Ultrastructure of Intrauterine Eggs: Evidence of Early Ovoviviparity in the Caryophyllidean Cestode Wenyonia Virilis Woodland, 1923

DOI: 10.2478/s11686-010-0044-0 © W. Stefan´ski Institute of Parasitology, PAS Acta Parasitologica, 2010, 55(4), 349–358; ISSN 1230-2821 Ultrastructure of intrauterine eggs: Evidence of early ovoviviparity in the caryophyllidean cestode Wenyonia virilis Woodland, 1923

Daniel Młocicki1,2*, Zdzisław S´widerski1,2, John S. Mackiewicz3 and Mohammed H. Ibraheem4 1W. Stefański Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warsaw, Poland; 2Department of General Biology and Parasitology, Medical University of Warsaw, 5 Chałubińskiego Street, Poland; 3Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12222, U.S.A.; 4Department of Zoology, Faculty of Science, Minia University, El-Minia 61519, Egypt

Abstract Ultrastructural evidence for early intraurerine embryonic development of Wenyonia virilis is presented. At the initial stage of egg formation, the fertilized oocyte or ovum is surrounded by numerous vitellocytes and newly formed eggshell. Individual vitellocytes undergo progressive fusion into a vitelline syncytium. During cleavage divisions, three types of blastomeres are formed: macromeres, mesomeres and micromeres. Two large macromeres contain a large nucleus with spherical nucleolus and numerous small heterochromatin islands dispersed in moderately electron-dense nucleoplasm. The granular cytoplasm shows a few large mitochondria. Medium-sized mesomeres contain a spherical nucleus with numerous heterochromatin islands, adjacent to the nuclear envelope, and a prominent electron-dense nucleolus. Their nuclei are embedded in granular cytoplasm with a few large and numerous small mitochondria and Golgi complexes. The small micromeres are characterized by presence of spherical nucleoli with large areas of highly condensed heterochromatin and a few islands of granular electron-lucent nucleoplasm. Their granular cytoplasm shows a few small lipid droplets and several spherical mitochondria. Majority of micromeres give rise to the hexacanth but many of them also undergo degeneration or apoptosis. Both mesomeres and macromeres are engaged in the formation of the oncospheral envelopes. The outer envelope is formed by a fusion of two macromeres whereas the inner envelope originates from a fusion of mesomeres. The intrauterine eggs of W. virilis usually contain an embryo at the early pre- oncopheral phase of development and possesses three primary envelopes: (1) thick eggshell; (2) thin cytoplasmic layer of the outer envelope and (3) inner envelope. Based on embryonic development, egg type and life-cycle characteristics, caryophyllideans tend to show closer affinities to spathebothriideans than to the former pseudophyllideans.

Keywords Caryophyllidea, Wenyonia virilis, egg formation, intrauterine embryonated eggs, ultrastructure

Introduction (Woodland 1926) and larval development “exhibits less of a tendency toward progenesis than of any other caryophyllid” Wenyonia virilis Woodland, 1923 has unusual morphological (Ibraheem and Mackiewicz 2006, p. 56). In addition, the pat- and developmental characteristics when compared to other tern of vitellogenesis in Wenyonia is different from that of caryophyllideans (Miquel et al. 2008, Świderski et al. 2009). other caryophyllideans (Świderski et al. 2009). Earlier Miquel Furthermore, the morphology-based analysis of the phyloge- et al. (2008) describing process of spermiogenesis in Wenyo- netic interrelationships among monopleuroid cestodes shows nia concluded that despite of the great similarity to that of that Wenyonia forms a sister group with gyrocotylideans rather Glaridacris and Khawia, the unusual flagellar rotation ob- than with the monophyletic group of caryophyllideans (Oros served in Wenyonia suggests that the genus may stand apart et al. 2008). The rugomonobothriate scolex and the pre-equa- from all other caryophyllaeids. torial position of gonopores set it apart from all other caryo- Except for limited light-microscopy data on the egg for- phyllidean genera. Also, uterine glands are absent (Woodland mation, embryology and mature eggs (Mackiewicz 1972, 1926, Oros et al. 2008), eggs may begin embryonation in utero 1981, 2003; Scholz 1991, 1993), there is no information on the

*Corresponding author: [email protected] 350 Daniel Młocicki et al.

Fig. 1. Light-microscopy picture of the newly laid eggs of Wenyonia virilis containing embryos composed of numerous blastomeres (white stars). Note: (a) a thick eggshell with well-defined operculum; (b) two large macromere nuclei localized at the operculate pole of the egg. Inset: enlarged operculate part of the egg showing a narrow line of separation between the two parts of the eggshell. Fig. 2. TEM micrograph illustrating early intrauterine egg. Note: (a) a thick and electron-dense eggshell; (b) numerous vitellocytes forming vitelline syncytium still containing: large shell-globules, mitochondria and small glycogen islands. Abbreviations used in all figures: D – discontinuous eggshell, DVC – degenerating vitelline cells, Em – embryo, ES – eggshell, FCD – areas of focal cytoplasmic degradation, GC – Golgi complexes, GER – granular endoplasmic reticulum, gl – glycogen-like particles, IE – inner envelope, L – lipid droplets, m –mitochondria, Ma – macromeres, MaN – macromere nucleus, Me – mesomeres, MeN – mesomere nucleus, Mi – micromeres, MiN – micromere nucleus, n – nucleolus, N – nucleus, OE – outer envelope, OP – operculum, SG – shell globule, SGC – shell globule cluster, UW –uterine wall, VC – vitelline cell TEM of intrauterine embryonic development of Wenyonia virilis 351

Figs 3 and 4. High power TEM micrograph showing advanced stages of eggshell formation and vitellocyte degeneration. On Fig. 3 note: (a) a shell-globule undergoing fusion with the inner layer of the eggshell; (b) two well-preserved vitellocytes with their cell organelles and inclusions; and (c) one vitellocyte showing three areas of focal cytoplasmic degradation. On Fig. 4 note: (a) more advanced stages of vitellocyte degeneration with enlarged areas of focal cytoplasmic degradation, shell-globules, lipid droplets and GER within a vitellocytes cytoplasm; (b) discontinuous part of the eggshell with enlarge protuberance probably corresponding to the operculum region. Inset: high power magnification showing details of the vitellocyte cytoplasm containing shell-globules, lipid droplets and GER 352 Daniel Młocicki et al. ultrastructure of embryonic development of any caryophyl- Materials and methods lidean. All previous studies on the embryogenesis in caryophyl- Adult specimens of the caryophyllidean cestode Wenyonia lideans have been at the light-microscopy level. That of Mo- virilis Woodland, 1923 were obtained from the small intes- tomura (1929) on Archigetes sp., in which the eggs embryo- tine of naturally infected catfish Synodontis schall (Bloch et nate in utero, was on cleavage and early embryonic develop- Schneider, 1801) collected by fishermen in the Nile River at ment of the preoncosphere. The examinations of the ex utero El-Minia, Upper Egypt. For light microscopical studies the development of Khawia sinensis and K. baltica by Scholtz living cestodes isolated from the intestine, were stored in (1991, 1993) dealt with hexacanth differentiation and postem- Petri dishes with river water where they released eggs spon- bryonic development of procercoids. taneously. Remaining eggs were pressed out from the uterus The aim of the present study is to describe the ultrastruc- of gravid worm. Eggs were observed under the light micro- ture of the intrauterine eggs of W. virilis and relate it to vitel- scope by means of the CCD camera combined with com- logenesis as described by Świderski et al. (2009). puter.

Fig. 5. Early intrauterine egg. Note: (a) degenerating vitellocytes undergoing fusion into vitellocyte syncytium; (b) large macromere with electron-lucent nucleus and cytoplasm; (c) smaller mesomere containing spherical nucleus with prominent electron-dense nucleolus, surrounded by granular cytoplasm with a few large mitochondria; (d) two smallest dense nuclei that may represent degenerating micromeres. Inset: part of the vitellocyte cytoplasm showing areas of focal cytoplasmic degradation TEM of intrauterine embryonic development of Wenyonia virilis 353

For transmission electron microscopical analyses gravid buffer. Material was dehydrated in a graded alcohol series tapeworms were washed in saline solution and cut into and in propylene oxide, then samples were embedded in pieces. Tissue samples were fixed in cold (4°C) 2.5% glu- Spurr’s epoxy resin. Ultrathin sections, double stained with taraldehyde in 0.1 M cacodylate buffer (pH 7.2), washed in uranyl acetate and lead citrate, were examined under a JEM the same buffer, postfixed in cold (4°C) 1% OsO4 in 0.1 M 100 B electron microscope operated at an accelerating volt- cacodylate buffer (pH 7.2) for 1 hour and washed in the same age of 80 kV.

Figs 6–8. TEM micrographs illustrating ultrastructure of three types of blastomeres: micromeres (Figs 6–7), mesomeres and macromeres (Fig. 8). Note: (a) small micromeres containing characteristic spherical nuclei with large areas of highly condensed heterochromatine and a few islands of electron-lucent nucleoplasm; granular cytoplasm of micromeres shows a few lipid droplets and several mitochondria; (b) medium sized mesomeres containing spherical nuclei with large heterochromatine islands adjacent to the nuclear envelope; granular cytoplasm rich in small mitochondria and Golgi complexes; (c) two large macromeres, undergoing fusion into the outer envelope, each contains large nucleus with spherical nucleolus and numerous small heterochromatine islands dispersed in moderately electron-dense nucleoplasm; a few large mitochondria are visible in their granular cytoplasm 354 Daniel Młocicki et al.

Results bryonic envelopes (Figs 1, 9 and 10) while many small micromeres were undergoing early degeneration or apoptosis The released eggs were smooth, thick-shelled and contained (Fig. 5). The outer envelope was formed by the fusion of two embryos composed of numerous symmetrically arranged cells macromeres, the elongate nuclei of which became sometimes (Fig. 1). Each ripe egg had a well-defined operculum (see: Inset) infolded, giving the impression of having three separate com- and two large macromere nuclei localized at the opercular pole partments (Fig. 9). This envelope of cellular origin was situ- of the embryo. ated directly beneath the eggshell. Its granular cytoplasmic At the ultrastructural level, early intrauterine eggs had a layer contained two types of mictochondria of different size thick electron-dense eggshell of slightly varying thickness in and electron-density: a large one of moderate electron-density the different regions (Fig. 2). Numerous vitellocytes were in and a much smaller one that was more electron-dense and had a process of a progressive fusion, thus forming a vitelline syn- a few lipid droplets (Figs 9–10 and Inset). In a more advanced cytium (Fig. 2). However, they still contained shell-globule stage of outer envelope differentiation, numerous multilamel- clusters, mitochondria and small electron-dense islands of late structures or areas of focal cytoplasmic degradation were glycogen-like material (Figs 2–5). A discontinuity of the frequently observed in this layer (Inset to Fig. 9 and Fig. 10). eggshell near an enlarge protuberance on the inner surface The inner envelope originated from a fusion of mesomeres. (Fig. 4) might correspond to the region of the operculum. Its granular cytoplasm contained numerous free ribosomes, The electron-dense layer of the eggshell was reinforced by long profiles of GER, Golgi complexes and moderately elec- shell-globule material released from the progressive degener- tron-dense mesomere nuclei (Fig. 10). ation of vitellocytes (Figs 2–4). At this initial stage, however, some of the individual vitellocytes still remained well-preserved and their cell organelles and inclusions were easily dis- Discussion tinguished (Figs 3–4 and Inset). They contained shell-globules, lipid droplets and GER embedded in the granular cyto- In utero embryo development in relation to the type of vitello- plasm and were very rich in free ribosomes (Fig. 4). With the genesis in W. virilis progressive fusion of individual vitelline cells into a vitelline syncytium, degeneration of their cell organelles and inclusions In his extended review on the order Caryophyllidea, Mac- was observed. The first evidence of this process was the ap- kiewicz (1972) mentioned that “Except for Archigetes in which pearance of areas of focal cytoplasmic degeneration (Fig. 3), embryonation is usually completed in utero, or at least begun which progressively increased in number and volume and be- in utero and finished after they are released (Wiśniewski 1930, came much enlarged in the later stages (Figs 4–5 and Inset). Kennedy 1965a) the eggs of most other caryophyllids are un- Early intrauterine embryos were surrounded by a hard embryonated when shed”. However, he added that some ex- eggshell and vitelline syncytium. They were composed of ceptions from this typical oviparity do exist and, among others, three types of blastomeres: micromeres, mesomeres and in Wenyonia spp. embryonated eggs were found in uterus by macromeres (Figs 5–8). The micromeres were characterized Woodland (1923). Because Woodland’s (1923) observations not only by their small size but also by their spherical nuclei, were at the light-microscopy level, it was desirable to verify them which contained large areas of highly condensed heterochro- and get more specific details of the nature of this embryo at the matin and a few islands of granular electron-lucent nucleo- ultrastructural level. Our results corroborate those of Woodland plasm (Figs 6–7). Their granular cytoplasm, rich in free (1923) that the early embryonic development of caryophyllidean ribosomes, had several spherical mitochondria and a few small cestode W. virilis does indeed take place in utero. lipid droplets (Figs 6–7). Some micromeres were rapidly un- The smaller amount of cytoplasmic and nuclear glycogen dergoing apoptosis and were visible as dense pycnotic nuclei in vitellocytes of W. virilis (Świderski et al. 2009) in compar- (Fig. 5). Medium-sized blastomeres, i.e. mesomeres, con- ison with five other previously examined caryophyllideans tained spherical nuclei with numerous heterochromatin islands (Mackiewicz 1968; Świderski and Mackiewicz 1976; Świder- (mainly adjacent to the nuclear envelope) and prominent elec- ski et al. 2004a, b) seems to be directly related with the in- tron-dense nucleoli (Fig. 5). Their nuclei were embedded in trauterine or extrauterine type of embryonic development, or granular cytoplasm with Golgi complexes and numerous mi- with oviparity, or with different degrees of ovoviviparity. The tochondria of various sizes (Figs 5 and 8). The two macro- caryophyllidean eggs are polylecithal and show various num- meres were the largest blastomeres. They contained promi- bers of vitellocytes per ovum, which is 8–12 in W. virilis and nent nuclei with spherical nucleoli and numerous small het- 3–11 in five other caryophyllidean genera (Świderski et al. erochromatin islands, more or less randomly dispersed in 2009). In comparison with bothriocephalideans represented moderately electron-dense nucleoplasm (Fig. 8). Their granu- by Bothriocephalus clavibothrium (Świderski 1994; Świder- lar cytoplasm was rich of free ribosomes and showed only a ski and Mackiewicz 2007a, b), they contain much fewer vitel- few large mitochondria (Fig. 8). locytes per fertilized ovum. During in utero development both Both macromeres and mesomeres seemed functional, nutritive and protective functions of the vitelline cells and em- probably being engaged in the formation of the primordial embryonic envelopes are taken over by the uterus. This may ex- TEM of intrauterine embryonic development of Wenyonia virilis 355

Fig. 9. Part of the intrauterine egg showing ultrastructure of the outer envelope. Note a granular layer of cytoplasm of the outer envelope containing a few lipid droplets, mictochondria and infolded macromere nuclei with prominent nucleoli. Inset: multilamellate structure of a focal cytoplasmic degradation, frequently observed in the cytoplasm, in an advanced stages of the outer envelope formation. Fig. 10. Peripheral part of the egg illustrating details of three egg envelopes surrounding the embryo: (a) a thick eggshell; (b) a thin cytoplasmic layer of the outer envelope, rich in numerous free ribosomes, containing a few large mitochondria and numerous multilamellate structures of focal cytoplasmic degradation; (c) underlying cytoplasmic layer of the inner envelope, which contains numerous free ribosomes, long profiles of GER, Golgi complexes and mesomere nuclei. Inset: high power magnification of a large mitochondrion localized within the cytoplasm of the outer envelope and surrounded by numerous free ribosomes 356 Daniel Młocicki et al.

plain why these two functions of vitellocytes may be reduced possible that degenerating vitellocytes and vitelline syncytium to some extent in W. virilis. are sites of autolysis and progressively accumulate the waste In W. virilis, the intrauterine eggs contain embryos com- metabolic products of developing embryos. The arguments posed of numerous blastomeres belonging to three different and facts supporting such interpretation is the complete lack of types. However, typical mitotic division has never been ob- lipids in differentiating and mature vitellocytes and their ap- served. It is astonishing because caryophyllideans are consid- pearance only in the egg enclosed degenerating vitellocytes. ered as a material for karyological studies as shown in nu- Verification of this hypothesis requires, however, experimen- merous light-microscopy (Jones and Mackiewicz 1969, Grey tal or at least cytochemical confirmation. and Mackiewicz 1980, Petkevičiute 1992, Petkevičiute and Kuperman 1992, Kralova-Hromadova et al. 2010) and ultra- Significance of embryonic envelope formation structural (Świderski and Mackiewicz 2002) studies. The only evidence for cleavage divisions remains, in this case, the in- Our results show that the early embryo within the intrauterine creasing number of blastomeres observed in advanced stages egg of W. virilis is surrounded by three primary envelopes: (1) of the embryonic development. relatively thick eggshell, (2) cytoplasmic layer of the outer envelope with macromere nuclei and (3) the inner envelope con- Significance of lipid occurrence in egg degenerating vitel- taining mesomere nuclei (Fig. 10). Their ultrastructure indi- locytes: a hypothesis cates different functions but the common function for them is their protective role for the differentiating embryo. Though lipid droplets have not been present in mature vitel- The terminology of the egg or embryonic envelopes used locytes of caryophyllideans (Mackiewicz 1968; Świderski and in this study follows the terms used in previous papers (Ry- Mackiewicz 1976; Świderski et al. 2004a, b), including vitel- bicka 1966; Świderski 1994; Świderski et al. 2001, 2004c, locytes of W. virilis (Świderski et al. 2009), they have been Młocicki et al. 2005). While the eggshell is formed from the observed in degenerating vitellocytes in eggs of this species. shell materials released from vitellocytes and has mainly pro- It is not known when lipids first appear. It would be supposed tective function, both outer and inner envelopes are cellular that lipids do not appear in the vitellocytes until they have lost in origin and, in addition to protection, they also store and pro- their vitelline material and the eggshell is formed. Since shell vide nutrients for the differentiating hexacanth. They may also formation is not initiated until the post-fertilization stage of probably accumulate waste products of metabolic processes lipid formation, the latter may be associated with the initial occurring during embryogenesis. All these structures, how- divisions of the ovum. According to Smyth and McManus ever, are surrounding the invasive hexacanth and, at this ter- (1989), there are two theories of the functions of lipids in ces- minal stage, their role is greatly reduced, mainly to their pro- todes; they may play an important role as a source of energy tective function and thus forming the basis for a classification or they may represent accumulation of waste metabolic prod- of the eggshell, outer and inner envelope as egg envelopes. ucts. Similar interpretation was discussed in an early TEM The mode of embryonic envelope formation in W. virilis paper by Świderski (1972) concerning functional ultrastruc- generally resembles that previously described in two species ture of egg envelopes and hexacanths of Catenotaenia pusilla. of the former pseudophyllideans, i.e. in Bothriocephalus clavi- The arguments supporting the role of lipids as an energy bothrium by Świderski (1994) and Eubothrium salvelini by source are described by Read and Simmons (1963) on the Świderski et al. (2005). In these two species, the number of basis of a decrease in the lipid amounts in the tissue of Hy- vitellocytes per egg is 20–30 while there are only about 8–12 menolepis diminuta during prolonged starvation. Similar re- vitelline cells per ovum in W. virilis. The main difference be- lationships occur during the egg envelope differentiation in tween the bothriocephalidean and caryophyllidean eggs con- Mosgovoyia ctenoides (Młocicki et al. 2005), especially cerns not only the number of vitellocytes but also the amount within the intraembryophoral layer of the inner envelope, and the character of their cellular inclusions. According to where evident reduction in sizes and number of lipid droplets Świderski and Xylander (2000), there are two main functions can be easily observed. The same relationship may be con- of cestode vitellocytes: eggshell formation and nourishment cluded from three reports on a decrease of the amount of lipid of the developing embryo. These two functions can be reduced droplets in coracidia of different pseudophyllideans (Grabiec or intensified depending on the oviparity or different degrees et al. 1965, Kuperman 1988, Świderski 1994, Świderski and of ovoviviparity and cestode life cycle. In general, a thick Mackiewicz 2004). On the other hand, Smyth and McManus eggshell is formed in these cestode species, in which numer- (1989) described an increase in the amount of lipids during ous vitellocytes, rich in shell-globules and nutrients, are pres- maturation of Schistocephalus and Ligula, which clearly in- ent. Examples of cestodes with such eggs are Caryophyllidea dicates that the large lipid deposits represent waste metabolic (Mackiewicz 1968; Świderski and Mackiewicz 1976; Świder- products. In our opinion, the only plausible explanation of ski et al. 2004a, b, 2009), former Pseudophyllidea (Świderski their late presence in degenerating vitellocytes of the W. viri- 1994, Świderski and Mackiewicz 2004), Trypanorhyncha lis egg could be our working hypothesis that they may repre- (McKerr 1985) and Spathebothriidea (Sandeman and Burt sent the waste metabolic products of the early embryos. It is 1972, Poddubnaya et al. 2006, Świderski et al. 2010). In other TEM of intrauterine embryonic development of Wenyonia virilis 357

cestode orders such as Proteocephalidea (Świderski and Su- Glaridacris catostomi. International Journal for Parasitol- bilia 1978, Bruňanska 1999, Młocicki et al. 2010) and Tetra- ogy, 10, 397–407. DOI: 10.1016/0020-7519(80)90041-7. phyllidea (Euzet and Mokhtar-Maamouri 1975, 1976; Mokhtar- Ibraheem M.H., Mackiewicz J.S. 2006. Scolex development, morphology and mode of attachment of Wenyonia virilis Wood- Maamouri 1976), in spite of their aquatic life cycles, all stages land, 1923 (Cestoidea, Caryophyllidea). Acta Parasitologica, of embryonic development take place in the uterus where ma- 51, 51–58. DOI: 10.2478/s11686-006-0007-7. ture hexacanths are formed. All cestodes belonging to these Jones A.W., Mackiewicz J.S. 1969. Naturally occurring triploidy and two orders have only one or two vitellocytes per ovum and parthenogenesis in Atractolytocestus huronensis Anthony nutritive and protective roles of their vitelline cells and em- (Cestoidea: Caryophyllidea) from Cyprinus carpio L. in North America. Journal of Parasitology, 55, 1105–1118. bryonic envelopes are greatly reduced due to intrauterine em- [Kennedy C.R. 1965a. The life history of Archigetes limnodrili bryonic development. (Yamaguti) (Cestoda: Caryophyllidea) and its development in The intrauterine development of the early embryos of invertebrate host. Parasitology, 55, 427–437. DOI: 10.1017/ W. virilis, consisting of several blastomeres, is similar to the S0031182000069146]. embryo development of the spathebothriidean cestode Didy- Kralova-Hromadova I., Štefka J., Špakulova M., Orosova M., Bom- barova M., Hanzelova V., Bazsalovicsova E., Scholz T. 2010. mobothrium rudolphii (Świderski et al. 2010). 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(Accepted July 07, 2010)