Chromatographic Separations of Ecdysone Acyl Esters and Their Application to the Distribution and Identification of Ecdysteroids in Adult House Crickets, Acheta Domesticus
Apolar ecdysteroid conjugates (acetates and long-chain fatty acyl esters) are being identified from a wide variety of invertebrate sources. It is apparent that they form a major class of ecdysteroid conjugates which can serve either as inactivation products prior to excretion or as a storage form of ecdysteroid. We have assessed the suitability of reversed-phase (RP) high performance liquid chromatography (HPLC) on a Cis-stationary phase and normal phase (NP) HPLC on silica for the separation and identification of such conjugates. When used in conjunction, these systems provide adequate resolution and selectivity for the preliminary identification of ecdysteroid acetates and fatty acyl esters.
We have compared the quantities of ecdysteroids present in mature male, and both immature and mature female house crickets (Acheta domesticus) by differential radioimmunoassay (RIA). Ecdysteroids were detected in all three adult samples, regardless of which of three antisera was used. Titres in mature adult females were significantly higher than in immature females, which in turn were higher than in adult males. We have analyzed the distribution of ecdysteroids in mature adult female insects and find that they are present in all tissues in relatively high concentrations. Further, evidence is provided that hydrolysable apolar conjugates are present in all tissues. Much lower concentrations of hydrolysable polar conjugates are also present in some tissues (ovaries and haemolymph). The chromatographic behaviour of the free and apolar conjugated ecdysteroids has been analyzed by RP and NP HPLC/RIA. The major free ecdysteroid present in all extracts was ecdysone; no evidence has been found for the presence of 20-hydroxyecdysone in adult house crickets. The proportion of the total ecdysteroids found in the form of apolar conjugates varied considerably from organ to organ, and it is clear that several different apolar conjugates occurred in some of the extracts. In addition to the previously identified ecdysone 22-fatty acyl esters, which were present in all tissues of mature adult females, preliminary evidence is presented for the occurrence of ecdysone 2,22-diacetate in extracts of fat-body, gut and haemolymph. The nature and distribution of ecdysteroids found in vivo is compared to the previously determined metabolism of [3H]ecdysone by organs from mature adult females in vitro.
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