Development of a Penis from the Vestigial Penis - Department of ...

Reference: Biol. Bull. 199: 316–320. (December 2000)
Development of a Penis from the Vestigial Penis in the
Female Apple Snail, Pomacea canaliculata
NAOKUNI TAKEDA
Brainway Group, Brain Science Institute, The Institute of Physical and Chemical Research,
2-1 Wako, Saitama 351-0198, Japan
In the apple snail (Pomacea canaliculata), females have
an undifferentiated mass of tissue near the anus. Although
this mass is called the vestigial penis, there are no signs of
a hermaphroditic gonad or any structure that represents a
transition from one sex to the other. Based on considerations
of the steroid hormone theory of reproduction and in
view of disruption of endocrine systems in molluscs by
organotins, a study was made of the effects of tributyltin on
female snails. Exposure to tributyltin resulted in the socalled
imposex phenomenon, and both a penis and a penis
sheath were newly generated from the so-called vestigial
penis. The same phenomenon was also induced by testosterone.
Thus the vestigial penis, named more than one
hundred years ago, has been demonstrated for the first time
to be a rudiment of the penis itself.
The Ampullariidae (Gastropoda, Prosobranchia, Architaenioglossa)
is a family of freshwater prosobranchs that are
widely distributed in Asia, Africa, and South America (1). It
is well known that some molluscs exhibit unusual sexual
diversity and hermaphroditism (2, 3). One of the most
interesting features of members of the Ampullariidae is that
females in 5 of the 10 genera—namely, Pila (4, 5, 6, 7, 8,
9, 10, 11, 12), Pomacea (13, 14, 15, 16), Lanistes (13, 17,
18), Afropomus (19), and Turbinicola (13)—have a socalled
vestigial penis in addition to the normal reproductive
system (Fig. 1). The vestigial penis was first described by
Bouvier (4, 5), a skilled neuroanatomist, in Ampullaria
(Pila) polita more than 100 years ago. Since then, many
molluscan researchers have used that term for this tissue
without further explanation. The question examined here is
this: Is the organ historically described as a vestigial penis
a remnant of a penis—a degenerate structure that lacks the
Received 7 December 1999; accepted 10 August 2000.
E-mail: takeda@brainway.riken.go.jp
316
capacity to develop further—or is it a rudiment, or precursor—an
incipient structure that, under the proper conditions,
could develop into a penis?
The vestigial penis is a tongue-like structure lying inside
the elevated mantle skirt, near the anus (Fig. 2A). Histologically,
it consists of connective tissue, and no differentiation
of the structure is apparent during the life cycle of the
female (Fig. 2B).
To my knowledge, no experimental evidence has been presented
to justify the designation of the elevated tissue near the
anus in some female snails as a “vestigial penis” (4, 5). No
clear evidence of hermaphroditism has yet been shown in any
extant species of Ampullariidae. In Pomacea canaliculata, the
apple snail, the positions of the gonads are basically different:
the testis is located at the tip of the spiral, and the ovary is
spread over the surface of the hepatopancreas at a location
similar to that of the testis. It has also been confirmed that there
is no apparent precursor or vestige of a hermaphroditic condition
in any part of the reproductive system throughout the life
history. As Andrews (13) stated, the copulatory apparatus
appears to develop at the same rate in both sexes until the
gonad becomes active, when its growth is arrested in the
female. Andrews hypothesized that the gonad might produce a
hormone responsible for the cessation of growth, but in the
early 1960s, when this work was published, the chemical
nature of reproductive hormones in molluscs had not yet been
established. In 1991, Berthold (20) proposed that the so-called
vestigial organ be designated an “oriment”—a term implying
that the tissue is a precursor with the potential to develop
into an adult organ. The question then arose as to whether
such a designation might be appropriate. As a basis for such
a designation, at the very least, some experiments involving
implantation of testes into females should be performed to
determine whether a true penis might develop from the
tissue mass.

As an approach to this problem, I examined the effects of
an endocrine disruptor that appears to be associated with as
yet unresolved environmental problems. Organotins, and in
particular tributyltin, which is a component of some antifouling
paints, induce a condition known as “imposex” in
prosobranch gastropods. Imposex, in which a penis and vas
deferens develop in females (21), has been widely observed
in marine snails that belong to the Caenogastropoda; Nucella
and Littorina are common examples (22). Females of
these species lack a vestigial penis, but the capacity exists
for induction of a penis and vas deferens. Pomacea canaliculata
has been proposed as a potential bioindicator for
tributyltin (22), which is used as a biocidal agent against
molluscs, in fungicides (23) and in anti-fouling paints in
freshwater environments. Anticipating possible endocrine
disruption by tributyltin, I examined its effects to see
whether the vestigial penis in this species might develop
IMPOSEX IN THE APPLE SNAIL, P. CANALICULATA
Figure 1. General appearance of the vestigial penis in a female apple snail (Pomacea canaliculata). The
shell was removed and the region to the left of the head was dissected to reveal the vestigial penis (1), rectum
(2), and oviduct (3), which are enclosed in a square. The siphon (4), pulmonary sac (5), albumen gland (6), and
ovary (7) are also indicated. Scale bar represents 1 cm.
317
further after female snails were exposed to this compound
and, if such a penis did develop, how would it differentiate?
Female specimens of P. canaliculata were reared in water
that contained 30 ng/l tributyltin. About 3 months after the
start of treatment, the outside of the elevated tissue of the
vestigial penis began to form a long process that resembled
a penis, and its interior developed as a thick mass. These
structures grew gradually and reached a maximum size after
about 6 months of treatment with tributyltin (Fig. 3A).
Histological staining revealed that the inside tissue mass
contained a penis; the cross section of a penis was also
found within the tissue mass (Fig. 3B). Thus, the outside
structure appeared to be a penis sheath. Within about one
further month, a complete penis had developed from the
tissue of the vestigial penis (Fig. 4).
In P. canaliculata, the copulatory system of the male
consists of a stout penis sheath and a long, slender penis

318 N. TAKEDA
Figure 2. (A) General appearance of the vestigial penis in a female
apple snail (Pomacea canaliculata: control) reared by artificial mass culture.
Scale bar represents 1 mm. (B) Histological appearance of the
vestigial penis in A. The regions containing a vestigial penis were fixed in
Bouin’s fluid and embedded in paraffin wax by the standard method.
Sections were stained with hematoxylin and eosin. Scale bar represents 200
m. A, anus; C, ctenidium; OD, oviduct; VP, vestigial penis.
within it (24). The penis and the penis sheath are located
together to the left of the extreme right margin of the mantle
cavity. In treated females, the arrangement of these male
copulatory organs was similar but differed in the distance
between the penis sheath and the penis: the penis sheath in
females was located at the edge of the ctenidium at the
mantle skirt, at a distance from the penis.
It has been suggested that tributyltin inhibits cytochrome
P450 aromatase, which converts testosterone to estradiol in
females (25, 26). Inhibition of aromatase activity thus increases
levels of testosterone which induces imposex, with
the development of male copulatory organs. Development
of the imposex phenomenon in P. canaliculata was also
confirmed by direct treatment with testosterone. Female
snails reared in water that contained 500 ng/l testosterone
exhibited changes similar to those induced by tributyltin,
including the development of a penis sheath and a penis.
Therefore, these observations support the proposed mechanism
of action of tributyltin.
It is difficult to explain the unusual phenomenon of a
rudimentary penis in females; however, I propose the following
hypothesis. In the early stages of development, both
sexual rudiments develop as an undifferentiated tissue mass.
Once the sex of the snail is determined genetically (27),
however, these rudiments differentiate in response to the
secretion of specific sex steroid hormones. The undifferentiated
tissue mass that develops into a penis in males is left
as an arrested rudiment in females. The vestigial penis
develops into a complete copulatory organ only if the anlage
of the gonad becomes a testis.
Figure 3. (A) Morphology of the imposex induced by tributyltin in a
female apple snail (Pomacea canaliculata). Female snails (n 100) for
experiments were reared in a freshwater tank that contained tributyltin
(Tokyo Kasei, Co. Ltd., Tokyo, Japan) at 30 ng/l for about 6 months. The
state of imposex was checked at weekly intervals. Scale bar represents 1
mm. Similar results were also obtained in female snails (n 100) reared
with testosterone (Wako, Co. Ltd., Osaka, Japan) at 500 ng/l for about 7
months. (B) Histological appearance of the vestigial penis in A. The
arrowhead indicates the cross section of a penis. Hematoxylin and eosin
stain. See legend to Fig. 2B for methods. Scale bar represents 200 m. P,
penis; PS, penis sheath; VD, vas deferens (see legend to Fig. 2 for other
abbreviations).

The “steroid hormone theory,” which I proposed previously
(28, 29) for the reproduction of terrestrial pulmonates, apparently
also applies to prosobranch snails. This theory states
basically that the development of accessory sex organs is
controlled by steroid hormones secreted by the gonad. This
concept of the effects of hormones on snail reproduction,
together with the effects of endocrine disruption in molluscs,
allowed me to demonstrate, for the first time, that the so-called
“vestigial penis,” named more than one hundred years ago (4,
5), is a rudiment of the penis itself.
Literature Cited
1. Perera, G., and J. G. Walls. 1996. Apple Snails in the Aquarium,
T. F. H. Publications, Jersey City, NJ. 121 pp.
2. Fioroni, P., J. Oehlmann, and E. Stroben. 1991. The pseudohermaphroditism
of prosobranchs: morphological aspects. Zool. Anz. 226:
1–26.
3. Takeda, N. 2000. Mollusca. Pp. 93–147 in Reproductive Biology of
Invertebrates, Vol. 10, Part A, Progress in Developmental Endocrinology,
A. Dorn, ed. John Wiley, New York.
4. Bouvier, E. L. 1888. Sur l’anatomie de l’Ampullaire. Bull. Soc.
Philom. Paris 12: 5–70.
5. Bouvier, E. L. 1888. Étude sur l’organisation des Ampullaires.
Mem. Soc. Philom. Paris (centenary vol.) 63–85.
6. Sachwatkin, V. A. 1920. Das Urogenitalsystem von Ampullaria
gigans Spix. Acta Zool. (Stockh.) 1: 67–130.
7. Hagler, K. 1923. Anatomie von Pachylabra (Ampullaria) cinerea
(Reeve). 1. Teil. Mit Einsch ass einer burzen Besprechung der wichtigsten
Literatur über die Ampullariidae. Acta Zool. (Stockh.) 4: 314–
410.
8. Franc, A. 1968. Sous-classe des Prosobranches (Prosobranchia
Milne Edwards 1848 Streptoneura). Pp. 40–324 in Traite de Zoologie
V (III), P.-P. Grasse, ed. Masson, Paris.
9. Kaewjam, R. S. 1987. The apple snails of Thailand: aspects of
comparative anatomy. Malacol. Rev. 20: 69–89.
IMPOSEX IN THE APPLE SNAIL, P. CANALICULATA
Figure 4. (A) A penis that arose from the vestigial penis of a female apple snail (Pomacea canaliculata)
after treatment with tributyltin for about 7 months. The same phenomenon was also seen in female snails reared
with testosterone for about 8 months. See legend to Fig. 3A for methods. (B) The extirpated penis from the
vestigial penis. See legends to Fig. 2 and 3 for other abbreviations. Scale represents 1 mm.
319
10. Keawjam, R., and E. S. Upatham. 1990. Shell morphology, reproductive
anatomy and genetic patterns of three species of apple snails of
the genus Pomacea in Thailand. J. Med. Appl. Malacol. 2: 45–57.
11. Prashad, B. 1925. Anatomy of the common Indian apple-snail, Pila
globosa. Mem. Indian Mus. 8: 91–154.
12. Scott, M. I. H. 1957. Estudio morfologica y taxonomica de los
Ampullaridos de la Republica Argentina. Rev. Mus. Argent. Cienc.
Nat. (Zool). 3: 233–333.
13. Andrews, E. B. 1964. The functional anatomy and histology of the
reproductive system of some pilid gastropod molluscs. Proc. Malacol.
Soc. Lond. 36: 121–140.
14. Thiengo, S. C. 1987. Observations on the morphology of Pomacea
lineata (Spix, 1827) (Mollusca: Ampullariidae). Mem. Inst. Oswaldo
Cruz Rio J. 82: 563–570.
15. Thiengo, S. C. 1989. On Pomacea sordida (Swaison, 1823) (Prosobranchia,
Ampullariidae). Mem. Inst. Oswaldo Cruz Rio J. 84: 351–
355.
16. Thiengo, S. C., C. E. Borda, and J. L. Barros Araujo. 1993. On
Pomacea canaliculata (Lamarck, 1822) (Mollusca; Pilidae: Ampullariidae).
Mem. Inst. Oswaldo Cruz Rio J. 88: 67–71.
17. Aboul-Ela, I. A., and E. A. Beddiny. 1969. Morphological and
histological studies on the genitalia of Lanistes bolteni Chemnitz,
1786. Ain Shams Sci. Bull. 13: 145–176.
18. Berthold, T. 1990. Phylogenetic relationships, adaptations and biogeographic
origin of the Ampullariidae (Mollusca, Gastropoda) endemic
to Lake Malawi, Africa. Abh. Naturwiss. Ver. Hamburg 31/32:
47–84.
19. Berthold, T. 1988. Anatomy of Afropomus balanoideus (Mollusca,
Gastropoda, Ampullariidae) and its implications for phylogeny and
ecology. Zoomorphology 108: 149–159.
20. Berthold, T. 1991. Vergleichende Anatomie, Phylogenie und historische
Biogeographie der Ampullariidae (Mollusca, Gastropoda). Abh.
Naturwiss. Ver. Hamburg. 29: 1–256.
21. Matthiessen, P., and P. E. Gibbs. 1998. Critical appraisal of the
evidence for tributyltin-mediated endocrine disruption in mollusks.
Environ. Toxicol. Chem. 17: 37–43.
22. Takeda, N. 2000. Endocrine disruption of reproduction by organotins.
Pp. 259–285 in Reproductive Biology of Invertebrates, Vol.

320 N. TAKEDA
10, Part A, Progress in Developmental Endocrinology, A. Dorn, ed.
John Wiley, New York.
23. Duncan, J. 1980. The toxicology of molluscicides. The organotins.
Pharmacol. Ther. 10: 407–429.
24. Takeda, N. 1999. Histological studies on the maturation of the
reproductive system in the apple snail, Pomacea canaliculata. J. Anal.
Biosci. 22: 425–432.
25. Spooner, N., P. E. Gibbs, G. W. Bryan, and L. J. Goad. 1991. The
effects of tributyltin upon steroid titres in the female dogwhelk, Nucella
lapillus, and the development of imposex. Mar. Environ. Res. 32:
37–49.
26. Bettin, C., J. Oehlmann, and E. Stroben. 1996. TBT-induced
imposex in marine neogastropods is mediated by an increasing androgen
level. Helgol. Meeresunters. 50: 299–317.
27. Brand, E., T. Yokosawa, and Y. Fujio. 1990. Chromosome analysis
of apple snail Pomacea canaliculata. Tohoku J. Agric. Res. 40:
81–89.
28. Takeda, N. 1983. Endocrine regulation of reproduction in the snail,
Euhadra peliomphala. Pp. 106–110 in Molluscan Neuro-Endocrinology,
J. Lever and H. H. Boer, eds. North-Holland, Amsterdam.
29. Takeda, N. 1989. Hormonal control of reproduction in land snails.
Venus 48: 99–139.