/ . Embryol. exp. Morph. Vol. 60, pp. 57-69, 1980
Printed in Great Britain © Company of Biologists Limited 1980
57
Hormonal control of head-wart development in the
snail, Euhadra peliomphala
By NAOKUNI TAKEDA 1
From the Department of Biology, Toho University
SUMMARY
The terrestrial snail, Euhadra peliomphala, has a peculiar organ between the optic tentacles
named the head-wart, which releases a sex pheromone just before courtship. The development
of the head-wart was closely correlated with the sexual maturity of the snail. Castration led
to the atrophy of the head-wart. Subsequent injection of hermaphrodite gland homogenate
into the body cavity of castrated individuals induced the development of the head-wart.
The peripheral regions of the acini in the hermaphrodite gland showed a positive reaction
for 3yff-hydroxy steroid dehydrogenase. Furthermore, preliminary radioimmunoassay also
showed that the hermaphrodite gland of the snail contained testosterone and estradiol.
When the immature head-wart was cultured for 10 days in a medium containing testosterone
and estradiol respectively, development of the head-wart was found only in mediumcontaining
testosterone. From these results, it is concluded that head-wart development in the snail,
Euhadra peliomphala, is under direct control from the hermaphrodite gland, probably by
means of testosterone.
INTRODUCTION
An irregular polygonal pattern, named the head-wart, exists between the
optic tentacles in some terrestrial snails (Taki, 1935). In a previous paper, we
demonstrated that the head-wart released a sex pheromone just before courtship
(Takeda & Tsuruoka, 1979). The development of the head-wart seems to be
closely correlated with the sexual maturity of the snail, because it parallels
progress of maturation of the reproductive system. Our knowledge of the
endocrinology of pulmonate gastropods has progressed considerably during
recent years (Joosse, 1975; Boer & Joosse, 1975). Since the discovery of estrogen
and progesterone in the ovaries of Pecten hericius (Boticelli, Hisaw & Wotiz, 1961)
many steroid hormones have been detected in the reproductive systems of
molluscs (Lehoux & Sandor, 1970; Longcamp, Lubet & Drosdowsky, 1974;
Nomura, 1978; Takeda & Kobayashi, 1980). The pulmonate gonad has also been
shown to synthesize steroids related to those of the vertebrate gonadal system
(Gottfried & Dorfman, 1970a, b). Although the endocrine role of these steroid
hormones has not been proved fully in pulmonates, the presence of these
substances seems to be related to the reproductive function. In the stylommato1
Author's address: Department of Biology, Facutly of Science, Toho University, Funabashi,
274, Japan.
58
N. TAKEDA
phoran Mollusca, many experiments have suggested that the growth and
differentiation of the accessory sex organs, such as the albumen gland and the
common duct, are under the hormonal control of the hermaphrodite gland,
e.g. in the slugs, Limaxflavus (Abeloos, 1943; Laviolette, 1954) and Agriolimax
reticulatus (Bailey, 1973; Runham, Bailey & Laryea, 1973) and in the snails
Helix aspersa (Gomot, 1973, 1976) and Taphius giabratus (Harry, 1965). The
head-wart is an accessory sexual character and the development of the headwart appears to be under hormonal control by the gonad as are the other
accessory sex organs. In the present paper we attempt to clarify the hormonal
control of head-wart development in the snail, Euhadra peliomphala.
MATERIALS AND METHODS
The Euhadra peliomphala used in these experiments are commonly distributed
terrestrial snails in Japan.
Castration. As the hermaphrodite gland is located in the apex of the shell, the
apical part of the shell was crushed for castration. The hermaphrodite gland
resembles bunches of grapes which are partially embedded in the hepatopancreas. The hermaphrodite gland was removed with forceps after cutting the
hermaphrodite duct and separating it from the hepatopancreas. As the castration is highly lethal, the operation resulted in partial castration. The opening was
sealed with Spongel (Sankyo). Control snails were sham operated.
Injections of hermaphrodite-gland homogenate and steroid hormones. An
extract of the hermaphrodite gland was made using 30 adult snails. Pieces of
hermaphrodite gland (5 mg) were homogenized in isotonic salt solution yielding
a total of 10 ml. Injections of 0-1 ml were made into the body cavity by means of
hypodermic needle. Control snails were injected with physiological salt solution
only. 100/tg of the steroid hormones, testosterone and estradiol, were dissolved in one drop of acetone and ethyl alcohol respectively. Each was diluted
with 100 ml of double distilled water. Four injections were given on days 7, 9,
11, and 13 after castration.
3/1-hydroxy steroid dehydrogenase (3/1-HSD) activity. Fresh-frozen sections
(15-20/£m) were made with a cryostat. They were picked up on cover glasses
and dried at room temperature. The following incubation medium was used:
Dehydroepiandrosterone (2-8 mg/ml acetone) 0-5 ml; nicotinamide (5 mg/ml
distilled water) 1-0 ml; nicotinamide adenine dinucleotide (0-8 mg/ml of 0-1 M
phosphate buffer at pH 8-0) 7-5 ml; nitro-blue tetrazolium chloride (1 mg/ml
distilled water) 1-0 ml; phenazine methosulfate (0-1 mg/ml distilled water) 0-1 ml.
Control sections were treated with the same medium without the substrate,
dehydroepiandrosterone. Sections were incubated at 37 °C for 3 h, fixed in 10 %
formalin and then mounted in Kaiser's glycerin jelly.
Radioimmunoassay. Preliminary radioimmunoassays for estradiol and testosterone in the hermaphrodite glands were carried out by the methods of
Control of head-wart development
in the snail
59
Nakamura, Shodono & Tanabe (1974) and Hirano, Nakamura & Tanabe
(1978). [2,4,6,7-H3]estradiol (specific activity, 105 Ci/m-mole) and [1,2,6,7-H3]testosterone (specific activity, 100 Ci/m-mole) were used as antigens. Rabbit
anti-estradiol-6-(O-carboxymethyl) oxine bovine serum albumen was used at
1:9000 dilution. Rabbit anti-testosterone-11-hemisuccinate bovine serum
albumen was used at 1:20000. The diluted antisera were mixed with the etherextracted hermaphrodite glands and [2,4,6,7-H3]estradiol or [1,2,6,7-H3]testosterone and the mixture was incubated at 4°C overnight. Separation of the
free from bound steroid was done by adding a suspension of 0-024 % charcoal in
phosphate buffer. The radioactivity was measured with a liquid scintillation
counter (Packard).
Organ culture of the head-wart. The snails were starved for 3 days before use.
They were washed several times with sterilized water after removing the shells.
A piece of the immature head-wart epidermis was cut out and immersed in
sterile Hedon-Fleig salt solution (Lockwood, 1961). This isolated epidermis was
placed on Maximov slides in sterile Hedon-Fleig salt solution and exposed to
ultraviolet irradiation (50 cm from the source) for 2 min for each side. Each
explant was divided into two to four parts. Each part was transferred into a
glass-ring chamber containing Hedon-Fleig salt solution. This solution was
replaced with the culture medium. The culture medium used was developed for
the snail, Helix aspersa by Burch & Cuadros (1965). For the experiments pieces
of epidermis were cultured in media containing hermaphrodite-gland homogenate, testosterone or estradiol. Cultures were kept at 25 °C for 10 days. The
medium was changed every 2 days.
Histology of the head-wart: The head warts, following both injections and
culture, were fixed with Bouin's fluid, embedded in paraffin wax and sectioned
at 8 //m. The sections were stained with hematoxylin and eosin.
RESULTS
As the head-wart attains full size with the progress of sexual maturation, the
development of the reproductive system and the head-wart was compared. In
the infantile stage, the reproductive system consists of a simple elongated tube,
the proximal end of which is connected to a rudimentary hermaphrodite gland,
and the distal part connected to the small penial sheath. The other accessory sex
organs are still rudimentary or entirely lacking. During growth of the shell the
reproductive system remains small. With the development of the reversed
periostome, the growth of the reproductive system was rapidly completed. The
abrupt growth and differentiation of the reproductive system was related to the
maturation of the gonad; the development of the head-wart appears to be
closely correlated with this sexual maturation.
Following castration, many snails died within 7 days but about 30 snails
out of the 250 operated individuals survived for nearly 3 months. After the
60
N . TAKEDA
Fig. 1. Effects of castration on the epithelial cells of the head-wart in the adult
snail, Euhadra peliomphala. (A) Epithelial cells of the head-wart in a control snail.
Scale 25 /tm. (B) Epithelial cells of the head-wart in a castrated snail. Scale 25 /tm.
operation, no sexual excitement was observed in such snails, movement became
sluggish and the snails tend to withdraw into the shell. The appetite of the snail,
decreased and the body weight, including shell, was gradually reduced. However,
behavioural activity recovered about 6 days after the operation. The swelling
of the head-wart was no longer observed. A complete atrophy of the genital
orifice was seen in some snails about 10 days after the operation.
The epithelial cells of the head-wart underwent regressive changes following
castration (Fig. 1). A few days after castration, the height of the cells and the
nuclear volume decreased and this inactive appearance persisted. However,
with the injection of hermaphrodite-gland homogenate, the long axis elongated
and the nuclear volume increased so that by about 20 days after castration the
head-wart had recovered completely to appear normal (Figs. 2 and 3). These
castration experiments suggest that the swelling of the head-wart is intimately
related to the function of the hermaphrodite gland and that the development of
the head-wart is controlled by substances secreted from the hermaphrodite
gland.
3/?-hydroxy steroid dehydrogenase is one of the most important enzymes in
the steroidogenic pathway. As shown in Fig. 4, 3/?-hydroxy steroid dehydrogenase activity was clearly detected in the hermaphrodite gland, especially in the
61
Control of head-wart development in the snail
70
-
60
50
40
AT
I I I I I I I I I I I 1 1 I 1 1 I I I I 1 I 1 I I I I I I I I I 1 I1
10
15
20
Days after castration
25
30
Fig. 2. Effects of castration and injection on the development of the epithelial cells
of the head-wart in the snail, Euhadrapeliomphala. • , Control snails; O, castrated
snails; ©, injection of hermaphrodite gland homogenate; • , injection of testosterone; A, injection of estradiol; $., injection: See text.
\ i i i i I i I i i I i i i I i i i i I i i i I I i i i I i I I I I I I
Fig. 3. Effects of castration and injection on the nuclear volume of the epithelial
cells of the head-wart in the snail, Euhadra peliomphala. # , Control snails; O, castrated snails; ©3 injection of the hermaphrodite gland homogenate; • , injection of
testosterone; A, injection of estradiol; <j>, injection: See text. * As the nuclei
approximated to a spherule or ellipsoid, the volume of each nucleus was calculated
by the formulae, V = TTD3/6 for spherical nuclei and V = nLl2/6 for ellipsoidal
nuclei. The diameter (D) of spherical, and short (/) and long (L) axes of ellipsoidal
nuclei were measured microscopically. See text.
EMB 60
62
N.
TAKEDA
Fig. 4. Occurrence of histochemical activity of 3/?-hydroxy steroid dehydrogenase
in the hermaphrodite gland in the snail, Euhadra peliomphala. A, Frozen section of
the hermaphrodite gland, demonstrating the reaction for 3/?-hydroxy steroid
dehydrogenase in the peripheral region of the acini. Arrow. Scale 25 /tm. B, Crosssection of the hermaphrodite gland, showing sperm and eggs with germinal vesicles.
Scale 25 /tm.
Table 1. Detection of steroid hormones (testosterone and estradiol) in the hermaphrodite gland of the snail, Euhadra peliomphala. Experiments were performed
in January which was the diapause period in all snails and the post-reproductive
stage in adult snails
Snails
(g)
Infantile (1-8)
Juvenile (5-8)
Adult (8 0)
Hermaphrodite
glands*
(mg)
131
262
650
Testosteronef
Esttadiol
(pg/mg tissue)
2-70
219
0-20
* The total amount of 13 individuals.
t Mean of the amount in 13 pooled gonads.
3-82
1 97
1-30
Control of head-wart development in the snail
63
Fig. 5. Effects of hermaphrodite-gland homogenate on the development of the
epithelial cells of the head-wart in the juvenile snail, Euhadia peliomphala in vitro.
(1) Control, scale 25 /tm, (2) Hermaphrodite gland homogenate, see text, scale 25/mi.
Cultures were performed for 10 days at 25 °C.
cells at the periphery of each acinus. The hermaphrodite gland was shown to
have the ability to synthesize steroid hormones.
Furthermore, testosterone and estradiol were demonstrated by radioimmunoassay within the hermaphrodite gland (Table 1). The amount of steroid hormones
was much greater in infantile snails than in juvenile and adult snails. More
estradiol was present than testosterone. The adult snails in the post-reproductive
condition contained low levels of these steroid hormones.
5-2
64
N . TAKEDA
"">'
**
m
'\
FIGURE 6
Effects of steroid hormones (estradiol and testosterone) on the development of the
epithelial cells of the head-wart in the juvenile snail, Euhadra peliomphala in vitro.
(1) Control, scale 25 /tm. (2) Estradiol (50/tg/dl medium), scale 25 /*m. (3) Testosterone plus estradiol (each 50 /*g/dl medium), scale 25 /tm. (4) Testosterone (50 [i%/
dl medium), scale 25 fim. Cultures were performed for 10 days at 25 °C.
Control of head-wart development in the snail
65
Table 2. Effects of hermaphrodite-gland homogenate and steroid hormones
{testosterone and estradiol) on the development of the epithelial cells of the headwart in the snail, Euhadra peliomphala. As the nuclei during cultivation were
approximately ellipsoid, the volume of each nucleus was calculated by the formula,
V = nLl2/6. See Fig. 3
Length (/tm)
,
Treatments
A
^
Nuclear volume
Long axis
Short axis
(/t3)
Hermaphrodite gland
homogenate
Estradiol + Testosterone
Control
68-8±15-4
5-6±0-8
171-5±570
48-8±6-3
431 ±8-8
6-5±l-3
6-7±l-9
153-8±43-2
151-5±60-2
Testosterone
Estradiol
Control
69-3 ±13-3
31-8 + 15-3
35-8 ±10-3
5-8 + 1-4
7-4±3-2
6-8 + 1-6
172-4 ±460
117-0±4M
119 3±46-5
Concentration of steroid hormones; 50/<g/dl medium, mean±s.D. The cultures were
maintained for 10 days.
As the hermaphrodite gland was shown to be able to synthesize steroid hormones, the biological activity of these hormones was examined. When testosterone was injected into the body cavity of castrated snails, the head-wart
developed gradually and recovered from castration within a few days (Figs. 2
and 3). However, no clear effect of estradiol injection was found. These substances were also injected into intact snails. The development of the head-wart
was also stimulated by injection of hermaphrodite gland homogenate and
testosterone but no effect was found following estradiol injection. These effects
were not as clear as in castrated snails.
When the immature head-wart was cultured in a medium containing hermaphrodite-gland homogenate, it developed enormously (Fig. 5). The epithelial
cells of the head-wart cultured with testosterone developed similarly (Fig. 6).
However, no effect was found in the head-wart cultured with estradiol. Antagonistic effects of estradiol and testosterone were found in the head-wart
cultured with testosterone plus estradiol. The development of the head-wart was
reflected not only in the nuclear volume change but also in the elongation of the
epithelial cells. Following 10 days cultivation, the development of the head-wart
was not as advanced as during the breeding season of the snails. These results
are summarized in Table 2. From the results, it is suggested that the development
of the head-wart is controlled by testosterone secreted from the hermaphrodite
gland of the snail.
66
N. TAKEDA
DISCUSSION
The head-wart is present in the Pleurodontidae, genera Satsuma and Chlorites,
and in the Cepolidae, genera Helicostyla, Aegista, Bradybaena, Euhadra,
Doiicheulota and Trishoprita (Taki, 1935). Several species of the African genus
Gymnarion carry on their head a similar organ called the frontal organ (Binder,
1969). These organs are only complete in adult snails during the mating season.
Furthermore, their existence implies a very particular mating behaviour (Binder,
1977; Takeda & Tsuruoka, 1979). The complete development of these organs
has been suggested to be correlated with that of the reproductive system and
thus an external indicator of their endocrinological state.
In some terrestrial pulmonate Stylommatophora, the growth and differentiation of the accessory sexual organs have been known to be under the hormonal
control of the hermaphrodite gland. Castration of the adult slugs, Avion ater
and Limax maximus (Abeloos, 1943; Laviolette, 1954) led to the regression of
the common duct and the albumen gland. Castration of shell-bearing snails in
which hermaphrodite glands are embedded in the hepatopancreas has been
known to be highly lethal. In Euhadra peliomphala, the survival time was also
short and the operation resulted in only partial castration. However, following
even this partial castration, the head-wart failed to develop. As in many gland
cells (Gabe & Arby, 1961), the increase and decrease in the nuclear volume of
the head-wart epithelial cells is also considered to represent the increasing and
decreasing activity of the head-wart. As the injection of the hermaphroditegland homogenate into castrated snails induced the development of the headwart, it is suggested that the hormones involved with the development of the
head-wart originate from the hermaphrodite gland. In Gymnarion, accidental
castration also led to the atrophy of the frontal organ (Binder, 1969). From
castration experiments it has been suggested that maintenance of the structural
characteristics of active secretion in the multifid gland in the snail, Helix aspersa
is partially dependent on a hormonal factor secreted from the hermaphrodite
gland (Gomot, 1973, 1976). The hormonal role of the hermaphrodite gland in
regulating accessory sexual gland development has therefore been established.
These changes are similar to those of the accessory reproductive glands of
mammals such as the prostate and seminal vesicles. However, no effect of
castration on the growth of the accessory sexual organs was found in the snail,
Bulinus truncatus (Brisson, 1971; de Jong-Brink, Borg, Bergamin-Sassen &
Boer, 1979) and Australobis glabratus (Vianey-Liaud, 1972).
There is tentative evidence that brain hormones may influence the reproductive organs via the gonad. Bailey (1973) proved with organ culture that the
cerebral ganglia and gonad in association, but not when cultured separately,
produce hormones that affect the male line in the gonad and secreting cells in the
prostate gland. In the snail, Lymnaea stagnalis, the dorsal body is known to
produce a hormone that stimulates vitellogenesis and growth of the female
Control of head-wart development in the snail
67
accessory sexual organs (Geraerts & Joosse, 1975; Geraerts & Algera, 1976). In
the slug, Agliolimax reticulatus, dorsal body hormone also controls the growth
and differentiation of the oviducal and albumen glands (Wijdenes & Runham,
1976). In these animals, the effect of the dorsal body hormone on the female
organ could be mediated by the hermaphrodite gland.
The detection of a steroid-synthesizing enzyme such as 3/?-hydroxy steroid
dehydrogenase in the hermaphrodite gland in Euhadra peliomphala has shown
that the synthesizing system for steroid hormones is present. In the pulmonate
Basommatophora, the hermaphrodite gland is not known to have an endocrine
function (Boer & Joosse, 1975; Joosse, 1975). However, recent experiments on
Lymnaea stagnalis have shown that its hermaphrodite gland also possesses
3/?-hydroxy steroid dehydrogenase (de Jong-Brink, Boer & Schot, 1978). This
probably indicates that the gonad is capable of producing steroid hormones,
although the endocrine role has not yet been proved.
Experimental evidence shows that the pulmonate hermaphrodite gland produces one or two sexual hormones. However, the cells involved in the synthesis
of these hormones are as yet not identified. Gottfried & Dorfman (1968, 1970 a,
b) have shown that steroid hormones, related to the vertebrate gonadal hormones, are synthesized by the hermaphrodite gland of Ariolimax californicus.
In this species, steroid hormones are implicated in modifying the eye-tentacle
effect upon the hermaphrodite gland. In the slugs, Deroceras reticulatum and
Limaxflavus, estrogen stimulated egg-laying in spite of the low rate of development, whereas androgen enhanced the rate of development rather than the
number of eggs laid (Takeda, 1979). Furthermore, in Umax flavus, spermatogenesis is controlled by testosterone secreted from the hermaphrodite gland
(Takeda &Tejima, 1979).
In the present experiments, it was demonstrated that the head-wart in the
snail, Euhadra peliomphala, is the target organ of the steroid hormone, testosterone, secreted from the hermaphrodite gland. It is interesting to note the
resemblance between the roles of steroid hormones in vertebrates and in
invertebrates.
The author wishes to express his thanks to Prof. J. Ishida, Toho University, for his encouragement of the present work, to former Prof. Iw. Taki, Hiroshima University, for his
many helpful suggestions and encouragement, to Prof. Y. Tanabe and Dr T. Nakamura,
Gifu University, for their teaching radioimmunoassay, to Teikoku Hormone Manufacturing
Co. Ltd., for supplying steroid hormones and to Miss H. Tsuruoka, Mr Y. Katohno and
Miss K. Nogami for their technical assistance. Special thanks are also made to Dr N. W.
Runham, University College of North Wales, who read the manuscript and gave many suggestions and encouragement. A part of this work was supported by the Itoh Science Foundation.
68
N. TAKEDA
REFERENCES
M. (1943). Effets de la castration chez un mollusque, Litnax maximus L. C. r. hebd.
Seanc. Acad. Sci., Paris. 216,90-91.
BAILEY, T. G. (1973). The in vitro culture of reproductive organs of the slug Agriolimax
reticulatus (Mull.) Neth. J. Zool. 23,72-85.
BINDER, E. (1969). Cephalic accessory sexual organ of Gymnarion: speciation and phylogeny
(Pulmonata, Helicarinodae). Malacologia. 9, 59-64.
BINDER, E. (1977). La pariade chez le genre Gymnarion Pilsbry 1919; role de l'organe frontal.
Arch Molluskenk. 107,249-255.
BOER, H. H. & JOOSSE, J. (1975). inPulmonates, Vol. I (ed. V. Fretter & J. Peake), pp. 245-307.
London, New York and San Francisco: Endocrinology, Academic Press.
BOTTICELLI, C. R., HISAW, F. L. & WOTIZ, H. H. JR. (1961). Estrogens and progesterone in
the sea urchin (Strongylocentrotus franciscanus) and Pecten (Pecten hericius). Proc. Soc.
exp. BioL Med. 106, 887-889.
BRISSON, P. (1971). Castration chirurgicale et regeneration gonadique che, quelques planorbides (Gastropodes Pulmones). Annals Embryol. Morph. Fr. 4, 189-210.
BURCH, J. B. & CUADROS, C. (1965). A culture medium for snail cells and tissues. Nature,
Lond. 206, 673-638.
ABELOOS,
DE JONG-BRINK, M., BORG, J. P., BERGAMIN-SASSEN, M. J. M. & BOER, H. H. (1979). Histology
and histochemistry of the reproductive tract of the pulmonate snail, Bulinus truncatus,
with observations on the effects of castration on its growth and histology. Int. J. Invert.
Reprod. 1,41-56.
DE JONG-BRINK, M., BOER, H. H. & SCHOT, P. (1978). The endocrine function of the ovotestis
in fresh water snail. In Comparative Endocrinology (ed. P. J. Gaillard & H. H. Boer), pp. 81.
Amsterdam: Elsevier/North-HollandBiomedial Press.
GABE, M. & ARVY, L. (1961). In The Cell, Vol. 5. (ed. J. Brachet & A. E. Mirsky), pp. 1-88.
Gland cells. New York and London: Academic Press.
GERAERTS. W. P. M. & ALGERA, L. H. (1976). The stimulating effect of the dorsal body hormone on cell differentiation in the female accessory sex organs of the hermaphrodite
freshwater snail. Lymnaea stagnalis. Gen. comp. Endocr. 29, 109-118.
GERAERTS, W. P. M. & JOOSSE, J. (1975). Control of vitellogenesis and of growth of female
accessory sex organs by the dorsal body hormone (DBH) in the hermaphrodite freshwater
snail, Lymnaea stagnalis. Gen. comp. Endocr. 27,450-464.
GOMOT, L. (1973). fitude de fonctionnement de l'appareil genital de l'escargot Helix aspersa
par la methode de cultures d'organs. Archs Anat. Histol. Embryol. norm, et exp. 56,
131-160.
GOMOT, L. (1976). Ultrastructural changes in the multifid glands of the snail, Helix aspersa
following castration. Gen. comp. Endocr. 29,298.
GOTTFRIED, H. & DORFMAN, R. I. (1968). The steroid biochemistry of the molluscan ovotestis:
a general concept of reproductive control mechanisms. In Progress in Endocrinology
(ed. C. Gual). Excerpta Med. Int. Cong. Ser. 184, 368-376.
GOTTFRIED, H. & DORFMAN, R. I. (1970a). Steroids in invertebrates. IV. On the optic tentaclegonadal axis in the control of the male phase ovotestis in the slug Ariolimax califomicus.
Gen. comp. Endocr. 15,101-119.
GOTTFRIED, H. & DORFMAN, R. I. (19706). Steroids in invertebrates. V. The in vitro biosynthesis of steroids by the male phase ovotestis of the slug Ariolimax califomicus. Gen. comp.
Endocr. 15,120-138.
HARRY, H. W. (1965). Evidence of a gonadal hormone controlling the development of the
accessory reproductive organs in Taphius glabratus Say (Gastropoda, Basommatophora).
Trans. Am. microsc. Soc. 84,157.
HIRANO, H., NAKAMURA, T. & TANABE, Y. (1978). Changes in plasma LH and testosterone
concentration of Japanese Quail from hatching to sexual maturity. Jap. Poultry Sci. 15,
242-247.
Control of head-wart development in the snail
69
J. (1975). Structural and endocrinological aspects of hermaphroditism in pulmonate
snails, with particular reference to Lymnaea stagnalis (L.). In Intersexuality in the Animal
Kingdom, (ed. R. Reinboth), pp. 158-169. Berlin, Heidelberg and New York: SpringerVerlag.
LAVIOLETTE, P. (1954). Role de la gonade dans le determinisms humoral de la maturite
glandulaire du tractus genital chez quelques gasteropodes Arionidae et Limacidae. Bull.
Biol. Fr. Belg. 88, 310-332.
LEHOUX, J. G. & SANDOR. T. (1970). The occurrence of steroid metabolizing enzyme systems
in invertebrates. A review. Steriod. 16,141-171.
LCCKWOOD, A. P. M. (1961). Ringer solutions and some notes on the physiological basis of
the ionic composition. Comp. Biochem. Physiol. 2,241-289.
LONGCAMP, D., LUBET, P. & DROSDOWSKY, M. (1974). The in vitro biosynthesis of steroids by
the gonad of the mussel (Mytilus edulis). Gen. comp. Endocr. 22, 116-127.
NAKAMURA, T. SHODONO, M. & TANABE, T. (1974). A manual of radio-immunoassay of
estradiol and progesterone in animal tissues andfluids.Res. Bull. Fac. Agr. Gifu Univ. 36,
319-328.
NOMURA, T. (1978). Biological Active Substances Produced by Marine Organisms, pp. 223.
Tokyo :Nankodo.
JOOSSE,
RUNHAM, N. W., BAILEY, T. G. & LARYEA, A. A. (1973). Studies on the endocrine control of
the reproductive tract of the greyfieldslug Agriolimax reticulatus. Malacologia 14,135-142.
N. (1979). Induction of egg-laying by steroid hormones in slugs. Comp. Biochem.
Physiol. 62A, 273-278.
TAKEDA, N. & KOBAYASHI, H. (1980). Animal hormones. Clin. Endocrinol. 28, 336-351.
TAKEDA, N. & TEJIMA, M. (1979). Hormonal control of the maturation of the hermaphrodite
gland in the slug, Limaxflavus. The Fourth Annual Meeting of Japanese Society ofComparative Endocr biologists. Abstract, pp. 13.
TAKEDA, N. & TSURUOKA, H. (1979). A sex pheromone secreting land in the terrestrial snail.
Euhadra peliomphala. J. exp. Zool. 207,17-26.
TAKI, IW. (1935). Notes on a warty growth on the head of some land snails. /. Sci. Hiroshima
Univ. Ser. B, Div. 1,3,159-183.
VIANEY-LIAXJD, M. (1972). £tude de controle de la maturite des tractus genitaux et de la
ponte par castration chirurgicale chez le planorbe Australorbis glabratus Say (Pulmonata,
Basommatophora). Bull. Biol. Soc. Zool. Fr. 97,675-690.
WUDENES, J. & RUNHAM,. N. W. (1976). Studies on the function of dorsal bodies of Agriolimax
reticulatus (Mollusca; Pulmonata). Gen. comp. Endocr. 29, 545-554.
TAKEDA,
{Received 11 December 1979, revised 1 May 1980)