Chasing the Cruciform Morphotype in Phaeodactylum tricornutum

P. tricornutum Pleomorphism

​One of the more interesting microalgae that we grow at Aquaholic Aquaculture is Phaeodactylum tricornutum. P. tricornutum is a pennate diatom that has a unique pleomorphic nature. Unlike most diatoms, P. tricornutum is weakly silicified, and, because of this, its cell wall has exceptional plasticity, enabling the diatom to change its shape (Tesson et al., 2009). P. tricornutum has four distinct morphotypes: (1) Ovoid (also referred to as Oval or Round), (2) Fusiform, (3) Triradiate, and (4) Cruciform (He et al., 2014; Lewin et al., 1958).

1. Ovoid

​The ovoid morphotype is either oval or round in appearance and possesses a raphe that enables motility (Tesson et al., 2009). This shape is preferentially benthic, with ovoid cells tending to clump together and sink to the bottom (Lewin et al., 1958). In laboratory cultures the ovoid morph usually predominates cultures grown on solid media (e.g., agar plates) or is found adhering to the walls of its culture vessel (Barker, 1935; Lewin et al., 1958; Tesson et al., 2009). Research has shown that the elongated versus rounded shape of the ovoid morphotype is dependent upon culture conditions, with a tendency for ovoid cells to take on a more circular shape when stressed (e.g., nutrient depleted) and an elongated shape when healthy and thriving (Tesson et al., 2009).

2. Fusiform

​The fusiform morphotype is best described as spindle or peapod shaped. In laboratory cultures grown in liquid media, the fusiform morph usually predominates (Barker, 1935; DeMartino, 2007; Lewin et al., 1958). The large peripheral vacuoles of the fusiform morph make this cellular shape significantly more buoyant than ovoid cells (Lewin et al., 1958; Tesson et al., 2009). Therefore, in an actively growing laboratory culture, fusiform cells will be found floating throughout the liquid media (Lewin et al., 1958).

Fusiform ​P. tricornutum
​© Aquaholic Aquaculture

3. Triradiate

​The triradiate morphotype is three-rayed in appearance and resembles the shape of a three-pointed star. Like the fusiform morphotype, the triradiate morphotype is very buoyant and therefore best suited for a planktonic lifestyle (DeMartino et al., 2007; Tesson et al., 2009). This morph usually thrives in standard laboratory cultures, and occasionally even outcompetes fusiform cells, especially if the culture is static. It is theorized in these instances that the triradiate cells were favored by selection due to their increased buoyancy (and decreased rate of sinking) enabling them to outcompete other cellular shapes (Lewin et al., 1958).

Triradiate ​P. tricornutum
​© Aquaholic Aquaculture

4. Cruciform

​The cruciform morphotype is an irregular four-armed cell in the shape of a perfect cross (Wilson, 1946). This morphotype is extremely rare (He et al., 2014; Wilson, 1946). The cruciform morphotype was first described in depth by Douglas Wilson in 1946 (under the name of Nitzschia closterium f. minutissima). Since Wilson's publication in 1946, this morphotype has rarely been reported being observed in laboratory cultures (He et al., 2014; Wilson, 1946). Wilson noted that while this morphotype is extremely rare, it is relatively stable and reproduces true to type (e.g., asexual clonal division) (Wilson, 1946).

Morphological Shifts in Response to Environment

​While it was previously thought that P. tricornutum morphology may be due to passive cellular mutation, recent research has demonstrated that morphological shifts are an active response to external factors that activate P. tricornutum’s morphogenetic mechanisms (Tesson et al., 2009). Current research now suggests that P. tricornutum’s pleomorphism is an adaptation that evolved in response to the ever-changing environmental conditions of the coastal waters where P. tricornutum is predominately found, with each of the four morphotypes representing distinct ecophenotypes adapted for specific environmental conditions (He et al., 2014; Tesson et al., 2009). In support of this theory, research by He et al. (2014) demonstrated that morphological shifts in P. tricornutum could be triggered in the laboratory by manipulating culture conditions such as temperature, salinity, light, and culture media.

Nutrition in Relation to Morphology

​The morphology of P. tricornutum cells impacts their biomacromolecule contents, with nutritional composition (e.g., lipid, protein dry weight, and carbohydrate content) varying depending on which of the four morphotypes the cell exhibits (He et al., 2014; Lewin et al., 1958). Of the four P. tricornutum morphotypes, He et al. (2014) demonstrated the cruciform morphotype to be the most nutritious, with an exceptional fatty acid profile. In their research, He et al., (2014) found that an abundance of cruciform morphotypes correlated with a significant increase in lipid content, with their predominately cruciform culture demonstrating both the maximum content of neutral lipid in a single cell and total yield.

Chasing Cruciform

​We are always looking to improve the nutritional qualities of our live feeds, and after learning of the cruciform’s superior lipid profile and nutritional value, it became a goal of ours to see if we could manipulate the conditions of our P. tricornutum cultures to stimulate the formation of cruciform cells in our cultures.

While reports of the cruciform morphotype in P. tricornutum laboratory cultures are sparse, prior research indicated that there may be a correlation between low culture temperature and the increased formation of cruciform cells (He et al., 2014). Gradually we started acclimating some of our P. tricornutum cultures to lower temperatures to see if this change effected morphology. We decreased the temperature on these experimental cultures incrementally by about 2F per month, checking periodically for the manifestation of cruciform cells.

For months, we had no success eliciting the formation of cruciform cells; our cultures continued to consist solely of fusiform and triradiate cells. However, we did start to notice that as we continued to decrease the temperature of the cultures that there was an increase in the proportion of triradiate cells to fusiform cells.

Finally! It happened! In February 2025, we pulled a sample from our experimental cultures to examine under the microscope, and we found our first cruciform cells!

Cruciform ​P. tricornutum
​​© Aquaholic Aquaculture

​After months of manipulating culture temperatures, we have found that the ‘sweet spot’ for eliciting the formation of cruciform cells is between 60-65F, with closer to 60F being optimal. By continuing to keep our P. tricornutum cultures in this temperature range, we have been able to slowly increase the ratio of cruciform cells in our cultures. Currently, cruciform cells comprise about 25% of each of our cultures.

​The highest reported ratio of cruciform cells in culture was recorded at approximately 50% (Wilson, 1946). This success was attributed to a number of factors, including good culture conditions, low temperatures, and repeated isolation of exclusively cruciform cells to use as the basis for new cultures (Wilson, 1946). We hope that by implementing these practices at Aquaholic Aquaculture that we can continue to increase the proportion of cruciform cells in our P. tricornutum cultures, thereby enhancing their fatty acid profile and thus increasing the nutritional value of our commercial products that contain P. tricornutum (e.g., REEFreshments®: Live Phytoplankton & REVIVE™ Live Zooxanthellae).

Pleomorphism in ​P. tricornutum
​© Aquaholic Aquaculture

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References

​[1] Barker, H. A. (1935). Photosynthesis in diatoms. Arch. Mikrobiol. 6. 141.
 
[2] DeMartino, A., Meichenin, A., Shi, J., Pan, K. H., & Bowler, C. (2007). Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accession. J Phycol., 43. 992-1009.
 
[3] He, L., Han, X., & Yu, Z. (2014). A rare Phaeodactylum tricornutum cruciform morphotype: culture conditions, transformation and unique fatty acid characteristics. PLoS One, 9(4).
 
[4] Lewin, J. C., Lewin, R. A., & Philpott, D. E. (1958). Observations on Phaeodactylum tricornutum. J. Gen Microbiol., 18(2).
 
[5] Tesson, B., Gaillard, C., & Martin-Jezequel, V. (2009). Insights into the polymorphism of the diatom Phaeodactylum tricornutum Bohlin. Botanica Marina, 52. 104-116.
 
[6] Wilson, D. P. (1946). The triradiate and other forms of Nitzschia. Journal of the Marine Biological Association of the United Kingdom, 26(3), 235-270.