The results of the transfer to 10- and 50-ppt salinities indicate that G. depressus is able to osmoregulate and survive at least for 10 days in these salinities as a hyper-hypoosmoregulating marine crab, and are consistent with the previous reports (Charmantier et al. 1998, 2002) on hemolymph osmolality in the other grapsid crabs (Armases miersii and Chasmagnathus granulata) as a function of the ambient salinity. Together with the results of the terrestrial exposure showing that hemolymph osmolality was in the same physiological range after 2 days despite dehydration, G. depressus can be considered as an appropriate model to study the osmotic/ionic regulatory mechanisms supporting salinity/terrestrial acclimation. In this investigation, it was considered necessary to obtain more basic information concerning the ionic status in the hemolymph as well as the branchial Na+/K+-ATPase activity as a prerequisite for further studies.
Regarding ionic changes in the hemolymph, changes in Na+ and Cl− generally paralleled those in osmotic concentrations in response to high and low salinity exposure as well as to water deprivation, and it is likely that altered osmolality of the hemolymph under varying environmental conditions was based for the most part on altered levels of Na+ and Cl− (Wilder et al. 1998). An interesting finding of this study, however, is the pattern of hemolymph calcium levels, particularly for those of complexed calcium. Slightly lower levels of hemolymph free Ca2+ in crabs kept out of water seems to be related to the fasted condition since intermolt terrestrial crabs regulate hemolymph calcium by controlling intake of dietary calcium (Wheatly 1999; Zanotto and Wheatly 2002). In crabs acclimated to 10-ppt salinity, free Ca2+ was maintained at twofold higher values than concentrations of the medium, and complexed calcium disappeared from the hemolymph after 10 days. In crabs exposed to 50-ppt salinity, free Ca2+ was regulated to the lower levels than those in the medium through the experiment while total calcium increased to higher levels after 2 days. These responses may indicate that hemolymph complexed calcium could serve as an internal reserve for maintaining free Ca2+ levels in the hemolymph. On the other hand, the complexed calcium decreased dramatically and disappeared from the hemolymph after 10 days in 50 ppt, and we speculate that this represents a surplus (unnecessary) calcium reservoir in the hemolymph for prolonged period in higher Ca2+ environment. Calcium regulation in various environments has been studied in crustaceans, mostly with respect to the control of epithelial calcium transport (Freire et al. 2008; Ahearn et al. 2004; Wheatly 1999; Zanotto and Wheatly 2002), and the role of complexed calcium in the hemolymph as a reserve for free Ca2+ is unknown. Our findings, therefore, suggest a new control mechanism of hemolymph free Ca2+ and imply that hemolymph concentrations of both total and free calcium need to be analyzed. At any rate, it appears that it is necessary to regulate free Ca2+ to a specific range and that this control is separate from the osmoregulatory mechanisms.
One of the ion transporters that has received the most intensive studies in osmoregulating crustaceans is Na+/K+-ATPase (Bianchini et al. 2008; Lucu and Towle 2003; Pequeux 1995; Henry et al. 2002; Towle et al. 1997, 2011; Ahearn et al. 1999; Serrano and Henry 2008; Towle and Weihrauch 2001). In addition to the Ca2+ channel, Ca2+-ATPase, and Na+/Ca2+ exchanger, the transportation of Ca2+ in the hemolymph of crustaceans is also affected by the potential energy of the Na+ gradient, established by Na+/K+ ATPase activity (Roer and Dillaman 1993). In this study, we observed higher Na+/K+-ATPase activity in the posterior gills when compared with the anterior gills (see Figure 3a), consistent with the molecular biological and physiological studies on many crab species (Bianchini et al. 2008; Lucu and Towle 2003; Pequeux 1995; Towle and Weihrauch 2001; Freire et al. 2008; Charmantier et al. 2009; Siebers et al. 1982; Onken and Putzenlechner 1995), which have designated the posterior gill epithelium, with its high abundance of Na+/K+-ATPase activity, as the principal site of osmoregulatory ion transport. These differences constitute the basis of the paradigm that anterior gills are structurally and functionally specialized for respiratory gas exchange, while the posterior gills have become specialized for active ion absorption counterbalancing passive losses in dilute media (Freire et al. 2008; Charmantier et al. 2009) as reflected in the significant increases in the Na+/K+-ATPase activity of G6 and G7 after acclimation to 10-ppt salinity (Figure 3b).
Following these observations, the present investigation provides an interesting finding that acclimation to 50-ppt salinity for 10 days, in which hypo-ionoregulation occurred, was accompanied by increased Na+/K+-ATPase activity exclusively in G8 (Figure 3b), suggesting that G8 may participate in ion excretion into the concentrated media. These different responses of the enzyme activity among the individual gills indicate a gill-specific pattern of the regulation and a higher degree of specialization in gill function in G. depressus. Together with the differences between the terrestrial condition and 50-ppt salinity, the increased Na+/K+-ATPase activity is not simply part of a cellular regulation since the cells were exposed to the similar osmo/ionic stresses. A study with the marble shore crab (Pachygrapsus marmoratus) also showed that the abundance of Na+/K+-ATPase mRNA induced in all nine gills following the transfer of crabs to low salinity but increased only in G7 after being transferred to high salinity (Jayasundara et al. 2007). Our enzyme activity results for G. depressus support the notion that individual gills do indeed play distinct osmoregulatory roles in euryhaline crustaceans. Other transport proteins and transport-related enzymes in gills, including a Na+/H+ antiporter, carbonic anhydrase, and Na+/K+/2Cl− cotransporters (Bianchini et al. 2008; Pequeux 1995; Henry et al. 2002; Towle et al. 1997, 2011; Serrano and Henry 2008; Jillette et al. 2011; Ahearn et al. 2004; Wheatly 1999), might be involved in the specificity of function. For example, a basolateral Na+/K+/2Cl− cotransporter involved in NaCl excretion appears to be induced during acclimation to concentrated seawater (Freire et al. 2008; Luquet et al. 2005). To further develop G. depressus as a new model for the study of salinity acclimation in crabs, future investigations will examine the role of these transporters and possible ionocytes in the acclimation responses of this euryhaline species.