A novel parasitoid, Pseudopirsonia chaetoceri n. sp. (Imbricatea, Cercozoa), of marine diatoms discovered in Korean coastal waters
Article information
Abstract
The cercozoan parasitoid Pseudopirsonia infects marine diatoms. Currently, only one species, P. mucosa, in the genus has been formally described in the North Sea. In June 2022 and 2023, we found that the diatom Chaetoceros constrictus was infected by a novel cercozoan parasitoid nanoflagellate during regular monitoring at Yongho Harbor, Korea. We established two strains of the parasitoid and their hosts in culture and performed morphological, biological, and molecular analyses. Molecular phylogeny inferred from small subunit ribosomal RNA (SSU rRNA) gene sequences demonstrated that the novel parasitoid clustered tightly with P. mucosa with a very low genetic distance. However, substantial morphological differences were identified between the two parasitoids. In the zoospore stage, two flagella of the novel parasitoid were subapically inserted and never completely retracted after attachment to the host cell. In contrast, P. mucosa has median- or submedian-inserted flagella, which disappear soon after attachment to the host cell. The auxosome of P. mucosa was surrounded by a mucilaginous envelope, and its division resulted in a morula-shaped accumulation of four flagellate mother cells (FMCs), producing eight zoospores. In contrast, the novel parasitoid auxosome does not cover a mucilaginous coat and produces FMCs via binary fission, which immediately detached from the auxosome and divided to produce two motile zoospores. The novel parasitoid presented a high degree of host specificity, infecting only Chaetoceraos species, whereas P. mucosa exhibited a broad host range, infecting species of four diatom genera without the genus Chaetoceros. We, therefore, suggest that this novel parasitoid is a new species of the genus Pseudopirsonia.
INTRODUCTION
Diatoms are major primary producers in the ocean and play a significant role in the global carbon cycle by efficiently sequestering atmospheric carbon dioxide into the ocean interior through their participation in the biological carbon pump (Smetacek 1999, Armbrust 2009, Tréguer et al. 2017). Diatom production may be transferred to higher trophic levels by micro- and mesozooplankton grazing in a classical grazing food chain. Alternatively, it may also be transformed into parasitoid nanoflagellates by parasite epidemics, which are then transferred to higher trophic levels through a zoosporic loop (Calbet 2001, Suzuki et al. 2002, Scholz et al. 2016). Diatoms are susceptible to various parasitoids, including aphelids, bigyromonadeans, cercozoans, chytrids, dinoflagellates, euglenoids, labyrinthuloids, oomycetes, and phytomyxeans (Drebes and Schnepf 1988, Kühn et al. 1996, Schweikert and Schnepf 1996, Schnepf and Kühn 2000, Kim et al. 2017, Yoo and Kim 2020, Catlett et al. 2023, Danz and Quandt 2023).
Several cercozoan species are known to either phagotrophically feed on or parasitize diatoms in various habitats, such as seawater, sediment, freshwater, and soil (Hoppenrath and Leander 2006, Howe et al. 2011). To date, three cercozoan parasitoids, Cryothecomonas aestivalis, Protaspa (ex Cryothecomonas) longipes, and Pseudopirsonia mucosa, have been documented to infect various marine diatoms (Drebes et al. 1996, Schnef and Kühn 2000, Kühn et al. 2004). The two species, C. aestivalis and P. longipes are closely related to each other and belong to the order Cryomonadida of the class Thecofilosea, whereas P. mucosa is distantly related to the former two species, nesting within the recently established order Marimonadida in the class Imbricatea (Howe et al. 2011). They resemble one another under light microscopy in that they are oval- to oblong-shaped biflagellates and show a gliding movement with possessing pseudopodia (Drebes et al. 1996, Schnef and Kühn 2000, Kühn et al. 2004). However, they can be distinguished from one another in developmental and infection processes and exhibit different host ranges and specificity although all three species shared the host species Guinardia delicatula. The parasitoid C. aestivalis infects specifically the diatom species G. delicatula by invading its whole body into the diatom frustule and then feeding on the cytoplasm of the host diatom using a short pseudopodium (Drebes et al. 1996). In contrast, P. longipes parasitized species across nine marine diatom genera: Cerataulina, Chaetoceros, Coscinodiscus, Guinardia, Leptocylindrus, Rhizosolenia, Navicula, Pleurosigma, and Thalassiosira (Schnef and Kühn 2000). P. longipes penetrates a pseudopodium into the host cell, and the pseudopodium pinches off portions of the diatom protoplast and transports the food back to the external cell body where the digestion takes place (Schnef and Kühn 2000). The infection process of P. mucosa is more similar to that of P. longipes than C. aestivalis. Pseudopirsonia mucosa has a broad host range that infects four diatom genera: Cerataulina, Guinardia, Leptocylindrus, and Rhizosolenia (Kühn et al. 2004). The parasitoid infects the host cells by penetrating a pseudopodium into the diatom frustule. The main body of P. mucosa remains outside the host frustule, becoming an auxosome, and the pseudopodium becomes a trophosome that digests the host cytoplasm and transports it to the auxosome (Kühn et al. 1996, 2004).
During regular monitoring in the Yongho Harbor of Busan, we found that the marine diatom Chaetoceros constrictus cells, which are periodically dominant in the system from June to July, were infected by a novel cercozoan parasitoid that presented gliding movements and infected the diatoms with a pseudopodium under light microscopic observations. We established two culture strains (MPL-CcPpc01 and MPL-CcPpc 02) of the novel parasitoid and host diatom systems in June 2022 and 2023, respectively. Using the host and parasitoid systems, we described their detailed morphological features using light and scanning electron microscopy, determined the host range and specificity and reconstructed the molecular phylogeny inferred from small subunit ribosomal RNA (SSU rRNA) gene sequences. A comparison of the morphological and developmental features and the molecular phylogenetic positions of the novel parasitoid with those of other known cercozoan parasitoids revealed that the novel parasitoid is a new species belonging to the genus Pseudopirsonia.
MATERIALS AND METHODS
Sample collection and cultivation
Samples were collected using a 20 μm plankton net through vertical towing from the bottom to the surface water at the Yongho Harbor (35°08′00″ N, 129°06′55″ E) of Busan in the Republic of Korea on Jun 18, 2022 and Jun 29, 2023. Water temperature and salinity were measured in situ using a YSI instrument (YSI Inc., Yellow Springs, OH, USA). The diatom C. constrictus cells infected by a novel parasitoid were isolated using a Pasteur pipette (Hilgenberg, Münnerstadt, Germany) under an inverted microscope (Axio Vert. A1; Carl Zeiss Inc., Oberkochen, Germany), gently washed four to five times with sea surface water passed through a 0.2 μm cellulose acetate syringe filter (Toyo Roshi Kaisha Ltd., Tokyo, Japan), and transferred to a 96-well plate (SPL Life Sciences, Pocheon, Korea) containing the filtered sea surface water. Two Pseudopirsonia strains were successfully established in culture. Uninfected C. constrictus cells were isolated as previously described. Parasitoid cultures were propagated through the stepwise transfer of samples from infected host stock cultures to uninfected host stock cultures cultivated in salinity 30 f/2 + Si medium (Guillard and Ryther 1962) every 4 to 5 d. Parasitoid and host cultures were maintained at 15 and 20°C, respectively, with a 14 : 10 light : dark cycle of cool-white fluorescent light at 100–130 μmol photons m−2 s−1.
Light and scanning electron microscopy
The morphology of the cultured cells was observed under an inverted microscope (Axio Vert. A1; Carl Zeiss Inc.) equipped with epifluorescence and differential interference contrast optics. The development and life history of the parasitoids were recorded at ×630 to ×1,000 magnifications using a Zeiss Axio Imager2 with a digital cinema camera (Canon Eos R5C, Tokyo, Japan) coupled with the microscope.
Glutaraldehyde-fixed cells (1% final concentration) were examined to observe nuclear division stained with SYBR green at 8°C for 1 h under blue light (excitation of 450–490 nm and emission of 515 nm). The cell length, width, flagellar length, and auxosome diameter were measured using an inverted microscope (Axio Vert A1; Carl Zeiss Inc.) equipped with a full HD box camera (MediCAM-K/2; Comart System Co., Ltd., Seoul, Korea) at ×400 magnification.
For scanning electron microscopy observations, the cells were fixed with glutaraldehyde (2% final concentration) at 8°C for 24 h, after which they were filtered onto Isopore membrane filters (3 μm pore-size; Millipore, Cork, Ireland), washed in seawater for 30 min, and dehydrated in a graded ethanol series (10, 50, 70, 90, 100% ×2) for 15 min at each step. The dehydrated samples were critical point dried in liquid CO2 using an Autosamdri-815, Series A (Tousimis, Rockville, MD, USA). Finally, the specimens were coated with 3 nm thickness of palladium and examined using an MIRA3 LMH (TESCAN, Brno, Czech Republic).
Cross-infection experiment
To assess the host range and susceptibility of the novel parasitoid, exponentially growing marine diatoms (34 strains, 32 species, 16 genera, 11 families, 10 orders, and three classes) isolated from Yongho Harbor were used. Each diatom strain (concentration of 1 × 102 cells mL−1) was incubated with recently formed zoospores (concentration of 1 × 103 cells mL−1) in 48-well plates, yielding a final volume of 1 mL in triplicate, and incubated for 72 h under the growth conditions described above. After incubation, the samples were examined under an inverted microscope. Parasite prevalence (i.e., the percentage of infected cells) was estimated as the percentage of infected cells out of the total remaining host cells at each time point. It was determined by scoring at least 100 host cells per sample as infected or uninfected. Infection was determined by the formation of an auxosome and / or a trophosome.
Parasitoid-host DNA extraction, polymerase chain reaction, and sequencing
The parasitoid and host cells were isolated using a Pasteur pipette, washed several times with sterilized filtered seawater, and approximately 100 cells were put about into polymerase chain reaction (PCR) tubes. The DNA was extracted using the Chelex extraction method (Kim and Park 2014). PCR amplifications were performed with the universal eukaryotic primers 1F and 1528R (Medlin et al. 1988) for SSU rRNA region, and primers D1R and D3B (Nunn et al. 1996) for large subunit ribosomal RNA (LSU rRNA) region, and primers ITSA and ITSB (Adachi et al. 1994) for internal transcribed spacer (ITS) rRNA region. PCRs were performed by adding 2 μL of template DNA, 2 μL of each forward and reverse primer, and 14 μL of deionized sterile water to an AccuPower PCR premix (Bioneer, Daejeon, Korea). The reactions were run using a C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA) with the following PCR protocols: an initial denaturation at 94°C for 2 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min 15 s, and final extension at 72°C for 7 min for the SSU rRNA region; an initial denaturation at 94°C for 3 min, 35 cycles of denaturing at 95°C for 45 s, annealing at 52°C for 45 s, and extension at 72°C for 1 min, and final extension at 72°C for 7 min for ITS and the LSU rRNA regions. The PCR products were visualized using EcoDye (SolGent Co., Daejeon, Korea) stained 1% agarose gels, incubated using an ExoSAP-ITTM (Thermo Fisher Scientific, Wilmington, DE, USA), and sequenced with primers using a Big-Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3730xl Analyzer (Applied Biosystems). The resulting sequence reads were assembled, and low-quality regions were edited using a ContigExpress (Vector NTI v. 10.1; Invitrogen, Grand Island, NY, USA). The assembled sequences were verified by comparison using BLASTN search in the NCBI database and deposited in GenBank (PQ590050–PQ590051).
Alignment and phylogenetic analyses
A total of 80 cercozoan and endomyxean SSU rRNA sequences, including two strains of P. chaetoceri n. sp. (MPL-CcPpc01 and MPL-CcPpc02), and Cryothecomonas aestivalis (MPL-GdCa01, Serial No. HNIBRPR42) obtained from this study, and 71 cercozoans, and six endomyxeans retrieved from GenBank (http://www.ncbi.nlm.nih.gov) of NCBI (National Center for Biotechnology Information), were used for molecular phylogenetic analysis. The sequences were aligned using MAFFT v7.10 (Katoh and Standley 2013, Katoh et al. 2019). Ambiguous sites were excluded using MEGA11 (Tamura et al. 2021) and 2,334 bp positions were used for further phylogenetic analyses. Maximum likelihood analysis was performed with RAxML 8.0.0 (Stamatakis 2014) using the GTRGAMMA evolution model and 2,000 replicates with the rapid bootstrapping option.
RESULTS
Infections of Pseudopirsonia chaetoceri n. sp. from field observations
During a two-year monitoring period from 2022 to 2023, infections of the diatom C. constrictus by a novel parasitoid, Pseudopirsonia chaetoceri n. sp. were detected at the Yongho Harbor in Busan, Korea. The cell abundances of the diatom C. constrictus ranged from 740 to 24,096 cells L−1 with parasite prevalence ranging from <0.03 to 3.0 (%) when the surface seawater temperatures ranged from 17.1–23.7°C (Table 1). The host abundance decreased rapidly when the highest prevalence was recorded on Jun 30, 2023. At the same time, another diatom species, C. curvisetus was also infected by the same parasitoid, but with a lower infection rate (0.1%) than the infection rates of the diatom C. constrictus (3.0%) (Table 1).
Morphology of Pseudopirsonia chaetoceri Cho et Kim sp. nov
Zoospores of Pseudopirsonia chaetoceri n. sp. were 8.4 (7.1–10.0) μm in length and 3.8 (3.1–5.0) μm in width (n = 40) (Table 2). They were oval and oblong in cell shape with an apical snout in the lateral view (Fig. 1A & B). They contained a large nucleus located in the central part of the cell, with a conspicuously large nucleolus (Fig. 1A & C). Numerous refractile granules were scattered near the nucleus, primarily in the posterior part of the cell (Fig. 1 A & C). In the ventral view, they had a prominent ventral groove running vertically across the cell and emitting pseudopodia from the ventral groove (Figs 1D, E, 2A & B). They had two unequal flagella inserted subapically (Fig. 1B). The length of the anterior flagellum ranged from 10.8–15.9 μm (13.6 ± 0.2 [mean ± standard error (SE)], n = 40), and the posterior flagellum ranged from 23.2–34.4 μm (29.0 ± 0.4, n = 40) (Table 2). The zoospores slowly glided along the substrate with their ventral sides (Supplementary Video S1).

Light micrographs with differential interference contrast (DIC) of Pseudopirsonia chaetoceri n. sp. (A & B) Lateral view of living cells, showing an apical snout. An anterior (Af) and a posterior flagellum (Pf) are subapically inserted (black arrow). Several refractile granules are shown in the posterior part. (C) Dorsal view. A spherical nucleus (nc) containing a prominent nucleolus (n). (D & E) Ventral view. Thin pseudopods (white arrows) emitted from a ventral groove (vg). Scale bars represent: A–E, 10 μm.

Scanning electron micrographs of Pseudopirsonia chaetoceri n. sp. on the diatom Chaetoceros constrictus. (A & B) Lateral and latroventral view. Zoospore with a ventral groove (vg) and anterior (Af) and posterior flagella (Pf). (C) An auxosome attached to the diatom with its pseudopodium penetrating the seta hole of the diatom. (D) The dividing auxosome with four flagella. Scale bars represent: A–C, 2 μm; D, 5 μm.
Infection and development process
The zoospore approached the diatom frustule by stretching several thin pseudopodia from the ventral groove to infect the host diatom C. constrictus (Fig. 3A). The parasitoid penetrated the pseudopodium into the host cell through poroids near the base of the diatom seta (Figs 2C & 3B). Occasionally, the parasitoid attached to the valve face of the diatom and penetrated the pseudopodium into the host cell through the rimopotulae on the diatom valve (Fig. 3C). After attachment of the zoospore, the pseudopodium transformed into a trophosome inside the host cell, and its main body remained outside the host frustule, becoming an auxosome (Fig. 3C & D). Two flagella of the parasitoid were wound around the auxosome and never completely retracted throughout its life cycle (Fig. 3C & D). The auxosome grew with the digested host materials supplied from the trophosome and reached up to 10.8 μm (9.0 ± 0.2, n = 10) in diameter (Table 2, Figs 3D, 4A & G). When the auxosomes began to divide, they had four flagella and underwent nuclear fission, followed by cytokinesis (Figs 2D, 3E, 4B–D, H & J). The daughter cell with two flagella (i.e., flagellate mother cell, FMC) immediately detached from the auxosome (Fig. 3F). The detached FMC divided once and produced two motile zoospores (Fig. 3G). The FMC maturation of the parasitoid proceeded asynchronously (Figs 3F–H, 4E, F, K & L). Typically, a single infection of the novel parasitoid with the diatom C. constrictus produced two FMCs, resulting in four motile zoospores within 8 h after attachment to the host (n = 10) (Table 2).

Light micrographs with differential interference contrast of the life cycle and developmental process of Pseudopirsonia chaetoceri n. sp. on the diatom Chaetoceros constrictus. (A) Zoospore attached to the frustule of C. constrictus by emitting their thin pseudopods (black arrowheads). (B) Pseudopod (ps) of the zoospore invades the host cytoplasm through the hole of seta of the diatom. (C) The main body of the parasitoid remains outside of the host frustule and becomes an auxosome (ax), and the pseudopod becomes a trophosome (tr). Another parasitoid (black arrow) attaches to the valve face of the host. (D) The auxosome (ax) grows with host materials supplied by the trophosome (tr). The two flagella (white arrows) did not completely retract throughout the life cycle. (E) The first division of the auxosome having four flagella (black double arrowheads). (F) Flagellate mother cell (FMC) detached from the auxosome. (G) The second division of the auxosome and the division of FMC produce two motile zoospores (white arrowheads). (H) The auxosome (double white arrowheads) leaving from the empty host. Scale bars represent: A–H, 10 μm.

Nuclear fission of Pseudopirsonia chaetoceri n. sp. during auxosome division. (A–F) Bright-field micrographs of glutaraldehyde-fixed specimens with differential interference contrast. (G–L) Epifluorescence micrographs of SYBR-stained specimens. Each cell in the upper panel corresponds to the same cell in the lower panels. Scale bars represent: A–L, 10 μm.
Host range and specificity
Cross-infection experiments revealed that the novel parasitoid P. chaetoceri, exhibited a high degree of host specificity. Among the tested diatoms of the 16 genera, only species belonging to the genus Chaetoceros was susceptible to the parasitoid (Fig. 5). Five Chaetoceros species, C. affinis, C. constrictus (MPL-Cc01 and MPL-Cc02), C. curvisetus, C. cf. lauderi, and C. rotosporus, were highly susceptible to the parasitoid, as they were completely consumed within 72 h. Two different strains of C. constrictus (MPL-Cc01 and MPL-Cc02) exhibited different susceptibilities to parasitoid infection. While the strain MPL-Cc01 of C. constrictus reached a prevalence of up to 90% within 8 h after inoculation of the parasitoid and rapidly consumed within 48 h, approximately half of the cells of the strain MPL-Cc02 remained uninfected at the end of the experiment (72 h).

Host range and prevalence of Pseudopirsonia chaetoceri n. sp. Parasite prevalence (% infected cells) was classified into four categories, representing a gradient grey scale from white to black. On the left side, a maximum likelihood tree inferred from small subunit ribosomal RNA gene sequences showing the phylogenetic relationships of 34 diatom species belonging to the three classes including Mediophyceae (Med), Bacillariophyceae (Bac), and Coscinodiscophyceae (Cos).
The seven Chaetoceros species, C. costatus, C. danicus, C. decipiens, C. mitra, Chaetoceros sp. 1, sp. 2, sp. 3, and Hemiaulus chinensis, were moderately susceptible (less than 60%) to the novel parasitoid infections during the incubation period (72 h) (Fig. 5). Two strains of C. didymus (MPL-Cd01 and MPL-Cd02) and Bacteriastrum hyalinum were moderately resistant to the parasitoid. The parasite prevalence remained at a low level (less than 30%) until 48 h and failed to infect subsequently thereafter. The other 13 tested diatom genera were highly resistant to infection by the novel parasitoid, and no infected cells were observed during the experiment.
SSU rRNA gene sequences and molecular phylogeny
Partial SSU rRNA sequences of the two strains, P. chaetoceri n. sp. (MPL-CcPp c01 and MPL-CcPpc02) isolated on the different sampling dates, were successfully obtained at a length of 1,701 bp (Supplementary Table S1). Pairwise comparisons of the sequences revealed no variation between the two Pseudopirsonia strains (Supplementary Table S1). Genetic distances among Pseudopirsonia species based on SSU rRNA gene sequences (1,647 bp) ranged from 0.68 to 7.71 (%). The highest similarity was observed between P. chaetoceri and P. mucosa (AJ561116), whereas the most distant species were between P. mucosa and the unidentified Pseudopirsonia species (MF615236).
The maximum likelihood phylogenetic analysis inferred from the SSU rRNA gene sequences revealed that all known Pseudopirsonia species including P. chaetoceri n.sp., P. mucosa, and unidentified Pseudopirsonia species formed a monophyly (Fig. 6). The two Korean strains of P. chaetoceri clustered tightly together with P. mucosa (AJ561116) isolated from Germany with a robust bootstrap support (100%), whereas the unidentified Pseudopirsonia species isolate PsND01 (MF615236) from Korea was distantly related to the former two species with a moderate bootstrap support of 63% (Fig. 6). The Pseudopirsonia clade was placed as a sister lineage of the two cercozoans Auranticordis quadriverberis and Cyranomonas australis. Abolifer globosa branched as a basal lineage of all clades in the order Marimonadia (Fig. 6).

Maximum likelihood tree inferred from small subunit ribosomal RNA gene sequences of 74 cercozoans and six endomyxans (2,334 positions). Values above nodes are shown ≥50% bootstrap support. Closed circles indicate robust statistical supports (100% bootstrap). All Pseudopirsonia species are shown in a grey box and the two strains of Pseudopirsonia chaetoceri n. sp. are represented in bold.
DISCUSSION
Morphological and developmental differences
To date, the genus Pseudopirsonia has been formally described as the only species, P. mucosa, in the genus (Kühn et al. 2004). The morphological characteristics of Pseudopirsonia species are summarized in Table 2. Pseudopirsonia species share morphological and developmental characteristics, such as the gliding movements of the free-living stage, formation of a globular auxosome and a trophosome, fusion of trophosomes in multiple infections, and asynchronous FMC maturation (Table 2). Nonetheless, several characteristics of the novel parasitoid P. chaetoceri were distinct from those of the most closely related species, P. mucosa (Fig. 7). First, the novel parasitoid P. chaetoceri had subapically inserted flagella, whereas the two flagella of P. mucosa were submedianly or medianly inserted. Second, the two flagella of the novel parasitoid never completely retracted during their life cycle, whereas the flagella of P. mucosa retracted soon after attachment to the host. Third, the division of auxosomes in P. mucosa generates a morula-shaped accumulation of daughter cells covered by a mucilaginous coat. In contrast, the auxosomes of P. chaetoceri undergo binary fission, producing FMCs that are immediately detached. After detachment, the FMCs of both species were divided once; however, the maximum number of offspring was four for P. chaetoceri and eight for P. mucosa. Nevertheless, during our cross-infection experiments, we found that the maximum number of offspring varied, depending on the host species. When P. chaetoceri infected the diatom Chaetoceros cf. lauderi, the maximum number of offspring reached up to 18 zoospores per infection (data not shown). This indicates that the maximum number of offspring might not be a good characteristic for distinguishing Pseudopirsonia species.

Schematic illustration of the life cycle and development process of Pseudopirsonia chaetoceri n. sp. on the diatom Chaetoceros constrictus. A, a motile stage, the zoospore of the novel parasitoid; B, attachment using its thin pseudopod and penetration the seta hole of the diatom; C, formation of the auxosome and the trophosome outside and inside the host cell without flagella retraction; D, the globular auxosome grows with host materials supplied by the trophosome. Additional pair of flagella generated before the auxosome division; E, the first division of the auxosome producing a flagellated mother cell (FMC, arrow); F, the FMC (arrow) detached from the auxosome; G, the division of the detached FMC (arrow) and the second division of the auxosome; H, the two motile zoospores (arrowheads) produced from the division of the detached FMC. The two FMCs (arrow) produced from the second division of the auxosome. The morphology and developmental characteristics of P. chaetoceri were compared with those of P. mucosa at each developmental stage.
Host range and specificity
One of the interesting findings from our results was the interstrain variation in susceptibility to parasitoid infections. This suggests that when host populations with relatively lower susceptibility predominate, biological control through parasitoid infections in the host population may be weakened, potentially leading to changes in host population dynamics as they interact with other environmental factors.
The two Pseudopirsonia species, P. chaetoceri and P. mucosa, exhibited different host ranges (Supplementary Table S2). According to a previous study, P. mucosa has a wide host range, infecting diatoms across six species, four genera, three families, three orders, and two classes (Kühn et al. 2004). Our cross-infection results showed that P. chaetoceri infected several Chaetoceros species and its phylogenetically close relative of the genus Chaetoceros, H. chinesis. The phylogenetically related hosts may share traits because they have inherited them from a common ancestor, making them more susceptible to the parasitoid infections (Ives and Godfray 2006, Heimpel et al. 2021). Although it remains unknown whether the diatoms C. constrictus and H. chinesis are susceptible to the parasitoid P. mucosa, P. chaetoceri did not infect the five diatom species susceptible to the parasitoid P. mucosa, such as Guinardia delicatula, G. flagccida, Leptocylindrus danicus, Rhisozolenia imbricata, and R. setigera. In addition, the diatom Coscinodiscus wailesii was reported to be the host species of the unidentified Pseudopirsonia sp. from field observations; however, both parasitoids, P. chaetoceri and P. mucosa, could not infect the diatom species (Kim et al. 2017). Thus, all three Pseudopirsonia species exhibited different host ranges and specificities, supporting that they are different species. This suggested that host range and specificity could serve as useful traits for distinguishing between Pseudopirsonia species.
Genetic distance of SSU rDNA
Despite the substantial differences between P. chaetoceri and P. mucosa in terms of morphology, development, and host range, the genetic distance of SSU rRNA gene sequences between the two species presented only a subtle difference (0.7% dissimilarity). As previously reported for several other free-living protists (Weisse 2007, Scoble and Cavalier-Smith 2014, Kudryavtsev and Gladkikh 2017), the SSU rRNA gene region may not be sufficiently variable to distinguish Pseudopirsonia species. To obtain better resolution at the species level, alternative molecular markers or genetic regions, such as the identification of compensatory base changes from predicted ITS2 secondary structures (Bachvaroff et al. 2012, Scoble and Cavalier-Smith 2014) and mitochondrial genes (i.e., cox) possessing highly variable informative regions (Heger et al. 2011, Lee et al. 2015), may be required to distinguish Pseudopirsonia species.
The unidentified Pseudopirsonia sp. isolate PsND01 from Korea shared some developmental traits with P. chaetoceri and P. mucosa, in that it formed auxosomes and trophosomes and fused adjacent trophosomes when multiple zoospores infected a single host cell, as presented in P. chaetoceri (Kim et al. 2017). However, the unidentified Pseudopirsonia sp. (MF615236) showed far greater genetic distance from the former two Pseudopirsonia species, as it exhibited 7.3 and 7.7% dissimilarity with SSU rRNA sequences of P. chaetoceri and P. mucosa, respectively. Furthermore, Pseudopirsonia sp. branched a sister lineage to the clade of P. chaetoceri and P. mucosa in maximum likelihood (ML) phylogeny but supported by a low bootstrap value (ML 63%). In the case of the two cercozoan parasite genera of marine diatoms, Protaspa and Cryothecomonas, belonging to Cryomonadida, they showed only 3.3% dissimilarity in SSU rRNA gene sequences (Chantangsi and Leander 2010). Therefore, the large genetic distance between Pseudopirsonia sp. and the other two Pseudopirsonia species may be sufficient to be considered at a different genus level. Further investigation of detailed morphological and developmental features and the host range of the unidentified Pseudopirsonia species is required to delimit species or genus boundaries to prove this.
Phylogenetic relationships of the order Marimonadida
The order Marimonadida (Imbricatea, Cercoza) encompasses four nominal genera of marine flagellates, Abollifer, Auranticordis, Cyranomonas, and Pseudopirsonia (Howe et al. 2011, Cavallier-Smith et al. 2018). They are non-scaly, non-thecate marine zooflagellates with a gliding movement that thrive in marine habitats (Kühn et al. 2004, Chantangsi et al. 2008, Shiratori et al. 2014, Lee and Park 2016). Our phylogenetic analyses inferred from SSU rRNA gene sequences revealed that Pseudopirsonia species fell within the order Marimonarida, forming a monophyletic clade and branching as a sister lineage to the cercozoans A. quadriverberis and C. australis with a high bootstrap support (ML 85%). The cercozoan Abollifer globosa branched into the basal lineage of a clade of Marimonadida. Despite their monophyletic close relationships, the morphology and feeding behavior of Pseudopirsonia species are distinct from those of other cercozoans in Marimonadida. The marine diatom parasitoid Pseudopirsonia is a gliding biflagellate with two subapically or medianly inserted flagella and a ventral groove that emits pseudopodia (Kühn et al. 2004). In contrast, A. quadriverberis is a large grooved gliding marine interstitial tetraflagellate with four recurrent posterior-directed flagella that contains photosynthetic endosymbionts derived from cyanobacterial prey (Chantangsi et al. 2008). The cercozoan C. australis has two unequally thickened flagella, shows a jerking process during gliding, and lacks pseudopodia (Lee and Park 2016). The latter two species feed on prokaryotes and lack pseudopodia (Chantangsi et al. 2008, Howe et al. 2011, Lee and Park 2016). The basal species, Abollifer cells, have two unequal flagella that emerge from a deep subapical flagellar pit and sometimes produce a lobose pseudopodium (Shiratori et al. 2014). They feed on marine diatoms, such as Skeletonema costatum, Thalassiosira sp., and Cyclotella sp., through a phagotrophic process (Kashiyama et al. 2012, Shiratori et al. 2014). The four genera of Marimonadida have not been found to have a prominent morphological synapomorphy, but the two cercozoan genera Pseudopirsonia and the basal Abollifer share some morphological features in that they are gliding posteriorly directed biflagellates with having a ventral groove and pseudopodia and feed on marine diatoms. Further detailed ultrastructural studies, including the reconstruction of the flagellar apparatus in other cercozoans, will provide a better understanding of the evolution of Marimonadida.
Revision of diagnosis of the genus Pseudopirsonia Kühn, Medlin, & Eller 2004
Obligate parasitoid nanoflagellates that infect marine diatoms, cells with an apical nose, two unequal flagella, attachment using a pseudopodium, feed using a trophosome and the auxosomes are globular, movement gliding, and refractive granules in the cytoplasm.
Pseudopirsonia chaetoceri Cho et Kim sp. nov. (Figs 1 & 2)
Description
Zoospore oval to oblong shaped, 7.1–10.0 μm long and 3.1–5.0 μm wide, two flagella subapically inserted, anterior flagellum 10.8–15.9 μm long and posterior 23.2–34.4 μm long, flagella never completely retracted throughout the life cycle, pseudopodium emitting from a ventral groove, penetrated poroids of seta or a rimoportulae of valve face of diatoms Hemiaulus chinensis and Chaetoceros spp.
Etymology
This species epithet chaetoceri refered to the host genus name Chaetoceros.
Habitat
Marine.
Type locality
Yongho Harbor, Busan, Republic of Korea (35°08′00″ N, 129°06′55″ E).
Type material
Glutaraldehyde-fixed material prepared from clonal strain MPL-CcPpc01 was deposited in the Nakdonggang National Institute of Biological Resources, Sangju, Republic of Korea (accession No. NNIBRPR27202).
Type host
Chaetoceros constrictus gran, 1897.
ACKNOWLEDGEMENTS
This research was supported by Korea Institute of Marine Science & Technology (KIMST) funded by the Ministry of Oceans and Fisheries (RS-2023-00256330; development of risk managing technology tackling ocean and fisheries crisis around Korean Peninsula by Kuroshio Current) and by grant from the National Research Foundation (NRF) (2022M316A1085991, RS-2024-00351443). This research was also supported by a grant from the Honam National Institute of Biological Resources (HNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (HNIBR202101111).
Notes
The authors declare that they have no potential conflicts of interest.
SUPPLEMENTARY MATERIALS
Genetic distance (number of base difference) between Pseudopirsonia species based on 1,647 positions of SSU rRNA gene region (https://www.e-algae.org)
Host range of Pseudopirsonia chaetoceri n. sp. and P. mucosa (https://www.e-algae.org)
Infection process of Pseudopirsonia chaetoceri n. sp. (https://www.e-algae.org).