10. Include the following controls for each cross:
(a) Virginity/self-fertility test: Place ten virgin females without
males on individual six lipid agar plates.
(b) Mating test/self-cross: Place ten virgin females and ten males
of the similar strain or even species on lipid agar plates, and
add ten more males 3 days later.
Note:
The outcome of a cross between dissimilar isolates is considered
valid if:
(a) There is no progeny in the virginity test.
(b) There is progeny in the self-cross.
Since IJs of Heterorhabditis species often mature into hermaphroditic females in the first adult generation, the second-generation
amphimictic adults are required for the cross-mating tests [171].
For this purpose, the technique to collect second-generation
adults is as follows:
1. Set up IJ infections of the suitable strain or species.
2. It is desirable to infect a series of G. mellonella larvae at 2-day
intervals to guarantee that female nematodes are available at a
suitable stage for cross-breeding.
3. Acquire second-generation adults from cadavers between 6 and
8 days post-infection by dissecting the G. mellonella cadavers in
saline solution (Ringer’s solution).
4. Choose female nematodes with immature gonads with the help
of a stereomicroscope.
5. Hoard immature females using a needle or by aspiration with a
micro capillary pipette which is drawn out to give an outer
diameter of 150–250 mm.
6. Collect males in the similar way as females; however, their age
or state of maturation is not critical.
Note:
(a) For a supplementary measure, gather only virgin females from
cadavers of which no more than 5–10% of the secondgeneration females have begun oogenesis.
(b) Virgin females may be used instantly for cross breeding or may
be placed on a lipid agar plate for 1–2 days to approve that they
are producing only unfertilized eggs.
86
Laboratory Techniques for Entomopathogenic Nematodes
(a) Virginity/self-fertility test: Place ten virgin females without
males on individual six lipid agar plates.
(b) Mating test/self-cross: Place ten virgin females and ten males
of the similar strain or even species on lipid agar plates, and
add ten more males 3 days later.
Note:
The outcome of a cross between dissimilar isolates is considered
valid if:
(a) There is no progeny in the virginity test.
(b) There is progeny in the self-cross.
Since IJs of Heterorhabditis species often mature into hermaphroditic females in the first adult generation, the second-generation
amphimictic adults are required for the cross-mating tests [171].
For this purpose, the technique to collect second-generation
adults is as follows:
1. Set up IJ infections of the suitable strain or species.
2. It is desirable to infect a series of G. mellonella larvae at 2-day
intervals to guarantee that female nematodes are available at a
suitable stage for cross-breeding.
3. Acquire second-generation adults from cadavers between 6 and
8 days post-infection by dissecting the G. mellonella cadavers in
saline solution (Ringer’s solution).
4. Choose female nematodes with immature gonads with the help
of a stereomicroscope.
5. Hoard immature females using a needle or by aspiration with a
micro capillary pipette which is drawn out to give an outer
diameter of 150–250 mm.
6. Collect males in the similar way as females; however, their age
or state of maturation is not critical.
Note:
(a) For a supplementary measure, gather only virgin females from
cadavers of which no more than 5–10% of the secondgeneration females have begun oogenesis.
(b) Virgin females may be used instantly for cross breeding or may
be placed on a lipid agar plate for 1–2 days to approve that they
are producing only unfertilized eggs.
86
Laboratory Techniques for Entomopathogenic Nematodes