Mineralogical approaches to the study of ... - IngentaConnect

Mineralogical Magazine, April 2008, Vol. 72(2), pp. 627–637

Mineralogical approaches to the study of biomineralization in fish otoliths S. MELANCON1,*, B. J. FRYER1, J. E. GAGNON1

AND

S. A. LUDSIN2

1

Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario N9B 3P4, Canada Aquatic Ecology Laboratory, Department of Evolution, Ecology and Organismal Biology, The Ohio State University, 1314 Kinnear Rd., Columbus, Ohio 43212, USA

2

[Received 13 November 2007; Accepted 4 June 2008]

ABSTR ACT

This paper highlights new research on the biomineralization of otoliths and uses a mineralogical approach to understand mechanisms of crystal growth and metal incorporation into otoliths. Petrographic observations of the nucleation of otolith growth in the core for several fish species reveals that sagittal otoliths appear to nucleate around a few or many nucleation sites (primordia) and that these sites vary in size (ranging in diameter from 1 to 20 mm), depending on the species. Spectroscopic data show a large Mn-enrichment in the primordia within the core but the reasons for this enrichment are still unclear (e.g. organic matter or possibly another material other than CaCO3). This study also provides the first multi trace-element data for endolymph fluid and the growing otolith; we found large enrichments (Ca and Sr) and depletions (Na, K, Zn and Rb) of elements in the otolith relative to the endolymph. The last part of this paper examines the effect of crystal structure on the microchemistry of otoliths. Our investigation helps understand how the chemical characteristics of the metal ions (i.e. ionic radii) and the crystalline structure interact to cause differential trace-metal uptake between the CaCO3 polymorphs, aragonite and vaterite. K EY WORDS : otoliths, trace metals, endolymph, nucleation, biomineralization, CaCO3 polymorphs.

Introduction TELEOST (bony) fish have three pairs of otoliths (sagittae, asteriscii and lapilli) that are used for balance and hearing. Although otoliths are mainly composed of calcium carbonate (CaCO 3 ; 97 99%), other metals can also be incorporated in the crystalline structure as impurities. Otoliths crystallize from fluid (endolymph) within the endolymphatic canal of the inner ear. This fluid predominantly contains ions of calcium, carbonate and bicarbonate, dissolved inorganic carbon (DIC), and trace metals. A small, but nonnegligible, presence of proteins and other

* E-mail: [email protected] DOI: 10.1180/minmag.2008.072.2.627

# 2008 The Mineralogical Society

organic materials is also found in the endolymph and otoliths (Payan et al., 1997). Otoliths have three properties that have made them valuable for fisheries-related research and management. First, the continual growth of otoliths is recognizable as concentric rings of alternating opaque and translucent zones (Fig. 1), which allows for individuals to be aged on both daily and annual time scales. Second, otoliths are not subject to resorption or chemical reworking, unlike other fish calcified hard-parts (e.g. scales, bones). Third, the microchemical composition of otoliths can reflect the ambient chemical environment in which an individual resides. Within the past decade, fishery scientists have used these time-keeping, structural and microchemical properties for numerous fisheries-related purposes, including helping to understand stock (subpopulation) structure and mixing, temperature

S. MELANCON ET AL.

found that the elemental composition of the otolith core can be fundamentally different than in other (non-core) parts of the otolith (Ruttenberg et al., 2005; Brophy et al., 2004). More specifically, the chemistry of the core has been characterized by a Mn enrichment, which could be related to the amount of organic matrix present at the beginning of biomineralization, the presence of another mineral at the core, and/or a maternal transfer during spawning (Brophy et al., 2004; Ludsin et al., 2006). This current conceptualization of how otoliths grow and biomineralize would suggest that the microchemical composition of the otolith core (during egg sac resorption) should be out of equilibrium with the ambient environment. In turn, this could be problematic for fisheries management investigations that attempt to apply otolith microchemical approaches, given that most use the otolith core to define the natal environment (Campana et al., 2000; Secor et al., 2001; Brophy et al., 2003; Warner et al., 2005; Chittaro et al., 2006). Herein, we document and present examples of: (1) the spatial distribution of the regions of large Mn concentrations in relation to the position of multiple primordia in the otolith core (initial nucleation); (2) the first multi trace-element data of endolymph fluid and growing otoliths; and (3) the effect of crystal structure on the microchemistry of otoliths. Ultimately, by increasing our understanding of the mechanisms underlying otolith structure and growth, we seek to improve the ability of scientists to use otolith microchemical investigations for fisheries-related research and management.

FIG. 1. Larval yellow perch otolith showing daily growth increments and multiple nucleation sites (primordia) in the core. The core is defined as the region before the first ring (hatch check).

and salinity histories of both modern and ancient oceans, migratory patterns of individuals and populations, and variability in life-history strategies (see reviews by Campana, 1999 and Thresher, 1999). Despite the recent, widespread use of otolith microchemistry in fisheries-related investigations, some unresolved issues remain, which have limited use of this tool to its fullest potential. For example, a complete explanation of physiological and chemical processes that determine the microchemical composition of otoliths is lacking. Thus, while previous research has identified that metals in the water must pass through many biological barriers (e.g. water-gills, gills-blood system, blood system-endolymph, endolymphotolith) before being incorporated into the otolith (Campana, 2005, 1999; Begg et al., 2005), our understanding of how metals are partitioned (fractionated) at each barrier remains enigmatic. In turn, this knowledge gap has limited our ability to understand why imbalances exist between the otoliths and their ambient environment for some elements. A significant knowledge gap also exists with regard to the processes of otolith nucleation and growth, including how otolith biomineralization is initiated. Our current belief is that a cluster of particles, originally of an organic-matrix origin, sets the first stage for otolith biomineralization (Nicolson, 2004). Previous studies have also

Materials and methods Part 1: nucleation of otolith growth Larval fish used in this study were collected throughout the Laurentian Great Lakes Basin as part of other ongoing studies. Larval rainbow trout (Oncorhynchus mykiss) were collected in 2006 from tributaries of Lake Huron. Larval yellow perch (Perca flavescens) were collected in the western Lake Erie during 2004 2006 (see Ludsin et al., 2006 for details). Lake cisco larvae (Coregonus artedi) from Lake Superior were provided by the USGS Great Lakes Science Centre. Specifically, lake cisco larvae were collected from the Apostle Islands region and in Minnesota waters (western arm) during 2005, and Thunder and Black bays during 2006 (Table 1).

628

BIOMINERALIZATION IN FISH OTOLITHS

TABLE 1. Biological data for this study including the number of fish analysed for each species (N), their life stage and the approximate number of nucleation sites (primordia) in the core. We also specify for which section of the paper each fish/es were used: 1 for nucleation of otolith growth; 2 for flux of metals to the growing otolith; and 3 for effect of crystal structure on element partitioning. ————— Fish ————— Common name Scientific name Lake cisco Lake trout Rainbow trout Yellow perch Walleye

Coregonus artedi Salvelinus namaycush Oncorhynchus mykiss Perca flavescens Sander vitreus

N

Life stage

Primodia

Section

20 13 10 >50 8

Larval Adult Larval Larval Adult

many N/A 6 11 2 4 N/A

1 3 1 1 2

Otoliths from larvae were removed and cleaned in a Class 100 clean room following the procedures of Ludsin et al. (2006), which consisted of multiple cleaning steps using only acid-washed glass instruments. In brief, otoliths were removed with glass probes while immersed in a drop of ultrapure Milli-Q water on a glass microscope slide. Afterwards, otoliths were rinsed with Milli-Q water and sonicated for 5 min in an acid-washed Petrie dish floating on top of a Milli-Q water bath located within an ULTRAsonik cleaner (model 57X; Ney Dental, Inc., Bloomfield, Connecticut). Afterwards, otoliths were rinsed in triplicate with ultra-pure Milli-Q water and mounted to double-sided tape on an acid-washed microscope slide for analysis. Part 2: flux of metals to the growing otolith Endolymph analysis Heads from eight frozen Walleye (Sander vitreus), collected by commercial fishers in western Lake Erie during June 2005, were used to explore metal partitioning among ambient surface water, endolymphatic fluid and otoliths (Table 1). Endolymph and otoliths were removed in the laboratory from partially frozen heads (~5 h of thawing). The skull was opened with a frontal incision behind the eyes and the saccular membrane was exposed. Liquid (mixture of body fluids and blood) surrounding the saccular sacs were removed using Kimwipes1 and water before the membrane was perforated. We then collected fluid from both the left and right endolymphatic sacs using 10 ml micropipettes (Kimble#) and extracted the sagittal otoliths. The fluid was transferred into an acid-washed Teflon1 PFA container for heavy metals analysis, digested to remove the organic fraction using a mixture of 629

trace-metal grade 8N HNO3 and H2O2 (30%), and then diluted with an internal standard (Be-, Inand Tl-spiked matrix diluted in 1% HNO3). Both left and right endolymph samples were analysed with solution-based inductively coupled plasma-mass spectrometry (SO-ICP-MS), using a Thermo-Elemental1 X71 ICP mass spectrometer. A paired t test and a Boneferroni correction were performed for each element to see if differences existed between the two saccular endolymph sacs. All solution samples and standards were spiked with our internal standard solutions (1% HNO3 +Be, In, Tl) to correct for matrix and drift effects on an individual sample basis. Using SO-ICP-MS, we measured the following isotopes: 23Na, 24Mg, 25Mg, 39K, 43 Ca, 44Ca, 55Mn, 66Zn, 85Rb, 86Sr, 88Sr, 137Ba and 138Ba. Otolith analysis Walleye otoliths were embedded in epoxy resin (West Coast Marine1) and transverse sections (~350 mm wide) were cut using a Buehler ISOMETTM saw. After mounting sections to a piece of overhead transparency sheet with Krazy Glue1, the upper surface was polished using a combination of 20-, 12-, 1- and 0.3-mm aluminium oxide 3M1 lapping film. Sections were then mounted (with a small piece of the transparency sheet) onto acid-washed glass slides with Krazy Glue1 and left to dry for 24 h. Slides were then sonicated for 10 min in ultrapure Milli-Q water, rinsed three times in Milli-Q water, dried for 24 h in a HEPA-filtered laminar flow hood and then stored in a covered Petrie dish until analysis. All post-polishing processing (including storage) occurred in a Class 100 clean room, and only non-metallic instruments were used to handle otoliths.

S. MELANCON ET AL.

Elemental concentrations of the otoliths were quantified by LA-ICP-MS using a purpose-built laser sampling system comprised of a Continuum1 Surelite1 I solid state Nd:YAG laser at a wavelength of 266 nm (maximum power: 40 mJ; pulse rate: 20 Hz; pulse width: 4 6 ns; laser spot diameter: 32 mm) coupled to the Thermo-Elemental1 X71 ICP-MS (peakjumping mode, 10 ms dwell time per isotope). We analysed the elements mentioned previously using SO-ICP-MS. A series of ablations were performed at the growing otolith edges which were in contact with the endolymph and represent the last 30 60 of the animals life (last ring). Calcium was used as an internal standard to correct for variations in the amount of material ablated (i.e. ablation yield).

Results and discussion Part 1: nucleation of otolith growth Microscopic observations Transmitted light microscopic observations of the otolith cores for all of our species demonstrate that sagittal otoliths appear to nucleate around a few (Fig. 1) or many (Fig. 2) nucleation sites or primordia (see also Campana and Neilson, 1985 for a review). These nucleation sites can vary in size both within and among species, ranging in diameter between ~1 and 20 mm. They are also optically different from the surrounding CaCO3 (aragonite) material, with the primordium appearing much darker. Nicolson (2004) described the beginning of these nucleation sites as clusters of particles attaching to the first sensory hair cells, with these clusters setting the first stage of biomineralization by incorporating organic-matrix molecules and starting to crystallize CaCO3. In a cross section through a yellow perch otolith core (Fig. 1), three to four primordia were observed, each no larger than 1 mm3. They appeared suspended in the CaCO 3 matrix deposited before the larval fish began to feed (represented by the first growth ring). The first daily ring was deposited ~10 mm from the nearest primordia leading to a shape that was not concentric. For several days after growth was initiated, the effect of the original primordia can be seen in the daily growth rings. Rainbow trout otolith cores were larger than yellow perch cores, being distributed across a

Water chemistry analysis During summer 2002, we collected surface water samples in five locations within the Western Basin of Lake Erie to characterize Na, Mg, K, Ca, Mn, Zn, Sr and Ba concentrations (Rb was not analysed). Water samples were collected in duplicate at each site and stored in acid-washed bottles after being filtered through a 0.45 mm membrane. The filtered samples were then acidified at a ratio of 1% with trace-metal grade concentrated HNO 3 , and refrigerated until analysis. All water analyses were conducted using IRIS Advantage inductively coupled plasma-optical emission spectroscopy (ICP-OES). The analytical wavelengths used for Mn, Mg, Zn, Fe, Ca, Na, Sr, Ba and K were 257.16, 285.213, 213.856, 259.940, 194.006, 589.592, 421.552, 455.403 and 766.490 nm, respectively. Three on-peak integrations with 10 s intervals and one off-peak integration were used. The internal calibration standards were certified ICP single-element standards traceable to a NIST Standard Reference Material. Calibration standards and calibration blanks were run every 10 samples. Part 3: effect of crystal structure on element partitioning Analyses were conducted on 13 adult lake trout (Salvelinus namaycush) (age 11) that spent their entire existence in the Allegheny National Fish Hatchery in Warren, Pennsylvania (Table 1). The techniques used to polish otoliths and configurations for the LA-ICP-MS are the same as for Part 2. Raman spectroscopic information can be found in Melancon et al. (2005).

FIG. 2. Rainbow trout otolith showing multiple nucleation sites (primordia) in the core, as well as subsequent growth initiated around the individual primordia. Some of the primordia are identified.

630


band ~100 mm wide and 20 mm across in plan view (in between the arrows in Fig. 2). This particular otolith shows 6 to 9 superposed primordia that range from 10 to 20 mm in diameter, with the first 5 to 10 daily rings clearly growing concentrically around the multiple original nucleation sites (Fig. 2). All of our rainbow trout otolith samples presented this phenomenon and exhibited 6 to 11 observable primordia in the sections of the core (Table 1). Because of the significant lateral (roughly planar) distribution of primordia in rainbow trout otolith cores, the otolith shape was disclike for rainbow trout larvae, unlike larval yellow perch otoliths which were spherical (Fig. 1). These differences between species in terms of shape and numbers of primordia were observed for all the otolith samples for both species (Table 1). Microchemistry of the otolith core The cores of otoliths are comprised of material deposited prior to active feeding and are considered to represent the portion of the otolith formed at the spawning site while the egg sac is being absorbed (Ruttenberg et al., 2005). Hence, otolith cores should be fundamentally different to other parts of the otolith in terms of microchemical composition. Indeed, Mn enrichment of the core, attributed to a maternal signature, has been observed by various researchers for many freshwater and marine fish species (Hedges, 2002; Brophy et al., 2004; Bartnik, 2005; Ruttenberg et al., 2005; Ludsin et al., 2006). Most of these studies used LA-ICP-MS spot analyses of 30 to 50 mm diameter which corresponded to simultaneous sampling of all or most of the core material. Our more detailed analysis of otolith cores, using a 6 mm laser spot size and laser traverse rate of 4 mm/s, allowed us to obtain higher spatial resolution data of Mn concentrations. Our LAICP-MS results for yellow perch otoliths show a single, sharp Mn concentration spike in the core centre (Fig. 3a), with no obvious changes being apparent for other elements such as Ba and Sr (Ludsin et al., 2006). For yellow perch otolith cores, the few primordia were closely spaced and much smaller (~1 mm3 each) than the traversing laser spot diameter (6 mm). Considering a laser sampling depth of ~10 mm and a 6 mm spot size, the volume of material being sampled (V = pR2h) by the laser is substantially greater than a single primordia. In turn, our calculated Mn concentration at the centre of the core (207 ppm; Fig. 3a)

would be significantly underestimated (100 to 300 fold). We also found that the cores of larval lake cisco otoliths were enriched in Mn (Fig. 3b). The Mn concentrations exhibit three maxima in the core area and a broader zone of Mn enrichment (relative to yellow perch), with a maximum concentration calculated at 4130 ppm. The prevalence of multiple, large and widelydispersed primordia in lake cisco correspond to

FIG. 3. Representative concentration profiles of Mn (ppm) in otoliths of (a) larval yellow perch and (b) larval cisco. Distance is from one edge of the otolith to the other with the laser transect passing through the middle of the otolith (Mn peaks in core).

631

S. MELANCON ET AL.

what we (and others) have found for other salmonines, e.g. rainbow trout (Fig. 2). Unfortunately, we did not achieve the resolution needed to present laser data for the core (and multiple primordia) of rainbow trout. Based on the coherence of the petrographic and microchemical observations in otolith cores of numerous unrelated species, we suggest that the seemingly anomalous Mn peaks observed are related to the formation, mineralization and micro-distribution of primordia rather than environmental factors or the natal core region in general. The strong Mn enrichment in the primordia could be associated with the large amount of organic matrix present at the beginning of aragonite biomineralization or perhaps the initial formation of a separate mineral phase (e.g. rhodochrosite, MnCO3), within the primordia. No spectroscopic information is currently available to test these hypotheses; however, our observed (large) Mn concentrations in otolith primordia does rule out Brophy et al.’s (2004) hypothesis that a calcite precursor is the source of nucleation in otoliths. Our deductions are supported by conclusions from Ferna`ndez-Dıáz et al. (2006), which indicate that smaller ions than calcium (e.g. Mn2+) can replace Ca2+ in the rhombohedral crystal structure of calcite; however, this can only occur in small amounts (not thousands of ppm) or it would strongly change the morphology and surface structure of the crystal (Temmam et al., 2000). The seeding of otolith growth has been studied by several groups (e.g. Riley et al., 1997; Pisam et al., 2002; Nicolson, 2004), and has determined that, for zebrafish (Danio rerio) larvae, otolith growth is initiated at 18 18.5 hours (post egg fertilization) by the local accretion of precursor glycogen particles at the ends of developing tether hair cells, usually 2 per otolith (Riley et al., 1997). This accretion of precursor particles, which fuse to form the early otolith material, is completed within 24 h. These otolith seed particles are presumably the primordia observed petrographically in otolith cores (Figs 1 and 2), which appear to be associated with the Mn enrichment. The localized Mn enrichment must only be associated with the earliest stage(s) of biomineralization in the core. Interestingly, certain otoconial deficiencies in mice caused by mutations can be corrected by dietary supplements of Mn and Zn (Riley et al., 1997). This result suggests that there may be a link between Mn segregation with the primordia and the beginning of aragonite crystallization in the otolith.

Part 2: flux of metals to the growing otolith Water-endolymph-otolith equilibrium in Walleye (Sander vitreus)

632

All post-larval otolith growth occurs within the endolymphatic canal of the inner ear, with the otolith crystallizing from a fluid (endolymph) that is chemically connected to the environment (water/food) through the blood system. The endolymph contains ions of Na, Ca, carbonate, bicarbonate, trace metals and organic forms of carbon (e.g. proteins) from which the growing otolith extracts these trace elements. Despite its obvious importance in understanding otolith microchemistry, few studies have considered the relationships between otolith chemistry and endolymph (-water) chemistry (see Kalish, 1991). All the previous investigations on endolymph chemistry focused on organic compounds (proteins, glucose, phosphate, triglycerides, etc.) and non-metals (C, CO2, Cl, S). Metal analyses were seldom incorporated in these studies, with the exception of Na, Mg, K, Ca and Sr (Payan et al., 1997, 2002; Guibbolini et al., 2006). Our analysis of Lake Erie Walleye found all elements to be statistically identical in both left and right endolymph fluids (paired t test: all p 50.30), supporting similar findings by Payan et al. (1997). As such, we averaged left and right endolymph data for each individual in subsequent analyses. We also found no linear relationship between fish age and the endolymph and otolith composition for any elements (p 50.27; not shown). The following results, however, need to be viewed with some caution, owing to potential issues with preservation effects. Milton and Chenery (1998), for example, found that Na, Mg and Ba concentrations of otoliths left in heads could be altered when those heads were preserved in ethanol for several weeks. These authors also found that otoliths removed after the fish were frozen for a period of 24 h had larger Na, Mg, Co and Ba concentrations as compared to the other storage treatments. However, endolymph was not analysed in that study and we are unaware of other papers that have investigated how preservation method affects endolymph composition. Even if endolymph and otolith composition were affected, we are optimistic that the effects would be minor, given that our fish were processed within 3 days post-collection and stored frozen (not in ethanol). Observations with juvenile Walleye otoliths support this conclusion, finding


little difference between otolith elemental (Sr, Ba) concentrations for otoliths stored frozen vs. in ethanol for prolonged periods of time (Hedges et al., 2002). Partition coefficients were calculated for each metal between water, endolymph and otoliths: KD KD KD

= [Metal]endolymph/[Metal]water = [Metal]otolith/[Metal]endolymph (o/w) = [Metal]otolith/[Metal]water (e/w) (o/e)

(1) (2) (3)

Our results show strong enrichment of Na, K, Zn and Ba from the water to the endolymph, all with KD (e/w) >> 1 (Table 2). Na, K and Zn are highly-regulated elements in fish (Campana, 1999), which helps to account for their large KD values in freshwater fishes (small aqueous concentrations). However, the strong enrichment of Ba is surprising. Other elements including Mg, Ca and Mn (except for Sr) had similar concentrations in water and endolymph (KD (e/w) & 1; i.e. they were not enriched). Partition coefficients between the growing otolith edge (Fig. 4) and water for all elements (except Mg) also indicated that otoliths were enriched relative to water (KD (o/w) >> 1). The partition coefficients between the otolith and the endolymph (KD (o/e)) were close to 1 for all elements except Ca and Sr. Ca and Sr were equally enriched in the otolith relative to the endolymph in which they crystallize (KD (o/e) = 4290 and 3590, respectively). Barium was much more concentrated than Sr in the endolymph relative to the water despite the fact that both these elements have been shown to directly reflect water chemistry in otolith microchemistry studies (Bath et al., 2000; Milton and Chenery, 2001;

FIG. 4. Sagittal otolith from an age-11 Walleye. Yearly growth bands are indicated by white dots and LA-ICPMS analyses were conducted on the edge of the year 11 growth band.

Wells et al., 2003). This deviation may be due to the extremely large strontium KD (o/e), which can deplete endolymph in Sr compared to Ba (Melancon et al., 2005). Na and K had similar fractionation patterns (i.e. similar K D (o/e) , K D (e /w ) and K D (o / w) values), presumably because both elements are alkali metals found in the blood and are highly physiologically regulated. The concentrations of K, Ca and Na in the endolymph fluid of individual fish were all strongly positively correlated (all R2 50.95 and p 40.0001; Figs. 5a c). Interestingly, Zn and Cu also showed a similar strong correlation (R2 = 0.93 and p = 0.0001; Fig. 5d) in endolymph samples from different individuals. This finding might indicate strong physiological control as these two elements are essential nutrients and may be regulated in fish the same way as Na, K and Ca (Roesijadi, 1992). Their cation radii are also ˚ for Zn and 0.87 A ˚ for Cu, similar, 0.88 A suggesting a similar role in the fish’s body.

TABLE 2. Mean metal concentrations (ppm) in western Lake Erie surface water, and the endolymph fluid and growing edges of otoliths from walleye collected in western Lake Erie. Values of partition coefficients KD (e/w), KD (o/e) and KD (o/w) are also shown, calculated from equations (1) to (3).

Na Mg K Ca Mn Zn Rb Sr Ba

Water (n = 10)

Endolymph (n = 8)

Otoliths (n = 8)

KD (e/w) (endolymph/ water)

KD (o/e) (otolith/ endolymph)

KD (o/w) (otolith/water)

19X5 21X1 3.9X0.6 69X6 0.04X0.01 0.009X0.001 N/A 0.9X0.4 0.048X0.003

1819X306 21X3 571X75 102X11 0.09X0.03 3.2X0.7 1.1X0.1 0.12X0.01 0.83X0.1

1366X260 8X5 277X39 400432 0.13X0.09 4X2 0.22X0.04 432X24 4.7X0.6

96 0.98 145 1.5 2.2 350 N/A 0.13 17

0.75 0.37 0.49 4290 1.75 1.3 0.20 3590 5.7

72 0.37 71 6305 3.1 450 N/A 480 98

633

S. MELANCON ET AL.

FIG. 5. Comparison of metal concentrations of the endolymph of Walleye. (a) Ca/K; (b) Ca/Na; (c) K/Na; and (d) Zn/Cu.

Part 3: effect of crystal structure on element partitioning Simultaneous vaterite-aragonite growth in lake trout otoliths Previous work has shown that more than one crystal polymorph of CaCO3 can be found in otoliths (Campana, 1983; Gauldie, 1986), the most common being aragonite and vaterite. Our microscopic analysis of otoliths from lake trout indicated that aragonite and vaterite can grow simultaneously within the endolymphatic sac (Fig. 6). Melancon et al. (2005) showed that aragonite and vaterite can have different growth rates and that generally vaterite growth rates exceeded that of co-precipitating aragonite. They 634

demonstrated that elemental concentrations were not affected by the growth rate of the polymorphs, as vaterite and aragonite, co-precipitating in the same otolith, produced similar results for most elements regardless of whether vaterite or aragonite was growing faster. The characteristics of the structural forms of CaCO3 in lake trout sagittal otoliths were investigated by Raman spectroscopy and LAICP-MS. The Raman spectra present three distinct characteristics associated with group vibration: lattice mode, symmetric stretching and in-plane bending (Gans, 1971; Truchet et al., 1995). These were used by Gauldie et al. (1997) to distinguish


TABLE 3. Mean metal concentrations (ppm) in the growing edges of mature lake trout otoliths reared in a hatchery.

Mn Mg Zn Li Ca Sr Ba Rb 1

FIG. 6. Adult lake trout otoliths showing the simultaneous growth of aragonite and vaterite. The ‘waviness’ of the vaterite growth bands can be easily differentiated petrographically when compared to the regular aragonite growth bands.

vaterite from aragonite in otoliths of Coho salmon (Oncorhynchus kisutch). We used the symmetric stretching of C O from carbonate anion bonded to calcium, n1, to differentiate vaterite from aragonite. Triplet bands (1075 cm 1, 1081 cm 1 and 1090 cm 1) characterized vaterite whereas aragonite has only a single band at 1084 cm 1 (Fig. 7). Table 3 presents a summary of mean metal concentrations in co-precipitated aragonite and

Cation radii1 ˚) (A

Aragonite (n = 25)

Vaterite (n = 25)

0.81 0.86 0.88 0.90 1.14 1.32 1.49 1.66

1.0X0.2 16X2 11X2 0.08X0.03 400432 780X27 7.3X0.4 0.12X0.01

9.4X0.7 596X29 8X2 0.2X0.1 400432 45X2 0.39X0.04 0.08X0.01

Shannon (1976)

vaterite in adult lake trout otoliths (data derived from Melancon et al., 2005). There is extreme elemental fractionation between the two crystal polymorphs with aragonite portions of otoliths being characterized by high Sr and Ba and low Mg and Mn, whereas vaterite portions have the opposite trends. Zn, Li and Rb have approximately equal concentrations for the two phases. Based on these data, the proportions of elements being removed by crystallization of otolith material from the endolymph fluid will be very different if one or the other (or both) CaCO3 polymorphs are forming at a particular time. This result suggests that great care will need to be taken when using and comparing otolith chem-

FIG. 7. Wavenumbers from Raman spectroscopy analysis of aragonite and vaterite in lake trout otolith edges in arbitrary units plotted against Raman shift in cm 1. The vaterite signal is on top and aragonite is at the bottom. Symmetric stretching (n1) values are presented on the Raman graph for both polymorphs.

635

S. MELANCON ET AL.

Ohio Department of Natural Resources Division of Wildlife, and the University of Windsor’s Great Lakes Institute for Environmental Research for supporting this research.

istry from species that are known to grow otoliths comprising multiple polymorphs. Conclusions The three examples presented in this paper indicate there are both similarities and differences with regard to biomineralization processes in teleost fish otoliths. Given that these processes can influence the ability of otoliths to incorporate trace metals into their crystalline structure, we feel it is critical that we improve our understanding of biomineralization processes, including the mechanisms of crystal nucleation and growth in these complex systems. While these types of studies certainly are of interest from a basicscience standpoint, they may also prove invaluable in the application of otolith microchemistry in fisheries management. There is a pressing need in the study of biomineralization, for more than just the application of geochemical methods to ecological questions. For example, our newly gained basic knowledge that core concentrations (primordia) of an element such as Mn are influenced primarily by maternal/early developmental processes rather than the environment. The microchemical concentrations can also vary considerably in an otolith depending on whether the crystalline structure is comprised of aragonite or vaterite. It could influence how fisheries researchers interpret otolith microchemical data, which are currently being use to help identify natal origins, life-history patterns and habitat-use requirements of both freshwater and marine fishes. Acknowledgements Walleye heads were provided by Presteve Foods Limited, Wheatley, Ontario. Lake Erie lake trout otoliths were graciously provided by Jim Markham, Lake Erie Fisheries Unit, New York State Department of Environmental Conservation (NYSDEC). Cisco larvae from Lake Superior were provided by Jason Stockwell (formerly of USGS, Great Lakes Science Centre, Ashland, WI). Rainbow trout fish were provided by Yolanda Morbey from the Ontario Ministry of Natural Resources (OMNR). We thank the Great Lakes Fishery Commission (Fisheries Research Program grant to B. Fryer et al.), the National Sciences and Engineering Research Council (Strategic Research Grant to P. Sale et al. and Discovery Grants to B. Fryer and J. Gagnon), the

References Bartnik, S.E.H. (2005) Population dynamics of age-0 walleye in western Lake Erie. M.Sc Thesis, University of Windsor, Windsor, Canada. Bath, G.E., Thorrold, S.R., Jones, C.M., Campana, S.E., McLaren, J.W. and Lam, J.W.H. (2000) Strontium and barium uptake in aragonitic otoliths of marine fish. Geochimica et Cosmochimica Acta, 64, 1705 1714. Begg, G.A., Campana, S.E., Fowler, A.J. and Suthers, I.M. (2005) Otolith research and application: current directions in innovation and implementation. Marine and Freshwater Research, 56, 477 483. Brophy, D., Danilowicz, B.S. and Jeffries, T.E. (2003) The detection of elements in larval otoliths from Atlantic herring using laser ablation ICP-MS. Journal of Fish Biology, 63, 990 1007. Brophy, D., Jeffries, T.E. and Danilowicz, B.S. (2004) Elevated manganese concentrations at the cores of clupeid otoliths: possible environmental, physiological, or structural origins. Marine Biology, 144, 779 786. Campana, S.E. (1983) Feeding periodicity and the production of daily growth increment in otoliths of steelhead trout (Salmo gairdneri) and starry flounder (Platichthys stellatus). Canadian Journal of Zoology, 61, 1591 1597. Campana, S.E. (1999) Chemistry and composition of fish otoliths: pathways, mechanisms and applications. Marine and Freshwater Research, 188, 263 297. Campana, S.E. (2005) Otolith science entering the 21st century. Marine and Freshwater Research, 56, 485 495. Campana, S.E. and Neilson, J.D. (1985) Microstructure of fish otoliths. Canadian Journal of Fisheries and Aquatic Sciences, 42, 1014 1032. Campana, S.E., Chouinard, G.A., Hanson, J.M., Frećhet, A. and Brattey, J. (2000) Otolith elemental fingerprints as biological tracers of fish stocks. Fisheries Research, 46, 343 357. Chittaro, P.M., Hogan, J.D., Gagnon, J., Fryer, B.J. and Sale, P.F. (2006) In situ experiment of ontogenetic variability in the otolith chemistry of Stegastes partitus. Marine Biology, 149, 1227 1235. Ferna`ndez-Dıáz, L., Astilleros, J.M. and Pina, C.M. (2006) The morphology of calcite crystals grown in a porous medium doped with divalent cations. Chemical Geology, 225, 314 321.

636


Gans, P. (1971) General considerations. Pp. 1 25 in: Vibrating Molecules. An Introduction to the Interpretation of Infrared and Raman Spectra. Edited by Williams Clowes & Sons Limited. London, Colchester and Beccles. Gauldie, R.W. (1986) Vaterite otoliths from chinook salmon (Oncorhynchus tshawytscha). New Zealand Journal of Marine and Freshwater Research, 20, 209 217. Gauldie, R.W., Sharma, S.K. and Volk, E. (1997) MicroRaman spectral study of vaterite and aragonite otoliths of the coho salmon, Oncorhynchus kisutch. Comparative Biochemistry and Physiology, A118, 753 757. Guibbolini, M., Borelli, G., Mayer-Gostan, N., Priouzeau, F., De Pontual, H., Allemand, D. and Payan, P. (2006) Characterization and variations of organic parameters in teleost fish endolymph during day-night cycle, starvation and stress conditions. Comparative Biochemistry and Physiology, A145, 99 107. Hedges, K.J. (2002) Use of calcified structures for stock discrimination in Great Lakes walleye (Stizostedion vitreum). M.Sc Thesis. University of Windsor, Windsor, Canada. Kalish, J.M. (1991) Determinants of otolith chemistry: seasonal variation in the composition of blood plasma, endolymph and otoliths of bearded rock cod Pseudophycis babatus. Marine Ecology Progress Series, 74, 137 159. Ludsin, S.A., Fryer, B.J. and Gagnon, J.E. (2006) Comparison of solution-based versus laser-ablation ICP-MS for analysis of larval fish otoliths. Transactions of the American Fisheries Society, 135, 218 231. Melancon, S., Fryer, B.J., Gagnon, J.E., Ludsin, S.A. and Yang, Z. (2005) Effects of crystal structure on the uptake of metals by lake trout (Salvelinus namaycush) otoliths. Canadian Journal of Fisheries and Aquatic Sciences, 62, 2609 2619. Milton, D.A. and Chenery, S.R. (1998) The effect of otolith storage methods on the concentrations of elements detected by laser-ablation ICP-MS. Journal of Fish Biology, 53, 785 794. Milton, D.A. and Chenery, S.R. (2001) Sources and uptake of trace metals in otoliths of juvenile barramundi (Lates calcarifer). Journal of Experimental Marine Biology and Ecology, 264, 47 65. Nicolson, T. (2004) Control of crystal growth in biology: a molecular biological approach using zebrafish. Crystal Growth and Design, 4, 667 669. Payan, P., Kossmann, H., Watrin, A., Mayer-Gostan, N. and Buf, G. (1997) Ionic composition of endolymph in teleosts: Origin and importance of endolymph alkalinity. The Journal of Experimental Biology,

200, 1905 1912. Payan, P., Borelli, G., Priouzeau, F., De Pontual, H., Boeuf, G. and Mayer-Gostan, N. (2002) Otolith growth in trout (Oncorhynchus mykiss): supply of Ca2+ and Sr2+ to the saccular endolymph. The Journal of Experimental Biology, 205, 2687 2695. Pisam, M., Jammet, C. and Laurent, D. (2002) First steps of otolith formation of the zebrafish: role of glycogen? Cell and Tissue Research, 310, 163 168. Riley, B.B., Zhu, C., Janetopoulos, C. and Aufderheide, K.J. (1997) A critical period of ear development controlled by distinct populations of ciliated cells in the zebrafish. Developmental Biology, 191, 191 201. Roesijadi, G. (1992) Metallothioneins in metal regulation and toxicity in aquatic animals. Aquatic Toxicology, 22, 81 114. Ruttenberg, B.I., Hamilton, S.L., Hickford, M.J.H., Paradis, G.L., Sheehy, M.S., Standish, J.D., BenTzvi, O. and Warner, R.R. (2005) Elevated levels of trace elements in cores of otoliths and their potential for use as natural tags. Marine Ecology Progress Series, 297, 273 281. Secor, D.H., Rooker, J.R., Zlokovitz, E. and Zdanowicz, V.S. (2001) Identification of riverine, estuarine, and coastal contingents of Hudson River striped bass based upon otolith elemental fingerprints. Marine Ecology Progress Series, 211, 245 253. Shannon, R.D. (1976) Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallographica, A32, 751. Temmam, M., Pauqette, J. and Vali, H. (2000) Mn and Zn incorporation into calcite as a function of chloride aqueous concentration. Geochimica et Cosmochimica Acta, 64, 2417 2421. Thresher, R.E. (1999) Elemental composition of otoliths as a stock delineator in fishes. Fisheries Research, 43, 165 204. Truchet, M., Delhaye, M. and Beny, C. (1995) Identification des carbonates de calcium, calcite, aragonite et vaterite par microsonde RamanCastaing. Application aux biomine´ ralisations. Analusis, 23, 516 518. Warner, R.R., Swearer, S.E., Caselle, J.E., Sheehy, M. and Paradis, G. (2005) Natal trace-elemental signatures in the otoliths of an open-coast fish. Limnology and Oceanography, 50, 1529 1542. Wells, B.K., Rieman, B.E., Clayton, J.L., Horan, D.L. and Jones, C.M. (2003) Relationships between water, otolith, and scale chemistries of Westlope Cutthroat Trout from the Coeur D’Alene River, Idaho: the potential application of hard-part chemistry to describe movements in freshwater. Transactions of the American Fisheries Society, 132, 409 424.

637