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1 Marine Biological Laboratory, Woods Hole, MA
2 University of California, Davis, CA
* Corresponding author: pbarmstrong{at}ucdavis.edu
Colonization by sessile fouling organisms (epibionts) is the usual fate for solid surfaces in the marine environment. The American horseshoe crab, Limulus polyphemus, ceases molting upon reaching maturity and lives several years as an adult in an epibiont-rich milieu, yet it typically maintains a cuticle that is largely free of macroscopic flora and fauna. Indeed, it is in the interest of the animal to maintain its carapace free from such organisms, as colonization of the cuticle by green and blue-green algae can be fatal (1). The mechanisms by which Limulus maintains a clean carapace are not well understood. We have investigated the antibiological properties of a potential anti-fouling system of Limulus, a viscous secretion of a system of dermal glands that discharge their product onto the surface of the carapace (2). We propose that this substance, dermal exudate (DE), contributes to maintaining the cleanliness, and thus the integrity, of the cuticle. In the past we have identified and partially characterized a hemolytic activity present in DE (3, 4). It was shown that the presence of macromolecular osmolites in the hemolysis assay medium, dextran-8 and to a lesser extent dextran-4, prevented lysis of the red blood cells. This effect is attributed to the ability of these macromolecular osmolites to establish an osmotic balance between the interior and exterior of the cell so that no net water flow into the cell occurs, protecting the cell from cytolysis. The inhibitory effect of dextrans suggests that DE-induced lysis results from hydrophilic pore formation in the lipid bilayer of the red blood cell rather than from a detergent-like disruption of lipid packing or from phospholipase activity. To investigate the potential interactions of DE directly with lipid bilayers, we have utilized a model system in which a self-quenching fluorescent dye has been trapped inside liposomes; an increase in the fluorescence of the dye is a marker for membrane permeation. Here we report the ability of an acid-precipitable constituent of DE to permeabilize liposomes. The agent or agents responsible for these hemolytic and liposome-permeating activities may function as deterrents against potential colonizers of the Limulus cuticle.
The secretion of DE can be stimulated by housing Limulus in stressful conditions such as a cage stocked with decaying fish material. Dermal exudate was collected by scraping the dorsal carapace. The exudate was stored at -20 °C or with 0.02% NaN3 to prevent bacterial growth. Liposomes were prepared with soybean type II phosphatidylcholine from Sigma. Lyophilized lipid was dissolved in chloroform and dried under a nitrogen stream to a thin film. The film was placed under vacuum overnight to remove any traces of solvent. The lipids were hydrated in 20 mM Tris pH 7.4, 100 mM carboxyfluorescein (CF), a self-quenching fluorescent dye. The resulting multilamellar liposomes were made unilamellar through five successive freeze-thaw cycles. Untrapped CF was removed from the liposome suspension by passage over a Sephadex G-50 column. The release of CF from the interior of liposomes was monitored as an indicator of lipid bilayer permeation. Assays were performed with a 1:1000 dilution of liposomes in 20 mM Tris, pH 7.2. Fluorescence intensity was observed with a Photon technologies fluorometer. Excitation and emission wavelengths were 460 nm and 550 nm respectively. The slit widths were adjusted such that the Raman scatter peak of distilled, deionized water at 397 nm gave an intensity of approximately 350,000 counts/s when excited at 350 nm. Release of entrapped CF from liposomes was seen as an increase in fluorescence intensity. A unit of activity is arbitrarily defined as an initial increase in fluorescent intensity of 5% per minute. Intensity values corresponding to 100% lysis were obtained with the addition of Triton X-100.
Crude dermal exudate showed potent liposome-permeating activity at a 1:100 dilution. Addition of 10 mM CaCl2 markedly reduced activity, as did acidic pH. The membrane-permeating activity of crude DE was retained by a 10-kDa cutoff filter (Amicon P-10) and was precipitated by 1 M HCl, suggesting that the responsible agent is a large molecule. When, however, the material that precipitated during the acid extraction was resuspended in an equivalent volume of 20 mM Tris, pH 7.2, a 71-fold increase in liposome-permeating activity was seen. Presumably, acid treatment removes an inhibitor. The activity of the resuspended material was, like that of crude DE, inactive at low pH. The active agent present in the acid-induced precipitate also failed to pass through a 10,000 molecular weight cutoff filter. Addition of NaCl or CaCl2 attenuated the activity in a concentration-dependant fashion. This suggests that the lytic agent might initially bind the lipid bilayer through ionic interactions.
The secretion is most likely the product of transdermal glands whose ducts open onto the surface of the carapace (2, 5). The viscosity of DE may provide for a matrix that slows the diffusion of cytolytic effector molecules into the surrounding aqueous environment. Direct cytolytic attack is a method of defense from potential pathogens that is used by many animal phyla. A voluminous literature exists documenting the diversity and mechanisms of proteins and peptides capable of permeabilizing lipid bilayers (6). We propose that the ancient arthropod Limulus polyphemus may employ just such a method for maintaining a carapace free from colonizing organisms. We have, in past publications, documented the ability of DE to lyse red blood cells and presented evidence for a mechanism of pore formation in the lipid bilayer of the target cell. Here we present further evidence for direct interaction of a lytic agent in DE with lipid bilayers in such a manner as to make them permeable. We propose that these lytic activities are components of a defensive system that operates at the surface of the carapace of Limulus to maintain the cleanliness and integrity of the animal’s cuticle.
This research was supported by Grant No. MCB-26771 from the National Science Foundation. We thank Mr. Louis Kerr for important assistance with use of the fluorometer and Dr. David Gadsby for donation of striped bass heads for the stimulation of DE production by caged horseshoe crabs.
Literature Cited
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