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A study in the light and electron microscope of the extruded peristome and related structures of the rumen ciliate entodinium caudatum
3 I S S U E & C F L I |971 3 (3) ",71~380 P u b h s h e d b2 Longman Group L t d Printed m Gleat B r t l a m
G S COLEMAN and P J. HALL'~
A S T U D Y IN THE LIGHT AND ELECTRON M I C R O S C O P E OF THE E X T R U D E D P E R I S T O M E AND RELATED S T R U C T U R E S OF THE R U M E N CILIATE E N T O D I N I U M CA UDA T U M A B S T R A C T A s t u d y m the h g h t a n d electron m ~ c m s c o p e o f sectmn~ o f the t u r e e n ciliate Entodzmum calulatum w a s u n d e r t a k e n m an a t t e m p t to e l u c i d a t e the s t r u c t m e o f the p e r l s t o m e when m the e x t r u d e d state T h e c o n f o r m a t m n o f t h e c d l a r y b a n d on the p e r l s t o m e a n d the s t r u c t u r e o f t h e h m n g o f the buccal c a w t y a r e described T h e s t r u c t u r e a n d position o f a tuft o f s p e c m h z e d sheet h k e e x t e n s m n s o f the cell surface ~s also described
Introduetmn
resting p r o t o z o a m w h i c h the p e r l s t o m e was r e t r a c t e d Howeve~ they did describe the s t r u c t u r e of lndlwdua[ cdJa and their basal bodies m detail a n d thJs will n o t be cons~dered m the papel The p u r p o s e o f the present p a p e r is to describe the structure of the a n t e r i o r end of the r u m e n cflmte Ento d#uum caudatum t h a t has been m a i n t a i n e d m v~tro for ovel e~ght years The use o f th~s c u l t u r e h a d the a d v a n t a g e t h a t firstly all the p r o t o z o a were the same secondly t h a t httle of the m o r p h o l o g y of tb~s o r g a m s m h a d b e e n described and thirdly t h a t the o r g a m s m s could b e easily harvested w i t h the p e n s t o m e e x t r u d e d as in s w i m m i n g a n d feeding protozoa
Entodmtum caudatum is an E n t o d m l o m o r p h l d p r o t o z o o n c o m m o n l y f o u n d m the r u m e n of sheep cattle and other r u m i n a n t s It ~s o n e of the simplest p r o t o z o a of thin o r d e r and possesses n o dorsal zone o f m e m b t a nelles T h e a d o r a l zone of m e m b r a n e l l e s is s i t u a t e d on a retractile p e n s t o m e which c a n exther be e x t r u d e d as m the s w i m m i n g or feeding o r g a m s m or retracted w h e n the p r o t o z o o n ~s resting A t Jts base the p e n s t o m e ~s circular m transverse s e c n o n w~th the o e s o p h a g u s m the m i d d l e T h e r e h a v e been several descriptions of the structures of the m e m b r a n e l l e s s u r r o u n d i n g the o e s o p h a g u s a n d sJtuated o n the perxstome of the r u m e n E n t o d m l o m o r p h t d p r o t o z o a (Bretschne~der 1960a b 1962 N o l r o t T l m o t h e e 1960 R u t h a n d Shlgenaka 1964) U n f o r t u n a t e l y these a u t h o r s considered t h a t all these p r o t o z o a have the same basic m o r p h o l o g y wathout d e m o n s t r a t i n g t h a t th~s was true a n d h a v e described only the structure of
Methods Source of p r o t o z o a a n d p r e p a r a t i o n o f p r o t o z o a l suspensions were as described b y C o l e m a n (1964) A t least h a l f of the p r o t o z o a in the suspensions wele actively s w i m m i n g even at r o o m t e m p e r a t u r e
Pr~paranon of sectzons o f protozoa for rmcl oscopy
* A R C Institute o f A m m a l P h y s i o l o g y 13abra ham Cambridge M a n u s c r i p t received 14 D e c e m b e r 1970 R e w s e d m a n u s c r i p t received 6 A p r i l 1971 A
T h e m e t h o d used was b a s e d on t h o s e o f K e l l e n b e r g e r R y t e r a n d Sechaud (1958) a n d 371
372
COLEMAN AND HALL
Sabatini, Bensch and Barrnett (1963). The protozoal suspension was fixed initially in 3 ~ glutaraldehyde dissolved in 0 - 1 M phosphate buffer p H 6"8-7.0 for 30 min. The pellet was then washed for 2 hr in three or four changes of phosphate buffer. The subsequent fixation in osmium tetroxide and preparation of sections for electron microscopy was as described by Coleman and Hall (1969). Sections for light microscopy were cut 0.2 0"3tz thick from the Araldite blocks used to prepare sections for electron microscopy and were stained for 3-5 min with 2 ~ methylene blue dissolved in 2~ borax prepared by heating at 60~'~C. The sections were dehydrated in solutions of increasing ethanol concentration, cleared in xylene and mounted in D.P.X.
Results Figs. 1--10 are a series of photomicrographs of serial transverse sections through the anterior end (and running from the anterior end) of an Entodinium caudatum organism in which the peristome was extruded. In Figs. 1~5 the U-shaped peristome (p) was free although in Figs, 4-5 at least it was surrounded by other parts of the protozoon. In Figs, 7-10 the peristome was joined to the remainder of the protozoon on all sides although the buccal cavity (bc) and its associated cilia (c) are still visible. An electron micrograph of a transverse section through the region where the peristome was attached to the rest of the organism is shown in Fig. 21 (e). Figs..11-20 are photomicrographs of serial longitudinal sections of E. caudalum and show as before that the peristome was free except at the base (at x in Fig. 15). The distribution o f cilia The distribution of cilia over the surface of the peristome was not uniform, At the posterior end cilia were present in a band down the dorsal side of the oesophagus shown as
c in Figs. 7 and 16 and at y in Fig. 22. This band was 3--4 cilia wide at the posterior end as is showh in Fig. 23 (z). The remainder of the oesophagus was bare of cilia and in many sections, e.g, Fig, 23, no cilia are visible over most of the length of the oesophagus. There is no evidence for a spiral of cilia as was suggested for Epidinium ecaudatum by Sharpe (1914). This band of cilia Jn the oesophagus was joined to the cilia situated on only one arm of the U-shaped peristome, i.e. at a in Fig. 2, at d in Fig. 22 and at g in Fig. 17. The ciliary band then continued round the convex surface of the peristome and in some of the more anterior transverse sections (Figs. 2-3) cilia were present principally on the convex surface of the peristome. However, in the most anterior section (Fig. 1 at h) and in sections through other protozoa it is apparent that the tip of the peristome was not ciliated. The bare tip of the peristome is also shown at f i n Fig. 12. A diagrammatic representation of the peristome is shown in Fig. 27. Inner and outer lips When the peristome was retracted it was covered by the inner lips and an organism with a semi-retracted peristome is shown in Fig. 23. The inner lips (i) did not form a complete ring round the peristome at the posterior end and there was a gap where this joined the protozoon and on the opposite side (Fig. 21). The outer lips (o) were just part of the remainder of the protozoon and frequently contained rnany protozoal polysaccharide granules. These outer lips disappeared as such when the peristome retracted and the inner lips joined together, In some sections (Fig, 21) the surface of the lips were corrugated and it is possible that these corrugations meshed together to make a seal when the peristome was retracted. However, they may have been the result of the folding of the walls of the lips when the peristome was retracted although this is considered to be unlikely.
Figs. 1-10. Photomicrographs of successive serial transverse sections through tile anterior end of Entodiniton cat,datum fixed with its peristome extruded. The sections run posteriorly from Fig. l to Fig. 10 and were approximately 0.2-0.3~o thick. • 1000. Figs. 11-20. Photomicrographs of successive serial longitudinal sections through Entodinium cau&ttum. The dorsal surface of the protozoon is on the botton3 right-hand side. The sections were approximatcly 0.2-0.3p~ thick. • 800.
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11
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374
COLEMAN
AND
HALL
Fig. 21. Electron m i c r o g r a p h of a transverse section t h r o u g h the anterior end of Etttodi~Tium caudatum, This section was cut in a p p r o x i m a t e l y the same place as Fig. 4. • 4000, Fig. 22. Elcctron micrograph of a l o n g i t u d i n a l section t h r o u g h the anterior end of Etttodillium caudatum with an extruded peristome. Note tlne cilia lining one side of the oesophagus only (y) and the continuity (d) of this band with those cilia at the anterior end of the peristome. • 6000. Fig. 23. Electron micrograph of a longitudinal section through the anterior end of Entodinium caudatum with a semi-retracted peristome. Note that in this section most of the walls of tl~e o e s o p h a g u s (10 are devoid of cilia a l t h o u g h four cilia presumably representing the end of the band down the side of the oesophagus are visible at the b o t t o m of the picture (z). Also note the position of the specialized sheet-like extension o f cell surface (s) on the opposite side of the organism to the nucleus (n). x 4400, Fig. 24. Electron m i c r o g r a p h of a section through the inner (posterior) end of the oesophagus showing a vesic]e (v) opening into the cavity and the rows of microtubules (m) between the vesicles. Two bacteria (b) t h a t have p r o b a b l y been recently engulfed are shown at the b o t t o m of the picture. • 30,000. Fig. 25. Electron m i c r o g r a p h of a longitudinal section through the anterior end o f Entodinium caudatum showing the specialized sheet-like extensions of the cell surface (s) which are a p p a r e n t l y interdigitated with cilia. • 24,200. Fig. 26, Electron m i c r o g r a p h of a transverse section through the anterior end o f L)ltodinium caudatum showing the position of the specialized sheet-like extensions of the celt surface relative to the inner lips and the peristome. N o t e that these sheet-like extensions appear similar in longitudinal and transverse section, x 20,000. K E Y TO A B B R E V I A T I O N S b bc c i m ~ o p s rv
bacterium. oesophagus (buccal cavity) cilia inner lips microtubulcs macronucleus outer lips peristome sheet-like extensions of cell surface osmiophilic structure at base of a cilium (see C o l e m a n and Hall, 1971)
|
0
DELACHAMBRE
500 filaments sera discut6 dans ce travail, mais il convient d6s maintenant de prdciser la terminologie utilisde ici; les 'pore-canals' des auteurs de langue anglaise est traduit par le terme 'canal cuticulaire', les filaments comparables aux 'pore-canal filaments' sont appel~s 'filaments axiaux', les filaments d6crits par Locke (1961) c o m m e 'wax-canal filaments' chez la nymphe de Tenebrio sont appel~s 'filaments multiples', les filaments en forme de tubes clairs entour6s d'un m a n c h o n dense, sont appelds 'filaments tubulaires'.
Mat&iel et Techniques
i
- - d ' u n e solution aqueuse de papaine (2X cristallis6e, Koch-Light) ~t 1% ~ 60~ de 24 ~t 72 heures, renouvel~e plusieurs fois; - - d ' u n e solution aqueuse de pronase (45000 P U K units, B.D.H.) ",k 5 % A 40~ de 4/~ 72 heures. Dans certains cas des pi~ces d6j~t d6prot6inisdes par la soude ont ~t6 soumises '~ l'action de la pronase. (3) Des essais de digestion enzymatique sur coupes incluses en resine, selon la technique de M o n n e r o n et Bernhard (1966) ont 6t6 effectu6s avec ces solutions de pronase, de trypsine ou de pepsine. Histochimie ultrastructurale:
Les conditions d'dlevage, la chronologie des diffdrents 6vdnements de la vie nymphale et la preparation du tdgument pour l'6tude ultrastructurale ont 6t6 ddcrits prdc6demment (Delachambre, 1970). Extractions lipidiques: des fragments non fixds de sternites abdominaux de nymphes et d'adultes h diffdrents gtges ont 6t6 plong6s dans plusieurs solvants des lipides: chloroforme, benz6ne-chloroforme (1/1), chloroforme-m& thanol (1 / I) ou pyridine pendant 6 h 24 heures tempdrature ambiante ou A 60~ Ces fragments ont dt6 ensuite fixds et trait& pour l'observation au microscope 61ectronique.
Certaines coupes r6cup6r~es sur grilles en or ou en or-palladium ont 6t6 traitdes par l'argent-m6th6namine selon la technique de Marinozzi (1961) modifi6e par R a m b o u r g et Leblond (1967) ou par la thiocarbohydrazideprot6inate d'argent selon Thierry (1967) avec ou sans oxydation par l'acide periodique ou l'eau oxyg6nde ~ 10%. Ces traitements ont ~t6 effectu6s sur des pi6ces fix6es soit par la glutarald6hyde suivie de t&roxyde d'osmium, soit par le tdtroxyde d ' o s m i u m seul, soit non fixdes.
Ddprotdinisation :
I. C A N A U X DE LA C U T I C U L E NYMPHALE
(I) Soude: des segments abdominaux entiers sont placds dans des r6cipients contenant une solution de soude 0,5 N pendant 4, 18, 24, 48 heures it temp6rature ambiante ou dans des tubes scellds contenant de la soude 0,5 N pendant 2, 6, 12, 24 heures /~ 100~ Apr~s action de la soude, le t6gument est rinc6 plusieurs heures /l l'eau courante puis dans plusieurs bains d'eau distill6e avant prdparation p o u r l'6tude ultrastructurale. (2) Enzymes prot6olytiques: des fragments de t6gument sternal frais ont 6t6 soumis ~t l'actJon:
L a cuticule nymphale de T. molitor (Fig. 1) est form6e d'une 6picuticule, d'une m6socuticule pr6-exuviale d'une trentaine de lamelles et d'une endocuticule post-exuviale plus fine (12 5. 14 lamelles) (Delachambre, 1967). Les canaux cuticulaires de cette cuticule nymphale ont ~t6 d~crits par Locke (1961) et ses observations ont 6t6 confirm6es par Caveney (1970). La forme de ces canaux est d6terminde par l'orientation des microfibres cuticulaires selon le sch6ma de Bouligand (1965), et Neville et al. (1969) les consid6rent c o m m e des rubans creux qui suivent la rotation des
R6sultats
Fig. 1. Coupe de cuticule sternale nymphale. Les canaux cuticulaires montrent un seul filament axial saul dans la r6gion apicale o~1 apparaissent les filaments multiples (ep: 6picuticule, meso: mdsocuticule, endo: endocuticule post-exuviale). • 12 600. Fig. 2. Coupe longitudinale d'un canal cuticulaire nymphal. Le filament axial est reli6 aux parois du canal par de fins filaments (fl6ches). • 80 000. Fig. 3. Insertion d'un filament axial nymphal sur l'6piderme par une zone it microtubules. La membrane plasmique ne p6n6tre pas dans le canal. Le filament axial est extra-cel/ulaire. On notera 6galement la densification de la membrane plasmique au contact du filament (fl6cfie). • 22 000.
378
Structure of the oesophagus Those parts of the peristome and oesophagus that were not covered with cilia also have a characteristic structure (Fig. 23). The surface was covered by a unit membrane 9 m~ thick underneath which were rows (70-200 mf~ apart) of 13 microtubules (m) each of which was 18-20 m~ in diameter. The rows ran approximately at right angles to the inner surface of the oesophagus and the individual tubules ran parallel to the surface of the oesophagus so as to partially encircle the lumen. Similar microtubules are commonly found in the cytostomial and cytopharyngeal walls of other protozoa, e.g. Gymnostomatida and Trichostomatida (Grain, 1966; Faurd-Fremiet, 1961). At the bottom of the oesophagus the rows of tubules were further (up to 250 mr0 apart and vesicles (v), some of which opened into the lumen, were present between the rows (Fig. 24). Entodinium caudatum rapidly engulfed bacteria and other particulate matter into vesicles in the endoplasm (Coleman, 1964; Coleman and Hall, 1969) and this is considered to be the site where the materials entered the cytoplasm. Although no bacteria have ever been lbund in these vesicles, even in protozoa that were actively engulfing bacteria when fixed, some (b) have been observed in closed vesicles just below the surface. As many bacteria were occasionally present in a single vesicle, the size of the vesicle opening into the oesophagus may also be variable.
Specialized sheet-like extensions of cell surface On the ventral side of the peristome and usually situated approximately midway along its length when in the extruded state was a tuft of specialized organelles (s) (Figs. 25 and 26). This tuft, which also contained apparently normal cilia that interdigitated with the specialized structures, and which was similar to the 'Paralabialorgan' described in Ophryoscolecidae by Bretschneider (1962), usually arose from a protuberance at the side of the peristome but more rarely these organelles were attached to the inner lips or part of the protozoon next to the contractile vacuole. When the peristome was extruded these organelles just protruded beyond the inner lips. They are considered to be 'sheet-like' rather than cylindrical
CO LEMA N AND HALL because in section they appear to be similar in. length and thickness in all planes of cut and because much more of any one is always visible than for the normal cilia which are cylindrical. These specialized sheet-like extensions of the cell surface were not of uniform thickness. The innermost which were 140-180m~ thick contained two sets of microtubules (20 m~ in diameter and 40 m~ apart) which did not run parallel to each other. The outer sheet-like extensions were only 90 m~ thick and contained a single set of tubules.
The absence of membrane-bound groups of cilia Bretschneider (1960a, b) and NoirotTimothde (1960) both considered whether or not the individual cilia were bound together as a cirrus or syncilium and the former drew groups of cilia surrounded by a membrane. To determine if there was a membrane surrounding cilia whole protozoa with extruded peristomes were negatively stained with potassium phosphotungstate (Brenner and Home, 1959). No evidence for any such membrane or rigid grouping of cilia was found under the electron microscope.
Discussion As far as we are aware the present investigation represents the first systematic study in the light and electron microscope on the ciliature of the extruded peristome of Entodinium caudatum. Fig. 27 is a diagrammatic representation of the anterior end of the protozoon when viewed from the sides and shows the position of the ciliary band on the peristome and the specialized sheet-like extensions of the cell surface relative to the nucleus. The function of the specialized sheet-like extensions is unknown as there is little evidence available. However, Roth and Shigenaka (1964) who showed the existence of a tuft of similar but different structure to that described above, attached to the peristome of a Diplodinium sp., considered that it was of importance in cell division and that the cilia were in an arrested state of development. Bretschneider (1962) described the structure of a 'Paralabialorgan' in Entodinium and Epidinium spp. and considered it to have a sensory function. Although this 'Paralabialorgan' was similar to
PERISTOME STRUCTURE OF
379
ENTODINIUM
the sheet-like extensions described above, it was larger, contained more cilia and was usually situated in the axil between the peristome and the inner lip. Unfortunately an exact comparison is not possible as Bretschneider worked with mixed protozoal species. The present authors have also found no evidence for any normal cilia of larger diameter than the rnajority in Entodinium caudatum although a single row of large
cilia was found in diplodinia by Roth and Shigenaka (1964) and Noirot-Timothde (1960). Acknowledgements We wish to thank Drs R. W. Cox and F. B. P. Wooding for helpful advice and discussion and Mrs B. C. Barker and Miss J. I. Davies for valuable technical assistance.
Ciliary Band Outer Lip
-
Position of 'Sheetqikfe;~~tsn;If:21l-
~
~
(
~
--
Inner Lip
Macronucleus
Oesophagus-
1 II
LATERAL VIEW
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V E N T R A L VIEW
LATERAL VIEW
Fig. 27. Diagrammatic representation of the peristome from three sides.
380
COLEMAN
AND
HALL
References
BRENNER, S. and HORNE, R. W. (1959). A negative staining method for high resolution microscopy of viruses. Biochim. biophys. Acta, 34, 103 110. BRETSCHNE~DER, L. H. /960a. EIektronenmikroskopische Untersuchung des Peristomapparates ciniger Ophryoscoleciden I. Proc. K, ned. Akad. Wet. c, 63, 291-305. B~E'rSCHNEmER, L. H. 1960b. Elektronenmikroskopische Untersuchung des Peristomapparates einiger Ophryoscoleciden I1. Proc. K. ned. Akad. Wet. c, 63, 306 317. BREd'SCHNEiDER, L. H, 1962. Das Paralabialorgan der Ophryoscoleciden. Proc. K. ned. Akad. I/Vet. c, 65, 423 452. COLEMAN, G- S. 1964. The metabolism of Escherichia coli and other bacteria by Entodinium caudatum. J. gen. Microbiol., 37, 209-223. COLEMAN, G. S. and HALt,, F. J. 1969. Electron microscopy of the rumen ciliate Entodinium caudatum, with special rcference to the engulfment of bacteria and other particulate matter. Tissue & Cell, 1,607-618. COLEMAN,G. S. and HALL, ]=. J. 1971. Studies in the electron microscope of the adoral zone of membranelles of the rumen ciliate Entodinium caudatum using the technique of negative staining. Tissue & Cell, 3, 381388. FAURt%FREM1EE, E. /96/. Le cytoplasme stomo-pharyngien des Cilids Cyrtophores. C.r. hebd. St:anc. Acad. Sci., Paris, 253, 357-362. GI',AIN, J. 1966. l~!tude cytologique de quelques cilids holotriches endocommensaux des ruminants et des dquidds. Protistologica, 2 (2) 5 47. KELLENBERGE~, E., RYTER, A. and Sf,CHAUL~, J. 1958. Electron microscopic study of DNA-containing plasma 2. Vegetative and mature phage DNAwith normal bacterial nucleoids in difi'erent physiological states. J. biophys, biochem, Cytol., 4, 671--676. NOIROT-TIMOTHF,E, C. 1960. Etude d'une famille de cili~s: Ies Ophryoscolecidae. Structures et ultrastructures, AHn/s. Sci. nat. (Zool.), 12e sdrie, 2, 533 718. ROTf{, L. E. and SHIGENAKA,Y. 1964. The structure and formatioll of cilia and filaments in rumeu protozoa. J. Cell Biol., 20, 208~213. SABATINhD. D., BrNSCH, K. and BARRNETT, R. J. /963. Cytochemistry and electron-microscopy. The preservation ol" cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17, 19 -58. SnAaPE, R. G. 1914. Diplodir~ium ecalMatum with an account of its neuromotor apparatus, Univ. CahT/i Ptcbls. Zool., 13, 43-122.
G S COLEMAN and P J. HALL'~
A S T U D Y IN THE LIGHT AND ELECTRON M I C R O S C O P E OF THE E X T R U D E D P E R I S T O M E AND RELATED S T R U C T U R E S OF THE R U M E N CILIATE E N T O D I N I U M CA UDA T U M A B S T R A C T A s t u d y m the h g h t a n d electron m ~ c m s c o p e o f sectmn~ o f the t u r e e n ciliate Entodzmum calulatum w a s u n d e r t a k e n m an a t t e m p t to e l u c i d a t e the s t r u c t m e o f the p e r l s t o m e when m the e x t r u d e d state T h e c o n f o r m a t m n o f t h e c d l a r y b a n d on the p e r l s t o m e a n d the s t r u c t u r e o f t h e h m n g o f the buccal c a w t y a r e described T h e s t r u c t u r e a n d position o f a tuft o f s p e c m h z e d sheet h k e e x t e n s m n s o f the cell surface ~s also described
Introduetmn
resting p r o t o z o a m w h i c h the p e r l s t o m e was r e t r a c t e d Howeve~ they did describe the s t r u c t u r e of lndlwdua[ cdJa and their basal bodies m detail a n d thJs will n o t be cons~dered m the papel The p u r p o s e o f the present p a p e r is to describe the structure of the a n t e r i o r end of the r u m e n cflmte Ento d#uum caudatum t h a t has been m a i n t a i n e d m v~tro for ovel e~ght years The use o f th~s c u l t u r e h a d the a d v a n t a g e t h a t firstly all the p r o t o z o a were the same secondly t h a t httle of the m o r p h o l o g y of tb~s o r g a m s m h a d b e e n described and thirdly t h a t the o r g a m s m s could b e easily harvested w i t h the p e n s t o m e e x t r u d e d as in s w i m m i n g a n d feeding protozoa
Entodmtum caudatum is an E n t o d m l o m o r p h l d p r o t o z o o n c o m m o n l y f o u n d m the r u m e n of sheep cattle and other r u m i n a n t s It ~s o n e of the simplest p r o t o z o a of thin o r d e r and possesses n o dorsal zone o f m e m b t a nelles T h e a d o r a l zone of m e m b r a n e l l e s is s i t u a t e d on a retractile p e n s t o m e which c a n exther be e x t r u d e d as m the s w i m m i n g or feeding o r g a m s m or retracted w h e n the p r o t o z o o n ~s resting A t Jts base the p e n s t o m e ~s circular m transverse s e c n o n w~th the o e s o p h a g u s m the m i d d l e T h e r e h a v e been several descriptions of the structures of the m e m b r a n e l l e s s u r r o u n d i n g the o e s o p h a g u s a n d sJtuated o n the perxstome of the r u m e n E n t o d m l o m o r p h t d p r o t o z o a (Bretschne~der 1960a b 1962 N o l r o t T l m o t h e e 1960 R u t h a n d Shlgenaka 1964) U n f o r t u n a t e l y these a u t h o r s considered t h a t all these p r o t o z o a have the same basic m o r p h o l o g y wathout d e m o n s t r a t i n g t h a t th~s was true a n d h a v e described only the structure of
Methods Source of p r o t o z o a a n d p r e p a r a t i o n o f p r o t o z o a l suspensions were as described b y C o l e m a n (1964) A t least h a l f of the p r o t o z o a in the suspensions wele actively s w i m m i n g even at r o o m t e m p e r a t u r e
Pr~paranon of sectzons o f protozoa for rmcl oscopy
* A R C Institute o f A m m a l P h y s i o l o g y 13abra ham Cambridge M a n u s c r i p t received 14 D e c e m b e r 1970 R e w s e d m a n u s c r i p t received 6 A p r i l 1971 A
T h e m e t h o d used was b a s e d on t h o s e o f K e l l e n b e r g e r R y t e r a n d Sechaud (1958) a n d 371
372
COLEMAN AND HALL
Sabatini, Bensch and Barrnett (1963). The protozoal suspension was fixed initially in 3 ~ glutaraldehyde dissolved in 0 - 1 M phosphate buffer p H 6"8-7.0 for 30 min. The pellet was then washed for 2 hr in three or four changes of phosphate buffer. The subsequent fixation in osmium tetroxide and preparation of sections for electron microscopy was as described by Coleman and Hall (1969). Sections for light microscopy were cut 0.2 0"3tz thick from the Araldite blocks used to prepare sections for electron microscopy and were stained for 3-5 min with 2 ~ methylene blue dissolved in 2~ borax prepared by heating at 60~'~C. The sections were dehydrated in solutions of increasing ethanol concentration, cleared in xylene and mounted in D.P.X.
Results Figs. 1--10 are a series of photomicrographs of serial transverse sections through the anterior end (and running from the anterior end) of an Entodinium caudatum organism in which the peristome was extruded. In Figs. 1~5 the U-shaped peristome (p) was free although in Figs, 4-5 at least it was surrounded by other parts of the protozoon. In Figs, 7-10 the peristome was joined to the remainder of the protozoon on all sides although the buccal cavity (bc) and its associated cilia (c) are still visible. An electron micrograph of a transverse section through the region where the peristome was attached to the rest of the organism is shown in Fig. 21 (e). Figs..11-20 are photomicrographs of serial longitudinal sections of E. caudalum and show as before that the peristome was free except at the base (at x in Fig. 15). The distribution o f cilia The distribution of cilia over the surface of the peristome was not uniform, At the posterior end cilia were present in a band down the dorsal side of the oesophagus shown as
c in Figs. 7 and 16 and at y in Fig. 22. This band was 3--4 cilia wide at the posterior end as is showh in Fig. 23 (z). The remainder of the oesophagus was bare of cilia and in many sections, e.g, Fig, 23, no cilia are visible over most of the length of the oesophagus. There is no evidence for a spiral of cilia as was suggested for Epidinium ecaudatum by Sharpe (1914). This band of cilia Jn the oesophagus was joined to the cilia situated on only one arm of the U-shaped peristome, i.e. at a in Fig. 2, at d in Fig. 22 and at g in Fig. 17. The ciliary band then continued round the convex surface of the peristome and in some of the more anterior transverse sections (Figs. 2-3) cilia were present principally on the convex surface of the peristome. However, in the most anterior section (Fig. 1 at h) and in sections through other protozoa it is apparent that the tip of the peristome was not ciliated. The bare tip of the peristome is also shown at f i n Fig. 12. A diagrammatic representation of the peristome is shown in Fig. 27. Inner and outer lips When the peristome was retracted it was covered by the inner lips and an organism with a semi-retracted peristome is shown in Fig. 23. The inner lips (i) did not form a complete ring round the peristome at the posterior end and there was a gap where this joined the protozoon and on the opposite side (Fig. 21). The outer lips (o) were just part of the remainder of the protozoon and frequently contained rnany protozoal polysaccharide granules. These outer lips disappeared as such when the peristome retracted and the inner lips joined together, In some sections (Fig, 21) the surface of the lips were corrugated and it is possible that these corrugations meshed together to make a seal when the peristome was retracted. However, they may have been the result of the folding of the walls of the lips when the peristome was retracted although this is considered to be unlikely.
Figs. 1-10. Photomicrographs of successive serial transverse sections through tile anterior end of Entodiniton cat,datum fixed with its peristome extruded. The sections run posteriorly from Fig. l to Fig. 10 and were approximately 0.2-0.3~o thick. • 1000. Figs. 11-20. Photomicrographs of successive serial longitudinal sections through Entodinium cau&ttum. The dorsal surface of the protozoon is on the botton3 right-hand side. The sections were approximatcly 0.2-0.3p~ thick. • 800.
~
bc
g
,#J-
11
;~-i '~,
~ ;
374
COLEMAN
AND
HALL
Fig. 21. Electron m i c r o g r a p h of a transverse section t h r o u g h the anterior end of Etttodi~Tium caudatum, This section was cut in a p p r o x i m a t e l y the same place as Fig. 4. • 4000, Fig. 22. Elcctron micrograph of a l o n g i t u d i n a l section t h r o u g h the anterior end of Etttodillium caudatum with an extruded peristome. Note tlne cilia lining one side of the oesophagus only (y) and the continuity (d) of this band with those cilia at the anterior end of the peristome. • 6000. Fig. 23. Electron micrograph of a longitudinal section through the anterior end of Entodinium caudatum with a semi-retracted peristome. Note that in this section most of the walls of tl~e o e s o p h a g u s (10 are devoid of cilia a l t h o u g h four cilia presumably representing the end of the band down the side of the oesophagus are visible at the b o t t o m of the picture (z). Also note the position of the specialized sheet-like extension o f cell surface (s) on the opposite side of the organism to the nucleus (n). x 4400, Fig. 24. Electron m i c r o g r a p h of a section through the inner (posterior) end of the oesophagus showing a vesic]e (v) opening into the cavity and the rows of microtubules (m) between the vesicles. Two bacteria (b) t h a t have p r o b a b l y been recently engulfed are shown at the b o t t o m of the picture. • 30,000. Fig. 25. Electron m i c r o g r a p h of a longitudinal section through the anterior end o f Entodinium caudatum showing the specialized sheet-like extensions of the cell surface (s) which are a p p a r e n t l y interdigitated with cilia. • 24,200. Fig. 26, Electron m i c r o g r a p h of a transverse section through the anterior end o f L)ltodinium caudatum showing the position of the specialized sheet-like extensions of the celt surface relative to the inner lips and the peristome. N o t e that these sheet-like extensions appear similar in longitudinal and transverse section, x 20,000. K E Y TO A B B R E V I A T I O N S b bc c i m ~ o p s rv
bacterium. oesophagus (buccal cavity) cilia inner lips microtubulcs macronucleus outer lips peristome sheet-like extensions of cell surface osmiophilic structure at base of a cilium (see C o l e m a n and Hall, 1971)
|
0
DELACHAMBRE
500 filaments sera discut6 dans ce travail, mais il convient d6s maintenant de prdciser la terminologie utilisde ici; les 'pore-canals' des auteurs de langue anglaise est traduit par le terme 'canal cuticulaire', les filaments comparables aux 'pore-canal filaments' sont appel~s 'filaments axiaux', les filaments d6crits par Locke (1961) c o m m e 'wax-canal filaments' chez la nymphe de Tenebrio sont appel~s 'filaments multiples', les filaments en forme de tubes clairs entour6s d'un m a n c h o n dense, sont appelds 'filaments tubulaires'.
Mat&iel et Techniques
i
- - d ' u n e solution aqueuse de papaine (2X cristallis6e, Koch-Light) ~t 1% ~ 60~ de 24 ~t 72 heures, renouvel~e plusieurs fois; - - d ' u n e solution aqueuse de pronase (45000 P U K units, B.D.H.) ",k 5 % A 40~ de 4/~ 72 heures. Dans certains cas des pi~ces d6j~t d6prot6inisdes par la soude ont ~t6 soumises '~ l'action de la pronase. (3) Des essais de digestion enzymatique sur coupes incluses en resine, selon la technique de M o n n e r o n et Bernhard (1966) ont 6t6 effectu6s avec ces solutions de pronase, de trypsine ou de pepsine. Histochimie ultrastructurale:
Les conditions d'dlevage, la chronologie des diffdrents 6vdnements de la vie nymphale et la preparation du tdgument pour l'6tude ultrastructurale ont 6t6 ddcrits prdc6demment (Delachambre, 1970). Extractions lipidiques: des fragments non fixds de sternites abdominaux de nymphes et d'adultes h diffdrents gtges ont 6t6 plong6s dans plusieurs solvants des lipides: chloroforme, benz6ne-chloroforme (1/1), chloroforme-m& thanol (1 / I) ou pyridine pendant 6 h 24 heures tempdrature ambiante ou A 60~ Ces fragments ont dt6 ensuite fixds et trait& pour l'observation au microscope 61ectronique.
Certaines coupes r6cup6r~es sur grilles en or ou en or-palladium ont 6t6 traitdes par l'argent-m6th6namine selon la technique de Marinozzi (1961) modifi6e par R a m b o u r g et Leblond (1967) ou par la thiocarbohydrazideprot6inate d'argent selon Thierry (1967) avec ou sans oxydation par l'acide periodique ou l'eau oxyg6nde ~ 10%. Ces traitements ont ~t6 effectu6s sur des pi6ces fix6es soit par la glutarald6hyde suivie de t&roxyde d'osmium, soit par le tdtroxyde d ' o s m i u m seul, soit non fixdes.
Ddprotdinisation :
I. C A N A U X DE LA C U T I C U L E NYMPHALE
(I) Soude: des segments abdominaux entiers sont placds dans des r6cipients contenant une solution de soude 0,5 N pendant 4, 18, 24, 48 heures it temp6rature ambiante ou dans des tubes scellds contenant de la soude 0,5 N pendant 2, 6, 12, 24 heures /~ 100~ Apr~s action de la soude, le t6gument est rinc6 plusieurs heures /l l'eau courante puis dans plusieurs bains d'eau distill6e avant prdparation p o u r l'6tude ultrastructurale. (2) Enzymes prot6olytiques: des fragments de t6gument sternal frais ont 6t6 soumis ~t l'actJon:
L a cuticule nymphale de T. molitor (Fig. 1) est form6e d'une 6picuticule, d'une m6socuticule pr6-exuviale d'une trentaine de lamelles et d'une endocuticule post-exuviale plus fine (12 5. 14 lamelles) (Delachambre, 1967). Les canaux cuticulaires de cette cuticule nymphale ont ~t6 d~crits par Locke (1961) et ses observations ont 6t6 confirm6es par Caveney (1970). La forme de ces canaux est d6terminde par l'orientation des microfibres cuticulaires selon le sch6ma de Bouligand (1965), et Neville et al. (1969) les consid6rent c o m m e des rubans creux qui suivent la rotation des
R6sultats
Fig. 1. Coupe de cuticule sternale nymphale. Les canaux cuticulaires montrent un seul filament axial saul dans la r6gion apicale o~1 apparaissent les filaments multiples (ep: 6picuticule, meso: mdsocuticule, endo: endocuticule post-exuviale). • 12 600. Fig. 2. Coupe longitudinale d'un canal cuticulaire nymphal. Le filament axial est reli6 aux parois du canal par de fins filaments (fl6ches). • 80 000. Fig. 3. Insertion d'un filament axial nymphal sur l'6piderme par une zone it microtubules. La membrane plasmique ne p6n6tre pas dans le canal. Le filament axial est extra-cel/ulaire. On notera 6galement la densification de la membrane plasmique au contact du filament (fl6cfie). • 22 000.
378
Structure of the oesophagus Those parts of the peristome and oesophagus that were not covered with cilia also have a characteristic structure (Fig. 23). The surface was covered by a unit membrane 9 m~ thick underneath which were rows (70-200 mf~ apart) of 13 microtubules (m) each of which was 18-20 m~ in diameter. The rows ran approximately at right angles to the inner surface of the oesophagus and the individual tubules ran parallel to the surface of the oesophagus so as to partially encircle the lumen. Similar microtubules are commonly found in the cytostomial and cytopharyngeal walls of other protozoa, e.g. Gymnostomatida and Trichostomatida (Grain, 1966; Faurd-Fremiet, 1961). At the bottom of the oesophagus the rows of tubules were further (up to 250 mr0 apart and vesicles (v), some of which opened into the lumen, were present between the rows (Fig. 24). Entodinium caudatum rapidly engulfed bacteria and other particulate matter into vesicles in the endoplasm (Coleman, 1964; Coleman and Hall, 1969) and this is considered to be the site where the materials entered the cytoplasm. Although no bacteria have ever been lbund in these vesicles, even in protozoa that were actively engulfing bacteria when fixed, some (b) have been observed in closed vesicles just below the surface. As many bacteria were occasionally present in a single vesicle, the size of the vesicle opening into the oesophagus may also be variable.
Specialized sheet-like extensions of cell surface On the ventral side of the peristome and usually situated approximately midway along its length when in the extruded state was a tuft of specialized organelles (s) (Figs. 25 and 26). This tuft, which also contained apparently normal cilia that interdigitated with the specialized structures, and which was similar to the 'Paralabialorgan' described in Ophryoscolecidae by Bretschneider (1962), usually arose from a protuberance at the side of the peristome but more rarely these organelles were attached to the inner lips or part of the protozoon next to the contractile vacuole. When the peristome was extruded these organelles just protruded beyond the inner lips. They are considered to be 'sheet-like' rather than cylindrical
CO LEMA N AND HALL because in section they appear to be similar in. length and thickness in all planes of cut and because much more of any one is always visible than for the normal cilia which are cylindrical. These specialized sheet-like extensions of the cell surface were not of uniform thickness. The innermost which were 140-180m~ thick contained two sets of microtubules (20 m~ in diameter and 40 m~ apart) which did not run parallel to each other. The outer sheet-like extensions were only 90 m~ thick and contained a single set of tubules.
The absence of membrane-bound groups of cilia Bretschneider (1960a, b) and NoirotTimothde (1960) both considered whether or not the individual cilia were bound together as a cirrus or syncilium and the former drew groups of cilia surrounded by a membrane. To determine if there was a membrane surrounding cilia whole protozoa with extruded peristomes were negatively stained with potassium phosphotungstate (Brenner and Home, 1959). No evidence for any such membrane or rigid grouping of cilia was found under the electron microscope.
Discussion As far as we are aware the present investigation represents the first systematic study in the light and electron microscope on the ciliature of the extruded peristome of Entodinium caudatum. Fig. 27 is a diagrammatic representation of the anterior end of the protozoon when viewed from the sides and shows the position of the ciliary band on the peristome and the specialized sheet-like extensions of the cell surface relative to the nucleus. The function of the specialized sheet-like extensions is unknown as there is little evidence available. However, Roth and Shigenaka (1964) who showed the existence of a tuft of similar but different structure to that described above, attached to the peristome of a Diplodinium sp., considered that it was of importance in cell division and that the cilia were in an arrested state of development. Bretschneider (1962) described the structure of a 'Paralabialorgan' in Entodinium and Epidinium spp. and considered it to have a sensory function. Although this 'Paralabialorgan' was similar to
PERISTOME STRUCTURE OF
379
ENTODINIUM
the sheet-like extensions described above, it was larger, contained more cilia and was usually situated in the axil between the peristome and the inner lip. Unfortunately an exact comparison is not possible as Bretschneider worked with mixed protozoal species. The present authors have also found no evidence for any normal cilia of larger diameter than the rnajority in Entodinium caudatum although a single row of large
cilia was found in diplodinia by Roth and Shigenaka (1964) and Noirot-Timothde (1960). Acknowledgements We wish to thank Drs R. W. Cox and F. B. P. Wooding for helpful advice and discussion and Mrs B. C. Barker and Miss J. I. Davies for valuable technical assistance.
Ciliary Band Outer Lip
-
Position of 'Sheetqikfe;~~tsn;If:21l-
~
~
(
~
--
Inner Lip
Macronucleus
Oesophagus-
1 II
LATERAL VIEW
\
V E N T R A L VIEW
LATERAL VIEW
Fig. 27. Diagrammatic representation of the peristome from three sides.
380
COLEMAN
AND
HALL
References
BRENNER, S. and HORNE, R. W. (1959). A negative staining method for high resolution microscopy of viruses. Biochim. biophys. Acta, 34, 103 110. BRETSCHNE~DER, L. H. /960a. EIektronenmikroskopische Untersuchung des Peristomapparates ciniger Ophryoscoleciden I. Proc. K, ned. Akad. Wet. c, 63, 291-305. B~E'rSCHNEmER, L. H. 1960b. Elektronenmikroskopische Untersuchung des Peristomapparates einiger Ophryoscoleciden I1. Proc. K. ned. Akad. Wet. c, 63, 306 317. BREd'SCHNEiDER, L. H, 1962. Das Paralabialorgan der Ophryoscoleciden. Proc. K. ned. Akad. I/Vet. c, 65, 423 452. COLEMAN, G- S. 1964. The metabolism of Escherichia coli and other bacteria by Entodinium caudatum. J. gen. Microbiol., 37, 209-223. COLEMAN, G. S. and HALt,, F. J. 1969. Electron microscopy of the rumen ciliate Entodinium caudatum, with special rcference to the engulfment of bacteria and other particulate matter. Tissue & Cell, 1,607-618. COLEMAN,G. S. and HALL, ]=. J. 1971. Studies in the electron microscope of the adoral zone of membranelles of the rumen ciliate Entodinium caudatum using the technique of negative staining. Tissue & Cell, 3, 381388. FAURt%FREM1EE, E. /96/. Le cytoplasme stomo-pharyngien des Cilids Cyrtophores. C.r. hebd. St:anc. Acad. Sci., Paris, 253, 357-362. GI',AIN, J. 1966. l~!tude cytologique de quelques cilids holotriches endocommensaux des ruminants et des dquidds. Protistologica, 2 (2) 5 47. KELLENBERGE~, E., RYTER, A. and Sf,CHAUL~, J. 1958. Electron microscopic study of DNA-containing plasma 2. Vegetative and mature phage DNAwith normal bacterial nucleoids in difi'erent physiological states. J. biophys, biochem, Cytol., 4, 671--676. NOIROT-TIMOTHF,E, C. 1960. Etude d'une famille de cili~s: Ies Ophryoscolecidae. Structures et ultrastructures, AHn/s. Sci. nat. (Zool.), 12e sdrie, 2, 533 718. ROTf{, L. E. and SHIGENAKA,Y. 1964. The structure and formatioll of cilia and filaments in rumeu protozoa. J. Cell Biol., 20, 208~213. SABATINhD. D., BrNSCH, K. and BARRNETT, R. J. /963. Cytochemistry and electron-microscopy. The preservation ol" cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17, 19 -58. SnAaPE, R. G. 1914. Diplodir~ium ecalMatum with an account of its neuromotor apparatus, Univ. CahT/i Ptcbls. Zool., 13, 43-122.