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Kirstine Drumm:
Functional biology, photophysiology and nutrient dynamics of non-constitutive mixotrophic protists

Date: 14-02-2020    Supervisor: Per Juel Hansen

Mixotrophy is the combination of phototrophic and heterotrophic nutrition in a single organism. Mixotrophy is no longer confined to phototrophic protists (i.e. organisms with “built-in” chloroplasts, constitutive mixotrophs), but also includes protists that lack built-in chloroplasts, and instead host symbionts or retain functional chloroplasts from their prey (non-constitutive mixotrophs). This thesis has studied specialist non-constitutive mixotrophs (SNCM), referring to the selective behaviour were only a special type of prey can be used for C-fixation, as appose to generalist (GNCM) where a Cfixation can be acquired from different types of prey organisms. SCMNs are found in both marine and freshwater systems. In this thesis I studied one freshwater dinoflagellate, Nusuttodinium aeruginosum, and three marine species, one dinoflagellate, Dinophysis acuminata, and two ciliates, Mesodinium rubrum and M. major.

Light response curves were done on N. aeruginosum and M. major and illustrated a high dependence of light for cell growth and photosynthetic activity. Their light responses were very similar to that of a phototrophic organism. M. major was able to increase the cellular content of chlorophyll a (Chl a) at low irradiances, which is similar to what has been shown previously for M. rubrum, indicating abilities of photoacclimation. Chl a was not measured in N. aeruginosum, so it is unknown if this species has the ability of photoacclimation. Inorganic carbon fixation accounted for up to 26% and 95% of the total C need for N. aeruginosum and M. major, respectively. When these two organisms were subjection to prey deprivation they continued to grow, going through 4-6 cell divisions over a period of 20-25 days, hereafter the cells just sustained themselves. So, N. aeruginosum and M. major both displayed a close relation to the retained prey chloroplasts.

The two ciliates, M. rubrum and M. major, keep some genetic material from the prey. In well-fed cultures the ciliates keep a single prey nucleus, which was termed the Centered Prey Nucleus (CPN). This nucleus is placed in close proximity to the ciliates macro and micro nuclei. The CPN nucleus is an enlarged version of a single prey nuclei, which the ciliates sequester from their prey. Besides this enlarged prey nucleus, well-fed ciliates keep some additional prey nuclei, termed “Extra Prey Nuclei” (EPN). In prey starved ciliate cultures only one of the daughter cells receives the CPN. In the other daughter cell one of the EPNs moved into close proximity to the ciliate nuclei and enlarges. This phenomenon was found for both M. rubrum and M. major. A direct correlation between the percentage of cells with a CPN and photosynthetic activity was found, stressing the importance of retaining the CPN. This was also confirm through gene expressing studies, where none of the genes related to photosynthesis were expressed by M. rubrum. The exact relationships between the photosynthetic prey and the host have in many aspects been clarified, but some discussions are still going on with regard to the stability of the symbiotic relationships. Paper IV was part of such dispute, where the conclusion of Qui et al. 2016 that M. rubrum “farms” endosymbiotic T. amphioxeia, was questioned. Here it is was argued that M. rubrum does not have a permanent endosymbiont, but rather rely on prey ingestion for prey chloroplasts, prey nuclei and other cell organelles.

Effects of nitrogen (N) availability and the N-form (NO3-, NH4+ and urea) was investigated for the SNCMs, D. acuminata and M. rubrum, and compared to their chloroplasts donor, the cryptophyte T. amphioxeia. Uptake and utilization of NH4+ by D. acuminata was demonstrated, which led to increased cell growth and photosynthetic activity. Uptake of NO3- was shown, but only at very low rates, and D. acuminata could not utilize NO3- for cellular growth. M. rubrum was able to take up all forms of inorganic N, and cell growth responded positively until N-depletion, very similar to the prey, T. amphioxeia. Also, additions of NH4+ to M. rubrum and T. amphioxeia cultures did not affect photosynthetic activity. Common for all tree species was the repressive effect by NH4+ additions on the uptake rate of NO3- and urea, and an increased uptake rate of NO3- with increasing ambient concentration – similar to a phototroph. Finally, the effect of time of prey deprivation was shown to have no effect on potential N-uptake in M. rubrum, while it had some effect on D. acuminata.