Gas chromatographic analysis of ecdysteroids in the desert locust Schistocerca gregaria

Abstract

The ecdysteroids present in 3rd, 4th, and 5th instar Schistocerca gregaria phase gregaria, and 5th instar Schistocerca gregaria, phase solitaria have been determined by gas chromatography with electron capture detection of their trimethylsilyl ethers. Both ecdysone and 20-hydroxy- ecdysone were detected with 20-hydroxyecdysone forming the major component. Titres of 20-hydroxyecdysone rose to a maximum of over 1 yg/insect during the 5th instar. Evidence of faecal excretion of ecdysteroids was sought, and small quantities of 20-hydroxyecdysone and polar conjugates of it were detected. These were not present in sufficient quantity to explain the observed fall in ecdysteroid levels in the insect. Both ecdysone and 20-hydroxyecdysone were present in the haemolymph of 5th instar insects ( 96 ng ml * and 432 ng ml * respectively).
Ecdysteroids in adult female insects were determined from the fifth-to-adult ecdysis to oviposition. The major ecdysteroids present were ecdysone and 20-hydroxyecdysone in the form of polar conjugates, the major component was ecdysone. These compounds were first detected a few days before oviposition and rose to 2-3 yg/insect at oviposition.
These ecdysteroids could then be detected in the newly laid eggs.
Structural requirements for the detection of ecdysteroids using the electron capture detector were investigated. The tri-TMS ether of the model compound 2(3,3g,l4a-trihydroxy-56-cholest-7-en-6-one was found to be approximately ten times more sensitive to electron capture than the 2#3^*di- 14ot-hydroxy compound. More complex ecdysteroids with modifications to the side chain did not show this increase in electron capturing ability upon silylation of the 14a-hydroxyl group.
Chemical and enzymic procedures for cleaving polar ecdysteroid conjugates were investigated. Enzymic methods involving the use of Helix pomatia digestive juice were found to cleave ecdysteroid conjugates.
No success was seen using chemical methods involving heating in dioxan or pyridine, or incubating in tetrahydrofuran, or ethyl acetate containing perchloric acid.
The model compounds 38-hydroxy*5a-cholestan-6-one, 30-hydroxy- -5a-cholest-7-en-6-one, and 38,14a-dihydroxy-5a-cholest-7-en-6-one were synthesised from cholesterol. The syntheses of 3-dehydroecdysone and 3-dehydro-20-hydroxyecdysone were attempted without success. The solvent partition and gas chromatographic properties of the model compounds were determined.