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Organ Culture
Subprotocol 10
Prothoracic Glands of Lepidopteran Insects (Lepidoptera)
Prothoracic glands of insects have been cultured by some investigators
for approximately a week in order to ascertain the secretion of ecdysteroid
from these glands. However, the culture conditions required for continuous culture of pro thoracic glands have not been examined properly. The
following method has been developed in order to maintain the function
of the pro thoracic glands of lepidopteran insects as long as possible
(Komiya et aI., 1998).
Materials
Equipment - Dissecting microscope and dissecting tray (see chapter 1, section 3
"Tools")
- Scissors
- Forceps
- Needles
- Maximov slides
- Pasteur pipettes
- Insect pins
- Plastic petri dish (35 mm diameter; no. 1008; Falcon; Becton-Dickinson)
- 96-Well test plate (no. 3072; Falcon; Becton-Dickinson)
Solutions - MGM-450 medium without serum (see Appendix 1)
- Carlson's solution (see Appendix 1)
Animals - The common armyworm Pseudaletia separata (final instar larvae)
Miscellaneous - Parafilm (American National Can)
Procedure
Sterilization 1. Surface sterilize the larvae by submersion in 70% ethanol for 5 min.
2. Wash the larvae twice in sterilized distilled water.
Dissection 1. Fix one larva in a dissecting tray, dorsal side up, using several insect
pins.
2. Fill the tray with Carlson's solution so as to sink the larva in the solution.
3. Make an incision in the larva along the dorsal midline.
4. Excise the prothoracic glands using needles and forceps, and place
the glands in Carlson's solution in the depression of a Maximov slide
(Fig. 6).