Journal of Fish Biology (2003) 63, 1383–1401 doi:10.1046/j.1095-8649.2003.00245.x,availableonlineathttp://www.blackwell-synergy.com
Morphometry and composition of aragonite and vaterite otoliths of deformed laboratory reared juvenile herring from two populations
J. TOMA´S* AND A. J. GEFFEN Port Erin Marine Laboratory, School of Biological Sciences, University of Liverpool, Port Erin, Isle of Man, IM9 5AP U.K.
(Received 24 March 2003, Accepted 19 August 2003)
Vaterite otoliths were sampled from two reared populations (Celtic and Clyde Seas) of juvenile herring Clupea harengus. The crystallography, elemental composition and morphometry were analysed and compared with those of normal aragonite otoliths. The incidence of vaterite otoliths in the juveniles sampled (n ¼ 601) ranged from 7 8% in the Clyde population to 13 9% in the Celtic Sea population, and was 5 5% in the small sample (n ¼ 36) of wild adults examined. In all but one case fish had only one vaterite otolith; the corresponding otolith of the pair was completely aragonite. Although the majority of the juveniles sampled showed cranio- facial deformities, there was no link between the skull or jaw malformation and the incidence of vaterite otoliths. All vaterite otoliths had an aragonite inner area, and vaterite deposition began sometime after the age of 90 days. The vaterite otoliths were larger and lighter than their corresponding aragonite partners, and were less dense as a consequence of the vaterite crystal structure. The vaterite areas of the otoliths were depleted in Sr, Na and K. Concentrations of Mn were higher in the vaterite areas. The transition between the aragonite inner areas and the vaterite areas was sharply delineated. Within a small spatial scale (20 mm3) in the vaterite areas, however, there was co-precipitation of both vaterite and aragonite. The composition of the aragonite cores in the vaterite otoliths was the same as in the cores of the normal aragonite otoliths indicating that the composition of the aragonite cores did not seed the shift to vaterite. Vaterite is less dense than aragonite, yet the concentrations of Ca analysed with wavelength- dispersive spectrometry (WDS) were the same between the two polymorphs, indicating that Ca concentrations measured with WDS are not a good indicator of hypermineralized zones with high mineral density. The asymmetry in density and size of the otoliths may cause disruptions of hearing and pressure sensitivity for individual fish with one vaterite otolith, however, the presence of vaterite otoliths did not seem to affect the growth of these laboratory reared juvenile herring. # 2003 The Fisheries Society of the British Isles Key words: biomineralisation; fish hearing; ICPMS; otolith development; Raman spectrometry.
*Author to whom correspondence should be addressed at present address. Grupo de Oceanografı´a Interdisciplinar (GOI), Institut Mediterrani d’Estudis Avanc¸ats (CSIC/UIB), Miguel Marque´s 21, 07190 Esporles, Illes Balears, Spain. Tel.: þ 34 971 61 17 21; fax: þ 34 971 61 17 61; email: [email protected] 1383 # 2003 The Fisheries Society of the British Isles 1384 J. TOMA´S AND A. J. GEFFEN
INTRODUCTION Otoliths are involved in the perception of sound and the maintenance of postural equilibrium in fishes. The perception of sound is a fundamental sense, providing information from the environment (Popper & Fay, 1999) for predator avoidance, food availability, and the location of other individuals for mating (Zelick et al., 1999). Otoliths are composed of CaCO3 that normally precipitates as aragonite. In aberrant otoliths the CaCO3 precipitates as calcite or vaterite. These three polymorphs of CaCO3 differ in the geometry of the crystal: calcite is trigonal, aragonite is orthorhombic and vaterite is hexagonal. The formation of calcite or vaterite otoliths has been reported in a number of marine and freshwater species from different environments (Strong et al., 1986; Gauldie, 1993; Bowen II et al., 1999). Otoliths may be completely or partially composed of calcite or vaterite but the mechanisms governing the switch between polymorphs are unknown. The presence of vaterite otoliths may affect the functioning of the inner ear and the growth and survival of the fish, and may lead to compensations in otolith size or shape, but these aspects have yet to be examined. Juvenile herring Clupea harengus L. with deformed heads were examined in this study after evidence was found in the zebrafish Danio rerio (Hamilton) that mutations affecting the development of jaws, gills and cranium resulted in the malformation of otoliths (Malicki et al., 1996; Whitfield et al., 1996). The crystallography of the malformed zebrafish otoliths was not reported in these studies, but it is possible that the macrostructural changes were accompanied by changes at the molecular level. Several reports of vaterite otoliths in wild fishes (Morales-Nin, 1985; Strong et al., 1986; Gauldie, 1993; Brown & Severin, 1999) have commented on the aberrant appearance of these otoliths. Populations of juvenile herring originating from fish spawning in the Firth of Clyde and the Celtic Sea were raised in the laboratory, and fish with jaw and cranial deformities were selected, and compared with normal juveniles, in order to study the relationships between otolith morphometry, crystal form and otolith and fish growth. Normal and aberrant otoliths were compared to explore the relationship between composition and otolith structure. The mor- phometry of aberrant otoliths was also analysed to provide information about the relationship between otolith growth and otolith shape.
MATERIALS AND METHODS
SOURCE OF MATERIAL One year-old juvenile herring were sampled from populations originating from gametes collected from the Celtic Sea (January spawning) and the Clyde Sea (spring spawning) herring populations. Eggs from several females were distributed on glass plates, and artificially fertilized with sperm from several males. After hatching, the larvae were reared in flowing sea water in 2000 l circular black tanks. Temperatures were not controlled and followed seasonal fluctuations. The light regime was maintained with fluorescent lighting, controlled with time clocks to follow the seasonal changes in photo- period. Larvae were fed with rotifers and subsequently Artemia sp. and were weaned at c. day 30 after hatching onto formulated dry food.
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1385
The juvenile fish sampled ranged from 3–14 months in age (Table I). Adult Celtic Sea herring were also sampled to assess the frequency of vaterite otoliths in the wild popula- tion. Every fish was measured (total length, LT), weighed and the otoliths removed. Otolith dissection was carried out using acid washed glass probes and distilled water to prevent otolith contamination. Once extracted, otoliths were double rinsed in distilled water, air dried and stored in acid washed polypropylene vials. Preliminary observations had indicated that deformed individuals had a high incidence of crystalline otoliths, presumed to be composed of vaterite and easily distinguishable from normal aragonite otoliths (Fig. 1). To test this, a random sample of 112 juveniles from the Celtic Sea stock and 109 juveniles from the Clyde were collected in 1998 to provide a control sample of normal fish to investigate the relationship between skull deformities and the incidence of vaterite otoliths (Table I).
OTOLITH MORPHOMETRY Otoliths were weighed using a Cahn G-2 electrobalance (precision 0 001 mg) and the otolith dimensions (mm) were measured using an image analysis system [Fig. 1(b)]. The otolith length was the distance between the anterior and posterior edges of the otolith; the width was the longest distance between the dorsal and ventral edges of the otolith, perpendicular to the length of the otolith. The perimeter of the otolith was traced automatically using an edge-recognition sub-routine, and the total otolith area (mm2) calculated by the image analysis software. All of the vaterite otoliths contained a central region that appeared to be normal aragonite. This aragonite area varied in shape and size between individuals [Fig. 1(b), (c), (d)]. The perimeter of the aragonite area of the vaterite otoliths was outlined manually, and the area calculated by the image analysis software [Fig. 1(b), (c), (d)].
CRYSTALLOGRAPHY X-ray diffraction spectrometry (XRD) was used to identify the polymorph composi- tion of the normal and apparently vaterite otoliths. Six otolith samples (three vaterite and three normal) were crushed and mixed for bulk crystallographic analysis. The spatial distribution of the different CaCO3 polymorphs within vaterite otoliths was examined by Raman spectrometry, which provides information about the metal and carbonate bonds within the crystal lattice. A laser was used to excite the sample surface and the Raman effect and the distortion of the crystal lattice suite was recorded as energy spectra which were characteristic for each polymorph of CaCO3. A vaterite otolith was mounted proximal side up on a glass microscope slide and analysed with a Renishaw Raman imaging microscope using the 1064 nm NIR line for excitation. The laser power at the surface of the samples was 3 mW. The scattering volume, which is the area encompassed in a single analysis spot, was c.20mm3. The spectra were collected in the range 100–1500 cm 1 with a spectral resolution of 2 cm 1. The data were collected 40 times with an exposure time of 10 s. Additional instrument details are given by Williams et al.(1994).
ELEMENTAL COMPOSITION OF NORMAL AND VATERITE OTOLITHS Aragonite and vaterite differ in the structure and spacing of the crystal, and this can lead to differences in elemental composition. The trace element composition of whole otoliths was analysed with solution based inductively coupled mass spectrometry (SB-ICPMS). Twenty-two otolith pairs (11 pairs from each the Celtic and Clyde Sea stocks) were analysed to assess the extent of elemental variation within normal pairs. Another two pairs of otoliths, each with a vaterite and a normal otolith were then analysed to examine the differences in composition related to vaterite. Individual otoliths were dissolved in 100 ml of concentrated nitric acid and diluted in 1% nitric acid to a final dilution ratio of 1 : 5000. The dissolved samples were analysed with a PlasmaQuad 3 (VG Elemental) ICPMS. The concentrations of Sr, Ba, Mn, Li, Pb, Ni, Co and Sc were
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1386 # 03TeFseisSceyo h rts Isles, British the of Society Fisheries The 2003
TABLE I. Number of cases of vaterite otoliths in normal herring and in fish displaying head deformities (Celtic and Clyde Seas)
Number of fish Number of fish with vaterite otoliths TOMA J.
Population, year class Age range (in days) Normal Deformed Normal Deformed (% of total) ´ N .J GEFFEN J. A. AND S Celtic 1997 390 0 61 0 13 21 Celtic 1998 348–410 112 107 17 9 11 In left otolith: 20, in right otolith: 19 Clyde 1997 317 0 29 0 2 7 ora fFs Biology Fish of Journal Clyde 1998 93–351 109 183 13 10 8 In left otolith: 9, in right otolith: 14 Celtic 1999 Wild adults 36 0 2 0 5 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1387
(a) (b)
(c) (d)
FIG. 1. Aragonite and vaterite otoliths of reared juvenile herring (scale bar ¼ 1 mm) Note the similarity in shape of these otoliths, regardless of the morph or extent of vaterite area. (a) Normal aragonite otolith, (b) otolith with vaterite areas, showing the dimensions measured for morphometry analysis and (c), (d) examples of the variation between individual fish in the extent of vaterite areas. measured following the spike method. Elemental concentrations were calculated based on the standards addition method. Any elements that failed to give a linear response at any dilution or failed to give a correct spike concentration were discarded from further analysis. Due to interferences with the argon gas, Ca had to be measured with atomic absorption spectrophotometry (AAS) instead of SB-ICPMS. Samples were analysed in a Perkin-Elmer 5000 atomic absorption spectrophotometer. The final dilution ratio for the measurement of calcium was 1:50. Standards were SB-ICPMS and AAS grade from AldrichÒ. One pair of otoliths, containing a normal and a vaterite otolith, from a fish from each population (Celtic and Clyde Seas) was analysed by wavelength-dispersive spectrometry (WDS) to assess the spatial variation in composition (Ca, Sr, Na and K) between aragonite and vaterite areas of the otoliths. The composition of the inner aragonite area of the primarily vaterite otoliths was also compared to the composition of the inner aragonite sector from the normal aragonite otoliths in order to test whether initial chemical differences in the inner area of an otolith could explain the shift from aragonite to vaterite. Otoliths were individually placed on glass slides, embedded in epoxy resin and ground and polished along the sagittal plane using diamond suspensions. Samples were ultrasonically cleaned between each polishing stage and before carbon coating. Samples were analysed with a Cameca Camebax Microbeam WDS microprobe fitted with four spectrometers (Table II). Quantitative analysis of each element was achieved by discri- minating the count rates at the peak of the element’s characteristic X-rays from the continuous spectrum, before a PAP (Pouchou and Pichoir) correction. The electron beam was used in raster mode, covering an area of 8 5 8 5 mm to avoid sample damage
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1388 J. TOMA´S AND A. J. GEFFEN
TABLE II. Analytical conditions of the WDS. The limits of detection (LOD)qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi of the WDS 1 1 for each element were calculated using the formula: LOD ¼ 3m RBTB , where 1 1 1 m ¼ counts s % element in the standard ,RB ¼ count rate on background (counts s ), and TB ¼ count time on background (s, half time of count time on peak). The precision of measurement (relative S.D., %ffiffiffiffi) of each element was calculated for each sample using ÂÃp ÀÁpffiffiffiffiffiffi pffiffiffiffiffiffiffi 1 the formula: Precision ¼ 100 T RP RB ,whereT¼ count time on peak (s), 1 1 RP ¼ count rate on peak (counts s )andRB ¼ count rate on background (counts s ) Electron beam operated in raster mode 8 5 8 5 mm
Gun potential: 15 keV
Beam current: 10 nA
Distance between spots: 25 mm
Element Counting times at peak (s) Spectrometer Crystal LOD (ppm)
Calcium 30 3 PET 338 Strontium 90 2 TAP 239 Sodium 90 1 TAP 112 Potassium 60 3 PET 219
(Kalish, 1989, 1991). Otoliths were all analysed along four transects between the nucleus and the outer edge of the otolith in the posterior, ventral and dorsal sides. Concentrations of the oxide form of each element were transformed into elemental composition using the oxide : element mass ratio. Final elemental concentration was expressed in parts per million (ppm).
DATA ANALYSIS Fish growth and mass–length relationships were analysed for each population sepa- rately using regression and ANCOVA techniques, after appropriate data transforma- tions. The incidence of vaterite otoliths was compared for each population separately. The w2 statistic was corrected with the Yates correction of continuity (Scherrer, 1984) to compensate for n < 40. When n < 20 Fisher’s exact test was used instead. Otolith pairs were categorized as normal if both right and left otoliths were aragonite, and vaterite if one or both otoliths contained vaterite. All comparisons between the individual otoliths of otolith pairs (normal aragonite v. normal aragonite; normal aragonite v. vaterite) were made for all fish pooled, since the level of comparison was within individual fish and because the Celtic and Clyde Sea fish were not significantly different in the proportion of fish with vaterite otoliths. The dimensions of left and right otoliths within each pair (normal v. normal, normal v. vaterite) were compared with the t-test for paired samples to test the relationship between aberrant crystallization and otolith size.
RESULTS
CRYSTALLOGRAPHY The XRD bulk crystallographic analysis revealed that the abnormal crystal- line otoliths were composed of vaterite and aragonite. Surface analysis of the spatial distribution of the different CaCO3 polymorphs within vaterite otoliths
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1389 with Raman spectrometry indicated that both aragonite and vaterite poly- morphs were present in close proximity, within a volume of 20 mm3 (Fig. 2).
INCIDENCE OF VATERITE OTOLITHS The juvenile herring with deformed heads or jaws were generally small compared to the rest of the population. These abnormalities were observed in fish as young as 3 months old (Table I). At c. 1 year old, the normal fish were significantly longer (ANOVA, Celtic Sea: d.f. ¼ 1 and 107, P < 0 001; Clyde Sea: d.f. ¼ 1 and 90, P < 0 001) and heavier (Celtic Sea: d.f. ¼ 1 and 107, P < 0 001; Clyde Sea: d.f. ¼ 1 and 90, P < 0 001) than the deformed fish (Fig. 3). Initially it appeared that the fish with skull deformities also had a high incidence of vaterite otoliths, but in fact most had normal aragonite otoliths. The proportion of fish with vaterite otoliths was the same in both populations (w2, P > 0 05). Taking as a reference the fish sampled in 1998 when both normal and deformed fish were sampled, there were no significant differences between healthy and deformed fish in the number of cases with vaterite otoliths (w2, Celtic and Clyde Seas both P > 0 05). Vaterite deposition occurred with equal frequency in right and left otoliths of both the Celtic and Clyde Seas fish (Fisher’s exact test, both P > 0 05) showing that the phenomenon affected equally left and right otoliths in fish from both stocks. None of the youngest fish dissected (Clyde Sea, 93 days old) had vaterite otoliths, suggesting that the change in otolith formation occurred in fish >3 months old, well after metamorphosis and the development of schooling behaviour. The length–mass relationship of fish with vaterite otoliths did not differ significantly from fish with normal otoliths in Celtic
2500 35 000 Vaterite with aragonite traces Vaterite Aragonite 30 000 aragonite) 2000 +
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Relative frequency (vaterite and vaterite 0 0 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
Raman shift (cm–1)
FIG. 2. Raman spectra from the vaterite otolith of herring analysed with the Raman spectrometer. Despite displaying the characteristic peaks of the vaterite at 106, 266 and 301 cm 1, the Raman spectra from a different location on the same otolith also showed the existence of two very clear 1 3 peaks at 152 and 204 cm characteristic of the aragonite (!ç). The scattering volume was 20 mm and indicates the spatial scale at which both aragonite and vaterite may co-precipitate.
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1390 J. TOMA´S AND A. J. GEFFEN
25 (a) 20 15 10 5 Fish mass (g) 0 Healthy Deformed Healthy Deformed Celtic Clyde
160 (b) 120
(mm) 80 T
L 40 0 Healthy Deformed Healthy Deformed Celtic Clyde
FIG. 3. Differences in (a) mass and (b) total length of healthy and deformed reared juvenile herring from the Celtic and Clyde populations (mean S.D.).
(ANCOVA, d.f. ¼ 1 and 168, P > 0 05) and Clyde fish (ANCOVA, d.f. ¼ 1 and 144, P > 0 05). Thus, among the deformed fish examined, there was no further effect of otolith crystallisation on fish condition. Among the adult herring sampled in the wild in the Celtic Sea, two adults out of 36 had vaterite in their otoliths (Table I).
MORPHOMETRY OF ARAGONITE AND VATERITE OTOLITHS Left and right otoliths of normal aragonite pairs differed significantly in length only; left otoliths were longer than right otoliths (Table III). In vaterite pairs, the vaterite otoliths were significantly larger in length, perimeter and area, and were also significantly lighter than their aragonite partners (Table III). The extent of vaterite deposition was highly variable and ranged from 12 to 91% of the total otolith area (Figs 1 and 4). There was a significant relationship between
TABLE III. Summary of the statistical differences between normal (both aragonite) and vaterite (one vaterite, one aragonite) pairs of otoliths of juvenile herring from the Celtic and Clyde Sea populations. Significant differences are highlighted in bold. td, statistic of the t-test for paired samples; L, left; R, right; A, aragonite; V, vaterite
Area (mm2) Perimeter (mm) Length (mm) Width (mm) Mass (mg)
Normal pairs xL ¼ 2 276 106 xL ¼ 7566 6 xL ¼ 2504 8 xL ¼ 1307 5 xL ¼ 1 218 n ¼ 101 xR ¼ 2 277 341 xR ¼ 7237 1 xR ¼ 2490 6 xR ¼ 1310 6 xR ¼ 1 214 td ¼ 0 16 td ¼ 1 41 td ¼ 2 57 td ¼ 0 67 td ¼ 0 52 P > 0 05 P > 0 05 P < 0.05 P > 0 05 P > 0 05
Vaterite pairs xA ¼ 2 408 805 xA ¼ 7654 8 xA ¼ 2582 8 xA ¼ 1338 6 xA ¼ 1 298 n ¼ 26 xV ¼ 2 536 751 xV ¼ 9140 1 xV ¼ 2643 5 xV ¼ 1354 7 xV ¼ 1 073 td ¼ 3 18 td ¼ 6 71 td ¼ 2 56 td ¼ 1 41 td ¼ 7 58 P < 0.05 P < 0.05 P < 0.05 P > 0 05 P < 0.001
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1391 the proportion of the total otolith area occupied by vaterite and the difference in weight between the vaterite and its aragonite pair (ANOVA, d.f. ¼ 1 and 23, P < 0 05; Fig. 4). Overall, 66% of the difference in otolith mass was explained by the extent of the vaterite area. The absolute size and outer boundary of the aragonite inner area of vaterite otoliths varied between individuals, indicating that the shift from aragonite to vaterite deposition occurred at different times for each individual fish. Based on the calculated relationship between the otolith area and fish age for fish with normal otoliths, vaterite deposition commenced between 4–10 months of age (Fig. 5). No vaterite otoliths were found in 3 month old fish that were examined. There was no relationship between the size of the inner aragonite sector and the total size of vaterite otoliths, so total otolith growth was independent of when the shift to vaterite deposition occurred. Regardless of the differences in the dimensions of vaterite otoliths when compared with their respective aragonite pairs, the overall shape of the vaterite otolith was the same as that of the aragonite otolith, indicating that otolith shape and polymorph precipitation were not controlled by the same mechan- isms. The dimensions of the aragonite otoliths from each vaterite pair were also compared to the dimensions of aragonite otoliths from normal pairs to estalish whether the presence of a vaterite otolith had any effect on the growth and development of its aragonite partner. There were no significant differences in dimension between the aragonite otoliths from normal pairs compared to those from vaterite pairs (Table IV) except for the otolith perimeter, probably because the outer edge was more crenulated in some otoliths than in others. The shift from aragonite to vaterite deposition was a local phenomenon since vaterite deposition in one otolith did not affect the dimensions of the otolith growing normally in the other sacculus.
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1 otolith and its normal pair (mg)
Difference in mass between the vateritic 0.05
0 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
Per cent of vaterite replacement
FIG. 4. Relationship between the extent of vaterite areas and the difference in mass between the vaterite otolith and its aragonite pair. The curve was fitted by y ¼ 0 0036x 0 0176 ( r2 ¼ 0 66).
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1392 J. TOMA´S AND A. J. GEFFEN
450
400
350
300
250
200
150
Age (in days after hatching) 100
50
0 0.00E + 00 5.00E + 05 1.00E + 06 1.50E + 06 2.00E + 06 2.50E + 06 3.00E + 06 3.50E + 06
Otolith area (µm2)
FIG. 5. Relationship between the total otolith area and the age of juvenile herring from the Clyde Sea population only. ^, aragonite otoliths; &, inner aragonite area in vaterite otoliths. This relationship was used to estimate the age at which the vaterite starts precipitating in Clyde Sea fish. The curve was fitted by y ¼ 0 0001x þ 94 66 (r2 ¼ 0 83).
COMPOSITION OF VATERITE AND NORMAL OTOLITH PAIRS The concentrations of Ca, Sr, Co, Ni, Mn and Sc were measured using SB-ICPMS and compared between vaterite and normal otoliths. There were no significant differences in composition between the two otoliths of normal pairs. The normal and vaterite otoliths within vaterite pairs had similar con- centrations of Ca, Co, Ni and Sc, but Sr concentrations were much lower and Mn concentrations much higher in the vaterite otoliths compared to the normal aragonite otoliths (Fig. 6). These results indicate the relevance of the crystal structure in determining the final composition of the otolith.
TABLE IV. One-way ANOVA between the different otolith dimensions of the aragonite otoliths from normal pairs and from vaterite pairs. The data used to characterise aragonite pairs came from the left otoliths. A, aragonite; V, vaterite
d.f. source MS source d.f. error MS error FPSNK test
Area 1 364 101 106 125 181 253 106 2 00 >0 05 – Perimeter 1 3 117 559 125 767 860 4 06 <0 05 V > A Length 1 125 867 125 82 429 1 52 >0 05 – Width 1 20 068 125 12 681 1 58 >0 05 – Mass 1 0 13 125 0 08 1 48 >0 05 –
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1393
50 000 1000 60 (a) (b) (c) 45 000 50 40 000 800 ) –1 ) ) g 35 000 –1 –1 g 40 g g
µ g g 30 000 600 µ µ
25 000 30
20 000 400 20 Calcium ( 15 000 Nickel 60 ( Strontium 88 ( 10 000 200 10 5000 0 0 0 Left Right Left Right Left Right Left Right Left Right Left Right Normal Vaterite Normal Vaterite Normal Vaterite otoliths otoliths otoliths otoliths otoliths otoliths
0.04 7 0.8 (d) (e) (f) . 6 0 7 ) ) 0.03 –1 0.6 –1 ) g 5 g
–1 g g g µ . µ
g 0 5
µ 4 0.02 0.4 3 0.3
Cobalt 59 ( 2 0.01 0.2 Scandium 45 ( Manganese 55 ( 1 0.1
0.00 0 0.0 Left Right Left Right Left Right Left Right Left Right Left Right Normal Vaterite Normal Vaterite Normal Vaterite otoliths otoliths otoliths otoliths otoliths otoliths
FIG. 6. SB-ICPMS results of the analysis of aragonite and vaterite herring otoliths (means S.D.). Vaterite samples are presented as left otoliths for convenience of representation. (a) Calcium, (b) strontium, (c) nickel, (d) cobalt, (e) manganese and (f) scandium.
SPATIAL VARIATION IN THE ELEMENTAL COMPOSITION OF VATERITE OTOLITHS The chemical transition between the aragonite part and the vaterite part of the otoliths was sharply delineated. WDS analyses along transects from the core to the outer edge of the otolith showed conclusive evidence that Sr, Na and K concentrations were much lower in the vaterite part of the otolith. Only 51% of the Sr measurements and 86% of the K measurements were above the analytical detection limits (Table II) in the vaterite area of the otoliths (Figs 7 and 8). The concentrations of Ca, Sr, Na and K at 15 randomly chosen spots within each of the aragonite and vaterite parts of the otoliths were compared. Vaterite areas of the otoliths had significantly lower concentrations of Sr, Na and K than aragonite areas (ANOVA, Sr: d.f. ¼ 1 and 58, P < 0 001; Na: d.f. ¼ 1 and 58, P < 0 001; K: d.f. ¼ 1 and 58, P < 0 001) whereas Ca concentrations did not show significant differences (d.f. ¼ 1 and 58, P > 0 05) (Fig. 9). There were no significant differences in Ca, Sr, Na or K concentrations between the inner aragonite part of the vaterite otoliths and the equivalent inner part of the normal otoliths (ANOVA, d.f. ¼ 1 and 61, all P > 0 05), indicating that the shift from aragonite to vaterite is independent of the elemental composition of the inner otolith area.
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1394 J. TOMA´S AND A. J. GEFFEN
(a) 2000 400 000
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(b) 2000 400 000
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1600 325 000 Sr, Na and K concentrations (ppm)
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Distance from the nucleus (µm)
& ~ FIG. 7. WDS results of the analysis ( &, Na; , Sr; &,K; , Ca) of an otolith pair from a reared herring from the Celtic Sea population. (a) Aragonite and, (b) vaterite otoliths. Horizontal lines represent the limits of detection (LOD) of the WDS measurements for Ca, Sr, K and Na from top to bottom under the analytical conditions detailed in Table II.
DISCUSSION
The occurrence of different CaCO3 polymorphs in otoliths of teleosts is well documented (Gauldie, 1993), but this is the first report of vaterite otoliths in C. harengus. Vaterite and calcite otoliths have been observed both in reared
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1395
(a) 3000 400 000
350 000 2500
300 000 2000 250 000
1500 200 000
150 000 1000
100 000 500 50 000
0 0 0 100 200 300 400 500 600 700 800 900
(b) 3000 400 000
350 000
2500 Ca concentration (ppm)
300 000 Sr, Na and K concentrations (ppm) 2000 250 000
1500 200 000
150 000 1000
100 000 500 50 000
0 0 0 100 200 300 400 500 600 700 800 900 Distance from the nucleus (µm)
& ~ FIG. 8. WDS results of the analysis ( &, Na; , Sr; &,K; , Ca) of an otolith pair from a reared herring from the Clyde Sea population. (a) Aragonite and, (b) vaterite otoliths. Horizontal lines represent the limits of detection (LOD) of the WDS measurements for Ca, Sr, K and Na from top to bottom under the analytical conditions detailed in Table II. species such as chinook salmon Oncorhynchus tshawytscha (Walbaum) (Gauldie, 1986, 1996) and lake trout Salvelinus namaycush (Walbaum) (Bowen II et al., 1999), and in fishes from wild populations (Morales-Nin, 1985; Strong et al., 1986; Gauldie, 1993; Brown & Severin, 1999). In the present study, the frequen- cies of vaterite otoliths of juvenile herring (13 9% in the Celtic Sea population and 7 8% in the Clyde Sea population) were lower than those reported in studies of other reared species [34% for juvenile chinook salmon (Gauldie, 1986, 1996), 26–41% for stocked lake trout (Bowen II et al., 1999)] but still
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1396 J. TOMA´S AND A. J. GEFFEN
800 4.5e5 (a) (b) 700 4e5 . 600 3 5e5 3e5 500 2.5e5 400 * 2e5
K (ppm) 300 Ca (ppm) 1.5e5 200 1e5
100 50 000 0 0 Aragonite Vaterite Aragonite Vaterite
2500 2000 (c) (d) 2000 1500
1500 * 1000 1000 Sr (ppm) * Na (ppm) 500 500
0 0 Aragonite Vaterite Aragonite Vaterite
FIG. 9. Comparisons of the composition (a) K, (b) Ca, (c) Sr and (d) Na (means S.D.) of the inner aragonite area and the outer vaterite area in the otolith pairs analysed with the WDS. *, Significant differences (P ¼ 0 05) between means. higher than the range reported for samples of fishes caught in the wild [2–5% in pollock Pollachius virens (L.) (Strong et al., 1986), 3% in Genypterus capensis (Smith) (Morales-Nin, 1985) and between 0 4 and 14% in a variety of species (Gauldie, 1993)]. Vaterite otoliths were found in reared red drum Sciaenops ocellatus (L.), but not in wild juveniles (David et al., 1994). In the present study, only two of the 36 (5 5%) adult herring from the wild caught in the Celtic Sea had vaterite otoliths. This was a smaller percentage than found in the juvenile herring of the same population reared in the laboratory (13 9%), but could simply be due to the selective mortality between juvenile and adult stages rather than the result of rearing conditions. Otolith malformations ultimately express otic malformations, which could also be the expression of malfunctioning genes (Gauldie, 1986). Complications during the development of the neural crest could result in otolith malformations together with deformities in the head of the fish. Gene loci mutations that cause malformations in the vestibular and auditory apparatus, including the otoliths, have been mapped on the zebrafish genome (Malicki et al., 1996; Whitfield et al., 1996). These zebrafish malformations were expressed during embryonic devel- opment, but in this herring study most vaterite otoliths had an aragonite core and juveniles sampled 93 days after hatching all had normal otoliths. The individual variation in age at the shift to vaterite deposition indicates that it is not the result of a pre-programmed disruption associated with ontogeny. External factors including stress, food quality, or viral infection might induce the expression of mutated genes. The fact that the composition of the aragonite portion of the vaterite otoliths did not differ from the same sector in normal
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1397 otoliths strongly suggests that the development of the otolith had been normal up to that point.
EFFECT ON FISH The vaterite otoliths were bigger in area, perimeter and length than their aragonite pairs, probably because the vaterite crystal system is larger than the aragonite crystal system. Wardlaw et al. (1978) calculated that a molecule-by- molecule replacement of aragonite by calcite in the shells of a marine gastropod, Strombus gigas resulted in an 8% increase in volume. The vaterite otoliths still held the characteristic herring shape, as observed in the vaterite and calcite otoliths of other species (Morales-Nin, 1985; Gauldie, 1986; Strong et al., 1986). The different CaCO3 polymorphs may affect the functioning of the inner ear since otolith function is determined by density, size and shape. Vaterite deposi- tion resulted in lighter, but larger, otoliths. A normal otolith has a density of c. 3gcm 3 (Fay & Simmons, 1999), corresponding to the density of aragonite. Inorganic vaterite is less dense (2 54 g cm 3) than inorganic aragonite (2 94–2 95 g cm 3) and the results of this study confirm that biovaterite is also less dense than bioaragonite. The lower density of vaterite otoliths could pro- duce a delayed response of lower intensity compared to an aragonite otolith. Density differences between the left and right otoliths could impair the detection of sound (Popper & Lu, 2000). In reality, there was little influence of aberrant otoliths on the growth of juvenile herring used in this study, since the growth of deformed fish with normal otoliths was no different from that of deformed fish with aberrant otoliths. Deformed fish grew more slowly that the normal looking fish, but the frequency of occurrence of vaterite otoliths was not different between normal and deformed fish. This indicates that the crystal form of the otolith was not directly linked to fish growth, nor did skull deformities directly result in vaterite deposition. Low growth was caused by the deformities in the head rather than by otolith deformities, probably because the deformities in the mouth, gills and jaws prevented the fish from eating properly. Herring use both visual and acoustic signals for feeding and predator avoidance (Blaxter & Fuiman, 1990). Schooling behaviour may also counteract any effects of asymmetry in the otolith density. Herring schools in the wild display a direc- tional avoidance away from a sound source (Olsen, 1976). Individual herring with vaterite otoliths may rely on visual cues and the response of the school to access food and avoid danger. It may be that fish survival is not dependant on the saccular otoliths, and the fishes have other sensory organs to sense the surrounding environment and may learn to compensate for the differences in the information received by one of the two ears (Lombarte et al., 1993). In some fish species there is a significant relationship between the area of the otolith and the area of the sensory epithelia (Lombarte & Fortun˜o, 1992; Arellano et al., 1995), indicating that certain otolith dimensions are important for the correct functioning of the inner ear. Moreover, the heterogeneity of hair cells in the sensory epithelia has led some authors to consider that different parts of the otolith are involved in the perception of sound of different frequencies (Popper et al., 1993). Vaterite
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 1398 J. TOMA´S AND A. J. GEFFEN otoliths have probably more ‘to say’ about the mechanisms of otolith mineral- ization than about functional anatomy.
DIFFERENCE BETWEEN VATERITE AND ARAGONITE In terms of the microchemistry of the otoliths, there was a dramatic change in composition between the aragonite and vaterite areas of the otoliths. The concentrations of Na, Sr and K were significantly depleted in the vaterite areas, though the decreases in Na were less marked. These elements were also depleted in the vaterite otoliths of Stenodus leucichthys (Gu¨ldensta¨dt) (Brown & Severin, 1999) and chinook salmon (Gauldie, 1996). The change in composition of the vaterite compared to aragonite otolith sections could be explained by the geometry of the crystal, resulting in molecular conformations with little room for inclusion of other elements (Curti, 1999). Sr (1 13 A˚) and K (1 33 A˚) have larger ionic radiuses than Ca (0 99 A˚) and would be included less readily, whereas Na (0 95 A˚) has a smaller ionic radius and its inclusion may be less affected by the shift from aragonite to vaterite. Sodium ions may be trapped in vaterite crystals defects (Gauldie, 1986; Brown & Severin, 1999) rather than substituting for Caþ ions (Busenberg & Plummer, 1985). Manganese was more concentrated in the vaterite otoliths than in the aragonite otoliths and has been observed to be strongly associated with vaterite precipitation and is used as a tracer of vaterite to calcite transformation (Nassrallah-Aboukais et al., 1996, 1998). Manganese is incorporated into the vaterite crystal lattice (Brecevic et al., 1996) by adsorption followed by surface precipitation, forming a ‘manganese coating’ that decreases the solubility of the vaterite (Nassrallah-Aboukais et al., 1998) preventing its dissolution. It is soluble at low pH (Fraser & Harvey, 1982; Moreau et al., 1983) and its presence in the vaterite otoliths may indicate pH and osmoregulatory changes that could also facilitate a reduction in Ca influx and in Na efflux (Reader & Morris, 1988). There was no evidence to suggest that changes in the composition of the aragonite areas of the otolith caused the shift to vaterite deposition since the concentrations of Ca, Sr, Na and K in the inner aragonite sector of vaterite otoliths were the same as the equivalent sector of the aragonite pair. The shift was a localized phenomenon because it happened generally only in one ear and sometimes only in a certain region of the otolith. The physico-chemical condi- tions of the endolymph may be variable around the otolith and allow vaterite and aragonite to precipitate concurrently at the small spatial scale revealed by the Raman analyses. In most fishes with aragonite otoliths the asteriscii are entirely vaterite (Chesney et al., 1998; Campana, 1999), indicating that the shared endolymph (Gauldie, 1993) is capable of producing the aragonite sagitta and vaterite asteriscus simultaneously, even though controlled by independent mechanisms (Riley & Grunwald, 1996). In conditions of high solubility vaterite is precipitated in preference to aragonite or calcite (Dalas et al., 2000). Loca- lized changes in the composition of the endolymph may affect the solubility of the solution at the surface of the growing otolith. In vitro experiments (Falini et al., 1996) have shown that vaterite co-precipitates with aragonite in the presence of aragonite-coding proteins, when the ion flux at the surface of
# 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1383–1401 HERRING VATERITE OTOLITHS 1399 precipitation is reduced resulting in small scale spatial differences similar in scale to those observed in the otolith using the Raman spectrometer. Since the shape of the otoliths was unaltered by polymorph replacement, the possibility that the vaterite replacement had started from the outside of the otolith to the inside was considered, so that diagenesis (a process of recrystal- lisation) explained the presence of vaterite or calcite. Diagenesis (of aragonite into calcite) occurs in fossils subject to pressure, temperature or pH influence over a geological time scale (Wardlaw et al., 1978), but also as an instantaneous process as demonstrated by immersing fossil and living coral in a solution of sodium chloride (Yoshioka et al., 1985). Nonetheless, since diagenesis can be traced by the unequivocal high Sr concentrations that remain in the aragonite when it changes to calcite (Wardlaw et al., 1978) and the present results showed that vaterite areas had very low Sr concentrations, diagenesis was not likely to be the cause of vaterite in the herring otoliths. The comparisons between vaterite and aragonite otolith areas also demon- strated problems inherent with WDS sampling of CaCO3 of different densities. Vaterite and aragonite otoliths differed in density, yet the WDS was not able to show this difference in absolute Ca concentrations. This is explained by the fact that concentrations are reported in ppm in mass not in volume, and so the amount of calcium in the vaterite is the same as in the aragonite (40%), as is the amount of carbonate (60%). For a given accelerating voltage, electrons penetrate deeper into a vaterite sample than in an aragonite sample and the volume sampled is greater in vaterite than in aragonite. The method of correc- tion of count rates of the WDS used in this study (the Pouchou & Pichoir method and by extension any ZAF correction procedure) cancels out any effects of sample density, and thus is insensitive to differences between vaterite and aragonite. Consequently, the analytical results from surface analysis techniques such as the WDS cannot be used to study differences in otolith density.
We are indebted to P. Hill (Faculty of Geology and Geophysics of the University of Edinburgh, U.K.) for access to the NERC funded WDS facility and to the Faculty XRD facility, and for valuable discussions on the use of the WDS. We thank R. Johnston (Strix Ltd., Castletown, Isle of Man) for the Raman analyses. Thanks are also due to N. Fullerton for supplying the reared fish from the Larval Rearing Centre of the Port Erin Marine Laboratory and to F. McArdle (Royal Hospital, University of Liverpool) for the ICPMS analyses. The manuscript was considerably improved by the constructive comments made by G. Pilling.
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