Morphometry and Composition of Aragonite and Vaterite Otoliths of Deformed Laboratory Reared Juvenile Herring from Two Populations
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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 7Á8% in the Clyde population to 13Á9% in the Celtic Sea population, and was 5Á5% 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 Æ 0Á001 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 cmÀ1 with a spectral resolution of 2 cmÀ1. The data were collected 40 times with