Otolith Matrix Research Articles

Immunohistochemical and ultrastructural characterization of the inner ear epithelial cells of splitnose rockfish (Sebastes diploproa).

The inner ear of teleost fish regulates the ionic and acid-base chemistry and secretes protein matrix into the endolymph to facilitate otolith biomineralization, which is used to maintain vestibular and auditory functions. The otolith is biomineralized in a concentric ring pattern corresponding to seasonal growth, and this calcium carbonate (CaCO3) polycrystal has become a vital aging and life-history tool for fishery managers, ecologists, and conservation biologists. Moreover, biomineralization patterns are sensitive to environmental variability including climate change, thereby threatening the accuracy and relevance of otolith-reliant toolkits. However, the cellular biology of the inner ear is poorly characterized, which is a hurdle for a mechanistic understanding of the underlying processes. This study provides a systematic characterization of the cell types in the inner ear of splitnose rockfish (Sebastes diploproa). Scanning electron microscopy revealed the apical morphologies of six inner ear cell types. In addition, immunostaining and confocal microscopy characterized the expression and subcellular localization of the proteins Na+-K+-ATPase, carbonic anhydrase, V-type H+-ATPase, Na+-K+-2Cl--cotransporter, otolith matrix protein 1, and otolin-1 in six inner ear cell types bordering the endolymph. This fundamental cytological characterization of the rockfish inner ear epithelium illustrates the intricate physiological processes involved in otolith biomineralization and highlights how greater mechanistic understanding is necessary to predict their multistressor responses to future climate change.

Qualitative Shotgun Proteomics Strategy for Protein Expression Profiling of Fish Otoliths

Despite decades of research on fish otoliths and their capacity to serve as biochronological recorders, much remains unknown about their protein composition, the mechanisms by which proteins are incorporated into the otolith matrix, or the potential for using otolith proteins to provide insight into aspects of fish life history. We examined the protein composition of Atlantic cod (Gadus morhua) otoliths using a state-of-the-art shotgun proteomics approach with liquid chromatography coupled to an electrospray ionization-orbitrap tandem mass spectrometer. In addition to previously known otolith matrix proteins, we discovered over 2000 proteins not previously identified in cod otoliths and more than 1500 proteins not previously identified in any fish otoliths. These included three novel proteins (Somatolactin, F-actin-capping protein subunit beta, Annexin) primarily involved in binding calcium ions and likely mediating crystal nucleation. However, most of the otolith proteins were not necessarily related to otolith formation but rather to other aspects of fish physiology. For example, we identified sex-related biomarkers for males (SPATA6 protein) and females (Vitellogenin-2-like protein). We highlight some noteworthy classes of proteins having diverse functions; however, the primary goal here is not to discuss each protein separately. The number and diverse roles of the proteins discovered in the otoliths suggest that proteomics could reveal critical life history information from archived otolith collections that could be invaluable for understanding aspects of fish biology and population ecology. This proof-of-concept methodology paper provides a novel methodology whereby otolith proteomics can be further explored.

Advances in otolith-related protein research.

Otoliths are biological crystals formed by a layer of calcium carbonate crystal that adhere to the ciliary surface of the utricular and saccular receptors in the vestibule of all vertebrates inner ear, enabling the utricle and saccule to better perceive the changes in linear and gravitational acceleration. However, the molecular etiology of otolith related diseases is still unclear. In this review, we have summarized the recent findings and provided an overview of the proteins that play important roles in otolith formation and maintenance (Otoconin-90, Otolin-1, Otolith Matrix Protein-1, Cochlin, Otogelin, α-Tectorin, β-Tectorin, Otopetrin-1, and Otopetrin-2, PMCA2, etc.), providing new insight for the prevention and management of benign paroxysmal positional vertigo (BPPV) with basis for otolith-related proteins as potential biomarkers of vestibular disease.

Varying frequency of vateritic otoliths in the Baltic herring Clupea harengus membras.

We report observations of vateritic crystallization in the sagittal otoliths of the Baltic herring Clupea harengus membras in the northern Baltic Sea. While the existence of vaterite in the calcium carbonate matrix of sagittal otoliths has been observed in various species globally, reports from the brackish Baltic Sea are few in number. Large variation in the frequency of vaterite in 1984, 1988, 1997, 2010 and 2017 was observed, suggesting that the phenomenon is not static and more long‐term studies should be conducted in search of the ultimate causing factors.

N′-terminal- and Ca2+-induced stabilization of high-order oligomers of full-length Danio rerio and Homo sapiens otolin-1

Otolin-1 is a C1q family member and a major component of the organic matrix of fish otoliths and human otoconia. To date, the protein molecular properties have not been characterized. In this work, we describe biochemical characterization and comparative studies on saccular-specific otolin-1 derived from Danio rerio and Homo sapiens. Due to the low abundance of proteins in the otoconial matrix, we developed a production and purification method for both recombinant homologues of otolin-1. Danio rerio and Homo sapiens otolin-1 forms higher-order oligomers that can be partially disrupted under reducing conditions. The presence of Ca2+ stabilizes the oligomers and significantly increases the thermal stability of the proteins. Despite the high sequence coverage, the oligomerization of Danio rerio otolin-1 is more affected by the reducing conditions and presence of Ca2+ than the human homologue. The results show differences in molecular behaviour, which may be reflected in Danio rerio and Homo sapiens otolin-1 role in otolith and otoconia formation.

Spatial and temporal variation in otolith elemental signatures of age-0 Pacific cod (Gadus macrocephalus) in the Gulf of Alaska

Shallow coastal waters of the Gulf of Alaska (GOA) serve as nursery habitats for young-of-year Pacific cod (Gadus macrocephalus). However, little is known regarding the relative contribution of these areas to the adult offshore stock. Trace elements incorporated into the otolith matrix can reflect the environmental conditions to which a fish has been exposed during its lifetime. When analyzed together, a suite of elements can serve as a natural marker, characteristic of a particular environment. We evaluated the potential of otolith elemental signatures to identify nursery habitats of age-0 Pacific cod, focusing on spatial patterns within and across the eastern and western GOA. Fish were collected from shallow nearshore areas within five large embayments in summer and fall, and element:calcium ratios were measured from two different time stanzas within the otolith. Elemental ratios were found to change over short (2 month) time periods and were related to seasonal changes in temperature and salinity. Fish were classified to nursery habitats using quadratic discriminant analysis based on their otolith elemental signatures; classification accuracy to individual bays ranged from 30% to 95%, with an overall success rate of 59%. Classification accuracy improved to 78% at greater spatial scales (eastern versus western GOA). Our results point out the limitations of this application to Gulf of Alaska Pacific cod and other widely distributed species residing in coastal embayments. While some nursery habitats impart unique chemical signatures to otoliths, it may not be possible to distinguish among specific areas without considering additional factors. However, our work demonstrates that otolith microchemistry may be a useful tool for understanding source contributions to the Pacific cod population at larger regional scales within the GOA.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64)

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed.

Polyketide Synthase is Required for Zebrafish Otolith Biomineralization

ObjectiveDeflecting biomineralized crystals attached to vestibular hair cells is necessary for maintaining the body's balance. Zebrafish (Danio rerio) are useful organisms to study these biomineralized crystals called otoliths as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a pair of genetically‐linked mutants, no content (nco) and corkscrew (csr), that fail to develop otoliths during early ear development.MethodsMutant nco embryos and wild‐type clutchmates (WT) were collected during the critical period of otolith nucleation (24 hours post fertilization, hpf) and submitted for RNA sequencing. Analysis was completed using MMAPPR (Mutation Mapping Analysis Pipeline for Pooled RNA‐seq). Whole genome sequencing of csr was performed and analyzed using MegaMapper. WT mRNA was synthesized using mMessage Machine from a clone provided by Dr. Hiroyuki Takeda (University of Tokyo), cleaned on an RNeasy column, and subsequently injected into single‐cell csr and nco embryos. For immunofluorescence (IF), csr embryos were collected during key stages in ear development, fixed with hydrogel and washed in CLARITY‐clearing solution. Embryos were decalcified with EDTA before blocking, incubating in primary and secondary antibodies, and imaging by confocal microscopy. To test the effects of exogenous calcium ions on otolith formation, embryos were kept in E3 Medium until early gastrulation. Embryos were washed and transferred to 1X Basic Solution (58 mM NaCl, 0.4 mM MgSO4 and 5 mM HEPES) supplemented with 0.7 mM potassium chloride [0 mM Ca2+], 0.6 mM calcium nitrate [0.15 mM Ca2+] or 0.6 mM calcium chloride [0.22 mM Ca2+]. Embryos were scored by the presence or absence of otoliths at 27 hpf. Statistical significance was calculated using Fisher's Exact Test and Chi‐Squared Distribution or by Linear Regression.ResultsTo positionally clone the gene responsible for nco and csr, we used complementary approaches for each strain. MMAPPR analysis of nco‐derived RNA sequencing and MegaMapper analysis of csr‐derived whole genome sequencing both identified a genomic region surrounding polyketide synthase 1 (pks1; current gene name wu:fc01d11), a candidate gene that was recently identified as a key factor of otolith biomineralization in Japanese medaka (Oryzias latipes, Ol). Microinjection of OI pks1 mRNA rescued otolith biomineralization in both csr (p<0.0001; χ2<0.0001) and nco (p=0.0032; χ2=0.0022) mutants. Using IF, an otoconial seeding protein, Otoconin‐90, and an otolith matrix protein, Starmaker, show diffuse expression inside the otocyst of csr mutant embryos. Expression of Otogelin, a primary component of the otolithic membrane, is localized near the apical surfaces of sensory hair cells in both csr and WT. Additionally, exogenous calcium concentrations correlate with penetrance of otolith formation in csr embryos (R2=0.9978); however, nco embryos were unaffected (R2=0.7802).ConclusionsOtolith nucleation is impaired in the genetically‐linked zebrafish mutants csr and nco. IF of csr embryos demonstrated that expression of a critical otoconial seeding protein, Otoconin‐90, within the otocyst is not sufficient for otolith biomineralization in the presence of the otolithic membrane. While WT Ol pks1 rescues otolith biomineralization in csr and nco, differences in penetrance of calcium ions on otolith formation suggest the nature of each mutation is fundamentally different.Support or Funding InformationThe work was supported by grants from the National Institutes of Health (8P20GM103471) and the Creighton Health Science Strategic Investment Fund.

Rapid growth causes abnormal vaterite formation in farmed fish otoliths.

Sagittal otoliths are essential components of the sensory organs that enable all teleost fish to hear and maintain balance, and are primarily composed of calcium carbonate. A deformity, where aragonite (the normal crystal form) is replaced with vaterite, was first noted over 50 years ago but its underlying cause is unresolved. We evaluated the prevalence of vateritic otoliths from two captive rearing studies which suggested that fast growth, due to environmental rather than genetic control, led to vaterite development. We then tested this by varying light and temperature to create phenotypes with different growth rates, which resulted in fast growers (5times larger) having 3times more vaterite than slow growers. A decrease in either the ratio of otolith matrix proteins (otolin-1/OMM-64) or [Ca2+]/[CO32-] may explain why fast growth causes vaterite deposition. As vaterite decreases hearing sensitivity, reducing growth rates in hatcheries may improve the welfare of farmed fish and increase the success of conservation efforts.

Not All Inner Ears are the Same: Otolith Matrix Proteins in the Inner Ear of Sub-Adult Cichlid Fish, Oreochromis Mossambicus, Reveal Insights Into the Biomineralization Process.

The fish ear stones (otoliths) consist mainly of calcium carbonate and have lower amounts of a proteinous matrix. This matrix consists of macromolecules, which directly control the biomineralization process. We analyzed the composition of this proteinous matrix by mass spectrometry in a shotgun approach. For this purpose, an enhanced protein purification technique was developed that excludes any potential contamination of proteins from body fluids. Using this method we identified eight proteins in the inner ear of Oreochromis mossambicus. These include the common otolith matrix proteins (OMP-1, otolin-1, neuroserpin, SPARC and otoconin), and three proteins (alpha tectorin, otogelin and transferrin) not previously localized to the otoliths. Moreover, we were able to exclude the occurrence of two matrix proteins (starmaker and pre-cerebellin-like protein) known from other fish species. In further analyses, we show that the absence of the OMP starmaker corresponds to calcitic otoliths and that pre-cerebellin-like protein is not present at any stage during the development of the otoliths of the inner ear. This study shows O. mossambicus does not have all of the known otolith proteins indicating that the matrix proteins in the inner ear of fish are not the same across species. Further functional studies of the novel proteins we identified during otolith development are required.

Spatial Expression of Otolith Matrix Protein-1 and Otolin-1 in Normally and Kinetotically Swimming Fish.

Kinetosis (motion sickness) has been repeatedly shown to affect some fish of a given clutch following the transition from 1g to microgravity or from hypergravity to 1g. This susceptibility to kinetosis may be correlated with irregular inner ear otolith growth. Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Here, we examine the morphology of otoliths and the expression pattern of the major otolith proteins OMP-1 and otolin-1 in a series of hypergravity experiments. In the utricle, OMP-1 is present in centripetal (medial) and centrifugal (lateral) regions of the meshwork area. In the saccule, OMP-1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the sulcus acusticus. In normal animals, the spatial expression pattern of OMP-1 reaches more posteriorly in the centrifugal aspect and is considerably broader in the centripetal portion of the utricle compared to kinetotic animals. However, otolin-1 was not expressed in the utricule. In the saccule, no differences were observed for either gene when comparing normal and kinetotically behaving fish. The difference in the utricular OMP-1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, the aberrant morphology of the utricular otoliths, based on OMP-1 expression, likely leads to the observed kinetotic behavior.

Intra-specific and inter-specific variability of the sulcus acusticus of sagittal otoliths in two gurnard species (Scorpaeniformes, Triglidae)

Morphology, morphometries and compounds of sulcus acusticus of sagittal otoliths of two gurnard species (Aspitrigla cuculus and Eutrigla gurnardus) from north-central Adriatic Sea (north-eastern Mediterranean) were investigated. The study objectives were to find morphometric variables and shape variability of sulcus acusticus of these two closely related sound producers species linked to developmental changes from juvenile to adult stage and species-specific pattern which influenced ecomorphological adaptations of the auditory system. The method used was a combination of multivariate techniques, X-ray diffractions pattern and ultrastructural investigation by SEM. Multivariate analysis (PCA, LDA and PERMANOVA) performed on standardized morphometrics of a total of 202 individuals, significantly separated four groups: A. cuculus juveniles, A. cuculus adults, E. gurnardus juveniles and E. gurnardus adults due to different life history characteristics. At intra-specific levels higher mean values of sulcus area/otoltih area (S:O) ratio of adults than the juveniles were observed within A. cuculus and not for E. gurnardus. At inter-specific levels significant differences were seen between adults and not between juvenile groups. Considering all the groups, the main variability on sulcus area were observed for caudal part of the sensorial macula. Although no differences in compounds were shown by X-ray diffraction at intra-specific level, the crystals on sulcus acusticus were different in shape, size and arrangement between juveniles and adults of each gurnard. The crystals of juveniles were quite enmeshed into otolith matrix and showed a rod/smooth-shape. The crystalline arrangement of adults became more complex with larger, rod/rectangular-shape and well-formed faces. Ontogenetic and specie-specific differences might be related to specie-specific physiological factor (i.e sexual maturity), depth distribution and feeding patterns that change during gurnards growth. These changes, relating to both endogenous and exogenous factors, were marked by the sulcus acusticus which provided a useful tool for fish ontogenetic, physiological and ecological studies.

Induction of carbonic anhydrase in SaOS-2 cells, exposed to bicarbonate and consequences for calcium phosphate crystal formation

Ca-phosphate/hydroxyapatite crystals constitute the mineralic matrix of vertebrate bones, while Ca-carbonate dominates the inorganic matrix of otoliths. In addition, Ca-carbonate has been identified in lower percentage in apatite crystals. By using the human osteogenic SaOS-2 cells it could be shown that after exposure of the cells to Ca-bicarbonate in vitro, at concentrations between 1 and 10 mm, a significant increase of Ca-deposit formation results. The crystallite nodules formed on the surfaces of SaOS-2 cells become denser and larger in the presence of bicarbonate if simultaneously added together with the mineralization activation cocktail (β-glycerophosphate/ascorbic acid/dexamethasone). In parallel, with the increase of Ca-deposit formation, the expression of the carbonic anhydrase-II (CA-II) gene becomes upregulated. This effect, measured on transcriptional level is also substantiated by immunohistological studies. The stimulatory effect of bicarbonate on Ca-deposit formation is prevented if the carbonic anhydrase inhibitor acetazolamide is added to the cultures. Mapping the surface of the Ca-deposit producing SaOS-2 cells by scanning electron microscopy coupled with energy-dispersive X-ray analysis revealed an accumulation of the signals for the element carbon and, as expected, also for phosphorus. Finally, it is shown that ortho-phosphate and hydrolysis products of polyphosphate inhibit CA-II activity, suggesting a feedback regulatory system between the CA-driven Ca-carbonate deposition and a subsequent inactivation of this process by ortho-phosphate. Based on the presented data we suggest that Ca-carbonate deposits act as bioseeds for a downstream Ca-phosphate deposition process. We propose that activators for CA, especially for CA-II, might be beneficial for the treatment of bone deficiency diseases.

Stable N and C isotopes in the organic matrix of fish otoliths: validation of a new approach for studying spatial and temporal changes in the trophic structure of aquatic ecosystems

Stable isotope studies of long-term ecosystem change are often hampered by lack of archived tissue samples. Here, we provide a reliable method for extracting the organic matrix from fish otoliths, demonstrate differences in isotope values between the soluble and insoluble organic fractions, and provide the trophic enrichment factors (ε). The analyses were performed on otoliths from wild-caught adult Atlantic cod (Gadus morhua) and otoliths from a 6-month diet-switch experiment with juvenile cod. Acid hydrolysis of otolith powder followed by ultrafiltration enabled us to purify and separate the soluble and insoluble organic material. There was a significant 1.72‰ and 0.15‰ difference in δ15N and δ13C, respectively, between the soluble and insoluble organic fraction of the otolith. This emphasizes the need for separation of the two fractions before analysis. The diet-specific trophic enrichment (εotolith-diet) in the soluble fraction varied between −0.20‰ and 0.31‰ for nitrogen and between 0.09‰ and 0.23‰ for carbon. In conclusion, the organic matrix of otoliths provides an attractive archive of organic material for ecological stable isotope analysis that has the potential to provide important insights into historic changes in the trophic structure of aquatic ecosystems.

Thyroid hormone-responsive genes mediate otolith growth and development during flatfish metamorphosis

Flatfish begin life as up-right swimming, bilaterally symmetrical larvae that metamorphose into asymmetrically shaped juveniles that swim with a highly lateralized posture. We have previously shown that TH induces abrupt growth and mineralization of one component of the vestibular system, the otoliths, during early larval development and metamorphosis. Here we report that four of five vestibular-specific genes that we tested ( alpha-tectorin, otogelin, otolith matrix protein, and otopetrins 1 and 2 that are known to be associated with otolith development in other vertebrates are up-regulated 1.5- to 7-fold in larval flatfish during spontaneous metamorphosis and/or following 72 h of TH treatment. These findings suggest that otolith growth and development are mediated by diverse TH-responsive genes during flatfish metamorphosis.

Simultaneous use of strontium:calcium and barium:calcium ratios in otoliths as markers of habitat: Application to the European eel ( Anguilla anguilla) in the Adour basin, South West France

Sr:Ca and Ba:Ca ratios in water from the Adour estuary show a clear relationship with the salinity of the surrounding water for salinities <20, while ratios are almost constant above this level of salinity. A positive relationship was observed for the Sr:Ca ratio, whereas it was inverse for the Ba:Ca ratio. These two elemental ratios were measured in the otoliths of the European eels ( Anguilla anguilla L.) using femtosecond laser ablation linked to an ICP-MS (fs-LA–ICP-MS). There was a direct relationship between the elemental ratios recorded in eel otoliths and those found in water from fresh and marine areas, suggesting that Sr:Ca and Ba:Ca ratios in eel otoliths can be used as markers of habitat in this estuary. Continuous profiling allowed the determination of three behaviour patterns in terms of habitat: freshwater, estuary and migratory individuals. Finally, the above results support the simultaneous use of both ratios for a better understanding of the migratory contingents and also as a relevant method to avoid a misidentification of environmental migratory history due to the presence of vaterite crystal in the otolith matrix.

Control of Polymorphism and Morphology of Calcium Carbonate Crystals by a Matrix Protein Aggregate in Fish Otoliths

We show that the high-molecular weight (HMW) protein aggregate of fish otolith matrix is involved in the regulation of crystal polymorphs during otolith biomineralization. Teleost fish otoliths are...

Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths

In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral-associated proteins. To identify such proteins, we have searched for proteins that may form high-molecular-weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule-64 (OMM-64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu-rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [Söllner C, Burghammer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282-286]. (45)Ca overlay analysis revealed that the Glu-rich region has calcium-binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM-64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM-64 is expressed specifically in otolith matrix-producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear-specific short-chain collagen otolin-1, and the resulting complex forms ring-like structures in the otolith matrix. Overall, OMM-64, by forming a calcium-binding aggregate that binds to otolin-1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization.

Otoc1: A novel otoconin‐90 ortholog required for otolith mineralization in zebrafish

Within the vestibular system of virtually all vertebrate species, gravity and linear acceleration are detected via coupling of calcified masses to the cilia of mechanosensory hair cells. The mammalian ear contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while otolith matrix protein (OMP) is the most abundant matrix protein found in the otoliths of teleost fish. We have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 was detected in the ear between 15 hpf and 72 hpf, and was restricted primarily to the macula and the developing epithelial pillars of the semicircular canals. Expression of otoc1 was also detected in epiphysis, optic stalk, midbrain, diencephalon, flexural organ, and spinal cord. During embryogenesis, expression of otoc1 mRNA preceded the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produced a variety of aberrant otolith phenotypes. Our results suggest that Otoc1 may serve to nucleate calcium carbonate mineralization of aragonitic otoliths.

Transition metal binding to cod otolith proteins

Otolith microchemistry can be very useful in identifying fish populations and reconstructing fish movements. Recent attempts have been made to evaluate otoliths as proxies of ambient levels of transition metals, but findings have been inconsistent. Some of the difficulty with obtaining a definitive answer stems from an incomplete understanding of the biological control of transition metal speciation in otoliths. Metals may be incorporated as part of the calcium carbonate phase, trapped in interstitial spaces within the crystal, or associated with the protein matrix. Metal binding to the protein phase may be inferred from its structural and biochemical properties but has not been observed previously. Inherent difficulties with the extraction of metal-binding proteins in their native state from the calcium carbonate phase make them extraordinarily difficult to measure. We have developed a method that facilitates the extraction of otolith proteins without total disruption of transition metal binding. Chelating agents such as EDTA, used in the decalcification of otoliths, can demetallate the proteins if allowed to reach equilibrium; however, if the reaction is halted prior to equilibration, intact metal–protein complexes can be obtained. Using such an approach, we have confirmed the presence of copper and zinc in the soluble portion of the protein matrix of cod otoliths, and we have established that between 70% and 100% of copper and 40% to 60% of zinc found in whole otoliths are associated with the soluble part of the protein matrix. Manganese was not observed to be associated with the protein, indicating that it is either weakly bound or that no binding is present. Our results, combined with an understanding of the biological control of these metals, suggest that otoliths are not likely to be reliable indicators of copper and zinc exposure, but they may provide useful insight into fish growth and physiological development.