Steroid hormone signaling: What we can learn from insect models.

Ecdysteroids are a group of steroid hormones in arthropods with pleiotropic functions throughout their life history. Ecdysteroid research in insects has made a significant contribution to our current understanding of steroid hormone signaling in metazoans, but how far can we extrapolate our findings in insects to other systems, such as mammals? In this chapter, we compare steroid hormone signaling in insects and mammals from multiple perspectives and discuss similarities and differences between the two lineages. We also highlight a few understudied areas and remaining questions of steroid hormone biology in metazoans and propose potential future research directions.

The plant-derived triterpenoid, cucurbitacin B, but not cucurbitacin E, inhibits the developmental transition associated with ecdysone biosynthesis in Drosophila melanogaster.

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis.

Twenty-hydroxyecdysone produced by dephosphorylation and ecdysteroidogenesis regulates early embryonic development in the silkmoth, Bombyx mori.

Ecdysteroids are key regulators of embryonic development as well as molting and metamorphosis in insects. Although an active form of ecdysteroids, 20-hydroxyecdysone (20E) is known to be produced through ecdysteroidogenesis from cholesterol and dephosphorylation of 20E-phosphate during embryogenesis in Lepidoptera, the importance of these production mechanisms in embryonic development has been unclear. Here, we investigated the activation timing of ecdysteroidogenesis from cholesterol and 20E-phosphate dephosphorylation during early embryogenesis in non-diapause eggs of the silkmoth Bombyx mori by observing morphological development, quantifying 20E and 20E-phosphate, measuring transcripts of enzymes involved in 20E production, and detecting activity of these enzymes using egg extracts. Stage-dependent 20E fluctuation and changes in mRNA amounts of enzymes suggest that the two 20E-producing mechanisms are activated at different stages during embryogenesis. Furthermore, knockdown of a dephosphorylation enzyme delayed development at early embryogenesis, whereas knockdown of an ecdysteroidogenic enzyme delayed development at early-middle embryogenesis. These results suggest that 20E is primarily produced initially by dephosphorylation of 20E-phosphate, and then by ecdysteroidogenesis from cholesterol to induce progression of embryonic development in B. mori.

Functional analysis of ecdysteroid biosynthetic enzymes of the rice planthopper, Nilaparvata lugens.

Ecdysteroids, insect steroid hormones, play key roles in regulating insect development and reproduction. Hemipteran insects require ecdysteroids for egg production; however, ecdysteroid synthesis (ecdysteroidogenesis) details have not been elucidated. We identified all known genes encoding ecdysteroidogenic enzymes in Nilaparvata lugens and clarified their necessity during nymphal and ovarian development. We confirmed that N. lugens utilized 20-hydroxyecdysone as an active hormone. Assays using heterologous expression of enzymes in Drosophila S2 cells showed conserved functions of enzymes Neverland, CYP306A2, CYP314A1 and CYP315A1, but not CYP302A1. RNA interference and rescue analysis using 20-hydroxyecdysone demonstrated that most of the genes were necessary for nymphal development. The identified N. lugens enzymes showed conserved functions and pathways for ecdysteroidogenesis. Knockdown of ecdysteroidogenic enzyme genes in newly molted females caused failure of egg production: less vitellogenic and mature eggs in ovaries, fewer laid eggs and embryonic development deficiency of laid eggs. Considering the high expressions of ecdysteroidogenic enzyme genes in adults and ovaries, ecdysteroidogenesis in ovaries was critical for N. lugens ovarian development. Our study presents initial evidence that hemipteran insects require ecdysteroidogenesis for ovarian development.

An insulin-like growth factor-like peptide promotes ovarian development in the silkmoth Bombyx mori.

Insulin family peptides are known to be key regulators of growth and metabolism in insects and vertebrates. Insects have two types of insulin family peptides: insulin-like peptides and insulin-like growth factor (IGF)-like peptides (IGFLPs). We recently demonstrated that an IGFLP in the silkmoth, Bombyx mori (BIGFLP) promotes the growth of the genital imaginal disc ex vivo. However, the role of BIGFLP in the regulation of insect growth remains unclear because no in vivo study has been performed. Therefore, we analysed the functions of BIGFLP in vivo by constructing BIGFLP knock-out (KO) B. mori using the clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR-Cas9) system. The KO moths exhibited decreased body weights and size of the appendages compared wild-type (wt) moths. Interestingly, KO females also had drastically lower ovary weights and number of eggs than wt females. However, mutant ovaries that were transplanted into wt host pupae reached a similar weight to wt ovaries that were transplanted into the wt hosts, suggesting that IGFLP in the haemolymph promotes ovarian development. These findings show that BIGFLP regulates the growth and development of adult organs, particularly the ovaries, in B. mori.

Antagonistic Effect of Short Neuropeptide F on Allatotropin-Inhibited Feeding Motivation of the Silkworm Larva, Bombyx mori.

Here, we demonstrated an antagonistic effect of short neuropeptide F (sNPF) in modulating feeding motivation in the silkworm Bombyx mori; sNPF reduced the feeding-delaying effects caused by administration of an inhibitory peptide, allatotropin (AT). In situ hybridization and MALDI-TOF MS analysis revealed the presence of three subtypes of sNPFs (sNPF-1, -2, and -3) in the midgut enteroendocrine cells. Ca2+-imaging analyses revealed that three subtypes of sNPF receptors (sNPFRs) (BNGR-A7, -A10, and -A11) showed different affinities with the three subtypes of sNPFs. In addition, sNPF activated its signaling via ERK phosphorylation in the midgut, while mixture of sNPF and AT reduced the phosphorylation level, agreeing with the results of behavioral assay. Together, our current findings suggest that intestinal sNPF positively modulates the feeding motivation by reducing the inhibitory effects by AT within the midgut.

Insulin-like growth factor (IGF)-like peptide and 20-hydroxyecdysone regulate the growth and development of the male genital disk through different mechanisms in the silkmoth, Bombyx mori.

It is well established that ecdysteroids play pivotal roles in the regulation of insect molting and metamorphosis. However, the mechanisms by which ecdysteroids regulate the growth and development of adult organs after pupation are poorly understood. Recently, we have identified insulin-like growth factor (IGF)-like peptides (IGFLPs), which are secreted after pupation under the control of 20-hydroxyecdysone (20E). In the silkmoth, Bombyx mori, massive amounts of Bombyx-IGFLP (BIGFLP) are present in the hemolymph during pupal-adult development, suggesting its importance in the regulation of adult tissue growth. Thus, we hypothesized that the growth and development of adult tissues including imaginal disks are regulated by the combined effects of BIGFLP and 20E. In this study, we investigated the growth-promoting effects of BIGFLP and 20E using the male genital disks of B. mori cultured ex vivo, and further analyzed the cell signaling pathways mediating hormone actions. We demonstrate that 20E induces the elongation of genital disks, that both hormones stimulate protein synthesis in an additive manner, and that BIGFLP and 20E exert their effects through the insulin/IGF signaling pathway and mitogen-activated protein kinase pathway, respectively. These results show that the growth and development of the genital disk are coordinately regulated by both BIGFLP and 20E.