Initial characterization of a potential anti-fouling system in the American horseshoe crab, Limulus polyphemus.

Although the surfaces of most solid objects are rapidly colonized by sessile fouling organisms within a short time after immersion in the ocean, macroscopic fouling organisms are surprisingly sparse on the cuticle of healthy adults of the American horseshoe crab, Limulus polyphemus. One of the major identified causes of mortality of adult horseshoe crabs is the erosion of the cuticle that follows its colonization by green or blue-green algae (1). Thus it is in the interest of the animal to maintain the cuticle clear of fouling species. The mechanism(s) responsible for keeping the cuticle clean in an environment liberally stocked with fouling species have not been identified. One likely candidate is a mucous secretion from the hypodermal glands, a system of glands that discharge onto the surface of the cuticle (2). Here we show that this secretion has antibiological activities that may contribute to its ability to deter the colonization of the cuticle of Limulus by fouling organisms. The cuticular secretion can be provoked by housing healthy adults in the presence of decaying fish (3). The secretion from the hypodermal glands was scraped from the dorsal cuticle of the Limulus shell with a rubber scraper and stored at 4°C in the presence of 0.2 mg/ml NaN3 to prevent bacterial contamination. Antibodies to whole cuticular secretion were prepared in rabbits. Immunostaining utilized a 1:1000 dilution of the primary antiserum, a 1:1000 dilution of HRP-anti rabbit IgG, and 4-chloronaphthol using standard methods for staining Western and dot blots (4). Stimulated animals renewed the layer of cuticular secretion within 1–2 h after the cuticle had been scraped clean. Although most of the animals in the Marine Resources Center of the Marine Biological Laboratory, which are collected from the pristine waters of Pleasant Bay, lacked the thick layer of cuticular secretion of the pollution-challenged animal, the secretion was present as a thin layer on their surfaces as well. Anti-cuticular secretion antisera specifically immunostained swipes of the surface of the cephalothorax of these animals, indicating the presence of the secreted products of the hypodermal glands. Control swipes immunostained with non-immune rabbit IgG in place of immune serum failed to stain. It has been proposed that the cuticular secretion contains the secreted products of the Limulus blood cells (2). This apparently is not true because the principal secreted product of the blood cells, the clotting protein coagulogen, is absent from the secretion. Antibodies to coagulogen failed to immunostain dot blots of cuticular secretion, and the anti-cuticular secretion antiserum failed to stain coagulogen or the collective secretion of the blood cells. This latter material was prepared as described previously (5, 6). The anti-coagulogen antiserum did stain dot blots of coagulogen, and the anti-cuticular secretion antiserum did stain dot blots of cuticular secretion. The cuticular secretion showed 15 protein bands by SDS-PAGE (reducing conditions, silver-staining) in the molecular mass range of 143–20 kDa. The anti-biological activity of the cuticular secretion was tested by its cytolytic actions on target cells. Cuticular secretion hemolysed sheep red blood cells at a 1:16 dilution in a standard hemolysis assay (7) (Fig. 1A). Hemolysis was judged to be divalent cation-dependent because the divalent cation chelator, ethylenediaminetetraacetic acid, reduced hemolysis (Fig. 1A). The macromolecular osmolites dextran-8 (Mr 8 –12 kDa) and, to a lesser extent, dextran-4 (Mr 4 – 6 kDa) reduced the extent of hemolysis (Fig. 1B). This suggests that hemolysis is the result of hydrophilic membrane channels established in the plasma membrane of the target red blood cell by the hemolytic protein of the cuticular secretion. Protection by the dextrans is suggested to result from their ability to balance the osmotic pressure across the permeabilized cell membrane, which will reduce the flow of water through the hemolytic pore and into the cell and will prevent swelling and lysis (8). It is difficult to envision ways for macromolecular osmolites to protect the cell if the hemolytic process featured such other possible mechanisms as phospholipase action or detergent-mediated membrane reorganization. It is proposed that the cuticular secretion is one agent that helps maintain the cleanliness of the cuticle of Limulus. Its