Protist 173 (2022) 125854

http://www.elsevier.de/protis
Published online date 17 December 2021 Protist
PROTIST Reviews

The Vampyrellid Amoebae (Vampyrellida,

Rhizaria)1
Sebastian Hess 2, and Andreas Suthaus

Institute for Zoology, University of Cologne, Zülpicher Str. 47b, D-50674 Cologne, Germany
Submitted September 30, 2021; Accepted December 13, 2021
Monitoring Editor: Alastair Simpson

The Vampyrellida (Endomyxa, Rhizaria) is a group of free-living, predatory amoebae, which is most
closely related to the Phytomyxea (plasmodiophorids and phagomyxids). It encompasses about 50
credibly described species that have a characteristic life history with the regular alternation of trophic
amoebae and immobile digestive cysts. All known vampyrellid amoebae are naked and filose, but the
different species display a broad morphological variety. Vampyrellids also vary greatly in their feeding
habits, and range from generalist predators to specialized ‘protoplast feeders’ that exclusively feed on
the cell contents of eukaryotic prey. They can be found in freshwater, soil and marine habitats, and
appear to be globally distributed. Yet, the phenotypic diversity and ecological roles of the Vampyrel-
lida are still poorly explored. Currently, there are eight well-recognized subclades that comprise four
families (Vampyrellidae, Leptophryidae, Placopodidae and Sericomyxidae) as well as some lineages
without any phenotypic information. Research on vampyrellids is challenging due to their cryptic
occurrence in nature, intricate feeding habits that complicate cultivation, and a convoluted taxonomic
history. Here, we review available information about cell structure, diversity, ecology, taxonomy and
phylogenetics, and provide an up-to-date introduction to the Vampyrellida that may facilitate future
research.
Ó 2021 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Key words: Cercozoa; rhizopod; protoplast feeder; vampire amoeba.

Contents

Introduction and Historical Sketch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Cellular Stages and Life History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Morphology and Cellular Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Trophic Specialization and Feeding Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Free Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1
This review is one in a series on the biology of selected protist groups (see https://doi.org/10.1016/j.protis.2021.125818)
2
Corresponding author;
e-mail sebastian.hess@uni-koeln.de (S. Hess).

https://doi.org/10.1016/j.protis.2021.125854
1434-4610/Ó 2021 The Authors. Published by Elsevier GmbH.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2 S. Hess, A. Suthaus

Colony Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Protoplast Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Prey Infiltration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cell Wall Penetration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Distribution and Ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Effects on Algal Blooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Vampyrellids as Prey and Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Systematics and Evolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Vampyrellids in the Lab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Challenges and Benefits of Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Detection, Enrichment and Isolation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Molecular Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
State of Experimental Research and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Appendix A. Supplementary Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Introduction and Historical Sketch
The vampyrellids (Order Vampyrellida) represent
one of the major groups of free-living amoebae.
They are phylogenetically distinct from the Amoebo-
zoa, Heterolobosea and Nucleariidae, and, instead,
form an isolated clade within the Rhizaria (Bass
et al. 2009). Most likely, they are the closest, pheno-
typically characterized relatives of the phytomyxid
parasites of plants and algae (e.g. Plasmodiophora;
Bass et al. 2009; Sierra et al. 2016), yet clearly dis-
tinct from them in terms of cellular structure, life his-
tory and nutrition. All known vampyrellids, except
Lateromyxa gallica (Hülsmann 1993), display a
free-living life style and – as far as we can judge –
lack flagellate stages. In contrast, the phytomyxids
(which include the plant pathogenic plasmodio-
phorids and the phagomyxids) spend most of their
active life inside host cells as trophic plasmodia,
and disperse by producing flagellate swarmers
and spores (Hittorf et al. 2020; Schwelm et al.
2018). Although both vampyrellids and phytomyxids
thrive on other eukaryotes, they apply different
strategies to overcome the prey/host cell wall (cell
wall dissolution vs. penetration by adhesorium and
‘stachel apparatus’) and to take up food (phagotro-
phy vs. osmotrophy) (Aist and Williams 1971;
Maier et al. 2000; Schwelm et al. 2018).
The captivating colloquial name ‘vampire amoe-
bae’ originates from the most popular genus of
these amoebae, Vampyrella CIENKOWSKI, 1865. The
meaning of ‘Vampyrella’ (Latin for ‘little vampire’)
points to the fascinating feeding habit of several
vampyrellid amoebae, which pierce the cell walls
of other eukaryotes and feed specifically on their cell
contents (Hoogenraad 1907; Hoogenraad and de
Groot 1942; Hülsmann 1985; Lloyd 1926; More
et al. 2019). However, these ‘protoplast feeders’
only represent a fraction of the known vampyrellid
species, and the true diversity of the vampyrellids
in terms of morphology, feeding strategies and ecol-
ogy is much larger. As detailed in this review,
vampyrellids display at least four distinct feeding
strategies to either engulf entire prey organisms or
to devour the cell contents of other eukaryotes.
Thus, many vampyrellids have adopted strategies
to handle relatively large or otherwise difficult-to-
consume prey, which is an exceptional evolutionary
path for a lineage of this size. Vampyrellids occur in
marine, brackish and freshwater habitats, and have
been frequently isolated from soil samples (e.g.
Anderson and Patrick 1980; Berney et al. 2013;
Hess et al. 2012). In these ecosystems, they prey
on organisms of diverse phylogenetic affinity, struc-
ture and size (diverse algae, protists, fungi, and
micrometazoa), and presumably act as widespread
microbial predators and parasitoids. Despite the
trophic diversification found in the vampyrellids, all
known species share a common life history feature:
the immobile, but highly active digestive cyst stage
that is formed after food uptake.
The vampyrellids have a long history of research,
with some noteworthy episodes. The description of
Amoeba lateritia by Fresenius in the mid-19th cen-

tury is probably one of the earliest unambiguous
reports of a vampyrellid, yet without any details on
its life history (Fresenius 1856). The first compre-
hensive documentation of the life history and the
feeding behaviour of algivorous freshwater
vampyrellids was provided by Cienkowski (1865).
In his often-cited work, he created the genus Vam-
pyrella with three species (V. spirogyrae, V. pen-
dula, V. vorax) and classified them in a subgroup
of his ‘monads’, a polyphyletic assemblage of pro-
tists united by parasitoid tendencies and superficial
similarities of their life histories. This work appears
to be a starting point of the ‘accidental’ vampyrellid
discoveries in the 19th and early 20th century –
based on observations of natural samples. Several
scientists, including Cienkowski, Dangeard,
Haeckel, Hertwig and Lesser, Klein, Penard, Soro-
kin, Zopf, and others described numerous species
(listed in Supplementary Material Table S1). The
monographs by Zopf deserve special attention, as
they contain numerous valuable observations con-
cerning the behaviour and ecology of vampyrellid
amoebae, the proof of nuclei in vampyrellids, and
the erection of the family Vampyrellidae ZOPF, 1885
(Zopf 1885a,b). One hundred years of research
after Cienkowski’s publication resulted in about 40
descriptions of vampyrellid species, all based
entirely on natural material and of varying quality –
several species remain ambiguous today. In the
mid of the 20th century the first vampyrellid labora-
tory culture was established (Weber et al. 1952).
Interestingly, this amoeba, Theratromyxa weberi,
was isolated from soil and fed on nematodes,
thereby deviating from the previously described
aquatic species of vampyrellids. The culture was
used for a detailed description of the species, includ-
ing its feeding habits and life history, but as in many
other cases has not survived until today. The discov-
ery of this soil-dwelling, ‘carnivorous’ amoeba was
followed by further studies on similar amoebae iso-
lated from soil, and by experiments testing the
potential of such amoebae for biological pest control
directed against phytopathogenic nematodes
(Sayre 1973; Van der Laan 1954; Winslow and
Williams 1957). Independent of this, soil scientists
documented peculiar perforations and annular sur-
face depressions in fungal spores that have been
incubated in natural soil (Old 1969; Old and
Robertson 1969). The ‘spore perforating agent’
was finally identified as another giant soil vampyrel-
lid (Old 1977; Old and Darbyshire 1978), and subse-
quent studies revealed the diversity of
The Vampyrellid Amoebae (Vampyrellida) 3
‘mycophagous’ vampyrellids (on the basis of perfo-
ration size), their wide geographic distribution, and
some ultrastructural details of the perforation pro-
cess (e.g. Anderson and Patrick 1978,1980;
Homma et al. 1979; Homma and Ishii 1984; Old
1978). In the early 1980s, Hülsmann started some
culture-based research on algivorous freshwater
vampyrellids and published movies of the feeding
process and life history of Vampyrella lateritia
(Hülsmann 1983, 1985). These films and the sup-
plementary publications (in German) still represent
an unsurpassed analysis of Vampyrella’s feeding
process. Later Hülsmann and colleagues described
and studied the parasitoid freshwater vampyrellid
Lateromyxa gallica, and compared the ultrastructure
of vampyrellid nuclei in various mitotic stages to
resolve evolutionary relationships (Hülsmann
1993; Röpstorf et al. 1993, 1994). The latter
approach did not work out, but still yielded valuable
structural information about the cultivated strains.
More or less in parallel, Grell discovered large, plas-
modial amoebae in marine waters from Australia,
Jamaica and Tenerife, and established the genus
Thalassomyxa (Grell 1985, 1992, 1994). These
predatory amoebae fed on diatoms and flagellate
algae, and displayed a remarkable rhythmicity in
the cultures when switching between ‘motile’ and
‘immotile’ phases. The connection of this genus to
(other) vampyrellid amoebae was, however, not
established at that time. In fact, the taxonomic com-
position and placement of vampyrellids have always
been difficult in the pre-genetic era, and there exists
a long and convoluted taxonomic history. Vampyrel-
lid species have been regarded as relatives of myx-
omycete slime moulds (Cienkowski 1865; Zopf
1885a), Haeckel’s monera (Haeckel 1870), heliozoa
(Loeblich and Loeblich 1965; Penard 1904), pro-
teomyxids (Honigberg et al. 1964; Rolleston and
Jackson 1888), and filose rhizopods (Leidy 1879;
Page 1987; Page and Siemensma 1991; West
1901). The mystery about their evolutionary origin
was finally resolved by phylogenies of small subunit
ribosomal RNA (SSU rRNA, also known as 18S)
genes, which were able to place Vampyrellida within
Rhizaria (Bass et al. 2009; Hess et al. 2012). Bass
et al. (2009) demonstrated the position of a soil-
dwelling mycophagous vampyrellid (Platyreta ger-
manica) in a distinct clade of the Endomyxa, Rhi-
zaria. This was followed by the phylogenetic
placement of the three freshwater vampyrellids ini-
tially described by Cienkowski in the same clade,
and a revised vampyrellid taxonomy, including the
4 S. Hess, A. Suthaus

Figure 1. Life history stages, feeding cycle and cell division of vampyrellid amoebae. A Schematic feeding
cycle (left) illustrating the alternation of motile trophozoites and digestive cysts. Karyokinesis (K!) occurs in the
late digestive cyst stage. Under unfavorable conditions, vampyrellids can form inactive stages (right) such as
hypnocysts, secondary cysts and true resting cysts (=spores); B Scheme of ‘internal plasmotomy’ (P!) right
after karyokinesis (K!); C Scheme of ‘external plasmotomy’ that happens in free trophozoites after or during
leaving the digestive cyst. Arrows in B and C indicate sequence of events.

reconstitution of the order Vampyrellida WEST, 1901
(Hess et al. 2012). Since then, good progress was
made in further exploring the phylogenetic and phe-
notypic diversity of vampyrellids. Berney et al.,
detected an unexpected vampyrellid diversity in
marine habitats, localised Thalassomyxa-like amoe-
bae in the vampyrellid tree, and established the
existence of eight major genetic subclades of
vampyrellids (Berney et al. 2013). Further studies
gradually explored some of these subclades, and
today there are five vampyrellid clades with pheno-
typic data, four of which are formally described as
families (Hess 2017a; More et al. 2019, 2021).
Yet, most of the vampyrellid diversity is still unknown

or incompletely described, and the overall picture of
the order Vampyrellida still has to emerge. Only 12
out of > 40 credibly described species have been
placed on the vampyrellid tree by molecular phylo-
genetic approaches, while at least three family level
subclades lack any phenotypic data.
The most significant hurdles in exploring and
studying the vampyrellid amoebae are their cryptic
occurrence in nature, the complications of cultiva-
tion given their intricate feeding habits, and a convo-
luted taxonomic history. Apart from a few
monographs from the pre-genetic era (Gobi 1915;
Gromov 1976; Zopf 1885a,b), most information
about vampyrellid amoebae is widely scattered
and there is currently no work drawing a compre-
hensive, phylogenetically-informed picture of
vampyrellid diversity and biology. This review is a
first attempt to compile relevant information, and to
provide an up-to-date introduction to the Vampyrel-
lida that may facilitate future research about these
intriguing protists.
Cellular Stages and Life History
The central trait of the vampyrellid life history is a
‘feeding cycle’ characterised by the obligatory and
regular alternation of mobile and immobile cellular
stages (Fig. 1A) (e.g. Anderson and Patrick 1980;
Grell 1985; Hülsmann 1985; Zopf 1885a). The
mobile, amoeboid cells are here termed ‘tropho-
zoites’ as their main activity is to search, acquire,
and incorporate food. In the more classical litera-
ture, the trophozoites were referred to as ‘swarmers’
(Hülsmann 1985; Klein 1882) or ‘motile stages’
(Grell 1985, 1992, 1994), emphasizing their role in
dispersal. However, the terms ‘amoebae’ or ‘tropho-
zoites’ appear to be more appropriate, as the term
‘swarmer’ is often specifically used for ciliated,
swimming cells, sometimes even for gametes
(Röttger 2001). The vampyrellid trophozoites can
show several strategies to handle prey, but all
known species incorporate particulate matter by
phagocytosis (see ‘Trophic Specialization and
Feeding Strategies’). After one or more feeding
events the trophozoite transforms into a ‘digestive
cyst’, a walled cellular stage of high metabolic activ-
ity (Fig. 1A). In some species, this stage was
referred to as ‘resting phase’ (Grell 1985, 1992,
1994), but it has to be emphasized that the digestive
cyst of vampyrellids does not correspond to a true
‘resting cyst’, i.e. a metabolically reduced stage to
survive adverse conditions. During digestive cyst
formation, the trophozoite of most species retracts
The Vampyrellid Amoebae (Vampyrellida) 5
the filopodia, and secretes a cell wall comprised of
one or two discernible layers (see ‘Morphology
and Cellular Organization’). In most known species,
the digestive cysts are firmly attached to other
objects or to the substrate (e.g. detritus, algal fila-
ments, the bottom of a Petri dish), but there are also
reports of free-floating cyst stages (Asterocaelum
spp.; Canter 1973; Kobayashi et al. 2018) and cyst
formation within prey cells (Lateromyxa gallica;
Hülsmann 1993). In the digestive cyst stage, the
digestion of incorporated food takes place, which
can involve the formation of a central main vacuole
(e.g. in Leptophrys vorax, Thalassomyxa spp.).
This, however, appears to be a species-specific
trait, as other species (e.g. Vampyrella lateritia, Pla-
copus flabellus) retain separate food vacuoles dur-
ing the entire digestive phase (e.g. Hess et al.
2012; Hess 2017a; Hülsmann 1985). Besides the
visual change of the food itself, the digestive phase
may be accompanied by a marked colour change of
the vampyrellid cytoplasm. Some species develop a
bright red, orange or yellowish colour (e.g. Arachno-
myxa, Lateromyxa, Placopus, Planctomyxa, Vam-
pyrella; Hess 2017a, b; Hülsmann 1985, 1993;
More et al. 2019), while others remain colourless
(e.g. Platyreta, Sericomyxa, Thalassomyxa; Bass
et al. 2009; Grell 1985; More et al. 2021; Pakzad
2003). Hence, the digestive cyst colour may be a
good indicator for the stages of digestive cyst matu-
ration. When the digestion is completed, one or sev-
eral trophozoites hatch out of the digestive cyst
through circular holes created by local cell wall dis-
solution (Fig. 1A). This process is typically associ-
ated with the exocytosis of food remnants, which
are either expelled as numerous brown particles
during and shortly after the hatching process (e.g.
Vampyrella lateritia; Hülsmann 1985), or are left
behind within the cyst (e.g. Leptophrys vorax; Zopf
1885b).
In some vampyrellids, the digestive cyst stage is
tightly linked to asexual reproduction. Species of the
genera Vampyrella, Placopus and Sericomyxa, for
example, divide within the late cyst stage when
digestion is completed – here termed ‘internal plas-
motomy’ (Hess 2017a; Hess et al. 2012; Hülsmann
1985; More et al. 2019, 2021). This generally results
in 2–4 daughter cells (sometimes more), which dis-
play a characteristic cruciate or tripartite pattern
(Fig. 1B). These daughter cells are then released
from the cyst as ‘young trophozoites’, often through
separate holes. Other species, e.g. some lep-
tophryids and Thalassomyxa spp. do not divide
6 S. Hess, A. Suthaus
within the closed cyst, but show ‘external plasmo-
tomy’ during or after the hatching process (Bass
et al. 2009; Grell 1985; Röpstorf et al. 1994; Zopf
1885b). Interestingly, these species still may pro-
duce more than one hole into the cyst wall. Different
‘domains’ of the trophozoite then leave the cyst at
different sites and may stretch the cell until the
domains split into two or more individuals
(Fig. 1C). A rather unusual mode of plasmotomy is
found in the infiltrating vampyrellid Lateromyxa gal-
lica (Hülsmann 1993). During feeding on algal cell
contents, the plasmodia of this species shed well-
fed cell portions that develop into digestive cysts.
However, ultrastructural data suggest that the dupli-
cation of genetic material (karyokinesis) typically
occurs in the late digestive cyst stage, irrespective
of the type of plasmotomy (see ‘Morphology and
Cellular Organization’ for details on karyokinesis).
Indeed, many vampyrellid species contain multi-
ple nuclei and behave like plasmodial organisms.
They can easily fuse upon cell-to-cell contact, or
split when two domains of the cell move into oppo-
site directions (e.g. Bass et al. 2009; Grell 1985;
Hess et al. 2012; Hülsmann 1985). This is why
vampyrellid cells of a given population can vary
greatly in size; even very small cell fragments of a
few micrometers can survive and continue with the
typical life history, provided they have the size nec-
essary for food uptake. However, the ability to fuse
and split, and the extent of plasmodia formation, var-
ies among vampyrellid taxa. Species of Leptophrys,
Platyreta, Sericomyxa, Thalassomyxa, and Thera-
tromyxa, for example, readily form plasmodia,
sometimes of virtually unlimited size, e.g. covering
large areas of a Petri dish (Bass et al. 2009; Grell
1985; Hess et al. 2012; More et al. 2021). The ‘fu-
sion plasmodia’ of other species (e.g. Arachnomyxa
cryptophaga, Planctomyxa polycarya, Vampyrella
lateritia) mainly occur in well-grown late stage cul-
tures, when the cell density is high and little food
available (Hess 2017b; Hülsmann 1985). Thus, it
is somewhat uncertain to what extent these species
would form plasmodia under natural conditions.
Fusion plasmodia are least common among the
known Placopus species. The trophozoites of P. fla-
bellus, P. melkoniani, P. pusillus, and P. rubicundus
tend to remain as uni- or binucleate individuals
(Hess 2017a; More et al. 2019; Röpstorf et al. 1994).
Under adverse conditions, vampyrellids can form
several types of resting stages, hypnocysts, sec-
ondary cysts and true resting cysts (Fig. 1A). Similar
to digestive cysts, the hypnocysts and secondary
cysts are relatively thin-walled, yet devoid of any
food content. Hypnocysts can be formed when the
trophozoite is disturbed by physical or chemical
cues, e.g. during cell isolation with a micropipette
(Hülsmann 1985; unpubl. observ. S.H.), while the
secondary cysts are a result of starvation, i.e. the
cell shrinks and produces several cyst walls
(Pakzad 2003). The true resting cysts (also referred
to as ‘sporocyst’ or ‘spore’ in the older literature) can
be found in natural samples or old cultures, when
food is depleted or conditions rendered otherwise
unfavorable (Canter 1973; Hülsmann 1985;
Röpstorf et al. 1993; Zopf 1885a, b). Resting cysts
bear several cyst walls and condensed contents.
As shown for Vampyrella lateritia, they can survive
up to at least three years, including desiccation
and freezing events (Hülsmann 1985). The develop-
ment of trophic Vampyrella cells could be induced
by the addition of fresh medium and prey organisms
but – to the knowledge of the authors – details about
this process have not been published. While the
highly active digestive cyst is an obligatory and
recurring phase of every growing vampyrellid, the
resting cyst has only been reported for a fraction
of species, and not every laboratory strain
can be induced to form resting cysts. It seems that
some vampyrellids lose the ability to form resting
cysts during longer times of cultivation (Canter
1973).
The obligatory digestive cyst stage and the non-
uniform cellular morphology caused by their propen-
sity to fuse and split are both characteristic of
vampyrellids. The only example known to the
authors that shows a close resemblance in this
respect is the aplanospore-forming, heterokont
amoeba Chlamydomyxa labyrinthuloides, which,
indeed, was erroneously assigned to the vampyrel-
lids in former times (Valkanov 1940; Wenderoth
et al. 1999). When suitable food is abundant, most
of the vampyrellid cells are typically found in the
digestive cyst stage, as the digestion takes much
longer than the feeding process (e.g. 1–2 days vs.
several minutes in Vampyrella lateritia; Hülsmann
1985). This also distinguishes vampyrellids from
almost all other major groups of amoebae, which
grow and multiply in the motile, amoeboid stage,
and whose immobile stages are typically associated
with reduced activity (Page and Siemensma 1991).
There is very little evidence for sexuality in
vampyrellids. An ultrastructural study revealed
some meiotic stages in forming resting cysts of
Lateromyxa gallica (Röpstorf et al. 1993).
 The Vampyrellid Amoebae (Vampyrellida) 7

Figure 2. Trophozoites of vampyrellid amoebae. A Expanded cell of Leptophrys vorax with clear filopodia
(DIC); B Highly branched cell of Thalassomyxa sp. with granular filopodia (phase contrast). Inset displays
magnified filopodium with granules and dilatations; C Network of Platyreta germanica with emptied yeast cells
(phase contrast); D Two isodiametric trophozoites of Vampyrella lateritia (DIC). Note the refractive
membranosomes on the filopodia; E Vampyrella lateritia with frontal accumulation of filopodia (phase
contrast). Black arrow indicates direction of movement; F Scheme illustrating two modes of locomotion in
isodiametric vampyrellid cells (rolling and rowing); G Isodiametric floating form of Leptophrys vorax (phase
contrast); H Moving trophozoite of Placopus melkoniani in top view with prominent granules in the lamella and
very fine frontal filopodia (phase contrast); I Moving trophozoite of Placopus flabellus with posterior cell hump
in top view (DIC); J Trophozoite of Placopus flabellus in side view (DIC). The cell moves to the right as
indicated by the hyaline frontal lamella; K Scheme illustrating the rolling motion of Placopus species in
truncated side view (Reprinted from Hess 2017a (1434–4610/Ó 2016 Elsevier GmbH) with permission from
Elsevier). Red arrows indicate filopodial growth and membrane flow. Scale bars in A, B, D, H-J = 10 mm,
C = 50 mm, E, G = 20 mm.
8 S. Hess, A. Suthaus
Morphology and Cellular Organization
In classical terms, the vampyrellids are considered
to be filose, naked amoebae. All known taxa exhibit
slender pseudopodia that are generally referred to
as ‘filopodia’, but differ markedly in structure and
motility (see below for details). In contrast to many
other amoebae, the plasma membrane of vampyrel-
lids seems to be devoid of external structures or
coverings such as scales, cell coats or a discernible
glycocalyx; there are, however, a few observations
indicating a (temporary?) extracellular mucilage
coat in the trophozoite stage (Canter 1973; More
et al. 2019). As depicted in Figure 2, the tropho-
zoites of different vampyrellid species vary greatly
in shape, size and colour. They can be roughly
grouped into three general morphotypes: isodiamet-
ric, expanded and filoflabellate. It is important to
understand that these morphotypes are more a cell
state than a stable character of a given taxon. Some
species can transition between morphotypes
depending on the surrounding conditions. This
applies, in particular, to expanded vampyrellids,
some of which form floating, isodiametric cells when
food is scarce – a potential dispersal strategy (e.g.
Leptophrys vorax; Hess et al. 2012).
The expanded, surface-bound morphotype
seems to be the most common among the known
vampyrellid amoebae. It can be found in three dis-
tinct phylogenetic lineages, specifically the families
Leptophryidae and Sericomyxidae, and the Thalas-
somyxa-clade. Expanded cells attach to the sub-
strate and change their overall shape frequently.
Depending on the species, they may differ drasti-
cally in their morphological details and motility.
Some members of the Leptophryidae (e.g. Lep-
tophrys vorax, Vernalophrys algivore) form fan-
shaped or branched cells, which move slowly by
the formation and extension of filopodia-bearing
fringes (Fig. 2A) (Gong et al. 2015; Hess et al.
2012). The size of such hyaline, lamella-like fringes
varies strongly among expanded cells. The marine
vampyrellid Sericomyxa perlucida (Sericomyxidae),
for example, forms relatively large hyaline lamellae
with emerging thread-like filopodia (More et al.
2021), while Arachnomyxa cryptophaga (Lep-
tophryidae) tends to attach only with long, mainly
unbranched filopodia (Hess 2017b). Other
expanded vampyrellids (e.g. Platyreta and Thalas-
somyxa species) readily form highly branched or
reticulate cells (Fig. 2B, C) (Bass et al. 2009; Grell
1985). The degree of reticulation, however, appears
to depend on cell size in some species.
The isodiametric morphotype is common in the
algivorous species of Vampyrella (e.g. V. lateritia,
V. pendula) and Lateromyxa gallica (Hess et al.
2012; Hülsmann 1993). The cells are almost spher-
ical with radiating filopodia, and typically float in the
water column, or locomote on substrates by filopo-
dial movement while the compact cell body remains
rather constant in shape. Floating trophozoites, in
particular those of V. lateritia, can attain an almost
heliozoa-like morphology, while crawling cells often
show a pronounced accumulation of frontal filopodia
(Fig. 2D, E). After their formation at the anterior cell
region, the fairly stiff filopodia attach to the sub-
strate, retract and move towards the posterior cell
region. In V. lateritia, the filopodia move under the
globular cell body, which results in a slow rolling
motion of the entire cell (Fig. 2F) (Hülsmann
1985). Instead, the filopodia of crawling Lateromyxa
trophozoites move towards the lateral sides, then
bend backwards and fuse with the cell’s posterior,
resulting in a rowing-like motion (Fig. 2F)
(Hülsmann 1993). The suspended, isodiametric
cells found in food-deprived cultures of some
expanded vampyrellids (e.g. Leptophrys vorax,
Planctomyxa polycarya) show a remarkable similar-
ity to the floating cells of the above-mentioned Vam-
pyrella species (Fig. 2G).
Filoflabellate cells have so far only been found in
members of the genus Placopus (family Placopodi-
dae), a valid junior synonym of Hyalodiscus (see
‘Systematics and Evolution’ for nomenclatural
details). These cells are flattened, with elliptic, circu-
lar or fan-shaped outlines, and exhibit a clear sepa-
ration into hyaloplasmic lamellae and a
granuloplasmic cell hump (Fig. 2H-J). The lamellae
are sometimes referred to as ‘lamellipodia’, but
there are clear-cut differences from the well-
studied lamellipodia of animal cell models, e.g. ker-
atocytes (Kucik et al. 1990). The lamella of filoflabel-
late vampyrellids can either be restricted to the
frontal or frontolateral region, or run around the
entire cell; then the cell hump occupies an almost
central position (Fig. 2H, I). Hence, some filoflabel-
late vampyrellids somewhat resemble lens- or fan-
shaped amoebozoans such as vannellids
(Smirnov et al. 2007). However, as the name sug-
gests, they are truly filose and bear numerous deli-
cate filopodia on their ventral side. As documented
for Placopus flabellus, these filopodia originate at
the edge of the frontal lamella, move to the ventral
side of the cell and then establish contact to the sub-
strate (Fig. 2K). The cell then rolls over the anchored

filopodia, which can be nicely seen by the antero-
grade movement of refractive granules situated in
the dorsal plasma membrane (Hess 2017a;
Hülsmann and Haberey 1973). In an excellent report
on locomotion in Placopus rubicundus, Hertwig and
Lesser (1874) also described the circular movement
of granuloplasmic inclusions, which can be best
ascertained in profile view. Hence, the motility of
filoflabellate cells bears some parallels to the much
more inconspicuous rolling motion of isodiametric
Vampyrella species (compare Fig. 2F and K), and
might be mechanistically related.
The pseudopodial processes of vampyrellid spe-
cies are diverse in terms of structure and motility,
and are important traits for species recognition.
They are all filose in nature, but vary greatly in size,
branching pattern and their propensity to anasto-
mose. For convenience, we refer to all of them as
‘filopodia’, even though in some instances they
may show extensive branching and fusion. In the
filoflabellate Placopus species, filopodia are so thin
and short that they are easily overlooked, while
those of the isodiametric Vampyrella cells can far
surpass the cell diameter. Some vampyrellids (e.g.
species of Arachnomyxa, Asterocaelum, Latero-
myxa, Leptophrys, and Vampyrella) bear hyaline,
tapering filopodia that rarely branch, but can origi-
nate in clusters (Canter 1973; Hess 2017b; Hess
et al. 2012; Hülsmann 1993). These filopodia are
relatively rigid, and ultrastructural data from Vam-
pyrella lateritia and Lateromyxa gallica suggest that
they are supported by bundled actin filaments
(Fig. 3A, B) (Hülsmann 1985, 1993). The filopodia
of Sericomyxa perlucida (Sericomyxidae) are clear
as well, but are rather thread-like, i.e. they do not
taper much, and originate from thin, lamellar struc-
tures (More et al. 2021). The clear filopodia and
lamellae of certain Vampyrella and Placopus spe-
cies contain conspicuous refractive granules that
can show independent motility (Fig. 2D, H, I and
3C). Transmission electron micrographs of the gran-
ules of V. lateritia show them as osmiophilic and
composed of dense stacks of membrane material
(Fig. 3B, D) (Hülsmann 1985). They were tentatively
suggested to represent a membrane reservoir for
the rapid formation of food vacuoles and, hence, ter-
med ‘membranosomes’. However, their exact func-
tion is still unknown. It might well be that the content
and function of such refractive granules differs
among vampyrellid taxa, as indicated by variations
in shape, size and ultrastructure between species
(Hess 2017a; Hülsmann 1985; More et al. 2021).
The Vampyrellid Amoebae (Vampyrellida) 9
In contrast to the above-mentioned examples,
members of the genera Arachnula and Thalas-
somyxa do not show a clear cytoplasmic differentia-
tion between their pseudopodial structures and the
main cell cytoplasm. Their filopodia exhibit a finely
granular texture, more irregular outlines and are sur-
prisingly dynamic, i.e. they quickly branch, anasto-
mose and collapse (Cienkowski 1876; Grell 1985,
1987). They show internal streaming of particles,
and can rapidly form straight outgrowths and local
dilations, both of which can move along the filopodia
(Fig. 2B and inset). With that, these filopodia more
closely resemble the reticulopodia of certain
foraminiferans than the rigid and clear filopodia of
other vampyrellids (Koonce et al. 1986). Using cry-
ofixation and freeze substitution, Frowerk (2010)
revealed the presence of mitochondria, vesicles
and – more rarely – arrays of microtubules in the
filopodia of Thalassomyxa australis. The ultrastruc-
tural differences found among vampyrellid filopodia
reflect the great variation in motility. Some species
are extremely slow and can even appear motion-
less, while others show mesmerizing cellular
activity.
Vampyrellids also vary in terms of the gross cyto-
plasmic appearance, but all known representatives
have a fairly granular and opaque cytoplasm. In
addition, certain species may contain numerous
vacuoles that result in a foam-like appearance. Con-
tractile vacuoles typically form at undefined sites of
the cell and cycle slowly, i.e. best observed in
time-lapse footage. One of the most prominent char-
acters of certain vampyrellids is the intense yellow-
ish, orange or reddish colour of the granuloplasm
(Fig. 2D, E, I, J and Fig. 3E). Some species (e.g.
V. lateritia, V. pendula, P. flabellus, P. rubicundus)
are always pigmented (Hess 2017a; Hess et al.
2012), while the colour of others (e.g. Leptophrys
vorax) depends on the algal diet (Cienkowski
1865, 1876; Dobell 1913). Although this indicates
that the reddish pigments (probably carotenoids)
stem from the prey, other vampyrellids will feed on
similar algae, but remain totally colourless (e.g. Pla-
tyreta germanica, Theratromyxa weberi; unpubl.
observ. S.H.).
The nuclei of vampyrellids are almost always dif-
ficult to discern in intact cells with standard light
microscopy, as they provide poor contrast and are
often obscured by the granular cytoplasm. This is
why vampyrellids were once thought to be devoid
of nuclei and assigned to Haeckel’s monera
(Haeckel 1870) – later, however, nuclei were
10 S. Hess, A. Suthaus

Figure 3. Cellular details and ultrastructure of vampyrellid amoebae. A Section through filopodium of
Lateromyxa gallica with parallel arrays of microfilaments (TEM, reprinted from Hülsmann 1993 (Ó 1993 by the
Society of Protozoologists) with permission from John Wiley and Sons); B Section through filopodium of
Vampyrella lateritia with microfilaments (black asterisks) and an osmiophilic membranosome (TEM,
micrograph by Norbert Hülsmann published by the Encyclopedia of Life); C Membranosomes (white
arrowheads) on filopodia of Vampyrella lateritia (DIC); D Detail of bloated membranosome of Vampyrella
lateritia revealing membrane layers (TEM, micrograph by Norbert Hülsmann published by the Encyclopedia of
Life); E Compressed cell of Placopus flabellus revealing colourful globules and vacuoles as well as a vesicular
nucleus (DIC); F Section through a nucleus of Leptophrys vorax with acentral nucleolus (TEM); G Mitochondria
of Leptophrys vorax with tubular cristae (TEM); H Polyhelical bodies (potential polyribosomes) in the cytoplasm
of Leptophrys vorax seen in cross and longitudinal section (TEM); I Intracellular bacteria (“Ca. Megaira
polyxenophila”) in the cytoplasm of Placopus flabellus (TEM); J, K Unidentified objects (viruses?) in the
cytoplasm of the marine Placopus pusillus (J) and an undescribed Placopus species (K) from freshwater
(TEM). Scale bars in A, H, J, K = 100 nm, B, D, F, G, I = 500 nm, C, E = 10 mm.

revealed with staining techniques (Zopf 1885b).
Most known species are multinucleate, but there
are exceptions, e.g. Arachnomyxa cryptophaga
and the uninucleate Placopus species. Vampyrellid
nuclei are often ‘vesicular’, i.e. with a clearly dis-
cernible, central (sometimes acentral) nucleolus
(Fig. 3E, F). Yet, they can also be deformed (Seri-
comyxa perlucida; More et al. 2021), with granular
content (Placopus melkoniani; More et al. 2019),
elongate (Platyreta germanica; Röpstorf et al.
1994), or fusiform (Thalassomyxa sp.; unpubl.
observ. S.H.). Ultrastructural data from the nuclei
of three phylogenetic lineages (Vampyrellidae, Lep-
tophryidae, Placopodidae) revealed that vampyrel-
lids perform a closed orthomitosis in late-stage
digestive cysts (Röpstorf et al. 1993, 1994). Despite
some differences in nuclear envelope retention, all
studied nuclei displayed arrays of intranuclear
microtubules, but no distinct microtubule organizing
centers (MTOCs). This is in line with the current
view that vampyrellids do to not produce flagella,
nor centrioles, in any part of their life history. Apart
from that, vampyrellid amoebae do not exhibit many
ultrastructural peculiarities. They typically contain
numerous roundish or elongate mitochondria with
tubular cristae (Fig. 3G), which distinguishes them
from the nucleariid amoebae with discoid cristae
(Dyková et al. 2013; Patterson 1983). In the cyto-
plasm of some vampyrellids (e.g. Lateromyxa gal-
lica, Leptophrys vorax, Vampyrella lateritia),
parallel arrays of helically arranged particles (poten-
tial polyribosomes) have been observed (Fig. 3H),
and referred to as ‘polyhelical bodies’ (Röpstorf
et al. 1993, 1994). In general, vampyrellids are still
rather poorly understood on the ultrastructural level,
partly due to difficulties in preserving these cells well
with chemical fixatives (Frowerk 2010; unpubl.
observ. S.H.). However, they offer much to explore,
e.g. intracellular bacteria and cylindrical objects of
unknown identity (Fig. 3I-K).
The digestive cyst is an important stage in the life
history of vampyrellids and comes in different sizes
and shapes. As the structural details of the cyst
depend on the type of food, they can reveal some-
thing about the potential life style of the vampyrellid
strain. The digestive cysts of vampyrellids that feed
on the cell contents of other eukaryotes (‘protoplast
feeders’) usually display a roundish outline, as the
engulfed food is relatively soft and does not deform
the trophozoite during cyst formation (Fig. 4A). Iden-
tifiable remains of prey cells are never found inside
those cysts. In contrast, the cyst morphology of
The Vampyrellid Amoebae (Vampyrellida) 11
vampyrellids performing free capture of entire prey
organisms varies strongly with the shape and size
of the food items, and the prey type might be still
identified after digestion or excystment. The diges-
tive cysts of Leptophrys vorax, for example, can
assume the shape of long diatom or desmid cells
(Fig. 4B). In the infiltrating Lateromyxa gallica, the
cyst morphology is constrained by the cell walls of
the prey, and hence cylindrical (Fig. 4C), while large
expanded vampyrellids such as Thalassomyxa spe-
cies can form irregular cysts, sometimes resembling
the rough outline of a branched trophozoite but with-
out filopodia (e.g. Grell 1985). Vampyrellid digestive
cysts bear an envelope of organic, extracellular
material (Fig. 4D). This envelope varies in thickness
and complexity depending on the species. In the
marine Thalassomyxa australis, it is very inconspic-
uous and potentially evanescent. The fibrous cyst
envelope of this species was imaged by transmis-
sion electron microscopy (Frowerk 2010), but does
not seem to remain after excystation (Grell 1985).
In other Thalassomyxa-like strains, the cyst envel-
ope remains visible after excystation (unpubl.
observ. S.H.). The algivorous freshwater species
show a clear differentiation into a thin primary cyst
wall or ‘velum’ (representing the outline of the
encysting trophozoite), and a thicker and smooth
secondary cyst wall that closely surrounds the proto-
plast. Hence, the secondary cyst wall is best seen
during and after the hatching process (Fig. 4D, E).
Most digestive cysts are broadly attached to the
substrate and somewhat flattened (Fig. 4F). There
are, however, also stalked digestive cysts found in
Vampyrella pendula and Placopus pedatus
(Cienkowski 1865; Hess et al. 2012; Klein 1882).
The stalk is formed by an arm-like extension of the
encysting trophozoite, which leaves behind the
outer, primary cyst wall extending into the emptied
algal cell (Fig. 4G, H). Free-floating digestive cysts
are known from Asterocaelum species (Canter
1973; Cook et al. 1974). These cysts are spherical
and bear several spiny projections covered by muci-
lage – potential floatation devices (Fig. 4I). We know
little about the composition of the digestive cyst
walls, but they seem to be fibrous and have been
suspected to be cellulosic (Cienkowski 1865; Lloyd
1927).
Trophic Specialization and Feeding
Strategies
All known vampyrellids are entirely heterotrophic
and feed by phagocytosis on live or recently killed
12 S. Hess, A. Suthaus

Figure 4. Digestive cysts of vampyrellid amoebae. A Compact digestive cysts of Placopus flabellus at different
maturation stages (DIC). The young green cyst contains numerous food inclusions as typical for extracting
vampyrellids; B Elongate digestive cysts of Leptophrys vorax feeding on a Closterium species (oblique
illumination); C Cylindrical cyst of the infiltrating Lateromyxa gallica in a filament of an Oedogonium species
(phase contrast; micrograph by Norbert Hülsmann published by the Encyclopedia of Life); D Planctomyxa
polycarya leaving a digestive cyst. Note the two visible openings and the compact food remnant left behind
(DIC); E Detail of digestive cyst envelope of Planctomyxa polycarya with two discernible walls (DIC); F
Digestive cyst of an undescribed vampyrellid broadly attached to an emptied filament of Oedogonium (SEM;
micrograph by Norbert Hülsmann published by the Encyclopedia of Life); G Vampyrella pendula during
digestive cyst formation with a pseudopodium running into the algal cell (SEM); H Stalked digestive cyst of
Vampyrella pendula on Oedogonium stellatum (DIC). The primary, outer cyst wall (=velum) extends into the
algal cell; I Planktonic digestive cysts of Asterocaelum algophilum in Indian ink revealing their mucilaginous
spines (brightfield; micrograph by Hilda Canter-Lund, published by the Freshwater Biological Association,
permalink: http://www.environmentdata.org/archive/fbaia:3117). Scale bars = 10 mm, except B (100 mm).

organisms. The list of potential prey is long and
includes chlorophyte and streptophyte green algae
(unicellular coccoids, flagellates, gelatinous colo-
nies, filamentous forms and desmids), motile, ses-
sile and planktonic diatoms, chrysophytes,
cryptophytes, euglenids, heterotrophic flagellates,
ciliate cysts, fungal hyphae and spores, yeasts,
and even micrometazoa such as nematodes and
rotifer eggs. Detailed information on the proven prey
of different vampyrellid taxa can be found in Supple-
mentary Material Table S1. Accounts of bacterivory
are rare and mostly involve filamentous cyanobacte-
ria such as Anabaena, Dolichospermum, Oscillato-
ria and others. The only vampyrellids documented
to feed on these cyanobacteria are species of Aste-
rocaelum, a Leptophrys-like organism, and a giant
soil amoeba (Cook et al. 1974; Dobell 1913;
Kobayashi et al. 2018; Old and Darbyshire 1978).
Unfortunately, in several reports on predator–prey
interactions from the pre-genetic era, the actual
identity of the vampyrellid remains unclear due to
the lack of strain names and available cultures
(e.g. the ‘giant soil amoebae’ and ‘mycophagous
amoebae’ studied in Old and Darbyshire 1978; Old
and Oros 1980; Old and Patrick 1979). In other
cases, the amoebae have likely been misidentified,
which can cause an incorrect picture of a species’
autecology (e.g. the ‘Arachnula impatiens’ studied
by Dobell 1913 rather matches the descriptions of
Leptophrys species). Hence, many of these histori-
cal reports on vampyrellid feeding habits require
careful taxonomic re-evaluation.
Vampyrellid species show different degrees of
specialization, i.e. the breadth of the food range.
Some algivorous representatives of Vampyrella (V.
lateritia, V. pendula, V. closterii) and Placopus (P.
flabellus) appear to be restricted to a few tough-
walled green algal species, hence are considered
specialist predators (Cienkowski 1865; Hess
2017a; Hülsmann 1985; Poisson and Mangenot
1933). Similar observations have been made for
the leptophryids Arachnomyxa cryptophaga and
Planctomyxa polycarya, which prefer volvocalean
green algae and euglenids, respectively (Hess
2017b). By contrast Leptophrys vorax is an omnivo-
rous generalist predator (Hess 2017b; Zopf 1885a).
The different degrees of specialization found in the
vampyrellids do not align with their phylogenetic
placement and thus no generalizations about certain
clades and families can be made (see ‘Systematics
and Evolution’). Furthermore, it has to be empha-
sized that assessing the food range specificity is a
The Vampyrellid Amoebae (Vampyrellida) 13
laborious effort and requires access to numerous
live cultures of eukaryotes. It is thus virtually impos-
sible to obtain a complete picture for a given strain,
and generally much easier to demonstrate general-
ist predation than to prove specificity.
The above listed prey types are very diverse in
terms of size, motility, cell structure and surface bio-
chemistry. Vampyrellids have evolved a number of
astonishing feeding strategies and variations of their
general life history, which allow them to consume
difficult-to-engulf prey such as tough-walled, fila-
mentous or bulky organisms. Here we define four
distinct feeding strategies observed in vampyrellid
amoebae (Fig. 5A; listed for specific species in Sup-
plementary Material Table S1).
Free Capture
Vampyrellids feeding by ‘free capture’ behave simi-
larly to amoebae of other phylogenetic groups. They
catch or collect prey cells and enclose them in a food
vacuole by conventional phagocytosis (Fig. 5A, B).
There are observations that some vampyrellids
can paralyse or adhere to motile prey cells before
phagocytosis (Grell 1985, 1987), but this is not
always the case. The motions of ‘metabolic’ eugle-
nids, for example, can effectively hinder engulfment
by certain vampyrellids that otherwise feed readily
on non-metabolic species and immobile prey
(Hess 2017b). Prey sizes for ‘free capture’ show
extreme variation. Some vampyrellids collect
numerous small prey cells before entering the diges-
tive cyst stage (More et al. 2021), while some
expanded species (Theratromyxa weberi, Lep-
tophrys vorax) can envelop entire nematodes or
large (sometimes colonial) green algae (Esser
1983; Hess 2017b; Weber et al. 1952). In some
cases, the vampyrellid cell is nothing more than a
thin sheath of cytoplasm encapsulating the prey
when entering the digestive phase. The cysts of
vampyrellids performing ‘free capture’ oftentimes
contain recognizable prey (in contrast to ‘protoplast
feeders’, see below) and form their cysts at non-
specific locations, e.g. attached to detritus.
Colony Invasion
Some vampyrellid species (Arachnomyxa cryp-
tophaga, Leptophrys vorax) also feed on cells
embedded in the colonies of certain microalgae by
‘colony invasion’ (Fig. 5A, C). The trophozoites
attach to the colonies of volvocalean algae (e.g.
species of Eudorina, Pleodorina, Volvox), penetrate
14 S. Hess, A. Suthaus

Figure 5. Feeding strategies of vampyrellid amoebae and remaining cell wall perforations. A Scheme of four
feeding strategies defined in this work that illustrate the feeding process and the position of the resulting digestive
cysts. The vampyrellid cell is reddish, the algal prey green; B Free capture in Leptophrys vorax. The trophozoite
envelops a cell of Euglena deses. Note the other two phagocytosed euglenids in the cytoplasm (DIC); C Colony
invasion in Arachnomyxa cryptophaga. The trophozoite devours daughter colonies inside Eudorina elegans
(DIC); D Protoplast extraction in Sericomyxa perlucida. The marine predator extracts the protoplast of the diatom
Attheya sp. with a feeding pseudopodium (DIC); E Protoplast extraction in an undescribed freshwater
vampyrellid feeding on a thin-celled Mougeotia species (DIC). The sequence of micrographs shows the release
of fluid into a large vampyrellid vacuole (top), lifting of an ‘excised’ cell wall disc resulting from type II perforation
(middle; arrowhead), and uptake of prey cell contents by thin feeding pseudopodia (bottom; double arrowheads);
F Theca of Tetraselmis sp. (Chlorodendrales, Chlorophyta) with circular cell wall perforation produced by
Placopus pusillus (SEM); G Complete and attempted perforations in a spore of Bipolaris sp. (=Cochliobolus)
produced by Platyreta germanica (SEM; micrograph by Norbert Hülsmann published by the Encyclopedia of
Life). Note the annular erosion (middle) and the loose cell wall disc (left). Scale bars = 10 mm, except F (1 mm).

the gelatinous matrix and then phagocytose individ-
ual algal cells (and daughter colonies) inside the col-
ony (Hess 2017b). The incorporation of prey
appears to be identical with that observed outside
of colonies, e.g. when the same vampyrellids devour
unicellular prey (e.g. Chlamydomonas). However,
‘colony invasion’ still requires the dissolution of
extracellular algal mucilage and might represent a
somewhat more specialised form of the engulfment
of entire cells. Both A. cryptophaga and L. vorax
form their digestive cysts within the algal colonies.
For A. cryptophaga, this behavior was repeatedly
observed in natural populations (Hess 2017b;
Mayer and Kreutz 2003), suggesting that it provides
some benefits for the vampyrellid (e.g. protection
from predators, remaining close to potential prey).
Protoplast Extraction
The most famous feeding strategy of vampyrellids is
‘protoplast extraction’, i.e. the specific removal,
ingestion and digestion of the cellular contents of
prey cells (Fig. 5A, D, E). Not much is known about
the cell biological details, but there exists excellent
documentation of the process and of the resulting
cell wall perforations from live cell imaging and scan-
ning electron microscopy (Anderson and Patrick
1978,1980; Homma and Ishii 1984; Hülsmann
1983, 1985; Lloyd 1927; More et al. 2019). Protoplast
extraction is always mediated by invading pseudopo-
dia, while the cell body stays outside of the prey cell
(Fig. 5D, E). This is preceded by the local, pre-
phagocytotic dissolution of the prey cell wall (in prey
with organic walls) or the displacement of silicified
cell wall components (in diatoms). Despite some
species-specific variations of these processes, the
disintegration of the prey cell is inevitable. The isodi-
ametric cells of Vampyrella and Placopus attach to
the prey in a compact, sometimes hemispherical
shape, while branched or reticulate species (e.g. Pla-
tyreta germanica) do not necessarily change their
overall morphology. The latter develop one or sev-
eral cellular domains that perform the perforation
process (Hülsmann 1985; unpubl. observ. S.H.). In
some freshwater species (V. lateritia, V. closterii),
the perforation process is followed by the pressure-
driven ejection of fluid and/or prey cytoplasm after
weakening the cell wall, also referred to as ‘plasmop-
tysis’ (Hülsmann 1985). This plasmoptysis leads to
the rapid formation of a large (food) vacuole, which
resembles a sucking motion and is maybe the reason
why Cienkowski created the name Vampyrella
The Vampyrellid Amoebae (Vampyrellida) 15
(meaning ‘small vampire’ in Latin; Cienkowski
1865). Plasmoptypsis can also have severe effects
on the attacker, clearly arguing against any suction
created by the vampyrellid cell. In some instances,
the ejecting algal cytoplasm shoots through the
trophozoite and injures the cell (Hülsmann 1985).
After the uptake of ejected protoplast material in a
central cavity, the vampyrellid attacker sends one
or two inconspicuous feeding pseudopodia (also
referred to as ‘calyculopodia’) into the prey cell and
removes the cell contents, often by pseudopodial
retraction after the feeding pseudopodium enclosed
the entire cell contents (Hoogenraad 1907;
Hülsmann 1985; Lloyd 1926, 1927). Similarly, some
mycophagous vampyrellids extract fungal spores
(Old 1978). In marine protoplast extractors (P.
melkoniani, P. pusillus), plasmoptysis was not
observed (More et al. 2019, 2021). These species
seem to rely entirely on the action of invading pseu-
dopodia, suggesting that the salinity (=osmotic pres-
sure) and the resulting turgor of the prey cell are
important factors for plasmoptysis.
Prey Infiltration
‘Prey infiltration’ is another variety of protoplast
feeding, but is only rarely found in vampyrellids.
Lateromyxa gallica perforates the cell wall of its algal
prey (Oedogonium, Chlorophyta) similarly to the
protoplast extractors mentioned above, but then
invades the algal cell and completes its feeding
cycle in the filaments of the prey (Hülsmann
1993). Lateromyxa is also able to perforate the
cross walls of the algal filaments and move laterally
through the alga – a behaviour that appears to be
common among infiltrating protoplast feeders
(Hess and Melkonian 2013; Hess et al. 2019). Dur-
ing food uptake, the plasmodial vampyrellid frag-
ments into smaller portions, which then turn into
digestive cysts, often arranged like a string of beads
in the algal filament (Hülsmann 1993; Röpstorf et al.
1993). Prey infiltration was also observed in
vampyrellids that attack unicellular prey, namely
Vampyrella multiformis (invading Cosmarium; Zopf
1885a), and an undescribed vampyrellid (invading
Closterium; unpubl. observ. A.S.).
Cell Wall Penetration
To access the prey cell contents, vampyrellid amoe-
bae penetrate cell walls of diverse biochemistry.
This includes the cellulose- and pectin-rich cell walls
16 S. Hess, A. Suthaus
of chlorophyte and streptophyte green algae
(Cienkowski 1865; Hülsmann 1983; Hess 2017a;
Hess et al. 2012), fungal cell and spore walls with
beta-glucans, chitin and melanin (Bass et al. 2009;
Old 1977, 1978), the thecae of chlorodendralean
green algae, which are composed of very unusual
keto-sugar acids (More et al. 2019), and the silic-
eous frustules of diatoms (More et al. 2021). Except
for the diatom frustules, vampyrellid amoebae pro-
duce well-defined perforations of circular or elliptic
outline (Fig. 5F, G). These perforations vary in the
pattern of local cell wall dissolution: ‘Type I’ perfora-
tions result from a uniform cell wall erosion and rep-
resent a simple hole, while ‘type II’ perforations are
caused by an annular erosion and result in an ‘ex-
cised’ lid (Fig. 5E, G). The type of cell wall perfora-
tion does not depend on the cell wall biochemistry,
but rather on the vampyrellid species. For instance,
both Vampyrella lateritia and V. closterii feed on
conjugating green algae, but show different types
of cell wall perforation. Annular perforations were
also frequently documented in fungal cells after con-
tact with terrestrial vampyrellids (Fig. 5G) (e.g.
Anderson and Patrick 1980; Homma and Ishii
1984; Old and Darbyshire 1978). The biochemical
background of the cell wall dissolution in vampyrel-
lids is still unknown, but some authors suggested
the action of lytic enzymes (Hülsmann 1982; Lloyd
1927; Old 1978; Old et al. 1985). The close contact
between the vampyrellid plasma membrane and the
eroded cell wall observed by transmission electron
microscopy led Old (1978) to assume contact diges-
tion, similar to that suspected for other rhizarian pro-
toplast feeders (Busch and Hess 2017).
The different feeding strategies found in
vampyrellid amoebae are not mutually exclusive
for a given species, and can vary with the type of
prey. The marine Sericomyxa perlucida, for exam-
ple, grazes on small diatoms by free capture, while
larger species (e.g. Attheya sp., Amphiprora sp.)
are extracted (Fig. 5D) (More et al. 2021). A similar
flexibility was observed for the ‘colony invaders’ (A.
cryptophaga, L. vorax), which also feed by free cap-
ture, if the prey size allows for it (Hess 2017b).
Hence, the feeding strategies of vampyrellid species
should be assessed by careful observation – ideally
with a broad selection of prey types.
Distribution and Ecology
So far, vampyrellid amoebae have been reported
from six continents (Africa, Asia, Australia, Europe,
North and South America) and marine ecosystems
across the world, indicating that they are globally dis-
tributed (Berney et al. 2013; Gong et al. 2015; Hess
et al. 2012; Homma and Kegasawa 1984; Lentendu
et al. 2018; Old and Oros 1980; Vimercati et al.
2019). Vampyrellids inhabit diverse natural and artifi-
cial systems, and can be found in freshwater, soil and
the sea including brackish waters. Most accounts
come from freshwater ponds, lakes and moorlands
(Canter 1973; Cienkowski 1865; Hess et al. 2012;
Klein 1882; Zopf 1885a), while vampyrellids are rela-
tively rarely reported from running waters. ‘Terrestrial’
vampyrellids could be obtained from agricultural and
forest soils by baiting techniques using fungal spores
(Homma and Kegasawa 1984; Old 1977; Old 1978),
and were also detected in close association with root
assemblages (Geisen et al. 2016; Yurgel et al. 2018).
In marine and brackish waters, vampyrellids were
mostly found in benthic habitats, e.g. tidal pools, dia-
tom lawns, in association with red algae (Grell 1994;
More et al. 2021; Schepotieff 1912); yet there are
exceptions (see below). In addition, environmental
sequencing data demonstrated that marine systems
hold a surprising vampyrellid diversity (Berney et al.
2013), and that vampyrellids colonize neotropical soil
(Lentendu et al. 2018), glacial cryoconite systems
(Vimercati et al. 2019), Brassicaceae leaves (Ploch
et al. 2016), Sphagnum-associated water bodies
(Lara et al. 2011), hydrothermal sediment (Berney
et al. 2013), and the deep sea (Schoenle et al.
2021). Due to our fragmentary knowledge of the phe-
notypic diversity of the Vampyrellida (see ‘Systemat-
ics and Evolution’), such environmental data are still
difficult to interpret.
Despite their wide distribution, vampyrellids are
rarely seen in large numbers in natural samples.
Yet, when environmental factors change after sam-
pling or after enriching samples with suitable food,
vampyrellids can exhibit strong population growth
in a short period of time. This is in line with the rapid
development observed in laboratory cultures and
indicates that the abundance of vampyrellid cells is
highly dependent on the availability and density of
suitable prey species. Indeed, there are a number
of studies that report mass infestations of algal
blooms by vampyrellid amoebae, sometimes with
pronounced consequences for the species commu-
nity, as discussed below.
Effects on Algal Blooms
The infiltrating freshwater vampyrellid Lateromyxa
gallica was reported to develop macroscopic mass

infestations of filamentous green algae (Hülsmann
1993). It fed on Oedogonium species that formed
massive floating mats on the two oligotrophic crater
lakes Lac Pavin and Lac Chauvet in France. Due to
the vampyrellid growth, these algal mats could turn
reddish during the summer months, sometimes with
Oedogonium filaments showing an infectivity of
100%. As revealed by observations over 16 years
(1973–1989) the extent of infestation by L. gallica
was dependent on the species composition of the
algal mats, as L. gallica exclusively fed on Oedogo-
nium species.
A study of diatom blooms in Loch Leven, Scot-
land revealed that the putative vampyrellid Astero-
caelum algophilum fed on the planktonic centric
diatom Stephanodiscus rotula, thereby contributing
to its decline as the dominant phytoplankton species
(Bailey-Watts and Lund 1973). Over a period of
more than two weeks, a significant fraction of all
S. rotula cells counted were found in the digestive
cysts of A. algophilum, with a maximum of 37%
(mid-September 1970). Due to the tendency of
diatom-filled cysts to sink, the obtained values were
probably underestimates. The effect of A. algo-
philum on the dominant diatom led to the cyanobac-
terium Synechococcus sp. replacing S. rotula as the
most abundant species. Asterocaelum algophilum
did not feed on Synechococcus sp. and other
cyanobacteria, which was confirmed by cultivation
experiments (Bailey-Watts and Lund 1973).
Interestingly, there are other species of Astero-
caelum that readily consume planktonic cyanobac-
teria. This was reported for two geographically
distant lakes, namely Lake Oshimma-ohnumma
(Hokkaido, Japan) and Lake Sidney Lanier (Geor-
gia, USA) (Cook et al. 1974; Kobayashi et al.
2018). The Asterocaelum species fed on the
cyanobacteria Dolichospermum planctonicum
(Lake Oshimma-ohnumma) and Anabaena planc-
tonica (Lake Sidney Lanier), and transformed the
blue green cyanobacterial blooms in milky white
slick within a short period of time. In Lake
Oshimma-ohnumma, the decline of Dolichosper-
mum planctonicum was followed by an increased
growth of Microcystis aeruginosa, another, poten-
tially toxic cyanobacterium (Harke et al. 2016).
While the Asterocaelum bloom in Lake Oshimma-
ohnumma was an isolated event (Kobayashi et al.
2018), the formation of milky white slick in Lake Sid-
ney Lanier was reported almost annually from 1968
until 1973 (Cook et al. 1974).
The Vampyrellid Amoebae (Vampyrellida) 17
Vampyrellids can also impact marine plankton
communities, as shown in the Rodrigo de Freitas
Lagoon, Rio de Janeiro, Brazil. During a three-
month period, a yet-undescribed Placopus species
was found to devour the dominant pelagic
picoplankton, the diatom Chaetoceros minimus
(Alves-de-Souza et al. 2019). During the bloom of
the diatom the predator and prey populations under-
went classical Lotka-Volterra dynamics. Previous
and subsequent blooms of C. minimus were unaf-
fected by Placopus sp., and it was suggested that
specific abiotic conditions (temperature, salinity
and water stratification) facilitated the rare
vampyrellid occurrence.
The aforementioned reports from planktonic sys-
tems are remarkable, as most known vampyrellid
amoebae are rather associated with surfaces, e.g.
benthic zones or floating algal mats. It might well
be that plankton infestations by vampyrellids are
occurrences of benthic-pelagic coupling, wherein
processes in the benthos (the vampyrellids) affect
the planktonic algae due to increased mixing
(Rowe et al. 1975). It is noteworthy that the Astero-
caelum bloom in Loch Leven, Scotland was pre-
ceded by rough weather, which presumably
disturbed the sediment, and that the vampyrellid in
culture grew best when undisturbed, surface-
bound grazing was possible (Bailey-Watts and
Lund 1973). This is why A. algophilum was regarded
as ‘pseudoplanktonic’. Likewise, Alves-de-Souza
et al. (2019) supposed that the marine, diatom-
devouring Placopus species found in the Rodrigo
de Freitas Lagoon resides in benthic zones during
non-bloom phases.
Vampyrellids can also cause fatal mass infesta-
tions in artificial systems. Investigations at the Ari-
zona State University revealed that the leptophryid
Vernalophrys algivore caused crashes of Scene-
desmus dimorphus cultures grown in raceway
ponds and photobioreactors (Gong et al. 2015).
Grazing rates of V. algivore were found to be as high
as 154 Scenedesmus cells per day per individual.
High throughput environmental sequencing of race-
way pond microbiomes revealed that vampyrellids
are common contaminants and must be considered
potential hazards for algal biomass production
(Carney and Lane 2014; Carney et al. 2016; Day
et al. 2017).
Given the ability of some terrestrial vampyrellids
to consume plant pathogenic fungi (e.g. Gaemanno-
myces gamini tritici) and nematodes (e.g. Meloidog-
18 S. Hess, A. Suthaus
yne incognita), their role as potential biological pest
control agents has been discussed (Esser 1983;
Homma and Kegasawa 1984; Old and Patrick
1979; Sayre 1973; Van der Laan 1954; Weber
et al. 1952; Winslow and Williams 1957). Initial small
pot experiments using Theratromyxa weberi against
Heterodera rostochiensis infestation were per-
formed with no success (Van der Laan 1954). Sim-
ilarly, greenhouse tests using Theratromyxa weberi
to control root-knot nematode disease in tomatoes
proved ineffective (Sayre 1973). The complexity of
microbial food webs and top-down control of thriving
vampyrellid populations might be reasons for the
low success in pest control, and for the rare mass
infestations in nature in opposition to cultures.
Vampyrellids as Prey and Hosts
There are indications that vampyrellids serve as prey
and hosts for parasites. The omnivorous Leptophrys
vorax, for example, was reported to engulf other
vampyrellids such as Vampyrella pendula (Zopf
1885a). Vampyrellid digestive cysts can also suffer
from infections by parasitoid nanoflagellates such
as Pseudospora species (Hülsmann 1985; Zopf
1885a; unpubl. observ. S.H.) or phycomycetes
(Bailey-Watts and Lund 1973). These trophic interac-
tions, sometimes referred to as ‘hyperparasitism’, are
poorly explored, however.
In addition, there is evidence that vampyrellids
are common hosts of intracellular bacteria
(Fig. 3I), which in some cases seem to cause an
increased mortality in the digestive cyst stage. Intra-
cellular bacteria have been detected in Vamyprella
lateritia (Hausmann 1977), V. pendula (Timpano
and Pfiester 1986), Placopus flabellus (Hess
2017a), Thalassomyxa australis (Frowerk 2010),
Sericomyxa perlucida (More et al. 2021), and Arach-
nomyxa cryptophaga (unpubl. observ. S.H.), and
thus occur in all studied vampyrellid family-level
clades. Yet, only for Placopus flabellus the intracel-
lular bacteria have been identified; they belong to
the Rickettsiales, Alphaproteobacteria (“Ca. Mega-
ira polyxenophila”; Hess 2017a). Finally, there is
also an account of virus-like particles in the cyto-
plasm of vampyrellid cells (Hausmann 1977), but
this needs further investigation.
The current data about vampyrellid distribution,
mass infestations and interactions with other
microbes suggest that vampyrellid amoebae may
play various ecological roles. From the above-
mentioned studies, we know that when conditions
are suitable, vampyrellid blooms can develop
quickly and function as ecological regulators or even
cause species replacement by selective grazing.
However, most of the time this appears to take place
secretly and, so far, has rarely been captured by
sophisticated ecological studies.
Systematics and Evolution
There are at least 45 credibly described species that
are either proved or likely to belong to the vampyrel-
lid clade within the Rhizaria. Most of these species
were found in freshwater habitats (34 species),
and they vary greatly in their feeding strategies;
most common are free capture and protoplast
extraction (for details see Supplementary Material
Table S1). The species herein accepted as
vampyrellids fall into 14 genera, which are listed in
Table 1 with their respective type species. The num-
ber of accepted species might increase in the future,
as some original descriptions could not be obtained
and evaluated (Vampyrella peritrichophaga, V.
helioproteus, Hyalodiscus limax, H. lomnickii). Fur-
thermore, there are taxa of ambiguous identity such
as Vampyrelloides roseus and Hyalodiscus korot-
newi that require a reinvestigation before a decision
can be made (see Supplementary Material
Table S1). Besides a few unjustified synonyms
and replacement names, there are also some spe-
cies that have been associated with vampyrellid
genera, but likely belong to other groups of amoe-
bae. This applies to some Hyalodiscus species that
might be Amoebozoa (listed in Hess 2017a), the
putative nucleariid amoebae Vampyrellidium
vagans and Vampyrellidium perforans (Patterson
et al. 1987), and Gobiella borealis, whose original
description closely matches the amoeboid hetero-
kont alga Chlamydomyxa labyrinthuloides
(Cienkowski 1881; Wenderoth et al. 1999).
Misidentifications and difficulties in assigning
species to particular genera on a purely morpholog-
ical basis are other prominent issues in the
vampyrellids. In particular, more than 20 species
names have been introduced for the genus Vampyr-
ella, including synonyms. Some of these taxa were
already moved to other genera, e.g. Vampyrella
vorax (Leptophrys) and Vampyrella pedata (Placo-
pus), and this might happen to many more Vampyr-
ella species once additional molecular evidence is
available (see below). Some taxonomic problems
have been acknowledged for many years (e.g.
Dobell 1913), and are unlikely to ever be entirely

Table 1. List of genera proven or likely to belong to the Vampyrellida with relevant information. 1 = genetically
characterized, PE = protoplast extraction, FC = free capture, Inf = prey cell infiltration, CI = colony invasion.
Genus Type species
Arachnomyxa HESS, 2017 1 A. cryptophaga
Arachnula CIENKOWSKI, 1876 A. impatiens
Asterocaelum CANTER, 1973 A. algophilum
Lateromyxa HÜLSMANN, 1993 L. gallica
Leptophrys HERTWIG & LESSER, 1874 1 L. cinerea
Monadopsis KLEIN, 1882 M. vampyrelloides
Placopus SCHULZE, 1875 1 P. ruber
= Hyalodiscus HERTWIG & LESSER, 1874
Planctomyxa HESS, 2017 1 P. polycarya
Platyreta CAVALIER-SMITH & BASS, 2008 1 P. germanica
Sericomyxa MORE et al. 2021 1 S. perlucida
Thalassomyxa GRELL, 1985 1 T. australis
Theratromyxa ZWILLENBERG, 1952 1 T. weberi
Vampyrella CIENKOWSKI, 1865 1 V. pendula
Vernalophrys GONG et al. 2015 1 V. algivore
resolved. Yet, the historical descriptions still provide
valuable insight into vampyrellid diversity and ecol-
ogy (see Supplementary Material Table S1).
So far, only SSU rRNA gene phylogenies are
available for the Vampyrellida, due to a lack of other
established genetic markers. In these phylogenies,
the vampyrellids that have been sequenced so far
form a monophyletic group, most closely related to
the Phytomyxea and ‘novel clade 90 within Endo-
myxa (Bass et al. 2009; Berney et al. 2013). The
most up-to-date phylogeny of the vampyrellids con-
tains 12 species from 9 genera, plus a number of
sequences from uncultivated vampyrellid cells
loosely assigned to Thalassomyxa (Fig. 6). The vast
majority of available vampyrellid sequences stem
from environmental sequencing approaches, which
have detected hundreds of vampyrellid phylotypes
that lack any phenotypic information (Berney et al.
2013). The vampyrellid tree was divided into sub-
clades A-C, but shows poor resolution in its deeper
branches (Berney et al. 2013). In particular, sub-
clade B does not appear to be monophyletic in
recent published phylogenies (e.g. More et al.
2019, 2021). However, there are at least eight lin-
eages that are consistently retrieved (Fig. 6). So
far, four of these lineages could be assigned to for-
mal vampyrellid families; some new, others recon-
stituted. Short outlines of the vampyrellid family-
level clades are given below.
Vampyrellidae ZOPF, 1885: The Vampyrellidae is a
relatively small clade that currently comprises the
genus Vampyrella with two sequenced morphos-
The Vampyrellid Amoebae (Vampyrellida) 19
No. of Habitat Feeding
species mode
1 Freshwater CI
2 Freshwater N/A
2 Freshwater FC
1 Freshwater Inf
3(4) Freshwater and soil FC, CI
1 Freshwater FC
6 Freshwater and marine PE, FC
1 Freshwater FC
1 Soil PE, FC
1 Brackish FC, PE
3 Marine FC
1 Soil FC
20 Freshwater PE, FC, Inf
1 Freshwater FC
pecies, V. lateritia and V. pendula. Both species
feed by protoplast extraction of filamentous green
algae, occur in stagnant freshwater, and display a
vibrant orange color. In the present phylogeny, there
are at least 1–2 additional species represented by
environmental sequences. This agrees with the
observations by Hülsmann (Hülsmann 1985), who
found strains resembling Vampyrella lateritia with
pronounced differences in size, behavior and prey
preference. However, it can also be expected that
some of the  20 traditionally described Vampyrella
species cluster outside of the Vampyrellidae.
Leptophryidae HESS, SAUSEN & MELKONIAN, 2012:
Together with the Vampyrellidae, this family robustly
forms ‘Clade A’ in the vampyrellid phylogeny. The
Leptophryidae were introduced on the basis of Lep-
tophrys vorax and the expanded soil vampyrellids
Platyreta germanica and Theratromyxa weberi
(Hess et al. 2012). Later, the monotypic genera
Arachnomyxa, Planctomyxa and Vernalophrys were
added (Hess 2017b; Gong et al. 2015), making the
Leptophryidae more heterogeneous than previously
thought. Although they have a marked tendency to
appear as expanded, surface-bound cells, the
known taxa vary in their feeding strategies (free cap-
ture, protoplast extraction and colony invasion) and
cytoplasmic color (from colorless to bright orange).
As in the aforementioned Vampyrella species, the
filopodia of leptophryids are hyaline and most known
species are multinucleate. The leptophryids seem to
be relatively common and diverse, as indicated by
their presence in environmental sequencing studies
20 S. Hess, A. Suthaus
(Geisen et al. 2016; Ploch et al. 2016; Schoenle
et al. 2021). Yet, all described leptophryids inhabit
freshwater and/or soil.
Placopodidae JAHN, 1928: The Placopodidae (syn.
Hyalodiscidae POCHE, 1913) are a clade of relatively
long branches in SSU rRNA gene phylogenies. Cur-
rently, the family only comprises the genus Placo-
pus SCHULZE, 1875 with five sequenced strains.
Placopus is a long-standing junior synonym of
Hyalodiscus HERTWIG & LESSER, 1874, a polyphyletic
genus of lens- and fan-shaped amoebae, which
was thought to have priority for long time (see
Hess 2017a; More et al. 2019 for details). However,
the homonymy of Hyalodiscus HERTWIG & LESSER, 1874
with the diatom genus Hyalodiscus EHRENBERG, 1845
(first described as a zoological taxon) was in conflict
with the code of the International Commission on
Zoological Nomenclature (ICZN). This finally
resulted in the adoption of the valid junior synonym
Placopus for the amoebae in question, and of the
respective family Placopodidae JAHN, 1928 (instead
of Hyalodiscidae POCHE, 1913; More et al. 2019). The
known representatives of the Placopodidae exhibit
trophozoites with a characteristic filoflabellate mor-
phology, rolling locomotion, and orange or reddish
cytoplasm. The cells are mostly uni- or binucleate
and have a limited tendency to form plasmodia.
Some Placopus species feed on protoplasts of
green algae (e.g. Hess 2017a; More et al. 2019),
but there are also species that consume chryso-
phyte flagellates (unpubl. observ. S.H.), planktonic
and benthic diatoms (Alves-de-Souza et al. 2019;
pers. comm. N. Hülsmann) and rotifer eggs
(Röpstorf et al. 1994). The genus Placopus appears
to have a wide ecological range, with species occur-
ring in both freshwater and marine habitats, includ-
ing brackish waters.
Sericomyxidae MORE, SIMPSON & HESS, 2021: The
Sericomyxidae represent the deepest-branching
clade of the Vampyrellida. The only phenotypic
information available comes from the brackish/-
marine Sericomyxa perlucida and the uncultivated
strain “NVam1” from brackish sediment. Both are
surface-inhabiting, colorless predators. S. perlucida
feeds on diatoms and other algae by free capture
and protoplast extraction.
The Thalassomyxa clade: Thalassomyxa-like
amoebae from marine habitats have been docu-
mented and sequenced, yet without any species
identification. They are part of ‘lineage B50 (= ‘Tha-
lassomyxa clade’), together with several, genetically
diverse environmental sequences from marine sam-
ples. The plasmodial amoebae of the Thalassomyxa
clade display finely granular filopodia and a huge
variation in size and overall morphology (branching,
anastomoses), which makes the identification and
delineation of species extremely difficult. They are
known to prey on diatoms and unicellular green
algae (Berney et al. 2013; unpubl. observ. S.H.).
These amoebae show some similarities to the
fast-moving, colorless freshwater vampyrellid
Arachnula impatiens (Cienkowski 1876), which is
the type of the family Arachnulidae PAGE, 1987. Arach-
nula, however, has not yet found its place in the
molecular phylogeny and due to the potential syn-
onymy with Thalassomyxa, we have refrained from
assigning any formal family name to ‘lineage B50
(e.g. More et al. 2019, 2021).
Furthermore, there are at least three, relatively
distinct vampyrellid lineages (B1, B2, B4) that are
only known from environmental sequencing, plus
some sequences that branch between the well-
supported Leptophryidae branch and the Vampyrel-
lidae (Fig. 6). These parts of the vampyrellid phy-
logeny still lack any morphological and ecological
information, except the sample origin (Berney
et al. 2013). Given this uncharacterized genetic
diversity and the numerous traditional vampyrellid
species yet awaiting genetic examination, our evolu-
tionary picture of the Vampyrellida is still very frag-
mentary. So far, there is evidence for multiple
transitions between marine habitats and freshwater,
including within family level clades such as the Seri-
comyxidae and Placopodidae (Fig. 6). The trophic
specialization to certain prey types and the evolution
of the feeding strategies are probably the most inter-
esting aspects. Surprisingly, there is currently no
apparent pattern. Protoplast feeding can be found
in three distantly related families, the Vampyrellidae,
Placopodidae and Sericomyxidae (Fig. 6). Within
the latter two clades, prey types vary greatly, from
diatoms to flagellate algae. An astonishing similarity
across families can be found in the two sequenced
Vampyrella species (V. lateritia, V. pendula) and
two Placopus species (P. flabellus, Placopus sp.
NCBI accession OK381883), all of which extract
the cells of filamentous green algae (Oedogonium,
zygnematophytes). Similarly, diatom-extractors
can be found in the two families Sericomyxidae
and Placopodidae. This indicates a broad and
ancestral genetic ‘toolkit’ that allowed the vampyrel-
lids of diverse subclades to attack (and specialize
on) various eukaryotic prey. This, however, requires
future genomic analyses.
The Vampyrellid Amoebae (Vampyrellida) 21
22 S. Hess, A. Suthaus

Vampyrellids in the Lab natural samples. They may be detected by regular

Challenges and Benefits of Cultures
The cultures of vampyrellids require a constant sup-
ply of live (mostly) eukaryotic prey (algae, fungi,
micrometazoa), and usually more attention than
those of many other microorganisms. However, as
shown by several examples in the literature, cultures
are crucial for an in-depth characterisation of a
vampyrellid species (Canter 1973; Grell 1985;
Hess 2017a, b; Hülsmann 1993; More et al. 2019,
2021; Zwillenberg 1953). This is because vampyrel-
lid cells are morphologically variable and morpho-
logical differences between taxa are sometimes
relatively indistinct. The entire morphological reper-
toire of a vampyrellid amoeba only becomes evident
in culture, when cells in various life history stages
and situations can be observed. Furthermore, simi-
lar vampyrellid species can differ markedly in their
autecology, especially prey range and feeding
strategies. Laboratory cultures can be used for feed-
ing experiments, and the resulting data contribute
relevant information to the descriptions of vampyrel-
lids. Cultures also allow for detailed studies on the
process of food acquisition and cell division (Hess
2017b; Hülsmann 1983, 1985; More et al. 2019,
2021), ultrastructure (Frowerk 2010; Hess 2017a;
Röpstorf et al. 1994), and the bacterial endosym-
bionts of vampyrellids (Hess 2017a). For any com-
prehensive description of a vampyrellid species a
culture is essential, and enables follow-up studies.
Vampyrellid strains available in public culture collec-
tions and the laboratory of the corresponding author
are listed in Supplementary Material Table S2.
Detection, Enrichment and Isolation
screening of natural samples (e.g. in Petri dishes),
and sometimes increase in number depending on
the conditions in the lab – this, however, is some-
times only a short period of increased visibility. It is
useful to specifically screen for digestive cysts; most
cells will be in this digestive phase when suitable
food is abundant. The identification of the natural
prey can be challenging, but is important for estab-
lishing laboratory culture of specialised vampyrel-
lids. Here, remains of prey in empty digestive
cysts (in case of free capture), and data from time-
lapse microscopy at low magnification can help to
solve this question. Other strategies to obtain
vampyrellids from natural samples are baiting tech-
niques and enrichment cultures. Terrestrial
vampyrellids were successfully baited with fungal
spores sandwiched between two filters and buried
in soil (Old 1977; Old and Darbyshire 1978;
Homma et al. 1979; Homma and Kegasawa 1984;
Pakzad and Schlösser 1998). Aquatic vampyrellids
have been enriched by mixing natural samples with
a suspension of microalgae or yeasts, followed by
incubation at dim light (Berney et al. 2013; Grell
1985; Hess 2017b; More et al. 2019, 2021). Even
in Winogradsky columns, which are typically set up
for the enrichment of anoxygenic phototrophic bac-
teria, a diversity of vampyrellids developed – though
only studied by amplicon sequencing (Lalla et al.
2021). In parallel to bait enrichment, the addition of
culture media (e.g. F/2, Waris-H) to the samples
can be used to promote growth of indigenous prey.
Once suitable prey is identified, vampyrellid cells
can be isolated in a prey suspension with a micro-
pipette, as done for many other protists.
Cultivation

As mentioned previously (see ‘Distribution and Ecol- Vampyrellids are kept in co-culture with their prey, in
ogy’), vampyrellids often occur at low abundance in distilled water or various mineral media, including
3
Figure 6. Maximum likelihood phylogeny of the Vampyrellida inferred from SSU rRNA gene sequences. The
sequence alignment (88 sequences) was based on the dataset published in More et al. (2021) and enriched
with environmental sequences from NCBI and 13 sequences from unpublished vampyrellid cultures (see
Supplementary Text S1 for details). New sequences were aligned with muscle (Edgar 2004) and 1,493 sites
were manually selected for the inferences with RAxML under the GTR + G + I model (Stamatakis 2014). The
best tree of 100 inferences was rooted with the earliest-branching clade (Sericomyxidae) according to previous
studies (e.g. Berney et al. 2013, Hess 2017a, More et al. 2021) and shows bootstrap support values (1,000
replicates, RAxML) at the branches, except for nodes that have full support (100%; bold) or support < 50%
(omitted). Bolded sequences are connected to phenotypic information and colour indicates origin
(green = freshwater/soil; blue = marine/brackish). Information about feeding habits is provided next to the
genus names in grey boxes (see legend top left for details). The scale bar represents 0.1 expected
substitutions per site.

commercial table water, Erdschreiber’s solution,
half strength Waris-H and F/8, a dilution of F/2
(Grell 1985; Hess 2017a, b; Hess et al. 2012;
More et al. 2019, 2021; Röpstorf et al. 1994;
Weber et al. 1952). For marine strains, artificial
sea water works well. Most known vampyrellids
grow well between 10 °C and room temperature,
and have to be subcultured every 2 to 3 weeks, by
transferring trophozoites or digestive cysts to a fresh
prey suspension. Most vampyrellid cultures contain
bacteria, which can show enhanced growth after
release of organic matter from prey as the vampyrel-
lid population grows. Excessive bacterial growth can
impair vampyrellid health and axenic cultures of
some leptophryids grow much more reliably (un-
publ. observ. S.H.). However, despite several
attempts, some other species do not grow under
bacteria-free conditions (e.g. V. lateritia); these cells
repeatedly died off after a few feeding events (un-
publ. observ. S.H.).
Molecular Techniques
The fact that most known vampyrellids exclusively
feed on eukaryotic prey complicates molecular
sequencing protocols based on isolated bulk DNA
from cultures, including polymerase chain reaction
(PCR) with universal eukaryotic primers. Single-
cell PCR of unfed trophozoites and late-stage diges-
tive cysts, followed by a semi-nested secondary
PCR worked well to amplify the SSU rRNA gene
for Sanger sequencing in several cases (Hess
et al. 2012; Hess 2017a,b). Single vampyrellid cells
can be picked with glass micropipettes, placed
in  10 ml nuclease-free water, and stored frozen
until the PCR master mix (minus 10 ml water) is
added. Primers that proved suitable for this protocol
include the forward primers EAF3 and EUK A, and
the reverse primers BR, ITS055R and EUK B (for
primer sequences see Marin et al. 2003; Medlin
et al. 1988). Alternatively, the primary PCR can be
replaced by a multiple displacement amplification
with random primers as used in the illustra Ready-
To-Go GenomiPhi V3 DNA Amplification Kit (Cytiva,
Marlborough, MA, USA) and the REPLI-g Advanced
DNA Single Cell Kit (Qiagen, Hilden Germany), both
successfully applied to vampyrellids (More et al.
2019, 2021, unpubl. work).
Vampyrellids were detected in several environ-
mental sequencing studies, including a comprehen-
sive vampyrellid-focused study by Berney at al. (see
‘Distribution and Ecology’ and references therein for
The Vampyrellid Amoebae (Vampyrellida) 23
technical details). However, careful primer selection
is crucial. Certain Cercozoa-favouring primers for
the V4 region of the SSU rRNA gene used in envi-
ronmental molecular approaches do not capture
vampyrellids and phytomyxeans (Fiore-Donno
et al. 2018). This led to the development of addi-
tional primers that show improved binding to phyto-
myxean and vampyrellid sequences (Fiore-Donno
et al. 2020).
State of Experimental Research and
Future Perspectives
Experimental research on vampyrellids is not as
advanced as in many other protist groups. Much of
their cell biology, biochemistry, and genetics is still
unknown, and there are currently no genomic data
available (only one transcriptome of Leptophrys
vorax; Sierra et al. 2016). Hence, there are many
exciting open questions, for example concerning
vampyrellid sexuality, enzymes and binding pro-
teins, cytoskeletal components (including the pres-
ence of flagella), physiology and metabolism. In
particular, the processes behind the penetration of
prey cell walls are of great interest. As revealed by
our vampyrellid phylogeny, the protoplast feeders
of the Vampyrellida show a complex evolutionary
pattern. A culture collection of vampyrellids (main-
tained by the authors) and the technical advances
in single-cell genomics and analytical techniques,
promise to provide great opportunities to explore
vampyrellid cell biology and evolution in the near
future. First efforts are currently underway in a
nationally funded project focused on these fascinat-
ing predatory amoebae (as part of the “Taxon-
omics” priority programme of the German Research
Foundation).
Declaration of Competing Interest
The authors declare that they have no known com-
peting financial interests or personal relationships
that could have appeared to influence the work
reported in this paper.
Acknowledgements
This work was supported by the Priority Programme
SPP 1991 ‘Taxon-omics’ [grant number 447190101]
and by the Emmy Noether Programme [grant number
417585753] of the German Research Foundation. It
is dedicated to the memory of Norbert Hülsmann
(1945–2020; Freie Universität Berlin), an esteemed
24 S. Hess, A. Suthaus
colleague with a strong passion for vampyrellid
amoebae. We also thank Alastair Simpson (Dal-
housie University, Halifax) for valuable comments
on the manuscript.
Appendix A. Supplementary Data
Supplementary data to this article can be found
online at https://doi.org/10.1016/j.protis.2021.
125854.
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