Through analyses of the ability of the Takifugu species to adapt to FW, we have demonstrated that only T. obscurus exhibits a high adaptability to both FW and SW. This observation is consistent with their natural anadromous habitats (Table 1). In our analyses, we used sexually immature fish (~10 g) and large fish (~350 g) with well developed testis or ovary. All the T. obscurus survived in both FW and SW for more than 10 days and they looked healthy, suggesting that size and sexual maturation do not affect their adaptability. Recently, Yan et al. reported the effect of salinity on food intake, growth, and survival of T. obscurus (~45 g) . They cultured T. obscurus in FW, BW, and SW for 54 days and compare their level of food-intake and growth rates. The fish survived and grew under all conditions tested, and the growth rates in low-salinity BW (23% SW) were better than those in FW, SW and high-salinity BW (51% SW). Their observation demonstrated that T. obscurus grows under a wide range of salinities and low-salinity BW is the best condition for young T. obscurus to grow.
Our analyses also demonstrated that many other Takifugu species exhibit a relatively high ability to cope with salinity changes. T. niphobles, T. rubripes, T. pardalis, and T. poecilonotus can survive in FW for several days and in BW for more than 10 days, suggesting that the Takifugu species are potentially euryhaline. These results are consistent with their natural brackish/marine habitats; they are sometimes found in brackish river mouths (Table 1). It is known that T. rubripes spawn in the entrance of bays. The fingerlings grow in shallow and river mouths of bays for one year, and then go to the broad ocean . Han et al. demonstrated that the best growing salinity of T. rubripes weighting ~0.02, ~1.2 and ~25 g were 73–91%, 29%, and 43% SW, respectively . Thus change of the environmental salinity is important for the growth of the fingerlings of T. rubripes.
During the acclimation to FW, serum Cl- of T. obscurus decreased although Na+ and osmolarity remained unchanged. In T. niphobles the decrease in serum Cl- was more extensive than that in serum Na+. These results suggest that the mechanisms whereby Cl- and Na+ are regulated differ. The decrease in serum Cl- during FW acclimation has also been observed in Japanese eel (Anguilla japonica)  and spotted green pufferfish (Tetraodon nigroviridis) . In Tetraodon and Takifugu species, the other electrolytes that compensate for Cl- were not determined. In the case of Japanese eel, serum SO42- concentration increases from ~1 to ~19 mM during acclimation from SW to FW . The expressions of kidney sulfate transporters are drastically induced during FW acclimation, suggesting that the serum SO42- reabsorbed by the kidney compensates for Cl- and helps improve the survival of eel in FW .
Some reports have categorized pufferfish as aglomerular [25, 26]. However, glomerular nephrons was observed in the species of from four genuses of the Tetraodontidae family, namely, Canthigaster rivulatus , Tetraodon nigroviridis , Sphoeroides testudineus , two Takifugu species reported by Ogawa , and six Takifugu species in this study (Figure 4). We think that many of the Tetraodontidae species are glomerular. The increase in size of the glomerulus after transferring to FW (Figure 4D–E) was also found in the threespine stickleback (Gasterosteus aculeatus L.) . In general, the largest difference between FW fish and glomerular SW fish regarding structure of the renal tubules is the presence or absence of a distal segment, which acts as a urine-diluting segment in FW fish . Most of the euryhaline fish have a FW-fish type of nephron such as the European eel (Anguilla vulgaris), Pacific pink salmon (Oncorhynchus gorbuscha), rainbow trout (Oncorhynchus mykiss), southern flounder (Paralichthys lethostigma), armored sculpin (Leptocottus armatus), medaka (Oryzias latipes), and spotted green pufferfish [20, 30]. In the Takifugu species, we demonstrated that only mefugu (T. obscurus) has the FW-fish type of nephron with a distal segment, and the other species have a SW-fish type of nephron lacking a distal segment (Figure 4F–J). These results are completely consistent with the ability of those species to adapt to FW, thus the presence of a distal segment is one of the most important factors that allow T. obscurus to be highly adaptalbe to a wide range of salinities.
Tetraodon nigroviridis (spotted green pufferfish) is a small pufferfish less than 10 cm in length that lives in brackish river and estuaries of Southeast Asia. T. nigroviridis also has a compact genome like the Takifugu species, and the whole genome was sequenced in 2004 . Recently, Lin et al. have demonstrated the strong adaptability of T. nigroviridis to FW, BW, and SW and its use in studies on osmoregulation . We think that both T. nigroviridis and T. obscurus are good models for studying osmoregulation. The advantage with T. nigroviridis is that it is readily available. The advantage with T. obscurus is that it can be used in a wide range of size (2–20 cm) and compare the functions of the gill and kidney with those of other Takifugu species that can not adapt to FW.
Many molecules have been identified as components of the chloride cells (or mitochondria-rich cells), the major site of ion regulation in the gill: transporters, channels, and pumps for Na+, K+, Cl-, HCO3-, H+, Ca2+, water, and urea; carbonic anhydrase; and hormone receptors . However, the complete physiological function of the chloride cells cannot be explaned by those components alone, and identification of further players is necessary. Furthermore, little is known of the molecular biology of osmoregulation by the kidney and intestine of teleost fish: NKA , sulphate transporters , urea transporter , chloride channel , Ca2+-sensing receptor , V-type H+-ATPases  in the kidney; Na-Pi cotransporter  and aquaporin water channels in both the kidney and intestine [38–40]; and Na+/K+/2Cl- cotransporter in the stomach and intestine . By determining the differences in gene expression patterns in the gill, intestine, and kidney of FW- and SW-acclimated mefugu (T. obscurus), we would be able to identify the genes that are important for osmoregulatory adaptation.