P. donghaiense cells in the later stage of exponential growth were centrifuged at 4000 g, the excess nutrients on the cell surface were washed off with sterilized seawater three times, and the cells were inoculated into normal f/2 medium with a nitrogen concentration of 883 μM and f/2 medium without a nitrogen source. The container was a 25 cm² breathable cell culture flask (Nest), and the initial density was 1×10⁴ cells/mL. The culture temperature was 20°C in a light:dark cycle of 12h:12h with a light intensity of 4000 lux. When the cells grew to the later stage of exponential growth in normal f/2 medium, the samples of the two groups were taken for sample determination.

For each sample, 2 ml algal solution was added to the same volume of 4% osmic acid for fixation for 50 min, and then algal cells were collected, dehydrated in acetone solutions of different concentrations (10%, 30%, 50%, 70%, 90%, 100%, each concentration for 15 min), dried by a CO₂ critical point dryer (EM cpd300, Leica), and sprayed with gold by a coating instrument (sputter/carbon thread, EM ace200, Leica). The morphology of P. donghaiense was observed by scanning electron microscopy (Hitachi S-4800, Japan) and measured by electron microscopy software (Veltkamp et al., 1994).

Cells were immobilized with the method used for Staphylococcus aureus cells (Pasquina-Lemonche et al., 2020), while details were modified, and polycarbonate membrane was used instead of NuNano silicon grids. P. donghaiense cells have flagella and different degrees of activity, so it is difficult to fix them when they are completely submerged in the liquid medium. In this study, a polycarbonate membrane (Millipore Isopore, with a thickness of 16 µm to trap the moisture) with a pore size similar to cell size was considered the matrix for fixation so that the cells could be properly “stuck” in the pores and kept moist. A total of 10 µL algal liquid without any treatment was directly dropped onto a 10 µm pore diameter polycarbonate membrane and scanned with atomic force microscopy (Bruker Bioscope Resolve) at room temperature 20°C with air humidity ranging from 50% to 70%. The images should be obtained while the cells are kept moist in approximately physiological conditions. When the cells are dry, salt separates out on the surface, which is also a way to judge the state of the cells. Both automatic mode and contact mode were employed in this study. The probes used are SCANASYST-AIR and DNP. The scanning frequency was 0.5 Hz, and the resolution was 512×512. The mechanical parameters were measured using the ramp mode, and cell surface areas of different sizes such as 5 µm×5 µm were selected for testing.

The image analysis software used was NanoScope Analysis 1.8. Adhesion and elasticity were calculated by this software. After the images were scanned by atomic force microscopy, they were uniformly processed by first-order flatten, and the cell surface roughness was analyzed by a rough module. The height and spacing of the cell surface ultrastructure were analyzed by the section module, and the original data were redrawn in Excel. Similarly, after the original mechanical analysis data were exported, they were replotted and analyzed in Excel. The roughness (R_a) is the arithmetic mean of the absolute height of the cell surface in the selected area. Roughness of cells under nitrogen limitation and under normal culture conditions were counted by five cells, respectively. The roughness calculation formula is as follows:

where Z is the absolute height of the cell surface and N is the number of calculation points.

In this study, images of P. donghaiense cells from different angles and at different scales were obtained by atomic force microscopy ( Figures 1A–F ). Figure 1B shows the overall appearance from the left valve view. Figure 1C shows depression structure in the flagellum area at the top of the cell, and the flagellum of P. donghaiense cells can be observed in more detail in Figure 1D . Figure 1E shows the collar structure around the flagellum pores at the top of the cell. Figure 1F is the megacytic zone of the cell, and Figures 1G, H show the knob-like spines and valve pores on the shell surface of the cell. These morphological characteristics of the cell surface of P. donghaiense revealed in this study are similar to those obtained by Lu et al. through SEM (Lu et al., 2005), which indicates that AFM can be used to characterize the cell surface ultrastructure of P. donghaiense.

Further high-resolution imaging of the unique structure on the cell surface of P. donghaiense showed clear knob-like spines and valve pores ( Figure 2A ). The height measurement results show that the height of knob-like spines relative to the cell surface is approximately 100 nm, the spacing between them is approximately 400 nm, the diameter of valve pores is approximately 200 nm, and the height is 120 nm ( Figures 2B, C ). Similarly, by measuring the height of the indirect zone of P. donghaiense cells ( Figure 2D ), it can be seen that the height of the bulge on the surface of the megacytic zone is not the same, and the height ranges from approximately 100-200 nm ( Figures 2E, F ).

Nitrogen limitation can affect the physiological activities of P. donghaiense cells, which include photosynthesis, carbon fixation, and protein and lipid synthesis. Compared with cells under normal culture conditions ( Figures 3A, C ), the cell surface under nitrogen limitation seemed to lack some “reticular substances” ( Figures 3B, D ).

Using AFM to observe moist P. donghaiense cells under two culture conditions, clear knob-like spines and valve pores were observed on the cell surface under normal culture conditions ( Figure 4A ), while the pores on the cell surface of P. donghaiense under nitrogen limitation were not sufficiently clear ( Figure 4B ). Further analysis of the height of knob-like spines showed that there were differences in the height of knob-like spines on the surface of the two cells. The height of knob-like spines on the cell surface under normal conditions was approximately 99 nm, while that under nitrogen limited conditions was approximately 75 nm. The cell surface protrusion angle under normal culture conditions was “sharp”, while the cell surface protrusion angle under nitrogen limitation was “blunt”.

Further roughness analysis was carried out on 30 areas of different sizes ranging from 0.3-43.7 µm² on the cell surface of P. donghaiense. The results showed that the variation range of cell surface roughness of P. donghaiense under normal culture conditions was 25-350 nm, while the value and range of cell surface roughness of P. donghaiense under nitrogen limitation were 25-40 nm ( Figures 5A, B ). Both the value and range of cell surface roughness of P. donghaiense under normal culture conditions were larger than those under nitrogen limitation. The difference was that the cell surface roughness under normal culture conditions was positively correlated with the surface area (R²= 0.858), and the cell surface roughness under nitrogen restriction was negatively correlated with the surface area (R²= 0.564) ( Figures 5C, D ).

Many important physiological functions of cells depend on their mechanical properties, such as adhesion and elastic modulus. This study found that the cell surface adhesion of P. donghaiense under normal culture conditions ranged from 0.03 to 2.11 nN, with an average of 0.65 nN ( Figures 6A, C ). Under nitrogen limitation, the cell surface adhesion of P. donghaiense ranged from 0.04 to 0.71 nN, with an average of 0.43 nN ( Figures 6B, D ). The surface adhesion of P. donghaiense cells under normal culture conditions is generally greater than that under nitrogen limitation, and the distribution range is wider.

The elastic modulus can be regarded as an index to measure the difficulty of elastic deformation to occur in materials. The greater its value is, the greater the stress causing certain elastic deformations in materials; that is, the greater the material stiffness is, the smaller the elastic deformation under the action of certain stress. This study found that the surface elastic modulus of P. donghaiense cells under normal culture conditions ranged from 0.17 to 0.88 MPa, with an average value of 0.60 MPa ( Figures 7A, C ). Under nitrogen limitation, the cell surface elastic modulus of P. donghaiense ranged from 0.14 to 3.91 MPa, with an average of 3.42 MPa ( Figures 7B, D ). The surface elastic modulus of P. donghaiense cells under normal culture conditions is less than that under nitrogen limitation, and the whole entity is “softer”.

For a long time, there has been a dispute about the species identification of Prorocentrum in the East China Sea. Lu et al. described the taxonomic characteristics of this species in detail, identified it as P. donghaiense, and found evenly distributed knob-like spines and valve pores on its surface by using scanning electron microscopy (Lu et al., 2005). In this paper, atomic force microscopy was employed to study moist cells in the harmful algal bloom species P. donghaiense for the first time. Typical ultrastructures such as knob-like spines and valve pores on the cell surface had similar characteristics compared with those observed on scanning electron microscopy images. What is more, the height and spacing of a typical nanostructure were quantified by AFM to reflect the microenvironment on the cell surface. Besides morphology characters, adhesion and elasticity were also revealed in this study, which is the first mechanical research of P. donghaiense.

One major advantage of AFM is that it can characterize cells under physiological conditions, which can more accurately reflect the original characteristics of cells (Demir-Yilmaz et al., 2021). Due to the lack of movement ability, diatoms can more easily adhere to the substrate to grow and fix themselves compared with other microalgae equipped with flagella, which avoids the requirements of atomic force microscopy for sample fixation (Gebeshuber et al., 2003). At present, atomic force microscopy in microalgae is mainly used in diatoms (Luis et al., 2017; Demir-Yilmaz et al., 2021). However, there are few reports on atomic force microscopy in the field of red tide algae.

For red tide algae, cell immobilization is an important challenge for the application of atomic force microscopy in the study of living cells, partly due to their strong movement ability. At present, the main methods for flagellated cell immobilization include fixed by glutaraldehyde and then adhered to the substrate (Gunther et al., 2014; Pillet et al., 2019). Unlike diatom cells that can adhere to the substrate for growth, red tide algae such as dinoflagellates have flagella and motion ability, so it is difficult to fix them. In this study, several fixation methods were compared. For dinoflagellates with different particle sizes, a polycarbonate membrane with a pore size similar to its cell size was considered the matrix for fixation so that the cells could be properly “stuck” in the pores and kept moist, and this method is similar to that used for S. aureus cells (Pasquina-Lemonche et al., 2020) and Emiliania huxleyi cells (Evans et al., 2021). In the 10 µm pore size polycarbonate membrane selected in this study, there are several types of “stuck slots”: a single 10 µm hole ( Figure 8A ) and two 10 µm connected holes ( Figure 8B ) which can immobilize P. donghaiense cells. Atomic force microscopy is an important method to study the biological mechanism of cells. Studies based on animal cells show that the elastic modulus E of cells is closely related to their life activities and health status. For example, the elastic modulus E of human cancerous cells is much lower than that of normal cells, and by directly measuring the elastic modulus E of biological cells, the cancerous state can be reflected (Cross et al., 2007). Cell differentiation is usually accompanied by changes in shape to realize special functions. To produce different shapes, cells need to change the mechanical properties of their surface (Bergert et al., 2021). By using a cell magnetic twisting instrument, Wang et al. found that integrin, a cell adhesion protein, is a cell mechanical receptor (Wang et al., 1993); moreover, mechanical stimulation can be directly transmitted to the nucleus through the cytoskeleton and nuclear membrane and directly activate gene expression. Directly stretching chromatin with mechanical force can upregulate transcription (Tajik et al., 2016).

The instrument can image microalgae cells with nanoscale resolution and probe the nanomechanical properties and nanoadhesive properties of microalgae cells (Demir-Yilmaz et al., 2021). Mechanical factors can play a key role in the response and adaptation of different levels of functions, such as mechanical regulation and transportation, tissue deformation, cell growth and movement, intermolecular interactions, and signal pathways (Cross et al., 2007; Tajik et al., 2016; Bergert et al., 2021). Through studying the surface ultrastructure and biomechanics, AFM is expected to make a new breakthrough in the environmental adaptation mechanism of microalgae.

Previous studies have shown that nitrogen limitation inhibited the growth of P. donghaiense, maintained its cell density, chlorophyll a content and particulate organic nitrogen content at a low level, and caused the downregulation of proteins involved in photosynthesis, carbon fixation, and protein and lipid synthesis (Zhang et al., 2015; Ou et al., 2019). In this study, the effect of nitrogen limitation on the surface morphology and mechanical characteristics of P. donghaiense cells under physiological conditions was further uncovered. Details of morphology parameters are shown in Table 1 . Compared with cells under normal culture conditions, the value and range of cell surface roughness under nitrogen limitation decreased, the value and range of cell surface adhesion decreased, and the value and range of elastic modulus increased. The function of cell surface is very complex. In addition to supporting and protecting cells, the cell surface is closely related to the behavior, physiological activities, mutual recognition, adhesion, material transportation, signal transduction, cell movement, growth, differentiation, aging, and pathological process of the whole cell (Ludwig et al., 2021). The cell surface is responsible for the material exchange and energy exchange inside and outside the cell, and carries out cell recognition, information reception and transmission, cell movement, and maintenance of various cell forms through the surface structure.