Ecdysteroids, acknowledged as the principal steroid hormones in arthropods, are commonly known as “molting hormones” in insect physiology [1]. Ecdysone serves as a prohormone crucial for synthesizing the primary insect molting hormone, 20E. Once ecdysone is released, it undergoes conversion into the more active form, 20E. This hormone is renowned for its central role in regulating various life stages in insects, facilitating the smooth transition from larval stages to pupation and eventually to maturity. It exerts a significant influence on cellular processes such as autophagy and apoptosis, which are essential for the developmental changes that occur during molting [2]. These cellular events are integral to insect development, growth, and survival, especially in species like D. melanogaster (Drosophila melanogaster), B. mori (Bombyx mori), H. armigera (Helicoverpa armigera), and others [3,4,5,6].

PCD is a vital cellular mechanism in insects, ensuring the proper development, maintenance, and survival [7,8]. Processes such as apoptosis, necrosis, and autophagy play pivotal roles in tissue remodeling and the removal of damaged cells [9]. In advanced eukaryotes, three main autophagy types have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy. Both microautophagy and chaperone-mediated autophagy direct cytosol segments and proteins to lysosomes, either autonomously or with assistance by chaperones [10]. Macroautophagy, often referred to as autophagy, is a recycling mechanism crucial for cellular homeostasis, guiding developmental shifts and addressing environmental stresses, especially in insects [11]. Apoptosis emerges as a pivotal process-shaping development, homeostasis, and defense [12]. Autophagy and apoptosis in insects have been extensively studied, not only in the midgut epithelium of Acheta domesticus (Insecta, Orthoptera, and Gryllidae), but also in the context of insect infections [13,14]. This indicates that both autophagy and apoptosis play indispensable roles in cellular development. Furthermore, 20E emerges as a key regulator of autophagy and apoptosis. In the silk-producing B. mori, a lepidopteran species, 20E’s regulation of autophagy and apoptosis are vital for its unique life cycle stages [15]. Similarly, the malaria vector, Anopheles gambiae, as an Diptera species depends on the processes regulated by 20E for its rapid larval development [16]. On the other hand, in the cotton bollworm, H. armigera, the interplay between 20E and other signaling pathways modulates PCD, which is crucial for its adaptability to diverse environments [6]. Collectively, 20E regulates autophagy, apoptosis, immune function, and the central nervous system, indicating high antioxidant activities and other functions [17]. This evolutionary pattern highlights the unique biological responses across different life phases. Therefore, it is crucial to assess regulatory mechanisms through the lens of age and developmental transitions.

The understanding between 20E, autophagy, and apoptosis holds profound implications beyond basic biology. In agriculture, a comprehensive understanding of this triad could pave the way for innovative pest control methods, reducing the reliance on environmentally harmful pesticides. This synergistic connection between 20E and autophagy/apoptosis in insects not only underscores the complexity of nature but also presents a fertile ground for potential advancements in both sustainable agriculture and medicine.

From a biochemical perspective, the sterol hormone 20E, which belongs to the ecdysteroid family, plays a pivotal role in orchestrating insect growth, development, and reproduction [18,19]. Its biosynthesis begins with the assimilation of dietary cholesterol, serving as the foundational precursor. The process of 20E biosynthesis in insects holds a central process governing the development and metamorphosis [20]. Biosynthesis of 20E involves a series of enzymatic reactions that convert cholesterol into the active hormone. Initially, cholesterol undergoes a hydroxylation process, which is catalyzed by several cytochrome P450 enzymes, leading to the production of precursors such as ecdysone. Subsequent enzymatic steps, including further hydroxylation and oxidation, result in the formation of 20E. Upon synthesis, 20E targets specific nuclear receptors, mainly the EcR (ecdysone receptor), to regulate gene expression essential for developmental processes [21]. This bioactive form of ecdysone then participates in the regulation of gene expression by forming a heterodimer with USP (ultraspiracle protein), which is essential for initiating the developmental processes [22]. The genes EcR-B1 (ecdysone receptor B1 isoform) and USP1 (ubiquitin specific peptidase 1) have been predominantly associated with 20E regulation [23,24]. Intriguingly, other pivotal factors such as E75B and Ha-eIF5c have also been identified to influence 20E regulation. It was elucidated that the genes modulated by 20E, namely EcR-B1, USP1, E75B, BR-CZ2, HHR3, and Ha-eIF5c, are influenced by the suppressive actions of both Ha-Ntf2 and Ha-Ran [25,26,27]. Taken together, these evidences suggest that the regulation of 20E exhibits conserved mechanisms across diverse species. These information provide a coherent framework, tracing from its formation, through its process, and to its ultimate effects. Therefore, a comprehensive understanding of 20E biosynthesis yields profound insights into its subsequent roles in biological metabolism.

E (Ecdysone), which is the immediate precursor of 20E, is synthesized and released via specialized structures in insects, such as the prothoracic glands or ovaries. After secretion, it circulates in the hemolymph and is transformed into the biologically active 20E in peripheral tissues. This conversion primarily occurs in the fat body, midgut, and malpighian tubules, particularly during the larval stage [28]. Illustratively, in Drosophila and other members of the Diptera order, ecdysone is synthesized and secreted via the prothoracic glands, acting as the seminal trigger for the initiation of the molting cascade [29]. Subsequently, E undergoes hydroxylated at the 20th position by the enzyme, shade, leading to the formation of the biologically activated 20E being released from the fat body and transported to different cells and tissues as needed. In the adults of some insects, the presence of ecdysteroids in the ovary was discovered 40 years ago, and now it is well established that ovarian follicular cells synthesize ecdysone de novo [30,31]. Intriguingly, in certain lepidopteran species, it has been documented that male gonads release significant quantities of ecdysone in vitro [32].

The biosynthetic cascade is triggered when the PTTH (neuropeptide Prothoracicotropic Hormone) binds to its receptor, Torso, on prothoracic gland cells [33]. This interaction initiates the Ras/Raf/ERK signaling pathway, activating genes crucial for ecdysone biosynthesis [34]. Simultaneously, the Insulin/IIS (IGF Signaling) pathway, responsive to nutritional cues, enhances ecdysone synthesis by increasing the expression of biosynthetic enzymes and by augmenting the prothoracic glands’ sensitivity to PTTH [35]. The biosynthetic process is further modulated by biogenic amines, particularly octopamine, and neuropeptides such as B. mori, although their roles may vary across different species. Significantly, external factors such as photoperiodicity and ambient temperature subtly influence this biosynthetic pathway [36,37,38,39]. This biosynthesis involves cytochrome P450 enzymes, with six identified as playing roles in the 20E pathway [40]. While the cytochrome P450 Cyp6t3 has been shown not to be essential for ecdysone biosynthesis in D. melanogaster, CYP18A1 is still crucial for its metamorphosis [41,42]. The biosynthesis of 20E involves multiple cytochrome P450 enzymes, including CYP306A1, which facilitates the production of the arthropod molting hormone [43]. These findings provide a new perspective, suggesting that P450 may have a more pronounced role in regulating the sterol hormone related to cellular processes.

Moreover, JH (Juvenile Hormone) adds an additional layer of regulation, often acting antagonistically to 20E. Elevated JH concentrations typically suppress 20E synthesis [44]. In the captivating world of beetles, as part of the Coleoptera order, the JH takes center stage, driving vitellogenesis within the fat body [45,46]. Vitellogenesis involves the synthesis and accumulation of yolk proteins in maturing oocytes, which are crucial for nourishing the embryo post-fertilization [47]. This crucial process primarily occurs in the fat body, an organ comparable to the mammalian liver. Interestingly, in the adult females of Hemimetabola (Dictyoptera to Hemiptera) and Coleoptera, JH serves as the main regulator, exerting pleiotropic controls over various aspects of female reproduction [48,49]. Given the diversity of Coleoptera, there is variability in how different beetle species respond to JH, reflecting their specific reproductive strategies and ecological niches. Furthermore, the coordinated interplay of these PCD forms, observed in the ovarian tropharia of certain beetles, underscores the delicate equilibrium insects establish to ensure their survival and adaptability amidst constantly evolving environments [50]. While ecdysone, 20E, and JH each govern insect growth and reproduction, an intriguing question arises: Could 20E also have a pivotal role in the insect reproductive processes? If so, what are the underlying mechanisms through which these hormones operate? Unraveling these questions could provide fresh insights into the multifaceted roles of steroid hormones in insect biology.

In the silkworm B. mori, the enzymatic activity of ecdysteroid-phosphate phosphatase plays a crucial role in the 20E biosynthetic pathway within eggs, catalyzing the conversion of maternal-conjugated ecdysteroids [5]. This process is paralleled in the species Drosophila, belonging to Diptera, where studies have uncovered a reciprocal regulatory relationship between 20E and JH in larvae [51]. Each hormone intricately influences the synthesis of the other, showcasing the complexity of hormonal interactions during development. Recent studies have revealed that 20E, which is specifically derived from cholesterol via the catalytic activity of a series of cytochrome P450 enzymes that hydroxylate E at carbon 20, particularly CYP314a1, is essential for insect molting and metamorphosis [52]. This underscores the significance of Drosophila E-20-monooxygenase, which is identified as the product of the shade locus. In a recent investigation focused on H. armigera, a representative of the Lepidoptera order, four cytochrome P450 homologs-HarmCYP302A1, HarmCYP306A1, HarmCYP314A1, and HarmCYP315A1, were identified. These homologs demonstrated evolutionary conservation across lepidopterans. Intriguingly, while HarmCYP302A1 and HarmCYP315A1 were predominantly expressed in the larval prothoracic glands, such consistent expression patterns were absent for HarmCYP306A1 and CYP314A1 [53]. It is evident that, at different times, the biological activity and expression of different enzymes can serve as indicators of biological activity to some extent. However, the question remains: Is the emergence of biological activity necessarily related to these enzymes?

In conclusion, the intricate process of 20E biosynthesis serves as a cornerstone in the field of insect physiology and development. A comprehensive grasp of this pathway not only sheds light into the evolutionary intricacies of insect life, but also offers promising opportunities for transformative applications in pest management, medicine, and biotechnology. As researchers unravel the steps and intricacies of 20E biosynthesis, researchers gain a deeper comprehension of insect biology and open avenues for sustainable and innovative solutions to challenges in agriculture and health.