Note:
To identify species of Steinernema and Heterorhabditis, the
following should be considered:
(a) IJ morphometrics are insufficient for species identification,
and male and female characteristics must be considered.
(b) IJ produced on artificial media (laboratory reared or commercial products) are shorter (rarely longer) than those produced
in vivo and do not meet the criteria of the original description.
Males and females collected 4 or 5 days after the host dies, and
IJ collected for 1 week after they first appear from cadavers,
usually meet original species descriptions.
(c) Measurements of at least ten individuals should be obtained
before trying to identify the species.
(d) Morphological and morphometric characteristics of different
stages of the identified nematode should be verified with the
original description to confirm the identification.
4.2 Scanning
Electron Microscopy
Studies
The use of SEM has substantially improved morphological and
taxonomic nematode studies. The descriptions of species and the
outer surface structure of nematode body of specimens increasingly
include scanning electron micrographs.
4.2.1 Materials Required
l
M9 buffer solution [167].
– Monopotassium phosphate (KH 2 PO 4 ): 15 g.
– Disodium hydrogen phosphate (Na 2 HPO 4 ): 30 g.
– Sodium chloride (NaCl): 25 g.
– Magnesium sulfate (MgSO 4 ) (1M): 5 ml.
– DH 2 O: 1000 ml.
l
Glutaraldehyde.
l
Formalin.
l
Sodium cacodylate.
l
Osmium tetroxide.
l
Ethanol.
4.2.2 Procedure
1. Rinse adults dissected from G. mellonella larvae three times in
M9 buffer for 5 min per rinse.
2. Collect IJs from White traps 3 days after emergence and triplerinse with M9 buffer for 5 min per rinse.
3. Relax all nematodes and heat-kill, next fix in a mixture of 4%
glutaraldehyde and 2% formalin buffered with 0.1 M sodium
cacodylate, pH 7.2, at 4
C for 24 h.
4. Fix them next in 2% osmium tetroxide solution at 4
C for 4 h
and dehydrate at 15-min intervals in a graded ethanol series.
82
Laboratory Techniques for Entomopathogenic Nematodes
To identify species of Steinernema and Heterorhabditis, the
following should be considered:
(a) IJ morphometrics are insufficient for species identification,
and male and female characteristics must be considered.
(b) IJ produced on artificial media (laboratory reared or commercial products) are shorter (rarely longer) than those produced
in vivo and do not meet the criteria of the original description.
Males and females collected 4 or 5 days after the host dies, and
IJ collected for 1 week after they first appear from cadavers,
usually meet original species descriptions.
(c) Measurements of at least ten individuals should be obtained
before trying to identify the species.
(d) Morphological and morphometric characteristics of different
stages of the identified nematode should be verified with the
original description to confirm the identification.
4.2 Scanning
Electron Microscopy
Studies
The use of SEM has substantially improved morphological and
taxonomic nematode studies. The descriptions of species and the
outer surface structure of nematode body of specimens increasingly
include scanning electron micrographs.
4.2.1 Materials Required
l
M9 buffer solution [167].
– Monopotassium phosphate (KH 2 PO 4 ): 15 g.
– Disodium hydrogen phosphate (Na 2 HPO 4 ): 30 g.
– Sodium chloride (NaCl): 25 g.
– Magnesium sulfate (MgSO 4 ) (1M): 5 ml.
– DH 2 O: 1000 ml.
l
Glutaraldehyde.
l
Formalin.
l
Sodium cacodylate.
l
Osmium tetroxide.
l
Ethanol.
4.2.2 Procedure
1. Rinse adults dissected from G. mellonella larvae three times in
M9 buffer for 5 min per rinse.
2. Collect IJs from White traps 3 days after emergence and triplerinse with M9 buffer for 5 min per rinse.
3. Relax all nematodes and heat-kill, next fix in a mixture of 4%
glutaraldehyde and 2% formalin buffered with 0.1 M sodium
cacodylate, pH 7.2, at 4
C for 24 h.
4. Fix them next in 2% osmium tetroxide solution at 4
C for 4 h
and dehydrate at 15-min intervals in a graded ethanol series.
82
Laboratory Techniques for Entomopathogenic Nematodes