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<I>Conus Spurius Atlanticus</I>

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<I>Conus Spurius Atlanticus</I> THE VENOM AND VENOM APPARATUS OF THE ATLANTIC CONE, CONUS SPURlUS ATLANTlCUS (CLENCH) JOHN H. SONGDAHU Department of Zoology, University of Miami ABSTRACT Some of the chemical characteristics of the venom and physical proper- ties of the radular teeth of Conus spurius atlanticus (Clench) are described. A mechanism of venom propulsion is proposed, and evidence is presented which indicates that this species probably includes mollusks among the prey species. INTRODUCTION The alphabet cone, Conus spurius atlanticus Clench, is an inhabitant of the muddy and sandy bottoms in shallow waters along the coast of Florida and the West Indies. Members of the family Conidae are characterized by a well-developed venom apparatus. Kohn et al. (1960) summarized the history of our knowledge of the venoms of various species of cones and described typical organization of the venom apparatus and some chemical and physical properties of the venom. Subsequent studies by other investigators have described some pharmacological and toxicological properties of the venom. However, previous studies have been almost entirely concerned with Indo-Pacific or eastern Atlantic species. Few data are available describing the venom and venom apparatus of western Atlantic cones. This work provides some information on the characteristics of the venom and venom apparatus of the alphabet cone. I wish to express my sincere gratitude to Dr. Charles E. Lane of the Rosenstiel School of Marine and Atmospheric Science, University of Miami, for his helpful suggestions and guidance. I also wish to thank Dr. James D. Stidham, Postdoctoral Fellow, University of Miami, for per- forming the free-amino-acid analysis and Dr. Francis Horne, Postdoctoral Fellow, University of Miami, for performing the nitrogen determination. MATERIALS AND METHODS Alphabet cones, Conus spurius atlanticus, were collected in Biscayne Bay, Miami, Florida. The venom was extracted and prepared by the method of Songdahl & Lane (1970). Crude venom extracts were tested for activity against the gastropods Ficus communis and Busycon contrarium, the fish Poecilia latipinna and 1 Present address: The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138. 1973J Songdahl: Venom of the A tlantic Cone 601 Cyprinodon variegatus, and juvenile white rats. Crude venom extract equivalent to a single cone was tested against each animal. A crude extract from several cones was also tested on a smooth-muscle preparation of rat ileum. Lyophilized venom was used in all chemical determinations. Nitrogen Determination.-A micro-Kjeldahl nitrogen determination was performed on 11.3 mg of lyophilized venom. Analysis of Free Amino Acids.-Twenty-five milligrams of venom were suspended in 3.0 ml of 70 per cent ethanol. The suspension was centri- fuged, and the clear supernatant removed. The procedure was repeated with the residue, and the two supernatant fractions were combined and dried in a vacuum desiccator. The residue was suspended in 2.0 ml of 10 per cent ethanol and acidified with 1.0 ml of 1.0N HCl. An internal standard of 0.1 ml of norleucine was added to the extract. Amino acids in the extract were identified qualitatively and quantitatively in a Technicon autoanal yzer. Lipid Analysis.-Twenty-five milligrams of venom were suspended in 3.0 ml of petroleum ether, shaken thoroughly, and centrifuged. The supernatant was evaporated in a vacuum desiccator to approximately 0.1 ml. A thin layer chromatogram was prepared by the method of Mangold (1961). The adsorbent was silica gel applied in a 250p.,-275p., layer on a glass plate. Samples of known lipids and lipid extracts from the venom were spotted in 2-lambda aliquots, to a total of 8 lambda per spot. The chromatogram was developed for 40 minutes in a solution of petroleum ether, diethyl ether, and glacial acetic acid (90:10:1). The plate was dried at room temperature and exposed to iodine vapors for several minutes to reveal unsaturated lipids. The plate was then sprayed with a solution of Rhodamine B, 0.05 per cent in ethanol, 96 per cent. The completed chromatogram was observed under ultraviolet light and the spots outlined. The lipid-free venom was dried in a vacuum desiccator and the residue weighed. Carbohydrate Analysis.-Determination of carbohydrates was by the spectrophotometric method of Dische (1955). By this method it is possible to detect hexoses, pentoses, and methyl pentoses in the same sample. For this determination, 3.0 mg of venom was suspended in 3.0 ml of distilled water to give a 100 mg per cent solution of venom. To 0.1 ml of the venom suspension, 0.9 ml of distilled water, and 4.0 ml of concentrated H2S04 were added while the test tubes were partially im- mersed in a cold-water bath. A standard was prepared by adding 0.5 ml of water and 4.0 ml of H2S04 to 0.5 m! of standard galactose, 10 mg 602 Bulletin of Marine Science [23(3) per cent. A blank was also prepared. After one hour, 0.1 ml of a 3 per cent cysteine solution was added to each of the test solutions. After 20 minutes, the solutions were read on a Beckman Model DU spectrophotom- eter at 390 mfL for pentose, and at 440 mfL for hexose. A chromatographic determination of hexoses and hexoseamines was performed by the method of Chargaff et al. (1948). For the determination, 4.22 mg of venom was hydrolyzed by suspending the venom in 4.0 ml of 2N HCI and placing the mixture in a boiling-water bath for 2 hours. The hydrolyzed mixture was then filtered, and the filter paper and container were washed with distilled water. The filtrate was evaporated to dryness and the residue dissolved in 0.5 ml of water. The venom hydrolyzate was then spotted on chromatograph paper in 10-lambda aliquots, to a total of 300 lambda. Standard solutions of glucose, mannose, galactose, fucose, glucosamine, and galactoseamine were prepared and spotted on the paper in 10-lambda aliquots, to a total of 60 lambda. The solvent was 30 ml of N-butanol, 20 ml of pyridine, and 15 ml of water (6: 4: 3 ), which had been placed in a chromatocab to saturate the ambient air. The chromatogram was run for 18 hours in a descending solvent system. A developing solution was prepared by dissolving 10 g of silver nitrate in 10 ml of water and adding the solution to 1 liter of acetone. The chromato- gram was developed and dried at room temperature. A second solution was prepared by dissolving 40.0 grams of sodium hydroxide in 40 ml of water and combining the NaOH with 800 ml of isopropanol and 1200 ml of ethanol. The developed and dried chromatogram was then rolled through this solution, which helped decrease the background color and accentuate the spots. Ash Content.-An ash determination was performed on 35.1 mg of venom. The venom was placed in a covered crucible and incinerated for 24 hours at 375°C in a Thermolyne muffle furnace Type A1500. The ashed venom was removed from the furnace and placed in a desiccator for 1 hour, to prevent hydration. The crucible and ashed venom were weighed, and the percentage remaining as ash was calculated. Preparation of Radular Teeth.-The radular apparatus was dissected from a number of cones and prepared for observation by the method of Hine- gardner (1958). The radular sheath was placed in concentrated NaOH (10 g of NaOH in 10.0 ml of water) for several days. The solution was gently agitated and the teeth were removed. The teeth were rinsed in dis- tilled water and placed in a saturated solution of silver nitrate for 3-5 minutes, rinsed in distilled water, rinsed in 95 per cent ethanol to remove excess water, and immediately immersed in xylene. They were not allowed to dry in air and were transferred directly from the xylene to a glass slide and mounted in Kleermount, a xylene-base mounting medium. 1973] Songdahl: Venom of the A tlantic Cone 603 TABLE 1 ANALYSISOF FREE AMINOACIDS Micro- Micrograms/ Amino acid moles/mg mg of venom Serine 0.022 2.31 Glutamic Acid 0.013 1.89 Glycine 0.007 0.525 Alanine 0.009 0.801 Valine 0.006 0.702 Isoleucine 0.004 0.524 Leucine 0.008 1.049 Norleucine* 0.038 4.98 Tyrosine 0.005 0.905 Phenylalanine 0.008 1.32 NHR 0.021 0.357 Lysine 0.002 0.448 Histidine 0.005 0.775 Arginine 0.009 1.567 TOTAL(less standard) 13.173 • Used as internal standard. RESULTS The Venom.-The venom of C. spurius atlanticus was a viscous milky fluid in the anterior section of the venom duct. The posterior section of the duct, nearest the venom bulb, contained venom which was frequently orange or yellow. The venom in the posterior section was more viscous and often had a jellylike consistency. The venom dried rapidly in air and attained a pastel ike texture after a few minutes in air. The venom had a pH of 8.2-8.3 and was granular under microscopic examination. The granules were from 0.511--3.011- in diameter. A micro-Kjeldahl determination showed 1.078 mg of nitrogen in a 11.3- mg sample of lyophilized venom. This indicated that the dried venom was 9.6 per cent nitrogen. At least 13 different free amino acids were detected in the venom (Table 1). Proline appeared only in small amounts and, due to the small peak, could not be accurately estimated. One peak appeared which was interpreted as either serine or serine, threonine, and aspartic acid occurring under the same peak. This acid, or group of acids, appeared to be the most abundant of the free acids in the venom.
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