Changes in valve morphology of two pennate diatom species during long-term culture
expand article infoDarya P. Petrova, Yekaterina D. Bedoshvili, Yulia R. Zakharova, Nadejda A. Volokitina, Yelena V. Likhoshway, Michael A. Grachev
‡ Limnological Institute, Siberian Branch, Russian Academy of Sciences, Irkutsk, Russia
Open Access


The morphology of diatom siliceous is a primary basis for their species identification. This study aims to measure the range of morphological changes induced in the monoclonal cultures of Fragilaria radians strains 280 and A6 and Ulnaria danica strain BK17 by cultivation in the lab for a year or more. The scanning electron microscopy revealed that the number of abnormal valves increases during the first year of culture maintenance. Specific abnormalities observed include curved valves and apices, axial areas and rimoportulae shifted from their normal positions, disordered or otherwise abnormal striae, and various growths on the valves. Similar morphological abnormalities are known to occur in diatoms exposed to microtubule inhibitors. These results show the limits of morphological variance in studied species and could be used to estimate the effect of toxic agents in natural and experimental conditions.


Diatom cultivation, monoclonal culture, pennate diatoms, valve abnormality


Diatom algae (Bacillariophyta) are a group of free-living or colonial eukaryotic microalgae. Their most distinctive feature is a siliceous cell; its overall layout and various micro- and nanoscale elements are species-specific. According to the recent works, there is somewhere between 12,000 and 30,000 extant diatom species (Guiry 2012; Mann and Vanormalingen 2013; Malviya et al. 2016; Guiry and Guiry 2020).

Before the advent of molecular genetics, diatom taxonomy was based solely on the valve morphology observed with light or electron microscopy (Schütt 1896; Round and Crawford 1981; Round et al. 1990; Kaczmarska et al. 2013; Mann et al. 2017). The difficulties in species identification are often caused by the phenotypic variance which lets a single species have multiple distinct morphotypes (Cerino et al. 2005; Glemser et al. 2019). On the other hand, there are groups of cryptic species that are morphologically very similar and cannot be reliably distinguished without genetic data (Sorhannus 2007; Alverson 2008; Cox 2014).

Fragilaria radians (Kützing) D.M. Williams & Round (Synedra acus subsp. radians (Kützing) Scabitchevsky) and Ulnaria danica (Kützing) Compère & Bukhtiyarova (S. ulna var. danica (Kützing) Grunow) are widespread in the freshwater basins of the world (Guiry and Guiry 2020). Monoclonal cultures of these two species isolated from Lake Baikal are relatively easy to culture, which has made them an object of various studies in genomics (Galachyants et al. 2019), molecular biology (Petrova et al. 2007; Marchenkov et al. 2018), and cytology (Annenkov et al. 2013; Shishlyannikov et al. 2014; Kharitonenko et al. 2015). The existence of an axenic culture protocol (Shishlyannikov et al. 2011) and high-volume biomass production technology (Vereshchagin et al. 2008) have also broadened the range of possible studies on these cultures. Our aim was to study the effects of long-term culture on F. radians and U. danica valve morphology and to estimate the abnormal accumulation rates.

Material and methods

This work was performed on Fragilaria radians strains A6 and A280, and Ulnaria danica strain BK17. All strains were isolated from Lake Baikal according to the previously published protocol (Shishlyannikov et al. 2011), and have been maintained in the lab for more than a year. Cells were grown on the DM medium (Thomson et al 1988) in flasks at 10°C and 16 μmol/m2*sec of light intensity using 12/12 h artificial light/dark cycle. For the present study, clonal cultures grown from one cell were used after several months of cultivation, as soon as their number in a clonal culture became sufficient to clear the valves.

To study the cell morphology, cultures were sedimented on the Mini Spin centrifuge (Eppendorf) for 6 minutes at 3500 g. The cell pellet was washed for 30 minutes at 95°C in 6% sodium dodecyl sulfate solution. After that, the cells were again centrifuged and the supernatant was removed; this stage was repeated three times. The sediment was washed in distilled water five times and treated with concentrated nitric acid for 1 hour at 95°C. After that, it was thrice washed in ethanol and treated with 36% hydrochloric acid for 24 hours at room temperature. After the final treatment, valves were washed in distilled water at least five times, resuspended in 100 μl 70% ethanol, and placed on SEM stubs. These plates were dried at room temperature and coated with gold.

Microscopy was performed using Quanta 200 scanning electron microscope. All counts were taken on 200 randomly chosen valves.

Results and discussion

Abnormalities were classified based on the data of Kharitonenko et al. (2015) and our results (Fig. 1, Table 1). We registered that after producing the monoclonal culture, 3% of F. radians cells and 15% of U. danica cells had some abnormalities; in both species, the proportion of malformed cells increased during culture maintenance (Figs 2, 3).

Figure 1.

The structure of normal (A–E) and abnormal (D–K) F. radians valves strain A280 (SEM). A – an overall view; B – an axial area, inside view; C – an axial area, outside view; D – valve apex with a rimoportula (arrow), outside view; E – valve apex with a rimoportula (arrow), inside view; F – a curved valve; G – local displacement of the axial area; H – an altered shape of the valve apex, rimoportula is marked with a white arrow, non-formed or overgrown stria is marked with a black arrow; I – a displacement of the rimoportula; J – misalignment of the rows of areolae; K – merged areolae within a stria. Scale bars: A – 50 µm; B, C, G – 5 µm; D, E, H, I, J – 2 µm; F – 10 µm; K – 1 µm.

Table 1.

Classification of valve abnormalities.

Abnormality type Description Kharitonenko et al. 2015 Figure, this study
I Valve curvature 4b 1F
II Local displacement of the axial area 4c 1G
III Altered shape of the valve apex 4e 1H
IV Displacement of the rimoportula 4f 1I
V Misalignment of the rows of areolae 4h 1J
VI Oversized areolae/striae not composed from individual areolae 1K
VII Areolae occlusion 1i 1H

During the first year of culture, most valves conformed to the curved shapes (Fig. 2). This anomaly becomes common after a year of culture maintenance. For F. radians strain 280, this change was often accompanied by displacement rimoportulae and axial areas, as well as the altered shape of apices (Fig. 1F-I). Unlike U. danica strain KB17, misalignment of the rows of areolae (Fig. 1J) was uncommon.

Figure 2.

The prevalence of valve abnormalities in U. danica KB17 from 2017 to 2018 (A) and in F. radians 280 from 2015 to 2016 (B, C). Abnormalities are numbered according to Table 1.

Morphological observations on F. radians strain A6 (which was cultured for more than 7 years) suggest that all cells will eventually become curved after more than a year of culture in a sufficiently old culture (Fig. 3).

Figure 3.

The morphology of F. radians A6 valve cells in 2018 (after seven years of culture isolation). Abnormalities are numbered according to Table 1.

Phenotypic anomalies in diatoms are known to be caused by various environmental stresses (Falasco et al. 2009), including anthropogenic pressure (Roubeix et al. 2011; Pandey et al. 2014). It was shown that pennate diatoms are prone to deformation (Cantonati et al. 2014; Pandey et al. 2016). Based on the literature data (despite its scarcity), some species of diatoms seem to be more prone to shell deformations than others. Thus, the single-sutured species Achnanthidium minutissimum (Cantonati et al. 2014), as it turned out, is less sensitive to stress conditions, at least this does not affect the structure of valves, unlike other species of pennate diatoms (eg, Cymbella tumida, Gomphonema rosenstockianum, and Nitzschia linearis - Falasco et al., 2009b). Interestingly, the last three sensitive species turned out to be biraphid. For araphid species Fragilaria and Ulnaria, the most characteristic deformation of the shell is a change in its shape, according to a 2014 study (Pandey et al. 2014). The present study shows that the last statement is true not only for the cultivation of species of these genera in unfavorable conditions but also for long-term cultivation of monoclonal cultures in laboratory conditions. Although diatom morphogenesis has been studied for several decades, only a few works have documented morphological abnormalities observed in diatom cultures (Estes and Dute 1994), including ones induced artificially by the exposure to microtubule inhibitors (Pickett-Heaps et al. 1979; Blank and Sullivan 1983; Van de Meene and Pickett-Heaps 2002; Tesson and Hildebrand 2010; Bedoshvili et al. 2018). According to the previously published data, structural abnormalities similar to the ones described in this work could be induced in F. radians and U. danica by microtubule inhibitors (Kharitonenko et al. 2015; Bedoshvili et al. 2017).

This raises a question of the mechanism(s) behind the increase in abnormality prevalence during long-term culture. First of all, culture conditions are not identical to the natural water requirements. A limited medium volume and a lack of mixing could cause most cells to attach themselves to flask walls and bottom with the mucus normally secreted on the outer surface of the valve (Higgins et al. 2003; Edgar 1983). This, in turn, can make normal valve disjunction during the doubling of cell volume before division more difficult, and thus affect the vegetative division process

Second, new valves are formed within the cell and are therefore limited by the shape of mother cell. It means that any deviations in valve shape are propagated to all descendants of the originally malformed cell. It is known that a curved sternum (the first stage of valve formation) could cause abnormalities in the structures that form later, such as striae, axial and apical fields, and others (Kharitonenko et al. 2015).

Third, it is known that cell cultures generally accumulate somatic mutations (for example, Kim et al. 2017). Since, as we have noted before, the abnormalities observed in old cultures are similar to the ones induced by cytoskeleton inhibition, diatom cells may accumulate deleterious mutations in cytoskeleton-related genes. Unlike mesenchyme cells, which were shown to accumulate somatic mutations without changes in the phenotype (Kim et al. 2017), cytoskeleton mutations could immediately lead to valve abnormalities in diatoms, since valve formation is highly dependent on the cytoskeleton (Van de Meene and Pickett-Heaps 2002).

Fourth, valve abnormalities are not selected against in culture conditions, since they do not directly kill the cells or prevent asexual reproduction. In natural populations, on the other hand, they are quickly eliminated perhaps through sexual reproduction.

These data on the accumulation of morphological changes in monoclonal cultures F. radians and U. danica extend the range of known variations in diatom valve structure of these genera and can be used for estimating the toxic effects of various agents in the laboratory and environmental conditions.


The work is done within the State Assignments of Limnological Institute, Siberian Branch of Russian Academy of Sciences (0345-2019-0001) (microscopy) and of the Russian Foundation for Basic Research grant #17-29-05030 (cultivation). Microscopic studies were carried out in the Electronmicroscopy center of collective instrumental center “Ultramicroanalysis” Limnological Institute of the Siberian Branchof the Russian Academy of Sciences (


  • Annenkov VV, Basharina TN, Danilovtseva EN, Grachev MA (2013) Putative silicon transport vesicles in the cytoplasm of the diatom Synedra acus during surge uptake of silicon. Protoplasma 250: 1147–1155.
  • Bedoshvili Ye, Gneusheva K, Likhoshway Ye (2017) Changing of silica valves of diatom Synedra acus subsp. radians influenced by paclitaxel. Tsitologia 59: 53–61. [In Russian]
  • Bedoshvili Ye, Gneusheva K, Popova M, Morozov A, Likhoshway Ye (2018) Anomalies in the valve morphogenesis of the centric diatom alga Aulacoseira islandica caused by microtubule inhibitors. Biology Open 7:bio035519.
  • Cantonati M, Angeli N, Virtanen L, Wojtal AZ, Gabriell J, Falasco E, Lavoie I, Morin S, Marchetoo A, Fortin C, Smirnova S (2014) Achnanthidium minutissimum (Bacillariophyta) valve deformities as indicators of metal enrichment in diverse widely-distributed freshwater habitats. Sci Total Environ 75: 201–215.
  • Cerino F, Orsini L, Sarno D, Dell’Aversano C, Tartaglione L, Zingon A (2005) The alternation of different morphotypes in the seasonal cycle of the toxic diatom Pseudo-nitzschia galaxia. Harmful Algae 4: 33–48.
  • Cox EJ (2014) Diatom identification in the face of changing species concepts and evidence of phenotypic plasticity. Journal of Micropalaeontology 33: 111–120.
  • Falasco E, Bona F, Ginepro M, Hlúbiková D, Hoffmann L, Ector L (2009) Morphological abnormalities of diatom silica walls in relation to heavy metal contamination and artificial growth conditions. Water SA 35(5): 595–606.
  • Galachyants YP, Zakharova YR, Volokitina NA, Morozov AA, Likhoshway YV, Grachev MA (2019) De novo transcriptome assembly and analysis of the freshwater araphid diatom Fragilaria radians, Lake Baikal. Scientific Data 6: 183.
  • Glemser B, Kloster M, Esper O, Eggers SL, Kauer G, Beszteri B (2019) Biogeographic differentiation between two morphotypes of the Southern Ocean diatom Fragilariopsis kerguelensis. Polar Biology 42: 1369–1376.
  • Guiry MD, Guiry GM (2020) AlgaeBase. World-wide electronic publication, National University of Ireland, Galway.
  • Higgins MJ, Molino P, Mulvaney P, Wetherbee R (2003) The structure and nanomechanical properties of the adhesive mucilage that mediates diatom-substratum adhesion and motility. J Phycol 39: 1181–1193.
  • Kaczmarska I, Poulíčková A, Sato S, Edlund MB, Idei M, Watanabe T, Mann DG (2013) Proposals for a terminology for diatom sexual reproduction, auxospores and resting stages. Diatom Res 28: 263–294.
  • Kharitonenko KV, Bedoshvili YeD, Likhoshway YeV (2015) Changes in the micro- and nanostructure of siliceous frustule valves in the diatom Synedra acus under the effect of colchicine treatment at different stages of the cell cycle. Journal of Structural Biology 190: 73–80.
  • Kim M, Rhee JK, Choi H, Kwon A, Kim J, Lee GD, Jekarl DW, Lee S, Kim Y, Kim TM (2017) Passage-dependent accumulation of somatic mutations in mesenchymal stromal cells during in vitro culture revealed by whole genome sequencing. Scientific Reports 7: 14508.
  • Malviya S, Scalco E, Audic S, Vincent F, Veluchamy A, Poulaind J, Wincker P, Ludicone D, de Vargas C, Bittnera L, Zingone A, Bowler C (2016) Insights into global diatom distribution and diversity in the world's ocean. PNAS. E1516–25.
  • Mann DG, Vanormelingen P (2013) An inordinate fondness? The number, distributions, and origins of diatom species. J Eukaryot Microbiol 60(4): 414–420.
  • Marchenkov AM, Petrova DP, Morozov AA, Zakharova YR, Grachev MA, Bondar AA (2018) A family of silicon transporter structural genes in a pennate diatom Synedra ulna subsp. danica (Kütz.) Skabitsch. PLoS ONE 13: 8 e0203161.
  • Nakov T, Ruck EC, Galachyants Yu, Spaulding SA, Theriot EC (2014) Molecular phylogeny of the Cymbellales (Bacillariophyceae, Heterokontophyta) with a comparison of models for accommodating rate variation across sites. Phycologia 53: 359–373.
  • Pandey LK, Bergey EA (2016) Exploring the status of motility, lipid bodies, deformities and size reduction in periphytic diatom community from chronically metal (Cu, Zn) polluted waterbodies as a biomonitoring tool. Sci Total Environ 550: 372–381.
  • Pandey LK, Kumar D, Yadav A, Rai J, Gaur JP (2014) Morphological abnormalities in periphytic diatoms as a tool for biomonitoring of heavy metal pollution in a river. Ecol Indic 36: 272–279.
  • Petrova DP, Bedoshvili YeD, Shelukhina IV, Samukov VV, Korneva ES, Vereshagin AL, Popkova TP, Karpyshev NN, Lebedeva DV, Klimenkov IV, Likhoshway YeV, Grachev MA (2007) Detection of the silicic acid transport protein in the freshwater diatom Synedra acus by immunoblotting and immunoelectron microscopy. Doklady Biochemistry and Biophysics 417: 113–116.
  • Pickett-Heaps JD, Tippit DH, Andreozi IA (1979) Cell division in the pennate diatom Pinnularia. IV – Valve morphogenesis. Biologie Cellulaire 35: 199–206.
  • Roubeix V, Mazzella N, Schouler L, Fauvelle V, Morin S, Coste M, Delmas F, Margoum C (2011) Variations of periphytic diatom sensitivity to the herbicide diuron and relation to species distribution in a contamination gradient: implications for biomonitoring. J Environ Monit 13: 1768–1774.
  • Round F, Crawford R, Mann D (1990) The diatoms: biology and morphology of the genera. Cambridge Univ. Press, Bath.
  • Schütt F (1896) Bacillariales (Diatomeae). Die naturlichen Pflanzen familien 1(1b): 31–150.
  • Shishlyannikov SM, Klimenkov IV, Bedoshvili YD, Mikhailov IS, Gorshkov AG (2014) Effect of mixotrophic growth on the ultrastructure and fatty acid composition of the diatom Synedra acus from Lake Baikal. Journal of Biological Research-Thessaloniki 21: 15.
  • Shishlyannikov SM, Zakharova YR, Volokitina NA, Mikhailov IS, Petrova DP, Likhoshway YV (2011) A procedure for establishing an axenic culture of the diatom Synedra acus subsp. radians (Kutz.) Skabibitsch. from Lake Baikal. Limnol Oceanogr: Methods 9: 478–484.
  • Tesson B, Hildebrand M (2010) Dynamics of silica cell wall morphogenesis in the diatom Cyclotella cryptica: substructure formation and the role of microfilaments. J Struct Biol 169: 62–74.
  • Thomson AS, Rhodes JC, Pettman I (1988) Culture collection of algae and protozoa, catalogue of strains. Freshwater Biological Association, Ambleside, Cumbria.
  • Vereshchagin AL, Glyzina OYu, Basharina TN, Safonova TA, Latyshev NA, Lyubochko SA, Korneva ES, Petrova DP, Annenkov VV, Danilovtseva EN, Chebykin EP, Volokitina NA, Grachev MA (2008) Cultivation of the freshwater diatom Synedra acus in a photobioreactor and assessment of the composition of the obtained biomass. Biotechnology. 4: 55–63. [In Russian]
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