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ABSTRACT Decapod crustaceans regulate molting through steroid molting hormones (ecdysteroids) synthesized by the molting gland (Y-organ, YO). Molt-inhibiting hormone (MIH), a neuropeptide synthesized and secreted by the eyestalk ganglia, negatively regulates YO ecdysteroidogenesis. MIH signaling is mediated by cyclic nucleotide second messengers. cGMP-dependent protein kinase (PKG) is the presumed effector of MIH signaling by inhibiting mechanistic Target of Rapamycin Complex 1 (mTORC1)-dependent ecdysteroidogenesis. Phylogenetic analysis of PKG contiguous sequences in CrusTome, as well as 35 additional species in NCBI RefSeq, identified 206 PKG1 sequences in 108 species and 59 PKG2 sequences in 53 species. These included four PKG1α splice variants in the N-terminal region that were unique to decapods, as well as PKG1β and PKG2 homologs. In vitro assays using YOs from the blackback land crab (Gecarcinus lateralis) and green shore crab (Carcinus maenas) determined the effects of MIH±PKG inhibitors on ecdysteroid secretion. A general PKG inhibitor, Rp-8-Br-PET-cGMPS, countered the effects of MIH, as ecdysteroid secretion increased in PKG-inhibited YOs compared with C. maenas YOs incubated with MIH alone. By contrast, a PKG2-specific inhibitor, AP-C5 {4-(4-[1H-imidazol-1-yl]phenyl)-N-2-propyn-1-yl-2-pyrimidinamine}, enhanced the effects of MIH, as ecdysteroid secretion decreased in G. lateralis and C. maenas YOs incubated with AP-C5 and MIH compared with YOs incubated with MIH alone. These data suggest that both PKG1 and PKG2 are activated by MIH, but have opposing effects on mTORC1-dependent ecdysteroidogenesis. A model is proposed in which the dominant role of PKG1 is countered by PKG2, resulting in low ecdysteroid production by the basal YO during intermolt. 

Abstract Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans. 

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed. 

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. 

Synopsis The 2020 SICB Society-wide Symposium “Building Bridges from Genome to Phenome: Molecules, Methods and Models” brought together a diverse group of scientists to discuss recent progress in linking phenotype plasticity to changes at the level of the genome, epigenome, and proteome, while exploring the boundaries between variation and speciation. In a follow-up workshop, participants were asked to assess strengths and weaknesses of current approaches, to identify common barriers inhibiting their progress, and to outline the resources needed to overcome those barriers. Discussion groups generally recognized the absence of any overarching theoretical framework underlying current genome to phenome research and, therefore, called for a new emphasis on the development of conceptual models as well as the interdisciplinary collaborations needed to create and test those models. Participants also recognized a critical need for new and improved molecular and bioinformatic approaches to assist in describing function/phenotypes across phylogeny. Additionally, like all scientific endeavors, progress in genome to phenome research will be enhanced by improvements in science education and communication both within and among working groups.