Fish otolith biomineralization process: first investigations about organic matrix and growth of Triglidae (Scorpaeniformes) otoliths.

Otolith formation involves rhythmic variations in the deposition and size of organic matrix framework and carbonate crystals, resulting in the formation of macroscopic translucent and opaque rings and microscopic zonations (growth increments) (Morales Nin, 2000). As in most biominerals, the otolith matrix forms only 2-3 % of its weight, but it is admitted that it has a considerable importance in the otolith crystallization processes of nucleation, growth, orientation and growth control. The goal of this study is to characterize the matrix protein composition in the otoliths of Triglidae (Scorpaeniformes) as a first step to understand molecular mechanisms of otolith formation according to biology and ecology of the species. In particular 500 sagittal otoliths from six gurnard species were analysed: Chelidonichthys cuculus, C. lucerna, Eutrigla gurnardus, Lepidotrigla cavillone, L. dieuzeidei and Trigloporus lastoviza. Protein contents were estimated by Bradford method and the urea 8 M extracts were loaded into a polyacrylamide gel, separated by SDS page and detected by Silver staining (Sigma) followed the protocol of Borelli et al. (2001) with some modifications regarding protein precipitation that was enhanced by using TCA, trichloroacetic acid, 100% w/v. The urea soluble fractions revealed a unique large band around 50-55 kDa. Another common clear band was visible at the top of the separating gel (proteins >300/350 kDa) unable to enter into the pores of polyacrylamide gels (12%). The complexity of the protein mixtures was investigated by 2-D electrophoresis (Gel TGX 4-20%); proteins were separated on the basis of both isoelectric point (pI) and molecular size. A common protein pattern of 50-75 kDa were found in all gurnards showing a similar composition of organic matter even if the 2-D maps of otolith samples showed specie-specific variation in acid protein fractions in all the pairwise comparison. This result confirmed that the amino acid composition influenced matrix content and its control on crystals growth and biomineralization process according to previous studies (Morales Nin, 1986; Vallisneri et al., 2014). Further investigation will be focused on comparing endolymph and otolith protein matter to related endogenous and exogenous elements with otolith growth.