Green algal symbionts are stably retained and provisioned in the dark despite a clear physiological cost

Photosymbioses, or associations between heterotrophs and photoautotrophs, are widespread and indispensable in today's ecosystems. The chloroplasts of algae and land plants, which are at the heart of most of earth's primary production, stem from ancient photosymbioses. Photosymbioses often combine heterotrophy and autotrophy and must thus efficiently allocate resources between these two costly cellular processes. We currently lack a clear picture of how photosymbioses allocate their valuable cellular resources in response to environmental change. In this study, we combine growth assays, automated fluorescence microscopy, transmission electron microscopy, and mass spectrometry-based proteomics to explore the physiology and cellular resource allocation of the ciliate-green alga photosymbiosis of Paramecium bursaria. In nutrient-rich environments that resemble P. bursaria's natural habitat, the maximum growth rate attained saturates regardless of light intensity. The green algae thus do not provide a benefit in nutrient-replete conditions, and the photosymbiosis primarily functions heterotrophically. The green algae occupy a remarkably similar and constant volume fractions across contrasting light environments despite displaying clear photo-physiological adaptation. This is true regardless of a clear physiological cost of the photosymbionts; aposymbiotic hosts always display higher growth rates in the dark. The host does not decrease 'symbiont load' in environments where green algae are not beneficial. Moreover, in the dark, the green algae are fully dependent on their hosts and take up a larger proteome mass fraction that increases with prey abundance. Differential protein expression analyses suggest that acetate and amino acids are the preferred sources of carbon and nitrogen for the green algae in the dark. The stable persistence and higher resource uptake by the photosymbionts in the dark argue against a view where hosts have full control over and selfishly exploit their symbionts.