Abstract

1. 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 ink release vesicles are largely devoid of ink. Successive activation of individual ink release vesicles by ink motor neurons cause them to secrete about 55% of their remaining ink -- similar to the percentage of ink reserves released from the intact gland (Nolen and Johnson, 1997). 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 ACh at concentrations < 0.5 mM.

2. Ultrastructural analysis commonly found three cell types in the ink gland: the RER cell, the most numerous, was characterized by an extensive rough endoplasmic reticulum with greatly distended cisternae. This cell type is probably the site for synthesis of the high molecular protein of secreted ink. The granulate cell, less common than RER cells, had nuclear and cell areas significantly greater than those of RER cells. In addition, granulate cells of red algal-fed snails had 4 to 14 vacuoles that contained electron dense material with staining characteristics similar to that of ink in mature ink release vesicles. In romaine lettuce-fed and green seaweed-fed snails these vacuoles were present but empty. Regardless of diet, 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 prevents large particles in the hemolymph either from being taken up by, or occluding the coated vesicles. Electron dense particles within coated vesicles were similar in size to those in granulate vacuoles but larger (on average by about 1nm) than those that compose ink. The third cell type, the ink release vesicle itself, originates from a single cell whose nucleus expands 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 one to three layers of cells of unknown origin. 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.

3. 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 6 to 11% of that for the digestive vacuoles of rhodoplast digestive cells lining the tubules of the digestive gland (Coelho et al., 1997). Our observations suggest that: a) the main form of the ink pigment in the gland is either phycoerythrobilin or/and a non-antigenic form of phycoerythrin, and b) 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 rhodoplast digestive cells of the digestive gland.

4. 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 weight, non-algal protein (whose function is unknown), which comprises 35% of the dry weight of secreted ink.