New Insights into the Apoptotic Process in Mollusks: Characterization of Caspase Genes in Mytilus galloprovincialis

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes’ tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature

Assessment of heavy metals bioconcentration factor (BCF) and genotoxicity response induced by metal mixture in Salmo salar tissues

The aim of this study was to evaluate metals bioconcentration factor (BCF) in gills, liver, kidneys and muscle in relation with genotoxicity effects of metal mixture in peripheral blood, kidneys, gills and liver erythrocytes of the Atlantic salmon (Salmo salar). Fish were exposed to maximum-permissible waterborne concentrations of Zn – 0.1, Cu – 0.01, Ni – 0.01, Cr – 0.01, Pb – 0.005 and Cd – 0.005 mg/L, respectively for 7 and 14 days. Genotoxicity was studied using the micronucleus test. In addition, erythrocyte nuclear abnormalities (ENAs) were analysed. Our study indicates that metal BCF in Atlantic salmon is tissue-dependent. Based on the BCF classification scale, the relatively low values of metals bioconcentration were assessed, except for Zn (gills) and Cu (liver) (359.6 and 594.0, respectively). Zn intensively concentrated in fish tissues, while Pb – least of all. Overall, metals were concentrated mostly in the liver, least – in the muscle. Significant differences among BCF values of Pb in gills and muscle and Cd in gills were measured between 7 and 14 d exposure groups. Treatment with metal mixture significantly increased micronucleus frequencies after 7 d of exposure in liver and peripheral blood erythrocytes. Significant genotoxicity response was not observed after 14 d treatment. The erythrocytic nuclei abnormalities determined in S. salar blood were nuclear bud on filament (NBf), nuclear bud (NB), blebbed (BL), kidney shaped, vacuolated (VacNuc), 8-shaped nuclei and fragmented-apoptotic (FA) erythrocytes. Significant elevation in total ENAs level was detected in kidneys and liver erythrocytes after 7 d treatment, while after 14 d – in gills and kidneys erythrocytes. No significant differences among analysed responses were measured between 7 and 14 d exposure groups, except total ENAs level in liver erythrocytes