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J-STAGE Advance Publication: August 12, 2020 Journal of Oral Science

Original article The teeth and dentition of the filefish (Stephanolepis cirrhifer) revisited tomographically Hirofumi Kanazawa1,2), Maki Yuguchi1,2,3), Yosuke Yamazaki1,2,3), and Keitaro Isokawa1,2,3)

1) Division of Oral Structural and Functional Biology, Nihon University Graduate School of Dentistry, Tokyo, Japan 2) Department of Anatomy, Nihon University School of Dentistry, Tokyo, Japan 3) Division of Functional Morphology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan (Received October 31, 2019; Accepted November 26, 2019)

Abstract: The upper and lower tooth-bearing jaws of the filefish (Stepha- Teeth of Non-Mammalian Vertebrates, Elsevier, 2017). nolepis cirrhifer) were scanned using a micro-CT system in order to With regard to filefish dentition in Japan, an early morphologic and address the existing gaps between the traditional pictures of the morphol- histologic study of Monocanthus cirrhifer and Cantherines modestus ogy and histology. 2D tomograms, reconstructed 3D models and virtual (synonyms Stephanolepis cirrhifer and Thamnaconus modestus, respec- dissection were employed to examine and evaluate the in situ geometry of tively) was carried out by Sohiti Isokawa (Isokawa, Zool Mag 64, 194-197, tooth implantation and the mode of tooth attachment both separately and 1955). Phylogenetic interrelationships in the balistoids were examined collectively. No distinct sockets comparable to those in mammals were extensively by Matsuura [4], based on many anatomical characteristics evident, but shallow depressions were observed in the premaxillary and including the tooth-bearing jaws, which were the premaxillary and the the dentary. The opening of the tooth pulp cavity was not simply oriented dentary. He showed that the balistids and monacanthids were clearly towards the apparent tooth base in a direction opposite to the tooth apex. separable by the number of teeth, and that a larger or smaller number The opening was distorted basoposteriorly or basoanteriorly depending of teeth in the balistoids was likely to represent a more primitive or an on the position of the tooth, and the edge of the pulp cavity opening was advanced systematic position, respectively; i.e. balistids for the former and barely ankylosed; i.e. the sites of pleurodont ankylosis along the basopos- monacanthids for the latter. A study of the upper jaw of S. cirrhifer at the terior or basoanterior edge of the opening appeared to closely match the ultrastructural level by Uehara & Miyoshi [5] made a significant contribu- contour of the shallow depression in the bone. These 3D findings appear to tion to histological knowledge of the tooth attachment. They demonstrated be very informative when considering the phylogeny of tooth attachment, a fibrous attachment of the tooth to bone, but also stated: “The teeth appear suggesting that micro-CT would be a useful modality concurrent with or in to attach directly to the bone at the middle region of the labial and lingual advance of histological investigations. components of the teeth”. These descriptions indicate that, despite accu- mulating observations, uncertainties still remain regarding the morphology Keywords; alveolar sockets, ankylosis, fish teeth, 3D analysis, tooth and histology of the tooth-bone interface in filefish, partly arising from attachment limitations in the morphological and histological methods employed, and also the historical, or conventional, definitions and usage of terminology such as gomphosis, periodontium, alveolar socket, fibrous attachment, etc. Introduction Therefore, it appears important to employ a new methodological approach for addressing the existing gaps in the current traditional picture The thread-sail filefish, Stephanolepis cirrhifer (Japanese: “kawahagi”), is of the morphology and histology. Fortunately, this can now be undertaken a well-known food fish widely served as “sashimi” in Japan. The filefish very efficiently and reliably using the recently introduced modality of family (Monacanthidae) includes 102 species within 27 genera and has a micro-CT. The three-dimensional visualization made possible by this tech- close taxonomic relationship with the triggerfish family (Balistidae) [1]. nique also allows consideration of the in situ geometry of tooth implantation These two sister families are therefore placed together within the suborder and the mode of tooth attachment both separately and collectively, which Balistoidei [2] in the Tetraodontiformes (Order#85; Nelson et al., Fishes of is significant because these two aspects of the tooth are not necessarily the World 5th ed., John Wiley & Sons, 2016). dependent on each other for supporting the tooth [6]. The present study In the mid-19th century, Sir Richard Owen published “Odontography” utilized a micro-CT system to examine the jaw teeth and dentition (exclud- (Owen, A Treatise on the Comparative Anatomy of the Teeth, Hippolyte ing pharyngeal teeth) of S. cirrhifer in order to compare the findings of Bailliere Publisher, 1840-1845), and described in detail the teeth and tomography with those of previous morphological and histological studies, dentition of the grey triggerfish (Balistes forcipatus; synonym Balistes focusing also on the development (and successional replacement) of teeth capriscus). He compared his findings with those of the queen triggerfish and the occlusal relationship of the upper and lower teeth. (Balistes vetula), which had been reported in a personal communication by the Swede, Anders Retzius, translated into German, and filed in Mül- Materials and Methods lers Archiv (Retzius, Arch Anat Physiol wiss Med, 486-566, 1837). Both Retzius and Owen made precise and valuable observations of the number, Specimens morphology and certain histologic details of the teeth and dentition of trig- Four thread-sail filefish (Stephanolepis cirrhifer), all approximately 24 cm gerfishes. Among the observations of B. capriscus, the tooth attachment in total length, were fixed with 10% buffered formalin and labeled serially was notably interpreted as a double gomphosis. In fact, after examining as Sc#1, 2, 3 and 4 for this study. These fish, from Goto, Nagasaki, were Balistes bursa (synonym Sufflamen bursa) and two other balistids, Soule obtained through a local distributor via the Metropolitan Central Whole- [3] reported that connective tissue comparable to the mammalian peri- sale Market (Tsukiji, Tokyo) with the generous help of Professor Noriaki odontal ligament connected the teeth and bone in shallow alveolar sockets. Koshikawa (Nihon University School of Dentistry; NUSD). Processing of However, the presence of a periodontal ligament in triggerfish or other the four specimens was carried out in accordance with the guidelines of actinopterygians has not been commonly accepted (Berkovitz et al., The the Animal Experimentation Committee of NUSD, which comply with the National Welfare and Management of Animals Act. Some of the histological specimens of the teeth of S. cirrhifer, which Correspondence to: Dr. Keitaro Isokawa, Department of Anatomy, Nihon University School of had been examined in the early 1950s by Sohiti Isokawa, were still stored at Dentistry, 1-8-13 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan Fax: +81-3-3219-8319 E-mail: [email protected] the Department of Anatomy of NUSD. These specimens were re-examined Color figures can be viewed in the online issue at J-STAGE. using a modern microscope, and several images from those sections are doi.org/10.2334/josnusd.19-0457 incorporated into figures and cited in the Discussion. DN/JST.JSTAGE/josnusd/19-0457 2

Fig. 1 The tooth-bearing jaws of specimen Sc#1 with (A-C) and without (D-F) mucosal integu- Fig. 2 The upper jaw of Sc#1 viewed in 3D-VR from different directions (A-C) and in horizontal ment. The frontal (A-E) and lateral (F) views of the 3D-VR of jaws are shown with the jaws open tomograms of the upper dental arch in MPR (D-K). Arrowheads in A and B indicate a fusion line (A-D) and closed (E, F); upper and lower dentitions are better demonstrated by tilting the VR image between the maxillary and the tooth-bearing premaxillary. Tomograms are shown intermittently slightly upward and downward in B and C, respectively. An outline of the integument surface is from the apices of o1 (D) to the deeper area where the bone between the outer and inner row of superimposed in yellow in F. For symbols of teeth and their positions, see “Materials and Methods” teeth (H-J) and some of the successional teeth (J, K) are observable. All images in D-K are at the and Fig. 6. Lingual cingulum (cg) is shown in each of teeth b1 and b2 in C. Asterisks indicate same magnification. successional teeth. All images are at the same magnification.

CT scanning and observations Results The upper and lower tooth-bearing jaws were scanned using a micro-CT system (R_mCT, Rigaku, Tokyo, Japan) with an X-ray exposure of 2 min, Ten teeth in total were observed in the upper jaw: 3 each (o1-3) in the right 90 kV, 100 µA, at magnifications of ×2, ×4, and ×6.7. Volume data were and left sides in the outer row of dentition, and 2 each (i1-2) in the right resliced with I-View software (J. Morita, Kyoto, Japan) and exported to and left sides in the inner row. In the lower jaw, 6 teeth – 3 each (b1-3) on Dicom viewers; the final isotropic voxel sizes were 100, 50 and 30 µm the right and left – were aligned in a single row (Fig. 1). at the three respective magnifications. Most of the observations were The labial surface of o1-3 and b1-3 was basically a smooth rectangle performed using multiplanar reconstruction (MPR) and 3-dimensional with an obtuse apex (Fig. 1D-F). There was evidence of apparent wear at volume rendering (3D-VR) provided in a RadiAnt DICOM Viewer (ver- the apex of some mesial teeth. Teeth in a row were closely aligned with sion 4.6.9; Medixant, Poznan, Poland). Another DICOM viewer, Mango each other, and the mesial pair (o1 or b1) was larger than the others except (version 4.1; Research Imaging Institute, University of Texas Health Sci- for o3, which appeared wider than o1 when examined without mucosa ence Center, San Antonio, TX, USA), was used to generate 3D-surface VR (Figs. 1F, 2A). images, which were useful for examining the occlusal relationship of the The lingual surface of i1-2 was exposed more widely over the oral upper and lower teeth. mucosa (Fig. 1B). The surface consisted of a rectangular part with a The “Scalpel” tool in the RadiAnt was used preferentially to extract a rounded edge at the base and a triangular part with an apex bent slightly tooth from the 3D-VR image, or to remove tissues other than teeth to a con- towards the labial side (Fig. 2B, C). The rectangular part had a series of siderable extent. This work was aided greatly by controlling the window perikymata-like characteristic linear grooves. The apex of the triangular level (WL) and window width (WW) and also made it possible to measure part in i1 and i2 projected towards gaps in o1-2 and o2-3, respectively, and reliably the maximum mesiodistal width of a functional tooth (predeces- thus the apical parts of the outer and inner rows of the upper teeth were sor) and its successional tooth, since in doing so both measurements was intercalated, forming a continuous dental arch (Figs. 1B, 2A-C). done on their surfaces of interest in an identical 3D image generated in the In the lower dentition, there was a lingual cingulum in each of b1-3, RadiAnt. making the lingual surface of the teeth curved. The size of this shallow bowl-like concavity was largely proportional to the size of the “bowl” Symbols for teeth and their positions teeth themselves (Figs. 1C, 3B, C). An array of these bowl-like concavities The teeth and their positions within the jaws were expressed with symbols: immediately behind the labial apical edge appeared to aid durophagy in any tooth in the outer and inner rows of the upper dentition in the premax- this fish, since the “bowl” was exposed entirely over the oral mucosa (Figs. illary was expressed as “o” and “i”, respectively, and any bowl-shaped 1C, 3C). tooth in the lower dentition in the dentary as “b”, followed by a number Observation of upper jaw tomograms (Fig. 2D-K) yielded further details to indicate its order from the mesial end in the mid-sagittal plane. Suc- of the filefish dentition. The o1-3 teeth were subtrihedral in shape. At the cessional teeth were assigned a suffix “s”; for example, a b3 successional apex, the tooth was a solid trihedron (typically in o1, Fig. 2D-F) and all tooth was denoted as b3s. Successional teeth in tomograms in MPR were three surfaces were covered with highly calcified tissue, which appeared to identified and scored as “calcified”, “detectable”, or “undetectable (radio- be enameloid. Further away from the apex, a hollow cavity appeared (Figs. graphically)”. These notation rules and scoring are illustrated elsewhere 2G-I, 4A-D), and this changed to a groove which opened lingually, becom- in this article. ing wider towards the tooth base (facing the supporting bone) (Figs. 2J, K, 4D-F). In tomograms at these levels, the labial surface of o1-3 appeared wider, and its radiopaque surface layer extended towards the tooth base for a longer distance than the other two surfaces (Figs. 2F-K, 4A-E). The i1-2 3

Fig. 3 The lower jaw of Sc#1 viewed in 3D-VR from different directions (A-C), in horizontal (D-K) and axial (L-Q) tomograms of the lower dental arch in MPR. Horizontal tomograms are shown intermittently from the apices of b1 (D) to the deeper area where the supporting bone (F-H) and some successional teeth (I-K) are observable. Axial tomograms are shown intermittently from a Fig. 5 The functional teeth (predecessors) and their successors revealed by removing most tissues rostral (L) to caudal (Q) direction. All images in D-Q are at the same magnification. other than teeth from a 3D-VR image (A-F) and shallow depressions for tooth attachment (G, H). The o1-3 teeth and their successors in Sc#3 are shown in the labial aspect, and i1-2 in the lingual aspect (A); no successors for i1-2 were found in this 3D-VR image. The b1-2 teeth and their suc- cessors in Sc#3 are shown in the labial and lingual aspects (B); since this specimen lacked b3s, the antecedent b3 is not included in this VR image. In addition, o1 (C) and o1s (D) in the right upper jaw and i1(E) and i1s (F) in the left upper jaw in Sc#4 are shown individually in four or two differ- ent aspects. Asterisks indicate the apical side of an individual tooth. The basal edge of o1 (double arrowheads in C) and the basal one third on the ridges of o1 and i1 (arrowheads in C and E) are both part of the pulp cavity opening and represent a detached surface which had been ankylosed to the bone. When a tooth was extracted digitally, rough surfaces were inevitably generated (diamonds in C and E) due to the close interdigitation of the apices of o1 and i1. Shallow depressions in bone, where o1 in Sc#3 (G) and b1 in Sc#4 (H) rested, are shown. About three- to four-fifths of the teeth are removed and the remaining U-shaped stumps seated on the depressions are shown. The ridge of supporting bone (ro) protrudes on the lingual side of o1 and b1. Images on the right are slightly tilted distally (G) or shown in their mesial aspect (H). In this figure, the images vary in size, since each has been enlarged arbitrarily to show as much detail as possible.

Table 1 The ratio in the maximum mesiodistal width of the successional teeth and their predecessors

Successors Predecessors* Sc# & Side tooth width** width** S/P ratio symbol*** (mm) (mm) 1 left o1s 2.54 2.25 1.13 1 right o2s 2.32 2.22 1.05 2 right o1s 2.25 2.01 1.12 3 left o1s 2.03 1.91**** 1.07 4 right o1s 2.50 2.32 1.08 4 left i1s 2.75 2.33 1.18 Fig. 4 Closer tomographic views of the jaws in Sc#4. A parasagittal tomogram of the left upper 1 left b1s 2.22 2.18 1.02 jaw (A), horizontal tomograms of the right upper jaw (B-G) and axial tomograms of the lower jaw (H-J) were shown at higher magnification. The higher degree of calcification in the enameloid is 1 right b2s 2.45 2.31 1.06 better delineated. Asterisks indicate supporting bone between o1-3 and i1-2 (A, D-F) and at the base 3 right b1s 2.80 2.46 1.14 of b1-3 (H-J). Arrowheads point to sites of ankylosis between the tooth and its supporting bone. All 3 left b1s 2.66 2.66 1.00 images are at the same magnification. 4 left b1s 3.18 2.84 1.12 *Erupted and functioning teeth; **An average of three-time measurements; ***Consult Materials & Methods and/or Fig. 6; ****Right o1 was measured, since left o1 was exfoliated. teeth showed a highly calcified plate-like morphology with an apical ridge ing teeth and their supporting bone. The bone did not come close to the (Figs. 2F-H, 4C, D), the latter giving rise to one central and two peripheral apices in any of the three types (o, i, b) of functional teeth. In the upper ridges facing the bone; the radiopacity of these ridges was lower than the jaw, the o1-3 and i1-2 teeth were barely ankylosed to the supporting bone plate-like part (Figs. 2I-K, 4D-F). at the tooth base and/or at the basal one third in the ridges facing the bone Tomograms of the lower jaw showed that the b1-3 teeth and their (Fig. 4A, F). In the lower jaw, the b1-3 teeth were again slightly ankylosed successors were trihedral. A solid apex and transition of the pulp cavity to the bone at the tooth base (Fig. 4H-J). In contrast, no bony ankylosis was from tubular to groove-like were also observed in b1-3, and the enameloid found in the developing successional teeth. covering (Fig. 3D-K) was basically similar to that in o1-3. In addition, the The successional teeth appeared to develop beneath the functioning surfaces of the lingual bowl-like concavities in b1-3 and in their successors predecessors. The erupted teeth and their successors were aligned almost were covered with a radiopaque layer of enameloid (Figs. 3L-Q, 4H-J). coaxially, but the axis of o3-o3s was tilted considerably distally compared Tomograms at higher magnification revealed the link between function- to those of o1-o1s and o2-o2s (Fig. 5A, B). The width of successional teeth 4

Fig. 6 Schematic summary of the functional teeth and their successors in the four specimens (Sc#1-4) examined in this study. Teeth and their positions are denoted by roman letters and numer- als. The development of successors is evaluated tomographically and represented as explained above.

Fig. 7 The occlusal relationship of the upper and lower left teeth in Sc#1. The 3D-surface VR Fig. 8 Histological images in the attachment tissues of teeth to the dentary bone in S. cirrhifer; images are shown in four sagittal planes from the next to mid-sagittal (B), then sequentially lateral images were photographed during re-examination of the specimens that had been prepared by Sohiti (C, D), and finally to the distal-most of o1 (E). An ordinary tomogram (A) is shown at the next to Isokawa in the early 1950s. In a section through the mesiodistal plane of the lower dentition (A), mid-sagittal plane (identical to B). All images are at the same magnification. interdental bundles of collagen fibers (asterisks) and the site of ankylosis (boxed) between b2 and its supporting bone (bo) are shown. In a parasagittal section (B, labial side is oriented right, mu; labial mucosa), the bundles of collagen fibers connecting b1 and bo are shown. Boxed areas in A and B are enlarged in C and D, respectively. The arrowhead indicates the site of ankylosis between the dentin of b2 (d.b2) and bo, both of which are located directly next to predentin (pd). In d.b2, cells are entrapped in dentin, implying an osteodentin nature. The arrow indicates the transition measured in this study was approximately 10% larger than the correspond- of the odontoblastic cell layer (od) to osteoblasts in the surface of the trabeculated bo. Diamonds ing predecessor teeth (Table 1), although the former might have developed indicate dentin or its equivalents in the outer surfaces of the dentin. Sections are stained with further before their eruption. hematoxylin-eosin (A-D). The image of a specimen stained by silver impregnation (E) is histologi- cally comparable to D. The development of successional teeth was asynchronous. They varied in both size and tomographic signal intensity, and therefore close examina- tion of multiple tomograms was necessary in order to trace the developing successors. In addition, some of the developing teeth detectable in tomo- comparing it with the apex of the unerupted successor (compare Fig. 5C grams could not be visualized in 3D-VR images (e.g. right i2s of Sc#3 in and D); in addition, a worn apex appeared indented at its tip, indicating a Figs. 5A, 6). In this series of four samples (Sc#1-4), only one tooth (left o1 difference in hardness between the outer enameloid and the inner dentin in Sc#3) had been exfoliated among 64 functional teeth examined, and the (apical aspect in Fig. 5C). Differences in the basal aspects evident in Fig. number of successors evaluated as “calcified”, “detectable” and “undetect- 5C and D indicated that the thickness of the dentin was still increasing able (radiographically)” was 41, 7 and 16, respectively. The distribution in the successor (Fig. 5D). Moreover, perikymata-like linear grooves had of successors in these three developmental categories is illustrated in a been developing on the lingual surface of the unerupted successor (i1s in recording chart in Fig. 6. Fig. 5F), where the bone-side morphology was still incomplete compared Observations of individual teeth extracted digitally from the 3D-VR to that of the erupted tooth (compare Fig. 5E and F). were very informative. The transition of the pulp cavity from tubular to The occlusion of the upper and lower teeth was well delineated by groove-like in o1-2 (Fig. 4D-F) was clarified in detail from the 3D mor- 3D-surface VR images (Fig. 7). When the jaws were closed, the apex and phology. Indeed, the changes in the number of labial ridges in i1-2 and i1s the distal nipping edge of b1 were in close contact with the lingual surface in tomograms (Fig. 4C-G) became easily discernible (Fig. 5E, F). More of i1, but not with o1. Similarly, b2 was in contact with both i1and i2, and importantly, it was clarified that the pulp cavity was not simply open at the b3 was in contact with i2. Thus, an arch-shaped area of contact was seen apparent tooth base but became elongated and widely open in a basoposte- between the i1-2 and b1-3 dentitions. Just behind this arch-shaped contact, rior direction in o1and o1s (Fig. 5C, D) and in a basoanterior direction in i1 the mucosal membrane of the oral cavity was protected with hard tissue and i1s (Fig. 5E, F), and thus the lingual ridges in o1 and the labial ridges armor, i.e. a roof provided by four inner teeth in the upper, and a basal ter- in i1 were in fact part of the edges of the pulp cavity opening. The edge of race formed from an array of bowl-like concavities in the lower dentition. the cavity opening, specifically in about a half or the basal two thirds, was the site of ankylosis between the tooth and its supporting bone (arrowheads Discussion in Fig. 5C, E). This part of the cavity opening was located on the bone, where we observed shallow depressions (Fig. 5G, H). The present study proved that it was possible to investigate and describe Wear was evident on the apex of an individual functional tooth by the highly elaborate morphologies of the tooth and dentition of S. cirrhifer 5 in greater detail using a modern micro-CT system. Some of the features that “predentin” was evident in the boundary area of the teeth and bone described had already been reported by Sohiti Isokawa in 1955, Matsuura of S. cirrhifer. This was confirmed by re-examination of the histological [4] and Uehara and Miyoshi [5] through perceptive observations based specimens he had prepared in the early 1950s (Fig. 8C). It was also rec- on morphological and histological approaches. However, the present ognized that predentin or its equivalents extended to the outer surface of approach employing 2D tomograms and reconstructed 3D models was the edge of pulp cavity opening, thus providing an anchorage for fibers undoubtedly advantageous, even though histological investigations at the connecting tooth to bone and tooth to tooth. These two types of fibers were light and electron microscopy levels are still indispensable for studies of also confirmed in the histological specimens (Fig. 8) and also reported in tooth morphology. an electron microscopy study by Uehara and Miyoshi [5]. The tooth-to- Since analysis of datasets generated by micro-CT allows non-destruc- tooth fibers should not be described as “periodontal”, and the tooth-to-bone tive observations, the in vivo geometry of teeth in the specimens can be fibers were considered to be part of the “fibrous attachment” relatively interrogated serially back and forth as 3D models from various directions, common in Osteichthyes [8], indicating that the fibrous attachment coex- allowing identification of overall functional and successional teeth in situ ists with ankylosis in the tooth of this species, S. cirrhifer, reinforcing the (Figs. 1-4). In addition, “virtual dissection”, described as “digital extrac- tooth attachment cooperatively. tion from 3D-VR” was possible using a scalpel tool (Fig. 5). This approach The surface rendering in this study showed how the upper and lower yielded a number of valuable findings, which were beneficial for in-depth teeth of S. cirrhifer meet together (Fig. 7), i.e. forming a continuous arch understanding of the tomographic images. The smallest voxel size in the consisting of the apex and its distal nipping edge of b1-3 coming completely original images was 30 μm3, which was feasible for evaluating the link into contact with the lingual surface of i1-2. Since the dentary is articulated between the teeth and bone (Fig. 4) and for measuring the width of devel- with the quadrate, and the maxillary and premaxillary are articulated with oping teeth in situ in the 3D models (Table 1). Kazzazi and Kranioti [7] the palatine, the upper and lower jaws are both able to rotate in species of reported that measurements on 3D-VR images were more accurate and this family [4,9]. Thus, the flat and wide lingual surfaces of teeth i1-2 (Fig. reliable than traditional odontometrics carried out on real teeth with digital 7) would serve as a cutting or crushing board after pecking and captur- calipers. ing prey. The feeding habits of the filefish may vary from herbivorous to The mode of tooth attachment in balistoids has long been a controver- carnivorous, with many being omnivorous [2,4,10]. The stomach contents sial topic. Owen described this as a “double gomphosis” in B. capriscus, of S. cirrhifer caught in the southern Shikoku, Japan, were reported to but appeared to imply that the teeth were implanted in alveolar sockets, include amphipods, barnacles and sea urchins [11], most of which would while bony struts projecting from alveolar bone were implanted in the have hard shells and/or calcified structures. Thus, the four inner teeth as narrow spaces between the teeth. Isokawa (1955) reported that the teeth in well as an array of bowl-like concavities in the lower dentition, both being S. cirrhifer were barely ankylosed on the bone margin via predentin (Fig. covered with enameloid tissue, might be functionally relevant as an armor 8). Soule [3] reported that in S. bursa and two other balistids the tooth for protecting the mucosal membrane during biting. attachment comprised a shallow alveolar socket, a periodontal ligament and acellular cementum. Berkovitz and Shellis (as stated in “The Teeth of Acknowledgments Non-Mammalian Vertebrates”, 2017) appeared to be unconvinced by the The authors wish to dedicate this article to Dr. Sohiti Isokawa (1927-1975), latter results and was more inclined to favor an attachment by ankylosis to who started histological studies of fish teeth at Nihon University School of shallow bony depressions. Uehara and Miyoshi [5] showed that the upper Dentistry, where he chaired the Department of Anatomy (1968-1975). This teeth of S. cirrhifer were tightly fixed to the jawbone by bundles of fibrils study was supported in part by the Sato Fund (2017-2019) and the Dental and also that the teeth appeared to attach directly to the bone at the middle Research Center (2017-2019), Nihon University School of Dentistry. portion of the labial and lingual components of the teeth. The present study examined and visualized three-dimensionally the tooth shape and related Conflict of interest geometry in the jaws of S. cirrhifer, and this revealed unequivocally that None. there were no distinct sockets comparable to those in mammals, whereas instead shallow bone depressions were observed (Fig. 5G, H), the edge References of the pulp cavity opening being barely ankylosed in these depressions 1. Matsuura K (2015) Taxonomy and systematics of tetraodontiform fishes: a review focusing (arrowheads in Figs. 4, 5 and 8). primarily on progress in the period from 1980 to 2014. Ichthyol Res 62, 72-113. The present 3D-VR resolved the uncertainty of how the filefish teeth 2. Tyler JC (1980) Osteology, phylogeny, and higher classification of the fishes of the order were ankylosed to shallow depressions in the bone. The opening of the Plectognathi (Tetraodontiformes). NOAA Tech Rep NMFS Circular 434, 1-422. 3. Soule JD (1969) Tooth attachment by means of a periodontium in the trigger-fish (Balisti- tooth pulp cavity was not simply oriented towards the apparent tooth base dae). J Morphol 127, 1-5. in a direction opposite to that of the tooth apex. The opening was distorted 4. Matsuura K (1979) Phylogeny of the superfamily Balistoidea (Pisces: Tetraodontiformes). basoposteriorly in o1-3 and b1-3, and basoanteriorly in i1-2, and therefore Mem Fac Fish Hokkaido Univ 26, 49-169. part of the basoposterior or basoanterior edge of the opening was ankylosed 5. Uehara K, Miyoshi S (1987) Structure of the upper teeth of the filefish, Stephanolepis cirrhifer. Anat Rec 217, 16-22. to the bone (Figs. 4, 5C, E). This meant that the tooth was pleurodont, and 6. Bertin TJC, Thivichon-Prince B, LeBlanc ARH, Caldwell MW, Viriot L (2018) Current not ankylosed to the bone by the actual labial or lingual walls. Pleurodonty perspectives on tooth implantation, attachment, and replacement in Amniota. Front Physiol is a mode whereby the labial (or lingual) surface of a tooth is set against the 9, 1630. 7. Kazzazi SM, Kranioti EF (2018) Applicability of 3D-dental reconstruction in cervical labial (or lingual) side of the tooth-bearing bone. The distribution of such odontometrics. Am J Phys Anthropol 165, 370-377. pleurodont ankylosis along the edge of the pulp cavity opening appeared 8. Fink WL (1981) Ontogeny and phylogeny of tooth attachment modes in actinopterygian to match well with the contour of a shallow depression in the supporting fishes. J Morphol 167, 167-184. 9. Konstantinidis P, Johnson GD (2012) Ontogeny of the jaw apparatus and suspensorium of bone (Fig. 5G, H). the Tetraodontiformes. Acta Zool 93, 351-366. The presence of a periodontal ligament and cementum as an attach- 10. Akagawa I, Okiyama M (1997) Reproductive and feeding ecology of Rudarius ercodes in ment apparatus for the teeth of balistids has been reported [3], but these different environments. Ichthyol Res 44, 82-88. 11. Kawase H, Nakazono A (1996) Two alternative female tactics in the polygynous mating two tissues are considered to be peculiar to the Crocodilia and Mammalia, system of the threadsail filefish, Stephanolepis cirrhifer (Monacanthidae). Ichthyol Res 43, being non-existent in almost all Osteichthyes. Indeed, Isokawa reported 315-323. in 1955 that nothing that could be regarded as cementum was present, but