3.1.7.2.1. In vitro infestation of wandering larvae with an aqueous suspension of nematodes

Nematode infective juveniles (IJs) often are suspended in water by the company from which the nematodes are obtained. It is such suspension that the following method is based on to infest SHB larvae.

  1. Place a piece of filter paper in the bottom of a petri dish.
  2. The filter paper should cover the bottom of the dish entirely.
  3. Determine the density of IJs suspended in the water solution.
  4. Add the desired number of IJs to the filter paper using a pipette. One should gently invert the container of IJs to ensure even dispersal of nematodes throughout the liquid.
    The total volume of water including the suspended IJs added to the filter paper should be 1.5 ml.
    For example, suppose that you are trying to infect wandering beetle larvae with 200 IJs per larvae. If you plan to add 10 beetle larvae to the petri dish, you will need to add 2,000 IJs to the dish. If you determine that there are 2,000 IJs per 0.5 ml of water, pipette 0.5 ml of the water/nematode suspension and 1 ml of distilled water to the paper for a total of 1.5 ml solution added to the paper.
  5. Add desired number of wandering SHB larvae to the petri dish.
  6. Replace the lid to the petri dish and secure the lid to the bottom using a tight rubber band.
  7. SHB larvae can push up the lid of a petri dish and escape so a rubber band is necessary to keep this from occurring.
  8. Place the dishes in an incubator at 25°C and no light.
  9. Assess mortality in the SHB larvae at any time period though resolution is greater the more often one views the dishes.
  10. Experimental controls include adding nothing to the dish (no paper, water or nematodes), adding only filter paper, adding only 1.5 ml of distilled water, or adding filter paper and 1.5 ml of distilled water.