5.7.1. Infecting single greater wax moth larvae with entomopathogenic nematodes

Per Molyneux (1985) and Fan and Hominick (1991). The method below can be used to screen for entomopathogenic nematodes that show action against wax moths. Though nematodes possibly can be used in wax moth control programs, the methods outlined below are also useful for nematologists who need an effective method for rearing nematode species of interest.

  1. Wash sand with distilled water.
  2. Autoclave
  3. Oven-dry.
  4. Filter through a 1.18 mm sieve.
  5. Moisten the filtered sand with 1 ml of distilled water for every 25 ml of sand (4 % V/V).
  6. Place 25 ml of moistened sand in a 30 ml plastic tube.
  7. Pipette nematodes diluted in 1 ml of water (per producer’s instructions or experimental needs) into the sand in the tube.
    The nematode/water solution brings the V/V content to 8 %. Any desired number of nematodes can be introduced to the soil in this way, though including more infective juveniles in the inoculum typically results in greater infestation with nematodes.
  8. Invert (turn upside down) the tube multiple times to disperse the nematodes in the sand.
  9. Place a single wax moth larva on the sand surface in the tube (late instar larvae are 250-350 mg).
  10. Replace the tube lid and invert the tube.
  11. Leave the tube inverted for set time periods and temperatures per the needs of the study.
  12. Recover the wax moth larva and wash it three times with distilled water.
  13. Process (dissect, etc.) the larvae immediately or maintain on moistened filter paper at 20ºC for a period of time before use.