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The new chytridiomycete Paradinomyces triforaminorum gen. et sp. nov. co-

occurs with other parasitoids during a Kryptoperidinium foliaceum
(Dinophyceae) bloom in the Baltic Sea

Article  in  Harmful Algae · December 2022

DOI: 10.1016/j.hal.2022.102352

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 Harmful Algae 120 (2022) 102352

Contents lists available at ScienceDirect

Harmful Algae
journal homepage: www.elsevier.com/locate/hal

The new chytridiomycete Paradinomyces triforaminorum gen. et sp. nov.

co-occurs with other parasitoids during a Kryptoperidinium foliaceum
(Dinophyceae) bloom in the Baltic Sea
Albert Reñé a, *, Elisabet Alacid b, Andrey E. Vishnyakov c, Kensuke Seto d,
Victoria S. Tcvetkova c, Jordina Gordi a, Maiko Kagami d, Anke Kremp e, Esther Garcés a,
Sergey A. Karpov c, f, g
a
Departament de Biologia Marina i Oceanografia. Institut de Ciències del Mar (CSIC). Passeig Marítim de la Barceloneta, 37-49 08003 Barcelona, Catalonia, Spain
b
Department of Zoology. University of Oxford. 11a Mansfield Rd, Oxford, OX1 3SZ, United Kingdom
c
Department of Invertebrate Zoology, Biological Faculty, St Petersburg State University, Universitetskaya nab. 7/9, St Petersburg, 199034, Russia
d
Yokohama National University, Faculty of Environment and Information Sciences, Tokiwadai 79-7, Hodogayaku, Yokohama, Kanagawa, 240-8501, Japan
e
Biological Oceanography, Leibniz Institute for Baltic Sea Research Warnemuende, Seestrasse 15 Rostock, 18119, Germany
f
Zoological Institute of Russian Academy of Sciences, Universitetskaya nab. 1, St Petersburg, 199034, Russia
g
North-Western State Medical University named after I.I. Mechnikov, Kirochnaya st. 41, St Petersburg, 191015, Russia

A R T I C L E I N F O A B S T R A C T

Edited by: Dr. C. Gobler A new chytrid genus and species was isolated and cultured from samples obtained in the Baltic Sea during a
dinoflagellate bloom event. This species is characterized by having a spherical sporangium without papillae and
Keywords: zoospores of 2–3 µm in diameter that are released through 3 discharge pores. Molecular phylogeny based on
Marine chytrids ribosomal operon showed its sister position to the Dinomyces cluster in Rhizophydiales. Zoospores lack fenes­
Dinomycetaceae
trated cisternae but contain a paracrystalline inclusion, found in a Rhizophydiales representative for the first
Ultrastructure
time. Additionally, the kinetid features are uncommon for Rhizophydiales and only observed in Dinomyces
Molecular phylogeny
Paracrystalline inclusion representatives so far. These morphological features and its phylogenetic relationships justify the description of
Host specificity the new genus and speciesParadinomyces triforaminorum gen. nov. sp. nov. belonging to the family Dinomyce­
taceae. The chytrid was detected during a high-biomass bloom of the dinoflagellate Kryptoperidinium foliaceum.
Laboratory experiments suggest this species is highly specific and demonstrate the impact it can have on HAB
development. The chytrid co-occurred with three other parasites belonging to Chytridiomycota (Fungi) and
Perkinsea (Alveolata), highlighting that parasitic interactions are common during HABs in brackish and marine
systems, and these multiple parasites compete for similar hosts.

1. Introduction
Abbreviations
EM electron microscopy Protists play a major role in aquatic food webs, interacting with each
ER endoplasmic reticulum other in many different ways, such as via predation, competition, sym­
HAB harmful algal bloom biosis, or parasitism (Worden et al., 2015). Among protists, zoosporic
KAS kinetosome-associated structures parasites play significant ecological roles by infecting a wide range of
MLC microbody-lipid globule complex phytoplankton groups (Gleason et al., 2014; Jephcott et al., 2016).
PI paracrystalline inclusion Zoosporic parasites belong to different taxonomic groups (e.g. Perkin­
sea, Syndiniales, Chytridiomycota), all of them producing motile infec­
tive stages along their life cycle. Their infections directly impact
microalgal populations, including harmful algal blooms, causing a

* Corresponding author.
E-mail address: albertrene@icm.csic.es (A. Reñé).

https://doi.org/10.1016/j.hal.2022.102352
Received 8 June 2022; Received in revised form 14 October 2022; Accepted 10 November 2022
Available online 24 November 2022
1568-9883/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A. Reñé et al.
decline in their abundances (Alacid et al., 2017; Chambouvet et al.,
2008; Frenken et al., 2017; Jephcott et al., 2016). Further, they can play
other functions in aquatic protist communities. For instance, fungal
chytrids facilitate the energy transfer from inedible large algae to
zooplankton by efficiently utilizing algal carbon and being grazed by
zooplankton (Kagami et al., 2014; Klawonn et al., 2021). Additionally,
those parasitic interactions can be beneficial for the overall health of
host populations by selective parasitism on weaker, unhealthy host in­
dividuals (Laundon et al., 2021). To better understand the functioning,
nature and impact of such interactions on the protist community and the
dynamics of HABs, the diversity and host preferences of these largely
unknown organisms needs to be determined.
The importance and diversity of chytrids in aquatic systems has been
studied for a long time in freshwater environments, e.g. lakes, rivers
(Shearer et al., 2007; Sime-Ngando, 2012; Van den Wyngaert and
Kagami, 2021), and more recently, molecular environmental studies
pointed out the importance of basal fungi in marine systems (Amend
et al., 2019; Comeau et al., 2016; Richards et al., 2015). However, the
diversity of these parasitic organisms in marine and brackish waters still
remains mostly unexplored and only a few species, most of them of
uncertain affiliation, have been described so far (Frenken et al., 2017;
Gleason et al., 2011; Ilicic and Grossart, 2022). A recent exploration of
public metabarcoding datasets obtained from contrasting marine envi­
ronments observed that chytrids are found at higher abundances in
environments with salinity values deviating from the global mean, e.g.
sea ice, Baltic Sea or Red Sea (Ha1ssett et al. 2020). Consequently, efforts
to observe, culture and characterize different parasitic organisms
infecting dinoflagellates were conducted in the Northern Baltic Sea, an
area where their diversity remains mostly unexplored. Those efforts
resulted in the previous characterization of two new Perkinsea species,
Parvilucifera catillosa Alacid & Reñé and Parvilucifera sp. infecting the
dinoflagellate Kryptoperidinium foliaceum (F. Stein) Lindemann during a
bloom event (Alacid et al., 2020). Likewise, those parasites co-occurred
with a new chytrid species, described as Ericiomyces syringoforeus Karpov
& Reñé, which also infected K. foliaceum (Karpov et al., 2021). In this
study, we describe a second chytrid species isolated and cultured during
the same sampling campaign conducted in the Baltic Sea and determine
the potential impact of this parasitic species on the development of
harmful algal blooms.
2. Material and methods
2.1. Sampling, isolation and cultivation
Samples were taken from Kökar Island, in the Åland Archipelago (SW
Finland, northern Baltic Sea) during summer 2016 (Alacid et al., 2020).
The sampling depth was 2–3m, and the salinity was 6–7. Surface water
was taken and integrated water samples from bottom to the surface (net
samples) were obtained using a plankton net (10-µm mesh). The
phytoplankton community was dominated by dinoflagellates, mainly
Heterocapsa rotundata (Lohmann) Loeblich and K. foliaceum, reaching
abundances of 106 and 105 cells L− 1, respectively (Alacid et al., 2020).
2–3mL of natural seawater and net samples were transferred to 12-well
culture plates and incubated for 6 days. The plates were observed daily
with an inverted optical microscope (Leica DMI3000B) to detect mi­
croorganisms parasitizing dinoflagellates. Kryptoperidinium foliaceum
cells infected by chytrids were observed. Those cells were manually
isolated by micropipetting and transferred to 96-well tissue culture
plates. Additional K. foliaceum cells from strain KFF1002 (FINNMARI
culture collection) were added to propagate infections. The established
host-parasitoid pairs were maintained in culture (as C7 strain) by a
1
Hassett, B. T., Vonnahme, T. R., Peng, X., Jones, E. B. G., Heuzé, C.. 2020.
Global diversity and geography of planktonic marine fungi. Bot. Mar. 63, (2),
121-39. https://doi.org/10.1515/bot-2018-0113.
2
Harmful Algae 120 (2022) 102352
weekly transfer into new wells and the addition of a volume of healthy
exponentially growing dinoflagellate culture. The well plates were
maintained in culture chambers at 20◦ C and 14:10 light:dark cycle until
date. A detailed description of the procedure can be found in Alacid
et al. (2020).
2.2. Microscopy analyses
Different stages of the infection development were observed in order
to characterize the morphology and ultrastructure of the chytrid. 2mL of
live sample were transferred to Utermöhl settling chambers and
observed with a Leica DM-IRB inverted microscope (Leica Microsystems,
Wetzlar, Germany) connected to a digital camera ProgRes C10
(JENOPTIK Laser, Optik, Systeme GmbH, Jena, Germany), and a Zeiss
Axioplan microscope (Zeiss GmbH, Jena, Germany) equipped with a
color Zeiss MRm Axiocam camera. For scanning electron microscopy
(SEM), 5mL of chytrid culture was fixed with 10% formaldehyde (v:v)
and filtered by gravity onto an 8-μm pore size polycarbonate filter. The
filters were then washed by soaking in filtered seawater for 15min and in
distilled water for 15min. Subsequent dehydration was carried out in a
25, 50, 75, 90, 96, and 100% ethanol series for ca. 10min. The final step
of 100% ethanol was repeated twice by 10min. The filters were critical-
point dried and mounted on stubs, sputter-coated with gold-palladium,
and examined under a HITACHI S-3500N scanning electron microscope
(Hitachi High Technologies Corp., Tokyo, Japan) at the Servei de
Microscopia Electrònica (ICM-CSIC).
For transmission electron microscopy, zoospores were fixed with a
mixture of 2% glutaraldehyde and 1% OsO4 prepared on the 0.05M
cacodylate buffer for 40–50min on ice in the dark. The fixed sample was
concentrated by filtration through a polycarbonate filter, embedded in
1.0% agar, and rinsed in cacodylate buffer twice for 5min. The sample
was then washed twice in the culture medium and embedded in Spurr’s
resin (Sigma Aldrich, St. Louis, MO) after dehydration in ethanol series
and propylene oxide. Ultrathin sections were prepared using a Leica
Ultracut ultratome with a diamond knife. After double staining, the
sections were observed using a JEM 1400 (JEOL, Tokyo, Japan) mi­
croscope equipped with a digital camera Olympus Veleta (Olympus,
Hamburg, Germany).
2.3. Amplification, sequencing and phylogenetic analyses
Single sporangia were manually isolated with a glass micropipette,
transferred to 0.2-μm PCR tubes and immediately stored at -80◦ C until
processed. Then, a first amplification of the whole rDNA operon was
amplified using the primers pair EK-82F and 28S-1611R primers
(López-García et al., 2001; Moreira et al., 2007) with an amplification
mixture containing 2 µL of 10X buffer (TaKaRa Bio), 1.5mM MgCl2, 1U
of TaKaRa Taq DNA polymerase (TaKaRa Bio, Shiga, Japan), 0.2mM of
each dNTP, and 0.4mM of each primer. PCR amplification conditions
were as follows: initial denaturation for 3min at 95◦ C, followed by 6
cycles of 15s at 95◦ C, 30s at 58–53◦ C, decreasing 1◦ C each cycle, and
2min at 72◦ C, and 34 additional cycles at annealing temperature of
52◦ C, followed by a final extension step for 5min at 72◦ C. Then, 1 μL of
the PCR product was used as template for a successive PCR using D1R
and D3B primers (Hansen et al., 2000; Scholin and Anderson, 1994)
targeting the first fragment of LSU rDNA following the same PCR con­
ditions described in Karpov et al. (2021). For ITS, a PCR amplification
was directly conducted with the fungal primers ITS1 and ITS4 (White
et al., 1990) using previously described conditions for LSU rDNA.
Because all attempts to obtain the SSU from the single sporangia were
unsuccessful, an additional 15-mL sample of host-parasite co-culture
was harvested and immediately centrifuged at 3000rpm for 15min. The
supernatant was removed and the remaining 1mL volume was centri­
fuged at 10,000rpm for 5min. The pellet obtained was used to extract
genomic DNA using the DNeasy Blood & Tissue (Qiagen, Barcelona,
Spain) extraction kit, following the manufacturer’s instructions. The
A. Reñé et al.
resulting DNA was used as template to amplify the chytrid SSU rDNA
sequence using the specific primers CRYPTO2–2F – AU4v2 (Lazarus and
James, 2015) under the same conditions than previously used for LSU
rDNA. PCR purification and Sanger sequencing was performed on a
3730XL DNA sequencer (Genoscreen, Lille, France) using both forward
and reverse primers. Resulting sequences were deposited in GenBank
under the following Accession Numbers: SSU rDNA: ON118448; ITS:
ON118449; LSU rDNA: ON118553.
Additional available sequences for each gene were selected from
public repositories to construct the dataset covering the molecular di­
versity of Rhizophydiales and an additional dataset was constructed for
the concatenated analysis (Supplementary Table 1). Each dataset was
independently aligned using MAFFT v7 (Katoh and Standley, 2013),
manually curated, and poorly aligned regions were trimmed using Tri­
mAl v1.2 (Capella-Gutierrez et al., 2009) under the –automated1 option.
The final alignments had a length of 4084 (concatenated), 1534 (SSU
rDNA), 847 (LSU rDNA) and 364 (ITS) positions. Maximum-likelihood
phylogenetic trees were constructed with IQ-TREE v2.1.2 (Minh et al.,
2020). The best-fit substitution model was estimated by jModelTest
implemented in IQ-TREE and in the case of the concatenated alignment,
it resulted in GTR+F+I+G4 for all partitions. The model parameters
were estimated separately for each of them. Statistical support was
computed with 1000 ultrafast bootstrap UFBoot replicates (Hoang et al.,
2018) and a posterior analysis of SH-like approximate likelihood ratio
branch test (SH-aLRT) with 1000 replicates on the generated consensus
tree. For these evaluators, a high support is only assumed when UFBoot
≥95% and SH-aLRT ≥80%.
2.4. Determination of infection susceptibility and prevalence
Four different cultured strains of dinoflagellate species, including
Kryptoperidinium foliaceum KFF1002 from FINNMARI culture collection,
Alexandrium ostenfeldii (Paulsen) Balech & Tangen strain AOTVC4 from
CCVIEO culture collection, Prorocentrum cordatum (Ostenfeld) J.D.
Dodge (=P. minimum) CCMP1329 and Alexandrium minutum Halim
AMP4, both from ICM-CSIC culture collection, were used for the ex­
periments. The two latter cultures were available at salinity 36, which
was gradually adjusted during one month to salinity 10 to represent
Baltic Sea conditions, where cultures of the other species were grown.
The susceptibility of the dinoflagellate strains to infection by the chytrid
strain C7 was tested in 24-well plates in a final volume of 2mL culture
medium at a host cell:zoospore ratio of 1:10. One mL of each dinofla­
gellate culture was fixed in Lugol’s iodine solution (2%) and counted
using a Sedgewick-Rafter chamber to determine host cell concentra­
tions. A living subsample from each potential host dinoflagellate species
was then added to each well at a final concentration of 106 cells L− 1. The
supernatant of the chytrid culture growing on K. foliaceum, containing
mostly chytrid zoospores, was harvested and a 1-mL subsample was
fixed with Lugol’s iodine solution (2%). Chytrid zoospores were counted
as previously described, and added to each well at a 1:10 host:zoospore
ratio. Filtered seawater was then added to the well to reach a final
volume of 2mL. Two replicates were prepared for each parasite – host
species combination. All plates were incubated for 30h at 21◦ C in culture
chambers with a L:D cycle of 10:14h at 100mmol photons m2 s− 1.
Finally, 1mL of each sample was preserved with 10% (v:v) formalin and
the infection prevalence, defined here as the proportion of infected host
cells, was determined under light microscopy using a Sedgewick-Rafter
counting chamber.
An additional experiment was performed to confirm the suscepti­
bility or resistance of studied hosts and determine the infection preva­
lence under high parasitic inoculum conditions. For that purpose, 5mL
of healthy host cells from the 4 cultured dinoflagellate strains (>106
cells L− 1) were transferred to a 6-well culture plate, and high numbers of
sporangia and infected K. foliaceum cells from the stock culture were
added. Two mL subsamples were harvested 3–4 days after parasites
addition onto healthy host cultures. This time was required for the
3
Harmful Algae 120 (2022) 102352
parasite to complete its life cycle. The subsamples were fixed with 10%
(v:v) formalin, and the number of infected cells were quantified under
light microscopy as previously detailed.
3. Results
3.1. Morphology of life-cycle stages
Many free-living zoospores can attack, and adhere to the host cell
(Fig. 1A). Then, they encyst and germinate into the dinoflagellate
(Fig. 1A–C). However, not all of them succeed and only one to six
sporangia normally progress on each infected cell of Kryptoperidinium
foliaceum, being closely attached to the host (Fig. 1B–F). The rhizoid is
developed, but the stem is relatively thin, and sometimes hardly visible
between the sporangium and the host cell (Fig. 1B–D). The rhizoid
branches into the host cytoplasm, and can be traced in the damaged host
cell (Fig. 1E). The chytrid feeds through the rhizoids (trophic stage), and
gradually grows becoming the young spherical sporangium (Fig. 1C–E).
When single infections occur and the sporangium is mature, its bio­
volume is comparable to that of the host cell, but depending on the
number of sporangia simultaneously infecting the host, sporangia bio­
volume is variable (Fig. 1D–F). The sporangium develops a thick cell
wall enveloping a multinuclear plasmodium with numerous equal lipid
droplets which will probably be included in each newly formed future
zoospore (Fig. 1G). Finally, the plasmodium divides on the numerous
small zoospores (Fig. 1H) that are released through three inoperculate
discharge pores (Fig. 1I, J, L). The discharge pores seem to develop
gradually at the opposite side of the host cell, as seen at the SEM images
(Fig. 1K, L).
Zoospores of strain C7 are spherical (1.9–3.1µm in diameter, mean
2.5 µm, n=35 cells) with a posterior flagellum (20–27µm long, mean
22.5µm, n=29 cells) and a posterior lipid globule (Fig. 1M–Q). The
flagellum of most zoospores coils around the cell, and its base often
emerges laterally (Fig. 1M–Q). Similar pictures are present at EM sec­
tions (see below). At the same time, we observed zoospores swimming
with a posteriorly directed flagellum, which seems to represent a normal
state of the zoospore. When attached to the host, the zoospores retract
the flagellum and produce a cyst wall.
3.2. Zoospore ultrastructure
Among the life-cycle stages of chytrids, the ultrastructural characters
of zoospores are key to determine their taxonomic position. The general
ultrastructural organization of C7 zoospores is shown in Fig. 2A–C and
Fig. 4. The nucleus lies near the center of the cell, slightly shifted to­
wards one side (Fig. 2A, B). The ribosomal core occupies the central part
of the zoospores separating the nucleus from the MLC (Microbody-Lipid
globule Complex). The latter contains a branching flat microbody partly
enclosing the anterior surface of the lipid globule, which separates it
from the mitochondrion (Fig. 2A, B). A fenestrated cisterna (rumpo­
some) was not found in the zoospores. One or two mitochondrial profiles
with flat cristae attach to the ribosomal core, which is outlined by the
endoplasmic reticulum (ER) (Fig. 2A). Small dense bodies are scattered
through the cytoplasm (Fig. 2A–C). The zoospore contains a rather
prominent vacuole (Fig. 2A) and small vesicles of different shape at the
posterior end of the cell (Fig. 2B).
The kinetid is composed by a flagellar kinetosome and a short non-
flagellar centriole (here called centriole) inclined at 60⁰ to each other
(Fig. 2C). The centriole lies opposite to a lipid globule and is connected
to the so-called paracrystalline inclusion (PI) located under the plasma
membrane (Fig. 2C–E). In C7 zoospores, this PI is connected to the non-
flagellar centriole of the kinetid (Fig. 2C–E), and can be oriented either
parallel to kinetosome-centriole plane (Fig. 2D), or tangentially and
even perpendicular to it (Fig. 2E).
A. Reñé et al. Harmful Algae 120 (2022) 102352

Fig. 1. Light microscopy (A-J, M-Q) and SEM (K, L) images of the life-cycle stages of Paradinomyces triforaminorum developed on the host algae Kryptoperidinium
foliaceum.
A – encysted zoospores (cy) on the host cell surface. B – two young sporangia; C,D – young sporangia with rhizoids (arrows); E – sporangium with branching rhizoids
(arrows) in degraded host cell; F – six sporangia growing on a host cell; G – multinucleate immature sporangium just before the multiple division; H – mature
sporangium with formed zoospores; I – empty sporangium with three discharge pores after zoospore release; J – empty sporangium with two discharge pores; K –
sporangium with three small (developing) discharge pores; L – sporangium with three developed discharge pores; M-Q – images of released zoospores. A, F, H, I, M-Q
– bright field, B-E, G – differential interference contrast; J – stained with calcofluor white. Scales bars: A-D, F-K – 10 µm, E – 20 µm, L, M-Q – 5 µm. Abbreviations: ar-
anterior root of 5 microtubules, br-bridge connecting centriole to kinetosome, c-centriole, ct- central tubules of the axoneme, cy-cyst, dp-discharge pore, dv-dense
vesicles, er-endoplasmic reticulum, fl-flagellum, h-host, k-kinetosome, kas-kinetosome-associated fibrillar structure, l-lipid globule, m-mitochondrion, mi-microbody,
n-nucleus, pi-paracrystalline inclusion, rc-ribosomal core, sh-sheath around the kinetosome, sp-sporangium, t-root-associated fibrillar teeth, tf-transitional filaments
(props), tp-transverse plate, v-vacuole, ve-small vesicles at the cell posterior, zo-zoospore.

4
A. Reñé et al.
Fig. 2. Released zoospore structure of Paradinomyces triforaminorum at ultrathin sections. A-B – general zoospore structure showing organelle disposition. C-E – main
elements of kinetid: kinetosome, centriole, and centriole-associated paracrystalline inclusion. Scale bars: A-B – 400 nm, C – 300 nm, D-E – 200 nm.
3.3. Kinetid structure
The kinetid structure of the C7 parasitoid strain is the typical
described for Rhizophydiales in the following characters. The kineto­
some has a spiral filament in the transition zone (Fig. 3C), an incon­
spicuous transverse plate and transitional fibers (Fig. 3D, E). The
kinetosome is formed by triplets, about 400nm long, producing an
anterior root of five microtubules (Fig. 3G–J). The microtubular root
passes along the kinetosome from its base (Fig. 3H) to the distal end
(Fig. 3I), then follows the flagellum underlying the plasma membrane in
the direction of the lipid globule (Fig. 3J). A short centriole (up to
100nm) lies on the opposite side of the kinetosome and is connected to
the proximal end of the latter through a fibrillar bridge (Fig. 2C). A zone
of convergence is not visible in the bridge. The kinetosome is covered by
a thin layer of an electron dense sheath (Fig. 3E–G, I). It produces two
fibrillar teeth at the base of an anterior root (Fig. 3E, F). Remarkably, a
paracrystalline inclusion is connected to the kinetid. A general scheme
of the zoospore structure is presented in Fig. 4.
3.4. Phylogeny
The 18S rDNA sequence obtained for C7 strain shows a highest
identity of 98.6% to ON081641 Rhizophydiales sp. ICMB1107 sequence.
5
Harmful Algae 120 (2022) 102352
It shows 97.4% identity to sequence KY980264 Uebelmesseromyces har­
deri M.J. Powell & Letcher, 96.9% to sequence ON081632 Dinomyces
arenysensis Karpov & Guillou, and 96.3% to ON081639 Rhizophydiales
sp. ICMB1106 sequence. The 28S rDNA sequence has an 88.8% identity
with sequence EF634248 Coralloidiomyces digitatus Letcher, 88.4% with
KJ027539 D. arenysensis PL233 and 87.9% with KJ027549 Dinomyces sp.
PL242. The blast results of the ITS sequence shows a sequence query
cover of 30–40% due to the high variability of ITS1 and ITS2 regions.
Highest identities range between 95.7% to 94% with environmental
sequences and described species like Collimyces mutans K. Seto & Y.
Degawa, U. harderi and D. arenysensis. The phylogenetic position and
relationships of the cultured chytrid was evaluated with concatenated
18S, ITS and 28S rDNA molecular information obtained (Fig. 5). The C7
strain clusters together with Rhizophydiales sp. ICMB1107 (UFBoot
100/SH-aLRT 98.1) and forms a sister clade (97%/98.9) with Dinomyces
representatives, including D. arenysensis, Dinomyces sp. P242 and Rhi­
zophydiales sp. ICMB1106 (100%/99.8). The sequence of C. digitatus
clusters at the base of this group of sequences, however, without sta­
tistical support.
The phylogenetic relationships of the C7 strain were also evaluated
for 18S and 28S rDNA independently. The 18S rDNA phylogeny shows a
similar topology as the concatenated one (Fig. S1). The sequence of the
C7 strain clusters with ON081641 Rhizophydiales sp. ICMB1107 (99%/
A. Reñé et al.
Fig. 3. Flagellar apparatus structure in zoospore of Paradinomyces triforaminorum. A-G – consecutive transversal sections of flagellum (A-D) and kinetosome (E-G)
centriole in direction from distal to proximal end. White arrows point a fibrillar sheet around kinetosome. H-J – selected serial transversal sections of kinetosome (H,
I) and flagellum (J) in direction from proximal to distal end tracing the anterior root of 5 microtubules (marked by white bracket). Scale bars: A-J – 200 nm.
92.8) and forms a sister clade lacking of statistical support with
D. arenysensis sequences and ON081639 Rhizophydiales sp. ICMB1106,
which clusters together (-/99.8). The 18S rDNA sequence of Cor­
alloidiomyces representatives is not available. By contrast, the 28S rDNA
phylogeny does not show a close relationship between the sequences of
Dinomyces and C7 (Fig. S2). Dinomyces arenysensis P234 and Dinomyces
sp. P242 cluster together (100%/99.8) and form a sister clade with
C. digitatus PL163L with no statistical support. The C7 sequence clusters
unrelated to any other sequence, occupying a basal position in a cluster
containing a large number of species, including the previously discussed.
The phylogenetic reconstruction using ITS sequences confirms the re­
lationships of the concatenated and 18S rDNA phylogenies (Fig. S3). The
sequences of D. arenysensis and Dinomyces sp. P242 form a cluster with
high support (98%/90.9). The sequence of C7 forms a sister branch, but
showing low statistical support. In none of the phylogenies constructed,
the sequences of the C7 strain show a close relationship with
E. syringoforeus strain E4, also isolated from the Baltic Sea and infecting
the same dinoflagellate host K. foliaceum.
3.5. Infection susceptibility of harmful algal species
Under the conditions used during the infection experiment, i.e.
initial host abundance of 106 cells L− 1 and a 1:10 host:zoospores ratio,
Alexandrium minutum, A. ostenfeldii and P. cordatum were not infected by
the chytrid strain. However, infected Kryptoperidinium foliaceum cells
were observed, showing a prevalence of 0.49 ± 0.16% after 30 h of
incubation. The additional experiment using a much higher inoculum of
parasites, and including not only zoospores, but developing sporangia,
showed infections of the chytrid strain on K. foliaceum with a prevalence
of 51% after 4 days from inoculation. Around 30% of dinoflagellate cells
were infected by a single sporangium. Moreover, multiple infections
were common and 17% of cells were infected by two sporangia, 13% by
three sporangia, and successively with gradually decreasing percent­
ages. Up to 14 sporangia were observed developing on the same host cell
(n=153). The presence of infections was not observed in infection ex­
periments performed with the other three dinoflagellate species,
A. minutum, A. ostenfeldii and P. cordatum.
6
Harmful Algae 120 (2022) 102352
4. Discussion
The sequences of C7 strain form a sister lineage to Dinomyces are­
nysensis in our phylogenetic trees (Fig. 4, Fig. S1-S3). However, based on
18S rDNA and ITS phylogenies, it could not be assumed to belong to the
genus Dinomyces with certainty. Furthermore, 28S rDNA phylogeny did
not support the close position with Dinomyces. Also, the morphology and
ultrastructure of the sporangium and zoospore of C7 differed essentially
from those of the type species D. arenysensis (see below). Consequently,
the C7 strain is described as belonging to a new genus in the family
Dinomycetaceae.
4.1. Taxonomy
Order Rhizophydiales
Family Dinomycetaceae Karpov & Guillou, 2014
Genus Paradinomyces gen. nov. Reñé & Karpov
Mycobank MB846461
Parasitoid of marine dinoflagellates with simple thallus, and
inoperculate, monocentric, epibiotic sporangium having branching
rhizoidal axis. Mature sporangia have a few discharge pores for zoospore
release. Zoospore with central ribosomal aggregation separating the
nucleus from the Microbody-Lipid-Complex, which contains a branched
microbody enveloping a large lipid globule without fenestrated cisterna.
The kinetid is adjacent to the ribosomal core. Flagellar transition zone
contains a spiral fiber. Centriole located at approximately 60◦ to the
kinetosome; both connected to each other by a narrow fibrillar bridge.
The centriole is connected with a paracrystalline inclusion. Anterior
five-microtubular root-associated with a few fibrillar teeth passes from
the kinetosome to the lipid globule.
Etymology: From Ancient Greek para- (close to) and -dinomyces
(chytrid genus), referring to the close relationship between both genera.
Type species: Paradinomyces triforaminorum
Paradinomyces triforaminorum sp. nov. Reñé et Karpov
MycoBank MB846462 GenBank ON118448; ON118449; ON118553;
Figs. 1–4.
Parasitoid of dinoflagellates, with a likely host preference for Kryp­
toperidinium species. Spherical epibiotic mature sporangium of 20–25µm
in diameter without papillae. Zoospores are spherical, 2–3µm in
A. Reñé et al.
Fig. 4. General scheme of the Paradinomyces triforaminorum zoospore ultrastructure.
diameter, containing a single posterior lipid globule. Zoospores are
released from the sporangium through 3 discharge pores.
Etymology: From Latin tri- (three) and foramina (pores), referring to
the three holes (discharge pores) present in the sporangium.
Type strain: C7, isolated on the host Kryptoperidinium foliaceum from
samples collected in Kökar Island, located in the Åland Archipelago (SW
coast of Finland), Northern Baltic Sea, in June 2016.
Holotype: a fixed specimen derived from the strain C7 embedded in
a resin block for electron microscopy deposited in the CCPP ZIN RAS
under the No X-136.
4.2. Comparison of P. triforaminorum with closest relatives
The closest phylogenetic relationship of P. triforaminorum was ob­
tained with 18S rDNA sequences belonging to Rhizophydiales sp.
ICMB1107 and they formed a sister lineage to the cluster of two species
of Dinomyces. Rhizophydiales sp. ICMB1107 sequence belongs to a
cultured strain from the Catalan Coast (Mediterranean Sea). It was iso­
lated during a bloom of the harmful A. minutum dinoflagellate occurring
in Arenys de Mar harbor (Fernández-Valero et al., 2022), which is the
same type locality of D. arenysensis. However, only a few morphological
7
Harmful Algae 120 (2022) 102352
features are shown, basically the presence of a lipid globule along the
early sporangium development, and its detailed characterization is
pending, preventing from making further comparisons. Likewise, no
morphological information of Dinomyces sp. strain P242 was provided
(Lepelletier et al., 2014), preventing any comparison with D. arenysensis
or P. triforaminorum features. Regardless of their close phylogenetic
relationship, P. triforaminorum is morphologically dissimilar to
D. arenysensis (Lepelletier et al., 2014). Zoospores of P. triforaminorum
are slightly smaller than those of D. arenysensis (2–3 vs. 2–4µm), but
have a much longer flagellum (20–27 vs. 12–13µm). Paradinomyces tri­
foraminorum lipid globule is located at the base of the flagellum while it
is found in the middle of D. arenysensis zoospore. The sporangium of
P. triforaminorum is spherical, but pyriform to spherical in D. arenysensis,
and does not have an apophysis-rhizoid swelling. Zoospores of
P. triforaminorum are released through 3 discharge pores, while in
D. arenysensis they simply break the sporangium wall. The core of ri­
bosomes occupies the center of the zoospores in both species, but in
contrast to D. arenysensis, the ER cisterna is not contained inside the
ribosomal core in P. triforaminorum. The MLC of P. triforaminorum has a
branched flat microbody, while D. arenysensis contains a simple flat
microbody. P. triforaminorum has no fenestrated cisterna (rumposome),
A. Reñé et al.
Fig. 5. Maximum likelihood phylogenetic tree of concatenated 18S, ITS, and 28S rDNA sequences representing the diversity of Rhizophydiales order. The sequence
of Paradinomyces triforaminorum is in bold. Statistical support of the nodes is presented by the ultrafast bootstrap value (%) and the likelihood ratio branch test. Only
values >90% are shown. Black dots indicate maximum statistical support.
which is rather prominent in D. arenysensis.
The kinetid features of P. triforaminorum are uncommon for Rhizo­
phydiales because a flagellar kinetosome and a non-flagellar centriole
are acutely angled, while they are generally parallel or nearly so in
Rhizophydiales. However, this character is also observed in
D. arenysensis, suggesting it is characteristic of Dinomycetaceae. Like in
D. arenysensis it has a spiral filament in the transition zone, an incon­
spicuous transverse plate, a short centriole forms an acute angle with the
kinetosome, and a root of five stacked microtubules passing in the lipid
globule direction. At the same time, P. triforaminorum has weaker
transitional fibers compared to those of D. arenysensis. A characteristic
spur at the distal end of D. arenysensis kinetosome is absent in
P. triforaminorum. At the same time, the kinetosome of P. triforaminorum
has two kinetosome-associated structures (KAS): lateral fibrils, here
called teeth, and a fibrillary sheet enveloping the kinetosome.
The paracrystalline inclusion is a common structure for many Chy­
tridiales (Davis et al., 2015; Letcher and Powell, 2014), but it was not
found in the Rhizophydiales representatives before. It is normally
located under the plasma membrane and not connected to the kinetid
like as it has been found for P. triforaminae zoospores. This structure
seems to have a variable position around the kinetid, but it is always
connected to the centriole.
Another close relative of P. triforaminorum, Coralloidiomyces digitatus,
is a saprotrophic fungus described by Letcher et al. (2008). Its sporan­
gium has a very peculiar highly variable “coralloid” shape with
branched rhizoids. The zoospore structure is typical for Rhizophydiales,
but has some differences with P. triforaminorum. The central ribosomal
core is crossed with ER and the MLC has a fenestrated cistern. The zone
of convergence is present in a relatively broad bridge between the
kinetosome and the centriole, and the anterior root is composed of four
8
Harmful Algae 120 (2022) 102352
microtubules. Thus, C. digitatus has significant differences from
P. triforaminorum. Even though it forms a sister branch to the Dinomy­
cetaceae with low support (Fig. 5), it still remains of uncertain position
in Rhizophydiales.
Another parasitic chytrid, E. syringoforeus, was described occurring
during the same dinoflagellate bloom (Karpov et al., 2021). It was
characterized by having spiny sporangia with smooth lateral papilla,
and by the formation of a syringe-like structure that penetrates the host
cell. Those features are clearly distinctive of those of P. triforaminorum
and they show a distant phylogenetic position. Thus, their co-occurrence
and their ability to infect the same host species do not imply a close
relationship.
4.3. Paracrystalline inclusion
The paracrystalline inclusion in Chytridiomycetes has been found in
Chytridiales zoospores only (Letcher and Powell, 2014). The PI presence
in P. triforaminorum represents the first observation among Rhizophy­
diales. This inclusion is normally intact and locates at the anterior end of
zoospores under the plasma membrane. It is located posteriorly in
P. triforaminorum and it is definitely connected with the centriole. At the
same time, paracrystalline inclusions are observed in zoospores of
Blastocladiomycota: Coelomomyces punctatus (Couch) Couch & H. R.
Dodge (Martin, 1971) and Callimastix cyclopis Weissenb. (Karpov and
Voronin, 1985; Vavra and Joyon, 1966). The nature of PI is not defi­
nitely known, but might be represented by crystalline proteins or lipo­
protein aggregates (Threadgold, 1976). The PI connection with the
kinetid in P. triforaminorum may suggest its role in the signal system of
zoospores as proposed by Letcher and Powell (2014).
A. Reñé et al.
4.4. Parasitic chytrids of algae in marine waters
Chytrids (zoosporic true fungi) include a diverse group of organisms
that produce motile spores and require an aquatic environment to
complete their life cycles. They grow as saprobes and/or parasites in
many freshwater and soil ecosystems (Barr, 2001; Powell, 1993; Shearer
et al., 2007; Sparrow, 1960). Chytrids infecting dinoflagellates have
been well known in lakes for a long time. The dinoflagellate Peridinium
gatunense Nygaard was reported to be chronically infected by Phlycto­
chytrium sp., Spizellomycetales (Alster and Zohary, 2007), whereas
Ceratium hirundinella Schrank was found to be infected by Amphicypellus
elegans Ingold, Chytridiales (Canter and Lund, 1951; Ingold, 1944). Only
four genera of chytrids have been reported in brackish and marine
ecosystems so far (Gleason et al., 2011; Ilicic and Grossart, 2022).
Among them, Rhizophydium spp. (Rhizophydiales) and Olpidium spp.
(Chytridiales) are reported to infect marine diatoms (Elbrächter and
Schnepf, 1998; Hanic et al., 2009). Garvetto et al. (2019) reported a
Rhizophydiales species infecting the marine diatom Skeletonema sp.
However, the species was not formally described due to the lack of
cultures. Besides D. arenysensis infecting A. minutum (Lepelletier et al.,
2014), another brackish - marine chytrid species was described,
E. syringoforeus infecting the dinoflagellate K. foliaceum (Karpov et al.,
2021). Consequently, this is the third report of a chytrid species
infecting marine dinoflagellates. The fact that two different species
(E. syringoforeus and P. triforaminorum) were isolated during the same
dinoflagellate bloom points out that chytrid parasitoids are still largely
unknown in marine waters. In fact, those two chytrid species and two
additional Parvilucifera species (Perkinsea) were observed to be infecting
the dinoflagellate populations in the same area (Alacid et al., 2020). A
study conducted in the Mediterranean Sea to determine the diversity of
parasites and host-parasite interactions during dinoflagellate blooms
also allowed to observe different co-occurring Perkinsea and chytrids
competing for the same resource (Reñé et al., 2021; Fernández-Valero
et al., 2022). It highlights that parasitism is common on marine phyto­
plankton, especially dinoflagellates and diatoms, but their diversity is
far from being known.
The presence and impact of parasitic infections during HABs has
been repeatedly reported in the literature, showing their role in the
control of transient algal blooms and the effects of parasites selection,
based on their specificity and host preferences, on host populations
(Alacid et al., 2017; Blanquart et al., 2016; Park et al., 2013; Reñé et al.,
2021). Those studies focus on Perkinsea or Amoebophrya, but little is
known about the effects of fungal infections on marine phytoplankton in
general, and dinoflagellates in particular (Fernández-Valero et al., 2022;
Gleason et al., 2015; Jephcott et al., 2016).
Phytoplankton communities dominated by dinoflagellates are com­
mon in shallow waters of the coastal Northern Baltic archipelago areas.
Besides A. ostenfeldii, which forms toxic blooms in late summer,
K. foliaceum, P. cordatum and Levanderina fissa (Levander) Moestrup,
Hakanen, Gert Hansen, Daugbjerg & M. Ellegaard regularly occur and
may locally grow to high cell concentrations (Hakanen et al., 2012;
Jerney et al., 2022). The results from the experiments conducted in this
study determined different susceptibility of harmful and potentially
harmful dinoflagellate species present in Baltic Sea waters (and also
A. minutum, a toxic species forming blooms in Mediterranean and North
Atlantic waters) to infection by the chytrid P. triforaminorum. While
K. foliaceum was highly susceptible to infections, the other tested species
were resistant to P. triforaminorum. It implies that in nature, the effects of
the parasite on the preferred host could control its growth during bloom
events and lead to shifts of the community, promoting the dominance of
non-susceptible dinoflagellates (Alacid et al., 2016). Actually,
P. triforaminorum co-occurred with other parasitoids of dinoflagellates
during the studied event. Kryptoperidinium foliaceum also showed a high
susceptibility to the chytrid E. syringoforeus while A. ostenfeldii or Pro­
rocentrum sp. among others were resistant (Karpov et al., 2021). The
same observations were obtained for the perkinsozoan Parvilucifera
9
Harmful Algae 120 (2022) 102352
catillosa (Alacid et al., 2020). By contrast, Parvilucifera sp. was able to
infect different dinoflagellate species including A. ostenfeldii, K. folia­
ceum, Heterocapsa triquetra (Ehrenberg) F. Stein and L. fissa. It demon­
strates that the different parasitoid species coexist and compete for
K. foliaceum, which shows a high susceptibility for most of them. Even
though initial experimental conditions mimicked those from natural
HABs, highly divergent infection prevalence was observed between the
two experiments using a different parasite inoculum size. Parasitoids
with a direct life cycle usually present density-dependence of their hosts,
where high host densities increase the contact rates between hosts and
parasites, facilitating the infection and transmission. Therefore, if high
abundances of hosts are present in nature for a sufficient period of time,
as is the case of bloom conditions, parasitic infections can reach higher
prevalence and impact on the dinoflagellate bloom development.
In conclusion, parasitism play an important role that cannot be
overlooked in the dynamics of harmful algal blooms (Gleason et al.,
2015). The discovered diversity allows to suggest that a multi-parasite –
multi-host system occurs in nature, leading to continuous evolution of
host and parasite populations, competition between parasites, with in­
teractions strength driven by host preference, specificity and virulence
of parasites. All those complex processes may result in changes in the
phytoplankton community. A great effort in studying these co-occurring
systems is needed if we are to better understand their role and impact in
aquatic environments in general, and dinoflagellate blooms in
particular.
Data availability of data and materials
Molecular sequences generated in this study are publicly available in
GenBank. Phylogenetic alignments analysed during the current study
are available from the corresponding author on request. Type material of
studied culture strain is deposited in the Culture Collection of Parasitic
Protists of the Zoological Institute RAS and available upon request.
Funding
AR and EG were funded by the Spanish MICINN Project SMART
PID2020–112978GB-I00. This work was partly supported by the Russian
Science Foundation grant no. 21–74–20089 (writing, microscopy anal­
ysis), the infrastructure of SAK’s laboratory was supported by ZIN RAS
program 1021051402849–1, and the C7 strain was incorporated in
CCPP ZIN RAS collection supported by a grant 075–15–2021–1069 of
the Ministry of Science and Higher Education of the Russian Federation.
AK was supported by the Academy of Finland, grant 310,449. MK was
supported by JSPS KAKENHI 19H05667. EA was supported by The
Royal Society (UK) under the Newton International Fellowship (grant
number NF170346, call 2017).
Authors’ contributions
The first draft of the manuscript was written by AR and SAK and all
authors commented on previous versions of the manuscript. All authors
read and approved the final manuscript. Contributions: Sampling: EA,
AK and EG; Microscopy analyses: AR, EA, AEV, KS, VST and SAK; Mo­
lecular and phylogenetic analyses: AR and EA; Experimental procedures:
JG; Funding acquisition: MK, AK, EG and SAK.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
A. Reñé et al.
Acknowledgements
Aurora Paloheimo is acknowledged for her contribution to sampling
and isolation of the chytrid strain, and Rachele Gallisai (ICM-CSIC) for
the maintenance of the culture. We thank the Spanish MICINN Project
SMART (PID2020–112978GB-I00), and the institutional support of the
“Severo Ochoa centre of Excellence” accreditation (CEX2019–000928-
S) and Scientific Park of Saint Petersburg State University for access to
TEM JEM 1400.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.hal.2022.102352.
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