Crustacean molt-inhibiting hormone: Structure,function, and cellular mode of action

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.     

Ecdysone and retinoid-X receptors of the blue crab, Callinectes sapidus: Cloning and their expression patterns in eyestalks and Y-organs during the molt cycle

Crustacean molting is known to be regulated largely by ecdysteroids and crustacean hyperglycemic hormone (CHH) neuropeptide family including molt-inhibiting hormone (MIH) and CHH. The surge of 20-OH ecdysone and/or ponasterone A initiates the molting process through binding to its conserved heterodimeric nuclear receptor: Ecdysone Receptor (EcR) and Ultraspiracle (USP)/Retinoid-X Receptor (RXR). To better understand the role of ecdysteroids in the molt regulation, the full-length cDNAs of the blue crab, Callinectes sapidus EcR1 and RXR1 were isolated from the Y-organs and their expression levels were determined in both Y-organs and eyestalks at various molt stages. Y-organs show the expression of four putative isoforms of CasEcRs and CasRXRs which differ in the length of the open reading frame but share the same domain structures as in typical nuclear receptors: AF1, DBD, HR, LBD, and AF2. The putative CasEcR isoforms are derived from a 27-aa insert in the HR and a 49-aa residue substitution in the LBD. In contrast, an insertion of a 5-aa and/or a 45-aa in the DBD and LBD gives rise to CasRXR isoforms. The eyestalks and Y-organs show the co-expression of CasEcRs and CasRXRs but at the different levels. In the eyestalks, the expression levels of CasRXRs are 3–5 times higher than those of CasEcRs, while in Y-organs, CasRXRs are 2.5–4 times higher than CasEcRs. A tissue-specific response to the changes in the levels of hemolymphatic ecdysteroids indicates that these tissues may have differences in the sensitivity or responsiveness to ecdysteroids. The presence of upstream open reading frame and internal ribosome entry site in 5′ UTR sequences of C. sapidus and other arthropod EcR/RXR/USP analyzed by in silico indicates a plausible, strong control(s) of the translation of these receptors.     

Ontogeny of neuroendocrine centers in the eyestalk of Homarus gammarus embryos: an anatomical and hormonal approach

Summary

The ontogeny of the eyestalk neuroendocrine centers of the European lobster, Homarus gammarus, throughout embryonic development has been studied using light and electron microscopy, and the localization of specific neuroendocrine substances has been identified by immunocytochemistry. The procephalic lobes, which are the prospective eyestalks, develop progressively during embryonic development. In the nauplius stage two neuron masses are well defined. The visual structure originates from one of them and the neuroendocrine structure from the other. The four definitive optic ganglia are present at the mid-metanauplius stage and retain their appearance and location in larvae and adults. The organ of Bellonci, an internal sensory structure, appears at the mid-metanauplius stage and is mainly characterized by onion bodies. The medulla terminalis X-organ complex, an important neuroendocrine system, is present and already functional at the beginning of the embryonic metanauplius stage. Two neurohormones have been visualized immunocytochemically: the crustacean hyperglycemic hormone (CHH) and the gonad inhibiting hormone (GIH). Both neuropeptides are localized in the perikarya of neuroendocrine cells of the X-organ as well as in their tracts joining the presumptive sinus gland. However, the sinus gland has only been observed in the early larval stages just after hatching.     

中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达

根据实验室分离自中华绒螯蟹(Eriocheir sinensis)的一种蜕皮抑制激素(Molting-inhibiting hormone,MIH)N端氨基酸测序结果设计简并引物,采用RACE方法,首次从中华绒螯蟹眼柄中克隆到蜕皮抑制激素基因全长cDNA(Es-MIH,GenBank登录号:DQ341280),该基因全长为1457 bp,开放阅读框为330 bp,编码110个氨基酸(含有35个氨基酸的信号肽);其成熟肽包含C7-C44、C24-C40和C27-C53三个二硫键,有典型的CHH家族结构域。该cDNA编码的氨基酸序列与地蟹(Gecarcinus lateralis)MIH同源性最高,达到了85%。Northern杂交和半定量RT-PCR显示蜕皮间期成体蟹仅在眼柄中有MIH基因表达,提示该基因的表达具有一定组织特异性。利用pCR T7/NT TOPO TA系统重组表达MIH成熟肽,纯化的重组蛋白得率为0.3 g/L,纯化产物经质谱鉴定为中华绒螯蟹MIH。研究解决了CHH家族神经肽在机体中的表达量少,直接纯化较难的问题,为深入研究MIH的作用机制和在生产上控制中华绒螯蟹蜕皮和生长奠定了基础。     

Regulation of protein synthesis in Y-organs of the blue crab (Callinectes sapidus): involvement of cyclic AMP

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-Organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide produced and released by the X-organ/sinus gland complex of the eyestalks. In the present studies, crab (Callinectes sapidus) Y-organs were incubated in vitro in the presence of [(35)S]methionine, and cyclic nucleotide analogs or experimental agents that influence the cAMP signaling pathway. In 4-hr incubations, 8-Br-cAMP and db-cAMP (but not 8-Br-cGMP) suppressed incorporation of [(35)S]methionine into Y-organ proteins; the effect of 8-Br-cAMP was concentration-dependent. Autoradiograms of radiolabeled Y-organ proteins separated on SDS-PAGE gels indicated the effect of 8-Br-cAMP was general (as opposed to selective) suppression of protein synthesis. Addition of both forskolin (an adenylyl cyclase activator) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) likewise suppressed incorporation of [(35)S]methionine into Y-organ proteins. Cycloheximide (a protein synthesis inhibitor) suppressed incorporation of [(35)S]methionine into Y-organ proteins and secretion of ecdysteroids. The combined results suggest that cAMP is involved in regulation of protein synthesis in C. sapidus Y-organs. We are currently investigating the link of protein synthesis to ecdysteroid production, and the possibility of cross-talk between cAMP and other cellular signaling pathways in Y-organs.     

Effects of bilateral and unilateral eyestalk ablation on vitellogenin synthesis in immature female kuruma prawns, Marsupenaeus japonicus

In penaeid shrimp species, vitellogenin (VTG) synthesis in the ovary and hepatopancreas is under the inhibitory regulation of a neuroendocrine system, the X-organ/sinus gland complex in the paired eyestalks, and eyestalk ablation (removal of the X-organ/sinus gland complex) is widely used for inducing ovarian development. However, the difference in effects of bilateral and unilateral ablation on VTG gene expression has not been clarified so far. In the present study, VTG synthesis was monitored over a 16-day period after ablation and compared between replicates of immature female kuruma prawns, Marsupenaeus (Penaeus) japonicus, that had been bilaterally or unilaterally ablated and control specimens. After bilateral ablation, ovarian development was induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary increased significantly. Significant VTG mRNA increase was detected 12 h after bilateral ablation. In contrast, after unilateral ablation, ovarian development was not induced, and the ovarian weight, hemolymph VTG levels, and VTG mRNA levels in the ovary did not change significantly from the control. These results indicate that in immature female prawns, unilateral ablation does not induce VTG gene expression, whereas bilateral ablation induces rapid VTG gene expression (<12 h). The ineffectiveness of unilateral ablation suggests that the remaining X-organ/sinus gland complex in the unilaterally ablated female prawns may secrete sufficient VIH to suppress VTG synthesis.     

Neurosecretory systems in decapod Crustacea

Summary The sinus glands of the Brachyura are composed of swollen nerve fiber endings, which store and release secretory material synthesized within cells of the eyestalks, the brain and probably the thoracic ganglionic mass. Removal of the sinus glands from the land crab, Gecarcinus lateralis, does not induce molt, because sinus glands are reservoirs, not sources, of molt-inhibiting hormone. Increase in respiratory rate and fall in respiratory quotient, which follow eyestalk removal and signify the approach of molt, do not occur after sinus gland removal.From recent studies on the eyestalks and brain of the crayfish, Cambarus virilis, it is clear that morphologically the neurosecretory system of this crayfish is similar to that of the land crab. There is a marked resemblance in arrangement of neurosecretory cells and in pathways followed by the fibers, the endings of which form the sinus glands. Maps of eyestalks and brain of Cambarus and Gecarcinus emphasize this fundamental likeness between an astacuran and a brachyuran. Regions in which neurosecretory cells are found have been numbered so that these maps may guide cytological and physiological study on these species and on other decapod Crustacea.This paper was delivered at the Symposium on Neurosecretion held May 11–16, 1953, at the Stazione Zoologica, Naples, Italy. An abstract of this paper. together with summaries of other papers presented at the Symposium, is appearing in the Pubblicazioni della Stazione Zoologica di Napoli.The preparation of this paper was aided by a National Science Foundation (USA.) grant to the first author and by the use of the American Table at the Naples Zoological Station.     

Evidence for matrotrophy in the viviparous sea cucumber Leptosynapta clarki: A role for the genital haemal sinus?

Summary

Viviparity, where the embryos develop in the female reproductive system, is a rare form of reproduction in marine invertebrates, being described in only 14 species of echinoderm. In the intraovarian brooding sea cucumber, Leptosynapta clarki Heding 1928 (cf., Sewell et al. 1995), we used direct evidence (changes in energetic content) to show that significant additional nutrients are provided to the embryos during viviparous development (matrotrophy). In the transition from a structure used to produce gametes to a long-term brooding structure there are visual, histological and transmission electron microscopy (TEM) changes in the structure of the ovarian wall. Changes occur primarily in the cells of the visceral peritoneum and involve an increase in size of the connective tissue/genital haemal sinus (CT/GHS). In the latter part of the brooding period the visceral peritoneum returns to a flattened form, and new oocytes develop along the tubule wall. Similar changes in the intraovarian brooding sea cucumber Oneirophanta mutabilis affinis lead us to suggest that there is a role for the genital haemal sinus in providing nutrition during the brooding period in viviparous echinoderms. Future research is suggested to focus on changes in the ovarian wall structure during the different phases of reproduction (gamete production/brooding) in these species.     

Development of pigment-dispersing hormone-immunoreactive neurons in the American lobster: homology to the insect circadian pacemaker system?

We have examined the development of pigment-dispersing hormone (PDH)-immunoreactive neurons in embryos of the American lobster Homarus americanus Milne Edwards, 1837 (Decapoda, Reptantia, Homarida) by using an antiserum against β-PDH. This peptide is detectable in the terminal medulla of the eyestalks and the protocerebrum where PDH immunoreactivity is present as early as 20% of embryonic development. During ontogenesis, an elaborate system of PDH-immunoreactive neurons and fibres develops in the eyestalks and the protocerebrum, whereas less labelling is present in the deuto- and tritocerebrum and the ventral nerve cord. The sinus gland is innervated by PDH neurites at hatching. This pattern of PDH immunoreactivity has been compared with that found in various insect species. Neurons immunoreactive to pigment-dispersing factor in the medulla have been shown to be a central component of the system that generates the circadian rhythm in insects. Our results indicate that, in view of the position of the neuronal somata and projection patterns of their neurites, the immunolabelled medulla neurons in insects have homologous counterparts in the crustacean eyestalk. Since locomotory and other activities in crustaceans follow distinct circadian rhythms comparable with those observed in insects, we suggest that PDH-immunoreactive medulla neurons in crustaceans are involved in the generation of these rhythms. This study was supported by Deutsche Forschungsgemeinschaft (DFG) grant Ha 2540 and National Science Foundation grant IBN 0344448. S.H. was a Heisenberg Fellow of the DFG during the experimental part of this study. Bill Hansson and the Max Planck Society provided support during the final period of work reported in this paper.     

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks

In decapod crustaceans, molt hormone (ecdysone) production by Y-organs is suppressed by an eyestalk neurosecretory product, molt-inhibiting hormone (MIH). Environmental stressors are known to delay or prevent molting in crabs. The present study assesses the function of the MIH-Y-organ neuroendocrine system in the crab Cancer antennarius under conditions of daily handling stress. After three days, stressed crabs showed significant suppression of hemolymph ecdysteroid levels, which continued to fall to 20% of controls by day 14. Ecdysteroid titers of stressed crabs returned to prestress levels seven days after stress termination. Ecdysteroid levels in de-eyestalked (DES) crabs rose 160% within 48 hr post-DES. Stressing DES crabs over 16 subsequent days did not significantly alter ecdysteroid levels compared with unstressed DES controls. Handling stress thus depresses hemolymph ecdysteroid levels in the crab, a response that is mediated by eyestalks and appears to result from stress-induced MIH release.     

Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture and a bioassay for MIH activity

Ecdysteroid secretion in vitro by gland quarters and dispersed cells of ecdysial glands (Y-organs) of the crab, Cancer antennarius Stimpson, was characterized. Optimum culture conditions are reported for maximum, sustained (72 hr) secretion and maintenance of cell viability in activated Y-organs obtained from de-eyestalked donors. Addition in vitro of eyestalk ganglia extracts containing the putative molt-inhibiting hormone (MIH) inhibited ecdysteroid production dose-dependently in the range of 0.1-4.0 and 0.01-4.0 eyestalk equivalents of MIH for gland quarters and dispersed cells, respectively. Inhibition by MIH was reversible, tissue specific as to source of MIH activity, and did not affect cell viability relative to controls. The results of replicate incubations of gland quarters with MIH were analyzed with formal statistics of parallel-line assay. The inhibitory action on ecdysteroid secretion is shown to be reproducibly linear and parallel in the dosage range, 0.1-4.0 eyestalk equivalents, amenable to calculation of relative potency among successive extracts, and of sufficiently high precision to serve as an MIH bioassay. Also, the results of these studies support the hypothesis that control of Y-organs by the eyestalks is physiologically direct.     

Isolation and use of embryonic stem cells from livestock species

Abstract

Embryonic stem (ES) cells are pluripotent cells isolated from early embryos. They proliferate in culture and retain the capacity to differentiate both in vitro and In vivo, including contributing to chimeric tissues after injection into normal blastocysts. Over the past decade ES cells have been used extensively as a model for embryogenesis. More recently they have been shown to be capable of stable integration of exogenous DNA and used for numerous studies involving genomic manipulation. ES cells provide many opportunities for genetic engineering of domestic livestock species, but to date their isolation from embryos has been documented only for the mouse and perhaps the hamster. Efforts to isolate pluripotent ES cells from embryos of domestic livestock species are described, including some of the problems encountered.     

In vitro secretion of ecdysteroids by Y-organs and lack of secretion by mandibular organs of the crayfish following molt induction

Summary The in vitro secretion of ecdysteroids by Y-organs taken from crayfish at different times after eyestalk removal was analyzed by radioimmunoassay. Analysis by thin layer chromatography of the immunoreactive secretion products suggests the presence of both ecdysone and ecdysterone, the former probably being the predominant product. The secretion rate exhibits a significant increase after 24 h and reaches a maximum 10–13 days after destalking. A marked decrease is observed after 17 days. A close correlation between the in vitro biosynthetic activity of the Y-organs and the in vivo level of hemolymph ecdysteroid exists until day 13. The level is maintained through day 17, in contrast with the decrease of the in vitro secretion rate of the Y-organs. Under the same conditions, mandibular organs do not secrete ecdysteroids detectable by radioimmunoassay. Selective sinus gland removal instead of destalking does not result in an activation of the Y-organs. Gastrolith deposition starts about 7 days after eyestalk removal and apolysis is completed after 12 days in most animals. At day 17, most animals were in the late D₂-stage. Occasionally, the first ecdyses were observed at this time.Dedicated to Professor P. Karlson on the occasion of his 60th birthday     

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y-organs: relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5?ng/mL on D0 to 49.6?ng/mL on D6 and 87.2?ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).     

The Foregut of Gecarcinus lateralis as an Organ of Salt and Water Balance

The permeability of the foregut of the land crab, Gecarcinuslateralis, to tritiated water (THO), Na²², and Cl³⁶ was studiedin vitro during the intermolt period and after ecdysis. In crabswith eyestalks, the foregut is permeable to water and ions inthe direction hemolymph-to-lumen and lumen-to-hemolymph, bothduring the intermolt period and after ecdysis. However, theforegut of animals without eyestalks is impermeable after ecdysis. The movement of THO always follows the movement of Na²² acrossthe wall of the foregut, while the movement of Cl³⁶ may or maynot be correlated with the movement of Na²² and THO. Comparisonof the ratio of water to ions in the hemolymph with the ratiocalculated from radioisotope flux indicates that Na⁺ and waterare probably moving isosmotically, although not necessarilyaccompanied by Cl^– When an extract of the thoracic ganglionic mass of G. lateralisis added to the "hemolymph side" of the foregut in vitro, thereis immediately a large increase in permeability to water andsalts. This occurs in the foregut of crabs with eyestalks duringintermolt and also in eyestalkless crabs after ecdysis. Thus, not only is the foregut of Gecarcinus lateralis permeableto water and salts in both directions, but also the extent ofits permeability is under neuroendocrine control. As a consequence,the animal may be able to move water and salts into the foregutor out of it into the hemolymph as needed. This may be an importantadaptation for a terrestrial crab that must conserve water,especially at the critical time of ecdysis.     

Ultrastructure of the y-organs of Cancer antennarius in normal and de-eyestalked crabs

The paired Y-organs of crustaceans control the molting process. In males of C. antennarius, these glands are opalescent, lobulated, epithelioid structures embedded in brown fatty tissue. Cells in the periphery extend processes to the connective tissue capsule, an arrangement that suggests increased surface area for metabolic exchange. The processes contain mitochondria and are tipped distally with electron dense material. The cytoplasm, scarce relative to nuclear volume, contains vesicles, polymorphic mitochondria with tubular cristae, and numerous free ribosomes, but little in way of smooth or rough endoplasmic reticulum or Golgi complexes. Progressing from intermolt to the premolt stage, mitochondria, as well as vesicles, and electron-dense particles in peripheral processes increase somewhat in number. Also, heterochromatin masses concentrate adjacent to the nuclear envelope. Eyestalk removal, which induces premolt stages in some species, did not produce consistent change in Y-organ substructure in C. antennarius. Although evidence is accumulating that Y-organs secrete a steroid molting hormone during late intermolt-premolt, the substructure of the glands exhibits neither (a) striking changes with the molt cycle, nor (b) all the characteristics typical of vertebrate steroid hormone synthesizing glands. Nevertheless, the structural features, respectively, are consistent with biochemical evidence that Y-organs (a) rapidly take up and convert sterol precursor and secrete a product without its accumulation or change in total sterol pool size, and (b) apparently cannot synthesize the sterol precursor. Y-organ cytology closely resembles that of some vertebrate steroid hormone secreting glands in which this synthetic capacity is minimal.     

Molt-inhibiting hormone immunoreactive neurons in the eyestalk neuroendocrine system of the blue crab, Callinectes sapidus

The production of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated by a neuropeptide, molt-inhibiting hormone. It is generally agreed that molt-inhibiting hormone is produced and released by the eyestalk neuroendocrine system. In the present study, immunocytochemical methods were used to detect molt-inhibiting hormone immunoreactive neurons in eyestalk ganglia of the blue crab, Callinectes sapidus. The primary antiserum used was generated against molt-inhibiting hormone of the green shore crab, Carcinus maenas. A preliminary Western blot analysis indicated the antiserum binds molt-inhibiting hormone of Callinectes sapidus. Using confocal and conventional immunofluorescence microscopy, molt-inhibiting hormone immunoreactivity was visualized in whole mounts and thin sections of Callinectes sapidus eyestalk ganglia. Immunoreactivity was detected in 15-25 neurosecretory cell bodies in the medulla terminalis X-organ, their associated axons and collateral branches, and their axon terminals in the neurohemal sinus gland. The cellular organization of molt-inhibiting hormone immunoreactive neurons in blue crabs is generally similar to that reported for other crab species. The combined results suggest the cellular structure of the molt-inhibiting hormone neuroendocrine system is highly conserved among brachyurans.     

Reciprocal neural influences upon the period length of the ERG circadian rhythm in the crayfish

Abstract

The relationship between left and right circadian oscillations in the ERG of the crayfishProcambarus bouvieri have been studied in intact animals and in animals with cerebral ganglion damage. Except in split‐brain preparations, the ERG oscillations of both sides were always in phase. Surgical bisection of the cerebral ganglion, also results in a shortening of the period length; this suggests that ERG circadian pacemakers normally receive a bilateral input of light stimulation. The neural connections between the eyestalks at the protocerebrum seem to have the most important role in the synchronization of the ERG oscillations of both sides.     

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

Summary The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D₂ of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.