Ultrastructure of vitellocytes in the cestode Progrillotia pastinacae Dollfus, 1946 (Trypanorhyncha, Progrillotiidae)

Ultrastructure of vitellocytes in the cestode Progrillotiapastinacae Dollfus, 1946 (Trypanorhyncha, Progrillotiidae)

Zdzis³aw Œwiderski1,2*, Jordi Miquel3, Daniel M³ocicki1, Lassad Neifar4, Barbara Grytner-Ziêcina2

and John S. Mackiewicz5

1W. Stefañski Institute of Parasitology, Polish Academy of Sciences, Warsaw, Poland; 2Department of General Biology and Parasitology,Warsaw Medical University, Warsaw, Poland; 3Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia SanitBries,Facultat de FarmBcia, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 Barcelona, Spain; 4Laboratoire de Bioécologie Animale,

Departement des Sciences de la Vie, Faculté des Sciences de Sfax, 3018 Sfax, Tunisia; 5Department of Biological Sciences,State University of New York at Albany, Albany, N.Y. 12222, U.S.A.

AbstractThe present study describes the ultrastructure of mature vitellocytes of the trypanorhynch cestode Progrillotia pastinacaeDollfus, 1946 (Progrillotiidae), a parasite of the common stingray Dasyatis pastinaca (Linnaeus, 1758) (Dasyatidae). Thevitelline cells of this species measure about 24 µm in length and about 20 µm in width. They have small, elongated, slightly lob-ulated nuclei, about 4�5 µm in length, with large dense elongated nucleoli and numerous irregularly-shaped dense clumps ofheterochromatin. The extensive cytoplasm is rich in numerous cell organelles and cell inclusions. While the perinuclear cyto-plasm contains numerous long parallel cisternae of GER, ribo- and polyribosomes, several Golgi complexes and mitochon-dria, the peripheral cytoplasm contains predominantly three types of cell inclusions: a great number of large lipid droplets, sev-eral shell globule clusters, and a very small amount of glycogen-like particles. The most characteristic features of vitellocytesin P. pastinacae are having almost no traces of glycogen and the great number of large, highly osmiophobic lipid droplets rep-resenting saturated fatty acids. The presence of large amounts of lipids also in two other trypanorhynchs, Grillotia erinaceus(Beneden, 1858) Guiart, 1927 and Dollfusiella spinulifera (Beveridge et Jones, 2000) Beveridge, Neifar et Euzet, 2004, is instrong contrast to the condition in the most evolved cestodes, Cyclophyllidea, that usually show no trace of lipids.

Key wordsTrypanorhyncha, Progrillotiidae, Progrillotia pastinacae, vitellocytes, ultrastructure, TEM

*Corresponding author: [email protected]

Introduction

Except for the recent detailed study on the ultrastructure andcytochemistry of vitellogenesis in Dollfusiella spinuliferaBeveridge, Neifar et Euzet, 2004 (�widerski et al. 2006), whichbelongs to family Eutetrarhynchidae, there are no other pub-lished data on vitellogenesis in trypanorhynch cestodes, amuch neglected group in ultrastructural and cytochemical stud-dies (Palm 2004). The extensive review on vitellogenesis inother cestode orders (�widerski and Xylander 2000), indicatesthat vitellocytes have 2 important functions in embryogenesis:(1) formation of a hard eggshell (e.g. Pseudophyllidea) or adelicate capsule (e.g. Cyclophyllidea), and (2) supplying nu-tritive reserves for the developing embryos. During cestodeevolution any of these two functions have been intensified orreduced in different taxa depending on the type of their em-bryonic development, degree of ovoviviparity and life cycles(�widerski and Mokhtar 1974; Mokhtar-Maamouri and �wi-derski 1976; �widerski and Mackiewicz 1976; �widerski et

al. 1978, 2004, 2005, 2006; �widerski and Xylander 1998,2000).

The aim of this study is to provide additional comparativeinformation on the ultrastructure of mature vitellocytes and, toa lesser extent, on vitellogenesis of another trypanorhynchcestode, Progrillotia pastinacae Dollfus, 1946 from a secondtrypanorhynchid family Progrillotiidae Palm, 2004.

Material and methods

Mature specimens of Progrillotia pastinacae Dollfus, 1946(Trypanorhyncha, Progrillotiidae) were obtained from the spi-ral valve of the common stingray Dasyatis pastinaca (L.,1758) (Dasyatidae) collected in the Tunisian coastal area offSidi Mansour.

Living cestodes were placed in a 0.9% NaCl solution anddifferent portions of mature proglottids were routinely proc-essed for TEM examination. Specimens were fixed in cold

Skóra

Stefañski

DOI: 10.2478/s11686-006-0030-8 © 2006 W. Stefañski Institute of Parasitology, PASActa Parasitologica, 2006, 51(3), 194–199; ISSN 1230-2821

Ultrastructure of vitellocytes in Progrillotia pastinacae 195

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Figs 1 and 2. Low power electron micrographs of the parts of the vitelline follicle showing perinuclear and peripheral parts of mature vitel-locytes of Progrillotia pastinacae. Note: (1) large nuclei with large electron-dense heterochromatin islands; (2) few mitochodria; (3) long pro-files of GER composed of parallel cisternae; (4) minute vesicles of Golgi complexes; (5) numerous large lipid droplets of very low electrondensity situated mainly in the peripheral cytoplasm; (6) several glycogen-like particles, in form of single granules or rosettes, localised fre-quently around the lipid droplets; and (7) several shell globule clusters. Inset: Detail of the parallel cisternae of GER and small Golgi vesi-cles at their terminal parts on the right upper corner. Abbreviations to all figures: GC � Golgi complex; GER � granular endoplasmic retic-ulum; gl � glycogen-like particles; Hch � heterochromatin islands; L � lipid droplet; m � mitochondrion; n � nucleolus; N � nucleus; sgc �shell globule cluster

Zdzis³aw �widerski et al.196

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Figs 3 and 4. Higher power electron micrographs showing ultrastructural details of the perinuclear regions of the vitellocytes. Note: (1) largenuclei with nucleoli and large electron-dense heterochromatin islands; (2) long profiles of GER composed of parallel cisternae; (3) few mito-chodria; (4) minute vesicles of Golgi complexes; (5) numerous large lipid droplets of very low electron density situated mainly in the periph-eral cytoplasm; (6) several glycogen-like particles, in form of single granules or rosettes, localised frequently around the lipid droplets; and(7) several shell globule clusters

Ultrastructure of vitellocytes in Progrillotia pastinacae 197

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Figs 5 and 6. High power magnification showing ultrastructural details of the peripheral cytoplasmic regions of the vitellocytes. Note: (1) welldeveloped concentric profiles of GER, surrounded by large and numerous lipid droplets; (2) osmiophilic shell globule clusters during differentstages of their development, observe that they consist of two different types of granules: the first homogeneous electron-dense, small, spher-ical granules and the second heterogeneous, large spherical granules with several electron-lucent areas; (3) β-glycogen-like particles localisednear the lipid droplets and shell globule clusters surface; (4) elliptical mitochondrion with β-glycogen-like particles closely connected to itsouter membrane; (5) numerous ribosomes dispersed in the cytoplasm

Zdzis³aw �widerski et al.

(4°C) 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buff-er at pH 7.4 for 2 h, rinsed in a 0.1 M sodium cacodylate bufferat pH 7.4, postfixed in cold (4°C) 1% osmium tetroxide in thesame buffer for 1 h, rinsed in a 0.1 M sodium cacodylatebuffer at pH 7.4, dehydrated in an ethanol series and propyl-ene oxide, and finally embedded in Spurr�s resin. Ultrathinsections were obtained using a Reichert-Jung Ultracut E ultra-microtome, placed on copper grids and double-stained withuranyl acetate and lead citrate. Ultrathin sections were exam-ined using a JEOL 1010 transmission electron microscopeoperated at 75 kV.

Results

Vitelline follicles of Progrillotia pastinacae are numerous andsituated in the cortical parenchyma. Each follicle from matureproglottids is composed of vitelline cells at different develop-mental stages and the so-called �interstitial syncytium�.

Mature vitellocytes of P. pastinacae (Figs 1�4) are large,elongated cells which measure about 24 µm in length andabout 20 µm in width. They have elongated, slightly lobulat-ed nuclei, which is about 4�5 µm long (Figs 1�4), with large(about 2.5 µm long) centrally situated, electron-dense elon-gated nucleoli (Fig. 4) and numerous dense areas of irregular-ly-shaped heterochromatin islands at the periphery of the gran-ular karyoplasm, usually adjacent to the nuclear membrane(Figs 1�4). The extensive cytoplasm is rich in numerous cellorganelles and three different types of cell inclusions (Figs1�6).

The perinuclear cytoplasm (Figs 1 and inset, 2�6) exhibitsnumerous long parallel cisternae of granular endoplasmic retic-ulum (GER), many ribo- and polyribosomes, several Golgicomplexes and mitochondria (Figs 1 and inset, 2�6). Detailsof Golgi complexes and their association with the smallestshell granules are shown on Figures 1 and inset, and 3.

The peripheral cytoplasm (Figs 1�6) mainly contains threetypes of cell inclusions: large lipid droplets, shell globule clus-ters, and few β-glycogen-like particles. Details of these struc-tures, are shown on Figures 5 and 6, where it is also possibleto observe the well developed GER. The most characteristicfeature of the vitellocytes are the numerous large, highly os-miophobic lipid droplets representing saturated fatty acids(Figs 1�6). These droplets, of variable sizes, appear lightlystained, and sometimes it is possible to observe their forma-tion (Fig. 5).

Other large membrane-bound vesicles contain clusters ofnumerous shell globules of different sizes (Figs 1�6). Thenumber and size of individual shell globules or units variesgreatly within one cluster depending on the level of section-ing. Some of the vesicles are asymmetrical or appear partial-ly empty (Figs 1�6). However, higher power electron micro-graphs show that they are filled with an almost amorphous,flocculent material of very low density (Figs 4 and 6). Clustersof vitelline granules often consist of two different types ofgranules: the first type is composed of homogeneous elec-

tron-dense, small, spherical granules and the second typerefers to heterogeneous, large spherical granules with severalelectron-lucent areas (Fig. 5). A few scattered β-glycogen-likeparticles, representing the third type of vitelline cell inclusion,appear associated with these vesicles (Figs 5 and 6).

Discussion

Data on the ultrastructural organization of vitellocytes of try-panorhynch cestodes are presently available for three species:Grillotia erinaceus by McKerr (1985), Dollfusiella spinuli-fera by �widerski et al. (2006), and Progrillotia pastinacaein the present study. Common features of mature vitelline cellsof these species are: (1) the great number of large lipid drop-lets; (2) the moderate amount of shell globule clusters; and (3)the total absence of α-glycogen rosettes and only a small traceof β-glycogen particles. In spite of these general similarities,the vitellocytes of these three species show some small andinconspicuous differences, which are characteristic for eachspecies: (1) the chemical nature of lipids (saturated or unsat-urated fatty acids), that appear osmiophobic or osmiophilic onthin sections; (2) a great variety of shapes, sizes and mode oftransformation of shell globule clusters; and (3) a stronglypositive cytochemical reaction for membrane-bound glyco-proteins in all membraneous structures, which is very charac-teristic for vitellocytes of D. spinulifera (�widerski et al.2006).

The cytochemical tests for polysaccharides or more speci-fically for glycogen were not made for vitellocytes of P. pasti-nacae (present study) or for those of G. erinaceus (McKerr1985). However, very characteristic rosettes of α-glycogen, orindividual isometric particles of β-glycogen (average size150�300 C), which are much larger than ribosomes and moredensely stained with lead ions, can easily be distinguished on electron micrographs even without using Thiery�s (1973)method.

The chemical nature of the lipid droplets in mature vitel-locytes varies among these three species: in the lacistorhyn-chid G. erinaceus and in the progrillotiid P. pastinacae, thelipids appear saturated (McKerr 1985; present study, respec-tively) while in the eutetrarhynchid D. spinulifera the lipidsare highly osmiophilic and therefore unsaturated (�widerski etal. 2006). In G. erinaceus, however, the lipid droplets are oflow saturated chemical nature and therefore slightly osmio-philic, while in P. pastinacae they are clearly saturated andevidently osmiophobic. The characteristic appearance of li-pids in electron micrographs is determined during fixationwith osmium tetroxide when triglyceride droplets rich in un-saturated fatty acids become blackened by reduction of osmi-um. The degree of blackening by osmium varies with the com-position of the lipids. The presence of a great number of largelipid droplets of saturated or usaturated chemical nature, is themost common character for trypanorhynch vitellocytes. Var-iation in the nature of the lipid has also been reported in thePseudophyllidea by �widerski and Mokhtar (1974) and Kor-

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Ultrastructure of vitellocytes in Progrillotia pastinacae

neva (2001), in the Nippotaeniidae by Korneva (2002) and inthe Spathobothriidea by Bruòanská et al. (2005). On the otherhand, lipids are usually absent from vitellocytes of the mostevolved cestodes, Cyclophyllidea, (�widerski et al. 1970a, b,2000, 2005; �widerski 1973) and they are also absent from alower cestode order, Caryophyllidea (Mackiewicz 1968; �wi-derski and Mackiewicz 1976).

Apart from the variation in the chemical nature of the lipiddroplets, differences were also found among the shell globulestructures in the three assessed trypanorhynch species. UnlikeG. erinaceus and P. pastinacae that retain the integrity of indi-vidual shell globule clusters, mature vitellocytes of D. spinu-lifera have a very characteristic single large vesicle filled withalmost amorphous, flocculant material of very low density.This large central vesicle is surrounded by (1) a significantamount of moderately electron-dense proteinaceus granules,showing consecutive stages of their growth and differentiationinto typical shell globule clusters, and (2) highly osmiophiliclipid droplets of unsaturated chemical character.

Both P. pastinacae and D. spinulifera represent insuffi-ciently known tapeworms with regard to the type of embry-onic development, degree of oviparity and life cycle and therefore it is premature to discuss functional ultrastructure of their vitellocytes during and after egg formation.

The discovery that lipids vary in the vitellocytes of differ-ent families of the Trypanorhyncha raises important questionsregarding the factors determining lipid type, functional sig-nificance, and what role they might have in assessing evolu-tionary relationships at any level.

Acknowledgements. Dr George McKerr (Faculty of Life and HealthScience, University of Ulster, North Ireland, UK) kindly provided acopy of his PhD thesis on Grillotia erinaceus for comparative pur-poses for this study. Authors wish to thank the �Serveis Cientifico-tPcnics� (University of Barcelona, Spain) for their support in the prep-aration of samples. This study was financially supported by theProject 2005-SGR-00576 from the �DURSI, Generalitat de Cata-lunya� and by the Project A/2390/05 from the �Programa Intercamp-us de Cooperación Científica e Investigación Interuniversitaria entreEspaZa y Túnez, Agencia EspaZola de Cooperación Internacional,Ministerio de Asuntos Exteriores� of Spain. The completion ofresults, analysis of TEM micrographs and preparation of thismanuscript for publication was supported by a Sabbatical Grant(SAB2005-0068) from the �Secretaría de Estado de Universidadese Investigación, Ministerio de Educación y Ciencia� of Spain to Z.�.

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(Accepted June 27, 2006)

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