Sterol Regulation of Development and 20-Hydroxyecdysone Biosynthetic and Signaling Genes in Drosophila melanogaster

Abstract

Ecdysteroids are crucial in regulating the growth and development of insects. In the fruit fly Drosophila melanogaster, both C₂₇ and C₂₈ ecdysteroids have been identified. While the biosynthetic pathway of the C₂₇ ecdysteroid 20-hydroxyecdysone (20E) from cholesterol is relatively well understood, the biosynthetic pathway of C₂₈ ecdysteroids from C₂₈ or C₂₉ dietary sterols remains unknown. In this study, we found that different dietary sterols (including the C₂₇ sterols cholesterol and 7-dehydrocholesterol, the C₂₈ sterols brassicasterol, campesterol, and ergosterol, and the C₂₉ sterols β-sitosterol, α-spinasterol, and stigmasterol) differentially affected the expression of 20E biosynthetic genes to varying degrees, but similarly activated 20E primary response gene expression in D. melanogaster Kc cells. We also found that a single dietary sterol was sufficient to support D. melanogaster growth and development. Furthermore, the expression levels of some 20E biosynthetic genes were significantly altered, whereas the expression of 20E signaling primary response genes remained unaffected when flies were reared on lipid-depleted diets supplemented with single sterol types. Overall, our study provided preliminary clues to suggest that the same enzymatic system responsible for the classical C₂₇ ecdysteroid 20E biosynthetic pathway also participated in the conversion of C₂₈ and C₂₉ dietary sterols into C₂₈ ecdysteroids.

Keywords: 20-hydroxyecdysone; Drosophila melanogaster; biosynthesis; dietary sterol.

Figure 1

Structures of different sterols and ecdysteroids. (A) Structures of C₂₇ sterols and ecdysteroids. (B) Structures of C₂₈ and C₂₉ dietary sterols. (C) Structures of C₂₈ ecdysteroids.

Figure 2

The comparison of the uptake efficiency of Kc cells to chloroform-dissolved 22-NBD cholesterol and HβC-dissolved 22-NBD cholesterol.

Figure 3

A heatmap of the relative expression of 20E biosynthetic and response genes in Kc cells treated with different sterols. Cholesterol, 7-dehydrocholesterol (7-dhC), brassicasterol, campesterol, ergosterol, β-sitosterol, stigmasterol, and α-spinasterol belong to dietary sterols. E, 20E, and MaA were used to compare the effects of different ecdysteroids. The control group in (A) was treated with chloroform, that in (B) was treated with 45% HβC, and that in (C) was treated with DMSO. Green indicates the lowest expression and red indicates the highest expression. The fold changes in gene expression levels were log2 transformed.

Figure 4

Development of Drosophila reared on diets with controlled composition of sterols. (A) Dietary sterol depletion arrests larvae growth and development. LDM with chloroform is the control group. The red cross means death. (B) Developmental timing and percentage of pupariation. In each group, 100 animals were measured. Asterisks indicate a significant difference, as calculated using two-tailed unpaired Student’s t-test (* p < 0.05). “ns” means “no significant difference”. (C) Total cholesterol content of whole body of Drosophila reared on LDM supplemented with individual sterols.

Figure 5

Expression changes in Drosophila 20E biosynthetic genes upon different dietary sterol treatments. Cholesterol was added as a control (black), while other dietary sterols (blue) were used to be compared with it. Data are reported as means ± standard deviation of three independent biological replications. The relative expression was calculated based on the value of the reference genes. Asterisks indicate a significant difference, as calculated using two-tailed unpaired Student’s t-test (* p < 0.05).

Figure 6

Immunostaining of 20E biosynthetic genes in the prothoracic gland (Nvd and Sad) and fat body (Shd).

Figure 7

Expression changes in Drosophila 20E signaling pathway genes upon different dietary sterol treatments. (A) The gene expression changes in the first instar larvae, about 48 h after egg laying. The chloroform-fed group was the control (black). The bars labeled with different lowercase letters are significantly different (p < 0.05) (B) The gene expression changes in the white prepupal stage. Cholesterol was added as a control (black), while other dietary sterols (green) were used to be compared with it. Data are reported as means ± standard deviation of three independent biological replications. The relative expression was calculated based on the value of the reference genes. “ns” means “no significant difference”.

Figure 8

Schematic description of hypothesis for C28 ecdysteroid biosynthesis and signaling pathway in Drosophila. Proposed pathway for biosynthesis of C28 ecdysteroid, catalyzed by Halloween genes.