J. Phycol.
53, 629641 (2017)
2017 Phycological Society of America
DOI: 10.1111/jpy.12525
THE FUNCTION OF THE OCELLOID AND PISTON IN THE DINOFLAGELLATE
ERYTHROPSIDINIUM (GYMNODINIALES, DINOPHYCEAE)1
o mez 2
Fernando G
Carmen Campos Panisse 3, E-11500 Puerto de Santa Mar
a, Spain
The marine dinoflagellate Erythropsidinium
possesses an ocelloid, the most elaborate
photoreceptor organelle known in a unicellular
organism, and a piston, a fast contractile appendage
unknown in any other organism. The ocelloid is
able to rotate, often before the cell swims. The
ocelloid contains lenses that function to concentrate
light. The flagellar propulsion is atrophied, and the
piston is responsible for locomotion through
successive extensions and contractions. During the
locomotion mode, the contraction is ~4 times
faster than the extension. The piston attained up to
50 mm
s1 and the cell jumps backwards at
4 mm
s1, while during the piston extension the
cell moves forwards. The net speed of
~1 mm
s1 is faster than other dinoflagellates.
The piston usually moved in the static mode
without significant cell swimming. This study
suggests that the piston is also a tactile organelle
that scans the surrounding waters for prey.
Erythropsidinium feeds on copepod eggs by
engulfing. The end of the piston possesses a
suction cup able to attach the prey and place it
into the posterior cavity for engulfing. The
cylindrical shape of Erythropsidinium, and the
anterior position of the ocelloid and nucleus, are
morphological adaptations that leave space for the
large vacuole. Observations are provided on
morphological development during cell division.
Most of the described species of Erythropsidinium
apparently correspond to distinct life stages of
known species, and the genus Greuetodinium
(=Leucopsis) corresponds to an earlier division stage.
Key index words: Dinoflagellata; Erythropsis; eyespot;
motile behavior; motility; Nematodinium; peduncle
feeding; photoreceptor organelle; protist locomo-
tion; Warnowia
Abbreviations: fps, frames per second; ms, millisec-
ond; TEM, transmission electron microscopy
The warnowiid dinoflagellates, Erythropsidinium P.
C. Silva, Warnowia Er. Lindemann, Nematodinium
Kofoid et Swezy and Proterythropsis Kofoid et Swezy,
1
Received 24 July 2016. Accepted 7 February 2017.
2
Author for correspondence: e-mail fernando.gomez@fitoplanc
ton.com.
Editorial Responsibility: J. Raven (Associate Editor)
629
are a group of unarmored heterotrophic species
that possess a complex photoreceptor organelle
named the ocelloid. Erythropsidinium (=Erythropsis
Hertwig) possesses the largest and most elaborate
ocelloid among all the unicellular organisms (Colley
and Nilsson 2016). Hertwig (1884) described Ery-
thropsidinium agile (Hertwig) P. C. Silva (as Erythropsis
agilis Hertwig) with an eyespot composed of a pig-
ment mass and lens, and a sophisticated body exten-
sion capable of extraordinary rapid extension and
contraction. This contractile organelle, named pis-
ton, dart, prod or tentacle, is unknown in any other
organism (Kofoid and Swezy 1921, Greuet 1967).
The unusual degree of structural complexity of the
ocelloid and the piston generated disbelief among
earlier protozoologists. Greuet (1965, 1967, 1968a,b,
1969, 1973, 1977, 1987) carried out numerous
observations, mainly ultrastructural studies based on
transmission electron microscopy (TEM). The ocel-
loid is a highly developed photoreceptor organelle
with cornea-, lense-, pigment cup and retina-like
structures consisting of stacked membranes (Greuet
1968a, 1987, Gavelis et al. 2015). The ocelloid exhi-
bits a parallelism to the structure of metazoan eyes,
with all of the major functional components except
for the conduction of signals, constructed entirely
at the subcellular level. Greuet (1977) considered
that the ocelloid derived from a chloroplast and is
homologous with the eyespot or stigma. Francis
(1967) investigated the optical properties of the lens
in Nematodinium. He reported that in the pyriform
lens there is no single point of focus but rather a
zone of focus, and only objects ~50 lm distant are
focused in the retinoid. Francis (1967) suggested
that the ocelloid functions as an image-forming eye.
Erythropsidinium, together with the other ocelloid-
bearing dinoflagellates, belongs to the clade of
Gymnodinium F. Stein (G
omez et al. 2009). Relatives
such as Polykrikos Buetschli are able to shoot ejectile
bodies, nematocysts or taeniocysts, to narcotize and
immobilize prey such as fast-swimming red-tide
dinoflagellates (Matsuoka et al. 2000, Lee et al.
2015). Warnowiid dinoflagellates such as Nemato-
dinium also possess nematocyst-taeniocyst complexes
(Greuet 1987), and it has been suggested that the
ocelloid plays a role in the prey selection and cap-
ture, and the cell fires its ejectile bodies, explosive
stingers, only when the target is at the right direc-
tion and distance (Omodeo 1980, Taylor 1980).
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F E R N A N D O G OM
Hayakawa et al. (2015) and Gavelis et al. (2015) car-
ried out the immune-cytological and genomic stud-
ies, and new TEM studies in warnowiid
dinoflagellates. These results concluded that the
ocelloid is a chimeric structure, incorporating orga-
nelles with different endosymbiotic histories. The
rhodopsin sequence of the retina-like structure of
Erythropsidinium branched with haptophytes, and
that supported the chloroplast-derived origin sug-
gested by Greuet (1977). From the observation of
live cells, Hayakawa et al. (2015) concluded that the
lenses can extend the range of light sensitivity by
concentrating dim light.
Evolutionists such as Gehring (2005, 2014)
hypothesized that the ocelloid of Erythropsidinium
may be the origin of the eyes in metazoans. He pro-
posed that the dinoflagellates may have transferred
their photoreceptor genes to cnidarians (Gehring
2005, 2014). For many, the elaborate ocelloid of Ery-
thropsidinium is considered a model of the smallest
eye on Earth (Schwab 2004, Colley and Nilsson
2016). The delicacy and the difficulties in culture of
these heterotrophic species make it necessary to
work with recently collected wild cells. Erythropsi-
dinium is widespread in the euphotic zone of the
warm oceans (G
omez 2008), while the specialized
laboratories are mostly located in high latitudes.
The role of the ocelloid in the ecology of Erythropsi-
dinium, tentatively used in prey selection and preda-
tor avoidance, remain unresolved. The piston is a
unique organelle whose mechanism of propulsion
and function has not been investigated.
This study reports observations of live cells of Ery-
thropsidinium with the aim to shed light on the func-
tions of the ocelloid and piston, and the influence of
these unique organelles in the morphology and behav-
ior of the entire cell. This study provides data on the
behavior, cell and ocelloid division, with new insights
in to the taxonomy and synonymy of Erythropsidinium.
This study provides the first data on the type of prey
and feeding as a first step for culturing under labora-
tory conditions, and the first data on the mechanism
of cell propulsion and piston velocity and function.
MATERIALS AND METHODS
Live cells were collected from four coastal sites in the
Mediterranean Sea in France and Spain (Marseilles, Banyuls-
sur-Mer, Villefranche-sur-Mer and Valencia) and two sites in
the South Atlantic Ocean in Brazil (S~ao Sebasti~ao Channel
and Ubatuba). Methods of collection and observation were
described in G
omez et al. (2016) and in Appendix S1 in the
Supporting Information. The aim of the observations at Mar-
seilles and Banyuls-sur-Mer was to obtain the first molecular
data of Erythropsidinium in order to resolve its phylogenetic
affiliation (G

omez et al. 2009). The cells were immediately
isolated for PCR analysis to prevent cell lysis, and conse-
quently the behavioral observations were scarce. Samples in
the Bay of Villefranche-sur-Mer were collected from hauls
from 80 m depth to the surface with a 53 lm mesh size
plankton net. This method damages the delicate cells and
the poor living conditions in a dense plankton concentrate

EZ
were unfavorable for unarmored dinoflagellates. The observa-
tions were also scarce from samples collected in the surface
waters of the port of Valencia, due to pollution and the dis-
tance between the sampling area and the laboratory that
delayed the observations more than 1.5 h after collection.
The observations of Erythropsidinium were more numerous in
the tropical coasts of Brazil, and the mild weather conditions
favored the sampling almost the entire year. In this case, the
results were limited due to the deficiencies in the microscope
and camera facilities. Sampling was carried out using a phyto-
plankton net (20 lm mesh size) between March and Decem-
ber 2013 in the surface waters of S~ao Sebasti~ao Channel
(23500 4.05 S; 45240 28.82 W, 40 m depth). The live
plankton concentrate (35 L) was collected in the early
morning. During the day, aliquots of 50 mL were serially set-
tled and examined (Appendix S1). Cells of Erythropsidinium
were found in the first hours of observations, and very rarely
at the end of the day. This suggests that the cells lysed as a
consequence of the unfavorable conditions in the plankton
concentrate. The samples were examined in Uterm ohl cham-
bers at 9100 and 9200 magnifications with an inverted
microscope Diaphot-300 (Nikon Inc., Tokyo, Japan). Cells
were photographed with a digital camera (Cyber-shot DSC-
W300; Sony, Tokyo, Japan) mounted on the microscopes
eyepiece. The camera also captured movies with time resolu-
tion of 30 frames per second (fps). The frames were
extracted using Free Video to JPG converter v. 5.0.28
(DVDVideoSoft Ltd, London, U.K.). An inverted microscope
Eclipse TS-100 (Nikon Inc.) with higher magnification objec-
tives was available from November 2013. Cells were pho-
tographed with the same digital camera mounted on the
microscopes eyepiece. This procedure was not optimal for
recording swimming organisms because while the camera was
mounted, the target cell moved out the observation field and
the camera needed to be removed in order to search again
to locate the cell. From the December 2013 to December
2015 samplings were carried out off Ubatuba (23320 20.15
S; 4550 58.94 W, 15 m depth), and using the same inverted
microscope Eclipse TS-100 and the Sony Cyber-shot camera.
From May 2015 an inverted microscope Olympus IX73
(Olympus Inc., Tokyo, Japan) was available. A digital camera
with a standard C-mount adapter (EUREKAM 3.0; BEL Pho-
tonics, Monza, Italy) was connected to the lateral camera
port, and operated with BEL View software. The video option
was not useful for swimming cells because the time resolution
of video recordings was limited to 6 fps. A high-speed camera
(Photron FASTCAM SA2 model 86K-C3; Photron, San Diego,
CA, USA) connected to the Olympus IX73 inverted micro-
scope was available for several days in late August 2015.
Despite winter being the most unfavorable season, several
active cells were recorded at 1,500 fps and 60 fps, and oper-
ated with Photron FASTCAM viewer. It should be noted that
at 1,500 fps, a recording file of a few seconds requires tens of
minutes for saving. During that period the target cell became
inactive or lysed. It was difficult to record a cell that was
already swimming because when using high magnification it
easily moved out of observation field or out of focus. When
using low magnification, the thin hyaline piston was hardly
visible. At the beginning of each observation, the cell was on
focus because it is still close to the bottom of the settling
chamber, and consequently there is wall effect until the
cell is suspended. Consequently, the speed values are proba-
bly underestimated.
RESULTS
Morphology of the ocelloid. The warnowiid dinoflag-
ellates are characterized by the presence of an
 O C E L L O I D A N D P I S T O N I N ER YT H R O P S I D I N I U M
ocelloid (see Video S1 in the Supporting Informa-
tion). The melanosome, dark retinoid-like body,
shows the red autofluorescence of chlorophyll a
when examined with epifluorescence microscopy
(Fig. 1, a and b). The cell body of Erythropsidinium
is slightly laterally compressed, with the ocelloid in
the left anterior side of the ventral view (Fig. S1 in
the Supporting Information). The dorsal contour is
convex, while the anterior ventral side is concave
with the sulcus and intercingular region in a
depressed area along the anterior longitudinal axis
(Fig. S2 in the Supporting Information). The ocel-
loid was sometimes laterally directed (Fig. 1c), and
more commonly anteriorly directed (Fig. 1, dm).
The entire ocelloid rotated up to 45 from its ante-
rior position towards the left lateral side, and
quickly returned to the anterior position (Fig. 1, d
and e). The ocelloid rotation often preceded cell
swimming (Video S1). The lenses of the hyalosome
concentrate the light on the melanosome (Fig. 1,
fj). The melanosome is chevron-like or kidney-
shaped in lateral view (Fig. 1i), and circular in api-
cal view (Fig. 1k). The hyalosome is hemispherical,
located on the concave side of the melanosome,
and composed of lenses forming concentric layers,
with a cornea-like external envelope. The inner
lenses overlap laterally in the wedge-shaped pattern
(Fig. 1m). When the cell lysed, the ocelloid
remained as an independent organelle (Fig. 1, or).
The melanosome remained intact for some time,
while the lenses immediately became disorganized,
but they maintained the capacity to concentrate
light (Fig. 1r).
Morphology of the piston and body exten-
sions. Warnowiid dinoflagellates are characterized
by a convex end of the hyposome or a pointed anta-
pex (Fig. 2, a and b). Proterythropsis showed a poste-
rior body extension (Fig. 2, c and d), and
sometimes Nematodinium showed a hand-like lateral
body extension with distal finger-like projections
that were moveable (Fig. 2e; Video S1). Some cells
whose morphology corresponded to Erythropsidinium
scarlatinum (Kofoid et Swezy) P. C. Silva showed an
elongated and thin lateral body extension with dis-
persed pigmented granules (Fig. 2, fh, m and n).
Other cells whose morphology corresponded to
E. extrudens (Kofoid et Swezy) P. C. Silva (Fig. 2,
ik) showed a shorter body extension, with a more
or less pointed end, and the same coloration of the
cell. The pigmented posterior or postero-lateral
body extensions of E. scarlatinum and E. extrudens
were likely mistaken for the piston in their original
descriptions. The piston was always hyaline and cov-
ered by papillae (Fig. 2, jm). The piston may pos-
sess one nodule or several nodules in different
positions along its length. It showed a distal swelling
of lanceolate (Fig. 2, k and l) or rounded shapes
(Fig. 2m). The piston extended and contracted in a
straight line, and it was sometimes bent (Fig. 2n).
The piston retracted into a bulb located at the base
631
of a posterior cavity or invagination (Fig. 2o). This
hemispherical posterior cavity showed a slit (Fig. 2p,
Fig. S2). A part of the piston can lyse, while the
other part may continue beating (Video S1). A fully
lysed piston was able to regenerate in half an hour.
If the cell lysed when the piston was beating, the
isolated piston may continue beating for 1 min, and
finally retract into the bulb before lysing. The swel-
ling at the end of piston acted as a suction cup
that adhered to the bottom glass of the settling
chamber (Fig. 2q, Fig. S3 in the Supporting Infor-
mation; Video S1). As the cell was unable to pull
the bottom glass, during the phases of contraction
and extension, the cell moved towards and then
and moved away from it. The piston contraction was
about three or four times faster than its extension
(Fig. 2q, Fig. S3; Video S1).
Cell and piston motility. The piston activity and cell
swimming were examined with a high-speed camera
(1,500 and 60 fps), and more frequently with a stan-
dard camera with the time resolution of ordinary
video recordings (30 fps). The activity of the piston
showed a phase of extension, contraction and then
remained in the posterior cavity before the next
extension. The time interval between these phases,
especially between the extension and the contrac-
tion suggests two modes: a static mode, without
significant cell swimming, and a locomotion
mode, where the cell swam usually backwards (in
the direction of the cell antapex) and more rarely
forwards (towards the cell apex). Here the back-
wards speed is considered as negative.
During the static mode, the phase of extension
showed a variable duration and once extended, the
piston may remain extended for a variable period.
The duration of the contraction was more regular
and faster than the extension. The piston retracted
into the posterior cavity in 16 ms. This implied that
the piston attained a peak speed of 36 mm
s1
(Figs. 3a, S4 and S5 in the Supporting Informa-
tion). The piston remained in the posterior cavity
for a variable period before the next extension. Dur-
ing piston extension, the cell body moved forwards
at ~0.1 mm
s1, while during the piston contrac-
tion moved backwards at ~0.4 mm
s1 (Figs. 3a,
S4 and S5). The balance between the extension and
contraction did not necessarily yield a significant
cell displacement. The cell remained motionless at
the bottom of the settling chamber, with some rota-
tion of the cell body (Fig. S6 in the Supporting
Information). It should be noted that the cell body
was in contact with the bottom glass of the settling
chamber, and consequently there is a friction and/
or wall-effect that may reduce the movement.
Observations of the locomotion mode were car-
ried out when a settled cell that was in focus began
to swim. Recordings were difficult when the cell was
already suspended and swimming because it easily
went out of focus or the observation field. When
compared to the static mode, in the locomotion
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F E R N A N D O G OM

FIG. 1. Light micrographs of warnowiid dinoflagellates and their ocelloids. (a and b) Warnowia sp. (b) Note the autofluorescence of
the melanosome. (c) Erythropsidinium sp. with laterally directed ocelloid. (dr) Erythropsidinium agile. (d and e) Lateral rotation of the ocel-
loid. (fj) Right lateral view in differences focuses. (j and r) Note the light concentration by the lenses. (k) Apical view. (l and m) Detail
of the ocelloid. (m) Note the overlapping of the lenses in the hyalosome (or) Ocelloid before and after cell lysis. ci, cingulum; lf, longitu-
dinal flagellum; hy, hyalosome (lenses); me, melanosome (pigment cup); nu, nucleus; oc, ocelloid; pi, piston; su, sulcus; tf, transversal flagel-
lum; scale bars, 20 lm. [Color figure can be viewed at wileyonlinelibrary.com]

mode: (i) the piston immediately retracted after piston during the static and locomotion modes
the extension, and (ii) the piston attained a greater attained 500 and 700 lm long, respectively. A phase
length during the extension. For the same cell, the of extension and contraction lasted for ~120 ms.
 O C E L L O I D A N D P I S T O N I N ER YT H R O P S I D I N I U M 633

FIG. 2. Light micrographs of warnowiid dinoflagellates and their body extensions. (a and b) Warnowia sp. with pointed antapex. (c and
d) Proterythropsis. (e) Nematodinium. Note the moveable lateral extension with finger-like projections. (fh, m and n, q) Erythropsidinium scar-
latinum. (il) E. extrudens. (o and p) E. agile. (km) Note the different outline of the distal end of the piston. (l and m) Note the papillae
in the piston surface. (n) Note the bended piston. (o) Note the posterior cavity. (p) The arrow points a slit. (q) Time-lapse sequence of
the piston end attached onto the bottom glass of the settling chamber. be, body extension; ci, cingulum; lf, longitudinal flagellum; nu,
nucleus; oc, ocelloid; pi, piston; st, surface striae; su, sulcus; scale bars, 20 lm. [Color figure can be viewed at wileyonlinelibrary.com]

During that time, the piston was extended for
~70 ms, the contraction lasted for 16 ms, and it
remained retracted in the posterior cavity for
~30 ms. A piston with a length of 700 lm that
retracted in 16 ms implied an average speed of
~50 mm
s1, and a higher peak speed in the mid-
dle of the contraction. During the extension phase
of ~70 ms, the cell swam forwards at ~0.5
0.6 mm
s1, and during the contraction the cell
jumped backwards at ~3 to 4 mm
s1 (Fig. S7 in
the Supporting Information). This was a peculiar
swimming pattern in a straight line. In order to
swim in a given direction, the cell was moving for
more time in the opposite direction. The balance
during a phase of contraction and extension was
~1 mm
s1. The cell swam usually backwards, but
also after a stop the same cell was able to reverse
the direction and swam forwards (Fig. S8 in the
Supporting Information).
Feeding behavior and morphology. Erythropsidinium is
the unicellular organism with the most elaborate
eye-like organelle, and a piston that is unknown in
any other organism. The type of prey and the feed-
ing mechanism may help to understand the adap-
tive advantages of these unique organelles. The
morphology of Erythropsidinium is different from
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F E R N A N D O G OM

FIG. 3. Time-lapse sequence of the piston activity of Erythropsidinium. (a) Phases of extension and retraction during the static mode
recorded at 1,500 fps. Only one each five consecutive frames is illustrated. The interval between two micrographs is 3.33 ms (See Fig. S4
for the complete sequence). (b) Several phases of extension and retraction during the locomotion mode recorded at 60 fps. The inter-
val between two micrographs is 16.66 ms (See Fig. S5 for the complete sequence). The lateral size scale corresponds to the stage microme-
ter with divisions each 10 lm. [Color figure can be viewed at wileyonlinelibrary.com]
 O C E L L O I D A N D P I S T O N I N ER YT H R O P S I D I N I U M
other warnowiid dinoflagellates. Erythropsidinium
shows a very anterior cingulum in a reduced epi-
some, and a short longitudinal flagellum (Fig. 2, j
and m). The flagella appear insufficient to propel
the cell, and Erythropsidinium remained motionless
in the static mode, moving the piston without sig-
nificant cell swimming (Fig. 3a). In contrast, the
cingulum of other warnowiid dinoflagellates turns
several times around the cell and that implied
higher propulsion by the transversal flagellum
(Fig. 4, ae). When the other warnowiid dinoflagel-
lates were not enclosed in a hyaline membrane
(Figs. 1a and 4, a and d), they were actively swim-
ming (Fig. 4, b and c; Video S1). The ocelloid and
nucleus are located in an anterior position in Ery-
thropsidinium (Figs. 1, dg; 2, fk; and 4, fq). The
antapex shows a large posterior cavity with a slit
(Figs. 2, o and p; S2). In contrast, other warnowiid
dinoflagellates show a convex antapex, more or less
pointed, and lacked the posterior cavity. The ocel-
loid is median or posterior and that may interfere
with harboring a large vacuole (Fig. 4, ae). In con-
trast, the cylindrical shape of Erythropsidinium per-
mits harboring a large vacuole that will not
interfere with the anterior organelles such as the
ocelloid that protrude from the cell and the nucleus
protected by a perinuclear envelope (Fig. 4, fk).
Nematodinium and Warnowia show spherical or
ellipsoidal food vacuoles (Fig. 4, ae). The vacuoles
of Nematodinium show brown pigmentation, evidence
of the capture of motile prey such as chain-forming
photosynthetic dinoflagellates (Fig. 4a). The species
of Warnowia with a median ocelloid also fed on pho-
tosynthetic dinoflagellates (Fig. 4, b and c). Species
of Warnowia with a more anterior ocelloid show vac-
uoles with a hyaline content that may correspond to
those of heterotrophic dinoflagellates (Fig. 4, d and
e). Erythropsidinium shows a large vacuole that occu-
pies the hyposome with a lateral hump and it is able
to engulf large prey items with a diameter greater
than its cell depth (Fig. 4, fk). The prey was spheri-
cal (~60 lm in diameter) with thick covering and a
brownish pigmentation. The prey contents were pro-
gressively consumed (Fig. 4, j and l), and residual
matter released through the posterior cavity in a
process that lasted for ten seconds (Fig. 4, mq;
Video S1). The product of the exocytosis was an
empty copepod egg with the envelope collapsed or
pierced in some areas (Fig. 4r).
Cell and ocelloid division. The division of the ocel-
loid began before the cytokinesis, and the ocelloid
remained incomplete even after the cytokinesis and
separation of the two daughter cells. The mecha-
nism of division is similar among warnowiid genera
(Fig. 5). A difference is that Nematodinium and War-
nowia divide inside a hyaline capsule (Fig. 5, a and
b) that is absent in Erythropsidinium (Fig. 5, cv). Ery-
thropsidinium develops a more elongated shape and
the ocelloid that usually protruded from the cell
surface, begins to migrate to the inner cell (Fig. 5,
635
cf). The hyalosome and melanosome divide inde-
pendently as two dissociated elements. The concen-
tric lenses of the hyalosome decompose and formed
810 globular hyaline masses (Fig. 5, e and f). At
this step, cells with an elongated shape and several
hyaline globules in an apical position corresponded
to the morphology described as the genus Greueto-
dinium A. R. Loeblich (Fig. 5g). The melanosome
loses the chevron-like shape and develops an elon-
gated ellipsoidal shape (Fig. 5, c and d). The hya-
line globules formed a lobulated sac formed of two
lots of about six overlapping globules (Fig. 5, h and
i). The nucleus migrates to the middle of the cell
and develops an elongated shape (Fig. 5, j and k).
The cell loses the red coloration in some parts
and the pigments concentrated into granules
(Fig. 5, hk). Five hours later, the cell changes to a
globular shape. The cingulum encircles the cell in a
single turn and in a more posterior position than
the non-dividing cell (Fig. 5, l and m). At this stage,
only the piston and the red coloration show that
the cell corresponds to Erythropsidinium (Fig. 5m).
The melanosome divided into two dark masses, and
the hyalosome is hardly visible. One hour later,
cytokinesis begins with a transversal constriction at
the middle of the cell body (Fig. 5n). Each daugh-
ter cell received a melanosome mass. The hyalo-
some is located between the two daughter cells that
still remained joined (Fig. 5, oq). The sulcus is
already visible in both daughter cells (Fig. 5r). After
cytokinesis, the daughter cells split (Fig. 5s), while
in other cases they remained joined for a longer
period with the apex of the posterior daughter cell,
attached to the basis of the posterior cavity of the
anterior daughter cell (Fig. 5t). The melanosome
and hyalosome began to migrate towards the apex,
and they will assemble to form a new ocelloid in
each daughter cell (Fig. 5, tv).
The posterior daughter cell keeps the piston from
the parent cell (Fig. 5, o and p), while the anterior
daughter cell must form a new bulb and piston.
The bulb is small, hyaline and internal, and it was
difficult to track its formation in the anterior daugh-
ter cell based on light microscopy. The new piston
in the anterior daughter cell appeared before the
cell split and the assembly of the new ocelloids
(Fig. 5s).
DISCUSSION
Speciation and synonymy in Erythropsidinium. The
unarmored dinoflagellates tend to show a high mor-
phological plasticity, and the cells often deformed
during observations. This is important to recognize
because the species richness of the warnowiid
dinoflagellates is likely overestimated; most species
have been described from a single or a few individu-
als, often moribund cells (Kofoid and Swezy 1921).
The genus Erythropsidinium (=Erythropsis) was
restricted to the type species E. agile, until Kofoid
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FIG. 4. Light micrographs of warnowiid dinoflagellates with food vacuoles. (a) Nematodinium. (b and c) Warnowia with antero-median
ocelloid. Note the brown vacuole. (d and e) Warnowia with posterior-median ocelloid and a vacuole near the melanosome. (fi) Erythropsi-
dinium scarlatinum with an ingested egg. (f) Note the longitudinal striae and deformation. (jq) Another individual during the absorption
and exocytosis of a crustacean egg. (lq) Sequential images of the exocytosis. (r) Empty egg after exocytosis. ci, cingulum; lf, longitudinal
flagellum; nu, nucleus; oc, ocelloid; st, surface striae; su, sulcus; va, vacuole; scale bars, 20 lm. [Color figure can be viewed at wileyonlineli-
brary.com]

and Swezy (1921) transferred into Erythropsis two
species originally described without the piston, Pou-
chetia cochlea F. Schutt and P. cornuta F. Schutt, and
E. agilis sensu Pavillard as E. pavillardii. Kofoid and
Swezy also described six new species and since then,
the number of species of Erythropsidinium has
remained unchanged. Kofoid and Swezy (1921)
divided the genus into the subgenus Erythropsis for
species with complete ocelloid [E. agile, E. cochlea
(F. Sch utt) P.C. Silva, E. cornutum (F. Sch utt) P.C.
Silva, E. pavillardii (Kofoid et Swezy) P. C. Silva,
E. extrudens and E. minus (Kofoid et Swezy) P. C.
Silva], and the subgenus Polyopsidella for species with
hyalosome divided in several globules [E. scarlat-
inum, E. hispidum (Kofoid et Swezy) P. C. Silva,
E. labrum (Kofoid et Swezy) P. C. Silva and
E. richardii (Kofoid et Swezy) P. C. Silva]. The obser-
vations reported here suggest that Kofoid and Swezy
(1921) likely erected a subgenus and new species
for life stages during cell division of erythropsisid
species. The species descriptions of the polyopsidel-
lid species were based on instable diagnostic
 O C E L L O I D A N D P I S T O N I N ER YT H R O P S I D I N I U M 637

FIG. 5. Light micrographs of warnowiid dinoflagellates during the cell division. (a and b) Warnowia with dissociated melanosome and
hyalosome. (b) Two daughter cells of Warnowia sp. (cv). Cells of Erythropsidinium spp. during the division. (c and d) Elongated cells at
the beginning of the cell division. Note the separation of the hyalosome into globules. (g) Greuetodinium with apical globular hyalosome
masses. (hr) Sequential images of a dividing cell. (h and i) Note the separation of the hyalosome and melanosome. (j and k) Note the
perinuclear envelope. (l and m) After five h, the cell acquired a globular shape. (n) After six h, the cell beginning of the cytokinesis. (o
q) Cell after seven h. (r) The two daughter cells after split. (sv) Other recently divided cells. ci, cingulum; hy, hyalosome; me, melano-
some; nu, nucleus; pi, piston; su, sulcus; scale bars, 20 lm. [Color figure can be viewed at wileyonlinelibrary.com]

characters such as the ocelloid morphology of divid-
ing cells, the cell coloration and the presence or
absence of an apical horn, or a distal stylet in the
piston. Greuet (1973) considered that all the warno-
wiid genera were probably monotypic. Elbrachter
(1979) proposed E. pavillardii, E. cornutum, and spe-
cies of Polyopsidella (E. hispidum, E. labrum, E. scarlat-
inum, E. richardii) as synonyms of E. agile. Kofoid
and Swezy (1921) illustrated E. extrudens with a later-
ally oriented piston with the same pigmentation of
the cell, and they even speculated on the cell
motility with that anomalous lateral propulsion.
Kofoid and Swezy (1921) also described E. scarlat-
inum with a short, slightly laterally oriented piston
that possesses pigmented granules. However, the
piston of Erythropsidinium is a hyaline structure and
always directed posteriorly, except in cells with
duplicated pistons (figs. 1315 in G
omez 2008).
Kofoid and Swezy (1921) possibly mistook the left
posterior body extension with the piston. Cells with
a posterior body extension have been reported from
the Pacific Ocean (fig. 16 in G
omez 2008), and in
638
F E R N A N D O G OM
the coasts of Brazil (Fig. 2, fk). These cells with a
body extension constitute a distinct species,
described as E. extrudens or E. scarlatinum. The co-
occurrence of these cells in the same sample, and
the intergradation in the position and shape of the
body extension suggested that they are morphotypes
of a single species. The original description of
E. scarlatinum fits better with the most common
morphotype.
Greuet (1968b) described Greuetodinium (as Leu-
copsis Greuet) for large cells with an elongated cylin-
drical body with an ocelloid dissociated into several
hyaline globules in an anterior position. Greueto-
dinium was not subsequently reported, with the
exception of Meave del Castillo et al. (2012). Later,
Greuet (1977) described the life stages during the
cell division of Erythropsidinium. He did not realize
that Greuetodinium is probably a life stage during the
division of Erythropsidinium (Fig. 5g). In conclusion,
Erythropsidinium (=Greuetodinium) is composed of, at
least, three species, E. minus and E. scarlatinum
(=E. extrudens) and the type species, E. agile with
many synonyms. Unfortunately, the speciation
cannot be tested by molecular phylogeny because
the sequences of Erythropsidinium are restricted to a
single isolate (G
omez et al. 2009).
Motility and function of the piston. Erythropsidinium
differs from other warnowiid dinoflagellates in the
piston, and position and the degree of complexity
of the ocelloid. The cingulum and the transversal
flagellum are reduced, located in a very anterior
position (Fig. 1, dj) and the longitudinal flagellum
is short (Fig. 2, j and m). In contrast, the cingulum
of other warnowiid dinoflagellates trace several
turns around the cell (Fig. 4, ae). This is com-
monly associated with higher flagellar propulsion in
dinoflagellates (Jeong et al. 1999). The develop-
ment of the piston may induce the atrophy of flagel-
lar propulsion. While other warnowiid
dinoflagellates are active swimmers, Erythropsidinium
usually remains motionless, with the piston inactive
or in the static mode (Fig. 3a), and only occasion-
ally swims in the locomotion mode (Fig. 3b).
TABLE 1. Swimming peak speed (mm
s1) of some dinoflagellates and ciliates.
Taxon Speed Reference
Erythropsidinium scarlatinum 4* This study
Erythropsidinium 50 Taylor (1987, p. 136)
Akashiwo sanguinea 0.28 Jeong et al. (1999)
Scrippsiella trochoidea 0.34 Jeong et al. (1999)
Cochlodinium polykrikoides 1.45 Jeong et al. (1999)
Cochlodinium polykrikoides 0.39 Sohn et al. (2011)
(single cell)
Cochlodinium polykrikoides 0.85 Sohn et al. (2011)
(8-cell chain)
Alexandrium affine 0.41 Fraga et al. (1989)
Gymnodinium catenatum 0.24 Fraga et al. (1989)
*Jump during the contraction.

EZ
Hausmann and H ulsmann (2010) reported micro-
graphs of the piston activity with a high-speed cam-
era. They commented that the contraction is about
three times faster than the extension, with no addi-
tional information. Taylor (1987, p. 136) reported:
Erythropsidinium travels extremely quickly and jerkly
in a straight line (5 cm
s1). Based on our obser-
vations this speed of 50 mm
s1 should be
reduced for, at least, one order of magnitude. The
piston attained up to 50 mm
s1 during the con-
traction phase. However, only a small fraction of the
piston movement is transmitted for the cell locomo-
tion. Our observations showed that during the con-
traction the cell jumped with a peak speed of
4 mm
s1 (Figs. 3b, S7 and S8). However, in the
balance between a phase of extension and retrac-
tion the cell swims at ~1 mm
s1. This is due to
the peculiar propulsion mechanism, with several for-
wards steps during the piston extension, and a
quick backwards jump during the contraction
(Figs. 3b, S7 and S8).
Table 1 summarizes the swimming speed of some
dinoflagellates and ciliates. The most typical dinoflag-
ellates swim in helicoidal trajectories propelled by the
flagella. Dinoflagellates such as Akashiwo Gert Hansen
et Moestrup, Prorocentrum Ehrenberg, Scrippsiella
Balech or Lingulodinium D. Wall do not attained
speeds higher than 0.4 mm
s1 (Jeong et al. 1999).
Other red-tide colony-forming species do not
attained speeds higher than 0.85 mm
s1 (Table 1).
During the locomotion mode, Erythropsidinium is
faster than most of the dinoflagellates. It is should be
noted that Erythropsidinium swims occasionally, while
the red-tide dinoflagellates are active swimmers for
longer periods.
The cilia-driven propulsion of the ciliates is faster
than the bi-flagellated dinoflagellates (Table 1).
There is a group of ciliates (Tontonia Faur
e-Fremiet,
Pseudotontonia Agatha) that possesses an appendix
or tail, capable of fast contraction reminiscent of
the piston. Greuet (1967) and Greuet et al. (1986)
investigated ultrastructure of the piston and the
contractile appendix of Tontonia, respectively.
Taxon Speed Reference
Gymnodinium catenatum (4-cell chain) 0.35 Lee et al. (2015)
Polykrikos hartmannii (2 zooids) 0.63 Lee et al. (2015)
Protoperidinium bipes 8.2 Jeong et al. (2004)
Myrionecta rubra 8 Crawford and
Lindholm (1997)
Mesodinium pulex 2.1 Jakobsen (2001)
Strobilidium sp. 5.2 Jakobsen (2001)
Strobilidium sp. 27 Gemmell et al. (2015)
Tontonia sp. 88 Gemmell et al. (2015)
Pseudotontonia sp. 55 Gemmell et al. (2015)
 O C E L L O I D A N D P I S T O N I N ER YT H R O P S I D I N I U M
Greuet et al. (1986) concluded that organization
structure of both organelles was similar. It is uncer-
tain whether the dinoflagellate piston and ciliate tail
have a common evolutionary origin, or there is only
a morphological convergence. The piston may also
derive from the posterior or lateral appendices
observed in other warnowiid dinoflagellates (Fig. 2,
ae). The ciliate Tontonia sp. possesses a tail of
310 lm that contracts up to 225 mm
s1 and the
cell jumps up to 88 mm
s1 (Gemmell et al.
2015). It should be noted that the quick jump of
the ciliates is restricted to a few ms, enough time to
escape from the aspiration current of the predator.
In contrast, the occasional swimming during the lo-
comotion mode of Erythropsidinium may last for
tens of seconds.
Predation and the piston. Greuet (1967) considered
that the piston is exclusively used for locomotion.
However, the piston shows a distal swelling or suc-
tion cup that resembles the tentacular clubs of the
squid, and sometimes including a distal stylet
(Kofoid and Swezy 1921, G
omez 2008, figs. 12, 17).
The suction cup is able to change its shape and
attach to objects or tentatively a prey (Figs. 2q, S3;
Video S1). The morphological adaptation of the pis-
ton does not seem to be directly related to the loco-
motion. During most of the time the piston moves
in the static mode, without a net cell swimming
(Fig. 3a). These features suggest that locomotion is
not the exclusive function of the piston.
This study documents an intracellular digestion
by Erythropsidinium. It is able to engulf a large prey,
of a diameter larger than feeding cell (Fig. 4, fk).
Important organelles as the nucleus and the ocel-
loid are placed in an anterior position, leaving space
for the large vacuole. The cylindrical cell shape and
the posterior cavity also facilitates phagocytosis of
large prey. The members of the Gymnodinium clade,
which Erythropsidinium belongs (G
omez et al. 2009),
are mostly constituted of species with a smooth cell
surface. In contrast, Erythropsidinium possesses a cell
surface with longitudinal striae (Fig. 4f) and a per-
inuclear envelope (Fig. 2, f and g) that also
occurred in unarmored dinoflagellates that experi-
ence considerable deformation after engulfing large
prey [i.e., Gyrodinium spirale (Bergh) Kofoid et
Swezy]. The longitudinal striae of Erythropsidinium
may help to control cell deformation (Fig. 4f). All
these features are adaptations to engulf a large prey
in an internal vacuole. Consequently, it is highly
improbable that Erythropsidinium will have a dupli-
cated feeding mechanism and developed additional
structures for an extracellular digestion.
Based on TEM observations, Greuet (1969)
described a harpoon-like organelle, the stomopod,
that he supposed to protrude from the cell and
injected lysing substances in the prey for an extra-
cellular digestion. Greuet never observed the use of
the stomopod, vacuoles or the engulfing or exocyto-
sis mechanisms, and then he assumed an
639
extracellular digestion in Erythropsidinium. Greuet
was perhaps influenced by the contemporaneous lit-
erature and co-workers that showed dinoflagellates
with peduncles to suck the prey contents (Cachon
and Cachon 1968). In this mechanism of extracellu-
lar nutrition known as myzocytosis, the prey is
pierced by means of an extensible, tube-like pedun-
cle and the contents are sucked out, including
both dissolved and particulate organic substances
(Schnepf and Deichgraber 1984). A thick envelope
protects the crustacean egg, and after engulfing the
harpoon-like stomopod may be an internal orga-
nelle used to perforate the prey cell covering and to
inject the lysing substances.
This study reveals that the post-digestion residuals
of prey items are released from the antapex (Fig. 4l;
Video S1). The exocytosis does not take place from
the intercingular region or the sulcus. It is difficult
to assume that Erythropsidinium would develop inde-
pendent structures for ingestion and egestion. Con-
sequently, it can be assumed that the prey was
ingested though the posterior cavity where the pis-
ton retracts (Fig. 2o). The observation revealed that
the piston is able to attach to objects using the dis-
tal suction cup (Fig. 2q). During the retraction, a
prey attached or pierced by the distal end of the pis-
ton will be placed into the posterior cavity for
engulfing. In the animal world, the suction cup of
the piston may act as the chameleon tongue used to
capture insects. Despite the lack of direct observa-
tions, this appears as the most probable mechanism
for prey capture (Video S1).
The piston activity showed two modes: The loco-
motion mode is occasional, while the piston moved
usually in the static mode without cell swimming,
except for some cell rotation (Fig. 3a). Conse-
quently, during most of the time the function of pis-
ton is not locomotion. When a piston is extended
500700 lm in a straight line, it contacts with
objects located in a sphere of a diameter of 1,000
1,400 lm around the cell. This is equivalent to a
radar-like beam that scans the surrounding waters.
Consequently, the piston may act as a tactile orga-
nelle or mechano-receptor for prey search. In fact, I
hypothesize that the static mode is a prey search
mode.
Function of the ocelloid. If the earlier protozoolo-
gists disbelief of the existence of Erythropsidinium,
further authors have speculated on the function of
the ocelloid (Colley and Nilsson 2016). Omodeo
(1980, p. 150) reported Nematodinium fires its bat-
teries only when the target is at the right direction
and distance. . .. It is interesting to note that Ery-
thropsidinium does not catch the prey with the
nematocysts as Nematodinium does but by means of
a wounding appendix, the stomopod, which
injects lysing substances into the prey. Polykrikos
shoots nematocysts to narcotize and immobilize the
fast-swimming prey before engulfing it in an intra-
cellular digestion (Matsuoka et al. 2000, Lee et al.
640
F E R N A N D O G OM
2015). The nematocyst probably does not contain
lysing substances, which will be injected after prey
engulfment. It makes little sense to inject lysing
substances if later the prey will be intracellularly
consumed.
This study reveals that Erythropsidinium can feed
on a copepod egg and it is consumed in an inter-
nal vacuole (Fig. 4, fk). A copepod egg is a high-
quality food item for a cell with the characteristics
of Erythropsidinium. Eggs are rich in lipids, useful
to maintain the high energy costs of the piston
activity, and rich in carotenoids (astaxanthin and
xanthophyll esters) that are precursors of the mel-
anosome components. An egg is a non-motile prey
and Erythropsidinium does not have the ejectile
bodies that other warnowiid dinoflagellates possess
to capture swimming dinoflagellates (Fig. 4, ae).
A crustacean egg is an optimal prey for Erythropsi-
dinium, as well as for other dinoflagellates and
many other predators (Cachon and Cachon 1968,
G
omez and Skovgaard 2015). Consequently, the
problem for the consumer is to find the egg. The
prey is captured and ingested from the posterior
end of the cell, and the ocelloid is directed in a
diametrically opposed direction. In the animal
world, this is like having a lion with the eyes in
the tail. The use then of the ocelloid for a visually
guided predation is questionable. The ocelloid
cannot project images because it lacks a conver-
gent lens, and even in that case, a unicellular
organism cannot interpret images. The high devel-
oped lenses are able to concentrate the light and
increase the sensitivity of the retinoid body (Fig. 1,
fr). Light is a key factor in the vertical (depth)
distribution of plankton organisms and copepod
spawning occurred at discrete depths (Melle and
Skjoldal 1989). The ocelloid, an eyespot with
highly developed lenses, may be used for photo-
taxis in order to select a habitat (i.e., optimal
depth for prey availability). Although the ocelloid
reaches the highest size and complexity in Erythrop-
sidinium, its function is similar than in other war-
nowiid dinoflagellates. The piston is a unique and
key organelle that has influenced on the morphol-
ogy and behavior of Erythropsidinium. Experiments
are limited by the availability of cells under labora-
tory conditions. This study reveals that Erythropsi-
dinium feeds on eggs (~6070 lm in diameter) of
broadcast spawning copepods. This information is
essential to establish cultures and further experi-
ments to test the exceptional evolutionary adapta-
tions of Erythropsidinium.
F.G. was supported by the contract JCI-2010-08492 of the
Ministerio Espa~ nol de Ciencia y Tecnolog
a and the Brazilian
Conselho Nacional de Desenvolvimento Cient
fico e Tec-
nol
ogico (grant no. BJT 370646/2013-14). This is a contribu-
tion to the ANR Biodiversity program (ANR BDIV 07 00402
Aquaparadox). I thank J.R. Dolan for the editing the Eng-
lish, R.M. Lopes for the loan of the high-speed camera and
J.R. Strickler for the advices in use.

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Supporting Information
Additional Supporting Information may be
found in the online version of this article at the
publishers web site:
641
Figure S1. Line drawings of Erythropsidinium. ag,
apical groove; be, body extension; ci, cingulum;
hy, hyalosome; lf, longitudinal flagellum; me, mel-
anosome; pi, piston; su, sulcus; tf, transversal flag-
ellum.
Figure S2. Light micrographs of Erythropsi-
dinium spp. in different views and focus levels.
Figure S3. Time-lapse sequence of Erythropsi-
dinium with the distal end of the piston adhered
to the bottom glass. Recording at 30 frames
s1
(fps). Frame number below the piston. The lat-
eral size scale corresponds to the stage microme-
ter with divisions each 10 lm.
Figure S4. Time-lapse sequence of the piston
retraction of Erythropsidinium during the static
mode recorded at 1,500 frames
s1 (fps).
Frame number in the upper left hand corner.
The lateral size scale corresponds to the stage
micrometer with divisions each 10 lm.
Figure S5. Time-lapse sequence of the piston
activity of Erythropsidinium during the static
mode recorded at 1,500 frames
s1 (fps).
Frame number in the upper left hand corner.
The lateral size scale corresponds to the stage
micrometer with divisions each 10 lm.
Figure S6. Time-lapse sequence of the piston
activity of Erythropsidinium during the static
mode recorded at 1,500 frames
s1 (fps).
Frame number in the upper left hand corner.
The lateral size scale corresponds to the stage
micrometer with divisions each 10 lm.
Figure S7. Time-lapse sequence of the piston
activity of Erythropsidinium during the locomotion
mode recorded at 60 frames
s1 (fps). Frame
number in the upper left hand corner. The lat-
eral size scale corresponds to the stage microme-
ter with divisions each 10 lm.
Figure S8. Time-lapse sequence of the piston
activity of Erythropsidinium during the locomotion
mode recorded at 30 frames
s1 (fps). Frame
number below the upper border of each micro-
graph. The lateral size scale corresponds to the
stage micrometer with divisions each 10 lm.
Appendix S1. Sampling and observation meth-
ods.
Video S1. Erythropsidinium and other warnowiid
dinoflagellates. Available at: https://youtu.be/ki
h8m3x-0eo.