FIGURE LEGENDS
FIGURE 1 Survival rates of (A) Helice tridens , (B)Macrophthalmus japonicus , and (C) Chiromantes dehaani at various NaCl concentrations and in natural seawater 48 h after initiation of rearing. The survival rate was determined in 8.0, 16.0, 32.1, 64.2, 128.3, 256.7, and 513.3 mmol/L NaCl solutions and seawater. Horizontal axes are scaled logarithmically. Values represent means of three experiments, and error bars represent standard errors. Significant differences were observed among different ambient NaCl concentrations in all three species (ANOVA, P < 0.05). *Significantly different values compared to seawater (P < 0.05; Williams’ test). Different letters indicate significantly different values among three species within the same NaCl concentration (P< 0.05; Tukey’s test). (SW), seawater.
FIGURE 2 (A, B) Survival rates of (A) Helice tridens and (B) Macrophthalmus japonicus in 513.3 mmol/L NaCl solution, 513.3 mmol/L NaCl+MMCK solution, and 513.3 mmol/L NaCl+MMCK solution minus any one of the four additional salts. Values represent means of three experiments, and error bars represent standard errors. Significant differences were observed among different compositions of bathing media in both H. tridens and M. japonicus (ANOVA, P< 0.05). Different letters indicate significantly different values (P < 0.05; Tukey’s test). (C, D) Survival rates of (C) H. tridens and (D) M. japonicus in 513.3 mmol/L NaCl, 513.3 mmol/L NaCl+K, and 513.3 mmol/L NaCl+K solutions supplemented with MgCl2 or CaCl2. Values represent means of three experiments, and error bars represent standard errors. Significant differences were observed among different compositions of bathing media in both H. tridens and M. japonicus (ANOVA, P < 0.05). Different letters indicate significantly different values (P < 0.05; Tukey’s test). (MMCK), 27.4 mmol/L MgSO4, 25.2 mmol/L MgCl2, 9.9 mmol/L CaCl2, and 10.7 mmol/L KCl; (K), 10.7 mmol/L KCl.
FIGURE 3 Hemolymph Na+ and K+ concentrations in (A, D) Helice tridens , (B, E) Macrophthalmus japonicus , and (C, F) Chiromantes dehaani 6 h after initiation of incubation. Hemolymph (A–C) Na+ and (D–F) K+ concentrations in crabs held in 8.6 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 513.3 mmol/L NaCl solution. Values represent means (number of analyzed crabs shown above each bar), and error bars represent standard errors. Significant differences of both Na+ and K+concentrations were observed among different compositions of bathing media in H. tridens and M. japonicus (ANOVA, P< 0.05), but not in C. dehaani (ANOVA, P> 0.05). Different letters indicate significantly different values (P < 0.05; Student’s t -test with Bonferroni correction). (MCK), 25.2 mmol/L MgCl2, 9.9 mmol/L CaCl2, and 10.7 mmol/L KCl.
FIGURE 4 Amino acid sequences of (A) NKA α subunit, (B) CAc, (C) NHE, and (D) NKCC of Helice tridens , Macrophthalmus japonicus , and Chiromantes dehaani deduced from cDNA fragments and alignments with those of the shore crab C. maenas.
FIGURE 5 Expression of genes encoding NKA α subunit, CAc, and NHE in the most posterior gills of Helice tridens ,Macrophthalmus japonicus , and Chiromantes dehaani 6 h after initiation of incubation in 513.3 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 8.6 mmol/L NaCl solutions. All gene expression was detected using Northern blotting, except the expression of CAc inM. japonicus , which was detected using RNase protection assay. The relative expression level was shown in each band as the expression in 513.3 mmol/L NaCl+MCK solution is 1.0. (NKA), Na+/K+ ATPase; (CAc), cytoplasmic carbonic anhydrase; (NHE), Na+/H+exchanger; (MCK), 25.5 mmol/L MgCl2, 9.9 mmol/L CaCl2, and 10.7 mmol/L KCl.
FIGURE 6 Survival rates of (A, C) Helice tridens and (B, D) Macrophthalmus japonicus in 513.3 mmol/L NaCl solution with or without acetazolamide, an inhibitor of CA (A and B), or amiloride hydrochloride hydrate, an inhibitor of NHE (C and D), 24 h after initiation of rearing. Values represent means of three (for H. tridens ) or four (for M. japonicus ) experiments, and error bars represent standard errors. Different letters indicate significantly different values (P < 0.05; Student’s t-test). (CA), carbonic anhydrase; (NHE), Na+/H+exchanger.
FIGURE 7 Survival rates of (A) Helice tridens , (B)Macrophthalmus japonicus , and (C) Chiromantes dehaanireared in 4.3 mmol/L NaCl, 4.3 mmol/L NaCl+1.2 MCK, and 17.1 mmol/L NaCl+1.2 MCK solutions. Values represent means of four (for H. tridens and M. japonicas ) or three (for C. dehaani ) experiments, and error bars represent standard errors. Significant differences were observed among different compositions of bathing media in M. japonicus (ANOVA, P < 0.05), but not inH. tridens and C. dehaani (ANOVA, P > 0.05). Different letters indicate significantly different values (P < 0.05; Student’s t-test with Bonferroni correction). (D) Survival rate of M. japonicus in 4.3 mmol/L NaCl, 4.3 mmol/L NaCl+1.2 MCK, and 4.3 mmol/L NaCl solutions supplemented with 30.5 mmol/L MgCl2 and 11.7 mmol/L CaCl2 (1.2 MC) or 13.4 mmol/L KCl (1.2 K). Values represent means of three experiments, and error bars represent standard errors. Significant differences were observed among different compositions of bathing media (ANOVA, P < 0.05). Different letters indicate significantly different values (P< 0.05; Tukey’s test). (E) Survival rate of M. japonicus in 4.3 mmol/L NaCl solution supplemented with 0, 2.7, 5.4, and 10.7 mmol/L KCl 24 h after initiation of rearing. Values represent means of four experiments, and error bars represent standard errors. *Significantly different values from that in 4.3 mmol/L NaCl solution without KCl. (P < 0.05; Williams’ test). (F) Survival rate of M. japonicus reared in 4.3 mmol/L NaCl (circles), 4.3 mmol/L NaCl+1.2 MCK (triangles), and 4.3 mmol/L NaCl+1.2 K (squares) solutions 15, 24, 39, and 48 h after initiation of experiments. Values represent means of three experiments, and error bars represent standard errors. Different letters indicate significantly different values in each time point (P < 0.05; Tukey’s test). *Significantly different values from the beginning point (P< 0.05; Williams’ test). (1.2 MCK), 30.5 mmol/L MgCl2, 11.7 mmol/L CaCl2, and 13.4 mmol/L KCl; (1.2 MC), 30.5 mmol/L MgCl2 and 11.7 mmol/L CaCl2; (1.2 K), 13.4 mmol/L KCl.
FIGURE 8 Hemolymph (A–D) Na+ and (E–H) K+ concentrations in (A, E) Helice tridens , (B, D, F, H) Macrophthalmus japonicus , and (C, G) Chiromantes dehaani incubated in (A–C, E–G) 4.3 mmol/L NaCl and 4.3 mmol/L NaCl+1.2 MCK solutions or in (D, H) 4.3 mmol/L NaCl, 4.3 mmol/L NaCl+K, and 4.3 mmol/L NaCl+MCK solutions 6 h after initiation of incubation. Values represent means (number of analyzed crabs shown above each bar), and error bars represent standard errors. Significant differences were observed among different compositions of bathing media in (D, H) (ANOVA,P < 0.05). Different letters indicate significantly different values (P < 0.05; Student’s t -test for A–C and E–G, and Student’s t -test with Bonferroni correction for D and H). (1.2 MCK), 30.5 mmol/L MgCl2, 11.7 mmol/L CaCl2, and 13.4 mmol/L KCl; (MCK), 25.2 mmol/L MgCl2, 9.9 mmol/L CaCl2, and 10.7 mmol/L KCl; (K), 10.7 mmol/L KCl.
FIGURE 9 Expression of genes encoding NKA α subunit and NKCC in the most posterior gills of Helice tridens , Macrophthalmus japonicus , and Chiromantes dehaani 6 h after initiation of incubation in 4.3 mmol/L NaCl and 4.3 mmol/L NaCl+1.2 MCK solutions. All expressions were detected using Northern blotting. The relative expression level was shown in each band as the expression in 4.3 mmol/L NaCl solution is 1.0. (NKA), Na+/K+ATPase; (NKCC), Na+/K+/2Clcotransporter; (1.2 MCK), 30.5 mmol/L MgCl2, 11.7 mmol/L CaCl2, and 13.4 mmol/L KCl.