THE VENOM AND VENOM APPARATUS OF THE ATLANTIC CONE, 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 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 . 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. Glutamic acid and ammonia were also present in substantial amounts. Spectrophotometric determinations showed that the venom contained 604 Bulletin of Marine Science [23(3) 0 0 0 0 0 0 0

Gluc-}lli2 Galactose Mannose Venom Fucose Gal-NH2 Glucose FIGURE 1. Chromatogram of venom of Conus spurius atlanticus and some carbohydrate standards.

approximately 16 per cent carbohydrate. The majority of the carbohydrate was identified as glucose and comprised 15.5 per cent of the venom. Less than 1.0 per cent of the venom was fucose, a methyl pentose. The presence of hexose was determined by spectrophotometric analysis and glucose and fucose were identified by chromatography (Fig. 1). 1973] Songdahl: Venom of the A tlantic Cone 605

g 0 ~ 8 Q 0 0 0 0 G 0 ([) e G) 0 9

Oleic Venom VenOM Wesson Oil Venom Venom Venom Caproic Acid in Ether Palmitic Oleic Behenic Cholesterol

FIGURE 2. Thin layer chromatogram of lipid extract of venom of Conus spurius at/anticus and some lipid standards.

The lipid content was determined to be 11.2 per cent. Three classes of lipids were detected by thin layer chromatography (Fig. 2). A spot which remained at the source probably represents a phospholipid, as they do not migrate in the solvent system used in the analysis. A second spot with Rp values of 0.218 and 0.258 probably represents fatty acids with chain lengths of 14 to 18 carbon atoms. All spots were stained by Rhodamine B and were also visible in the presence of iodine vapors, which indicated unsaturation. Triglycerides or diglycerides were not detected. Incineration of lyophilized venom showed 2.3 per cent ash. The venom was active against the gastropods Ficus and Busycon. Within 30 seconds of injection of crude venom extract into the mantle of Ficus, the animal showed darkening and mottling of the mantle. Within 24 hours, the animal was dead and the foot extended from the shell. A hole 1.32 mm in diameter was drilled into the operculum, and an equal amount of 606 Bulletin of Marine Science [23(3) crude extract was injected through the hole into the foot of a large Busycon. The whelk was placed upside down in an aquarium to test for a righting action. The animal made rectractile responses to sound and tactile stimuli, but did not attempt to complete the righting action. Ten days after injection, the whelk righted itself and appeared to resume normal activity. The crude venom, equivalent to that of one cone, had no visible effect on the fish Poecilia and Cyprinodon, or the juvenile white rats. No effect was observed on the smooth-muscle preparation of rat ileum. The Venom Apparatus.-The venom apparatus was composed of the proboscis, venom duct which produces the venom, the venom bulb, and the radular sheath which contained the radular teeth. The venom duct was an elongated, twisting tube that originated at the end of the venom bulb and occupied much of the body cavity. The distal end of the duct entered the pharynx. In this species the duct was two to six times the length of the shell. Generally the specimens less than 35 mm in shell length had a lower ratio of venom-duct length to shell length. The venom duct could usually be delineated into two sections due to color differences. The venom in the anterior section was always lighter in color and less viscous than the venom in the posterior section of the duct. The duct was frequently as large as 1.0 mm in diameter in the larger , but was less than 0.5 mm in smaller specimens. In the 50 animals used, the length of the duct ranged from 51 mm in a specimen with a shell length of 33 mm to a duct length of 388 mm in a specimen with a 57-rom shell length. The venom bulb was 4-22 mm long and had a diameter of 2-5 mm, depending on the size of the animal. The bulb was a white, slightly translucent, sausage-shaped organ and was transversely oriented, dorsal and anterior to the viscera. The radular apparatus was anterior to the venom bulb and consisted of a bilobed sheath containing a long dorsal arm, a short ventral arm, and a ligament sac. The longer lobe contained 15-30 teeth oriented with points directed toward the blind end of the sheath. These teeth were found in various stages of development. The shorter lobe contained 7-10 teeth, all of which pointed toward the pharynx, or in opposite direction to those in the longer lobe. The radular sheath was frequently translucent, and the teeth were occasionally visible through the sheath. The radular teeth were 2.1-3.0 mm long in the mature animal. The tip of each tooth was barbed on both sides, with one barb below and posterior to the other (Fig. 3). A third barb was situated approximately halfway along the shaft and was directed toward the base. The teeth have small serrations from the anterior barb to approximately half the length of the tooth. A fourth barb, or posterior barb, was located at the base of the 19731 Songdahl: Venom of the A tlantic Cone 607 .Q•.. en~

~•... eJ) r:: ~ -"0 ..r:: I ..•...• I I 0 I t;'~:;1 ..r:: ,~ -0 2

C'd 3 "0 ~'" ,...; ~ C( ;l 0 ~ 608 Bulletin of Marine Science [23(3) shaft and was pointed toward the tip of the tooth. The radular teeth were affixed to the ligament sac by a hyaline ligament. The teeth were asym- metrical and difficult to present in a line drawing or photograph. However, the distinguishing features, which cannot be seen from one view, are presented in a composite drawing in Figure 3.

DISCUSSION The venom of C. spurius atlanticus was apparently effective against certain animals. Songdahl & Lane (1970) showed that the venom was effective against the crabs Uca sp. and Cardisoma guanhumi. They attributed possible neurotoxic properties to the venom. The present study shows that the venom is also active against the mollusks Ficus and Busycon, but not against rats or fish. Darkening and mottling of the mantle was apparent shortly after the Ficus was envenomated. This was probably due to expansion of the chromatophores as a result of flaccid paralysis. I suggest that the venom is possibly ineffective against verte- brates, but probably quite active against invertebrate prey species. There is a paucity of data on the ecology of the Conidae of the Atlantic Ocean, and to my knowledge there are no publications describing prey species of C. spurius atlanticus. However, Lowell Thomas (pers. commun.) has seen a cone of this species eating a nemertine worm. Kohn (1959) reported that the Condiae have specific prey organisms and classified them as piscivorous, molluscivorous, or vermivorous. Endean & Rudkin (1965) related tooth type to prey, according to the morphological classification of Peile (1939). They concluded that "it seems possible to distinguish between piscivorous, molluscivorous, and vermivorous Condiae on the basis of marked differences in structure of radular teeth possessed by Conidae belonging to these three groups." Nybakken (1970) showed correlation of tooth structure with food type. This strict specificity is probably not absolute. Saunders & Wolfson (1961) found that under aquarium con- ditions C. californicus fed on a variety of dead gastropods, pelecypods, polychaetes, and fishes and killed and ate certain gastropods and a number of species of polychaetes. On the basis of some of the following evidence I suggest that C. spurius atlanticus probably preys on molluscs, if not primarily, at least occasionally. Several of these cones were placed in an aquarium with the pelecypods Chione cancellata and Donax variabilis, which were subsequently eaten. However, I did not observe the animals feeding. The venom was toxic to Busycon and Ficus, but not to fish. Endean & Rudkin (1965) showed that the venoms of the piscivorous Conidae used in their study quickly paralyzed blennies, but did not paralyze the gastropod2 Melarhaphe (= Littorina) scahra. These in-

2 The authors reported the genus and species as Melarapha scabra. 1973] Songdahl: Venom of the A tlantic Cone 609 vestigators also stated that the venoms of molluscivorous Conidae will paralyze Melarhaphe but not blennics and, of the molluscivorous species studied, only the venom of C. textile paralyzed the polychaete Phyllodoce. The venoms of most vermivorous species studied will paralyze Phyllodoce, but will not paralyze Melarhaphe. Furthermore, the structure of the radular tooth of C. spurius atlanticus has a general configuration and characteristics similar to known molluscivorous Conidae. Endean & Rudkin (1965) noted that the teeth of the molluscivorous species used in their study were slightly enlarged at the base of the elongated shaft, were minutely serrated, and bore a posteriorly directed spur approximately halfway along the shaft (compare with Fig. 3). This evidence does not preclude the possibility that the species is also vermivorous. Although the radular teeth of this species are not of the squat, sturdy type frequently observed in vermivorous species, they possess a "cone" or posterior barb which projects anteriorly. Kohn (1959) has shown this basal or posterior barb to be generally correlated with feeding on eunicid and tube-dwelling polychaetes. The venom bulb is the most conspicuous component of the venom apparatus and has been credited with several functions. Some workers (Bergh, 1896; Shaw, 1914) suggested that the bulb was the source of the venom, whereas others (Bouvier, 1887; Hermitte, 1946; Hinegardner, 1957) considered it improbable that the venom was produced in the bulb. Bouvier (1887) suggested that the bulb functioned as an expulsive organ for the venom, and Hinegardner (1957) expanded on this sug- gestion and proposed that the bulb filled with some liquid other than the venom, which served as a pressure source for venom expulsion. Hermitte (1946) indicated that the bulb functioned as a reservoir for the venom and also the propulsive force. It is unlikely that the bulb has any function of storage or production of venom in C. spurius atlanticus. In most instances the bulb was filled with a clear fluid which could be expressed when pressure was applied to the bulb. I suggest that the bulb is probably an accessory organ for venom expulsion. Due to the extreme length and natural elasticity of the venom duct, it is unlikely that bulb pressure alone is capable of ejecting appreciable quantities of venom. On several occasions I attempted to extract venom from the duct by applying pressure to the bulb. The pressure is barely capable of expelling venom from the duct 10.0 mm from the bulb and the pressure appeared to be dissipated well prior to the end of the duct. This species is capable of expelling more than the trace amounts of venom which could be forced out by contraction of the bulb. This capability was observed when an animal with a shell length of 47 mm was placed in a saturated solution of magnesium sulfate. Shortly after immersion, several drops of venom were slowly expelled. Contraction of the venom bulb perhaps provides a small amount of 610 Bulletin ot Marine Science [23(3) pressure, and the venom duct, possibly aided by a peristaltic action, moves the venom to the proboscis. Contraction of the proboscis probably expels any venom that is to be used at the time and utilizes only the venom in the proboscis. Data on the chemistry of Conus venom is sparse. Kohn et ai. (1960) reported on some preliminary studies of the venom and showed the presence of protein, carbohydrate, and quaternary ammonium compounds. The current study shows the presence of carbohydrates, lipids, and a substantial amount of nitrogen. The high nitrogen content suggests that the venom contains a large amount of protein. However, a significant portion of the nitrogen may be in quaternary ammonium compounds or amines. Kohn et al. (1960) showed the presence of the quaternary ammonium compounds N-methylpyridinium, homarine, and gamma-butyrobetaine, and possibly amines. This work provides some additional information on the characteristics of the venom and, hopefully, will stimulate further studies and identification of the active principle of Conus venom.

SUMMARY The venom of C. spurius atlanticus was effective against the mollusks Ficus sp. and Busycon sp., but not against fish or rats. The lyophilized venom contained 9.6 per cent nitrogen. Some of the nitrogen was in the form of free amino acids, and at least 13 amino acids were detected. The venom also contained 11.25 per cent lipid in the form of two fatty acids with carbon chain lengths of 14 to 18 carbon atoms, a sterol, and possibly a phospholipid. The lipid fractions were colored in the presence of iodine vapors, which indicated unsaturation. The venom was 2.3 per cent ash. Carbohydrate was present and comprised approxi- mately 16 per cent of the total composition. Hexose, in the form of glucose, was the most common sugar and comprised 15.5 per cent of the total composition. Fucose was present as less than 1.0 per cent of the total venom. The radular teeth were 2.1-3.0 mm long in the mature animal, and a complement of 28-35 teeth was common. The teeth had features which are common to known molluscivorous species and which included an elongated shaft enlarged at the base, serrations, and a posteriorly directed barb approximately halfway along the shaft.

SUMARIO VENENO Y APARATO VENENOSO DEL CONO ATL.\NTICO Conus spurius atianticus (CLENCH) £1 veneno de Conus spurius atianticus fue efectivo contra los moluscos Ficus sp. y Busycon sp., pero no contra peces 0 ratas. 1973] Songdahl: Venom of the Atlantic Cone 611 £1 veneno liofilizado contenia 9.6 por ciento de nitr6geno. Parte del nitr6geno estaba en forma de amino acidos libres y por 10 menos fueron detectados 13 amino acidos. £1 veneno tambien contenia 11.25 por ciento de Hpidos en forma de dos acidos fMidos con cadena de carbona de 14 a 18 atom os de carbono de largo, un esterol y posiblemente un fosfollpido. Las fracciones de llpido se colorearon en presencia de vapores de yodo, 10 cual indic6 no saturaci6n. £1 veneno era 2.3 por ciento ceniza. Se encontr6 carbohidrato y comprendi6 aproximadamente el 16 por ciento de la composici6n total. La hexosa, en forma de glucosa, fue el azucar mas comun y comprendi6 el 15.5 por ciento de la composici6n total. La fucosa estuvo presente como menos del 1.0 por ciento del total del veneno. Los dientes radulares median 2.1-3.0 mm de longitud en el animal maduro, siendo comun un complemento de 28-35 dientes. Los dientes tenian aspectos que son comunes en especies conocidas como comedoras de moluscos, incluyendo un eje elongado engrosado en la base, endentaduras y una lengtieta dirigida posteriormente, aproximadamente en la mitad de la longitud del eje.

LITERATURE CITED BERGH, R. 1896. Beitrage zur Kenntnis der Coniden. Nova Acta, 65: 67-214. BOUVIER, E. L. 1887. Systeme nerveux, morphologie generale et classification des Gastro- podes Prosobranchs. Annis Sci. nat., Zool., Ser. 7, 3: 1-510. CHARGAFF, E., C. LEVINE, AND C. GREEN 1948. Chromatographic determination of hexose and hexosamines. J. bioI. Chern., 175: 65-71. DISCHE, Z. 1955. New color reactions for determination of sugars in polysaccharides. Pp. 313-358 in Gluck, D. (Ed.), Methods of biochemical analysis. Vol. 2. lnterscience Publishers, Inc., New York. ENDEAN, R. AND C. RUDKIN 1965. Studies of the venoms of Conidae. Toxicon, 2(4): 225-249. HERMITTE, L. C. D. 1946. Venomous marine molluscs of the genus Conus. Trans. R. Soc. trop. Med. Hyg., 39: 485-512. HINEGARDNER, R. T. 1957. The anatomy and histology of the venom apparatus in several gastro- pods of the genus Conus. M.S. Thesis, University of Southern Cali- fornia. KOHN, A. J. 1959. The ecology of Conus in Hawaii. Eco!' Monogr., 29: 47-90. KOHN, A. J., P. R. SAUNDERS, AND S. WEINER 1960. Preliminary studies on the venom of the marine snail Conus. Ann. N.Y. Acad. ScL, 90: 706-725. MANGOLD, H. K. 1961. Thin layer chromatography of lipids. J. Am. Oil Chern. Soc., 38(12): 708-727. 612 Bulletin of Marine Science [23(3)

NVBAKKEN, J. 1970. Radular anatomy and systematics of the West American Conidae (, ). Am. Mus. Novit., No. 2414: 1-29. PElLE, A. J. 1939. Radular Notes, VIII. Proc. malac. Soc. Lond., 23: 348-356. SAUNDERS, P. R. AND F. WOLFSON 1961. Food and feeding behavior in Conus cali/omicus Hinds, 1844. Veliger, 3: 73-76. SHAW, H. O. N. 1914. On the anatomy of Conus tulipa Linn., and Conus textile Linn. Q. 11.microsc. ScL, 60: 1-60. SONGDAHL, J. H. AND C. E. LANE 1970. Some pharmacological characteristics of the venom of the alphabet cone, Conus spurius atlanticus. Toxicon, 8(4): 289-292.