Carbonic anhydrase is not the only factor regulating otolith mineralization in fish in dependence of the gravitational environment

Abstract

Earlier experiments have shown, that fish otolith growth and mineralization is slowed down by hypergravity (hg). The enzyme carbonic anhydrase (CAH) provides carbonate and, thus, plays a major role in otolith calcification. Indeed, CAH reactivity in inner ear maculae is downregulated by hg.

The following experiment was designed in order to elucidate as of whether CAH is the only factor regulating otolith mineralization in dependence of the gravity vector: A first group of larval cichlid fish (Oreochromis mossambicus) was reared in normal aquarium water at 1g (1g-Aq). A second group received hg (3g, 7 days) as a physical factor to decrease CAH reactivity (3g-Aq). A third group (1g-AZ) was (at 1g) treated with azetazolamide (AZ; 1 g/l), an inhibitor of CAH (the AZ-concentration used resulted in a complete inhibition of CAH as had been proven by a biochemical assessment of enzyme activity). The last group was maintained both in AZ and at hg (3g-AZ).

Both the saccular and utricular otoliths (sagittae and lapilli, respectively) of the 1g-AZ group showed a decrease in otolith growth (surface area) as compared to the 1g-Aq animals (1g-AZ < 1g-Aq). Similar results were obtained when comparing 3g-Aq with 1g-Aq samples (3g-Aq < 1g-Aq). Regarding sagittae, AZ treatment had no significant additional effect on otolith mineralization under hg (3g-AZ = 1g-AZ). In case of lapilli, however, growth received a further reduction when reared in 3g-AZ (i.e., 3g-AZ < 1g-AZ). Thus, in lapilli, hg and AZ added their effects on otolith growth.

This finding clearly indicates that hg does not only act on otolith growth via a regulation of CAH activity.

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.