Relation Between Fish Habitat and the Periodicity of Incremental Lines in the Fossil Otoliths

crystals

Article Relation between Fish Habitat and the Periodicity of Incremental Lines in the Fossil Otoliths

Hiroyuki Mishima 1,*, Yasuo Kondo 2, Fumio Ohe 3, Yasuo Miake 4 and Tohru Hayakawa 1

1 Department of Dental Engineering, Tsurumi University of Dental Medicine, Yokohama 230-8501, Japan; [email protected] 2 Research and Education Faculty, Natural Sciences Cluster, Science and Technology Unit, Kochi University, Kochi 780-8520, Japan; [email protected] 3 Nara National Research Institute of Cultural Properties, Nara 630-8577, Japan; fumi-ohe-ﬁ[email protected] 4 Department of Oral Anatomy, Tsurumi University of Dental Medicine, Yokohama 230-8501, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-45-580-8369

Received: 28 July 2020; Accepted: 14 September 2020; Published: 16 September 2020

Abstract: There are few research reports on the relationship between fish habitats and the periodicity of the fishes’ incremental lines of otolith fossils. The present study examines this relationship through histological and analytical studies on otolith fossils from Nobori Formation, Pliocene, Japan. The specimens were observed and analyzed using light microscopy, polarizing microscopy, Miniscopy, Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) analysis, electron probe X-ray microanalyzer (EPMA), Raman spectroscopy, and XRD. The otolith crystals were aragonite according to XRD and Raman analysis. The incremental lines contained C, O, and Ca, with Si as a trace element. In the layer between the incremental lines, Si was not detected. The circadian incremental lines were unclear and irregularly observed in both Lobianchia gemellarii and Diaphus gigas. Their behavioral pattern included a diurnal vertical movement. By comparison, for Cetonurus noboriensis, Ventrifossa sp., Sebastes scythropus, and Congriscus megastomus, the circadian incremental lines were evident. The habitat of the fishes that live exclusively on the continental slope is kept constant, and the circadian incremental lines are formed regularly. However, for fishes that spend the day in the deep sea and ascend to the shallow sea at night, the ecosystem, such as seawater temperature and pressure, fluctuates, and the circadian incremental lines become unclear and irregular. The period of the circadian incremental lines of otolith may vary due to differences in the ecosystems.

Keywords: otolith; incremental lines; circadian rhythm; fossil; aragonite

1. Introduction The otolith is present in the bony fish’s internal ear and is the mineralized structure consisting of calcium carbonate with a small number of organic matrices [1–3]. The main component of the otolith crystal is calcium carbonate (CaCO3)[1–3]. The otoliths crystals of fish and amphibians are mainly aragonite, while the reptiles, birds, and mammals have calcite [2]. In fish otolith matrix proteins, two matrix proteins (OMP-1, Otolin-1) have been identified [4,5]. Otolith matrix proteins OMP-1 and Otolin-1 are necessary for healthy otolith growth [6,7]. There are incremental lines with several types of periodicity in the otolith’s internal structure [1,8–10]. The daily incremental lines of otoliths are considered to be 1–4 µm [8,10]. The age of living fish is estimated by utilizing the annual incremental lines [11,12]. There have also been studies of the environmental history analysis of the fish [2].

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The otoliths are long preserved in the stratum due to the mineralized structures and are found as otolith fossils. The otolith fossils are used to identify the fish species and estimate the paleoecology or the paleoenvironment [13–16]. The body of vertebrates, including fish, have different mineralized structures as compared with otoliths. These mineralized structures are bone and tooth. The crystals of bone and tooth are biological apatite crystals, which are different from otolith crystals. However, bone and tooth have some periodic incremental lines similar to otoliths [17,18]. It was reported that the periodicity of the incremental lines in tooth dentin has a daily periodicity (circadian rhythm), about a 14-day cycle (tidal periodicity, new moon to full moon), about a 28-day cycle (lunar periodicity: new moon to new moon), a seasonal periodicity, and an annual periodicity (circannual), respectively [17–19]. It is thought that the periodicity of incremental lines in otoliths and dentin is synchronized with the body’s biological rhythms [9,20,21]. The biological rhythms are influenced by the periodic changes in the external environment [10,16,18,21,22]. The otolith serves as a part of the hearing and balance systems [1–3]. It is thought that otolith-forming cells (the transition epithelial cells) are affected by the fishes’ environment, such as the depth of sea habitat [2,23]. Therefore, the periodicity of the incremental lines may be different. The careful study of otoliths’ incremental lines may reveal a whole range of physiological incidents that have occurred to the fish and affected its growth rate [2]. There are few research reports on the relationship between the fish habitat and the periodicity of the incremental line of otolith fossils of fishes [10,16]. Little information is available regarding the ultrastructure, chemical composition, and the periodicity of the incremental lines of otolith fossils of fish [10,11]. The purpose of the present study is to examine the structure, the composition, and the periodicity of incremental lines in the otolith fossils due to differences in fish habitat through histological and analytical studies.

2. Materials and Methods

2.1. Materials In this research, the otolith fossils of fish (Nobori Formation of Tonohama Group, Pliocene, Nobori, Kochi prefecture, about 3.2 2.8 million years ago [24]) were used. The rock facies of the Nobori − Formation consisted of sandstone, siltstone and mudstone layers. The sedimentary environment was from the continental shelf to the continental lower slope [24,25]. The samples were otolith fossils of different fishes from different habitats using the following samples. On each species, five otolith fossils samples were examined.

2.1.1. Myctophidae (1) Myctophidae spp., (2) Lobianchia gemellarii (Cocco) (Cocco’s lanternﬁsh), (3) Diaphus gigas Gilbert (Suitô lanternﬁsh). The main habitats were the deep sea of the continental shelf rim with a depth of 200 m or deeper and diurnal vertical movement.

2.1.2. Macrouridae (1) Macrouridae spp., (2) Cetonurus noboriensis (Aoki and Baba) (Nonbori grenadier), (3) Macrouridae, Ventrifossa sp. (Grenadier), (4) Coryphaenoides cinereus (Gilbert) (Pop eye grenadier). The habitat was the continental shelf and slope at a depth of 200 m or deeper. On Coryhaenoides cinereus, it was a cold-current system; the ecology was diﬀerent from other species and dwelled more deeply.

2.1.3. Scorpaenidae Sebastes scythropus (Jordan and Snyder) (Ukeguchi rockﬁsh). The habitat was the continental shelf to the upper slope (water depth 300 m). Crystals 2020, 10, 820 3 of 10

2.1.4. Congridae Congriscus megastomus (Günther) (Oki-anago conger). The habitat was the continental shelf to the upper slope.

2.2. Methods Polyester resin-embedded samples and non-embedded samples were used in this study. After embedding, these samples were cut into two sections or cross-section specimens with a thickness of about 200 µm using the cutting device (Isomet, Bueller, USA). On the ground sections, one single polishing and double-side polishing was carried out with a grinding stone, a diamond lapping film (final particle size 3 µm), and a diamond paste (final particle size 0.25 µm). These ground specimens were observed and analyzed using light microscopy (Eclipse 80i, Nikon, Japan), digital microscopy (VHX-5000, Keyence, Japan), polarizing microscopy (Eclipse LV100N, Nikon, Japan), scanning electron microscopy (SEM, JSM-6500F, JEOL, Japan; Miniscope, TM3000, Hitachi, Japan), electron probe X-ray microanalyzer (EPMA, JXA-8200, JEOL, Japan), laser Raman microprobe spectrometry (Raman, NRS-3100, Jasco, Japan), and X-ray diffraction method (XRD, RINT2000, RIGAKU, Japan). The polished surface to be analyzed was carbon deposited. Each sample was observed and analyzed at five regions by the point analysis using SEM-energy dispersive X-ray spectroscopy (SEM-EDS) and EPMA. The chemical compositions were analyzed with an accelerating voltage of 15 kV, and the measuring time was 60 s. In the quantitative analysis of EPMA, the quantity of each element was presented in percent weight. The XRD analysis was performed under 40 kV conditions, 200 mA, measurement angle = 20–90◦, STEP = 0.02, STEP time = 0.1 s, using a filter kβ. The irradiation time of the X-ray was 500 s. The collimator diameter was 100 µm. Raman’s analysis was analyzed under the following conditions: laser wavelength = 532 nm, laser output = 0.5 mW, analysis area = 1 µm, analysis time = 20 s. The measurement was analyzed in several points.

2.3. Statistical Analyses Statistical analysis was performed using the Statistical Package for the Social Sciences version 19 software (SPSS; IBM Corp., USA). Seven species of fish (Lobianchia gemellarii, Diaphus gigas, Cetonurus noboriensis, Ventrifossa sp., Coryphaenoides cinereus, Sebastes scythropus, and Congriscus megastomus) were used for the statistical analysis on Ca content. The statistical significance among the Ca content of the incremental lines and the Ca of the layer between the incremental lines was assessed by an independent t-test. In all cases, the selected significance level was p < 0.05. It was analyzed by assuming equal variance as a precondition.

3. Results On the Macrouridae spp. otolith fossil cross-sections, the inside of the otolith was found to consist of needle-like crystals crossed by some kind of incremental lines. Needle crystals of about 0.9–1.3 µm (diameter) were observed. The incremental lines contained C, O, and Ca, with Si as a trace element. Moreover, the layer between the incremental lines contained C, O, and Ca. Na, Mg, Al, S, and K were locally detected in some samples. By the result of the XRD analysis of otolith fossils, the crystal of otolith fossils showed aragonite. On the Raman analysis of the otolith crystals, the Raman band 1 showed aragonite (1084 cm− ). The layer between the incremental lines had better crystallinity than the incremental lines (Figure1). Crystals 2020, 10, x FOR PEER REVIEW 4 of 10

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(A) (B)

Figure 1. Raman spectrum of otolith fossil (Myctophidae spp.). The intensity of Raman band in the layer between the incremental lines (A) was more conspicuous than that of the incremental lines (B). Vertical axis: Raman intensity, horizontal axis: Raman shift (cm−1).

3.1. Myctophidae

With the otolith of Lobianchia(A) gemellarii (Cocco), the narrower incremental(B) lines were unclear, and long-period incremental lines were observed (average 41 μm and average 66 μm) (Figure 2). By FigureFigure 1.1. RamanRaman spectrumspectrum ofof otolithotolith fossilfossil (Myctophidae(Myctophidae spp.).spp.). TheThe intensityintensity ofof RamanRaman bandband inin thethe the backscattered electron image of SEM, the incremental lines were irregularly observed (Figure 2D, layerlayer betweenbetween thethe incrementalincremental lineslines ((AA)) waswas moremore conspicuousconspicuous thanthan thatthat ofof thethe incrementalincremental lineslines ((BB).). yellow lines). The composition of the incremental line consisted of C: 24.64 ± 2.39%, O: 40.44 ± 1.76%, VerticalVertical axis: axis: RamanRaman intensity, intensity, horizontal horizontal axis: axis: Raman Raman shift shift (cm (cm−11).). Ca: 34.73 ± 0.94%, and Si: 0.20 ± 0.07%. The layer's composition− between the incremental lines consisted3.1.3.1. MyctophidaeMyctophidae of C: 14.30 ± 0.79%, O: 49.40 ± 0.75%, and Ca: 36.30 ± 0.40%. The incremental line Ca content was significantly lower than that of the layer between the incremental lines (p < 0.05, n = 5). With the otolith of Lobianchia gemellarii (Cocco), the narrower incremental lines were unclear, and OnWith the the otolith otolith of Diaphusof Lobianchia gigas Gilbert,gemellarii the (Cocco), narrower the incrementalnarrower incremental lines were unclear,lines were and unclear, long- long-period incremental lines were observed (average 41 µm and average 66 µm) (Figure2). By the periodand long-period incremental incremental lines were lines observed were (averageobserved 49 (average μm) (Figure 41 μm 3). and Other average long-period 66 μm) (Figure incremental 2). By backscattered electron image of SEM, the incremental lines were irregularly observed (Figure2D, linesthe backscattered (average 730 μelectronm) were image observed of SEM, (Figure the incremental3A). The composition lines were of irregularly the incremental observed line (Figure consisted 2D, yellow lines). The composition of the incremental line consisted of C: 24.64 2.39%, O: 40.44 1.76%, ofyellow C: 21.31 lines). ± 1.99%, The composition O: 42.04 ± 0.91%, of the Ca: incremental 36.40 ± 1.43%, line consistedand Si: 0.25 of C:± 0.03%. 24.64± ±The 2.39%, layer's O: 40.44composition± ± 1.76%, Ca: 34.73 0.94%, and Si: 0.20 0.07%. The layer’s composition between the incremental lines betweenCa: 34.73 the ±± incremental0.94%, and linesSi: 0.20 consisted ±± 0.07%. of C:The 11.46 laye ± r's0.34%, composition O: 46.66 ±between 0.74%, and the Ca:incremental 41.89 ± 0.73%. lines consisted of C: 14.30 0.79%, O: 49.40 0.75%, and Ca: 36.30 0.40%. The incremental line Ca content Theconsisted incremental of C: 14.30 line± ± 0.79%,Ca content O: 49.40 was± ± 0.75%,significan and tlyCa: lower36.30 ±± than0.40%. that The ofincremental the layer linebetween Ca content the was signiﬁcantly lower than that of the layer between the incremental lines (p < 0.05, n = 5). incrementalwas significantly lines (p lower < 0.05, than n = that 5). of the layer between the incremental lines (p < 0.05, n = 5). On the otolith of Diaphus gigas Gilbert, the narrower incremental lines were unclear, and long- period incremental lines were observed (average 49 μm) (Figure 3). Other long-period incremental lines (average 730 μm) were observed (Figure 3A). The composition of the incremental line consisted of C: 21.31 ± 1.99%, O: 42.04 ± 0.91%, Ca: 36.40 ± 1.43%, and Si: 0.25 ± 0.03%. The layer's composition between the incremental lines consisted of C: 11.46 ± 0.34%, O: 46.66 ± 0.74%, and Ca: 41.89 ± 0.73%. The incremental line Ca content was significantly lower than that of the layer between the incremental lines (p < 0.05, n = 5).

Figure 2. Micrograph of Lobianchia gemellarii (Cocco). The narrower incremental lines were unclear Figure 2. Micrograph of Lobianchia gemellarii (Cocco). The narrower incremental lines were unclear and irregularly observed (B–D). The long-period incremental lines (average 41 µm: yellow arrows and and irregularly observed (B–D). The long-period incremental lines (average 41 μm: yellow arrows average 66 µm: black arrows) were observed (B,C). Digital microscopy images (A: otolith surface, B: and average 66 μm: black arrows) were observed (B,C). Digital microscopy images (A: otolith surface, cross-sectional image). Polarizing micrograph of the ground specimen (C). SEM micrograph (D). Scale B: cross-sectional image). Polarizing micrograph of the ground specimen (C). SEM micrograph (D). bar: 500 µm (A), 100 µm (B), 50 µm (C,D). Scale bar: 500 μm (A), 100 μm (B), 50 μm (C,D). On the otolith of Diaphus gigas Gilbert, the narrower incremental lines were unclear, and long-period incremental lines were observed (average 49 µm) (Figure3). Other long-period incremental lines (averageFigure 730 2.µ Micrographm) were observed of Lobianchia (Figure gemellarii3A). The(Cocco). composition The narrower of the incremental incremental lines line were consisted unclear of C: 21.31and irregularly1.99%, O: observed 42.04 0.91%,(B–D). Ca:The 36.40long-period1.43%, incremental and Si: 0.25lines (average0.03%. 41 The μm: layer’s yellow composition arrows ± ± ± ± betweenand theaverage incremental 66 μm: black lines arrows) consisted were ofobserved C: 11.46 (B,C0.34%,). Digital O: microscopy 46.66 0.74%, images and (A: Ca:otolith 41.89 surface,0.73%. ± ± ± The incrementalB: cross-sectional line Ca image). content Polarizing was signiﬁcantly micrograph lower of the than ground that specimen of the layer (C). betweenSEM micrograph the incremental (D). Scale bar: 500 μm (A), 100 μm (B), 50 μm (C,D). lines (p < 0.05, n = 5).

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Figure 3. Micrograph of Diaphus gigas Gilbert. The longer period of incremental lines was observed (A, blue arrows). The narrower incremental lines were unclear, as well as the long-period incremental lines (B,C). The long-period of incremental lines (average 49 μm: yellow arrows) were observed (C). Digital microscopy images (A: otolith surface, B: cross-sectional image). Polarizing micrograph of the ground specimen (C). Scale bar: 500 μm (A), 100 μm (B), 50 μm (C).

3.2. MacrouridaeFigure 3. Micrograph of Diaphus gigas Gilbert. The longer period of incremental lines was observed Figure 3. Micrograph of Diaphus gigas Gilbert. The longer period of incremental lines was observed (A, blue arrows). The narrower incremental lines were unclear, as well as the long-period incremental On(A, bluethe otolitharrows). of The Cetonurus narrower noboriensis incremental (Aoki lines wereand Baba),unclear, narrower as well as theincremental long-period lines incremental (average 3.3 lines (B,C). The long-period of incremental lines (average 49 µm: yellow arrows) were observed (C). μm) werelines ( Bobserved,C). The long-period (Figure 4). ofBy incremental backscattered lines elec(averagetron 49images μm: yellow of SEM, arrows) the incremental were observed lines (C). were Digital microscopy images (A: otolith surface, B: cross-sectional image). Polarizing micrograph of the regularlyDigital observed microscopy (Figure images 4D). (A :The otolith long surface, period B :of cross-sectional incremental image). lines (average Polarizing 63 micrograph μm and average of the 118 ground specimen (C). Scale bar: 500 µm (A), 100 µm (B), 50 µm (C). μm) wasground observed specimen (Figure (C). Scale 4B,C). bar: The500 μincrementalm (A), 100 μ mline (B ),composition 50 μm (C). consisted of C: 19.46 ± 3.04%, O: 44.773.2. Macrouridae ± 0.90%, Ca: 35.76 ± 2.31%. The layer's composition between the incremental lines consisted of C:3.2. 11.91 Macrouridae ± 0.37%, O: 48.20 ± 0.45%, and Ca: 39.89 ± 0.44%. The Ca content of the incremental lines was On the otolith of Cetonurus noboriensis (Aoki and Baba), narrower incremental lines (average 3.3 µm) significantly lower than that of the layer between the incremental lines (p < 0.05, n = 5). wereOn observed the otolith (Figure of 4Cetonurus). By backscattered noboriensis electron (Aoki and images Baba), of SEM, narrower the incremental incremental lines lines were (average regularly 3.3 On the otolith of Ventrifossa sp., the narrower incremental lines (average 3.5 μm) were observed, μobservedm) were (Figureobserved4D). (Figure The long 4). periodBy backscattered of incremental electron lines images (average of 63SEM,µm the and incremental average 118 linesµm) were was as well as the long-period incremental lines (average 78 μm) (Figure 5). The composition of the regularlyobserved observed (Figure4B,C). (Figure The 4D). incremental The long period line composition of incremental consisted lines (average of C: 19.46 63 μm3.04%, and average O: 44.77 118 incremental line consisted of C: 27.31 ± 2.28%, O: 42.21 ± 1.16%, Ca: 29.72 ± 1.47%, and± Si: 0.75 ± 0.12%.± μ0.90%,m) was Ca: observed 35.76 (Figure2.31%. 4B,C). The layer’s The incremental composition line between composition the incremental consisted of linesC: 19.46 consisted ± 3.04%, of O: C: The layer's composition± between the incremental lines consisted of C: 15.19 ± 0.52%, O: 48.26 ± 0.38%, 44.7711.91 ± 0.90%,0.37%, Ca: O: 48.2035.76 ± 0.45%,2.31%. andThe Ca:layer's 39.89 composition0.44%. The between Ca content the incremental of the incremental lines consisted lines was of and Ca:± 36.55 ± 0.27%. The± incremental line Ca ±content was significantly lower than that of the layer C:signiﬁcantly 11.91 ± 0.37%, lower O: than48.20 that ± 0.45%, of the and layer Ca: between 39.89 ± the0.44%. incremental The Ca content lines (p of< the0.05, incremental n = 5). lines was betweensignificantly the incrementallower than that lines of (p the < 0.05,layer n between = 5). the incremental lines (p < 0.05, n = 5). On the otolith of Ventrifossa sp., the narrower incremental lines (average 3.5 μm) were observed, as well as the long-period incremental lines (average 78 μm) (Figure 5). The composition of the incremental line consisted of C: 27.31 ± 2.28%, O: 42.21 ± 1.16%, Ca: 29.72 ± 1.47%, and Si: 0.75 ± 0.12%. The layer's composition between the incremental lines consisted of C: 15.19 ± 0.52%, O: 48.26 ± 0.38%, and Ca: 36.55 ± 0.27%. The incremental line Ca content was significantly lower than that of the layer between the incremental lines (p < 0.05, n = 5).

Figure 4. Micrograph of Cetonurus noboriensis (Aoki and Baba). The narrower incremental lines were Figure 4. Micrograph of Cetonurus noboriensis (Aoki and Baba). The narrower incremental lines were regularly observed (C, red arrows). The narrower incremental lines were spaced 1.5–3.4 µm (D, yellow regularly observed (C, red arrows). The narrower incremental lines were spaced 1.5–3.4 μm (D, arrows). The long-period incremental lines (average 63 µm: yellow arrows and average 118 µm: black yellow arrows). The long-period incremental lines (average 63 μm: yellow arrows and average 118 arrows) were observed (B,C). Digital microscopy images (A: otolith surface, B: cross-sectional image). μm: black arrows) were observed (B,C). Digital microscopy images (A: otolith surface, B: cross- Ground specimen of polarizing micrograph (C). SEM micrograph (D). Scale bar: 500 µm (A), 100 µm sectional image). Ground specimen of polarizing micrograph (C). SEM micrograph (D). Scale bar: 500 (B), 50 µm (C,D). μm (A), 100 μm (B), 50 μm (C,D).

FigureOn the 4 otolith. Micrograph of Ventrifossa of Cetonurussp., noboriensis the narrower (Aoki incremental and Baba). The lines narro (averagewer incremental 3.5 µm) werelines observed,were as wellregularly as the observed long-period (C, red incremental arrows). The lines narro (averagewer incremental 78 µm) lines (Figure were5). spaced The composition 1.5–3.4 μm (D of, the

incremental line consisted of C: 27.31 2.28%, O: 42.21 1.16%, Ca: 29.72 1.47%, and Si: 0.75 yellow arrows). The long-period incremental± lines (average± 63 μm: yellow arrows± and average 118 ± 0.12%.μm: The black layer’s arrows) composition were observed between (B,C the). Digital incremental microscopy lines consistedimages (A of: otolith C: 15.19 surface,0.52%, B: cross- O: 48.26 ± ± 0.38%,sectional and Ca: image). 36.55 Ground0.27%. specimen The incremental of polarizing line micrograph Ca content (C was). SEM signiﬁcantly micrograph lower(D). Scale than bar: that 500 of the ± layerμ betweenm (A), 100 the μm incremental (B), 50 μm (C lines,D). (p < 0.05, n = 5).

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Figure 5. Micrograph of Ventrifossa sp. The narrower incremental lines (C, red arrow) were observed, Figure 5. Micrograph of Ventrifossa sp. The narrower incremental lines (C, red arrow) were observed, asFigure well 5as. Micrographthe long-per ofiod Ventrifossa incremental sp. Thelines narrower (B,C, average incremental 78 μm: lines black (C arrows)., red arrow) Digital were microscopy observed, as well as the long-period incremental lines (B,C, average 78 µm: black arrows). Digital microscopy imagesas well (asA: theotolith long-per surface,iod Bincremental: cross-sectional lines image).(B,C, average Ground 78 specimen μm: black of arrows). polarizing Digital micrograph microscopy (C). images (A: otolith surface, B: cross-sectional image). Ground specimen of polarizing micrograph (C). Scaleimages bar: (A 500: otolith μm ( Asurface,), 100 μ Bm: cross-sectional (B), 50 μm (C). image). Ground specimen of polarizing micrograph (C). Scale bar: 500 µm (A), 100 µm (B), 50 µm (C). Scale bar: 500 μm (A), 100 μm (B), 50 μm (C). WithWith the the otolith otolith of of CoryphaenoidesCoryphaenoides cinereus cinereus (Gilbert),(Gilbert), the the narrower narrower incremental incremental lines lines were were unclear. unclear. With the otolith of Coryphaenoides cinereus (Gilbert), the narrower incremental lines were unclear. TwoTwo typestypes of of long-period long-period incremental incremental lines lines (average (average 33 µ m33 and μm average and average 97 µm) were97 μm) observed were observed (Figure6). Two types of long-period incremental lines (average 33 μm and average 97 μm) were observed (FigureThe incremental 6). The incremental line composition line composition consisted consisted of C: 26.62 of C:0.64%, 26.62 O:± 0.64%, 39.69 O:0.75%, 39.69 ± and 0.75%, Ca: and 33.69 Ca: (Figure 6). The incremental line composition consisted of± C: 26.62 ± 0.64%, ±O: 39.69 ± 0.75%, and Ca:± 33.690.31%. ± The0.31%. composition The composition of the layer of the between layer between the incremental the incremental lines consisted lines consisted of C: 12.61 of C:0.32%, 12.61 O:± 33.69 ± 0.31%. The composition of the layer between the incremental lines consisted of± C: 12.61 ± 0.32%,47.06 O:0.53%, 47.06 and ± 0.53%, Ca: 39.84 and Ca:0.42%. 39.84 The ± 0.42%. incremental The incremental line Ca content line Ca was content signiﬁcantly was significantly lower than 0.32%,± O: 47.06 ± 0.53%, and± Ca: 39.84 ± 0.42%. The incremental line Ca content was significantly lowerthat of than the layerthat of between the layer the between incremental the incremental lines (p < 0.05, lines n (p= 5).< 0.05, n = 5). lower than that of the layer between the incremental lines (p < 0.05, n = 5).

Figure 6. Micrograph of Coryphaenoides cinereus (Gilbert). The narrower incremental lines were unclear. Figure 6. Micrograph of Coryphaenoides cinereus (Gilbert). The narrower incremental lines were The long-period incremental lines (yellow arrows: average 33 µm, and black arrows: average 97 µm) unclear.Figure 6.The Micrograph long-period of incrementa Coryphaenoidesl lines (yellowcinereus arrows:(Gilbert). average The narrower33 μm, and incremental black arrows: lines average were were observed. Digital microscopy images (A: otolith surface, B: cross-sectional image). Ground 97unclear. μm) wereThe long-period observed. Digitalincrementa microscopyl lines (yellow images arrows: (A: otolith average surface, 33 μm, B and: cross-sectional black arrows: image).average specimen of polarizing micrograph (C). Scale bar: 500 µm (A), 100 µm (B), 50 µm (C). Ground97 μm) specimenwere observed. of polarizing Digital micrograph microscopy (C images). Scale bar:(A: otolith500 μm surface,(A), 100 Bμ:m cross-sectional (B), 50 μm (C). image). 3.3. ScorpaenidaeGround specimen of polarizing micrograph (C). Scale bar: 500 μm (A), 100 μm (B), 50 μm (C). 3.3. Scorpaenidae 3.3. ScorpaenidaeOn the otolith of Sebastes scythropus (Jordan and Snyder), the narrower incremental lines (average 4.6 µOnm) werethe otolith observed. of Sebastes The two scythropus types of long-period (Jordan and incremental Snyder), linesthe narrower (average 46incrementalµm and average lines On the otolith of Sebastes scythropus (Jordan and Snyder), the narrower incremental lines (average107 µm) were4.6 μm) observed. were observed. The composition The two oftypes the incrementalof long-period line incremental consisted of lines C: 26.41 (average2.52%, 46 μ O:m 42.42 and (average 4.6 μm) were observed. The two types of long-period incremental lines (average± 46 μm and average1.16%, 107 Ca: μm) 31.00 were1.43%, observed. and The Si: 0.17composition0.14%. of The the layer’sincremental composition line consisted between of C: the 26.41 incremental ± 2.52%, ±average 107 μm) were± observed. The composition± of the incremental line consisted of C: 26.41 ± 2.52%, O:lines 42.42 consisted ± 1.16%, of C:Ca: 11.82 31.00 0.36%,± 1.43%, O: 48.17and Si:0.56%, 0.17 ± and 0.14%. Ca: 40.02The layer's0.27%. composition The incremental between line the Ca O: 42.42 ± 1.16%, Ca: 31.00± ± 1.43%, and ±Si: 0.17 ± 0.14%. The layer's± composition between the incrementalcontent was signiﬁcantlylines consisted lower of thanC: 11.82 that of± 0.36%, the layer O: between 48.17 ± the0.56%, incremental and Ca: lines 40.02 (p <± 0.05,0.27%. n =The5). incrementalincremental linelines Ca consisted content wasof C:significantly 11.82 ± 0.36%, lowe rO: than 48.17 that ± of0.56%, the layer and between Ca: 40.02 the ± incremental0.27%. The linesincremental (p < 0.05, line n = Ca 5). content was significantly lower than that of the layer between the incremental lines (p < 0.05, n = 5).

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3.4. Congridae On the otolith of Congriscus megastomus (Günther), the narrower incremental lines (average 5.9 µm) were observed. The two types of the long-period incremental lines (average 43 µm and average 96 µm) were observed. The composition of the incremental line consisted of C: 27.18 1.09%, O: 42.19 1.09%, ± ± Ca: 29.95 1.05%, and Si: 0.67 0.09%. The composition of the layer between the incremental lines ± ± consisted of C: 12.78 1.19%, O: 48.48 0.80%, and Ca: 38.69 0.69%. The incremental line Ca content ± ± ± was signiﬁcantly lower than that of the layer between the incremental lines (p < 0.05, n = 5).

4. Discussion The crystals of otoliths fossils were aragonite by the XRD and the Raman analysis. This result supports the results of previous studies [1,2]. The incremental lines were less crystalline by the Raman analyses than the layer between the incremental lines of otolith fossils. The Ca content in the incremental lines of otoliths fossils was significantly low by SEM-EDS and EPMA analysis in all samples (p < 0.05). The incremental lines detected C, O, and Ca, with Si as a trace element. As for trace elements, Si was detected in all samples. Si is contained in the phytoplankton, and it is possible that the phytoplankton entered the body as food and was contained in the otoliths. Si is also contained in the deep-sea water [26]. Furthermore, Si exists in the strata. Therefore, it is considered that Si may have invaded the otolith during fossilization in the stratum. There are some possibilities, and further studies will be necessary to explain the presence of Si. Cuif et al. [1] reported that Mg, S, and Sr exist as trace elements in otoliths. Sr was not detected in this study. The origin of Sr is considered to be the seawater. When taken into the body, it accumulates in the otolith instead of Ca during the growth process. However, it is presumed that Sr dissolves during fossilization and flows out the stratum. The trace elements (Na, Mg, Al, S, and K) may be widely involved in the nucleation and growth of crystal in the mineralized structures [2,27,28]. Four types of incremental lines were confirmed in the otolith fossils of fish of the Nobori Formation. From the short-period incremental line: (1) 3–6 µm, (2) 33–78 µm, (3) 96–118 µm, (4) 730 µm. From previous studies, the periods of these incremental lines correspond to the following periodic intervals: (1) circadian incremental lines, (2) about 14-day periodicity: tidal incremental lines, (3) about 28-day periodicity: lunar incremental lines, (4) annual incremental lines [8–10,13,16,29]. In fish otoliths, the lunar periodicity is the common feature. The lunar periodicity is related to the swimming and feeding activity for fish in the tidal environment [16]. The periodicity of the otolith incremental lines was similar to the incremental lines in the tooth dentin of animals such as crocodiles and mammals [17,18]. The formation of incremental lines in dentin is determined by the change in the deposition of mineralization and the change in the deposition of the organic matrix [17,19,30]. The intervals of incremental lines in otoliths may be related to the rhythm of the transition epithelial cells function and the fish’s fundamental biological rhythm. Otolith matrix proteins OMP-1 and Otolin-1 are the organic matrix-rich part of the incremental lines [6,7,12]. Carbonic anhydrase (CA) controls the diurnal variation of pH in the endolymph. The inorganic environments controlled by CA may contribute to the mineralization process of the otoliths [23]. These organic matrices and CA may be involved in the formation of the incremental line periodicity. The circadian incremental lines were unclear or observed to be irregular in Lobinachia gemellarii and Diaphus gigas. Their habits were diurnal vertical movements (Figure7, see O5 ). By comparison, Cetonurus noboriensis, Ventrifossa sp., Sebastes scythropus, and Congriscus megastomus circadian incremental lines were evident. Their habitats were the upper slope of the continental shelf (Figure7, see O3 ). In fish otoliths, the increments are deposited at a daily rate [29]. The growth pattern must either be directly linked to cyclic environmental changes or caused by an endogenous clock, which could, in turn, be entrained by these external stimuli [2,8,29]. The rate of otolith growth has been shown to be generally controlled by external factors such as temperature and may vary depending on physiological circumstance [16]. Due to the diurnal vertical movement, the temperature and pressure of the Crystals 2020, 10, 820 8 of 10

Crystalsseawater 2020 surrounding, 10, x FOR PEER the REVIEW fish change considerably. It is considered that the rapid change in seawater8 of 10 temperature causes a change in the otolith formation [16,20]. Furthermore, we are led to speculate ledthat to the speculate abrupt that change the abrupt in water change pressure, in water which pressure, is associated which is with associated the diurnal with the vertical diurnal movement, vertical movement,affects the balanceaffects the system balance of the system fish and of the further fish changesand further the functionchanges the of the function epithelial of the cells epithelial forming cellsthe otolithsforming [ 2the,16 ].otoliths It is possible [2,16]. It that is possible the periodicity that the of periodicity the incremental of the linesincremental of otoliths lines varies of otoliths along varieswith the along ecosystems. with the With ecosystems.Coryphaenoides With Coryphaenoides cinereus, it is a cold-currentcinereus, it is system a cold-current and a deeper system habitat and and a deeperthe ecology habitat is and different the ecology from other is different species from of Macrouridae other species (Figure of Macrouridae7, see O4 ). (Figure Imai and 7, see Nonaka ④). Imai [20] andreported Nonaka otolith [20] dailyreported incremental otolith daily lines incremental were observed lines inwere Shimonoseki, observed in Japan, Shimonoseki, and noincremental Japan, and nolines incremental were observed lines inwere the observed otolith of inTakifugu the otolith niphobles of Takifugu(Grass puniphoblesffer) in (Grass winter. puffer) The temperature in winter. The and temperaturesea depth of and the fishsea habitatdepth aofff ectedthe fish the functionhabitat affe of transitioncted the function epithelial of cells transition forming epithelial the otoliths cells of formingthe internal the ear,otoliths suggesting of the internal that the circadianear, suggesting incremental that the lines circadian might beincremental irregular and lines unclear. might Webe irregularconsider thatand theunclear. environment We consider changes that surrounding the environment the epithelial changes cells surrounding influence both the the epithelial deposition cells of influencemineralization both the and deposition the deposition of mineralization of the organic and matrix the deposition in the otolith. of the We organic presume matrix that the in the fish otolith. habitat Weaffected presume the rhythmthat the of fish the habitat central affected clock in the the rhythm body and of atheffected central the clock periodicity in the body of the and formation affected of the the periodicitytransition epithelialof the formation cells forming of the thetransition incremental epithelial lines. cells forming the incremental lines.

Figure 7. Environmental reconstruction of Nobori fish fauna. Figure 7. Environmental reconstruction of Nobori fish fauna. 5. Conclusions 5. Conclusions It is possible that the periodicity of the incremental lines of otolith changed with the ecosystems. We speculateIt is possible that that the the circadian periodicity and of other the incremen long-periodtal lines incremental of otolith lines changed may with be independent the ecosystems. and Wedriven speculate by diff erentthat the oscillatory circadian mechanisms. and other long-p More dataeriod concerning incremental the lines implication may be of independent these findings and are drivenneeded by to different elucidate oscillatory this phenomenon. mechanisms. More data concerning the implication of these findings are needed to elucidate this phenomenon. Author Contributions: Conceptualization, H.M. and Y.K.; methodology, H.M.; formal analysis, H.M., F.O. and Y.M.; investigation, H.M.; resources, Y.K.; data curation, H.M.; writing—original draft preparation, H.M.; Authorwriting—review Contributions: and editing, Conceptualization, Y.K., Y.M. and H.M. T.H.; and funding Y.K.; methodolo acquisition,gy, T.H. H.M.; All authorsformal analysis, have read H.M., and agreedF.O. and to Y.M.;the published investigation, version H.M.; of the resources, manuscript. Y.K.; data curation, H.M.; writing—original draft preparation, H.M.; writing—review and editing, Y.K., Y.M. and T.H.; funding acquisition, T.H. All authors have read and agreed Funding: This research received no external funding. to the published version of the manuscript. Acknowledgments: This study was performed under the cooperative research program of center for Advanced Funding:Marine Core This Research research (CMCR),received Kochino external University funding. (Accept No. 15A021, 15B018, 16A0006, 16B006, 17A009, 17B009, 18A009, 18B008, 19A003, 19B002) (with the supported of JAMSTEC). Acknowledgments: This study was performed under the cooperative research program of center for Advanced MarineConflicts Core of Interest: ResearchThe (CMCR), authors declare Kochi no University conflict of interest.(Accept No. 15A021 ， 15B018, 16A0006, 16B006, 17A009,17B009, 18A009, 18B008, 19A003, 19B002) (with the supported of JAMSTEC).

Conflicts of Interest: The authors declare no conflict of interest.

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