- Email: [email protected]
Stealth breathing of the angelshark
Zoology 130 (2018) 1–5
Contents lists available at ScienceDirect
Zoology journal homepage: www.elsevier.com/locate/zool
Stealth breathing of the angelshark Taketeru Tomita a b
a,b,⁎
b
, Minoru Toda , Kiyomi Murakumo
T b
Okinawa Churashima Research Center, Okinawa Churashima Foundation, 888, Motobu, Okinawa, 905-0206, Japan Okinawa Churaumi Aquarium, 424, Motobu, Okinawa, 905-0206, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Buccal pumping Crypsis Hyoid Oropharyngeal cavity Respiration Squatina japonica
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.
1. Introduction Generation of a respiratory water current over the gills is essential for effective gas exchange across the gill surface in fish. Previous studies have shown that the majority of fish, including benthic elasmobranchs (sharks and batoids), achieve this function by buccal pumping (e.g., Brainerd and Ferry-Graham, 2006). Buccal pumping is a mechanism that utilizes rhythmical volume changes of the oropharyngeal cavity to generate a respiratory water current. As the oropharyngeal cavity expands, water is drawn in through the mouth and/or spiracles and forced over the gills by oropharyngeal compression, which involves closure of the mouth/spiracles and expansion of the parabranchial cavity (Hughes, 1960, 1965; Ferry-Graham, 1999; Brainerd and FerryGraham, 2006). Hitherto, this mechanism has only been documented for a small number of cartilaginous species, such as Scyliorhinus canicula, Cephaloscyllium ventriosum, Raja clavata, and Leucoraja erinacea (Hughes, 1960, 1965; Ferry-Graham, 1999; Summers and FerryGraham, 2001). The present study aimed to investigate a newly observed respiratory behavior in the Japanese angelshark (Squatina japonica). The angelshark is a benthic specialist with a dorso-ventrally compressed, batoidlike body morphology (Compagno, 1984). One of the characteristics of its respiration system is its apparent lack of movement. A captive angelshark at Okinawa Churaumi Aquarium, Okinawa, Japan, has been observed to exhibit no respiratory motions that characterize buccal pumping (e.g., rhythmic movements of the mouth, spiracle valve, and
⁎
pharyngeal region) when resting on the bottom of the tank. This observation suggests that respiration in angelsharks does not employ buccal pumping. The ostensibly motionless respiration system of the angelshark was previously documented by Darbishire (1907), although technological limitations prevented an internal kinematical observation, thus prohibiting a detailed analysis on the breathing mechanism of the species. The purpose of the present study was firstly to describe the internal and external respiratory behavior of the angelshark, and secondly to discuss the ecological significance of this species. 2. Materials and methods 2.1. Kinematics Two captive Japanese angelsharks, specimen A and specimen B (51.5 cm and 96.0 cm total length (TL), respectively), were used for the kinematical observations. Both specimen A (male), and specimen B (female), were kept in a tank at Okinawa Churaumi Aquarium for more than a year before the experiment. Specimen A was transferred from the original tank to an acrylic aquarium (170 liters) one hour prior to the experiment. The sides and bottom of the aquarium were transparent, allowing for video monitoring of the fish’s oral and opercular motions using two digital video cameras, Panasonic HC-W870 M (Panasonic Co., Osaka, Japan) and Sony DCR-SR220 (Sony Co., Tokyo, Japan). Specimen B was transferred from the original tank to a portable
Corresponding author at: Okinawa Churaumi Aquarium, 424, Motobu, Okinawa, 905-0206, Japan. E-mail address: [email protected] (T. Tomita).
https://doi.org/10.1016/j.zool.2018.07.003 Received 1 March 2018; Received in revised form 26 June 2018; Accepted 4 July 2018 0944-2006/ © 2018 Elsevier GmbH. All rights reserved.
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Fig. 1. Gill flap and jaw kinematics of the angelshark during respiration. A. Right-side gill regions of specimen A, showing the posteriormost point of the third gill flap (point a), which was used for kinematical analysis. B. Left lateral view of the head of specimen A showing the anterior tip of the lower jaw (point b). C. Displacement of points a and b over time.
(Fig. 3A). Morphometric measurements were obtained using a hand caliper. For comparison, we also looked at the gills of five benthic/ benthopelagic shark species: Centrophorus moluccensis (TL = 74 cm), Cephaloscyllium umbratile (TL = 84 cm) and Orectolobus japonicus (TL = 102 cm), Proscyllium venustum (TL = 66 cm), Squalus cf. mitsukurii (TL = 55 cm). To obtain imagery of the angelshark’s skeletal anatomy, we used computed tomography (CT) data. CT setting and specimen information can be found in Tomita et al. (2012). Three-dimensional reconstructions were prepared from CT-slices (1.25 mm interval) using DICOM editing software, AZE VirtualPlace (AZE Ltd., Tokyo, Japan). The anatomical terminology follows that described by Shirai (1992).
container (500 liters). A transparent acrylic panel was set at a depth of 30 cm, upon which the fish was placed. A mirror was placed on the bottom of the container, which enabled observation of the ventral side of the fish. Video monitoring of the motion of the mouth, pharyngeal area, and gill flaps, was carried out using a digital video camera, Olympus Tough TG-4 (Olympus Imaging Co., Tokyo, Japan). In order to track the flow of water through the respiratory tract, a syringe was used to deliver water-diluted squid ink and cow milk adjacent to the mouth, spiracle, and gill slits of each specimen. During the experiments, the water temperature was maintained at 14 °C in keeping with that of the original tank. Following video recording, still images were captured from the footage for specimen A using the movie editing software, KMplayer 2.9.4.1.1435 (Jelsoft Enterprises Ltd., Pangbourne, UK). The capture rate was 29 and 29.5 frames per second for mouth and gill flap motions, respectively. From each still image, coordinates of the distal-most point of the third gill flap (point a in Fig. 1) and anterior tip of the lower jaw (point b in Fig. 1) were acquired using ImageJ (US National Institutes of Health, Bethesda, MD, USA). In addition to visual monitoring, sonographic experiments were carried out to observe internal movements in each fish during respiration. Each specimen was placed in a plastic tray with seawater and fitted with an ultrasonic transducer on the dorsal and ventral surfaces. We used ARIETTA Prologue (Hitachi-Aloka Medial Ltd., Tokyo, Japan) and FAZONE M (Fujifilm Co., Tokyo, Japan) sonographic diagnostic imaging systems for specimens A and B, respectively. Sonographic data were imported to ImageJ, and coordinates of the anterior-most point of the lower jaw (Meckel’s cartilage; point a in Fig. 2) and the anteriormost point of the hyoid apparatus (basihyal cartilage; point b in Fig. 2) were obtained. In sonography, the outline of internal structures is sometimes vague, and thus the landmark collection from these structures involves some uncertainty. Therefore, the precision of our sampling method was estimated by calculating the standard deviation for 30 repeated coordinate samples for points a and b from the single still image captured from the ultrasound.
3. Results 3.1. Kinematics External observation of breathing motions in specimens A and B showed that their mouths remained stationary and slightly open (Movie S1 in the supplementary online Appendix). Spiracles also remained open during respiration. Gill flaps actively moved and gill slits rhythmically opened and closed (Movies S2 and S3 in the supplementary online Appendix). One or two pulse waves were continuously transmitted from the proximal to the distal portion of the gill flaps (Fig. 4; Movie S3 in the supplementary online Appendix). The average respiratory frequencies ( ± SD) were 57.4 ( ± 10.5, based on 7 times the measurement) min-1 and 51.7 ( ± 4.1, based on 6 times the measurement) min-1 for specimens A and B, respectively. Flow visualization with water-diluted milk or squid ink showed that water was continuously taken in by both specimens through the mouth and spiracles and ejected through the gill slits. Upon expulsion, the water passed over the ventral side of the pectoral fin in a tail-ward direction and diffused into the surrounding water around the posterior margin of the pectoral fin. Ultrasound footage for each specimen showed that the jaws (palatoquadrate and Meckel’s cartilage) and hyoid apparatus (basihyal cartilage) were immobile during the experimental trials (Fig. 2; Movie S4 in the supplementary online Appendix), with jaw and hyoid cartilage coordinates remaining almost unchanged, barring minor fluctuations (Fig. 2D). These fluctuations were probably due to sampling error during landmark collection, because: 1) more than 95% of all points were between ± 1 pixels ( ± 0.15 mm), a range much smaller than that found in other species with buccal pumping (e.g., > 10 pixels in a 70 cm TL neonate tiger shark, Tomita, unpublished data); and 2) the standard deviation of the fluctuations (0.82 and 0.76 pixels, in the lower jaw and hyoid cartilage, respectively) does not significantly
2.2. Anatomy After the kinematical observations, specimens were euthanized with phenoxyethanol and placed in formalin for anatomical analysis. We calculated the relative gill flap length (r) using the equation: r = Lgf / Ls where Ls (length of the interbranchial septum) is defined as the linear distance between the gill-arch articulation and the distal margin of gill filaments; Lgf (length of gill flap) as the linear distance between the distal margin of gill filaments and the free margin of the gill slit 2
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Fig. 2. Hyoid and jaw kinematics of the angelshark. A. Computed tomography (CT) image of the angelshark in ventral view, showing the arrangement of the lower jaw (Meckel’s cartilage, red) and anterior-most point of the hyoid apparatus (basihyal, yellow). B. Mid-sagittal cross section (cut along the plain X-X’ in panel A) of the CT image of the angelshark, showing the arrangement of the Meckel’s cartilage (red) and basihyal cartilage (yellow) as in the sonogram. C. A still image from the sonogram. Point a marks the anterior-most point of the symphysis of the right and left Meckel’s cartilages; point b marks the anterior-most point of the mid-sagittal plain of the basihyal cartilage. D. Displacement of points a and b over time.
exceed the precision of our sampling method (0.61 pixels, p 0.05, Ftest). Gill flap motion was the only evidence of active breathing, during which the pulse wave was transmitted in a distal to proximal direction over the gill flaps (Movie S5 in the supplementary online Appendix).
0.54 in Cephaloscyllium umbratile; 0.46 in Orectolobus japonicus; 0.46 in Proscyllium venustum, and 0.50 in Squalus cf. mitsukurii).
3.2. Anatomy
The most striking feature of angelshark respiration is the absence of water-pumping in the oropharyngeal cavity. This is highly contrasted with the buccal pumping system exhibited by other shark species, in which oropharyngeal motion is among the main mechanisms generating respiratory water current over the gills (Hughes, 1960, 1965; Ferry-Graham, 1999; Summers and Ferry-Graham, 2001; Brainerd and Ferry-Graham, 2006). The absence of oropharyngeal pumping in the
4. Discussion
The angelshark is characterized by proximodistally long gill flaps (Fig. 3B). In the present study, the proportional length of the gill flap against the interbranchial septum (r) was 0.95 and 0.96, for specimens A and B, respectively. These values were nearly twice those of other shark species examined in this study (0.47 in Centrophorus moluccensis;
Fig. 3. Comparison of the relative gill-flap size between the angelshark and other sharks. A. Schematic representation of the single gill structure, showing the location of morphometric measurements. Abbreviation: Ls, length of interbranchial septum; Lgf, length of gill flap (see text for details). B. Posterior side of the first gill structure of the Japanese angelshark (left) and Centrophorus moluccensis (right). Scale bars = 1 cm.
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other benthic fishes, including all examined benthic sharks and batoids (e.g., Hughes, 1960; Hughes and Ballintijn, 1965; Tomita et al., 2013). It is still not known how the angelshark creates a respiratory water current without oropharyngeal motion. We suspect that the motion of the gill flaps plays a central role, because the gill flaps were the only structures that exhibited rhythmic motion in our sonographic experiment. The gill flap of the angelshark is larger than those of other species and exhibits continuous wave transmissions from the proximal to the distal portion of the flaps. These observations indicate that the gill flap of the angelshark is not the “passive valve” previously described by Hughes (1960) for that of the catshark, but probably functions as an active pump to expel the respiratory water through the gill slits. This hypothesis can be further tested by measuring pressure change in the oropharyngeal and branchial cavities, and the activity patterns of respiratory muscles; although the collection of these data were beyond the scope of this study. The discrete mode of respiration found in the angelshark, in which breathing motion is restricted to the gill flaps on the ventral side of the body, may have evolved as a means of avoiding detection by prey and predators. Hydrodynamic crypsis is potentially important for aquatic vertebrates considering that many aquatic animals use hydrodynamic stimuli to detect prey and predators (Hanke, 2014). For example, harbor seals (Phoca vitulina) use water disturbance caused by respiration to detect and localize benthic prey (Niesterok et al., 2017). In situ observation of the Pacific angelshark (S. californica) showed that these sharks usually bury themselves in the seafloor sediment and rapidly lunge at passing prey. Indeed, the crypsis exhibited by angelsharks is crucial for surviving in their relatively exposed habitat (Fouts and Nelson, 1999). Previous studies have suggested that crypsis in benthic elasmobranchs is generally achieved by: 1) hiding in substrates; 2) modification of its body shape (e.g., dorsoventrally compressed body); and 3) camouflage (Fouts and Nelson, 1999; Theiss et al., 2011; Garla et al., 2015). The present study shows that the respiratory mode of the angelshark may also facilitate visual and hydrodynamic crypsis, which previously has not been reported in elasmobranchs. Acknowledgements We thank Atsushi Kaneko, Hiroko Takaoka, Makio Yanagisawa, and other staff members at the Okinawa Churaumi Aquarium for their assistance with our experiments. We extend our gratitude to Kei Miyamoto (Okinawa Churashima Research Center) for photographing the shark gills. This work was supported by the admission fees for the Okinawa Churaumi Aquarium. Appendix A. Supplementary data Fig. 4. Pulse wave transmission through gill flaps of specimen A. A. Line drawing of the ventral view of the angelshark showing the location of the left gill flaps that were used for kinematic observation. B. Serial images (left) of gill flaps and their sketches (right), showing a pulse wave (black arrowhead) moved from the proximal to the distal portion of the gill flaps.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.zool.2018.07.003. References Brainerd, E.L., Ferry-Graham, L.A., 2006. Mechanics of respiratory pumps. In: Lauder, G.V., Shadwick, R.E. (Eds.), Biomechanics: A Volume of the Fish Physiology Series. Elsevier Science, San Diego, CA USA, pp. 1–29. Compagno, L.J.V., 1984. FAO Species Catalogue, vol. 4, Sharks of the World: An Annotated and Illustrated Catalogue of Shark Species Known to Date, part 2, Carcharhiniformes. FAO Fisheries Synopsis 125, 251–655. Darbishire, A.D., 1907. On the direction of the aqueous current in the spiracle of the dogfish; together with some observations on the respiratory mechanism in other elasmobranch fishes. J. Linn. Soc. Lond. Zool. 30, 86–94. Ferry-Graham, L.A., 1999. Mechanics of ventilation in swellsharks, Cephaloscyllium ventriosum (Scyliorhinidae). J. Exp. Biol. 202, 1501–1510. Fouts, W.R., Nelson, D.R., 1999. Prey capture by the Pacific angel shark, Squatina californica: visually mediated strikes and ambush-site characteristics. Copeia 1999, 304–312. Garla, R.C., Garrone-Neto, D., Gadig, O.B.F., 2015. Defensive strategies of neonate nurse sharks, Ginglymostoma cirratum, in an oceanic archipelago of the Western Central Atlantic. Acta Etholog. 18, 167–171. Hanke, W., 2014. Natural hydrodynamic stimuli. In: Bleckmann, H., Coombs, S.,
angelshark is supported by two lines of evidence: 1) absence of hyoid motion. In buccal pumping, expansion and compression of the oropharyngeal cavity is enabled by the depression and elevation of the hyoid apparatus. However, our sonographic data showed that the hyoid apparatus does not move during respiration (Fig. 2). 2) The spiracles and mouth stayed open during respiration. In buccal pumping, the spiracle and mouth are rhythmically opened and closed. It is widely accepted that this process is important to prevent water reversal and expulsion through these openings during oropharyngeal compression (e.g., Ferry-Graham, 1999). However, in the angelshark, no water reversal or expulsion through the spiracle or mouth was observed, even though these openings remained open. To the best of our knowledge, the absence of oropharyngeal movement has never been found in any 4
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Related Taxa. Hokkaido University Press, Sapporo. Summers, A.P., Ferry-Graham, L.A., 2001. Ventilatory modes and mechanics of the hedgehog skate (Leucoraja erinacea): testing the continuous flow model. J. Exp. Biol. 204, 1577–1587. Theiss, S.M., Collin, S.P., Hart, N.S., 2011. Morphology and distribution of the ampullary electroreceptors in wobbegong sharks: implications for feeding behavior. Mar. Biol. 158, 723–735. Tomita, T., Toda, M., Yamamoto, Y., Sato, K., Uchida, S., Nakaya, K., 2013. A novel pharyngeal expansion mechanism in the yellow-spotted fanray, Platyrhina tangi (Elasmobranchii: Batoidea), with special reference to the function of the fifth ceratobranchial cartilage in batoids. Zoomorphol 132, 317–324.
Mogdans, J. (Eds.), Flow Sensing in Air and Water. Springer, Heidelberg, pp. 3–29. Hughes, G.M., 1960. The mechanism of gill ventilation in the dogfish and skate. J. Exp. Biol. 37, 11–27. Hughes, G.M., 1965. Comparative Physiology of Vertebrate Respiration, vol. 2 Harvard University Press, Cambridge, MA. Hughes, G.M., Ballintijn, C.M., 1965. The muscular basis of the respiratory pumps in the dogfish (Scyliorhinus canicula L.). J. Exp. Biol. 57, 363–383. Niesterok, B., Krüger, Y., Wieskotten, S., Dehnhardt, G., Hanke, W., 2017. Hydrodynamic detection and localization of artificial flatfish breathing current by harbor seals (Phoca vitulina). J. Exp. Biol. 220, 174–185. Shirai, S., 1992. Squalean Phylogeny: A New Framework of “Squaloid” Sharks and
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Contents lists available at ScienceDirect
Zoology journal homepage: www.elsevier.com/locate/zool
Stealth breathing of the angelshark Taketeru Tomita a b
a,b,⁎
b
, Minoru Toda , Kiyomi Murakumo
T b
Okinawa Churashima Research Center, Okinawa Churashima Foundation, 888, Motobu, Okinawa, 905-0206, Japan Okinawa Churaumi Aquarium, 424, Motobu, Okinawa, 905-0206, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Buccal pumping Crypsis Hyoid Oropharyngeal cavity Respiration Squatina japonica
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.
1. Introduction Generation of a respiratory water current over the gills is essential for effective gas exchange across the gill surface in fish. Previous studies have shown that the majority of fish, including benthic elasmobranchs (sharks and batoids), achieve this function by buccal pumping (e.g., Brainerd and Ferry-Graham, 2006). Buccal pumping is a mechanism that utilizes rhythmical volume changes of the oropharyngeal cavity to generate a respiratory water current. As the oropharyngeal cavity expands, water is drawn in through the mouth and/or spiracles and forced over the gills by oropharyngeal compression, which involves closure of the mouth/spiracles and expansion of the parabranchial cavity (Hughes, 1960, 1965; Ferry-Graham, 1999; Brainerd and FerryGraham, 2006). Hitherto, this mechanism has only been documented for a small number of cartilaginous species, such as Scyliorhinus canicula, Cephaloscyllium ventriosum, Raja clavata, and Leucoraja erinacea (Hughes, 1960, 1965; Ferry-Graham, 1999; Summers and FerryGraham, 2001). The present study aimed to investigate a newly observed respiratory behavior in the Japanese angelshark (Squatina japonica). The angelshark is a benthic specialist with a dorso-ventrally compressed, batoidlike body morphology (Compagno, 1984). One of the characteristics of its respiration system is its apparent lack of movement. A captive angelshark at Okinawa Churaumi Aquarium, Okinawa, Japan, has been observed to exhibit no respiratory motions that characterize buccal pumping (e.g., rhythmic movements of the mouth, spiracle valve, and
⁎
pharyngeal region) when resting on the bottom of the tank. This observation suggests that respiration in angelsharks does not employ buccal pumping. The ostensibly motionless respiration system of the angelshark was previously documented by Darbishire (1907), although technological limitations prevented an internal kinematical observation, thus prohibiting a detailed analysis on the breathing mechanism of the species. The purpose of the present study was firstly to describe the internal and external respiratory behavior of the angelshark, and secondly to discuss the ecological significance of this species. 2. Materials and methods 2.1. Kinematics Two captive Japanese angelsharks, specimen A and specimen B (51.5 cm and 96.0 cm total length (TL), respectively), were used for the kinematical observations. Both specimen A (male), and specimen B (female), were kept in a tank at Okinawa Churaumi Aquarium for more than a year before the experiment. Specimen A was transferred from the original tank to an acrylic aquarium (170 liters) one hour prior to the experiment. The sides and bottom of the aquarium were transparent, allowing for video monitoring of the fish’s oral and opercular motions using two digital video cameras, Panasonic HC-W870 M (Panasonic Co., Osaka, Japan) and Sony DCR-SR220 (Sony Co., Tokyo, Japan). Specimen B was transferred from the original tank to a portable
Corresponding author at: Okinawa Churaumi Aquarium, 424, Motobu, Okinawa, 905-0206, Japan. E-mail address: [email protected] (T. Tomita).
https://doi.org/10.1016/j.zool.2018.07.003 Received 1 March 2018; Received in revised form 26 June 2018; Accepted 4 July 2018 0944-2006/ © 2018 Elsevier GmbH. All rights reserved.
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Fig. 1. Gill flap and jaw kinematics of the angelshark during respiration. A. Right-side gill regions of specimen A, showing the posteriormost point of the third gill flap (point a), which was used for kinematical analysis. B. Left lateral view of the head of specimen A showing the anterior tip of the lower jaw (point b). C. Displacement of points a and b over time.
(Fig. 3A). Morphometric measurements were obtained using a hand caliper. For comparison, we also looked at the gills of five benthic/ benthopelagic shark species: Centrophorus moluccensis (TL = 74 cm), Cephaloscyllium umbratile (TL = 84 cm) and Orectolobus japonicus (TL = 102 cm), Proscyllium venustum (TL = 66 cm), Squalus cf. mitsukurii (TL = 55 cm). To obtain imagery of the angelshark’s skeletal anatomy, we used computed tomography (CT) data. CT setting and specimen information can be found in Tomita et al. (2012). Three-dimensional reconstructions were prepared from CT-slices (1.25 mm interval) using DICOM editing software, AZE VirtualPlace (AZE Ltd., Tokyo, Japan). The anatomical terminology follows that described by Shirai (1992).
container (500 liters). A transparent acrylic panel was set at a depth of 30 cm, upon which the fish was placed. A mirror was placed on the bottom of the container, which enabled observation of the ventral side of the fish. Video monitoring of the motion of the mouth, pharyngeal area, and gill flaps, was carried out using a digital video camera, Olympus Tough TG-4 (Olympus Imaging Co., Tokyo, Japan). In order to track the flow of water through the respiratory tract, a syringe was used to deliver water-diluted squid ink and cow milk adjacent to the mouth, spiracle, and gill slits of each specimen. During the experiments, the water temperature was maintained at 14 °C in keeping with that of the original tank. Following video recording, still images were captured from the footage for specimen A using the movie editing software, KMplayer 2.9.4.1.1435 (Jelsoft Enterprises Ltd., Pangbourne, UK). The capture rate was 29 and 29.5 frames per second for mouth and gill flap motions, respectively. From each still image, coordinates of the distal-most point of the third gill flap (point a in Fig. 1) and anterior tip of the lower jaw (point b in Fig. 1) were acquired using ImageJ (US National Institutes of Health, Bethesda, MD, USA). In addition to visual monitoring, sonographic experiments were carried out to observe internal movements in each fish during respiration. Each specimen was placed in a plastic tray with seawater and fitted with an ultrasonic transducer on the dorsal and ventral surfaces. We used ARIETTA Prologue (Hitachi-Aloka Medial Ltd., Tokyo, Japan) and FAZONE M (Fujifilm Co., Tokyo, Japan) sonographic diagnostic imaging systems for specimens A and B, respectively. Sonographic data were imported to ImageJ, and coordinates of the anterior-most point of the lower jaw (Meckel’s cartilage; point a in Fig. 2) and the anteriormost point of the hyoid apparatus (basihyal cartilage; point b in Fig. 2) were obtained. In sonography, the outline of internal structures is sometimes vague, and thus the landmark collection from these structures involves some uncertainty. Therefore, the precision of our sampling method was estimated by calculating the standard deviation for 30 repeated coordinate samples for points a and b from the single still image captured from the ultrasound.
3. Results 3.1. Kinematics External observation of breathing motions in specimens A and B showed that their mouths remained stationary and slightly open (Movie S1 in the supplementary online Appendix). Spiracles also remained open during respiration. Gill flaps actively moved and gill slits rhythmically opened and closed (Movies S2 and S3 in the supplementary online Appendix). One or two pulse waves were continuously transmitted from the proximal to the distal portion of the gill flaps (Fig. 4; Movie S3 in the supplementary online Appendix). The average respiratory frequencies ( ± SD) were 57.4 ( ± 10.5, based on 7 times the measurement) min-1 and 51.7 ( ± 4.1, based on 6 times the measurement) min-1 for specimens A and B, respectively. Flow visualization with water-diluted milk or squid ink showed that water was continuously taken in by both specimens through the mouth and spiracles and ejected through the gill slits. Upon expulsion, the water passed over the ventral side of the pectoral fin in a tail-ward direction and diffused into the surrounding water around the posterior margin of the pectoral fin. Ultrasound footage for each specimen showed that the jaws (palatoquadrate and Meckel’s cartilage) and hyoid apparatus (basihyal cartilage) were immobile during the experimental trials (Fig. 2; Movie S4 in the supplementary online Appendix), with jaw and hyoid cartilage coordinates remaining almost unchanged, barring minor fluctuations (Fig. 2D). These fluctuations were probably due to sampling error during landmark collection, because: 1) more than 95% of all points were between ± 1 pixels ( ± 0.15 mm), a range much smaller than that found in other species with buccal pumping (e.g., > 10 pixels in a 70 cm TL neonate tiger shark, Tomita, unpublished data); and 2) the standard deviation of the fluctuations (0.82 and 0.76 pixels, in the lower jaw and hyoid cartilage, respectively) does not significantly
2.2. Anatomy After the kinematical observations, specimens were euthanized with phenoxyethanol and placed in formalin for anatomical analysis. We calculated the relative gill flap length (r) using the equation: r = Lgf / Ls where Ls (length of the interbranchial septum) is defined as the linear distance between the gill-arch articulation and the distal margin of gill filaments; Lgf (length of gill flap) as the linear distance between the distal margin of gill filaments and the free margin of the gill slit 2
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Fig. 2. Hyoid and jaw kinematics of the angelshark. A. Computed tomography (CT) image of the angelshark in ventral view, showing the arrangement of the lower jaw (Meckel’s cartilage, red) and anterior-most point of the hyoid apparatus (basihyal, yellow). B. Mid-sagittal cross section (cut along the plain X-X’ in panel A) of the CT image of the angelshark, showing the arrangement of the Meckel’s cartilage (red) and basihyal cartilage (yellow) as in the sonogram. C. A still image from the sonogram. Point a marks the anterior-most point of the symphysis of the right and left Meckel’s cartilages; point b marks the anterior-most point of the mid-sagittal plain of the basihyal cartilage. D. Displacement of points a and b over time.
exceed the precision of our sampling method (0.61 pixels, p 0.05, Ftest). Gill flap motion was the only evidence of active breathing, during which the pulse wave was transmitted in a distal to proximal direction over the gill flaps (Movie S5 in the supplementary online Appendix).
0.54 in Cephaloscyllium umbratile; 0.46 in Orectolobus japonicus; 0.46 in Proscyllium venustum, and 0.50 in Squalus cf. mitsukurii).
3.2. Anatomy
The most striking feature of angelshark respiration is the absence of water-pumping in the oropharyngeal cavity. This is highly contrasted with the buccal pumping system exhibited by other shark species, in which oropharyngeal motion is among the main mechanisms generating respiratory water current over the gills (Hughes, 1960, 1965; Ferry-Graham, 1999; Summers and Ferry-Graham, 2001; Brainerd and Ferry-Graham, 2006). The absence of oropharyngeal pumping in the
4. Discussion
The angelshark is characterized by proximodistally long gill flaps (Fig. 3B). In the present study, the proportional length of the gill flap against the interbranchial septum (r) was 0.95 and 0.96, for specimens A and B, respectively. These values were nearly twice those of other shark species examined in this study (0.47 in Centrophorus moluccensis;
Fig. 3. Comparison of the relative gill-flap size between the angelshark and other sharks. A. Schematic representation of the single gill structure, showing the location of morphometric measurements. Abbreviation: Ls, length of interbranchial septum; Lgf, length of gill flap (see text for details). B. Posterior side of the first gill structure of the Japanese angelshark (left) and Centrophorus moluccensis (right). Scale bars = 1 cm.
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other benthic fishes, including all examined benthic sharks and batoids (e.g., Hughes, 1960; Hughes and Ballintijn, 1965; Tomita et al., 2013). It is still not known how the angelshark creates a respiratory water current without oropharyngeal motion. We suspect that the motion of the gill flaps plays a central role, because the gill flaps were the only structures that exhibited rhythmic motion in our sonographic experiment. The gill flap of the angelshark is larger than those of other species and exhibits continuous wave transmissions from the proximal to the distal portion of the flaps. These observations indicate that the gill flap of the angelshark is not the “passive valve” previously described by Hughes (1960) for that of the catshark, but probably functions as an active pump to expel the respiratory water through the gill slits. This hypothesis can be further tested by measuring pressure change in the oropharyngeal and branchial cavities, and the activity patterns of respiratory muscles; although the collection of these data were beyond the scope of this study. The discrete mode of respiration found in the angelshark, in which breathing motion is restricted to the gill flaps on the ventral side of the body, may have evolved as a means of avoiding detection by prey and predators. Hydrodynamic crypsis is potentially important for aquatic vertebrates considering that many aquatic animals use hydrodynamic stimuli to detect prey and predators (Hanke, 2014). For example, harbor seals (Phoca vitulina) use water disturbance caused by respiration to detect and localize benthic prey (Niesterok et al., 2017). In situ observation of the Pacific angelshark (S. californica) showed that these sharks usually bury themselves in the seafloor sediment and rapidly lunge at passing prey. Indeed, the crypsis exhibited by angelsharks is crucial for surviving in their relatively exposed habitat (Fouts and Nelson, 1999). Previous studies have suggested that crypsis in benthic elasmobranchs is generally achieved by: 1) hiding in substrates; 2) modification of its body shape (e.g., dorsoventrally compressed body); and 3) camouflage (Fouts and Nelson, 1999; Theiss et al., 2011; Garla et al., 2015). The present study shows that the respiratory mode of the angelshark may also facilitate visual and hydrodynamic crypsis, which previously has not been reported in elasmobranchs. Acknowledgements We thank Atsushi Kaneko, Hiroko Takaoka, Makio Yanagisawa, and other staff members at the Okinawa Churaumi Aquarium for their assistance with our experiments. We extend our gratitude to Kei Miyamoto (Okinawa Churashima Research Center) for photographing the shark gills. This work was supported by the admission fees for the Okinawa Churaumi Aquarium. Appendix A. Supplementary data Fig. 4. Pulse wave transmission through gill flaps of specimen A. A. Line drawing of the ventral view of the angelshark showing the location of the left gill flaps that were used for kinematic observation. B. Serial images (left) of gill flaps and their sketches (right), showing a pulse wave (black arrowhead) moved from the proximal to the distal portion of the gill flaps.
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.zool.2018.07.003. References Brainerd, E.L., Ferry-Graham, L.A., 2006. Mechanics of respiratory pumps. In: Lauder, G.V., Shadwick, R.E. (Eds.), Biomechanics: A Volume of the Fish Physiology Series. Elsevier Science, San Diego, CA USA, pp. 1–29. Compagno, L.J.V., 1984. FAO Species Catalogue, vol. 4, Sharks of the World: An Annotated and Illustrated Catalogue of Shark Species Known to Date, part 2, Carcharhiniformes. FAO Fisheries Synopsis 125, 251–655. Darbishire, A.D., 1907. On the direction of the aqueous current in the spiracle of the dogfish; together with some observations on the respiratory mechanism in other elasmobranch fishes. J. Linn. Soc. Lond. Zool. 30, 86–94. Ferry-Graham, L.A., 1999. Mechanics of ventilation in swellsharks, Cephaloscyllium ventriosum (Scyliorhinidae). J. Exp. Biol. 202, 1501–1510. Fouts, W.R., Nelson, D.R., 1999. Prey capture by the Pacific angel shark, Squatina californica: visually mediated strikes and ambush-site characteristics. Copeia 1999, 304–312. Garla, R.C., Garrone-Neto, D., Gadig, O.B.F., 2015. Defensive strategies of neonate nurse sharks, Ginglymostoma cirratum, in an oceanic archipelago of the Western Central Atlantic. Acta Etholog. 18, 167–171. Hanke, W., 2014. Natural hydrodynamic stimuli. In: Bleckmann, H., Coombs, S.,
angelshark is supported by two lines of evidence: 1) absence of hyoid motion. In buccal pumping, expansion and compression of the oropharyngeal cavity is enabled by the depression and elevation of the hyoid apparatus. However, our sonographic data showed that the hyoid apparatus does not move during respiration (Fig. 2). 2) The spiracles and mouth stayed open during respiration. In buccal pumping, the spiracle and mouth are rhythmically opened and closed. It is widely accepted that this process is important to prevent water reversal and expulsion through these openings during oropharyngeal compression (e.g., Ferry-Graham, 1999). However, in the angelshark, no water reversal or expulsion through the spiracle or mouth was observed, even though these openings remained open. To the best of our knowledge, the absence of oropharyngeal movement has never been found in any 4
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Related Taxa. Hokkaido University Press, Sapporo. Summers, A.P., Ferry-Graham, L.A., 2001. Ventilatory modes and mechanics of the hedgehog skate (Leucoraja erinacea): testing the continuous flow model. J. Exp. Biol. 204, 1577–1587. Theiss, S.M., Collin, S.P., Hart, N.S., 2011. Morphology and distribution of the ampullary electroreceptors in wobbegong sharks: implications for feeding behavior. Mar. Biol. 158, 723–735. Tomita, T., Toda, M., Yamamoto, Y., Sato, K., Uchida, S., Nakaya, K., 2013. A novel pharyngeal expansion mechanism in the yellow-spotted fanray, Platyrhina tangi (Elasmobranchii: Batoidea), with special reference to the function of the fifth ceratobranchial cartilage in batoids. Zoomorphol 132, 317–324.
Mogdans, J. (Eds.), Flow Sensing in Air and Water. Springer, Heidelberg, pp. 3–29. Hughes, G.M., 1960. The mechanism of gill ventilation in the dogfish and skate. J. Exp. Biol. 37, 11–27. Hughes, G.M., 1965. Comparative Physiology of Vertebrate Respiration, vol. 2 Harvard University Press, Cambridge, MA. Hughes, G.M., Ballintijn, C.M., 1965. The muscular basis of the respiratory pumps in the dogfish (Scyliorhinus canicula L.). J. Exp. Biol. 57, 363–383. Niesterok, B., Krüger, Y., Wieskotten, S., Dehnhardt, G., Hanke, W., 2017. Hydrodynamic detection and localization of artificial flatfish breathing current by harbor seals (Phoca vitulina). J. Exp. Biol. 220, 174–185. Shirai, S., 1992. Squalean Phylogeny: A New Framework of “Squaloid” Sharks and
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