Effect of blocking transforming growth factor-β/Activin-Myostatin signaling on the expression of ecdysteroid metabolism and responsive genes in the crustacean molting gland (Y-organ)

Molting in decapod crustaceans is controlled by ecdysteroids synthesized and secreted by the molting gland, or Y-organ (YO). The YO undergoes phenotypic changes in ecdysteroid production that drive molt cycle stage transitions; these are the basal, activated, committed, and repressed states in the intermolt, early premolt, mid- and late premolt, and postmolt stages, respectively. Reduced secretion of molt-inhibiting hormone (MIH) by a neurosecretory center in the eyestalk ganglia activates the YO and the animal transitions to early premolt. During premolt, transforming growth factor-beta (TGFβ)/Activin-Myostatin (Mstn) signaling mediates the transition of the YO from the activated to the committed state, as SB431542 blocks this transition. In the blackback land crab, Gecarcinus lateralis, the YO expresses genes involved in ecdysteroid synthesis (Gl-NADK, Gl-ALAS and Halloween genes Gl-Nvd, Gl-Spo, Gl-Phm, Gl-Dib, and Gl-Sad) and catabolism (Gl-CYP18a1); ecdysteroid signaling (ecdysteroid responsive genes Gl-EcR, Gl-RXR, Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, and Gl-Ftz-f1); and Gl-FOXO. Intermolt adult G. lateralis were induced to molt by eyestalk ablation (ESA) and injected with either dimethyl sulfoxide (DMSO) vehicle (control) or SB431542 in DMSO (experimental) at Day 0. ESA increased hemolymph ecdysteroid titer at 1, 3, and 5 days post-ESA in both control and experimental groups, indicating that SB431542 had no effect on YO activation. Ecdysteroid titer did not increase further in the experimental group at 7 and 14 days post-ESA, indicating that SB431542 prevented transition of the YO to the committed state. ESA with or without SB431542 had no effect on the mRNA levels of the eight ecdysteroid metabolism genes, seven of the eight ecdysteroid responsive genes (the only exception was Gl-E74 at 1 day post-ESA), and Gl-FOXO at 1, 3, and 5 days post-ESA. Compared to the control group, SB431542 lowered the mRNA level of Gl-Nvd at 7 and 14 days post-ESA and mRNA levels of Gl-Spo, Gl-Phm, Gl-Dib, Gl-Sad, Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-EcR, Gl-RXR, Gl-Br-C, and Gl-FOXO at 14 days post-ESA. SB431542 had no effect on the mRNA levels of Gl-HR3 Gl-HR4, Gl-E74, Gl-E75 and Gl-Ftz-f1. These results suggest that TGFβ/Activin-Mstn signaling maintains the mRNA levels of genes needed for increased ecdysteroid synthesis and signaling in the committed YO during mid- and late premolt.