Table Of ContentZOOSYSTEMATICA 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
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