Carbonic anhydrase is not the only factor regulating otolith mineralization in fish in dependence of the gravitational environment
Introduction
Teleost inner ear stones (otoliths) are calcified structures involved in hearing and maintenance of equilibrium. Regarding their sensory function, the growth of otoliths (and thus their weight) must be regulated (Campana and Neilson, 1985; for review see Anken and Rahmann, 1999, Anken, 2006). In the course of earlier studies, it was found that the “physical capacity” (i.e., weight and size) of an otolith on the sensory epithelium is a regulating factor in controlling the growth of the otolith (Anken et al., 1998, Anken et al., 2001). Indeed, a neuronally guided (Edelmann et al., 2004) feedback mechanism slows down otolith growth at hypergravity (hg), so that an otolith formed at hg will possibly cause about the same shearing forces during tilts as a respective normally sized otolith at 1g earth gravity (Anken et al., 2002). Since calcium carbonate (CaCO3) constitutes the main component of otoliths regarding their weight (Farrell and Campana, 1996, Borelli et al., 2001) and carbonic anhydrase (CAH), an enzyme being functionally involved in carbonate deposition in otoliths (Fermin et al., 1998, Tohse and Mugiya, 2001) was shown to be influenced by hg (Beier et al., 2002, Beier et al., 2004), the carbonate status seems to be one of the factors regulated by the feedback mechanism.
We were thus prompted to investigate by inhibition of CAH with azetazolamide (AZ), if CAH reactivity and with this the carbonate deposition is the only factor regulating otolith mineralization in dependence of the gravity vector.
The results obtained provide clear evidence that the effect of hg on otolith growth is not necessarily limited to an alteration of the provision of the amount of CAH based carbonate production, but that (concerning lapilli, the utricular otoliths) hg affects additional, hitherto unknown mechanisms involved in the crystallization process of the respective otoliths.
Section snippets
Materials and methods
Larval cichlid fish siblings (Oreochromis mossambicus PETERS, Perciformes) of stage 16 (some 8 days after fertilization at 28 °C, fin ray primordia in dorsal fin bud; Anken et al., 1993) were kept for 7 days at 22 °C in the dark either in normal aquarium water at 1g normal earth gravity (1g-Aq) or at hypergravity (hg; 3g, centrifuge) as a physical factor to decrease CAH reactivity (3g-Aq). A third and a fourth group of sibling animals was treated with azetazolamide (Sigma Aldrich, Germany; AZ; 1
Results
Both the hg-exposed and the AZ-treated animals developed normally regarding outer features (for an atlas of normal stages, see Anken et al., 1993). Regarding the total length of the larvae, no differences were obtained (p > 0.2; not shown).
AC applied before the experiment was incorporated into all otoliths examined and the incorporation resulted in a clearly visible bright red shining fluorescence band on the otoliths. After the experiment, AC was only poorly incorporated in the otoliths of
Discussion
The actual mass of an otolith depends basically on its size and its density concerning CaCO3, which is incorporated in an organic protein matrix that only adds minimally to the total weight (Farrell and Campana, 1996). The carbon status of the endolymphatic fluid, which surrounds the otoliths, is maintained by the conversion of CO2 to via CAH (Fermin et al., 1998, Tohse and Mugiya, 2001) and significantly affects otolith calcification (Tohse and Mugiya, 2001). Interestingly, CAH (as well
Acknowledgements
This work was financially supported by the German Aerospace Center DLR e.V. (FKZ: 50 WB 9997). Marion Beier was financially supported by a stipendium of the Landesgraduiertenförderung Baden-Württemberg.
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