Otoliths developed in microgravity

Little is known about mechanisms that regulate the development of the otoliths in the gravity-sensing organs. Several reported experiments suggest that the growth of the otoliths is adjusted to produce a test mass of the appropriate weight. If this is the case, larger than normal otoliths would be expected in animals reared in reduced gravity and a reduced mass, relative to 1-g controls, would be expected in animals reared at elevated g. In gastropod mollusks, the gravity-sensing organ is the statocyst, a spherical organ whose wall is made largely of sensory receptor cells with motile cilia facing the lumen. Dense statoconia in the cyst lumen interact with cilia of receptor cells at the bottom of the cyst and action potentials in their axons carry information on direction and magnitude of gravity and linear acceleration. In the marine mollusk, Aplysia californica, larvae reared at 2 to 5-g, the volume of statoconia was reduced in a graded manner, compared to 1-g control animals. In the statocyst of the fresh-water pond snail, Biomphalaria glabrata, reared in space in the Closed Equilibrated Biological Aquatic System (CEBAS), the number and total volume of statoconia was increased approximately 50%, relative to ground-reared controls. Lychakov found the utricular otolith to be 30% larger in space-reared Xenopus, whereas we found the saccular otolith to be significantly larger in newt larvae reared in space. In cichlid fish reared on a centrifuge, the saccular otolith was smaller than in 1-g controls. Here, we demonstrate that the otoliths of late-stage embryos of the swordtail fish, Xiphophorus helleri, reared in space on STS-89 and STS-90 (Neurolab) were significantly larger than those of ground-controls reared in functionally identical hardware.     

Effect of hypergravity on the Ca/Sr composition of developing otoliths of larval cichlid fish (Oreochromis mossambicus)

The amounts of calcium and strontium were measured by inductively coupled plasma mass spectrometry (ICP-MS) in saccular and utricular inner ear otoliths (sagittae and lapilli, respectively) of developing cichlid fish. These fish had been maintained for 22 days at 3-g hypergravity conditions within a centrifuge. During this time-span, the animals completed their ontogenetic development from hatch to the free-swimming stage. Neither the morphogenetic development nor the timely onset and gain of performance of the swimming behaviour was impaired by the experimental conditions. Experimental and control animals also did not differ concerning their size (total length). ICP-MS revealed that the otoliths contained significantly less calcium (in microg/otolith) after hyper-g exposure compared to parallelly raised 1-g control specimens (lapilli: 0.74+/-0.21 vs. 1.16+/-0.41; sagittae: 2.09+/-0.49 vs. 2.76+/-0.47). The content of strontium (in microg/otolith: lapilli: 0.0044+/-0.0023 vs. 0.0022+/-0.0013; sagittae: 0.0094+/-0.0026 vs. 0.0081+/-0.0016) and, consequently, the Sr/Ca ratio (Sr/Cax100) was increased (lapilli: 0.607+/-0.267 vs. 0.201+/-0.12; sagittae: 0.439+/-0.093 vs. 0.301+/-0.086). Since the calcium content can be taken as a proxy for otolith weight, and because parallelly undertaken morphometric investigations revealed smaller otoliths (maximum radius and surface area) due to hyper-g exposure, the results suggest that the growth of otoliths at hyper-g is slowed down. Since the concentration of trace elements incorporated into otoliths is likely based on the composition of the respective protein matrix, our findings suggest that the protein metabolism is affected by hypergravity.     

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.     

Sperm motility-initiating activity in the egg jelly of the externally-fertilizing urodele amphibian, Hynobius lichenatus

Low osmolality initiates sperm motility during the external fertilization of aquatic anuran amphibians. It is thought that this process occurs also in urodeles, but this has not been fully examined in these species. We report here that fertilization was achieved in the externally fertilizing hynobiid, Hynobius lichenatus, by direct insemination onto the egg jelly surface without initial exposure of the sperm to a hypoosmotic solution. To identify the factors in addition to low osmolality that initiate sperm motility in Hynobius, we suspended the sperm of this amphibian in egg jelly extract (JE), and about 90% began to move within 1 min. This indicated the presence of a substance in JE that promotes motility initiation, as is also the case in the newt, Cynops pyrrhogaster. To examine whether this JE factor is homologous to the sperm motility-initiating substance (SMIS) in the newt, we tested for possible inter-species cross-reactivity of the JE. The percentage of moving Cynops sperm was increased to 67% in Hynobius JE at 5 min, and 65% of the Hynobius sperm began to move in Cynops JE within 1 min, indicating that JE is indeed cross-reactive between these species of salamander and newt. Concomitantly, pretreatment of Hynobius JE with Fab fragments of a Cynops SMIS monoclonal antibody resulted in a decreased number of moving Hynobius sperm. Immunoblotting further suggested that the substance in Hynobius JE responsible for motility initiation has an 18 kDa molecular mass, with an isoelectric point at 7.5.     

Distribution of the Germinal Vesicle Material during Progesterone-Induced Oocyte Maturation in Xenopus and in Cynops

The distribution of the germinal vesicle material in the oocyte during progesterone-induced maturation was studied in Xenopus and in Cynops. In both species, two distinctive masses of yolkfree cytoplasm appear in specific areas of the oocyte and at definite stages of maturation. One, the primary cytoplasmic mass, is formed at the basal side of the germinal vesicle during early maturation and is very RNA-rich. In Xenopus , a large part of the primary cytoplasmic mass persists as a mass during maturation and ends up as a thin disk at the boundary between the animal and the vegetal hemisphere in the mature oocyte. In Cynops , a rod-like primary cytoplasmic mass extends near to the equatorial zone and becomes indistinct in the mature oocyte. The other, the secondary cytoplasmic mass, is formed at or prior to germinal vesicle breakdown in areas around the germinal vesicle and is also RNA-rich. The secondary cytoplasmic mass is dispersed and constitutes the RNA-rich animal hemisphere cytoplasm in the mature oocyte. Observed results suggest that the primary and the secondary cytoplasmic mass contain different germinal vesicle materials.     

Otolith elemental signatures reflect residency in coastal water masses

We examined variability in otolith chemistry of wild caught fish in relation to in situ temperature and salinity within the California Current System. Barium, magnesium, and iron from the most recent growth zone in otoliths differentiated pelagic juvenile shortbelly rockfish (Sebastes jordani) residing in water masses with distinct temperature and salinity properties from central and southern California spanning nearly 500 km of coastline. The 3-element signature also discriminated fish that resided in different water masses that were associated with mesoscale cyclonic eddy circulation in the Santa Barbara Channel. Variability in otolith chemistry reflected the spatial patterns of both horizontal gradients and vertical gradients in water mass properties related to circulation. Although we found that the concentrations of particular elements in otoliths were correlated to ambient temperature or salinity, we suggest that these parameters are more useful as an identifying signature of distinct water masses associated with unique otolith signatures rather than as factors directly affecting otolith chemistry. Other factors varying among the water masses or among the fish populations residing in the water masses may also affect otolith chemistry. We discuss how oceanographic phenomena associated with the 1997–1998 El Niño and the persistent, recirculating eddy in the Channel may have affected coastal ocean conditions and variation in otolith chemistry of fish in the study area.     

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.     

Temperature effects on otolith pattern formation in Atlantic cod Gadus morhua

The effect of food intake and temperature on otolith macrostructure and microstructure was examined experimentally in Atlantic cod Gadus morhua. Daily increment formation was validated and otolith accretion rate and optical density quantified using image analysis. Two‐week periods of starvation had no discernable effect on otolith increment width or optical density, despite having negative effects on somatic growth. In contrast, temperature had a strong positive effect on otolith accretion rate and clear effects on optical density with the otolith becoming more translucent at higher temperatures. Somatic growth, otolith accretion and otolith optical density each had a significantly different response curve to temperature. No relationship was detected between individual somatic growth rates and the accretion rate or optical properties of the otolith. The experimental manipulation of temperature‐induced otolith patterns similar to the ‘false ring’ secondary structures sometimes observed in the otoliths of wild fish. The results suggest that otolith pattern arises from a combination of temperature and seasonal effects, but not directly from individual variation in somatic growth.     

Effects of starvation on the formation of daily growth increments in the otoliths of milkfish, Chanos chanos (Forsskål), larvae

The effects of starvation on daily growth and increment formation in the otolith were examined using a double oxytetracycline-labelling method on larval milkfish, Chanos chanos (Forsskål), reared under different feeding regimes. The results indicated that the differences in body and otolith growth between the larvae fed once and three times a day were not significant, and that the otolith growth increment was deposited daily in both groups of fed larvae. In contrast, the starved larvae grew at a slower rate than fed larvae in body length and otolith dimensions, and the otolith growth increment in the starved larvae was not deposited on a daily basis. After undergoing starvation, the larvae were unable to recover their normal growth either in otolith increment deposition or in body and otolith growth even though they were fed. Therefore, the application of ageing techniques based on counting otolith growth increments seems to be inaccurate for starved larvae.     

Growth of North Alboran Sea sardine larvae estimated by otolith microstructure, nucleic acids and protein content

Wet mass and DNA, RNA and protein content increased significantly with standard length ( L _S) of sardine Sardina pilchardus larvae, collected in January 1995, in the Bay of Málaga, North Alboran Sea. L _S, wet mass and DNA, RNA and protein content were closely related allometrically to otolith radius ( R ). Larval daily length increments decreased but DNA, protein and wet mass daily increments increased with larval age. Daily length increments showed a negative and poor relationship with long-term otolith growth. In contrast, DNA, protein and wet mass daily increments were positively correlated. Differences between observed and back-calculated otolith radius-at-age indicated that larvae with slow otolith growth were under represented in older age groups, suggesting the existence of growth-selective mortality. Recent otolith growth, estimated from the mean widths of the last six increments, increased with age and R . Individual RNA: DNA and protein: DNA ratios were correlated significantly, although weakly, with L _S and larval growth.     

Femtosecond laser ablation ICP-MS measurement of otolith Sr:Ca and Ba:Ca composition reveal differential use of freshwater habitats for three amphidromous Sicyopterus (Teleostei: Gobioidei: Sicydiinae) species

The use of freshwater habitats was examined in three amphidromous goby species of the genus Sicyopterus using otolith microchemistry. Two species were endemic to either New Caledonia or Vanuatu whilst the other was widely distributed. Depositional patterns of strontium (Sr) and barium (Ba) in the otolith of adults were analysed with femtosecond laser ablation inductively coupled plasma mass spectrometry (ICP-MS). The Sr:Ca and Ba:Ca results uncovered three different adult behaviours within the freshwater habitat. Some fishes stayed in elevated locations (square profile); others undertook back-and-forth migrations between higher and lower reaches (up-and-down profile), and finally, others stayed in the lower reaches (constant profile). The consequences of these movements to larval survival or competition for food and territory are discussed. This work brings new knowledge on amphidromous behaviour, and it highlights the necessity of multi-elemental analysis to study amphidromy in freshwater systems.     

Lesser Sandplover,Charadrius mongolus,in Turkey

Sagittal otoliths are widely used to determine taxon, age and size of the teleost fishes, and are useful tools for studies of prey-predator relationships, population dynamics and ichthyo-archaeology. They can also be used to estimate the size of the prey. We examined the relationships between otolith measurements (length, height and weight) and fish size (total length and weight) for two species of Argentinidae (Argentina sphyraena and Glossanodon leioglossus) from the Southern Aegean Sea, Turkey. Length, height and mass of sagittae were shown to be good indicators for the length and weight of fish in both species. Glossanodon leioglossus has relatively larger sagittae than Argentina sphyraena. Linear and exponential functions provided the best fit for relations between otolith and fish measurements. No significant differences were found between left and right otolith sizes.     

长江口鮻矢耳石形态特征及质量与年龄的关系

为研究长江口鮻矢耳石形态特征及其质量与年龄的关系,2017年2—6月从长江口水域采集鮻358尾,解剖并提取耳石324对,并对其形态学参数进行测量分析.结果表明:鮻矢耳石具有基叶和翼叶,中央听沟明显,矢耳石的大小和形状随个体生长变化明显,矢耳石由边缘规则的圆润瓜子状渐变为褊窄的叶片状,边缘波浪状突起逐渐变多.矢耳石平均密度为1.52 mg·mm^-2,平均矩形趋近率为0.68,耳石长占其体长的0.021%~0.047%,耳石宽占其体长的0.009%~0.021%,耳石质量占其体质量的0.045‰~0.731‰.鮻矢耳石长(OL)、宽(OW)、质量(OM)均与体长呈显著相关,耳石宽与耳石质量函数关系式的决定系数(R^2)最高(0.928).耳石质量与体长(BL)最佳拟合方程为幂函数:OM=0.0009BL1.8737(R2=0.967);耳石质量与年龄(A)以及体长与年龄的最佳拟合方程为多项式:OM=2.9262A^2+4.8437A+2.1894(R^2=0.847);BL=-3.2248A^2+102.54A+38.373(R^2=0.858),矢耳石质量与年龄呈显著线性相关,用耳石质量与年龄关系估算的年龄与从耳石上直接读取的年龄无显著差异,矢耳石质量可以作为鉴定鮻年龄的有效辅助手段.     

Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou

Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.     

Otolith mass marking techniques for aquaculture and restocking: benefits and limitations

The use of farmed and restocked fish to supplement the worldwide human consumption of fish, recreational fishing stocks, and conservation efforts, is growing at a rapid rate. Yet, monitoring the benefits of using hatchery-raised fish for supplementation is lacking, often due to hatcheries not marking or tagging all fish prior to release, despite a range of easy to apply, cost effective and accurate mass-marking methods being available to mark farmed and restocked fish en masse. Here we review otolith marking techniques that have the capability of mass marking millions of hatchery-reared fish that are, or could be, used for monitoring and compliance purposes. The otolith mass marking methods consist of otolith thermal marking and a range of otolith chemical marking methods (tetracyclines, alizarin compounds, calcein, strontium chloride, stable isotopes of Ba and Sr, and rare earth elements). We assessed and compared marking technique in terms of (1) ease of application, (2) cost of application, (3) mark retention and detectability, and (4) fish welfare. In addition, we determine the suitability of different otolith marking techniques for mass marking entire hatchery populations whether it be for restocking purposes, or for identifying and tracing escapees from aquaculture facilities. We conclude that although some techniques have restricted use due to regulations, the majority of otolith mass marking techniques are simple, easy to apply, cost effective and highly suitable for long term monitoring of hatchery produced fish.     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

Sex-specific somatic–otolith growth relationship in two Gadidae

Otolith and somatic mass of two Gadidae ( Merlangius merlangus and Trisopterus minutus ) were compared in order to analyse the sex-specific relationship between otolith and somatic growth at age. In the present study, otolith mass appeared a reliable indicator of age in both species. Otolith growth reflected somatic growth, but the relationship between these characters varied and differed between species and sexes.     

cost-effective method of preparing larval fish otoliths for reading using enzyme digestion and staining

A fast and cost-effective method for examining otoliths in fish larvae was developed whereby the otolith remains in situ . Whole fish of the clownfish Amphiprion melanopus were enzymecleared using a laundry pre-soak and then stained using the Von Kossa silver staining method for calcium. The otolith nucleus, daily rings and the otolith edge were all clearly visible and were suitable for a variety of age and growth analyses. The total 'hands on' time required to process these otoliths was c . 3 min, and multiple samples could be processed simultaneously. The reduction in labour of this method to produce clear daily rings in the otolith lends itself to broad use in fish biology where large quantities of otoliths need to be examined in a cost- and time-efficient manner.