Despite being surrounded by highly nutrient-poor (oligotrophic) waters, tropical coral reefs are among the most productive marine ecosystems (Hatcher, 1988). Reef ecosystems are sustained by an efficient uptake, retention, and reuse of nutrients on all levels of biological organization (Hatcher, 1990; Wild et al., 2004). In particular, the symbiosis between cnidarian hosts and endosymbiotic algae of the genus Symbiodinium facilitates the recycling of nutrients as it sustains primary productivity in the absence of major nutrient sources (Muscatine and Porter, 1977; Rädecker et al., 2015). In this symbiosis, Symbiodinium translocate most of their photosynthates to the cnidarian host that in turn provides inorganic nutrients derived from its metabolism (Muscatine et al., 1989). Thereby, this tight nutrient-exchange relationship, particularly in stony corals, is the functional basis for the ecological success of tropical coral reefs over millions of years.

Being surrounded by host membranes, Symbiodinium rely on their host to fulfill their photosynthetic carbon dioxide (CO₂) requirements. The supply of CO₂ to the symbiont is controlled by two major processes: (1) CO₂ is produced during holobiont respiration (Muscatine et al., 1989). (2) Active carbon concentrating mechanisms (CCMs) by the host facilitate the uptake of dissolved inorganic carbon from surrounding seawater (Furla et al., 2000).

Despite these processes, several studies suggest that productivity in Symbiodinium may be carbon-limited even in stable symbiotic systems (Muscatine et al., 1989; Herfort et al., 2008; Klein et al., 2017). Hence, understanding the processes and environmental controls of in hospite CO₂ availability is crucial for our understanding of the cnidarian—alga symbiosis.

To address this issue, we experimentally tested whether photosynthesis of Symbiodinium in hospite is carbon-limited. Specifically, we investigated photosynthetic activity during glucose-stimulated holobiont respiration in the upside-down jellyfish Cassiopeia sp. Unlike most other Scyphozoa, Cassiopeia spp. are mixotrophic, i.e. draw energy and nutrients from both heterotrophic and autotrophic sources (Rahav et al., 1989; Muscatine, 1990), as they form a close endosymbiotic relationship with Symbiodinium. Thereby, Cassiopeia spp. offer distinct advantages for the study of the cnidarian—alga symbiosis, similar to the Aiptasia model system (Baumgarten et al., 2015). For instance, they are easy to rear in aquaria cultures, are non-calcifying, have motile medusa stages and can be infected with various algal symbionts (Klein et al., 2017). Using this emerging cnidarian model system allowed us to tackle the issue of CO₂ limitation in the cnidarian—Symbiodinium symbiosis in a straightforward experiment.

Glucose enrichment stimulated respiration rates in seawater during both light and dark incubations (Supplementary Table S1). Still, seawater respiration rates were ~5-fold below Cassiopeia sp. respiration rates at all times. Holobiont respiration rates of Cassiopeia sp. increased by ~80% under glucose-enriched conditions compared to untreated controls [t₍₁₃₎ = 5.27, P < 0.001, Figure 1]. Despite this increase in respiratory O₂ consumption, net photosynthesis rates during glucose-enriched conditions showed a significant increase of nearly 400% compared to controls [t₍₁₃₎ = 3.08, P = 0.008]. Consequently, gross photosynthesis rates increased by ~140% under glucose-enriched conditions compared to untreated controls [t₍₁₃₎ = 4.94, P < 0.001].

Glucose enrichment, hence, not only stimulated respiration rates but also caused a stark increase in photosynthetic activity in the mixotrophic cnidarian holobiont Cassiopeia sp. The increase in respiration rates indicates that glucose was rapidly taken up and consumed (i.e., respired) within the holobiont (Pogoreutz et al., 2017). Given our current understanding of cnidarian holobionts, there is no reason to assume that glucose enrichment directly affected photosynthetic activity in Symbiodinium. Rather, the observed increase in net and gross photosynthesis can be attributed to an increase in CO₂ availability in hospite, stemming from increased respiration in the Cassiopeia holobiont and seawater planktonic communities within the incubation chamber. In the case of Cassiopeia sp. this CO₂ limitation may be potentially attenuated by their continuous pumping motion facilitating increased gas exchange with the surrounding seawater (Wild and Naumann, 2013).

On a broader scale, these results could have implications for our understanding of the mechanisms underlying the cnidarian—alga symbiosis. The observation of glucose-stimulated photosynthesis implies that productivity of Symbiodinium in hospite may be tightly limited by CO₂ derived from holobiont metabolism.

Wooldridge (2009) proposed that a failure of coral CCMs during heat stress may ultimately result in a CO₂ limitation of photosynthetic dark reactions in Symbiodinium, ultimately leading to coral bleaching. Direct empirical evidence for this theory is missing to date. Our results, therefore, add to a growing emerging body of work suggesting that Symbiodinium may be CO₂-limited even in stable symbiotic systems (Muscatine et al., 1989; Herfort et al., 2008; Buxton et al., 2009; Klein et al., 2017). Hence, environmental stressors which alter metabolic processes in the holobiont may indeed lead to severe CO₂ limitation as predicted by Wooldridge (2009). Furthermore, we could show that the stimulation of host heterotrophy may attenuate CO₂ limitation in Symbiodinium. In this context, several studies reported that increased heterotrophic feeding may mitigate the effects of thermal stress in reef-building corals, resulting in increased bleaching resilience (Grottoli et al., 2006; Baird et al., 2009; Houlbrèque and Ferrier-Pagès, 2009; Ezzat et al., 2016). While this effect was mostly attributed to a compensation of autotrophic with heterotrophic energy sources by the host, here we show that heterotrophy may also increase bleaching resilience by increasing CO₂ availability in hospite.

Taken together, our study highlights that the role of CO₂ availability within the cnidarian—algae symbiosis deserves further in-depth assessment. Further work will be necessary to understand the effects of environmental conditions on CO₂ availability in hospite, along with their implications for the cnidarian—alga symbiosis.

NR and CP designed and conducted the experiment. All authors analyzed the data and wrote and revised the manuscript.