Akira Kato

Reef-building corals are increasingly threatened by environmental stressors, including ocean warming, acidification, and osmotic stress caused by fluctuations in seawater salinity, highlighting the need for a better understanding of coral physiology. However, the molecular mechanisms by which corals regulate water and solute transport remain poorly understood. To investigate the role of aquaporins (Aqps) in coral physiology, we examined the evolutionary history and transport properties of the Aqp family in reef-building corals. Comparative genomic analyses across cnidarians revealed that the nine-member core hexacorallian aqp repertoire, comprising aqp4L1.1 , aqp4L1.2 , aqp4L1.3 , aqp4L2 , aqp4L3 , glp1 , glp2.1 , glp2.2 , and aqp12L1 , has been highly conserved since the common ancestor of reef-building corals and sea anemones, whereas aqp8L was consistently absent across all examined hexacorallian species. Conservation of synteny and exon–intron structures further confirmed that major components of the aqp family architecture originated deep in metazoan evolution. Functional characterization of Acropora millepora Aqp4L and Glp proteins demonstrated that most transport properties were consistent with phylogenetic predictions, with the majority of Aqp4L proteins exhibiting water-selective permeability, whereas Glp proteins transported both water and small solutes such as glycerol. Unexpectedly, Aqp4L1.2, which belongs to the water-specific aquaporin lineage, transported not only water but also glycerol, urea, and boric acid, suggesting functional co-option after gene duplication and underscoring the evolutionary flexibility of the Aqp4L lineage. These findings suggest that the hexacorallian aqp family evolved through a combination of deep evolutionary conservation and lineage-specific functional innovation, providing new insight into the molecular foundations of coral physiology. Significance Statement: How reef-building corals regulate water and solute transport at the molecular level remains largely unknown. Here, comparative genomic and functional analyses revealed the evolutionary history and transport properties of the aqp family in reef-building corals. Reef-building corals retain a conserved aqp repertoire shared with sea anemones, comprising the aqp4-like , aquaglyceroporin , and aqp12-like subfamilies, whereas the aqp8-like subfamily is entirely absent. Phylogenetic analyses further showed that these genes form hexacorallian-specific clades distinct from homologs in other cnidarian lineages, indicating that the fundamental architecture of the hexacorallian aqp repertoire was established in their common ancestor. Unexpectedly, we identified a duplicated water-channel gene that evolved the capacity to transport multiple solutes, revealing a previously unrecognized example of functional co-option in coral aquaporins. These findings show that coral aquaporin evolution has been driven by both long-term conservation and lineage-specific innovation, providing a genomic and functional framework for understanding the molecular basis of coral physiology.

Nkcc2 plays an important role in salt (re)absorption in the intestines and kidneys of seawater teleosts. However, the K ⁺ channels that recycle K ⁺ for the Nkcc2 function remain unknown. This study revealed that Kcnj15 is localized to the apical region of Nkcc2-positive nephron segments and intestinal epithelial cells and is also present at the basolateral region with Nka. Our results provide insights into the mechanisms underlying water-electrolyte metabolism in teleosts.

Aquaporin-10 (AQP10) is an aquaglyceroporin that transports small, uncharged molecules, such as water and glycerol. In some species, such as mice and cattle, the AQP10 gene ( Aqp10 ) is pseudogenized. In rodents, pseudogenization of Aqp10 is limited to the Myomorpha suborder, while Aqp10 remains intact in other rodent lineages. The order Cetartiodactyla encompasses diverse species, including Suina, Tylopoda, Ruminantia, and Whippomorpha. The distribution of intact Aqp10 across the entire Cetartiodactyla and analyses estimating the timing of Aqp10 pseudogenization have not been sufficiently investigated. In this study, we identified species within Cetartiodactyla that retain intact Aqp10 and analyzed the detailed patterns of pseudogenization to elucidate the specifics of pseudogenization within this order. We collected Aqp10 loci by BLAST and synteny analyses of publicly available genome databases of 51 cetartiodactyl species from 17 families. Dot plot and alignment analyses revealed that seudogenization of Aqp10 occurred in 42 Cetartiodactyla species, including the Camelidae, warthog (Suidae), Antilocapridae, okapi (Giraffidae), Cervidae, Bovidae, Hippopotamidae, and Cetacea. In contrast, nine species retained an intact Aqp10 : pig and babirusa (Suidae), peccary (Tayassuidae), mouse deeres (Tragulidae), giraffes (Giraffidae), and musk deers (Moschidae). The timing of pseudogenization was estimated by comparing patterns of exon deletions, frameshifts, and nonsense mutations in Aqp10 pseudogenes. RT-PCR analysis of cattle and sheep tissues revealed that these species express Aqp3 and Aqp7 in their intestines, but not Aqp10 . Collectively, these findings indicate that Aqp10 pseudogenization occurred independently in multiple cetartiodactyl lineages and provide new insights into the evolutionary history of Aqp10 loss within this order.

In marine teleosts, gill ionocytes play central roles in the excretion of excess monovalent ions (Na ⁺ , Cl ⁻ , and K ⁺ ). Basolateral K ⁺ channels are considered essential for sustaining ion secretion by maintaining K ⁺ homeostasis and membrane potential; however, the molecular identity of basolateral K ⁺ channels in teleost gill ionocytes remains incompletely understood. Kcnj15 is a K ⁺ channel expressed in multiple osmoregulatory tissues of the Japanese pufferfish ( Takifugu rubripes ), and its localization changes depending on tissue type and osmotic conditions; however, its localization in the gills has remained unresolved. Here, we determined the cellular localization of Kcnj15 in the gills and examined its expression in response to changes in environmental salinity. In seawater-acclimated fish, Kcnj15 was localized to the basolateral membrane of Nka- and Nkcc1-positive ionocytes. Following acclimation to hypoosmotic brackish water (1 ppt), transcript levels of cftr , nkcc1 , and kcnj1a decreased, whereas kcnj15 expression remained unchanged. Under this condition, Kcnj15 was localized to the basolateral membrane of Nka-positive ionocytes. These results demonstrate that Kcnj15 is localized to the basolateral membrane of gill ionocytes and suggest that it may function as a basolateral K ⁺ leak channel supporting ionocyte homeostasis under both seawater and low-salinity conditions.

Regulation of water permeability in the collecting duct is important for osmoregulatory acclimation in teleost fish. In hyperosmotic environments such as seawater (SW), the teleost kidney functions as a site of divalent ion excretion. The collecting ducts reabsorb Na ⁺ , Cl ⁻ , and water, thereby reducing urine volume and producing small amounts of isotonic urine with high concentrations of divalent ions. In hypoosmotic environments such as freshwater (FW) or low-salinity brackish water (BW), the kidney produces large volumes of hypotonic urine and serves as a site of water excretion; under these conditions, the collecting ducts reabsorb Na ⁺ and Cl ⁻ but not water. To identify aquaporins (Aqps) involved in regulating water permeability in the collecting ducts of teleosts, we analyzed renal Aqp expression in a euryhaline marine fish, the Japanese pufferfish ( Takifugu rubripes ), which possesses 16 Aqp genes in its genome, seven of which (Aqp1aa, 1ab, 3a, 4a, 7, 8bb, and 11a) are expressed in the kidney. Quantitative RT-PCR analysis showed that Aqp1aa and Aqp4a were highly expressed in collecting duct tissues, and that Aqp1aa expression was markedly reduced in fish acclimated to BW. Immunohistochemistry revealed apical localization of Aqp1aa and basolateral localization of Aqp4 in collecting duct cells, with apical Aqp1aa downregulated in BW. These results suggest that Aqp1aa and Aqp4 mediate water reabsorption in SW and that downregulation of Aqp1aa contributes to hypotonic urine production in BW. NEW & NOTEWORTHY: Regulation of water permeability in the collecting duct is important for osmoregulation in teleost fish. Expression analyses of aquaporins (Aqps) in the marine pufferfish Takifugu rubripes showed that Aqp1aa and Aqp4a are highly expressed in the collecting duct and localized to the apical and basolateral membranes, respectively. Renal Aqp1aa expression was markedly reduced in fish acclimated to hypoosmotic brackish water. These results indicate that collecting duct water permeability is regulated by Aqp1aa expression.

The aquaporin genes ( aqps ) are widely distributed among vertebrates. In cartilaginous fish, aqp3 and aqp10 are duplicated, whereas many species have lost aqp8 . While the solute permeability of aquaglyceroporins and Aqp8 exhibits significant diversity, systematic evaluations of their activity within species are limited. Consequently, we conducted a comprehensive analysis of the aqp s in various cartilaginous fish genome databases. Our analysis revealed that the sharpnose sevengill shark ( Heptranchias perlo ) and North Pacific spiny dogfish ( Squalus suckleyi ) possess all aqp genes found in cartilaginous fish, rendering them suitable for comparative analysis of Aqp activity. The activity of all aquaglyceroporins and Aqp8 in the sharpnose sevengill shark was analyzed using Xenopus oocyte swelling assays. The water permeability of Aqp3c2, Aqp8, Aqp9, Aqp10c1, and Aqp10c2; the glycerol permeability of Aqp3c2, Aqp9, and Aqp10c2; the urea permeability of Aqp3c2, Aqp8, Aqp9, and Aqp10c2; and the boric acid permeability of Aqp8 and Aqp9 were determined. The urea and boric acid permeabilities of Aqp9 were higher than those of other Aqps. Our results provide genomic evidence for the loss of aqp7 and further elucidate the diversification of aqp8 in cartilaginous fishes. Moreover, the urea permeability of Aqp9 suggests a functional role in their unique urea‐based osmoregulation strategy.