Molting in arthropods is a complex process governed by regulatory mechanisms that have evolved and adapted over millennia to allow these animals to grow, despite being confined by a hardened exoskeleton. We isolated the molt-regulating Y-organs (YO) from the common shore crab Carcinus maenas at molt stages B, C1-3, C4, and D0 to assess how changes in protein abundances might underline the unique physiology of each of these stages. We found that changes in protein abundance were most notable in the postmolt stages (B and C1-3), where an increase in energy metabolism and the reactive oxygen species stress (ROS) response proteins was observed. An increase in triosephosphate isomerase and transketolase suggest that the postmolt YO is participating in triglycerides storage and is also actively recycling excess ribose sugars manufactured during the YO’s previously activated state. We also propose as mechanism through which ROS-induced release of cyclophilin A may contribute to YO atrophy during postmolt through the remodeling of structural proteins such as collagen. We support the standing observation of YO atrophy during postmolt by drawing attention to hemolymph protein abundances, especially those of cryptocyanin isoforms, which dropped precipitously in intermolt (C4) and remained at low abundances into early premolt (D0). Finally, though our evidence is preliminary, we propose that future investigations into the YO proteome address the significance of the protein glutamate dehydrogenase. Glutamate dehydrogenase, a key enzyme involved in the formation of glutamate, represents a potential nutrient-sensing checkpoint that might be involved in YO activation. Historically, most attention has gone to the acute molt stages, where signaling mechanisms involved in the activation of the YO have been the focus. Here, we present data suggesting that other regulatory mechanism may be governing the atrophy the postmolt YO. A better understanding of crustacean physiology has the potential to benefit ecosystems and economies worldwide.

Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of the molting hormones, ecdysteroids, by the YO. Analysis of gene expression in the XOs and YOs has led to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting in crustaceans. In this study, changes in protein abundance in the YO were characterized over the course of a molt cycle (intermolt, early premolt, mid premolt, and late premolt) induced by multiple leg autotomy (MLA) in the blackback land crab, Gecarcinus lateralis. In all, 457 distinct protein spots were detected in the molting gland using two-dimensional gel electrophoresis, of which 230 (50%) changed significantly in abundance over the course of the molt cycle (one-way permutation ANOVA, p≤0.05). Changes in protein abundance were most notable between the intermolt and the three premolt stages, indicative of a biological ‘on-off’ switch in the Y-organ. Several hemolymph species proteins, including hemocyanin, cryptocyanin, and transglutaminase, were identified which characterized physiological changes associated with molting beyond the Y-organ. An abundance of cytoskeletal proteins were identified which correspond with glandular hypertrophy and are indicative of vesicular-mediated exocytosis, possibly of ecdysteroids. Further, several proteins involved in the immune, proteostasis, and oxidative stress response are characteristic of supporting the dynamic and demanding cellular changes associated with ecdysteroidogenesis and the transition of the Y-organ from the basal to the highly active state. Many proteins involved in energetic pathways including glycolysis, the citric acid cycle, amino acid metabolism, and one-carbon metabolism changed in abundance in response to both the higher energy demands and the requirement for precursors of macromolecular synthesis of the YO over the molt cycle. Taken together, these changes in diverse physiological pathways represent the complexity involved with regulation of the Y-organ, even with just the single proposed physiological purpose of ecdysteroidogenesis.