Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?

Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.     

Exposure to thyroid hormone in ovo affects otolith crystallization in rainbow trout Oncorhynchus mykiss

Calcareous otoliths in the inner ears of fishes are necessary for proper hearing and vestibular function. Sagittal otoliths are usually composed of the calcium carbonate polymorph aragonite but may contain the polymorph vaterite, a phenomenon called otolith crystallization. The causes of otolith crystallization are poorly understood. Thyroid hormone (TH) can influence the chemical microenvironment and structure of the inner ear, suggesting that TH may influence otolith crystallization. The present study examined the effect of exogenous TH treatment on sagittal otolith crystallization and growth in larval and juvenile rainbow trout, Oncorhynchus mykiss. In the first experiment, 110?C179?day-old fish raised from TH-treated oocytes had significantly fewer sagittal otoliths containing the crystalline calcium carbonate polymorph vaterite as compared to untreated fish. Vaterite-containing otoliths were significantly longer than those containing the typical polymorph aragonite, although there was no effect of TH treatment on otolith length. In the second experiment, juveniles immersed in an exogenous solution of TH for 6?weeks had slightly longer otoliths (relative to fish length) than age-matched controls, but this effect was not significant. This juvenile population had a very high percentage (88.3?%) of vaterite sagittae overall and this percentage did not change significantly with treatment, suggesting the switch from aragonite to vaterite occurred prior to inclusion of the fish in the study. These results suggest that early manipulation of TH levels may affect calcium carbonate deposition on the otolith but that later TH exposure is unable to restore typical otolith composition.     

Vaterite Otoliths from the Opah,Lampris immaculatus,and Two Species of Sunfish,Mola mola and M. ramsayi

The cosmopolitan opah Lampris immaculatus (also known as the moonfish or mariposa) occurs commonly in New Zealand waters. The otoliths of 23 individuals were examined by light microscopy. All three of the otoliths of the endolymphatic sac of the opah have the characteristic appearance of the Stolkowski crystal form of the vaterite morph of calcium carbonate. Five randomly chosen pairs of asterici and sagittae and two lapilli otoliths were shown by X-ray diffraction to be composed of vaterite. In addition to their mineral composition, the sagitta and astericus of the opah present an unusual combination of anatomical features found in both what are generally regarded as primitive and as advanced fish. Vaterite otoliths also occur in the sunfish Mola mola and M. ramsayi, but with a different crystalline habit to that found in the opah, and in a distinctively different anatomical arrangement of the endolymphatic sac. It is argued that, based on their otolith mineralogy and morphology, the opah and the sunfish are each side of one of the biochemical and anatomical boundaries involved in the separation of the teleosts from other actinopterygian fish (sturgeons, paddle fish, gars and bowfins).     

Morphology and microchemistry of abnormal otoliths in the ayu, <Emphasis Type="Italic">Plecoglossus altivelis</Emphasis>

The sagittal otolith morphology and microchemistry of reared juvenile ayu, Plecoglossus altivelis, were examined to describe the occurrence and microchemical characteristics of the abnormal otoliths in this species. Juvenile ayu (N = 31) were collected in June 2004 at three different locations, Wakayama, Kumamoto, and Biwa Lake in Japan, where they were being reared in freshwater aquaculture ponds after having been collected in the wild as larvae. Otolith abnormality was found in the sagittae of 26% (N = 8) of the individuals examined, of which five fish had abnormal otoliths only on one side, while the otolith on the other side was normal. Abnormal otoliths were more transparent and crystalline in appearance with irregular shapes compared to normal ones that were more opaque and less irregular. Abnormal otoliths were divided into two types, semi-abnormal (Type 1) with a normal part in the center, and fully-abnormal (Type 2) that were completely crystalline in appearance. The line transects and whole otolith concentration maps showed that the contents of Sr, Na and K were lower in the abnormal otolith regions compared to the normal ones, while those of Ca and S were almost constant in both. The appearance and microchemical properties of the abnormal ayu otoliths were similar to the abnormal otoliths in other species in which vaterite replaces the aragonite. Abnormal formation of otoliths occurred in ayu from Biwa Lake (30%) and Kumamoto (45%), while the Wakayama samples had no abnormality. The microchemical analyses of the normal and abnormal otoliths indicated that some abnormal otoliths had formed before the fish were captured and transferred to the hatchery, so the possible causes of otolith abnormality in ayu are discussed.     

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.     

Strontium:cakium concentration ratios in otoliths of herring larvae as indicators of environmental histories

Synopsis Elemental analyses, using wave-length dispersive electron microprobe techniques on otoliths from reared Atlantic herring larvae, Clupea harengus, showed trace quantities of strontium relative to that of calcium, and an inverse relationship between Sr/Ca concentration ratios and rearing temperature. These data are consistent with those for coral aragonite, in that there appears to be an inverse temperature effect on physiological incorporation of strontium in the otolith aragonite. Our determinations of Sr/Ca concentration ratios of lab-reared herring larvae showed that the deposition of strontium relative to calcium and the rearing temperature were related, where: T (° C) = −2.955 [Sr/Ca] × 1000 ± 19.172. This principle thus makes it possible to use Sr/Ca concentration ratios in fish otoliths to delineate past temperatures experienced by an individual. Further, combining electron microprobe analyses with scanning electron microscope (SEM) examinations of daily increments in the same otolith makes it possible to reconstruct the temperature history for an individual fish on a time scale of days. An example of the application of the technique to an approximately six-month-old field-caught herring larva is given, and the limitations of the technique are discussed.     

Crystalline otoliths in teleosts: Comparisons between hatchery and wild coho salmon (<Emphasis Type="Italic">Oncorhynchus kisutch</Emphasis>) in the Strait of Georgia

Otoliths, or ‘ear stones’, are calcium carbonate structures found in all vertebrates. In teleosts, they have a number of sensory functions, including hearing. Daily growth increments of these structures have permitted advanced age and population studies of teleosts. Whereas ‘normal’ otoliths are composed of crystals imbedded within a protein matrix as aragonite, a ‘crystalline’ form of calcium carbonate termed vaterite is also found. A review of the otolith literature demonstrates a significant level of understanding of the structure and function of otoliths, but the cause for crystalline otolith structure remains speculative. Pairs of otoliths from hatchery and wild juvenile and adult coho salmon (Oncorhynchus kisutch) were examined visually for determination of otolith microstructure type. The vateritic or crystalline otoliths were found in much higher percentages in juvenile hatchery-reared coho salmon than in juvenile wild coho salmon, supporting previous studies. There did not seem to be any negative impact on size or survival. There was also no correlation between crystalline otoliths and premature maturation in coho males. A preliminary study of adult coho salmon returning to Big Qualicum and Chilliwack hatcheries showed even higher ratios of vateritic otoliths than observed in juveniles.     

High incidence of otolith abnormality in juvenile European flounder Platichthys flesus from a tidal freshwater area

Otolith abnormality has been reported for a wide range of freshwater and marine fish species. In this study, the sagittal otolith morphology and mineralogy of juvenile European flounder, Platichthys flesus, were examined to describe the incidence and types of structural deformities in this species. Juvenile flounder were collected over the years 2013–2015 in the tidal freshwater section of an estuarine nursery (Minho estuary, NW Portugal). Otolith abnormality was found in 43% of the individuals and, in most cases, occurred in both otoliths. Despite an abnormal mineralogy confirmed by scanning electron microscopy, the morphometry and the mineral polymorph composition of abnormal otoliths did not differ from normal ones (i.e. aragonite). This contrasts with other studies where vaterite and/or calcite were found to replace aragonite in abnormal otoliths. Further studies are needed to elucidate whether abrupt salinity changes associated with habitat transitions may have played a role in the abnormal otolith biomineralization in this species.     

Significance of otolith calcium carbonate crystal structure diversity to microchemistry studies

Otoliths, calcium carbonate (CaCO₃) ear stones of fish, contain a wealth of information about fish life and environmental history yet the CaCO₃ polymorph form the otolith is made of is a critical, but seldom considered, piece of information during otolith analysis. Otolith trace element chemistry data increasingly informs management decisions, but recent work has shown that CaCO₃ polymorphs—aragonite, vaterite, and calcite—can bear on incorporation of trace elements in a non-trivial way. Most fishes are thought to have otoliths of the aragonite CaCO₃ form, but this construct is potentially outdated with many recent literature reports showing otherwise. Our study used previously unpublished neutron diffraction data and reports from published literature to address three objectives: (1) summarize the relative effects of otolith CaCO₃ polymorphism on otolith microchemistry, (2) summarize reports of otolith polymorphs to gain a better understanding of the extent of non-aragonite otoliths among fishes, (3) outline future research needed to align interpretations of microchemistry with our current understanding of otolith polymorph diversity. We found that while aragonite otoliths are the most common, so are exceptions. For example, the ostensibly rare (among species) CaCO₃ form vaterite was reported in at least some otoliths of 40% of the species surveyed. Our work suggests that examination of the CaCO₃ polymorph composition of otoliths should become more common particularly in studies where results will or may be used to inform management decisions. Future research should work to attribute controls on otolith CaCO₃ polymorph expression using a combination of -omics and material characterization approaches to enrich the life history and environmental information output from otoliths and increase our understanding of the assumptions made in otolith trace element chemistry studies.

     

Otoliths of sub-adult Lake Sturgeon Acipenser fulvescens contain aragonite and vaterite calcium carbonate polymorphs

In this study we quantified the percent CaCO₃ polymorph composition in otoliths of larval and juvenile Lake Sturgeon Acipenser fulvescens via X-ray microdiffraction. Sagittal otoliths of sub-adults were primarily composed of aragonite (> 90%) while the lapilli otoliths were 100% vaterite. This is the first time the presence of aragonite in otoliths has been reported in an acipenseriform and is surprising given that the ability to form aragonite otoliths was not thought to have evolved until the separation of teleost and holostean species from other Actinopterygian fishes (e.g., sturgeon, paddlefish, gar).     

Seasonal changes in the physiology of brook trout, Salvelinus fontinalis (Mitchill), in a sub-Arctic river system

Wild herring larvae were sampled from the Firth of Clyde, Scotland in 1980 and 1981 to assess the suitability of otolith ageing methods for herring larval studies. The ages and growth rates were estimated directly from otolith ring counts, by comparison of ring counts with the results of validation studies on reared larvae of known age, and by back-calculation. Growth rates based on otolith data were compared with indirect calculations based on length at capture. The assumption of daily ring deposition, even allowing for a period of lag before the initiation of daily ring deposition, led to incorrect predictions for age at yolk-sac absorption, and back-calculations led to overestimated growth rates, indicating that ring deposition does not begin at hatching and that light microscope counts give a rate different from one ring per day in wild herring larvae.     

石首鱼科(Sciaenidae)三种鱼耳石CaCO_3晶相结构分析

软骨及硬骨鱼以及其它高等脊椎动物耳石中,CaCO_3占绝大部分.本文主要以X射线衍射分析对白姑鱼,大黄鱼,小黄鱼三种石首鱼科鱼类耳石作了分析,发现CaCO_3结晶均为霰石结构.依文献,对耳石晶相结构在动物分类上的意义作了初步讨论.     

Using otolith chemical and structural analysis to investigate reservoir habitat use by juvenile Chinook salmon Oncorhynchus tshawytscha

Isotopic composition of ⁸⁷Sr:⁸⁶Sr and natural elemental tracers (Sr, Ba, Mg, Mn and Ca) were quantified from otoliths in juvenile and adult Chinook salmon Oncorhynchus tshawytscha to assess the ability of otolith microchemistry and microstructure to reconstruct juvenile O. tshawytscha rearing habitat and growth. Daily increments were measured to assess relative growth between natal rearing habitats. Otolith microchemistry was able to resolve juvenile habitat use between reservoir and natal tributary rearing habitats (within headwater basins), but not among catchments. Results suggest that 90% (n = 18) of sampled non‐hatchery adults returning to the Middle Fork Willamette River were reared in a reservoir and 10% (n = 2) in natal tributary habitat upstream from the reservoir. Juveniles collected in reservoirs had higher growth rates than juveniles reared in natal streams. The results demonstrate the utility of otolith microchemistry and microstructure to distinguish among rearing habitats, including habitats in highly altered systems.     

Asteriscus v. lapillus: comparing the chemistry of two otolith types and their ability to delineate riverine populations of common carp Cyprinus carpio

The chemical composition of common carp Cyprinus carpio asteriscus (vaterite) and lapillus (aragonite) otoliths from the same individual and reflecting the same growth period was measured to (1) determine whether there are differences in the uptake of trace metals (Mg:Ca, Mn:Ca, Sr:Ca and Ba:Ca ) and Sr isotope ratios (⁸⁷Sr:⁸⁶Sr) in co‐precipitating lapilli and asterisci and (2) compare the ability of multi‐element and isotopic signatures from lapilli, asterisci and both otolith types combined to discriminate C. carpio populations over a large spatial scale within a river basin. Depth profile analyses at the otolith edge using laser‐ablation inductively coupled plasma mass spectrometry showed that asterisci were enriched in Mg and Mn and depleted in Sr and Ba relative to lapilli, whilst ⁸⁷Sr:⁸⁶Sr values were nearly identical in both otolith types. Significant spatial differences among capture locations were found when all trace element and Sr isotope ratio data were aggregated into a multi‐element and isotopic signature, regardless of which otolith type was used or if they were used in combination. Discriminatory power was enhanced, however, when data for both otolith types were combined, suggesting that analysis of multiple otolith types may be useful for studies attempting to delineate C. carpio populations at finer spatial or temporal scales.     

Population structure of the black seabream Spondyliosoma cantharus along the south-west Portuguese coast inferred from otolith chemistry

The chemistry of black seabream Spondyliosoma cantharus otoliths from three main fishery grounds (Olhão, Sagres and Sesimbra) located along c. 400 km of the Portuguese south and west coasts was examined. Element:Ca ratios were determined in whole otoliths and otolith cores of young adult specimens of 2–3 years of age. Using the data from whole otoliths, it was possible to discriminate among S. cantharus from the three fishing grounds with an average accuracy of 91%. Differences among fishing grounds were significant for all element:Ca ratios, and otoliths from Sagres had significantly higher levels of all ratios compared to the other fishing grounds. In contrast, the chemical composition of the otolith core, representative of the larval stage, showed limited variation among the fishing grounds, with an average discrimination accuracy of only 44%, although the Mg:Ca ratio of the otolith cores was also significantly higher for the Sagres samples. The data suggest that larval stages experienced a homogenous environment consistent with an offshore oceanic spawning. Juveniles appeared to display local residency on the inshore fishing grounds, areas probably characterized by greater environmental heterogeneity. Spondyliosoma cantharus population structure is consistent with distinct local population units that share a spawning ground providing recruits to different coastal fishery areas.     

Differences in Lateral Line Morphology between Hatchery- and Wild-Origin Steelhead

Despite identification of multiple factors mediating salmon survival, significant disparities in survival-to-adulthood among hatchery- versus wild-origin juveniles persist. In the present report, we explore the hypothesis that hatchery-reared juveniles might exhibit morphological defects in vulnerable mechanosensory systems prior to release from the hatchery, potentiating reduced survival after release. Juvenile steelhead (Oncorhynchus mykiss) from two different hatcheries were compared to wild-origin juveniles on several morphological traits including lateral line structure, otolith composition (a proxy for auditory function), and brain weight. Wild juveniles were found to possess significantly more superficial lateral line neuromasts than hatchery-reared juveniles, although the number of hair cells within individual neuromasts was not significantly different across groups. Wild juveniles were also found to possess primarily normal, aragonite-containing otoliths, while hatchery-reared juveniles possessed a high proportion of crystallized (vaterite) otoliths. Finally, wild juveniles were found to have significantly larger brains than hatchery-reared juveniles. These differences together predict reduced sensitivity to biologically important hydrodynamic and acoustic signals from natural biotic (predator, prey, conspecific) and abiotic (turbulent flow, current) sources among hatchery-reared steelhead, in turn predicting reduced survival fitness after release. Physiological and behavioral studies are required to establish the functional significance of these morphological differences.     

Otolith accretion rates: Does size really matter?

This study examined the relationship between otolith size and growth in juvenile cod (Gadus morhua L.). Two groups of juvenile cod were reared under different food ration and temperature regimes to obtain fish of similar somatic size but with different sized otoliths. The two groups were subjected to alternating temperature regimes and intermediate ration levels. Large otoliths grew significantly faster than the small ones and variation between individuals was extensive. The ratio of otolith growth during cold and warm temperature exposure did not differ between groups, and the observed growth pattern is therefore not attributable to differential growth within individual temperature periods. The ratio decreased with otolith size, presumably as a result of ontogenetic decrease in otolith protein composition. These results suggest that processes coupled to the metabolic rate of the endolymphatic epithelium are the key driver behind otolith growth.     

Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles

Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles were investigated. Under transmitted light, translucent (W(t)) and opaque otoliths (W(o)) were detected in juveniles collected from Wakasa Bay between July 2005 and April 2006, whereas only opaque otoliths (G(o)) were detected in Goto-nada Sea individuals between May and June 2006. Three groups of juveniles were distinguished based on differences in hatch season, otolith size and growth history, and body morphometrics. As T. japonicus has different spawning seasons according to spawning grounds, each group was estimated to hatch in different waters. Juveniles with W(t) otoliths were considered to have stayed in coastal habitat longer, as the hatch area was estimated to be near Wakasa Bay. Juveniles with W(o) and G(o) otoliths appear to recruit to coastal waters at larger size, since their hatch areas were estimated to be far from each collection area. Larger otoliths of W(t) were attributed to otolith accretion after the second growth flexion, which was observed only for W(t) . Standard length of W(t) fish at the second otolith growth flexion was estimated to correspond to recruitment size to coastal rocky reefs in Wakasa Bay. Body morphometrics were correlated with otolith size after removing body size effect, suggesting that morphological variations of T. japonicus juveniles were also associated with the timing of recruitment to coastal habitat.     

Seasonal Stable Isotope Records of Otoliths from Ocean-pen Reared and Wild Cod, Gadus morhua

Otoliths of ocean-pen reared cod, Gadus morhua, provide a unique opportunity to examine the lifetime history of the fish. Here we report 18 analyses of such otoliths on seasonal (winter and summer) stable oxygen and carbon isotope ratios. The calculated isotopic temperatures from otoliths of reared cod were essentially in agreement with the temperature record during rearing, suggesting that temperature is a dominant factor in the precipitation of otolith aragonite. Carbon isotope ratios increased with age and leveled off at 4 years old, presumed to correlate with sexual maturity of cod. As compared with otoliths of wild-caught cod, significant differences were found in isotope variation and the correlation between ¹³C and ¹⁸O. These differences were probably attributable to the different environmental constraints and food supply.     

Microstructural growth in otoliths of black rockfish,sebastes schlegeli, from prenatal larval to early juvenile stages

Microstructural growth in the sagittae ofSeastes schlegeli, a viviparous scorpaenid, is described from prenatal larval to early juvenile stages, and related to morphological changes. Embryos and prenatal larvae were extruded from a gravid female from 21 d prior to birth onwards, and released larvae reared and sampled up to 58 d after birth. Eggs hatched in the ovary 14 d prior to birth. At this time, otoliths consisted of a core surrounded by a prominent check, similar to the otolith structure seen in oviparous fishes. Fourteen growth increments had been deposited by birth. The parturition mark it-self comprised a prominent check and narrow growth increment Growth increments were deposited daily from hatching up to 58 d after birth, whereas accessory primordia first appeared in otoliths by ca. 32 d after birth, at a specimen total length of ca. 13 mm. This corresponded to the period during which the larvae metamorphosed into juveniles. Otoliths grew exponentially during the larval stage and linearly during the juvenile stage. when plotted against total length. Growth in total length from hatching to 58 d after birth could be represented by the Gompertz curve.