Neuropeptides encoded by the genomes of the Akoya pearl oyster Pinctata fucata and Pacific oyster Crassostrea gigas: a bioinformatic and peptidomic survey

Abstract

Background: Oysters impart significant socio-ecological benefits from primary production of food supply, to estuarine ecosystems via reduction of water column nutrients, plankton and seston biomass. Little though is known at the molecular level of what genes are responsible for how oysters reproduce, filter nutrients, survive stressful physiological events and form reef communities. Neuropeptides represent a diverse class of chemical messengers, instrumental in orchestrating these complex physiological events in other species.

Results: By a combination of in silico data mining and peptide analysis of ganglia, 74 putative neuropeptide genes were identified from genome and transcriptome databases of the Akoya pearl oyster, Pinctata fucata and the Pacific oyster, Crassostrea gigas, encoding precursors for over 300 predicted bioactive peptide products, including three newly identified neuropeptide precursors PFGx8amide, RxIamide and Wx3Yamide. Our findings also include a gene for the gonadotropin-releasing hormone (GnRH) and two egg-laying hormones (ELH) which were identified from both oysters. Multiple sequence alignments and phylogenetic analysis supports similar global organization of these mature peptides. Computer-based peptide modeling of the molecular tertiary structures of ELH highlights the structural homologies within ELH family, which may facilitate ELH activity leading to the release of gametes.

Conclusion: Our analysis demonstrates that oysters possess conserved molluscan neuropeptide domains and overall precursor organization whilst highlighting many previously unrecognized bivalve idiosyncrasies. This genomic analysis provides a solid foundation from which further studies aimed at the functional characterization of these molluscan neuropeptides can be conducted to further stimulate advances in understanding the ecology and cultivation of oysters.

Figure 1

Summary of identified genes encoding putative full-length or partial-length neuropeptide precursors from the Pinctada fucata and Crassostrea gigas genome, and transcriptome databases . For each neuropeptide we highlight whether a cDNA clone, a full-length ORF sequence, and MS evidence are available. We also indicate the numbers of peptides that are cleaved from the mature neuropeptide precursor, if they are amidated, acetylated, or pyroglutaminated peptides, and if a leader signal peptide is encoded.

Figure 2

Identification and characterization of Pinctata fucata and Crassostrea gigas APGW and FMRF precursors. (A) Amino acid sequence of APGW precursors and schematics showing organization of precursors. (B) Amino acid sequence of FMRF precursors and schematics showing organization of precursors.

Figure 3

Identification and characterization of Pinctata fucata and Crassostrea gigas GnRH-like peptides. (A) Amino acid sequence of GnRH precursors. (B) Schematics showing organization of precursors within molluscs and other invertebrates and vertebrates. (C) Comparative sequence alignment of GnRH peptide and (D) phylogenetic analysis. Sequence logo is shown above alignments.

Figure 4

Identification and characterization of P. fucata and C. gigas ELH-like peptides. (A) Amino acid sequences of P. fucata and C.gigas ELH precursors. (B) Schematics showing ELH precursors identified in molluscs. (C) Comparative sequence alignment and (D) Phylogenetic analysis of bioactive ELH/DH44/CRH region between species. Sequence logo is shown above alignments. (E) Protein models of oyster ELH1 and ELH2 assembled from molecular dynamics simulation. Secondary structures: blue, helix; purple, alpha-helix; cyan, turn; white, random coil; dark yellow, isolated beta bridge. (F) Off-line nLC-MALDI tandem MS analysis of Crassotrea gigas cerebral ganglia. MS/MS spectrum of the egg-laying hormone Cg-ELH2 m/z 3939. Immonium, a-, b- and y-ions detected are marked.

Figure 5

Identification of Pinctata fucata and Crassostrea gigas neuropeptides. Comparative sequence alignment and schematic representation of precursors for P. fucata (Pfu) and C. gigas (Cgi). Full-length precursor sequences were identified in both for achatin, allatotropin, CCK/SK and conopressin, elevenin, NKY, NPF/Y and LFRFa, showing high amino acid identity within bioactive peptides. A sequence logo is shown above alignments, where the height of each letter is proportional to the observed frequency of the corresponding amino acid in the alignment column.

Figure 6

Identification of GBP5 and GPA2 in oysters. Comparative sequence alignments of GBP5 and GPA2 precursors for Pinctada fucata and Crassotrea gigas with the gastropods Aplysia californica and Lottia. gigantea. Sequence logo is shown above alignments.