Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland
Date
2012
Authors
Abuhagr, Ali Moftah M., author
Mykles, Donald L., advisor
Garrity, Deborah M., committee member
Reddy, Anireddy N., committee member
Curthoys, Norman P., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Regulation of the molt cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). Molt-inhibiting hormone (MIH), secreted by the XO/SG complex, suppresses production of molting hormone (ecdysteroids) by a pair of YOs. In the blackback land crab, Gecarcinus lateralis, molting can be induced by eyestalk ablation (ESA) or autotomy of 5 or more walking legs (multiple leg autotomy or MLA). During the molt cycle, the YO transitions through four physiological states: "basal" state at postmolt and intermolt; "activated" state at early premolt; "committed" state at mid premolt and "repressed" state at late premolt. The basal to activated state transition is triggered by a transient reduction in MIH; the YOs hypertrophy, but remain sensitive to MIH. The main hypothesis is that up-regulation of mechanistic Target of Rapamycin (mTOR) signaling, which controls global translation of mRNA into protein, is necessary for YO hypertrophy and ecdysteroidogenesis. cDNAs encoding mTOR, Rheb, Akt (protein kinase B) and p70 S6 kinase (S6k) were cloned from blackback land crab, G. lateralis, and green shore crab, Carcinus maenas. All four genes were expressed in all tissues examined. mTOR appears to be involved in YO activation in early premolt, as rapamycin inhibited YO ecdysteroidogenesis in vivo and in vitro. In addition, the expression of Gl-elongation factor 2 (EF2), Gl-mTOR, and Gl-Akt increased significantly in YOs from premolt, suggesting that an increase in protein synthetic capacity is necessary for YO activation. A putative transforming growth factor-beta (TGFâ) appeared to be involved in the transition of the YO from the activated to committed state, as SB431542, an Activin receptor antagonist, lowered hemolymph ecdysteroid titers in mid premolt animals and abrogated the premolt increases in Gl-EF2, Gl-mTOR, and Gl-Akt mRNA levels. By contrast, molting had no effect on Cm-EF2, Cm-mTOR, Cm-Rheb, Cm-Akt, and Cm-S6k expression in C. maenas YOs. Unlike G. lateralis, adult C. maenas was refractory to ESA. ESA caused a small increase in hemolymph ecdysteroid titers, but animals did not immediately enter premolt. Some ES-ablated animals molted after many months, but most failed to molt at all. We hypothesized that other regions of the nervous system, specifically the brain and/or thoracic ganglion, were secondary source(s) of MIH. Nested endpoint RT-PCR showed that MIH transcript was present in brain and thoracic ganglion of intermolt crabs. Sequencing of the PCR product confirmed its identity as MIH. Real time PCR was used to quantify the effects of ESA on MIH expression in brain and thoracic ganglion on C. maenas red and green color morphs. ESA had little effect on MIH transcript levels, indicating that MIH was not regulated transcriptionally by the loss of the eyestalks. The data suggest that MIH secreted by neurons in the brain and thoracic ganglion is sufficient to prevent molt induction when the primary source of MIH is removed by ESA. There was also no effect of ESA on the expression of Gl-EF2 and mTOR signaling components in C. maenas YOs.
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Subject
molting cycle in crustaceans
protein synthesis
mTOR signaling
molting in decapod crustaceans