Sequencing the large genomes of sharks. We focused on the brownbanded bamboo shark Chiloscyllium punctatum, for which we recently tabled embryonic stages 8 , and the cloudy catshark Scyliorhinus torazame. Their whole genomes, measured to be approximately 4.7 and 6.7 Gbp, respectively, were sequenced de novo to obtain assemblies including megabase-long scaffolds (Supplementary Note 1.1). We also assembled the genome of the whale shark Rhincodon typus using short sequence reads previously generated 3 (Supplementary Note 1.2). Using these genome assemblies, we performed genome-wide gene prediction, assisted by transcript evidence and protein-level homology to other vertebrates. The obtained genome assemblies and gene models exhibit high coverage (Supplementary Fig. 1), and of these, the bamboo shark genome assembly achieved the highest continuity (N50 scaffold length, 1.9 Mbp) and completeness (97% of reference orthologues identified at least partially). Using the novel gene models, we constructed orthologue groups encompassing a diverse array of vertebrate species (see below). Our products outperform existing

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

Captive breeding of whale sharks is one of the great challenges for aquariums. However, there is limited information available related to reproductive physiology due to the difficulty of sampling and long-term observation. In the present report, we provide information on the reproductive physiology of female whale sharks, which were incidentally captured as bycatch in a set-net off the coast of Okinawa, Japan. Total lengths of three captured female whale sharks were 403, 665, and 761 cm, respectively, at the time of their death. Collected paired ovaries differed in size between right and left. However, it seems not to determine which side of ovary becomes developed. Histological observations revealed that oocytes surrounded by follicle cell layers localized in the developed ovary, and most developed oocytes exhibited yolk vesicle stage. Additionally, in the largest specimen, there were low levels of three steroid hormones (Testosterone, Dihydrotestosterone, and Estradiol-17ß) that did not show seasonal variation. The present results indicate that even the whale shark over 7 m in TL are still histologically and endocrinologically immature. We expect that the present data will provide fundamental information related to reproductive physiology of female whale sharks, and will contribute to protection activities and increased success in captive breeding of whale sharks.

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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 ABSTRACT Most cartilaginous fishes live principally in seawater (SW) environments, but a limited number of species including the bull shark, Carcharhinus leucas, inhabit both SW and freshwater (FW) environments during their life cycle. Euryhaline elasmobranchs maintain high internal urea and ion levels even in FW environments, but little is known about the osmoregulatory mechanisms that enable them to maintain internal homeostasis in hypoosmotic environments. In the present study, we focused on the kidney because this is the only organ that can excrete excess water from the body in a hypoosmotic environment. We conducted a transfer experiment of bull sharks from SW to FW and performed differential gene expression analysis between the two conditions using RNA-sequencing. A search for genes upregulated in the FW-acclimated bull shark kidney indicated that the expression of the Na + -Cl − cotransporter (NCC; Slc12a3) was 10 times higher in the FW-acclimated sharks compared with that in SW sharks. In the kidney, apically located NCC was observed in the late distal tubule and in the anterior half of the collecting tubule, where basolateral Na + /K + -ATPase was also expressed, implying that these segments contribute to NaCl reabsorption from the filtrate for diluting the urine. This expression pattern was not observed in the houndshark, Triakis scyllium, which had been transferred to 30% SW; this species cannot survive in FW environments. The salinity transfer experiment combined with a comprehensive gene screening approach demonstrates that NCC is a key renal protein that contributes to the remarkable euryhaline ability of the bull shark.