# Oxygen as a primary selective pressure for photosymbiosis evolution

Loïc Quevarec, Rachel Bonnarde, Christophe Robaglia, Gaël Brasseur

► To cite this version:

Loïc Quevarec, Rachel Bonnarde, Christophe Robaglia, Gaël Brasseur. Oxygen as a primary selective pressure for photosymbiosis evolution. Current Biology, 2026, 36 (4), pp.928-939.e2.
10.1016/j.cub.2026.01.010. hal-05534503

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# Oxygen as a primary selective pressure for photosymbiosis evolution

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Loïc Quevarec¹,², Rachel Bonnardе¹,²,³, Christophe Robaglia¹,*, and Gaël Brasseur²,⁴,*

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¹Aix Marseille Université, CEA, CNRS, BIAM, Luminy Génétique et Biophysique des Plantes,
13009 Marseille, France

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²Laboratoire de Chimie Bactérienne, IMM, CNRS, Aix-Marseille Université, 13009 Marseille,
France.

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³Present address: Laboratoire Physiologie Cellulaire & Végétale, CEA, 17 Avenue des martyrs,
38000 Grenoble France.

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⁴Lead contact

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*Co-corresponding authors: christophe.robaglia@univ-amu.fr, brasseur@imm.cnrs.fr

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# Summary

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Photosymbioses provide carbon and oxygen to the biosphere, yet the mechanisms underlying their evolution remain poorly understood. We develop a naive system based on the predatory ciliate *Tetrahymena thermophila*, not known for hosting symbionts, to recapitulate early events of photosymbiosis evolution. *Tetrahymena thermophila* readily phagocytoses eukaryotic algae (*Chlorella variabilis*) or cyanobacteria (*Synechococcus elongatus*). Feeding on either prey in a low-carbon medium provided little or no growth advantage. In contrast, in a hypoxic environment, both intracellular *C. variabilis* or *S. elongatus* can support temporarily growth of *T. thermophila*. These results suggest that oxygen supply within the host could represent a more plausible initial advantage supporting photosymbiosis evolution than carbon metabolites. While
25 most extant photosymbiosis are based on carbon supply to the host cell, we therefore propose
26 that this would be a secondary event occurring from initial evolution in anoxic or hypoxic
27 conditions, where O₂ production is crucial for establishing the initial steps of photosymbiosis.

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Keywords

29 *Tetrahymena thermophila*, *Synechococcus elongatus*, *Chlorella variabilis*, photosymbiosis,
30 evolution, oxygen, carbon, phagocytosis.

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# Introduction

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Oxygenic photosynthesis appeared in cyanobacteria probably around 3 Byr ago¹. Using light energy, it provides the chemical energy to fix atmospheric CO₂ into building block of life, supporting the proliferation of photosynthetic organisms and indirectly the organisms that feed on them, and producing O₂ as a by-product. Primary photosynthetic endosymbiosis is defined as the acquisition of photosynthesis by eukaryotes following the engulfment of cyanobacteria.

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One event occurred around 1.6 Byr ago²,³ leading to the Archaeplastida group containing the Glaucophyta, the Rhodophyta (red algae) and the Viridiplantae (green algae and land plants)⁴.

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The only known other event occurred around 90–140 million years ago and involves the amoeba *Paulinella chromatophora*⁴,⁵. Further spreading of photosynthesis occurred by integration of photosynthetic eukaryotes within eukaryotes and are known as secondary and higher order endosymbiosis events. These events concern many eukaryotic groups⁴,⁶. Photosymbiosis is thus a ubiquitous phenomenon on Earth and its impacts on the environment are considerable, contributing to the production of oxygen and the fixation of carbon. Numerous species benefit from this trait, making it highly advantageous and leading to its recurrent and independent selection throughout evolution⁷,⁸.

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Acquisition of symbionts often occurs through predation, particularly via phagocytosis⁹⁻¹¹.

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Extant stable photosymbiosis in plants and algae are clearly based on the advantage provided by carbon autotrophy¹²,¹³ and it is often assumed to be the evolutionary force promoting photosymbiosis. By comparison, the advantage provided by oxygen production has received far less consideration, even though it is ecologically relevant. Due to organic matter mineralization, water stratification and low oxygen solubility, hypoxia is a very common condition in a wide range of warm, shallow, illuminated, aquatic environments such as estuaries¹⁴, flooded soils, shallow lakes¹⁵, coral reefs¹⁶ and exerts a pressure on organism that

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57 populate them. For example, the photosymbiotic ciliate *Tetrahymena utricularia* lives in
58 symbiosis with the alga *Micractinium tetrahymenae* within the anoxic bladder-like traps of the
59 aquatic carnivorous plant *Utricularia reflexa*. In this ecosystem, the abundance of dissolved
60 carbon and other nutrients suggests that oxygen might be the primary benefit¹⁷. Furthermore,
61 the primary endosymbiosis (~1.6 Byr) that gave rise to chloroplasts occurred in globally anoxic
62 aquatic environments and secondary endosymbiosis events began to emerge before
63 Neoproterozoic Oxygenation Event (NOE; -800 to -500 Myr) and diversified during this
64 period¹⁸, when aquatic environments were still highly hypoxic¹⁹. The percentage of O₂ in the
65 atmosphere before the NOE is estimated to be low, between 0.02 and 2%¹⁹.

66 In this work, using a predator-prey model system, we experimentally evaluate what could be
67 the primary selective force for photosymbiosis establishment. We used the ciliate *T.
68 thermophila* as a model of predatory unicellular host, the cyanobacterium *Synechococcus
69 elongatus* and the eukaryotic green algae *Chlorella variabilis* as prey. Those can recapitulate
70 early events leading to either primary (*S. elongatus*) or secondary (*C. variabilis*)
71 photosymbiosis. *Tetrahymena thermophila* is a widely used model organism, that can grow by
72 osmotrophy (i.e., type of nutrition for an organism using osmosis to transport soluble 'food
73 particles' or metabolites into a cell) or phagocytosis of various prey²⁰,²¹. Despite being studied
74 for many years, it has never been found to stably host photosynthetic symbionts in the wild.
75 However, related species such as *Paramecium bursaria*²² or *T. utricularia*¹⁷ and many other
76 ciliates are known to host algal symbionts²³. The cyanobacterium *S. elongatus* is a model
77 organism for photosynthesis²⁴ living as a possible symbiont within the tissues of the oyster
78 *Crassostrea gigas*²⁵. The *Prochlorococcus* or *Synechococcus* genus has the greatest genetic
79 proximity to the *P. chromatophora* chromatophore, which results from a recent primary
80 endosymbiosis⁴,⁵,²⁶. The model organism *C. variabilis* is a species of photosynthetic unicellular
81 algae²⁷, which together with others members of its genus, are symbiont in several
82 photosymbioses (e.g., with the ciliates *P. bursaria*²⁸ or *Stentor pyriformis*²⁹, the heliozoan
83 *Acanthocystis surfacea*³⁰ or the cnidarian *Hydra viridissima*³¹).

84 We show that in an initial predatory-prey state, the supply of carbon by photosynthetic prey
85 support little growth of the predator, while the supply of oxygen in hypoxic environment leads
86 to a readily observable growth advantage, making it a possible proximal selective pressure for
87 initiation of photosymbiosis.

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# Results

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## Phagocytosis dynamics

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In coculture of *T. thermophila* (Figure 1A) with cyanobacterium *S. elongatus* UTEX 2973 (Figure 1B) or the algae *C. variabilis* (Figure 1C), *T. thermophila* multiplied by asexual division (doubling time at 30°C ~7 h (see Figure 5A)) and the resulting two daughter cells share the prey (*S. elongatus* or *C. variabilis*) during mitosis showing a transgenerational transmission of the prey cells (Figures 1D and E). This observation was made at the first division following the contact between the *T. thermophila* and the prey. In presence of the cyanobacterium *S. elongatus*, *T. thermophila* phagosomes containing several red autofluorescent bacteria were readily observed (Figure 1F). Flow cytometry using both scatters (Side scatter (SSC) and Forward scatter (FSC); Figure 2A) and chlorophyll fluorescence signals was further used to quantify the rate and the amplitude of the phagocytosis process. This experiment showed a fast phase of bacterial phagocytosis leading to a maximum of 160 cyanobacteria accumulated per *T. thermophila* cell at a calculated maximum initial speed of about 750 cyanobacteria phagocytosed per hour (1 cyanobacteria phagocytosed every 4.5 s) (Figure S2B). This period is followed by a longer period during which cyanobacterial cells progressively disappeared from *T. thermophila* cells at a rate of about 20 cyanobacteria per hour (Figure 2B). This kind of phagocytic dynamics was previously reported for *Tetrahymena pyriformis*³². We noticed that around 40 min after the initiation of coculture, a new population of fluorescent particles appears with an intermediate size between *T. thermophila* and *S. elongatus*. Fluorescence microscopy shows that they were often localized at the cytoproct and were droppings (fecal pellets) ejected by *T. thermophila* in the medium and containing up to 4 cyanobacterial cells, based on the average fluorescence of a free *S. elongatus* (Figures 2A, C, D and 1G for droppings image).

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In a contact between *T. thermophila* and the algae *C. variabilis*, a first rapid phase of phagocytosis at a maximum rate of about 40 algae phagocytosed per hour (1 every 1.6 min)
succeeded a slower phase where *C. variabilis* cells progressively disappeared at a rate of 3.7 cells per hour (Figures 3A, B and S3). The average size of free remaining *C. variabilis* cells increased during the phagocytosis process, suggesting a selective preference by *T. thermophila* for smaller *C. variabilis* probably linked to the size of its oral apparatus (6-10 µm) compared to that of *C. variabilis* cells (heterogenous between 2-10 µm) (Figure S3B). We observed droppings containing *C. variabilis* cells under the microscope (Figure 1H), but we were unable to quantify them by flow cytometry because their size overlapped with large free-living *C. variabilis*.

In the presence of a GFP (Green Fluorescent Protein) labelled *Escherichia coli* bacterium, up to 700 bacterial cells were phagocytosed in one hour with a maximum initial rate at the onset of more than 3000 *E. coli* per hour (Figure S4B – D). After one hour of phagocytosis, droppings containing up to 7 *E. coli* per dropping appeared, based on the average fluorescence of a free *E. coli*-GFP (Figure S4A and E). The small size of *E. coli* (1–2 µm) compared to *S. elongatus* (4–7 µm) and *C. variabilis* (2–10 µm) may explain *T. thermophila* ability to phagocytose large numbers of them.

## 129 Carbon benefit for *T. thermophila* through phagocytosis of phototrophs

*Tetrahymena thermophila* is routinely cultured in sugar rich media containing amino-acids in the form of peptone and yeast extract (Neff medium). In this medium, the addition of either *S. elongatus* (Figure 4A; Table S1) or *C. variabilis* (Figure 4B; Table S2) did not modify *T. thermophila* growth in the dark. *Tetrahymena thermophila* did not grow in the mineral Tris Acetate Phosphate (TAP) medium in the dark, routinely used for the culture of phototrophs (Figure 4A and B; Tables S1c and S2c). This experiment in the dark allowed us to evaluate the contribution of carbon from prey digestion to the growth of *T. thermophila* from direct transfer of exudated carbon from photosynthesis in the presence of light. In TAP medium, the presence
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of *C. variabilis* has no effect on *T. thermophila* growth (Figure 4B; Table S2) while it was
slightly improved by *S. elongatus* (Figure 4A; Table S1).

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To evaluate the contribution of photosynthetic sugars to *T. thermophila* growth in the presence
of phototrophs, we developed a modified Proteose Peptone (PP) medium, devoid of sugars, and
allowing basal growth of *T. thermophila* compared to rich Neff medium. *Tetrahymena*
*thermophila* and phototrophs were grown independently then centrifuged, washed, and
resuspended in modified PP medium with an excess of phototrophs. Using this procedure, a
modest stimulation of *T. thermophila* growth with *S. elongatus* was observed in the dark that
was improved in the light (Figure 4C; Table S3). In contrast, *C. variabilis* did not improve and
even inhibits *T. thermophila* growth both in the light and in the dark, compared with a culture
of *T. thermophila* alone in modified PP medium (Figure 4D; Table S4). The non-photosynthetic
*E. coli* allowed a consistent growth of *T. thermophila* in modified PP medium (Figure 4E; Table
S5), although less efficiently than the rich Neff medium (Figure 4A).

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Oxygen benefit for *T. thermophila* through phagocytosis of phototrophs

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We probed the lower limit of oxygen concentration to allow *T. thermophila* growth (Figure 5A). Optimum growth in continuous oxygen bubbling occurs at atmospheric tension of 21% O₂, while significant growth occurs with as little as 0.05% oxygen compared with a totally anoxic environment (Figure 5A; Table S6).

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To evaluate the role of photosynthetic oxygen, we initiate anoxic cocultures by mixing *T. thermophila* and phototrophs cells in conditions where most phototrophs were phagocytosed by ciliates. In the dark and anoxic conditions, *T. thermophila* did not grow and even lysed in the presence of all kinds of prey (Figure 5B and C, Table S7). In contrast either *S. elongatus* or *C. variabilis* support *T. thermophila* survival for several days in initial anoxia in the light but not in the dark (Figure 5B and C, Table S7). In anoxic coculture with *T. thermophila*, the non-phototrophic *E. coli*, did not support nor growth nor survival in the light or in dark (Figure 5D,

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Table S7). In addition, experiments where *T. thermophila* and phototrophs cultures in initial anoxia were separated by a tubing and sterile gas filter further confirm that a gaseous component rather than other photosynthetic metabolites was involved in supporting *T. thermophila* growth in initial anoxia (Figure S6).

We next evaluated the influence of an initial anoxia on the phagocytosis competence of *T. thermophila* using flow cytometry. In this experiment *T. thermophila* and phototrophs were mixed in proportion defined by the number of cells that *T. thermophila* is able to phagocytose in oxic conditions (see methods). In anoxia in the dark, *T. thermophila* is unable to phagocytose either *S. elongatus* or *C. variabilis* whereas in anoxia and in the light, phagocytosis kinetics is delayed compared with oxic conditions but still results in an average 100 *S. elongatus* (max. 200) and 10 *C. variabilis* (max. 20) per *T. thermophila* cell (Figure 6A and B). The transit time of ingested cells appeared to have increased when under anoxia and light compared to oxic conditions (Figure 6A and B); this is particularly clear for *S. elongatus*.

## Prey O₂ production and predator O₂ consumption are compatible

We measured O₂ consumption by *S. elongatus*, *C. variabilis* and *T. thermophila* and O₂ production by *S. elongatus* and *C. variabilis* at their maximal rate in presence of saturating amount of substrate (glucose) in the light (100 µmol·m⁻²·s⁻¹) with a modified oxymeter device (Figures 7 and S5). *Tetrahymena thermophila* mitochondrial O₂ consumption amounts to 43 nmol O₂·min⁻¹·mg⁻¹ total protein (i.e. 430 x 10⁻⁷ nmol O₂·min⁻¹·cell⁻¹, calculated with the calibration curves in Figure S1) with endogenous substrates present in the cells (Figure S5B) and to 60 nmol O₂·min⁻¹·mg⁻¹ total protein (i.e. 600 x 10⁻⁷ nmol O₂·min⁻¹·cell⁻¹) using glucose as substrate (Figure 7). This activity is completely inhibited by potassium cyanide (KCN), a terminal oxygen reductase inhibitor (Figure S5B – C). *Chlorella variabilis* and *S. elongatus* exhibits an O₂ consumption activity in the dark of 67 and 20 nmol O₂·min⁻¹·mg⁻¹ total protein, respectively (Figures 7 and S5C). For O₂ production in the light, *C. variabilis* and *S. elongatus*
produced 190 and 105 nmol O₂·min⁻¹·mg⁻¹ total protein (i.e. around 2.3 and 0.4 x 10⁻⁷ nmol

O₂·min⁻¹·cell⁻¹) (Figures 7B and S5C), respectively. This implies that about 260 *C. variabilis*.

and 1500 *S. elongatus* per *T. thermophila* cell would be required to provide maximum O₂

consumption by *T. thermophila* at atmospheric oxygen concentration (21% O₂), while only a

fraction of these numbers would be required to provide the minimal O₂ concentration

supporting *T. thermophila* growth (0.05% O₂).
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# Discussion

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Stable photosymbiosis evolved independently in many species of ciliates. *T. thermophila* is not known to host photosynthetic symbionts in nature but is able to phagocytose different species of prokaryotic or eukaryotic phototrophs and long-term co-culture with algae and bacteria has been described<sup>12,33</sup>. Assuming that photosymbiosis evolved from predation, this makes *T. thermophila* a suitable evolutionary model to explore the early events that can support a prey to symbiont transition.

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Conceptually, from the predator's (host) point of view, the evolution of a mutualistic symbiosis requires them to benefit from a greater inheritable selective advantage by maintaining prey (symbionts) than by digesting them. Depending on the environment, the energetic cost of maintaining the symbiont can be compensated by the by-products of photosynthesis such as exudated sugars or dioxygen molecule fueling aerobic metabolism. We wished to test these scenarios using *T. thermophila* as a predator, *C. variabilis* and *S. elongatus* as photosynthetic eukaryotic and prokaryotic prey and *E. coli* as a non-photosynthetic prey.

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In co-culture with *T. thermophila*, *E. coli* is rapidly phagocytosed (Figure S4) and can support growth of *T. thermophila* in a low-carbon media (Figure 4E). Although droppings containing fluorescent cells can be observed suggesting incomplete digestion (Figure S4), this shows that *E. coli* is fairly susceptible to phagocytosis and digestion by *T. thermophila*<sup>34,35</sup>.

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In low carbon media, *T. thermophila* derives only a moderate growth advantage from the phagocytosis of *S. elongatus* in the dark that is slightly enhanced in the light (Figure 4C), showing a small positive effect of photosynthesis that can be due to a better nutrient supply from digesting cyanobacteria cells or by the utilization of exudates. *Synechococcus elongatus* is known to be digested by numerous groups of predatory unicellular eukaryotes<sup>36-38</sup>, although it is a relatively poor-quality food source<sup>39</sup>. The amount of autofluorescent droppings containing

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undigested *S. elongatus* cells shows that it is partially resistant to digestion (Figure 2C and D),
explaining the modest growth advantage provided by comparison to *E. coli*.

In contrast, *T. thermophila* is unable to benefit from phagocytosis of *C. variabilis*, either by
digestion or by the use of exudates (Figure 4B and D). A significant reduction in growth either
in the light or in the dark indicates the presence of a cost (Figure 4D) which can have several
causes. *Chlorella variabilis* is not known to release toxins, but a lethal effect of lectins produced
by *Chlorella vulgaris* on mosquito *Aedes aegypti* larvae was observed⁴⁰. Intracellular
accumulation without digestion of *C. variabilis* can inhibit *T. thermophila* growth or cell
division. *Chlorella variabilis* is progressively eliminated from *T. thermophila* cytoplasm as
fecal pellets. In our conditions, these results made it unlikely that carbon metabolites provided
by phototrophs (*S. elongatus* or *C. variabilis*) could fuel the early stages of evolution towards
photosymbiosis but may play a role later for its stabilization.

In sheer contrast to the modest or inexistent benefit provided by phagocytosed phototrophs in
low carbon media compared to *E. coli*, growth of *T. thermophila* was maintained in initial
anoxia in the presence of either *C. variabilis* or *S. elongatus*. Survival of *T. thermophila*
populations last for around 2 to 3 days with *S. elongatus* or *C. variabilis*, respectively (Figure
5). This effect was dependent on light, showing that photosynthesis was required.
Quantification of maximum oxygen production and consumption shows that the maximum
number of phagocytosed prey in initial anoxia (average 10 *C. variabilis* and 120 *S. elongatus*
per *T. thermophila* cell; Figure 6) are theoretically fully compatible with *T. thermophila*
maximum energy requirements in sub-atmospheric O₂ concentration (Figures 2, 3 and 7). The
maximum possible O₂ production is likely underestimated since it was measured at 100
µmol·m⁻²·s⁻¹, while some *Chlorella* species and *S. elongatus* can grow at significantly higher
light intensities⁴¹,⁴². In addition, an important biological variability is observed in the numbers
of phototrophs cells ingested by *T. thermophila* in anoxia (Figure 6) implying that some cells
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in the population can obtain more O₂ than others, opening the door to selection of intracellular retention of phototrophs. In our growth conditions and in oxic conditions, *T. thermophila* population decrease occurs around 72h, probably due to resources limitation. Therefore, the benefit of the prey in hypoxic conditions cannot be evaluated beyond this time limit, since resources limitations will still occur, even if hypoxia slows down the metabolism⁴³. Although prey can provide a transient advantage in hypoxia, they are progressively either digested, expelled or diluted by cell divisions. Prey stabilization would require further genetic or epigenetic events that are discussed below.

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Our experiments were performed in initial complete anoxia, which is an extreme condition, but, of course, cannot last for long in the presence of phototrophs with light. This raises the question of the contribution of non-phagocytosed prey in relieving the anoxia. Lavergne⁴⁴ demonstrates that a significant fraction (30%) of photosynthetic oxygen can be used by the mitochondria of the same algal cell (*Chlorella Sorokiniana*) before it diffuses, since the average distance between cells in suspension is several orders of magnitude larger than the distance between organelles within the cell. In *Chlorella* cells, the unique mitochondria occupy 3-5% of the cell volume⁴⁵. In *Tetrahymena* cells, which are far larger, about 1500 mitochondria occupy 11%-15% of the cell volume⁴⁶. Therefore, assuming that the position of the oxygen source is comparable, the probability of intercepting photosynthetic oxygen is actually higher in *Tetrahymena* and our calculations confirm that a small number of algal cells can support *Tetrahymena* growth in hypoxia. Inducing autophagy, hypoxia and other stresses can trigger relocation of mitochondria close to the phagosomes further increasing the use of photosynthetically evolved oxygen⁴⁷.

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## A model for the transition from prey to symbiont

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To establish itself within another cell following phagocytosis, a cell must first escape digestion to avoid degradation, and then evade the host's intracellular surveillance (immunity) to prevent

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rejection. These events can result from genetic changes in both partners. The prey may first
evolve a resistant and impermeable cell wall⁴⁸. In our system, this stage has probably already
been reached by *C. variabilis*, which cannot be digested and is expelled as droppings. The
moderate growth advantage provided by *S. elongatus* in low carbon media can be due to the use
of exudates by *T. thermophila* potentially paving the way towards symbiosis evolution. On the
other hand, *Tetrahymena* species like *T. pyriformis* can feed voraciously on *S. elongatus*⁴⁹.
Further experiments would be necessary to distinguish between digestion and the use of
exudated carbon compounds in our system. The next step requires either a transient or a stable
deficiency in the host cell's surveillance system preventing rejection of undigested prey. In
support of this, algae in cnidarians (corals and anemones) and stolen chloroplasts in
kleptoplastic sacoglossan slugs are stored in arrested phagosomes⁵⁰. We observed that, for *S.
elongatus*, while the rate of prey phagocytosis decreases in hypoxia (Figure 6A), the
cytoplasmic transit time increases (Figure 6A), extending the time window during which the
host can benefit from the intracellular presence of undigested prey. Indeed, hypoxia suppresses
or blunts immunity in a wide range of cells and organisms, even for hypoxia-tolerant species⁵¹–
⁵³. In addition, the dampening of host immunity in the establishment or maintenance of
photosymbiosis has been demonstrated⁵⁴,⁵⁵.

If these stabilizing events occur, even partially or transiently (incomplete digestion or slower
rejection) the resulting host will potentially be better adapted in its environment. The repeated
emergence of photosymbiosis suggests that significant advantages are gained rapidly at each
step to allow further evolution. Our experiments suggest that an immediate advantage provided
by carbon metabolites is small and would be further hampered by cell wall impermeability.
Photosymbiosis evolution supported by exported sugars is nevertheless possible and indeed
host and symbiont sugar transportome remodeling occurs during several oceanic
photosymbiosis⁵⁶,⁵⁷. Although it probably depends on prey abundance and nature, a host would
generally obtain a better nutritional advantage by digesting its prey rather than by relying on exported sugars. Therefore, under our scenario, photosymbiosis evolution supported by exported sugars would imply that the prey had already evolved indigestibility and is not expelled, which may be several steps downstream the initiation of prey to symbiont transition. In contrast, oxygen diffusion is unavoidable and can immediately benefit the host. This model could explain why species such as those of the genus *Chlorella*, despite their robust cell walls⁵⁸, are frequently involved in photosymbiosis²⁸⁻³¹,⁵⁹.

Ecological observations further support that photosymbionts provide advantages in hypoxia. In a stratified freshwater pond, 96% of ciliated protozoa living below the oxic-anoxic limit were found to host algae, whereas above this limit, the majority of ciliates do not possess symbiotic algae⁶⁰. The intracellular presence of phagocytosed photosynthetic prey, even transiently, can provide an extended phenotype allowing expansion of the ecological niche into hypoxic hunting zones, and possible refuge from competitors and predators, at least in the presence of daylight⁹,¹¹,⁶⁰. Oxygen produced by photosynthetic *Symbiodiniaceae* was also suggested to improve survival of host anemones *Anthopleura elegantissima* and *Anemonia viridis* in hypoxic environments⁶¹,⁶². In the salamander *Ambystoma maculatum*, oxygen produced by the symbiotic alga *Oophila amblystomatis* promotes embryo development by protecting them from hypoxia⁶³⁻⁶⁵. Even long evolved photosymbiotic organisms living in oxic conditions, such as plants, survive better in the light than in the dark under hypoxic stress caused by flooding, suggesting a protective role of photosynthetic oxygen⁶⁶,⁶⁷. Chronologically, the hosts of the first photosymbioses (~1.6 Byr) living in highly hypoxic environments¹⁹ were already able to use oxygen produced by their photosymbionts since terminal oxygen reductases were already present (~3 Byr)⁶⁸. We hypothesize that in hypoxic/anoxic environments, this makes O₂ a more parsimonious driving force stabilizing early events leading to photosymbiosis, while sugar supply would result from further and later molecular and cellular exaptations⁶⁹. However, we
318 cannot exclude the possibility that metabolites (other than O₂) produced by phototrophs may

319 provide context-dependent benefits to heterotrophic hosts, particularly in natural environments

320 different from our artificial co-cultures.
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**Resource availability**

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**Lead contact**

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Requests for further details and resources should be directed to and will be fulfilled by the lead contact, Gaël Brasseur (brasseur@imm.cnrs.fr).

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**Materials availability**

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This study did not generate new unique reagents.

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**Data and code availability**

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All data reported in this paper will be shared by the lead contact upon request.

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This paper does not report original code.

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Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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**Acknowledgments**

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We thank Dr. K. Mochizuki, T. Noto and J. Saksouk (IGH CNRS Montpellier, France), Aaron P. Turkewitz (Chicago University, U.S.A.) for the kind gift of *Tetrahymena thermophila* strains and precious advices. We thank the cyanobacteria team at LCB-Marseille (Prof. A. Latifi, V. Risoul, A. Scholivet, S. Champ) for the kind gift of *Synechococcus elongatus* and the use of equipments, Dr. B. Ezraty and Prof. L. Aussel for *E. coli* strains, Dr. Y. Li-Beisson and P. Auroy-Tarrago (BIAM) for *Chlorella variabilis*. We also thank Prof. D. Aragnol and S. Ridaoui for preliminary experiments and the students V. Faroux, N. Landrau and Y. Allio for their help with some experiments. Our IAM team is also acknowledged for support and Dr. L. Espinosa for microscopy advices. We thank all the LGBP members for stimulating discussions. We thank P. Notareschi and A. Grossi for 3D printing accessories. This project PHOCEE N° 21-CE20-0035 to CR and GB was financially supported by the ANR agency and by IM2B (Institut de

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344 Microbiologie, Bioénergies et Biotechnologie, Aix-Marseille University) for a fellowship to

345 RB.

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347

## Author contributions

348 LQ, CR and GB conceptualized the study. LQ, RB, CR and GB designed the experiments. LQ,

349 RB and GB performed the experiments and collected the data. LQ and GB performed the

350 statistical analyses. LQ, CR and GB wrote the manuscript. All authors read, commented and

351 approved the final manuscript.

352

353

## Declaration of interests

354 The authors declare no conflict of interest.

355

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357

# Tables and figures

358

<caption>**Figure 1: Optical microscopy representing the organisms of our predator-prey model.**</caption>

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(A) *Tetrahymena thermophila*, (B) *Synechococcus elongatus* and (C) *Chlorella variabilis* cells; (D) *S. elongatus* and (E) *C. variabilis* in *T. thermophila* cells undergoing the first mitosis following the contact between the *T. thermophila* and the prey, showing transgenerational transmission; (F) *C. variabilis* phagocytosed by *T. thermophila* shown by red fluorescence; (G) and (H) green droppings (d.) exocytosed by *T. thermophila* and containing *S. elongatus* and *C. variabilis* cells, respectively.

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<caption>**Figure 2: Phagocytosis of cyanobacteria *Synechococcus elongatus* (SE) by the ciliate *Tetrahymena thermophila* (TT).*</caption>

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(A) Flow cytometry scatter density plots (Side scatter (SSC) versus Forward Scatter (FSC)). Analysis of populations of cyanobacteria SE mixed with TT in oxic conditions at 30 °C (initial ratio of 400 SE/TT). The apparition of TT droppings composed of fluorescent SE is indicated. Color code from blue to red indicates low to high relative density of populations respectively (total events accumulated per time point, n = 8 x10⁵). (B) Kinetics SE transit inside of TT using the intrinsic red autofluorescence of SE. Shaded area corresponds to the standard deviation of TT fluorescence (total data accumulated per curve, n=1 x10⁶). (C) SSC histograms of free cyanobacteria SE and the new population corresponding to the formation of droppings (see Figure 2A). (D) Kinetics of droppings formation expressed as the number of SE per dropping as a function of time. Experiments were carried out with four independent biological samples. See also Figure S4.

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<caption>**Figure 3: Phagocytosis of *Chlorella variabilis* (CV) by *Tetrahymena thermophila* (TT).*</caption>
(A) Analysis of populations of CV mixed with TT in oxic conditions at 30 °C (initial ratio of 381 200 CV/TT). Flow cytometry scatter density plots (Side scatter (SSC) versus Forward Scatter 382 (FSC)) shows the larger size of TT, the smaller diffraction of the CV and the disappearing of 383 smaller CV as a function of time. Color code from blue to red indicates low to high relative 384 density of populations respectively (in a constant number of total events per time point, n = 8 x 385 10⁵). (B) Kinetics of the transit time of CV phagocytosed per TT using the intrinsic red 386 autofluorescence of CV. Shaded area corresponds to the standard deviation of fluorescence 387 (data points per curve n > 6.10⁵). Experiments were carried out with five independent cultures. 388 See also Figures S3 and S4. 389

390

391 **Figure 4: Contribution of prey microorganisms as carbon source for *Tetrahymena thermophila* (TT) growth.**

393 Shaded areas represent 95% confidence intervals. (A) and (B) Growth of TT in dark and in rich 394 Neff medium or mineral minimum TAP medium with (A) *Synechococcus elongatus* (SE) or 395 (B) *Chlorella variabilis* (CV) as prey; (C) and (D) Growth in modified PP medium in light or 396 dark with (C) SE or (D) CV; (E) Growth in modified PP medium with *Escherichia coli* (EC). 397 Each curve consists of 3 independent replicates estimated 10 times during the experiment based 398 on 10 counts (each curve is constructed with 3 x 10 x 10 = 300 counts). The "PP + light/TT" 399 curves in graphs (D) and (E) are the same control experiment. For greater clarity, we have 400 chosen to split the results in two panels according to the prey studied. The data were analyzed 401 using GAMM. All the results of the statistical test are shown in Table S1 - S5. Briefly, we 402 observed a significant growth of TT in the rich Neff medium with phototrophs ((A) SE p-value 403 < 0.001***; (B) CV p-value < 10⁻⁶*** ) or without phototrophs ((A) SE p-value < 0.001***; 404 (B) CV p-value < 10⁻⁶*** ) compared with a culture on minimum TAP medium. In TAP medium 405 (A), only SE increased slightly the growth of TT (p-value < 0.001***). In the modified PP
medium (C), SE stimulated the growth of TT in the light (p-value = 5.36 x 10⁻⁵***) but not in the dark (p-value = 0.556), while (D) CV decreased the growth of TT both in the light (p-value = 0.000324*** ) and in the dark (p-value = 0.000376***). (E). EC stimulated the growth of TT (p-value = 5.69 x 10⁻⁸***). ns: no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. See also Figure S1.

411

412 **Figure 5: Contribution of prey microorganisms to *Tetrahymena thermophila* (TT) growth**
413 *in limiting oxygen.*

414 The experiments were conducted in Neff medium. (A) Growth of TT by measuring the OD (λ = 600 nm) of the culture with flushing increasing oxygen concentrations ranging from 0% (pure N₂) to atmospheric level of 21% O₂. Each curve consists of 2-5 independent replicates with 12-21 measurements. The data were analyzed using a GAMM. Shaded areas represent 95% confidence intervals. All the results of the statistical test are shown in Table S6. Briefly, we observed a significant growth for 0.05% oxygen (p-value = 0.0138*), 0.1% (p-value = 0.0119*), 0.4% (p-value = 0.011*), 1.4% (p-value = 5.22 x 10⁻⁶***), 5% (p-value = 5.27 x 10⁻¹²***), 21% (p-value < 2 x 10⁻¹⁶***). (B) to (D) Growth of TT under anoxic conditions in light or dark, with or without prey ((B) *Synechococcus elongatus* (SE) (n = 2 independent replicates), (C) *Chlorella variabilis* (CV) (n = 4 independent replicates) and (D) *Escherichia coli* (EC). The data were analyzed using a Kruskal-Wallis rank sum test and a post hoc pairwise comparison (Dunn's test) with Holm correction. All the results of the statistical test are shown in Table S7. Briefly, we observed a significant growth of TT in anoxia only in the light with phototrophs compared with a culture in dark ((B) SE: adjusted p-value = 2.78 x 10⁻⁵***; (C) CV: adjusted p-value = 3.29 x 10⁻²⁰*** ) or without phototrophs ((B) SE: adjusted p-value = 5.43 x 10⁻¹⁶***; (C) CV: adjusted p-value = 4.31 x 10⁻¹³***). ns: no significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. See also Figures S1 and S6.

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**Figure 6: O₂ produced by the phototrophs allows *Tetrahymena thermophila* (TT) phagocytosis in anoxia.**

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The curves indicate the mean number of *Synechococcus elongatus* (SE) or *Chlorella variabilis* (CV) present inside TT cells ($n \ge 10^6$ data points) as a function of time. The shaded area corresponds to the standard deviation of fluorescence. (A) Kinetics of SE transit within TT in oxic conditions (atmospheric 21% O₂, dark cyan dots), in anoxic conditions in the dark (black triangles) or in anoxic conditions in the light (light cyan squares) at 30 °C (initial ratio of 500 SE/TT). (B) Kinetics of CV transit within TT in oxic conditions (atmospheric 21% O₂, dark green dots), in anoxic conditions in the dark (black triangles) or in anoxic conditions in the light (light green squares) at 30 °C (initial ratio of 150 CV/TT).

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**Figure 7: O₂ evolution by the photosynthetic prey *Synechococcus elongatus* and *Chlorella variabilis* is compatible with *Tetrahymena thermophila* aerobic respiration.**

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(A) O₂ consumption and production activities (nmol O₂·min⁻¹·mg⁻¹ protein, see Figure S5) corresponding to values with endogenous substrates, in saturation of substrate (glucose 20 mM) or in presence of light enabling optimal O₂ production for the phototrophs (60-100 µmol·m⁻²·s⁻¹) are shown for the three organisms. The dots on the histograms represent the number of replicates (4 ≤ n ≤ 11 independent replicates). (B) O₂ production and consumption calculated at the single cell level, based on the calibration curves presented in Figure S1. The results expressed per cell indicate that for maximum *T. thermophila* oxygen consumption rate, it requires the equivalent of about 260 *C. variabilis* and 1500 *S. elongatus* producing O₂ by photosynthesis.

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## Key resources table

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Bacterial and virus strains</td></tr><tr><td>Synechococcus elongatus UTEX 2973</td><td>UTEX culture collection of algae</td><td>UTEX 2973</td></tr><tr><td>Escherichia coli strain MG1655 sodA::gfp</td><td>Dr Ezraty's group (CNRS - LCB, Marseille)</td><td></td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Yeast extract</td><td>Biokar diagnostics</td><td>Cat#A1202</td></tr><tr><td>Bacteriological peptone Oxoid</td><td>Thermo Fisher Scientific Inc.</td><td>Cat#LP0037</td></tr><tr><td>Glucose</td><td>Merck millipore</td><td>Cat#108337</td></tr><tr><td>LB Broth Difco</td><td>BD Biosciences</td><td>Cat#244620</td></tr><tr><td colspan="3">Experimental models: Organisms/strains</td></tr><tr><td>Chlorella variabilis NC64A</td><td>Culture collection of algae and protozoa</td><td>CCAP 211/84</td></tr><tr><td>Tetrahymena thermophila CU427</td><td>Dr Mochizuki's group (CNRS - IGH, Montpellier)</td><td></td></tr><tr><td colspan="3">Software and algorithms</td></tr><tr><td>Prism8</td><td>GraphPad Software</td><td></td></tr><tr><td>Rstudio</td><td>Posit</td><td></td></tr><tr><td>NIS-Elements</td><td>Nikon</td><td></td></tr><tr><td>ProSort software</td><td>Bio-Rad</td><td></td></tr><tr><td>FlowJo v10.6</td><td>BD</td><td></td></tr><tr><td colspan="3">Other</td></tr><tr><td>S3e Cell Sorter</td><td>Bio-Rad</td><td></td></tr><tr><td>Cell density meter model 40</td><td>Fisher Scientific</td><td></td></tr><tr><td>Massic gas mixer Pegas 4000 MF</td><td>Columbus Instruments</td><td></td></tr><tr><td>Mettler-Toledo M700</td><td>Mettler-Toledo</td><td></td></tr><tr><td>Nikon Eclipse Ti microscope</td><td>Nikon</td><td></td></tr><tr><td>Motic B3 Professional microscope</td><td>Motic</td><td></td></tr><tr><td>Oxygraph Model K-ICC</td><td>Gilson</td><td></td></tr><tr><td>Optode based oxymeter</td><td>Presens</td><td></td></tr><tr><td>minitron shaker incubator</td><td>Infors HT</td><td></td></tr></tbody></table>

## Experimental Model and Study Participant Details

### Organisms and population maintenance

The experiments were carried out with the *T. thermophila* strain CU 427 (Figure 1A) obtained from Dr Mochizuki's group (CNRS - IGH, Montpellier). The ciliate *T. thermophila* is a strictly aerobic, mobile unicellular organism with an oral apparatus and a cytoprot for phagocytosis
463 of prey and elimination of phagocytosis waste, respectively. As it is able to grow by
464 osmotrophy, *T. thermophila* were routinely cultured in Neff medium⁷⁰ at 30 °C with orbital
465 shaking (~90 rpm). *S. elongatus* UTEX 2973 (Figure 1B) was routinely cultured in Blue Green
466 11 (BG11) medium⁷¹ at 28 °C in light (30-50 µmol·m²·s⁻¹) with orbital shaking (~70 rpm).
467 *Chlorella variabilis* NC64A (Figure 1C) was cultured in TAP medium⁷² at 28 °C in light (30-
468 50 µmol·m²·s⁻¹) with orbital shaking (~70 rpm). *E. coli* strain MG1655 sodA::gfp with the sodA
469 gene interrupted by the gfp gene was grown in Lysogeny Broth (LB) media.

470

471 Growth of *Tetrahymena thermophila*

472 We performed a calibration curve to estimate the number of individuals as a function of the
473 optical density (OD) measured with the cell density meter model 40 (Fisher Scientific) (λ = 600
474 nm) (Figure S1). The number of individuals was measured using a Malassez counting chamber
475 by microscopy.

476 Co-cultures of *T. thermophila* with prey (*S. elongatus*, *C. variabilis* or *E. coli*) was performed
477 in rich Neff medium, in modified PP medium (33.3 µM FeCl₃, 0.25% proteose peptone)⁷⁰
478 allowing very low growth or in minimum TAP medium⁷². The 'carbon' experiments were
479 carried out in Hungate tubes (gas-tight glass tubes with rubber cap for anaerobic culture) at 30
480 °C with orbital shaking (~90 rpm) in the dark or in the light (150 µmol·m²·s⁻¹) (Figure 4). To
481 test the advantage provided by oxygen from phagocytosed phototrophs, we co-cultured *T.
482 thermophila* in Neff medium at 30 °C under shaking with the different prey (*S. elongatus*, *C.
483 variabilis* or *E. coli*) in oxic or in anoxic sealed Hungate tubes in the dark or in the light (150
484 µmol·m²·s⁻¹) (Figure 5B–D). For anoxic conditions, cultures of *T. thermophila* in Neff medium
485 were bubbled with N₂ for 30 minutes, and phototrophs were added to the tubes at half-time. In
486 oxic condition, the septum seal was replaced with a cotton seal to allow gas exchange. To
487 measure *T. thermophila* growth in different oxygen concentrations (Figure 5A), the low oxygen
content was maintained by a massic gas mixer Pegas 4000 MF (Columbus Instruments) with a
constant flux of 25 mL·min⁻¹ using a mix of pure N₂ and synthetic air (20% O₂ and 80% N₂).

Concentration of O₂ in the continuously flushed Hungate tubes was measured with a Mettler-Toledo M700 recorder equipped with an O₂ module ppb 4700 and a nanomolar range sensitive Inpro 6900 O₂ probe calibrated with two points 0 and saturated air (237 µM O₂ dissolved at 30 °C). All cultures are in axenic conditions in Neff medium (Figure 5A).

Microscopy (Figure 1) were performed using an inverted Nikon Eclipse Ti microscope and a Motic B3 Professional microscope supplemented with a Moticam 4000 camera.

Method details
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Oxygen production and consumption by the different species
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O₂ consumption and production activities were measured polarographically with a Clark-type electrode in a stirred volume of 1.8 mL of corresponding medium saturated with air (237 µM O₂ at 30 °C) (Figure S5). Activities are expressed in nmol O₂·min⁻¹·mg⁻¹ total protein (Figure 7A) (proteins content from whole cells were measured with Bradford solution + 0.1 M NaOH using BSA as standard) or in 10⁻⁷ nmol O₂·min⁻¹·cell⁻¹ (Figure 7B) using calibration curves for the corresponding microorganism (Figure S1). Samples containing phototrophs were illuminated at light intensity of 100 µmol·m⁻²·s⁻¹. All experiments were carried out at 30 °C with 4 ≤ n ≤ 11 independent replicates (Figure 7A).

Experiment of cultures connected by a tubing: we cultivate the ciliate in a Neff medium under anoxic condition (except for positive control in oxic condition) at 30 °C with orbital shaking (~90 rpm). The *T. thermophila* culture is connected to the culture of phototrophs (*S. elongatus* or *C. variabilis*) via sterile tubing and gas filter (0.2 µM) in the light (100 µmol·m⁻²·s⁻¹) or in the dark (Figure S6). *Tetrahymena thermophila* growth was measured directly without
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withdrawing sample with OD ($\lambda$ = 600 nm). To facilitate comparison between experiments, the
growth was expressed as net growth ($\Delta$ OD).

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Phagocytosis dynamics followed by flow cytometry

517

We measured the predation level of *T. thermophila* on the prey *S. elongatus*, *C. variabilis* or *E. coli* - GFP using flow cytometry with the measure of internal fluorescence accumulated in *T. thermophila* over time (Figures 2, 3, 6, S2, S3 and S4). Thanks to the red autofluorescence of the phototrophs due to some photosynthetic pigments including chlorophyll (Figure 1F), we were able to timely and quantitatively follow the phagocytosis process knowing the level of fluorescence of the free partners. In addition, after exocytosis by cytoproct, we were also able to determine the number of prey in each dropping over time. For flow cytometry, the scatter density plots obtained (small-angle scattering or forward scatter (FSC) versus wide angle scattering or side scatter (SSC) signals) were gated on the population of interest, filtered to remove multiple events and then analysed for the autofluorescence intensity of the phototrophs (filter 655/Longpass) or the GFP fluorescence (filter 525/30) of *E. coli* strain MG1655 sodA::gfp. Samples were run in the low-pressure mode and a total number between 3 x 10⁵ and 1 x 10⁶ events were collected per sample and to construct the kinetics curves of phagocytosis.
The cytometry experiments were carried out between three and six times. Data were acquired with a S3e cells sorter (Bio-rad) using 488 and 561 nm lasers and were analysed and plotted using FlowJo v10.6.

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Statistical analysis

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Before the analysis, we square root-transformed data of OD or number of *T. thermophila*·µl⁻¹ for the growth curves in order to obtain normality and homoscedasticity of the data (Figures 4 and 5). For *T. thermophila* – *E. coli* and *T. thermophila* – *S. elongatus* in modified PP medium

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experiments, we used raw data. We used a Generalized Additive Mixed Model (GAMM) with

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R software<sup>73</sup> and the Mgcv package<sup>74</sup> to analyze and compare the curves of several experiments

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with gaussian distribution. No overdispersion of the data was observed. The residuals for each

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model were inspected using the "gam.check" function from the Mgcv package to verify that the

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conditions of normality and homoscedasticity were satisfied. We analyzed the number of T.

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thermophila as a function of time (in hours) (a continuous variable), culture conditions (i.e.,

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medium, prey, presence of oxygen) as fixed effects. Replicates were added as random effects.

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The smoothing was performed on the variable time in function of culture condition. A contrast

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matrix is created for pairwise comparison of each culture condition with the glht function of the

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multcomp package<sup>75</sup> for 'carbon' experiments with at least 4 different treatments (Table S1 and

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S2C). We used Kruskal-Wallis rank sum test with R software and the stats package<sup>73</sup> to compare

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the growth of *T. thermophila* in anoxia with phototrophs (*S. elongatus*, *C. variabilis*) or *E. coli*

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in light and dark (Figure 5, Table S7). Then, a post hoc pairwise comparison (Dunn's test) with

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Holm correction was realized between each treatment with rstatix package<sup>76</sup>.

552
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<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>T = 3h</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>T = 6h</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_16.png" /></figure>

SSC intensity of free SE and Droppings
(Arbitrary unit)
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_23.png" /></figure>
A

<figure><img src="image_24.png" /></figure>

B

<figure><img src="image_25.png" /></figure>
<figure><img src="image_26.png" /></figure>

<table><thead><tr><th></th><th>O<sub>2</sub> consumption<br>(nmol O<sub>2</sub>-min<sup>-1</sup>-cell<sup>-1</sup>)</th><th>O<sub>2</sub> production<br>(nmol O<sub>2</sub>-min<sup>-1</sup>-cell<sup>-1</sup>)</th><th>T. thermophila O<sub>2</sub> consumption/<br>phototroph O<sub>2</sub> production</th></tr></thead><tbody><tr><td><i>Thermosynechus thermophilus</i></td><td>600 x 10<sup>-3</sup></td><td>n.a.</td><td>n.a.</td></tr><tr><td><i>Chlorococcus variabilis</i></td><td>0.82 x 10<sup>-3</sup></td><td>2.3 x 10<sup>-3</sup></td><td>200</td></tr><tr><td><i>Synothococcus elongatus</i></td><td>0.073 x 10<sup>-3</sup></td><td>0.40 x 10<sup>-3</sup></td><td>1600</td></tr></tbody></table>
<figure><img src="image_27.png" /><figcaption>**Figure S1:** Calibration curves for (A) *Tetrahymena thermophila*, (B) *Synechococcus elongatus* and (C) *Chlorella variabilis*, related to Figures 4, 5 and 7.</figcaption></figure>

Optical density (OD, λ = 600 nm ± 20 nm) as a function of the number of individuals counted on a Malassez counting chamber (5 ≤ n ≤ 10 for each dilution).
<figure><img src="image_28.png" /><figcaption>Figure S2: Phagocytosis of cyanobacteria *Synechococcus elongatus* (SE) by the ciliate *Tetrahymena thermophila* (TT), related to Figure 2.</figcaption></figure>

(A) Histograms of the intensity of red fluorescence of TT accumulated by phagocyting autofluorescent SE as a function of time measured by flow cytometry. X axis represents the intensity of TT red fluorescence in log scale. Y axis is the mode of number of events measured in the gated population of TT. (B) First derivative kinetic of data in Figure 2B representing the number of SE phagocytosed/TT/hour as a function of time showing the fastest phagocytosis at the beginning and a decreasing kinetic over time until negative values corresponding to the digestion process. Maximum rate at the beginning reached about 750 SE phagocytosed/TT/hour. Experiments were carried out four times.
<figure><img src="image_29.png" /><figcaption>Figure S3: Phagocytosis of *Chlorella variabilis* (CV) by *Tetrahymena thermophila* (TT), related to Figure 3.</figcaption></figure>

(A) Histograms of the intensity of red fluorescence of TT accumulated by phagocyting autofluorescent CV as a function of time. The X axis represents the intensity of TT red fluorescence in log scale. Y axis is the mode of number of events measured in the gated population of TT. (B) Histograms of the intensity of side scatter (SSC) signal gated on the population of free CV showing the size selectivity of TT which first phagocytoses medium and small size CV, larger CV remaining not phagocytosed (see also Figure 3A). Experiments were carried out five times.
<figure><img src="image_30.png" /><figcaption>Figure S4: Phagocytosis of *Escherichia coli*-GFP (EC) by *Tetrahymena thermophila* (TT), related to Figures 2 and 3.</figcaption></figure>

(A) Analysis of populations of EC-GFP mixed with TT in oxic conditions at 30 °C (initial ratio of 10.000 EC/TT). Flow cytometry scatter density plots (Side scatter (SSC) versus Forward Scatter (FSC)) showing TT, the smaller diffraction of the EC cells and the apparition of a density region corresponding to the formation of TT droppings made of EC as a function of time. Color code from blue to red indicates low to high relative density of populations
respectively (in a constant number of total events per time point, n = 1 x 10⁶). (B) Histograms of the intensity of GFP fluorescence of TT accumulated by phagocyting EC as a function of time. X axis represents the intensity of TT green fluorescence in log scale and Y axis is the mode of number of events measured in the gated population of TT. (C) Kinetics of the number of EC phagocytosed per TT during fast phagocytosis (first phase) and digestion (second decreasing phase) using the green fluorescence of EC. The shaded area corresponds to the standard deviation of fluorescence. (D) First derivative kinetic of data in (C) representing the number of EC phagocytosed/TT/hour as a function of time showing the fast phagocytosis at the beginning and a decreasing kinetic over time until negative values corresponding to the digestion process. The maximum rate at the beginning reached about 3500 phagocytosed EC/TT/hour. (E) Kinetics of droppings formation with the number of EC per dropping as a function of time. Experiments were carried out 3 times.
<figure><img src="image_31.png" /><figcaption>**Figure S5:** O₂ consumption by *Tetrahymena thermophila* (TT) and O₂ production by the phototroph *Chlorella variabilis* (CV), related to Figure 7.</figcaption></figure>

(A) Device used to measure O₂ level in aqueous solution (TAP media for CV) using a Clark type electrode, a closed 1.8 mL temperature-controlled cuvette with an injection capillary and an optic fiber to illuminate the sample when needed with white lamp (intensity between 50-100 µmol·m⁻²·s⁻¹ enabling optimal O₂ production). (B) Example of O₂ consumption by TT cells (see Figure 7 for the mean and standard deviation of 11 replicates) completely inhibited by KCN (1 mM). (C) Production of O₂ by CV in the light and O₂ consumption in total darkness (see Figure 7 for quantification).
<figure><img src="image_32.png" /><figcaption>Figure S6: Experiments involving *Tetrahymena thermophila* (TT) and *Synechococcus elongatus* (SE) or *Chlorella variabilis* (CV) cultures separated by a tubing and gas filter, related to Figure 5.</figcaption></figure>

Photographs of tubbing experiments in light (A) and in the dark (B). (C) Net growth of TT alone (Δ OD; λ = 600 nm) in anoxia (grey dots and line) or in oxia (orange dots and line); (D) Net growth of TT (Δ OD; λ = 600 nm) connected to a SE culture by a tubing and gas filter in the dark (black dots and line, the small increase is probably due to a microleak of air in the system) or in the light (blue dots and line); (E) Net growth of TT (Δ OD; λ = 600 nm) connected
to a CV culture by a tubing and gas filter in the dark (black dots and line) or in the light (green dots and line). Growth was measured during more than 4 days.
## Parametric coefficients :

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>21.315</td><td>0.427</td><td>49.913</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>Neff/TT + SE</td><td>-0.723</td><td>0.603</td><td>-1.197</td><td>0.232</td><td>ns</td></tr><tr><td>TAP/TT</td><td>-16.321</td><td>0.623</td><td>-26.164</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>TAP/TT + SE</td><td>-13.709</td><td>0.641</td><td>-21.377</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr></tbody></table>

## Approximate significance of smooth terms:

<table><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): Neff/TT</td><td>6.357</td><td>6.357</td><td>64.905</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>s(time): Neff/TT + SE</td><td>5.458</td><td>5.458</td><td>72.329</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>s(time): TAP/TT</td><td>1.000</td><td>1.000</td><td>0.348</td><td>0.555</td><td>ns</td></tr><tr><td>s(time): TAP/TT + SE</td><td>2.889</td><td>2.889</td><td>22.172</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr></tbody></table>

## Pair-wise comparison:

<table><caption>**Table S1: Statistical analysis of the effect of medium richness and *Synechococcus elongatus* (SE) on the growth of *Tetrahymena thermophila* (TT), related to Figure 4A.**</caption><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>z value</th><th>Pr(&gt;|z|)</th><th></th></tr></thead><tbody><tr><td>Neff/TT + SE - Neff/TT</td><td>-0.722</td><td>0.604</td><td>-1.197</td><td>0.628</td><td>ns</td></tr><tr><td>TAP/TT - Neff/TT</td><td>-16.321</td><td>0.624</td><td>-26.164</td><td>&lt; 0.001</td><td>***</td></tr><tr><td>TAP/TT + SE - Neff/TT</td><td>-13.709</td><td>0.641</td><td>-21.377</td><td>&lt; 0.001</td><td>***</td></tr><tr><td>TAP/TT - Neff/TT + SE</td><td>-15.599</td><td>0.624</td><td>-25.015</td><td>&lt; 0.001</td><td>***</td></tr><tr><td>TAP/TT + SE - Neff/TT + SE</td><td>-12.986</td><td>0.641</td><td>-20.258</td><td>&lt; 0.001</td><td>***</td></tr><tr><td>TAP/TT + SE - TAP/TT</td><td>2.612</td><td>0.660</td><td>3.958</td><td>&lt; 0.001</td><td>***</td></tr></tbody></table>

Effects of culture conditions (minimum TAP medium or rich Neff medium with or without *S. elongatus* (SE)) (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT). A pairwise comparison was carried out. The data were analyzed using Generalized Additive Mixed Model (GAMM). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
## Parametric coefficients:

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>21.467</td><td>1.327</td><td>16.183</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>Neff/TT + CV</td><td>-0.954</td><td>1.865</td><td>-0.512</td><td>0.609</td><td>ns</td></tr><tr><td>TAP/TT</td><td>-15.865</td><td>1.829</td><td>-8.676</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>TAP/TT + CV</td><td>-15.161</td><td>1.836</td><td>-8.259</td><td>4.11 x 10<sup>-16</sup></td><td>***</td></tr></tbody></table>

## Approximate significance of smooth terms:

<table><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): Neff/TT</td><td>6.241</td><td>6.241</td><td>270.405</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>s(time): Neff/TT + CV</td><td>5.994</td><td>5.994</td><td>245.674</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr><tr><td>s(time): TAP/TT</td><td>1.000</td><td>1.000</td><td>0.165</td><td>0.684</td><td>ns</td></tr><tr><td>s(time): TAP/TT + CV</td><td>3.208</td><td>3.208</td><td>31.705</td><td>&lt; 2 x 10<sup>-16</sup></td><td>***</td></tr></tbody></table>

## Pair-wise comparison:

<table><caption>Table S2: Statistical analysis of the effect of medium richness and *Chlorella variabilis* (CV) on the growth of *Tetrahymena thermophila* (TT), related to Figure 4B.</caption><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>z value</th><th>Pr(&gt;|z|)</th><th></th></tr></thead><tbody><tr><td>Neff/TT + CV - Neff/TT</td><td>-0.954</td><td>1.865</td><td>-0.512</td><td>0.956</td><td>ns</td></tr><tr><td>TAP/TT - Neff/TT</td><td>-15.865</td><td>1.829</td><td>-8.676</td><td>&lt; 10<sup>-6</sup></td><td>***</td></tr><tr><td>TAP/TT + CV - Neff/TT</td><td>-15.161</td><td>1.836</td><td>-8.259</td><td>&lt; 10<sup>-6</sup></td><td>***</td></tr><tr><td>TAP/TT - Neff/TT + CV</td><td>-14.911</td><td>1.817</td><td>-8.204</td><td>&lt; 10<sup>-6</sup></td><td>***</td></tr><tr><td>TAP/TT + CV - Neff/TT + CV</td><td>-14.207</td><td>1.825</td><td>-7.786</td><td>&lt; 10<sup>-6</sup></td><td>***</td></tr><tr><td>TAP/TT + CV - TAP/TT</td><td>0.704</td><td>1.787</td><td>0.394</td><td>0.979</td><td>ns</td></tr></tbody></table>

Effects of culture conditions (minimum TAP medium or rich Neff medium with or without *C. variabilis* (CV)) (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT). A pairwise comparison was carried out. The data were analyzed using Generalized Additive Mixed Model (GAMM). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
## Parametric coefficients:

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>51.045</td><td>8.555</td><td>5.967</td><td>3.94e-09</td><td>***</td></tr><tr><td>PP + light/TT + SE</td><td>49.182</td><td>12.096</td><td>4.066</td><td>5.36e-05</td><td>***</td></tr><tr><td>PP + dark/TT + SE</td><td>7.128</td><td>12.096</td><td>0.589</td><td>0.556</td><td>ns</td></tr></tbody></table>

## Approximate significance of smooth terms:

<table><caption>**Table S3: Statistical analysis of the effect of light and *Synechococcus elongatus* (SE) on the growth of *Tetrahymena thermophila* (TT), related to Figure 4C.**</caption><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): PP + light/TT</td><td>5.153</td><td>5.153</td><td>13.14</td><td>&lt;2e-16</td><td>***</td></tr><tr><td>s(time): PP + light/TT + SE</td><td>4.917</td><td>4.917</td><td>15.33</td><td>&lt;2e-16</td><td>***</td></tr><tr><td>s(time): PP + dark/TT + SE</td><td>4.965</td><td>4.965</td><td>19.14</td><td>&lt;2e-16</td><td>***</td></tr></tbody></table>

Effects of culture conditions (light or dark; with or without *S. elongatus* (SE)) (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT) in modified PP medium (PP). The data were analyzed using Generalized Additive Mixed Model (GAMM). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
## Parametric coefficients:

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>8.756</td><td>0.691</td><td>12.671</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>PP + light/TT + CV</td><td>-3.524</td><td>0.976</td><td>-3.612</td><td>0.000324</td><td>***</td></tr><tr><td>PP + dark/TT + CV</td><td>-3.491</td><td>0.977</td><td>-3.573</td><td>0.000376</td><td>***</td></tr></tbody></table>

## Approximate significance of smooth terms:

<table><caption>**Table S4: Statistical analysis of the effect of light and *Chlorella variabilis* (CV) on the growth of *Tetrahymena thermophila* (TT), related to Figure 4D.**</caption><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): PP + light/TT</td><td>3.896</td><td>3.896</td><td>29.146</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>s(time): PP + light/TT + CV</td><td>2.351</td><td>2.351</td><td>2.538</td><td>0.149</td><td>ns</td></tr><tr><td>s(time): PP + dark/TT + CV</td><td>3.855</td><td>3.855</td><td>9.919</td><td>7.48e-07</td><td>***</td></tr></tbody></table>

Effects of culture conditions (light or dark; with or without *C. variabilis* (CV)) (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT) in modified PP medium (PP). The data were analyzed using Generalized Additive Mixed Model (GAMM). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Parametric coefficients:

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>93.943</td><td>6.972</td><td>13.474</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>PP + light/TT + EC</td><td>54.437</td><td>9.872</td><td>5.514</td><td>5.69e-08</td><td>***</td></tr></tbody></table>

Approximate significance of smooth terms:

<table><caption>Table S5: Statistical analysis of the effect of *Escherichia coli* (EC) on the growth of *Tetrahymena thermophila* (TT), related to Figure 4E.</caption><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): PP + light/TT</td><td>3.825</td><td>3.825</td><td>19.81</td><td>&lt;2e-16</td><td>***</td></tr><tr><td>s(time): PP + light/TT + EC</td><td>6.090</td><td>6.090</td><td>94.93</td><td>&lt;2e-16</td><td>***</td></tr></tbody></table>

Effects of the presence of *E. coli* (EC) (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT) in modified PP medium (PP). The data were analyzed using Generalized Additive Mixed Model (GAMM). ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
## Parametric coefficients:

<table><thead><tr><th></th><th>Estimate</th><th>Std. Error</th><th>t value</th><th>Pr(&gt;|t|)</th><th></th></tr></thead><tbody><tr><td>(Intercept)</td><td>0.0381</td><td>0.0672</td><td>0.567</td><td>0.572</td><td>ns</td></tr><tr><td>0.05% oxygen</td><td>0.1926</td><td>0.0776</td><td>2.481</td><td>0.0138</td><td>*</td></tr><tr><td>0.1% oxygen</td><td>0.1966</td><td>0.0776</td><td>2.534</td><td>0.0119</td><td>*</td></tr><tr><td>0.4% oxygen</td><td>0.1987</td><td>0.0775</td><td>2.564</td><td>0.0110</td><td>*</td></tr><tr><td>1.4% oxygen</td><td>0.3838</td><td>0.0823</td><td>4.663</td><td>5.22e-06</td><td>***</td></tr><tr><td>5% oxygen</td><td>0.5994</td><td>0.0824</td><td>7.271</td><td>5.27e-12</td><td>***</td></tr><tr><td>21% oxygen</td><td>0.7869</td><td>0.0774</td><td>10.170</td><td>&lt; 2e-16</td><td>***</td></tr></tbody></table>

## Approximate significance of smooth terms:

<table><caption>**Table S6: Statistical analysis of the effect of oxygen on the growth of *Tetrahymena thermophila* (TT)*, related to Figure 5A.</caption><thead><tr><th></th><th>edf</th><th>Ref.df</th><th>F</th><th>p-value</th><th></th></tr></thead><tbody><tr><td>s(time): 0% oxygen (N2)</td><td>1.828</td><td>1.828</td><td>1.201</td><td>0.365</td><td>ns</td></tr><tr><td>s(time): 0.05% oxygen</td><td>1.000</td><td>1.000</td><td>15.254</td><td>0.000123</td><td>***</td></tr><tr><td>s(time): 0.1% oxygen</td><td>1.000</td><td>1.000</td><td>72.543</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>s(time): 0.4% oxygen</td><td>3.106</td><td>3.106</td><td>138.045</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>s(time): 1.4% oxygen</td><td>3.463</td><td>3.463</td><td>110.232</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>s(time): 5% oxygen</td><td>3.699</td><td>3.699</td><td>295.663</td><td>&lt; 2e-16</td><td>***</td></tr><tr><td>s(time): 21% oxygen</td><td>3.984</td><td>3.984</td><td>1131.154</td><td>&lt; 2e-16</td><td>***</td></tr></tbody></table>

Effects of the oxygen concentration (Parametric coefficients table) and time (Approximate significance of smooth terms table, EDF: effective degrees of freedom) on the growth of *T. thermophila* (TT). The data were analyzed using Generalized Additive Mixed Model (GAMM).

ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
## *Tetrahymena thermophila* (TT) and *Synechococcus elongatus* (SE) experiment

### Kruskal-Wallis rank sum test

Kruskal-Wallis chi-squared = 68.647, df = 2, p-value = 1.24 x 10⁻¹⁵ ***

### Dunn's test (with Holm correction)

<table><thead><tr><th>group 1</th><th>group 2</th><th>n 1</th><th>n 2</th><th>statistic</th><th>p</th><th>p.adj</th></tr></thead><tbody><tr><td>light + TT</td><td>light + TT + SE</td><td>42</td><td>79</td><td>8.23</td><td>1.81e-16</td><td>5.43e-16 ***</td></tr><tr><td>light + TT</td><td>dark + TT + SE</td><td>42</td><td>72</td><td>4.58</td><td>4.61e-6</td><td>9.21e-6 ***</td></tr><tr><td>light + TT + SE</td><td>dark + TT + SE</td><td>79</td><td>72</td><td>-4.19</td><td>2.78e-5</td><td>2.78e-5 ***</td></tr></tbody></table>

## *Tetrahymena thermophila* (TT) and *Chlorella variabilis* (CV) experiment

### Kruskal-Wallis rank sum test

Kruskal-Wallis chi-squared = 106.93, df = 2, p-value < 2.2 x 10⁻¹⁶ ***

### Dunn's test (with Holm correction)

<table><thead><tr><th>group 1</th><th>group 2</th><th>n 1</th><th>n 2</th><th>statistic</th><th>p</th><th>p.adj</th></tr></thead><tbody><tr><td>light + TT + CV</td><td>dark + TT + CV</td><td>154</td><td>108</td><td>-9.33</td><td>1.10e-20</td><td>3.29e-20 ***</td></tr><tr><td>light + TT + CV</td><td>light + TT</td><td>154</td><td>58</td><td>-7.34</td><td>2.15e-13</td><td>4.31e-13 ***</td></tr><tr><td>dark + TT + CV</td><td>light + TT</td><td>108</td><td>58</td><td>0.245</td><td>8.06e-1</td><td>8.06e-1 ns</td></tr></tbody></table>

## *Tetrahymena thermophila* (TT) and *Escherichia coli* (EC) experiment

### Kruskal-Wallis rank sum test

Dark + TT + EC vs light + TT + EC: Kruskal-Wallis chi-squared = 0.3375, df = 1, p-value = 0.5613 ns

<caption>**Table S7: Statistical analysis of the effect of light and different prey on the growth of *Tetrahymena thermophila* (TT) in anoxia, related to Figure 5B – D.**</caption>

Effects of the presence of *Synechococcus elongatus* (SE), *Chlorella variabilis* (CV) or *Escherichia coli* (EC) on the growth of *Tetrahymena thermophila* (TT) in anoxia with or without light. The data were analyzed using a Kruskal-Wallis rank sum test and a post hoc pairwise comparison (Dunn's test) with Holm correction. ns: not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.