| Summary: | Accumulation of anthropogenic CO₂ is fuelling the decline of coral reef ecosystems. Increasing sea surface temperatures disrupt the endosymbiotic relationship between cnidarians and their single-celled dinoflagellate partners (genus Symbiodinium), while ocean acidification is known to impede calcification. At the cellular level, however, ocean acidification also has the potential to cause acidosis, with negative impacts on cell structure and function. Yet, despite the importance of intracellular pH (pHᵢ), the mechanisms involved in pH regulation and the buffering capacity within coral cells are not well understood. Combining pH-sensitive fluorescent dyes with either confocal microscopy or flow cytometry enables the measurement of pHᵢ within live cells. Here, I employed these techniques to determine the relationship between symbiont photosynthesis and host- and symbiont pHᵢ under ocean acidification and thermal stress. The specific aims of the study were: (1) to design a protocol for measuring the pHᵢ of the Symbiodinium cell and to quantify the effect of the diel light cycle on the pHᵢ of both members of the endosymbiosis; (2) to determine the role of the symbiont in modifying host cellular responses to short-term CO₂-induced acidification; (3) to quantify how exposure to elevated temperature changes the responses of the host and the symbiont pHᵢ to short-term CO₂-induced acidification; and (4) to establish the relationship between photo-physiology and pHᵢ after longerterm exposure to CO₂-induced acidification. In Chapter 2, I used flow cytometry in conjunction with the ratiometric fluorescent dye BCECF to quantify pHᵢ in Symbiodinium cells and to monitor the effect of the diel light/dark cycle on pHᵢ. The pHᵢ of ITS2 type B1 cells (freshly isolated from the sea anemone Aiptasia pulchella) was 7.25 ± 0.01 (mean ± S.E.M) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHᵢ 7.21–7.39 in the light and 7.06–7.14 in the dark. Of note, there was a significant genotypic difference in pHᵢ, irrespective of irradiance, with this parameter being lowest in types E2 and F1. The pHᵢ of A. pulchella host cells was then measured using the SNARF-4F probe and confocal microscopy. Light-induced alkalinisation observed in the dinoflagellate cells was reflected in the pHᵢ of the host cells, with pHᵢ increasing from 6.86 ± 0.04 in the dark, to 7.02 ± 0.06 in the light. The inter-dependence of host cell pHᵢ on its symbiont is described in Chapter 3 using cells isolated from the coral Pocillopora damicornis. BCECF was used in conjunction with confocal microscopy to determine how host- and symbiont pHᵢ responds to pCO₂-driven seawater acidification under saturating irradiance, in symbiotic and nonsymbiotic states, with and without the photosynthetic inhibitor DCMU. Each treatment was run under control (pH 7.8) and CO₂-acidified seawater conditions (decreasing pH from 7.8 - 6.8). After 105 min of CO₂ addition, by which time the external pH (pHₑ) had declined to 6.8, the dinoflagellate symbionts had increased their pHᵢ 0.5 pH units above control levels. In contrast, in both symbiotic and nonsymbiotic host cells, 15 min of CO₂ addition (0.2 pH unit drop in pHₑ) in the presence and absence of DCMU led to cytoplasmic acidosis equivalent to 0.4 pH units. Despite further seawater acidification over the duration of the experiment, the pHᵢ of nonsymbiotic coral cells did not change, though in host cells containing a symbiont cell the pHᵢ recovered to control levels. This recovery was negated when cells were incubated with DCMU, revealing that the photosynthetic activity of the endosymbiont is tightly coupled with the ability of the host cell to recover from cellular acidosis after exposure to high CO₂ / low pH. This raised the interesting possibility that bleached corals may be more sensitive to cellular acidosis than are their non-bleached counterparts, a hypothesis that was tested in Chapter 4. I exposed P. damicornis (a thermally sensitive coral) and Montipora capitata (a thermally resilient coral) fragments to four temperature treatments: 23.8 (ambient), 25.5, 28 and 31°C. Host coral cells containing their symbionts were then isolated and subjected to CO₂-addition, designed to mimic predicted the VI CO₂ stabilisation scenario (pHₑ 7.6) provided by the Intergovernmental Panel on Climate Change (IPCC, 2014). Host cells in P. damicornis were much more susceptible to cellular acidosis under the highest temperature treatment (7.40 ± 0.07 at 23°C to 6.56 ± 0.03 at 31°C) than their counterparts in M. capitata (7.35 ± 0.07 at 23°C to 6.95 ± 0.05 at 31°C). A similar decrease was observed in the Symbiodinium cell, with pHᵢ dropping from 7.45 ± 0.02 to 6.86 ± 0.01 in P. damicornis, and from 7.43 ± 0.04 to 7.14 ± 0.03 in M. capitata after CO₂-addition, suggesting that thermally sensitive corals may be at the highest risk of cellular acidosis. Finally in Chapter 5, I describe the relationship between CO₂-driven acidification, photo-physiological performance and pHᵢ in A. pulchella. I exposed anemones to ambient (289.94 ± 12.54 μatm), intermediate (687.40 ± 25.10 μatm) or high (1459.92 ± 65.51 μatm) CO₂ conditions for two months, conditions that represent the IPCC IV and VI stabilisation scenarios (IPCC, 2014). At regular intervals I measured the maximum dark-adapted fluorescent yield of PSII (Fv/Fm), the gross photosynthetic rate, respiration rate, symbiont population density, and the light-adapted pHᵢ of both the symbiont and the host cell. I observed increases in all but one photo-physiological parameter (Pgross: R ratio). Increases in light-adapted symbiont pHᵢ were observed under both intermediate and high CO₂ treatments, relative to control conditions (pHᵢ 7.35 ± 0.03 and 7.46 ± 0.06 versus pHᵢ 7.25 ± 0.05, respectively). The response of light-adapted host pHᵢ was more complex, with no change observed under the intermediate CO₂ treatment, but a 0.3 pH-unit increase under the highest CO₂ treatment (pHᵢ 7.19 ± 0.01 and 7.48 ± 0.02, respectively). This suggests that, rather than causing cellular acidosis, the addition of CO₂ will enhance the organism’s photosynthetic performance, enabling the host-symbiont association to withstand the predicted ocean acidification scenarios. Overall, the results from this study provide new insight into the cellular interactions that underpin cnidarian-dinoflagellate symbiosis. Moreover, they highlight the dynamic and species-specific nature of the cellular responses, reinforcing the need to incorporate species-specific acidification/warming interactions into models that are designed to predict the response of marine organisms to global climate change.
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