Sagittal otolith morphogenesis asymmetry in marine fishes.

This study investigated and compared asymmetry in sagittal otolith shape and length between left and right inner ears in four roundfish and four flatfish species of commercial interest. For each species, the effects of ontogenetic changes (individual age and total body length), sexual dimorphism (individual sex) and the otolith's location on the right or left side of the head, on the shape and length of paired otoliths (between 143 and 702 pairs according to species) were evaluated. Ontogenetic changes in otolith shape and length were observed for all species. Sexual dimorphism, either in otolith shape and length or in their ontogenetic changes, was detected for half of the species, be they round or flat. Significant directional asymmetry in otolith shape and length was detected in one roundfish species each, but its inconsistency across species and its small average amplitude (6·17% for shape and 1·99% for length) suggested that it has barely any biological relevance. Significant directional asymmetry in otolith shape and length was found for all flatfish species except otolith length for one species. Its average amplitude varied between 2·06 and 17·50% for shape and between 0·00 and 11·83% for length and increased significantly throughout ontogeny for two species, one dextral and one sinistral. The longer (length) and rounder otolith (shape) appeared to be always on the blind side whatever the species. These results suggest differential biomineralization between the blind and ocular inner ears in flatfish species that could result from perturbations of the proximal-distal gradient of otolith precursors in the endolymph and the otolith position relative to the geometry of the saccular epithelium due to body morphology asymmetry and lateralized behaviour. The fact that asymmetry never exceeded 18% even at the individual level suggests an evolutionary canalization of otolith shape symmetry to avoid negative effects on fish hearing and balance. Technically, asymmetry should be accounted for in future studies based on otolith shape.

Diel vertical migration of European hake Merluccius merluccius and associated temperature histories: insights from a pilot data-storage tagging (DST) experiment.

A pioneering experiment of archival tagging of European hake Merluccius merluccius provided evidence of a diel vertical migration pattern which was analysed using an automatic method, developed and validated through time-frequency analyses. Frequent vertical movements across the thermocline were observed and fish experienced rapid temperature changes >7° C. These tagging-recapture data also suggested a homing behaviour to feeding grounds.

Composition of biomineral organic matrices with special emphasis on turbot (Psetta maxima) otolith and endolymph.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout.

Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO(2) concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO(2), total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca(2+) increases during the dark period and was maximal by the end of the night when the total CO(2) concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO(3) precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO(3) precipitation starts once the solubility of CaCO(3) is exceeded.

Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph.

Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. 45Ca2+influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1)microl(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200 % h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.

Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima).

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity.

Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.