Gene co-option, duplication and divergence of cement proteins underpin the evolution of bioadhesives across barnacle life histories.

Acquisition of new genes often results in the emergence of novel functions and is a key step in lineage-specific adaptation. As a group of sessile crustaceans, barnacles establish permanent attachment through initial cement secretion at the larval phase followed by continuous cement secretion in juveniles and adults. However, the origins and evolution of barnacle larval and adult cement proteins remain poorly understood. By performing microdissection of larval cement glands, transcriptome and shotgun proteomics and immunohistochemistry validation, we identified 30 larval and 27 adult cement proteins of the epibiotic turtle barnacle Chelonibia testudinaria, of which the majority are stage- and barnacle-specific. While only two proteins, SIPC and CP100K, were expressed in both larvae and adults, detection of protease inhibitors and the cross-linking enzyme lysyl oxidase paralogs in larvae and adult cement. Other barnacle-specific cement proteins such as CP100k and CP52k likely share a common origin dating back at least to the divergence of Rhizocephala and Thoracica. Different CP52k paralogues could be detected in larval and adult cement, suggesting stage-specific cement proteins may arise from duplication followed by changes in expression timing of the duplicates. Interestingly, the biochemical properties of larval- and adult-specific CP52k paralogues exhibited remarkable differences. We conclude that barnacle larval and adult cement systems evolved independently, and both emerged from co-option of existing genes and de novo formation, duplication and functional divergence of lineage-specific cement protein genes. Our findings provide important insights into the evolutionary mechanisms of bioadhesives in sessile marine invertebrates.