First study of vitellogenesis of the broad fish tapeworm

Parasitology International 63 (2014) 747–753

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First study of vitellogenesis of the broad fish tapeworm Diphyllobothrium latum (Cestoda, Diphyllobothriidea), a human parasite with extreme fecundity Aneta Yoneva a,b,⁎, Roman Kuchta b, Tomáš Scholz b a b

Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05, České Budějovice, Czech Republic

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Article history: Received 7 March 2014 Received in revised form 11 June 2014 Accepted 5 July 2014 Available online 12 July 2014 Keywords: Ultrastructure Vitellogenesis Diphyllobothrium latum Cytochemistry Diphyllobothriosis

a b s t r a c t In the present study, the process of vitellogenesis of one of the most prolific organisms, the broad tapeworm, Diphyllobothrium latum, the causative agent of human diphyllobothriosis, was studied for the first time using transmission electron microscopy. Cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for detection of glycogen was applied. Starting from the periphery toward the center of the vitelline follicle four stages of vitellocytes are differentiated: immature vitellocytes, early maturing vitellocytes, advanced maturing and mature vitellocytes. Differentiation into mature vitellocytes involves the formation of shell globule clusters containing shell globules, large amount of saturated lipid droplets and glycogen. A peculiar ultrastructural feature of D. latum vitellogenesis is the presence of lamellar bodies in the cytoplasm of mature vitellocytes. This feature is similar to that present in the closely related caryophyllideans and spathebothriideans. Despite the great similarity observed in the embryonic development of diphylobothriideans, caryophyllideans and spathebothriideans, and the fact that their vitellocytes share a feature not reported from other cestode groups, there are substantial differences in the morphology of vitelline clusters, types, amount and localization of their nutritive reserves. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The Neoophora, i.e. a group that includes most flatworms (Lophotrochozoa: Platyhelminthes), is unique among animals in having a female germ cell lineage, which divides into two cell lines – ovarium and vitellarium – which become spatially and functionally segregated into separate organs [1]. The vitellarium line forms vitellocytes, which generally possess glycogen, yolk and shell globules needed for later development [1]. Fecundity, i.e. reproductive potential, most easily measured as daily production of eggs, of human-infecting tapeworms (Cestoda) is extremely high, being one of the highest within all animals. Besides strobilization, i.e. serial repetition of a large number (up to several thousands) of proglottids, i.e. sets of genital organs, this extraordinary fecundity is facilitated by effective production of vitellocytes. The vitellocytes have two important functions in the embryogenesis of cestodes: (1) protein synthesis for the formation of a thick and hard egg-shell or a delicate vitelline capsule and (2) lipid accumulation and glycogenesis as a food source for the developing embryo [2,3]. ⁎ Corresponding author at: Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria. Tel.: + 359 2 9792328; fax: + 359 2 8705 498. E-mail address: [email protected] (A. Yoneva).

http://dx.doi.org/10.1016/j.parint.2014.07.002 1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.

Broad fish tapeworm, Diphyllobothrium latum (Linnaeus, 1758), is the most important causative agent of diphyllobothriosis, which itself is not a life-threatening disease, but is considered the most important fish-borne zoonosis caused by a cestode parasite, with up to 20 million persons estimated to be infected worldwide [4]. This cestode has extremely high fecundity and one worm is estimated to produce up to 1 million eggs per day [5]. Ultrastructure of its genital system is only partly known, even though the first ultrastructural studies were carried out in the 1960s [6–8]. However, no ultrastructural data exist on a very important component of the process of reproduction, i.e. vitellogenesis. In fact, little is known about the vitellogenesis in the whole order Diphyllobothriidea, which includes usually large-sized parasites of mammals, including man, birds, reptiles and amphibians [9]. The only study of vitelline cells and their role in the formation of the egg-shell, based on light microscopical observation, was published more than 50 years ago [10]. However, no details of the process of maturation of vitelline cells of broad fish tapeworms are presented in this pioneer, but now much outdated study. In the present study, a complete ultrastructural description of the process of vitellogenesis in the broad fish tapeworm is provided, with the main goals to characterize features that may have contributed to extraordinary fecundity of this humaninfecting parasite and to provide ultrastructural characters that might unravel the evolution of morphological and physiological adaptations of cestodes related to their reproductive biology.

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A. Yoneva et al. / Parasitology International 63 (2014) 747–753

2. Materials and methods Adult specimens of D. latum studied by TEM were collected from the intestine of golden hamster (Mesocricetus auratus) experimentally infected with plerocercoids from the musculature of naturally infected perch (Perca fluviatilis) collected from Lake Iseo, Italy, on February 5, 2013. The tapeworms from freshly killed host were rinsed in 0.9% NaCl solution. Fragments of strobila containing mature and gravid proglottids were fixed in cold (4 °C) 1.5% glutaraldehyde and 1.5% paraformaldehyde solutions in 0.1 M Hepes (pH 7.4) and stored for 4 weeks at 4 °C. After washing with 0.1 M Hepes (pH 7.4), they were post-fixed in cold (4 °C) 1% osmium tetraoxide (OsO4) in the same buffer for 1 h, dehydrated in a graded series of acetone, embedded in Spurr's epoxy resin and polymerized at 62 °C for 48 h. Ultrathin sections (60–90 nm in thickness) were cut on a Leica Ultracut UCT ultramicrotome, placed on copper grids and stained sequentially with uranyl acetate and lead citrate according to Reynolds [11]. The Thiéry technique [12] was applied for detection of glycogen. Gold grids with ultrathin sections were treated in periodic acid, thiosemicarbazide and silver proteinate (PA-TSC-SP) as follows: (1) 10% PA for 30 min, rinsed in distilled water, (2) TSC for 24 h, rinsed in acetic solutions and distilled water, (3) 1% SP for 30 min in the dark, and rinsed in distilled water. Grids with ultrathin sections were examined under a JEOL 1010 transmission electron microscope at 80 kV (Laboratory of Electron Microscopy, Institute of Parasitology, České Budějovice). The voucher material (whole mounts of the same specimen) is deposited at Helminthological Collection of the Institute of Parasitology, Academy of Sciences of the Czech Republic (IPCAS No. C-650). 3. Results Vitellarium of D. latum is formed by numerous follicles situated in two lateral bands in the cortical parenchyma. Each vitelline follicle is composed of vitelline cells in various stages of maturation that are surrounded by interstitial tissue consisting of cytoplasmic processes and nuclei (Fig. 1A). The interstitial nuclei are situated in the central part or at the periphery of the follicle and contain electron-dense clumps of heterochromatin. The cytoplasm of the interstitial tissue is filled with a few small mitochondria and vesicular inclusions (Fig. 1B). The follicles are enveloped by a basal lamina (Fig. 1A). Four stages of vitellocyte development can be differentiated: immature vitellocytes (stage I), early stage of maturation (stage II), advanced stage of maturation (stage III) and mature vitellocytes (stage IV). 3.1. Immature vitellocytes (Figs. 1A,C and 4I) Immature vitellocytes of D. latum possess a large, centrally situated nucleus and a small amount of cytoplasm (Figs. 1C; 4I). They occur predominantly at the periphery of the vitelline follicle and progressively mature toward the center of the follicle (Fig. 1A). The nucleus of immature vitelline cells exhibits irregular dense clumps of heterochromatin. At this stage of development, the nucleolus is not observed. Numerous free ribosomes and mitochondria of different sizes are visible in the perinuclear cytoplasm, and there are no evident endoplasmic reticulum or Golgi complexes. 3.2. Early stage of maturation (Figs. 1D–F, 2A,B,E and 4IIA,B) At this stage of maturation, vitelline cells are distinguished by increase of cytoplasm volume, development of cytoplasmic organelles and appearance of individual shell globules. The cells contain a large nucleus with numerous clumps of heterochromatin and a roundish nucleolus (Figs. 1D; 4IIA). Cytoplasm matrix possesses Golgi complex, which is made up of a few flattened membrane-enclosed sacs and long parallel cisternae of granular endoplasmic reticulum (GER) (Figs. 1E, F; 2A, B,E).

These organelles are associated with the formation of shell globules and shell globule clusters. Initially, small round individual shell globules (ca. 0.3 μm in diameter) appear inside the electron-translucent vesicles of Golgi origin. Usually, they are confined to the cell periphery (Fig. 1D). The vesicles further gradually increases in size and fuse, thus forming larger membrane-bound shell globule clusters embedded in an electron-lucent matrix (Fig. 1F). The number of shell globules within clusters varies widely. At this stage, the clusters are made up of 2–5 shell globules (Figs. 1F; 2A, B). In more mature cells, the number of shell globules in clusters reach up to 30. A few electron-lucent lipid droplets are present in the cytoplasm (Figs. 2A; 4IIB).

3.3. Advanced stage of maturation (Figs. 2C,D, F, G and 4III) During this stage the cytoplasm is abundant with cellular organelles such as granular endoplasmic reticulum, lipid inclusions and glycogen granules, which denotes high secretory activity of the vitellocytes. Vitelline cells present also the nucleus with electron dense clumps of heterochromatin (Figs. 2C, F; 4III). Shell globule clusters are composed of numerous individual shell globules of various sizes (from 0.4 μm to 0.9 μm in diameter) (Fig. 2D). The concentric rows of granular endoplasmic reticulum are often found in close association with the shell globule clusters and lipid droplets (Fig. 2D, G). The cytoplasm of vitellocytes is provided with large amount of glycogen granules scattered among the lipid droplets (Fig. 2G).

3.4. Mature vitellocytes (Figs. 2H, 3A–F and 4IV) Mature vitellocytes are large ovoid in shape and their cytoplasm is rich in shell globule clusters, electron-lucent lipid droplets, glycogen granules and lamellar bodies. Shell globule clusters can consist of more than 35 loosely packed shell globules (Figs. 2H; 3A, F; 4IV). Large amount of glycogen granules and lipid droplets (up to 1.4 μm in diameter) are observed throughout the cytoplasm (Figs. 2H; 3F). The presence of glycogen in vitelline cells has been revealed using PATSC-SP cytochemical technique (Fig. 3E). A remarkable feature of some mature vitelline cells is the presence of dark lamellar bodies (ca. 0.6 μm) (Fig. 3A–D). 4. Discussion One of the most remarkable features of the reproductive system of flatworms (Neoophora) is the presence of specialized cells, vitellocytes, participating in the process of egg-shell formation. The embryonic development in cestodes of the recently established order Diphyllobothriidea [9] is polylecithal and occurs in external aquatic environment, similar to other lower eucestodes, i.e. caryophyllideans, bothriocephalideans, spathebothriideans, diphyllideans and trypanorhynchs. Diphyllobothriidean cestodes produce a large number of polylecithal eggs, which are characterized by a thick and hard egg-shell. A great amount of nutritive reserves (lipids and glycogen) is required during embryonic development of these cestodes, which allows them to remain in the water until the embryonation is complete [3]. Therefore, the ultrastructural aspects of the maturation of vitelline cells participating in the egg-shell formation are considered an important step toward better understanding of the reproductive biology of cestodes. The first detailed study on vitellogenesis in broad fish tapeworm, D. latum, which is a human parasite with extreme reproductive potential, demonstrated that the process follows the general pattern previously described in other investigated representatives of evolutionary more basal cestodes [13–27]. However, numerous studies in the past 10 years have indicated that there are some important distinction regarding the morphology, chemical nature and amounts of vitelline inclusions.


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Fig. 1. (A–F) Vitellogenesis of Diphyllobothrium latum. (A) Vitelline follicle showing vitelline cells in various stages of maturation. (B) Interstitial tissue between vitellocytes. (C) Immature vitellocyte. (D) Early maturing vitellocyte with few shell globules. (E) Detail of Golgi complex. (F) Golgi complex participates in formation of shell globule clusters. Abbreviations: BL, basal lamina; GC, Golgi complex; IT, interstitial tissue; L, lipid droplets; M, mitochondrion; N, nucleus; Nu, nucleolus; SG, shell globules; SGC, shell globule clusters; V, vesicles; I, immature vitellocyte; II, early stage of vitellocyte maturation, III, advanced stage of vitellocyte maturation; IV, mature vitellocyte. Scale bars: A = 4 μm; B and C = 0.1 μm; D = 1 μm; E = 0.8 μm; F = 0.2 μm.

The vitellocytes of D. latum exhibit four types of vitelline material in their cytoplasm, i.e., shell globules and shell globule clusters, lipid droplets, glycogen granules and lamellar bodies. The immature vitelline cells of D. latum have a large nucleus and cytoplasm with high content of free ribosomes and mitochondria. They undergo maturation, which is characterized by increase of cytoplasm volume and the appearance of extensive Golgi complexes and the granular endoplasmic reticulum (GER). The amounts of these organelles increase greatly in the early stage of maturation of the vitellocytes. These organelles are responsible for the secretion and packaging of shell globules and the formation of membrane-bound shell globule clusters [28], which play a key role in the egg-shell formation. Two types of shell globule clusters can be observed in the cytoplasm of vitelline cells: shell globule clusters consisting of loosely packed

dense globules embedded in electron-lucent matrix and shell globule clusters consisting of tightly packed dense shell globules in a matrix of moderate density. Our observations show that the globule clusters in D. latum are loosely packed and embedded in electron lucent matrix. In addition to D. latum and the most basal cestodes, i.e. amphilinideans and gyrocotylideans [29,30], such arrangement of shell globules is present in some members of basal eucestodes, i.e., Spathebothriidea, Caryophyllidea and Bothriocephalidea [13,15,22,26,27,31,32]. In contrast, tightly packed electron dense globules embedded in a matrix of moderate density have been illustrated in three other caryophyllideans studied [14,23,25]. This character is also visualized in two spathebothriideans studied [22,26]. Both types of clusters are reported in Atractolytocestus huronensis, a caryophyllidean cestode with parthenogenetic type of reproduction [33]. Moreover, variations in the number, size and

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Fig. 2. (A–H) Vitellogenesis of Diphyllobothrium latum. (A) and (B) Early stages of vitellocyte maturation. (C) Advanced stage of vitellocyte maturation. (D) Shell globule cluster associated with concentric rows of granular endoplasmic reticulum. (E) Detail of granular endoplasmic reticulum with long parallel cisternae. (F) Lipid droplets associated with granular endoplasmic reticulum. (G) Advanced maturing vitellocyte with shell globule clusters, lipid droplets and glycogen granules. (H) Mature vitellocyte. Abbreviations: GER, granular endoplasmic reticulum; Gl, glycogen; L, lipid droplets; N, nucleus; Nu, nucleolus; SG, shell globules; SGC, shell globule clusters. Scale bars: A, B and G = 0.1 μm; C = 3 μm; D and F = 1 μm; E = 0.5 μm; H = 0.2 μm.

arrangement of shell globules within the clusters have been observed. As maturation of vitelline cells continues shell globule clusters become packed with lipid droplets and glycogen. The role of these inclusions is very important as a source of energy for developing embryo. In spite of the great similarity observed in the embryonic development of diphylobothriideans, caryophyllideans and spathebothriideans, their

vitellocytes differ in the chemical compounds and amount of their nutritive reserves. In cestode vitellocytes, lipids of different chemical nature were observed [3]. The ultrastructural examination of the mature vitelline cells in D. latum shows the presence of a large amount of electronlucent lipid droplets containing a high level of saturated fatty acids. Recent studies on the Caryophyllidea have shown the occurrence of


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Fig. 3. (A–D) Vitellogenesis of Diphyllobothrium latum. (A) Mature vitellocyte with shell globule clusters, lipid droplets, glycogen and lamellar bodies. (B) Mature vitellocyte with numerous lamellar bodies. (C) Mature vitellocyte with shell globule cluster and lamellar body. (D) Detail of lamellar body. (E) Glycogen granules revealed by PA-TSC-SP cytochemical technique. (F) Mature vitellocyte. Abbreviations: Gl, glycogen; L, lipid droplets; LB, lamellar bodies; SGC, shell globule clusters. Scale bars: A, B and E = 0.1 μm; C = 1 μm; D = 0.1 μm; F = 2 μm.

the same type of lipid droplets in the vitellocytes of five species studied [23–25,34,35]. Interestingly, the chemical nature of the lipid droplets in the spathebothriidean vitellocytes varies among the species studied. As in D. latum and the above-mentioned caryophyllideans, saturated lipid droplets have been observed in the spathebothriidean Didymobothrium rudolphii [26]. Electron-dense lipid droplets containing a high level of unsaturated fatty acids have been found in Diplocotyle olrikii, whereas both types of lipid droplets were detected in Cyathocephalus truncatus [22]. In addition, a very high amount of saturated lipids was described in the rhinebothriidean Echeneibothrium beauchampi, a parasite of rays, in which large lipid droplets were accumulated also in the nucleus [36]. In D. latum, similar to the spathebothriideans studied [22,26], a large amount of glycogen granules have been detected in the cytoplasm of mature vitellocytes. In the Caryophyllidea, whose reproductive capacity is much lower compared to that of D. latum, glycogen is stored in both the cytoplasm and the nucleus [14,15,23–25,33,37,38]. Poddubnaya et al. [26] suggested the presence of vitelline inclusions in the nucleus as a plesiomorphic character in cestodes.

Our study shows that dark lamellar bodies are present in the cytoplasm of mature vitellocytes of D. latum. Inclusions with similar structure have had several different terms throughout the literature including labyrinthine shell globules, endoplasmic reticulum whorls, yolk globules, glycan vesicles, lamellar granules or dark concentric bodies [22,26,39–42]. Two theories have been proposed to explain the origin of lamellar inclusions in mature vitellocytes. These may be associated with either the transformation of shell globule clusters during the process of egg formation or the breakdown of granular endoplasmic reticulum [26,43]. More recently, lamellar GER-bodies composed of balls of GER embedded in dense cytoplasm have been found in vitellocytes within intrauterine eggs of the caryophyllidean cestode Wenyonia virilis [34]. The latter authors consider the engagement of GER-bodies in the synthesis of glycoproteins or in the formation of areas of focal cytoplasmic degradation. Among cestodes, lamellar bodies were described for the first time in mature vitellocytes of representatives of the Spathebothriidea, namely C. truncatus and D. rudolphii [22,26]. More recent data on five species

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Fig. 4. (I–IV) Schematic diagram of the development of vitellocytes of Diphyllobothrium latum. I, immature vitellocyte; IIA and IIB, early stages of vitellocyte maturation; III, advanced stage of vitellocyte maturation; IV, mature vitellocyte. Abbreviations: GC, Golgi complex; GER, granular endoplasmic reticulum; Gl, glycogen; L, lipid droplets; LB, lamellar bodies; M, mitochondrion; N, nucleus; Nu, nucleous; SG, shell globules; SGC, shell globule clusters.

of caryophyllideans also provided evidence for the presence of such a structure [23–25,33]. In contrast, the absence of lamellar bodies was reported in other species of the Spathebothriidea, namely D. olrikii, and three other species of the Caryophyllidea [14,15,22,35]. The function of this structure and its phylogenetic importance cannot be clarified until more extensive studies on more species are carried out. In conclusion, the process of vitellogenesis in D. latum follows a similar pattern to that observed in all evolutionary basal cestodes studied so far, with most of them having considerably lower fecundity. It thus seems that the extraordinary reproductive capacity of broad fish tapeworm is not directly related to the unique process of vitellocyte formation and that other morphological characteristics such as large-sized body containing huge number of proglottids and physiological adaptations related to the speed of cell division of reproductive and somatic cells play a key role in attaining such a high level of fecundity, with daily production of hundreds of thousands of eggs.

Acknowledgments The authors would like to thank the staff of the Laboratory of Electron Microscopy, Institute of Parasitology, BC, AS CR, and to Michaela Kotková, also from the Institute of Parasitology, BC, AS CR for technical assistance. Perch infected with plerocercoids of D. latum were kindly provided by Eleonora Bernardoni and Francesca Barbieri (supervisors Andrea Gustinelli and Maria-Letizia Fioravanti), University of Bologna, Italy. This study was supported by the Grant Agency of the Czech Republic (project No. P506/12/1632) and the Institute of Parasitology (RVO: 60077344).

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