Peter A. Smith page 2 Abstract 1- Salivary glands of the marine carnivorous snail Nucella emarginata con- tain a substance which causes positive chronotropic and ionotropic effects on the hearts of Mercenaria mercenaria and Mytilus californianus. 2- Comparison of the effects of salivary gland homogenate with the action of known doses of serotonin (5HT) on Mytilus hearts showed the glands con¬ tain the equivalent of 23-85/g. 5HT/ g. fresh tissue. Thin layer chroma- tography demonstrated that the glands contain less than 150Ag. 5HT /g. fresh tissue. 3-Mucous secreted from the hypobranchial gland of Nuc lla caused a marked increase in tonus and eventual cessation of beat of! tilus hearts, effects similar to those produced by large concentrations of acetylcholine. Peter A. Smith page 3 Introduction Nucella emarginata inhabits exposed, wave-swept outcroppings of rock along the North American west coast, where it feeds mainly on mussels and barnacles. The snail can drill through the shells or plates of prey with its particularly efficient radula aided by an acessory boring organ on the sole of its foot (Abbot,in print).However, observations have shown that Nucella paralyses its prey before eating, and may not require drilling to gain acess (Gunter, 1968). Nucella emarginata contains a pharmacologically active choline ester, tentatively identified as N- methylmurexine, in its hypobranchial gland (Bender et. al.1974). Keyl and Whittaker have shown that choline esters block neuromuscular transmission (cited in Endean,1972). Espéramer (1956) has also found large concentrations of serotonin (5- hydroxytryptamine, 5H along with choline esters in the hypobranchial glands of some prosobranchs of the superfamily Muricacea, to which Nucella belongs. The hypobranchial gland, however, contains no duct to deliver these possible toxins to the prey of the snail; it secretes à mucous into the mantle cavity which is generally supposed to aid in excretion (Fretter and Graham, 1962). Con¬ sequently several authors have doubted this glands efficacy as an offensive apparatus.(Endean, 1972: Halstead,1978). Huang and Mir (1972) assayed salivary gland extracts from Thais (=Nucella) haemastoma on various vertebrates and discovered that the gland contained a powerful vasodilator and hypotensive agent. They suggested that the salivary glands were the main poison gland of the animal. The glands contain ducts which lead along the proboscis to the lower channel of the buccal cavity. Peter A.Smith page 4 This study sought to investigate the possible toxicity and pharmaco- logical activity of the salivary gands of Nucella emarginata, and to further elaborate the role of the hypobranchial gland - particularly to see if the choline ester is included in secreted mucous. Hopefully this would add to the knowledge about the feeding behavior and pharmacology of this snail. Materials and Methods Specimens -- 160 snails were collected over the course of 6 weeks, at approx¬ imately two week intervals. Nucella were obtained from near Fisherman's Wharf, Monterey and at Point Joe, Carmel. Mercenaria mercenaria (cherry stone clams) were bought from a seafood stand on Fisherman's Wharf. Mytilus californianuswere collected at Point Joe and Point Cabrillo, Pacific Grove. All specimens were stored in aquaria with running seawater at 14 C for not longer than two weeks before use. Assay -- Hearts of the clam, Mercenaria mercenaria and the mussel, Mytilus californianus, were prepared in the method of Welsh and Taub (1948) with several differences. The junction of each auricle with the central ventricle was tied off with a length of thread. The heart was immersed in a 4 ml, bath filled with seawater, and then stretched by means of the threads between a glass hook at the bath bottom and the input of of a force-displacement trans¬ ducer (Grass FT-03). The transducer was hooked up to a polygraph (Grass 5A) which recorded heartbeat on one of the channels. Air was bubbled up through the bottom of the bath, and seawater circulating through the water jacket maintained a temperature of 14 - 15 C. gracts - The snails" shells were cracked with a pliers and the salivary Peter A. Smith page 5 glands dissected out under a dissecting microscope. Glands were weighed, wrapped in parafilm, and stored in a freezer until used. Usually fresh prep arations of glands were made twice weekly, each froma dissection of 10 snails (avg. weight glands.14 g./ 10 glands). On the day of an experiment glands to be assayed were homogenized with a glass homogenizer in i ml. of seawater. Known quantities of homogenate were added to the top of the heart bath with a micropipette,or a plastic syringe with the needle bent at right angles. Hypobranchial gland secretion was collected on two separate occasions. It was noted that snails prepared for dissection often secreted a colorless mucous from their hypobranchial gland, which rapidly turned bright green and then a deep purple with exposure to light. On each occasion, the mucous from 4 snails was collected. The mantle was lifted carefully to expose the mantle cavity ; mucous was collected with a fine forceps until approximately 1 ml. had been obtained. The secretion collected was dried on a watch glass overnight, reconstituted in .5 ml. seawater. and added to the heart bath. E - Thin layer chromatography plates (silica gel) were run in isopropanol: NHOH : H20 (18:1:1) for twelve hours. .15 g. of tissue was homogenized in 5ml. of a mixture of acetone (10 volumes) and ethanol (3 volumes) to precipitate salts and proteins, according to the method outlined by Lovenberg and Engel- man (1971). The extract was filtered and the solvents evaporated off in a flash evaporator at room temperature. The residue was dissolved in 100) meth¬ anol, and 75)were spotted. Plates were dried overnight, sprayed with Ehrlich's reagent (Sigma Chem. Co.), and heated in an oven at 100 C for visualization. e Peter A. Smith page 6 Results Salivary Gland Extracts Hearts of Mercenaria showed a marked response to both salivary gland extracts and 5—hydroxytryptamine. Both substances caused an increase in both tension (amplitude) and frequency of heartbeat (fig. 1). Doses as low as .3 mg. gland / ml. in the bath produced significant responses. Unfor¬ tunately the hearts proved too irregular for quantitative comparison of salivary gland homogenates to known doses of 5HT. Mytilus hearts showed positive chronotropic and ionotropic effects when salivary gland homogenate was added to the bath. Several hearts were regular and responsive enough,so that it was possible to obtain an estimate of the amount of serotonin equivalent per gram of fresh salivary gland tissue. (fig. 2). A dose of 5HT was added and the response noted. The bath was then washed, and an amount of salivary gland homogenate was added that hopefully matched the former response. This wassuccessful on three occasions (three different hearts). Calculations show readings of the equivalent of 23Ag., WOAg., and 85Ag. of 5HT per gram of fresh tissue. Extracts of salivary gand spotted next to known concentrations of 5HT on TLC plates showed that there was not more than 150/g. 3HT/ g. fresh tissue. A more sophisticated method of purification was necessary for more precise determinations. pobranchial Gland Extracts Dried mucous secretion reconstituted in seawater caused an increase in tonus and cessation of beat in the two Mytilus hearts studied.(fig. 3) These effects are comparable to those produced by large concentrations of acetylcholine (fig. 4). Peter A. Smith page 7 Discussion Salivary Gland The discovery of a cardioactive substance in relatively high concen¬ rginata suggests that these trations in the salivary glands of Nucella em glands may play a role in the observed paralysis of prey. The substance may be serotonin. While it does not appear to be very toxic to vertebrates, high levels of serotonin have been found in the salivary gland of Octopus vulgaris and in the venom glands of many arthropods (Welsh and Moorehead, 1960). The salivary glands of the carnivorous whelk Buccinum also contain a cardio- excitatory substance which may be serotonin (Welsh, 1956). Serotonin might serve to relax the prey of Nucella; for instance, it may cause flaccid paralysis of the adductor muscle of Mytilus. Twarog (1960) has shown that 5HT acts as an antagonist to acetylcholine in the anterior byssus retractor muscle of Mytilus, causing relaxation. Welsh (1960) has speculated that 5HT may facilitate the absorbtion and distribution of true toxic substances. Nucella may useserotonin to enhance the activity of the toxic choline esters which occurr in its hypobranchial gland. However, it is possible that the salivary gland of Nucella is not used to poison its prey; possibly the glands are highly innervated,with a sub- sequently high concentration of neurotransmitter present. Kater et. al.(1977) have shown that the cells in the salivary gland of a mollusk display action potentials and are highly electrically coupled. Welsh and Moorehead (1960) have demonstrated that the ganglia of some molluscs contain high concentrations of serotonin, up to 60/g. 5HT / g. fresh tissue, and Lent (1977) has shown that serotonin plays a major role in stimulating the activity of mucous and/ or secretory structures such as the hypobranchial gland in gastropods. Finally, the cardioactive substance in Nucella salivary glands may not be serotonin. Four cardioexcitatory fractions have been resolved from homo¬ 0 Peter A. Smith page 8 genates of Mercenaria, the most abundant one being a tetrapeptide amide. Two of the others seem to be peptides also (Price and Greenberg, 1977). Obviously a more sophisticated assay of various tissues of Nucella for 5HT needs to be done before any conclusions can be reached. Several simple methods for determining levels of serotonin in tissue have been described (Lovenberg and Engelman 1972). Hypobranchial Gland Secretion The acetylcholine—like effect of hypobranchial gland mucous seems to indicate that choline esters are present along with the purple dye in secre¬ tions of Nucella. This may explain the function of the gland. Several authors have noted the presence of a purple stain around drill holes (Gunter 1968. Hemmingway, 1973). The absence of a duct with which the hypobranchial gland can serve to directly inject toxins into prey may at first seem to undermine the theory that Nucella uses this gland to paralyze the molluscs it feeds upon. But the mucous secreted has several remarkable features. Detailed analysis of the hypobranchial mucous of the whelk, Buccinum undatum has shown that it has highly viscoelastic and hydrophobic properties. Picking out a strand of mucous from an aggregate and transferring it to another nearby dish will cause subsequent complete transferral of the entire mass (Hunt and Jevons, 1967). Personal observations of Nucella mucous confirm these findings. Obviously hypobranchial gland secretions could be transported by the cilia of the propodium to the drill hole site of Nucella prey without a diffusion of the toxin. However, more work needs to be done before this theory is confirmed. including collection and assay of the mucous under more natural conditions, and a study of the effects of choline esters on invertebrates. Peter A. Smith page 9 Acknowledgements Much thanks to Dr. Fred Fuhrman for for his constant support and optimism. I also appreciate the efforts of Dr. Arnold Eskin, who introduced me to real science, and Lani West, who was always willing to help. Peter A. Smith page 10 Literature cited Bender, J. A., DeRiemer, K., Roberts, T. E., Rushton, R., Boothe, P.. Mosher, H.S., and Fuhrman, F. A. 1974. Choline esters in the marine gastropods Nucella emarginata and Acanthina spirata: a new choline ester, tentatively identified as N-methylmurexine. Comp Gen. Pharmac.. 5. 191-198 Endean,R. 1972. Aspects of molluscan pharmacology. In Chemical Zoology 7 421-466 Fretter, V. and Graham,A. 1962. British prosobranch molluscs. Bernard Quaritch, Ltd., London. 755p. Gunter, G. 1968. Some factors concerning the drilling apparatus and the feeding and predation of prosobranchiate gastropods, especially on other molluscs. Proc. Symposium on Mollusca 1, 370.- 3 4 Halstead B. W. 1978. Poisonous and venomous marine organisms of the world, Science Press, Ephrata Pennsylvania Hunt S. and Jevons, F. R. 1966. The hypobranchial mucin of the whelk, Buccinum undatum L. Biochem J. 98 p. 522 Lent, C. M. 1977. The retzius cells within the central nervous system of leeches. Progress in Neurobiology 8,81-117 Lovenberg, W. and Engelman, K. 1971. Assay of serotonin, related metabolites, and enzymes. In Methods of biochemical analysis, Supplemental volume: Analysis of biogenic amines and their related enzymes. Glick, ed. J. Wiley and Sons, N.Y. Price, D. A, and Greenberg, M. J. 1977. Structure of a molluscan cardio¬ excitatory peptide. Science. 197, 670-671. C Peter A. Smith page 11 Literatur cited (cont'd) Twarog, B. M. 1960. Effects of acetylcholine and 5- hydroxytryptamine on the contraction of molluscan smooth muscle. J. Physiol. 152, 236. Welsh J. H. 1956.Neurohormones of invertebrates : I cardio-regulators of prina and Buccinum. J. Mar. Biol. Assoc. UK 35, 193 Welsh, J. H. and Moorehead, M. 1960. The quantitative distribution of 5- hydroxytryptamine in the invertebrates, especially in their nervous systems. J. Neurochem. 6, 146-169 Welsh, J. H. and Taub, R. 1948. The action of choline and related compounds on the heart of Venus mercenaria. Bio. Bul.,Woods Hole 102, 48-57 Kater dal, 1722 Faunl Peter A. Smith pae 12 A 2 ske ei ee e eeee iei 1O B. K C. " W -:- O LP Osec. Peter A. Smith page 13 ââ O O .. ..... O -A A O —7 2 — a PAS. G is laienen e B 1 11: eeeeeetoeebeelbe — --- ose A -. — ebe Werbe A -6 BCM .O 10M 8 ACsenteten -6 5 0 Peter A. Smith page 15 Captions Figure + 1. Effect of serotonin and salivary gland extracts on the heart of Mercenaria. Arrows show time of application of the dose to the bath. A.- Addition of 5HT to a final concentration of 10-° Molar in the bath. B.— Effect of 1.5 mg. salivary gland/ ml. in the bath. C.-- Response to .3 mg S. G./ ml. Figure f 2. An attempt to match the effects of a dose of salivary gland homogenate with a known concentration of serotonin. A.- The effect of two large doses of serotonin on the heart of a Mytilus. B.— A similar response obtainedby adding the shown amounts of salivary gland homogenate. C.-- The addition of S.G. to the same final concentration as in B., show¬ ing that results are somewhat repeatable. This particular match showed that the glands contained the equivalent of 85/g. 5HT/ g. fresh tissue. The cycling behavior was common, and a nuisance. Amplitude and frequency (max) are in parentheses above each characteristic segment. Doses rep¬ resent the final concentration of substance in the bath. Figure + 3. The action of hypobranchial gland mucous (approx. .1 ml. reconstituted in .5 ml. seawater) when assayed on the heart of Mytilus on two separate occassions. Note the increase in tonus. Figure + 4. Several responses to various doses of acetylcholine shown by two mytilus hearts. A.- response of the same heart as in B., fig. 3 above. B.-- Response of a different heart, showing a slight difference in reaction from the same dose applied in A. (.5X 10-° M.) C