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.

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.

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.

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.

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.

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.

Aerobic Dechlorination of Dichloromethane Using Biostimulation Agent BD-C in Continuous and Batch Cultures of Xanthobacter autotrophicus GJ10.

It is important to construct microbiological treatment systems for organic solvent-contaminated water. We developed a continuous culture supplemented with a biostimulation agent named BD-C, which is formulated from canola oil, and Xanthobacter autotrophicus strain GJ10 for an aerobic dichloromethane (DCM)-dechlorinating microorganism. The continuous culture was a chemostat constructed using a 1 L screw-capped bottle containing artificial wastewater medium with 2.0 mM DCM and 1.0% (v/v) BD-C. The expression of genes for DCM metabolism in the dechlorinating aerobe was monitored and analyzed by reverse transcription-quantitative PCR. Strain GJ10 was able to dechlorinate approximately 74% of the DCM in medium supplemented with BD-C during 12 days of incubation. The DCM dechlorination rate was calculated to be 0.11 mM/day. The ΔΔCT method showed that expression of haloalkane dehalogenase increased 5.4 times in the presence of BD-C. Based on batch culture growth tests conducted with mineral salt medium containing three DCM concentrations (0.07, 0.20, 0.43 and 0.65 mM) with BD-C, the apparent maximum specific consumption rate (νmax) and the saturation constant (Ks) determined for DCM degradation in this test were 19.0 nmol/h/CFU and 0.44 mM, respectively. In conclusion, BD-C enhanced the aerobic degradation of DCM by strain GJ10.

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.

How great white sharks nourish their embryos to a large size: evidence of lipid histotrophy in lamnoid shark reproduction.

The great white shark (Carcharodon carcharias) exhibits viviparous and oophagous reproduction. A 4950 mm total length (TL) gravid female accidentally caught by fishermen in the Okinawa Prefecture, Southern Japan carried six embryos (543-624 mm TL, three in each uterus). Both uteri contained copious amounts of yellowish viscous uterine fluid (over 79.2 litres in the left uterus), nutrient eggs and broken egg cases. The embryos had yolk stomachs that had ruptured, the mean volume of which was approximately 197.9 ml. Embryos had about 20 rows of potentially functional teeth in the upper and lower jaws. Periodic acid Schiff (PAS)-positive substances were observed on the surface and in the cytoplasm of the epithelial cells, and large, secretory, OsO4-oxidized lipid droplets of various sizes were distributed on the surface of the villous string epithelium on the uterine wall. Histological examination of the uterine wall showed it to consist of villi, similar to the trophonemata of Dasyatidae rays, suggesting that the large amount of fluid found in the uterus of the white shark was likely required for embryo nutrition. We conclude that: (1) the lipid-rich fluid is secreted from the uterine epithelium only in early gestation before the onset of oophagy, (2) the embryos probably use the abundant uterine fluid and encased nutrient eggs for nutrition at this stage of their development, and (3) the uterine fluid is the major source of embryonic nutrition before oophagy onset. This is the first record of the lipid histotrophy of reproduction among all shark species.

Ultrasound and physical models shed light on the respiratory system of embryonic dogfishes.

Embryos of live-bearing elasmobranchs (sharks, skates, and rays) must acquire oxygen in the uterus for several months to more than a year, but the mechanisms of delivery and uptake are still largely unknown. Diagnostic sonography performed on a captive Japanese dogfish (Squalus japonicus) showed that a late-stage embryo used buccal movement to pump uterine fluid, suggesting that the embryo acquires oxygen from uterine fluid via gill ventilation. It has been assumed that embryonic respiration in aplacental sharks depends on oxygen supplied by the uterine wall. To test this hypothesis, the rate of oxygen diffusion was estimated by applying a physical model to the uterine wall of two dogfish species (Squalus cf. mitsukurii and Squalus cubensis). The model calculations indicate that the supply of oxygen via diffusion through the uterine villi contributes less than 15-30% of the total oxygen demand of late-stage embryos. Some previous authors have suggested that pregnant dogfish intermittently exchange uterine fluid with external seawater during late gestation. Thus, late-stage embryos may acquire oxygen primarily from uterine seawater introduced from the external environment.

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.

Onset of buccal pumping in catshark embryos: how breathing develops in the egg capsule.

Respiration in fishes involves buccal pumping, which is characterized by the generation of nearly continuous water flow over the gills because of the rhythmic expansion/compression of the pharyngeal cavity. This mechanism is achieved by the functions of the vascular, skeletal, and muscular systems. However, the process by which the embryo establishes the mechanism remains a mystery. Morphological and kinematical observations on captive cloudy catsharks, Scyliorhinus torazame, have suggested that the embryo starts buccal pumping just before the respiratory slits open on the egg capsule. During the pre-opening period, the embryo acquires oxygen mainly via the external gill filaments. After slit opening, respiration of the embryo involves buccal pumping to pass water over the "internal gills." The onset of buccal pumping accompanies four morphological changes: (1) regression of the external gill filaments, (2) development of blood vessels within the "internal gills," (3) completion of the development of hyoid skeletal and muscular elements, and (4) development of the oral valve. A previous study showed that buccal pumping allows the embryo to actively regulate oxygen intake by changing the pumping frequency. Thus, establishment of buccal pumping in the egg capsule is probably important for embryo survival in the unstable oxygen environment of the egg capsule after slit opening.

Live-bearing manta ray: how the embryo acquires oxygen without placenta and umbilical cord.

We conducted an ultrasonographic experiment on a pregnant manta ray, Manta alfredi (Chondrichthyes, Batoidea). This study showed how the embryo of the live-bearing elasmobranchs respires in the body of the female. In the embryonic stage, the manta ray embryo takes in uterine fluid by buccal-pumping. After birth, the manta ray shifts its respiratory mode from buccal-pumping to ram-ventilation. The rapid reduction of the spiracle size in the young manta ray may reflect this shift of respiratory mode. Unlike mammals or some carcharhinid sharks that acquire oxygen through a placenta and umbilical cord, the manta ray embryo does not have a direct connection with the mother. Thus, the manta ray embryo obtains oxygen by buccal-pumping of the uterine fluid, in the same way that the embryos of egg-laying species obtain oxygen from the water in the egg case. This finding extends our understanding of the diversity of embryonic respiratory systems in live-bearing vertebrates.

Rapid reductive dechlorination of trichloroethene in contaminated ground water using biostimulation agent, BD-1, formulated from canola oil.

A case study of the bioremediation of groundwater contaminated with trichloroethene (TCE) was conducted using the biostimulation agent, BD-1. TCE levels were monitored by gas chromatography-mass spectroscopy. Total organic carbon (TOC) and volatile fatty acids (VFAs) were analyzed to investigate the environmental fate of BD-1. The effects of BD-1 on microbial activity were investigated using 16S rRNA gene-based quantitative polymerase chain reaction (qPCR) analysis. The biodegradation of BD-1 was accompanied by a reduction in TCE, and the initially high TOC levels decreased rapidly as BD-1 was transformed into VFAs. qPCR analysis showed that the genus Dehalobacter became progressively dominant through the experiment. These results suggested that BD-1 might dechlorinate TCE by activating dechlorinating bacteria.

Detailed investigations of polymer/metal multilayer matching layer and backing absorber structures for wideband ultrasonic transducers.

Detailed investigations of multilayer front and back matching layers and a novel backing absorber have been conducted, the detailed theory for which was presented in a previous paper. To design useful structures using the simple proposed equations, the material parameters of the constituent layers must be identified. Therefore, polyimide (for the matching layer) and adhesive-backed copper tape (for the absorber) were characterized by bonding them to polyvinylidene fluoride-trifluoroethylene P(VDF-TrFE) copolymer ultrasonic transducers and then applying a parameter-fitting algorithm to the resulting impedance data. A double matching layer was designed using an 11-µm PVDF (inner) and 23-µm copper (outer) multilayer construction in the first matching section followed by a 75-µm polyimide layer as a typical quarter-wavelength material in the second (outermost) matching section. This structure was bonded to 330-µm PZT with air backing and the reflection waveform from a short pulse was captured. The FFT frequency response showed a 3.1-MHz bandwidth centered at 6.4 MHz, which agreed with the Mason's model analysis. The use of multiple layers of copper tape as a backing absorber was also investigated. At 3 MHz, the measured impedance was 4 MRayl, attenuation was 220 dB/cm, and velocity was 890 m/s, which agreed with the design theory. The 4-MRayl copper-tape structure was bonded to a back matching structure made from one layer of polyimide and one layer of brass (multilayer matching), and the effectiveness of the backing absorber made of 10 layers of copper tape on a 3-MHz transducer was confirmed.

Novel multi-layer polymer-metal structures for use in ultrasonic transducer impedance matching and backing absorber applications.

This paper presents a novel design principle for designing multilayer polymer-metal structures which are well suited for front surface impedance conversion (matching) and for back surface acoustic absorption. It is shown that a polymer layer with an outer metal layer, when loaded by a low impedance propagation medium, acts as an efficient impedance converter. The resulting impedance seen at the inner polymer surface is increased and the structure provides the same performance as a traditional quarter-wavelength matching layer. Experimental evidence is also shown for a double-matching scheme for a lead zirconate titanate (PZT) transducer using an inner polymer-metal multilayer and an outer polymer quarterwavelength layer, resulting in a 55% bandwidth at 2.6 MHz with air backing. Also, it is theoretically shown that multiple layers of a lossy polymer adhesive-metal structure produce low propagation velocity and high absorption. Experimental proof of this ultrasonic multilayer backing absorber is provided. Design theories based on both a simplified mass and spring model and a rigorous one-dimensional wave model have been developed and show fair agreement.

New symmetric reflector ultrasonic transducers (SRUT).

This paper proposes a new structure composed of reflector plates mounted in front and in back of the transducer at an angle of 45 degrees, so that acoustic waves from front and back of the transducer are combined to form a single main beam. This principle is applicable to both transmitters and receivers. Because the propagation path lengths of the 2 beams are identical, they constructively add at all frequencies. Theoretical investigations revealed that far field directivity becomes sharper and acoustic pressure output is doubled. Experiments using an air ultrasonic transducer constructed of 50-kHz polyvinylidene fluoride corrugated film has shown doubled acoustic pressure in the far field, and the predicted sharper directivity has been observed. The reflector structure has been also applied to a 2.6-MHz lead zirconate titanate transducer in water with matching layers at both surfaces which has shown almost doubled bandwidth compared with an air backing structure, and doubled output power in continuous drive. Also pulse echo experiments have shown the effectiveness of this scheme.

Narrowband impedance matching layer for high efficiency thickness mode ultrasonic transducers.

A new matching layer design concept has been proposed for narrowband continuous wave (CW) devices. Analysis has shown that the mechanical impedance of a resonant-type transducer in thickness mode CW operation does not equal its acoustic impedance rhoVs but roughly equals rhoVs/Q, where p is density, Vs is acoustic velocity, and Q is the mechanical quality factor. The value of rhoVs/Q is much lower than the acoustic impedance of water for any transducer material, including lead zirconium titanate (PZT), single crystals, or polyvinylidene fluoride (PVDF). With this new approach, the impedance of the matching layer must also be between water and pVs/Q, but there are few such practical low impedance materials. To realize equivalent low impedance structure, a novel double layer design is presented: a relatively low impedance material (such as polyethylene or polyurethane) on the inside and a relatively high impedance material (such as polyester or metal) on the outside. A high power CW transducer structure was designed and fabricated with PVDF-TrFE (polyvinylidene fluoride trifluoroethylene) to operate at 1.4 MHz. The basic quarter wavelength resonator structure is 0.7-mm alumina/0.2-mm piezo-polymer/0.25-mm polyester, and the matching section is 0.2-mm polyurethane and 0.25-mm polyester. A maximum power output of 6 to 9 W/cm2 with conversion efficiency of 30 to 35% was observed. For the transducer without matching section, the observed power was 3 to 4 W/cm2. Mason's model analyses (1) predict that the traditional matching layer is for broadband purposes and reduces output power both for PZT and PVDF-TrFE (2); this new matching scheme can be applied to PZT high power transducer. This high efficiency technique has application in various CW systems, such as Doppler sensors, interferometry, phase-sensitive imaging, or high energy focused beam systems.