Abstract
Acorn barnacles are major marine fouling organisms. Their success is largely due to an ability to adhere to diverse substrates via a sub-micron thick proteinaceous adhesive layer that develops as the organism molts and expands its base. Recent work has expanded the set of proteins identified within the adhesive interface, but one outstanding question concerns their spatial distribution throughout the organism. Here, we employ immunological analysis of Amphibalanus amphitrite tissue sections and identify the presence of two cement proteins, AaCP19-1 and AaCP43-1, in areas far removed from the adhesive interface. Confocal imaging reveals specific staining along different tissue linings of the organism as well as other non-cementing regions. Additionally, we employ a modified, pressure cycling technology approach to recover protein from histological tissue sections to perform proteomics analysis. Mass spectrometry analysis of proteins recovered from transverse histological sections of the upper portion of barnacles indicates the presence of these same proteins, complementing the immunostaining observations. The proteomics analysis also revealed the presence of other proteins first identified in the adhesive layer. While some proteins are clearly enriched at the surface interface, our findings challenge the concept that cement proteins are exclusive to the substrate interface and suggest they may have an expanded physiological role beyond substrate adhesion-related processes of A. amphitrite.
Highlights
As a major marine macrofouler, acorn barnacles adhere to a variety of surfaces via a proteinaceous adhesive, commonly referred to as barnacle cement
With an abundance of proteins identified from the cement, we seek answers to fundamental questions regarding these materials: are proteins first identified in the adhesive interface in A. amphitrite unique to the adhesive layer? If not, what is their distribution within the barnacle? Using a combination of immunofluorescence confocal microscopy, western blotting, and proteome profiling of formalin fixed paraffin embedded (FFPE) sections, we primarily focus on the distribution of two prominent proteins found in barnacle cement, AaCP19-1 and AaCP43-1, and expand our findings to other proteins associated with the cement layer of A. amphitrite
Histological FFPE sections were prepared to observe the interior anatomy of A. amphitrite and to assess the spatial distribution of two cement proteins, AaCP19-1 and AaCP43-1, using fluorescent-labeled, custom polyclonal antibodies with confocal microscopy
Summary
As a major marine macrofouler, acorn barnacles adhere to a variety of surfaces via a proteinaceous adhesive, commonly referred to as barnacle cement. This cement is a secreted material (Dickinson et al, 2009) that transitions to a recalcitrant and permanent solid in the mature form (Naldrett, 1993; Naldrett and Kaplan, 1997; Kamino et al, 2000). Understanding the distribution of these proteins could shed light on whether they have an expanded biological role in cirripedes
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