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First published online December 14, 2005
Journal of Experimental Biology 209, 78-88 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.01972

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Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica

Paul M. Johnson^1,2, Cynthia E. Kicklighter¹, Manfred Schmidt¹, Michiya Kamio¹, Hsiuchin Yang¹, Dimitry Elkin³, William C. Michel³, Phang C. Tai¹ and Charles D. Derby^1,*

¹ Department of Biology, Center for Behavioral Neuroscience, and Brains and Behavior Program, Georgia State University, Atlanta, GA 30303 USA
² Department of Biology, University of Washington, Seattle, WA 98195 USA
³ Department of Physiology, University of Utah School of Medicine, Salt Lake City, UT 84108 USA

View larger version (26K): [in a new window]   Fig. 1. Western blot shows that escapin is expressed only in the ink gland. The tissues were tested from adult Aplysia californica, with the addition of albumen gland complex from juvenile animals. An anti-escapin antibody labels purified protein and a 60 kDa protein in ink and the ink gland.

View larger version (20K): [in a new window]   Fig. 2. Concentration of the 60 kDa protein in ink. The 60 kDa protein is escapin from Aplysia californica and dactylomelin-P from Aplysia dactylomela, which are close homologues (Yang et al., 2005). For each species, two methods of collecting ink were used. `Ink released', ink collected from animals that were removed from the aquarium, blotted dry, handled until they inked and then ink was pipetted from the mantle cavity. `Ink from squeezed glands', ink squeezed from dissected ink glands. Values are mean ± s.e.m., N=6 animals for each group.

View larger version (104K): [in a new window]   Fig. 3. Western blot shows that escapin is present only in amber vesicles of the ink gland. This SDS-PAGE of Aplysia californica ink and vesicles shows that the 60 kDa protein escapin is present in ink and the amber vesicles of the ink gland but not in the red-purple vesicles.

View larger version (66K): [in a new window]   Fig. 4. Immunostaining with anti-escapin shows that escapin in the ink gland is expressed only in amber vesicles. Tissue is 100 µm thick vibrating microtome sections of ink gland of Aplysia californica. (A) Transmitted light illumination reveals the presence of amber (black asterisks), red-purple (white asterisk) vesicles, and an uncolored vesicle-like structure (arrow, highlighted in C). (B) Fluorescence image showing anti-escapin immunoreactivity. Note that only the amber vesicles are immunoreactive. The additional small area of immunoreactivity (arrow) might represent the cap of a vesicle whose bulk is located in the adjacent section. (C) Overlay of A and B. Note that red-purple vesicle and the uncolored vesicle-like structure (highlighted by dotted line) are unlabeled.

View larger version (139K): [in a new window]   Fig. 5. Antimicrobial assay shows bioactivity only in amber vesicles of Aplysia californica. Clear spots on the plate represent antibacterial activity and are seen only for spots 1, 3 and 5. Spot 1, pure ink (note that this spot is free of bacteria, but appears dark because of the purple pigment of the ink); 2, buffer control; 3, red-purple and amber vesicle extracts together; 4, opaline (note that this spot is dark because it has bacterial growth greater than the control), because opaline is a very good growth medium; 5, amber vesicle extract alone; 6, red-purple vesicle extract alone.

View larger version (98K): [in a new window]   Fig. 6. Immunostaining with antisera against amino acids and escapin in ink gland of Aplysia californica. Tissue is 0.5 µm thick plastic sections. A, B and C are from three different ink glands, each a set of four sections from the same region of tissue labeled with different antisera, i.e. antiserum against either escapin, alanine, taurine, arginine, cysteine or aspartate. `Preimmune' is the control, being labeled with preimmune serum. Vesicles labeled `a' are strongly immunopositive for many amino acids but not escapin; vesicles labeled `e' are intensely immunopositive for escapin but not for amino acids; vesicles labeled `c' are clear and not strongly immunopositive either for amino acids or escapin.

View larger version (105K): [in a new window]   Fig. 7. Immunostaining with antisera against amino acids in an opaline gland of Aplysia californica. Shown is a set of 0.5 µm thick plastic sections from the same region of tissue labeled with different antisera, i.e. antiserum against (A) aspartate, (B) alanine, (C) lysine, (D) cysteine or (E) taurine, or (F) preimmune serum. Vesicles labeled `1' are strongly immunopositive for taurine and have some, though usually less, immunoreactivity for other amino acids. Vesicles labeled `2' are less immunoreactive to taurine than to most another amino acids, particularly aspartate and alanine.