Reliability of melanomacrophage centres as indicators of stress response in teleost fishes

Melanomacrophage centres (MMCs) are aggregates of pigmented phagocytes, characterized by heterogeneous inclusions and located in hemolymphopoietic organs of various non-mammalian vertebrates [1]. In the present work, data from published and ongoing studies on MMCs were analysed in order to get insights on MMC reliability as response biomarkers to stress and environmental pollution. Liver samples from 31 wild and captive reared Atlantic bluefin tuna (Thunnus thynnus) [2, 3] and from 90 wild European anchovies (Engraulis encrasicolus) caught in areas differently exposed to industrial and agricultural pollutants [4], and liver and spleen samples from 47 wild and captive reared greater amberjack (Seriola dumerili) [5, unpublished data] were fixed in 10% buffered formalin and embedded in paraffin wax. Deparaffinized sections were stained with haematoxylin-eosin; Mallory’s basic fucshin (Merck) and Perls VanGieson (Bio-Optica) stainings were used to identify lipofuscin–ceroids and ferric iron respectively; peroxidase detection was performed by a Leukocyte Peroxidase kit (Sigma). The terminal deoxynucleotidyl transferase-mediated d’UTP nick-end labelling (TUNEL) method was used to identify apoptotic cells and the immunohistochemical detection of cytochrome P450 monooxygenase 1A (CYP1A) was performed by means of polyclonal antibodies anti-fish CYP1A (Biosense Laboratories). Lipofuscin–ceroids and ferric iron were detected in MMCs of all the three examined species, whereas peroxidase was mainly detected in free macrophages. In Atlantic bluefin tuna, a high density of MMCs and liver apoptotic cells, and a strong CYP1A immunostaining were observed in young individuals reared in the central Adriatic Sea compared with adults caught from the wild or reared in the western Mediterranean. In European anchovy, a high density of MMCs and a strong CYP1A immunostaining were observed in fish sampled in the Gulf of Gela, a marine area dramatically affected by environmental pollution. In greater amberjack, MMC density was higher in spleen than in liver sections. Confinement in captivity did not affect MMC density, whereas differences were found between males and females and among fish in different reproductive conditions. The present study confirms that MMCs represent a useful biomarker of fish exposure to environmental pollution; however, sex and reproductive state may affect MMC density, possibly leading to data misinterpretations.