3.2| Isolation and expression analysis of genes encoding Na+/K+ ATPase α subunit, cytoplasmic carbonic anhydrase, and Na+/H+ exchanger
To analyze the possibility that changes in the expression of genes involved in Na+ uptake disturbed hemolymph ionic composition and ultimately led to the death of crabs in rearing experiments, cDNA fragments of NKA α subunit, CAc, and NHE were amplified by PCR and isolated, and gene expression was analyzed in the most posterior gills of H. tridens , M. japonicus , andC. dehaani. The lengths of isolated cDNA fragments for the NKA α subunit, CAc, and NHE genes were 700, 300, and 800 bp, respectively. Sequencing results showed that the cDNA fragment sequences of these genes were highly similar to those in other crab species. The deduced amino acid sequences of the NKA α subunit of H. tridens , M. japonicus , and C. dehaani had 95%, 95%, and 94% similarity, respectively, to that of C. maenas ; those of CAc of H. tridens , M. japonicus , and C. dehaani had 76%, 78%, and 75% similarity, respectively, to that of C. maenas; and those of the NHE of H. tridens , M. japonicus , and C. dehaanihad 94%, 93%, and 92% similarity, respectively, to that of C. maenas (Fig. 4A–C) (Towle et al., 1997; Serrano and Henry, 2008).
The expression of these genes in the most posterior gills of crabs held in the 8.6 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 513.3 mmol/L NaCl solutions for 6 h were analyzed by Northern blotting. However, the expression levels of CAc gene was not sufficiently high in M. japonicus to be detected by Northern blotting; therefore, RNase protection assay was carried out to detect the expression of this gene in M. japonicus . These analyses revealed that the expression of all genes in the most posterior gills in H. tridens and M. japonicas was higher in 8.6 mmol/L NaCl solution compared to 513.3 mmol/L NaCl+MCK solution except CAc in M. japonicus . Furthermore, the expression of all genes in the most posterior gills was also higher in 513.3 mmol/L NaCl solution compared to 513.3 mmol/L NaCl+MCK solution as in 8.6 mmol/L NaCl solution (Fig. 5). On the other hand, the expression of these genes was different in C. dehaani. The expression of genes encoding NKA α subunit was enhanced in 513.3 mmol/L NaCl solution but was less drastic compared to H. tridens andM. japonicus. In addition, the expression of CAc gene was not induced in 513.3 mmol/L NaCl solution but was in 8.6 mmol/L NaCl solution. In contrast, the expression of NHE gene was enhanced in 513.3 mmol/L NaCl solution but not in 8.6 mmol/L NaCl solution (Fig. 5).