Alex R. Quijada-Rodriguez

Aim: To determine whether the crustacean Rh1 protein functions as a dual CO₂/ammonia transporter and investigate its role in branchial ammonia excretion and acid–base regulation.Methods: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO₂ binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO₂ transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site‐directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO₂. Further, immunohistochemistry was used to assess sub‐cellular localization of CmRh1 and a membrane‐bound carbonic anhydrase (CmCAg).Results: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO₂ binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO₂ transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un‐ionized ammonia/methylamine species. CmRh1 was co‐localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia.Conclusion: CmRh1 is the first identified apical transporter‐mediated route for ammonia and CO₂ excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO₂ transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organism and ecosystem levels. In comparison, freshwater acidification has received less attention, rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a crustacean model to investigate the impact of CO₂-mediated freshwater acidification. Our integrative approach, investigating changes in the animal's acid–base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate, indicates that this economically relevant crustacean will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid–base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion, reducing the animal's overall fitness and increasing its mortality. Results indicate that present-day Chinese mitten crab could be heavily affected by freshwater acidification like their marine counterparts and emphasize the importance of understanding the long-term implications of freshwater acidification on species' fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

Dissolution of anthropogenic CO₂ is chronically acidifying aquatic ecosystems. Studies indicate that ocean acidification will cause marine life, especially calcifying species, to suffer at the organismal and ecosystem levels. In comparison, freshwater acidification has received less attention rendering its consequences unclear. Here, juvenile Chinese mitten crabs, Eriocheir sinensis, were used as a calcifying model to investigate the impacts of CO₂-mediated freshwater acidification. Our integrative approach investigating changes in the animal's acid-base homeostasis, metabolism, calcification, locomotory behaviour, and survival rate indicate that the crab will face energetic consequences from future freshwater acidification. These energetic trade-offs allow the animal to maintain its acid-base homeostasis at the cost of reduced metabolic activity, exoskeletal calcification, and locomotion reducing the animal's overall fitness and increasing its mortality. Results suggest that present-day calcifying invertebrates could be heavily affected by freshwater acidification similar to their marine organisms and emphasizes the importance in understanding the long-term implications of freshwater acidification on species fitness.

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.