Clock-controlled rhythm of ecdysteroid levels in the haemolymph and testes, and its relation to sperm release in the Egyptian cotton leafworm, Spodoptera littoralis

In Spodoptera littoralis, testicular sperm release occurs in a daily rhythm, which is controlled by endogenous circadian oscillator located in the male reproductive system. Although this rhythm is essential for male fertility, factors that initiate and maintain daily sperm release are not understood. In this study, we investigated a modulatory role for ecdysteroids in the sperm release rhythm and identified the source of ecdysteroids in adult males. We found that the onset of sperm release occurs two days pre-eclosion and coincides with a significant decrease in haemolymph ecdysteroids levels. 20-HE injection into the pupae prior to the first sperm release delayed its initiation and disrupted the developing rhythm in a dose dependent manner. 20-HE injection into adults depressed the number of sperm bundles leaving the testes. A day before the initial sperm release, ecdysteroid levels in the haemolymph and testes begin to oscillate in a circadian fashion. Ecdysteroid rhythms continue throughout imaginal life and correlate with the rhythm of sperm release. In each cycle, testicular sperm release coincides with a trough in testicular ecdysteroid concentration. Rhythmic changes in ecdysteroid levels are regulated by an endogenous circadian oscillator that continues to function in decapitated males. The generation of a complete cycle of ecdysteroid release by testes cultured in vitro indicates that this oscillator is located in the gonads. The haemolymph ecdysteroid levels are significantly lower and arrhythmic in males with removed testes, indicating that the testes are an important ecdysteroid source that may contribute to oscillations in haemolymph ecdysteroid levels.