Defensive Ink Pigment Processing and Secretion in Aplysia Californica: Concentration and Storage of Phycoerythrobilin in the Ink Gland
Authors: Prince J. , Nolen T. G. , Coelho L. 
Published: April 2021

ABSTRACT
The marine snail Aplysia californica obtains its defensive ink exclusively from a diet of red seaweed. It stores the pigment (phycoerythrobilin, the red algal photosynthetic pigment, r-phycoerythrin, minus its protein) in muscular ink-release vesicles within the ink gland. Snails fed a diet of green seaweed or romaine lettuce do not secrete ink and their ink-release vesicles are largely devoid of ink. Successive activation of individual ink-release vesicles by ink motor neurons causes them to secrete approximately 55 % of their remaining ink (similar to the percentage of ink reserves released from the intact gland). The peripheral activation of vesicles appears to be cholinergic: 70 % of isolated vesicles were induced to squeeze ink from their valved end by solutions of acetylcholine at concentrations of 0.5 mmol l−1 or below.
Ultrastructural analysis commonly found three cell types in the ink gland. The RER cells, the most numerous, were characterized by an extensive rough endoplasmic reticulum with greatly distended cisternae. This cell type is probably the site for synthesis of the high molecular mass protein of secreted ink. The granulate cells, less common than RER cells, had nuclear and cell areas significantly larger than those of RER cells. In addition, granulate cells of red-algal-fed snails had 4−14 vacuoles that contained electron-dense material with staining characteristics similar to that of ink in mature ink-release vesicles. The granulate cell’s plasma membrane was regularly modified into grated areas, which both localized and expanded the surface area for coated vesicle formation and provided a sieve structure that prevented large particles in the hemolymph either from being taken up by, or from occluding, the coated vesicles. Electron-dense particles within coated vesicles were similar in size to those in granulate vacuoles but larger (on average by approximately 1 nm) than those that make up the ink. In green-seaweed-fed snails, granulate cells and their vacuoles were present but the vacuoles were empty. The third cell type, the vesicle cell, expands markedly, with its nucleus enlarging concurrent with cell growth until it is on average 50 times larger in cross-sectional area than the nuclei of either RER or granulate cells; the cytoplasm eventually becomes filled with ink, which obscures the mitochondria, vacuoles and nucleus. Continued cell expansion ceases with the appearance of an encircling layer of muscle and 1−3 layers of cells of unknown origin, thereby becoming the ink-release vesicle itself. The absorption spectra of the soluble contents of mature ink-release vesicles from snails fed red algae had peaks characteristic of the red algal pigment r-phycoerythrin or/and phycoerythrobilin.
Immunogold localization of r-phycoerythrin showed no statistical difference in the amount of label within the ink-release vesicles, RER or granulate cell types. Furthermore, there was no localization of phycoerythrin immunoreactivity within the various cellular compartments of either the RER or granulate cells (nucleus, endoplasmic reticulum, mitochondria, vacuoles). Immunogold labeling in the ink gland ranged from 11 to 16 % of that for the digestive vacuoles of the rhodoplast digestive cells lining the tubules of the digestive gland. Our observations suggest (a) that the main form of the ink pigment in the gland is phycoerythrobilin or/and a non-antigenic form of phycoerythrin, and (b) that separation of the bilin from phycoerythrin (or its modification so that it is no longer antigenic) occurs before it reaches the ink gland, probably within the vacuoles of the rhodoplast digestive cells of the digestive gland.
We propose the following model. The ink pigment, phycoerythrobilin, is cleaved from its protein in rhodoplast digestive vacuoles in the digestive gland. Phycoerythrobilin is carried in the hemolymph to the ink gland, where granulate cells take it up and transport it via coated vesicles to membrane-bound vacuoles for long- or short-term storage; later, the ink is incorporated into developing ink-release vesicles. RER cells synthesize the high molecular mass, non-algal protein (whose function is unknown), which constitutes 35 % of the dry mass of secreted ink.