The Parvilucifera infectans strain PAinf-LOHABE01 and its athecate dinoflagellate host A. sanguinea strain As-LOHABE07 were isolated from plankton samples (20 μm mesh net) taken in Masan Bay, Korea (35°12′ N, 128°34′ E) on Apr 28, 2015. The concentrated samples were examined by using an inverted microscope (Leica DM LED; Leica Microsystems, Wetzlar, Germany) to find dinoflagellates infected by P. infectans. An infected A. sanguinea cell was isolated using a drawn glass micropipette, washed six times in syringe filtered sample water (0.45 μm pore size; Advantec, Tokyo, Japan), and then transferred to a well of a 24-well plate (SPL Life Sciences, Pocheon, Korea) containing 1 mL of stock culture of A. sanguinea strain As-LOHABE03. The P. infectans was propagated by sequentially transferring either aliquots of infected A. sanguinea cells or sporangia to uninfected host culture. Stock culture of the parasite was maintained in plant culture dishes (SPL Life Sciences) and grown in 30 psu f/2-Si medium (Guillard and Ryther 1962) at 20°C under a 14 : 10 h light : dark cycle with cool-white fluorescent light providing 100 μmol photons m⁻² s⁻¹. Later, host culture of the parasite was replaced with host culture of A. sanguinea strain As-LOHABE07, which co-occurred with the parasite in the original field sample. Uninfected A. sanguinea strain As-LOHABE07 was also isolated using the same protocol described above and maintained as host stock culture under the same growth condition described above.

For light microscopic observations of parasite morphology and development, A. sanguinea stock cultures in exponential growth were inoculated with recently formed zoospores and incubated for 3 d under growth conditions described above. To obtain recently formed zoospores, stock parasite culture consisting only of mature sporangia was induced to germinate by addition of syringe filtered (0.45 μm pore size; Advantec), host culture medium. Germinated zoospores accumulated near the surface of the culture dish 1 h after addition of host culture medium were harvested using a pipette. Light microscopic images of live infected host cells and mature sporangia were taken using an AxioCam HRc (Carl Zeiss Inc., Hallbergmoos, Germany) photomicrographic system coupled to an Axio Imager A2 (Carl Zeiss Inc.) equipped with differential interference contrast optics and epifluorescence capability. Images were obtained at 12, 24, 36, 49, and 70 h after inoculation. At each time, aliquots of infected host culture were fixed with 2% glutaraldehyde (final concentration), and stained with 5% SYBR Gold (final concentration) (Molecular Probes, Eugene, OR, USA) for 1 h at 4°C, and photographed using epifluorescence microscopy with blue light excitation (Filter Set 09; excitation BP 450–490, beam splitter FT 510, emission LP 515).

Cultures infected by recently formed zoospores at each time were fixed in 2% glutaraldehyde (final concentration) with 0.2 M cacodylate buffer at pH 7.4 for 1 h at 4°C. Cells were then filtered onto Isopore membrane filters (10 μm pore size for sporangia and 0.8 μm for zoospores; Millipore, Cork, Ireland), washed in distilled water for 1 h and dehydrated in a graded ethanol series (25, 50, 70, and 99%) for 12 min at each step, and then rinsed three times in absolute ethanol for each 12 min. Samples were critical point dried in liquid CO₂ using a HCP-2 (Hitachi, Tokyo, Japan). Filters were subsequently glued to SEM stubs with carbon tape, sputter-coated with platinum and examined with a Hitachi FE-SEM (model S-4700; Hitachi) scanning electron microscope operating at 15 kV.

For whole mount, germinated zoospores from single sporangium into droplet were loaded onto formvar coated copper grids using a pipette. These were deposited at the gird, rinsed in distilled water, dried and examined using a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). For TEM, aliquots of sample at each time were mixed 1 : 1 with cold 4% glutaraldehyde in f/2-Si culture medium buffered with 0.2 M cacodylate buffer at pH 7.4 at 4°C and centrifuged for 5 min at 2,000 ×g to form pellets and remove the supernatant. The samples were rinsed three times in filtered seawater, post-fixed in 1% OsO₄ for 1 h at 4°C and rinsed three times in sterile deionized distilled water. Dehydration was carried out in a graded ethanol series of 50, 60, 70, 80, and 90% for 10 min each and then rinsed three times in absolute ethanol at 4°C. Pellets were then brought to room temperature and transferred through propylene oxide for 5 min, 50% Spurr’s embedding resins (Electron Microscopy Sciences, Hatfield, England) in propylene oxide for 1 h, 75% for 1 h, and 100% overnight. At the following day, pellets were transfer to new pure resin and polymerized at 70°C. Blocks were thin-sectioned on a PT-X ultramicrotome (RMC Products; Boeckeler Instruments, Tucson, AZ, USA). Sections were collected on slot grids, stained with 3% uranyl acetate and Reynold’s lead citrate, and examined using a JEM-1010 transmission electron microscope (JEOL).

Recently formed (≤1 h) zoospores were added to a 24-well plate containing exponentially growing A. sanguinea host (1 × 10³ mL⁻¹) at zoospore: host ratio of 10 : 1 to induce multiple infections. After incubation of 2 d under the same growth condition described above, the diameter and number of every sporangium (n = 51) inside host were determined at ×400 magnification using an AxioCam HRc photomicrographic system coupled to an Axio Imager A2.

For measurement of the number and diameter of aperture in sporangium, 7-day-old empty sporangia (n = 14), which already completed the germination of zoospores, were individually picked up using a micropipette and those parameters were determined using the same photomicrographic system above at 1,000 magnification.

Germination of mature sporangia was induced by the addition of syringe-filtered (0.45 μm pore size; Advantec), host culture medium. The number of zoospores that escaped from individual sporangia was determined either from video recordings of each specimen in vivo (n = 31), or direct enumeration of zoospores following fixation (n = 7). For in vivo observations, germination of single mature sporangia was recorded using an eXcope X3 digital camera (DIXI Optics, Daejeon, Korea) coupled to an inverted microscope (Leica DM LED). The zoospores were then counted while replaying the video at 0.5× normal speed. For enumeration following fixation, single mature sporangia were put into a hemocytometer and allowed to germinate. Shortly after the completion of germination, zoospores were fixed with acid Lugol’s solution and counted under an inverted microscope (BX51; Olympus, Tokyo, Japan) at ×200 or ×400 magnifications. In addition, the diameter of every sporangium was measured using eXcope X3 digital camera coupled to an inverted microscope.

A single, mature parasite sporangium was isolated from stock culture using a micropipette, washed six times with sterile filtered seawater, crushed manually using the tip of the pipette, placed into a polymerase chain reaction (PCR) tube, and stored at about −80°C for 1 h. Sample was thawed at room temperature for 5 min, and DNA was extracted using Chelex 100 resin (100–200 mesh, sodium form; Bio-Rad Laboratories, Hercules, CA, USA). Fifty microliters of 10% Chelex solution was added into the PCR tube containing the sporangium and incubated at 95°C for 1 h. The tube was then centrifuged at 8,000 rpm at 4°C for 1 min. DNA present in the supernatant was transferred to a new PCR tube and stored at −20°C until analyzed.

PCR amplifications were performed with eukaryotic primers EukA and EukB (Medlin et al. 1988) for SSU rRNA gene, primers D1R and 28-1483R (Scholin et al. 1994, Daugbjerg et al. 2000) for LSU rRNA gene, primers ITS1 and ITS4 (White et al. 1990) for ITS region, and primers β-tubulinF and β-tubulinR for β-tubulin gene (Turon et al. 2015). PCRs were run in 20 μL of reaction solution containing 2 μL of DNA as a template, 2 μL of 10× Taq reaction buffer (containing 25 mM MgCl₂), 0.4 μL of dNTP mix (10 mM), 2 μL of each primer (3 pmole μL⁻¹), 0.1 μL of Taq DNA polymerase (5 U μL⁻¹, DT16-R500; SolGent Co., Daejeon, Korea). The reactions were conducted using an automated thermocycler (C1000 Touch Thermal Cycler; Bio-Rad Laboratories) with the following conditions: for the SSU rRNA gene, an initial denaturing step at 94°C for 5 min followed by 35 cycles (95°C for 45 s, 55°C for 1 min, and 72°C for 3 min), with a final extension at 72°C for 10 min; for the LSU rRNA gene, an initial denaturing step at 95°C for 4 min followed by 40 cycles (95°C for 45 s, 52°C for 45 s, and 72°C for 2 min), with a final extension at 72°C for 7 min; for the ITS gene, an initial denaturing step at 95°C for 5 min followed by 40 cycles (95°C for 1 min, 55°C for 45 s, and 72°C for 1 min 15 s), with a final extension at 72°C for 7 min. PCR for β-tubulin were run in 20 μL of 2× TOPsimple PreMIX-HOT (Enzynomics, Daejeon, Korea) containing 5 μL of DNA as a template, 1 μL of each primer (5 pmole μL⁻¹). PCR condition was conducted as initial denaturing step at 95°C for 10 min followed by 35 cycles (95°C for 30 s, 51.6°C for 45 s, and 72°C for 1 min), with a final extension at 72°C for 5 min. Aliquots of the amplified PCR products were electrophoresed for 25 min at 100 V in a EcoDye (SolGent Co.) stained 1% agarose gels and then visualized under UV illumination. Amplified PCR products were purified with a PCR purification kit (Bioneer, Daejeon, Korea) and sequenced with primers (EukA and EukB for SSU rRNA genes, D1R and 28-1483R for LSU rRNA gene, ITS1 and ITS4 for ITS gene, and β-tubulinF and β-tubulinR for β-tubulin gene) using a Big-Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3730xl Analyzer (Applied Biosystems), according to manufacturer’s protocols at Macrogen Corp. (Seoul, Korea). The amplicons were sequenced until double stranded coverage was reached. ContigExpress (Vector NTI version 10.1; Invitrogen, Grand Island, NY, USA) was used to assemble the individual sequence reads and edit out low quality regions. The assembled sequences were verified by comparison using BLASTN search in the NCBI database and deposited in GenBank (accession No. MG189592 for SSU rDNA; MG189902 for ITS; MG209369 for LSU rDNA; MG229892 for β-tubulin).

The obtained sequences, which are SSU, LSU, ITS, and β-tubulin region, were aligned with related sequences from GenBank database using ClustalW 1.6 (Thompson et al. 1994) and were manually refined using MacGDE 2.4 (Linton 2005). Ambiguously aligned positions were removed and final alignments of 1,499 positions for the SSU region, 815 positions for LSU region, and 2,398 positions for SSU + LSU region were selected. For β-tubulin dataset, both noncoding DNA sequences and ambiguously aligned positions were excluded, and final alignments of 660 positions were obtained. Phylogenetic trees were inferred using the maximum likelihood (ML) and Bayesian inference methods. Modeltest v.3.7 (Posada and Crandall 1998) was performed to select the most appropriate model of substitution for the ML method in PAUP. ML analyses were performed using RAxML (Stamatakis 2006) with the rapid bootstrapping option and 2,000 replicates. The GTRGAMMA evolution model was selected from MODELTEST using RAxML. TrN + I + G (−ln L = 13,743.2119), TrN + I + G (−ln L = 7115.9648), TrN + I + G (−ln L = 18996.6191), and TrNef + I + G (−ln L = 3138.8420) models were selected for SSU, LSU, SSU + LSU and β-tubulin datasets, respectively (Posada and Crandall 1998). Bayesian analysis was performed with MrBayes 3.1.1 (Ronquist et al. 2012) running four simultaneous Monte Carlo Markov Chains for 2,000,000 generations and sampling every 100 generations, following a burn in of 2,000 generations.

The complete life-cycle and development of the Korean isolate of Parvilucifera were studies under light microscopy, SEM and TEM using A. sanguinea strain As-LOHABE07 as a host.

Infection occurred when a free-living zoospore penetrated into the host cell through the flagellar pore region (Fig. 1), during which the host lost its flagella and sank to the bottom of the culture flask. When exposed to zoospores, the penetration of several zoospores into a host was often observed, subsequently resulting in multiple infections (see below). During the penetration of zoospores, cell shape of the host A. sanguinea changed rapidly and became round. Early stages of infection were recognizable at 12 h after penetration by the presence of a round body in the cytoplasm of host cell (Fig. 2B). The round body continued to grow by consuming the host cytoplasm until it occupied most of the host cell and contained numerous peripheral lipid-like structures (Fig. 2C). At early trophocyte, it was hard to clear observe the nucleus of parasite when stained SYBR gold (Fig. 2I), due probably to the shortage of condensed genetic materials. At the late trophocyte, sporangium was released from the host, which was almost completely consumed (Fig. 2D), and at this time nucleus of the parasite was invisible when stained with SYBR gold (Fig. 2J). At the early sporocyte (49 h), a large vacuole was observed in the center of the sporangium and peripheral site was filled with zoospores as clearly seen in SYBR gold-stained sporangium (Fig. 2E & K). During the late stage of infection (70 h), the sporangium was full of numerous zoospores and the mature sporangium was dark-colored under the light microscope (Fig. 2F & L). Under the growth conditions described above, it took 3 days from addition of Parvilucifera zoospores to A. sanguinea to development of mature sporangia. In a single A. sanguinea cell, even up to 9 sporangia were observed to develop. When more than 1 sporangium developed, their size decreased with increasing number of sporangia (Fig. 3). The sporangium diameter ranged from 12.3 μm in case of multiple infections to 50.4 μm in single infection. When the zoospores were fully developed and ready to be escaped, they swam within the sporangium and escaped from the sporangium through several apertures. The number and diameter of aperture appeared to be dependent on sporangium diameter (Fig. 4). The number of aperture ranged from 3 to 17. The aperture was round-shaped and measured from 1.7 to 4.3 μm in diameter. Each aperture was covered by an operculum until release of the zoospores. The number of zoospores produced per sporangium was related with sporangium size (Fig. 5). When infecting A. sanguinea, minimum and maximum numbers of zoospores produced were 34 and 2,300 zoospores per sporangium, respectively.

Early trophocyte was visible inside the host cytoplasm 12 h after inoculation of zoospores (Fig. 6A). Early trophocyte grew inside the parasitophorous vacuole and contained vacuoles, a Golgi apparatus, and lipid inclusions. The mitochondria of the host were frequently observed outside parasitophorous vacuole (Fig. 7A). The trophocyte gradually grew, increasing in size while consuming the host cytoplasm and contained vacuoles, starch granules, and lipid globules at 24 h (Fig. 6B). At this time, the parasitoid plasma membrane with folded surface was visible and was covered with parasitophorous vacuole membrane. Developing processes were also found, embedding between parasitoid plasma membrane and parasitophorous vacuole membrane (Fig. 7B). At 36 h, the trophocyte occupied most part of the host body and sometimes released from the completely disintegrated host. The trophocyte was characteristic of the presence of a large vacuole in the central part, together with several starch granules, lipid globules, and a large nucleus (Fig. 6C). The outer layer was still connected to the distal ends of processes. At this time, sporangium wall was observed to be clearly consisting of four-layers (i.e., innermost, middle, transparent, and outer layers). The transparent layer became more evident at 36 h, compared to that at 24 h. Invagination of the pellicle underneath innermost layer started to occur, probably for formation of operculum (Fig. 7C). At this stage, the released sporangium was characterized by covering with an intact outer layer as well as remnant of host materials (Fig. 7F). At 49 h, the division of nuclei and cellular organelles was observed (Fig. 6D & E). The outer layer of sporangium started to degrade at this time (Fig. 7G). At 70 h, sporangium was filled with numerous immature zoospores and pellicles were observed between zoospores (Fig. 6F & G). The innermost layer was cut off to form operculum (Fig. 7D). At this time, the outer layer continued to degrade and it was easy to observe processes on the surface of sporangium (Fig. 7H). Mature sporangium was filled with fully developed zoospores and residual body (Fig. 6H). At 1-week-old sporangium, the outer layer fully disappeared and even processes were detached (Fig. 7E & I).

The zooids just germinated from sporangium had an elongated body (5.31 ± 0.35 μm in length, 1.38 ± 0.09 μm in width, n = 14 using SEM). Swimming zoospores, which had a large refractile body and a long flagellum, were easily visible under the light microscope (Fig. 8B). SEM and whole mounts clearly revealed the presence of two flagella of unequal size (Fig. 8A, D & E). The short flagellum measured 2.79 ± 0.19 μm (n = 7), was bare, and had a proximal swelling (Fig. 8D). The short flagellum was located quite close to the cell body and was difficult to observe under light microscope. The long flagellum measured 12.77 ± 0.67 μm (n = 3) and terminated in a short conical tip (Fig. 8D & E). The long flagellum had hairs (Fig. 8E), but its hairs were rarely visible in the whole mounts, unlike the absence in SEM. The two flagella were located at near the central part of the body (Fig. 8A & D). Zoospore had a large mitochondrion and an elongated nucleus at ventral side in the middle of the cell. A large refractile body was located at the posterior end of zoospore and lipid globules were observed in zoospore (Fig. 8C).

From 10 individually isolated single sporangium, partial SSU rDNA sequences of 1,421 bp, partial ITS rDNA sequences of 463 bp, partial LSU rDNA sequences of 1,176 bp, and β-tubulin sequences of 957 bp (319 amino acids) were obtained, with 100% identity among the parasites. Phylogenies based on the SSU rDNA (Fig. 9), LSU rDNA (Fig. 10), SSU rDNA + LSU rDNA (Supplementary Fig. S1), and β-tubulin (Fig. 11) gene sequences provided overall congruent tree topologies that coincided with the ones already published for those genes (Figueroa et al. 2008, Lepelletier et al. 2014b, Turon et al. 2015, Reñé et al. 2017a, 2017b) although phylogenetic position among Parvilucifera species changed in the LSU rDNA phylogeny. The Korean isolate of Parvilucifera was included in a clade that contained the sequences of P. infectans / P. sinerae species complex in all phylogenies, with a high bootstrap support (ML, 100%) or a posterior probabilities (PP) of 1. In all trees, all members of Parviluciferaceae (Parvilucifera, Snorkelia, and Dinovorax) known so far and Perkinsus species clustered together as a clade corresponding to Perkinsozoa, but had low statistical supports. In the case of β-tubulin, the Korean isolate grouped with the sequence of P. sinerae, with strong bootstrap support (ML, 100%) or a PP of 1 (Fig. 11).

The percentage of genetic similarities between the rDNA and β-tubulin sequences of Korean isolate of Parvilucifera and those of members of Parviluciferaceae is shown in Table 1. The SSU rDNA sequence (AF133909) of P. infectans in GenBank deposited by Norén et al. (1999) had 100% identity with that of P. infectans specimen collected at the same location (Kristineberg Marine Research Station, Sweden) as the type species (F. Norén personal communication). The SSU rDNA sequence of the Korean isolate had 99.1% similarity (23 nucleotides difference) with those of P. infectans (AF133909) reported by Norén et al. (1999) as well as P. infectans specimen from type locality and 99.5% similarity (7 nucleotides difference) with the P. infectans sequence (KF359485) reported by Lepelletier et al. (2014b), but it was 100% identical to the P. sinerae sequence (KM878667) reported by Turon et al. (2015). The SSU rDNA sequence of the Korean isolate, however, differed from those of P. rostrata (KF359483), P. corolla (KX519760), Snorkelia prorocentri (FJ424512), and Dinovorax pyriformis (MF197551), with 87.7, 85.7, 80.3, and 80.5% similarities, respectively. The LSU rDNA sequence of the Korean isolate was all 100% identical to those of P. infectans (KF359486), P. infectans specimen collected from the same area as type locality (F. Norén personal communication), and P. sinerae (KM878666), but it differed from the sequences of P. rostrata (KF359484) (68% similarity), P. corolla (MF197555) (49.9% similarity), and D. pyriformis (MF197553) (49% similarity). The ITS region of the Korean isolate sequence had 100% similarity with the sequences of both P. infectans (KF359485) published by Lepelletier et al. (2014b) and P. sinerae (KM878665) published by Turon et al. (2015), but had 54% similarity with the sequence of P. rostrata (KF359483). In the case of β-tubulin, the sequence of the Korean isolate was 100% identical to that of P. sinerae (KM878668) reported by Turon et al. (2015).