THE JOURNAL OF EXPERIMENTAL ZOOLOGY 238:129-139 (1986) Stimulation of Genital Eversion in the Land Snail Helix aspersa by Extracts of the Glands of the Dart Apparatus DANIEL J.D. CHUNG Division of Biological Sciences, and Museum of Zoology, University of Michigan, Ann Arbor, MI 48109 ABSTRACT The dart apparatus, used during courtship in some groups of hermaphroditic land snails, has long been assumed to have a “stimulatory” effect on the mating partner, though how stimulation occurs and exactly what function it serves has never been determined. In this study, extracts of the mucous glands of the dart apparatus of the land snail Helix aspersa were injected into conspecifics and into a related snail, Cepaea nemoralis, in order to test the hypothesis that the dart is used to achieve inflow of bioactive mucous gland secretions into the darted snail. Helix aspersa injected with the extract responded by everting their terminal genitals; eversion normally takes place during courtship and mating. Boiling the extract increased the bioactiv- ity. Pronase-treated extract lost bioactivity, and gel filtration of the boiled extract indicated that the active substance has a molecular weight of about 5,000. The active substance may be a polypeptide. Cepaea nemoralis also everted their genitals when injected with the boiled Helix extract. The active substance appears to be a contact sex pheromone, the second such pheromone in a pulmonate land snail for which experimental evidence has been obtained. Accessory organs in the terminal genitalia dart might cause increased tonus of the pen- of the hermaphroditic land snail order Sty- ial muscles. lommatophora (reviewed by Tompa, ’84) in- The hypothesis that the mucous glands as- clude calcareous or chitinous spears or darts sociated with the dart apparatus (= dart that are thrust into the mating partner dur- glands or multifid glands) might secrete ing courtship. About 11 of the 65 families of stimulatory substances during courtship has pulmonate land snails possess a dart appa- been advanced by Dorello (‘25) and Bornchen ratus, comprising a dart held in a dart sac (’67). Dorello (’25) found that H. aspersa in- and associated glands (Tompa, ’80). In addi- jected with secretions of the mucous glands tion, at least two families of opisthobranchs contorted their bodies and showed, after dis- possess darts (see Pruvot-Fol, ’60; Boss, ’82). section, increased stiffening of the penis. In the Euthyneura (pulmonate snails and sea Bornchen (‘67) found that extract of the mu- slugs), a dart apparatus may have evolved cous glands of H. pomatia increased the fre- independently in at least seven different ma- quency and amplitude of the heart beat. jor lineages. Because of its use in courtship, However, Goddard (’62) found no effect of it has long been assumed that the dart is a mucous gland extract on contraction of a pen- “stimulatory” device in courtship and mat- ial sheath preparation of H. aspersa. Other ing (see Dorello, ’25; Bornchen, ’67; Tompa, hypotheses on the function of the mucous ’84 for reviews), though this has never been glands include: lubrication for the dart and proven. genitals, formation of the egg shell, and for- The hypothesis that the dart is used to mation of the dart (Meisenheimer, ’12; Mo- stimulate the courting partner mechanically quin-Tandon, 1855; also see Bornchen, ’67 and thereby induce mating is very old (e.g. and Tompa, ’84 for reviews). It is now known see Moquin-Tandon, 1855; Cooke, 1895; Mei- that the mucous glands do not form either senheimer, ’07). Goddard (‘62) has suggested egg shell or the dart. from experiments on isolated penis sheaths In recent observations on mating behavior of Helix aspersa that damage of tissue by the in helicids, Lind (‘761, Jeppesen (’761, and 0 1986 ALAN R. LISS, INC. 130 D.J.D. CHUNG Giusti and Lepri (’80) did not observe any then ground in water or in either Bohuslav’s stimulation of courtship or any facilitation of (‘33) or Roach’s (‘63) saline in a glass tissue mating by receipt of a dart. Lind (‘76) ob- grinder. The unboiled homogenates were in- served that snails receiving a dart were more jected either fresh or after centrifuging for likely to break off courtship than those not five minutes in a clinical centrifuge to re- receiving a dart, and he suggested that dart move large cellular debris. Boiled mucous shooting might test the mating tendency of gland extract was prepared by boiling for five the partner. minutes after centrifugation and was in- Though darts may wound a partner, the jected after it had cooled to room tempera- elaborate structure of the dart apparatus ture. Injections were made into the hemocoel suggests that it serves some adaptive func- of crawling snails through the upper left side tion or functions. Tompa (‘80, ’84) suggested of the headfoot with a 25 gauge needle. Injec- that dart receipt might stimulate egg matur- tion of the “nuchal” region of the snail mini- ation and release in a snail; dart shooting mized wound response (withdrawal into the might thereby help the dart shooter to fertil- shell and bleeding). ize the eggs of its partner. Diver (’40) be- lieved that darts evolved as a reproductive Preliminary characterization of isolation mechanism. Though there is little active substance direct evidence for Diver’s hypothesis, Webb Unboiled mucous gland extract and boiled (’51) cited a single instance of the death of a extracts of dart sac, penis, mantle collar, and Helminthoglypta caused by receiving a dart brain were injected, and the results were from a non-conspecific when they attempted compared to the results of boiled mucous to mate. gland injections. Non-tissue control injec- This paper presents experiments involving tions of distilled water, or Bohuslav’s or injections of whole mucous gland extract and Roach’s saline, and of solutions of the neuro- also of fractionated components of the gland transmitters acetylcholine and 5-hydroxy- extract into Helix aspersa in order to test the tryptamine were also performed. Acetylcho- hypothesis, first proposed by Dorello (’25), line and 5-HT occur naturally in the brain of that the dart is used for inoculation of a Helix aspersa (Kerkut and Cottrell, ’63). At- stimulatory substance from the mucous tempted extraction of the active substance glands into the mating partner. with chloroform and treatment of the mu- cous gland extract with nonspecific protease MATERIALS AND METHODS (Pronase), DNase, and RNase were also car- Materials ried out. Live adults of Helix aspersa were obtained Tissues were extracted from adult donor from California through College Biological snails that had been kept in isolation for at Supply Co. (Escondido, California). Individ- least two weeks. The tissues were excised, ual snails were isolated in small plastic con- collected, ground; and the extracts were in- tainers lined with moist soil and fed carrot jected as described above. The “dart sac” tis- slices ad libitum. The snails were kept at 21” sue used included only the inner tissue lining to 26°C on a 12-hr light:12-hr dark cycle of the dart sac and the calcareous dart. Doses throughout the year. Only healthy active of the different tissue extracts were chosen adults isolated for at least two weeks were so that the results from the different extracts used for injections. would be comparable. The “standard dose” (Sd)of an organ extract was that which would Preliminary assays be equivalent to injecting a snail of average Unboiled and boiled mucous gland extracts weight with an average-sized organ from a and unboiled extracts of dart sac, penis, and donor. For mucous glands, the standard dose mantle collar were injected in volumes of 0.1 was 28.5 mg mucous gland (wet wt tissue to 0.7 ml and at doses varying from 0.9 to extracted) per 6.3 g snail (including shell), or 23.7 mg wet wt- of tissue extractedg snail 4.5 mglg. Average weights of penis, dart sac (including shell) to determine differences be- (inner lining + dart), and brain were ob- tween the effects of mucous gland extract tained to give the standard doses shown in and other tissue extracts. Mucous glands, Table 1. The dose for boiled mantle collar was penes, dart sacs (plus dart), and pieces of the only dose not standardized in this way mantle collar were excised from donors. col- and was less than one mantle collar Der in- lected on ice and weighed. The tissues were jected snail; a full dose would have bGen too FUNCTION OF HELIX ASPERSA DART APPARATUS 131 TABLE 1. Results of second series of bioassay injections on Helix aspersa Response Extract or Dose >RO: solution (mgfg) RO (Rl-R2-R3-R4) Othera Mucous gland Boiled 4.5 4 26 P.D (1-4-20-1) Boiled, 112 volume 4.5 0 5 NSb (1-0-4-0) Unboiled 4.5 14 5 **c (4-1-0-0) Chloroform fraction 4.5 13 0 ** HzO fraction 4.5 6 9 NS (2-0-5-2) + Pronase 4.5 12 0 ** + DNase and 4.5 2 3 NS RNase (0-1-2-0) Darts + secretions 0.6 3 12 NS shot from mating (1-4-6-1) snails Other tissues, boiled Penis 7.9 7 4 ** (1-3-0-0) Dart sac 5.2 14 2 ** (1-1-0-0) Mantle collar 6.8 5 0 ** P Brain 5.1 8 0 ** P,D Non-tissue injections HzO or saline - 10 0 ** NR Pronase (boiled) 0.04-6.3 12 0 ** NR ACh 5-50 7 0 ** P 5-HT 0.50-50 5 0 ** P “P = tetany and paralysis. D = some genital pore swelling.