Contrasting Mixotrophic Lifestyles Reveal Different Ecological Niches in Two Closely Related Marine Protists

Abstract

Many marine microbial eukaryotes combine photosynthetic with phagotrophic nutrition, but incomplete understanding of such mixotrophic protists, their functional diversity, and underlying physiological mechanisms limits the assessment and modeling of their roles in present and future ocean ecosystems. We developed an experimental system to study responses of mixotrophic protists to availability of living prey and light, and used it to characterize contrasting physiological strategies in two stramenopiles in the genus Ochromonas. We show that oceanic isolate CCMP1393 is an obligate mixotroph, requiring both light and prey as complementary resources. Interdependence of photosynthesis and heterotrophy in CCMP1393 comprises a significant role of mitochondrial respiration in photosynthetic electron transport. In contrast, coastal isolate CCMP2951 is a facultative mixotroph that can substitute photosynthesis by phagotrophy and hence grow purely heterotrophically in darkness. In contrast to CCMP1393, CCMP2951 also exhibits a marked photoprotection response that integrates non-photochemical quenching and mitochondrial respiration as electron sink for photosynthetically produced reducing equivalents. Facultative mixotrophs similar to CCMP2951 might be well adapted to variable environments, while obligate mixotrophs similar to CCMP1393 appear capable of resource efficient growth in oligotrophic ocean environments. Thus, the responses of these phylogenetically close protists to the availability of different resources reveals niche differentiation that influences impacts in food webs and leads to opposing carbon cycle roles.

Keywords: chrysophytes; microbial food web; mixotrophy; phagotrophy; phytoplankton.

Figure 1

Phylogenetic reconstruction of chrysophytes based on 18S rRNA gene sequences. A total of 1,444 positions from 132 near full‐length sequences were used. Tree topology is based on maximum likelihood inference and node statistical supports are indicated based on Bayesian posterior node probabilities, and percent of bootstrap replicates from two maximum likelihood methods (1,000 replicates in RAxML and 100 replicates in PhyML). Columns of colored squares indicate the nutritional capacities of described genera for photosynthesis (left column: green—known phototrophic potential, white—potential not reported in cultured isolates, grey—unknown potential) and for phagotrophic ingestion of prey (right column: black—known phagotrophic potential, white—potential not reported in cultured isolates, grey—unknown potential). [Color figure can be viewed at http://www.wileyonlinelibrary.com]

Figure 2

Growth responses to light under prey‐amended conditions in the two Ochromonas isolates. (a and b) Light–response curves and cellular chlorophyll a fluorescence of CCMP1393 and CCMP2951 during prey‐amended growth in the light (mixotrophic growth; Experiment 1). Cultures were maintained semi‐continuously with daily additions of Vibrio fischeri as prey. (c and d) Growth curves of CCMP1393 and CCMP2951 (Experiment 2) with daily prey‐amendments in the light (mixotrophic growth), with daily prey‐amendments in darkness (heterotrophic growth), or after ceasing prey‐amendments in the light (autotrophic growth). Asterisk indicates significant difference from mixotrophic conditions (P < 0.05; Holm‐Sidak test following a two‐way repeated measures ANOVA). Error bars indicate standard deviation among biological triplicates. [Color figure can be viewed at http://www.wileyonlinelibrary.com]

Figure 3

Carbon acquisition and growth of the two Ochromonas isolates under prey‐amended and deplete conditions. Rates of (a) net primary production, (b) ingestion, and (c) growth in CCMP1393 and CCMP2951 acclimated to 15 (LL) or 100 (HL) μmol photons · m⁻² · s⁻¹ under either prey‐amended or prey‐deplete conditions. Error bars indicate standard deviation among biological triplicates. For figure simplicity, results of statistical comparisons are shown in Table 1.

Figure 4

Pigmentation of the two Ochromonas isolates under prey‐amended and prey‐deplete conditions. (a) Cellular chlorophyll content and relative content of (b) carotene, (c) fucoxanthin, and (c) Violaxanthin cycle pigments (VAZ) in CCMP1393 and CCMP2951 acclimated to 15 (LL) or 100 (HL) μmol photons · m⁻² · s⁻¹ under either prey‐amended or prey‐deplete conditions. Relative pigment contents are expressed as molar ratios to Chl a. Error bars indicate standard deviation among biological triplicates. For figure simplicity, results of statistical comparisons are shown in Table 1.

Figure 5

Electron transport in the two Ochromonas isolates under prey‐amended and deplete conditions. (a and b) Rapid light–response curves of electron transport through photosystem II. Lines represent photosynthesis–irradiance curves fitted to the data (see Table 2 for parameter values). (c and d) Non‐photochemical quenching (NPQ) in CCMP1393 and CCMP2951 pre‐acclimated to low‐light (15 μmol photons · m⁻² · s⁻¹) and high‐light (100 μmol photons · m⁻² · s⁻¹) intensities under either prey‐amended conditions or after depletion of prey for 3 d. (e and f) Effect of inhibition of mitochondrial respiratory complex III (C_III), mitochondrial alternative oxidase (AOX), both C_III and AOX, or the plastid terminal oxidase (PTOX) involved in chlororespiration on effective photochemical yield of PSII in CCMP1393 and CCMP2951. Measurements were performed with mixotrophic, prey‐amended cultures acclimated to 100 μmol photons · m⁻² · s⁻¹ and exposed to a sub‐saturating light intensity, the acclimation intensity, or a super‐saturating intensity for a period of 3 min prior to measurements. Error bars indicate standard deviation among biological triplicates. [Color figure can be viewed at http://www.wileyonlinelibrary.com]

Figure 6

Responses of the two mixotrophic Ochromonas isolates to different environmental conditions. Both isolates reach highest growth rates under saturating light and prey availability. The obligate mixotroph (CCMP1393, top row) has higher chlorophyll content under low‐light compared to high‐light conditions and when prey is available compared to prey‐deplete conditions. Rates of both carbon fixation in the Calvin–Benson cycle (CBC) and ingestion of prey are highest under mixotrophic conditions under high light. Reducing equivalents are exchanged between the plastid and mitochondria linking the photosynthetic with the respirational electron transport chain. Growth is most strongly reduced at low‐light intensities. In contrast, the facultative mixotroph (CCMP2951, bottom row) increases its chlorophyll content when prey is depleted at high light, while in the presence of prey light has little impact on chlorophyll content. Rates of ingestion are highest under low‐light condition and maximum rates of carbon fixation are reached when prey is depleted. Export of reducing equivalents from the chloroplast to mitochondria only acts as overflow during high‐light condition. Growth is most strongly reduced in the absence of prey. AOX, alternative oxidase; TCA, tricarboxylic acid cycle; C_I, respiratory complex I; C_III, respiratory complex III; PSII, photosystem II; PTOX, plastid terminal oxidase; PSI, photosystem I; CBC, Calvin–Benson cycle. [Color figure can be viewed at http://www.wileyonlinelibrary.com]