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.

An integrative phylogenetic approach for inferring relationships of fossil gobioids (Teleostei: Gobiiformes).

The suborder Gobioidei is among the most diverse groups of vertebrates, comprising about 2310 species. In the fossil record gobioids date back to the early Eocene (c. 50 m.y. ago), and a considerable increase in numbers of described species is evident since the middle Miocene (c. 16 m.y. ago). About 40 skeleton-based gobioid species and > 100 otolith-based species have been described until to date. However, assignment of a fossil gobioid species to specific families has often remained tentative, even if well preserved complete specimens are available. The reasons are that synapomorphies that can be recognized in a fossil skeleton are rare (or absent) and that no phylogenetic framework applicable to gobioid fossils exists. Here we aim to overcome this problem by developing a phylogenetic total evidence framework that is suitable to place a fossil skeleton-based gobioid at family level. Using both literature and newly collected data we assembled a morphological character matrix (48 characters) for 29 extant species, representing all extant gobioid families, and ten fossil gobioid species, and we compiled a multi-gene concatenated alignment (supermatrix; 6271 bp) of published molecular sequence data for the extant species. Bayesian and Maximum Parsimony analyses revealed that our selection of extant species was sufficient to achieve a molecular 'backbone' that fully conforms to previous molecular work. Our data revealed that inclusion of all fossil species simultaneously produced very poorly resolved trees, even for some extant taxa. In contrast, addition of a single fossil species to the total evidence data set of the extant species provided new insight in its possible placement at family level, especially in a Bayesian framework. Five out of the ten fossil species were recovered in the same family as had been suggested in previous works based on comparative morphology. The remaining five fossil species had hitherto been left as family incertae sedis. Now, based on our phylogenetic framework, new and mostly well supported hypotheses to which clades they could belong can be presented. We conclude that the total evidence framework presented here will be beneficial for all future work dealing with the phylogenetic placement of a fossil skeleton-based gobioid and thus will help to improve our understanding of the evolutionary history of these fascinating fishes. Moreover, our data highlight that increased sampling of fossil taxa in a total-evidence context is not universally beneficial, as might be expected, but strongly depends on the study group and peculiarities of the morphological data.

Morphology and composition of Goldeye (Hiodontidae; Hiodon alosoides) otoliths.

We provide up-to-date morphological and compositional data on otoliths of the osteoglossomorph Goldeye (Hiodon alosoides). Using computed tomography (CT) X-ray, we documented the location of each of the three pairs of otoliths (lapilli, sagittae, and asterisci) in relation to the swim bladder, which extended forward in close proximity to the sagittae and asterisci. The lappili were the largest otoliths in terms of surface area and volume, but the sagittae were highly modified, appearing spiral in shape when viewed dorsally, with a surface area to volume ratio more than double that of the lapilli. Using scanning electron microscopy, the surface of each otolith was viewable in great detail, and small otoconia (~10.5 µm diameter) were observed on each, but were most numerous on the sagittae. On scanning electron micrographs, the sagittae appeared to be bi-lobed, with asymmetrical lobes each oriented in the same general direction. Using neutron and X-ray diffraction methods, we found three polymorphs of calcium carbonate crystals (aragonite, vaterite, and calcite), sometimes all within the same otolith. However, in general, lapilli and sagittae were composed predominately of aragonite whereas asterisci were composed chiefly of vaterite. With these results, we provide information on a unique species, whose inclusion in future studies would benefit our understanding of fish hearing, fish evolution, and fisheries ecology.

[Ultrastructural study of ampulla of mouse inner ear].

Objective: To observe the ultrastructure of the ampulla, and analyze its physiological and pathological significance. Methods: In this study, 20 Kunming mice were used, and scanning electron microscopy was used to observe the ultrastructure of the ampulla of inner ear. Results: Otoconia was found among the cilia bundles of different haircell(intercilla otoconia of ampulla). The cupula was attached to the lateral wall of the ampulla, and easily to be separated; after separated, a kind of slender crystal(surface otoconia of ampulla) could be seen between the cupula and lateral wall of the ampulla, both sides of ampullary crest were covered with slender crystals too. On the canal side of the ampulla wall, there was more particulate matter attached to the wall near the bottom of ampullary crest, partially embedded in the wall, and less on the utricle side of the ampulla wall. Conclusions: The observation of the ultrastructure of the ampulla is helpful for better understanding the physiological functions of the semicircular canals and the ampulla, and better understanding the pathogenesis and solution of some vertigo diseases.

Age and growth of slender tuna (Allothunnus fallai) in an unexploited temperate population.

Many large predatory fishes are in decline and tuna sustainability is high on the global agenda. Slender tuna (SLT), Allothunnus fallai, is data-poor and a rare contemporary example of a globally unexploited temperate tuna. This study analysed 214 otoliths for age and growth of fish collected in the South Atlantic. Observed ages varied between 9 and 42 years for a size range of 68-90 cm fork length. We reveal important life history data for SLT before exploitation and underline the relevance of data-poor stocks in understanding wider questions for exploited tuna.

Quantitative reconstruction of salinity history by otolith oxygen stable isotopes: An example of a euryhaline fish Lateolabrax japonicus.

RATIONALE: Otolith strontium:calcium (Sr:Ca) ratios have been extensively used to study fish migration across a river, estuary and ocean at each life stage. However, otolith Sr:Ca ratios only explain partial variations in salinity and quantitative reconstruction of salinity history of fishes remains a challenge. Using a euryhaline fish Lateolabrax japonicus as an example, this study demonstrated an isotopic method of quantitatively reconstructing the salinity histories and habitat uses of the fish. METHODS: Otolith oxygen stable isotopic ratios (δ18 Ooto values) were measured using a mass spectrometer for subsamples sequentially milled from the otolith core to the edge, and otolith Sr:Ca ratios were measured by an electron probe microanalyzer for the comparison. The mean water temperature within the studied area in the time period of each milled subsample was estimated from the Copernicus database. Based on an isotopic fractionation equation, each δ18 Ooto value and water temperature pair determined the water δ18 O value, which was then converted into salinity using published linear equations or an equation that was newly generated for this study. RESULTS: Individual fish clearly revealed different preferences in habitat use. The retrospectively reconstructed salinity history indicated that most L. japonicus inhabit and can spawn in seawater as well as in brackish estuaries. Few fish lived in freshwater during the young stage based on the analysis of δ18 Ooto profiles; however, otolith Sr:Ca ratios can only reveal freshwater residence and cannot distinguish residence in brackish water or seawater. CONCLUSIONS: This study demonstrated that otolith temporal microstructure and δ18 Ooto values are well-suited approaches for quantitative reconstruction of salinity histories of the fish. This method can improve the understanding of the habitat uses of other fishes inhabiting diverse habitats among the rivers, estuaries and oceans.

Magnesium as an intrinsic component of human otoconia.

OBJECTIVES: To investigate morphology changes of artificial otoconia (CGC) in the presence of magnesium during growth under in vitro conditions. METHODS: Investigating human otoconia by environmental scanning electron microscope and determining their magnesium content by energy-dispersive X-ray microanalysis (EDX). Comparing structural and morphological data of human and artificial otoconia (CGC, Ca1Mg0) without and with magnesium substitution (Ca1-xMgx). RESULTS: EDX- and X-ray data reveals that the inorganic component in human otoconia consists of calcite containing a minor amount of magnesium substitution (Ca1-xMgx). CGC containing magnesium (length 397.0 ± 146.4 µm, diameter 325.6 ± 100.1 µm) are slimmer and significantly smaller (p < .01) than pure CGC (length 548.6 ± 160 µm, diameter 373.0 ± 110.4 µm) and reveal a significant influence on the final morphology. The length/diameter ratio is significantly higher by incorporation of magnesium into CGC (1.84 ± 0.25 µm versus 1.48 ± 0.11 µm in pure CGC, p < .01), which brings the overall shape to a close relationship with human otoconia (1.98 ± 0.08 µm). CONCLUSIONS: Magnesium is an intrinsic component of human otoconia by partial substitution of calcium in the calcite crystal structure (Ca1-xMgx) and affects the development of the shape of artificial otoconia (calcite gelatin composites, CGC).

Morphology of the utricular otolith organ in the toadfish, Opsanus tau.

The utricle provides the vestibular reflex pathways with the sensory codes of inertial acceleration of self-motion and head orientation with respect to gravity to control balance and equilibrium. Here we present an anatomical description of this structure in the adult oyster toadfish and establish a morphological basis for interpretation of subsequent functional studies. Light, scanning, and transmission electron microscopy techniques were applied to visualize the sensory epithelium at varying levels of detail, its neural innervation and its synaptic organization. Scanning electron microscopy was used to visualize otolith mass and morphological polarization patterns of hair cells. Afferent nerve fibers were visualized following labeling with biocytin, and light microscope images were used to make three-dimensional (3-D) reconstructions of individual labeled afferents to identify dendritic morphology with respect to epithelial location. Transmission electron micrographs were compiled to create a serial 3-D reconstruction of a labeled afferent over a segment of its dendritic field and to examine the cell-afferent synaptic contacts. Major observations are: a well-defined striola, medial and lateral extra-striolar regions with a zonal organization of hair bundles; prominent lacinia projecting laterally; dependence of hair cell density on macular location; narrow afferent dendritic fields that follow the hair bundle polarization; synaptic specializations issued by afferents are typically directed towards a limited number of 7-13 hair cells, but larger dendritic fields in the medial extra-striola can be associated with > 20 hair cells also; and hair cell synaptic bodies can be confined to only an individual afferent or can synapse upon several afferents.

The sense of balance in humans: Structural features of otoconia and their response to linear acceleration.

We explored the functional role of individual otoconia within the otolith system of mammalians responsible for the detection of linear accelerations and head tilts in relation to the gravity vector. Details of the inner structure and the shape of intact human and artificial otoconia were studied using environmental scanning electron microscopy (ESEM), including decalcification by ethylenediaminetetraacetic acid (EDTA) to discriminate local calcium carbonate density. Considerable differences between the rhombohedral faces of human and artificial otoconia already indicate that the inner architecture of otoconia is not consistent with the point group -3m. This is clearly confirmed by decalcified otoconia specimen which are characterized by a non-centrosymmetric volume distribution of the compact 3+3 branches. This structural evidence for asymmetric mass distribution was further supported by light microscopy in combination with a high speed camera showing the movement of single otoconia specimen (artificial specimen) under gravitational influence within a viscous medium (artificial endolymph). Moreover, the response of otoconia to linear acceleration forces was investigated by particle dynamics simulations. Both, time-resolved microscopy and computer simulations of otoconia acceleration show that the dislocation of otoconia include significant rotational movement stemming from density asymmetry. Based on these findings, we suggest an otolith membrane expansion/stiffening mechanism for enhanced response to linear acceleration transmitted to the vestibular hair cells.

[The ultrastructure of utricular maculae of mouse].

Objective: Using scanning electron microscope to observe the ultrastructure of utricular maculae of mouse. Methods: Ten young (6 to 8 weeks) and ten old (>12 months) mice were executed, and their utricles were harvested and the specimens were processed, using scanning electron microscope to observe the structures of the utricles from the surface of otoconia layer to the roots of hair cell cilia. Results: Under the scanning electron microscope, several ultrastructures were observed, including otoconia layer, unstructured gelatinous extracellular matrix layer, honeycomb-like gelatinous extracellular matrix layer, inter-cilia otoconia and hair cell cilia associated with these structures. When compared with young mouse, the otoconia surface of aged mouse was smoother, the gelatinous extracellular matrix between the adjacent otoconias was thinner. Conclusions: Using SEM, ultrastructures can be clearly observed from surface otoconia layer to the roots of hair cell cilia. By the analysis of the ultrastructure of utricular maculae, it is helpful for investigation of the pathological mechanisms of vestibular diseases, such as otolith diseases.

Otoconia and otolithic membrane fragments within the posterior semicircular canal in benign paroxysmal positional vertigo.

OBJECTIVES/HYPOTHESIS: Benign paroxysmal positional vertigo (BPPV) is the most common vestibular disorder with an incidence between 10.7 and 17.3 per 100,000 persons per year. The mechanism for BPPV has been postulated to involve displaced otoconia resulting in canalithiasis. Although particulate matter has been observed in the endolymph of affected patients undergoing posterior canal occlusion surgery, an otoconial origin for the disease is still questioned. STUDY DESIGN: In this study, particulate matter was extracted from the posterior semicircular canal of two patients and examined with scanning electron microscopy. METHODS: The samples were obtained from two patients intraoperatively during posterior semicircular canal occlusion. The particles were fixed, stored in ethanol, and chemically dehydrated. The samples were sputter coated and viewed under a scanning electron microscope. Digital images were obtained. RESULTS: Intact and degenerating otoconia with and without linking filaments were found attached to amorphous particulate matter. Many otoconia appeared to be partially embedded in a gel matrix, presumably that which encases and anchors the otoconia within the otolith membrane, whereas others stood alone with no attached filaments and matrix. The otoconia measured roughly 2 to 8 µm in length and displayed a uniform outer shape with a cylindrical bulbous body and a 3 + 3 rhombohedral plane at each end. CONCLUSIONS: These findings suggest that the source of the particulate matter in the semicircular canals of patients with BPPV is broken off fragments of the utricular otolithic membrane with attached and detached otoconia. LEVEL OF EVIDENCE: NA Laryngoscope, 127:709-714, 2017.

[Otoconia : Current aspects of research]. / Otokonien : Aktuelle Aspekte aus der Forschung.

Otoconia are calcite-based nanocomposites containing >90 % calcite and <10 % organic material. The mean size is approximately 10 µm. The external structure of all otoconia in the utricle and saccule is similar, with a cylindrical bulbous body with a slightly hexagonal contour. The internal structure consists of a composite with varying volume thickness, dense branching structures (branches) and less dense surrounding areas (bellies). Intact otoconia can be clearly identified only by scanning electron microscopy. In the case of morphological changes (e.g. due to "degeneration") the origin of even very small particles of otoconia can be assigned using physical and chemical analytical methods. The inorganic component of otoconia (calcite) is extremely sensitive to chemical influences, which leads to morphological alterations. A "degeneration" of otoconia can be objectively accomplished in vitro by alterations in pH, electrolyte imbalance and by the influence of complex formation. These three main processes then lead to irreversible morphological alterations. Artificial (biomimetic) otoconia serve as a suitable model system for detailed investigation of growth and degenerative processes.

Morphological and biometrical characteristics on otolith of Barbus tauricus Kessler, 1877 on light and scanning electron microscope / Características morfológicas y biométricas de los otolitos de Barbus tauricus Kessler, 1877 bajo microscopio de luz y electrónico de barrido

The otolith morphology, biometry and the relationship between the otolith size and fish length were examined. The weight, length, width, area and perimeter were recorded for each pair of otoliths. The values of form factor (FF), roundness (RD), aspect ratio (AR), circularity (C), rectangularity (R) and elipticity (E) were calculated and the relationships between otolith length (OL) and shape indices were showed with graphics. According to paired t-test results, difference between right and left otoliths was important statistically (P<0.05), except otolith length. It is the first time, the difference of otolith variables were observed not only right and left pairs but also females and males of fish a member of Cyprinidae. While the otolith weight had the lowest coefficient of determination (r2) such as 0.69; 0.59; 0.65, otolith length had the highest 0.80; 0.81; 0.80, for female, male and all specimens, respectively. In addition, SEM images were firstly shown for otoliths of B. tauricus to determine the otolith morphology.

Se examinó la morfología de los otolitos, su biometría y la relación entre el tamaño de ellos y la longitud de los peces. Se registró el peso, longitud, grosor, área y perímetro para cada par de otolitos. Se calcularon y se identificaron en los gráficos los valores de factor de forma, redondez, relación de aspecto, circularidad, rectangularidad, elipticidad y las relaciones entre la longitud del otolito y los índices de la forma. De acuerdo con los resultados, la diferencia entre otolitos derechos e izquierdos fue estadísticamente significativo (P<0,05), excepto la longitud del otolito. Esta es la primera vez que se observa la diferencia de las variables de otolitos, no sólo ente los pares de derecha e izquierda, sino también entre peces hembras y machos. Mientras que el peso de los otolitos tuvo el menor coeficiente de determinación (R2), tales como 0,69; 0,59; 0,65, la longitud de los otolitos presentó un R2 más alto: 0,80; 0,81; 0.80, para hembras, machos y todas las muestras, respectivamente. Además, las imágenes de miscroscopía electrónica de barrido son las primeras en mostrar los otolitos de B. tauricus para determinar la morfología de éstos.

Latitudinal Gradient in Otolith Shape among Local Populations of Atlantic Herring (Clupea harengus L.) in Norway.

Otolith shape analysis of Atlantic herring (Clupea harengus) in Norwegian waters shows significant differentiation among fjords and a latitudinal gradient along the coast where neighbouring populations are more similar to each other than to those sampled at larger distances. The otolith shape was obtained using quantitative shape analysis, the outlines were transformed with Wavelet and analysed with multivariate methods. The observed morphological differences are likely to reflect environmental differences but indicate low dispersal among the local herring populations. Otolith shape variation suggests also limited exchange between the local populations and their oceanic counterparts, which could be due to differences in spawning behaviour. Herring from the most northerly location (69°N) in Balsfjord, which is genetically more similar to Pacific herring (Clupea pallasii), differed in otolith shape from all the other populations. Our results suggest that the semi-enclosed systems, where the local populations live and breed, are efficient barriers for dispersal. Otolith shape can thus serve as a marker to identify the origin of herring along the coast of Norway.

Close encounters with eddies: oceanographic features increase growth of larval reef fishes during their journey to the reef.

Like most benthic marine organisms, coral reef fishes produce larvae that traverse open ocean waters before settling and metamorphosing into juveniles. Where larvae are transported and how they survive is a central question in marine and fisheries ecology. While there is increasing success in modelling potential larval trajectories, our knowledge of the physical and biological processes contributing to larval survivorship during dispersal remains relatively poor. Mesoscale eddies (MEs) are ubiquitous throughout the world's oceans and their propagation is often accompanied by upwelling and increased productivity. Enhanced production suggests that eddies may serve as important habitat for the larval stages of marine organisms, yet there is a lack of empirical data on the growth rates of larvae associated with these eddies. During three cruises in the Straits of Florida, we sampled larval fishes inside and outside five cyclonic MEs. Otolith microstructure analysis revealed that four of five species of reef fish examined had consistently faster growth inside these eddies. Because increased larval growth often leads to higher survivorship, larvae that encounter MEs during transit are more likely to contribute to reef populations. Successful dispersal in oligotrophic waters may rely on larval encounter with such oceanographic features.

Principles of calcite dissolution in human and artificial otoconia.

Human otoconia provide mechanical stimuli to deflect hair cells of the vestibular sensory epithelium for purposes of detecting linear acceleration and head tilts. During lifetime, the volume and number of otoconia are gradually reduced. In a process of degeneration morphological changes occur. Structural changes in human otoconia are assumed to cause vertigo and balance disorders such as benign paroxysmal positional vertigo (BPPV). The aim of this study was to investigate the main principles of morphological changes in human otoconia in dissolution experiments by exposure to hydrochloric acid, EDTA, demineralized water and completely purified water respectively. For comparison reasons artificial (biomimetic) otoconia (calcite gelatin nanocomposits) and natural calcite were used. Morphological changes were detected in time steps by the use of environmental scanning electron microscopy (ESEM). Under in vitro conditions three main dissolution mechanisms were identified as causing characteristic morphological changes of the specimen under consideration: pH drops in the acidic range, complex formation with calcium ions and changes of ion concentrations in the vicinity of otoconia. Shifts in pH cause a more uniform reduction of otoconia size (isotropic dissolution) whereas complexation reactions and changes of the ionic concentrations within the surrounding medium bring about preferred attacks at specific areas (anisotropic dissolution) of human and artificial otoconia. Owing to successive reduction of material, all the dissolution mechanisms finally produce fragments and remnants of otoconia. It can be assumed that the organic component of otoconia is not significantly attacked under the given conditions. Artificial otoconia serve as a suitable model system mimicking chemical attacks on biogenic specimens. The underlying principles of calcite dissolution under in vitro conditions may play a role in otoconia degeneration processes such as BPPV.

Crystallinity and microchemistry of Nassarius reticulatus (Caenogastropoda) statoliths: towards their structure stability and homogeneity.

Gastropod statoliths are spherical biocarbonates formed during their lifespan. The stability and homogeneity of these structures' mineral matrix was characterised along their radiuses, using Nassarius reticulatus as a model. Generally, they were proved to be bimineralic. Two of the three CaCO3 crystalline polymorphs occurring in biocarbonates - aragonite and calcite - coexist along statolith radiuses, aragonite being unequivocally the most abundant phase. The presence of a diffuse organic matrix was also perceived by the detection of a weak Raman band between 2800 and 3000 cm(-1) consistently observed along radiuses. Beyond the apparent stability and homogeneity, different crystalline orientations were disclosed by Raman spectroscopy. A change in the intensity pattern of the features related to the lattice and bending modes of aragonite between different radiuses give new insights for a possible spherulitic-like growth of these structures. As expected from the relative homogeneity of both mineral and organic signals, there was no pattern on the distribution of Ca, O, Na and S along radiuses. However, a higher concentration of Sr occurs in growth rings (known as winter tags), corroborating the already described negative correlation between the concentration of this element in statoliths and temperature. Despite the apparent stability and homogeneity of the matrix during its lifespan, the periodic distribution of Sr potentially influences a dissimilar incorporation of trace elements in increments and growth rings. Since gastropod statolith elemental fingerprinting was recently suggested as a new tool to monitor marine environmental changes, the pressing need for further studies on the incorporation of traces in these structures is highlighted.

Detection of human utricular otoconia degeneration in vital specimen and implications for benign paroxysmal positional vertigo.

Otoconia are assumed to be involved in inner ear disorders such as benign paroxysmal positional vertigo (BPPV). Up to now, the distinct structure and morphology of intact and degenerate human utricular otoconia has been only poorly investigated on vital specimen. In this study, human otoconia were obtained from the utricle in five patients undergoing translabyrinthine vestibular schwannoma surgery. Specimens were examined by environmental scanning electron microscopy. Intact and degenerate otoconia as well as fracture particles of otoconia and bone were analyzed by energy dispersive X-ray microanalysis (EDX) and powder X-ray diffraction (XRD). Intact otoconia reveal a uniform size showing characteristic symmetry properties. Degenerative changes can be observed at several stages with gradual minor and major changes in their morphology including fragment formation. EDX analyses reveal the characteristic chemical composition also for otoconia remnants. XRD shows that intact and degenerate otoconia as well as remnants consist of the calcite modification. In conclusion, electron microscopy serves as a standard method for morphological investigations of otoconia. Human utricular otoconia show a uniform outer morphology corresponding to a calcite-based nanocomposite. Morphological changes provide further evidence for degeneration of utricular otoconia in humans, which might be a preconditioning factor causing BPPV. In case of uncertain origin, particles can be clearly assigned to otoconial origin using EDX and XRD analyses.

Gentamicin-induced structural damage of human and artificial (biomimetic) otoconia.

CONCLUSIONS: Gentamicin causes irreversible structural damage of human and artificial otoconia by progressive dissolution of calcite. The inner architecture of otoconia is strongly affected by degradation scenarios during gentamicin exposure. Artificial otoconia can be used as a model system mimicking the chemical attacks for detailed investigations. OBJECTIVES: To investigate the chemical interactions of gentamicin with natural calcite and human and artificial otoconia under in vivo conditions. METHODS: Pure calcite crystals and artificial and human otoconia were exposed to gentamicin injection solutions at various concentrations. Morphological changes were observed in time steps by the use of environmental scanning electron microscopy (ESEM). RESULTS: Dissolution of pure calcite crystals results in the formation of well oriented nanoshoots indicating an irreversible chemical reaction with gentamicin. Human and artificial otoconia reveal irreversible structural changes of their surface areas as well as of their inner structure, resulting in characteristic changes at different gentamicin concentrations. Minor changes are first observed by surface alterations and dissolution of calcite in the belly region. Major changes result in further reduction of the belly area reaching the center of symmetry. Finally, a complete dissolution of the branches takes place. Artificial otoconia provide detailed insight into surface alterations.

Texture and nano-scale internal microstructure of otoliths in the Atlantic molly, Poecilia mexicana: a high-resolution EBSD study.

Otoliths of modern bony fishes are massive polycrystalline structures consisting mainly of calcium carbonate (primarily aragonite), and 1-10% organic residuals. Unlike other biomineralisates like shells, teeth and bones, they are not optimized for mechanical loads but serve the senses of hearing and balance in the inner ear. We examined internal structural variation of otoliths through microstructural and texture analyses. Our study applied the electron backscattered diffraction technique (EBSD) to whole sections of saccular otoliths on cave- and surface-dwelling fish. Application of high spatial resolution EBSD on otoliths of the livebearing fish Poecilia mexicana allowed for an investigation of crystal orientation despite the small size (<150 nm) of aragonite crystallites. Crystallites at the rims of otoliths had a higher structural organization than those situated near the center, where no dominant orientation pattern was discernible. Moreover, the medial (sulcal) face of otoliths, which makes contact with the sensory epithelium, was more structured than the lateral (antisulcal) face.