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

Abstract

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.

Biological investigations on the freshwater snail Pirenella conica (Blainville, 1829) infected with the developmental stages of Heterophyes sp.

“…Apoptotic processes are characterized by many morphological features, which have been stated by Sweet, Passino-Reader, Meier, and Omann (1999). Furthermore, Romero, Estévez-Calvar, Dios, Figueras, and Novoa (2011) stated that the adequate clearance of damaged, senescent, and infested cells without inflammation are supported by apoptosis. It is well known that the molluscan immune system is based on innate mechanisms, whereas humoral and cellular processes approve cytotoxic and antimicrobial roles (Auffret, Rousseau, Boutet, Tanguy, & Baron, 2006).…”

The effects of zinc nanooxide on cellular stress responses of the freshwater mussels Unio tumidus are modulated by elevated temperature and organic pollutants

“…The opposite effects of n-ZnO and Zn 2+ exposures on cathepsin D and caspase-3 activities may reflect the roles of these enzymes in alternative apoptotic pathways differentially regulated by different environmental stressors (Conus et al, 2008). Caspase-3 is a key executor caspase of the extrinsic apoptotic pathway (Romero et al, 2011), whereas cathepsin D is an aspartic protease involved in lysosomal proteolysis as well as the apoptotic pathways (Conus et al, 2008;Venugopal and Siva Kumar, 2014). In mammalian neutrophils, cathepsin D was released from azurophilic granules in a caspase-independent but reactive oxygen species -dependent manner (Conus et al, 2008).…”

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“…Despite the scant information concerning the activity of caspases in bivalve molluscs, caspase-3 activity seems to represent a valuable marker for the determination of toxic effects, as confirmed by CART analysis for the mussels from sites A and N ( Figure 6). The important finding of Romero et al (2011), that caspase genes in Mytilus galloprovincialis have extremely high expression levels within the gland and gills due to the clearance of damaged cells, contributes to the rationale for including this assay in the set of biomarkers of the effect of warming on molluscs.…”