SCANNING ELECTRON MICROSCOPE STUDY OF EGGSHELL DEVELOPMENT IN CANCRIFORMIS (BOSC) (CRUSTACEA, NOTOSTRACA) S Tommasini, F Scanabissisabelli, M Trentini

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S Tommasini, F Scanabissisabelli, M Trentini. SCANNING ELECTRON MICROSCOPE STUDY OF EGGSHELL DEVELOPMENT IN TRIOPS CANCRIFORMIS (BOSC) (CRUSTACEA, NOTO- STRACA). Vie et Milieu / Life & Environment, Observatoire Océanologique - Laboratoire Arago, 1989, pp.29-32. ￿hal-03033599￿

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. VIE MILIEU, 1989, 39 (1) : 29-32

SCANNING ELECTRON MICROSCOPE STUDY OF EGGSHELL DEVELOPMENT IN TRIOPS CANCRIFORMIS (BOSC) (CRUSTACEA, NOTOSTRACA)

S. TOMMASINI, F. SCANABISSISABELLI and M. TRENTINI

Department of Expérimental Evolutionary Biology, University ofBologna, Via Belmeloro, 8 40 126 Bologna, Italy

OEUF RESUME - Le développement de l'enveloppe tertiaire de l'oeuf du Notostracé Triops can- COQUE criformis a été étudié au microscope électronique à balayage. La coque est sécrétée comme MICROSCOPE A BALAYAGE MORPHOLOGIE une strate unique qui se délamine en deux, puis en trois strates définitives. La cuticule em- NOTOSTRACA bryonnaire a été identifiée au-dessous de la couche interne de la coque.

EGGSHELL ABSTRACT - The development of the eggshell of Notostracan Triops cancriformis is exa- SCANNING ELECTRON MICROSCOPY mined with the SEM. The eggshell is initially composed of one layer, then, this layer is dela- MORPHOLOGY NOTOSTRACA minated in two and finally three layers. The embryonic cuticule is recognized beneath the inner layer of eggshell.

INTRODUCTION in order to check any possible structural différences be- tween the supposed subitaneous and resting eggs (Lin- der, 1960 ; Hempel-Zawitkowska, 1967; Belk and Cole, Différent species of Phyllopods (Anostra- 1975; Bandu and Bowland, 1985). ca, Notostraca and Conchostraca) live in temporary pools of freshwater. The animais living in such an environment, subject to abrupt variations like dehydration, drying up MATERIAL AND METHODS and wide thermie excursions, have to face numerous adaptations, one of the most significant consists of rather long cryptobiosis (even a number of years) during which The spécimens were collected in the spring of 1986 the larval stage survives. This capacity is greatly due to and 1987 in rice-fields, flooded for almost a month, in the particular type of shell enveloping the embryo (Maz- the Province of Ferrara, from maleless populations (Zaf- zini et al., 1984 for a review). fagnini and Trentini, 1980) and laboratory bred. Eggshell formation in Crustacean Phyllopods has The génital System of rudimentary hermaphrodite fe- been studied with a Transmission électron microscope, males consists of two ovaries and an utérus which run above ail in (Linder, i960; Morris and Afze- along the 's body ; the utérus, by means of two lius, 1967; Anderson et al., 1970; Garreau de Loubresse, short ducts, leads into the ovigerous pockets, a modifica- 1974; Mazzini, 1978); the structure and morphology of tion of the eleventhy pair of feet (fig. 1). the shell by SEM has been observed mainly in relation to their (Mura et al., 1978; Gilchrist, 1978; The eggs examined were taken : 1) from the ducts, Alonsoand Alcaraz, 1984; Mura, 1986; Mura and Thiery, 2) from the ovigerous pockets, 3) outside, at various âges 1986). As to the Notostraca by SEM, already laid and (hours, days, up to over a year) from déposition, in water dried eggs have been studied (Gilchrist, 1978; Alonso and dry. and Alcaraz, 1984; Thiery, 1985), while our research is The eggs received no treatment either with ultrasound aimed at studying the development of the Triops cancri- or with solvent since they showed no traces of surface formis eggshell, whose follicular duct cell ultrastructure, dirt, having always been taken either from the animal's which synthetizes the material (Trentini and Sabelli Sca- body or in water; only those preserved in dry places re- nabissi, 1982), has already been studied by us. So much ceived brief ultrasound treatment. They were mounted 30 S. TOMMASINI, F. SCANABISSISABELLI and M. TRENTINI

structure; in section it is like a layer of about 9 um shape- less and compact (Fig. 2a; Plate 1,1). In the subséquent 10-12 hours it delaminates into two distinct layers, the outer one of about 7 um and the inner about 2,5um (Fig. 2b; Plate 1,2). The maturing process continues when numerous circular craters, up to 20(xm in diameter, open on the surface (Plate I, 3); at the same time numerous al- veoli form in the outer layer and thèse stratify in suc- cessive planes from the outside inwards, thus causing an increase in the shell of up to 25um (Plate 1,5). The inner layer, instead, is not at ail affected by the formation of the alveoli and remains compact and constant in thick- ness (Plate 1,7). When the formation of alveoli is begun, the typical craters of the preceding phase are obliterated (Plate 1,4) and the surface appears uniformly covered with pores (Plate 1,6) which affect only a new external Fig. 1. - Section of reproductive apparatus in Triops cancrifor- layer of about 1 m, which can only now observed and mis. d = duct ; op = ovigerous pocket ; u = utérus. which may delaminate in dried eggs, while still remain- ing perforated (Plate 1,8). In the final stage, then, the sec- tioned shell is formed of three layers : 1) perforated outer and sectioned in order to observe the internai structure layer (Plate I, 6,8); 2) alveolar layer (no longer three on a two-sided adhesive tape, metalized with gold and séries of alveoli with holes passing between one cham- observed with a Philips 515 SEM. ber and another (Plate 1,5,8,10); 3) inner layer of 2,5um, which may frequently detach itself from the alveolar layer (Fig. 2c; Plate 1,9,10). The time required for the RESULTS shell to mature is estimated, for eggs laid in water, at about 36-48 hours. From ours observations it cornes out Observation of the eggshell of T. cancriformis by that the shell maturation process starts, and may even be SEM was carried out at various stages of maturation in completed, in the ovigerous pockets. The eggs recently order to verify their formation. The oocytes, which hav- passed into the ovigerous pockets are deposited in water, ing matured in the follicles, on moving down the utérus the shell not completely developed, but this in no way meet the eggshell material secreted by the tubule cells préjudices their survival since they, too, continue shell (Trentini and Sabelli Scanabissi, 1982). The eggs are development in the same manner. If the eggs deposited massed together in the utérus and completely enveloped in water meet with drying, eggshell maturity is reached by the sécrétion, a reddish fluid (Longhurst, 1955), then, more rapidly, presumably due to direct exposure to the along a short duct, deposited in the ovigerous pockets air. (fig. 1 ), where they remain for the whole intermoult, last- With the déposition, the eggs begin the segmentation ing about a week, in the laboratory, after which they are until the embryo is formed and, subsequently, the larval deposited outside with the moult. stages. Our expérimental data demonstrate that the em- In the earliest phase, the shell is still soft and sticky, bryonic development is unrelated to eggshell formation the surface smooth and it has no particular pattern or and it is stopped by drying at any stage (i.e. : egg, gastru- la, nauplius, metanauplius). The successful hatching and its rapidity are related to the embryonic stage reached be- fore drying (Hempel-Zawitkowska, 1967). The embryo produces, in the first hours after déposi- tion, a cuticular membrane (Plate 1,2,7); at the hatching the metanauplius (Longhurst, 1955) is enclosed in an- other larval envelope (hatching membrane) (Plate 1,9), which continues to envelope the metanauplius even for some hours after the émergence from the eggshell.

DISCUSSION

Fig. 2. - Three-dimensional représentation of three layers de- Our scanning studies on the T. cancriformis eggshell, velopment of Triops cancriformis eggshell. a = newformed and observed during the maturing process up to hatching, unitratified eggshell; b = inner layer formation; c = final egg- have shown, partially rectifying known data (Gilchrist, shell. 1978; Trentini and Sabelli Scanabissi, 1982; Thiery, TRIOPS CANCRIFORMIS EGGSHELL DEVELOPMENT 31

Plate I. - 1, unistratified eggshell; 2, inner layer formation; 3, eggshell surface with craters; 4, eggshell with obliterated craters; 5, cross section of increasing eggshell thickness after alveolar layer formation; 6, eggshell surface with characteristic pores of final stage; 7,8, cross sections of three layers and embryonic cuticle. Arrow = hole in alveolar layer, 9, cross section of embryonated egg showing alveolar and inner layer and hatching membrane. Arrow = embryonic cuticle; 10, gênerai view of final eggshell showing the three typi- cal layers, after metanauplius hatching. al = alveolar layer; ec = embryonic cuticle; hm = hatching membrane; il = inner layer ; ol = outer layer. 32 S. TOMMASINI, F. SCANABISSI SABELLI and M. TRENTINI

1985), the présence not of two but three layers forming BANDU V.H. & A.E. BOWLAND, 1985. Transmission and the eggshell. The inner layer, identif ied by the same auth- scanning électron microscopy of tadpole eggs. Proc. ors as embryonic cuticle, is really the inner layer of the Electr. Micr. Soc. South Afr., 15 : 123. BELK D. & G.A. COLE, 1975. Adaptation biology of désert eggshell. In fact, we have observed that this originates at temporary-pond inhabitants. In N.F. Hadley éd., Environ- the beginning of shell maturation and is preserved up to mental physiology of désert organisms. Dowden, Hutchin- hatching and, when the embryo develops, the true em- son and Ross, Inc., Stroudsburg, Pa. : 207-226. bryonic cuticle is clearly évident beneath it. The study of GARREAU de LOUBRESSE N., 1974. Etude chronologique de embryonic cuticle and other larval membranes (i.e. la mise en place des enveloppes de l'oeuf d'un Crustacé hatching membrane) will be the object of following in- Phyllopode : Tanymastix lacunae. J. Microscopie, 20 : 21- vestigations. 38. GILCHRIST B.M., 1978. Scanning électron microscope studies A second aspect is the relationship one can see be- of the eggshell in some Anostraca (Crustacea : Branchiopo- tween the deep craters appearing at the beginning of al- da). CellTiss. Res., 193 : 337-351. veolar layer formation and the passage of air inside the HEMPEL-ZAWITKOWSKA J., 1967. Natural history of Triops compact layer. Confirming this, such craters later disap- cancriformis (Bosc). Zool. Pol., 17(3) : 173-239. pear, while only pores on the surface and wide openings LINDER H.J., 1960. Studies on the freshwater fairy shrimp CM- between the alveoli maintain the air passage in the alve- rocephalopsis bundyi (Forbes). II. Histochemistry of egg- olar layer. shell formation. X Morphol., 107 : 259-284. LONGHURST A.R., 1955. A review of the Notostraca. Bull. The particular architecture of the eggshell in T. can- British Mus. Zool., 3 : 1-57. criformis is, as in other Phyllopods living in astatic wa- MAZZINI M., 1978. Scanning électron microscope morphology ters, highly adaptative (Belk and Cole, 1975; Mazzini et and amino-acid analysis of the egg-shell of encysted brine al., 1984). In fact, the shell is protective in function, thus shrimp, Artemia salina Leach (Crustacea, Anostraca). Monit. maintaining survival in spécial conditions like drying up, zool. ital. (N.S.), 12 : 243-252. sudden changes of température, ultraviolet radiation ; the MAZZINI M., G. CALLAINI & C. MENCARELLI, 1984. A embryo can also remain in cryptobiosis for many years comparative analysis of the évolution of the egg envelopes and the eggs may be scattered by wild fauna. The cyclic and the origin of the yolk. Boll. Zool., 51 : 35-101. MORRIS J.E. & B.A. AFZELIUS, 1967. The structure of the infestations of T. cancriformis on rice-fields are also ex- shell and outer membranes in encysted Artemia salina em- plainable even after antiparasitic treatment or early ex- bryos during cryptobiosis and development. /. Ultrastrct. siccation. Res., 20:244-259. As to the supposed existence in T. cancriformis of MURA G., 1986. Scanning électron microscope morphological subitaneous or resting eggs (Linder, 1960; Belk and Cole, survey on the egg shell in the italian Anostracans (Crustacea, 1975), in the material examined we found no morpho- ). Hydrobiologia, 134 (3) : 273-286. logical différences in the eggshell. The différent hatch- MURA G., F. ACCORDI & M. RAMPINI, 1978. Studies on the resting eggs of some fresh water fairy of the genus ing times, which probably induced some authors to Chirocephalus : biometry and scanning électron microscopie suppose the existence of two types of eggs, are probably morphology (Branchiopoda, Anostraca). Crustaceana, 35 caused by the différent maturing stages of the embryo (2) : 190-194. and, in the second time, by laid eggshells. MURA G. & A. THIERY, 1986. Taxonomical significance of scanning électron microscopie morphology of the Euphyllo- pods' resting eggs from Morocco. Vie Milieu, 36 (2) : 125- REFERENCES 131. THIERY A., 1985. Ponte et ultrastructure de l'oeuf chez Triops granarius Lucas (Crustacea, Notostraca) : adaptations à l'as- ALONSO M. & M. ALCARAZ, 1984. Huevos resistentes de sèchement de l'habitat. Verh. Internat. Verein. Limnol., 22 : crustâceos eufilôpodos no cladôceros de la pem'nsula ibéri- 3024-3028. ca : Observaciôn de la morfologfa externa mediante técnicas TRENTINI M. & F. SABELLI SCANABISSI, 1982. Follicle de microscopîa electrônica de barrido. Oecologia aquatica, duct cell ultrastructure and eggshell formation in Triops can- 1:73-78. criformis (Crustacea, Notostraca). /. Morphol., 172 : 113- ANDERSON E., J.H. LOCHHEAD, M.S. LOCHHEAD & E. 121. HUEBNER, 1970. The origin and structure of the tertiary en- ZAFFAGNINI F. & M. TRENTINI, 1980. The distribution and velope in thick-shelled eggs of the , Artemia. J. reproduction oîTriops cancriformis (Bosc) in Europe (Crus- Ultrastruct. Res., 32 : 497-525. tacea, Notostraca). Monitore zool. Ital. (N.S.), 14 : 1-8. Reçu le 4 janvier 1988; received January 4,1988 Accepté le 12 février 1988; accepted February 12,1988