Transcriptomic analysis of crustacean molting gland (Y-organ) regulation via the mTOR signaling pathway

S Shyamal,D L Mykles,S Das,A Guruacharya,D S Durica

doi:10.1038/s41598-018-25368-x

S Shyamal, D L Mykles + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-25368-x

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Abstract

The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-β signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.

Highlights

The Y-organ (YO), or molting gland, is the source of steroid hormone production and consequent molt cycle regulation in decapod crustaceans, and is responsive to both external environmental and internal physiological signals[1,2,3]
Previous studies have implicated the mechanistic target of rapamycin (mTOR) pathway in mediating Y organs (YOs) transcriptional responses associated with YO activation during premolt[13,14] and we examined in detail mTOR signaling genes within the Kyoto Encyclopedia for Genes and Genomes (KEGG) orthology (KO) reference pathway
We focused on other pathways that interact with mTOR signaling, the Wnt, AMPK, insulin signaling, and insect biosynthetic hormone pathways

Summary

Introduction

The Y-organ (YO), or molting gland, is the source of steroid hormone production and consequent molt cycle regulation in decapod crustaceans, and is responsive to both external environmental and internal physiological signals[1,2,3]. In addition to translational control, increases in G. lateralis mRNA levels of mTOR signaling components Gl-mTOR and Gl-Akt coincide with increased YO ecdysteroid production during mid and late premolt stages[13]. We hypothesize that Gl-Mstn may have a similar function in crustaceans, as the YO shows decreased ecdysteroid production when TGFβ signaling is inhibited[14] and shows a continuous increase in ecdysteroid synthesis in response to reduced sensitivity to MIH5,7. Taken together, these data suggest that mTOR and TGFβ/Smad pathways play essential roles in the regulation of both crustacean and insect molting glands by neuropeptides. The mTOR pathway has been associated with regulation at the translational level, this analysis indicates mTOR pathway interference contributes to both increases and decreases in gene transcription associated with genes encoding putative regulatory pathway components, as well as the down-regulation of ecdysteroid biosynthetic pathway genes

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