Johnson Paul M, Kicklighter Cynthia E, Schmidt Manfred, Kamio Michiya, Yang Hsiuchin, Elkin Dimitry, Michel William C, Tai Phang C, Derby Charles D
Department of Biology, Center for Behavioral Neuroscience, and Brains and Behavior Program, Georgia State University, Atlanta, GA 30303 USA.
J Exp Biol. 2006 Jan;209(Pt 1):78-88. doi: 10.1242/jeb.01972.
Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an L-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates--L-lysine and L-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml(-1), or 30 micromol ml(-1), which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l(-1) and lysine is present in ink at <1 mmol l(-1) but in opaline at 65 mmol l(-1). Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin's antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l(-1), our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapin's antipredatory functions remains to be determined.
海兔通过多种化学防御方式来保护自己免受捕食者的攻击。其中一种方式是喷墨，即在受到捕食者攻击时主动释放一种保护液。在许多海兔中，包括加州海兔和指状海兔，这种液体是来自两个不同腺体的两种分泌物的混合物，通常会共同释放：墨水，一种来自墨腺的紫色液体；以及乳白液，一种来自乳白腺的白色粘性分泌物。这两种分泌物在鳃腔中混合，并导向攻击的捕食者。这些分泌物中的一些化学物质及其作用机制已经被确定。在我们的研究中，我们使用蛋白质免疫印迹法、免疫细胞化学、氨基酸分析和生物测定来研究这些成分的分布：（1）一种名为逃避素的L-氨基酸氧化酶（对于加州海兔）和指状海兔素-P（对于指状海兔），它具有抗菌活性，但我们认为其主要功能是保护海兔免受引发其释放的捕食者的侵害；（2）逃避素的主要氨基酸底物——L-赖氨酸和L-精氨酸。逃避素仅在墨腺中产生，不存在于任何其他组织或分泌物中。此外，逃避素仅被隔离在墨腺的琥珀色囊泡中，而不在红紫色囊泡中，红紫色囊泡中含有赋予墨水独特紫色的藻类衍生发色团。墨腺中以及释放后墨水中逃避素和指状海兔素-P的浓度高达2毫克/毫升（或30微摩尔/毫升），远高于其抗菌阈值。赖氨酸和精氨酸（以及其他氨基酸）被包装在墨腺和乳白腺的囊泡中，但精氨酸在墨水和乳白液中的含量<1毫摩尔/升，赖氨酸在墨水中的含量<1毫摩尔/升，但在乳白液中的含量为65毫摩尔/升。我们之前的结果表明，赖氨酸和精氨酸都介导逃避素的抑菌作用，但只有赖氨酸介导其杀菌作用。鉴于逃避素的抗菌作用需要赖氨酸和/或精氨酸的浓度>1毫摩尔/升，我们的数据使我们得出结论，乳白液中的赖氨酸是墨水中逃避素的主要天然底物。此外，将酶逃避素及其底物赖氨酸包装在两个单独的腺体中，并在捕食者攻击时共同释放和混合，使得能够在需要的精确时刻从无害前体生成生物活性防御化合物。赖氨酸和/或精氨酸是否是逃避素抗捕食功能的底物仍有待确定。
