THE ULTRASTRUCTURE OF SOME ENDOGENOUS STAGES OF THE COCCIDIAN ELMERIA BOVEROI CARINI & P I N T O , 1 9 2 6
IN THE GUT EPITHELIAL CELLS OF THE GECKO HEMIDACTYLUS MABOUIA FROM BRAZIL
PAPERNA I.* & LAINSON R.**
Summary:
The ultrastructure of intracytoplasmic meronts and macrogamonts of Eimeria s.l. boveroi in the small intestine of the gekkonid lizard Hemidactylus mabouio from Belem, Para north Brazil is described.
Young meronts, and some of the fully grown macrogamonts, are coated with a glycocalyx which, in cross-section, has the appearance of a series of fine tubules. The wall of the
parasitophorous vacuole (PV) is a single membrane, lined on the host-cytoplasm side with an endoplasmic reticulum (ER) which sometimes expands into large cisternae filled with electron-lucent, globular material. The membranal edges of the ER, canaliculi and cisternae have regularly spaced indentations filled with an electron-dense substance. Vesicular mitochondria are present in addition to those of the conventional type. Macrogamonts develop the characteristic type 1 and type 2 wall-forming bodies, and in some zygotes the cisternae containing the latter are grossly expanded. Both types of wall-forming bodies persist in young oocysts that have a distinct oocyst wall.
KEY WORDS : coccidia, Eimeria boveroi, gekkonid lizard, Hemidactylus mabouia, Brazil, ultrastructure.
Résumé : ULTRASTRUCTURE DE QUELQUES STADES ENDOGÈNES D'EIMERIA BOVEROI CARINI & PINTO, 1926, COCCIDIE PARASITE DE L'ÉPITHÉLIUM INTESTINAL DU GECKO HEMIDACTYLUS MABOUIA DU BRÉSIL Description ultrastructurale des mérontes et macrogamontes intra- cytoplasmiques a"E. boveroi de l'intestin grêle du gecko Hemidactylus mabouia de Bélém, Brésil. Les jeunes mérontes et certains macrogamontes totalement mûrs sont enveloppés d'un glycocalyx qui, en coupe transversale, est formé d'une série de fins tubules. La paroi de la vacuole parasitophore (PV) est une membrane simple doublée du côté du cytoplasme de la cellule hôte par un reticulum endoplasmique (ER) qui, parfois, se prolonge par de grandes citernes emplies d'un matériel globuleux peu dense. Les limites membranaires de l'ER des canalicules et des citernes présentent des indentations régulièrement espacées emplies d'une substance dense aux électrons. En plus des
mitochondries du type habituel, des mitochondries vésiculaires sont présentes. Les précurseurs de la membrane des macrogamontes présentent les caractères des types I et II et chez certains zygotes les citernes qui les contiennent sont hyperthrophiées. Les deux types de précurseurs de la paroi persistent chez les jeunes oocystes ayant une paroi bien définie.
MOTS CLES : coccidies, Eimeria boveroi, gecko, Hemidactylus mabouia, Brésil, ultrastructure.
INTRODUCTION
S
p e c i e s o f reptilian Eimeria ( s e n s u lato) differ from t h o s e o f m a m m a l s a n d birds in having s p o - rocysts w h i c h are devoid o f Stieda a n d substieda b o d i e s ( C a n n o n , 1967; Vetterling & Widmer, 1 9 6 8 ) . Paperna & Landsberg ( 1 9 8 9 ) p r o p o s e d the g e n e r i c n a m e o f Cboleoeimeria for s o m e o f t h o s e saurian parasites w h i c h d e v e l o p in hypertrophied epithelial cells o f the gall-bladder. T h e generic n a m e Acroeimeria w a s given to a n o t h e r g r o u p w h i c h b u l g e invested within a displaced host-cell b o r d e r o n t h e surface o f the intestinal mucosal epithelium. A third group o f rep- tilian eimeriids exists, the m e m b e r s o f which, like t h e Eimeria s p e c i e s (sensu stricto) o f birds a n d mammals, have a simple intracytoplasmic development in the epi-* Department of Animal Sciences, Faculty of Agriculture o f the Hebrew University of Jerusalem, Rehovot 76-100, Israel.
** Department o f Parasitology, Instituto Evandro Chagas, Caixa Postal 1128 Belem, Para, Brazil.
Correspondence: Ilan Paperna.
Tel.: 972-8-9481945 - Fax: 972-8-9465763.
thelial cells, without major c h a n g e s in the host cell o r strange d i s p l a c e m e n t s . T h e only saurian intracyto- plasmic c o c c i d i u m ultrastructurally studied thus far is E. gastrosauris, this s p e c i e s w h i c h develops in the sto- m a c h epithelium demonstrates a n u m b e r o f unique structural peculiarities (Paperna, 1 9 9 3 ) .
In a recent paper (Lainson & Paperna, 1 9 9 9 ) w e gave a redescription o f Eimeria boveroi Carini 6k Pinto, 1926 in t h e h o u s e - g e c k o Hemidactyius mabouia, a n d s h o w e d the e n d o g e n o u s stages d o undergo this latter, simple type o f intracytoplasmic development in the epi- thelial cells o f the small intestine. T h e purpose o f the present p a p e r is to describe the ultrastructure o f s o m e o f these stages: it w o u l d a p p e a r t o b e the first time that such a study h a s b e e n made o f a m e m b e r o f this group o f saurian eimeriids.
MATERIALS AND METHODS
M
aterial for transmission electron m i c r o s c o p y ( T E M ) w a s f r o m t h e s a m e s p e c i m e n o f H. mabouia u s e d in o u r light m i c r o s c o p eP a r a s i t e , 1 9 9 9 , 6, 2 3 7 - 2 4 2
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description o f the oocysts and e n d o g e n o u s stages o f Eimeria (s.l.) boveroi (Lainson & Paperna, 1 9 9 9 ) . T h e intestine w a s cut longitudinally and o n e half used to pinpoint the site o f development o f the parasite by exa
mination o f the gut s e g m e n t b e t w e e n a slide and a coverslip. Appropriate portions w e r e fixed in 2.5 % glutaraldehyde in cacodylate buffer (0.1M, pH 7 . 4 ) for 24 hours at 4 ° C , rinsed repeatedly in the s a m e buffer, post-fixed in 1.0 % o s m i u m tetroxide in the s a m e buffer for o n e hour and, after rinsing in the buffer, d e h y d r a t e d in g r a d e d ethyl a l c o h o l dilutions a n d e m b e d d e d in Agar 100 medium (Agar Scientific Ltd., U.K.). Thin sections, cut o n a Reichert Ultracut micro
t o m e with a diamond knife, w e r e stained o n grids with uranyl acetate and lead citrate, and e x a m i n e d with a J e o l 100CX TEM.
RESULTS
I
nfection in the epithelium o f the small intestine was scanty, and only young meronts (Figs 1-4) and young and mature macrogamonts (Figs 5-13) were traced on the grids examined. Parasitophorous vacuoles (PV) with young meronts and macrogamonts (Figs 1, 5 ) c o n t a i n e d aggregates o f globular particles, w h i c h c o u l d b e also traced in the host-cell cytoplasm adjoining the PV (Fig. 7 ) . T h e globular particles w e r e also present in variable quantities in PV's containing later stage macrogamonts (Figs 6, 7 ) and sometimes these also contained an e l e c t r o n - d e n s e precipitate (Figs 8, 1 1 ) . T h e PV p o s sessed a single m e m b r a n e (Figs 4, 6A) bordered on the side adjoining the host cytoplasm b y an e n d o p l a s m i c
Figs. 1-4. Fig. 1. Y o u n g m e r o n t (X a n d a r r o w h e a d m a r k s t h e a r e a s e n l a r g e d in Figs. 2 & 3 ) ( x 1 3 . 6 4 0 ) - F i g s . 2 a n d ) . E n l a r g e d v i e w o f t h e m e r o n t s rim s h o w i n g t h e g l y c o c a l y x - l i k e c o a t i n g o f t h e p e l l i c u l e a n d t h e l a r g e v e s i c u l a r m i t o c h o n d r i o n ( x 3 2 . 7 3 5 ) . Fig. 4 . F u r t h e r dif
f e r e n t i a t e d y o u n g m e r o n t ( x 1 0 . 0 0 0 ) .
Abbreviations to figures 1-13: A. A m y l o p e c t i n g r a n u l e - ad. A d n u c l e a r o r g a n e l l e s - dM. M i t o c h o n d r i o n filled w i t h e l e c t r o n d e n s e s u b s t a n c e - C. C i s t e r n a - Cn. C a n a l l i c u l i - f. F o o d v a c u o l e - G b . G r a n u l a r b o d i e s - g. I n t r a v a c u o l a r g l o b u l a r s u b s t a n c e - g x . c o n s o l i d a t i n g g r a n u l a r b o d i e s - L. Lipid v a c u o l e - M. M i t o c h o n d r i o n - p . Cell wall m e m b r a n e - n. N u c l e u s - R. E R l a y e r b e n e a t h t h e P V w a l l , w i t h regularly- s p a c e d e l e c t r o n - d e n s e d o t s - V C . E x p a n d e d c i s t e r n a c o n t a i n i n g t y p e 2 w a l l - f o r m i n g b o d y - vM. v e s i c u l a t e m i t o c h o n d r i o n - x . G l y c o c a l y x - l i k e l a y e r - w . C o n s o l i d a t i n g w a l l - f o r m i n g b o d y - W . W F B - W 1 . T y p e 1 w a l l - f o r m i n g b o d y - W 2 . T y p e 2 w a l l - f o r m i n g b o d y . S c a l e s = 1 µm.
ULTRASTRUCTURE OF EIMERIA BOVEROI OF A GECKO FROM BRAZIL
Figs. 5 - 8 . Fig. 5. Y o u n g m a c r o g a m o n t ( d e s m o s o m e - a r r o w e d ) x 1 0 . 8 3 0 - Fig. 6 . M a t u r i n g m a c r o g a m o n t ( x 6 . 3 6 4 ) - Fig. 7. Maturing m a c r o - g a m o n t , e n l a r g e d v i e w , star: c o n v o l u t e d m i t o c h o n d r i o n ( x 1 4 . 7 8 0 ) - Fig. 8. Maturing m a c r o g a m o n t a l r e a d y w i t h b o t h t y p e 1 a n d 2 W F B s ( x 1 2 . 8 5 2 ) .
P a r a s i t e , 1 9 9 9 , 6, 2 3 7 - 2 4 2
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reticulum ( E R ) which at times e x p a n d e d into very large cisternae filled with electron-lucent globular mate
rial. T h e m e m b r a n e e d g e s facing the PV wall o f both the ER canaliculi and the cisternae c o n t a i n e d regularly s p a c e d indentations filled with an e l e c t r o n - d e n s e sub
stance ( r i b o s o m e s ? ) (Figs 1, 4, 11, 1 2 ) . T h e host cells are epithelial, as evident b y the p r e s e n c e o f d e s m o - s o m e s (Fig. 5 ) : the cytoplasm o f s o m e infected cells was o f a d e n s e r nature than that o f the surrounding, non-infected host cells (Fig. 5 ) .
Y o u n g meronts w e r e already b o u n d e d by a single unit m e m b r a n e . T h e first o n e w e studied (Fig. 1) w a s c o v e r e d with what a p p e a r e d to b e a glycocalyx which, in cross-section, was s e e n as a tubular grid (Fig. 2 ) . Figure 3 gives a detailed view o f this glycocalyx-like coat over the cell wall. It was absent from the s e c o n d , s o m e w h a t m o r e d e v e l o p e d m e r o n t that w e e x a m i n e d (Fig. 4 ) , but w a s again s e e n covering the plasma- l e m m a o f a fully d e v e l o p e d m a c r o g a m o n t (Fig. 1 2 ) . In both o f the meronts the cytoplasm was fringed by elon
gate, vesicular mitochondria, with a single array o f tubules at their periphery (Fig. 2 ) . A very few micro- n e m e s w e r e present, a variable n u m b e r o f electron- d e n s e spheres ( w h i c h w e r e possibly the residue o f rhoptries), and amylopectin granules.
Y o u n g macrogamonts, measuring 7.0-8.0 x 4.0-5.0, were recognizable by their nucleus containing a very large, central nucleolus: only a few organelles could b e traced in the cytoplasm, including marginally positioned food vacuoles and vesicular mitochondria, segments o f the ER and a few m i c r o n e m e s (Fig. 5 ) . T h e mature macro
gamonts (Fig. 6 ) reach from 13.0-15.0 x 8.0-11.0 in size.
In the developing macrogamont the type 1 wall-forming b o d i e s ( W F B 1 ) appear first: they are double-walled, round b o d i e s w h i c h are filled either h o m o g e n o u s l y o r with clumps o f an electron-dense substance (Figs 7, 8 ) . T h e type 2 wall-forming b o d i e s ( W F B 2 ) develop sub
sequently and are larger, homogenously electron-dense spheres formed within ER cisternae (Figs 6, 8, 9 ) . Gro
wing macrogamonts also contain a few large, low-den
sity granular b o d i e s which are similar in size to the W F B 2 , and enclosed in a concentric formation o f rough ER (Fig. 7 ) . Small, granular bodies, s e e n emerging from a similar rough ER c o n c e n t r i c formation, s e e m to b e anlagens o f either W F B 2 or the low-density granular bodies. Vesicular mitochondria, with a single layer o f peripheral tubules, o c c u r together with regular tubular mitochondria which s o m e t i m e s appear to b e c o n v o luted (Fig. 7 ) . Also present in both the pre-mature and mature m a c r o g a m o n t s w e r e a few large lipid vacuoles, amylopectin granules and canaliculi (Figs 6, 8 ) . T h e nucleus o f these parasites is a c c o m p a n i e d by dense, granular adnuclear b o d i e s (Fig. 6 ) .
Images seen in Figures 9 and 10 are apparently zygotes.
T h e r e is n o sign, however, o f the onset o f o o c y s t wall
formation, and the parasites still contain both W F B 1 and W F B 2 . W F B 1 content is either h o m o g e n o u s o r in a variable stage o f disintegration: the W F B 2 remain intact (Fig. 9 ) . In o n e m a c r o g a m o n t (Fig. 1 0 ) the cis
ternae containing the W F B 2 are e x p a n d e d , and the e l e c t r o n - d e n s e wall-forming b o d y appears to b e floa
ting inside: part o f the mitochondrion, in such s p e c i m e n s , b e c o m e s filled with e l e c r o n - d e n s e d e p o s i t . Y o u n g oocysts w e r e generally resistant to processing for TEM, and only a few images (Fig. 1 3 ) e n a b l e d o n e to s e e formation o f the o o c y s t wall which, superim
p o s e d by a thin m e m b r a n e , c o v e r e d a similarly thin plasmalemma. Such zygotes still contain a few W F B 1 and a larger n u m b e r o f W F B 2 within their cisternae.
DISCUSSION
[
**% osition o f the young meronts and m a c r o g a m o n t s - J within their host cells, and the general arrangement o f internal structures, is similar to that usually s h o w n by avian and mammalian eimeriid para
sites. T h e r e are, h o w e v e r , s o m e noteworthy peculia
rities at the host/parasite interphase, as well as in the parasites themselves. ER and large cisternae lining the PV wall have, till n o w , b e e n found only in the piscine c o c c i d i u m E. vanasi (Paperna, 1 9 9 5 ) , and in m o u s e m a c r o p h a g e s infected with Toxoplasma gondii ( J o n e s
& Hirsch, 1 9 7 2 ) . In the latter c a s e the ER e d g e s , dotted at regular intervals with e l e c t r o n - d e n s e material des
cribed as r i b o s o m e s , are reminiscent o f the regularly s p a c e d indentations filled with electron-dense material s e e n along the ER and cisternae in the host cells o f E. (s.l.) boveroi, d e s c r i b e d a b o v e . In E. vanasi the ER structure is dependent o n structural changes which take place as the parasite d e v e l o p s (Paperna, 1 9 9 5 ) . It has b e e n suggested (Mauel, 1 9 9 6 ) that in m a c r o p h a g e s infected with Toxoplasma gondii the ER lining either provides protection against the m a c r o p h a g e ' s lyso
somal activity or is involved in providing the PV with 'factors necessary for parasite multiplication'.
T h e tubule-like layer (apparently a g l y c o c a l y x ) coating the y o u n g m e r o n t pellicle and the wall o f mature macrogamonts is an u n c o m m o n feature, but it has b e e n s e e n in a few c o c c i d i a n s such as Acroeimeria pintoi (Lainson & Paperna, 1999; Paperna & Lainson, unpu
blished). Again, vesicular mitochondria are unusual, but have b e e n s e e n in meronts o f the saurian coccidian Isospora cannoni ( P a p e r n a & F i n k e l m a n , 1 9 9 8 ) . Although wall-forming b o d i e s are found universally in all terrestrial-host s p e c i e s o f Eimeria, Isospora and related eimeriids s o far e x a m i n e d , the relative sizes o f W F B 1 and W F B 2 , and their configuration in the para
site, d o vary in different s p e c i e s . T h e e x p a n s i o n o f the cisternae holding the W F B 2 (Fig. 1 0 ) could have b e e n
ULTRASTRUCTURE OF EIMERIA BOVEROI OF A GECKO FROM BRAZIL
Figs 9 - 1 3 - Fig. 9 . M a t u r e m a c r o g a m o n t ( z y g o t e ) , e n l a r g e d v i e w ( x 1 4 . 8 8 2 ) - Fig. 1 0 . Z y g o t e with e x p a n d e d c i s t e m a e ( x 1 4 . 8 9 0 ) - Fig. 1 1 . E n l a r g e d s e c t o r from F i g u r e 8, o f a m a c r o g a m o n t , a n d P V w a l l a s s o c i a t e d s t r u c t u r e s ( d e n s e d e p o s i t e a r r o w e d ) ( x 2 6 . 8 6 1 ) - Fig. 12. E n l a r g e d s e c t o r o f F i g u r e 9, s h o w i n g m a c r o g a m o n t ' s w a l l c o a t e d w i t h a g l y c o c a l y x - l i k e l a y e r a n d t h e P V w i t h t h e a s s o c i a t e d ER s y s t e m ( x 3 9 . 8 8 3 ) - Fig. 13- Y o u n g o o c y s t s w i t h c o n s o l i d a t i n g wall: 1. o u t e r m e m b r a n e ; 2. t h e t h i c k wall; 3- t h e p l a s m a l e m m a ( x 1 8 . 5 7 1 ) .
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the result o f a processing fault, for such a fault is evi
dent in the parasite's nucleus; all m e m b r a n e s and other ultrastructural configurations in the parasite cell were, h o w e v e r , intact. A similar p r o c e s s involving cis- ternae e x p a n s i o n has also b e e n s e e n in Schellackia ptyodactyii (Paperna & Finkelman, 1996). T h e observed
three layered o o c y s t w a s apparently b e y o n d the five m e m b r a n e formation stage, characteristic to the wall formation p r o c e s s o f Eimeria spp. o f avian and mam
malian hosts ( C h o b o t a r et al, 1 9 8 0 : the outermost m e m b r a n e appears to b e c o m p r i s e d o f o o c y s t wall m e m b r a n e s 1 and 2, the thick middle layer as in other coccidia is wall 3, while wall 4 merges with 5 into the oocyst plasmalemma.
PAPERNA I. & FINKELMAN S. Ultrastructural studies on reptilian Isospura species with intranuclear endogenuous develop
ment. Parassitologia, 1998, 40, 283-296.
PAPERNA I. & LANDSBERG J.H. Description and taxonomic dis
cussion of eimerine coccidia from African and Levantine geckoes. South African Journal of Zoology, 1989, 24, 345- 335.
VETTERLING J . M . & WIDMER E.H. Eimeria cascabeli sp. n.
(Eimeridae, Sporozoa) from rattlesnakes, with a review of the species Eimeria from snakes. Journal of Parasitology, 1968, 54, 569-576.
Recu le 15 mars 1999 Accepté le 28 avril 1999
ACKNOWLEDGEMENTS
T o Marina Schein for the preparation o f the TEM grids.
Work supported by a grant from the W e l l c o m e Trust, London (to RL).
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