Molting in decapod crustaceans is controlled by molt-inhibiting hormone (MIH), an eyestalk neuropeptide that suppresses production of ecdysteroids by a pair of molting glands (Y-organs or YOs). Eyestalk ablation (ESA) activates the YOs, which hypertrophy and increase ecdysteroid secretion. At mid premolt, which occurs 7-14days post-ESA, the YO transitions to the committed state; hemolymph ecdysteroid titers increase further and the animal reaches ecdysis ~3weeks post-ESA. Two conserved signaling pathways, mechanistic target of rapamycin (mTOR) and transforming growth factor-β (TGF-β), are expressed in the Gecarcinus lateralis YO. Rapamycin, an mTOR antagonist, inhibits YO ecdysteroidogenesis in vitro. In this study, rapamycin lowered hemolymph ecdysteroid titer in ESA G. lateralis in vivo; levels were significantly lower than in control animals at all intervals (1-14days post-ESA). Injection of SB431542, an activin TGF-β receptor antagonist, lowered hemolymph ecdysteroid titers 7 and 14days post-ESA, but had no effect on ecdysteroid titers at 1 and 3days post-ESA. mRNA levels of mTOR signaling genes Gl-mTOR, Gl-Akt, and Gl-S6k were increased by 3days post-ESA; the increases in Gl-mTOR and Gl-Akt mRNA levels were blocked by SB431542. Gl-elongation factor 2 and Gl-Rheb mRNA levels were not affected by ESA, but SB431542 lowered mRNA levels at Days 3 and 7 post-ESA. The mRNA level of an activin TGF-β peptide, Gl-myostatin-like factor (Mstn), increased 5.5-fold from 0 to 3days post-ESA, followed by a 50-fold decrease from 3 to 7days post-ESA. These data suggest that (1) YO activation involves an up regulation of the mTOR signaling pathway; (2) mTOR is required for YO commitment; and (3) a Mstn-like factor mediates the transition of the YO from the activated to the committed state.

No abstract

There was an error published in J. Exp. Biol. 217, In Fig. 3, the letters and numbers indicating which means were significantly different are missing from panel A. The correct figure is printed below.We apologise to the authors and readers for this omission.

There was an error published in J. Exp. Biol. 217, In Fig. 3, the letters and numbers indicating which means were significantly different are missing from panel A. The correct figure is printed below.We apologise to the authors and readers for this omission. ) characteristic of late premolt animals and animals did not molt by 90 days post-ESA. There was no effect of ESA or MLA on the expression of Cm-elongation factor 2 (EF2), Cm-NOS, the beta subunit of GC-I (Cm-GC-Iβ), a membrane receptor GC (Cm-GC-II) and a soluble NO-insensitive GC (Cm-GC-III) in green morphs. Red morphs were affected by prolonged ESA and MLA treatments, as indicated by large decreases in Cm-EF2, Cm-GC-II and Cm-GC-III mRNA levels. ESA accelerated the transition of green morphs to the red phenotype in intermolt animals. ESA delayed molting in premolt green morphs, whereas intact and MLA animals molted by 30 days post treatment. There were significant effects on YO gene expression in intact animals: Cm-GC-Iβ mRNA increased during premolt and Cm-GC-III mRNA decreased during premolt and increased during postmolt. Cm-MIH transcripts were detected in eyestalk ganglia, the brain and the thoracic ganglion from green intermolt animals, suggesing that MIH in the brain and thoracic ganglion prevents molt induction in green ESA animals.

SUMMARY Molt-induced claw muscle atrophy in decapod crustaceans facilitates exuviation and is coordinated by ecdysteroid hormones. There is a 4-fold reduction in mass accompanied by remodeling of the contractile apparatus, which is associated with an 11-fold increase in myofibrillar protein synthesis by the end of the premolt period. Loss of a walking limb or claw causes a loss of mass in the associated thoracic musculature; this unweighting atrophy occurs in intermolt and is ecdysteroid independent. Myostatin (Mstn) is a negative regulator of muscle growth in mammals; it suppresses protein synthesis, in part, by inhibiting the insulin/metazoan target of rapamycin (mTOR) signaling pathway. Signaling via mTOR activates translation by phosphorylating ribosomal S6 kinase (s6k) and 4E-binding protein 1. Rheb (Ras homolog enriched in brain), a GTP-binding protein, is a key activator of mTOR and is inhibited by Rheb-GTPase-activating protein (GAP). Akt protein kinase inactivates Rheb-GAP, thus slowing Rheb-GTPase activity and maintaining mTOR in the active state. We hypothesized that the large increase in global protein synthesis in claw muscle was due to regulation of mTOR activity by ecdysteroids, caused either directly or indirectly via Mstn. In the blackback land crab, Gecarcinus lateralis, a Mstn-like gene (Gl-Mstn) is downregulated as much as 17-fold in claw muscle during premolt and upregulated 3-fold in unweighted thoracic muscle during intermolt. Gl-Mstn expression in claw muscle is negatively correlated with hemolymph ecdysteroid level. Full-length cDNAs encoding Rheb orthologs from three crustacean species (G. lateralis, Carcinus maenas and Homarus americanus), as well as partial cDNAs encoding Akt (Gl-Akt), mTOR (Gl-mTOR) and s6k (Gl-s6k) from G. lateralis, were cloned. The effects of molting on insulin/mTOR signaling components were quantified in claw closer, weighted thoracic and unweighted thoracic muscles using quantitative polymerase chain reaction. Gl-Rheb mRNA levels increased 3.4-fold and 3.9-fold during premolt in claw muscles from animals induced to molt by eyestalk ablation (ESA) and multiple leg autotomy (MLA), respectively, and mRNA levels were positively correlated with hemolymph ecdysteroids. There was little or no effect of molting on Gl-Rheb expression in weighted thoracic muscle and no correlation of Gl-Rheb mRNA with ecdysteroid titer. There were significant changes in Gl-Akt, Gl-mTOR and Gl-s6k expression with molt stage. These changes were transient and were not correlated with hemolymph ecdysteroids. The two muscles differed in terms of the relationship between Gl-Rheb and Gl-Mstn expression. In thoracic muscle, Gl-Rheb mRNA was positively correlated with Gl-Mstn mRNA in both ESA and MLA animals. By contrast, Gl-Rheb mRNA in claw muscle was negatively correlated with Gl-Mstn mRNA in ESA animals, and no correlation was observed in MLA animals. Unweighting increased Gl-Rheb expression in thoracic muscle at all molt stages; the greatest difference (2.2-fold) was observed in intermolt animals. There was also a 1.3-fold increase in Gl-s6k mRNA level in unweighted thoracic muscle. These data indicate that the mTOR pathway is upregulated in atrophic muscles. Gl-Rheb, in particular, appears to play a role in the molt-induced increase in protein synthesis in the claw muscle.