Active ion transport in the gill of the estuarine bivalve Crassostrea hongkongensis under salinity stress: molecular evidence from homologs of typical ion transporters NKAα, NKCC1 and NHE3

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Abstract
Whether marine bivalves, as osmoconformers, actively transport inorganic ions to counter osmotic stress remains unclear due to insufficient direct evidence. The estuarine bivalve Crassostrea hongkongensis offers an ideal model to address this issue, as our previous ultrastructural study hinted at the existence of ionocyte‑like cells in the gill. Here, single‑cell RNA sequencing revealed that the homologs of key ion transporters in oyster gill, Na⁺/K⁺-ATPase α subunit (CHNKAα), Na⁺/K⁺/2Cl⁻ cotransporter 1 (CHNKCC1) and Na⁺/H⁺ exchanger 3 (CHNHE3), lack the cluster‑specific expression typical of teleost homologs. Instead, their transcripts are broadly distributed across multiple gill epithelial cells, confirmed by RNA fluorescence in situ hybridization. At 8 h post‑salinity exposure, CHNKAα and CHNHE3 transcripts increased at salinity 30 (positively correlated with gill Na⁺, K⁺ and Cl⁻ contents), whereas CHNKCC1 was upregulated at salinity 6 (negatively correlated), implicating their involvement in active ion transport. Multiplex immunofluorescence revealed an interesting membrane distribution: CHNKAα retained a conserved basal membrane pattern in gill epithelial cells, CHNKCC1 showed an unusual apical localization, and CHNHE3 displayed a cell‑type‑specific dual (apical and basal) membrane distribution. RNA interference-mediated knockdown further indicated that CHNKAα drives Cl⁻ uptake while CHNKCC1 mediates Cl⁻ secretion, directly linking their active transport to salinity‑induced ion content changes. Notably, these observations, particularly membrane distribution and functional contribution to ion transport, do not completely conform to any existing ion transport model in aquatic animals. Our findings provide direct evidence for active ion transport in a euryhaline bivalve under salinity stress and indicate a potential ion transport system distinct from that observed in most teleosts, opening new avenues for understanding osmoregulation in marine invertebrates.

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