Molt-dependent transcriptomic analysis of cement proteins in the barnacle Amphibalanus amphitrite

Abstract

BackgroundA complete understanding of barnacle adhesion remains elusive as the process occurs within and beneath the confines of a rigid calcified shell. Barnacle cement is mainly proteinaceous and several individual proteins have been identified in the hardened cement at the barnacle-substrate interface. Little is known about the molt- and tissue-specific expression of cement protein genes but could offer valuable insight into the complex multi-step processes of barnacle growth and adhesion.MethodsThe main body and sub-mantle tissue of the barnacle Amphibalanus amphitrite (basionym Balanus amphitrite) were collected in pre- and post-molt stages. RNA-seq technology was used to analyze the transcriptome for differential gene expression at these two stages and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) was used to analyze the protein content of barnacle secretions.ResultsWe report on the transcriptomic analysis of barnacle cement gland tissue in pre- and post-molt growth stages and proteomic investigation of barnacle secretions. While no significant difference was found in the expression of cement proteins genes at pre- and post-molting stages, expression levels were highly elevated in the sub-mantle tissue (where the cement glands are located) compared to the main barnacle body. We report the discovery of a novel 114kD cement protein, which is identified in material secreted onto various surfaces by adult barnacles and with the encoding gene highly expressed in the sub-mantle tissue. Further differential gene expression analysis of the sub-mantle tissue samples reveals a limited number of genes highly expressed in pre-molt samples with a range of functions including cuticular development, biominerialization, and proteolytic activity.ConclusionsThe expression of cement protein genes appears to remain constant through the molt cycle and is largely confined to the sub-mantle tissue. Our results reveal a novel and potentially prominent protein to the mix of cement-related components in A. amphitrite. Despite the lack of a complete genome, sample collection allowed for extended transcriptomic analysis of pre- and post-molt barnacle samples and identified a number of highly-expressed genes. Our results highlight the complexities of this sessile marine organism as it grows via molt cycles and increases the area over which it exhibits robust adhesion to its substrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2076-1) contains supplementary material, which is available to authorized users.

Profiling of adhesive-related genes in the freshwater cnidarianHydra magnipapillataby transcriptomics and proteomics

“…In bioadhesion, peroxidases are known to participate in the catalysis of di-tyrosine formation to post-draw silk fibres produced by the caddisfly larvae (Wang et al 2014), and in the hardening of egg fertilisation membranes of sea urchin (Foerder & Shapiro 1977). It was also found to be present in the adhesive secretions of other organisms such as sea stars, sea urchins, and barnacles (Hennebert et al 2015; Wang et al 2015; Lebesgue et al 2016). The possible functions of peroxidases in the adhesive secreted by Hydra were discussed by Rodrigues et al (Forthcoming), and are similar to the ideas proposed for sea urchins by Lebesgue et al (2016).…”

“…About 40% of the transcripts expressed in the basal disc had no annotated function and even no orthologue in other metazoans. Novel adhesive proteins were discovered for several organisms, including barnacles, one of the most studied systems (Bacchetti De Gregoris et al 2009; Jonker et al 2014; Wang et al 2015). Already stressed by Jonker et al (2014), more sequence data spanning throughout the metazoan are necessary to identify conserved adhesive domains and gain insight into the relationship between sequence structure and protein function.…”

“…In the caddisfly larvae Hesperophylax sp., the silk gland-specific transcriptome was used to identify homologous H-fibroin contigs, an important component of proteins present in silk fibres (Ashton et al 2013; Wang et al 2014). It was also used to identify a novel 114 kDa cement protein in the barnacle Amphibalanus amphitrite (Wang et al 2015), and successfully implemented by Hennebert et al (2015) for identification of proteins secreted by the tube feet of the sea star Asterias rubens . In the scallop Chlamys farreri a comparison between several tissue specific transcriptomes and proteomes helped in identifying seven foot-specific proteins, which might be involved in adhesion (Miao et al 2015).…”

Rearing Temperature Affects the Expression of Proteins in The Adhesive of the Striped Acorn Barnacle, Balanus Amphitrite

“…Another study attempted to investigate a molt-related transcriptomic response of barnacle cement proteins in different tissues by employing a variety of collection methods and analytical techniques (Wang et al, 2015). Similar to the mass spectrometry (MS) analysis performed in our study, they too had difficulty detecting and identifying cement proteins in their MS analysis.…”

“…One proposed explanation was that the cement specific proteins were masked by the presence of more abundant household and scaffold proteins and that the inability to detect the putative adhesive proteins from the uncured material does not necessarily mean that they were not present (Wang et al, 2015). In addition to lacking a standard method to collect the liquid adhesive, they suggested that the limited amount of material that can be collected during a short time period, and the unknown possibility of cross-linking prior to enzymatic digestion for MS analysis (plus the absence of a fully annotated genome), may all contribute to the challenges for the detection of cement proteins in various barnacle secretions (Wang et al, 2015). These rationalizations illustrate the difficulties in detecting cement proteins and provide insight about the overall complexity of this unique and interesting material.…”