Search for: All records

Molting in decapod crustaceans is controlled by ecdysteroids synthesized and secreted by the molting gland, or Yorgan (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 (TGF beta)/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, GlBr-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 TGF beta/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. 

Meeting abstract 

Meeting abstract 

Meeting abstract 

Meeting abstract 

Meeting abstract 

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation. 

Ecdysteroid molting hormone synthesis is directed by a pair of molting glands or Y-organs (YOs), and this synthesis is inhibited by molt-inhibiting hormone (MIH). MIH is a member of the crustacean hyperglycemic hormone (CHH) neuropeptide superfamily, which includes CHH and insect ion transport peptide (ITP). It is hypothesized that the MIH receptor is a Class A (Rhodopsin-like) G protein-coupled receptor (GPCR). The YO of the blackback land crab,Gecarcinus lateralis, expresses 49 Class A GPCRs, three of which (Gl-CHHR-A9, -A10, and -A12) were provisionally assigned as CHH-like receptors. CrusTome, a transcriptome database assembled from 189 crustaceans and 12 ecdysozoan outgroups, was used to deorphanize candidate MIH/CHH GPCRs, relying on sequence homology to three functionally characterized ITP receptors (BNGR-A2, BNGR-A24, and BNGR-A34) in the silk moth,Bombyx mori. Phylogenetic analysis and multiple sequence alignments across major taxonomic groups revealed extensive expansion and diversification of crustacean A2, A24, and A34 receptors, designatedCHHFamilyReceptorCandidates (CFRCs). The A2 clade was divided into three subclades; A24 clade was divided into five subclades; and A34 was divided into six subclades. The subclades were distinguished by conserved motifs in extracellular loop (ECL) 2 and ECL3 in the ligand-binding region. Eleven of the 14 subclades occurred in decapod crustaceans. InG. lateralis, seven CFRC sequences, designated Gl-CFRC-A2α1, -A24α, -A24β1, -A24β2, -A34α2, -A34β1, and -A34β2, were identified; the three A34 sequences corresponded to Gl-GPCR-A12, -A9, and A10, respectively. ECL2 in all the CFRC sequences had a two-stranded β-sheet structure similar to human Class A GPCRs, whereas the ECL2 of decapod CFRC-A34β1/β2 had an additional two-stranded β-sheet. We hypothesize that this second β-sheet on ECL2 plays a role in MIH/CHH binding and activation, which will be investigated further with functional assays. 

A pair of Y-organs (YOs) synthesize ecdysteroids that initiate and coordinate molting processes in decapod crustaceans. The YO converts cholesterol to secreted products through a biosynthetic pathway involving a Rieske oxygenase encoded by Neverland (Nvd) and cytochrome P450 monooxygenases encoded by Halloween genes Spook (Spo; Cyp307a1), Phantom (Phm; Cyp306a1), Disembodied (Dib; Cyp302a1), and Shadow (Sad; Cyp315a1). NAD kinase (NADK) and 5-aminolevulinic acid synthase (ALAS) support ecdysteroid synthesis in insects. A 20-hydroxylase, encoded by Shed in decapods and Shade in insects, converts ecdysone to the active hormone 20-hydroxyecdysone (20E). 20E is inactivated by cytochrome P450 26-hydroxylase (Cyp18a1). Contigs encoding these eight proteins were extracted from a Gecarcinus lateralis YO transcriptome and their expression was quantified by quantitative polymerase chain reaction. mRNA levels of Gl-Spo and Gl-Phm were four orders of magnitude higher in YO than those in nine other tissues, while mRNA levels of Gl-NADK and Gl-ALAS were similar in all ten tissues. In G. lateralis induced to molt by multiple leg autotomy, YO mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-NADK, and Gl-ALAS were highest in intermolt and premolt stages and lower in postmolt. Gl-Dib mRNA level was not affected by molt stage. mRNA level of Gl-Sad, which converts 2-deoxyecdysone to ecdysone, was higher in mid-and late premolt stages, when YO ecdysteroidogenic capacity is greatest. Gl-Cyp18a1 mRNA level was highest in intermolt, decreased in premolt stages, and was lowest in postmolt. In animals induced to molt by eyestalk ablation, YO mRNA levels of all eight genes were not correlated with increased hemolymph 20E titers. These results suggest that YO ecdysteroidogenic genes are differentially regulated at transcriptional and translational levels.