Identification and Characterization of Neuropeptides by Transcriptome and Proteome Analyses in a Bivalve Mollusc Patinopecten yessoensis

Abstract

Neuropeptides play essential roles in regulation of reproduction and growth in marine molluscs. But their function in marine bivalves - a group of animals of commercial importance - is largely unexplored due to the lack of systematic identification of these molecules. In this study, we sequenced and analyzed the transcriptome of nerve ganglia of Yesso scallop Patinopecten yessoensis, from which 63 neuropeptide genes were identified based on BLAST and de novo prediction approaches, and 31 were confirmed by proteomic analysis using the liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty genes encode known neuropeptide precursors, of which 20 commonly exist in bilaterians and 30 are protostome specific. Three neuropeptides that have not yet been reported in bivalves were identified, including calcitonin/DH31, lymnokinin and pleurin. Characterization of glycoprotein hormones, insulin-like peptides, allatostatins, RFamides, and some reproduction, cardioactivity or feeding related neuropeptides reveals scallop neuropeptides have conserved molluscan neuropeptide domains, but some (e.g., GPB5, APGWamide and ELH) are characterized with bivalve-specific features. Thirteen potentially novel neuropeptides were identified, including 10 that may also exist in other protostomes, and 3 (GNamide, LRYamide, and Vamide) that may be scallop specific. In addition, we found neuropeptides potentially related to scallop shell growth and eye functioning. This study represents the first comprehensive identification of neuropeptides in scallop, and would contribute to a complete understanding on the roles of various neuropeptides in endocrine regulation in bivalve molluscs.

Keywords: bivalve mollusc; cardioactivity; eye functioning; ganglia transcriptome; mass spectrometry; neuropeptide; reproduction; shell growth.

FIGURE 1

Summary of identified genes encoding putative full- or partial-length neuropeptide precursors from the P. yessoensis nerve ganglia transcriptome and proteome. The 20 ancestral bilaterian neuropeptide precursors are in red; the 12 neuropeptide precursors which exist in all the major groups of protostomes are in green; the 7 neuropeptide precursors only found in Mollusca, Annelida, and Nematoda are in purple; the 11 neuropeptide precursors that were only characterized in Lophotrochozoa are in yellow; the 13 potentially novel neuropeptide precursors are in blue.

FIGURE 2

Alignment of glycoprotein hormone precursors. Conserved amino acid residues are highlighted in black, conservative replacements in gray, and other cysteine residues specifically conserved within bus icons in red. The information of sequences used in the figure is displayed in the Supplementary Table S3.

FIGURE 3

Alignment of A and B chains of insulin-like peptide (ISNL), insulin (ISN), and relaxin (REL) precursors. Conserved amino acid residues are highlighted in black, conservative replacements in gray, and other cysteine residues conserved between Brachiopoda and Mollusca in red. The information of sequences used in the figure is displayed in the Supplementary Table S3.

FIGURE 4

Schematic representation of scallop allatostatin family and alignment of potential bioactive peptides. (A) Schematic representation of allatostatin A/buccalin precursor and sequence alignment of LDAs among molluscs. Yellow, signal peptide (SP); orange, amidated peptides; green, non-amidated predicted peptides; blue, LDA. The biologically active peptides confirmed by MS are indicated in bold. (B) Schematic representation of allatostatin B precursor. (C) Sequence alignment of bioactive allatostatin C and somatostatin. Cysteine residues are highlighted in red, and conservative replacements in gray. All the information of sequences used in the figure is displayed in the Supplementary Table S3.

FIGURE 5

Comparison of the linear schematic organization of (A) FMRF, (B) LFRF, and (C) luqin and alignment of known orthologous peptides from (D) NPF/NPY and (E) CCK/SK. The height of each letter of the logo is proportional to the observed frequency of the corresponding amino acid in the alignment column. All the information of sequences used in the figure is displayed in the Supplementary Table S3.

FIGURE 6

Comparison of the linear schematic organization of (A) APGWamide and (B) ELH; (C) alignment of GnRH peptides among molluscs, vertebrates, and echinoderms. All the information of sequences used in the figure is displayed in the Supplementary Table S3.