Hydrophilic Shell Matrix Proteins of Nautilus pompilius and the Identification of a Core Set of Conchiferan Domains

Abstract

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the basally diverging Nautilids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautilid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we performed a multiomics study on the shell of N. pompilius, by conducting transcriptomics of its mantle tissue and proteomics of its shell matrix. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet and the Euhadra snail) revealed that three proteins and six protein domains were conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six protein domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were probably present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Even though further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be carried out in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

Keywords: Cephalopoda; Mollusca; biomineralization; multiomics; proteomics; shell evolution.

Figure 1

(A) Phylogeny of Conchiferans including Nautilus pompilius. (B) N. pompilius. (C) The microstructures of the shell of N. pompilius. In detail: (D) Outer prismatic layer, (E) Middle prismatic layer and (F) Inner prismatic layer.

Figure 2

Comparisons of the Shell Matrix Proteins in several Conchiferans for which the data are available using Search Settings 2. Detailed explanation of the settings is written in the main text. (A) Venn diagram showing the numbers of shared proteins identified through local BLASTp searches among the five Conchiferans. Red line enclosure is the number of conserved proteins among the Conchifera. (B) Homologous proteins of the five Conchiferans compared, plotted on to the phylogeny of the animals. (C) Homologous proteins colored deep green were conserved among all five species. (D) Homologous proteins colored Pale green were conserved among marine mollusks.

Figure 3

Comparisons of the domains contained in the Shell Matrix Proteins of several Conchiferans for which the data are available. (A) Schematic representations of the domains in the Shell Matrix Proteins of Nautilus pompilius. (B) Shared domains in the Shell Matrix Proteins of the five Conchiferans (N. pompilius, Pinctada fucata, Crassostrea gigas, Lottia gigantea and Euhadra quaesita) compared, mapped on to the phylogeny of the animals. The reconstructed Ancestral Conchiferans most likely had all of the shared domains.

Figure 4

Phylogenetic trees of selected Shell Matrix Proteins. (A) The maximum likelihood tree of the Pif/BMSP amino acid sequences, inferred using the LG + Γ model with 1000 bootstrap replicates. (B) The maximum likelihood phylogenetic tree of Tyrosinase inferred under the LG + Γ + I model with 1000 bootstrap replicates. Bootstraps values <40% are not shown, and a black square on a node indicates 100% bootstrap support. Abbreviations: Apca: Aplysia californica, Bigl: Biomphalaria glabrata, Bomo: Bombyx mori, Cael: Caenorhabditis elegans, Cene: Cepaea nemoralis, Crgi: Crassostrea gigas, Crvi: Crassostrea virginica, Drfi: Drosophila ficusphila, Drme: Drosophila melanogaster, Euqu: Euhadra quaesita, Gaga: Gallus gallus, Hala: Haliotis laevigata, Hosa: Homo sapiens, Ilar: Illex argentines, Lili: Littorina littorea, Logi: Lottia gigantea, Miye: Mizuhopecten yessoensis, Miga: Mytilus galloprovincialis, Mumu: Mus musculus, Pale: Pacifastacus leniusculus, Phau: Phoronis australis, Pimar: Pinctada margaritifera, Pimax: Pinctada maxima, Ptpe: Pteria penguin, Seof: Sepia officinalis, Hadi: Haliotis discus, Myco: Mytilus coruscus, Mytr: Mytilus trossulus, Ocvu: Octopus vulgaris, Pimarg: Pinctada margaritifera, Pifu: Pinctada fucata, Piim: Pinctada imbricata, Rano: Rattus norvegicus, Trps: Trichinella pseudospiralis, Toca: Toxocara canis. An OTU name started with the word “Contig” denotes the Nautilus pompilius sequence obtained in this study.