ParasiL hung. 24:81-87,1991 O Hungarian Society of Parasitologists

Nippostrongylus brasiliensis - Ultrastructure of the body wall

É. FOK1, Ya. MIZINSKA-BOEVSKA2 and O. POLYAKOVA-KRUSTEVA2

Abstract: The body wall of Nippostrongylus brasiliensis (Travassos 1914; Travassos and Darriba 1929) was studied by transmission electron microscopy. The basic layers of the body wall, i.e. three-zoned or layered cuticle, the hypodermis and the layer of muscle cells were studied with special attention to the outer part of the cuticle, the so-called epicuticle. Similarly to most cellular types, this layer is covered with a pofy- anionic coat, the glycocaryx, which was well visualised by the anionic dye ruthenium red (RR). The binding of RR demonstrates the surface of TV. brasiliensis more clear­ ly than does the traditional method.

Key words: Nippostrongylus brasiliensis, body wall, ruthenium red, glycocalyx, trans­ mission electron microscopy.

INTRODUCTION

The body wall plays an important role in the survival of nematodes by separa­ ting and protecting them from a hostile environment. Not only the structure but also the surface of the body wall of nematodes is important in the taxonomy, classifi­ cation and parasite-host relationships. The Chitwoods (1940) wrote the first comprehensive review of the body wall of some nematodes, based on the results of earlier workers and their own researches. Their findings have been reviewed by several authors, among others Lee (1966), Lee and Bonner (1982), Bird (1958,1971,1980) and Maggenti (1981). Lucker (1936) was the first to call attention to the different layers of the body wall of Nippostrongylus brasiliensis (Trichostrongyloidea, Heligmonellidae), the ne­ matode of rat small intestine. He found that this nematode has a simple cuticle ap­ parently made up of four layers. Since then some studies have been published about the ultrastructure of the body wall of this nematode (Lee 1965; Jamuar 1966; Bon­ ner and Weinstein 1972).

1 Helminthological Research Laboratory, Department of Parasitology and Zoology, University of Veterinary Science, Budapest, Hungary 2 Institute of Parasitology, Bulgarian Academy of Sciences, Sofia, Bulgaria 82 É. Fok, Ya. Mizinska-Boevska and O. Potyakova-Kmsteva

The present work is part of a series of morpho-functional studies on the body wall of N. brasiliensis which is a useful model (Bonner et al. 1972; Kassai 1982) also for this purpose. Our aim was to study the ultrastructure of the layers by trans­ mission electron microscopy after two different staining procedures.

loose^ dense'

glycocatyx

epicuticle

NOMENCLATURE ( SYNONYMS

epicutic le (external cortical envelope]

-'"^cortical I (internal cortical envelope, zone or layer exocuticle )

(matrix, mesocuticle) zone or layer

basal I zone or layer basal membrane

muscle cells layer and hypodermis (epidermis, subcuticle )

Fig. la. Diagram oîN. brasiliensis body wall. Fig. Ib. Transmission electron microscopy of the body wail of M brasiliensis. x 2500 (C - cortical layer; M - median layer; B - basal layer; Mu - muscle layer; H hypodermis) Fig. 2. Cortical (C) and median (M) layer with the epicuticle (EC), x 10000 Fig. 3. Epicuticle with the glycocalyx is well visualised by the dye ruthenium red. x 100000 Fig. 4. Ruthenium red is most strongly accumulated in the infolds of the epicuticle. x 100000 Fig. 5. The amorphous fluffy electron-dense matter within a translucent matrix can be seen in the median layer (M), x 16000 Fig 6 Postenor body region with the median layer (M) possessing electron-dense struts, arranged at regular intervals, x 8000 Fig. 7. Basal zone (B) of the cuticular layers, x 31 S(M) (BM basal membrane) Fig. 8. Lysosomes in the hypodermal layer. x 16000 Fig. 9. Mitochondria in the hypodermal layer. x 25000 (GER - Granular endoplasmic reticulum, G - Golgi apparatus) Fig. 10. The muscle layer (Mu) is built up of a contractile and a plasmatic part, x 16000 (CO - contractile part, PL - plasmatic part) MATERIALS AND METHODS

Female albino CFY rats (150-200 g) of a conventional colony maintained at the Institute of Laboratory Animals, Gödöllő, Hungary were used in the present study. Adult (6-day-old) N. brasiliensis were obtained as described by Takáts (1972).

Transmission electron microscopy The nematodes collected from the small intestine of rats were cut into small pieces (approximately 1 mm2). They were fixed in 4 % glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.4, for 4 h at 0-4 °C. This was followed by washing, post-fixa­ tion in 1% Os04 solution in the same buffer, dehydration through an ethanol series and embedding in Durcupan. Sections were prepared on Ultrotome LKB III and were stained with uranyl acetate and lead citrate. The sections were examined with an Opton 10 C electron microscope.

Staining with ruthenium red (RR) The procedure proposed by Luft (1971) was used. The small pieces of N. brasi­ liensis were rinsed for a short time in 0.1 M Na cacodylate buffer, pW 1A, and were fixed for 1 h at 0-4°C in a fixative freshly prepared by mixing of equal parts of aqueous RR stock solution (5 mg/ml), 4 % cacodylate buffered glutaraldehyde and cacodylate buffer. After thorough rinsing in three changes of buffer, the materials were transferred into a freshly prepared mixture of equal parts of aqueous RR stock solution (6 mg/ml), 2 % aqueous Os04 and cacodylate buffer. Post-fixation was per­ formed for 3 h at room temperature. The remaining part of the procedure was as de- cribed above.

RESULTS

Similarly to other nematodes, the body wall of TV. brasiliensis is characterized by three basic types of tissue, i.e. the three-zoned or layered cuticle, the hypodermis and the layer of muscle cells (Fig. la,b). According to the classification of Lee (1965) and Bird (1971), the cuticle may be divided into three basic layers, i.e. cortical, median and basal. The surface of the cortical layer is covered by a fine (0,02 pm) triple-layered membrane, the epicuticle. Due to its symmetrical segmentation, the surface layer is uneven. At equal intervals, the surface membrane folds into the cortical layer (Fig. 2). Externally, similarly to most cellular types, the epicuticle is covered with an an­ ionic coat which is well visualised by the dye ruthenium red (RR) (Fig. 3). This an­ ionic coat, called glycocalyx, demonstrates the following peculiarities: a dense con­ tinuous osmiophilic band marks the overall surface layer. This band has grainy structure and is twice as thick as the epicuticle itself, i.e. 0.04 pm. Over this thick glycocalyx component lies a looser layer comprising branched filaments of various length. They may have the form of antennae or of crossed thin nets. To sum up, N. 84 É. Fok, Ya. Mizinska-Boevska and O. Polyakova-Krusteva brasiliensis possesses a well-developed surface coat consisting of a dense and a loose layer. RR is most strongly accumulated in the infolds of the epicuticle, and those areas are often filled with the electron dense reaction product (Fig. 4). The three cuticular zones or layers are not sharply separated from each other but are rather distinct for their density and structure. The cortical layar is 1.5 urn thick. It has homogeneous density throughout the helminth body. Its structure is grainy (Figs 3,4). The median layer is the widest cuticular part (10 ^m) (Fig. 1). It is more elec­ tron transparent than the cortex. Higher magnifications reveal amorphous fluffy electron-dense matter within a translucent matrix (Fig. 5) which results in a more translucent electron-miscroscope image of the layer as a whole. In the posterior body region of the parasite, the median zone possesses also electron-dense struts, arranged at regular intervals (Fig. 6). They are biconcave, resembling the figure "8", and have a double outer contour. Their cortex is denser than their periphery. The basal layer is the thinnets of the cuticular layers (0.7 urn) and is situated over a basal membrane. This cuticular layer has a well-outlined, striated structure (Fig. 7). The latter is described in the current literature under various names, of which we prefer to call it "regularly spaced striations". Under these striations lies a light homogeneous strip which is internally well outlined by the basal membrane. This internal border is infolded due to the presence of evaginations of the under­ lying tissues. The basal membrane is underlined by either muscle or hypodermal cells. The hypodermal and muscle layers are thus intermingled, and not strictly delimited. The hypodermal cells are not distinctly divided between each other and can be assumed to form a syncytium. The hypodermis possesses most of the cellular organelles. Nu­ clei are rarely observed. Osmiophilic structures with the dimensions of primary lyso- somes can be seen in the cytoplasm. They are encircled by an unit membrane and have a dense, spotted matrix (Fig. 8). A lot of typically structured mitochondria are found. They are oval, with a double membrane, the matrix is of moderate density and the cristae are radially orientated. Mitochondria can be found close to the di­ lated cisternae of the granular endoplasmic reticulum, and to Golgi tubules and vesicles (Fig. 9). The muscle cell layer is built up of a contractile and a plasmatic part (Fig. 10). The contractile part comprises microfibrillar fields consisting of parallel thin and thick myofilaments. The plasmatic part possesses abundant mitochondria of oval, elongated or unusual form. The mitochondrial matrix is translucent and the cristae are well developed. The cytoplasm of the rest of the myocytes has grainy appearance due to the presence of ribosomes. The separate muscle cells are interbound by connective fibrils originating prob­ ably from the myocyte plasmalemma. DISCUSSION

The general concept (Lee 1966; Bird 1971 and 1980; Maggenti 1981; and No­ bles 1982) about the structure of the body wall of nematodes is that it consists of a three-zoned or layered cuticle, a hypodermis and a single layer of longitudinal muscle cells. The cuticle layers are of varying thickness in different species and they are often subdivided. According to Lucker (1936), N. brasiliensis has a simple cuticle, apparently made up of four layers. By light microscopic studies, he and Chitwoods (1940) found that the cuticle of this nematode is ridged longitudinally and the cortical layers of the cuticle are separated from the basal layers by a fluid-filled region. Under the transmission electron microscope the cuticle of N. brasiliensis was recognized by Lee (1965) and Jamuar (1966) as being composed of three major layers, the cortex, the matrix and the two (Lee 1965) or three fibre layers (Jamuar 1966). In our studies, the cuticular structure of N. brasiliensis can be recognized as being composed of a cortical, a median and a basal layer (Figs. la,b) as it was sug­ gested by the general concept mentioned above. The cortex is covered by a fine triple-layered membrane, the so-called epicu­ ticle (Figs. 2, 3) as described by Bird (1971). The outer part of this layer is covered with an anionic coat, the glycocalyx (Figs. 3 and 4) as it can be seen on the surface of other nematodes (Bird 1980). This surface layer is thought to be involved in molecu­ lar interactions between the body surface and the surrounding medium (Wharton 1986). The dye ruthenium red (RR) visualised the epicuticle with the glycocalyx (Figs 3 and 4) more clearly than did the traditional method (Fig. 2). The median zone (the fluid-filled region mentioned above) has an amorphous fluffy electron-dense matter with struts, arranged at regular intervals (Fig. 6) as de­ scribed by Lee (1965) and Jamuar (1966). It seems likely that these serve for making the cuticle more rigid and firm. The basal layer with the "regularly spaced striations" (Fig. 7) is equivalent to the fibre layer as was shown by Lee (1965) and Jamuar (1966). In this figure a light homogeneous strip can be seen under this striation which is internally well outlined by the basal membrane. According to our findings the basal membrane is underlain either by muscle or by hypodermal cells so these layers are not strictly delimited (Fig. 10). This differs from the observations of Lee (1965) and Jamuar (1966) and agrees with the state­ ment of Nobles (1986). The structure of the muscle cell layer of two parts (Fig. 10) is similar to that de­ scribed by Jamuar (1966). The contractile part contains microfibrillar fields and the plasmatic one has a mitochondrial matrix with different forms of mitochondria. The mitochondria are usually large and numerous, elongated and branched in shape. There are also many ribosome particles present. This suggests that the muscle cells carry on protein synthesis possibly in conjunction with the growth of the parasite. 86 É. Fok, Ya. Mizinska-Boevska and O. Poh/akova-Krusteva

ACKNOWLEDGEMENTS

We wish to thank Reny Datcheva (Institute of Parasitology B. A S., Sofia) for her help in preparing the samples for transmission electron microscopy. We are grateful to Ljubo Danailov (Institute of Microbiology, B. A S., Sofia) who helped us with the TEM.

Fok, É., Mizinska-Boevska, Y., Polyakova-Krusteva, O.: Nippostrongylus brasiliensis - a testfal ultrastruktúrája

A szerzők a Nippostrongylus brasiliensis (Travassos 1914; Travassos és Darriba 1929) testfalát elektronmikroszkóppal vizsgálták. Tanulmányozták a testfal fő rétegein (a három rétegű kutikula, a hypodermis és az izomsejtek rétege) kívül a kutikula külső rétegét, az ún. epikutikulát. Ez utóbbi, hasonlóan a legtöbb sejtes típusú réteghez, pozitívan festődő glikokalix-szal borított, mely jól láthatóvá tehető rutén vörös fes­ tékkel. A szerzők vizsgálata szerint ezzel a festési eljárással sokkal tisztább kép nyerhető a N brasiliensis felületéről, mint a hagyományos módszerekkel.

REFERENCES

Bird, A F. (1958): Further observations on the structure of nematode cuticle. - Parasitol, 48: 32-57. Bird. A F. (1971): The structure of nematodes. - Academic Press, New York and London. Bird, A F. (1980): The nematode cuticle and its surface. - In: Zuckerman B. M. (ed): Nematodes as biological models. Vol. II. Academic Press, London New York. Bonner, T. P. and Weinstein, P. P. (1972): Ultrastructure of cuticle formation in the nematodes Nippostrongylus brasiliensis and Nematospiroides dubius. - J. Ultra- struct. Res. 40: 261-271. Chitwood, B. G. and Chitwood, M. B. (1940): An introduction to nematodology. Ba­ bylon, New York. Jamuar, M. P. (1966): Electron microscope studies on the body wall of the nema­ tode Nippostrongylus brasiliensis. - J. Parasitol. 52: 209-232. Kassai, T. (1982): Handbook of Nippostrongylus brasiliensis (Nematode). Common­ wealth Agricultural Bureaux and Akadémiai Kiadó, Budapest. Lee, D. L. (1965): The cuticle of adult Nippostrongylus brasiliensis. - Parasitol. 55: 173-181. Lee, D. L, (1966): The structure and composition of the helminth cuticle. - In: Dawes B. (ed.): Advances in Parasitology. Vol. IV. - Academic Press, London New York. Lee, D. L, and Bonner, T. P. (1982): Freeze etch studies on nematode body wall. - Parasitology 84: xliv, Proc. Br. Soc. Parasitol., 12-16 April 1981, Noordwijkar- hoot (The Netherlands). Lucker, J. T. (1936): Preparasitic moults in Nippostrongylus muris, with remarks on the structure of the cuticle of trichostrongyles. -Parasitol. 28:161-171. Luft, J. H. (1971): Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanism of action. - Anatomical Record 171:347-368. Maggenti, A R. (1981): General Nematology. - Springer, Newyork and Heidelberg and Berlin. Noble, E. R. and Noble, G. A (1982): Parasitology. The biology of animal parasites. Vth edition, - Lea and Febiger, Philadelphia. Takáts, Cs. (1972): Screening of compounds for anthelmintic action in model ex­ periments. -Parasit hung., 5: 203-216. Wharton, D. A (1986): A Functional Biology of Nematodes. Functional biology series. Biddies Ltd, Guilford and King's Lynn.

Received: 12 May, 1991

Authors' address: Dr. Éva FOK, Helminthological Research Laboratory, Department of Parasitology and Zoology, University of Veterinary Science, P.O.Box 2., H-1040 Budapest HUNGARY

Dr. Yana MIZINSKA-BOEVSKA, and Dr. Olga POLYAKOVA-KRUSTEVA, Institute of Parasitology, Bulgarian Academy of Sciences, kv. Geo Milev, Acad. G. Bonchev str. bl. 25. B-1113 Sofia BULGARIA