Effects of acidic conditions on the physiology of a green alga (Micractinium sp.) before and after a 5-year interaction with Tetrahymena thermophila in an experimental microcosm

The mechanisms through which algae evolve physiological characteristics related to endosymbiotic associations with heterotrophic organisms remain unclear. We previously showed that a green alga (Micractinium sp.) was able to evolve a host (ciliate)-benefiting phenotype that prolonged the longevity of Tetrahymena thermophila in the absence of bacteria during a 5-year culture period in an experimental microcosm. Comparative experiments between the ancestral alga (i.e. original) and evolved algal clones of the same lineage can be performed to analyse the mechanisms that underlie algal evolution during interactions with ciliates. Here, we investigated the effects of acidic conditions on algal physiology because the acidic conditions within the food vacuoles of Tetrahymena could potentially affect algal evolution during long-term interactions. It might be expected that algal clones isolated from T. thermophila hosts would have developed the ability to resist acidic conditions in order to establish within the host, when compared with the ancestral strain. Unexpectedly, comparative analyses demonstrated that acidic conditions with pH values ranging from 3.9 to 4.3 limited the growth of the isolated algal clones SC9-1 and SC10-2, whereas the ancestral strain could grow under these conditions. Acidic conditions were responsible for significantly lower chlorophyll a/chlorophyll b ratios in the isolated algal clones, and the isolated clone SC9-1 exhibited a high cellular content of chlorophyllide a during a short exposure to acidic conditions, suggesting that degradation of chlorophyll a occurred. Furthermore, acidic conditions increased the release of extracellular glycerol and sucrose in the SC9-1 clone. These results indicated that the ancestral strain showed phenotypic changes related to acidic conditions, which may reflect the ongoing development of an algal phenotype suited to symbiosis. Hypotheses are proposed to explain the limited growth of the isolated clones, and implications for the Micractinium–Tetrahymena association are discussed.