Insights into the Synthesis, Secretion and Curing of Barnacle Cyprid Adhesive via Transcriptomic and Proteomic Analyses of the Cement Gland

Abstract

Barnacles represent one of the model organisms used for antifouling research, however, knowledge regarding the molecular mechanisms underlying barnacle cyprid cementation is relatively scarce. Here, RNA-seq was used to obtain the transcriptomes of the cement glands where adhesive is generated and the remaining carcasses of Megabalanus volcano cyprids. Comparative transcriptomic analysis identified 9060 differentially expressed genes, with 4383 upregulated in the cement glands. Four cement proteins, named Mvcp113k, Mvcp130k, Mvcp52k and Mvlcp1-122k, were detected in the cement glands. The salivary secretion pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes, implying that the secretion of cyprid adhesive might be analogous to that of saliva. Lysyl oxidase had a higher expression level in the cement glands and was speculated to function in the curing of cyprid adhesive. Furthermore, the KEGG enrichment analysis of the 352 proteins identified in the cement gland proteome partially confirmed the comparative transcriptomic results. These results present insights into the molecular mechanisms underlying the synthesis, secretion and curing of barnacle cyprid adhesive and provide potential molecular targets for the development of environmentally friendly antifouling compounds.

Keywords: barnacle; cement gland; cement protein; cyprid adhesive; transcriptome.

Figure 1

Dissected cement gland of a Megabalanus volcano cyprid.

Figure 2

Comparative analysis of unigene expression levels between cement glands and carcasses. A. Volcano plot of the Differentially Expressed Genes (DEGs) between cement glands and carcasses. The blue spots indicate unigenes that were upregulated in cement glands, the red spots indicate downregulated unigenes, and the black spots indicate unigenes that were not differentially expressed. B. Gene Ontology (GO) enrichment analysis of the upregulated DEGs in cement glands, terms related to protein synthesis and protein modification are in red. C. KEGG enrichment analysis of all the DEGs between cement glands and carcasses, salivary secretion pathway (ko04970) is in red.

Figure 3

Cement protein-100 kDa homologues expressed in cement glands. A. Sequence alignment of Mvcp113k (MK336236) and Mvcp130k (MK336237) of M. volcano, the homology level of the sequences = 100% are shaded in black. B. Expression levels (Fragments Per Kilobase of Transcript Per Million Mapped Reads (FPKM) values) of Mvcp113k and Mvcp130k in cement glands and carcasses.

Figure 4

Cement protein-52 kDa homologue expressed in cement glands. A. Sequence alignment of Mvcp52k (MK336235), Mrcp52k (BAL22342.1) from M. rosa and Aacp52k (AKZ20820.1) from A. amphitrite, the homology level of the sequences = 100% and ≥ 50% are shaded in black and blue; 4 repeat sequences are labeled as Rep-1, -2, -3 and -4, and boxed in yellow, purple, green and orange rectangles respectively. B. Sequence alignment of the four repeat sequences of Mvcp52k, the homology level of the sequences = 100%, ≥ 75% and ≥ 50% are shaded in black, pink and blue, locations of a Cys residues are boxed in red rectangle. C. Expression levels (FPKM values) of Mvcp52k in cement glands and carcasses.

Figure 5

Cement gland-specific protein 122 kDa homologues expressed in cement glands. A. Sequence alignment of Mvlcp1-122k (MT024661) and Mrlcp1-122k (MK490677) of M. rosa, the homology level of the sequences = 100% are shaded in black. Asn-Xaa-Ser/Thr sequon is boxed in red rectangle, and the Asparagine predicted to be N-glycosylation site is marked with blue asterisk, predicted mucin type GalNAc O-glycosylation sites are marked with inverted purple triangle. B. Expression levels (FPKM values) of Mvlcp1-122k in cement glands and carcasses.

Figure 6

Characterization of M. volcano lysyl oxidase. A. Sequence alignment of MvLOX and AaLOX-1 (AQY78507.1) from A. amphitrite, the homology level of the sequences = 100% are shaded in black, predicted signal peptide is boxed in yellow rectangle and conserved lysyl oxidase domain is boxed in red rectangle. B. Expression levels (FPKM values) of MvLOX in the cement glands and carcasses.

Figure 7

KEGG enrichment analysis of all the proteins identified in the cement gland proteome. The salivary secretion pathway (ko04970) is in red, and the PPAR signaling pathway (ko03320) is boxed in blue rectangle.