Endocrine network essential for reproductive success in Drosophila melanogaster

Abstract

Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.

Keywords: ecdysis triggering hormone; ecdysone; fecundity; juvenile hormone; oogenesis.

Fig. 1.

ETH signaling persists into the adult stage, evidenced by presence of Inka cells. (A) ETH immunoreactivity in ETH-Gal4 > UAS-RedStinger males and females and schematic diagram showing relative locations of Inka cells. (Scale bars, 20 µm.) (B and C) Patterns of ETH, ETHR-A, and ETHR-B expression detected by RT-PCR of females (B) and males (C) on days −2, −1, 1 h after eclosion (0), 1, 3, 5, 8, 10, 13, 16, and 20. Band intensity quantified and graphed below respective bands.

Fig. S1.

Images of Inka cells in vivo in ETH-Gal4 > UAS-RedStinger adults, Inka cell nuclei (red, indicated by white arrowheads) visible through the cuticle after 4 h of H₂O₂ treatment in an adult female, locations of thoracic (A and C) and abdominal (B and D) cells in color (A and B) and black and white (C and D). (Scale bar, 100 μm.) Higher-magnification images of Inka cells on trachea (E and F). (Scale bar, 50 μm.)

Fig. S2.

RT-PCR of ETH transcript deficiency in ETH-Gal4 > UAS-rpr flies. Inka cell-ablated flies showed no detectable levels of ETH mRNA on day 4 posteclosion.

Fig. 2.

ETH1 mobilizes calcium in the adult CA in a dose-dependent manner. (A) CA from a female (Upper) and male (Lower) before (Left) and after (Right) exposure to 1 µM ETH1. (Scale bars, 10 μm.) (B and C) Time course of calcium mobilization in the CA in response to 600 nM (red) and 2 µM (black) ETH1 exposure (Aug21-Gal4 > UAS-GCaMP3). (D) Latencies to response for males and females following exposure to increasing concentrations of ETH1. Female latency was longer than male latency at the tested concentrations (P < 0.0001) and latency was negatively correlated with ETH concentration (P < 0.0001), confirmed by factorial ANOVA (n = 8–10). (E) Knockdown of ETHR (JHAMT-Gal4 > UAS-GCaMP5;UAS-ETHR-Sym) decreased responsiveness to 5 µM ETH1 treatment, but among responders, both variance (**P < 0.01) and mean (*P < 0.05) latency were significantly increased in both sexes (F-test and Mann–Whitney u test, respectively) (n = 8–10). (F) ETHR transcript levels in males and females after silencing with JHAMT-Gal4 > UAS-ETHR-Sym compared with genetic controls, as well as ETHR levels of control males compared with females (n = 3). Error bars represent SEM. NS, P > 0.05; *P < 0.05; **P < 0.01.

Fig. 3.

Disruption of ETH signaling results in decreased juvenile hormone levels and reduced reproductive success in females. (A and B) Reduction of JH-III levels in both sexes following ETHR silencing in CA (A) (JHAMT-Gal4 > UAS-ETHR-Sym) or Inka cell-ablation (B) (ETH-Gal4 > tubulin-Gal80^ts/UAS-rpr; n = 100). (C and D) Reduced egg production by females following ETHR knockdown in the CA (C) ( JHAMT-Gal4 > UAS-ETHR-Sym) or Inka cell ablation (D) (ETH-Gal4;tubulin-Gal80^ts/UAS-rpr) is rescued by topical treatment with methoprene (n = 15–25). Error bars represent SEM. NS, P > 0.05; *P < 0.05; ****P < 0.0001.

Fig. 4.

Disruption of ETH signaling leads to reduced ovary size, decreased yolk deposition, and altered egg development. (A) Reduction of ovary width following knockdown of ETHR in the CA or (B) ablation of Inka cells and rescue with methoprene; one ovary per fly was examined (n = 35–55). (C) Changes in number of progressing (P) and degenerating (D) eggs following Inka cell ablation. Stage 8–13 eggs not undergoing apoptosis were classified as progressing, whereas those that were DAPI diffuse and TUNEL⁺ were labeled as degenerating. (D) Example of progressing eggs taken from day 5 ovaries of control (D) and those undergoing apoptosis (indicated by TUNEL⁺ red staining of fragmenting DNA) from Inka cell-ablated females (D′). Arrowheads call attention to progressing (D) or degenerating (D′) eggs. (Scale bar, 50 µm.) (E) Extreme example of yolk-deficient stage 14 eggs dissected from Inka cell-ablated females (Left) compared with normal controls (E′). (Scale bar, 200 μm.) (F) Protein solubilized from 100 stage 14 eggs following Inka cell ablation and ETHR knockdown in the CA (n = 6). (G) qPCR of yolk protein mRNA compared with multiple t tests; asterisks in each case represent lowest significance value of comparisons to controls (n = 4–5). Error bars represent SEM. NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. S3.

ETH-Gal4 > UAS-rpr (Inka cell-ablated flies) (A) but not CA-ETHR-silenced (B) flies showed an increase in mature eggs (stage 14) retained. Methoprene treatment had no effect on egg retention. Error bars represent SEM; NS, P > 0.05; ****P < 0.0001.

Fig. S4.

Ovarioles of day 5 females of Inka cell-ablated and control flies were separated from ovaries and DAPI stained. Stage 8–13 eggs including eggs degenerating at stage 8 and 9 (8D, 9D), from each ovary were totaled, defined as progressing (P) or degenerating (D) and compared (n = 15). Error bars represent SEM. NS, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. S5.

Impaired ETH signaling reduces reproductive potential of males. Reduced egg production following mating of Canton-S females with males following Inka cell ablation (A) (ETH-Gal4;tubulin-Gal80^ts/UAS-rpr) or ETHR-knockdown in the CA (B) (JHAMT-Gal4 > UAS-ETHR-Sym or UAS-ETHR-IR2) and rescue following topical treatment with methoprene (n = 15–25). (C) Impairment of male reproductive potential following suppression of EcR expression in Inka cells (n = 20–25). (D) Reproductive potential of Inka cell-ablated males returns to normal by day 10 (n = 10–15). (E) Hatch rate of females mated to low JH males (n = 20–25). Error bars represent SEM. NS, **P < 0.01; ****P < 0.0001.

Fig. 5.

Impairment of 20E signaling in Inka cells reduces expression of ETH signaling genes and reproductive performance. (A) RT-PCR of the ETH gene at 1 and 4 h following saline (S) or 20E injection (n = 3–4). (B) Fold-change in ETH and ETHR expression 1 h and 4 h after 20E injection measured by qPCR in females (pink bars) and males (blue bars) (n = 4–5), statistical differences in gene expression between treated and control groups were significant at P < 0.05, assessed by Mann–Whitney test. (C) Fecundity is impaired following reduction of EcR expression in Inka cells following expression of EcR-RNAi or an EcR dominant-negative (DN; n = 20–30) and rescue with methoprene (n = 15–20). Error bars represent SEM. *P < 0.05; ***P < 0.001.

Fig. S6.

CA-ablated flies show reproductive potentials reduced to levels comparable to those of ETH-JH interrupted flies (see Fig. 3). Number of eggs produced by Canton-S females mated to males of the indicated genotype in the 72 h following mating (left side) for CA-ablated flies (Aug21-Gal4 > UAS-NIPP1) as well as genetic controls (n = 15–25). Number of eggs produced in the 72 h following mating to a Canton-S male (right side) by CA-ablated flies (Aug21-Gal4 > UAS-NIPP1) and genetic controls (n = 15–25). Error bars represent SEM. P > 0.05; **P < 0.01; ****P < 0.0001.

Fig. 6.

Model for gonadotropic coregulation by the hormonal network consisting of 20E, ETH, and JH in Drosophila adults. (A) Ecdysone (20E) induces expression of ETH in Inka cells and ETHR in target tissues. The CA integrates ETH and other cues to determine JH level; high JH exerts negative feedback inhibition on 20E production. (B) Timing of 20E and JH release into the hemolymph. Ecdysone induces ETH synthesis, but inhibits its release, possibly through inhibition of the secretory competence factor βFTZ-F1. ETH release occurs as 20E levels decline.