Morphological characterization and functional immune response of the carpet shell clam (Ruditapes decussatus) haemocytes after bacterial stimulation

Abstract

The morphology and functionality of Ruditapes decussatus haemocytes have been characterized by light microscopy and flow cytometry, leading to the identification of three different cellular subpopulations. Granulocytes were the largest cells, the hyalinocytes were smaller and contained fewer granules and the intermediate cells showed a size similar to hyalinocytes and a higher number of granules. The phagocytosis of different particles and the associated production of oxygen radicals were measured by flow cytometric methods. Granulocytes were the most active cells, followed by the intermediate cells and hyalinocytes. The effect of stimulation of haemocytes with lipopolysaccharide (LPS), with a heat inactivated bacterial mixture or with the infection of Vibrio splendidus on the cell viability and the expression of selected immune-related genes were studied. While significant low levels of damaged cells were registered in LPS-stimulated cells, the treatment with dead bacteria or V. splendidus reduced cell viability 1 h, 3 h and 6 h after treatment. The stimulation of haemocytes with LPS and dead bacteria induced changes in the expression of defender against cell death (DAD-1), thrombin, prosaposin, inhibitor of apoptosis (IAP), factor B and C3 complement component.

Characterization of an inhibitor of apoptosis protein in Crassostrea gigas clarifies its role in apoptosis and immune defense

“…Invertebrate studies have revealed that IAPs play a similar role in this taxon. For example, both live V. splendidus treatments and treatments with a mixture of dead bacteria (Micrococcus luteus, V. splendidus, and V. anguillarum) increased IAP expression in the carpet shell clam (Ruditapes decussatus) (Prado-Alvarez et al, 2012). In addition, previous study demonstrated that CgIAP1 transcript levels increased soon after a V. anguillarum challenge and reached the highest expression at 12 h in the Pacific oyster (Zhang et al, 2011).…”

Genes of the Mitochondrial Apoptotic Pathway in Mytilus galloprovincialis

“…The other cells were treated with 1 mL of N-acetyl-L-cystein (NAC) and pyrrolidine dithiocarbamate (PDTC) from Sigma (1 mM and 50 µM, respectively) for 1 h before UV treatment, as these components were scavengers of oxygen free radicals that directly interacted with reactive oxygen species. Immediately after 45 min irradiation, the production of ROS was measured using a 2′,7′-dichlorofluorescein-diacetate probe (DCFH-DA, Molecular Probes) through flow cytometry according to the protocol of Prado-Alvarez et al [40]. Cell Quest software (B&D) was used to determine the mean of fluorescence of 10,000 events in the R1 (granulocytes) and R2 (hyalinocytes) regions.…”

Induction of apoptosis by UV in the flat oyster, Ostrea edulis

“…galloprovincialis and the clam Ruditapes philippinarum [17,55]. In our study, whatever was the origin of the oysters and the tested conditions, cells identified by flow cytometry as hyalinocytes were more abundant than cells identified as granulocytes.…”