Angus B. Thies

Reef-building corals maintain intracellular photosymbiosis with dinoflagellate algae, allowing them to use photosynthetic products as metabolic fuel. Carbon dioxide (CO ₂ ) is a key substrate for photosynthesis, but its rapid increase in the global ocean due to anthropogenic emissions (known as “ocean acidification”) may disrupt coral symbiosis. We examined the effects of elevated CO ₂ on photosynthetic O ₂ production, oxidative damage, and antioxidant systems in the Caribbean reef-building corals Porites astreoides and Orbicella franksi . Coral colonies collected at 3m depth in Bocas del Toro (Panama) were exposed to control (∼390 μatm CO ₂ , pH ∼8.00) or elevated CO ₂ (∼1,060 μatm CO ₂ , pH ∼7.70) conditions in outdoor flow-through aquaria for 10 days. Flowing seawater had higher O ₂ concentrations in elevated CO ₂ tanks, with the largest differences at higher irradiance. Microsensor measurements confirmed that acute and chronic exposure to elevated CO ₂ significantly stimulated photosynthetic O ₂ production in both species. Despite increased O ₂ production. neither species showed significant changes in lipid peroxidation, reduced glutathione, or superoxide dismutase, catalase, and glutathione-S- transferase activities. Protein oxidation was unchanged in O. franksi but significantly decreased in P. astreoides . These results are consistent with partial relief of carbon limitation in P. astreoides symbionts under elevated CO ₂ , potentially improving photosynthetic efficiency and decreasing reactive oxygen species production. These findings support the resilience of P. astreoides to OA and suggest that elevated CO ₂ may increase daytime O ₂ levels on future reefs, with implications for the aerobic metabolism of reef organisms.

The upside-down jellyfish Cassiopea spp. host their algal symbionts inside a subset of amoebocytes, phagocytic cells that also play innate immune functions akin to macrophages from vertebrate animals. Amoebocyte precursors phagocytose algae from the jellyfish gut and store them inside intracellular compartments called symbiosomes. Subsequently, the precursors migrate to the mesoglea, differentiate into symbiotic amoebocytes, and roam throughout the jellyfish body, where the algae remain photosynthetically active and supply the jellyfish host with a significant portion of their organic carbon needs. Here, we show that the amoebocyte symbiosome membrane contains V-H+-ATPase (VHA), the proton pump that acidifies phagosomes and lysosomes in all eukaryotes. Many symbiotic amoebocytes also abundantly express a carbonic anhydrase (CA), an enzyme that reversibly hydrates CO2 into H+ and HCO3−. Moreover, we found that the symbiosome lumen is pronouncedly acidic and that pharmacological inhibition of VHA or CA activities significantly decreases photosynthetic oxygen production in live jellyfish. These results point to a carbon concentrating mechanism (CCM) that co-opts VHA and CA from the phago-lysosomal machinery that ubiquitously mediates food digestion and innate immune responses. Analogous VHA-dependent CCMs have been previously described in reef-building corals, anemones and giant clams; however, these other two cnidarians host their dinoflagellate algae inside gastrodermal cells – not in amoebocytes – and the clam hosts theirs within the gut lumen. Thus, our study identifies an example of convergent evolution at the cellular level that might broadly apply to invertebrate–microbe photosymbioses while also providing evolutionary links with intracellular and extracellular food digestion and the immune system.

Animals often acquire their microbial symbionts from the environment, but the mechanisms underlying how specificity of the association is achieved are poorly understood. We demonstrate that the conserved proton pump, V-type ATPase (VHA), plays a key role in the establishment of the model light-organ symbiosis between the squid Euprymna scolopes and its bacterial partner, Vibrio fischeri. Recruitment of V. fischeri from the surrounding seawater is mediated by juvenile-specific ciliated fields on the organ’s surface. These epithelia produce acidic mucus containing antimicrobials with low-pH optima, creating a chemical environment fostering specific recruitment of V. fischeri. We provide evidence that this critical acidic landscape is created by activity of VHA. VHA inhibition abolished epithelial-cell acidity, resulting in increased mucus pH and inefficient symbiont colonization. Thus, VHA provides a mechanistic link between host modulation of microenvironmental acidity, immune function, and selection of microbial symbionts, a strategy for specificity that may govern other symbioses.

Aim: To identify the physiological role of the acid‐base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout.Methods: We used: (i) super‐resolution microscopy to determine the subcellular location of sAC protein; (ii) live‐cell imaging of RBC intracellular pH (pH_i) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid‐base regulation; (iii) spectrophotometric measurements of haemoglobin–oxygen (Hb‐O₂) binding in steady‐state conditions; and (iv) during simulated arterial‐venous transit, to determine the role of sAC in systemic O₂ transport.Results: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pH_i regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pH_i recovery after an acid‐base disturbance. RBC pH_i recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO₃⁻‐dependent sAC signaling. Inhibition of sAC decreased Hb‐O₂ affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O₂ binding. During in vitro simulations of arterial‐venous transit, sAC inhibition decreased the amount of O₂ that is unloaded by ~11%.Conclusion: sAC represents a novel acid‐base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pH_i and blood O₂ transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates.

Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH ₃ and CO ₂ diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH ₄ ⁺ -trapping mechanism that promotes nitrogen delivery to algae during the day—necessary to sustain photosynthesis—and restricts nitrogen delivery at night—to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO ₂ diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.

In reef-building corals (order Scleractinia) and giant clams (phylum Molluca), V-type H ⁺ -ATPase (VHA) in host cells is part of a carbon concentrating mechanism (CCM) that regulates photosynthetic rates of their symbiotic algae. Here, we show that VHA plays a similar role in the sea anemone Anemonia majano , a member of the order Actinaria and sister group to the Scleractinia, which in contrast to their colonial calcifying coral relatives is a solitary, soft-bodied taxa. Western blotting and immunofluorescence revealed that VHA was abundantly present in the host-derived symbiosome membrane surrounding the photosymbionts. Pharmacological inhibition of VHA activity in individual anemones resulted in an approximately 80% decrease of photosynthetic O ₂ production. These results extend the presence of a host-controlled VHA-dependent CCM to non-calcifying cnidarians of the order Actiniaria, suggesting it is widespread among photosymbiosis between aquatic invertebrates and Symbiodiniaceae algae.

Coral algal symbionts are hosted inside the symbiosome of gastrodermal cells, an intracellular compartment that isolates algae from the external environment and allows host cells to control the delivery of metabolites to their symbionts. However, the underlying molecular mechanisms are largely unknown. Here, we report the diel trafficking of NH ₃ -transporting Rhesus (Rh) channels between the cytoplasm and the symbiosome membrane in the coral Acropora yongei , which matches established patterns of nitrogen delivery to endosymbionts. Heterologous expression in Xenopus oocytes established that A. yongei Rh (ayRhp1) is a channel that facilitates NH ₃ diffusion across membranes following its partial pressure gradient. Immunostaining revealed ayRhp1 is widely distributed throughout coral tissues and most abundantly present in oral ectodermal cells, desmocytes, and gastrodermal cells. In the latter, ayRhp1 was observed in the symbiosome membrane of alga-containing cells. Together with V-type H ⁺ -ATPases that make the symbiosome highly acidic (pH~4), ayRhp1 constitutes an NH ₄ ⁺ -trapping mechanism analogous to that in mammalian renal tubule. Remarkably, ayRhp1 presence in the symbiosome membrane was higher during the day than the night. This indicates a regulatory mechanism that facilitates NH ₄ ⁺ delivery to alga during the day, likely to sustain high turnover rates of photosynthetic proteins, while restricting NH ₄ ⁺ delivery at night to maintain the endosymbiotic algae in a nitrogen-limited stage that stagnates their growth. The dynamic trafficking of proteins to and away from the symbiosome membrane is a previously unknown mechanism that contributes to metabolic regulation between symbiotic partners. Significance Statement: The endosymbiotic relationship between corals and algae relies on the coordinated exchange of metabolites. Disruption of these metabolic exchanges can result in interruption of the symbiosis; however, the underlying molecular mechanisms are poorly understood. Here we report that Acropora yongei coral host cells express ammonia-transporting channel proteins (ayRhp1), which traffic to and away from the symbiosome membrane surrounding the endosymbiotic algae. In conjunction with the acidic symbiosome microenvironment, this mechanism allows host cells to regulate nitrogen delivery to endosymbionts sustaining essential functions while restricting growth. This work provides novel mechanistic information about metabolic regulation of animal-algae symbioses, and advances our understanding of physiological mechanisms that might determine coral local adaptation, resilience, and vulnerability to environmental stress including climate change.