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.

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.

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.

Heterodonty and ontogenetic shift dynamics in the dentition of the tiger shark Galeocerdo cuvier (Chondrichthyes, Galeocerdidae).

The lifelong tooth replacement in elasmobranch fishes (sharks, rays and skates) has led to the assemblage of a great number of teeth from fossil and extant species, rendering tooth morphology an important character for taxonomic descriptions, analysing phylogenetic interrelationships and deciphering their evolutionary history (e.g. origination, divergence, extinction). Heterodonty (exhibition of different tooth morphologies) occurs in most elasmobranch species and has proven to be one of the main challenges for these analyses. Although numerous shark species are discovered and described every year, detailed descriptions of tooth morphologies and heterodonty patterns are lacking or are only insufficiently known for most species. Here, we use landmark-based 2D geometric morphometrics on teeth of the tiger shark Galeocerdo cuvier to analyse and describe dental heterodonties among four different ontogenetic stages ranging from embryo to adult. Our results reveal rather gradual and subtle ontogenetic shape changes, mostly characterized by increasing size and complexity of the teeth. We furthermore provide the first comprehensive description of embryonic dental morphologies in tiger sharks. Also, tooth shapes of tiger sharks in different ontogenetic stages are re-assessed and depicted in detail. Finally, multiple cases of tooth file reversal are described. This study, therefore, contributes to our knowledge of dental traits across ontogeny in the extant tiger shark G. cuvier and provides a baseline for further morphological and genetic studies on the dental variation in sharks. Therefore, it has the potential to assist elucidating the underlying developmental and evolutionary processes behind the vast dental diversity observed in elasmobranch fishes today and in deep time.

Mode of uterine milk secretion in the white shark.

Examination of the uterus of a dead female white shark (Carcharodon carcharias), which contained the earliest known white shark embryos, revealed that the uterine wall produces lipid-rich secretion (histotroph or "uterine milk") for embryonic nutrition. Uterine tissue was processed for light and electron microscopy, and immunohistochemical techniques to identify its secretory mechanism. Our results indicate that the white shark uterus secretes lipids via holocrine secretion. This type of secretion is characterized by the release of large lipid droplets accumulated in the epithelial cells into the uterine lumen through cell disintegration. The secretory epithelium of the uterus is stratified, and new surface epithelial cells are continuously supplied from deeper epithelial layers to replace the dead secretory cells at the surface. This vertical replacement possibly facilitates the active renewal of the surface epithelium, which is necessary for maintaining holocrine secretory mechanisms. These secretory mechanisms are different from those of myliobatiform stingrays, another elasmobranch taxon that exhibits lipid histotrophy. This may reflect the different origins of lipid histotrophy between these taxa.

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.

Morphological features of the nasal cavities of hawksbill, olive ridley, and black sea turtles: Comparative studies with green, loggerhead and leatherback sea turtles.

We analyzed the internal structure of the nasal cavities of hawksbill, olive ridley and black sea turtles from computed tomography images. The nasal cavities of all three species consisted of a vestibule, nasopharyngeal duct and cavum nasi proprium that included anterodorsal, posterodorsal and anteroventral diverticula, and a small posteroventral salience formed by a fossa of the wall. These findings were similar to those of green and loggerhead sea turtles (Cheloniidae), but differed from those of leatherback sea turtles (Dermochelyidae). Compared to the Cheloniidae species, the nasal cavity in leatherback sea turtles was relatively shorter, wider and larger in volume. Those structural features of the nasal cavity of leatherback sea turtles might help to suppress heat dissipation and reduce water pressure within the nasal cavity in cold and deep waters.

Iris-like eye closure of the fine-patterned pufferfish, Takifugu flavipterus.

Unlike many tetrapods and elasmobranchs, eye-closing ability is absent in bony fishes, with the single-known exception of the family Tetraodontidae. We observed the eye-closing response of the tetraodontid fine-patterned puffer, Takifugu flavipterus, which provides the first detailed data on the kinematics and mechanism of this ability in this family. During eye-closing behavior, the skin around the eye converges toward the center of the iris. This is very different to the reversing uni-directional (e.g., upward then downward) movement of the eyelids of other vertebrates. Electrical stimulation of a freshly dead specimen showed that this movement occurs due to the contraction of a sheet of muscle located just beneath the skin around the eye, which is characteristic of Family Tetraodontidae. Eye-closing is accompanied by simultaneous retraction of the eyeball away from the surface, which is initiated just before the skin of the eye begins to move. The eye-closing ability observed in this study appears to have been acquired independently in the Tetraodontidae.

The megamouth shark, Megachasma pelagios, is not a luminous species.

Despite its five meters length, the megamouth shark (Megachasma pelagios Taylor, Compagno & Struhsaker, 1983) is one of the rarest big sharks known in the world (117 specimens observed and documented so far). This filter-feeding shark has been assumed to be a luminous species, using its species-specific white band to produce bioluminescence as a lure trap. Another hypothesis was the use of the white band reflectivity to attract prey or for social recognition purposes. However, no histological study has ever been performed to confirm these assumptions so far. Two hypotheses about the megamouth shark's luminescence arose: firstly, the light emission may be intrinsically or extrinsically produced by specific light organs (photophores) located either on the upper jaw white band or inside the mouth; secondly, the luminous appearance might be a consequence of the reflection of prey luminescence on the white band during feeding events. Aims of the study were to test these hypotheses by highlighting the potential presence of specific photophores responsible for bioluminescence and to reveal and analyze the presence of specialized light-reflective structures in and around the mouth of the shark. By using different histological approaches (histological sections, fluorescent in situ hybridization, scanning electron microscopy) and spectrophotometry, this study allows to unravel these hypotheses and strongly supports that the megamouth shark does not emit bioluminescence, but might rather reflect the light produced by bioluminescent planktonic preys, thanks to the denticles of the white band.

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.

Etmopteridae bioluminescence: dorsal pattern specificity and aposematic use.

BACKGROUND: In the darkness of the ocean, an impressive number of taxa have evolved the capability to emit light. Many mesopelagic organisms emit a dim ventral glow that matches with the residual environmental light in order to camouflage themselves (counterillumination function). Sharks use their luminescence mainly for this purpose. Specific lateral marks have been observed in Etmopteridae sharks (one of the two known luminous shark families) suggesting an inter/intraspecific recognition. Conversely, dorsal luminescence patterns are rare within these deep-sea organisms. RESULTS: Here we report evidence that Etmopterus spinax, Etmopterus molleri and Etmopterus splendidus have dorsal luminescence patterns. These dorsal patterns consist of specific lines of luminous organs, called photophores, on the rostrum, dorsal area and at periphery of the spine. This dorsal light seems to be in contrast with the counterilluminating role of ventral photophores. However, skin photophores surrounding the defensive dorsal spines show a precise pattern supporting an aposematism function for this bioluminescence. Using in situ imaging, morphological and histological analysis, we reconstructed the dorsal light emission pattern on these species, with an emphasis on the photogenic skin associated with the spine. Analyses of video footage validated, for the first time, the defensive function of the dorsal spines. Finally, we did not find evidence that Etmopteridae possess venomous spine-associated glands, present in Squalidae and Heterondontidae, via MRI and CT scans. CONCLUSION: This work highlights for the first time a species-specific luminous dorsal pattern in three deep-sea lanternsharks. We suggest an aposematic use of luminescence to reveal the presence of the dorsal spines. Despite the absence of venom apparatus, the defensive use of spines is documented for the first time in situ by video recordings.

IFPA meeting 2018 workshop report I: Reproduction and placentation among ocean-living species; placental imaging; epigenetics and extracellular vesicles in pregnancy.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, four of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) viviparity in ocean-living species; 2) placental imaging; 3) epigenetics; and 4) extracellular vesicles in pregnancy.

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.

Heart Anatomy of Rhincodon typus: Three-Dimensional X-Ray Computed Tomography of Plastinated Specimens.

In this study, we examined the structure of the heart of the whale shark, Rhincodon typus, using a plastination technique and three-dimensional X-ray computer tomography (3DCT). Inspection of the atrium revealed a symmetric distribution of the pectinate muscles attached to the commissures of the sino-atrial valve, suggesting some functional advantages. The majority of the ventricular wall comprised spongiosa, and compacta accounted for only ~3% of the entire thickness. There were three major fiber orientations in the spongiosa: the fibers on the endocardial side formed trabeculae that were aligned with the blood flow tract, whereas those on the epicardial side formed a circular pattern around the flow tract. Transmural myofibers connected the inner and outer layers in the spongiosa, which may serve as an intraventricular conduction pathway. Plastination and 3DCT is a powerful combination that allowed for multifaceted visualization of the internal structure of rare heart specimens in a nondestructive manner. Anat Rec, 301:1801-1808, 2018. © 2018 Wiley Periodicals, Inc.

Morphology of a Hidden Tube: Resin Injection and CT Scanning Reveal the Three-dimensional Structure of the Spiracle in the Japanese Bullhead Shark Heterodontus japonicus (Chondrichthyes; Heterodontiformes; Heterodontidae).

The spiracle of elasmobranchs (sharks, skates, and rays) is a gill-slit-derived tube located behind the eye. Its inner structure was well studied in the late nineteenth to early twentieth century, but its entire morphology has rarely been characterized and is poorly understood. The present study shows the three-dimensional morphology of the spiracular tube for the first time, using resin injection and CT scanning, in the Japanese bullhead shark. The spiracular tube is characterized by the presence of two caeca (dorsal and ventral spiracular caeca) on the medial wall of the spiracular tube and the presence of a pseudobranch on the anterior wall. This study provides a basis for further studies on the morphological diversity, function, and evolution of spiracles in elasmobranch fishes. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Development of the Lunate-Shaped Caudal Fin in White Shark Embryos.

The lunate-shaped caudal fin in lamnid sharks is a morphological specialization for their thunniform mode of locomotion, but its developmental process during gestation has been poorly investigated. Observations of 21 embryonic specimens of the white shark (Carcharodon carcharias) revealed that their caudal fin morphology drastically changes from strongly heterocercal to lunate-shaped through ontogeny. This morphological change involves (1) rapid elongation of the ventral lobe, (2) increased upward curvature of the vertebra within the caudal fin, and (3) formation of keels at both lateral sides of the caudal fin base. These morphological changes are probably shared among the members of the family Lamnidae and are in contrast with the developmental process of the heterocercal tail in the lamniform Carcharias taurus, in which the caudal fin morphology is almost unchanged through the late gestation period. Anat Rec, 301:1068-1073, 2018. © 2018 Wiley Periodicals, Inc.

Live-bearing without placenta: Physical estimation indicates the high oxygen-supplying ability of white shark uterus to the embryo.

One of the mysteries of shark aplacental viviparity is the ability of the embryos to acquire oxygen from their mothers without a placental connection. It has been assumed that embryonic respiration in aplacental viviparous shark depends on oxygen from the uterine wall, although this hypothesis has not been confirmed quantitatively. Morphological observations of the uterine wall of white shark (Carcharodon carcharias) provided the first quantitative evidence to support the ability of the uterus to supply ample oxygen to the embryo of viviparous elasmobranchs. The uterine surface of the white shark is characterized by (1) uterine lamellae that develop perpendicular to the uterine wall, (2) uterine lamellae folded in an accordion-like fashion, and (3) numerous micro-ridges on the lamellar surface. These modifications result in increased uterine surface are to up to 56 folds compared to the uterus with a smooth surface. Histological observations revealed that the diffusion barrier of the uterine wall is approximately 12 µm. By using these values, the oxygen-diffusion capacity of 1 cm2 of the uterine wall of white shark was estimated to be 63.6 nmol·min-1·torr-1. This value is 250-400 times greater than that observed in other aplacental viviparous sharks (Squalus spp.) and is comparable with that of fish gills.

Dental ontogeny of a white shark embryo.

Unlike most viviparous vertebrates, lamniform sharks develop functional teeth during early gestation. This feature is considered to be related to their unique reproductive mode where the embryo grows to a large size via feeding on nutritive eggs in utero. However, the developmental process of embryonic teeth is largely uninvestigated. We conducted X-ray microcomputed tomography to observe the dentitions of early-, mid-, and full-term embryos of the white shark Carcharodon carcharias (Lamniformes, Lamnidae). These data reveal the ontogenetic change of embryonic dentition of the species for the first time. Dentition of the early-term embryos (∼45 cm precaudal length, PCL) is distinguished from adult dentition by 1) the presence of microscopic teeth in the distalmost region of the paratoquadrate, 2) a fang-like crown morphology, and 3) a lack of basal concavity of the tooth root. The "intermediate tooth" of early-term embryos is almost the same size as the adjacent teeth, suggesting that lamnoid-type heterodonty (lamnoid tooth pattern) has not yet been established. We also discovered that mid-term embryos (∼80 cm PCL) lack functional dentition. Previous studies have shown that the maternal supply of nutritive eggs in lamnoid sharks ceases during mid- to late-gestation. Thus, discontinuation of functional tooth development is likely associated with the completion of the oophagous (egg-eating) phase. Replacement teeth in mid-term embryos include both embryonic and adult-type teeth, suggesting that the embryo to adult transition in dental morphology occurs during this period. J. Morphol. 278:215-227, 2017. © 2016 Wiley Periodicals,Inc.

A Mighty Claw: Pinching Force of the Coconut Crab, the Largest Terrestrial Crustacean.

Crustaceans can exert a greater force using their claws than many animals can with other appendages. Furthermore, in decapods, the chela is a notable organ with multifunctional roles. The coconut crab, Birgus latro, is the largest terrestrial crustacean and has a remarkable ability to lift weights up to approximately 30 kg. However, the pinching force of this crab's chelae has not been previously investigated. In the present study, we measured the pinching force of the chelae in 29 wild coconut crabs (33-2,120 g in body weight). The maximum force ranged from 29.4 to 1,765.2 N, and showed a strong positive correlation with body mass. Based on the correlation between pinching force and body weight, the force potentially exerted by the largest crab (4 kg weight) reported in a previous study would be 3300 N, which greatly exceeds the pinching force of other crustaceans as well as the bite force of most terrestrial predators. The mighty claw is a terrestrial adaptation that is not only a weapon, which can be used to prevent predator attack and inhibit competitors, but is also a tool to hunt other terrestrial organisms with rigid exteriors, aiding in these organisms to be omnivores.