© 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 RESEARCH ARTICLE Comprehensive analysis of genes contributing to euryhalinity in the bull shark, Carcharhinus leucas;Na+-Cl− co-transporter is one of the key renal factors upregulated in acclimation to low-salinity environment Itaru Imaseki1,‡, Midori Wakabayashi1, Yuichiro Hara2,*, Taro Watanabe1, Souichirou Takabe1, Keigo Kakumura1, Yuki Honda1, Keiichi Ueda3, Kiyomi Murakumo3, Rui Matsumoto3, Yosuke Matsumoto3, Masaru Nakamura4, Wataru Takagi1, Shigehiro Kuraku2 and Susumu Hyodo1 ABSTRACT leucas, have the capacity to inhabit both seawater (SW) and Most cartilaginous fishes live principally in seawater (SW) freshwater (FW) environments (see Compagno, 1984; Hazon et al., environments, but a limited number of species including the bull 2003; Pillans and Franklin, 2004). Bull sharks spend most of their shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) lifetime in SW, but they utilize a wide range of salinities throughout environments during their life cycle. Euryhaline elasmobranchs their life cycle (Thorson, 1971; Montoya and Thorson, 1982; maintain high internal urea and ion levels even in FW environments, Simpfendorfer et al., 2005; Carlson et al., 2010). Female bull sharks but little is known about the osmoregulatory mechanisms that enable are thought to give birth in estuaries or river mouths, and neonates and them to maintain internal homeostasis in hypoosmotic environments. juveniles are thought to migrate upstream to occupy inshore rivers as In the present study, we focused on the kidney because this is the only nursery habitats (Ortega et al., 2009; Werry et al., 2012). The use of organ that can excrete excess water from the body in a hypoosmotic FW habitats by juvenile bull sharks has been reported in the southern environment. We conducted a transfer experiment of bull sharks from USA (Simpfendorfer et al., 2005; Ortega et al., 2009; Matich and SW to FW and performed differential gene expression analysis Heithaus, 2015), central America (Thorson, 1971; Montoya and between the two conditions using RNA-sequencing. A search for Thorson, 1982), Australia (Pillans and Franklin, 2004; Werry et al., genes upregulated in the FW-acclimated bull shark kidney indicated 2012) and South Africa (Cliff and Dudley, 1991; McCord and that the expression of the Na+-Cl− cotransporter (NCC; Slc12a3) was Lamberth, 2009). Once bull sharks reach sexual maturity – 10 times higher in the FW-acclimated sharks compared with that in SW [approximately 160 180 cm in total length (TL) in Florida], they sharks. In the kidney, apically located NCC was observed in the late move from riverine and estuarine environments into full-strength SW distal tubule and in the anterior half of the collecting tubule, where for further growth and breeding (Curtis et al., 2011). basolateral Na+/K+-ATPase was also expressed, implying that these It is well recognized that marine cartilaginous fishes, including segments contribute to NaCl reabsorption from the filtrate for diluting SW-adapted bull sharks, conduct urea-based osmoregulation. Plasma the urine. This expression pattern was not observed in the houndshark, ion levels are maintained at approximately half that of SW, whereas Triakis scyllium, which had been transferred to 30% SW; this species total plasma osmolality is slightly hyperosmotic to the surrounding cannot survive in FW environments. The salinity transfer experiment SW environment through the retention of urea. As a result, combined with a comprehensive gene screening approach cartilaginous fish do not suffer dehydration in a SW environment demonstrates that NCC is a key renal protein that contributes to the (Smith, 1936; Robertson, 1975; Hazon et al., 2003; Hyodo et al., remarkable euryhaline ability of the bull shark. 2004b). In FW environments, bull sharks and other euryhaline elasmobranch species maintain high internal NaCl and urea levels, KEY WORDS: Kidney, Ion transporters, Euryhalinity, resulting in a plasma osmolality of approximately 600 mOsm (Janech Osmoregulation, Freshwater adaptation, Cartilaginous fish and Piermarini, 2002; Pillans and Franklin, 2004) that is almost twice that of FW teleosts (Evans, 1993). This strategy is in contrast with FW INTRODUCTION stingrays (Potamotrygonidae), which, like FW teleosts, do not Most cartilaginous fishes are principally marine species, and only a accumulate urea (Wood et al., 2002). Therefore, it is likely that bull limited number of species, including the bull shark, Carcharhinus sharks face incipient water gain and solute loss in a FW environment by branchial diffusion and urinary loss. Recently, the contribution of + + + + 1Laboratory of Physiology, Atmosphere and Ocean Research Institute, The Na /K -ATPase (NKA) and Na /H exchanger 3 was reported for University of Tokyo, Kashiwa, Chiba 277-8564, Japan. 2RIKEN Center for branchial Na+ uptake in bull sharks in a FW environment (Reilly Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan. 3Okinawa Churaumi Aquarium, Motobu, Okinawa 905-0206, Japan. 4Okinawa Churashima et al., 2011). However, mechanisms enabling the euryhalinity in Foundation, Motobu, Okinawa 905-0206, Japan. cartilaginous fish are still largely unknown. *Present address: Research Institute of Environmental Medicine, Nagoya It is generally accepted that multiple organs are involved in University, Nagoya 464-8601, Japan. maintaining body–fluid homeostasis in vertebrates. Among them, ‡Author for correspondence ([email protected]) the kidney is one of the most important osmoregulatory organs in vertebrates including cartilaginous fishes. In a SW environment, I.I., 0000-0002-3003-926X; M.W., 0000-0003-0661-6052; K.K., 0000-0002- 0556-5110; W.T., 0000-0002-7882-0223 more than 90% of filtered urea is reabsorbed from the primary urine and returned to the blood system, thereby reducing the urinary loss Received 14 February 2019; Accepted 18 May 2019 of urea (Smith, 1936; Kempton, 1953; Boylan, 1967). Reflecting Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb201780. doi:10.1242/jeb.201780 Japanese banded houndsharks, Triakis scyllium Müller & Henle List of abbreviations 1839 (n=9, TL=68.1±1.0 cm, BM=1.0±0.05 kg), were collected in BM body mass Koajiro Bay, near the Misaki Marine Biological Station of the CT collecting tubule University of Tokyo. They were transported to the Atmosphere and EDT early distal tubule Ocean Research Institute at the University of Tokyo and kept in FDR false discovery rate 2×103 liter holding tanks (20–22°C) under a constant photoperiod FPKM fragments per kilobase of exon per million reads mapped FW freshwater (12 h:12 h light:dark). The houndsharks were fed on squid for at GFR glomerular filtration rate least 2 weeks before the experiments and were kept without feeding LDT late distal tubule during the experimental period (see Yamaguchi et al., 2009). logFC log2 fold change All animal experiments were conducted according to the Guidelines − NCC Na+-Cl co-transporter for Care and Use of Animals approved by the committees of the α + + α NKA 1Na/K ATPase subunit 1 University of Tokyo and the Okinawa Churaumi Aquarium. NKCC2 Na+-K+-Cl− cotransporter 2 ORF open reading frame RNA-seq RNA-sequencing Transfer experiments Slc solute carrier In March of 2012, three bull sharks kept in holding tanks were SW seawater moved to an experimental tank (15×103 liters, filled with full- UFR urine flow rate strength SW, 35 ppt) 3 days before the onset of salinity change. It UT urea transporter was ascertained that they could swim comfortably in the new tank. On day 1 of the experiment, the salinity was adjusted by adding FW to achieve a salinity of 80% SW. On day 2, the same degree of their crucial function, the renal tubules of marine cartilaginous fish salinity change was performed to produce 60% SW. On days 3–8, are highly elaborate and show unique features compared with those the salinity of the tank was reduced by 10% per day, and the salinity of other vertebrates (Lacy and Reale, 1985; Hentschel et al., 1998; was deemed to have reached nearly FW (less than 2 ppt) on day 8 Hyodo et al., 2014). We have conducted mapping of various pumps, (Fig. S1). Bull sharks were killed on days 8–10. Two SW control channels and transporters in the nephron segments, and proposed a sharks were maintained in the holding tank with running SW, and model for urea reabsorption in the kidney of SW-adapted were killed on days 8 and 9, and two further SW sharks were killed cartilaginous fishes (Hyodo et al., 2004a, 2014; Yamaguchi et al., in November 2011 and February 2013, respectively. For sampling, 2009; Kakumura et al., 2015; Hasegawa et al., 2016). In FW bull sharks were initially anesthetized with an intramuscular environments, in contrast, it is assumed that the kidney of the bull injection of midazolam (2.5 mg kg−1 BM) and then euthanized shark must excrete excess water caused by the osmotic influx of with an intravenous injection of 2,6-diisoprophylphenol (0.6– water, while concomitantly retaining ions and urea. Therefore, it is 1.6 mg kg−1 BM). Blood samples were collected from the dorsal highly probable that the expression of key proteins involved in vasculature by a syringe and were centrifuged at 10,000 g at 4°C for osmoregulation is altered in the kidney when bull sharks move from 10 min to obtain the plasma. Urine samples were collected using a SW to FW habitats. Pillans et al. (2005) reported that the expression urethral catheter that was inserted under anesthesia (All Silicone Foley level of NKA decreased in the kidney of bull sharks following Balloon Catheter, Create Medic Co. Ltd, Kanagawa, Japan). Plasma transfer from FW to SW, supporting our notion that mechanisms for and urine samples were stored at −80°C until analysis. Ion and urea retaining ions and urea are upregulated in a FW environment.