Aurantiochytrium limacinum –formerly Schizochytrium limacinum [2]– is a member of labyrinthulomycetes, a class [2] of marine heterokont protists characterized by the production of zoospores and ectoplasmic nets. They are involved in the carbon cycle as decomposers of organic material and in marine food webs as producers of metabolites such as carotenoids and omega-3 fatty acids.

Heterokonts are defined by the production of zoospores that move using two flagella, one of which has tripartite ‘hair’ like structures known as mastigonemes.  Figure 1 shows cells from an A. limacinum culture in log phase growth in liquid medium where sporulation has taken place. Zoospores can be seen in the bottom left corner with flailing flagella. The zoospores are somewhat ovoid in shape and can swim at high velocity. A great variability in cell size exists within the culture; the zoospores are 5-8 μm wide [1], whilst the largest vegetative cells are 20-32 μm wide. The time lapse represented in this image comprises a total of ~29 s.

 

 

When zoospores settle on a solid surface, they may form the ectoplasmic network: branched extensions of the cytoplasmic membrane that connect to the cell body through a unique organelle known as the bothrosome, sagenogenetosome, or sagenogen [4,5]. Figure 2 shows time-lapse video of this process in three  A. limacinum zoospores that have just settled on a glass slide. The ectoplasmic network not only attaches the vegetative cells to the substrate, but also excretes enzymes to break down complex organic molecules and takes up digestion products, delivering them to the cell body. In some laybrinthulomycetes the ectoplasmic network also allows the vegetative cells to move on the substrate or form colonies.

 

 

 

Immediately after the images in Figure 1 were captured, 1 μl of culture was used to inoculate 149 μl of liquid media in a 96 well plate. A 12 hour time lapse was taken at a magnification of 30X. Hours 1 – 6  are shown in Figure 3 and hours 6-12 are shown in Figure 4.

At the start of the timecourse we observe three microcolonies comprised of vegetative cells with smaller vegetative cells and motile zoospores dispersed throughout the field of view.  The three microcolonies increase in size through binary division [2], with the bottom-most colony  sporulating at 20 minutes after inoculation. At 62 minutes clonal cells start switching from a circular vegetative state to an amoebic limaciform state, moving across the surface of the plate in a slug-like manner [2]. Most of the cells from this time point onwards, both congregated and dispersed, transition from circular vegetative to motile limaciform cells, some several times, before finally settling into the vegetative state. Cells in the limaciform state can move at an average of ~8 μm/minute. Some cells through the entirety of the timecourse stay vegetative, never switching to the limaciform state, and increase in size.

The limaciform cells move away from the colony to empty spaces, eventually disassembling the original colonies; at 6 hours the two smaller microcolonies seen at the start of the timecourse are gone, and only a vestige of the bottom-most colony remains. Limaciform cells physically interact with each other as well as vegetative cells, pushing and pulling vegetative cells to new locations. In Figure 4 we see that this reorganization has led to the forming of new microcolonies.

This rapid and coordinated behavior is evidence of substantial cell-to-cell communication and a complex community structure. Other thraustochytrids also form amoeboid cells, most notably the Ulkenia genus. However all these species exhibit different and unique behaviors from each other and A. limacinum [3]. The loss and gain of these phenotypes amongst thraustochytrids may be an interesting evolutionary question offering ecological insights.

 

Figure 4. A. limacinum growth timecourse hours 6 to 12.

 

 

Original Images and Text by Kash Bandaralage, modified Dec 31 2020 by JL Collier

This work is funded by a grant from the Moore Foundation to  Collier, Rest, et al., to develop molecular genetic tools for the marine protist group labyrinthulomycetes. Our goal is to develop methods for the genetic manipulation of a broad diversity of labyrinthulomycetes as tools to elucidate their ecological roles and interactions with other marine organisms. We are developing tools for heterologous expression and gene deletion that can be utilized across the four distinct types of labyrinthulomycetes: thraustochytrids, oblongichytrids, labyrinthulids, and aplanochytrids. This grant is part of a larger effort by the Moore Foundation to accelerate the development of experimental model systems in marine microbial ecology.

References

1. Honda, Daiske et al. “Schizochytrium Limacinum Sp. Nov., a New Thraustochytrid from a Mangrove Area in the West Pacific Ocean.” Mycological Research 102.4 (1998): 439–448. Web.
2. Yokoyama, Rinka, and Daiske Honda. “Taxonomic Rearrangement of the Genus Schizochytrium Sensu Lato Based on Morphology, Chemotaxonomic Characteristics, and 18S RRNA Gene Phylogeny (Thraustochytriaceae, Labyrinthulomycetes): Emendation for Schizochytrium and Erection of Aurantiochytrium and Oblongichytrium Gen. Nov.”Mycoscience 48.4 (2007): 199–211. Web
3. Yokoyama, Rinka, Baharuddin Salleh, and Daiske Honda. “Taxonomic rearrangement of the genus Ulkenia sensu lato based on morphology, chemotaxonomical characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Ulkenia and erection of Botryochytrium, Parietichytrium, and Sicyoidochytrium gen. nov.” Mycoscience 48.6 (2007): 329-341.
4. Iwata, I., K. Kimura, Y. Tomaru, T. Motomura, K. Koike, K. Koike, & D. Honda (2017). “Bothrosome formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during zoospore settlement.” Protist, 168(2), 206-219.
5. Iwata, I. and D. Honda (2018). “Nutritional Intake by Ectoplasmic Nets of Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles).” Protist 169(5): 727-743.