3.5| Isolation of the gene encoding Na+/K+/2Clcotransporter and expression analysis of the gene and Na+/K+ ATPase α subunit under hypo-osmotic conditions
To analyze the possibility that changes in the expression of genes involved in K+ transport disturbed hemolymph K+ concentration and ultimately led to the death ofM. japonicus in 4.3 mmol/L NaCl+1.2 MCK solution, cDNA fragment of NKCC was amplified by PCR and isolated, and gene expression of it and NKA α subunit was analyzed in the most posterior gills of H. tridens , M. japonicus , and C. dehaani. The deduced amino acid sequence of NKCC of H. tridens , M. japonicus , andC. dehaani had 81%, 79%, and 77% similarity to that ofC. maenas (Fig. 4D). Northern blotting revealed that NKA α subunit expression in the most posterior gills of crabs incubated in 4.3 mmol/L NaCl+1.2 MCK solution for 6 h decreased compared to 4.3 mmol/L NaCl solution in all three species. In H. tridens and M. japonicus , NKCC expression also decreased in the presence of ambient minor cations, although the expression was relatively low (Fig. 9). In contrast, NKCC expression in C. dehaani was less affected by ambient minor cations (Fig. 9).