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.

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 predominantly localized to the basal membrane, CHNKCC1 showed an unusual apical localization, and CHNHE3 exhibited dual (apical and basal) localization in different cells. 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 suggest that a distributed, flexible network of gill epithelial cells contributes to ion transport in a euryhaline bivalve, revealing a potential ion transport system that is fundamentally distinct from the teleost paradigm and opening new avenues for understanding osmoregulation in marine invertebrate.