7.1.1 Calibrate
the System
in the Following Way
2. Centrifuge at 300 rpm for 1 min.
3. Divide the number of nematodes in the tube by the volume
(in ml) that the nematodes now fill to acquire the number of
nematodes/ml (V).
4. Estimate “V” several times from various samples to authenticate that the system is relatively consistent for the given species
and procedure.
5. Estimate the number of nematodes once the system is calibrated, by multiplying the volume taken up by the
appropriate V.
7.2 Ecological
Characterization
The virulence of the extracted nematode suspension is assessed
using last instar G. mellonella larvae.
7.2.1 Penetration
Bioassay [186]
Purpose: To assess the penetration efficacy of IJs into the host insect.
Materials Required
l
G. mellonella larva.
l
IJs.
l
24-Well plate.
l
Filter paper.
l
Stereoscopic microscope.
Procedure
1. Place a suspension of 200 IJs in 100 μl in each well plate
followed by a single G. mellonella larva.
2. Maintain control well plate with 250 μl of sterile water.
3. Record the mortality of larvae daily.
4. Wash the dead larvae in tap water to eliminate nematodes from
their surface and keep separately in 5-cm petri dishes with moist
filter paper.
5. Incubate for a further 2 days.
6. Dissect the insects under a stereoscopic microscope.
7. Count the number of males and females in each larva.
8. Evaluate the penetration rate as the percentage of the initial IJ
that have intruded the host insect.
7.2.2 Exposure Time
Bioassay [186]
Purpose: To assess the time required by the IJs to attain host insect
mortality.
Materials Required
l
G. mellonella larva.
l
IJs.
l
24-well tissue culture plate.
96
Laboratory Techniques for Entomopathogenic Nematodes
the System
in the Following Way
2. Centrifuge at 300 rpm for 1 min.
3. Divide the number of nematodes in the tube by the volume
(in ml) that the nematodes now fill to acquire the number of
nematodes/ml (V).
4. Estimate “V” several times from various samples to authenticate that the system is relatively consistent for the given species
and procedure.
5. Estimate the number of nematodes once the system is calibrated, by multiplying the volume taken up by the
appropriate V.
7.2 Ecological
Characterization
The virulence of the extracted nematode suspension is assessed
using last instar G. mellonella larvae.
7.2.1 Penetration
Bioassay [186]
Purpose: To assess the penetration efficacy of IJs into the host insect.
Materials Required
l
G. mellonella larva.
l
IJs.
l
24-Well plate.
l
Filter paper.
l
Stereoscopic microscope.
Procedure
1. Place a suspension of 200 IJs in 100 μl in each well plate
followed by a single G. mellonella larva.
2. Maintain control well plate with 250 μl of sterile water.
3. Record the mortality of larvae daily.
4. Wash the dead larvae in tap water to eliminate nematodes from
their surface and keep separately in 5-cm petri dishes with moist
filter paper.
5. Incubate for a further 2 days.
6. Dissect the insects under a stereoscopic microscope.
7. Count the number of males and females in each larva.
8. Evaluate the penetration rate as the percentage of the initial IJ
that have intruded the host insect.
7.2.2 Exposure Time
Bioassay [186]
Purpose: To assess the time required by the IJs to attain host insect
mortality.
Materials Required
l
G. mellonella larva.
l
IJs.
l
24-well tissue culture plate.
96
Laboratory Techniques for Entomopathogenic Nematodes