HELGOLANDER MEERESUNTERSUCHUNGEN
Helgol~nder Meeresunters. 50, 497-515, 1996
Cryothecomonas aesfivalis sp. n o v . , a c o l o u r l e s s
nanoflagellate
feeding on the marine centric diatom
Guinardia delicatula { C l e v e ) H a s l e
G. Drebes 1, S. F. Kfihn 2, A. Gmelch 3 & E. Schnepf 3
1 Biologische Anstalt Helgoland, Wattenmeerstation Sylt; D-25992 List/Sylt, Germany
2 Alfred Wegener Institut ffir Polar- und Meeresforschung; P. O. Box 120161,
D-27515 Bremerhaven, Germany
3 Zellenlehre, Fakult~t f~r Biologie, UniversitSt Heidelberg; Im Neuenheimer Feld 230,
D-69120 Heidelberg, Germany
ABSTRACT: The vegetative life cycle, host specificity, morphology, and ultrastructure of a new
phagotrophic nanoflagellate are described: Cryothecomonas aestivalis Drebes, Kfihn & Schnepf sp.
nov. During summer and autumn it is frequently found in the North Sea phytoplankton feeding on
the centric diatom Guinardia delicatula. The flagellate penetrates the diatom cell a n d phagocyfizes
the host cytoplasm by m e a n s of a pseudopodium that emerges from the posterior cell pole. The
mature trophont gives rise to eight or more biflagellate swarmers which leave the emptied diatom
frustule. Transmission electron microscopy revealed a delicate theca surrounding the swarmers. The
pseudopodium protrudes through a gap in the theca. The cytostome consists of a m e m b r a n o u s
labyrinth. The mitochondria are of the tubular type. The two apically inserted flagella are heretodynamic and of u n e q u a l length. They are smooth, a n d their basal bodies are surrounded by satellites
and fibrous strands ("transitional fibres" sensu Thomsen et al., 1990). In the trophonts a n d dividing
flagellates the transition region b e t w e e n the flagellum and the basal body ends apically with a
massive transitional cylinder instead of distinct microtubules, and is surrounded by a funnel of the
theca. The nuclear envelope disintegrates during mitosis. Due to the fine structural details the new
flagellate is placed in the genus Cryothecomonas Thomsen et al., a genus of still uncertain position.
INTRODUCTION
In t h e N o r t h S e a p l a n k t o n , Guinardia delicatula (Cleve) H a s l e is a c o m m o n c e n t r i c
d i a t o m ( D r e b e s , 1974) a n d , e s p e c i a l l y i n s u m m e r , o n e of t h e p r o m i n e n t p r i m a r y p r o d u c e r s i n t h e p h y t o p l a n k t o n of t h e G e r m a n B i g h t . F o r t h e p a s t I 0 0 y e a r s t h e s p e c i e s w a s
c o m m o n l y k n o w n as Rhizosolenia deh'catula C l e v e , b u t r e c e n t l y it h a s b e e n t r a n s f e r r e d to
t h e g e n u s Guinardia H. P e r a g a l l o b y H a s l e & S y v e r t s e n (1996). A c a r e f u l i n v i v o
i n s p e c t i o n of f r e s h l y c o l l e c t e d p l a n k t o n s a m p l e s as w e l l as of r a w c u l t u r e s r e v e a l e d t h a t
t h i s d i a t o m is f r e q u e n t l y a t t a c k e d b y a h i t h e r t o u n d e s c r i b e d f l a g e l l a t e , w h i c h i n v a d e s t h e
f r u s t u l e a n d p h a g o c y t i z e s t h e p r o t o p l a s t . W e s t u d i e d t h e v e g e t a t i v e h f e c y c l e of this
f l a g e l l a t e , e s p e c i a l l y its m o d e of f o o d u p t a k e , a n d its u l t r a s t r u c t u r e . A s a r e s u l t o u r n e w
f l a g e l l a t e is a s s i g n e d to t h e g e n u s Cryothecomonas, w h i c h h a s r e c e n t l y b e e n d e s c r i b e d
b y T h o m s e n et al. (1990). C. aestivalis sp. nov. b e l o n g s to t h e p a r a s i t i c / p a r a s i t o i d i c
9 Biologische Anstalt Helgoland, H a m b u r g
498
G. Drebes, S. F. Kfihn, A. G m e l c h & E. Schnepf
nanoflagellates, to which little attention has previously b e e n paid, M e a n w h i l e , however,
there is i n c r e a s i n g e v i d e n c e that acting as parasites or p r e d a t o r s they c a n c o n s i d e r a b l y
affect the d y n a m i c s of p h y t o p l a n k t o n populations (Ktihn et al., 1996).
MATERIALS AND METHODS
For this study, the flagellate Cryothecomonas aestivah's was i s o l a t e d in s u m m e r
(August 1993 a n d 1994) from littoral p l a n k t o n s a m p l e s collected in the W a d d e n S e a n e a r
List/Sylt ( G e r m a n Bight, North Sea). The clones w e r e grown in small Petri dishes,
s u p p l i e d with the F/2 m e d i u m (Guillard & Ryther, 1962), a n d n o r m a l l y k e p t at 16~
u n d e r a 14:10 h l i g h t : d a r k regime, t o g e t h e r with the host diatom, Guinardia delicatula
(Cleve) Hasle. Two or three times p e r w e e k the flagellates w e r e transferred to a n e w Petri
dish containing G. delicatula.
For live observations by light microscopy, w e used Leitz s e a - w a t e r immersion
objectives. C y t o c h a l a s i n D was u s e d to affect the actomyosin system d u r i n g infection.
The a g e n t was dissolved in dimethyl sulfoxide, a n d the stock solution d i l u t e d with the
culture m e d i u m to a final concentration of 5 ~g 9 m1-1.
For transmission electron microscopy, we u s e d a Philips SM 10. Cultures with
infected diatoms a n d free flagellates w e r e centrifuged a n d e m b e d d e d in 3 % Sea Prep
agarose (Biozym, Hameln, G e r m a n y ) (Reize & Melkonian, 1989). T h e y w e r e t h e n prefixed with 2 % g l u t a r a l d e h y d e in F/2 m e d i u m , rinsed, a n d postfixed with 2 % OsO4 in the
s a m e m e d i u m . Alternatively, w e u s e d 2 % g l u t a r a l d e h y d e in c a c o d y l a t e buffer (0.2 M,
p H 7.2, plus 0.25 M saccharose), a n d s u b s e q u e n t l y 2 % OsO4 as fixatives. After rinsing
with d e c r e a s i n g concentrations of saccharose we e m b e d d e d the s p e c i m e n s in S e a Prep
agarose. Both s a m p l e s were d e h y d r a t e d in an acetone series and e m b e d d e d in Spurr's
resin.
RESULTS
Occurrence
Cryothecomonas
aestivalis was discovered in plankton samples off Helgoland (North
Sea) in July 1970, and again recognized in August 1993 and in summer 1994 in plankton
samples collected in the Wadden Sea near List/Sylt. During the main occurrence of the
flagellate, the water temperatures ranged between 14-20 ~
So far, C. aestivalis was only found on Guinardia delicatula, d u r i n g the p a s t y e a r s
usually in August, occasionally as late as N o v e m b e r , in s a m p l e s collected from the
W a d d e n Sea. R e g a r d i n g the host range, f e e d i n g e x p e r i m e n t s with various o t h e r diatoms
w e r e not successful.
Life c y c l e
The v e g e t a t i v e hfe cycle of C. aestivalis comprises three stages: (1) the motile
flagellates, outside a n d inside the host before food u p t a k e ; (2) the trophic (nutritive)
phase, a n d (3) the p h a s e of digestion a n d cell division.
The colourless f a g e U a t e s (Pigs 1, 46) are oblong to oval, 9-12 [~m l o n g a n d 4-5 ~m
wide. Two h e t e r o d y n a m i c flagella of u n e q u a l l e n g t h are inserted apically, one b e i n g
anteriorly d i r e c t e d a n d up to 15 ~m long, a n d the other one posteriorly a n d up to 25 ~m
Cryothecomonas aestivalis sp. nov., a colourless nanoflagellate
499
long. Both flagella are shorter during the trophic p h a s e and in immature flagellates. The
relatively large nucleus lies in the apical r e g i o n of the cell. Flagellates in the motile p h a s e
are h y a l i n e a n d contain only a few refractive bodies.
The flagellate swims slowly, sometimes sliding, but usually slightly t u m b l i n g in large
curves. W h e n it a p p r o a c h e s a G. delicatula chain, the curves b e c o m e narrower, a n d
e v e n t u a l l y it moves b y gliding along the surface of the chain, while scanning it with the
a p e x of the anterior flagellum. Finally, it attaches with a p s e u d o p o d i u m e m e r g i n g from
the posterior pole to one of the diatom cells (Fig. 2), g e n e r a l l y close to the suture b e t w e e n
valva a n d cingulum. Becoming slightly a m e b o i d it s q u e e z e s into the interior of the diatom
frustule, inserting the posterior pole first (Fig. 3). For some minutes, the flagella r e m a i n
outside w h e n the cell b o d y has a l r e a d y e n t e r e d the diatom (Fig. 4), but e v e n t u a l l y they
are also d r a w n inside. Presumably, t h e y b e c o m e shorter at the s a m e time, for the
flagellate inside the frustule has only very short flagella which are difficult to d e t e c t in the
light microscope. The whole invasion process t a k e s 5-10 rain. Multiple infections are
possible, especially u n d e r culture conditions. Only v e g e t a t i v e cells of G. delicatula, but
no sexual s t a g e s (gametes, zygotes), are i n v a d e d by the flagellate.
Frequently, after having e n t e r e d the diatom cell, the C. aestivalis cell m i g r a t e s into a
corner of a valva (Fig. 5). The diatom p r o t o p l a s t p l a s m o l y z e s at first locally, a n d after 2-3 h
the C. aestivalis cell, s u r r o u n d e d b y a larger, e m p t y space, starts feeding.
The trophic p h a s e begins with the e m e r g e n c e of a p s e u d o p o d i u m at the posterior
pole of the cell. Portions of the diatom protoplast, frequently consisting of a chloroplast
a n d some cytoplasm, are gradually p h a g o c y t i z e d (Fig. 6) a n d individually i n c l u d e d in a
food vacuole, a process which t a k e s a b o u t 10 rain in each case. The p h a g o c y t i z i n g
p s e u d o p o d i u m varies in shape, at times b e i n g long (Fig. 7) or short and b r o a d (Fig. 8), a n d
contracts rhythmically during the ingestion phase.
The trophont grows considerably, filling a l a r g e part of the diatom cell w h e n the
cytoplasm has b e e n completely ingested. It t h e n b e a r s two short flagella, a n d several
food vacuoles fill the posterior region of the cell (Fig. 9). During digestion the chloroplasts
c h a n g e colour, b e c o m i n g brown. D e p e n d i n g on the size of an a t t a c k e d diatom cell the
globular, m a t u r e trophont m a y b e c o m e more t h a n 20 ~m tong a n d 10 ~tm wide. The food
u p t a k e finishes early w h e n the m e m b r a n e of the Guinardia protoplast is r u p t u r e d a n d the
r e m a i n i n g cell organelles disintegrate.
A b o u t 4 h after the infection, the division p h a s e b e g i n s with the formation of another
pair of flagella. The resulting d a u g h t e r ceils (Fig. 10) divide after further 4 h (Fig. 11), a n d
a g a i n 2 h later a third division (and later further ones) m a y follow. The food vacuoles are
e q u a l l y distributed a m o n g the d a u g h t e r cells. Defecation of b r o w n g r a n u l a r m a t e r i a l
(fecal bodies) t a k e s place before the last cell division (Fig. 12) a n d is not r e l a t e d with the
cytokinesis. The n u m b e r of offspring d e p e n d s on the a m o u n t of i n g e s t e d food. As a result
there are usually 8 (up to 32) swarmers. The offspring n u m b e r is lower in small Guinardia
cells or after multiple infections.
The new, slightly a m e b o i d flagellates slip with their posterior pole foremost into the
frustule (Fig. 13), p r e s u m a b l y at the sutures b e t w e e n valva a n d cingulum, or b e t w e e n the
o v e r l a p p i n g epi- a n d hypocingulum. T h e y are soon c a p a b l e of i n v a d i n g a n e w diatom
cell. The whole v e g e t a t i v e life cycle t a k e s 18-20 h. The b r o w n fecal b o d i e s inside the
otherwise e m p t y Guinardia cell r e m a i n as the v e s t i g e s of the p r e c e d i n g a t t a c k b y C.
aestivalis (Fig. 14). The cell chains of the diatom do not get fragile w h e n a t t a c k e d b y
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Cryothecomonas aestivalis sp. nov,, a colourless nanoflagellate
501
Cryothecomonas. E v e n w h e n all protoplasts are p h a g o c y t i z e d (Figs 11-14), both diatom
ceils and cell chains r e m a i n intact for a long time.
Occasionally, in old cultures, finely g r a n u l a t e d cyst-like cells of Cryothecomonas are
formed inside the diatom frustules (not illustrated).
A t r e a t m e n t with cytochalasin (5 ~g 9 m1-1) slows d o w n the m o v e m e n t of the una t t a c h e d s w i m m i n g flagellates within 20 min and stops it nearly completely after 35 min,
w h e r e a s an o t h e r unidentified, non-infective flagellate (Cafeteria ?), a c o n t a m i n a n t in the
s a m e culture, is not affected in its motility. U n d e r the influence of cytochalasin n e w
infections h a v e not b e e n observed; some Guinardia cells b e c o m e slightly plasmolyzed.
Fine structure
Using electron microscopy, w e i n v e s t i g a t e d mainly cells of C. aestivalis in the trophic
an d the r e p r o d u c t i v e phase w h i c h w e r e e n c l o s e d in Guinardia frustules. Th e fine
structure of the cells differed slightly in some details, mainly due to variations during
development.
A survey of a cell at the e n d of the trophic p h a s e is shown in Figure 15. Th e nucleus
lies close to the basal bodies of the flagella w h i c h are inserted apically. It is l e n s - s h a p e d
an d highly lobed. T h e prominent nucleolus is associated with b an d s of d en se h et er o ch r o matin. Occasionally, there are nuclei with two nucleoli. D en se h e t e r o c h r o m a t i n also
forms a conspicuous, peripheral layer at the n u cl ear e n v e l o p e (see Figs 15, 26, 39, 43).
Th e outer m e m b r a n e of the nuclear e n v e l o p e is s t u d d e d with ribosomes (Fig. 26). Th e cell
in Figure 15 contains several food vacuoles at different stages of digestion. T h e contents
of one r e s e m b l e s the fecal body shown in Figure 16 and seem s to be largely c o m p o s e d of
hpids. The a m o u n t of osmiophilic droplets in the cytoplasm is especially high at the e n d of
the trophic phase.
Th e flagellates are generally s u r r o u n d e d by a close-fitting, delicate theca. It consists
of an electron-dense, 8 nm thick basal layer w h i ch is c o v e r e d with a 50 n m thick fuzzy
coat (Figs 17, 19). A r e g u la r substructure in the form of a striation could not b e detected.
Th e t h eca is often incompletely p r e s e r v e d or e v e n nearly c o m p l e t e l y absent, especially in
m a t u r i n g trophonts and dividing cells. In the flagellar pit (Fig. 19) the theca is modified,
an d it is absent at the cytostome (see below).
T h e mitochondria of C. aestivalis h a v e small cristae, and b e l o n g to the tubular rather
than to the vesicular-discoid type (Fig. 18). T h e r e are a few, small dictyosomes (Fig. 17),
s cat t er ed mainly in the anterior region of the cell.
Figs 1-14. Cryothecomonas aestivafis, hve cells. Scale bar: 10 ~m. 1: Free-motile flagellate. 2:
Flagellate forming a pseudopodium (arrowhead) to attach to the surface of a host cell (Guinardia
deh'catula). 3: Flagellate squeezing, with its posterior end first, into the host cell. 4: Flagellate having
entered the diatom, with the flagella still outside (arrowhead). 5: Two Cryothecomonas cells under
the valvae of their host cells. 6: Young trophont engulfing a chloroplast. 7: Trophont with a very long
pseudopodium. 8: Double infection, left trophont with distinctly recognizable flagella and a short,
broad pseudopodium. 9: Trophont at the end of the nutritive phase; note the two short flagella. 10:
Two daughter ceils after the first division. 11: Four cells after the second division; note their short
flagella. 12: Many swarmers at the final stage of reproduction; numerous fecal bodies (dark), set free
before the last division, fill the host frustule. 13: A flagellate without digestion vacuoles leaves the
diatom frustule; fecal bodies are left behind. 14: Most of the swarmers have left the frustule; fecal
bodies remain as vestiges of a preceding attack by Cryothecomon'as
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Cryothecomonas aestivalis sp, nov., a c o l o u r l e s s n a n o f l a g e l l a t e
503
Figs 20, 21. Cryothecomonas aestivalis, transmission electron microscopy: food uptake. Scale bar:
0.5 gm. 20: Retracted pseudopodium. W, theca a b s e n t in the cytostome region (asterisk). Cytostomal
cytoplasm highly vesiculated, with coated vesicles (arrowheads), and many invaginations and
protrusions. 21: Protruded pseudopodium (above) in close contact with the diatom cytoplasm D,
numerous vesicles, in part with m e m b r a n o u s contents, fibrillar-granular cytoplasm in the contact
zone (asterisks)
In t h e b a s a l r e g i o n of t h e t r o p h o n t s , w h e r e t h e f o o d is p h a g o c y t i z e d , t h e t h e c a h a s a
g a p . T h e cell p e r i p h e r y c o n s i s t s h e r e of a l a b y r i n t h of c y t o p l a s m i c p r o c e s s e s a n d i n v a g i n a t i o n s - t h e c y t o s t o m e i n t h e t e r m i n o l o g y of T h o m s e n e t al. (1990) (Fig. 20). It c a n b e
t r a n s f o r m e d i n t o t h e p s e u d o p o d i u m as s h o w n i n F i g u r e 21. T h e c y t o p l a s m of t h e
p s e u d o p o d i u m a n d of t h e c y t o s t o m e a r e a is h i g h l y v e s i c u l a t e d . M a n y v e s i c l e s h a v e
m e m b r a n o u s c o n t e n t s , a n d s o m e of t h e m a r e c o a t e d . In t h e r e g i o n w h e r e t h e p s e u d o p o d i u m is i n c l o s e c o n t a c t w i t h t h e d i a t o m p r o t o p l a s t , it is v e s i c l e - f r e e a n d h a s a g r a n u / a r fibrillar a p p e a r a n c e .
Figs 15-19. Cryothecomonas aestivalis, transmission electron microscopy. 15: Flagellate at the end
of the trophic phase. V, several food vacuoles with contents at different stages of digestion; F, apical
flagellum; N, nucleus with nucleolus; O, osmiophilic bodies; D, part of the diatom frustule. Scale bar:
1 ~tm. 16: Fecal body; compare with Fig. 15. Scale bar: 0.5 [tm. 17: Cryothecomonas theca consisting
of two layers, a n d to the left a dictyosome. Scale bar: 0.5 [tm. 18: Mitochondrium; N, nucleus. Scale
bar: 0.5 ~tm. 19: Cell wail a n d its modification at the funnel in the flagellar pit, seen in tangential
section, with the coarse sleeve as continuation of the cell wall (arrowheads) and the h o m o g e n e o u s
cone (arrow); B, basal body in tangential section. Scale bar: 0.5 [tm
504
G. Drebes, S. F. Kfihn, A. G m e l c h & E. Schnepf
The flagella are both smooth; one ot them m ay be thicker at the basis, i m m e d i a t e l y
over a conspicuous funnel of the theca. A flagellar rod with a regular s u b s t r u c t u r e could
not be d e t e c t e d within the flagellar swelling.
Each fully d e v e l o p e d f l a g e l lu m inserts separately in a small pit, a n d e a c h is surr o u n d e d by a funnel consisting of two parts (see Figs 19, 22-25, 26, 27, 35 an d the
scheme, Fig. 31). T h e l o w e r part of the funnel, the "sleeve", is continuous w i t h the inner
d en s e layer of the theca, w h e n the latter is present, and has a relatively coarse structure.
It surrounds the u p p e r part, the "cone", w h i c h is densely h o m o g e n e o u s in structure.
The transition r e g i o n b e t w e e n the flagellum and the basal body v a r i e s in structure,
p r e s u m a b l y in connection with t h e d e v e l o p m e n t of the cell. It b e g i n s at t h e l e v e l of the
funnel. H e r e the flagellum b e c o m e s thinner (Figs 26, 27); the two cen t r al microtubules
e n d (Fig. 27), a n d the p e r i p h e r a l doublets of the a x o n e m e b e c o m e indistinct, m e r g i n g
into a dense, massive, 35 n m b r o a d "transitional cylinder". The latter a p p e a r s nearly
h o m o g e n e o u s in cross sections t h r o u g h the flagellum, and is in close co n t act with the
plasma m e m b r a n e (Figs 22-27). Longitudinal tangential sections t h r o u g h the flagella
r e v e a l the transitional c y h n d e r to consist of a stack of 2-7 subrings, visible as a fine
striation with a spacing of 7 n m (Figs 26, 32). In flagella with a basal t h i c k e n i n g the
transitional cylinder is g e n e r a l l y thicker than in n o n - t h i c k e n e d flagella. Below the
transitional cylinder 9 p e r i p h e r a l structures r e a p p e a r (Figs 24, 25). N e a r the g r o u n d of the
flagellar pit they are massive, about triangular in cross section, and s u r r o u n d a central
d en s e plug (Fig. 25). The structure of the basal b o d y proper is typical - with 9 microtubular triplets (Fig. 29). Basal body and transitional region are about 0.5 ~tm long.
The two basal bodies form an a n g le of 70~
~ (Fig. 30), and the insertion sites of the
flagella are s e p a r a t e d by a small papilla (Fig. 39). Th e proximal ends of t h e basal bodies
show a typical c a r t w h e e l structure (Figs 30, 38), and are c o n n e c t e d by a short striated
b a n d (Figs 30, 36, and the scheme, Fig. 37). The basal bodies are s u r r o u n d e d by m a n y
e l e c t r o n - d e n s e satellites of variable size and s h ap e (Figs 26, 28-30), w h i c h c o m e in close
contact with the microtubular triplets in the proximal region of the b a s a l bodies. The
Figs 22-30. Cryothecomonas aestivalis, transmission electron microscopy: flagella and flagellar
bases of trophonts and cells in the division phase. Scale bar: 0.5 ~m. Figs 22-25. Series of cross
sections through a transition zone level with the flagellar pit, from distal to proximal. 22: Nine
peripheral elements are still visible; the funnel consists here mainly of the cone (arrow). 23: A
massive, homogeneous transitional cyhnder instead of nine peripheral elements. 24: Nine peripheral
elements are indistinctly visible. Funnel with cone (arrow) and sleeve (arrowhead). 25: Nine
triangular peripheral elements and a central plug at the base of the flagellar pit. The funnel is absent
here. 26: Flagellum with basal swelling. Funnel with cone (arrows) and sleeve (arrowheads),
transitional cyhnder (big arrowhead) in tangential section showing fine striation. R, flagellar root; s,
satellites; group of microtubules in the apical papilla (double arrowheads); N, nucleus with
ribosomes close to the nuclear envelope. 27: Unswollen flagellum and basal body. Transitional
cyhnder less high than in Fig. 26. Funnel with cone (arrows) and sleeve (arrowheads) which is in
connection with the insufficiently preserved cell wall. Figs 28-30. Series of cross-sections through a
pair of basal bodies, from distal to proximal. 28: Cross-section through basal body 1 at the base of the
flagellar pit (asterisk). Radial "transitional fibres" to satellites (s). S: satellites of basal body 2. 29:
Ring of satelhtes (s), surrounding basal body 1 and partly connected by "transitional fibres" with the
microtubular triplets. S: satellites of basal body 2.30: Basal body 1 with cartwheel structure, basal
body 2 in longitudinal section, surrounded by satellites (S) in funnel-hke arrangement. A striated
band (white arrow) connects the proximal ends of the two basal bodies. Flagellar funnel with cone
and sleeve.
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G. D r e b e s , S. F. Kfihn, A. G m e l c h & E. S c h n e p f
Fig. 31. Cryothecomonas aestivalis, scheme of flagellar base at a stage containing numerous
microtubules. Funnel with cone (C) and sleeve (S) level with the transitional cyhnder (T) and
satellites as initiation sites of microtubules
Figs 32-36. Cryothecomonas aestivalis, transmission electron microscopy: flagellar root a n d division.
Scale bar: 0.5 ~m. 32: R, microtubular flagellar root at the basal bodies. Funnel, transitional cylinder
(white arrowhead) with fine striation; N, nucleus. 33: Distal part of the microtubules of the flagellar
root (arrowheads) running along the plasma m e m b r a n e . 34: Flagellar pit with two flagella, cross
sectioned at the level of the transitional cyhnder. This cell has another pair of flageha nearby. 35:
Basal body associated with m a n y microtubules (arrowheads) r u n n i n g in diverse directions, some
microtubules originating near satelhtes. 36: Two pairs of flagella, each with a striated connecting
b a n d (arrows) a n d surrounded by m a n y microtubules (arrowheads)
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Fig. 37. C r y o t h e c o m o n a s aestivalis, scheme of pair of flagellar bases with microtubular root and
striated connecting band
satellites of the two basal bodies m e r g e h e r e (Fig. 30). More apically the satellites are
a r r a n g e d in the form of a cylinder or cone. Some of t h e m are c o n n e c t e d by radial strands
("transitional fibres" sensu T h o m s e n et al., 1990) with the triplets of the b asal body (Figs
28, 29).
A microtubular flagetlar root originates in the r e g i o n w h e r e the two b asal bodies lie
close t o g e t h e r (Figs 26, 32, and the scheme, Fig. 37). It consists of a b a n d of microtubules
which are e m b e d d e d in an e l e c t r o n - d e n s e matrix. It is proximally associated with
satellites, an d runs to the cell surface w h e r e the matrix is reduced, and the microtubules
d i v e r g e to a c c o m p a n y the p l a s m a m e m b r a n e in the apical part of the cell (Fig. 33). Th e
apical papilla contains a n o th e r small group of microtubules (Fig. 26). O t h e r cytoskeletal
microtubules originate near the satellites, g e n e r a l l y b e t w e e n the basal b o d i e s (Fig. 35).
T h e y differ highly in n u m b e r and orientation. Some of t h e m run also t o w a r d and along
the plasma m e m b r a n e , others toward or along the nucleus. Th er e are cells w h i c h s e e m to
contain only very few of these microtubules (Figs 27, 29), while other cells, especially
those in the division phase, h a v e m a n y of them, often in a s e e m i n g l y i r r eg u l ar arrangem e n t (Fig. 36).
Mitosis and cytokinesis are initiated by the formation of a n e w pair of flagella.
Initially, the n e w pair is situated in a c o m m o n flagellar pit (Fig. 34). M a n y microtubules
radiate from the flagellar bases w h e n the pairs b e g i n to m o v e apart (Figs 36, 38). Most of
Figs 38-41. CQ, o t h e c o m o n a s aestivalis, transmission electron microscopy: division. Scale bar: 1 ~m.
38: Two pairs of basal bodies moving apart and connected by a fibrillar band (arrows). The upper
pair reveals the cartwheel structure in cross-section and longitudinal section. Microtubules (arrowheads) radiate from the basal bodies, but are absent in the region between the two pairs, R, flagellar
root; N, nucleus. 39: Flagellate with four flagella or basal bodies, respectively; in this section not all
of them are visible at each cell pole (F); N, lobed nucleus. 40: Nucleus in metaphase stage. D, part of
the diatom frustule. 41: Same metaphase as in Fig. 40. Bundles of spindle microtubules (arrowheads)
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G. Drebes, S. F. Kfihn, A. Gmelch & E. Schnepf
them r u n peripherally, some also into the interior of the cell, a n d they do n o t a p p e a r to be
well ordered. B e t w e e n the separating pairs of basal bodies microtubules are largely
absent, but they are transiently connected by a fibrous b a n d (Fig. 38). Later, t h e flageIla
are found at opposite poles of the cell (Fig. 39). At this stage, they are a l r e a d y associated
with n e w basal bodies, w h e n the cells are about to continue the divisions.
During prophase the nuclear envelope disintegrates completely. Figure 40 shows a
cell in m e t a p h a s e , with the chromosomes lined up in the equatorial plate. At higher
magnification the spindle microtubules can be seen (Fig. 41). Only diffuse kinetophores
seem to b e present. At early telophase the nuclear envelope b e g i n s to reconstitute at the
periphery of the chromosome mass, while polar spindle microtubules r e m a i n initially
p r e s e n t b e t w e e n the two d a u g h t e r nuclei (Fig. 42).
Cells with two nuclei are often found, indicating that there is a certain time span
b e t w e e n mitosis a n d cytokinesis. The cell division b e g i n s with a n a c c u m u l a t i o n of
vesicles in the region of the developing furrow, m a n y of them with m e m b r a n o u s contents
(Fig. 43). The n a r r o w i n g c o n n e c t i n g strand b e t w e e n the two d a u g h t e r ceils is traversed
by m a n y microtubules (Figs 44, 45). In contrast to a midbody a distinct overlap zone does
not seem to be p r e s e n t here. A peripheral contractile ring is not always clearly visible. It is
best seen in t a n g e n t i a l sections (Fig. 45).
DISCUSSION
There can be no doubt that the present flagellate is closely related with Cryothecomonas T h o m s e n et al., a g e n u s comprising marine, free-living, phagotrophic flagellates.
Unlike T h o m s e n et al. (1990), we had m a i n l y trophontic a n d s u b s e q u e n t reproductive
stages inside the diatom frustule at our disposal. Nevertheless, m a n y characters of C.
aestivalis are completely or largely identical with those of the four species described by
T h o m s e n et al. (1990), which also differ from one another in some details.
Similarities
and slight differences
are:
(1) The s h a p e of the cell with a n apical pair of smooth flagella of different length,
each inserted in its own flagellar pit a n d separated by a small apical papilla; further the
position a n d size of the food vacuoles.
(2) The position of the n u c l e u s close to the flagellar bases, the n u c l e a r structure with
the conspicuous heterochromatin along the nuclear envelope a n d in the form of irregular
b a n d s t h r o u g h o u t the interior of the nucleus, a n d a big nucleolus.
(3) The p r e s e n c e of a close-fitting theca which differs somewhat in the four species of
T h o m s e n et al. (1990). In our flagellate it consists of a thin electron-dense i n n e r layer, a n d
a thicker, looser outer layer which is, in contrast to the above m e n t i o n e d species, not
regularly ridged. A further deviation is the frequently msuffient p r e s e r v a t i o n of the theca,
perhaps b e c a u s e it is modified d u r i n g the trophic a n d reproductive phase. Obvious
Figs 42-45. Cryothecomonas aestivalis, transmission electron microscopy: division. Scale bar:
0.5 ~m. 42: Nucleus at telophase stage. Reconstitution of the nuclear envelope (asterisks), spindle
microtubules (arrowheads). 43: Beginning of cyt0kinesis. Accumulation of vesicles in the division
furrow. N, nucleus. 44: Final stage of cytokinesis. The narrowing connecting strand is traversed by
many microtubutes, cytoplasm vesiculated, fibrillar ring indistinctly visible (arrowheads). 45: Final
stage of cytokinesis. Tangential section through the narrowing connecting strand reveals the fibrillar
ring (arrows); microtubules
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512
G. Drebes, S. F. Kfihn, A. Gmelch & F.. Schnepf
similarities b e t w e e n the species are the presence of a gap in the posterior part of the cell
and, especially, the c o m p l i c a t e d structure of the f u n n e l through w h i c h the flagella
emerge.
(4) The thecal gap of all species is associated with a cytostome consisting of
cytoplasmic i n v a g i n a t i o n s a n d protrusions, a n d it can be transformed into a pseudopodium. The cytostomal cytoplasm is in every case filled with vesicles, in part with
m e m b r a n o u s contents, a n d contains a n organelle-free area of fine granular-fibrillar
appearance, p r e s u m a b l y r e p r e s e n t i n g actomyosin complexes.
(5) The flagelIar basal bodies a n d their s u r r o u n d i n g s with radial "transitional fibres"
a n d "satelhtes" (electron-dense a g g r e g a t i o n s sensu T h o m s e n et al., 1990) are likewise
very similar in all species. These electron-dense bodies are believed by T h o m s e n et al.
(1990) to represent glycogen. Our micrographs rather suggest that they are proteinaceous
a n d function as microtubule organizing centres in the phase of divison. According to
T h o m s e n et al. (1990), the transition region of their species contains a transitional helix
which is, however, indistinctly shown. Instead, we found in our species a massive
transitional cylinder at the distal e n d of the transitional region. Because of the lack of
exact data on the flagellar root system in the p a p e r of T h o m s e n et al. (1990), these
structures c a n n o t be compared in detail with those of our flagellate. As far one can see,
they seem to be similar. This applies also to the group of microtubules in the apical
papilla.
(6) The mitochondria likewise b e l o n g to the same type in that they h a v e indistinctly
tubular cristae.
Compared with Cryothecomonas armigera, our species does not c o n t a i n extrusomes,
a n d also no muciferous bodies as for instance in C. vesiculata. Furthermore, we could not
u n a m b i g u o u s l y identify lysosomes. It r e m a i n s to be confirmed whether the "lysosomes"
of T h o m s e n et al. (1990) are correctly characterized or are perhaps microbodies.
Major differences
are the following:
(1) The adaptation to cold t e m p e r a t u r e s of the four species described b y T h o m s e n et
al. (1990), which seem to have a world-wide occurrence in sea ice biota, as in the Arctic,
Antarctic, a n d the Baltic Sea (Ik~valko & Thomsen, 1995). It is reflected i n the n a m e of the
g e n u s (Cryo ...), but does not apply to our species, which occurs in late s u m m e r and
a u t u m n in the North Sea.
(2) Cryothecomonas aestivalis has a peculiar m o d e of nutrition a n d d e v e l o p m e n t : it
invades specifically cells of Guinardia delicatula, a n d gradually phagocytizes the host
cytoplasm.
(3) Presumably, only our species b e h a v e s as a "parasite", i. e., with a n e x t e n d e d
trophic p h a s e followed by a series of cell divisions, as k n o w n from o t h e r "parasitic"
flagellates (Gaines & Elbr~chter, 1987).
In conclusion, it seems justified to place our flagellate in the g e n u s Cryothecomonas
T h o m s e n et al., a n d to erect a n e w species, C. aestivalis.
The taxonomic position of Cryothecomonas is uncertain, as discussed in detail by
T h o m s e n et al. (1990). Our studies provide only few n e w data which m i g h t b e taxonomically relevant. The mitosis is a n open orthomitosis in that the n u c l e a r e n v e l o p e disintegrates completely, i. e., it is of the evolved type (Raikov, 1994). During cell division, a
b u n d l e of microtubules r e m a i n s in the n a r r o w i n g c o n n e c t i n g strand b e t w e e n the two
sister cells. The transitional cylinder at the base of the (developing?) flagella is perhaps of
Cryothecomonas aestivalis sp. nov., a colourless nanoflagellate
513
additional significance in this respect. But these further characters are not yet sufficient to
assign the g e n u s with absolute taxonomic certainty. Molecular biological methods, which
have b e e n initiated, will be more informative.
The flexibility of the cell body a n d of the theca allows C. aestivalis to slip through the
diatom frustule into the cell. This is done after the suitability of the host cell as food source
has b e e n tested, obviously by the tip of the anteriorly directed flagellum. A similar
b e h a v i o u r is displayed by Pirsonia Schnepf et al., another flagellate feeding on diatoms
(Schnepf et al., 1990). The attachment to the diatom frustule is perhaps not performed by
the secretion of a sticky material but by the sucking force of the pseudopodium.
The e n t r a n c e of the flagellate into the diatom cell a n d food uptake seems to be driven
b y the actomyosin system of the pseudopodium, as indicated by the inhibitory effects of
cytochalasin D, a n d the presence of a fibrillar-granular cytoplasm in the area where the
p s e u d o p o d i u m contacts with the host cytoplasm.
AF
PF
Fig. 46. Cryothecomonas aestivalis, sketch of a flagellate in the free, motile stage. AF, anteriorly
directed flagellum; PF, posteriorly directed flagellum; N, nucleus; n, nucleolus; refractive granula
(asterisks)
514
G. Drebes, S. F. Kfihn, A. G m e l c h & E. Schnepf
T h e n u m e r o u s vesicles in the cytostomal cytoplasm, m a n y of t h e m with m e m b r a nous contents (see Daley et al., 1973, for phagocytizing O c h r o m o n a s ) or with a coat,
s u g g es t an i n t e n s iv e m e m b r a n e flow and a high turnover rate of m e m b r a n e s d u r i n g food
uptake. It can be e x p e c t e d that Golgi a p p a r a t u s - d e r i v e d vesicles d e l i v e r digestive
e n z y m e s into the p h a g o s o m e s (Cole & Wynne, 1974), but w e did not o b s e r v e a conspicuous activity of the relatively f e w dictyosomes during f eed i n g and digestion, an d w e r e
not able to identify primary lysosomes with certainty.
Th e cytostome of C r y o t h e c o m o n a s a e s t i v a l i s differs from the c y t o s t o m e s of most
other p h a g o t r o p h i c flagellates in that it is not s h a p e d by a distinct m i c r o t u b u l a r cytoskeleton (see the r e v i e w by R a d e k & H a u s m a n n , 1994).
T h e eco l o g y of C. a e s t i v a l i s will be treated separately (K~ihn, in prep.).
Diagnosis
C r y o t h e c o m o n a s a e s t i v a l i s Drebes, Kfihn & Schnepf, sp. nov.
Colourless flagellates; in the free, motile stage, oblong to oval, 9-12 ~m long and 4 - 5
am wide. Two apically inserted flagella. Anteriorly directed flagellum 15 `am long;
posteriorly directed flagellum up to 25 ,~m. Feeds on the marine p l a n k t o n i c diatom
G u i n a r d i a d e l i c a t u l a . Flagellate p e n e t r a t e s the diatom frustule. Tr o p h o n t s gradually
p h a g o c y t i z e the host cytoplasm by m e a n s of a p s e u d o p o d i u m , which e m e r g e s posteriorly
through a g a p in the theca. T h e c a delicate, consisting of two layers, occasionally lacking.
Trophonts and division stages with shortened, in part basally t h i c k e n e d flagella, and a
massive transitional cylinder that lacks a distinct transitional helix. E x t r u s o m e s and
muciferous bodies absent. M a t u r e trophonts give rise to 8-32 n e w flagellates (swarmers).
Defecation before the last division.
D i s t r i b u t i o n : Plankton of the North Sea, J u l y - N o v e m b e r .
T y p e 1 o c a 1i t y : W a d d e n Sea at List/Sylt, G e r m a n Bight (North Sea).
Holotype:
Figs 15,46.
E t y m o 1 o g y : Species n a m e refers to the main occurrence of t h e flagellate in
summer.
A c k n o w l e d g e m e n t s . We are grateful to Ms H. Hailiger and Ms S. Gold for technical assistance, to Dr.
M. Elbr6chter for valuable discussions, and to the Deutsche Forschungsgemeinschaft for financial
support. This is contribution No. 1055 of the Alfred-Wegener-Institut fiir Polar- und Meeresforschung, Bremerhaven.
LITERATURE CITED
Cole, G. T. & Wynne, M. J., 1974. Endoclrtosis of Microcystis aeruginosa by Ochromonas danica. - J.
Phycol. I0, 397-410.
Daley, R. J., Morris, G. P. & Brown, S. R., 1973. Phagotrophic ingestion of a blue-green alga by
O c h r o m o n a s . - J. Protozool. 20, 58-6I.
Drebes, G., 1974. Marines Phytoplankton. Thieme, Stuttgart, 186 pp.
Gaines, G. & Elbr/ichter, M., 1987. Heterotrophic nutrition. In: The biology of dinoflagellates. Ed. by
F. J. R. Taylor. Blackwell, Oxford, 224-268.
Guillard, R. R. L. & Ryther, J. H., 1962. Studies on marine phytoplankton diatoms. 1. Cyclotella nana
Hustedt and Detonula confervacea (Cleve) Gran. - Can. J. Microbiol. 8, 229-239.
C r y o t h e c o m o n a s aestivalis sp. nov., a colourless nanoflagellate
515
Hasle, G. R. & S y v e r t s e n , E. E., 1996. M a r i n e diatoms. In: I d e n t i f y i n g m a r i n e d i a t o m s a n d dinoflagellates. Ed. b y C. R. T o m a s . Acad. Press, S a n Diego, 598 pp.
Ik~ivalko, J. & T h o m s e n , H. A., 1995. Baltic s e a ice biota (March I994): r e m a r k s on t h e o c c u r r e n c e
a n d a b u n d a n c e of Cryothecomonas armigera (Protista i n c e r t a sedis) a n d C. scybalophora. - Eur.
J. Protistol. 3I, 113.
Kiihn, S. F., D r e b e s , G. & Schnepf, E., 1996. Five n e w s p e c i e s of t h e n a n o f l a g e l l a t e Pirsonia in t h e
G e r m a n Bight, N o r t h Sea, f e e d i n g on p t a n k t i c diatoms. - H e l g o l a n d e r M e e r e s u n t e r s . 50,
205-222.
R a d e k , R. & H a u s m a n n , K., 1994. Endocytosis, digestion, a n d d e f e c a t i o n in flagellates. - Acta
Protozool. 33, 127-147.
Raikov, I. B., 1994. T h e diversity of forms of m i t o s i s in protozoa: a c o m p a r a t i v e review. - Eur. J.
Protistol. 30, 253-269.
Reize, I. B. & M e l k o n i a n , M., 1989. A n e w w a y to i n v e s t i g a t e living flagellated/ciliated cells in
a g a r o s e . - B o t a n i c a Acta 102, 145-151.
S c h n e p f , E., Drebes, G. & Elbrachter, M., 1990. Pirsonia guinardiae, g e n . et spec. nov.: A parasitic
flagellate o n t h e m a r i n e d i a t o m Guinardia flaccida with a n u n u s u a l m o d e of food u p t a k e . H e l g o l 5 n d e r M e e r e s u n t e r s . 44, 275-293.
T h o m s e n , H. A., Buck, K. R., Bolt, P. A. & Garrison, D. I., 1990. Fine s t r u c t u r e a n d biology of
Cryothecomonas g e n . nov. (Protista i n c e r t a sedis) from ice biota. - Can. J. Zoot. 69, 1048-1070.