Successful delivery of viviparous lantern shark from an artificial uterus and the self-production of lantern shark luciferin.

Our recent success in the long-term maintenance of lantern shark embryos in artificial uterine systems has provided a novel option for the medical treatment of premature embryos for captive viviparous elasmobranchs. The remaining issue with this system is that the embryos cannot survive the abrupt change in the chemical environment from artificial uterine fluid (AUF) to seawater during delivery. To overcome this issue, the present study developed a new protocol for seawater adaptation, which is characterized by a long-term and stepwise shift from AUF to seawater prior to delivery. This protocol was employed successfully, and the specimen survived for more than seven months after delivery, the longest captive record of the species. During the experiment, we unexpectedly detected bioluminescence of the embryonic lantern shark in the artificial uterus. This observation indicates that lantern sharks can produce luciferin, a substance for bioluminescence. This contradicts the recent hypothesis that lantern sharks lack the ability to produce luciferin and use luciferin obtained from food sources.

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization.

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.

Narrowing, twisting, and undulating: Complicated movement in shark spiral intestine inferred using ultrasound.

Shark intestine presents a complicated three-dimensional morphology, characterized by the development of a coiled internal septum. A basic question regarding the intestine is its movement. This lack of knowledge has prevented the testing of the hypothesis on its functional morphology. The present study, to our knowledge, for the first time, visualized the intestinal movement of three captive sharks using an "underwater ultrasound" system. The results indicated that the movement of the shark intestine involved strong twisting. We suspect that this motion is the mechanism that tightens the coiling of the internal septum, enhancing compression of the intestinal lumen. Our data also revealed the presence of active undulatory movement of the internal septum, of which the undulatory wave propagated in the opposite (anal-to-oral) direction. We hypothesize that this motion decreases the flow rate of the digesta and increases absorptive time. These observations indicate that the kinematics of the shark spiral intestine are more complicated than expected based on morphology, and the fluid flow in the intestine is likely highly regulated by intestinal muscular activity.

Squalomix: shark and ray genome analysis consortium and its data sharing platform.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

Long-term monitoring of egg-laying cycle using ultrasonography reveals the reproductive dynamics of circulating sex steroids in an oviparous catshark, Scyliorhinus torazame.

The many diverse reproductive strategies of elasmobranchs (sharks, skates and rays) from lecithotrophic oviparity to matrotrophic viviparity have attracted significant research attention. However, the endocrine control of elasmobranch reproduction is less well-documented largely due to their reproductive characteristics, such as a long reproductive cycle, and/or repeated internal fertilization using stored sperm in oviparous species. In the present study, for the first time, we succeeded in non-invasive monitoring of the continuing egg-laying cycle of the cloudy catshark Scyliorhinus torazame using portable ultrasound devices. Furthermore, long-term simultaneous monitoring of the egg-laying cycle and measurement of plasma sex steroids revealed cycling patterns of estradiol-17ß (E2), testosterone (T) and progesterone (P4). In particular, a decline in T followed by a reciprocal surge in plasma P4 were consistently observed prior to the appearance of the capsulated eggs, implying that P4 is likely associated with the ovulation and/or egg-case formation. While the cycling pattern of E2 was not as apparent as those of T and P4, threshold levels of E2 (>5 ng/mL) and T (>1 ng/mL) appeared to be crucial in the continuation of egg-laying cycle. The possibility to trace the dynamics of plasma sex steroids in a single individual throughout the reproductive cycles makes the catshark a useful model for regulatory and mechanistic studies of elasmobranch reproduction.

Fluorescent cacao oil: A material suited for highlighting blood vessel in macro-scale dissection.

This study describes a novel method to highlight vascular networks in animal tissue during macro-scale dissection using cacao oil and ultraviolet (UV) fluorescent dye. This is a three-step method: 1) injecting warmed cacao oil containing oil-based UV fluorescent dye ("fluorescent cacao oil" or FCO) into the blood vessels of a dead animal; 2) lowering the temperature to solidify the FCO in blood vessels; and 3) illuminating blood vessels with UV light when the specimen is dissected. This method uses the unique properties of cacao oil, which is solid at room temperature but becomes liquid at 40°C. Such a relatively low melting temperature meets two conflicting demands, i.e., maintaining low viscosity for better flow into the blood vessels and preventing damage of animal tissue by heat. This method is:•Practical, as blood vessel is strongly highlighted using handy UV light during dissection; therefore, a specific medical equipment is not required•Inexpensive, as FCO is made by mixing two commercially available produces (cacao oil and UV fluorescent dye)•Stable, as FCO-injected tissue can be fixed and preserved semi-permanently in formalin. The fluorescent ability of FCO is not affected by this process.

Volume of the whale shark and their mechanism of vertical feeding.

The present study provides a noninvasive method to estimate the body volume of sharks (Elasmobranchii, Selachii) using a computational geometric model. This method allows the volume of sharks to be estimated from lateral and ventral photographs assuming an elliptical body cross-sectional geometry. A comparison of the estimated and actual body volumes of several shark species showed that the estimation error was < 0.5%. The accuracy of the model decreased if photographs that were inclined to the orthogonal plane were used, although this error was on average < 2.3% if the inclination angle was 10° or less. Applying this model to captive whale sharks (Rhincodon typus) that were 8.0 and 8.8 m in total length revealed that their body volumes were 3.5 and 4.5 m3, respectively. These estimates allowed for the quantitative evaluation of our hypothesis, that the whale shark uses suctioned air for buoyancy control during vertical feeding-a behavior unique to this species among elasmobranchs. The volume estimates of the captive whale sharks, together with the density estimates from their liver proportions, revealed that the air occupying a part of oro-pharyngeal and branchial cavities can help the whale sharks to keep their body floating. This hypothesis may explain how the whale shark sometimes stays at the water surface without fin motion during vertical feeding, even though their body density is greater than that of seawater.

Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis.

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.

Armored eyes of the whale shark.

This report elaborates on adaptations of the eyes of the whale shark Rhincodon typus (Elasmobranchii, Rhincodontidae), including the discovery that they are covered with dermal denticles, which is a novel mechanism of eye protection in vertebrates. The eye denticle differs in morphology from that of the dermal denticles distributed over the rest of the body, consistent with a different function (abrasion resistance). We also demonstrate that the whale shark has a strong ability to retract the eyeball into the eye socket. The retraction distance was calculated to be approximately half the diameter of the eye, which is comparable to those of other vertebrates that are known to have highly retractable eyes. These highly protective features of the whale shark eye seem to emphasize the importance of vision for environmental perception, which contradicts the general, though poorly established, notion of low reliance on vision in this species.

Viviparous stingrays avoid contamination of the embryonic environment through faecal accumulation mechanisms.

In viviparous (live-bearing) animals, embryos face an embryo-specific defecation issue: faecal elimination in utero can cause fatal contamination of the embryonic environment. Our data from the viviparous red stingray (Hemitrygon akajei) reveals how viviparous elasmobranchs circumvent this issue. The exit of the embryonic intestine is maintained closed until close to birth, which allows the accumulation of faeces in the embryonic body. Faecal accumulation abilities are increased by (1) the large intestine size (represents about 400-600% of an adult intestine, proportionally), and (2) the modification in the intestinal inner wall structure, specialized to increase water uptake from the faecal matter. According to the literature, faecal accumulation may occur in embryos of the lamniform white shark as well. The reproductive biology of myliobatiform stingrays and lamniform sharks is characterized by the onset of oral feeding before birth (i.e. drinking of uterine milk and eating of sibling eggs, respectively), which is expected to result in the production of large amounts of faeces during gestation. The strong ability of faecal accumulation in these lineages is therefore likely an adaptation to their unique embryonic nutrition mechanism.

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.

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.

Stealth breathing of the angelshark.

For benthic fishes, breathing motion (e.g., oral, pharyngeal, and branchial movements) can result in detection by both prey and predators. Here we investigate the respiratory behavior of the angelshark Squatina japonica (Pisces: Squatiniformes: Squatinidae) to reveal how benthic elasmobranchs minimize this risk of detection. Sonographic analyses showed that the angelshark does not utilize water-pumping in the oropharyngeal cavity during respiration. This behavior is in contrast with most benthic fishes, which use the rhythmical expansion/contraction of the oropharyngeal cavity as the main pump to generate the respiratory water current. In the angelshark, breathing motion is restricted to the gill flaps located on the ventral side of the body. We suspect that the gill flaps function as an active pump to eject water through the gill slits. This respiratory mode allows conspicuous breathing motion to be concealed under the body, thereby increasing crypsis capacity.

Shark genomes provide insights into elasmobranch evolution and the origin of vertebrates.

Modern cartilaginous fishes are divided into elasmobranchs (sharks, rays and skates) and chimaeras, and the lack of established whole-genome sequences for the former has prevented our understanding of early vertebrate evolution and the unique phenotypes of elasmobranchs. Here we present de novo whole-genome assemblies of brownbanded bamboo shark and cloudy catshark and an improved assembly of the whale shark genome. These relatively large genomes (3.8-6.7 Gbp) contain sparse distributions of coding genes and regulatory elements and exhibit reduced molecular evolutionary rates. Our thorough genome annotation revealed Hox C genes previously hypothesized to have been lost, as well as distinct gene repertories of opsins and olfactory receptors that would be associated with adaptation to unique underwater niches. We also show the early establishment of the genetic machinery governing mammalian homoeostasis and reproduction at the jawed vertebrate ancestor. This study, supported by genomic, transcriptomic and epigenomic resources, provides a foundation for the comprehensive, molecular exploration of phenotypes unique to sharks and insights into the evolutionary origins of vertebrates.

Changes in sex steroid hormone levels reflect the reproductive status of captive female zebra sharks (Stegostoma fasciatum).

Captive breeding in aquaria is a useful means for ex situ preservation of threatened elasmobranch species. To promote captive breeding, it is important to determine the female reproductive status. However, information regarding reproductive status in female elasmobranchs is limited. Here, we used zebra sharks, Stegostoma fasciatum, as a model for elasmobranch reproduction in captivity. We investigated the relationships among changes in the sex steroid hormone levels, follicle size, and egg-laying period to develop indicators for the female reproductive status. We confirmed that mature female zebra sharks undergo an annual reproductive cycle. Additionally, we showed that the variations in sex steroid hormone levels correlated with reproductive status in mature female zebra sharks. Plasma estradiol-17ß (E2) concentrations increased two months before ovarian follicle development and decreased along with follicle regression. Interestingly, E2 levels were inversely correlated with water temperature (R = -0.901). Moreover, high levels of testosterone (T) correlated well with the laying period. These results strongly suggest that E2 is an indicator for ovarian follicle development, and that T is a useful indicator for both the onset and end of the egg-laying period in captive zebra sharks.

Eye retraction in the giant guitarfish, Rhynchobatus djiddensis (Elasmobranchii: Batoidea): a novel mechanism for eye protection in batoid fishes.

Eye retraction behavior has evolved independently in some vertebrate linages such as mudskippers (fish), frogs and salamanders (amphibians), and cetaceans (mammals). In this paper, we report the eye retraction behavior of the giant guitarfish (Rhynchobatus djiddensis) for the first time, and discuss its mechanism and function. The eye retraction distance was nearly the same as the diameter of the eyeball itself, indicating that eye retraction in the giant guitarfish is probably one of the largest among vertebrates. Eye retraction is achieved by unique arrangement of the eye muscle: one of the anterior eye muscles (the obliquus inferior) is directed ventrally from the eyeball and attaches to the ventral surface of the neurocranium. Due to such muscle arrangement, the obliquus inferior can pull the eyeball ventrally. This mechanism was also confirmed by electrical stimulation of the obliquus inferior. The eye retraction ability of the giant guitarfish likely represents a novel eye protection behavior of elasmobranch fishes.

Gonadal Morphology, Histology, and Endocrinological Characteristics of Immature Female Whale Sharks, Rhincodon typus.

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.