Pergamon
nnis of Infdve s venezuehsis (N ANA M. B. MARTINEZ*?
and WANDERLEY
DE SOUZA*$
*Laboratdrio de Ultraestrutura Celular Hertha Meyer, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941, Ilha do Fundrjb, Rio de Janeiro, BrasiI tDepartamento de Histologia e Embriologia, Instituto de Ci&cias BiomPdicas, Universidade Federal do Rio de Janeiro, 21941, Ilha do FundGo, Rio de *Janeiro,Brasil jLaborat6rio de Biologia Celular e Tecidual, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense, Au. Albert0 Lamego, 2000, CEP 28015320. Camposdos Goytacazes, Rio de Janeiro, Brasil (Received
I April
1996; accepted
16 Seplember
1996 1
and nmmcle cells alsO became evident with tbls technique. 8 1997 AustraIIaa Society for Psrsslt&gy. P~~blIshedby Etsevier Science Ltd. Key words: Nematode; Strongyloides freeze-fracture.
cuticle; hypodermis; ulrrastructure; deep-etching:
venezuelensis;
INTRODUCTION
The cuticle of nematodesis a highly ordered extracellularmultilayeredstructureformedby the assembly of macromolecules secretedby the hypodennal cells. Its structure varies in different speciesand even betweendifferent developmentalstagesof the same species (Bird&Bird, 1991;Wright, 1987).Biochemical studieshave shownthat the major structural elements of the nematode cuticles are collagens,which are $To whom correspondence should be addressed at: Laborat&o de Biologia Celular e Tecidual. Fax: 55-247-230160.
cross-linkedby disulphidebondsand cuticulin (Cox, 1992;Maizels et al., 1993).Recent studiesusingthe classicalfreeze-fracture techniquehave providd new information on the structural organization of the epicuticleof nematodes(De Souza~1al., 1993;Leeef al., 1993;Arahjo et al.. 1994;Peixoto & De Souza, 1994). The nematodeStrongyloidesvenezuelensis presents an adult form which iivesin the intestineof rats. and larval forms which are deveiouedfrom embrvonated eggsthat are hatchedin the soil. The 3rd~stagelarva is the infective form, which penetratesthe host’sskin to initiate a new life cycle. During recent years there
289
A. M. B. Martinez and W. De Soura
290
has been an increased interest in studies of infective forms of nematodes due to their importance as aetiological agents of various diseases that affect people, animals and plants. Quick-freeze followed by freeze-fracture. deep-etch and rotary replication has been used to analyse the organization of macromolecular assemblies such as the cytoskeleton network (Heuser & Kirschner, 1980), flagellar and ciliar axonemes (Goodnough & Heuser, 1982), the paraxial structure of trypanosomatids (Souto-Padron et al., 1984) the cell wall of plant cells (Fujino & Itoh, 1994). More recently we have used this approach to analyse the structural organization of the cuticle of adult forms of Caenorhabditis e/egans (Peixoto & De Souza, 1995) and Strongyloides venezuelensis (Martinez & De Souza, 1995). The results obtained showed for the first time that the cuticle is formed by a complex network of filamentous structures. The present work extends these studies to the infective larvae (3rd stage) of S. uenezuelensis. MATERIALS
AND
METHODS
Collection of larval,forms (3rd stage) of S. venezuelensis. The strain of S. venezuelensis we used was kindly given by Dr M. T. Ueta, University of Campinas, Sao Paulo. Faeces of rats infected with S. venezuelensis were collected and cultured in charcoal in covered Petri dishes which were left at a temperature of 27-28°C for 48 h. After that period, 3rd stage larvae were collected by the method of Rugai et al. (1954) and processed for electron microscopy. The strain was maintained by serial passage in Wistar rats. Transmission electron microscopy. Third stage larvae were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), rinsed in the same buffer and post-fixed in a solution containing 1% osmium tetroxide, 2mM CaCl,, 0.8% potassium ferricyanide in 0.1 M cacodylate buffer (pH 7.2) for 1 h. The specimens were then washed in the same buffer, dehydrated in acetone solution and embedded in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate. For localization of surface carbohydrates, specimens were incubated in a solution containing 0.05% ruthenium red and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 4 h at room temperature. Afterwards they were washed in the same buffer and post-fixed in a solution containing 0.05% ruthenium red and 1% osmium tetroxide in 0.1 M cacodylate buffer for 90min. Then they were dehydrated and embedded as described above. Ultrathin unstained sections were observed in a Zeiss 900 transmission electron microscope. Freeze-fracture. After being fixed in glutaraldehyde, larvae were washed in cacodylate buffer, kept in the same buffer, and infiltrated gradually (for 30 min) with a solution of 40% glycerol in the same buffer until a final concentration of 20% glycerol was achieved. They were then kept overnight in a 40% glycerol solution. Next day the material was mounted on Balzers support disks, frozen in liquid Freon 22 cooled by liquid nitrogen and fractured at - 115°C in Balzers freezefracture equipment. The specimens were shadowed with platinum at an angle of 45” at 2 x 1O-6 Torr. Replicas were obtained after digestion with sodium hypochloride. washed
in distilled water and collected on 200-mesh grids for electron microscope observation. QuickTf‘ree:ing, ,freeze-fkarturr and deep-etching. Specimens fixed in glutaraldehyde were washed in buffer. rinsed several times in distilled water and mounted on Balzers support disks. Then were quickly frozen in a MedVac rapid freezing apparatus by impact against a copper block which was cooled by liquid nitrogen to - 196-C. Fracture was performed in the Balzers equipment. and etching was achieved by raising the temperature to - 105C for 20 min. Platinum was evaporated on to the specimen at an angle of 15” as the specimen was rotated. Carbon was evaporated at an angle of 90’ Replicas were obtained after digestion with sodium hypochloride. washed in distilled water, collected on 200-mesh grids and observed in a Zeiss 900 electron microscope.
RESULTS
Figure 1 shows a transverse section of a 3rd~stage larva of Strongyloides venezuelensis displaying the cuticle, hypodermis and muscle layers. The cuticle presented the following 5 layers: epicuticle, cortical, medial, fibrous and basal. The epicuticle appeared as a trilaminate unit measuring 7.5nm in thickness. It was covered by a fuzzy material which represents the surface coat. The cortical layer was electron lucent and measured 30nm in thickness. The medial was more electron dense and had 2 distinct parts: an external part which was light and homogeneous (35 nm) and an internal part which was more heterogeneous (35nm). The fibrous layer was very prominent and was characteristically formed by parallel bars of 175 nm in length intercalated by clear spaces with a periodicity of 7Snm. The last layer was the basal which was very irregular, varying from 17.5 to 42.5 nm in thickness. The thinnest areas were very closely associated with indentations of the hypodermis. In these areas we could observe patches of dense material which project into the cuticle. The hypodermis, situated below the cuticle, is represented in Fig. 1 by a narrow layer, measuring 87.5 nm in thickness. Under the hypodermis we could observe the contractile part of the muscle cells represented by thick and thin myofilaments (Fig. 1). When the specimens were fixed in solutions containing ruthenium red, a strong reaction, represented by an electron-dense material overlying the surface of the epicuticle, was observed (Fig. 2). In freeze-fracture replicas of transverse fractured specimens we observed the cuticle, hypodermis and muscle layers (Figs 3 and 4). In the cuticle it was possible to distinguish an external row of particles measuring 21 nm in diameter, the epicuticle, followed by a layer (125 nm thick) in which there were few scattered particles (16.7 nm in diameter) embedded in an amorphous matrix. The fibrous layer was rep-
The cuticle of S. wneru&nsi.s
(L j)
uenemelensis, showing the cuticle ‘ig. 1. A transverse section through the body wall of 3rd~stagelarva of Strongyloides :‘). hypodermis (H) and muscle (M) layers. Observe the epicuticle(thick arrow) and the surface coat (thin arrow I Sc;tlc bar=0.2gm.
resented by parallel bars measuring 12.5 nm in thickness and 200nm in length. The basal layer was not clearly visualized in these preparations. In the hypodermis there were particles of 8.3nm in diameter. Below, we could observe part of a muscle cell exhibiting thick and thin globous elements measuring 20.8 nm and 4.2 nm, respectively. The surface of the nematode was usually covered by an amorphous material (Fig. 4). In longitudinally freeze-fractured specimens the body wall of the nematodes was commonly fractured at the level of the hypodermis (Figs 5-7). In this situation the fibrous layer of the cuticle showed a different aspect when compared to transversely sectioned specimens. Layers of particles arranged in rows were seen (Fig. 5). The P face of the outer hypodermal membrane (close to the cuticle) showed longitudinally orientated reticulated ridges with particles (42nm in diameter) concentrated at the periphery of the ridges and in the valleys, and transverse annulations (2.3 pm) presenting fewer particles on it. The corresponding E face showed longitudinally orientated reticulated ridges, where we observed fewer particles concentrated along the sides of the ridges. The P face of the inner hypodermal membrane displayed many particles (21 nm in diameter) concentrated in
depressed areas, sometimes arranged in small groupa. In between these depressed areas we observed elevated areas where there were fewer particles. The E face showed transverse depressed areas which matched with the cuticular annulations, with few particles on it. Raised areas. probably corresponding to the depressed areas of the P face, showed caveolac and scattered particles (Figs 6 and 7). When the specimens were submitted to quickfreeze, freeze-fracture and deep-etch techniques, the surface of the nematodes was exposed, revealing the cuticular annulations, particles of 13nm in diameter and a network of thin cord-like fibrous elements (Fig. 8). Sometimes this surface was covered by a very weilorganized material arranged in repetitive thin layers which resembles a crystalline pattern (Fig. 9). In deepetch preparations transversely sectioned through the body wall, the cytoskeleton of the cuticle, hypodermis and muscle layer became evident (Fig. 10). The cuticle showed 4 distinct regions: the first was composed of 2 rows of particles separated by an apparent free space. The particles (6.7 nm in diameter) were disposed very closely to each other and accompanied the cuticular annulations. The second region was formed by interconnected thick and thin fibrous elements and globous structures leaving empty spaces among them. These
292
A. M. B. Martinez
and W. De Souza
2 Fig, 2.
A transverse
section
through
the body wall to show the strong reaction by ruthenium red. Scale bar = 0.2pm.
(arrow)
of the surface
coat after
treatment
Fig. 3.. A-transverse fracture showing the cuticle (C), hypodermis (H) and muscle (M) layers. Note the presence of particles (thick arrow) arranged in rows at the epicuticle (thin arrow) at the level of the cortical and medial layers, and the parallel bars of the fibrous layer (curved arrow). Scale bar = 0.2 pm.
The cuticle
ig. 4.
Conventional
transverse
g. 5. Longitudinal fracture longitudinal rows (arrow). rows) which coincide with
fracture
showing
of S. clenezuelensk
the amorphous bar=0.2pm.
material
(L,)
(*) covering
the surface
of the larva.
Scale
through the hypodermis. The cuticle shows, at the level of the fibrous layers, particles arranged The E face of the inner hypodermal membrane (hEF‘) shows depressed areas (in between the cuticular annulations. and elevated areas with particles on them (arrow head). Sc:~le bar= I.Oycm.
A. M. B. Martinez and W. De Soma
294
Fig:, 6. Longitudinal fracture through the hypodermis showing the E face of the inner hypodermal membrane (hEF) and the P face of the outer hypodermal membrane (hPF) presenting reticulate ridges (arrow heads). transverse annulations (thick arrows) and particles (thin arrow). Scale bar= 1.Opm.
spaceswereprobably filled by water in vivo. The thick fibrous elementswere very uniform in sizeand measured48nm in length and 9 nm in diameter.The thin fibrous elementsmeasured17nm in lengthand 1.2nm in diameter.The globular elementshad a diameterof 17nm. The third region, which correspondsto the fibrous layer, wascomposedof parallel bars of thick fibrous elementshaving 253nm in length and 6.7nm in thickness. Each bar was apparently formed by globularelementswith a meandiameterof 19nm (Fig. 10). The fourth region (basallayer) was formed by interconnectedfibrous elementsof variable thickness and length. Eachfibre wasapparently formedby small subunits(insetto Fig. 10). The hypodermis is clearly formed by 2 distinct regions,1 being composedof interconnectedfibrous elementsleaving small spacesamong them, and the other by a delicatednet of filamentsof various size andthickness,leavinglargeempty spacesamongthem (Fig. 10). Fibrous elements(477nm long and 6nm thick) wereclearly seenconnectingthe hypodermisto the musclelayer (Fig. 10). DISCUSSION
thin sections showsthat it is a trilaminated structure, with which surfacecomponentsare associated.The presenceof a surfacecoat on larval stagesof animal parasitic nematodeshas been associatedwith the ability of infective stages to evade the immune responseof the host (Blaxter et al., 1992;Pageet al., 1992).Our resultsconfirm the presenceof a surface coat with a glycidic composition,aswasdemonstrated by other authorsin different species of nematodes(for reviewsseeLumsden,1975and Blaxter et al., 1992). In deep-etchpreparations,the surfaceof the larvae wasexposeddisplayingparticlesand fibrous elements which we believeare part of the surfacecoat already described.The crystalline-likematerialthat coversthe surface of the larvae, as seen in deep-etchpreparations,isvery similarto proteinaceousmaterialpresentin envelopesof bacteria(Sleytr, 1978). Recent studieshave shown the presenceof intramembranousparticlesin fractured facesof the epicutitle on different speciesof nematodes(Araujo et al., 1994;De Souzaet al., 1993;Lee et al., 1993;Peixoto & De Souza, 1994,1995;Martinez&De Souza, 1995). Unlike the adult forms of Strongyloides venezuelensis, we did not observefracture facesof the epicuticlein the larvae, despiteseveralexperimentsperformed. It
fact that the cuticle of nematodes The nature of the epicuticleof nematodeshasbeen is a well-known the subject of many controversies. Examination of changes according to its developmental stage fn bre-
The cuticle of S. rwnrzuelensis (L,)
‘C)i
Fig. 7. Transverse fracture showing the E face of the outer hypbder&al me&b&e (h&‘) witk fongitndinally oriented ridges (in between arrows) and the P face of the inner hypodermal membrane (hPF) with groups of particles (arrow head j Scale bar = 0.3 pm.
Fig. 8. Deep-etch view of the surface of the larva in which we can observe particles (thick arrow) and the cord-like fibrous elements (arrow heads). Scale bar=0.4pm.
Fig. 9. Deep-etch of the larva exhibiting the crystalline pattern of the well-organized material that covers its iurt’ace (*). Scale bar=0.3Ltm.
vious work (Peixoto et al., 1994), using the imidazoleosmium technique. we have shown the presence of 3rd-stage larvae of S. venelipids in the cuticle of 3rd~stage ~~elerrsis. and no reaction in adult forms. Put together
these results suggest the possibility that the epicuticle of 3rd-stage larvae of S. twxezuelmsis has a different composition from that of adults which does not allow it to be easily fractured.
296
A. M. B. Martinez and W. De Souza
Fig. 10. Deep-etch view of a transversely fractured specimen showing the skeleton of the cuticle (C). hypodermis (H) and muscle (M) layers. Note the double row of particles (arrow, heads), the thick (thick arrow), thin (thin arrow) and globous elements at the level of the cortical and medial layers. At the fibrous layer it is possible to visualize the parallel bars sometimes formed by subunits (arrow) and the presence of thin and short filaments cross-linking the bars. The basal layer exhibits fibrous interconnected elements which are apparently formed by small subunits (arrow-inset). Scale bar= 0.2 pm. Inset scale bar = 0.2 pm. Although transmission electron microscopy of thin sections of the cuticle has shown that it presents several layers, this approach is not adequate to analyse structures formed by a complex array of macromolecules. In contrast, replicas of quick-frozen, freeze-fractured, deep-etched and rotary replicated nematodes have revealed new information on the organization of the cuticle of these organisms. as shown previously for adult forms of C. elegans (Peixoto & De Souza, 1995) and S. r~enezuelensis (Martinez & De Souza, 1995). Our present observations show that the cuticle appeared to be formed by a complex array of interconnected fibrous and globous elements, giving this structure a certain rigidity compatible with the assumption that the cuticle is an extracellular matrix.
The main component of the cuticle is collagen, a fibrous protein cross-linked by covalent bonds (Blaxter et al., 1992; Cox, 1992). This collagen appears as fibres of 45 nm in length (Betschart & Wyss, 1990), which is very close to the value that we found. Therefore, the thick fibrous elements seen in our replicas probably represent collagen fibres. The apparently empty spaces seen among the fibrous components are probably filled by water in uivo which was sublimated during the etching procedure. This water material is probably associated with the diffusion of substances across the cuticle.
Acknowledgements-We are grateful to Mr Sebastiao Cruz and Geniiton Jose Vieira for excellent technical assistance.
The cuticle
of S. reneruelensis
and to Alessandra S. M. Gonzalez for her help in some experiments. and Mrs Alba Val&ia Peres for secretarial assistance. This work was supported by UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases. Conselho National de Desenvolvimento Cientifico e Tecnolbgico (CNPq) and Financiadora de Estudos e Projetos (FINEP).
REFERENCES Arabjo A., Souto-Padron T. & De Souza W. 1994. An ultrastructural. cytochemical and freeze-fracture study of the surface structures of Brugia mnlayi microfilariae. Internutional Journal.for Parasitology 24: 899-907. Betschart B. & Wyss K. 1990. Analysis of the cuticular collagens of Ascaris suum. Acta Tropica 47: 297-305. Bird A. F. & Bird J. 1991. The Structure qf Nematodes. Academic Press, San Diego. Blaxter M. L., Page A. P.. Rudin W. & Maizels R. M. 1992. Nematode surface coats: actively evading immunity. Parasitology Todq 8: 243-247. Cox G. N. 1992. Molecular and biochemical aspects ofnematode collagens. Journal qf’Parasifo/ogy 78: l-15. De Souza W.. Souto-Padron T., Dreyer G. & Andrade L. D. 1993. Wuchereria bancrofti: freeze-fracture study of the epicuticle of microfilariae. Experimental Parusitolog~~ 76: 287-290. Fujino T. & ltoh T. 1994. Architecture of the cell wall of a green alga OccJstis apiculata. Protoplasma 180: 39-48. Goodnough U. W. & Heuser J. E. 1982. Substructure of the outer dynein arm. Journal of Cell Biology 95: 798-815. Heuser J. E. & Kirschner M. W. 1980. Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. Journal of Cell Biologjl86: 2 12-234. Lee D. L.. Wright K. A. & Shivers R. R. 1993. A freezefracture study of the cuticle of adult Nippostrongylus bra.si/iensis (Nematoda). Parasitology 107: 545-552.
( L; )
:4-
Lumsden R. R. 1975. Surface ultrastructure and q!ochemistry of parasitic helminths. E.rperirnc,nral I’(II.Nsitology 37: 267-339. Maizels R. M.. Blaxter M. L. & Selkirk M. E. 1993. Forms and functions of nematl>de surfaces. E.~perinwntu/ PU:YIsitology 77: 380-384. Martinez A. M. B. & De Souza W. 1995. A quick-irolen. freeze-fracture, and deep-etch study of the cuticle of adult forms of StrongJloides venezuelensis (Nematoda). f’cirasitology 111: 523-529. Page A. P.. Rudin W., Fluri E., Blaxter M. L. & Ma~zeis R. M. 1992. Toxocura c&r: a labile antigenic coat overlymg the epicuticle of infective larvae. &perimmtcl/ I’crrrrsitology 75: 72-m86. Peixoto C. A. &De Souza W. 1994. Freeze-fracture chdr:icterization of the cuticle of adult and dauer forms ot’ Caenorhabditis elegons. Parasitology Research 80: 53 -57. Peixoto C. A. & De Souza W. 1995. Freeze-fracture and deep-etch view of the cuticle of adult forms of C’tre,rlorhubditis elegans. Tissue Cell 27: 561-568. Peixoto C. A., Martines A. M. B.. Souza M. I-‘.. De Souz.a W. 1994. Caenorhabditis elegans and Strong!+oide.\ wwxelensis. Ultrastructural visualization of lipids in the cutlcle of adults and larvae forms (Nematoda: Rhabditordca). Acta Microscopica 3: 107-I I 5. Rugdi E., Mattos T. & Brizola A. P. 1954. Nova ttcnica para isolar larvas de nematbides das fezes; modiiica@e\ do mCtodo de Baermann. Rr~ir~u &J Insfifzrtn Aridfh Lx/ 14 5-8. Sleytr U. B. 1978. Regular arrays of macromolecuic~ on bacterial cells wall: structure. chemistry. assembly .Ind function. International Review oj’Cyto1og.v 53: I 3? Souto-Pad& T., De Souza W. & Heuser J. E. 1984. Qmckfreeze, deep-etch. rotary replication of Trvpunosomo cruzi and Herpetomontrs mr~useliue. Journul of Cell Scrcw~ 69: 167-178. Wright K. A. 1987. The nematode’s cuticle---its surl’ace and the epidermis: function, homology. analogy~- -a current consensus. The Journui’ of’ Parasitology 6: 1077~ IOX?.