Original Contributions Immunolocalization of the Toxin Latrunculin B within the Red Sea Sponge Negombata magnifica (Demospongiae, Latrunculiidae) O. Gillor, 1 S. Carmeli, 2 Y. Rahamim, 2 Z. Fishelson, 3 and M. Ilan 1, * 1 Department of Zoology, 2 School of Chemistry, 3 Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Abstract: The location of latrunculin B, the major toxin of the Red Sea sponge Negombata magnifica, was revealed using specific antibodies. Antibodies from rabbits immunized with a conjugate of latrunculin B with keyhole limpet hemocyanin (KLH) were purified over a latrunculin B–Sepharose affinity column. Analysis of immunohistochemical and immunogold-stained sponge sections, using light and transmission electron mi- croscopy, revealed latrunculin B labeling mostly beneath the sponge cortex at the border between the external (ectosome) and internal (endosome) layers (ectosome-endosome border). The endosome was less labeled than the border. Immunogold localization revealed latrunculin B in the sponge cells but not in its prokaryotic symbionts. Archeocytes and choanocytes were significantly more labeled than other cells. The antibodies primarily labeled membrane-limited vacuoles within archeocytes and choanocytes that are perhaps latrunculin B secretory or storage vesicles. Peripheral latrunculin B may have a role in defense against external epibionts, predators, and competitors. Key words: Porifera, natural product, antibodies, secondary metabolite, defense. I NTRODUCTION Many exposed, sessile organisms have developed an array of secondary metabolites (natural products) with potent bio- toxic and cytotoxic properties (Ko ¨nig and Wright, 1996). It has been hypothesized that such compounds act in a variety of ways, mostly as a defensive mechanism against predators (Pawlik et al., 1995) and pathogens (Becerro et al., 1994), and as an aid to the organism in competition with neigh- boring benthic organisms (Sears et al., 1990; Willemsen, 1994). Sponges, the oldest and simplest metazoans (Wilkin- son, 1983), are one of the richest sources for such metabo- lites. Sponge natural products have proved to be an impor- tant source of new pharmaceuticals and experimental phar- maceutical compounds (Scheuer, 1990; Ko ¨nig and Wright, 1996). Although the structure and toxicity of sponge bio- active metabolites have been extensively studied (e.g., Higa et al., 1994; Ko ¨nig and Wright, 1996), their localization within the sponge has rarely been established (Table 1). Localization of metabolites within the sponge is a complex task for several reasons. First, sponges contain within them symbiotic microorganisms (bacteria, algae, fungi, and pro- Received December 21, 1999; accepted March 5, 2000. *Corresponding author: telephone 972-3-640-8613; fax 972-3-640-9403; e-mail milan@post.tau.ac.il Mar. Biotechnol. 2, 213–223, 2000 DOI: 10.1007/s101260000026 © 2000 Springer-Verlag New York Inc.