Italian Journal of Zoology

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Structure of the Reproductive Apparatus and Life

Cycle of Milax Gagates (Draparnaud). Mollusca
Gastropoda Pulmonata

Silvano Focardi & Diletto Quattrini

To cite this article: Silvano Focardi & Diletto Quattrini (1972) Structure of the Reproductive
Apparatus and Life Cycle of Milax Gagates (Draparnaud). Mollusca Gastropoda Pulmonata, Italian
Journal of Zoology, 39:1, 9-27, DOI: 10.1080/11250007209429172

To link to this article: https://doi.org/10.1080/11250007209429172

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Boll. Zool., 39: 9-27, 1072

STRUCTURE O F THE REPRODUCTIVE. PP. R. .TUS AND LIFE

CYCLE OF MILAX GAGATES (DRAPARNAUD). MOLLUSCA GA-
STROPODA PULiUONATA (*)

SILVANO FOCARDI and DIIZTTO QUATTRINI

Institute of Comparative Anatomy, Siena University

(received h'ocembet 9, 1971)

IKTRODUCTION

Wc began raising Milux gagafes (Draparnaud), a pulmonate gastro-

pod of the Limacidac family, in our Institute about 2 years ago. The
primary scopc of this activity was t o study thc structure of the nerve
cells (and in particular a group of neurons that sccm t o have a secretory
function) in the varying ages and physiological states of these animals
but i t was also possible to follow their entire life cycle, from the embryonic
development through hatching and reproduction until death, which is the
subject of the prcscnt work.
The complete life cycle of ill. gagates does not appear to have been
studied in detail by any other Authors. The available information mainly
concerns their reproductive- period and is often conflicting ; GERMAINin
u Faune de France D (1930, 1960 reprint), reports that this occurs once n
year in October while GALANCAU (1964)maintains that it occurs in 2 di-
stinct periods - autumn and spring. KARLIN and BACON(1961)examined
mating and oviposition in JI. gagafes and various other species of Lima-
cidae (Deroceras laeve, D. reticulatum, Litnax maximus, L. flavus and L.
poirieri).
Onc of us had prcviously studied the reproduction of ill. gagafes
with the chief aim of determining whether these, like other hermaphrodite
Gastropoda, are capable of self-fertilization (QUATTRINI,1070). These
laboratory specimens were always seen to reproduce only once in (L life-
time, cither in the spring or the autumn, and to die as soon as oviposition
terminated.

(*) This investigation was supported by n grant from the Consiglio Nazionale
delle Ricerclie.
10

AIATERIAL AND METHODS

Our observations were made on scveral specimens of ill. gugaies either born
and raised in the laboratory or collected from gardens in Siena wherc this species of
mollusk is quite common. An adult specimen is shown in fig. 1.
The laboratory specimens were raised at room temperature (18-25OC) in 10 x 18
or 18 x 25 em plastic containers lined on the bottom with 5-10 em of a mixture of mould
and sand : isolated individuals were kept in the smaller containers, groups of animals
in the larger ones. The specimens n-ere fed on lettucc and occasional carrot fragments,
renewed every 1 or 2 days. The earth layer, .kept very humid in order to avoid perilous.
dehydration, was changed every 15-20 days in order t o obviate tlic possibility of exees-
sive pollution from dejection, food remains, ete. 111. gagales survives very well under these
conditions :the mortality rate is very low (not more than 2% in adults and only slightly
higher in younger specimens, occurring particularly within the first month after hatching)
and there are no evident signs of any sufrering.
The morphology of the adult reproductive apparatus was studied in specimens
saerificed by immersion for about 10 hours in water that had been previously de-aera-
ted by boiling for 10-15 minutes. Under these conditions the specimens die in extension,
which greatly facilitates tlic study of the morpliology nnd reciprocal topopaphicalmp-
port of most of their organs.
. Thc histological examination w a s conducted mainly on adult specimens either
just before or just after oviposition, or immediately after death. The material for study
was fixed in Bouin, Sanfeliee, Carnoy or 10% formnlin, embedded in paralfn, sectioned
in 5-7 p slices and stained with lmernatoxylin-cosin, toluidinc blue, Unna-Pappenheim
or with the PAS-reaction (with or without 1% salivary diastase for 1 hour a t 87OC).

HISTO1\IORI’IIOLOGY OF THE REPRODUCTIVE APPARATUS

Much has been written on the morphology, and often on the histology,
of the reproductive apparatus of pulmonate and pseudopulmonate gastro-
pods. Among the more recent works we mention those by MALEK(1954),
FO OR TON (1955), BOUNOURE and AUBRY(195G), CHIA-TUNG-PAN (1958),
ALAPIIILIPPE (1959), DUNCAN (lOGO), QUICK (lOGO), RIGBY(1963), MAE-
TOJA (1964), LANZA and QUATTBINI (10G4), QUATTRIKI and LANZA (19G5),
FORCART (19G7), VAN BIOL (1968).
The sexual apparatus of Jf. gugates has been studied in particular
by LESSONA and POLLOXERA (1882), SIXROTII (1911) and, more recently,
by TAVIANI (1954). A summary of its anatomic conformation and fine
structure is givcn, dwelling a bit longer on some particulars that mere
not examined or which were less clearly illustrated in the other
works.
On the whole the reproductive apparatus of Jf. guguies is like that
of most pulmonate gastropods, having a single gonad and gonoducts
 11

with various connected organs. A semi-schematic illustration is seen in

fig. 2.
The gonad is situated very caudally, immersed in the liver and,
in animals sacrificed during reproduction, is 8-0 mm long and, at its max-
imum, 2-3 mm thick. Usually it has 3 lobes containing numerous folli-
cles of varying sizes and shapes which produce both the male and female
germinal cclls (figs. 2, 27, 28 and 29). According to GALANGAU(1964)) the
...
sexual cycle shows s un hermaphrodisme protandrique final, avcc sper-
matozoides mQrs expulses dans le canal hermaphrodite avant les oeuf o.
Our observations neither confirm nor deny such a finding. I n sexually ma-
ture animals the gonad follicles contain free sperms and oocytes in me-
taphase of the first meiotic division (the maximum stage of maturity
they reach within the gonad) which seems to give evidence of their con-
temporaneous maturation. In sexually immature but not much younger
specimens (about 3 months old) one normally finds very nearly full-grown
ovocytes next to a great number of male germinals cells, prevalently sper-
matogonia and spermatocytes (spermatids are always scarse and spcr-
matozon absent) suggesting, on the whole, a protogynous hermaphroditism.
But this could agree just the same with what GALAIWAU(loc. cit.) afirms
because she observes that, despite protandrous hermaphroditism being the
...
final phenomenon u au cours dc l'evolution dans les follicules hermaphro-
dites, cc sont Ics ovocytes qui se formcnt les premiers, Ies spermatogonics
ne ce diffhnciant que par la suite D, an observation also made by LAVIO-
LETTE (1950) in d r i o n rz1f2is.
As w e have mentioned the female germinal cells detach themselves
from the gonad walls -as ovocytes of the 1st order in metaphase of the
first meiotic division and remain in that stage until fecundation (fig. 3).
The gonoducts, of the diaulic type as in most pulmonates, are com-
posed of a small hermaphrodite duct, a large hermaphrodite duct, an ovi-
duct and a defcrens.
The small hermaphrodite duct, about 15 mm long, is almost straight
in the tract proximal to the gonad, very tmistcd distally and lined with
a simple, ciliated epithelium, containing cells that often have a volumi-
nous, chromatin rich nucleus (figs. 2, 4 and 5). Cilia appear either rare or
absent where such cells arc abundant ; in size and structure these resemble
the gonad nurse cells but do not appcar in the duct until later, The duct
itself contains similar cells that seem to be detached from its walls as well
as groups of cclls resembling various stages of spermatocyte evolution,
almost as if spermatogenesis where possible in such a site. I n adult spe-
cimens this duct is packed with sperm except in the short distal tract where
12

thcy arc rarc or absent. During the reproductive period a very limited
number of female germinal cells, always in metaphase of the 1st meiotic
division, are also found. We have never observed spermatozoa in their cy-
toplasm which could testify to a prior fecondation. The duct epithelial
cells contain glycogen.
Towards the distal end of the littlc hermaphrodite duct there is a
minuscule saclike diverticulum, about 1 mm long and lined with n simple
ciliated epithelium, which is difficult to locate because of its being rather
deeply embedded in the albumin gland (figs. 2 and G). Described by SI-
MROTII (lgll), its existence in- 31. gugates was not verificd by TAVIANI,
despite accurate research (1051). It could be thought that this organ exists
in some specimens and not in others but we deem it more likely that, due
to its minuscule size and particular location, i t can simply escape the
attention of even a very careful observor. This organ is found in many other
gastropods and is indicated varyongly as a fertilization pocket o, a seminal
vescicle)) or amaturation vesciclen. I n our specimens it did not contain
either ovocytes or sperm.
The large hermaphrodite duct, much more voluminous and very si-
nuous, is about 30 mm long with two furrows dividing its lumen: the
much larger one (thc uterus or uterine part of the duct) is reserved for the
passage of the eggs while the much smaller one (seminal or spermatic
furrow or prostatic part of the duct) is for the transit of the male gametes
(figs. 2 and 7). The gonoduct is lined with a simple ciliated epithelium
(with more developed and eyidcnt cilia in the spermatic furrow). The Ute-
rine part of the gonoduct has very thick Tvalls, rich in voluminous glandular
cells that secrete intensely PAS-positive products into the duct. An al-
bumin and a prostatic gland are connected to the gonoduct.
The albumin gland, which reaches its maximum size during the re-
productive period, opens into the gonoduet near the junction with the
small hermaphrodite duct (figs. 2 and 8).
The prostate consists of numerous tubules that open into the sper-
matic furrow along almost its entire length (figs. 2 and 7).
At the end.of the large hermaphrodite duct the uterinc and sper-
matic furrows close and separate into two canals, the oviduct and
deferens.
Tlic oviduct (or vagina), about 10 mm long, has thick connective
walls rich in muscular tissue and is lined internally with a simple ciliated
epithelium. Only its initial tract contains glandular cells, which secrete
intensely PAS-positive materials. It opens into a genital atrium situated
about 7 mm from the base of the right tentacle (figs. 2 and 9).
 Connected t o the oviduct is a copulatory sac (spermatheca or seminal
receptacle), a sac-like organ G-7 mm long and, a t its maximum, about 8 mm
wide, which receives the spermatophores during copulation (fig. 2). Distally
this organ has very thin, almost transparent walls while proximally it
is bulb shapcd with largely muscular, much thicker walls. The sac is lined
with a simple epithelium which has a striated PAS-positive cuticle; no
vibratile cilia were ever found in this cpithclium. Distally the same epithe-
lium is usually much thicker then in a11 the other zones of the reproductive
apparatus (figs. 11 and 12).
The deferens is about the same length as the oviduct and adheres
tightly to this until nearing the penis where they separate (figs. 2 and 9).
It has connective walls rich in muscular tissue and is lined with a simple
ciliated epithelium.
The retracted penis is almost cylindrical, about 3 mm long and
with a masimum diameter of about 1.5 mm (fig. 2). It has thick walls for-
med by connective and abundant musclc tissue, this latter running lon-
gitudinally, circularly, radially or irregularly, and containing numerous
blood lacunae which give it a cavernous aspect. Opening into the genital
atrium is an internal cavity, lined with a simple ciliated epithelium and
forming numerous indentations that penetrate more or less deeply into
the penis wall (figs. 13 and 15).
Tile penis prolongs into an epiphallus (<allled spermatophore sac by
SIMROTII, 1911) which is directly continuous with the deferens and which
resembles the penis in form and size (fig. 2). Histologically it is easily rc-
cognizable by the presence of a thick compact layer of usually very re-
gular, longitudinally-running muscle fibers in the peripheral wall region.
The male genital path, a t its maximum width in this site, is lined by a
simple ciliated epithelium which forms, even more so than in the penis
itself, a large number of complicated infoldings (fig. 14). This organ was
seen t o produce spermatophores and the complex conformation of its
cavity seems t o shape thc equally complicated design of their surfaces.
N.gagates presents 8 particular accessory genital organ that LEs-
SONA and POLLONERA (1882) named the corniform organ because of its
morphological aspect. This organ, found near the oviduct and copulatory
sac (on the side opposite the penis) and contained in a pocket that opens
into the gcnital atrium (fig. 2), represents a differentiation of the lateral
wall of the atrium itself (TAVIAKI,1954). I t s shape is that of a slightly
curved horn and, on the surface facing the atrium, there are about a dozen,
more or less voluminous protruding papillae. According t o different authors,
these papillac arc arranged in two (LESSONA and POLLONERA, 1882 ;GER-
MAIN, 1930, 1969) or three (TAVIAKI,
1954; RESZOPI’I,1963) series but,
in our material, they did not appear t o be arranged in any constant or
regular order. Considered as a stimulating organ, this herniates outside
the genital atrium during copulation and also in asphyxiated specimens,
even though, in this case, the copulating organ is completely retracted.
It is formed of connective and abundant muscular tissue and contains
numerous hemolymphatic spaces (fig. 16). It has no internal cavities. ‘
It is covered with a simple, occasionally very flattened epithelium (in which
cilia were never observed) which is continuous with that lining the genital
atrium (fig. 16). The organ is about 3 mm long and, at its maximum (to-
ward the base), is little more than 1 mm wide. Reaching the base itself
are a bundle of slender, delicate canaliculi which originate from the vesti-
bular gland on the other side (fig. 2).
The canaliculi, all independent from each other, are 5-43 nim long,
have a diameter of about 100 p and vary in quantity ; a control of three
specimens revealed the presence of 35, 38 and 42, respectively. Lined
inside with a non-ciliated cubic epithelium, their mall is completed by
a relatively thick layer of connective and abundant muscle tissue. No
secretory material has ever been found in them (fig. 17). They penetrate
at tlie base of the corniform organ and exit generally at its apex (some
seem t o exit laterally along the organ) after following a very sinuous path
between its connective and muscular tissue (fig. IG). At this point their
walls are much thinner (having lost the major part of their connective
and muscular components) while the lumen, always devoid of any secretory
products in all our specimens, does not undergo any notable changes
(figs. 16 and 17).
The vestibular gland, gencralIy formed as shown in fig. 2, is flattened
dorso-ventrally and reaches its maximum size in sesually mature indivi-
duals. It is shaped like a tubular gland (fig. 17) with sometimes very vo-
luminous tubules lined by a non-ciliated cubic epithelium. The tubule
walls contain numerous large glandular cells having abundant cytoplasm
and a nucleus that is frequently rich in chromatin. The gland is often packed
with intensely PAS-positive secretory material that does not present any
mctacliromatic phenomena and which does not stain like mucous (REF
ZOXI, 1963, indicates the vestibular gland as a n atrial, non-mucosal one).
The secreted material is poured inside tlie tubule lumen which are directly
Continuous with the excretory canaliculi described above : these latter
convey the secretion to the corniform organ. The tubules are immersed
in a n abundant layer of connective tissue which thins out in correspon-
dence with the excretory ducts (fig. 17).
 15

It should be rcmembered that the corniform organ is not present

in all species of JIiZax. LESSONA and POLLONERA (1882) divided the genus
according to this factor, distinguishing the two sub-genus of Pirainea and
Tandonia (with and without, respectively). GERMAIN (loc. cit.) distingui-
shed the gcnus Lalleiiznntia (JI. gngates and JI. ochracezts) and the genus
Tandoiiin (JI. marghatus and ill. sowerbyi).

STATE OF THE FRESIILY LAID EGGS

At the moment of deposition the eggs are subsphcric or ellipsoidal,

with an average diameter of about 8 mm (fig. 19). Their greater mass is
formed by various albumen layers covered externally by a mucous capsule
(figs. 24-2G). The albumen is produced by the homonymous gland. Two
layers are usually easily discernible : a central, thicker and very fluid
one and another, more consistent peripheral one, capable of maintaining
the form of the egg almost perfectly even after removal of the external
mucous membrane. This membrane is rather thin, very elastic and quite
transparent even though diffused with innumerable tiny calcite crystals
(figs. 24-26), The membrane is produced by the glands situated inside
the uterine part of the large hermaphrodite gonoduct. The crystals are
formed in the eggs in thc oviduct.
The true egg cell, still non-segmented and embedded in the albumen
mass, has a spheroidal form and a diameter that is generally between
100-110 p. The transparency of the albumen and mucous capsule makc it
possible to follow the ulterior phases of its evolution under the microscope.
Strictly speaking, this is not a mature egg ready to segment itself but
rather a fertilized oocyte of the 1st order, still in metaphase of the first
meiotic division.

FIRST CLEAVAGES, HATCIIIKG AND AGE AT WHICI1 REPRODUCTION

BEGINS

Each egg, upon deposition, contains an oocyte of the 1st order in

mctaphase of the first meiotic division ; i t is thus'in the same stage as
the female germinal cells when thcse detach themselves from tlic gonad
malls. After 1-2 hours the first polar body is formed,'followed, after ano-
thcr 2-8 hours, by the appearance of the second one (fig. 20). The first
polar body, never seen to divide itself, degenerates a bit before the second
onc. It is not uncommon to find them both still present even a t advanced
stages of cleavage (figs. 21 and 23). The two polar bodies arc usually the
same size.
Upon completing meiosis, caryogamy occurs and cleavage begins :
figs. 21, 22 and 23 show the embryo with 2, 4 and 8 blastomeres.
Four or five days after deposition the nutritive sac is formed (figs. 24-
26), a cephalic vesicle of endodermic derivation that communicates extcn-
sively with the stomach and represents an embryonic annex with a trophic
function. Towards the 10th day the podocyst (figs. 25 and 26), another
sac-shaped embryonic annex with thin, contractile walls, appears a t the
caudal portion of the foot and, though usually considered in rapport with
the circulation during the precoocius stages of development, probably expli-
cates various functions (QUATTRINI and BANI, 1071). Both these organs
rcach their maximum dcvelopment towards the 20th day of life, then
gradually regress and finally disappear upon hatching. According to FOL
(1880), the nutritive sac doesn’t completely disappear : a part of i t is used
to form the liver. The podocyst, instead, is completely reabsorbed so that
no trace remains at hatching: its structure, ultrastructure and functional
significance have been discussed recently by QUATTRINIand BANI
(loc. cit.).
Six or seven days after deposition crystals bcgin appearing in the
egg albumen, a t first scantily and then in ever-increasing quantity (fig. 26).
Usually very small, they can reach a.rclativcly notable size (their major
axis can measure 150 p). Appertaining to the tetragonal system they can-
not thus be of calcite that has been eventually dissolved by the mucous
capsule and then re-crystallized inside the albumen in order to be used
in some way by the organism during its dcvelopment. If considered as ca-
tabolytes which, expelled by the embryo, gradually precipitate ,in crystal-
line form as little by little their quantity increases and the same albumen
is at first saturated, then reabsorbed, this hypotesis seems to lack confir-
mation in the fact that a few days before hatching these begin t o diminish
and have completely disappeared by birth of the animals.
The albumen is completely reabsorbed by the timohatching occurs: of
the egg only the external mucous membrane remains and this no longer
contains any calcite crystals which have been utilized by the animal in the
course of its development.
I n order to establish the time elapsing between deposition and hat-
ching.10 groups of eggs, laid in the autumn, were examined with the fol-
lowing results :
 I

. I

L-- . I . -~ - i - - -___ - - A

PLATE I

Fig. 1 - X f h z gugufes (Draparnaud). A sesually mature spccimcn. x 1.5.

Fig. 2 - Jfilux gugufes. Scmiscliernatic drawing of the scsual apparatris : 1, Tentacle ;
2, JIantle ; 3, Epipliallus ; 4, Penis ; 5, Genital atrium ; G, Deferens ; 7, Cor-
niforni organ ; 8, Copulatory sac ; 9, Excretory canaliculi of the vestibular
gland ; 10, Oviduct ; 11, Vestibular gland ; 12, Prostate ; 13, Large herma-
phrodite duct ; 14, Albumin gland; 15, Sac-like diverticulum of the small
hermaphrodite duet ; lG, Small hermaphrodite duet ; 17, Gonad. X 2.5.

s. FOCAI~DK
and D. -
QUATTRISI Sfrttcfure of Ihe reproditctice uppurafus etc.
 PLATE I1

ilfilax gagates in reproduction (figs. 3-4) and imnicdiatcly after death (fig. 5).
Bouin ; haematosylin-cosin.
Fig. 3 - An oocytc in nictapliase of the 1st meiotic division, inside n gonad follicle.
x 500.
Pigs. 4, 5 - Oblique cross-sections of the small lierniapliroditc duct. Tlic duct is
packed witli sperms and contains an oocytc in fig. S(fig.4 x 2GO; fig. 5 x 130).
 P LATE 111
Milax gagales during reproduction.
Fig. G - Cross-scction of tlic sniall Iicrniapliroditc duct divcrticuluiii. I k l o ~ v ,riglit,
is n tubule of the albumin gland. Uouin ; PAS ; x OGO.
Fig. 7 - Cross-section of tlic large Iicrniapliroditc duct. ‘l%c arrow indicntcs tlic duct
spermatic furrow. Above, riglit, arc some prostntc tubulcs. Uouin ; PAS ;
x 40.
Pig. 8 - Cross-section of some of tlic albumin gland tubules. The tubule \valls arc w r y
thick, while the lunicn (indicated in some of tlic tubulcs b y arrows) is vcry
thin. Sanfelicc ; Iiaernatoxylin-cosin ; x ?GO.
 PLATE I\.
ilfilnx gngntrs during rcproduction. Bouin ; Iiacniatosylin-cosin.
Fig. 9 - Cross-section of tlic oviduct and dcfcrcns. x 50.
Fig. 10 - Cross-scction of tlic dcfcrcns. x 150.
Fig. 11 - Cross-section of tlic incdial portion of tlic copulatory sac. x 50.
Fig. If! - Cross-section of a bricf tract of tlic distal wall region of the copulatory sac.
x 400.
I

13

PLATE V
JZilaz gagates during reproduction. Bouin ; haematosylin-eosin.
Fig. 13 - Cross-section of the penis. Below, right, a section of the deferens. x 45.
Fig. 14 - Oblique cross-section of the epiphallus. x 50.
Fig. 15 - Part of a cross-section of the penis, more enlarged. X 130.
 PLATE VI
Milax gugafes during reproduction. Iiouin ; Iiaetiiatosylin-cosin ; X 110.
Fig. 16 - Oblique longitudinal section of the corniforni organ. The secretory canaliculi
of the vestibular gland are readily visible.
17 - Part of the vcstibular gland. To tlic lcft arc sonic of tlic gland’s cscrctory
canaliculi.
 PLATE VII
ilf ilax gagates.
Fig. 18 - Copulating animals. x 2.5.
Fig. 10 - A group of frcslily spawned eggs. x 5.
 .,-

' i
1i

2 31
r L A T E VIII
JIilax gagnfes. First cleavages pliotografed in albumen.
Fig. 20 - Oocyte a t the end of meiosis. x 180.
Fig. 21 - Embryo at first cleavage. x 400.
Fig. 22 - Embryo at second cleavage. x 100.
Fig. 23 - Embryo a t third cleavage. x 180.
In figs. 20, 21 and 23 the polar bodies are readily visible.
 . 25

P L A T E IS
J l i l u r gugufes. Various stages of development photograplied in albumen.
Fig. 2.4 - Four-five days after spawning. x 100.
Fig. 25 - Ten days after spawning. x 120.
Fig. 2G - Sixteen days after spawning. x 40.
a - Xutritivc sac ; b - Foot ; c - I'odocyst ; d - Jlantle.
 .. .. . .. ........ - r
-
. __
.
.. ... . .. ._ . .
~

~ I . , \ T E s
J I k z gagafes. Gonad sections from animals sacrificed during copulation (fig. 27), nfter
spawning the first eggs (fig.28) and inimediatdy aftcr death (fig. 29). Sanklire ; hae-
niatosylin-cosin ; x 40.
 17

-
Egg groups Oviposition date Number Days necessary
of eggs for hatching

1 10 /I 0/1970 15 . 30
2 27/10/1070 16 37
3 30/10/1970 14 38
4 6/11/1970 8 28
5 8/11 /lo70 15 20
6 9/11 11070 10 28
7 0/ll/l970 21 30
8 9/11/lS70 0 30
0 l o l l 1 /lo70 11 27
10 11 111 11070 20 20

Under our experimental conditions hatching occurs about 1 month

after oviposition. GERMAIN, in his B Faune de France D, recorded 55-40
days for the same species while a shorter period (20-25 days) was encoun-
tered by one of us in eggs deposed in the spring (QUATTRINI,1970). l n
great part the differences are doubtlessly due t o the different temperatures
of the experimental environment. Not all the animals of each group hatch
contemporaneously. The times reported above refer to tlic first born ani-
mals of each group ; there is usually a lapse of 4-5 days between the birth
of the first and last animal, a period of time that can greatly exceed that
between deposition of the first and last egg of each group (normally not more
than 10 hours). Aborted eggs sccm relatively rare; we found 2-3 a t most
per group.
At birth the animals are 4-5 mm long, in average extension, and still
almost transparent. The intensely dark pigmentation that distinguished
the adults (fig. 1)occurs much later and in degrees, above allafter the second
month of life. Reproduction begins a t the age of 4-5 months, alpost exclu-
sively in the autumn or spring. As GALANGAU(1964)revealed, not all the
animals reach gonad maturity a t the same time of the year and thus each
of their reproductive cycles can fall within varying periods of time.

NATING AND OVIPOSITION

ill. gugutes can reproduce itself in individual isolation for a t least

two generations (QUATTRINI,1970 ; ZANPI and QUATTRINI, 1971) but re- *
production normally takes place by mating. The mating animals assume
18

the position seen in fig. 18 and remain so for 30-40 hours (sometimes less,
rarely longer ; this factor w a s controlled in 10 couples with the following
results : 27-28-32-32-33-34-36-37-39-43 hrs). During mating illilnx' gn-
gufes remains almost immobile and neither eats nor emits the abundant
quantity of mucous that is seen in other Limaeidae. If disturbed they
separate and mill return to mating only after a certain period of time
which may be a few hours or some days ; otherwise, reproduction does not
seem to repeat itself.
I n order to observe the may whieli eggs are laid, 16 animals were
isolated in separate containers, immediately after mating, with the fol-
lowing results :

~~~ ~ ~~~~

Days from end Number of Length of

Animal of mating to Number of GTOUPS oviposition
beginning eggs laid of eggs in days
of oviposition

1 4 3G7 20 89
2 21 02 10 03
3 10 2 1 10
4 15 52 8 50 .
5 15 48 8 57
0 15 12 3 a0
7 5 39 3 10
8 4 140 11 50
9 14 211 15 75
10 5 242 21 90
11 a5 93 8 48
12 10 140 13 48
13 14 59 0 31
14 7 133 10 35
15 5 193 15 05
10 17 9G 9 45

The great variance between the data of each animal make any pre-
cise conclusions rather difiieult. The more notable differences are probably
due to thc fact that these are laboratory raised animals which do not
exactly repcat what occurs in nature. The following deductions, however,
seem possible :
. Oviposition never begins before the 4th day after mating, usually
occurring 5-15 days later and only very rarely, after a month. At least 4
 19

days arc ncccssary in order for tlie eggs to be fertilized and laid once the
sperm have penetrated the copulatory sac. As mating took place only once
in all our specimens, it is evident that the spermatozoa can survive and
function normally - prcsumably inside the same sac - u p until the end
of deposition, which can cndurc more than 3 months. The site of ferti-
lization has not yet been ascertained. It could take place inside tlic small
hermaphrodite duct, as is believed to occur in other pulmonate gastropods,
but we have never noted the presence of any sperm in thc fcw ovocytes
found there.
The total number of eggs laid, usually not more than a hundred,
can vary greatly. Also, notably differcnt quantities are sometimes laid by
two partners such as, for example, the couple formed by animals 3-4, one
of which laid 2 eggs and the other, 52. A similar occurrence was verified
in couples 7-8, 11-12 and 15-16. This factor is not always in rapport with
their differcnt life span and length of oviposition period. In some instances
one of tlic partners is probably self-fcrtilizcd, as would secm tlie case of
n. 8 ;its 2 anomalous eggs contained several oocytes, a phcnomenon verified
very rarely in normal rcproduction while very frequent in caws of sclf-
fertilization (QUATTRINI, 1970 ; ZAMPI and QUATTRINI, 1971).
The eggs, laid in little groups at varying depths in the soil of tlie
containers, are never united by the mucous masses or cords found in other
gastropods, for example in Veronicellidae (LAKZA and QUATTRINI,1964).
Each group rarely exceeds 20 eggs, is usually made up of about 15 and
can sometimes be much less. The various groups are usually laid from 4
to 40 days apart.
Oviposition is usually completed within a month or a month and a
half but can endure for 3 months. Notable individual differcnccs are cn-
countered, and between partners as well (compare the data relative t o num-
bers 3-4, 7-8, 11-12 and 15-16). Obviously, in many cascs the greater the
number of eggs laid the longer the period of oviposition will be but the
exceptions to this rule are not rare.

REIIAVIOR O F THE ANIMALS AFTER OVIPOSITION

After oviposition all the animals die. This fact, already noticcd
in a preceding work (QUATTRINI,1970), was observed in all our specimens
without exception. Precise observations were made on 20 animals after
their reproduction in the autumn, giving the following data :
20

Animal End of oviposition Dcatli Days between

1 30]in/i~70 1/11I1070 2
2 4/11/1070 13/12/1070 a0
3 28/11/1970 14/12/1070 16
4 2D/ll/l970 20/12/lD70 30
5 1/12/1070 S/ Ill071 38
0 2/12/1070 5 / 1/1071 34
7 G/12/1070 31/12/1070 25
8 0/12/1070 15/12/1070 G
0 0/12/1070 14/12/1070 5
10 11/12/1070 2G/12/1070 15
11 10/12/1070 2 8 / 1/1071 40
12 21/12/1U70 51 1/1071 15
13 24/11/1970 171 1/1071 24
14 2a/iqio7n l8/ 1/1071 28
15 2G/l2/1070 lo/ 1/1071 21
10 31/13/1070 4/ 2/107l 35
17 0 / Ill071 29/ 111071 20
18 13/ 111‘371 27/ Ill071 14
10 l3/ Ill071 a/ 2/1071 21
20 . 171 l/107l 51 “1071 10

The lifc cycle of 111. gugafes terminates with the cnd of oviposition,
in our experience a rule without any csceptions. Usually death occiirs
15-20 days after laying the last group of eggs but this pcriod can rcduce
itself to a couplc of days or esceed 8 month. On tlic wliole this species
of gastropod mollusk oKers a model of a bi-annual rcproductivc biological
cycle (autumn and spring) with a complete replacement of the animals
in that all the fecond individuals of each rcproductivc season nre the pro-
geny of tliosc of the preceding one.
According to our data tlie animals of the autumn cycle iisually begin
oviposition in October, reach a masimum in Novemnbcr, finish in Dcccinber
and dic the same month or in January.
The animals of the spring reproductivc pattern usually begin ovi-
position in March, reach a niaxiinum in April, end in May and die the
same month or in June.
Up until 2-3 days beforc their natural death the animal shows no
particular external signs of suffering. Then, ratlicr rapidly, they undergo
a marked dehydration, losing much of thcir volume and Iyeiglit (sometimes
more than half), their foot turns reddish in color, they grow thinner and
thinner and finally die in masimum extension, as wllcn they are sacrificcd
in dcaereated .water.
 21

Immediately following death, some animals were fixed and their

internal organs examined, particularly the gonad. I n all fivc specimens
studied the most notable alteration was presented by the liver whose
cells were abnormally pigmented. The gonad, still containing both male
and female germ cells, did not show any relevant changes. Howcver the
quantity of the female germinal cells is superior to that \generally found
in animals sacrificed during the course of reproduction while just the
opposite is true for the male germ cells (compare figs. 27, 28 and 20). Some
of these were oocytes in metaphase of the 1st meiotic division. The little
hermaphrodite duct was full'of sperm and a few oocytes (fig. 5 ) as well
as various other types of cells, some with LL voluminous nucleus rich in
chromatin like those described above for this organ but in much greater
quantity (compare figs. 4 and 5).
It is difficult to say wheter 31. gugates lias the same life cycle in na-
ture that is constantly repeated in the laboratory. That this possibility
exists seems to be indicated by the following observations :
I n zones continually controlled for this purpose me rarely found adult
animals in the late summer or winter while young specimens wcrc frequent
enough, as if all, or almost all, .the adults mere already dead.
The few adult specimens found in this period and brought back to
the laboratory all died without mating and only occasionally laid a very
limited number of eggs, iis if mating had long since occurred and ovipo-
sition had nearly terminated, analogous t o what is verified in the laboratory.

COKCLUSIOSS

The data on the anatomic conformation and structure of the re-

productive apparatus agree fundamentally with those already known from
the authors who first studied the problem. This consists of one gonad and
gonoducts, of the diaulic type, with various connected organs.
The gonad is made up of many hermaphrodite folliclcs which are at
their largest during the reproductive period. At that time each follicle
contains germ cells of both sexes which have matured as far as possible
within the gonad. An examination of this organ in sexually immature ani-
mals seems to indicate, as GALAKCAU revealed, that the femalc germ '

cells arc the first to appear. These latter detach themselves from the
gonad' walls at the oocyte stage' during metaphasc of the first meiotic
division, get into the small hermaphrodite gonodirct and complete the
final stages of meiosis only after being fertilized.
 22

The small hermaphrodite duct in adult specimens has an epithelial

lining which contains, next to common ciliate ones, a certain number of
morc voluminous poplyploid cells. Sometimes these arc present in the
samc duct which they seem t o reach after being detached from its mall.
They are very similar to the nurse cells of the male gonia, frequently found
in the folliclcs of the gonad. Inside thc small hermaphrodite duct, espc-
cially in the initial tract, groups of cells arc occasionally found that re-
semble various stages in the evolution of the male gonia. A similar picture
mas observed by AUBRYin Limiiaea stagnalis which suggests tliat this
site, commonly considered as sterile, may produce germ cells. This is ana-
logous, so i t seems, to what has been verified in other molluscan species
. in particular esperimental conditions, for example in Arim rtgus after re-
moval of the gonad (LAVIOLETTE). During the reproductive period the
small hermaphrodite duct is full OF sperms, except in a short distal tract
where they are rarc or absent. Near the junction with thc large hermaphro-
dite duct there is a minuscule diverticulum, doubtlessly homologous to
the socalled fertilization pocket in other species of Pulmonata, the esi-
stencc of mliicli has been confirmed or denied by various Authors (SIU-
ROTIT, TAVIANI).
The large hermaphrodite duct (distinguished by the presence of a
uterine and a spcrniatic furrow) receives the openings of the prostate and
albumin glands and numerous glandular cells, contained in the uterine
furrow mall, wliicli are destined, at least in part, t o elaborate the mucous
capsule covering the egg.
The oviduct contains scarse glands. Near the genital atrium is the
opening of the copulatory sac where thc spermatophores are deposited
during copulation.
The dcferens rarely contains sperms. There are no connected glan.
dular organs.
The penis prolongs into an epiphallus. Penis and epiphallus arc separa-
ted by a usually very evident constriction. The malc gonoduct terminates
in the epiphallus where the spcrmatophores are produced. These latter
seem to offer considerable protection to the spermatozoa as me have found
several of these still alive 8 days after fixing the animal in 10% formalin.
The corniform organ, a particular accessory structure of the sexual
apparatus of Jl. gagates, is formed of connective and abundant muscular
tissue. Present in only a few species of this genus of Gasteropods it is
contained in a pocket that opcns into the genital atrium. The organ is
crossed by the secrctory canals of about 40 simple glandular tubules that,
especially at the level of the adenomers are immersed in a thick connective
 23

layer and which form, altogether, tlie so-called vestibular gland. This gland
elaborates highly PAS-positive matkrial, apparently not a mucous se-
cretion (REXZONI) and the openings of its secretory canalicdi are in cor-
respondence with papillae situated in tlic eorniform organ.
The corniform organ and connected glands probably represent a
stimulative and excitatory apparatus with an essentially mechanical
function, auxiliary to copulation, such as is encountered in many other
Pulmonata, but with a structure among the more complcx a t least in the
group of the Limacidae. It lias been compared with the analogous appa-
ratus in T7eronicellidae (TAVIANI).
tit the momcnt of deposition tlic eggs are formed of a thick mass
of albumin covered by a mucous membrane diffused with calcite crystals.
Every egg contains an oocyte immersed in the internal mass of the al-
bumen and which is still in the metaphase stage of tlie first meiotic division.
It completes mciosis after 4-5 hours and begin the cleavagc. The extreme
transparency of the egg membranes make it very easy to follow all the
ulterior phases of development under the microscope.
Under our laboratory conditions the following results have becn ob-
tained :
1) 4 or 5 days after oviposition the nutritive sac - an embryonic
annex with a trophic function - is formed. Towards tlie l o t h day tlie
podocyst appears, another embryonic annex which is usually considered
in rapport with the circulation during the precocious stages of development
but which probably explicates other functions (gas eschange, secretion,
absorbtion of nutritive materials in the albumcn ; QUATTRINI& BANI).
2) 6 or 7 days after the beginning of the embryonic development
numerous crystals appear in the albumen which then gradually dissolve
so that, at hatching, no trace remains.
3) Upon hatching all the albumen appears to be reabsorbed, the
embryonic annexes have disappeared and only the mucous capsule of the
egg remains, though this is no longer diffused with the calcite crystals
which, thus have been utilized by the organism during development.
4) Usually the eggs hatch about a month after their deposition.
However this period of time can vary, above all in rapport with the tem-
perature of the environment where development occurs.
5 ) Reproduction generally begins 4-5 months after hatching and,
as the gonad is not in the same stage of maturity in all the animals at a
single period of the year, every reproductive cycle may fall within a va-
rying period of time. Though reproduction in individual isolation is pos-
24

sible (very probably by self-fertilization), in natural conditions this al-

ways occurs by reciprocal copulation.
0 ) Oviposition usually begins 15-20 days after the end of copulation
and continues for a month or a month and a half. The eggs are laid in little
groups (often 15 per group) at varying intervals of time. Generally the total
number doesn't exceed 100.
7 ) At the end of oviposition all the animals die, for the most part
within a month after laying the last group of eggs.
8) Jf. gugutes offers a model of a bi-annual reproductive cycle -
once in the spring and once in the autumn - with a complete replace-
ment of the animals in that the fecund animals of each reproductive season
are not those of the preceding one but rather their progeny.
9) Usually the oviposition of the animals that reproduce in the
spring begins in March, ends in May and the animals die this same month
or in June. I n the autumnal reproduction the first eggs are deposited in
October and the last in December and the animals die in December or in
January.
10) The natural death of the animals follows their extreme dehydra-
tion and a notable loss in volumc and weight, phenomena that seem to
initiate - or a t least are evident - only 2 or 3 days before death occurs.
At death the major part of the internal organs appear normal. Considera-
ble alterations are visible above all in the liver, whose cells'are abnormally
pigmented. The gonads do not prcscnt an anomalous picture of any im-
portance. This is still rich in both male and fernale germ cells in various
stages of gametogenesis. I n comparison to the gonads of animals sacrificed
during the period of sexual maturity the most important divergence is
represented by a greater quantity of female gonia. The hermaphrodite
duct is still packed with sperm and contains a few oocytes in the stage
of development that precedes fecondation.

SUMAIARY

The structure of the reproductive apparatus and life cycle of Jf. gagales liave
been studied in specimens collected in nature or raised in the laboratory. Tlic sexual ap-
paratus consists of a gonad, a small and a large hermaphrodite duct, deferens, oviduct,
copulatory sac, and the corniform organ (a complex stimulatory apparatus connected
to n vestibular gland).
The female germ cells break away from tlic gonad walls as oocytes in metapliasc
of the 1st meiotic division and remain in this stage until the fecondation.
 25

The small hermaphrodite duct of sexually mature specimens usually contains,

apart from a very large number of spermatozoa and some oocytes, various’other types
of cells. Some of these resemble the gonad nurse cells, others various stages of the male
germ cells, almost as if this site, generally considered as sterile, could produce germ cells.
A t deposition embedded in the albumen of each egg is a n oocyte still in me-
taplinsc of the 1st meiotic division, which then completes meiosis after a few hours and
begins cleavage.
The nutritive sac and podocyst (two embryonic ann-eses) arc formed during a pre-
cocious stage of development, then are gradually reabsorbed and completely disappear
before hatching.
Generally tlie eggs of laboratory raised animals (18-25%) hatch about a month
after spawning. They reach scsual maturity i n 4-5 months and usually reproduce b y co-
pulation. Spawning usually bcgins 15-20 days after copulation a n d endures a month
or a month and a half. At thc end of spawning t h e animals die.
Even after death tlie gonad is still rich in both malc and female germ cells in
various stagcs of gametogenesis. The female germ cells are more numerous here than in
the gonad of specimens sacrificed during tlic reproductive period.
I n our espcrimcntal conditions ill. gagates presents a bi-annual reproductive
pattern (spring and autumn) but with a complete substitution of thc animals in each cycle.

RIASSUNTO

e . s t a t a studiata la struttura dcll’apparato riproduttorc e il ciclo v i t d e di Nilax

gugales, in animali raccolti in natura o allcvati in laboratorio. L’apparato gcnitale consta
della gonade, dcl piccolo dotto errnnfrodito (con nnnesso un divcrticolo, omologo dells
cosiddetta tasca di fecondazione, prcsente in altrc specie di Gasteropodi), dcl grande
dotto ermafrodito (che riccvc gli sbocchi dclla prostata c della ghiandola dell’albumc),
dcl deferentc, dcll’ovidotto, della borsa copulatrice, del pcnc (che si prolunga distal-
mcnte in un epifallo) c di u n complesso apparato stimolatore, costituito da u n organ0
corniforme a1 quale f a capo una gliiandola vestibolare.
I gonociti femminili si staecano dalle pareti della gonade allo stadio di ovociti
nella metahse della prima divisione meiotica e in talc stadio rimangono bloccati fino
alla loro fecondazione.
Nel piccolo dotto crmafrodito degli animali scssualmcnte maturi, oltre a d un
grnndissimo nuniero di spermi e a qualche ovocita, si trovano non di rado anclic ccl-
lule di nltra natura, alcunc simili allc ccllulc nutrici dclla gonadc, altre che ricordano
vari stadi di evoluzione dei gonociti mascliili ; quasi clie anclie in qucsta sede, di norma
eonsiderata sterile, possa aver luogo una produzione di elemcnti germinali.
A1 momcnto della dcposizionc dellc uova, cntro l’albume E prcsentc un ovocita,
ancora nella metafase dclla prima divisione mciotica, clie dopo alcunc ore completa la
meiosi e inizia a segmcntarsi.
h’egli animali in via di sviluppo si forma u n sacco nutritivo e una podocisti, due
annessi embrionali elie vcngono poi gradatamentc riassorbiti c clie scompaiono alla schiusa
dellc uova.
Negli animali nllcvati in laboratorio, a tcmperatura ambicntc, fra i I8oC e i 2 P C ,
in generc le U O Y ~scliiudono dopo circa un mese dalla loro deposizione. Gli animali sono
 2G

sessualrncnte maturi n 4-5 tnesi di cth e, in condizioni normali, si riproducono solo pre-
vio rcciproco accoppiamento. Per lo pih la dcposizionc dclle uova h a inizio 15-20 giorni
dopo l'accoppiamento c si protrae per un mew - un mese e mezzo. A1 termine della
deposizione gli animali muoiono.
Subito dopo la morte, la gonade E aneora ricca di clcmenti germinali, sia ma-
scliili chc femminili, nei pih vari stadi d e l h gametogenesi. I gonociti femminili sono pih
nurnerosi di quelli presenti nella gonade degli animali sacrificati nel periodo della lor0
riproduzione.
11 Af. gagates, offre, almeno nellc nostre condizioni spcrimentali, u n modello di
ciclo biologico con due riproduzioni annuali, una a primavera e una in autunno; ma
con una completa sostituzione degli animali.

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 27

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