Takifugu obscurus is a euryhaline fugu species very close to Takifugu rubripes and suitable for studying osmoregulation

Background The genome sequence of the pufferfish Takifugu rubripes is an enormously useful tool in the molecular physiology of fish. Euryhaline fish that can survive both in freshwater (FW) and seawater (SW) are also very useful for studying fish physiology, especially osmoregulation. Recently we learned that there is a pufferfish, Takifugu obscurus, common name "mefugu" that migrates into FW to spawn. If T. obscurus is indeed a euryhaline fish and shares a high sequence homology with T. rubripes, it will become a superior animal model for studying the mechanism of osmoregulation. We have therefore determined its euryhalinity and phylogenetic relationship to the members of the Takifugu family. Results The following six Takifugu species were used for the analyses: T. obscurus, T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus. When transferred to FW, only T. obscurus could survive while the others could not survive more than ten days in FW. During this course of FW adaptation, serum Na+ concentration of T. obscurus decreased only slightly, but a rapid and large decrease occurred even in the case of T. niphobles, a peripheral fresh water species that is often seen in brackish river mouths. Phylogenetic analysis using nucleotide sequences of the mitochondrial 16S ribosomal RNA gene of each species indicated that the six Takifugu species are very closely related with each other. Conclusion T. obscurus is capable of adapting to both FW and SW. Its genomic sequence shares a very high homology with those of the other Takifugu species such that the existing Takifugu genomic information resources can be utilized. These properties make "mefugu", which has drawn little attention from animal physiologists until this study, a useful model animal for studying the molecular mechanism of maintaining body fluid homeostasis.


Background
Maintenance of a stable internal environment is important for vertebrate animals to survive in a variety of habi-tats. Even small changes in ionic balance, osmolarity, and pH of body fluid seriously affect the survival of the animals. Strategies for maintaining body fluid homeostasis are different depending on animals and their habitats. Freshwater (FW) teleosts (modern bony fish) maintain the osmolarity of extracellular fluid around 300 mOsM, while the osmolarity of the environmental freshwater is generally less than 10 mOsM. In order to balance passive loss of salts and gain of water, they take up salts from FW through the gills and excrete a lot of dilute urine from which most of the salts have been reabsorbed by the kidney [1]. Marine teleosts also maintain the osmolarity of extracellular fluid to a level similar to that of freshwater fish, despite that the osmolarity of seawater (SW) is approx. 1000 mOsM. In order to balance passive loss of water and gain of salts, they drink seawater, absorb salts and water both in the intestine, and excrete salts through the gills [1]. The systems used by teleosts to adapt to FW and SW differ not only in the direction of ion and water movements but also in their molecular components. Euryhaline fish adapts to both FW and SW by switching these systems.
To identify the molecular components involved in body fluid homeostasis, the change of expression of each gene during adaptation of euryhaline fishes to different salinities is a potential useful marker since the genes involved are expected to be drastically up-or down-regulated during the adaptation. In fact, several genes have been identified by this strategy using euryhaline fishes such as tilapia [2], salmon [3][4][5], killifish [6], and eel [7][8][9][10]. However, this systematic approach is very laborious because genome sequences are not available for the euryhaline fishes that are currently being used for molecular physiological studies.
Six Takifugu species used in this study Takifugu is a genus of puffer fish and belongs to the family Tetradontidae of teleost fish. It consists of approx. 20 species living in the Northwest Pacific Ocean around China, Korea, and Japan [11,12]. Takifugu [13]. In 2002, the genome project of T. rubripes was completed and the sequence information is now available for free on the websites [14].
Within the genus Takifugu, two species are known to be anadromous, namely, T. obscurus [15][16][17][18][19] and T. ocellatus [16]. T. obscurus ( Figure 1, Table 1) is found in the East China Sea, the South China Sea, and inland waters in China and the Korean Peninsula. It lives in the bottom layer of inshore and inland waters, and grows 20-40 cm in length. Most of the growth takes place in the sea but they spawn in brackish and fresh water. During the spawning season, which is from late spring to early summer, the sexually mature fish run into river estuaries and spawn in inland waters including rivers, lakes, and ponds. The fingerlings grow in the inland water and either return to the sea the next spring or they there for a few months before returning to the sea. In the sea they grow to sexually mature fish over several years, and then return to the inland water again to spawn. T. ocellatus is also found in an area similar to that of T. obscurus. T. ocellatus is a small species and grows to around 15 cm in length. The life cycle of T. ocellatus has not been well described but is expected to be similar to that of T. obscurus.
In this study, we focus on the suitability of T. obscurus as a novel animal model for studying the molecular mechanism of body fluid homeostasis. With the euryhalinity and applicability of the currently available fugu genome sequence, we conclude that T. obscurus is a useful animal model for studying the mechanism of osmoregulation.

Survival of Takifugu species in FW
A summary on six Takifugu species used in this study is shown in Figure 1 and Table 1. The survival rate of each species after transfering from SW to FW is shown in Figure  2A.  East Asia [12,22] * Adult fish are often seen in BW river mouths and sometimes seen in FW rivers. ** Adult fish are sometimes seen in BW river mouths [48]. *** Fingerlings are often seen in brackish river mouths [22].
The fishes that survived for 10 days in BW were transferred to FW and survival was monitored (time course data not shown). Mean survival in FW were: 3.1 ± 0.6 days, 4.6 ± 0.6 days, and 5.5 ± 0.7 days for T. niphobles (n = 7), T. poecilonotus (n = 8), and T. pardalis (n = 6), respectively. Mean survival in FW following the transfer from BW did not differ significantly between T. poecilonotus and T. pardalis, and was short for T. niphobles (P < 0.001) when compared to the survival of those that were transferred from SW to FW. These results indicate that 10 days' adaptation to BW does not improve the adaptability of T. poecilonotus, T. pardalis, and T. niphobles to FW.

Changes in serum osmolarity and concentrations of ions and urea during adaptation
To gain insights into the way that the Takifugu species adapt to different salinities, we sampled the blood from two species, T. obscurus and T. niphobles, and determined serum osmolarity and concentrations of ions and urea ( Table 2). In SW, serum osmolarity and ion concentration of T. obscurus and T. niphobles were similar to those reported for other teleost fish [1]. When transferred to FW, however, significant changes were observed in serum osmolarity and concentrations of Na + and Clfor T.
niphobles, whereas the changes were small for T. obscurus. The reductions in osmolarity during FW adaptation of T. obscurus and T. niphobles were -17 and -148 mOsM, respectively. The decrements of serum concentrations of Na + and Clduring FW adaptation were -13 and -16 mM in T. obscurus, and -69 and -52 mM in T. niphobles, respectively. These results suggest that T. obscurus has a much stronger ability to maintain body fluid homeostasis against salinity fluctuations and can survive in FW. Figure 3 shows the time course of changes in serum Na + concentration following exposure of T. obscurus and T. niphobles to low salinities. In the case of T. obscurus, a slightly decreased level that was observed on day 1, remained throughout the course, but in the case of T. niphobles, a relatively large decrease occurred continuously until death. In BW where T. niphobles exhibited 64% survival rate (Figure 2), a significant recovery of the decreased serum Na + levels was observed on day 9 ( Figure 3). The standard errors of serum Na + concentration of T. niphobles (7.6-32 mM) were much larger than those of T. obscurus (2.3-7.9 mM), suggesting that the individual differences of adaptability to FW and BW are large in T. niphobles. In T. niphobles the decrease in serum Clwas more extensive than that in serum Na + . In T. obscurus serum Cldecreased while Na + and osmolarity remained unchanged.

Comparison of nephron structure of Takifugu species
Kidney sections of the six Takifugu species were analyzed to compare their nephron structures. Under light microscope, a number of glomeruli were observed within all sections stained with hematoxylin-eosin, demonstrating that all six Takifugu species have glomerular nephrons ( Figure 4A-C). The glomeruli of FW-acclimated T. obscurus appeared to be loose compared to those of the SWacclimated fish ( Figure 4D-E). There was no clear difference between those species rich in glomeruli at the histological level.
To characterize the segments of the renal tubules, kidney sections of T. obscurus, T. rubripes, T. niphobles, T. pardalis, and T. poecilonotus were stained with anti-Na + -K + -ATPase (NKA) antibody, and observed under a fluorescence microscope. NKA is the most important molecule that provides a driving force for many transporting systems in the renal tubules, and the patterns of NKA localization are different among the segments of renal tubule ( Figure 4I-J) [20]. In both FW-and SW-acclimated T. obscurus, proximal and distal segments were clearly observed ( Figure  4F-G). In contrast, the distal segment is not found in T. rubripes, T. niphobles, T. pardalis, and T. poecilonotus ( Figure  4H).

Phylogeny of Takifugu species
To know the phylogenetic relationship of the Takifugu species, we isolated the mitochondrial 16S rRNA gene from each species and determined the sequence. Resulting data were compared with the sequences of the 16S rRNA genes of other species in databases, and a phylogenetic tree was constructed ( Figure 5). Surprisingly, the Takifugu species were very closely related each other. The identities of 16S rRNA within the Takifugu species are 99% whereas those between Takifugu and Tetraodon nigroviridi, Oryzias latipes, or Homo sapiens were 86%, 77%, and 63%, respectively. Our preliminary results of the nucleotide sequences of several cDNA clones for ion transporters (Na + /H + exchangers; accession numbers AB200326-AB200333) and hormone receptors (members of the adrenomedullin receptor family: accession numbers AB219765-AB219771, AB219835-AB219840) [21] of T. obscurus were 99% identical to those of T. rubripes including the non-coding sequences (data not shown). These results suggest that the Takifugu species diversified very recently and the genome resources of T. rubripes can be used for studying the T. obscurus genes and their products.

Discussion
Through analyses of the ability of the Takifugu species to adapt to FW, we have demonstrated that only T. obscurus Changes in serum Na + concentration during transfer from seawater (SW) to freshwater (FW) or to brackish water (BW) Figure 3 Changes in serum Na + concentration during transfer from seawater (SW) to freshwater (FW) or to brackish water (BW). Results of T. obscurus and T. niphobles are shown on the left and right, respectively. Serum Na + concentrations of the fish that adapted to SW, FW, and BW are shown as the black, white, and gray bars, respectively (n = 3-4). 14% SW was used as BW. *P < 0.01, **P < 0.001.  (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) [19]. 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 highsalinity 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 [22]. 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 [23]. Thus change of the environmental salinity is important for the growth of the fingerlings of T. rubripes.
During the acclimation to FW, serum Clof T. obscurus decreased although Na + and osmolarity remained unchanged. In T. niphobles the decrease in serum Clwas more extensive than that in serum Na + . These results suggest that the mechanisms whereby Cland Na + are regulated differ. The decrease in serum Clduring FW acclimation has also been observed in Japanese eel (Anguilla japonica) [24] and spotted green pufferfish (Tetraodon nigroviridis) [20]. In Tetraodon and Takifugu species, the other electrolytes that compensate for Clwere not determined. In the case of Japanese eel, serum SO 4 2concentration increases from ~1 to ~19 mM during acclimation from SW to FW [24]. The expressions of kidney sulfate transporters are drastically induced during FW acclimation, suggesting that the serum SO 4 2reabsorbed by the kidney compensates for Cland helps improve the survival of eel in FW [24].
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 [27], Tetraodon nigroviridis [20], Sphoeroides testudineus [28], two Takifugu species reported by Ogawa [27], 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.) [29]. 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 [30]. 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.
Phylogenetic relationship between Takifugu and other species  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 [31]. Recently, Lin et al. have demonstrated the strong adaptability of T. nigroviridis to FW, BW, and SW and its use in studies on osmoregulation [20]. 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 -, HCO 3 -, H + , Ca 2+ , water, and urea; carbonic anhydrase; and hormone receptors [32]. 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 [20], sulphate transporters [24], urea transporter [33], chloride channel [34], Ca 2+ -sensing receptor [35], Vtype H + -ATPases [36] in the kidney; Na-Pi cotransporter [37] and aquaporin water channels in both the kidney and intestine [38][39][40]; and Na + /K + /2Clcotransporter in the stomach and intestine [41]. 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.

Conclusion
• Mefugu (T. obscurus) is an anadromous fish of the genus Takifugu that has a strong ability to maintain body fluid homeostasis during adaptation to low and high environmental salinities and is fully adaptable to both FW and SW.
• Members of the genus Takifugu are very closely related and share ~99% sequence identities in their genomes as shown by a phylogenetic analysis using the mitochondrial DNA sequence for the 16S ribosomal RNA gene.
• The nephrons of FW-or SW-acclimated T. obscurus exhibit a structure that is typical of FW fish. On the other hand, T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus have nephrons of that are typical of SW glomerular fish.
• T. obscurus can be used as an animal model for studying the molecular mechanism of osmoregulation by exploiting the Takifugu genome resources.

Animals and transfer experiment
The animal protocols and procedures were approved by the Institutional Animal Care and Use Committee of Tokyo Institute of Technology and conformed to the American Physiological Society's Guiding Principles in the Care and Use of Laboratory Animals. T. obscurus (10-350 g) were cultured in a brackish river in Korea and China. The fish cultured in BW (14% SW) were transported to The Shimonoseki Marine Science Museum in Japan and kept in 150-2000-l tanks containing BW. The fish were then acclimated to SW for 7-14 days. None of the fish died during the acclimation to SW. To determe FW adaptability, the SW in the tank was gradually replaced with FW by pouring FW at a speed that allowed a complete replacement after 1-2 h. Some fish were transferred to FW directly. Survival was then monitored every 12 h for 10 days.
Other species were caught or cultured in seawater. T. rubripes (30-4200 g) were cultured and sampled at the Japan Sea. T. niphobles (18-128 g), T. pardalis (29-175 g), T. poecilonotus (18-43 g), and T. porphyreus (521-1000 g) were sampled at the Japan Sea. They were transported to the Aquarium and kept in 200-5700-l tanks containing SW. Their adaptability to FW and BW were determined as described above.
All fish used in the analyses were adult fish. The normal size of each species is shown in Table I. Most of the T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus were sexually mature adult fish. T. obscurus and T. rubripes were mixtures of mature and immature fish. The distinction between the species was performed according to Nakabo [11].

Blood analyses
T. obscurus and T. niphobles were maintained in SW and transferred to FW or BW (14% SW). Bloods were collected from the fish in SW and those in FW or BW after 1, 3, and 9 days of the transfer. Healthy fish that had adapted to in various conditions were anaesthetized by immersion in 0.1% ethyl m-aminobenzoate methanesulfonate, and blood was collected from the hepatic vein or heart. Serum from T. obscurus and T. niphobles were diluted in water at the ratio of 1:2 and 1:8, respectively, and used for the analyses. Serum osmolarity was measured by a cryoscopic method. Concentrations of Na + , K + , and Clwere measured by the established electrode methods. Ca 2+ and Mg 2+ concentrations were determined by the o-cresolphthalein complexone method and xylysine blue method, respec-tively. Urea nitrogen concentration in the serum was measured by standard urease assay. The dilution of serum in water did not affect the results (data not shown). These measurements were conducted by SRL Laboratories (Tokyo, Japan).
The following pufferfish were used for the analyses: T. obscurus acclimated to FW for 9 days; T. obscurus acclimated to SW for 9 days; T. rubripes, T. niphobles, T. pardalis, T. poecilonotus, and T. porphyreus maintained in SW.
For the evolutionary analyses, the nucleotide sequences were aligned using Clustal W software [43], and then a phylogenetic tree was constructed by the neighbor-joining method [44] using MEGA software [45] based on Jukes-Cantor evolutionary distances [44]. Statistical analysis was performed by bootstrap methods [44].

List of abbreviations
SW -seawater, FW -freshwater, BW -brackish water

Authors' contributions
AK and SH planned of and designed the study, and wrote the manuscript. HD planned the sections of the study, and performed the operations relating to the supply, transfer, and maintenance of the fish. HD and AK performed the salinity transfer analyses. HS cloned and sequenced genes for 16S rRNA, and collected information on ecobiology of the Takifugu species. AK performed blood assays and construction of the phylogenetic tree. TN performed the histochemical analyses. All authors read and approved the final manuscript.