9 Insecta (Hymenoptera)
105
2. Rinse the released eggs twice in tissue culture-grade distilled water.
3. Surface sterilize the eggs in 70% ethanol for 5 min.
4. Rinse the eggs three times with distilled water.
5. Place the eggs in 3 ml PCM supplemented with 1 % Ultracer G, 1 %
Nuserum, 9% FBS albumin, and 50 Ilg/ml gentamicin.
1. Open the host eggs with a microscalpel, and collect 20-50 parasitoid Dissection
eggs (see Comments).
2. Transfer the released parasitoid eggs to 1.0 ml PCM in a 35-mm tissue
culture petri dish.
1. Cut the parasitoid eggs into four to eight pieces with a microscalpel. Culture
2. Seal the petri dish with parafilm.
set-up
3. Incubate at 28°C.
4. After 4 days, cut the explants again into smaller pieces.
5. Replace between one-third and one-half of the medium at 1-2 week
intervals. Centrifuge (100 g, 5 min) the removed medium at the time
of replacement, and return sedimented cells to the original culture.
Comments
The first step of the dissection is difficult, because parasitoid eggs are
very small. It is possible to homogenize host eggs in which parasitoid
eggs are present, remove host egg chorions, and dispense the homogenate into culture vessels. The host cells will not multiply because they
have been irradiated. Thus, if some cells do grow in culture, they are
parasitoid cells.
Results
Lynn and Hung (1986) cultured embryonic cells of Trichogramma
pretiosum that were oviposited into the eggs of a host insect (Heliothis
zea). Cell growth is very slow in primary culture. However, after 24-30
weeks, it becomes possible to subculture regularly with a 1 : 2 split ratio
and, ultimately, Lynn and Hung (1986) obtained continuous cell lines.
References
Giauffret, A., Quiot, J.M., Vago, C. and Poutiers, P. (1968) Cultures cellulaires d'Apis mellifera. In:
Barigozzi, C. (ed) Proceeding of the Second International Colloq uium on Invertebrate Tissue
Culture, Villa Carlotta, 1967. Istituto Lombardo di Scienze e Lettre, Milano, pp 74-80.
Lynn, D.E. and Hung, A.C.P. (1986) Development of a continuous cell line from the insect egg parasitoid, Trichogramma pretiosum (Hymenoptera: Trichogrammatidae). In Vitro Cell. Dev. BioI.
22, 440-442.
Lynn, D. E. and Hung, A.C.P. (1991) Development of continuous cell lines from the egg parasitoids
Trichogramma confusum and T. exiguum. Arch. Insect Biochem. Physiol. 18,99-104.
Sohi, S.S. and Ennis, T.J. (1981) Chromosomal characterization of cell lines of Neodiprion lecontei
(Hymenoptera: Diprionidae). Proc. Entomol. Soc. Ontario 112,45-48.
105
2. Rinse the released eggs twice in tissue culture-grade distilled water.
3. Surface sterilize the eggs in 70% ethanol for 5 min.
4. Rinse the eggs three times with distilled water.
5. Place the eggs in 3 ml PCM supplemented with 1 % Ultracer G, 1 %
Nuserum, 9% FBS albumin, and 50 Ilg/ml gentamicin.
1. Open the host eggs with a microscalpel, and collect 20-50 parasitoid Dissection
eggs (see Comments).
2. Transfer the released parasitoid eggs to 1.0 ml PCM in a 35-mm tissue
culture petri dish.
1. Cut the parasitoid eggs into four to eight pieces with a microscalpel. Culture
2. Seal the petri dish with parafilm.
set-up
3. Incubate at 28°C.
4. After 4 days, cut the explants again into smaller pieces.
5. Replace between one-third and one-half of the medium at 1-2 week
intervals. Centrifuge (100 g, 5 min) the removed medium at the time
of replacement, and return sedimented cells to the original culture.
Comments
The first step of the dissection is difficult, because parasitoid eggs are
very small. It is possible to homogenize host eggs in which parasitoid
eggs are present, remove host egg chorions, and dispense the homogenate into culture vessels. The host cells will not multiply because they
have been irradiated. Thus, if some cells do grow in culture, they are
parasitoid cells.
Results
Lynn and Hung (1986) cultured embryonic cells of Trichogramma
pretiosum that were oviposited into the eggs of a host insect (Heliothis
zea). Cell growth is very slow in primary culture. However, after 24-30
weeks, it becomes possible to subculture regularly with a 1 : 2 split ratio
and, ultimately, Lynn and Hung (1986) obtained continuous cell lines.
References
Giauffret, A., Quiot, J.M., Vago, C. and Poutiers, P. (1968) Cultures cellulaires d'Apis mellifera. In:
Barigozzi, C. (ed) Proceeding of the Second International Colloq uium on Invertebrate Tissue
Culture, Villa Carlotta, 1967. Istituto Lombardo di Scienze e Lettre, Milano, pp 74-80.
Lynn, D.E. and Hung, A.C.P. (1986) Development of a continuous cell line from the insect egg parasitoid, Trichogramma pretiosum (Hymenoptera: Trichogrammatidae). In Vitro Cell. Dev. BioI.
22, 440-442.
Lynn, D. E. and Hung, A.C.P. (1991) Development of continuous cell lines from the egg parasitoids
Trichogramma confusum and T. exiguum. Arch. Insect Biochem. Physiol. 18,99-104.
Sohi, S.S. and Ennis, T.J. (1981) Chromosomal characterization of cell lines of Neodiprion lecontei
(Hymenoptera: Diprionidae). Proc. Entomol. Soc. Ontario 112,45-48.