Non-redundant functions of insulin/FOXO and EcR signaling pathways regulate accessory gland structure, function and male fertility in Drosophila.

Somatic reproductive tissues, such as the male accessory glands in insects, are critical for insect fertility. However, the signaling pathways that maintain accessory gland homeostasis remain poorly understood. Using tissue-specific knockdown or overexpression in Drosophila, we modulated insulin signaling pathway genes in male accessory glands and examined their effects on male fertility. The impact of FOXO overexpression on gland structure and function was studied by analyzing cellular integrity, expression of genes encoding accessory gland proteins, and autophagy markers. Additionally, we explored the interaction between insulin signaling and steroid signaling by simultaneously overexpressing FOXO and knocking down the Ecdysone receptor in male accessory glands. We find that normal levels of InR, PI3K, AKT & FOXO in the accessory glands are essential for male fertility. Males with FOXO overexpression in accessory glands were sterile with reduced, structurally and functionally defective glands, possibly due to a metabolic block characterized by disrupted upstream insulin signaling and impaired autophagy, as evidenced by non-lipidated Atg8a and increased lysosomes. Both FOXO overexpression and EcR knockdown elevated glucose levels through distinct effects on insulin signaling. Unlike FOXO overexpression, EcR knockdown reduced autophagy markers. FOXO overexpression in an EcR-deficient background partially rescued gland structure and glucose levels, although the glands remained underdeveloped. Our observations highlight interactive yet non-redundant regulatory roles for insulin/FOXO and EcR signaling in accessory gland structure and function. These findings suggest that male accessory glands in insects selectively deploy distinct metabolic/hormonal input-specific stress-response programs to maintain their structural and functional integrity, as well as male fertility.