l
Petri dishes.
l
Filter paper.
Procedure
1. Add 400 IJs in 100 μl in each well followed by a single G. mellonella larva.
2. Remove larvae randomly after 1, 2, and 3 h.
3. Rinse the insects and incubate in 5-cm petri dishes with moist
filter paper.
4. Record mortality at 24–48 h intervals post-exposure to
nematodes.
5. Calculate ET 50 value (nematode exposure time in minutes
required to attain 50% insect mortality).
7.2.3 One-on-One
Bioassay [187]
Purpose: To assess interactions of a single host with a given EPN
inoculum.
Materials Required
l
G. mellonella larva.
l
24-Well tissue culture plates.
l
IJs.
l
Filter paper/sand (1 g).
Procedure
1. Use a 24-well tissue culture plate with lid (8 Â 12.2 cm).
2. Place a piece of filter paper or sand (1 g) inside each well.
3. Place an IJ in each well using a 25-μl micropipette.
4. Place one last instar G. mellonella larva into each well.
5. Cover with the lid and incubate at 25
C.
6. Record mortality at 24 h intervals.
Note: Cut filter paper into circles to a diameter suitable to
the diameter of the well.
7.2.4 Sand Bioassay
[188]
Purpose: To assess EPN host foraging behavior.
Materials Required
l
Plastic tubes (45 mm height  40 mm diameter).
l
IJ suspension.
l
G. mellonella larva.
l
Moist sterile sand.
Procedure
1. Place a suspension of 100 IJs in 100 μl into each tube.
2. Fill the tube with moist sterile sand.
3. Place one last instar G. mellonella larva on top.
Ecological Characterization
97
Petri dishes.
l
Filter paper.
Procedure
1. Add 400 IJs in 100 μl in each well followed by a single G. mellonella larva.
2. Remove larvae randomly after 1, 2, and 3 h.
3. Rinse the insects and incubate in 5-cm petri dishes with moist
filter paper.
4. Record mortality at 24–48 h intervals post-exposure to
nematodes.
5. Calculate ET 50 value (nematode exposure time in minutes
required to attain 50% insect mortality).
7.2.3 One-on-One
Bioassay [187]
Purpose: To assess interactions of a single host with a given EPN
inoculum.
Materials Required
l
G. mellonella larva.
l
24-Well tissue culture plates.
l
IJs.
l
Filter paper/sand (1 g).
Procedure
1. Use a 24-well tissue culture plate with lid (8 Â 12.2 cm).
2. Place a piece of filter paper or sand (1 g) inside each well.
3. Place an IJ in each well using a 25-μl micropipette.
4. Place one last instar G. mellonella larva into each well.
5. Cover with the lid and incubate at 25
C.
6. Record mortality at 24 h intervals.
Note: Cut filter paper into circles to a diameter suitable to
the diameter of the well.
7.2.4 Sand Bioassay
[188]
Purpose: To assess EPN host foraging behavior.
Materials Required
l
Plastic tubes (45 mm height  40 mm diameter).
l
IJ suspension.
l
G. mellonella larva.
l
Moist sterile sand.
Procedure
1. Place a suspension of 100 IJs in 100 μl into each tube.
2. Fill the tube with moist sterile sand.
3. Place one last instar G. mellonella larva on top.
Ecological Characterization
97