Metabolic Reliance on Photosynthesis Depends on Both Irradiance and Prey Availability in the Mixotrophic Ciliate, Strombidium cf. basimorphum

Many species of the ciliate genus Strombidium can acquire functional chloroplasts from a wide range of algal prey and are thus classified as generalist non-constitutive mixotrophs. Little, however, is known about the influence of irradiance and prey availability on their ability to exploit the photosynthetic potential of the chloroplasts, and how this may explain their spatial and temporal distribution in nature. In this study, inorganic carbon uptake, growth, and ingestion rates were measured for S. cf. basimorphum under three different irradiances (10, 40, and 120 μmol photons m–2 s–1) when acclimated to three different prey densities (5 × 103, 1 × 104, and 4 × 104 cells mL–1), as well as when allowed to deplete the prey. After prey depletion, cultures survived without prey longest (∼6 days) at the medium irradiance treatment (40 μmol photons m–2 s–1), while ciliate density, inorganic carbon uptake rates, and cellular chl-a content declined fastest at the highest irradiance treatment. This indicates that the ciliates may be unable to maintain the chloroplasts functionally without replacement at high irradiances. Ingestion rates were not shown to be significantly influenced by irradiance. The maximum gross growth efficiency (GGE) in this study (1.1) was measured in cultures exposed to the medium test irradiance and lowest prey density treatment (5 × 103 cells mL–1). The relative contribution of inorganic carbon uptake to the ciliate carbon budget was also highest in this treatment (42%). A secondary GGE peak (0.99) occurred when cultures were exposed to the highest test irradiance and the medium prey density. These and other results suggest that S. cf. basimorphum, and other generalist non-constitutive mixotrophs, can flexibly exploit many different environmental conditions across the globe.

This preliminary experiment was carried out to give a rough estimate of how Strombidium cf. basimorphum may react to different light treatments and to inform the choice of which prey densities to use in Experiment 2. Prey density was not adjusted during the experiment, and thus results could be skewed by early prey depletion in experimental replicates that began at lower prey densities.
Six prey densities (0.5, 1, 2, 5, 10, and 15 ×10 3 cells mL -1 ) were tested in three different light conditions (10, 40, and 120 μmol photons m -2 s -1 , referred to as I10, I40, and I120, respectively). A sample of 20 mL of each experimental prey density was prepared by mixing concentrated algal cultures with filtered seawater. Then, each prepared algal culture was distributed into eight wells, which each contained 2 mL of the culture. The first four wells were assigned to be experimental mixed cultures, and 10 twicewashed ciliate cells were added to each of the four wells. The remaining four wells of the same prey density were designated as control T. amphioxeia monocultures. The experiment was initiated with the introduction and mixing of ciliates and prey in the plates as described and ended after three days when all cultures were fixed in Lugol's solution and counted to determine both the growth and ingestion rates of the ciliates. T. amphioxeia densities were determined using Sedgewick-Rafter chambers, counting a minimum of 200 cells.
The resulting response curve for growth rate was fitted to a Michaelis-Menten equation (Supplementary Equation 1), as modified by (Montagnes, 1996).
Sup. Eq. 1 V is the ciliate's growth rate, Vmax is their maximum possible growth rate, C is the algal/prey density, T refers to the prey density at which ciliate growth rate is 0, and Km is the prey density at which V is exactly half of Vmax.
The subsequent Michaelis-Menten Kinetics curves were compared between irradiances using an extrasum-of-squares F-test. This test separately compared the pooled sum of squares from the curves for each of the light treatments to the extra sum of squares for their combined data fit to a single common curve. This was also done separately for parameters Vmax, Km, and T. The null hypothesis is that a single curve or parameter estimate provides a better fit for the three data sets, rather than separate curves or parameters (Motulsky and Ransnas, 1987). A p-value of 0.05 was used for significance testing. Both the curve fitting and subsequent analysis were done utilizing GraphPad Prism version 8.4.2 for Mac.

Results
As these experiments were meant to give a rough estimation of light and prey effects, prey density was not as strongly controlled as in Experiment 2. The prey density over the three days of this experiment was quite variable (see Supp. With that in mind, prey density was shown to have a significant impact on both growth and ingestion rates, while light did not appear to affect either variable (p-value = 0.052). Growth rate (Supp. Figure  1A) increased from 0.03 d-1 at the lowest initial prey density (5 ng C mL -1 ) to 0.42 d -1 at the highest prey density (150 ng C mL -1 ). Ingestion rates ranged from 26.9 prey cells predator -1 d -1 at the lowest initial prey density and 481 prey cells predator -1 d -1 at the highest.
The growth rate data were fitted to a Michaelis-Menten kinetics curve for each of the three light levels (Supp. Figure 1A, Supp. Table 2). The resulting curves indicated that ciliates grown at 10 μmol photons m -2 s -1 required the highest prey threshold density (T) to sustain the population. Cultures grown at the highest light treatment, 120 μmol photons m -2 s -1 , required the lowest density of prey to achieve half their maximum growth (Km).  basimorphum across three different light treatments and six different prey densities. Growth rate response curves were numerically fitted to Michaelis-Menten kinetics, and curves in (A) represent the subsequent models. Curves in (B) connect average ingestion rates at each prey density to each other. Solid, dashed, and dotted lines denote light treatments I10, I40, and I120, respectively.

Experiment 2: Data controlled for biovolume
Upon finding large variability in the size of ciliates acclimated to different light and prey conditions (Supplementary Figure 2; Supplementary Table 3), it was clear that simply reporting cell-specific results for variables such as growth rate and chl-a content would not accurately capture the full implications of S. cf. basimorphum's physiologic changes (Supplementary Figure 2). Therefore, to determine the amount of additional volume gained by ciliate cells (in µm 3 d -1 ) the growth rate (in cellular divisions d -1 ) for each culture was multiplied by the average cell biovolume (Supplementary Figure 3A). Similarly, to compare chl-a content across cells of different sizes, volume-specific chl-a (in pg chl-a 10 -3 µm -3 ) was calculated by dividing the cell-specific chl-a content by the average cell biovolume (Supplementary Figure 3B).