mTOR signaling genes are involved in molt regulation during thermal acclimation of juvenile Dungeness crabs (Metacarcinus magister)

We hypothesize that the mTOR pathway and AMP-dependent protein kinase (AMPK) are involved in the regulation of the molt cycle in response to temperature in juvenile Dungeness crabs Metacarcinus magister. We used PCR cloning to obtain partial cDNA sequences of mTOR, Rheb, AKT, S6K and AMPK. We incubated crabs at 12 d, 18 d and 26 d postmolt at 5, 10, 15, 20, 25 and 30°C for up to 14 d to study survival and progression of the molt cycle. We determined gene expression in the molting gland of AMPK, AKT and Rheb (upstream of mTOR), mTOR, S6K (downstream of mTOR) and of the possible reference gene RbS3 by quantitative real-time PCR in crabs that had been held at 10, 15 or 20°C. Hemolymph molting hormone titers were determined by ecdysteroid ELISA. Survival was 97-100% at temperatures from 5-20°C, and was time-dependent, but 0% after 14 d at 25°C. All animals had died after 24 h at 30°C. Progression of the molt cycle was observed at 10, 15 and 20°C, but not at 5°C. Two-way ANOVA revealed a significant effect of molt stage on the expression of all investigated genes, and significant effects of temperature on AKT, Rheb and mTOR expression. The GTPase Rheb was most strongly regulated at the mRNA level, with an up to 2.9-fold increase between intermolt and late premolt, and a 2.7-fold decrease between 10 and 20°C of animals in intermolt. There was a significant positive correlation of Rheb expression with ecdysteroid titers at all temperatures. We conclude that the mTOR pathway, and especially Rheb, is involved in the regulation of the molt cycle during thermal acclimation by controlling protein synthesis necessary for the production and release of molting hormones. Funding: EU FP7 Marie Curie International Outgoing Fellowship PIOF-GA-2012-326483 to ACW, NSF grant IOS-1257732 to DLM.