Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland

Abstract

SUMMARY Two eyestalk (ES) neuropeptides, molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), increase intracellular cGMP levels in target tissues. Both MIH and CHH inhibit ecdysteroid secretion by the molting gland or Y-organ (YO), but apparently through different guanylyl cyclase(GC)-dependent pathways. MIH signaling may be mediated by nitric oxide synthase (NOS) and NO-sensitive GC. CHH binds to a membrane receptor GC. As molting affects neuropeptide signaling, the effects of ecdysteroid on the expression of the land crab Gecarcinus lateralis β subunit of a NO-sensitive GC (Gl-GC-Iβ), a membrane receptor GC (Gl-GC-II) and a NO-insensitive soluble GC (Gl-GC-III) were determined. Gl-GC-Iβ isoforms differing in the absence or presence of an N-terminal 32-amino acid sequence and Gl-GC-III were expressed at higher mRNA levels in ES ganglia, gill,hepatopancreas, ovary and testis, and at lower levels in YO, heart and skeletal muscle. Three Gl-GC-II isoforms, which vary in the length of insertions (+18, +9 and +0 amino acids) within the N-terminal ligand-binding domain, differed in tissue distribution. Gl-GC-II(+18) was expressed highly in striated muscle (skeletal and cardiac muscles); Gl-GC-II(+9) was expressed in all tissues examined (ES ganglia, YO, gill, hepatopancreas, striated muscles and gonads); and Gl-GC-II(+0) was expressed in most tissues and was the dominant isoform in ES and thoracic ganglia. ES ablation, which increased hemolymph ecdysteroid, increased Gl-GC-II(+18) mRNA level in claw muscle. Using real-time RT-PCR, ES ablation increased Gl-GC-Iβ, Gl-GC-III and ecdysone receptor mRNA levels in the YOs ∼ten-, ∼four- and∼twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A single injection of 20-hydroxyecdysone into intact animals transiently lowered Gl-GC-Iβ in hepatopancreas, testis and skeletal muscle, and certain Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other tissues can modulate responses to neuropeptides by altering GC expression.

Molt regulation in green and red color morphs of the crab,Carcinus maenas: gene expression of molt-inhibiting hormone signaling components

“…Although the increase in Cm-GC-Iβ expression during premolt (Fig. 8B) is not consistent with the hypothesis, it resembles the increase in Gl-GC-Iβ mRNA when the G. lateralis YO is activated by ESA (Lee et al, 2007a). The ecdysteroid titers and the structure of the integument indicated that 13 of the 18 premolt animals were in early premolt (Stage D 0 ; compare ecdysteroid titers in Fig.…”

“…Gene expression in YOs from C. maenas and G. lateralis differ in response to ESA. ESA increases Gl-NOS, Gl-GC-Iβ and Gl-GC-III expression in G. lateralis (Lee et al, 2007a;McDonald et al, 2011). By contrast, ESA had little or no effect on Cm-NOS, Cm-GC-Iβ or Cm-GC-III expression in C. maenas nor was there any correlation with hemolymph ecdysteroid levels (Fig.…”

“…3). In the land crab, Gl-NOS and Gl-GC-Iβ transcript copy numbers are correlated with hemolymph ecdysteroid levels (Lee et al, 2007a;McDonald et al, 2011), indicating that expression of these genes is associated with YO activation. The differences in the effect of ESA on NOS and GC-Iβ gene expression in the two species can be attributed to whether ESA induces molting.…”

“…MIH signaling pathway may involve a calmodulin (CaM)-dependent NO synthase (NOS) and NO-dependent guanylyl cyclase (GC-I) Covi et al, 2012;Webster et al, 2012). YOs express NOS, the beta subunit of GC-I (GC-Iβ), a receptor GC (GC-II) and a soluble NO-insensitive GC (GC-III) (Kim et al, 2004;Lee et al, 2007a;McDonald et al, 2011). Thus, CHH and MIH inhibit YO ecdysteroid biosynthesis through two distinct signaling pathways involving membrane receptor and NOdependent GCs, respectively.…”

“…8B). The Gl-GC-III is a constitutively active soluble GC and may act synergistically with Gl-GC-I to increase intracellular cGMP levels (Lee et al, 2007a). Although the increase in Cm-GC-Iβ expression during premolt (Fig.…”