Parasitol Res (2001) 87: 662 665
DOI 10.1007/s004360100423
O R I GI N A L P A P E R
Guy Brugerolle · Jean-Pierre Regnault
Ultrastructure of the enteromonad fagellate Caviomonas mobilis
Received: 27 February 2001 / Accepted: 19 March 2001 / Published online: 16 May 2001
© Springer Verlag 2001
Abstract Caviomonas mobilis was collected from the
caecum of mice harbouring a controlled fauna. Phase
contrast and immunofuorescence microscopy using an
anti­tubulin antibody and electron microscopy demon­
strated the presence of one basal body bearing a fagel­
lum and a second barren basal body, both inserted in the
face of two cup­like depressions in the nuclear surface,
as in other enteromonad/diplomonad genera. Three
microtubular fbres arise close to the main basal body:
the frst, composed of three microtubules cross­linked
with a dense structure, lies within a groove above the
nuclear surface; the second is oriented antero­dorsally
and corresponds to the peristyle as observed by light
microscopy; and the third is situated ventrally, below the
proximal part of the recurrent fagellum, and corre­
sponds to the funis. There is no mitochondrion, no
Golgi body, the endoplasmic reticulum is reduced, there
is no cytostome, the cell feeds by pinocytosis and
phagocytosis and the division spindle is intranuclear.
The cytological characters of Caviomonas are homo­
logous to those of genera which comprise the
enteromonad/diplomonad evolutionary lineage, as pre­
viously presumed.
lntroduction
Caviomonas mobilis is a very small fagellate which lives
in the caecum of guinea pigs and other rodents. It was
G. Brugerolle (L)
Biologie des Protistes, UMR CNRS 6023,
Universite Blaise Pascal de Clermont Ferrand,
63177 Aubiere Cedex, France
E mail: [email protected] bpclermont.fr
J. P. Regnault
Centre de Developpement des Techniques
Avancees pour l'experimentation animale,
CNRS, 45071 Orleans, France
very precisely described, after haematoxylin and
protargol staining, by Nie (1950), who suggested a close
relationship with Enteromonas, another intestinal
fagellate. The common features of Enteromonadidae,
defned by the electron microscopic studies of Entero­
monas and Trimitus (Brugerolle 1975, 1986), have shown
that these fagellates belong to diplomonads, despite
their mono­monad constitution. In several reviews, the
genus Caviomonas was assumed to belong to the
Enteromonadidae family (Brugerolle and Muller 2000;
Brugerolle and Taylor 1977; Honigberg et al. 1978;
Kulda and Nohynkova 1978; Lee et al. 1985; Vickerman
1990), but there were no electron microscopic or
molecular studies to confrm this viewpoint.
Materials and methods
Caviomonas mobilis was collected from the caecum of laboratory
mice which contained a selected fauna comprising only this fa
gellate and one bacteria of the Fusiformis genus. Cells, swimming in
Ringer solution, were aspirated with a thin pipette and concen
trated by centrifugation. They were fxed initially in 1% glutaral
dehyde in 0.1 M phosphate bufer for 1 h, then in 1% osmium
tetroxide in the bufer for 1 h. Washed cells were pre embedded in
1% agar, stained en bloc with saturated uranyl acetate in 70%
ethanol, then dehydrated in alcohol series and fnally embedded in
Epon 812. Ultrathin sections were obtained with a Reichert Ul
tracut S ultramicrotome, stained with lead citrate, carbon coated
and observed using a JEOL 1200EX electron microscope at 80 kV.
For light microscopy, cells were photographed after fxation in
glutaraldehyde/osmium with a phase contrast and a diferential
contrast Leica DMRH microscope. Immunofuorescence with an
anti tubulin antibody was used to decorate microtubules. Cells
were fxed in PBS containing 3.5% formaldehyde for 15 min at
room temperature, then permeabilized in 1% Triton X100 in PBS
for 10 min. After washing in PBS and treatment with 0.1 M glycine
for 15 min, cells were placed on slot slides coated with 1% poly
lysine solution. After blocking with 1% bovine serum albumin for
15 min, cells were incubated overnight at room temperature with
an anti tubulin monoclonal antibody produced in the laboratory
and washed 3 times in PBS prior to 1 h incubation with 1:200
dilution of an anti mouse Ig: IgG/M antibody conjugated with
fuorescein isothiocyanate (FITC) (Sigma). Slides were mounted
663
with 1:1 glycerol PBS containing 10 mg/ml DABCO (Sigma) and
then observed under a Leica DMRH indirect immunofuorescence
microscope. Control slides were treated with a 1:200 dilution of
pre immune mouse serum as the primary antibody.
(Fig. 1r). Pseudocysts containing an internalized fagel­
lum were present, but no true cysts with a cyst wall were
observed.
Results
Discussion
Light microscopy results confrm that Caviomonas has
one posteriorly oriented fagellum inserted on one side of
the cell, close to the nucleus situated in the apical area
(Fig. 1a-c). By immunofuorescence, two microtubular
fbres arising at the base of the fagellum were distin­
guished. One fbre, situated on the ventral face, extends
under the proximal part of the fagellum and then curves
inside the cell (Fig. 1d-h). The second fbre surmounts
the nucleus, then extends along the dorsal face of the
cell. These fbres subdivide into several smaller fbres
which follow the same orientation as the main fbre
(Fig. 1d, f).
Electron microscopy studies show the position of the
basal body (1) of the single fagellum, inserted orthog­
onally to the nucleus on the ventral side of the cell
(Figs. 1i, 2). They also reveal a second short barren
basal body (2) situated under the major one (Fig. 1j, k).
Interestingly, in front of the proximal end of each basal
body, there is a cup­like depression on the nuclear en­
velope (Fig. 1k). The fagellum has the typical 9+2
structure and does not adhere to the ventral face of the
cell (Fig. 1i, p). Three microtubular fbres, forming the
cytoskeleton, arise from a dense structure at the base of
the basal body (1) of the fagellum. One fbre, composed
of three microtubules, depresses the nuclear envelope
forming a narrow groove on the surface of the nucleus
(Fig. 1k-m). The three microtubules are cross­linked by
bridges to dense material which underlies the fbre
(Fig. 1j inset, k). The two other fbres have a common
origin on the dense structure associated with the anterior
side of the basal body (1). Among the 20-40 micro­
tubules which arise and fan out, a few are oriented
ventrally, forming the ventral fbre, while the others are
oriented dorsally, forming the dorsal fbre (Fig. 1m-o).
These fbres underlie the plasma membrane, but there is
no evident cross­link to the membrane or between the
microtubules.
There is no mitochondrion or a Golgi body. Endo­
plasmic reticulum profles are rather rare in the cyto­
plasm, which contains numerous small shuttle­like
vesicles which could represent smooth endoplasmic
reticulum (Fig. 1o). Glycogen granules are abundant.
Food vacuoles vary in size and some contain Fusiformis
bacteria (Fig. 1p). Absorption of food occurs by both
pinocytosis and phagocytosis, and there is no special­
ized cytostomal structure. Several sections of cells
showed an internal bundle of microtubules inside the
nucleus, which are obviously a part of the division
spindle, but no complete nuclear division was observed
Immunofuorescence and electron microscopy observa­
tions confrm the structures previously reported for
Caviomonas mobilis cells by Nie (1950). There is only one
fagellum, but a second barren basal body, which
probably develops at division, is present. Cytoskeletal
structures are composed mainly of a ventral microtu­
bular fbre previously named funis and by an antero­
dorsal fbre named peristyle by Nie (1950). The third
fbre, with a composite structure comprising three
microtubules forming a depression in the nuclear sur­
face, is only visible on electron microscopy; it is not
revealed by immunofuorescence staining and was not
mentioned in previous light microscopy studies (Kulda
and Nohynkova 1978; Nie 1950).
This study reveals that C. mobilis also shares
several characteristics present in enteromonads and
diplomonads (Brugerolle 1975, 1986; Brugerolle and
Muller 2000; Kulda and Nohynkova 1978). The basal
bodies inserted in front of cup­like depressions of the
nuclear envelope are typical of diplomonads in general
(Brugerolle 1974, 1975, 1986). The number of fagella is
reduced to one plus a barren basal body, difering from
Enteromonas and Trimitus which have two pairs of basal
bodies/fagella (Brugerolle 1975, 1986). In Caviomonas it
is difcult to recognize the three diferent fbres: supra­
nuclear, infra­nuclear and cytostomal fbres, which are
common characteristics in enteromonad or diplomonad
genera (Brugerolle 1975; Brugerolle and Muller 2000).
One dorsal fbre is represented by the three microtubules
depressing the nucleus surface, whose close association
with the nucleus could make it homologous to the
supranuclear fbre in other diplomonads. The second
dorsal fbre or peristyle has no strict homologue in
entromonads and diplomonads. The ventral fbre seems
to be homologous to the ventral or cytostomal fbre of
other diplomonads, but in Caviomonas there is no true
cytostome/cytopharynx. There is no infra­nuclear fbre
in Caviomonas in contrast to several other diplomonad
and enteromonad genera. The cell has no mitochondrion
or Golgi body, like other diplomonads. The endoplas­
mic reticulum structures and shuttle­like vesicles are
reminiscent of those found in Giardia which is nourished
by pinocytosis (McCafery and Gillin 1994). The pres­
ence of an internal spindle in the dividing nucleus, which
retains its nuclear envelope, is reminiscent of the
semi­open mitosis of diplomonads (Brugerolle 1974).
Caviomonas belongs to the enteromonad/diplomonad
lineage as presumed from previous light microscopic
studies.
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Fig. 1a h Photomicrographs of Caviomonas mobilis. a, b Phase
contrast micrographs; c diferential contrast micrograph of the cell
in b, showing the one fagellum inserted close to the anterior
nucleus (arrowhead), and food vacuoles containing bacteria
(arrow). d h Indirect immunofuorescence micrographs using an
anti tubulin antibody revealing the fagellum, the dorsal microtu
bular fbre (arrows) and the ventral microtubular fbre (arrow­
heads); e is a phase contrast micrograph of the cell in d. Bar 10 µm.
i r Electron micrographs of C. mobilis. i Longitudinal section
showing the recurrent fagellum (F) inserted at one side of the
nucleus (N), ventral microtubular fbre (vF) and dorsal microtubu
lar fbre (dF). j m Position of basal bodies and attached fbres. The
basal body (1) of the recurrent fagellum and a second barren basal
body (2) are inserted in front of two cup like depressions of the
nuclear envelope (arrowheads k). The nuclear fbre (nF), composed
of three microtubules cross linked to a subjacent dense structure, is
attached to the basal body (1) and is located in a groove on the
nucleus surface (j inset, k); the ventral fbre (vF) and the dorsal fbre
(dF) arise close to the basal body (1) (m). n p Transverse sections of
the cell at diferent levels showing the two basal bodies (1, 2) and
the recurrent fagellum (F), the nuclear fbre (nF), the ventral fbre
(vF), the dorsal fbre (dF) and a food vacuole (V). q Shuttle shaped
vesicles in the cytoplasm (arrowheads).r Dividing nucleus with its
nuclear envelope containing an internal bundle of microtubules
(arrows). Bar 1 µm
Fig. 2 Reconstruction diagram showing the arrangement of basal
bodies (1) and (2) and the attached fbres: nuclear fbre (nF), dorsal
fbre (dF) and ventral fbre (vF), close to the nucleus (N)
References
Brugerolle G (1974) Contribution aa l'etude cytologique et phyle
tique des diplozoaires (Zoomastigophorea, Diplozoa Dangeard
1910). III. Contribution aa l'etude du genre Hexamita (Dujardin
1910). Protistologica 10:83 90
Brugerolle G (1975) Etude du genre Enteromonas (Zoomastigo
phorea) et revision de l'Ordre des Diplomonadida (Wenyon).
J Protozool 22:468 475
Brugerolle G (1986) Separation des genres Trimitus (Diplomona
dida) et Tricercomitus (Trichomonadida) d'apreas leur ultra
structure. Protistologica 22:31 37
Brugerolle G, Muller M (2000) Amitochondriate fagellates. In:
Leadbeater BSC, Green JC (eds) The fagellates: unity, diversity
and evolution. (The Systematics Association, sp vol, ser 59)
Taylor and Francis, London, pp.166 189
Brugerolle G, Taylor FJR (1977) Taxonomy, cytology and evolu
tion of the Mastigophora. In: Hutner SH (ed) Proceedings of
the V International Congress of Protozoology, New York. Pace
University, New York, pp 14 18
Honigberg BM, Vickerman K, Kulda J, Brugerolle G (1978)
Cytology and taxonomy of parasitic fagellates. Review work
shop Sect B5, Proceedings of the IV International Congress of
Parasitology, Warsaw
Kulda J, Nohynkova E (1978) Flagellates of human intestine and
of intestines of other species. In: Kreier JP (ed) Parasitic pro
tozoa, vol 2. Academic Press, New York, pp 2 138
Lee JJ, Hutner SH, Bovee EC (1985) An illustrated guide to the
protozoa. Allen Press, Lawrence, Kan.
McCafery JM, Gillin F (1994) Giardia lamblia: ultrastructure basis
of protein transport during growth and expansion. Exp
Parasitol 79:220 235
Nie D (1950) Morphology and taxonomy of the intestinal protozoa
of the guinea pig Cavia porcella. J Morphol 86:381 493
Vickerman K (1990) Phylum Zoomastigina, Class Diplomona
dida. In: Margulis L, Corliss JO, Melkonian M, Chapman
DJ (eds) Handbook of protoctista. Jones & Bartlett, Boston,
pp 200 210