Introduction
Schizogony is an asexual mode of reproduction in which multiple daughter cells are produced by the mother cell. This mode is employed by many protozoan parasites (Batra and Suvorova, Reference Batra and Suvorova2025). The canonical eukaryotic cell cycle consists of the G1 phase for cell growth and preparation for DNA synthesis, the S phase for DNA replication, the G2 phase for checking the replicated DNA and preparing for the M phase, and the M phase for both karyokinesis and cytokinesis. However, in some species, this characteristic schizogony appears to lack a G2 phase due to the continual, cyclical use of the S phase (Gerald et al., Reference Gerald, Mahajan and Kumar2011; Guttery et al., Reference Guttery, Zeeshan, Holder and Tewari2024).
In some parasitic protozoan species, schizogony is regulated by host-derived factors. In malarial parasites such as Plasmodium falciparum, the transition from G1 to S phase depends on host-derived isoleucine, reminiscent of nutrient-dependent cell cycle checkpoints described in other organisms (Babbitt et al., Reference Babbitt, Altenhofen, Cobbold, Istvan, Fennell, Doerig, Llinas and Goldberg2012; McLean and Jacobs-Lorena, Reference McLean and Jacobs-Lorena2020). This may represent an adaptive evolutionary strategy that prevents unnecessary proliferation by allowing division only under suitable conditions or that enables long-term parasitism by suppressing proliferation when the host is in poor physiological conditions.
Protozoans of the genus Perkinsus, which infect marine molluscs and often cause serious adverse impacts, proliferate within their hosts by schizogony, generating parasitic-stage trophozoites (Mackin et al., Reference Mackin, Owen and Collier1950; Azevedo, Reference Azevedo1989; Dungan and Reece, Reference Dungan and Reece2006). A recent in vitro study showed that schizogony in Perkinsus species is promoted by glycine betaine (GB; Liu et al., Reference Liu, Ninomiya, Yoshinaga and Itoh2023). Notably, in Perkinsus olseni and P. beihaiensis, proliferation was not promoted by GB precursors such as choline chloride and betaine aldehyde chloride, suggesting that these organisms depend on the GB abundantly supplied by the host molluscs for proliferation (Liu et al., Reference Liu, Ninomiya, Yoshinaga and Itoh2023).
GB, also known as trimethylglycine, serves as an osmolyte and osmoprotectant for macromolecules in various organisms (Lever and Slow, Reference Lever and Slow2010; Fedotova and Kruchinin, Reference Fedotova and Kruchinin2017) as well as a carbon source for marine bacteria (Mausz et al., Reference Mausz, Airs, Dixon, Widdicombe, Tarran, Polimene, Dashfield, Beale, Scanlan and Chen2022). Moreover, GB acts as a methyl donor in homocysteine metabolism (Lever and Slow, Reference Lever and Slow2010), and GB-associated metabolites participate in various biochemical processes (Kathirvel et al., Reference Kathirvel, Morgan, Nandgiri, Sandoval, Jayapal, Zern and Sanyal2010; Li et al., Reference Li, Wang, Wang, Wang and Feng2017; Boysen et al., Reference Boysen, Durham, Kumler, Follows, Kujawinski and Moran2022). Therefore, the GB may be involved in various biological regulatory processes (Figueroa-Soto and Valenzuela-Soto, Reference Figueroa-Soto and Valenzuela-Soto2018). In the context of cell division, GB inhibits the proliferation of cancer cells (Gerile et al., Reference Gerile, Sogawa, Ohyama, Masuko, Kusama, Morita, Sogawa and Kitayama2012; Bingula et al., Reference Bingula, Dupuis, Pichon, Berthon, Filaire, Pigeon and Filaire2016; López-Castro et al., Reference López-Castro, Rosas-Rodríguez, Icedo-García, Stephens-Camacho and Gonzalez-Ochoa2025) and promotes the proliferation of intramuscular adipocytes, potentially through increased NADPH (nicotinamide adenine dinucleotide phosphate) levels (Liu et al., Reference Liu, Yang, Liu, Liu, Zhu, Huang and Wang2025). However, the involvement of the GB in the characteristic schizogonic proliferation of protozoan parasites has never been reported, except in the aforementioned study (Liu et al., Reference Liu, Ninomiya, Yoshinaga and Itoh2023), and the underlying mechanism remains unknown.
Therefore, the present study investigated the GB-mediated regulatory mechanism underlying schizogony in P. olseni, with the aim of elucidating which stage of the schizogony process – cell growth, DNA replication or daughter cell formation downstream of DNA replication – is regulated by GB.
Materials and methods
Parasite isolate and culture conditions
P. olseni isolate PRA-181, originating from the Manila clam Ruditapes philippinarum in Japan, was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The previous study showed that this isolate exhibited robust proliferation in media supplemented with FBS (Fetal Bovine Serum), host clam tissue extract or GB, whereas supplementation with the GB precursors, choline chloride and betaine aldehyde chloride, did not result in comparable proliferation (Liu et al., Reference Liu, Ninomiya, Yoshinaga and Itoh2023).
In addition to Perkinsus Broth Medium (PBM; ATCC medium 1886) for proliferation of the isolate, we prepared PBMΔF, a medium composed of PBM components without FBS (known to contain GB; Artegoitia et al., Reference Artegoitia, Middleton, Harte, Campagna and de Veth2014; Foroutan et al., Reference Foroutan, Fitzsimmons, Mandal, Piri-Moghadam, Zheng, Guo, Li, LL and DS2020), to evaluate the effect of GB on proliferation. In brief, PBM was prepared by adding 20 mL of FBS per litre, whereas PBMΔF was prepared identically except that FBS was not added. These media were sterilized using 0.22 μm filters, and 2% (v/v) antibiotic solution (50 mg mL−1 streptomycin and 50 000 units mL−1 benzylpenicillin potassium) was added before use.
To support parasite proliferation, the isolate was inoculated into 10 mL of PBM in 25 cm2 flasks maintained at 25 °C, and 200–300 μL of the old culture was transferred to 10 mL of fresh PBM every 2–3 weeks for subcultures.
Effects of GB on the trophozoite proliferation of P. olseni
To investigate the effects of GB, P. olseni cells were inoculated into non-GB-supplemented medium (PBMΔF) and GB-supplemented medium (PBMΔF + GB), and temporal changes in DNA content and cell division were compared.
Trophozoites of P. olseni were cultured in PBM at 25 °C and harvested during the logarithmic growth phase (approximately 3 weeks after inoculation). Cells were collected by centrifugation at 225 g for 5 min at room temperature, washed by resuspension in filtered-sterilized artificial seawater (FASW) composed of 36 g L−1 Daigo’s Artificial Seawater SP for Marine Microalgae Medium (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and centrifuged as described above. After repeating this washing step twice, the cell suspension was passed through a 27-G needle 5 times to release unicellular daughter cells from schizonts, and then transferred into 75-cm2 culture flasks containing 100 mL of the GB-deficient PBMΔF for the control, or PBMΔF supplemented with 15 µg mL−1 GB (CAS Number: 107-43-7; Wako 1st grade, Fujifilm Wako Pure Chemical; PBMΔF + GB) for the experimental group, at a density of 2.0 × 105 cells mL−1. Two technical replicates of the different cell batches were prepared for each group.
During the incubation at 25 °C for 8 days, 4.5 mL of the cell suspension was collected every 24 h, and the cells pelleted by centrifugation at 250 g for 10 min were resuspended in 200 µL of FASW and stained with 0.4 µL SYBR Green I Nucleic Acid Gel Stain (10 000× concentrate in DMSO (dimethyl sulfoxide); Lonza Rockland, USA) for 10 min at room temperature to stain nuclei for fluorescent microscopic observations.
Additionally, to evaluate DNA content by flow cytometry, cells in another 4.5 mL of the suspension were pelleted by centrifugation, resuspended in 200 µL of FASW containing 0.25% (v/v) glutaraldehyde for fixation and stored at 4 °C until use. The nuclei of the fixed trophozoites were stained with SYBR Green I overnight, and the cells were resuspended in 4 mL of FASW before being subjected to flow cytometry (CyFlow Space System, Sysmex-Partec GmbH, Görlitz, Germany) equipped with a 488 nm blue laser. Side-scatter parameters were used to discriminate single cells from the debris and aggregates. DNA content was quantified based on the green fluorescence detected in the FL1 channel (excitation: 488 nm). Raw FCS files obtained from flow cytometry were imported using the read.FCS function in the flowCore package (Bioconductor) in R (version 4.3.3), and preprocessed for fluorescence signal extraction. The DNA content of each sample was expressed as log10-transformed fluorescence intensity (FI) values. Subsequently, data visualization and density plotting were performed using the ggplot2 package in R.
Temporal analysis of proliferation affected by GB
To determine the time point at which GB exerts the greatest proliferative effect, a GB withdrawal assay was conducted. Briefly, trophozoites of P. olseni in the logarithmic growth phase of PBM were harvested, washed and dispersed as described above. The cells were suspended in 15 mL of PBMΔF + GB at a density of 2.0 × 105 cells mL−1, and incubated in 25-cm2 culture flasks at 25 °C in the dark for 8 days. Three flasks were used as technical replicates.
Every 12 h until 132 h after inoculation, 1 mL of the medium from each flask was collected and centrifuged at 250 g for 5 min to pellet the cells, which were then washed twice with FASW to remove the GB. These cells were resuspended in 1 mL of fresh PBMΔF and incubated in a 24-well plate at 25 °C in the dark. The total incubation period in PBMΔF + GB and PBMΔF was fixed at 192 h (8 days). As an example, cells incubated in PBMΔF + GB for the first 12 h were subsequently cultured in PBMΔF for the remaining 180 h. To evaluate the maximum cell proliferation in PBMΔF + GB, the experimental group incubated in PBMΔF + GB for the entire 192-h period was also prepared, whereas the group incubated in PBMΔF for the entire period (i.e. 0 h in PBMΔF + GB) was used to evaluate the minimum cell proliferation. After completion of the 192-h culture period, 100 µL of media from each well was subjected to Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assays to quantify cell proliferation as previously described (Liu et al., Reference Liu, Ninomiya, Yoshinaga and Itoh2023).
Differences in cell proliferation among multiple groups were evaluated using the Kruskal–Wallis test, followed by Dunn’s multiple comparison test with Bonferroni correction. Statistical analyses were performed using SPSS (IBM, Chicago, IL, USA).
Additionally, to quantitatively examine the timing of proliferation state changes following GB withdrawal, OD₄₅₀ values from CCK-8 assays were normalized to a 0–1 scale using min–max normalization based on the minimum and maximum mean OD values observed across the experiments. A 3-parameter logistic growth model was then fitted to the mean values of the replicates at each time point using nonlinear least-squares regression as follows:
\begin{equation*}y = \frac{{Asym}}{{1 + \exp \left( {\left( {xmid - x} \right)/scal} \right)}}\end{equation*}where Asym represents the maximum proliferation, xmid indicates the time required to reach 50% of the maximum proliferation (t 50) and scal represents the steepness of the curve. Curve fitting was performed in R using the NLS function with a self-starting SSlogis model. The standard error (SE) of t 50 was calculated from the parameter covariance matrix in the nonlinear regression summary.
Restoration of cell division in nutrient-deficient conditions by GB supplementation
To determine whether GB was involved in daughter cell formation downstream of DNA replication, P. olseni cells that had become enlarged and exhibited increased DNA content in PBMΔF were transferred to GB-supplemented seawater, and subsequent cellular changes were observed.
As described above, trophozoites of P. olseni in the logarithmic growth phase of PBM were harvested, washed and dispersed. After dispersion by passing through a 27-G needle 5 times, cells were incubated in 10 mL of PBMΔF at a density of 2.0 × 105 cells mL−1 for 12 days in 25-cm2 culture flasks.
After incubation, trophozoites were washed twice with FASW to remove PBMΔF, resuspended in 10 mL of FASW, and then 2.5 mL of the cell suspension was distributed into 4 wells of a 6-well plate. A volume of 7.5 µL of the GB solution in FASW (5 mg mL−1) was added to 2 wells at a final concentration of 15 µg mL−1 of GB, and the same volume of FASW was added to the other 2 negative control wells. The morphology and proliferation of trophozoites in both groups were assessed microscopically 4 days after incubation. At the end of the incubation, trophozoite membranes were stained with PlasMem Bright Green (Dojinbo Laboratories) and observed using differential interference contrast (DIC) and fluorescence microscopy (Olympus BX60; Olympus Corporation, Tokyo, Japan).
Time-course monitoring of exogenous GB uptake and schizogony progress
To analyse the process from external GB uptake to cell division, P. olseni cells incubated with PBMΔF were transferred to seawater containing GB, and changes in extracellular GB levels and cell division were monitored over time.
Trophozoite suspension of P. olseni pre-incubated in PBMΔF for 12 days was prepared as described above. The cell suspensions were transferred into 75-cm2 culture flasks containing 70 mL of FASW supplemented with GB at 15 µg mL−1, with the cell density adjusted to 2.0 × 105 cells mL−1. The same cell suspensions were inoculated into FASW without GB supplementation for the negative control. Three technical replicates were performed for each group.
To monitor the uptake of exogenous GB, 5 mL of FASW from the GB supplementation group was recovered at 0, 1, 4, 7, 10, 13, 16, 24, 32, 40, 48, 72, 96 and 120 h after inoculation, whereas FASW was recovered at 0, 13, 24, 72 and 120 h in the negative control group. After centrifugation at 250 g for 10 min, the supernatant was collected, passed through a 0.22 µm filter to remove P. olseni cells and stored at −20 °C until GB quantification by liquid chromatography–mass spectrometry (LC-MS).
For LC-MS, 5 mL of the FASW samples was dried in vacuo, and the residue was dissolved in 2–3 mL of methanol, loaded onto an octadecylsilyl silica gel column and eluted with methanol. After methanol was dried in vacuo, the remaining solid was dissolved in 500 µL of distilled water, and 50 µL of these samples was applied to an LC-20 HPLC system (Shimadzu, Kyoto, Japan) equipped with a CAPCELL PAK ADME column (2.1 mm I.D. × 150 mm; Shiseido, Tokyo, Japan) coupled to an amaZon SL mass spectrometer (Bruker, Billerica, MA, USA). Isocratic elution was performed with water containing 0.1% formic acid at a flow rate of 0.3 mL min−1, and GB was detected using the m/z 118 ion in positive ion mode. A solution of chemically synthesized GB (CAS Number: 107-43-7) in distilled water was used to verify retention time and mass identity, and to confirm the linearity between measured peak area and concentration.
LC-MS data were analysed based on extracted ion chromatograms of m/z 118. Peak areas were calculated using Compass DataAnalysis 4.1 software (Bruker), and the results are presented as relative signal intensities.
For morphological observations, the cells were recovered at 0, 13, 24, 48, 72, 96 and 120 h, pelleted by centrifugation and stained with PlasMem Bright Green to visualize the cell membrane, as described above.
Results
Effects of GB on the trophozoite proliferation of P. olseni
In PBMΔF + GB, microscopic observations revealed that trophozoites enlarged from Days 0 to 2, and schizonts with multiple intracellular single-nucleus daughter cells appeared on Day 3 and became dominant on Day 5. The release of daughter cells was observed from Days 6 to 8 (Figure 1). In this group, the trophozoite population exhibited a median log10 fluorescence intensity (MFI) of 2.92 immediately after inoculation (Day 0), which increased to 3.24 by Day 4. On Day 5, a subpopulation of cells with low DNA content, characterized by an FI of approximately 2.91, emerged, and by Day 6, the cell density of this low-DNA-content population exceeded that of the high-DNA-content population with MFI 3.23. By Day 8, the final day of the experiments, the overall population resembled that observed on Day 0 of inoculation, with an MFI of 2.95 (Figure 1).
Temporal changes in DNA content, cell density distribution and cell morphology of Perkinsus olseni PRA-181 in GB-supplemented (left) and GB-deficient (right) PBMΔF. The y-axis of the histogram represents the relative cell density, and the x-axis shows the fluorescence intensity (log₁₀ scale) measured in the FL1 channel. The images to the right of each histogram show differential interference contrast (DIC) and fluorescence (FL) micrographs of cells collected on each sampling day and stained with SYBR Green I. Scale bars = 20 µm.
Figure 1 Long description
Two columns labeled PBMDeltaF plus GB and PBMDeltaF, each containing eight rows representing days 0 to 8. Each row includes a histogram and two micrographs labeled DIC and FL. The histograms display relative cell density against fluorescence intensity on a logarithmic scale from 2.7 to 3.3. In the PBMDeltaF plus GB column, the histograms show changes in peak fluorescence intensity over time, with notable shifts on days 3, 5 and 6. The corresponding DIC and FL micrographs illustrate changes in cell morphology and fluorescence, with visible enlargement and increased fluorescence intensity over time. In the PBMDeltaF column, the histograms also show temporal changes in fluorescence intensity, but with different patterns compared to the supplemented group. The DIC and FL micrographs reveal variations in cell morphology and fluorescence intensity, with less pronounced changes than the supplemented group. Scale bars are present in the micrographs, indicating magnification consistency across the images.
In GB-deficient PBMΔF, trophozoites enlarged from Days 0 to 2, similarly to the trophozoites inoculated in PBMΔF + GB, and this enlargement slowly continued until the end of the experiments. Most of the cells remained unicellular until the end of the experiments, although schizonts with internal daughter cells were occasionally observed (Figure 1). The DNA content, as measured by MFI, increased from 2.92 on Day 0 to 3.14 on Day 4, then remained largely unchanged until Day 8 (MFI = 3.17). A cell population with lower DNA content did not emerge until the end of the experiments (Figure 1).
Temporal analysis of proliferation affected by GB
Trophozoites incubated in PBMΔF + GB for longer periods showed higher proliferation, and proliferation of trophozoites incubated in PBMΔF + GB longer than 96 h was comparable to that of the maximum proliferation of cells incubated in PBMΔF + GB for the entire 192 h (Figure 2A). In contrast, trophozoites incubated with PBMΔF + GB for 108 h showed significantly higher proliferation than those incubated for 0 or 24 h (p < 0.05; Figure 2A).
Proliferation of Perkinsus olseni PRA-181 pre-cultured in GB-supplemented PBMΔF for different durations. (A) Absorbance at 450 nm (OD₄₅₀), measured by CCK-8, after culturing the cells in GB-deficient PBMΔF for the remainder of the 192-h total cultivation period. The x-axis indicates the duration of pre-culture in GB-supplemented PBMΔF. Median values of the absorbance with range are shown (n = 3). (B) Logistic growth curve fitted to the relative proliferation data. Grey dots represent raw values; black dots represent means; the solid curve indicates the fitted logistic function; and the dashed line marks t₅₀.
Figure 2 Long description
A bar graph of Absorbance left parenthesis OD subscript 450 right parenthesis versus incubation time. The y axis label is, Absorbance left parenthesis OD subscript 450 right parenthesis, with a range from 0.0 to 2.0. The x axis label is, Incubation time in PBM delta F supplemented with glycine betaine left parenthesis h right parenthesis, with labeled values 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 192. Each timepoint has one bar with an error bar. The bar heights increase overall from the earliest timepoints to later timepoints. The tallest bar is at 108 h and the bars at 120 h, 132 h and 192 h are lower than 108 h and are similar to each other. The image B showing a scatter plot of Relative proliferation versus incubation time, with a fitted curve and a vertical dashed reference line. The y axis label is, Relative proliferation, with a range from 0.0 to 1.0. The x axis label is, Times incubated in PBM supplemented with glycine betaine left parenthesis h right parenthesis, with a range from 0 to 120. Text inside the plot area reads, t subscript 50 equals 68.5 h left parenthesis SE equals 9.5 h right parenthesis. A legend lists, Raw data, Mean plus fitting, Fitted curve. The plotted points and the fitted curve rise from low relative proliferation at early times to higher values at later times, with the curve flattening near the upper part of the y axis at the highest times. The vertical dashed line is placed near 70 h. Across both images, the time axis is in hours and the plotted values increase with longer incubation time, with image A showing absorbance values by timepoint and image B showing relative proliferation values with a fitted curve and the marked t subscript 50 value.
A 3-parameter logistic model provided a good fit to the proliferation data (pseudo-R 2 = 0.93; AIC (Akaike Information Criterion) = −16.45). The time at which the parasite population reached 50% of its maximal proliferation potential (t 50) was estimated as 68.5 ± 9.5 h (SE; Figure 2B).
Restoration of cell division in nutrient-deficient conditions supplemented with GB
P. olseni trophozoites immediately after incubation in PBMΔF were 6.27 ± 1.58 µm in diameter (n = 37) and had a single nucleus with a few granules (Figure 3A). After 12 days of incubation in PBMΔF, trophozoites were enlarged (12.87 ± 2.88 µm, n = 40) and swollen with prominent vacuoles, but most cells remained in a single-cell state and did not form schizonts (Figure 3B).
Restoration of schizogony of Perkinsus olseni PRA-181 pre-cultured in GB-deficient PBMΔF after transferring to GB-supplemented or GB-free FASW. (A) Trophozoites immediately after inoculation into GB-deficient PBMΔF. (B) Cells incubated in GB-deficient PBMΔF for 12 days. (C–E) Cells pre-cultured in GB-deficient PBMΔF for 12 days and subsequently cultured in GB-supplemented FASW for 4 days. Differential interference contrast image (D) and fluorescence micrograph of cells after staining the cell membrane with PlasMem Bright Green (E). (F–H) Cells pre-cultured in GB-deficient PBMΔF for 12 days and subsequently cultured in GB-free FASW for 4 days. Differential interference contrast (DIC) image (G) and fluorescence micrograph of cells after staining the cell membrane with PlasMem Bright Green (H). Scale bars = 20 µm.
These cells developed into schizonts with multiple intracellular daughter cells 4 days after transfer into FASW supplemented with GB (Figure 3C), and the daughter cell membranes were also observed within the schizonts by fluorescent membrane staining (Figure 3D and E). In contrast, the swollen trophozoites developed in PBMΔF did not exhibit morphological changes even 4 days after transfer to GB-free FASW (Figure 1F), and the formation of intracellular daughter cells with cell membranes was rarely observed in these cells (Figure 3G and H).
Time-course monitoring of exogenous GB uptake and trophozoite division
The concentration of GB in FASW drastically decreased during the first hour after inoculating trophozoites pre-incubated in PBMΔF for 12 days, then moderately declined until 13 h, and thereafter remained at low levels until the end of the observation at 120 h (Figure 4A).
Time-course monitoring of Perkinsus olseni PRA-181 pre-cultured in GB-deficient PBMΔF after exposure to GB-supplemented FASW. (A) GB concentration in FASW after inoculating P. olseni PRA-181 pre-cultured in GB-deficient PBMΔF. Points represent the mean GB concentration at each time point, with error bars indicating the standard deviation (n = 3). (B) Differential interference contrast (DIC) images and fluorescence micrographs of cells after staining the cell membrane with PlasMem Bright Green (FL) at each sampling point. The images of 120 h (-GB) were cells at 120 h after exposure to GB-free FASW. Scale bars = 20 µm.
Figure 4 Long description
A line graph with error bars and downward triangle markers above selected time points. The y-axis label is, EIC peak area, open parenthesis times 10 superscript 6, AU close parenthesis. The y-axis shows tick labels 0, 5, 10, 15, 20. The x-axis label is, Time after transfer to ASW supplemented with GB, open parenthesis h close parenthesis. The x-axis shows tick labels 0, 24, 48, 72, 96, 120. The plotted series starts near 18 at 0 h, drops to about 8 near the first point after 0 h, then declines to about 4 by around 13 h. From about 24 h through 120 h, the line stays between about 2 and 4 with small error bars. Eight time points arranged as paired micrographs per time point, each pair labeled DIC and FL. The time labels above the pairs are 0 h, 13 h, 24 h, 48 h, 72 h, 96 h, 120 h and 120 h open parenthesis minus GB close parenthesis. Each time point shows a left DIC micrograph with multiple round cells on a plain background and a right FL micrograph with multiple round bright spots on a dark background. Each micrograph includes a scale bar at the bottom right.
Schizonts containing a few intracellular daughter cells were first observed 48 h after transfer, whereas some schizonts contained numerous daughter cells (Figure 4B). After 72 h, most cells developed into schizonts with numerous daughter cells (Figure 4B).
In the negative control, in which cells were transferred to GB-free FASW, GB was not detected (data not shown), and most cells remained in a single-cell state and did not form schizonts throughout the experimental period (Figure 4B).
Discussion
The present study revealed that trophozoites of P. olseni PRA-181 increased in cell size and replicated intracellular DNA, but most cells did not form intracellular daughter cells in the GB-deficient medium PBMΔF. The effects of GB supplementation were strongest at 68.5 ± 9.5 h after exposure to GB, which roughly corresponded to the initiation timing of daughter cell formation. Trophozoites pre-incubated in PBMΔF for 12 days exhibited daughter cell formation after exposure to GB, even under nutrient-free seawater conditions. Trophozoites pre-incubated in PBMΔF incorporated GB from FASW within 13 h and initiated daughter cell formation within 48 h. Thereafter, daughter cell formation continued, whereas the surrounding GB was not further depleted, indicating that GB regulated the initiation of intracellular daughter cell formation but not nutrient uptake, cell enlargement or DNA replication in P. olseni.
The trophozoites of P. olseni showed an increase in cell size and intracellular DNA content by Day 3, regardless of the presence or absence of GB in PBMΔF. However, whereas an increase in DNA associated with daughter cell formation and a cell population with lower DNA content attributable to the release of daughter cells were observed in PBMΔF + GB, these phenomena were not observed in PBMΔF. Thus, it was suggested that GB was involved in the formation of internal daughter cells during schizogonic proliferation, but not in cell enlargement or DNA replication.
GB withdrawal assays showed that the effects of GB were most pronounced approximately 68.5 h after exposure to GB, which was later than the initiation of cell enlargement and increase in intracellular DNA content, but roughly corresponded to the timing of the initiation of daughter cell formation on Day 3 (72 h) in the previous experiments, further supporting that GB was not involved in the earlier steps of cell enlargement or DNA replication, but was involved in the initiation of internal cell division processes downstream of DNA replication (i.e. karyokinesis or cytokinesis) to produce daughter cells.
Trophozoites pre-incubated in PBMΔF for 12 days exhibited daughter cell formation within 4 days after transferring into GB-supplemented FASW. This result indicated that P. olseni cells completed the uptake of nutrients required for cell enlargement and DNA replication and remained alive in PBMΔF for at least 12 days, while halting daughter cell formation under GB-deficient conditions, further supporting the involvement of GB in the processes downstream of DNA replication that produce daughter cells. GB also played a role in cell division in parasite cells rather than in extracellular functions, such as stabilizing biomacromolecules present in the culture medium.
The concentration of GB in FASW decreased rapidly within 13 h when the parasite cells pre-cultured in PBMΔF for 12 days were transferred, and daughter cell formation was observed from 48 h, strongly indicating that the trophozoites rapidly took up extracellular GB and that GB regulated daughter cell formation. Moreover, because daughter cell formation continued even when the GB concentration remained low, GB was considered to critically regulate the initial cell division processes downstream of DNA replication to produce the first daughter cell.
The canonical eukaryotic cell cycle consists of G1, S, G2 and M phases, and checkpoints are established at each phase to halt the cycle, allow repair and inhibit abnormal cell division (Saqcena et al., Reference Saqcena, Menon, Patel, Mukhopadhyay, Chow and Foster2013; Barnum and O’Connell, Reference Barnum and O’Connell2014). Although schizogony appears to lack the G2 phase due to its continual and cyclical use of S phase (Gerald et al., Reference Gerald, Mahajan and Kumar2011; Guttery et al., Reference Guttery, Zeeshan, Holder and Tewari2024), cell cycle checkpoints in each phase are thought to regulate these complex processes, although genes encoding key checkpoint proteins have not yet been clearly identified (Matthews et al., Reference Matthews, Duffy and Merrick2018; McLean and Jacobs-Lorena, Reference McLean and Jacobs-Lorena2020). In the present study, GB was not required for cell growth or DNA replication, suggesting that GB regulated the checkpoint to allow progression into the M phase and produce the first daughter cells of P. olseni schizogonic proliferation. In addition, trophozoites pre-incubated in PBMΔF exhibited daughter cell formation within 48 h after absorbing exogenous GB, earlier than 72 h (3 days), when the initial daughter cell formation was first confirmed in PBMΔF + GB, suggesting that GB exerts its regulatory effect before completion of the S phase. To initiate proliferation as early as possible within the host, P. olseni may prepare for karyokinesis and cytokinesis in parallel with cell growth and DNA replication.
Because P. olseni PRA-181 probably lacks the capacity to biosynthesize GB, it depends on host-derived GB to regulate schizogonic proliferation. In general, marine bivalves contain abundant GB as an osmolyte (Konosu and Hayashi, Reference Konosu and Hayashi1975; Wright et al., Reference Wright, Wunz and Silva1992; Pierce et al., Reference Pierce, Rowland-Faux and Crombie1995; de Zwart et al., Reference de Zwart, Slow, Payne, Lever, George, Gerrard and Chambers2003), and in the Manila clam – the host of P. olseni PRA-181 – the GB content in tissues reaches 4.1 mg g−1 (Farabegoli et al., Reference Farabegoli, Zironi and Gazzotti2019). However, the amount of GB markedly decreases under low-salinity conditions (Pierce et al., Reference Pierce, Rowland-Faux and O’Brien1992; de Vooys and Geenevasen, Reference de Vooys and Geenevasen2002); therefore, it is possible that the proliferation of P. olseni PRA-181 may decline within the host inhabiting low-salinity environments. P. olseni PRA-181 shows reduced efficiency in developing hypnospores and forming zoospores under low-salinity conditions (Umeda et al., Reference Umeda, Shimokawa and Yoshinaga2013, Reference Umeda, Yang, Waki, Yoshinaga and Itoh2020). This host-mediated control of proliferation may prevent wasteful cell division under unfavourable environmental conditions.
Another possible adaptive explanation is that the coexistence strategy aims to minimize excessive damage to the host. Manila clams show high mortality within 96 h at salinities below 15 (practical salinity units; Matsuda et al., Reference Matsuda, Shinagawa, Higano, Fujii, Hirano and Ishimatsu2008). If the parasite proliferates actively under low-salinity conditions unsuitable for the host, it could inflict fatal damage before achieving sufficient population growth. Therefore, it is speculated that P. olseni PRA-181 may suppress schizogonic proliferation in response to a decline in the surrounding GB concentration of the host to prevent excessive harm to the host and maintain a parasitic relationship favourable for the parasite’s long-term survival. However, because P. olseni acquires GB directly from its host, severe infections may increase the risk that Manila clams fail to withstand sudden salinity changes in natural environments. Therefore, this strategy may not provide a fully balanced form of coexistence. To understand this adaptive explanation, studies are needed to investigate the relationships between GB concentration, host, surrounding salinity and parasitic intensity in this species.
To elucidate the regulatory mechanism mediated by GB, the present study examined the proliferation of P. olseni PRA-181 in the presence of PBMΔF supplemented with dimethylglycine, sarcosine, carnitine and trimethylamine, which are intracellular metabolic products of GB. However, none of these compounds promoted cell division or proliferation (data not shown). Likewise, the structural analogues of GB, trigonelline and stachydrine also failed to enhance proliferative activity (data not shown). Consequently, the mechanism underlying the GB-dependent effect remains unclear, and future studies using metabolomic analysis with radiolabelled GB to track its metabolic products and transcriptomic analyses are required.
In summary, this study demonstrated that host-derived GB played an essential role in the initiation of daughter cell formation during schizogony in P. olseni PRA-181. Because GB is an important osmolyte for host molluscs, these findings provide intriguing insight into the life history of this parasite. Moreover, identifying a factor that controls a specific step in cell division will enable more detailed analyses of cell division processes in this genus.
Acknowledgements
We would like to thank Associate Professor Taketoshi Kodama (Laboratory of Aquatic Biology and Environmental Science, The University of Tokyo) for his support and guidance in flow cytometric analyses. We would also like to thank Editage (www.editage.jp) for English language editing.
Author contributions
Y.L., Y.W. and N.I. conceived and designed the study. A.N. conducted the data gathering using LC-MS. Y.L. and N.I. performed statistical analyses and wrote the article.
Financial support
This study was financially supported by JSPS Kakenhi (grant number 23K27000) for A.N. and N.I. and the JST SPRING GX program (grant number JPMJSP2018) for Y.L.
Competing interests
The authors declare there are no conflicts of interest.
Ethical standards
Not applicable.
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