morphologically similar or closely associated (based on phylogenetic molecular evidence)] for comparisons.
4.2.6 Hemolymph
Hanging Drop Technique
This technique was developed by Poinar Jr and Thomas [170] and
modified by Kaya and Stock [163].
Materials Required
l
G. mellonella larvae.
l
70% ethanol.
l
Serum-free medium.
l
IJs.
Procedure
1. Surface-sterilize G. mellonella larvae in 70% ethanol.
2. Bleed larva by puncturing between the head and the first
prothoracic segment.
3. Cautiously place a drop of hemolymph on a coverslip. Make
sure it is hemolymph and not fat is collected.
4. Add 10 ml of serum-free medium for insect tissue culture
(SF-900 II SFM Gibco) to prevent hemolymph from
desiccating.
5. Keep 30–50 surface-sterilized IJs in the drop.
6. Swap the coverslip upside down, and smoothly position it on
deep concave slide.
7. Place the slide in a petri dish (100 Â 15 mm) consisting of one
filter paper disc drenched with water. Moist filter paper will
prevent the sample from drying.
8. Cover dish with the lid.
9. Place the dish inside a plastic carrier (to prevent water loss) and
incubate at 25–27
C.
10. Observe daily and eliminate pre-adult (J4) males and females.
11. Place male and female isolates to be tested in new hanging drop
slides with adults of the opposite sex of the other isolates
(a ratio of five males to five females is recommended).
Note: Assessment of the mating ought to be performed over a
period 10 days. Controls include crosses of the similar isolates. The
appearance of offspring is regarded positive, indicating the mated
pair is of the same species, whereas absence of progeny is a negative
result indicating the mated pair is different species. Crosses must
have adequate repetitions to authenticate the findings.
84
Laboratory Techniques for Entomopathogenic Nematodes
4.2.6 Hemolymph
Hanging Drop Technique
This technique was developed by Poinar Jr and Thomas [170] and
modified by Kaya and Stock [163].
Materials Required
l
G. mellonella larvae.
l
70% ethanol.
l
Serum-free medium.
l
IJs.
Procedure
1. Surface-sterilize G. mellonella larvae in 70% ethanol.
2. Bleed larva by puncturing between the head and the first
prothoracic segment.
3. Cautiously place a drop of hemolymph on a coverslip. Make
sure it is hemolymph and not fat is collected.
4. Add 10 ml of serum-free medium for insect tissue culture
(SF-900 II SFM Gibco) to prevent hemolymph from
desiccating.
5. Keep 30–50 surface-sterilized IJs in the drop.
6. Swap the coverslip upside down, and smoothly position it on
deep concave slide.
7. Place the slide in a petri dish (100 Â 15 mm) consisting of one
filter paper disc drenched with water. Moist filter paper will
prevent the sample from drying.
8. Cover dish with the lid.
9. Place the dish inside a plastic carrier (to prevent water loss) and
incubate at 25–27
C.
10. Observe daily and eliminate pre-adult (J4) males and females.
11. Place male and female isolates to be tested in new hanging drop
slides with adults of the opposite sex of the other isolates
(a ratio of five males to five females is recommended).
Note: Assessment of the mating ought to be performed over a
period 10 days. Controls include crosses of the similar isolates. The
appearance of offspring is regarded positive, indicating the mated
pair is of the same species, whereas absence of progeny is a negative
result indicating the mated pair is different species. Crosses must
have adequate repetitions to authenticate the findings.
84
Laboratory Techniques for Entomopathogenic Nematodes