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Observations on the ShellFormation in the Cocoon ofDugesia Lugubris s.lMarinella Marinelli aa Institute of Comparative Anatomy of the Universityof Perugia, ItalyPublished online: 12 Nov 2009.

To cite this article: Marinella Marinelli (1972) Observations on the Shell Formationin the Cocoon of Dugesia Lugubris s.l, Bolletino di zoologia, 39:3, 337-341, DOI:10.1080/11250007209432042

To link to this article: http://dx.doi.org/10.1080/11250007209432042

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Boll. ZOO^., 39: 337-841, 1072

OBSERVATIONS ON THE SHELL FORMATION IN THE COCOON OF DUGESIA LUGUBRIS s.1.

MARINELLA n u R m L L r Institute of Comparative Anatomy of the .University of Perugia (Italy)

(received April 26, 2972)

INTRODUCTION

Alike all the triclads, Dugesia lugubris s.1. lays cocoons containing one or more oocytes and a number of yolk cells. The outer shell is con- siderably tough. The cocoon is spherical and its diameter ranges from 0.8 to 2 mm; it is supplied with a peduncle exhibiting a discoidal end with ,which it sticks to the substrate.

At laying, the cocoon varies from yellow to orange in colour ; within one day it gradually grows harder and darker until it assumes its definitive dark-brown hue.

I ts shell consists of two different proteins, e.g. sclerotizcd and ke- ratinized ones (MINOANTI, 1957).

I n the flatworms sclerotized proteins arise through a step-wise process which unfolds as follows. Diphenol granules are oxidized to quinone by a polyphenoloxidase. The quinone becomes linked to the side amino groups of the protcin, whereby sclerotin is produced (NURSE, 1950 ; SYYTH, 1954).

VIALLI (1038), GERZELI and GERZELI PEDRAZZI (19G5) have studied the sequence of these phenomena in some triclad species (Dendrocoelutn Incteum, Polycelis nigra, Planaria torva, Dugesia Iugubris) bringing to light a diffuse polyphenoloxidase specific activity in the cytoplasm of the yolk cells. Evidence was provided by them of the presence of polyphcnol and oxindol granules and of a polyphenoloxidase system.

I n addition it was-noticed by the same workers that these materials are then carried by the yolk cells surrounding the oocytes as these, once shed from the ovaries travel down along the oviducts, and that on their being extruded, formation of thc shell sets in.

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The present observations in Dugesia lttgubris s.1. shorn that the material secreted by thc yolk cells contained in the cocoon contribute to a consi- derable extent to the shell formation.

MATERIAL AND METHODS

Dugesia fugubris s.1. species from the Bettona region (Pcrugia, Italy) which I have raised in the laboratory for a long time yere used.

Histological slides were prepared from animals in which the cocoon formation is at its beginning, which is indicated by the presence of a bulge in the ventral region, near the genital pore.

The Bouin-fixed matcrial was stained by the Mnsson method with silver-amine salts, which is speciflc for argcntaffine substances, polyphcnols included (LISON, 1060).

RESULTS

h the cocoon is being formed, it is contained in the genital antrum, and does not exhibit a spherical shape because it protrudes into the cavity of the antrum thc male copulatory organ (fig. 1). It mill become spherical at the:moment of its being shed, thanks to the shell plasticity.

Shortly after the oocytes and yolk cells havc gone down into the gcnital antrum, the forming shell, heavily stained by ammonia silver salts, is considerably variable in thickness (from a very few to about 35 micra). The shell thicker zones lie mostly adjacent to the opening of the genital antrum, whereas the thinner oncs are prevalently located in thc opposite zones, adhering to the antrum walls. I n histological sections, moreover, thc thicker zones which as a rule lie against the antrum wall, display a rather regular fragmented pattern made up of more or less squareshaped units. This fragmentation is likely to be due to sectioning of the material. .However, mention is deserved by the fact that thc fracture lines ge- nerally coincide with the boundaries of the underlying yolk cells (fig. 2).

The thinner zones never look fragmented. At this moment no uniformity in the cytoplasmic argentaffin granule

population is displayed by the cells contained in the cocoon. I n particular, it is possible to identify some territories where these granules are scanty while the intercellular spaces are extremely rich in argcntafh material. Generally, these territories are encountered near the thinner regions of the shell which at some points appear to be continuous with thc interccl- lular material (fig. 3).

Conversely, next to the thicker shell regions the yolk cells appear

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P L A T E 1

I\LinIsELLii JI,innm.LI - Observations on the shell formation, etc.

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ID 1. A T E I

Fig. 1 - Dirgesin loigitbris s.1. frontal scction. Cocoon within the riialc genital antrum. (p), ninlc copulntory organ. x 45.

Fig. 2 - Pragmcntation of the thickest slicll zonc. X 400. Fig. 3 - Plentiful argentamn nintcrial in thc intcrccllular spnccs ndjaccnt to a thin

Fig. 4 - Inncrniost cocoon yolk cclls with abundant cytoplasmic argcntaffin ninterinl. slicll zone. x 200.

Intcrccllular spaces devoid of argcntaflin mntcrial. x 480.

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P L A T E 11

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1’ 1. A T E I1

Fig. 5 - Estrcincly scanty argcntanin niatcri:il bctwccn the pcriplicral yolk cells in n ncwhid cocoon. X 200.

Fig. 0 - Cocoon one day after deposition. Absence of intcrccllular argcntallin niaterial. x 107.

Fig. 7 - Ccnicnt glands surrounding both the comnion genital antruni (age) and the lower portion of the nialc genital antruni (agni). x 70. In the insert : detail of tlic ccrncnt glands. x 020.

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830

dcvoid of granules, and the argentaffin material is abscnt from the inter- cellular spaces (fig. 2).

Furthermore, in some innermost cocoon regions the yolk cells may be crammed with grrrnules while granule-free intercellular spaces are found (fig. 4).

I n the newlaid cocoon the sheII exhibits a uniform thickness (somc 85 micra). In this case i t was also noticed that i t shows the tendency to get fragmented, but, unlike the shell in the making, fragmentation no longer occurs evenly. In the cocoon at this stage the yolk cells have a very uniform appearance. They no longer contain cytoplasmic granules and the intercellular material is missing, save for faint traces in the outermost cocoon portion (fig. 5).

Later, also this more peripheral intercellular material gets lost (fig. 6). I wish to recall here that when shedding of the cocoon occurs, the

cement glands surrounding the genital antrum and the lower portion of the male genital antrum (fig. 7 ) make up the peduncle and a thin sheath around the cocoon (MEISNER, 1928).

CONCLUSIONS

The above reported observations seem to provide evidence that also the yolk cells contained in the cocoon are definitely involved in the shell formation.

This process would unfold as follows. In the early developmental stages of the cocoon, when this is still contained in the genital antrum, the di- phenol-rich granules of the yolk cells are released into the intercellular spaces and migrate towards the shell, thereby increasing its thickness to completion.

The earliest yolk cells involved in this process arc the most peripheral ones, starting by those lying in the cocoon zonc nearer the antrum opening. I n fact, this is the earliest shell zone to attain its definitive thickness (35 p).

Thereafter also the yolk cells lying in the cocoon central zone take part in the shell construction, gradually releasing their granules into the intercellular spaces. These granules are also going to diffuse outward to the periphcry. Such process docs not appear to be achieved yct, when the cocoon is laid.

I n the following days, as sclerotization is fully attained, the shell takes on its definitive colour and toughness. Analogously to what was demonstrated for the egg envelopes in other flatworms (Smmr, 1954 ; GERZELI, 1908) I would suggest that also in Dugesia Zugtibris the sclero-

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tization process is likely to depend upon the action of a polyphenoloxidase. It is my purpose to further my investigations on this subject by more ri- gorous methods.

As to the appcarance of a regularly p-atterned fragmentation of the cocoon shell when it is still within the genital antrum (fragmentation which is likely to be due to the mechanical action of the microtome blade), one might speculate that it is an indication that the hardening promoted by polyphenoloxidme activity would occur not uniformly-initially a t least- a t the level of discrete yolk cells adjacent to the shell, For this reason the fracture lines would come to correspond to the intercellular zones.

The formation of Dugesia tugubris s.1. cocoon, still contained in the genital an- trum, is described from optical microscope observations. It is concluded that the yolk cells inside the cocoon contribute sensibly to the formation of the cocoon-shcll. I n fact, it is observed that the diphenol granules and the polyphcnoloxidase system, forming part of these yolk cells, are aet free in the intercellular spaces and migrate towards the forming cocoon-shell.

The first yolk cells to eliminate diphenol granules are the more pcripheral ones, followed by the 'central ones.

The proccss having begun in the genital antrum, becomes complete after the deposition with a find darkening and hardening of the cocoon.

RIASSUNTO

Vengono riferite osservazioni a1 microscopio ottico d a formazione del bozzolo in Dugesia lugubris s.1.. quando esso t ancora contenuto nell'atrio genitale. Si conclude che le cellule vitelline interne a1 bozzolo contribuiscono notevolmente alla formazione del guscio. Infatti si osserva che i granuli contenenti difenoli c il sistema polifenolossi- dasico, contenuti in dette cellule vitelline, vengono liberati negli spazi interccllulari e mipano verso il guscio in formazione.

I primi elementi che eliminano i granuli contenenti difenoli 8ono i pih periferici; successivamente anche quelli centrali riversano il lor0 contenuto negli spazi intercellulari.

I1 processo, iniziato nell'atrio genitde, si completa dopo la deposizione con il' deanitivo imbrunimento e indurimento del bo&olo.

REFERENCES

GERZEU G. e GERZELI PEDRAZZI G., 1005 - Aspelti istomorjologici e istochimid della dif- jerenziazione dei uiletlogeni e della jormmione del bonolo netle ptanarie. Arch. Zool. it., so: 1-19.

GERZELI G., 1908 - Aspetti istochimici della formmione degli incolum' ovulari in Aspi-

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dogaster conchicoln Von Baer (Trematoda). Istituto Lombard0 (Rend. Sc.), B 100 : 208-210.

LISON L., 1000 - Ilistochim'ie el cytochimie animales. Gauthier-Villards Ed., Paris, vol. 20, pp. 724-720.

MEIXNER J., 1028 - Der Genitalapparaf der Tricladen und seine Beziehungen zu ihrer allgemeinen Morphologie, Philogenie, t%ologie und Verbrcifung. Zeitschrift f r Morphologie und C)kologie der Tiere, 11 : 670-612.

BIlNGAN'M A,, 195'7 - Sulla costituzione chimica degli involveti ovulan' negli Animali. Boll. Zool., 25: 65-88.

NURSE F. R., 1950 - Quinonc Tanning i n the Cocoon-Shell of Dendrocoelum lacteum. Nature, 165: 570.

Sxma J. D., 1954 - A technique for fhe histochernfcal demonsfration of polyphenolariduse and its application to egg-shell formalion in hrlminths and bysms formation in Mytilus. Quart. J. Micr. Sci., 93: 189-162.

V r m M., 1083 - Ricerchc isfochimichc sui vitellogeni dei Platelminti. Boll. Zool., 4: 185-188.

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