5.1 Chemotaxis for
EPN Behavior [146,
151, 152]
This assay is carried out to ascertain if chemotaxis is species and
strain specific and to evaluate behavioral influence of EPNs toward
volatile compounds.
5.1.1 Materials Required
l
Infective juveniles.
l
9-cm diameter petri dishes.
l
1.6% Technical agar.
l
Potassium phosphate (pH 6.0).
l
CaCl 2 .
l
MgSO 4 .
l
95% Linalool.
l
98% α-caryophyllene or 98% β-caryophyllene.
l
M9 buffer.
l
Parafilm.
l
Dissecting microscope.
5.1.2 Procedure
1. Add 25 ml agar, 5 mM potassium phosphate, 1 mM CaCl 2 , and
1 mM MgSO 4 to the petri dish.
2. Make three spherical marks of 1 cm diameter on the underside
of the petri dish—one in the center, one on the right side, and
the other on the left side (1.5 cm from its edge).
3. Place 50 μl drop of 100 IJs in the midpoint of the agar surface.
4. Place 10 μl drop of linalool, α-caryophyllene, or
β-caryophyllene on the right side of the agar surface.
5. Place 10 μl of M9 buffer as control on the left side of the agar
surface.
6. Seal the petri dishes with parafilm.
7. Once sealed, keep the petri dishes within a rearing chamber at
25
C and 75% RH without light.
8. Allow the nematodes to freely wander for 3 or 22 h.
9. Place these petri dishes inside a freezer for 5 min at 20
C and
restrain the nematodes.
10. Count the total nematodes in the control and treatment part
under a dissection microscope.
11. Repeat the experiments three times.
12. Include five replicates for each treatment.
13. Calculate the specific chemotaxis index (CI) [153] as given:
(Number of nematodes in the treatment area À Number of
nematodes in the control area)/Total number of nematodes in
the assay.
40
Chemical Ecology
EPN Behavior [146,
151, 152]
This assay is carried out to ascertain if chemotaxis is species and
strain specific and to evaluate behavioral influence of EPNs toward
volatile compounds.
5.1.1 Materials Required
l
Infective juveniles.
l
9-cm diameter petri dishes.
l
1.6% Technical agar.
l
Potassium phosphate (pH 6.0).
l
CaCl 2 .
l
MgSO 4 .
l
95% Linalool.
l
98% α-caryophyllene or 98% β-caryophyllene.
l
M9 buffer.
l
Parafilm.
l
Dissecting microscope.
5.1.2 Procedure
1. Add 25 ml agar, 5 mM potassium phosphate, 1 mM CaCl 2 , and
1 mM MgSO 4 to the petri dish.
2. Make three spherical marks of 1 cm diameter on the underside
of the petri dish—one in the center, one on the right side, and
the other on the left side (1.5 cm from its edge).
3. Place 50 μl drop of 100 IJs in the midpoint of the agar surface.
4. Place 10 μl drop of linalool, α-caryophyllene, or
β-caryophyllene on the right side of the agar surface.
5. Place 10 μl of M9 buffer as control on the left side of the agar
surface.
6. Seal the petri dishes with parafilm.
7. Once sealed, keep the petri dishes within a rearing chamber at
25
C and 75% RH without light.
8. Allow the nematodes to freely wander for 3 or 22 h.
9. Place these petri dishes inside a freezer for 5 min at 20
C and
restrain the nematodes.
10. Count the total nematodes in the control and treatment part
under a dissection microscope.
11. Repeat the experiments three times.
12. Include five replicates for each treatment.
13. Calculate the specific chemotaxis index (CI) [153] as given:
(Number of nematodes in the treatment area À Number of
nematodes in the control area)/Total number of nematodes in
the assay.
40
Chemical Ecology