The ultrastructure of the cyst wall has been studied in severa! hypotrichous ciliates. In Oxytrichafallax (Grimes, 1973),Pleurotricha sp. (Matsusaka, 1976), Stylonychia mytilus (Walker et al., 1975), Gastrostyla steinii (Walker et al., 1980) and Laurentiella acuminata (Gutierrez et al., l 980), the cyst wall is composed of four layers called, from outside to inside :the ectocyst, meso­ cyst, endocyst and granular layers. In these ciliates a resorption of cilia and kinetosomes occurs during encystment, and the mature cyst is termed kinetosome resorbing. Diophrys scutum (Walker & Maugel , 1980) has a three layered cyst wall and the mature cyst is non kinetosome resorbing.
In the present work a structural and cytochemical study of the cyst wall in Histriculus similis
was carried out by means of light microscopy and transmission electron microscopy 

Calvo Ruiz, Purificación

Navas, P.

Pérez Silva, Julio

Torres Rueda, Antonio Ildefonso

idUS. Depósito de Investigación Universidad de Sevilla

   Jouma/ of General Microbiology (1983), 129, 829-832.   Printed in Great Britain 829    Complex Carbohydrates in the Cyst Wall of Histriculus similis By P. CA LV0 , 1  A . TO R R ES, 1 * P. N A V AS 2  AN D J. PE R E Z-SI LV A t Departamento de Microbiología 1 and Departam ento de Citología e Histología 2, Facultad de Biología , Universidad de Sevilla, Sevilla, Spain ( Received 8 Ju/y 1982; revised 8 September 1982) A cytochemical and structural study of the cyst wall of Histriculus similis has been carried out. Application of cytochemical methods for complex carbohydrates indicated that the mesocyst is rich in sulphated glycosaminoglycans. The endocyst and granular !ayer contained neutral and sulphated glycoproteins, respectively.    I NTRODU CTIO N The ultrastructure of the cyst wall has been studied in severa! hypotrichous ciliates. In Oxytrichafallax (Grimes, 1973),Pleurotricha sp. (Matsusaka, 1976), Stylonychia mytilus (Walker et al., 1975), Gastrostyla steinii (Walker et al., 1980) and Laurentiella acuminata (Gutierrez et al., l 980), the cyst wall is composed of four layers called, from outside to inside :the ectocyst, meso- cyst, endocyst and granular layers. In these ciliates a resorption of cilia and kinetosomes occurs during encystment , and the mature cyst is termed kinetosome resorbing. Diophrys scutum (Walker & Maugel , 1980) has a three layered cyst wall and the mature cyst is non kinetosome resorbing. In the present work a structural and cytochemical study of the cyst wall in Histriculus similis was carried out by means of light microscopy and transmission electron microscopy .  M ETHO DS Organism and culture conditions. Histric11lus similis (a hypotrichous ciliate) was cultured at 20 ± 1 ºC in Pring- sheim·s solution and fed on Chlorogonit1m sp. (Ammerma nn, 1974). The encystmen t was induced by placing the cell in Pringsheim's solution deprived of nutrients. Electron microscopy. The cysts were fixed in 2% (v/v) glutaraldehyde in O·I M-phosphate buffer pH 7·0 and then post fixed in 1% (w/ v) osmium tetroxide in the same buffer and embedded in Epon. Thin sections were contrast stained with uranyl acetate and lead citrate. Thick sections were stained with toluidine blue (Trump et al., 1961). Light microscopic cytochemistry. Thick sections of cysts fixed in 2 % glutaraldehyde in O·I M-phosphate buffer pH 7·0 and embedded in Epon were subjected to the following treatments: the periodic acid-Schiff test for neutral mucosubstance s (McManus, 1946): alcian blue 8GX pH 2·5 (Mowry, 1956); and 0·05%(w/ v) alcian blue pH 5·8 in the presence of graded concentration s of Mgl+ for the identification and differentiation of the three negatively charged  acidic groups of mucins (-COOH,  -P03H and  -S03 H) (Scott & Dorling, 1965). Ultrastrnct11ral cy 1ochemistry. Thin sectionsof cysts fixed in buffered 2% glutaraldehyde and embedded in Epon wcre mounted on gold gridsand stained by the pcriodic acid-thiocarbohydrazide-silver proteinatc (PA-TCH-SP) met hod of Thiery (1967). Oxidized control sections were treated either with silver proteinate without previou s thioca rbohydrazide treatment or with thiocarbohydrazide alone. Other sections were floated on 1% (w/ v) phos- photungstic acid in 1 M-HCI for 40 to 60 min according to Fléchon (1970) and washed with l ·25 M-HCI. Alternatively , cysts fixed in buffered 2% glutaraldehyde were stained with dialyzed iron (DI; Wetzel er al., 1966} or high iron diamidc (H ID: Spicer et al., 1978), dehydrated and embedded  in Epon.  R ESU LTS A fine structural analysis of the cyst wall of H . similis revealed striking similarities to other kinetosome resorbing cysts. The cyst wall consisted of four layers. The outermost !ayer (the ecto- Abbreriations : DI, dialysed iron ; HID, high iron diamide; PA-TCH-SP method , periodic acid-thiocarbo- hydrazidc-silver proteinate method.  0022-1287/83/0001-0680 $02.00 © 1983 SGM 830 P.  CA L VO  ANO  OTHE RS                                      En        Fig. l . Cyst wall of H . similis. (a) Photomicrograph of a thick section stained with toluidine blue. (b-f) Electron micrographs of ultrathin sect ions: (b) resting cyst wall; (e) cyst treated by the PA-TCH-SP method; (d) cyst wall staincd with phosphotungstic acid at low pH; (e) DI staining; (f) HID staining. Ec, ectocyst; M, mesocyst: En, endocyst: G. granular !ayer . The bar markers represen! 1 µm. cyst) presented an electron dense )amellar structure forming concentric plates, while the second )ayer (the mesocyst) was formed by a stratum of interwoven fibres. The endocyst was an electron dense fibrillar thin layer and the innermost layer (granular layer) had an extremely variable thickness and appeared to be composed of a granulated and electron dense material. (Fig. 1a,b).     Glycoconjugates in the Histricu/us cyst wal/ 831 Both the endocyst and granular layers had an intense periodic acid-Schiff reaction which d id not disappear after digestion with a-amylase. The mesocyst exhibited alcianophilia at pH 2·5 that was completely abolished after methylation at 60 ºC; this alcianophilia was reversed com- pletely after a methylation -saponification-alcian blue (pH 2·5) sequence of treatment. The staining with alcian blue at pH 5·8 in the presence of O· l M-MgCl 2 gave intense staining of the mesocyst ; this intense reaction also occurred in 0·5 M-MgCl 2 but decreased in 0·8 M- and 1 M- MgCl2. The intense alcianophilia of the mesocyst could mask any reaction of the endocyst with alcian blue; therefore, the reactivity of the endocyst towards cationic reagents was not deter- mined by light microscopy. The ectocyst appeared unreactive in techniques used for characterizing  glycoconjugates. In agreement with the above results, the PA-TCH-SP technique revealed the existence of glycoconjugates in both endocyst and granular layers (Fig. 1e). Treatment with phosphotungstic acid at low pH produced similar results: precipitates of phosphotungstic acid appeared in the endocyst and granular !ayer (Fig. 1 d). Finally, acidic mucosubstances were tested by electron microscopy using the DI and HID procedures. Both the mesocyst and granular layers showed DI and HID affinity (Fig. 1 e, J).  DISCUSSIO  N The characteristics of complex carbohydrates determined at the electron microscopic leve! agreed with those found by light microscopic cytochemistry . According to Spicer et al. (1979), mucosubstances which possess PA-TCH-SP reactivity but which lack DI and HID affinity are neutral glycoproteins, while mucosubstances which are DI or HID positive but which lack PA- TCH-SP reactivity can be regarded as glycosaminoglycans. Substances which possess PA- TCH-SP reactivity and also DI and/or HID affinity are acidic glycoproteins. These can be separated further into sulphated glycoconjugates which have affinity for both DI and HID, and carboxylated glycoproteins which have affinity for DI but not for HID. Cytochemical results obtained from the cyst wall of H .similis indicate that the mesocyst is rich in sulphated glycosaminoglycans, the endocyst contains neutral glycoproteins and the granular !ayer contains sulphated glycoproteins. The ectocyst was unreactive in the techniques used for characterizing glycoconjugates. However , the ectocyst precursors are PA-TCH-SP positive in the hypotrichous ciliate Laurentiella acuminata (J. C. Gutierrez & A. Torres, unpublished data). The lack of PA-TCH-SP staining in the ectocyst might be due to a blockage of vicinyl-glycol groups occurring during the encystment. Sulphated glycosaminoglycans in the mesocyst form a hydrophilic region that would play an important role in the transport of solutes and in the retention of water. Acidic glycoproteins of the granular !ayer, in contact with the cytoplasm of the cyst, are good candidates for mediating cyst-environment interactions.  R EFER E NCES  AMMERMANN , 0. (1974). The development of the macronucleus in the ciliated Protozoan Stylonychia mytilus. Chromosoma 45, 401--429. FLÉCHON, J. E. (1970). Nature glycoprotéique des granules corticaux de l"oeuf de lapine. Journal de Microscopie  9, 221-242. GRIMES, G. W. (1973). Differentiation during encyst- ment and excystment in Oxytrichafal/ax . Journal of Protozoology 20, 92-104. GUTJERREZ , J. C., TORRES, A . & PEREZ-SILVA , J. (1980). Transmission and scanning electron micros- copy, and solubility test of thc resting cyst walls of Laurentiel/a acuminata (Hypotrichida , Oxytrichi- dae). Joumal of Protozoo/ogy 27, 82A. MATSUSAKA, T. (1976). A structural study of encyst- ment in the hypotrichous ciliate Pleurotricha sp. Kumamoto Journal of Science Biol. 13, 13-26. MCMANUS, J. F. A. (1946). Histological demonstration of muein after periodie acid.Nature, London  IS, 202. MOWRY, R. W. (1956). Alcian blue techniques for the h1stochemical study of acid carbohydrates. Journal of Histoclremistry and Cytochemistry 4, 407--408 . Scon, J . E. & DORLING, J . (1965). Differential staining of acid glycosaminoglycans (mucopoly- saccharides) by Alcian blue in salt solutions. Histo- chemie 5, 221-223. SPICER, S. S., HARDIN, J. H. & SETSER, M. E. (1978). Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure . Histoclzemical Joumal 10, 435--452 . SPICER, S. S., SANNES, P. L. & KATSUYAMA, T. (1979). Cytochemical characterization of secretory and cell surface   glycoconjugates   by   light   and   electron 832 P . CAL YO  A NO  OTHE RS  microscopy. Journal o/ Histochemistry and Cyto- chemistrr 27, 1 182-1184. THIERY , J. P. (1967). Mise en evidence des poly- saccharides sur coupcs fines en microscopic électron- ique. Joumal de M icroscopie 6, 987-1O18. TRUMP, B. F., SMUCKLER, E. A. & BENDITT, E. P. (1961).A method for staining cpoxy sections for light microscopy. Journal o/ Ultrastmcture Research 5, 343-348. WALKER , G. K.& MAUGEL, T. K .(1980). Encystmenl and excystmenl in Hypotrich Ciliates. 11. Diophrys scut1m1 and remarks on comparative featurcs. Protis- tologica 16, 525-531.  WALKER, G. K., MAUGEL, T. K .& GOODE, D. (1975). Sorne ultrastructural observations on cncystment in Stylonyclria mytilus (Ciliophora ; Hypotrichida). Transactions of the American Microscopica/ Society 94,  147-154. WALKER, G. K., MAUGEL, T. K .& GOODE, D. (1980). Encystment and excystment in Hypotrich Ciliates. l. Gastrostyla steinii. Protistologica 16, 51 1-524. WETZEL, M. G., WETZEL , B. K . & SPICER , S. S. (1966). Ultras1ruclu ral localization of acid mucosubstances in the mouse colon with iron-containing stains. Journal o/ Ce// Bio/ogy 30, 299-315.  

Complex Carbohydrates in the Cyst Wall of Histriculus similis

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Complex Carbohydrates in the Cyst Wall of Histriculus similis

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idUS. Depósito de Investigación Universidad de Sevilla