Hemolymph ecdysteroid titer and midgut ecdysone 20-monooxygenase activity during the last larval stage of Diploptera punctata

Abstract

Hemolymph ecdysteroid titers during the last larval stage (lasting 20–21 days) of Diploptera punctata were determined by radioimmunoassay. A small peak (1 γM on day 10) preceded a larger peak (> 10 γM on day 17). Although ecdysone was the predominant circulating ecdysteroid in the beginning of the last larval stage, the ratio of 20-hydroxyecdysone to ecdysone increased to about 7 1 at the time of the large ecdysteroid peak. Ecdysone was converted to 20-hydroxyecdysone by fat body, midgut and Malpighian tubules incubated in vitro. Conditions for the measurement of ecdysone 20-monooxygenase activity were established for midgut homogenates. Maximal activity was found on days 5–9 and activity declined in the second half of the last larval stage. By using differential centrifugation and marker enzymes, most midgut ecdysone 20-monooxygenase activity was found to be associated with microsomes. Antibodies to NADPH-cytochrome P-450 reductase inhibited microsomal ecdysone 20-monooxygenase activity, thus confirming the cytochrome P-450 nature of this enzyme. The antibodies inhibited the ecdysone 20-monooxygenase activity associated with mitochondria as strongly as they inhibited the microsomal enzyme activity, thus showing that the mitochondrial activity was a microsomal contamination and that there was little or no intrinsically mitochondrial ecdysone 20-monooxygenase in the midgut.