| Abstract |
Chromatophores (melanophores, xanthophores, erythrophores, leucophores, iridophores, cyanophores) are the basis of the integumentary coloration in fish. They enable fish to change their coloration either through changes in their morphology and density (morphological color change), or through changes in the motility of their chromatosomes (physiological color change). Colour change of fish is controlled by both endogenous (nervous, endocrine) and exogenous (environmental) parameters. Red porgy, Pagrus pagrus, candidate for aquaculture, presents a number of advantages for studying the regulation of skin colour since it develops, under intensive rearing, a dark body coloration as opposed to the aesthetically pink colour of wild caught fish. The aim of the present dissertation was to investigate the basis of this difference through (i) qualitative and quantitative study of chromatophores, (ii) identification of the regulatory mechanisms involved in the control of melanophores' motility, and (iii) determination of melanophores' ontogenesis. In order to quantify the morphological differences between reared and wild adult populations, method for the measurement of melanophores density was developed. Three body areas (dorsal, lateral, ventral, all left side) each one divided in 5 sub-areas (along the anterior-posterior axis) and the tail area (5 random sub-areas) were chosen and the melanophores were counted, cell size was measured and the cell area coverage was calculated. Besides, an electron microscopy study was performed, to determine qualitative differences between the type of melanophored in the dorsal, lateral and ventral skin area of both reared and wild specimens The results showed a decrease in the number of melanophores from the dorsal (85 cells/mm2) to ventral area (15, 25 cells/mm2) in both reared and wild individuals. The melanophores of reared fish were larger than those of wild individuals due to pigment dispersion state, and this difference was more evident in the ventral area (up to four-times). As a result, melanophore coverage in different body areas (basic cause of darkening) ranged from two-fold (dorsal area: reared 85%; wild 30%) up to 6-fold higher (ventral area: reared 20%; wild 3.5%). The type of chromatophores in the dermis of reared red porgy was limited to melanophores and iridophores while wild fish dermis contained melanophores, iridophores and xanthophores. To identify the regulatory mechanisms involved in the control of melanophores' motility (melanin-aggregation vs. melanin-dispersion), the in vitro effect of various chemical factors (a- and β-adrenergic agonists and antagonists, hormones) in innervated scale melanophores of adult reared and wild red porgies was investigated. Both, α- agonists (norepinephrine HCl, clonidine HCl, L-phenylephrine HCl) and β agonists (metaproterenol hemisulfate, fenoterol HBr, terbutaline hemisulfate, isoproterenol bitartrate and salbutamol) were applied at a concentration of 10-5 M for 15 min, while β- antagonists (propranolol HCl, atenolol) at concentration of 10-6 M for 15 min. The previous treatments were immediately followed by another 15 min immersion in 10-5 M norepinephrine HCl to get maximum pigment aggregation to determine the melanophore receptors. The melanophores response on acetylcholine Cl, at concentrations of 10-4 M to 10- 15M, and on selected hormones was also studied. Four major hormones, two responsible for melanin-aggregation (MCH, melatonin) and two responsible for melanin- dispersion (α-MSH, ACTH) were applied in vitro for 15 min at concentrations of 10-6 M to 10-12 M. a-Agonists exerted melanin-aggregating effect on scale melanophores (max: 55% to phenylephrine), while a melanin-dispersing action (>95 %) was observed in the presence of all β-agonists, within 5 min of exposure. Both α- and β-antagonists were found to disperse melanosomes in more than 75 % of the melanophores within 5 min. The percentage of melanophore aggregation following norepinephrine HCl administration was ranged between 15% (fenoterol) and 42% (salbutamol) for "b-agonists" while between 59% (yohimbine) and 87% (atenolol) for antagonists, in 15 min. Concerning hormonal control, α-MSH and ACTH produced melanin-dispersion, while MCH and melatonin (MT) a moderate and weak melanin-aggregation, respectively. These results indicate that the adrenergic receptors that mediate melanin-aggregation response of the melanophores of red porgy (P. pagrus) are alpha in nature, while receptors which mediate melanin-dispersion response are beta-adrenergic and muscarinic in nature. The skin darkening of red porgy is also under hormonal control via activation of melanophore receptors. Finally, a preliminary study on melanophores ontogenesis was perfromed. The main developmental stages (prelarvae, larvae, postlarvae, juvenile (day 75)) were selected and skin melanophores development was monitored at macroscopically and ultrastructural level. The melanophores present on the red porgy body from embryonic and prelarvae stage were mainly in aggregated state. The complete skin darkening similar to reared adult individuals as result of melanin dispersion state was well developed on 75 day postfertilization may be due to nutritional and environmental reasons.
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