The relationship between seaweed diet and purple ink production in Aplysia dactylomela rang, 1828 from Northeastern Brazil

Journal of Shellfisheries Research, August, 2004 by Luis Ernesto Arruda Bezerra, Ana Fontenele Urano Carvalho, Luciana Aires Barreira, Vanessa Lucia Rodrigues Nogueira, Jose Roberto Feitosa Silva, Ilka Maria Vasconselos, Vania Maria Maciel Melo

Histologic Analysis of the Ink Gland

Specimens of A. dactylomela were collected during low tide and transported to the laboratory in a container with seawater. Some specimens were de-inked by squeezing them gently for a few minutes outside the water and then kept in seawater tanks for 15 days with unialgal diets (U. fasciata or Gracilaria sp.) to observe whether there was purple ink production. At the end of this period, sea hares were anaesthetized by storage in a refrigerator and then dissected carefully to remove the ink gland.

For light microscopy, ink gland samples were fixed with Bouin mixture (saturated picric acid solution, 40% formaldehyde, glacial acetic acid (15:5:1 v/v) and embedded in paraffin. Tissue sections (5 [micro]m) were stained with hematoxylin and eosin (Junqueira & Junqueira, 1983) and with bromophenol blue for detection of proteins (Pearse 1960). Histologic sections were observed under a microscope, and photographs were taken.

Chemical Composition of Purple Ink

Purple ink was analyzed for contents of water, protein (total nitrogen), reduced carbohydrate, lipid, and ash. For the water determination, 1g of ink was dehydrated in an oven at 100[degrees]C to 110[degrees]C to constant weight. Total nitrogen was determined in samples of freeze-dried purple ink by micro-Kjeldahl digestion (Baethgen & Alley 1989). The content of reduced carbohydrate was determined according to Dubois et al. (1956). Lipid content was determined by n-hexane Soxleht extraction, and ash was quantified by heating 1g of ink in a muffle furnace at 620[degrees]C for 18 h.

Amino Acid Analysis

Dry purple ink (1 mg) was hydrolyzed in 1 mL 6 M HC1 with 1% phenol (w/v), in a sealed glass tube under [N.sub.2], at 110[degrees]C for 22 h. After hydrolysis, HCl and phenol were removed by evaporation, and the residue was analyzed in a Biochrom 20 (Pharmacia-LKB) amino acid analyzer.

Electrophoretic Profile of Ink Proteins

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of beta-mercaptoethanol according to the method of Laemmli (1970). Proteins were stained with silver nitrate (Blum et al. 1987). Bovine serum albumin (66.0 kDa), egg albumin (45.0 kDa), porcine pepsin (34.7 kDa), bovine [beta]-lactoglobulin (18.4 kDa), and egg lysozyme (14.3 kDa) were used as standards (Sigma Co., USA).

Seaweed Protein Extraction and Determination

Proteins were extracted from fresh samples of the green seaweed U. fasciata and the red species H. musciformis and Gracilaria sp. The samples were ground with a mortar and pestle with the following buffers at 50 mM: Glycine-HCl, pH 2.6; Tris HCl, pH 7.0; and sodium borate, pH 9.0. The extracts of U. fasciata and H. musciformis were prepared at the proportion of 1:3 (m/v) and that of Gracilaria sp. at 1:5 (m/v). The extracts were filtered through a nylon tissue and centrifuged at 15,000 g for 10 rain at 4[degrees]C. The supernatants (crude extracts) had the protein content determined according to Bradford (1976) with bovine serum albumin (BSA) as the standard (purchased from Sigma Co., USA).