Enigmatic ear stones: what we know about the functional role and evolution of fish otoliths

Otoliths in bony fishes play an important role in the senses of balance and hearing. Otolith mass and shape are, among others, likely to be decisive factors influencing otolith motion and thus ear functioning. Yet our knowledge of how exactly these factors influence otolith motion is incomplete. In addition, experimental studies directly investigating the function of otoliths in the inner ear are scarce and yield partly conflicting results. Herein, we discuss questions and hypotheses on how otolith mass and shape, and the relationship between the sensory epithelium and overlying otolith, influence otolith motion. We discuss (i) the state‐of‐the‐art knowledge regarding otolith function, (ii) gaps in knowledge that remain to be filled, and (iii) future approaches that may improve our understanding of the role of otoliths in ear functioning. We further link these functional questions to the evolution of solid teleost otoliths instead of numerous tiny otoconia as found in most other vertebrates. Until now, the selective forces and/or constraints driving the evolution of solid calcareous otoliths and their diversity in shape in teleosts are largely unknown. Based on a data set on the structure of otoliths and otoconia in more than 160 species covering the main vertebrate groups, we present a hypothetical framework for teleost otolith evolution. We suggest that the advent of solid otoliths may have initially been a selectively neutral ‘by‐product’ of other key innovations during teleost evolution. The teleost‐specific genome duplication event may have paved the way for diversification in otolith shape. Otolith shapes may have evolved along with the considerable diversity of, and improvements in, auditory abilities in teleost fishes. However, phenotypic plasticity may also play an important role in the creation of different otolith types, and different portions of the otolith may show different degrees of phenotypic plasticity. Future studies should thus adopt a phylogenetic perspective and apply comparative and methodologically integrative approaches, including fossil otoliths, when investigating otoconia/otolith evolution and their function in the inner ear.     

The effect of otolith storage methods on the concentrations of elements detected by laser-ablation ICPMS

Otoliths of male (1+ year old) Terubok, tropical shad Tenualosa toli from the Lupar River, Sarawak, were treated in different ways to assess the effect of post-capture storage on element concentrations in the otolith core and edge detected by laser-ablation inductively coupled plasma mass spectrometry (ICPMS). Five treatments were compared: rapid removal and dry storage; fish kept 5 h after capture then otoliths removed; rapid removal and stored in alcohol; fish rozen for 24 h then otoliths removed; and storage of intact head in alcohol for several weeks. Seven elements were detected at the core and edge of otoliths (Li, Na, Mg, Mn, Co, Sr, Ba). Of these, Na, Mg, Co and Ba differed among treatments, being highest in treatments where the otolith remained in the head. Variation in element measurements due to treatment effects was similar to within-otolith variation for the other elements and was least variable in the fresh treatment. The variation in concentration between the core and edge was more significant than other sources of variation (up to 100 times) for some elements. These differences were probably due to physiological factors (ontogenetic) or to yearly changes in water chemistry. The results indicate that the method of otolith storage, especially freezing whole fish, can have a small, but measurable, effect on the concentrations of Na, Mg, Co and Ba.     

Saccular otolith mass asymmetry in adult flatfishes

A dimensionless measure of otolith mass asymmetry, χ, was calculated as the difference between the masses of the right and left paired otoliths divided by average otolith mass. Saccular otolith mass asymmetry was studied in eight flatfish species (110 otolith pairs) and compared with data from a previously published study on roundfishes. As in the case of symmetrical fishes, the absolute value of χin flatfishes does not depend on fish length and otolith growth rate, although otolith mass and the absolute value of otolith mass difference are correlated with fish length. The values of χwere between ?0·2 and +0·2 in 96·4% of flatfishes studied. The mean ±s .e . value of χin flatfishes was significantly larger than in standard bilaterally symmetrical marine fishes (‘roundfishes’), respectively 0·070 ± 0·006 and 0·040 ± 0·006. The most prominent distinction is the existence of downside prevalence of saccular otolith mass in flatfishes, which contrasts with no right–left prevalence in roundfishes found in a previous study. In the right‐eyed flatfishes (Soleidae), the left saccular otoliths are heavier than the right otoliths. In the left‐eyed flatfishes (Bothidae and Citharidae), the right saccular otoliths are heavier than the left otoliths. Not all flatfishes, however, fit in this design: 11·8% of flatfishes studied had the heavier saccular otoliths in the upside labyrinth and 5·4% of flatfishes had no otolith mass asymmetry (within the accuracy of the analysis). At the same time, the more mobile flatfishes (bothids and citharids) have more symmetrical and, hence, more precisely organized saccular otolith organs than the bottom‐associated flatfishes (soleids). It is possible to assume that the value of the otolith asymmetry is not only correlated with flatfish placement in a particular family, or position of eyes, but also may correlate with general aspects of their ecology. Mathematical modelling indicated that for most flatfishes one‐side saccular prevalence had no substantial significance for sound processing. On the other hand, calculations showed that 49% of flatfishes (but only 14·5% of roundfishes) have |χ| which exceed the critical level and, in principle, could sense the difference between the static displacement of the large and small paired otoliths. At that, the number of the soleids that could sense this difference is greater than the number of the bothids and citharids, 84 and 27%, respectively.     

Rapid larval growth predisposes sex change and sexual size dimorphism in a protogynous hermaphrodite, Parapercis snyderi Jordan & Starks 1905

The temporal relationship between growth history, sex-specific growth divergence and sex change was investigated in the haremic sandperch Parapercis snyderi using otolith microstructure and gonad histology. Parapercis synderi was found to display rapid near-linear growth with a maximum longevity of 303 days. All individuals matured first as female and later changed sex to become male (monandric protogynous hermaphroditism). Individual age-based growth histories obtained from otolith increment widths illustrated that males were larger than females at any given age. Males were found to diverge from the female growth trajectory during two ontogenetic periods; during the larval period and during the period that sex change took place. In addition, male otoliths contained a discontinuity, or 'check mark', associated with the rapid increase in otolith growth during the sex-change period. This microstructural feature was absent from all female otoliths. Accelerated growth in male otoliths lasted up to 25 days, following check-mark formation, after which time otolith growth returned to the pre-check-mark rate. Given the isometric relationship between otolith and somatic growth in P. synderi , and the temporal relationship between the time of check-mark formation and gonad condition, these results strongly suggest that individuals accelerate somatic growth during sex change to become the largest members of the population. Moreover, evidence suggests that the factors that determine the initial growth of larvae influence which individuals will later become males and achieve the highest reproductive success.     

An evaluation of the otolith characteristics of Conger conger during metamorphosis

A sample of 20 metamorphosing conger eel Conger conger leptocephali were collected from the Minho River, Portugal, in February 1999 and their sagittal otoliths were analysed by scanning electron microscopy. Four different etching agents were applied along both sagittal and frontal sections during otolith preparation to examine the microstructural growth in this species. Otolith growth increments were visible throughout the increment countable zone using all four treatments, but a permanent peripheral diffuse zone, where the daily increments were unclear, appeared on all otoliths, preventing accurate age estimation. To understand more about the nature of the diffuse zone, otoliths of 10 other metamorphosing leptocephali reared in aquaria were marked by immersion in tetracycline hydrochloride. The distance between the fluorescent marks and otolith edge, measured over a fixed period of time, was used to estimate the otolith growth rate. The application of this technique led to an anomalously high estimated otolith growth rate, probably as a result of the capture, marking and handling stress.     

Computed tomography scanning as a tool for linking the skeletal and otolith‐based fossil records of teleost fishes

Micro‐computed tomography (μCT) scanning now represents a standard tool for non‐destructive study of internal or concealed structure in fossils. Here we report on otoliths found in situ during routine μCT scanning of three‐dimensionally preserved skulls of Palaeogene and Cretaceous fishes. Comparisons are made with isolated otolith‐based taxa to attempt correlations between the body fossil and otolith fossil records. In situ otoliths previously extracted mechanically from specimens of Apogon macrolepis and Dentex laekeniensis match our μCT models. In some cases, we find a high degree of congruence between previously independent taxonomic placements for otolith and skeletal remains (Rhinocephalus, Osmeroides, Hoplopteryx). Unexpectedly, in situ otoliths of the aulopiform Apateodus match isolated otoliths of Late Cretaceous age previously interpreted as belonging to gempylids, a group of percomorph fishes that do not appear in the body fossil record until the Palaeogene. This striking example of convergence suggests constraints on otolith geometry in pelagic predators. The otoliths of Apateodus show a primitive geometry for aulopiforms and lack the derived features of Alepisauroidea, the lizardfish clade to which the genus is often attributed. In situ otoliths of Early Cretaceous fishes (Apsopelix and an unidentified taxon) are not well preserved, and we are unable to identify clear correlations with isolated otolith morphologies. We conclude that the preservation of otoliths suitable for μCT scanning appears to be intimately connected with the taphonomic history, lithological characteristics of surrounding matrix, and syn‐ and postdepositional diagenetic effects.     

基于椭圆傅里叶分析的次南极电灯鱼矢耳石形态多样性

次南极电灯鱼矢耳石形态特征具有多样性.为了深入研究其形态特征,利用南设得兰群岛外侧水域采集的456尾次南极电灯鱼(体长范围6.0～8.8 cm)样本,对其矢耳石形态进行分析和判别.根据形态特征将次南极电灯鱼耳石分为4种类型,并采用椭圆傅里叶分析法选取表征耳石类型的77个傅里叶特征系数进行了分析.结果表明: 对4种耳石类型两两比较后发现,具有显著性差异的傅里叶特征系数最多及最少分别占总体的61%和28.6%;对77个傅里叶系数进行主成分分析,前22个主成分解释了总变异的76.6%;选取了17个傅里叶特征系数进行判别分析,建立判别函数,总体判别率为87.2%;根据椭圆傅里叶分析重建的耳石轮廓反映了4种耳石类型间的差异.4种耳石类型在不同体长及体质量的次南极电灯鱼中皆有出现,表明耳石类型具有随机性,且左右耳石类型不一致,表明其左右耳石外形具有差异性.4种耳石类型中,Ⅰ型和Ⅱ型占总体的72.6%,为次南极电灯鱼耳石的主要形态;Ⅲ型和Ⅳ型占总体的27.4%,为次要形态.     

Comparison of spatial variation in otolith chemistry of two fish species and relationships with water chemistry and otolith growth

Spatial variation in the chemistry (Mg, Mn, Sr and Ba) of recently deposited otolith material (last 20–30 days of life) was compared between two demersal fish species; snapper Pagrus auratus (Sparidae) and sand flathead Platycephalus bassensis (Platycephalidae), that were collected simultaneously at 12 sites across three bays in Victoria, south-eastern Australia. Otolith chemistry was also compared with ambient water chemistry and among three sampling positions adjacent to the proximal otolith margin. For both species, variation in otolith chemistry among bays was significant for Ba, Mn and Sr; however, differences among bays were only similar between species for Ba and Mn. Only Ba showed significant variation at the site level. Across the 12 sites, mean otolith Ba levels were significantly positively correlated between species. Further, although incorporation rates differed, mean ambient Ba levels for both species were positively correlated with ambient Ba levels. Spatial variation in multi-element otolith chemistry was also broadly similar between species and with multi-element water chemistry. Partition coefficients clearly indicated species-specific incorporation of elements into otoliths. Mg and Mn were consistently higher in snapper than sand flathead otoliths (mean ±s .d ., Mg snapper 22·1 ± 3·8 and sand flathead 9·9 ± 1·5 μg g⁻¹, Mn snapper 4·4 ± 2·6 and sand flathead 0·5 ± 0·3 μg g⁻¹), Sr was generally higher in sand flathead otoliths (sand flathead 1570 ± 235 and snapper 1346 ± 104 μg g⁻¹) and Ba was generally higher in snapper otoliths (snapper 12·1 ± 12·8 and sand flathead 1·8 ± 1·4 μg g⁻¹). For both species, Mg and Mn were higher in the faster accreting regions of the otolith margin, Sr was lower in the slower accreting region and Ba showed negligible variation among the three sampling regions. This pattern was consistent with the higher Mg and Mn, and generally lower Sr observed in the faster accreting snapper otoliths. It is hypothesized that the differences between species in the incorporation of these elements may be at least partly related to differences in metabolic and otolith accretion rate. Although rates of elemental incorporation into otoliths appear species specific, for elements such as Ba where incorporation appears consistently related to ambient concentrations, spatial variation in otolith chemistry should show similarity among co-occurring species.     

Comparison of otolith growth and morphology with somatic growth and age in young-of-the-year bluefin tuna

Otolith morphological characteristics were studied using image analysis techniques and the relationships between otolith growth and somatic growth and age, as estimated from counting daily otolith increments, were examined in young-of-the-year (YOY) bluefin tuna Thunnus thynnus ranging in fork length ( L _F) from 8·5 to 55·5 cm. Whole otolith length, width, area and perimeter, and three shape indexes, circularity, E value and rectangularity, were extracted for each pair of sagittae. Since no statistical significant differences between left and right otolith morphometrics were found, only one otolith from each fish was used for correlations. Statistically significant relationships were observed between otoliths measurements and fish somatic growth when a linear regression was applied after logarithmic transformation of all variables tested. Among the variables, otolith length was the one that showed the highest correlation with L _F, followed by otolith area and perimeter, whereas otolith rectangularity exhibited the lowest correlation. Statistically significant relationships were also observed between the otolith variables tested and the age of the fish, which ranged from 20 to 129 days. The ages estimated using otolith mass were very close to those assessed using daily increment counts (bias ranged from 1 to 24 days). Therefore, otolith mass could represent a valuable criterion for age estimation in YOY bluefin tuna that is objective, economic and easy to perform compared to daily increment counting method.     

Validation of a simple and well‐suited chemical cleaning method for fish otoliths

In marine biology, many research fields are based on use of fish otoliths. All the studies dealing with otoliths need as starting point a perfectly clean otolith. Dissection is difficult when working on small or highly jagged otoliths. A common problem is that during otolith preparation some fish tissue may remain stuck to it, even after a mechanical cleaning. Then, supplementary cleaning with chemicals is needed. Classical methods are known to possibly alter otolith's structure and/or composition. Here, we present a chemical cleaning method using only sodium hydroxide. We have validated the method on two different fish species, Oblada melanura and Dicentrarchus labrax. Observation and analyses of morphological measurements have confirmed significant suppression of residual tissues. We propose this method as a good alternative to previously published mono‐ or multichemical methods.     

The detection of elements in larval otoliths from Atlantic herring using laser ablation ICP-MS

Trace element concentrations of otoliths from larval herring Clupea harengus collected from known spawning beds in the Celtic and Irish Seas, were investigated using laser ablation ICP-MS and compared with concentrations in the larval cores of juvenile otoliths from the same populations and year class. A range of elements (Mg, Zn, Sr, Ba and Pb) was detectable in early larval otoliths (20–40 µm diameter). Larval otolith concentrations exceeded the larval core concentrations of juvenile otoliths and also the concentrations reported in the literature, for Mg, Zn, Ba and Pb, indicating that the measurement of elements in larval otoliths was severely affected by post-mortem contamination, most likely due to adherence of tissue and endolymph residue on the otolith surface. Comparison of otolith composition between larvae from two freezing treatments showed that contamination from Mg and Zn was more serious in otoliths that had remained in frozen larvae for prolonged periods. Larval populations from the two seas showed significant differences in otolith Sr concentrations, which were consistent over two sampling years. Similar differences were seen in the corresponding juvenile populations. The results show that while early larval otoliths are extremely susceptible post-mortem contamination, Sr concentrations can be reliably measured using laser ablation ICP-MS and for this element, the detection of region specific differences is possible.     

Using the otolith sulcus to aid in prey identification and improve estimates of prey size in diet studies of a piscivorous predator

Diet studies are fundamental for understanding trophic connections in marine ecosystems. In the southeastern US, the common bottlenose dolphin Tursiops truncatus is the predominant marine mammal in coastal waters, but its role as a top predator has received little attention. Diet studies of piscivorous predators, like bottlenose dolphins, start with assessing prey otoliths recovered from stomachs or feces, but digestive erosion hampers species identification and underestimates fish weight (FW). To compensate, FW is often estimated from the least affected otoliths and scaled to other otoliths, which also introduces bias. The sulcus, an otolith surface feature, has a species‐specific shape of its ostium and caudal extents, which is within the otolith edge for some species. We explored whether the sulcus could improve species identification and estimation of prey size using a case study of four sciaenid species targeted by fisheries and bottlenose dolphins in North Carolina. Methods were assessed first on otoliths from a reference collection (n = 421) and applied to prey otoliths (n = 5,308) recovered from 120 stomachs of dead stranded dolphins. We demonstrated in reference‐collection otoliths that cauda to sulcus length (CL:SL) could discriminate between spotted seatrout (Cynoscion nebulosus) and weakfish (Cynoscion regalis) (classification accuracy = 0.98). This method confirmed for the first time predation of spotted seatrout by bottlenose dolphins in North Carolina. Using predictive models developed from reference‐collection otoliths, we provided evidence that digestion affects otolith length more than sulcus or cauda length, making the latter better predictors. Lastly, we explored scenarios of calculating total consumed biomass across degrees of digestion. A suggested approach was for the least digested otoliths to be scaled to other otoliths iteratively from within the same stomach, month, or season as samples allow. Using the otolith sulcus helped overcome challenges of species identification and fish size estimation, indicating their potential use in other diet studies.     

The differentiation of Stegastes partitus populations using lapillar and sagittal otolith chemistry

The comparison of elemental concentrations of sagittal and lapillar otoliths from the same individuals of Stegastes partitus indicated significant differences for several elements. Sagittal otoliths were superior at differentiating individuals, yet the differentiation of individuals was further improved when the elemental concentrations of both otolith types were used in the same analysis.     

Influence of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers: does otolith growth cease at low temperatures?

The influences of water temperature and feeding regime on otolith growth in Anguilla japonica glass eels and elvers were investigated using individuals reared at 5, 10, 15, 20, 25 and 30° C and in fed or unfed conditions at salinity 32 after their otoliths were marked with alizarin complexone (ALC). To eliminate the difficulty of observing the edges of otoliths with optical (OM) or scanning electron (SEM) microscopes, three to 10 individuals were sampled from each tank at 10, 20 and 30 days during the experiment and reared for an additional 10 days at 25° C after their otoliths were marked a second time. Otolith growth and the number of increments were measured using both OM and SEM. Most A. japonica commenced feeding after 10 days at 20–30° C or after 20 days at 15° C, but no feeding occurred at 5 and 10° C. No otolith growth occurred at 5 and 10° C except in two individuals with minimal increment deposition at 10° C. Otolith growth was proportional to water temperature within 15–25° C and not different between 25 and 30° C. At 15, 25 and 30° C, the mean otolith growth rate in fed conditions was higher than in unfed conditions. The number of increments per day was significantly different among water temperatures (0·00–0·01 day⁻¹ at 5 and 10° C, 0·43–0·48 day⁻¹ at 15° C and 0·94–1·07 day⁻¹ at 20–30° C). These results indicated that otolith growth in A. japonica glass eels and elvers was affected by temperature and ceased at ≤10° C under experimental conditions. Hence, future studies analysing the otoliths of wild-caught A. japonica glass eels and elvers need to carefully consider the water temperatures potentially experienced by the juveniles in the wild.     

Otolith size trends in marine fish communities from different depth strata

A comparison of 681 saccular otoliths (sagitta) from 134 species belonging to six demersal communities from different depth strata and the epipelagic community from the north-western Mediterranean Sea was made in order to study otolith relative size and function related morphologies. A relationship between otolith size composition, habitat and depth was found. The epipelagic community was characterized by species with very small and small otolith sizes (68% of the epipelagic species). In the demersal communities, the proportion of species with large sagitta increased with depth until 750 m (reached 50% of the species of the upper slope). The abyssal community (between 1000 and 2000 m), however, was characterized by a decrease in the mean otolith size and an increase in the proportion of species with very small otoliths. With exception of the abyssal community, endogenous causes (a mixture of geneaology, plesiomorph characters shared by the all species of the taxonomic group and recent adaptive ones) may be even more important than exogenous factors in determining the otolith relative size. Within the endogenous causes that condition sagitta size, the adaptive features associated with specialization in acoustic communication are relevant.     

Morphometry and composition of aragonite and vaterite otoliths of deformed laboratory reared juvenile herring from two populations

Vaterite otoliths were sampled from two reared populations (Celtic and Clyde Seas) of juvenile herring Clupea harengus. The crystallography, elemental composition and morphometry were analysed and compared with those of normal aragonite otoliths. The incidence of vaterite otoliths in the juveniles sampled (n = 601) ranged from 7·8% in the Clyde population to 13·9% in the Celtic Sea population, and was 5·5% in the small sample (n = 36) of wild adults examined. In all but one case fish had only one vaterite otolith; the corresponding otolith of the pair was completely aragonite. Although the majority of the juveniles sampled showed craniofacial deformities, there was no link between the skull or jaw malformation and the incidence of vaterite otoliths. All vaterite otoliths had an aragonite inner area, and vaterite deposition began sometime after the age of 90 days. The vaterite otoliths were larger and lighter than their corresponding aragonite partners, and were less dense as a consequence of the vaterite crystal structure. The vaterite areas of the otoliths were depleted in Sr, Na and K. Concentrations of Mn were higher in the vaterite areas. The transition between the aragonite inner areas and the vaterite areas was sharply delineated. Within a small spatial scale (20 μm³) in the vaterite areas, however, there was co‐precipitation of both vaterite and aragonite. The composition of the aragonite cores in the vaterite otoliths was the same as in the cores of the normal aragonite otoliths indicating that the composition of the aragonite cores did not seed the shift to vaterite. Vaterite is less dense than aragonite, yet the concentrations of Ca analysed with wavelength‐dispersive spectrometry (WDS) were the same between the two polymorphs, indicating that Ca concentrations measured with WDS are not a good indicator of hypermineralized zones with high mineral density. The asymmetry in density and size of the otoliths may cause disruptions of hearing and pressure sensitivity for individual fish with one vaterite otolith, however, the presence of vaterite otoliths did not seem to affect the growth of these laboratory reared juvenile herring.     

Scanning electron microscopic analysis of annulus microstructure in otolith of European eel, Anguilla anguilla

To validate the yearly periodicity of annulus formation in the otolith of the eel, the structure of annuli in otoliths of the European eel, Anguilla anguilla , stocked for 7 and 12 years in Lake Ommen on the east coast of southern Sweden, was examined. The population was stocked from elvers imported both from France (Bay of Biscay) in April 1979 and England (River Severn) in March–April 1984. The microstructure of an annulus consisted of single, double and/or composite tings depending on the location in the otolith. The counts of annuli in otoliths of these eels were approximately consistent with the expected age. However, supernumerary false annuli and/or annulus underestimation frequently occurred. The methodology for annulus discrimination with light and scanning electron microscopes is described.     

Estimating the timing of growth rings in Atlantic cod otoliths using stable oxygen isotopes

A technique involving micro‐scale sampling of otolith carbonate and analyses of stable oxygen isotope composition was used to relate the zone appearance of the otolith to the seasonal temperature cycle. Otolith opacity could then be related to the timing of zone formation. Otoliths from two groups of Atlantic cod Gadus morhua held under known temperature conditions over a period of 4 and 6 years were examined. The otolith translucency followed the same pattern as the estimated temperature (from otolith δ¹⁸O values) in the yearly increments three and four, meaning that the translucent zones were deposited at the seasonal highest temperature in late summer and early autumn. The relative light intensities of otolith yearly increments five and six of older fish (deposited in the same years), however, were not significantly correlated to the estimated temperatures since increased otolith translucency also occurred at low temperatures. This might have been caused by stress in connection with gonad development or starvation during the spawning period. The results showed that this method of coupling otolith opacity and stable oxygen isotope composition can be used to estimate the timing of zone formations in otoliths.     

Evidence of perturbations induced by reproduction on somatic growth and microincrement deposition in Oreochromis niloticus otoliths

The direct effects of reproduction (energy expenditure for gametogenesis) were studied in gonadectomized Oreochromis niloticus females, incapable of reproducing. The indirect effects of reproduction were studied in intact females that had either mouth brooded their eggs or not. In gonadectomized females the slowing of growth observed in the marginal zone of their otoliths coincided with the time of gonad regeneration. The responses were very varied in the group of intact females. In these females, there was less otolith growth and more checks were observed on the otoliths. The results showed that reproduction influenced the growth of fish and their otoliths leading to a significant underestimate of the number of primary increments during this period of life.