Figure 1.
Halloween gene expression during development and in adult Tribolium.
The relative mRNA distribution of the Halloween genes during development and in adults was measured by qPCR. Expression was normalized to rpS3 mRNA levels. The step in the ecdysteroid biosynthetic pathway catalyzed by each enzyme is shown on the right. Numbers on the x-axis indicate the day of each stage. E; embryo, F; females, O; ovaries, M; males, R; reproductive systems of males, 7dC; 7-dehydrocholesterol, 2,22,25-dE; 2,22,25-trideoxyecdysone (ketodiol), 2,22-dE; 2,22-dideoxyecdysone (ketotriol), 2-dE; 2-deoxyecdysone, 20E; 20-hydroxyecdysone. Note that the Black Box is believed to include multiple uncharacterized reactions converting 7dC to the ketodiol.
Figure 2.
Knock down of phm and spo delays development and reduces ecdysteroid levels in Tribolium.
Number of larvae that have undergone a molt after knock down of phm (phm-RNAi) and spo (spo-RNAi) expression 5 days (A) and 9 days (B) after injection with dsRNA. GFP dsRNA (GFP-RNAi) was used as a negative control. (C) Ecdysteroid levels in spo-RNAi and phm-RNAi larvae are reduced compared to the GFP control and to spot-RNAi 5 days after injection with dsRNA. Hemolymph ecdysteroid levels were below the limit of RIA detection (about 10 pg) in the hemolymph of spo-RNAi and phm-RNAi animals. L-L; larval-larval molt, L-P; larval-pupal molt.
Figure 3.
Tribolium male tubular accessory glands express the genes required for biosynthesis of E, but not 20E.
(A) qPCR analysis shows that the tubular accessory glands express spot and the biosynthetic enzymes to produce E but not shd, although E may be converted to 20E in other tissues as shd is expressed in carcass (n = 3–4). (B) In situ hybridization with a spot antisense probe showing expression in the TAGs. (C) Negative control in situ hybridization using a spot sense probe. (D) The fertility of males with reduced expression of key E biosynthetic enzymes was examined by the number of progeny produced per day of virgin females mated with males injected with dsRNA (n = 6–7). Error bars are SE. RS; reproductive system, RAG; rod-shaped accessory gland, TAG; tubular accessory gland.
Figure 4.
Adult ecdysteroid levels are regulated by PTTH that may target the accessory glands in Drosophila.
(A) GFP expression driven by a torso-Gal4 line in the bi-nucleated secondary cells of a male accessory gland of Drosophila. Inserts are magnifications showing the two nuclei of spherical secondary cells. Green: GFP, Magenta: DAPI. (B-E) Gene expression was analyzed by qPCR in adult Drosophila males overexpressing ptth (da>ptth) and in animals with reduced expression of torso (da>torso-RNAi) or ptth (da>ptth-RNAi). da>+ (da> crossed to w1118, genetic background used by VDRC for generating the RNAi lines) was used as a control and expression was normalized to rpL23 mRNA levels. (B and C) Ecdysteroid levels estimated as levels of expression of ecdysteroid target genes E74A, E74B and E75B. (D and E) Expression of Halloween genes phm, dib and sad.
Figure 5.
A model for sex-specific synthesis and action of ecdysteroids in adults.
The ovaries of female Tribolium express spo and the genes for the terminal hydroxylases, including shd, required for synthesis of 20E. Male accessory glands also express a spo-like gene, spot, and the genes for the terminal hydroxylases required for synthesis of E, but not shd. However, shd expression was detected in the carcass without the reproductive system indicating that E synthesized by the accessory gland might be converted to 20E in peripheral tissues, like during the larval stages. Alternatively, E produced by the accessory gland may be involved in male-specific hormone signaling or be transferred to females during mating. Multiple arrows indicate several steps in the biosynthetic pathway.