Role of the Digestive Gland in Ink Production in Four Species of Sea Hares: An Ultrastructural Comparison

The ultrastructure of the digestive gland of several sea hare species that produce different colored ink (Aplysia californica produces purple ink, A. juliana white ink, A. parvula both white and purple ink, while Dolabrifera dolabrifera produces no ink at all) was compared to determine the digestive gland’s role in the diet-derived ink production process. Rhodoplast digestive cells and their digestive vacuoles, the site of digestion of red algal chloroplast (i.e., rhodoplast) in A. californica, were present and had a similar ultrastructure in all four species. Rhodoplast digestive cell vacuoles either contained a whole rhodoplast or fragments of one or were empty. These results suggest that the inability to produce colored ink in some sea hare species is not due to either an absence of appropriate digestive machinery, that is, rhodoplast digestive cells, or an apparent failure of rhodoplast digestive cells to function. These results also propose that the digestive gland structure described herein occurred early in sea hare evolution, at least in the common ancestor to the genera Aplysia and Dolabrifera. Our data, however, do not support the hypothesis that the loss of purple inking is a synapomorphy of the white-ink-producing subgenus Aplysia. 1. Introduction The release of bright purple ink from the ink glands of many species of sea hares (Gastropoda: Opisthobranchia: Anaspidea) is a major component of their defensive arsenal against predators [1–4]. Though all sea hares possess ink glands in their mantle cavities, not all of these glands release purple ink and, in fact, some release no ink at all [3]. Ink released by sea hares is a mixture of compounds from two glands, the ink and opaline glands [3, 5]. That released from the ink gland has two major components: aplysioviolin (APV) which is 65% of the dry weight of the bright purple ink released by the best studied ink producing sea hare, Aplysia californica Cooper [6], and a high molecular mass protein that has been named escapin [7, 8]. APV is derived from the red algal photosynthetic pigment, r-phycoerythrin (PE), minus its low molecular mass protein. This produces phycoerythrobilin (PEB) that is then methylated in the ink gland to form APV [6, 7, 9–13]. APV may be a better storage form of red algal pigment than PEB and this could account for the conversion of PEB to APV. Escapin, like similar high molecular mass protein from related sea hare species, is capable of producing antipredator responses as well as provide both antimicrobial and antitumor activity ([4], see [14] for a review). This ink