- Email: [email protected]
Ultrastructural reconstruction of Taenia ovis oncospheres from serial sections
International Journal for Parasitology 40 (2010) 1419–1431
Contents lists available at ScienceDirect
International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Ultrastructural reconstruction of Taenia ovis oncospheres from serial sections Abdul Jabbar a,*, Simon Crawford b, Daniel Młocicki c,d, Zdzisław P. S´widerski c,e, David B. Conn f, Malcolm K. Jones g,h, Ian Beveridge a, Marshall W. Lightowlers a a
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia School of Botany, The University of Melbourne, Parkville, Victoria 3010, Australia c Witold Stefanski Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warsaw, Poland d Medical University of Warsaw, Department of Medical Biology, 73, Nowogrodzka Street, 02-018 Warsaw, Poland e Medical University of Warsaw, Department of General Biology and Parasitology, 5 Chalubinskiego Street, 02-004 Warsaw, Poland f School of Mathematical and Natural Sciences, Berry College, Mount Berry, GA 30149-5036, USA g School of Veterinary Sciences, The University of Queensland, Brisbane, Queensland 7072, Australia h Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia b
a r t i c l e
i n f o
Article history: Received 1 April 2010 Received in revised form 22 April 2010 Accepted 26 April 2010
Keywords: Taenia ovis Non-activated oncosphere Ultrastructure Serial sections Transmission electron microscopy
a b s t r a c t The cellular organisation of Taenia ovis oncospheres is interpreted from ultrathin serial sections and transmission electron microscopy following high pressure freezing and freeze-substitution. The surface of a hatched, non-activated T. ovis oncosphere is covered by an oncospheral membrane below which is the tegument bearing microvilli. The basal lamina of the tegument is underlain by broad bands of peripheral somatic musculature. Three pairs of hooks and associated muscles are present in the somatophoric third of the oncosphere. Approximately 19 cells of seven different types were identified which include: (i) a quadri-nucleated syncytium of penetration gland type 1 containing two lateral pairs of cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) a single-nucleated median mesophoric gland cell; (iv) 10 somatic cells; (v) two germinative cells; (vi) two nerve cells; and (vii) a pair of median somatophoric cells. This study provides a clear understanding of the morphology of T. ovis oncospheres and forms the basis for further investigations into the biology of taeniid oncospheres. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Taenia ovis is a taeniid cestode parasite infecting sheep and goats as intermediate hosts and canids as definitive hosts. Taenia ovis has played a prominent role in the study of immunity to cestode infections (Gemmell, 1962, 1964), particularly following its use in the development of the first recombinant anti-parasite vaccine (Johnson et al., 1989; Lightowlers, 2006). The host-protective antigens of T. ovis are uniquely associated with the oncosphere, which is the infective life cycle stage present within the parasite’s eggs (Lightowlers, 2006). The ultrastructural location of the hostprotective antigens is unknown and the available information on the structure of the T. ovis oncospheres is insufficient to allow accurate interpretation of the antigens’ localisation (Jabbar et al., 2010). The studies reported here sought to improve our understanding of the biology of T. ovis oncospheres by providing a detailed description of the parasite’s ultrastructure. A number of studies have been undertaken which describe the structure of taeniid (Platyhelminthes: Cestoda: Taeniidae) eggs * Corresponding author. Tel.: +61 (0) 3 9731 2022; fax: +61 (0) 3 9741 5461. E-mail address: [email protected] (A. Jabbar).
and/or oncospheres using light microscopic methods, including Taenia pisiformis (Janicki, 1907; Heath and Smyth, 1970); Taenia saginata (Skvortzov, 1942; Silverman, 1954), Taenia taeniaeformis (Wardle and McLeod, 1952; Heath and Smyth, 1970), T. ovis (Heath and Smyth, 1970), Taenia hydatigena (Heath and Smyth, 1970) and Echinococcus granulosus (Heath and Smyth, 1970; Heath and Lawrence, 1976). Following the advent of electron microscopy, further investigations were undertaken on oncosphere structure in taeniid cestodes including Taenia crassiceps (Chew, 1983), Taenia multiceps (Race et al., 1966), T. ovis (Harris et al., 1987), Taenia parva (S´widerski et al., 2007), T. saginata (Schramlova and Blazek, 1982; Schramlova et al., 1984), T. taeniaeformis (Nieland, 1968; Engelkirk and Williams, 1982, 1983) and E. granulosus (S´widerski, 1983; Harris et al., 1989; Holcman et al., 1994). The covering of the taeniid egg after it leaves the proglottid, the embryophore, has also been the subject of interest in a number of investigations (Inatomi, 1962; Morseth, 1965; Race et al., 1966). All of these studies describe the structure of oncosphere of the respective taeniid species based on sections taken at random, with the exception of S´widerski (1983) who described the structure of E. granulosus oncospheres based on ultrathin serial sections dealing mainly with the hook– muscle system. The cellular composition of oncospheres in other
0020-7519/$36.00 Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2010.04.011
1420
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
cestodes has also been studied including Hymenolepis citelli (Collin, 1969), Catenotaenia pusilla (S´widerski, 1972), Nematotaenia dispar (S´widerski and Tkach, 1997), Staphylocystoides stefanskii (S´widerski and Tkach, 1999), Inermicapsifer madagascariensis (S´widerski and Tkach, 2002), Bothriocephalus clavibothrium (Swiderski and Mackiewicz, 2004) and Mosgovoyia ctenoides (Młocicki et al., 2006) based on ultrathin serial sectioning. The only published report on the ultrastructure of T. ovis oncospheres is that of Harris et al. (1987). These authors investigated activated hexacanths (terminology adopted after Conn and S´widerski, 2008) and metacestodes cultured in vitro, and observed that there were four lobes and four nuclei in the penetration glands and that each of these lobes was filled with granules of varying electron densities. The study utilised sections taken at random rather than serial sections. Two reports describe the ultrastructure of activated hexacanths of E. granulosus and metacestodes cultured in vitro (Harris et al., 1989; Holcman et al., 1994), again based on examination of random sections. Although random sections allow general features of oncospheres to be described, reconstructions based on serial ultrathin sections are required to obtain comprehensive ultrastructural details about the relationship between various structures and the number of different cell types. Here we describe a comprehensive study using ultrathin serial sections and reconstruction of hatched, non-activated oncospheres of T. ovis. 2. Materials and methods 2.1. Collection of parasite material Taenia ovis was maintained in dogs and sheep as described by Coman and Rickard (1975) and Kyngdon et al. (2006). All of the procedures were performed in accordance with the requirements of the University of Melbourne Animal Ethics Committee. 2.2. Collection and hatching of oncospheres Mature eggs were collected from the terminal gravid proglottids of the adult worm which was obtained by purging an experimentally infected dog. Non-activated oncospheres were collected following hatching of the mature eggs as described by Lightowlers et al. (1984). This method has the advantage over conventional techniques (use of artificial gastric and intestinal fluids) as the immature oncospheres dissolve in sodium hypochlorite and only mature oncospheres are retained after the dissolution of embryophores (Lightowlers et al., 1984). In brief, sodium hypochlorite (1.25%) was used to obtain non-activated oncospheres from the eggs. Approximately 5–7 ml of sodium hypochlorite was added to a pre-determined number of eggs (400,000–500,000) and the suspension was mixed gently with a Pasteur pipette. The hatching process was monitored while mixing by examining oncospheres under a microscope. Following adequate hatching of eggs (80– 90%), the reaction was stopped by adding excess water (Milli-Q, Millipore, NSW, Australia). The hatched oncospheres were washed (centrifugation 194g, 10 min) at least five times with pre-heated (37 °C) RPMI-1640 (Invitrogen, Vic, Australia). These hatched but non-activated oncospheres were then processed for ultrastructural studies. 2.3. Cryofixation of oncospheres Oncospheres were gently pelleted (centrifugation 194g, 10 min) and the supernatant discarded. Approximately 2–3 lL droplets of the suspension were sandwiched between type A brass freezer hats (ProSciTech, Thuringowa, Qld, Australia). The enclosed onco-
sphere suspensions were frozen using a Leica EM High Pressure Freezer (Vienna, Austria). The freezer hats enclosing the frozen oncosphere suspensions were split apart and stored in liquid N2 in cryo-vials prior to freeze-substitution in a Leica EM automated freeze-substitution unit. 2.4. Freeze-substitution Frozen oncosphere pellets were freeze-substituted in 0.1% uranyl acetate in acetone at 90 °C for 48 h and the temperature raised to 50 °C at 6 °C/h. Pellets were scraped out of the freezer hats, rinsed in three 30 min changes of acetone and infiltrated with a graded series of Lowicryl HM20 low temperature resins (Polysciences, Warrington, PA, USA) in acetone consisting of 25% (8 h), 50% (overnight), 75% (8 h) and 100% resin (overnight). The infiltrated samples were placed in a fresh change of 100% resin in gelatin capsules, polymerised under UV light for 48 h at 50 °C, and brought to room temperature at 6 °C/h. The soft sample blocks were then hardened under UV light for a further 24 h at room temperature. 2.5. Transmission electron microscopy Oncospheres embedded in blocks were sectioned with a diamond knife on a Leica Ultracut R microtome (Vienna, Austria). For ultrathin serial sections, 90 nm thick sections were cut throughout the oncospheres and three consecutive sections in sequence, taken at 1 lm intervals, were selected for further processing. All of these sections were collected onto pioloform-coated slot copper grids (ProSciTech, Thuringowa, Qld, Australia). The grids were blot dried overnight on filter paper prior to staining. Ultrathin serial sections were double stained with 2% uranyl acetate for 10 min and Triple Lead Stain for 5 min (Sato, 1968) and viewed in a Phillips CM120 Biotwin transmission electron microscope at 120 kV. Images were captured with a Gatan Multiscan 600CW digital camera (Gatan Inc., CA, USA) using the appropriate software. For the purpose of orientation, the terminology proposed by Ogren (1971) and S´widerski (1983) was followed where the hook region is referred to as the anterior pole or somatophore, and the opposing posterior pole being the mesophore. 3. Results 3.1. Cellular composition of the oncospheres The general topography and symmetry of cellular organisation of the hatched non-activated oncospheres of T. ovis is revealed by the serial sections shown in Fig. 1. Taenia ovis oncospheres consist of approximately 19 cells and their arrangement was bilaterally symmetrical in a plane running in an anterio-posterior direction and through the dorsal and ventral poles. Based on the analysis of ultrathin serial sections of 15 T. ovis oncospheres, seven different cell types were distinguished: (i) a quadri-nucleated syncytium of penetration gland type 1 containing four cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) singlenucleated median mesophoric gland cell (MMGC); (iv) 10 somatic cells representing myocytons; (v) two germinative cells; (vi) two nerve cells in the somatophore; and (vii) a pair of median somatophoric cells. 3.2. Structures associated with membranes and hook region The outermost layer in hatched, non-activated T. ovis oncospheres is the oncospheral membrane (Fig. 2A and B). The next
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1421
Fig. 1. Selected ultrathin serial (1 lm apart) sections (A–L) showing the oncospheral and hook-region membranes, tegument, different cell types and hooks of a single Taenia ovis oncosphere. Note the major portion of the oncosphere occupied by the penetration gland cells. In all figures: BL, basal lamina; D1, duct-like structures of penetration gland type 1; D2, duct-like structures of penetration gland type 2; DMSC, duct of median somatophoric cell; GC, germinative cell; GER, granular endoplasmic reticulum; H, hook; HM, hook muscle; HC, heterochromatic; HD, hemidesmosomes; HRM, hook-region membrane; M, microtubules; MSC, median somatophoric cell; MMGC, median mesophoric gland cell; MT, mitochondria; Mv, microvilli; N, nucleus; NC, nerve cell; NCP, nerve cell process; NSG, neurosecretory granules; OM, oncospheral membrane; OT, oncospheral tegument; PG1, penetration gland type 1; PG2, penetration gland type 2; PM, plasma membrane; SC, somatic cell; SG1, secretory granules from penetration gland 1; SG2,secretory granules from penetration gland 2; SM, somatic muscle; 1, 2 and 3, three different layers of a hook.
1422
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 1 (continued)
layer is the oncospheral tegument which is anucleated and possesses characteristic microvillar projections on its surface (Fig. 2A). At the somatophore, these microvilli are particularly long
and are surrounded by the hook-region membrane which contains several laminae (Fig. 2B). The oncospheral tegument comprises two layers: (i) a basal lamina which is thinner, amorphous and less
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1423
Fig. 2. Transmission electron micrographs showing the details of oncospheral membranes and hooks. (A) High magnification image of the details of the oncospheral membrane, tegument and underlying somatic muscles. (B) Part of the somatophore of the oncosphere showing the ultrastructural details of the oncospheral tegument and hook-region membrane. Note the projections of basal lamina (arrowheads) and electron-dense inclusions of the tegument (arrows). (C) High magnification image of crosssection of hooks showing different layers of the hooks (1–3) and hook muscles. (D) An oblique section of the hook showing hook hemidesmosomes. (E) Longitudinal section of the hook showing its different parts. Details of abbreviations are given in Fig. 1.
electron-dense; and (ii) a thicker, electron-dense outer layer containing electron-dense inclusions (Fig. 2B). The basal lamina digitates into the upper tegumental layer near the hook-region membrane (Fig. 2B) and it is underlain by broad bands of the peripheral, somatic musculature (Fig. 2A). The somatophoric pole of the oncosphere is occupied by hooks, somatic cells, nerve cells and a pair of median somatophoric cells. There are three pair of hooks, one medial pair and two lateral ones (Fig. 1C–H) which are interconnected by a complex hook–muscle system (Figs. 1F and G; 2C and D). A hook consists of a base which is composed of a guard or collar and handle and a blade projecting out from the oncosphere. Near the guard, the blade of the hook is surrounded by a hook hemidesmosome (Conn, 1993), by which it is attached in the sheath (Fig. 2D). The hook muscles are attached to the guard (or collar) and base and these do not terminate directly on the hook, but are embedded into the basal lamina surrounding the base of the hook (Fig. 2D). In sections, the hooks appear as heterogeneous structures consisting of three layers of different electron densities (Fig. 2C–E). Highly electron-dense material forms the outermost covering or cortex, which is underlain by a thin layer comprised of discontinuous blocks of decreased electron opacity. The innermost layer or core consists of amorphous and moderately electron-dense material (Fig. 2D). A pair of nerve cells occupies the space between the medial and lateral hooks in the somatophoric pole of the oncosphere (Figs. 1D; 3A). The nerve cells have pro-
cesses which extend to various cells and these processes are numerous in the hook-region (Figs. 1C–G; 2D and E; 3A and C). The nerve cells are characterised by the presence of membranebound dense-cored neurosecretory granules in their cytoplasm (Fig. 3A and B). The two median somatophoric cells are located in the middle of the oncosphere between the hook region and the PG2 cell (Figs. 1F and G; 3C and D). These cells contain irregularly-shaped nuclei and have relatively little cytoplasm containing granules (Fig. 3E and F). Each median somatophoric cell has a duct which runs laterally separating the hook region (above) and the PG2 cell (below) (Fig. 3E). 3.3. Gland cells Three different types of glandular cells are present in oncospheres of T. ovis, based on their different locations and the characteristics of their granules. There is a syncytial PG1 cell containing what appears to be four interconnected cell bodies arranged with one pair located towards the dorsal pole and one located towards the ventral pole. The separate pairs of cell bodies are connected via a narrow isthmus which takes a path towards the mesophore, forming a U-shape (Figs. 1A, B and H–L, 4A, E and F). Each cell body is demarcated from its neighbour by a clearly defined plasma membrane (Fig. 4A–C). The PG1 cell bodies do not have a specific shape and contain various cytoplasmic processes emanating in dif-
1424
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 3. Transmission electron micrographs showing the details of nerve and median somatophoric cells. (A) A nerve cell showing the presence of characteristic granules in its cytoplasm. (B) High magnification image of a nerve cell process containing characteristic neurosecretory granules. (C) A pair of median somatophoric cells lying between the hook region and the penetration gland type 2 cell. (D) High magnification image of median somatophoric cells. (E) A median somatophoric cell showing its lateral duct (arrows). (F) High magnification image of a median somatophoric cell showing its nucleus, granular endoplasmic reticulum and granules (arrowheads). Details of abbreviations are given in Fig. 1.
ferent directions (Figs. 1A and I; 4A) which inter-digitate through the centrally located PG2 cell (see Fig. 1D, E, G, H, the cross-sections of PG1). The major part of each PG1 cell body is occupied by a large and irregularly-shaped nucleus containing islands of heterochromatin (Figs. 1A; 4A and B), perinuclear material rich in free ribosomes, a well-developed granular endoplasmic reticulum and numerous mitochondria (Fig. 4B). The granules of these cells are round to rod-shaped and have a well defined membrane containing amorphous electron-dense material (Fig. 4C and D). Typi-
cally, these granules lie individually in the tightly packed cytoplasm. The second type of gland cell (PG2) is also syncytial and occupies 50–60% of the hexacanth body (Fig. 1D–I). This cell contains four nuclei arranged in two lateral pairs (Fig. 5A). Evidence supporting this conclusion is based on images showing pairs of nuclei in very close proximity while other images show nuclei in the same cell but widely separated (Fig. 1C–F). No single image was obtained in which three or all four of the nuclei could be seen in the same
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1425
Fig. 4. Transmission electron micrographs showing the ultrastructural details of the penetration gland type 1 (PG1) cell. (A) Low magnification image of a lateral pair of the PG1 cell bodies showing their processes. (B) High magnification image of a PG1 cell body showing its nucleus and the perinuclear space filled with ribosomes, granular endoplasmic reticulum and mitochondria. (C) High magnification image of PG1 cell bodies showing that two neighbouring nuclei are separated from each other by a plasma membrane. (D) The cytoplasm of the PG1 cell containing characteristic secretory granules. (E) Low magnification image of an oncosphere showing the connection between two lateral pairs of PG1 cell bodies (arrows). (F) High magnification image of the connecting duct (isthmus) between PG1 cell bodies (arrows). Details of abbreviations are given in Fig. 1.
section. The size of the PG2 cell nucleus is smaller than that of the PG1 cell and contains heterochromatin islands (compare Figs. 4B and 5A). The nucleus is surrounded by a rough syncytial cytoplasm rich in free ribosomes, granular endoplasmic reticulum and numerous vesicles (Fig. 5A). Unlike the PG1 cell, there are no mitochondria in the perinuclear area and the cytoplasm of the PG2 cell is filled with granules (Fig. 5B). The granules of PG2 cells are larger,
less electron-dense and sparser than those in the cytoplasm of the PG1 cell (Fig. 5B). The PG1 and PG2 cells are separated from each other, at certain levels, by somatic muscles (Fig. 5C). A novel, possibly third type of gland-like cell (MMGC) is located centrally at the mesophore just underneath the oncospheral tegument and between two somatic cells (Figs. 1E–H; 6A–C). The MMGC is surrounded by the processes of another cell (Fig. 6D)
1426
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 5. Transmission electron micrographs showing the ultrastructural details of penetration gland type 2 (PG2) cell, somatic and germinative cells. (A) Note that the nuclei of the PG2 cell lie together without a plasma membrane and the perinuclear space is filled with free ribosomes, granular endoplasmic reticulum and vesicles. (B) High magnification image of the secretory granules of the PG2 cell. (C) Low magnification of an oncosphere showing the separation of PG1 and PG2 cells through a layer of somatic muscles. (D) Low magnification image of a somatic cell showing its nucleus and long lateral processes of myofibrils. (E) High magnification image of a somatic cell showing a cross-section of muscle fibres in its cytoplasm. (F) A germinative cell showing its characteristic large nucleus and granular cytoplasm containing mitochondria. Details of abbreviations are given in Fig. 1.
which has more regular-shaped less electron-dense granules (Fig. 6F) and it seems that the shape and electron density of these granules are similar to those of median somatophoric cells. This uninucleate gland cell is oval (Fig. 6B) and the cytoplasm contains irregularly-sized granules of varying electron densities (Fig. 6E). No duct was seen that clearly connected gland cells and the tegument. However, structures surrounded by microtubules were seen below the tegument between the medial and lateral pairs of hooks
(Fig. 7A). Similar structures were also observed in cross and tangential sections below the tegument posterior to the lateral pair of hooks near PG2 (Fig. 7B and C). 3.4. Cells other than glands There are two main types of cells in T. ovis oncospheres other than the secretory cells. These are the somatic and germinative
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1427
Fig. 6. Transmission electron micrographs showing the ultrastructural details of the median mesophoric gland-like cell (MMGC). (A and B) Low magnification image of the MMGC showing its nucleus and granular cytoplasm. (C) MMGC lies between a pair of somatic cells. (D) MMGC is surrounded by the processes of the median somatophoric cell containing the granules of different electron density. (E) High magnification of MMGC showing electron-dense as well as translucent granules in its cytoplasm. (F) High magnification image of the granules of MMGC and those of median somatophoric cell wrapping it. Details of abbreviations are given in Fig. 1.
cells. Somatic cells, which are most numerous, are located mainly in the somatophoric part of the hexacanth (Fig. 1B, E and H). These cells are myocytons, characterised by a direct connection with muscle fibres of both somatic and hook musculature (Fig. 5D and E). Somatic cells contain oval or spherical nuclei having abundant heterochromatin islands. The nucleus of a somatic cell is surrounded by a thin layer of cytoplasm (Fig. 5D and E). The two germinative cells are larger than the somatic cells and located in close proximity at either the dorsal or ventral pole (it was not possible to determine in which particular pole the cells were located). These cells have a higher nucleocytoplasmic ratio than somatic cells
(compare Fig. 5D, E and F). The nucleus of germinative cells has a large oval nucleus which contains granular nucleoplasm with numerous irregularly-shaped islands of heterochromatin and a relatively thin layer of cytoplasm comprising numerous free ribosomes and mitochondria (Fig. 5F).
4. Discussion A reconstruction of a T. ovis oncosphere is summarised and shown in Fig. 8. Many features seen in T. ovis were consistent with
1428
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 7. Transmission electron micrographs showing the ultrastructural details of duct-like structures. (A) Cross-section of a duct-like structure surrounded by microtubules. The structure is located adjacent to the oncospheral tegument, between the medial and lateral pairs of hooks and near the somatophoric pole of the hexacanth. (B) Cross-section of a similar duct-like structure located posterior to the lateral pairs of hooks near the penetration gland type 2 cell (arrow). Inset shows high magnification. (C) Tangential section of a duct-like structure posterior to the lateral pairs of hooks and adjacent to the penetration gland type 2 cell. Details of abbreviations are given in Fig. 1.
the ultrastructure of other cestode oncospheres, but a number of characteristics of T. ovis oncospheres were identified which clearly differed from previous descriptions of oncosphere structures. A single cell not previously described was found in T. ovis and was designated a median mesophoric gland cell. In addition, the characteristics of a previously described oncospheral cell from E. granulosus, the binucleate medullary cell (S´widerski, 1983), were different in T. ovis and this cell type was given the name median somatophoric cells. In other respects (germinative, nerve, penetration gland and somatic cells), the cell types identified in T. ovis oncospheres were consistent with the descriptions of cell types found in other cestode oncospheres (Collin, 1969; Schramlova and Blazek, 1982; S´widerski, 1983; Harris et al., 1989; Holcman et al., 1994; S´widerski and Tkach, 2002; Młocicki et al., 2006) and with the limited previous description of T. ovis oncospheres (Harris et al., 1987). We have chosen to adopt the same nomenclature for the cells as has been used by previous authors in the field. Taenia ovis oncospheres were found to be comprised of seven different cell types (germinative, median mesophoric gland, median somatophoric, nerve, two types of penetration gland and somatic cells). Major differences found in T. ovis were the number of some types of cell and the absence of clear evidence of penetration gland ducts. The number of cells comprising a taeniid oncosphere has only been estimated previously for a single species, E. granulosus (S´widerski, 1983). In E. granulosus 48 cells were enumerated by S´widerski (1983). In T. ovis oncospheres, we identified a total of 19 cells. The definition used here for the identification of a cell was recognition of the cell nucleus; multinucleated cells were counted as a single cell. Complete or near complete sets of serial sections were obtained from 15 different T. ovis oncospheres, providing sections at different orientations through the oncospheres. Comparisons of the results from different oncospheres allowed data on cell numbers, which may be equivocal in one plane of sectioning, to be resolved, for example in cases where two nuclei were in close proximity (Fig. 5A). We observed 25 nuclei (two cells were multinucleated) in total in T. ovis oncospheres. In E. granulosus, S´widerski (1983) found 54 nuclei associated with 48 different cells. A number of studies have described the ultrastructure of non-taeniid oncospheres from ultrathin serial sections (Collin 1969; S´widerski, 1972; S´widerski and Tkach, 1997, 2002; Młocicki et al., 2006). Collin (1969) described 44 nuclei in H. citelli while S´widerski (1972) identified only six nuclei in C. pusilla. Similarly, S´widerski and Tkach (1997, 2002) observed differences in the total number of nuclei in N. dispar (n = 52) and I. madagascariensis (n = 50), respectively. Recently, Młocicki et al. (2006) reported 26 nuclei in M. ctenoides. It would appear that there are major differences between cestode species with respect to the cellular contents of oncospheres (Collin, 1969; S´widerski, 1972, 1983; S´widerski and Tkach, 1997, 2002; Młocicki et al., 2006), even within the same family, Taeniidae. It could be argued that the procedures used in the work described here with T. ovis had the potential to underestimate the number of nuclei. The sequential 90 nm sections were collected and processed at intervals of 1 lm. Three sections were collected at each micrometer interval to minimise artefacts arising during the processing of the specimen, as it is common for either whole individual sections or parts of sections, to disappear from the sample grid. In this case, if the most distant numbers of two section sets were used in the ultrastructural reconstruction, these would have been a maximum of 1.45 lm apart. Hence structures of this size could theoretically fall completely between two serial sections and not be observed. However, we are confident that comparisons of sections from different oncospheres, sectioned in different planes allowed such potential ambiguities to be resolved unequivocally, adding precision to the ultrastructural reconstruction. The data included here as Fig. 1 is, to our knowledge, the first publication of one such complete series.
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1429
Fig. 8. Schematic diagram of Taenia ovis oncosphere illustrating the symmetry and cellular organisation. Details of abbreviations are given in Fig. 1.
There has been some inconsistency in the use of the term ‘‘oncospheral tegument” in different taeniid species. Nieland (1968) referred to it as a cytoplasmic layer in T. taeniaeformis while others have termed it an embryonic epithelium in T. crassiceps and E. granulosus (Chew, 1983; S´widerski, 1983). However, a consensus has developed over the past few years in favour of the use of the term ‘‘tegument” for a number of cestode species (S´widerski et al., 2001; S´widerski and Tkach, 2002; Młocicki et al., 2006). In the current study, we adopt the term ‘‘tegument” for the covering of the surface of the oncosphere which gives rise to long microvilli, particularly near the somatophore. Microvilli have also been reported on the tegumental surface of oncospheres of T. taeniaeformis by Nieland (1968) and other cestodes (Collin, 1968; Młocicki et al., 2006). In T. ovis oncospheres, the tegument is composed of two layers i.e., a basal lamina and an upper cytoplasmic layer. The basal lamina gives rise to many infoldings into the upper cytoplasmic layer. Similar infoldings had been reported for other cestodes, H. citelli (Collin, 1968) and Paricterotaenia porosa (Gabrion, 1981), but not previously for a taeniid cestode.
In T. ovis, an additional membrane covers the somatophore of the oncosphere just above the microvilli of the tegument. This is the so-called hook-region membrane which was described for the first time in C. pusilla (S´widerski, 1972) and subsequently in other species such as Anoplocephaloides dentata (S´widerski et al., 2001) and M. ctenoides (Młocicki et al., 2006), but has not been recorded previously in Taenia spp. A binucleate medullary cell was observed in E. granulosus oncospheres by S´widerski (1983). In T. ovis, two similar cells were identified; however, these were not binucleate and hence the term median somatophoric cell was adopted for this cell type in T. ovis. These cells have granules in their cytoplasm, suggesting they might be involved in secretory activity. Two nerve cells were observed in T. ovis oncospheres with their identification being based on the presence of characteristic granules. Initially these cells were identified as a third type of penetration gland cell in E. granulosus (S´widerski, 1983). However, Fairweather and Threadgold (1981) had described that cytoplasmic granules in these cells stained positively for paraldehyde-fuchsin (a stain for neurosecretory material) and subsequently these have
1430
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
been regarded as nerve cells in E. granulosus and Echinococcus multilocularis (Z. S´widerski, unpublished data). Somatic and germinative cells are regarded as being distributed symmetrically in the oncospheres of various cestode species (S´widerski, 1983; Młocicki et al., 2006). In relation to the number of these cell types, S´widerski (1983) described 34 muscle cells (16 somatic and 18 hook muscle cells) in E. granulosus oncospheres. However, in T. ovis, only five pairs of somatic cells with myocytons were associated with the somatic musculature. We were not able to identify any cell nuclei associated with the hook muscles. Only one pair of germinative cells was observed in T. ovis oncospheres in contrast with the five pairs of those in E. granulosus (S´widerski, 1983). In T. ovis the germinative cells are located in at either the dorsal or ventral pole. It was not possible to determine the curvature of the medial hooks in the sectioned oncospheres and hence it was not possible to designate either pole specifically according to the terminology developed by S´widerski (1983), which would have allowed us to be more specific about the location of the germinative cells. In this respect however, it appears that T. ovis is different to some other cestode species in which the germinative cells have been described as being located at the mesophore (Ogren, 1971; S´widerski, 1983). In various cestode species, glandular structures have been identified in oncospheres and referred to as penetration glands. The cells associated with these glands have been referred to as being binucleated (S´widerski et al., 2001; Młocicki et al., 2006), tetranucleated, hexa-nucleated (S´widerski and Tkach, 2002), multicellular (Chew, 1983), or unicellular (S´widerski et al., 2001; S´widerski and Tkach, 2002). In T. ovis oncospheres, we distinguished three different types of gland cells. Two of these gland cells (PG1 and PG2) contained four nuclei each, while the third (MMGC) had only one nucleus. S´widerski (1983) described two types of penetration gland cells in E. granulosus with both PG1 and PG2 cells being syncytial; the PG1 cell having four nuclei and the PG2 cell being binucleate. In T. ovis, four nuclei were found in the PG1 cell (the cell identified on the basis of its characteristic cytoplasmic granules). The four nuclei are associated with discrete cell bodies which are separated by cell membranes and distributed bilaterally in pairs, but having the various cell bodies connected by a narrow isthmus. The PG2 cell in T. ovis was found to contain four nuclei in contrast with E. granulosus which was described as binucleate (S´widerski, 1983). It is possible that this discrepancy may be due to ongoing nuclear division even at this late stage of oncosphere development but further investigations are required to test this hypothesis. The PG1 cellular components were found to be distributed bilaterally in T. ovis oncospheres, with the components connected by narrow cytoplasmic bridges, some of which appeared to extend as processes through the intervening PG2 cell (see Fig. 1D, E, G and H, the cross-sections of PG1). In activated hexacanths of T. ovis, Harris et al. (1987) reported that there were four ‘‘lobes” of a penetration gland cell each containing one nucleus. Inconsistency between our observations and those of Harris et al. (1987) may be due to differences in the methodologies used in the two studies. The cytoplasm of the penetration gland cells is filled with characteristic granules. We observed that the secretory granules of all glandular cells present in T. ovis oncospheres were of different sizes, shapes and electron densities. Previously, Harris et al. (1987), Fairweather and Threadgold (1981), Schramlova and Blazek (1982) and more recently, S´widerski and Tkach (2002) and Młocicki et al. (2006) also reported the different types of granules based on their shapes and electron densities. Secretions from penetration glands are believed to be involved in the transfer of granules from the penetration gland cells to the external cytoplasm of the oncospheral tegument and many authors have referred to ducts connecting the penetration gland(s) with the
tegument (Fairweather and Threadgold, 1981; Harris et al., 1987; Młocicki et al., 2006). In addition, it is believed that penetration gland ducts are usually supported by microtubules (Collin, 1969; Lethbridge and Gijsbers, 1974). We did observe structures surrounded by microtubules in non-activated oncospheres of T. ovis (Fig. 7). However, no unequivocal evidence was found which would confirm the hypothesis that these were ducts from the penetration gland cells. Harris et al. (1987) reported that they observed ducts in activated hexacanths of T. ovis. Use of the term ‘‘duct” in relation to oncosphere structures may be problematic and inaccurate. A thin cytoplasmic process (e.g., ‘‘internuncial processes” of adult cestode tegument) could carry intracellular granules from one cytoplasmic compartment to another as occurs in many taxa of animals. A ‘‘duct” comprises a cellular or syncytial tube enclosing an extracellular lumen through which materials pass extracellularly (but not typically through the extracellular matrix). In many animals, materials can even be transported through the extracellular matrix, after being secreted from the cells. This route of passage also would not be properly called a ‘‘duct”. It is not clear whether the precise use of the term ‘‘duct” in relation to structures in oncospheres has been consistent with the understood cytological use of the term. A number of research workers have proposed hypotheses for the mechanism of secretion from the penetration glands with secretion regarded as being merocrine (Lethbridge and Gijsbers, 1974), apocrine (Furukawa et al., 1977; Młocicki et al., 2006) and holocrine (Fairweather and Threadgold, 1981). Based on our studies of activated hexacanths of T. ovis (Jabbar et al., unpublished data) we hypothesise that the contents of penetration glands of T. ovis oncospheres may be secreted by an apocrine mode of secretion. Silverman (1955) speculated on the potential antigenic nature of penetration gland secretions, but there has been no direct evidence to support this hypothesis. Recently, Jabbar et al. (2010) have demonstrated the presence of host-protective antigens within what appeared to be penetration gland cells in T. ovis oncospheres. The present study identifies the antigen-containing cells described by Jabbar et al. (2010) as being the PG1 cell suggesting that this cell type may be responsible for the production of hostprotective antigens in T. ovis and probably in other taeniid oncospheres. However, comprehensive studies on ultrastructural immunolocalisation of host-protective antigens of taeniid oncospheres would be required to test this hypothesis. Acknowledgement Funding from National Health and Medical Research Council, Australia (Grants 350279, 400109 and 628320) is acknowledged. References Chew, M.W., 1983. Taenia crassiceps: ultrastructural observations on the oncosphere and associated structures. J. Helminthol. 57, 101–113. Collin, W.K., 1968. Electron microscope studies of the muscle and hook systems of hatched oncospheres of Hymenolepis citelli McLeod, 1933 (Cestoda: Cyclophyllidea). J. Parasitol. 54, 74–88. Collin, W.K., 1969. The cellular organization of hatched oncospheres of Hymenolepis citelli (Cestoda, Cyclophyllidea). J. Parasitol. 55, 149–166. Coman, B.J., Rickard, M.D., 1975. The location of Taenia pisiformis, Taenia ovis and Taenia hydatigena in the gut of the dog and its effect on net environmental contamination with ova. Z. Parasitenkd. 47, 237–248. Conn, D.B., 1993. The biology of flatworms (Platyhelminthes): parenchyma cells and extracellular matrices. Trans. Am. Microsc. Soc. 112, 241–261. Conn, D.B., S´widerski, Z., 2008. A standardised terminology of the embryonic envelopes and associated developmental stages of tapeworms (Platyhelminthes: Cestoda). Folia Parasitol. 55, 42–52. Engelkirk, P.G., Williams, J.F., 1982. Taenia taeniaeformis (Cestoda) in the rat: ultrastructure of the host–parasite interface on days 1 to 7 postinfection. J. Parasitol. 68, 620–633.
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431 Engelkirk, P.G., Williams, J.F., 1983. Taenia taeniaeformis (Cestoda) in the rat: ultrastructure of the host–parasite interface on days 8 to 22 postinfection. J. Parasitol. 69, 828–837. Fairweather, I., Threadgold, L.T., 1981. Hymenolepis nana: the fine structure of the ‘penetration gland’ and nerve cells within the oncosphere. Parasitology 82, 445–458. Furukawa, T., Miyazato, T., Okamoto, K., Nakai, Y., 1977. The fine structure of the hatched oncopsheres of Hymenolepis nana. Jpn. J. Parasitol. 26, 49–62. Gabrion, C., 1981. Recherches sur l’oncosphère des cestodes: origine et formation de la calotte recouvrant les crochets. Z. Parasitenkd. 65, 191–205. Gemmell, M.A., 1962. Natural and acquired immunity factors inhibiting penetration of some hexacanth embryos through the intestinal barrier. Nature 194, 701–702. Gemmell, M.A., 1964. Species specificity of the immunogenic complexes of the tapeworm hexacanth embryo. Nature 204, 705–707. Harris, A., Heath, D.D., Lawrence, S.B., Shaw, R.J., 1987. Ultrastructure of changes at the surface during the early development phases of Taenia ovis cysticerci in vitro. Int. J. Parasitol. 17, 903–910. Harris, A., Heath, D.D., Lawrence, S.B., Shaw, R.J., 1989. Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro. Int. J. Parasitol. 19, 621–629. Heath, D.D., Smyth, J.D., 1970. In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis from oncosphere to cystic larva. Parasitology 61, 329–343. Heath, D.D., Lawrence, S.B., 1976. Echinococcus granulosus: development in vitro from oncosphere to immature hydatid cyst. Parasitology 73, 417–423. Holcman, B., Heath, D.D., Shaw, R.J., 1994. Ultrastructure of oncosphere and early stages of metacestode development of Echinococcus granulosus. Int. J. Parasitol. 24, 623–635. Inatomi, S., 1962. Submicroscopic structure of the egg shell of helminths. Okayama Igakkai Zasshi 74, 31–81. Jabbar, A., Kyngdon, C.T., Gauci, C.G., Walduck, A.K., McCowan, C., Jones, M.K., Beveridge, I., Lightowlers, M.W., 2010. Localisation of three host-protective oncospheral antigens of Taenia ovis. Int. J. Parasitol. 40, 579–589. Janicki, K., 1907. Über die embryonalentwicklung von Taenia serrata Goeze. Z. Wiss. Zool. 87, 685–724. Johnson, K.S., Harrison, G.B., Lightowlers, M.W., O’Hoy, K.L., Cougle, W.G., Dempster, R.P., Lawrence, S.B., Vinton, J.G., Heath, D.D., Rickard, M.D., 1989. Vaccination against ovine cysticercosis using a defined recombinant antigen. Nature 338, 585–587. Kyngdon, C.T., Gauci, C.G., Rolfe, R.A., Velasquez Guzman, J.C., Farfan Salazar, M.J., Verastegui Pimentel, M.R., Gonzalez, A.E., Garcia, H.H., Gilmanl, R.H., Strugnell, R.A., Lightowlers, M.W., 2006. In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium. J. Parasitol. 92, 273–281. Lethbridge, R.C., Gijsbers, M.F., 1974. Penetration gland secretion by hexacanths of Hymenolepis diminuta. Parasitology 68, 303–311. Lightowlers, M.W., Mitchell, G.F., Bowtell, D.D., Anders, R.F., Rickard, M.D., 1984. Immunization against Taenia taeniaeformis in mice. studies on the characterization of antigens from oncospheres. Int. J. Parasitol. 14, 321–333. Lightowlers, M.W., 2006. Cestode vaccines: origins, current status and future prospects. Parasitology 133 (Suppl.), S27–S42.
1431
Młocicki, D., S´widerski, Z., Miquel, J., Eira, C., Conn, D.B., 2006. Cellular organization of the oncosphere of Mosgovoyia ctenoides (Cestoda: Anoplocephalidae). J. Parasitol. 92, 953–961. Morseth, D.J., 1965. Ultrastructure of developing taeniid embryophores and associated structures. Exp. Parasitol. 16, 207–216. Nieland, M.L., 1968. Electron microscope observations on the egg of Taenia taeniaeformis. J. Parasitol. 54, 957–969. Ogren, R.E., 1971. Criteria for anterior polarity and symmetry in tapeworm hexacanth embryos. Proc. Penn. Acad. Sci. 45, 40–41. Race, G.J., Martin, J.H., Larsh, J.E., Esch, G.W., 1966. A study of the adult stage of Taenia multiceps (Multiceps serialis) by electron microscopy. J. Elisha Mitchell Sci. Soc. 82, 44–57. Sato, T., 1968. A modified method for lead staining of thin sections. J. Electron Microsc. 17, 158–159. Schramlova, J., Blazek, K., 1982. Ultrastructure of the hatched and unhatched oncospheres of Taenia saginata. Folia Parasitol. 29, 45–50. Schramlova, J., Blazek, K., Marhoul, Z., Singhvi, A., 1984. Ultrastructure of Taenia saginata oncospheres cultivated in artificial media. Folia Parasitol. 31, 247–251. Silverman, P.H., 1954. Studies on the biology of some tapeworms of the genus Taenia II. The morphology and development of the taeniid hexacanth embryo and its enclosing membranes, with some notes on the state of development and propagation of gravid segment. Ann. Trop. Med. Parasitol. 48, 356–366. Silverman, P.H., 1955. A technique for studying the in vitro effect of serum on activated taeniid hexacanth embryos. Nature 176, 598–599. Skvortzov, A.A., 1942. Egg structure of Taeniarhynchus saginata and its control. Zool. Zhurnal. 21, 10–18. S´widerski, Z., 1972. La structure fine de l’oncosphere du cestode Catenotaenia pusilla (Goeze, 1782) (Cyclophyllidea: Catenotaenidae). Cellule 69, 207–237. S´widerski, Z., 1983. Echinococcus granulosus: hook–muscle systems and cellular organisation of infective oncospheres. Int. J. Parasitol. 13, 289–299. S´widerski, Z., Tkach, V.V., 1997. Ultrastructural studies on the cellular organisation of the oncospheres of the nematotaeniid cestode, Nematotaenia dispar (Goeze, 1782). Acta Parasitol. 42, 158–167. S´widerski, Z., Tkach, V.V., 1999. Electronmicroscopical studies on the cellular organisation of the oncospheres of the hymenolepidid cestode, Staphylocystoides stefanskii (Zarnowski, 1954). Acta Parasitol. 44, 31–38. S´widerski, Z., Ndiaye, P.I., Miquel, J., Tkach, V.V., Marchand, B., Chomicz, L., GrytnerZiecina, B., Sereda, M.J., 2001. Ultrastructural study of the embryonic development of the anoplocephalid cestode Anoplocephaloides dentata, an intestinal parasite of Arvicolidae rodents. II. Differentiation and cellular organisation of the oncosphere. Acta Parasitol. 46, 171–185. S´widerski, Z., Tkach, V.V., 2002. Ultrastructure of embryonic development of Inermicapsifer madagascariensis (Cestoda, Anoplocephalidae) with emphasis on the cellular organisation of the infective eggs. Acta Parasitol. 47, 105–120. S´widerski, Z., Mackiewicz, J.S., 2004. Ultrastructural studies on the cellular organisation of the coracidium of the cestode Bothriocephalus clawibothrium Ariola, 1899 (Pseudophyllidea; Bothriocephalidae). Acta Parasitol. 49, 116–139. S´widerski, Z., Miquel, J., Młocicki, D., Georgiev, B.B., Eira, C., Grytner-Ziecina, B., Feliu, C., 2007. Post-embryonic development and ultrastructural characteristics of the polycephalic larvae of Taenia parva Baer, 1926 (Cyclophyllidea, Taeniidae). Acta Parasitol. 52, 31–50. Wardle, R.A., McLeod, J.A., 1952. The Zoology of Tapeworms. University of Minnesota Press, Minneapolis, USA.
Contents lists available at ScienceDirect
International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Ultrastructural reconstruction of Taenia ovis oncospheres from serial sections Abdul Jabbar a,*, Simon Crawford b, Daniel Młocicki c,d, Zdzisław P. S´widerski c,e, David B. Conn f, Malcolm K. Jones g,h, Ian Beveridge a, Marshall W. Lightowlers a a
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia School of Botany, The University of Melbourne, Parkville, Victoria 3010, Australia c Witold Stefanski Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warsaw, Poland d Medical University of Warsaw, Department of Medical Biology, 73, Nowogrodzka Street, 02-018 Warsaw, Poland e Medical University of Warsaw, Department of General Biology and Parasitology, 5 Chalubinskiego Street, 02-004 Warsaw, Poland f School of Mathematical and Natural Sciences, Berry College, Mount Berry, GA 30149-5036, USA g School of Veterinary Sciences, The University of Queensland, Brisbane, Queensland 7072, Australia h Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia b
a r t i c l e
i n f o
Article history: Received 1 April 2010 Received in revised form 22 April 2010 Accepted 26 April 2010
Keywords: Taenia ovis Non-activated oncosphere Ultrastructure Serial sections Transmission electron microscopy
a b s t r a c t The cellular organisation of Taenia ovis oncospheres is interpreted from ultrathin serial sections and transmission electron microscopy following high pressure freezing and freeze-substitution. The surface of a hatched, non-activated T. ovis oncosphere is covered by an oncospheral membrane below which is the tegument bearing microvilli. The basal lamina of the tegument is underlain by broad bands of peripheral somatic musculature. Three pairs of hooks and associated muscles are present in the somatophoric third of the oncosphere. Approximately 19 cells of seven different types were identified which include: (i) a quadri-nucleated syncytium of penetration gland type 1 containing two lateral pairs of cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) a single-nucleated median mesophoric gland cell; (iv) 10 somatic cells; (v) two germinative cells; (vi) two nerve cells; and (vii) a pair of median somatophoric cells. This study provides a clear understanding of the morphology of T. ovis oncospheres and forms the basis for further investigations into the biology of taeniid oncospheres. Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Taenia ovis is a taeniid cestode parasite infecting sheep and goats as intermediate hosts and canids as definitive hosts. Taenia ovis has played a prominent role in the study of immunity to cestode infections (Gemmell, 1962, 1964), particularly following its use in the development of the first recombinant anti-parasite vaccine (Johnson et al., 1989; Lightowlers, 2006). The host-protective antigens of T. ovis are uniquely associated with the oncosphere, which is the infective life cycle stage present within the parasite’s eggs (Lightowlers, 2006). The ultrastructural location of the hostprotective antigens is unknown and the available information on the structure of the T. ovis oncospheres is insufficient to allow accurate interpretation of the antigens’ localisation (Jabbar et al., 2010). The studies reported here sought to improve our understanding of the biology of T. ovis oncospheres by providing a detailed description of the parasite’s ultrastructure. A number of studies have been undertaken which describe the structure of taeniid (Platyhelminthes: Cestoda: Taeniidae) eggs * Corresponding author. Tel.: +61 (0) 3 9731 2022; fax: +61 (0) 3 9741 5461. E-mail address: [email protected] (A. Jabbar).
and/or oncospheres using light microscopic methods, including Taenia pisiformis (Janicki, 1907; Heath and Smyth, 1970); Taenia saginata (Skvortzov, 1942; Silverman, 1954), Taenia taeniaeformis (Wardle and McLeod, 1952; Heath and Smyth, 1970), T. ovis (Heath and Smyth, 1970), Taenia hydatigena (Heath and Smyth, 1970) and Echinococcus granulosus (Heath and Smyth, 1970; Heath and Lawrence, 1976). Following the advent of electron microscopy, further investigations were undertaken on oncosphere structure in taeniid cestodes including Taenia crassiceps (Chew, 1983), Taenia multiceps (Race et al., 1966), T. ovis (Harris et al., 1987), Taenia parva (S´widerski et al., 2007), T. saginata (Schramlova and Blazek, 1982; Schramlova et al., 1984), T. taeniaeformis (Nieland, 1968; Engelkirk and Williams, 1982, 1983) and E. granulosus (S´widerski, 1983; Harris et al., 1989; Holcman et al., 1994). The covering of the taeniid egg after it leaves the proglottid, the embryophore, has also been the subject of interest in a number of investigations (Inatomi, 1962; Morseth, 1965; Race et al., 1966). All of these studies describe the structure of oncosphere of the respective taeniid species based on sections taken at random, with the exception of S´widerski (1983) who described the structure of E. granulosus oncospheres based on ultrathin serial sections dealing mainly with the hook– muscle system. The cellular composition of oncospheres in other
0020-7519/$36.00 Ó 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2010.04.011
1420
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
cestodes has also been studied including Hymenolepis citelli (Collin, 1969), Catenotaenia pusilla (S´widerski, 1972), Nematotaenia dispar (S´widerski and Tkach, 1997), Staphylocystoides stefanskii (S´widerski and Tkach, 1999), Inermicapsifer madagascariensis (S´widerski and Tkach, 2002), Bothriocephalus clavibothrium (Swiderski and Mackiewicz, 2004) and Mosgovoyia ctenoides (Młocicki et al., 2006) based on ultrathin serial sectioning. The only published report on the ultrastructure of T. ovis oncospheres is that of Harris et al. (1987). These authors investigated activated hexacanths (terminology adopted after Conn and S´widerski, 2008) and metacestodes cultured in vitro, and observed that there were four lobes and four nuclei in the penetration glands and that each of these lobes was filled with granules of varying electron densities. The study utilised sections taken at random rather than serial sections. Two reports describe the ultrastructure of activated hexacanths of E. granulosus and metacestodes cultured in vitro (Harris et al., 1989; Holcman et al., 1994), again based on examination of random sections. Although random sections allow general features of oncospheres to be described, reconstructions based on serial ultrathin sections are required to obtain comprehensive ultrastructural details about the relationship between various structures and the number of different cell types. Here we describe a comprehensive study using ultrathin serial sections and reconstruction of hatched, non-activated oncospheres of T. ovis. 2. Materials and methods 2.1. Collection of parasite material Taenia ovis was maintained in dogs and sheep as described by Coman and Rickard (1975) and Kyngdon et al. (2006). All of the procedures were performed in accordance with the requirements of the University of Melbourne Animal Ethics Committee. 2.2. Collection and hatching of oncospheres Mature eggs were collected from the terminal gravid proglottids of the adult worm which was obtained by purging an experimentally infected dog. Non-activated oncospheres were collected following hatching of the mature eggs as described by Lightowlers et al. (1984). This method has the advantage over conventional techniques (use of artificial gastric and intestinal fluids) as the immature oncospheres dissolve in sodium hypochlorite and only mature oncospheres are retained after the dissolution of embryophores (Lightowlers et al., 1984). In brief, sodium hypochlorite (1.25%) was used to obtain non-activated oncospheres from the eggs. Approximately 5–7 ml of sodium hypochlorite was added to a pre-determined number of eggs (400,000–500,000) and the suspension was mixed gently with a Pasteur pipette. The hatching process was monitored while mixing by examining oncospheres under a microscope. Following adequate hatching of eggs (80– 90%), the reaction was stopped by adding excess water (Milli-Q, Millipore, NSW, Australia). The hatched oncospheres were washed (centrifugation 194g, 10 min) at least five times with pre-heated (37 °C) RPMI-1640 (Invitrogen, Vic, Australia). These hatched but non-activated oncospheres were then processed for ultrastructural studies. 2.3. Cryofixation of oncospheres Oncospheres were gently pelleted (centrifugation 194g, 10 min) and the supernatant discarded. Approximately 2–3 lL droplets of the suspension were sandwiched between type A brass freezer hats (ProSciTech, Thuringowa, Qld, Australia). The enclosed onco-
sphere suspensions were frozen using a Leica EM High Pressure Freezer (Vienna, Austria). The freezer hats enclosing the frozen oncosphere suspensions were split apart and stored in liquid N2 in cryo-vials prior to freeze-substitution in a Leica EM automated freeze-substitution unit. 2.4. Freeze-substitution Frozen oncosphere pellets were freeze-substituted in 0.1% uranyl acetate in acetone at 90 °C for 48 h and the temperature raised to 50 °C at 6 °C/h. Pellets were scraped out of the freezer hats, rinsed in three 30 min changes of acetone and infiltrated with a graded series of Lowicryl HM20 low temperature resins (Polysciences, Warrington, PA, USA) in acetone consisting of 25% (8 h), 50% (overnight), 75% (8 h) and 100% resin (overnight). The infiltrated samples were placed in a fresh change of 100% resin in gelatin capsules, polymerised under UV light for 48 h at 50 °C, and brought to room temperature at 6 °C/h. The soft sample blocks were then hardened under UV light for a further 24 h at room temperature. 2.5. Transmission electron microscopy Oncospheres embedded in blocks were sectioned with a diamond knife on a Leica Ultracut R microtome (Vienna, Austria). For ultrathin serial sections, 90 nm thick sections were cut throughout the oncospheres and three consecutive sections in sequence, taken at 1 lm intervals, were selected for further processing. All of these sections were collected onto pioloform-coated slot copper grids (ProSciTech, Thuringowa, Qld, Australia). The grids were blot dried overnight on filter paper prior to staining. Ultrathin serial sections were double stained with 2% uranyl acetate for 10 min and Triple Lead Stain for 5 min (Sato, 1968) and viewed in a Phillips CM120 Biotwin transmission electron microscope at 120 kV. Images were captured with a Gatan Multiscan 600CW digital camera (Gatan Inc., CA, USA) using the appropriate software. For the purpose of orientation, the terminology proposed by Ogren (1971) and S´widerski (1983) was followed where the hook region is referred to as the anterior pole or somatophore, and the opposing posterior pole being the mesophore. 3. Results 3.1. Cellular composition of the oncospheres The general topography and symmetry of cellular organisation of the hatched non-activated oncospheres of T. ovis is revealed by the serial sections shown in Fig. 1. Taenia ovis oncospheres consist of approximately 19 cells and their arrangement was bilaterally symmetrical in a plane running in an anterio-posterior direction and through the dorsal and ventral poles. Based on the analysis of ultrathin serial sections of 15 T. ovis oncospheres, seven different cell types were distinguished: (i) a quadri-nucleated syncytium of penetration gland type 1 containing four cell bodies interconnected by narrow cytoplasmic bridges (PG1); (ii) a quadri-nucleated syncytium of penetration gland type 2 (PG2); (iii) singlenucleated median mesophoric gland cell (MMGC); (iv) 10 somatic cells representing myocytons; (v) two germinative cells; (vi) two nerve cells in the somatophore; and (vii) a pair of median somatophoric cells. 3.2. Structures associated with membranes and hook region The outermost layer in hatched, non-activated T. ovis oncospheres is the oncospheral membrane (Fig. 2A and B). The next
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1421
Fig. 1. Selected ultrathin serial (1 lm apart) sections (A–L) showing the oncospheral and hook-region membranes, tegument, different cell types and hooks of a single Taenia ovis oncosphere. Note the major portion of the oncosphere occupied by the penetration gland cells. In all figures: BL, basal lamina; D1, duct-like structures of penetration gland type 1; D2, duct-like structures of penetration gland type 2; DMSC, duct of median somatophoric cell; GC, germinative cell; GER, granular endoplasmic reticulum; H, hook; HM, hook muscle; HC, heterochromatic; HD, hemidesmosomes; HRM, hook-region membrane; M, microtubules; MSC, median somatophoric cell; MMGC, median mesophoric gland cell; MT, mitochondria; Mv, microvilli; N, nucleus; NC, nerve cell; NCP, nerve cell process; NSG, neurosecretory granules; OM, oncospheral membrane; OT, oncospheral tegument; PG1, penetration gland type 1; PG2, penetration gland type 2; PM, plasma membrane; SC, somatic cell; SG1, secretory granules from penetration gland 1; SG2,secretory granules from penetration gland 2; SM, somatic muscle; 1, 2 and 3, three different layers of a hook.
1422
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 1 (continued)
layer is the oncospheral tegument which is anucleated and possesses characteristic microvillar projections on its surface (Fig. 2A). At the somatophore, these microvilli are particularly long
and are surrounded by the hook-region membrane which contains several laminae (Fig. 2B). The oncospheral tegument comprises two layers: (i) a basal lamina which is thinner, amorphous and less
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1423
Fig. 2. Transmission electron micrographs showing the details of oncospheral membranes and hooks. (A) High magnification image of the details of the oncospheral membrane, tegument and underlying somatic muscles. (B) Part of the somatophore of the oncosphere showing the ultrastructural details of the oncospheral tegument and hook-region membrane. Note the projections of basal lamina (arrowheads) and electron-dense inclusions of the tegument (arrows). (C) High magnification image of crosssection of hooks showing different layers of the hooks (1–3) and hook muscles. (D) An oblique section of the hook showing hook hemidesmosomes. (E) Longitudinal section of the hook showing its different parts. Details of abbreviations are given in Fig. 1.
electron-dense; and (ii) a thicker, electron-dense outer layer containing electron-dense inclusions (Fig. 2B). The basal lamina digitates into the upper tegumental layer near the hook-region membrane (Fig. 2B) and it is underlain by broad bands of the peripheral, somatic musculature (Fig. 2A). The somatophoric pole of the oncosphere is occupied by hooks, somatic cells, nerve cells and a pair of median somatophoric cells. There are three pair of hooks, one medial pair and two lateral ones (Fig. 1C–H) which are interconnected by a complex hook–muscle system (Figs. 1F and G; 2C and D). A hook consists of a base which is composed of a guard or collar and handle and a blade projecting out from the oncosphere. Near the guard, the blade of the hook is surrounded by a hook hemidesmosome (Conn, 1993), by which it is attached in the sheath (Fig. 2D). The hook muscles are attached to the guard (or collar) and base and these do not terminate directly on the hook, but are embedded into the basal lamina surrounding the base of the hook (Fig. 2D). In sections, the hooks appear as heterogeneous structures consisting of three layers of different electron densities (Fig. 2C–E). Highly electron-dense material forms the outermost covering or cortex, which is underlain by a thin layer comprised of discontinuous blocks of decreased electron opacity. The innermost layer or core consists of amorphous and moderately electron-dense material (Fig. 2D). A pair of nerve cells occupies the space between the medial and lateral hooks in the somatophoric pole of the oncosphere (Figs. 1D; 3A). The nerve cells have pro-
cesses which extend to various cells and these processes are numerous in the hook-region (Figs. 1C–G; 2D and E; 3A and C). The nerve cells are characterised by the presence of membranebound dense-cored neurosecretory granules in their cytoplasm (Fig. 3A and B). The two median somatophoric cells are located in the middle of the oncosphere between the hook region and the PG2 cell (Figs. 1F and G; 3C and D). These cells contain irregularly-shaped nuclei and have relatively little cytoplasm containing granules (Fig. 3E and F). Each median somatophoric cell has a duct which runs laterally separating the hook region (above) and the PG2 cell (below) (Fig. 3E). 3.3. Gland cells Three different types of glandular cells are present in oncospheres of T. ovis, based on their different locations and the characteristics of their granules. There is a syncytial PG1 cell containing what appears to be four interconnected cell bodies arranged with one pair located towards the dorsal pole and one located towards the ventral pole. The separate pairs of cell bodies are connected via a narrow isthmus which takes a path towards the mesophore, forming a U-shape (Figs. 1A, B and H–L, 4A, E and F). Each cell body is demarcated from its neighbour by a clearly defined plasma membrane (Fig. 4A–C). The PG1 cell bodies do not have a specific shape and contain various cytoplasmic processes emanating in dif-
1424
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 3. Transmission electron micrographs showing the details of nerve and median somatophoric cells. (A) A nerve cell showing the presence of characteristic granules in its cytoplasm. (B) High magnification image of a nerve cell process containing characteristic neurosecretory granules. (C) A pair of median somatophoric cells lying between the hook region and the penetration gland type 2 cell. (D) High magnification image of median somatophoric cells. (E) A median somatophoric cell showing its lateral duct (arrows). (F) High magnification image of a median somatophoric cell showing its nucleus, granular endoplasmic reticulum and granules (arrowheads). Details of abbreviations are given in Fig. 1.
ferent directions (Figs. 1A and I; 4A) which inter-digitate through the centrally located PG2 cell (see Fig. 1D, E, G, H, the cross-sections of PG1). The major part of each PG1 cell body is occupied by a large and irregularly-shaped nucleus containing islands of heterochromatin (Figs. 1A; 4A and B), perinuclear material rich in free ribosomes, a well-developed granular endoplasmic reticulum and numerous mitochondria (Fig. 4B). The granules of these cells are round to rod-shaped and have a well defined membrane containing amorphous electron-dense material (Fig. 4C and D). Typi-
cally, these granules lie individually in the tightly packed cytoplasm. The second type of gland cell (PG2) is also syncytial and occupies 50–60% of the hexacanth body (Fig. 1D–I). This cell contains four nuclei arranged in two lateral pairs (Fig. 5A). Evidence supporting this conclusion is based on images showing pairs of nuclei in very close proximity while other images show nuclei in the same cell but widely separated (Fig. 1C–F). No single image was obtained in which three or all four of the nuclei could be seen in the same
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1425
Fig. 4. Transmission electron micrographs showing the ultrastructural details of the penetration gland type 1 (PG1) cell. (A) Low magnification image of a lateral pair of the PG1 cell bodies showing their processes. (B) High magnification image of a PG1 cell body showing its nucleus and the perinuclear space filled with ribosomes, granular endoplasmic reticulum and mitochondria. (C) High magnification image of PG1 cell bodies showing that two neighbouring nuclei are separated from each other by a plasma membrane. (D) The cytoplasm of the PG1 cell containing characteristic secretory granules. (E) Low magnification image of an oncosphere showing the connection between two lateral pairs of PG1 cell bodies (arrows). (F) High magnification image of the connecting duct (isthmus) between PG1 cell bodies (arrows). Details of abbreviations are given in Fig. 1.
section. The size of the PG2 cell nucleus is smaller than that of the PG1 cell and contains heterochromatin islands (compare Figs. 4B and 5A). The nucleus is surrounded by a rough syncytial cytoplasm rich in free ribosomes, granular endoplasmic reticulum and numerous vesicles (Fig. 5A). Unlike the PG1 cell, there are no mitochondria in the perinuclear area and the cytoplasm of the PG2 cell is filled with granules (Fig. 5B). The granules of PG2 cells are larger,
less electron-dense and sparser than those in the cytoplasm of the PG1 cell (Fig. 5B). The PG1 and PG2 cells are separated from each other, at certain levels, by somatic muscles (Fig. 5C). A novel, possibly third type of gland-like cell (MMGC) is located centrally at the mesophore just underneath the oncospheral tegument and between two somatic cells (Figs. 1E–H; 6A–C). The MMGC is surrounded by the processes of another cell (Fig. 6D)
1426
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 5. Transmission electron micrographs showing the ultrastructural details of penetration gland type 2 (PG2) cell, somatic and germinative cells. (A) Note that the nuclei of the PG2 cell lie together without a plasma membrane and the perinuclear space is filled with free ribosomes, granular endoplasmic reticulum and vesicles. (B) High magnification image of the secretory granules of the PG2 cell. (C) Low magnification of an oncosphere showing the separation of PG1 and PG2 cells through a layer of somatic muscles. (D) Low magnification image of a somatic cell showing its nucleus and long lateral processes of myofibrils. (E) High magnification image of a somatic cell showing a cross-section of muscle fibres in its cytoplasm. (F) A germinative cell showing its characteristic large nucleus and granular cytoplasm containing mitochondria. Details of abbreviations are given in Fig. 1.
which has more regular-shaped less electron-dense granules (Fig. 6F) and it seems that the shape and electron density of these granules are similar to those of median somatophoric cells. This uninucleate gland cell is oval (Fig. 6B) and the cytoplasm contains irregularly-sized granules of varying electron densities (Fig. 6E). No duct was seen that clearly connected gland cells and the tegument. However, structures surrounded by microtubules were seen below the tegument between the medial and lateral pairs of hooks
(Fig. 7A). Similar structures were also observed in cross and tangential sections below the tegument posterior to the lateral pair of hooks near PG2 (Fig. 7B and C). 3.4. Cells other than glands There are two main types of cells in T. ovis oncospheres other than the secretory cells. These are the somatic and germinative
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1427
Fig. 6. Transmission electron micrographs showing the ultrastructural details of the median mesophoric gland-like cell (MMGC). (A and B) Low magnification image of the MMGC showing its nucleus and granular cytoplasm. (C) MMGC lies between a pair of somatic cells. (D) MMGC is surrounded by the processes of the median somatophoric cell containing the granules of different electron density. (E) High magnification of MMGC showing electron-dense as well as translucent granules in its cytoplasm. (F) High magnification image of the granules of MMGC and those of median somatophoric cell wrapping it. Details of abbreviations are given in Fig. 1.
cells. Somatic cells, which are most numerous, are located mainly in the somatophoric part of the hexacanth (Fig. 1B, E and H). These cells are myocytons, characterised by a direct connection with muscle fibres of both somatic and hook musculature (Fig. 5D and E). Somatic cells contain oval or spherical nuclei having abundant heterochromatin islands. The nucleus of a somatic cell is surrounded by a thin layer of cytoplasm (Fig. 5D and E). The two germinative cells are larger than the somatic cells and located in close proximity at either the dorsal or ventral pole (it was not possible to determine in which particular pole the cells were located). These cells have a higher nucleocytoplasmic ratio than somatic cells
(compare Fig. 5D, E and F). The nucleus of germinative cells has a large oval nucleus which contains granular nucleoplasm with numerous irregularly-shaped islands of heterochromatin and a relatively thin layer of cytoplasm comprising numerous free ribosomes and mitochondria (Fig. 5F).
4. Discussion A reconstruction of a T. ovis oncosphere is summarised and shown in Fig. 8. Many features seen in T. ovis were consistent with
1428
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
Fig. 7. Transmission electron micrographs showing the ultrastructural details of duct-like structures. (A) Cross-section of a duct-like structure surrounded by microtubules. The structure is located adjacent to the oncospheral tegument, between the medial and lateral pairs of hooks and near the somatophoric pole of the hexacanth. (B) Cross-section of a similar duct-like structure located posterior to the lateral pairs of hooks near the penetration gland type 2 cell (arrow). Inset shows high magnification. (C) Tangential section of a duct-like structure posterior to the lateral pairs of hooks and adjacent to the penetration gland type 2 cell. Details of abbreviations are given in Fig. 1.
the ultrastructure of other cestode oncospheres, but a number of characteristics of T. ovis oncospheres were identified which clearly differed from previous descriptions of oncosphere structures. A single cell not previously described was found in T. ovis and was designated a median mesophoric gland cell. In addition, the characteristics of a previously described oncospheral cell from E. granulosus, the binucleate medullary cell (S´widerski, 1983), were different in T. ovis and this cell type was given the name median somatophoric cells. In other respects (germinative, nerve, penetration gland and somatic cells), the cell types identified in T. ovis oncospheres were consistent with the descriptions of cell types found in other cestode oncospheres (Collin, 1969; Schramlova and Blazek, 1982; S´widerski, 1983; Harris et al., 1989; Holcman et al., 1994; S´widerski and Tkach, 2002; Młocicki et al., 2006) and with the limited previous description of T. ovis oncospheres (Harris et al., 1987). We have chosen to adopt the same nomenclature for the cells as has been used by previous authors in the field. Taenia ovis oncospheres were found to be comprised of seven different cell types (germinative, median mesophoric gland, median somatophoric, nerve, two types of penetration gland and somatic cells). Major differences found in T. ovis were the number of some types of cell and the absence of clear evidence of penetration gland ducts. The number of cells comprising a taeniid oncosphere has only been estimated previously for a single species, E. granulosus (S´widerski, 1983). In E. granulosus 48 cells were enumerated by S´widerski (1983). In T. ovis oncospheres, we identified a total of 19 cells. The definition used here for the identification of a cell was recognition of the cell nucleus; multinucleated cells were counted as a single cell. Complete or near complete sets of serial sections were obtained from 15 different T. ovis oncospheres, providing sections at different orientations through the oncospheres. Comparisons of the results from different oncospheres allowed data on cell numbers, which may be equivocal in one plane of sectioning, to be resolved, for example in cases where two nuclei were in close proximity (Fig. 5A). We observed 25 nuclei (two cells were multinucleated) in total in T. ovis oncospheres. In E. granulosus, S´widerski (1983) found 54 nuclei associated with 48 different cells. A number of studies have described the ultrastructure of non-taeniid oncospheres from ultrathin serial sections (Collin 1969; S´widerski, 1972; S´widerski and Tkach, 1997, 2002; Młocicki et al., 2006). Collin (1969) described 44 nuclei in H. citelli while S´widerski (1972) identified only six nuclei in C. pusilla. Similarly, S´widerski and Tkach (1997, 2002) observed differences in the total number of nuclei in N. dispar (n = 52) and I. madagascariensis (n = 50), respectively. Recently, Młocicki et al. (2006) reported 26 nuclei in M. ctenoides. It would appear that there are major differences between cestode species with respect to the cellular contents of oncospheres (Collin, 1969; S´widerski, 1972, 1983; S´widerski and Tkach, 1997, 2002; Młocicki et al., 2006), even within the same family, Taeniidae. It could be argued that the procedures used in the work described here with T. ovis had the potential to underestimate the number of nuclei. The sequential 90 nm sections were collected and processed at intervals of 1 lm. Three sections were collected at each micrometer interval to minimise artefacts arising during the processing of the specimen, as it is common for either whole individual sections or parts of sections, to disappear from the sample grid. In this case, if the most distant numbers of two section sets were used in the ultrastructural reconstruction, these would have been a maximum of 1.45 lm apart. Hence structures of this size could theoretically fall completely between two serial sections and not be observed. However, we are confident that comparisons of sections from different oncospheres, sectioned in different planes allowed such potential ambiguities to be resolved unequivocally, adding precision to the ultrastructural reconstruction. The data included here as Fig. 1 is, to our knowledge, the first publication of one such complete series.
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
1429
Fig. 8. Schematic diagram of Taenia ovis oncosphere illustrating the symmetry and cellular organisation. Details of abbreviations are given in Fig. 1.
There has been some inconsistency in the use of the term ‘‘oncospheral tegument” in different taeniid species. Nieland (1968) referred to it as a cytoplasmic layer in T. taeniaeformis while others have termed it an embryonic epithelium in T. crassiceps and E. granulosus (Chew, 1983; S´widerski, 1983). However, a consensus has developed over the past few years in favour of the use of the term ‘‘tegument” for a number of cestode species (S´widerski et al., 2001; S´widerski and Tkach, 2002; Młocicki et al., 2006). In the current study, we adopt the term ‘‘tegument” for the covering of the surface of the oncosphere which gives rise to long microvilli, particularly near the somatophore. Microvilli have also been reported on the tegumental surface of oncospheres of T. taeniaeformis by Nieland (1968) and other cestodes (Collin, 1968; Młocicki et al., 2006). In T. ovis oncospheres, the tegument is composed of two layers i.e., a basal lamina and an upper cytoplasmic layer. The basal lamina gives rise to many infoldings into the upper cytoplasmic layer. Similar infoldings had been reported for other cestodes, H. citelli (Collin, 1968) and Paricterotaenia porosa (Gabrion, 1981), but not previously for a taeniid cestode.
In T. ovis, an additional membrane covers the somatophore of the oncosphere just above the microvilli of the tegument. This is the so-called hook-region membrane which was described for the first time in C. pusilla (S´widerski, 1972) and subsequently in other species such as Anoplocephaloides dentata (S´widerski et al., 2001) and M. ctenoides (Młocicki et al., 2006), but has not been recorded previously in Taenia spp. A binucleate medullary cell was observed in E. granulosus oncospheres by S´widerski (1983). In T. ovis, two similar cells were identified; however, these were not binucleate and hence the term median somatophoric cell was adopted for this cell type in T. ovis. These cells have granules in their cytoplasm, suggesting they might be involved in secretory activity. Two nerve cells were observed in T. ovis oncospheres with their identification being based on the presence of characteristic granules. Initially these cells were identified as a third type of penetration gland cell in E. granulosus (S´widerski, 1983). However, Fairweather and Threadgold (1981) had described that cytoplasmic granules in these cells stained positively for paraldehyde-fuchsin (a stain for neurosecretory material) and subsequently these have
1430
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431
been regarded as nerve cells in E. granulosus and Echinococcus multilocularis (Z. S´widerski, unpublished data). Somatic and germinative cells are regarded as being distributed symmetrically in the oncospheres of various cestode species (S´widerski, 1983; Młocicki et al., 2006). In relation to the number of these cell types, S´widerski (1983) described 34 muscle cells (16 somatic and 18 hook muscle cells) in E. granulosus oncospheres. However, in T. ovis, only five pairs of somatic cells with myocytons were associated with the somatic musculature. We were not able to identify any cell nuclei associated with the hook muscles. Only one pair of germinative cells was observed in T. ovis oncospheres in contrast with the five pairs of those in E. granulosus (S´widerski, 1983). In T. ovis the germinative cells are located in at either the dorsal or ventral pole. It was not possible to determine the curvature of the medial hooks in the sectioned oncospheres and hence it was not possible to designate either pole specifically according to the terminology developed by S´widerski (1983), which would have allowed us to be more specific about the location of the germinative cells. In this respect however, it appears that T. ovis is different to some other cestode species in which the germinative cells have been described as being located at the mesophore (Ogren, 1971; S´widerski, 1983). In various cestode species, glandular structures have been identified in oncospheres and referred to as penetration glands. The cells associated with these glands have been referred to as being binucleated (S´widerski et al., 2001; Młocicki et al., 2006), tetranucleated, hexa-nucleated (S´widerski and Tkach, 2002), multicellular (Chew, 1983), or unicellular (S´widerski et al., 2001; S´widerski and Tkach, 2002). In T. ovis oncospheres, we distinguished three different types of gland cells. Two of these gland cells (PG1 and PG2) contained four nuclei each, while the third (MMGC) had only one nucleus. S´widerski (1983) described two types of penetration gland cells in E. granulosus with both PG1 and PG2 cells being syncytial; the PG1 cell having four nuclei and the PG2 cell being binucleate. In T. ovis, four nuclei were found in the PG1 cell (the cell identified on the basis of its characteristic cytoplasmic granules). The four nuclei are associated with discrete cell bodies which are separated by cell membranes and distributed bilaterally in pairs, but having the various cell bodies connected by a narrow isthmus. The PG2 cell in T. ovis was found to contain four nuclei in contrast with E. granulosus which was described as binucleate (S´widerski, 1983). It is possible that this discrepancy may be due to ongoing nuclear division even at this late stage of oncosphere development but further investigations are required to test this hypothesis. The PG1 cellular components were found to be distributed bilaterally in T. ovis oncospheres, with the components connected by narrow cytoplasmic bridges, some of which appeared to extend as processes through the intervening PG2 cell (see Fig. 1D, E, G and H, the cross-sections of PG1). In activated hexacanths of T. ovis, Harris et al. (1987) reported that there were four ‘‘lobes” of a penetration gland cell each containing one nucleus. Inconsistency between our observations and those of Harris et al. (1987) may be due to differences in the methodologies used in the two studies. The cytoplasm of the penetration gland cells is filled with characteristic granules. We observed that the secretory granules of all glandular cells present in T. ovis oncospheres were of different sizes, shapes and electron densities. Previously, Harris et al. (1987), Fairweather and Threadgold (1981), Schramlova and Blazek (1982) and more recently, S´widerski and Tkach (2002) and Młocicki et al. (2006) also reported the different types of granules based on their shapes and electron densities. Secretions from penetration glands are believed to be involved in the transfer of granules from the penetration gland cells to the external cytoplasm of the oncospheral tegument and many authors have referred to ducts connecting the penetration gland(s) with the
tegument (Fairweather and Threadgold, 1981; Harris et al., 1987; Młocicki et al., 2006). In addition, it is believed that penetration gland ducts are usually supported by microtubules (Collin, 1969; Lethbridge and Gijsbers, 1974). We did observe structures surrounded by microtubules in non-activated oncospheres of T. ovis (Fig. 7). However, no unequivocal evidence was found which would confirm the hypothesis that these were ducts from the penetration gland cells. Harris et al. (1987) reported that they observed ducts in activated hexacanths of T. ovis. Use of the term ‘‘duct” in relation to oncosphere structures may be problematic and inaccurate. A thin cytoplasmic process (e.g., ‘‘internuncial processes” of adult cestode tegument) could carry intracellular granules from one cytoplasmic compartment to another as occurs in many taxa of animals. A ‘‘duct” comprises a cellular or syncytial tube enclosing an extracellular lumen through which materials pass extracellularly (but not typically through the extracellular matrix). In many animals, materials can even be transported through the extracellular matrix, after being secreted from the cells. This route of passage also would not be properly called a ‘‘duct”. It is not clear whether the precise use of the term ‘‘duct” in relation to structures in oncospheres has been consistent with the understood cytological use of the term. A number of research workers have proposed hypotheses for the mechanism of secretion from the penetration glands with secretion regarded as being merocrine (Lethbridge and Gijsbers, 1974), apocrine (Furukawa et al., 1977; Młocicki et al., 2006) and holocrine (Fairweather and Threadgold, 1981). Based on our studies of activated hexacanths of T. ovis (Jabbar et al., unpublished data) we hypothesise that the contents of penetration glands of T. ovis oncospheres may be secreted by an apocrine mode of secretion. Silverman (1955) speculated on the potential antigenic nature of penetration gland secretions, but there has been no direct evidence to support this hypothesis. Recently, Jabbar et al. (2010) have demonstrated the presence of host-protective antigens within what appeared to be penetration gland cells in T. ovis oncospheres. The present study identifies the antigen-containing cells described by Jabbar et al. (2010) as being the PG1 cell suggesting that this cell type may be responsible for the production of hostprotective antigens in T. ovis and probably in other taeniid oncospheres. However, comprehensive studies on ultrastructural immunolocalisation of host-protective antigens of taeniid oncospheres would be required to test this hypothesis. Acknowledgement Funding from National Health and Medical Research Council, Australia (Grants 350279, 400109 and 628320) is acknowledged. References Chew, M.W., 1983. Taenia crassiceps: ultrastructural observations on the oncosphere and associated structures. J. Helminthol. 57, 101–113. Collin, W.K., 1968. Electron microscope studies of the muscle and hook systems of hatched oncospheres of Hymenolepis citelli McLeod, 1933 (Cestoda: Cyclophyllidea). J. Parasitol. 54, 74–88. Collin, W.K., 1969. The cellular organization of hatched oncospheres of Hymenolepis citelli (Cestoda, Cyclophyllidea). J. Parasitol. 55, 149–166. Coman, B.J., Rickard, M.D., 1975. The location of Taenia pisiformis, Taenia ovis and Taenia hydatigena in the gut of the dog and its effect on net environmental contamination with ova. Z. Parasitenkd. 47, 237–248. Conn, D.B., 1993. The biology of flatworms (Platyhelminthes): parenchyma cells and extracellular matrices. Trans. Am. Microsc. Soc. 112, 241–261. Conn, D.B., S´widerski, Z., 2008. A standardised terminology of the embryonic envelopes and associated developmental stages of tapeworms (Platyhelminthes: Cestoda). Folia Parasitol. 55, 42–52. Engelkirk, P.G., Williams, J.F., 1982. Taenia taeniaeformis (Cestoda) in the rat: ultrastructure of the host–parasite interface on days 1 to 7 postinfection. J. Parasitol. 68, 620–633.
A. Jabbar et al. / International Journal for Parasitology 40 (2010) 1419–1431 Engelkirk, P.G., Williams, J.F., 1983. Taenia taeniaeformis (Cestoda) in the rat: ultrastructure of the host–parasite interface on days 8 to 22 postinfection. J. Parasitol. 69, 828–837. Fairweather, I., Threadgold, L.T., 1981. Hymenolepis nana: the fine structure of the ‘penetration gland’ and nerve cells within the oncosphere. Parasitology 82, 445–458. Furukawa, T., Miyazato, T., Okamoto, K., Nakai, Y., 1977. The fine structure of the hatched oncopsheres of Hymenolepis nana. Jpn. J. Parasitol. 26, 49–62. Gabrion, C., 1981. Recherches sur l’oncosphère des cestodes: origine et formation de la calotte recouvrant les crochets. Z. Parasitenkd. 65, 191–205. Gemmell, M.A., 1962. Natural and acquired immunity factors inhibiting penetration of some hexacanth embryos through the intestinal barrier. Nature 194, 701–702. Gemmell, M.A., 1964. Species specificity of the immunogenic complexes of the tapeworm hexacanth embryo. Nature 204, 705–707. Harris, A., Heath, D.D., Lawrence, S.B., Shaw, R.J., 1987. Ultrastructure of changes at the surface during the early development phases of Taenia ovis cysticerci in vitro. Int. J. Parasitol. 17, 903–910. Harris, A., Heath, D.D., Lawrence, S.B., Shaw, R.J., 1989. Echinococcus granulosus: ultrastructure of epithelial changes during the first 8 days of metacestode development in vitro. Int. J. Parasitol. 19, 621–629. Heath, D.D., Smyth, J.D., 1970. In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis from oncosphere to cystic larva. Parasitology 61, 329–343. Heath, D.D., Lawrence, S.B., 1976. Echinococcus granulosus: development in vitro from oncosphere to immature hydatid cyst. Parasitology 73, 417–423. Holcman, B., Heath, D.D., Shaw, R.J., 1994. Ultrastructure of oncosphere and early stages of metacestode development of Echinococcus granulosus. Int. J. Parasitol. 24, 623–635. Inatomi, S., 1962. Submicroscopic structure of the egg shell of helminths. Okayama Igakkai Zasshi 74, 31–81. Jabbar, A., Kyngdon, C.T., Gauci, C.G., Walduck, A.K., McCowan, C., Jones, M.K., Beveridge, I., Lightowlers, M.W., 2010. Localisation of three host-protective oncospheral antigens of Taenia ovis. Int. J. Parasitol. 40, 579–589. Janicki, K., 1907. Über die embryonalentwicklung von Taenia serrata Goeze. Z. Wiss. Zool. 87, 685–724. Johnson, K.S., Harrison, G.B., Lightowlers, M.W., O’Hoy, K.L., Cougle, W.G., Dempster, R.P., Lawrence, S.B., Vinton, J.G., Heath, D.D., Rickard, M.D., 1989. Vaccination against ovine cysticercosis using a defined recombinant antigen. Nature 338, 585–587. Kyngdon, C.T., Gauci, C.G., Rolfe, R.A., Velasquez Guzman, J.C., Farfan Salazar, M.J., Verastegui Pimentel, M.R., Gonzalez, A.E., Garcia, H.H., Gilmanl, R.H., Strugnell, R.A., Lightowlers, M.W., 2006. In vitro oncosphere-killing assays to determine immunity to the larvae of Taenia pisiformis, Taenia ovis, Taenia saginata, and Taenia solium. J. Parasitol. 92, 273–281. Lethbridge, R.C., Gijsbers, M.F., 1974. Penetration gland secretion by hexacanths of Hymenolepis diminuta. Parasitology 68, 303–311. Lightowlers, M.W., Mitchell, G.F., Bowtell, D.D., Anders, R.F., Rickard, M.D., 1984. Immunization against Taenia taeniaeformis in mice. studies on the characterization of antigens from oncospheres. Int. J. Parasitol. 14, 321–333. Lightowlers, M.W., 2006. Cestode vaccines: origins, current status and future prospects. Parasitology 133 (Suppl.), S27–S42.
1431
Młocicki, D., S´widerski, Z., Miquel, J., Eira, C., Conn, D.B., 2006. Cellular organization of the oncosphere of Mosgovoyia ctenoides (Cestoda: Anoplocephalidae). J. Parasitol. 92, 953–961. Morseth, D.J., 1965. Ultrastructure of developing taeniid embryophores and associated structures. Exp. Parasitol. 16, 207–216. Nieland, M.L., 1968. Electron microscope observations on the egg of Taenia taeniaeformis. J. Parasitol. 54, 957–969. Ogren, R.E., 1971. Criteria for anterior polarity and symmetry in tapeworm hexacanth embryos. Proc. Penn. Acad. Sci. 45, 40–41. Race, G.J., Martin, J.H., Larsh, J.E., Esch, G.W., 1966. A study of the adult stage of Taenia multiceps (Multiceps serialis) by electron microscopy. J. Elisha Mitchell Sci. Soc. 82, 44–57. Sato, T., 1968. A modified method for lead staining of thin sections. J. Electron Microsc. 17, 158–159. Schramlova, J., Blazek, K., 1982. Ultrastructure of the hatched and unhatched oncospheres of Taenia saginata. Folia Parasitol. 29, 45–50. Schramlova, J., Blazek, K., Marhoul, Z., Singhvi, A., 1984. Ultrastructure of Taenia saginata oncospheres cultivated in artificial media. Folia Parasitol. 31, 247–251. Silverman, P.H., 1954. Studies on the biology of some tapeworms of the genus Taenia II. The morphology and development of the taeniid hexacanth embryo and its enclosing membranes, with some notes on the state of development and propagation of gravid segment. Ann. Trop. Med. Parasitol. 48, 356–366. Silverman, P.H., 1955. A technique for studying the in vitro effect of serum on activated taeniid hexacanth embryos. Nature 176, 598–599. Skvortzov, A.A., 1942. Egg structure of Taeniarhynchus saginata and its control. Zool. Zhurnal. 21, 10–18. S´widerski, Z., 1972. La structure fine de l’oncosphere du cestode Catenotaenia pusilla (Goeze, 1782) (Cyclophyllidea: Catenotaenidae). Cellule 69, 207–237. S´widerski, Z., 1983. Echinococcus granulosus: hook–muscle systems and cellular organisation of infective oncospheres. Int. J. Parasitol. 13, 289–299. S´widerski, Z., Tkach, V.V., 1997. Ultrastructural studies on the cellular organisation of the oncospheres of the nematotaeniid cestode, Nematotaenia dispar (Goeze, 1782). Acta Parasitol. 42, 158–167. S´widerski, Z., Tkach, V.V., 1999. Electronmicroscopical studies on the cellular organisation of the oncospheres of the hymenolepidid cestode, Staphylocystoides stefanskii (Zarnowski, 1954). Acta Parasitol. 44, 31–38. S´widerski, Z., Ndiaye, P.I., Miquel, J., Tkach, V.V., Marchand, B., Chomicz, L., GrytnerZiecina, B., Sereda, M.J., 2001. Ultrastructural study of the embryonic development of the anoplocephalid cestode Anoplocephaloides dentata, an intestinal parasite of Arvicolidae rodents. II. Differentiation and cellular organisation of the oncosphere. Acta Parasitol. 46, 171–185. S´widerski, Z., Tkach, V.V., 2002. Ultrastructure of embryonic development of Inermicapsifer madagascariensis (Cestoda, Anoplocephalidae) with emphasis on the cellular organisation of the infective eggs. Acta Parasitol. 47, 105–120. S´widerski, Z., Mackiewicz, J.S., 2004. Ultrastructural studies on the cellular organisation of the coracidium of the cestode Bothriocephalus clawibothrium Ariola, 1899 (Pseudophyllidea; Bothriocephalidae). Acta Parasitol. 49, 116–139. S´widerski, Z., Miquel, J., Młocicki, D., Georgiev, B.B., Eira, C., Grytner-Ziecina, B., Feliu, C., 2007. Post-embryonic development and ultrastructural characteristics of the polycephalic larvae of Taenia parva Baer, 1926 (Cyclophyllidea, Taeniidae). Acta Parasitol. 52, 31–50. Wardle, R.A., McLeod, J.A., 1952. The Zoology of Tapeworms. University of Minnesota Press, Minneapolis, USA.