Gills of euryhaline fishes possess great physiological and structural plasticity to adapt to large</p><p>changes in external osmolality and to participate in ion uptake/excretion, which is critical for</p><p>the re-establishment of fluid and electrolyte homeostasis. The osmoregulatory plasticity of</p><p>gills provides an excellent model to study role of microRNAs (miRs) in adaptive osmotic</p><p>responses. The present study is to characterize an ex-vivo gill filament culture and using</p><p>omics approach, to decipher the interaction between tonicity-responsive miRs and gene</p><p>targets, in orchestrating the osmotic stress-induced responses. Ex-vivo gill filament culture</p><p>was exposed to Leibovitz’s L-15 medium (300 mOsmol l–1) or the medium with an adjusted</p><p>osmolality of 600 mOsmol l-1 for 4, 8 and 24 hr. Hypertonic responsive genes, including</p><p>osmotic stress transcriptional factor, Na+</p><p>/H+</p><p>exchange regulatory cofactor, with-no-lysine</p><p>kinase, inward rectifying K+</p><p>channel, cystic fibrosis transmembrane regulator, Na+</p><p>/Cl-</p><p>-</p><p>taurine transporter, Na+</p><p>/K+</p><p>-Atpase, and calcium-transporting ATPase were significantly</p><p>upregulated, while hypo-osmotic genes, like aquaporin-3 and V-type proton ATPase were</p><p>downregulated. The data illustrated that the ex-vivo gill filament culture exhibited distinctive</p><p>responses to hyperosmotic acclimation, as compared with the current primary gill cell culture</p><p>model. In the hyperosmotic treatment, four key factors (i.e. drosha RNase III endonuclease,</p><p>exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis were</p><p>dysregulated. MicroRNA-sequencing of gill filament samples at 4 and 8 hr identified two</p><p>downregulated miRs, miR-29b-3p and miR-200b-3p. Integrated miR-mRNA-omics analysis</p><p>predicted the functional implications of the miRs in calcium homeostasis, ion channel</p><p>activities and ATP production. Collectively, the ex-vivo gill filament culture would be a</p><p>useful alternative model to investigate underlying mechanisms of osmotic responses in fish</p><p>gills.
