An ecdysteroid-responsive gene in a lobster - a potential crustacean member of the steroid hormone receptor superfamily.

The role of ecdysteroids in modulating exoskeletal growth during the moult cycle of Crustacea has been well described. However, little is known about the action of ecdysteroids at the level of gene transcription and regulation in Crustacea. This paper reports the cloning of an ecdysteroid responsive gene, HHR3, a potential Manduca sexta MHR3 homologue in the American lobster, Homarus americanus. Levels of HHR3 expression are up-regulated in response to in vivo injections of premoult concentrations (10(-6) M) of 20-hydroxyecdysone in the epidermal and muscle tissue of the lobster after 6 h. Maximal mRNA levels are observed after 21 h before returning to basal levels. In muscle tissue, elevated levels of HHR3 mRNA follow a time course similar to elevated actin mRNA expression in response to hormonal injection. In contrast, in eyestalk tissue, the HHR3 levels decline up to 21 h post-injection before rising to basal levels after 48 h. Eyestalk, epidermal and leg muscle tissue was extracted over the moult cycle to determine the levels of expression. In muscle, HHR3 is high during the premoult period that corresponds to the period of the moult cycle when the ecdysteroid titre is high. In the epidermis, HHR3 levels are also high during the premoult with elevated levels maintained into the postmoult period. In the eyestalk, mRNA levels of HHR3 show an opposite pattern of expression with low levels during premoult and postmoult and high levels found during the intermoult period. Our results provide novel evidence for an ecdysteroid responsive gene in a crustacean that has many similarities to MHR3 in Manduca and DHR3 in Drosophila melanogaster. This raises the question of whether a similar cascade of ecdysteroid responsive genes exist in other members of Arthropoda such as the Crustacea, as has been demonstrated in Drosophila. In addition, we provide further evidence for negative feedback regulation of ecdysteroids at the site of moult-inhibiting hormone (MIH) production in the lobster eyestalk.

Identification and characterization of methyl farnesoate binding proteins from the crab, Cancer magister.

Methyl farnesoate (MF) binding proteins were identified in the hemolymph of male crabs, Cancer magister, using a tritium-labeled photoaffinity analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [3H]FDK in the presence of increasing amounts of unlabeled MF and the proteins were separated using SDS-PAGE. The associated fluorogram revealed the presence of two specific MF binding proteins with apparent molecular masses of 34 and 44 kDa. MF binding proteins were not detected in other tissues including testes, eyestalks, hepatopancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [3H]FDK from hemolymph MF binding proteins in a dose-dependent way. The apparent dissociation constant (Kd) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand binding assay followed by Scatchard analysis was used to determine a more accurate apparent Kd value of 145 +/- 10 nM. A single MF binding peak was demonstrated when hemolymph samples incubated with [3H]FDK were electrophoresed under nondenaturing conditions.

Methyl farnesoate stimulates ecdysteroid secretion from crab Y-organs in vitro.

Methyl farnesoate (MF) stimulation of ecdysteroid secretion by Y-organs of the crab, Cancer magister, is demonstrated. Isolated Y-organs that were incubated with a mandibular organ (MO) secreted significantly higher levels of ecdysteroids into the medium when compared to those incubated in the absence of a MO. MOs secrete both farnesoic acid (FA) and MF into the medium and are not themselves a source of ecdysteroids. To determine if MF has a role in the regulation of ecdysteroid secretion, Y-organs from C. magister were incubated with various concentrations of MF. Y-organs in the presence of MF secreted significantly more ecdysteroids into the medium after a 24-hr incubation when compared to controls (P < or = 0.05). The magnitude of this stimulation increased with higher concentrations of MF and increasing incubation times. The response was specific to (2E,6E)-MF. The cis,trans isomer of MF ((2Z,6E)-MF), FA, and farnesyl diazomethyl ketone, a photoaffinity analog of MF, did not have any effect on ecdysteroid secretion from Y-organs. The primary ecdysteroid secreted by C. magister Y-organs comigrates with authentic ecdysone and its secretion is stimulated by MF.