/. Embryol. exp. Morph. Vol. 30, 2, pp. 317-328, 1973
Printed in Great Britain
317
Genetic and physiologic control of fissioning
and sexuality in planarians
By MARIO GRASSO 1 AND MARIO BENAZZP
From the Irstitute of Zoology, University of Bologna, and the
Institute of Zoology, University of Pisa
SUMMARY
Several fissiparous strains of the planarian Dugesia gonocephala s.l. have been subjected
for many months to continuous feeding on crushed tissues of sexually mature specimens of
the planarians Polycelis nigra or (in a few cases) Dugesia lugubris. In a very high percentage
of cases (especially after feeding on Polycelis nigra crushed tissues) the fissioning phenomena
have been stopped and a sexual state has been induced. The sexualized specimens developed
hyperplasic ovaries, testicular follicles and a more or less complete copulatory apparatus;
some of them went so far as to lay sterile cocoons. As for the nature of sexualizing substances
contained in crushed tissues of sexual planarians, further researches are required; we suggest
they are neurosecretions stimulating sexuality and so inhibiting fissioning phenomena.
INTRODUCTION
Agamic reproduction by fission is a widespread phenomenon among many
freshwater triclads of the family Planariidae. In most species it consists of an
architomic mechanism ('architomy' means a fissioning phenomenon in
which the new organs of the dividing animals regenerate only after the fission
process is terminated) correlated with an asexual condition peculiar to the
fissiparous specimens. In fact, the genital pore is lacking in these animals and
histological examination shows that the genital apparatus is undeveloped,
although in some cases faint traces of gonads and copulatory organs are visible
(Benazzi, 1938, 1968).
Fissioning may enter the reproductive cycle in different ways. In some
planarian species there are either exclusively sexual or exclusively fissiparous
races, as well as races in which both sexual and fissiparous individuals may
coexist or alternate during the year.
The relationship between agamic reproduction and sexuality is therefore
rather complex and different interpretations have been advanced regarding the
importance of the genetic and environmental factors in determining these
phenomena. Benazzi's researches on several species of the 'Dugesia gonocephala
group' have shown that reproduction by fission, though influenced in its
rhythm by environmental conditions, depends primarily on genetic factors.
1
2
Author's address: Istituto di Zoologia, Via S. Giacomo 9, Bologna, Italy.
Author's address: Istituto di Zoologia, Via A. Volta 4, Pisa, Italy.
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M. GRASSO AND M. BENAZZI
These factors not only induce the reproduction by fission but they also inhibit
the development of the gonads and consequently of the copulatory apparatus.
In some fissiparous strains, however, a few specimens do occasionally become
sexual. They appear, in laboratory cultures, in the midst of many other specimens which, on the contrary, continue reproducing by fission. For this reason
any sexualizing influence among the more habitual environmental factors seems
to be excluded. Instead it is possible to suppose that the inhibiting action
normally exerted upon the development of sexual organs by the fission-controlling genetic factors, for unknown reasons, in some individuals is overcome at a
certain moment. The frequency of the appearance of these ex-fissiparous specimens, as well as their ability to lay fertile ovigerous capsules (cocoons), vary
greatly from strain to strain, maybe because the number or the penetrance of the
fission-controlling genes are different according to the various planarian populations. In the races or strains which reproduce exclusively by fission, the very
few individuals that become sexual are usually sterile; their ovaries are abnormally enlarged and therefore they occupy a very extensive area in the anterior
part of the animal body (Benazzi, 1968, 1973; Benazzi & Ball, 1972). This
anomalous structure of the ovaries was attributed to an increase in the number
of female germ cells. New investigations by Gremigni & Banchetti (19726) have
given experimental proof that ovarian hyperplasia originates above all through
an intense transformation of neoblasts into oogonia, which subsequently produce a very high number of oocytes. These oocytes, however, only rarely
succeed in completing their growth, as far as to leave the ovaries and become
enclosed in cocoons.
We are justified therefore in calling these anomalous structures 'hyperplasic
ovaries' with an abortive oogenesis. Their ultrastructure was recently studied
by Gremigni & Banchetti (1972a). The electron-microscope examination
showed that the oogonia and the youngest oocytes at the first stages of growth
keep normal, while the larger oocytes, which have reached the diplotene stage
and the beginning of the vitellogenesis, undergo a progressive nuclear vacuolization and an increase of the development of the lysosomal system, followed by
the destruction of the various cell structures. The vitellogenesis is present in the
oocytes of planarians, as was demonstrated by recent researches (Benazzi-Lentati
& Del Papa, 1965; Benazzi-Lentati & Gremigni, 1966; Gremigni, 1969a, b).
According to Gremigni & Banchetti (1912a, b), in the hyperplasic ovaries the
cause for the metabolic troubles culminating in the degeneration of almost all
the oocytes might reside in a blocking of their growth during the diplotene stage.
This blocking must take place very slowly: in fact, in every ovary many diplotenic
oocytes are recognizable at various progressive phases of their degeneration, but
the oocytes at an advanced phase of necrosis are very rare.
The male gonads of the ex-fissiparous specimens do not exhibit so anomalous
a structure, compared with the testes of normal sexual animals. Sometimes, in
fact, ripe sperms are present; in the main, however, the spermatogenesis has
Control of fissioning and sexuality in planar ians
319
stopped at the prophase of the first meiotic division, or even at the spermatogonium stage.
Obviously, therefore, the ex-fissiparous specimens having reached a sexual state
are not able to take part (or can only take part to a very limited extent) in the perpetuation of the strain to which they belong. This means they do not represent
a real sexual phase alternating with the fissioning ones in the reproductive
cycle; rather, they can be considered as an abnormal expression of the sexual
state. Benazzi believes that these features of an abortive sexuality are produced
by the same fission-controlling genetic factors we previously mentioned: even
when repressed, these factors would exert in this way their anti-sexual action.
To this genetic approach to the problem we may now add another, of a
physiological (perhaps endocrinological) nature. As communicated elsewhere in
a preliminary way, Grasso (1971) has been able to demonstrate that in fissiparous strains of Dugesia gonocephala s.l. a very high percentage of animals
attain the sexual state when fed on crushed tissues of sexually mature specimens
of Polycelis nigra - another planarian species which reproduces exclusively in a
sexual way.
In the present paper we intend to illustrate more extensively the results
obtained by Grasso, and also to expose further results basically corroborating
them, obtained by Benazzi on other fissiparous strains of the same species of
freshwater planarian.
Analogous attempts to induce sexuality by feeding planarians of fissiparous
strains on crushed tissues obtained from planarians of sexual strains had already
been made by Kenk (1941) and by Okugawa (1957), but had been unsuccessful.
Vandel's attempt (1921) to induce sexual state in agamic planarians by injecting
crushed tissues of sexually mature specimens into their parenchyma also gave a
negative result. Kenk (1941) and Okugawa (1957) had been able instead to induce
sexuality in planarians of fissiparous strains by grafting the cephalic anterior third
of sexual in dividuals on the posterior two thirds of the body of agamic individuals.
MATERIALS AND METHODS
For our experiments we used specimens of fissiparous strains of Dugesia
gonocephala originating from various localities; since all these races had already
been reared for a long period in our laboratories, it had already been ascertained
that their usual way of reproduction is the agamic one, with a rare or extremely
rare frequency of appearance of sexual individuals.
For each strain we studied, we established a comparison between the behaviour of control groups (which every week were fed either on live Tubifex,
beef liver, beef heart, or on crushed tissues of asexual specimens of the same
planarian fissiparous strain) and the behaviour of experimental groups, which
were nourished instead with a proportional quantity of crushed tissues of
sexually mature specimens of the planarian Polycelis nigra. Experiments were
320
M. GRASSO AND M. BENAZZI
also carried out with other groups that were fed on crushed tissues of sexually
mature specimens of Dugesia lugubris. The various control or experimental
groups were bred in separate containers placed in thermostatic cells at a
constant temperature of about 20 °C during periods of several months.
The planarians which were used for the histological investigation were killed
with a 5 % nitric acid solution, in order to obtain the utmost stretching out of
their body. They were then fixed in Serra's fluid (16 parts of ethyl alcohol, 8
parts of 40 % formol, 1 part of glacial acetic acid), paraffin-embedded and
cut in cross- or sagittal sections 8 /on thick. The sections were stained with
haemal um-eosin.
EXPERIMENTAL RESULTS
A first series of experiments was carried out by Grasso upon about 300
specimens of an agamic strain of Dugesia gonocephala originating from the
neighbourhood of Rome and bred for several years in the Institute of Zoology
of the University of Bologna, at a constant temperature of about 20 °C.
In all our control groups, either if normally fed on live Tubifex, beef liver,
beef heart, or if nourished with crushed tissues of asexual specimens of the
same fissiparous strain of Dugesia gonocephala, the animals continue to reproduce by fission, without any noticeable tendency to give rise to sexual
individuals. This situation drastically changes if the animals are nourished with
the crushed tissues of sexually mature specimens of Polycelis nigra. The specimens of Dugesia gonocephala, in fact, within about 2 months of treatment,
firstly reduce and later almost completely stop multiplying by fission; consequently their size increases to a great extent. In most cases this phenomenon is
followed by the appearance of the characteristic hyperplasic ovaries and of a
more or less developed copulatory apparatus often provided with genital pore;
these structure are visible also in vivo (Fig. 1 A, B).
Histological examination of these animals shows, in the retrocephalic region
of the planarian body, the typical structure of the hyperplasic ovaries, very rich
in oocytes which usually do not surpass a moderate stage of growth (Fig. 2 A,
B). At more caudal levels, a number of testicular nodes are generally also
present, in the main composed exclusively by spermatogonia (Fig. 2C, D). A
well-developed copulatory apparatus (Fig. 2E) is usually present in the
animals provided with testes. It is important to remark that in all these
animals which have attained a great size as a consequence of the stoppage
of agamic reproduction, and which are engaged in the morphogenesis of
the genital apparatus, the parenchyma always appears filled with neoblasts;
many of them seem to be migrating towards the region of neoformation of
gonads.
In many cases, after several months of treatment, a number of pedunculate
cocoons, typical of the species of the genus Dugesia, have been found stuck to
the walls of the containers in which the planarian experimental cultures were
Control of fissioning and sexuality in planarians
321
Fig. 1. Dorsal (A) and ventral (B) view of Dugesia gonocephala, for a comparison
between untreated and treated animals. The smaller specimen is an agamic untreated animal of a fissiparous strain; the larger one is an animal belonging to the same
fissiparous strain, sexualized by our feeding treatment, ov, Hyperplasic ovaries; gp,
genital pore, in which opens the copulatory apparatus;/?//, pharynx, x 6.
bred; but in our experiments these cocoons always turned out to be completely
sterile.
None of the above-described characteristics was ever found in specimens of
the control groups; they in fact went on reproducing regularly by fission and
kept their normal size without any trace of genital apparatus (Fig. 1 A, B).
A numerical valuation of the positive cases of induction of sexuality, obtained
in the aforesaid feeding experiments, may be reached keeping in mind that in
the experimental groups of planarians fed on crushed tissues of Polycelis nigra
(about 20 animals for each container), when fission occurred during the first
period of treatment the posterior pieces of the animals were eliminated from the
culture. The number of specimens under experiment therefore did not increase
with time. Under these conditions, after 5 months of treatment in almost 90 % of
the animals the induction of a sexual state was attained.
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M. GRASSO AND M. B E N A Z Z I
Fig. 2. From sexualized specimens of Dugesia gonocephala. A, Hyperplasic ovaries
(ov) in cross-section (x 140). B, Detail of hyperplasic ovary, showing oocytes
(arrow) stopped at a moderate stage of their growth (x 350). C, D, Cross-sections
of testes (0 rich in spermatogonia (x 350). E, Copulatory apparatus (c) in crosssection ( x 140).
In order to verify these findings, Benazzi has repeated the same feeding
treatment upon more than 100 specimens of other fissiparous strains of Dugesia
gonocephala taken from his rich and varied cultures originating from several
localities and bred in the Institute of Zoology of the University of Pisa for a
Control of fissioning and sexuality in planarians
323
period of many years, during which they showed different degrees of tendency
towards a spontaneous appearance of the sexual state. In order to avoid
exhausting such a precious collection of animals only a few individuals were
subjected to the feeding treatment and the posterior pieces of the animals were
not eliminated each time fission occurred as in Grasso's experiments. The
number of specimens under experiment therefore increased with time. This
undoubtedly resulted in a diminished percentage of responses to the treatment,
because the specimens recently born by fission are slower than the original ones
in assuming a sexual state. It is therefore highly significant to point out that even
in these conditions the nourishment with crushed tissues of sexually mature
specimens ofPolycelis nigra always gave rise to a more or less marked inhibition
of fissioning phenomena, followed by a more or less frequent acquisition of
sexuality.
Among the various fissiparous strains which underwent treatment, the specimens originating from Marettimo (Egadi islands) originally were 5 and later
became 10 after fissioning; 2 of them attained the sexual state within 70 days of
treatment. Among the specimens originating from San Nicholas (Gran Canada,
Canary islands), originally 10 and then 21 as a result of fissioning, 6 reached the
sexual state after 65 days of treatment. A higher degree of sexualization was
achieved by a strain from Camogli (Ligurian Riviera) and by one from Valsequillo (Gran Canada): as to the former, of 10 original specimens which became
52 through fission, 12 became sexual within 182 days; as to the latter, of 5
original specimens which became 8 through fission, 4 became sexual within 141
days. The highest tendency to undergo sexual induction was found in strains
from Meudon (environs of Paris), from the River Jordan (Israel), and from
Ponza island (Tyrrhenian Sea): as to the former, the 5 original specimens never
reproduced by fission, and within 68 days they had all attained the sexual state;
as to the second, the 5 original specimens became 6 after the fission of one of
them, then all became sexual within 127 days; as to the latter, the 10 original
specimens became 16 through fission, then all became sexual within 116 days of
treatment.
There seems to exist, therefore, differences between the various fissiparous
strains, not only as regards the percentage of specimens which respond to the
treatment by reducing or eliminating the processes of reproduction by fission
and acquiring a sexual state, but also as regards the length of time which is
necessary for the starting of these phenomena. It is to be noted that, among the
fissiparous strains we used, there are differences also as regards their natural
tendency to show phenomena of spontaneous sexualization: this tendency is
lacking in the San Nicholas, Camogli and Meudon strains, scarce in the Marettimo strain and comparatively strong in the Ponza strain. Perhaps a correlation
exists between the natural tendency of the various fissiparous strains to show
spontaneous sexualization and their more or less strong ability to attain the
experimentally induced sexual state.
324
M. GRASSO AND M. BENAZZI
Altogether, of the 118 specimens used in Benazzi's experiments, 51 became
sexual. If we consider that, as previously pointed out, in this experimental
series the feeding treatment was applied also to animals recently born by
fission, scarcely sensitive to the sex-inducing influence, the percentage of
positive responses, computable around 43 %, seems to be close to the about
90 % value attained in the above-described Grasso's experiments.
Two of the above-mentioned fissiparous strains of Dugesia gonocephala (the
one from Camogli and the one from Marettimo island) were also submitted, by
Benazzi, to the effects of a feeding consisting of crushed tissues of sexually
mature specimens of Dugesia lugubris. The induced sexualization in this case
turned out to be scarcer and slower than in treatments with crushed tissues of
Polycelis nigra. In the Camogli strain, of 5 original specimens which later became
13 through fission, only 3 became sexual within 110 days of treatment; in the
Marettimo strain, of the 5 specimens existing at the beginning, which later
became 7, only 2 became sexual within 112 days. Collectively, therefore, a
sexualization took place in about 25 % of cases. It is to be noted that the two
strains used have little or no natural tendency towards a spontaneous sexualization.
Our experiments are now going on, with the aim of determining the degree of
stability of the sexual state induced by our feeding treatment. At the moment
our observations concern only the fissiparous strain of Dugesia gonocephala
originating from the neighbourhood of Rome (Italy), reared by Grasso in
Bologna. In the experimental groups of this strain, the feeding on crushed
tissues of sexually mature specimens of Polycelis nigra was stopped after 5
months of treatment, and substituted by a normal feeding on live specimens of
Tubifex. Throughout the next 6 months of this normal feeding none of the
numerous planarians, which had become sexual during the previous period of
experimental feeding, has resumed reproduction by fissioning; their genital
apparatus remained present. Only at the seventh month of normal feeding did
a few previously sexualized specimens begin again, little by little, to reproduce
by fission. Their study is yet in progress.
This study is still in progress, using fissiparous strains of other planarian
species.
CONCLUSIONS
The findings we have illustrated above prove that a feeding consisting of
crushed tissues of sexual specimens of the planarian Polycelis nigra, and also (in
a more limited fashion) of the planarian Dugesia lugubris is capable of stopping
the fissioning processes and of inducing the development of the genital apparatus
in asexual specimens of fissiparous strains of another planarian species, Dugesia
gonocephala s.l.
After an increase in size of the animals, due to a cessation of fission, the first
sign of the induced sexualization (detectable even by an observation in vivo) is
Control of fissioning and sexuality in planarians
325
the appearance of ovaries which very rapidly grow and very soon assume the
characteristics of hyperplasic ovaries. In some individuals the sexualization
process may stop at this stage and be followed later by a return to multiplication
by fission; but in most cases the sexualization process proceeds with the appearance of testes, more or less numerous and more or less mature, and with the
development of a more or less complete copulatory apparatus, often provided
with a genital pore. Copulatory apparatus and genital pore are easily detectable
by observation in vivo. At the histological examination, the sexualized specimens
show characteristics very similar to those of the ex-fissiparous animals with
hyperplasic ovaries, which more or less rarely may spontaneously appear in
several fissiparous strains.
The specimens of Dugesia gonocephala which had become sexual after feeding
on Polycelis nigra crushed tissues maintained the sexual state for a long period,
even if such feeding was stopped and replaced by a normal nourishment. However, the sexualized individuals always proved to be completely sterile. Several
specimens went so far as to lay ovigerous capsules (cocoons), but until now
none of them have hatched young planarians. From this point of view, too, the
ex-fissiparous specimens whose sexualization was induced by feeding behave
exactly like the ex-fissiparous individuals with hyperplasic ovaries which, as
stated above, seldom appear spontaneously in agamic strains.
A physiogenetical interpretation of these facts may be suggested. The substances endowed with the power of inducing sexuality, which must be contained
in the crushed tissues of sexually mature specimens of planarians, obviously
cannot modify the genetic condition of the fissiparous specimens subjected to
the experimental feeding; clearly, they can only stimulate the expression of the
genes responsible for the developing of genital apparatus, probably after having
inhibited the expression of genetic factors responsible for the fissioning phenomena. As the histologic examination plainly demonstrates, the induced development of gonads and other parts of genital apparatus widely depends on activation of neoblasts.
As regards the origin and the chemical nature of the sexualizing substances
contained in the crushed tissues, further researches are required. For the present,
we suggest a working hypothesis according to which these substances might be
neurohormones. The abundance of neurosecretory cells in the very primitive
cordonal nervous system of planarians is now widely demonstrated (Lender &
Klein, 1961, 1962; Vendrix, 1963; Lender, 1964; Ude, 1964; Morita & Best,
1965; Grasso, \965a,b, 1966; Bondi & Pascolini, 1966; Kaplonska, 1967;
Liotti & Rosi, 1968; Lender & Zghal, 1969; Scharrer & Weitzman, 1970;
Grasso & Quaglia, 1970 a, b, 1971) and it has been correlated to\arious physiological and morphogenetic activities, as regeneration, agamic reproduction by
fission, ripening of genital apparatus, and probably also osmoregulation. It is
worth mentioning that in particular the relationship between neurosecretion
and gonad maturation is in full agreement witrTprevious experimental data,
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M. GRASSO AND M. BENAZZI
firstly obtained by Grasso (1959, 1963, 1964) and later confirmed by FedeckaBruner (1967, 1968) and especially by Teshirogi & Fujiwara (1970), which
definitely prove the importance of integrity of cerebral plexuses ('brain') and
posterior ventral nerve cords for development, ripening and preservation of
gonads and whole hermaphroditic genital apparatus in freshwater planarians.
More recently it has been also demonstrated (Budini, Grasso & Vannini,
1972) that the genital apparatus undergoes a rapid and complete regression in
planarians subjected to a treatment with LSD (lysergic acid diethylamide): this
drug acts upon the nervous system and probably interferes with the production
(or with the release within the tissues) of those neurosecretions which are
responsible for the development, ripening and preservation of the hermaphroditic genital apparatus.
Neurosecretory activities are present both in the sexual and in the fissiparous
strains of planarians. It seems reasonable therefore to suppose the existence of a
variety of neurosecretions endowed with different roles in the planarian life.
Probably the induction of sexuality, obtained in our feeding experiments,
depends upon neurohormones different from the ones which regulate other
physiological activities, such as fissioning, regeneration, and so forth. Also the
hormone produced by the testes, responsible for the development of the copulatory apparatus (Fedecka-Bruner, 1961), is probably different from the
neurosecretion responsible for the gonad maturation.
Some of our experiments suggest that the relative abundance of the substances
inducing sexuality might be different among the various species of planarians
endowed with sexual reproduction. The crushed tissues obtained from sexually
mature specimens ofPolycelis nigra proved in fact to be more effective than the
ones obtained from sexually mature specimens of Dugesia lugubris. Further
feeding experiments on crushed tissues obtained from sexual strains of other
planarian species are planned.
Another problem requiring further study is that of the different sensitivity
which seems to exist among the various planarian fissiparous strains in regard
to our feeding experiments. This problem will be dealt with in a further paper
dealing with the results of feeding experiments on fissiparous strains of other
planarian species, different from Dugesia gonocephala.
Probably the sexualizing substances, administered as food in our experiments,
exert their action by interfering with the genetic factors which in the agamic
planarian strains are responsible both for the inhibition of development
of genital apparatus and for activation of fissioning phenomena. If this is true,
it might be possible to suggest that the results obtained in our feeding experiments would derive, on the whole, from a resultant of two antagonistic forces:
the hereditary characteristics, which inhibit the sexual state and induce reproduction by fission, and the substances contained in the crushed tissue, which stimulates the development of genital apparatus and inhibit fission.
Control of fissioning and sexuality in planarians
327
RIASSUNTO
Un certo numero di ceppi agami della planaria Dugesia gonocephala s.l. e stato sottoposto
continuativamente per molti mesi ad una nutrizione consistente esclusivamente di una
poltiglia preparata sminuzzando esemplari sessualmente maturi delle planarie Polycelis nigra
o (piu raramente) Dugesia lugubris. In un'alta percentuale di casi (specialmente dopo trattamento con poltiglia di Polycelis nigra) si e arrestata la riproduzione per schizogenesi ed e
stato indotto lo sviluppo dell'apparato genitale ermafrodita. Gli esemplari sessualizzati
presentano ovari iperplasici, noduli testicolari e un apparato copulatore piu o meno completo; alcuni di essi hanno deposto capsule ovigere, risultate sterili. La natura delle sostanze
sessualizzanti contenute nella poltiglia ricavata da planarie sessuate e finora ignota; in attesa
di ulteriori ricerche, possiamo prospettare la possibility che si tratti di un neurosecreto
stimolante la sessualita e percio inibente i processi di schizogonia.
The present work has been financed by the Consiglio Nazionale delle Ricerche (Italy), to
which we extend our warm thanks.
REFERENCES
BENAZZI, M. (1938). Ricerche sulla riproduzione delle Planarie Tricladi Paludicole con
particolare riguardo alia moltiplicazione asessuale. Memorie Accad. Lincei (ser. VI), 7,
33-84.
BENAZZI, M. (1968). Ulteriori ricerche sul controllo genetico della scissiparita nelle planarie.
Boll. Zool. 35, 424.
BENAZZI, M. (1973). Fissioning in planarians from a genetic stand-point. In Biology of the
Turbellaria, Libbie H. Hyman Memorial Volume. New York: McGraw-Hill Book Company.
BENAZZI, M. & BALL, I. R. (1972). The reproductive apparatus of sexual specimens from
fissiparous populations of Fonticola morgani (Tricladida, Paludicola). Can. J. Zool. 50,
703-704.
BENAZZI-LENTATI, G. & DEL PAPA, R. (1965). Prime osservazioni su inclusi paraplasmatici
in ovociti della planaria Dugesia lugubris. R. C. Accad. Lincei 38, 945-947.
BENAZZI-LENTATI, G. & GREMIGNI, V. (1966). Ulteriori studi morfologici ed istochimici
sugli ovociti della planaria Dugesia lugubris. Atti Soc. Toscana Sc. Nat. B 72, 101-105.
BONDI, C. & PASCOLINI, R. (1966). Osservazioni sulla rigenerazione in Dugesia lugubris e
suoi rapporti con la neurosecrezione. Acta Medica Romana 4, 1-6.
BUDINI, R., GRASSO, M. & VANNINI, M. L. (1972). Regressione dell'apparato genitale in
Dugesia lugubris per effetto di LSD (dietilamide delFacido lisergico). Atti. Accad. Sci. 1st.
Bologna, ser. XII, 9, 74-80.
FEDECKA-BRUNER, B. (1961). La regeneration de l'appareil copulateur chez la planaire
Dugesia lugubris. Archs Anat. microsc. Morph. exp. 50, 221-231.
FEDECKA-BRUNER, B. (1967). Fvtudes sur la regeneration des organes genitaux chez la planaire
Dugesia lugubris. I. Regeneration des testicules apres destruction. Bull. biol. Fr. Belg.
101, 255-319.
FEDECKA-BRUNER, B. (1968). fitudes sur la regeneration des organes genitaux chez la
planaire Dugesia lubugris. II. Regeneration de l'appareil copulateur. Bull. biol. Fr. Belg.
102, 3-44.
GRASSO, M. (1959). Fenomeni rigenerativi e apparato genitale in Dugesia lugubris. Boll. Zool.
26, 523-526.
GRASSO, M. (1963). L'organizzazione dell'apparato genitale ermafrodita in esemplari
rigenerati e in esemplari bicefali di Dugesia lugubris. R. C. Accad. Lincei 35,101-104.
GRASSO, M. (1964). Rigenerazione cefalica e apparato genitale in Polycelis nigra. R. C.
Accad. Lincei 37, 203-206.
GRASSO, M. (1965a). Presenza e distribuzione delle cellule neurosecretrici in Dugesia lugubris.
Monitore zool. ital. 73, 182-187.
GRASSO, M. (19656). Dimostrazione di cellule neurosecretici in Dugesia tigrina. R. C. Accad.
Lincei 38, 712-714.
328
M. GRASSO AND M. BENAZZI
M. (1966). Rapporti fra sistema nervoso, gonadi e neurosecrezione in Polycelis
nigra. Riv. Biol. 59, 157-172.
GRASSO, M. (1971). Esperimenti sul controllo della maturazione sessuale in ceppi agami di
planarie. Boll. Zool. 38, 532.
GRASSO, M. & QUAGLIA, A. (1970a). Studies on neurosecretion in planarians. I. Neurosecretory fibres near testes on Dugesia lugubris. J. submicr. Cytol. 2, 119-125.
GRASSO, M. & QUAGLIA, A. (19706). Studies on neurosecretion in planarians. II. Observations on the ovaries of Dugesia lugubris. J. submicr. Cytol. 2, 127-132.
GRASSO, M. & QUAGLIA, A. (1971). Studies on neurosecretion in planarians. III. Neurosecretory fibres near the testes and ovaries of Polycelis nigra. J. submicr. Cytol. 3, 171-180.
GREMIGNI, V. (1969a). Ricerche istochimiche e ultrastrutturali sull'ovogenesi dei Tricladi.
I. Inclusi deutoplasmatici in Dugesia lugubris e Dugesia benazzii. R. C. Accad. Lincei
47, 101-108.
GREMiGNr, V. (19696). Origine del vitello negli ovociti di Dugesia lugubris e D. benazzii
(Tricladida, Paludicola). Atti Vllth Congr. Ital. Micr. Elettr., Modena, pp. 68-70.
GREMiGNr, V. & BANCHETTI, R. (1972a). Submicroscopic morphology of hyperplasic ovaries
of ex-fissiparous individuals in Dugesia gonocephala s.l. R. C. Accad. Lincei 52, 539-543.
GREMiGNr, V. & BANCHETTr, R. (19726). The origin of hyperplasia in the ovaries of exfissiparous specimens of Dugesia gonocephala s.l. R. C. Accad. Lincei (in the Press).
KAPLONSKA, J. (1967). Neurosecretion in Tricladida with reference to seasonal variations and
changes occurring in the course of regeneration. Zoological Pol. 17, 73-95.
KENK, R. (1941). Induction of sexuality in the asexual foim of Dugesia tigrina. J. exp. Zool.
87, 55-69.
LENDER, T. (1964). Mise en evidence et role de la neurosecretion chez les planaires d'eau
douce (Turbellaries, Triclades). Annl Endocr. Paris 25, 61-65.
LENDER, T. & KLEEN, N. (1961). Mise en evidence de cellules secretrices dans le cerveau de la
planaire Polycelis nigra. Variation de leur nombre au cours de la regeneration posterieure.
C. r. hebd. Seanc. Acad. Sci., Paris 253, 331-333.
LENDER, T. & KLEIN, N. (1962). Les cellules neurosecretrices de Dendrocoelum lacteum. Bull.
Soc. zool. Fr. 87, 380-381.
LENDER, T. & ZGHAL, F. (1969). Influence des conditions d'elevage et de la neurosecretion
sur les rythmes de scissiparite de la race asexuee de Dugesia gonocephala. Annls Embryol.
Morph. 2, 379-385.
LIOTTI, F. S. & Rosi, G. (1968). Osservazioni sui diversi stadi del ciclo neurosecretorio di
Dugesia lugubris. Riv. Biol. 61, 419-432.
MORITA, M. & BEST, J. B. (1965). Electron microscopic studies on Planaria. II. Fine structure
of the neurosecretory system in the planarian Dugesia dorotocephala. J. ultrastruct. Res.
13, 396-408.
OKUGAWA, K. J. (1957). An experimental study of sexual induction in the asexual form of
Japanese fresh-water planarian, Dugesia lugubris (Duges). Bull. Kyoto Gakugei Univ. B
II, 8-27.
SCHARRER, B. & WEITZMAN, M. (1970). Current problems in invertebrate neurosecretion. In
Aspects of Neuroendocrinology, pp. 1-2. Berlin: Springer.
TESHIROGI, W. & FUJIWARA, H. (1970). Some experiments on regression and differentiation
of genital organs in a fresh-water planarian, Bdellocephala brunnea. Scient. Rep. Hirosaki
Univ. 17, 38-49.
UDE, J. (1964). Untersuchungen zur Neurosekretion bei Dendrocoelum lacteum Oerst.
(Plathelminthes-Turbellaria). Z. wiss. Zool. 170, 223-255.
VANDEL, A. (1921). Recherches experimentales sur les modes de reproduction des planaires
triclades paludicoles. Bull. biol. Fr. Belg. 55, 343-518.
VENDRIX, J. J. (1963). Existence de cellules neurosecretrices chez Polycelis nigra Ehrenberg
et Dugesia gonocephala Duges (Triclades, Paludicoles). Caracteristiques cytologiques et
histochimiques. Bull. Soc. Sc. Liege 32, 293-303.
GRASSO,
{Received 9 November 1972, revised 8 March 1973)