This study investigates the effects of small-molecule nematicides on the motility of the plant-parasitic nematode Ditylenchus dipsaci. The methodology involves screening potential nematicides and assessing their impact on nematode movement and viability.
Plant-parasitic nematodes such as Ditylenchus dipsaci are microscopic worms. The living worms exhibit sinusoidal movements through muscle contractions, and their motility is an important determinant of their health in culture conditions.
Exposure to nematicides - chemical toxicants, can interfere with key biological processes, affecting the mobility of these cultured nematodes, leading to their death.
To screen small-molecule nematicides, take a distilled water-containing, multi-well plate. Using a clean pinning tool, transfer the potential small molecular nematicides from the chemical stock plate to the assay plate to ensure a transfer of consistent amounts of molecules into each well.
Dispense an equal number of nematodes to each well of the plate. Seal the plates while maintaining moist conditions to support nematode survival and mobility. Incubate the plate with mild agitation to prevent worms from settling.
During incubation, various toxic small molecules act on worms and kill them, while non-toxic ones have no impact. Observe the plate under a dissecting microscope to identify the wells with a non-motile worm population.
Supplement the assay plates with sodium hydroxide solution - end-point chemical stimulant. This triggers the movement of any resting worms and differentiates them from dead ones.
The presence of non-motile worms after sodium hydroxide treatment in some wells confirms the efficacy of corresponding small molecules as nematicides.
To prepare the assay plates, pour autoclaved distilled water into a sterile trough, and dispense 40 microliters of distilled water from the trough into each well of a flat-bottom, 96-well plate with a multichannel pipette. Add chemicals from the 96-well chemical stock plates to the assay plates, by pinning three times into the chemical plate. Then, transfer the pins 10 times into the assay plate. Blot onto paper in front of the cleaning solution.
To count the number of nematodes from the collection, first, resuspend the collection, and then, pipette 5 microliters of the solution using low-retention tips onto a slide. Count the number of nematodes in 5 microliters using a dissection microscope. Then, adjust the concentration to two worms per microliter using sterile distilled water.
Next, add 10 microliters of the sample to each well of the 96-well plates with a multichannel pipette and a trough. Wrap the plates with a damp paper towel and place them in a box. Then, add an extra damp paper towel to stabilize and ensure minimal movement of the plates, and affix on a sticky pad in a 20-degree Celsius shaking incubator set to 200 rpm.
Observe the plates on day 5 under a dissecting microscope. Count the number of mobile and total D. dipsaci in DMSO solvent controls and drug-treated wells.
If the worms are immobile, add 2 microliters of 1 molar sodium hydroxide to a final concentration of 40 millimolar to the well to stimulate movement. Calculate the proportion of mobile worms. In the D. dipsaci screens, wells that reproducibly yielded 0% mobile worms are categorized as strong hits.
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