Beyond the transect: An alternative microchemical imaging method for fine scale analysis of trace elements in fish otoliths during early life
Graphical abstract
Introduction
Fish otoliths provide an unparalleled source of demographic and ecological information in the animal kingdom. Otoliths are small (usually < 30 mm diameter) calcified structures used for hearing and balance in the heads of all teleost species (Popper and Fay, 2011; Fig. 1). They grow continuously throughout an individual's lifetime, depositing calcium carbonate (CaCO3) and proteinaceous tissue (otolin) in alternating bands (increments) that are visible when viewed under magnification (Campana and Neilson, 1985). Increments are deposited chonologically, allowing estimation of age with great precision (Campana, 2001). The width of increments can also be measured to estimate growth rates, as otolith and body growth are proportional (Casselman, 1990). Otolith-based assessments of annual age and growth are used to monitor the status of exploited fish stocks across the globe (Campana and Thorrold, 2001, Cushing, 1975, Maciena et al., 2007), while examination of daily increments in the juvenile region of the otolith is used to determine the growth performance and timing of critical early life stages (e.g. the larval phase) (Brothers et al., 1976, Suthers, 1998).
In addition to information on age and growth, otoliths can provide valuable insights into the connectivity of fish populations. Most reef fishes have a bipartite life cycle, with a highly mobile larval phase followed by a relatively sedentary adult phase (Leis, 1991, Roughgarden et al., 1988). The larval phase lasts days to months depending on the species, during which time ocean currents can transport larvae thousands of kilometers from the location where they were spawned (Cowen et al., 2006). The sources of larvae for populations are therefore rarely known, rendering conservation of important breeding areas a challenging task. Trace elements incorporated into the otolith from ambient water can identify the source locations of individuals, because elemental compositions (microchemical ‘signatures’) are unique to particular locations (Campana et al., 2000). Microchemical analysis can be used to compare signatures from the larval core of the otolith (source signature) to a range of potential source locations until a match is found. If a large enough sample of the population is examined, the extent of larval exchange between adjacent populations can be estimated. Analysis of the otolith region corresponding to settlement onto adult habitat (settlement zone) can also be used to determine the duration of the larval phase (pelagic larval duration, PLD), because water chemistry can differ greatly between pelagic and benthic habitats (Hamilton and Warner, 2009). PLD is an essential parameter in physical models used to predict larval dispersal distances and trajectories (Wilson and McCormick, 1999), and cannot always be obtained by counting daily increments from the time of hatching because the settlement mark is not always visible in otolith microstructure (Hamilton and Warner, 2009, Hoover and Jones, 2013). Microchemical analysis of settlement zones in the otolith may provide PLD estimates for many species for which such information is currently unavailable.
Despite the utility of otolith microchemistry for connectivity investigations, the spatial resolution of commonly used spectrometry techniques is often too coarse to resolve elemental concentrations over time-scales relevant to larval dispersal and settlement events (i.e. days). Spatial profiling of trace elements (e.g. Sr and Ba) in otoliths is usually conducted using laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), where a focused laser beam is directed onto the surface of a sectioned otolith to quantify elemental concentrations in specific growth regions. This technique is typically used to gather data on spots or transects across the otolith surface using beam diameters ranging from 20 to 50 μm (Ashford et al., 2006, Chang et al., 2012, DiMaria et al., 2010, Elsdon et al., 2008, Ferguson et al., 2011, Hamilton and Warner, 2009, Longmore et al., 2010). However, daily growth increments can be < 1 μm wide (generally 1–5 μm) depending on the species and developmental stage (DiMaria et al., 2010, Hamer and Jenkins, 2007). Narrow increments present an obvious limitation: data obtained using laser beam diameters greater than these increment widths means that daily resolution is often unachievable. Given that dispersal and settlement in reef fishes can occur over a time period of days (Hoover and Jones, 2013), such techniques limit the resolution of short-term events in early life, including identification of the settlement zone. Additionally, single spots or transects only provide a limited view of elemental changes throughout early life, because concentrations are known to vary spatially within the otolith (Durrant and Ward, 2005), even within increments of the same chronological period. A method that quantifies elemental concentrations in multiple directions is required to visualize gradients in elemental composition across key developmental boundaries (e.g. larval settlement).
Microchemical imaging by LA-ICP-MS, as opposed to single spot or transect analysis, has the capacity to resolve a greater number of microchemical features, whilst still retaining the ability to observe radial trends in otolith microchemistry. The crater diameter and depth produced by the solid-state Nd:YAG laser beam are dependent on beam energy, pulse frequency, duration and laser power output; and the volatility of the analyte, which determines the volume of material ablated and ultimately detected by the ICP-MS (Lear et al., 2012). Using a smaller laser beam diameter results in somewhat shallower depth penetration, and has the potential to provide more precise information about short-term life-history events. A smaller beam diameter results in higher detection and quantification limits, and decreases the precision of determined analyte concentrations in the specimen. Therefore, a compromise between laser beam diameter and power to achieve optimal imaging parameters is essential.
A further limitation to LA-ICP-MS analysis of materials such as otoliths is the lack of characterized matrix-matched reference materials (Campana, 1999, Hare et al., 2012). The ablation and ionization of particles from the sample surface are characteristic of that sample's chemical and physical composition, and the substitution of a non matrix-matched standard has the potential to generate inaccurate results. A small number of studies have achieved success using partially matrix-matched materials. NIST (National Institute of Standards and Technology) 1486 (bone meal) has been used as a hydroxyapatite matrix for quantifying trace metals in teeth (Arora et al., 2011, Austin et al., 2013, Hare et al., 2011). Two sagittal otolith standards (INMS FEBS-1 and NIES-022) are produced for bulk elemental analysis by solution nebulization ICP-MS (Sturgeon et al., 2005), but have not been characterized as reference materials for solid sampling.
This paper describes a new method for producing fine-scale quantitative images (maps) of trace element distribution in fish otoliths during early life using a matrix matched standard and NIST glasses 610 and 612. We demonstrate how the technique can be used to observe gradients in Sr and Ba concentrations during the early life history of a small reef fish (Pomacentrus coelestis) by correlating high resolution scanning electron microscopy (SEM) with images produced by LA-ICP-MS. We compare the results obtained using microchemical imaging to those achieved using a single ablation transect, which is a common method for microchemical analysis of fish otoliths during early life. The calibration method of Longerich et al. (1996) has been applied to microchemical imaging by LA-ICP-MS of a biological matrix here for the first time to test the suitability of readily available non matrix-matched standards NIST 610 and 612 for external calibration of the LA-ICP-MS system in the analysis of fish otoliths. Paul et al. (2014) recently used a similar approach for imaging multiphase assemblies in geological samples. Limburg et al. (2011) described a synchrotron microprobe-based X-ray florescence microscopy imaging approach, though this technique does not suffer the same quantitative pitfalls of LA-ICP-MS. The approach described here is a potential alternative to single line transects.
Section snippets
Standard preparation
Glass standards NIST 610 and 612 were prepared for analysis by washing the wafers in 30% ultrapure HNO3 (Merck, Kilsyth, Australia) for 5 min in a sonicator prior to analysis. Certified values for NIST 610 and 612 were taken from a comprehensive analysis and summary of both reference materials by Pearce et al. (1997). Pressed pellets of INMS FEBS-1 sagittal otolith reference material were prepared by transferring ca. 500 mg of the powdered standard into a stainless-steel Specac pellet die (0.5
Analytical suitability of Longerich's calibration method for imaging
According to Longerich's method of quantification for LA-ICP-MS (Longerich et al., 1996), Sr and Ba concentration in FEBS-1 returned recoveries of 99.9 ± 1.25 and 97.0 ± 2.7%, respectively. The limit of quantification for Ba and Sr was 1.9 ± 1.2; and 0.8 ± 0.6 μg g− 1, respectively.
Resolution of early life history features in the otolith
The ESEM images indicated distinguishable daily increments in both the larval and juvenile (post-settlement) regions of P. coelestis otoliths (Fig. 3, Fig. 4, Fig. 5). The settlement zone marking the end of the larval phase was
Conclusion
Investigations into the early life history of fishes are often hampered by an inability to resolve crucial events occurring over a timescale of days, including larval dispersal and settlement. The microchemical imaging method developed in this study can provide data on elemental concentrations in the otoliths of juvenile fish over time scales as short as 2 days. This allows microchemical signatures of spawning locations to be obtained from the larval core of the otolith with a high degree of
Acknowledgements
The authors wish to acknowledge financial support from the Australian Research Council (LP100200254), Agilent Technologies and Kennelec Scientific.
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