ZOOSYSTEMATICA ROSSICA, 18(2): 196–204 25 DECEMBER 2009 The characteristics of copulation in Segmentina oelandica (Westerlund, 1885) (Gastropoda: Pulmonata: Planorbidae) E.V. SOLDATENKO & A.A. PETROV E.V. Soldatenko, Zoological Institute, Russian Academy of Sciences, Universitetskaya Emb. 1, St. Petersburg 199034, Russia. E-mail: [email protected] A.A. Petrov, Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia. E-mail: [email protected] The paper examines the precopulatory behaviour and copulation in Segmentina oelandica. The morphological traits associated with this process are discussed. The anatomy of the copulatory apparatus is studied using histological and histochemical (phalloidin fluorescence) methods. The copulation is shown to be a complex process determined by a specific set of morphological, physiological, behavioural and ecological characteristics. Key words: molluscs, morphology, mating behaviour, copulation, Gastropoda, Pulmonata, Planorbidae, Segmentina INRODUCTION genera. The anatomical studies of the repro- ductive system, and, especially, of the copu- The first description of copulation in orb latory apparatus provided some additional snails was given in the publication of the insights about the copulation in Planor- field naturalist’s observations on the mol- bidae (Larambergue, 1939; Baker, 1945; luscan fauna of the outskirts of Budapest Malek, 1952; Pace, 1971) and, specifically, (Hazay, 1881). Subsequent studies (Col- about the correlation between the morpho- ton, 1918; Precht, 1936, Geldiay, 1956; Be- logical structure of reproductive organs and rezkina & Starobogatov, 1988) were mostly the copulatory mechanics. focused on other aspects of reproduction in The early authors (Simroth & Hoff- Planorbidae Rafinesque, 1815, while giving mann, 1908–1928: 1119; Precht, 1936), little attention to copulation. Precht (1936) who described the copulation in freshwa- examined some aspects of the mating mech- ter Planorbidae, claimed that a compli- anism in Аnisus leucostoma (Millet, 1813), cated pre-copulatory behaviour (foreplay, А. vortex (Linnaeus, 1758), Armiger crista epilogues, and mating dances), such as that (Linnaeus, 1758), Bathyomphalus сontortus known, for example, for Stylommatophora (Linnaeus, 1758), Gyraulus аlbus (Мüller, (Gerhardt, 1936), is generally lacking in 1774), Planorbis carinatus (Müller, 1774), orb snails. Subsequent studies (Pace, 1971; and P. planorbis (Linnaeus, 1758), and was Trigwell et al., 1997), however, challenged the first to describe the copulation in Seg- these conclusions. Pace (1971), who ob- mentina nitida (Мüller, 1774) by studying served the copulation in the members of the a single copulating pair. Bondesen (1950) genus Helisoma, was the first author who summarized somewhat conflicting available noticed that the snails follow “very similar data on mating behaviour in freshwater behaviour patterns” during the copulation. snails and distinguished three major modes He also observed that the mating is usually of copulation (unilateral, reciprocal and initiated by reciprocal tentacular explora- self-copulation); he also posited that some tion between prospective mates. Trigwell et features of mating mechanism in Planorbi- al. (1997) provided a detailed description of dae are specific to genera or the groups of the precopulatory behaviour in Biomphalar- © 2009 Zoological Institute, Russian Academy of Scienсes E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA 197 ia glabrata (Say, 1818) and divided the cop- ities. EVS; Russia, Smolensk Prov., Demi- ulatory process into several distinct phases. dovskiy Distr., outskirts of Przhevalskoye, Morphologists and taxonomists often a pool at Nikitenki Village; EVS; Rus- have to deal with preserved material and, sia, Smolensk Prov., Demidovskiy Distr., therefore, can only speculate about the at Przhevalskoye, temporary ponds in the functions of various structures. The study outskirts of the ‘Chistik’ Teaching Center of copulation, however, may help to uni- (Smolensk State University). fy the whole spectrum of morphological, The groups of sexually mature speci- physiological, behavioural and ecological mens from the both localities given above characteristics, to enable the assessment of were placed in 3 cm-deep containers, 20 correlation between various morphological specimens in each, for observation on the traits, to facilitate our understanding of the mating behaviour. The beneficial effect of relationships between morphological char- rising temperature on mating activity in acteristics and biological processes, and to freshwater snails (Duncan, 1978; Smith, improve the characterization of species and 1981) was used to induce mating: the water supraspecific taxa. The purpose of the pres- was heated with incandescent lamps to raise ent study is to describe the characteristics of temperature to 20–26°С. The copulating copulation in Segmentina oelandica (West- pairs were photographed; the copulation at- erlund, 1885) and to examine the relation- tempts were counted and the time intervals ship between the copulation mechanics, on were recorded. The observations were re- one hand, and the structure of the copula- peated, at least, for four groups per season. tory apparatus, physiological data and char- Some of the specimens were marked with acteristics of the habitat, on the other. colored nail polish to count the number of The particular type of copulatory behav- copulation attempts made by the same indi- iour observed in S. oelandica is determined vidual and to determine whether this indi- by a specific set of morphological characters vidual mates as a male or female, and wheth- of this species (smooth plano-convex shell, er or not the individual alternates sexual wide shell aperture, curved palatal edge of roles. Several times during the mating sea- the aperture, lamellae within the outer shell son the samples were replenished by freshly whorl, characters of the copulatory appara- collected specimens (newcomers), because tus, relative position of the genital openings, otherwise the mating stops after the end of and length of tentacles). Whereas the gross the active mating period (2–3 days). anatomy and the structure of the penial com- Three specimens of S. oelandica were plex in the genus Segmentina Fleming, 1818 is prepared as unstained whole mounts. These relatively well studied (Baker, 1945; Huben- whole-mount preparations were examined dick, 1955; Kruglov & Soldatenko, 1997), and photographed on a Leica DMLS-2 mi- the details of the histological structure and croscope equipped with a CCD camera; the the musculature of the copulatory apparatus line drawings were copied from photographs are much less known. These aspects of the according to their original proportions. copulatory apparatus are examined in the For confocal microscopy, two speci- present paper using histological techniques mens were fixed in 4% formaldehyde (pre- and phalloidin staining for muscle fibers. pared from paraformaldehyde powder) in 0.1M PBS for one hour at room tempera- ture, rinsed in 0.1M PBS (three times for MATERIAL AND METHODS 15 minutes), permeabilized for one hour in The specimens of S. oelandica were col- PBS containing 0.2% Triton X-100, rinsed lected and the experiments were conducted again shortly in the same buffer and then in 2008–2009 by the first co-author (EVS). transferred to phalloidin-TRITC (Sigma) The material came from the following local- for one hour. After that, the specimens were © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 198 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA washed again three times for 15 minutes in in response to shock caused by injury. Al- the same buffer, mounted in 80% glycerol though, in this case, the eversion reflex was on glass slides, and viewed on a Leica TSC not accompanied by physiological and be- SP5 microscope. havioural responses, it showed the structur- Seven specimens were taken for histo- al elements of the penial complex and their logical observations. The dissected copula- arrangement, typically obscured during the tory apparatus was fixed in 70% alcohol and copulation by the partner (Fig. 1b–c). after dehydration in xylene was embedded Although the individuals of S. oelandica in paraffin using standard histological tech- are simultaneous hermaphrodites, the recip- niques (Lillie, 1969). Serial sections 6 μm rocal non-simultaneous copulation, i.e. the thick were stained with hematoxylin and alternation of sexual roles in a pair, with- eosin. Whole mounts and histological sec- out any intermittent pauses (recognized by tions were examined and photographed on Kruglov, 1980 as a separate type of copula- a Leica DM LS-2 microscope. tion in pulmonates), was never observed in S. oelandica. The only mode of copulation we observed was unilateral copulation. For RESULTS this mode of copulation we adopt here the The copulatory behaviour in S. oelandica conventional terminology (Precht, 1936; was initially observed in a wounded speci- Bondesen, 1950; Smith, 1981; Berezkina & men that everted the copulatory apparatus Starobogatov, 1988) and refer to the copu- Fig. 1. Segmentina oelandica, diagrammatic representation of the copulatory apparatus (a) and dia- grams showing eversion of the copulatory organs (b, c); f, foot; fl, flagellum; pa, papilla; pe, penis; po, preputial organ; pr, preputium; ps, penial sac; r, retractor; t, tentacle; vas, vas deferens. Scale bars (a–c): 1 mm. © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA 199 Fig. 2. Copulation in Segmentina oelandica. Color codes: male: brown; female: grey; everted preputial organ: red; penis: pink. Figures e–f represent different views of the same stage. See text for further explanation. Scale bars (a–c): 1 mm. © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 200 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA Fig. 3. Anatomy of copulatory apparatus in Segmentina oelandica: confocal micrographs of phalloidin- stained whole mounts showing penial sac with penis (a, b), light micrograph of whole mount of papilla (c), microphotographs of histological sections through preputial organ (d); mp, muscular portion of the preputial organ; ics, inner circular layer of the penial sac muscles; lp, longitudinal muscles of penis; ls, longitudinal penial sac muscles; ocs, outer circular layer of penial sac muscles; ovd, opening of vas deferens; pa, papilla; pe, penis; po, preputial organ; pw, preputium wall; rp, radial muscles of penis; sp, secretory portion of preputial organ. Scale bars: 50 μm (a–c), 100 μm (d). © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA 201 lating individuals as males or females, de- false copulation, i.e. multiple attempts to pending on their behaviour. copulate without a successful sperm trans- The individuals of S. oelandica begin fer. This phenomenon is very common in copulating copiously, when the water tem- laboratory, when the size of the group is in- perature is raised above 20°С. The smaller creased by adding new individuals after 5–7 specimen in a pair is usually mating as a days of active mating period. Previously in- male. The male approaches the female, seminated females refuse to copulate with mounts the apical side of the partner’s shell newcomers; they often resort to biting and on the left side of the aperture, crawls a avoid true copulation. The male can un- full circle along the body whorl toward the dertake up to 25 attempts of false copula- aperture and finally takes up the position tion with the same partner (by following over the partner’s head (Fig. 2a). If the fe- the courtship ritual, described above), but, male changes the direction of motion, turn- without getting an ‘approval’ from the fe- ing toward the approaching male, the male male, leaves her to pursue another partner. mounts from the opposite side of the shell If the false copulation (including several (Fig. 2b), immediately turns around in a succeeding precopulatory rituals) lasts as small circle, and heads toward the partner’s long as 15 min to 1 h, a ring of pink secre- aperture. When the male is within a short tion is formed on the male’s left tentacle. distance of the shell margin (about 1/8 In some cases, a chain of 3–4 copulat- of the body whorl), he begins to evert the ing individuals is formed, in which the true preputial organ and then lowers the pink copulation between the successive members swollen left tentacle between the female’s of the chain almost never occurs simultane- tentacles (Fig. 2c). The male affixes himself ously: as the bottommost individual tries to the partner’s shell using the preputial to assume a vertical position on the wall organ as a hold-fast, without ever touching of the container, the upper specimens fail the mate’s body, and pulls his shell down to- to successfully complete the courtship and ward the foot thereby forcing the partner to the chain disintegrates. Throughout the pe- slow down (Fig. 2d e). The female continues riod of observations, the true simultaneous to move forward, albeit slower than before, copulation of all 3 specimens in a chain was and stretches her body to expose the female observed only once. pore. The distal end of the male’s inflated After the active spawning period (2–3 tentacle produces pink secretion, which he days), the individuals cease copulating. probably uses to attract the female. When Under experimental conditions, the males the male becomes firmly affixed to the part- never attempted copulation with more than ner’s shell, he inserts the penis into the fe- 2–3 different females. The females can mate male’s vagina (Fig. 2f). The penetration is again as females only after 3–4 weeks, and usually very rapid, without seeking move- only if the population is replenished with ments, and the final phase of the copulation potential males (small-sized specimens col- is accompanied by cramp-like contractions lected in cold water); however, over the of the male’s body. The intromission and half of the specimens would die during this insemination take up no more than 2.5 min, period. and then the male quickly leaves the part- The penial sac of S. oelandica (Fig. 1a) ner. Occasionally, the female can bite the is furnished with two glandular appendages, partner’s preputial organ with her jaw whose function is likely to be the produc- (Fig. 2g). The courtship ritual lasts no more tion of substances helping the mollusc to than 3.5 min, and, during the whole process, attach to the partner’s shell during the cop- the female never stops moving or feeding. ulation. The penial sac has relatively thin This sequence can be referred to as the walls, lined with several muscle layers (Fig. true copulation, but we also observed the 3a–b). The outermost muscle layer consists © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 202 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA of wide circular (sometimes diagonal) fi- ber true copulations in laboratory, but this bers (Fig. 3a–b, ocs); underneath this layer is probably not the case for individuals in are 4–5 layers of longitudinal fibers (Fig. nature. Copulating chains composed of 3b, ls); the innermost layer consists of the 3–4 specimens are also likely to be uncom- circular fibers (Fig. 3a–b, ocs). The latter mon in nature. The same copulatory phases are relatively widely spaced and have an L- described by Trigwell et al (1997) for B. shaped profile in cross-section. One or two glabrata, i.e. pre-copulation – copulation – inner layers of longitudinal muscles are par- insemination – fertilization, are observed in tially intertwined with subjacent circular S. oelandica as well. muscles. The penis is long and has a thick The morphological characteristics of the muscular wall comprising 2 layers of mus- shell and the position of the female genital cles (Fig. 3a–b): the outer layer composed pore necessitate the development of addi- of longitudinal fibers (Fig. 3a, lp), and the tional muscular (preputial organ) and glan- inner layer, composed of strong radial fibers dular structures (flagellums). The walls of (Fig. 3b, rp). The penis is tipped with the the preputium are relatively thin, especially muscular papilla (Fig. 3c, pa). The prepu- in its proximal portion, most likely because tium bears a well-developed preputial or- they function as a passive sheath and, like gan (Fig. 3d), which is a glandulo-muscular the walls of the penial sac, are not subjected adhesive organ that plays a significant role to mechanical stress during the copulation. in fixation of the male on the female’s shell The penis is a highly extensible organ due during the copulation. to a pronounced layer of radial muscles. The pointed soft penial papilla is not covered DISCUSSION with the cuticle and the additional protec- tion from the cuticle is not required, be- The comparison of the description made cause: 1) mechanical stress is minimal (our by Precht (1936) for the copulation of S. experiment shows that the males rarely in- nitida with our own observations shows seminate more than 3 females per season), that this author most likely observed the and the time interval allowed for intromis- false copulation, which occurred after an sion is very brief; 2) during the copulation, active mating period. The assumption (Pre- the penis bends over the outer margin of cht, 1936: 82) that the members of this ge- the shell aperture; in this case, a more rigid nus can copulate in a side-by-side position structure would have been less mobile or is incorrect. The misinterpretation of the sensitive, and, therefore, would have pro- reproductive morphology of S. nitida made vided less selective advantage. by this author can be also easily explained – As the phenotypic traits associated with we suppose that he apparently mistook the mating strategies are subject to strong selec- preputial organ for the penis due to the ex- tive pressures, it seems reasonable to assume tremely small size of the latter (Fig. 2f–g). that the mating behaviour in S. oelandica is The lack of any ritual behaviour (fore- also affected by the characteristics of the play) during the mating of freshwater snails habitat of this species. Segmentina oelandica reported by Simroth & Hoffmann (1908– inhabits shallow waters (during the active 1928) and Precht (1936) is inconsistent phase of the lifecycle the individuals of this with our observations. It was noted in our species are never found deeper than 0.2 m), experiment, that the “reference point” used favouring thick patches of duckweed and by the male to mount on the partner’s shell coarse plant detritus, as was also reported is defined by the position of the female’s by Frömming (1956) and Stadnichenko head and by tentacular contact. The same (1990) for other Segmentina species. Seg- precopulatory behaviour is exhibited by mentina oelandica is also found abundantly all individuals. False copulations outnum- on the underside of floating or submerged © 2009 Zoological Institute, Russian Academy of Scienсes, Zoosystematica Rossica 18(2): 196–204 E.V. SOLDATENKO & A.A. PETROV. COPULATION IN SEGMENTINA OELANDICA 203 tree branches. It may be concluded that REFERENCES the mating process in S. oelandica evolved Baker, F. C. 1945. 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