Figure 3: Spatial arrangement of lipidaceous and proteinaceous adhesive granules within the cyprid cement gland.

Using a combination of lipid-specific (FM 1-43 FX) and protein-specific (fluoresceinamine) dyes we confirm: (a) the presence of two granule types within separate columnar cells of the cement gland; lipidaceous granules (white arrows) and proteinaceous granules (black arrows) in this confocal micrograph. Cell nuclei are blue. (b) Lipidaceous granule-containing cells are present along the entire length of the gland and are not restricted to the dorso-ventral extremes, with their contents concentrated closest to the median cement-collecting duct. (c) Proteinaceous granule-containing cells are also distributed throughout the length of the gland. Lipidaceous granules appear to be larger than the proteinaceous granules and are packed more loosely. (d) TPEFM volume projection of cement gland stained with Nile-Red, showing that the lipidaceous granules (white arrow) in the lipid granule-containing cells are present only in the lower ventral half of the gland, closest to the median cement-collecting duct (arrow head). (e) Isosurface rendered view of the lipidaceous granule-containing cells superimposed onto the volume projection of the gland. (f) YZ orthogonal view of the Nile-Red-labelled cement gland. Since Nile-Red does not label the proteinaceous granules, the bulk of the cement gland appears empty (green arrow). (g) TPEFM volume projection of a cement gland stained specifically for phosphoproteins showing that proteinaceous granules constitute the bulk of the gland; also seen in YZ orthogonal view in h. Lipidaceous granules present around the collecting duct are not labelled by the phosphoprotein stain and appear as vacuoles (yellow arrow). Also see Supplementary Figs 6 and 7. Scale bars (μm), ~20 (a–c,d,e), ~30 μm (g,h).