J. Eukaryot. Microbiol., 48(1), 2001 pp. 95–101 q 2001 by the Society of Protozoologists
Trichites of Strombidium (Ciliophora, Oligotrichida) Are Extrusomes LETIZIA MODEO, GIULIO PETRONI, MICHELA BONALDI and GIOVANNA ROSATI Dipartimento di Etologia, Ecologia, Evoluzione via A. Volta 4-6, 56126 Pisa, Italy ABSTRACT. The trichites of Strombidium and related genera have been considered either as a cytoskeletal armature or as extrusomes. To demonstrate their true nature, a study was undertaken on two marine Strombidium species by ultrastructural and cytochemical analysis as well as in vivo experiments. Trichites, extending from the cortex into the cell, are rod-shaped, membrane-bounded, and have a complex structure. The following elements of the trichites, are distinguishable: an electron-transparent lumen, a laminated layer, and a compact layer. In trichites of one species, thin ‘‘rings’’ surround the lumen. Numerous short, curved tubules with a polysaccharide wall are present in the cytoplasm surrounding the trichites. At the cortical end, each trichite is enveloped by a ‘‘cap’’ of electron-dense proteinaceous material. In some cases, the cortical alveoli appear interrupted, forming a ‘‘hole’’ for trichite ejection. Ejection of rodshaped structures, up to 5 times longer than resting trichites, was obtained by in vivo treatments with dextran and aminoethyldextran. Negative staining indicated that these structures were transformed trichites. As no other possible extrusive structures were observed in the cytoplasm of Strombidium, trichites were considered extrusomes. Key Words. Cytochemistry, cytoskeletal elements, extrusive organelles, protozoa, Strombidiidae, ultrastructure.
T
RICHITES are unique structures of oligotrich ciliates belonging to the Strombidiidae. Although trichites were mentioned in the literature more than 100 years ago (Bu¨tschli 1873), their nature and function are still debated: they have been considered either as a cytoskeletal armature (Carey 1992; Corliss 1979; Faure´-Fremiet and Ganier 1970; Gourret and Roeser 1888; Maeda and Carey 1985) or as extrusomes (Entz 1884; Krainer 1991; Petz and Foissner 1992). Montagnes et al. (1988) reported that in Strombidium conicum, Strombidium sulcatum (now recognised as Strombidium emergens, Montagnes et al. 1990), and Strombidium capitatum, some of these structures were extruded from the cell; at times the extruded materials lengthened and disaggregated. Possibly, the extrusion was due either to fixation or to changes in the environment such as variations in salinity; extrusion of trichites of S. sulcatum occurred under both these conditions, but was apparently not accompanied by an increase in trichite length (Faure´-Fremiet and Ganier 1970), leading these authors to exclude any relation between trichites and extrusomes. The study by Faure´-Fremiet and Ganier (1970) on S. sulcatum is the only ultrastructural analysis on a Strombidium where trichites were described in detail. The possibility that at least some Strombidium species contain two types of structures, namely trichites as cytoskeletal rods and extrusomes, has been also considered (Montagnes et al. 1988; Montagnes & Humphrey 1998). In protargol-stained preparations of Strombidium lingulum, elongate tear-shaped extrusomes, apparently different from trichites, were observed (Montagnes and Humphrey 1998). We have examined two marine Strombidium species to investigate the nature of trichites. Trichite ultrastructure was analysed by electron microscopy, and trichite chemical composition was investigated by cytochemical treatments with different enzymes and specific contrast methods on thin sections. In vivo experiments were also performed to verify the ejection of trichites. Our results support the hypothesis that trichites are a unique category of extrusomes.
erotrophic flagellate Chilomonas sp. (S1) or with the green flagellate, Dunaliella salina (S3). Electron microscopy. For scanning electron microscopic (SEM) observations, specimens of S3 were fixed for 20 min in 2% OsO4 in seawater while S1 was fixed for the same time in a 1:1 mixture of 2% OsO4 and a saturated solution of HgCl2, both in seawater. All the specimens were processed as reported by Rosati et al. (1999). Transmission electron microscopy (TEM) was conducted on specimens fixed for 30 min at room temperature in a 1:1 combination of 2% OsO4 in distilled water and 5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4. Specimens were then ethanol-dehydrated, transferred to 100% acetone, and embedded in an Epon-araldite mixture. Thin sections were picked up on copper grids and stained with uranyl acetate and lead citrate. Cytochemical treatments. The chemical nature of trichites was investigated by cytochemical treatments on thin sections of S3, prepared by the technique described above. The sections were picked up on mylar rings, floated on 2% H2O2 for 20 min to remove the reduced osmium (as osmium could prevent the cytochemical reactions), rinsed in distilled water, and incubated as follows. The TSC-silver proteinate method (Thie´ry 1967) was used to test for polysaccharides. Enzymatic protein extraction was conducted by incubating the samples with either 0.5% protease (Sigma, St. Louis, MO) in water (brought to pH 7 with NaOH) or 0.5% pepsin (Sigma) in 0.1 M HCl. Both protease treatments were incubated for 4 h at 37 8C. As controls, some sections were incubated under the same conditions without the corresponding enzymes. Both treated and control sections were then mounted on grids and stained as above. In vivo treatments. Treatments were carried out using substances that trigger ejection of Paramecium trichocysts (Plattner et al. 1985). Some specimens of S3 (10–15) were transferred to a drop (; 200 ml) of sea water containing 3% dextran or 3% aminoethyldextran (AED), placed on a microscope slide, and covered with a coverslip (18 3 18 mm). A corresponding number of ciliates were transferred to a drop of pure seawater, and prepared as above. Both samples were immediately observed using differential interference contrast microscopy. This procedure was repeated 10 times; thus . 100 treated and control specimens were observed. Extraction and separation of trichites. Cells were concentrated and washed twice in pure seawater by centrifugation at 100 g for 10 min, and then broken by centrifugation at 15,000 g for 10 min at 4 8C. The obtained pellet was resuspended and concentrated in a microfuge at 20,000 g for 10 min at 4 8C, and stratified by centrifuging at 1,000 g for 15 min at 4 8C on a discontinuous 7% to14 % (w/v) sucrose gradient (Eperon and Peck 1988) made up in seawater. The phases were checked for
MATERIALS AND METHODS Collection and culturing. The Strombidium species are referred to as Strombidium S1 and S3 as both taxa appear to be new species or at least require redecription (Maeda and Carey 1985; Montagnes et al. 1990; Montagnes, D. J. S., pers. commun.). Both species were collected from tidal pools on a rocky shore in the Ligurian Sea; they were maintained in the laboratory in artificial seawater, periodically enriched with the hetCorresponding Author: G. Rosati—Telephone number: 139 050500840; FAX number: 139.05024653; E-mail:
[email protected]
95
96
J. EUKARYOT. MICROBIOL., VOL. 48, NO. 1, JANUARY–FEBRUARY 2001
MODEO ET AL.—TRICHITES OF STROMBIDIUM ARE EXTRUSOMES
Fig. 12–13.
Fig. 12–13. Detailed reconstruction of a portion of the trichite region of Strombidium S3 shown in Fig. 3. 12. Longitudinal view. 13. Cross view. C, cilium; SM, subcortical material; CSL, concentric sheet layer; CT, curved tubules; EL, electron-dense layer; ER, electron-dense rings; L, lumen; MT, microtubules. Bars 5 0.3 mm.
the presence of trichites employing a differential interference contrast microscope. Negative staining. A 10-ml droplet of the richest phase in trichites was deposited on a Formvar/carbon-coated copper grid and a 10-ml drop of 1% phosphotungstic acid brought to pH 7 with NaOH. After staining for 3–5 min the grid was blotted. RESULTS Specimens of Strombidium S3 are shown from the ventral and dorsal views, respectively (Fig. 1, 2) and in vivo (Fig. 4). The girdle and the trichite insertions extend around the equator of the cell (Fig. 3). Strombidium S1 is a larger species whose
97
Continued.
trichite zone is less conspicuous in fixed (Fig. 5, 6) and living specimens (Fig. 7). In SEM preparations a continuous band of protrusions above the girdle is never evident. Trichite general morphology and localisation. Strombidium S3 trichites are rod shaped, ; 11 mm long and 0.35 mm in diam. They form a funnel-like complex within the ciliate (Fig. 4). Trichites originate at the cortex, just above the ciliated girdle, and extend towards the posterior part of the cell. In SEM preparations, in the band corresponding to the trichite insertions, protrusions occur beneath the pellicle, regularly arranged in single or double rows, each consisting of 6–7 trichites (Fig. 2, 3). The trichites end in the cytoplasm without reaching the posterior of the cell (Fig. 4). Trichites in Strombidium S1 are less abundant than in S3; they are ; 16 mm long and 0.45 mm in diam. Their cortical ends occur just above the girdle of cilia, following its profile. The ciliary girdle in this species shows pronounced displacement of the two ends; starting from the left side of the ventral surface, it continues obliquely on the dorsal surface and ends on the right side of the ventral surface at a lower level (Fig. 5, 6). Consequently, trichite ends are at different levels. Ultrastructure. TEM observation of many specimens, some examined by serial sections, revealed that in both S1 and S3, no other extrusive organelles were present beside trichites. Trichites are delimited by a membrane and possess a complex structure in which three distinct compartments are recognised (Fig. 8, 9); the ultrastructure of S3 and S1 trichites differed slightly. In S3 (Fig. 8, 9), the compartments, from the exterior to the interior, are: 1) an amorphous electron-dense layer (EL) 0.08 mm thick, lining the membrane even at the anterior and posterior ends; 2) a layer (; 0.04 mm) consisting of concentric sheets (CSL); and 3) a lumen (L) 0.11 mm wide.
← Fig. 1–7. Specimens of Strombidium S3 and Strombidium S1. 1. Ventral view of S3 (arrow indicates the peristomal area) SEM. 2. Dorsal view of S3. G, ciliary girdle. SEM. Bars 5 10 mm. 3. Enlargement of trichite region of S3. C, cilia. Bar 5 5 mm. 4. S3 in vivo (Nomarski). Note the trichites as rod-like structures in the middle of the cell. 5. Ventral view of S1; arrowhead indicates the peristomal area; arrows indicate the two ends of girdle. 6. Dorsal view of S1. Note the oblique pattern of the girdle. 7. S1 in vivo (Nomarski); arrowhead indicates the oral cavity. Bars 5 10 mm. Fig. 8–11. Fine structure of the trichites of Strombidium S3 and S1. 8. Longitudinal section of trichites of S3 at their cortical end. Arrows indicate the caps formed by the electron-dense subcortical material. CSL, concentric sheet layer; CT, curved tubules; EL, electron-dense layer; ER, electron-dense rings; L, lumen; MT, microtubules. Bar 5 1 mm. 9. Cross-sectioned trichites of S3. Bar 5 0.5 mm. In the inset, a section at higher magnification. Bar 5 0.1 mm. 10. Longitudinal section of a trichite of S1. The ERs are not present. Bar 5 1 mm. 11. Cross-sectioned trichite of S1. The thickness of the various compartments is different in comparison with that of trichites of S3. Bar 5 0.5 mm.
98
J. EUKARYOT. MICROBIOL., VOL. 48, NO. 1, JANUARY–FEBRUARY 2001
Fig. 14–18. Cytochemical treatments on trichites of Strombidium S3. 14. Protease treatment. The EL is partially digested. Bar 5 0.5 mm. 15, 16. Pepsin treatment. 15. The EL is completely digested together with the ERs and the electron-dense material forming the subcortical ‘‘caps’’ at the trichite insertion (arrows). 16. In cross-section, six ‘‘arms’’ are visible, connecting the undigested CSL with the trichite membrane. 17. After TSC-silver proteinate treatment (Thie´ry 1967) the ERs, the trichite membrane, and the CTs are specifically stained. 18. The intense staining of paraglycogen granules (PG), the polysaccharide subcortical platelets (PP), and the perilemma (P) confirm the specificity of the reaction with polysaccharide components. Bars 5 0.5 mm.
In cross-sections the lumen is roughly six-sided and six small electron-transparent zones are evident in the CSL; extensions of this layer reach the trichite membrane and pass through the EL, which is therefore discontinuous (Fig. 9 inset). Electrondense ‘‘rings’’ (ER) 0.02 mm high, surround the lumen, at distances which become progressively lower towards the cortical end of the structure (Fig. 8). In the cytoplasm surrounding each trichite, in contact with their membranes, there are numerous, short, curved tubules (CT) (Fig. 8, 9). Electron-dense material is present under the cortex, associated with the trichite insertion. As demonstrated by serial
sections, this material forms a continuous layer under the pellicle and, associated with each trichite, becomes curved and covers its cortical end like a cap. Microtubules emerge from the caps and parallel the trichites along ; 20% of their length (Fig. 8). Crosssections indicate that these microtubules form sheets, separating trichite rows (Fig. 9). These features have been schematically illustrated for the trichite region of S3 (Fig. 12, 13). In S1 trichites, the EL is reduced (0.03 mm), and the CSL is wider than in S3 (0.13 mm). The trichite layers of S1 are more easily distinguishable than those of S3; six dense zones are visible in the CSL. The trichite lumen is 0.15 mm in diam., and
MODEO ET AL.—TRICHITES OF STROMBIDIUM ARE EXTRUSOMES
99
Fig. 19–26. The ejection of trichites of Strombidium S3. 19. The ejection in vivo: rod-like structures can be seen near a swimming specimen. Arrows indicate those with a length corresponding to resting trichites; arrowheads indicate the longer ones (see text). Bar 5 5 mm. 20. A presumed initial stage of the ejecting process. The ‘‘cap’’ material is thinner at the top of the trichite approaching the cortex (arrowhead). Bar 5 1 mm. 21. A site (arrow) at which the cortical alveoli appear interrupted; the corresponding trichite is absent. Bar 5 1 mm. 22. At the exterior of a cell, in the vicinity of the alveolar interruption, a section of an ejected trichite is visible; the CSL appears to be sliding out of the EL. The emergence is indicated by arrows. An electron-dense ring (ER) is visible. Bar 5 1 mm. 23. Negative contrast picture of a corresponding phase (see Fig. 22) of the trichite ejection. Bar 5 1 mm. 24. A later phase of trichite ejection. The more internal layers of CSL have slipped out from the external ones. Bar 5 1 mm. 25, 26. Trichites seen at the end of the ejecting process (SEM, 25) and after negative contrast (TEM, 26). In both preparations trichites appear as thin tubes with smooth surfaces and a constant diameter. Bars 5 10 mm.
100
J. EUKARYOT. MICROBIOL., VOL. 48, NO. 1, JANUARY–FEBRUARY 2001
electron-dense rings are not evident. The CTs surround each trichite, as in S3, and ribosomes are abundant in the neighbouring cytoplasm (Fig. 10, 11). Cytochemistry. The S3 trichite compartments reacted differently from each other following the various treatments. In particular, the EL was partially digested by protease (Fig. 14) and was completely removed by pepsin (Fig. 15). The latter enzyme also digested the rings and the subcortical electrondense material (Fig. 15). Following the removal of the EL, six ‘‘arms’’ became visible; these connected the undigested CSL with the trichite membrane (Fig. 16) and correspond to the CSL extensions described in untreated specimens (Fig. 9). Digestion did not occur in control specimens (data not shown). The TSC-silver proteinate staining methods gave positive results on the rings, the trichite membrane, and the CT in contact with the membrane (Fig. 17). As expected, the perilemma, the polysaccharide platelets at the posterior region of the cell body, and the large paraglycogen granules were also stained by this method specific for polysaccharides (Fig. 18). Extrusion. Living specimens of S1 and S3 were put in a seawater drop on a microscope slide, and observed under the microscope. Under these conditions the ciliate stopped swimming in 8–10 min. At this point some rod-like structures of different length were observed around the cell. Most of these structures had the typical length and shape of trichites. The longest ones, which appeared thinner, reached 50 mm in length (Fig. 19). Generally, a few seconds later the cell disintegrated, likely due to the increased salinity and temperature of the water. When Strombidium S3 and S1 were transferred to seawater containing 3% dextran or 3% AED, a few rod-like structures were immediately seen projected outside of the swimming cell. After ejection, the cell ‘‘jumped’’ and changed its direction. The ejected structures were 50 mm long. Ejection was not observed in the control specimens. Some minutes later, the treated and the control ciliates stopped their swimming and rod-like structures could be observed around the cells; the shorter rods (; 11 mm) appeared less numerous in the treated cells. The S3 specimens transferred to pure seawater from the AED treatment were able to swim similarly to untreated cells. Ultrastructural observations. In some thin sections the electron-dense material at the top of single trichites appeared thinner or even completely lacking at the anterior end (Fig. 20). In other cases, not only the electron-dense material, but also the cortical alveoli appeared interrupted and extended, possibly forming a suitable ‘‘hole’’ for trichite ejection. Where a hole existed, the corresponding trichite was missing (Fig. 21). In some cases, exterior to the cell, adjacent to the ‘‘hole’’, a hollow structure, delimited by thin layers was visible; this was interpreted as a section of an ejected trichite (Fig. 22). By centrifugation in sucrose gradients a fraction mainly consisting of 50-mm-long rod structures was obtained. TEM observations of negatively stained samples showed that these longer rod structures were ejected trichites. The increase in trichite length was apparently due to the sliding of the CSL out of the EL (Fig. 22, 23). The emerging portion is ; 0.20 mm wide (the whole CSL surrounding the lumen) and the various sheets of the CSL apparently successively slide apart (Fig. 24). The trichites at the end of the ejection process, as seen with SEM and TEM, are hollow tubes with a smooth surface and a constant diameter (Fig. 25, 26). DISCUSSION Our data suggest that trichites of Strombidium are extrusomes. In the two species considered, the trichites differ slightly in position and ultrastructural feature, but in both species, no other structures could be associated with extrusive organelles.
On the contrary, extrusion of trichites was repeatedly observed under standardised, although not natural, conditions. Extrusion occurred following in vivo treatments with substances that are known to trigger the trichocyst ejection in Paramecium (Plattner et al. 1985). Following the ejection, which takes place rapidly, Strombidium did not appear to be damaged and continued to swim normally, although in a different direction. The trichites elongated ; 5 times compared to the resting state. Ultrastructural analysis showed that they matched the definition of extrusomes: membrane-bounded structures, usually located in the cortical cytoplasm, which are discharged when subjected to suitable stimuli and which undergo characteristic morphological changes when ejected (Hausmann 1978). Nevertheless, trichites represent a new and puzzling kind of extrusome. They are complicated in structure and have a similar general architecture in all three Strombidium species analysed at the ultrastructural level: S. sulcatum (Faure´-Fremiet and Ganier 1970), S3, and S1 (present study). The cytochemical analysis revealed that the various compartments of trichites differ in morphology and chemical composition. The external electron-dense layer consists of proteins, especially pepsin-sensitive proteins; similar proteins are present in the ER together with polysaccharidic components. Nothing can be inferred about the composition of the CSL; in our experimental conditions it was not modified by any of the applied procedures. The cytoplasmic features accompanying the trichites are also peculiar: 1) the CT delimited by a polysaccharide wall, 2) the proteinaceous dense material, forming a cap where trichites approach the cortex, and 3) the external microtubular sheets. The functions of these features are unknown. The thickness of the cap material is perhaps indicative of the readiness of the corresponding trichite to eject. Only a few trichites readily eject after dextran or AED treatments. Although these are artificial and non-specific stimuli, it is also possible that only a few trichites are ready at one time. The ejection mechanism can be tentatively reconstructed only on the basis of static images. It appears to involve, at first, the emergence of the whole CSL from the EL, followed by the slipping of the CSL layers one into the other. As the final product of the ejection is a tube with a smooth surface and a constant diameter, it can be supposed that the very thin layers of CSL reorganize themselves and fuse together during the process. Extrusomes are organelles widely present in protists and have been interpreted as organelles for interspecific interactions (Miyake and Harumoto 1996). Indeed, a defensive function has been demonstrated for trichocysts in Paramecium spp. (Harumoto and Miyake 1991; Knoll et al. 1991; Miyake and Harumoto 1996). In ciliates, tube-like extrusomes (toxicysts, pexicysts, haptocysts) are generally found in predatory species and function as offensive organelles (Hausmann 1978). Most Strombidium species are filter feeders, and the extrusion of trichites was accompanied by a change of direction in the swimming. So a defensive function, like that supposed for the tubular ejectisomes of some flagellates (Kugrens et al. 1994) and the trichocysts of Paramecium spp. appears more credible. However, it is also possible that, being rigid structures, they actually function as cytoskeletal elements. ACKNOWLEDGMENTS The authors express gratitude to D.J.S. Montagnes for critically reviewing the manuscript. Thanks are due to S. Gabrielli for his assistance in photographic work. LITERATURE CITED Bu¨tschli, O. 1873. Einiges u¨ber Infusorien. Arch. Mikr. Anat., 9:657– 678.
MODEO ET AL.—TRICHITES OF STROMBIDIUM ARE EXTRUSOMES Carey, P. G. 1992. Marine interstitial ciliates. Chapman & Hall, London, UK. Corliss, J. O. 1979. The Ciliated Protozoa. Characterization, classification and guide to the literature, 2nd ed. Pergamon, Oxford, UK. ¨ ber Infusorien des Golfes von Neapel. Mitt. Zool. Stat. Entz, G. 1884. U Neapel, 5:289–444. Eperon, S. & Peck, R. K. 1988. Structural and biochemical characterization of isolated trichocysts of the ciliate Pseudomicrothorax dubius. J. Protozool., 35:280–286. Faure´-Fremiet, E. & Ganier, M. Cl. 1970. Structure fine du Strombidium sulcatum Cl. et L. (Ciliata, Oligotrichida). Protistologica, 6:207–223. Gourret, P. & Roeser, P. 1888. Contribution a` l’e´tude des Protozoaires de la Corse. Arch. Biol., 8:143–156. Harumoto, T. & Miyake, A. 1991. Defensive function of trichocysts in Paramecium. J. Exp. Zool., 260:84–92. Hausmann, K. 1978. Extrusive organelles in protists. Int. Rev. Cytol., 52:197–276. Knoll, G., Haacke-Bell, B. & Plattner, H. 1991. Local trichocyst exocytosis provides an efficient escape mechanism for Paramecium cells. Europ. J. Protistol., 27:381–385. Krainer, K.-H. 1991. Contributions to the morphology, infraciliature and ecology of the planktonic ciliates Strombidium pelagicum n. sp., Pelagostrombidium mirabile (Penard, 1916) n. g., n. comb., and Pelagostrombidium fallax (Zacharias, 1896) n. g., n. comb. (Ciliophora, Oligotrichida). Europ. J. Protistol., 27:60–70. Kugrens, P., Lee, R. E. & Corliss, J. O. 1994. Ultrastructure, biogenesis, and functions of extrusive organelles in selected non-ciliate protists. Protoplasma, 181:164–190. Maeda, M. & Carey, P. G. 1985. An illustrated guide to the species of the family Strombidiidae (Oligotrichida, Ciliophora), free swimming
101
protozoa common in aquatic environment. Bull. Ocean. Res. Inst. Univ. Tokyo, 19:1–68. Miyake, A. & Harumoto, T. 1996. Defensive function of trichocysts in Paramecium against the predatory ciliate Monodinium balbiani. Europ. J. Protistol., 32:128–133. Montagnes, D. J. S. & Humphrey, E. 1998. A description of occurrence and morphology of a new species of red-water forming Strombidium (Spirotrichea, Oligotrichia). J. Eukaryot. Microbiol., 45:502–506. Montagnes, D. J. S., Taylor, F. J. R. & Lynn, D. H. 1990. Strombidium inclinatum n. sp. and reassessment of Strombidium sulcatum Clapare`de and Lachmann (Ciliophora). J. Protozool., 37:318–323. Montagnes, D. J. S., Lynn, D. H., Stoecker, D. K. & Small, E. B. 1988. Taxonomic descriptions of one new species and redescription of four species in the family Strombidiidae (Ciliophora, Oligotrichida). J. Protozool., 35:189–197. Petz, W. & Foissner, W. 1992. Morphology and morphogenesis of Strobilidium caudatum (Fromentel), Meseres corlissi n. sp., Halteria grandinella (Mu¨ller), and Strombidium rehwaldi n. sp., and a proposed phylogenetic system for Oligotrich Ciliates (Protozoa, Ciliophora). J. Protozool., 39:159–176. Plattner, H., Su¨rzl, R. & Matt, H. 1985. Synchronous exocytosis in Paramecium cells. IV. Polyamino compounds as potent trigger agents for repeatable trigger-redocking cycles. Eur. J. Cell Biol., 36:32–37. Rosati, G., Petroni, G., Quochi, S., Modeo, L. & Verni, F. 1999. Epixenosomes, peculiar epibionts of the hypotrich ciliate Euplotidium itoi, defend their host against predators. J. Eukaryot. Microbiol., 45:278– 282. Thie´ry, J. P. 1967. Mise en e´vidence des polysaccharides sur coupes fines en microscopie e´lectronique. J. Microscopie, 6:987–1018. Received: 04-07-00, 07-18-00, 09-18-00; accepted 09-18-00
