Cloning of the Na+-K+-2Cl−(NKCC) transporter in Macrobrachium rosenbergii and its role in osmoregulation

Abstract

The full-length cDNA sequences of two Na-K-2Cl cotransporter (NKCC) isoforms, MrNKCC1 and MrNKCC2, were identified in Macrobrachium rosenbergii. MrNKCC1 contains a 3183 bp open reading frame (ORF) encoding 1060 amino acids, while MrNKCC2 has a 2679 bp ORF encoding 892 amino acids. Both isoforms share a similar predicted secondary structure, featuring 12 transmembrane domains and a C-terminal, and their amino acid sequences and domain arrangement are highly conserved. Phylogenetic analysis groups fish and crustacean NKCC isoforms into distinct branches, each containing two isoforms. Expression analysis using real-time quantitative PCR (qPCR) revealed distinct tissue distribution patterns. MrNKCC1 was broadly expressed, with the highest levels in the intestine, whereas MrNKCC2 was predominantly found in the gill, hepatopancreas, and epidermis, with peak expression in the gill. To examine their roles in osmoregulation, prawns were exposed to 24 ‰ seawater for 96 h. Hemolymph concentrations of Cl⁻, K⁺, and Na⁺ increased, peaking at 24, 24, and 12 h, respectively. During this period, MrNKCC1 expression was suppressed, while MrNKCC2 expression significantly increased at 2 h after seawater exposure and then returned to initial levels. Gill filaments showed significant histological changes under seawater exposure, including the expansion and proliferation of chloride-secreting cells. Following a 96-h seawater exposure, prawns were transferred to freshwater. This transition led to a decline in plasma Cl⁻, K⁺, and Na⁺ concentrations, accompanied by a marked increase in MrNKCC1 and MrNKCC2 expression. The rapid transcriptional response of both MrNKCC isoforms to salinity fluctuations highlights their critical role in the osmoregulatory mechanisms of M. rosenbergii.