plates on day 5 under a dissecting microscope. Count the number of mobile and total number of D. dipsaci in DMSO solvent controls and drug-treated wells.
   1. If the worms are relatively immobile, add 2 µL of 1 M NaOH to a final concentration of 40 mM to the well to stimulate movement.
      NOTE: After adding NaOH, worms will move instantly and need to be viewed within 5 min. The number of mobile worms and the total number of worms will be used to calculate the proportion of mobile worms. The length of the assay may change depending on the aim of the screen.
2. Calculate the proportion of mobile worms. In the D. dipsaci screens, wells that reproducibly yielded 0% mobile worms are categorized as strong hits.
   NOTE: Prolonged exposure of plates on the stage of dissection microscopes is avoided because the light source can heat the plates and induce variable effects on the bioassay.