Lumhol-dependent chemiluminescence by hemocytes of two marine bivalves, Ostrea edulis and Crassostrea gigas

Abstract

Microbicidal phagocytic function and oxidative metabolism of the hemocytes of 2 oyster species, Ostrea edulis and Crassostrea gigas, were investigated using a luminol-enhanced chemiluminescence (CL) technique. First, experimental parameters adapted to marine bivalve hemocytes were established on hemolymph pools of C. gigas in order to obtain qualitatively and quantitatively homogeneous samples and so to perform statistically comparable assays. The use of Modified Alsever Solution (MAS) allowed hemocytes to b e kept non-aggregated and in a non-stimulated state until starting the assays. A number of aliquots of 2 X lo5 hemocytes with a n MAS final concentration of 3.5 % showed high chemiluminescent response after stimulation by zymosan particles A partic1e:hemocyte ratio of 80:1 gave optimal CL activity. Activity was inhibited by cytochalasin B, a phagocyt~c inhibitor, and by sodlum azide, the latter indicating the involvement of oxygen products. Having thus defined suitable parameter values, the CL protocol was then applled to 0. edulis for qualitative a n d quantitative analyses of respiratory burst capacity at the species and individual levels. Results suggested inter-and intra-specific variability of CL responses. Under the same experimental conditions, 0. eduhs hemocytes generally displayed higher CL activities than C. gigas. Moreover, clear individual vanabllity was demonstrated. The same experimental number of hemocytes showed great differences in CL responses between individuals suggesting a posslble correlation with hemogram characteristics such a s cell type percentages.

Effect of a mono-specific algal diet on immune functions in two bivalve species -Crassostrea gigasandRuditapes philippinarum

“…Two main haemocyte functions can be evaluated using this technique: phagocytosis, which represents probably the most investigated functional aspect of haemocyte activity (Cheng, 1996), and the 'oxidative burst'. Generation of reactive oxygen species at the end of the oxidative burst process has been observed in several bivalves such as C. gigas (Bachère et al, 1991;Greger et al, 1995), C. virginica (Anderson et al, 1992), O. edulis (Hervio et al, 1989;Bachère et al, 1991), M. edulis (Pipe, 1992;Noël et al, 1993) and Mediterranean mussel Mytilus galloprovincialis (Arumugan et al, 2000) but has not been detected in clam species such as R. decussatus (López et al, 1994) and hard shell clam Mercenaria mercenaria (Cheng et al, 1975). Also, production of nitrite oxide, another active oxygen intermediate, has been recently demonstrated in C. gigas (Nakayama and Maruyama, 1998), M. edulis (Ottaviani et al, 1993), M. galloprovincialis (Arumugan et al, 2000) and R. decussatus (Taffala et al, 2003).…”

Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei

“…et al, 1989;Bachere et al, 1991b;Morvan et al, 1994 . The quantification of NBT reduction and the ability of haemocytes to produce ROIs can be considered as functional tests for the evaluation of immune potential at a cellular and organismal level.…”

“…Ž vertebrate blood cells Roschger et al, 1990 and marine bivalve haemocytes Fisher . and Newell, 1986;Bachere et al, 1991b . Seawater has been widely used for in vitrò assays when marine invertebrate haemocytes functionality is studied because it may provide the haemocytes with cations or other essential elements for their successful stimulation and activity.…”

Lipopolysaccharide and β-1,3-glucan binding protein in the hard clam (Meretrix meretrix): Molecular characterization and expression analysis

“…The gill represents the main interface between aquatic organisms and the external environment. Bivalve mollusk gills are one of the first lines of defense against bacterial infection; it has become increasingly evident that the hemocytes play a central role in the internal defense of bivalves (Pipe, 1990;Bachere et al, 1991;Wang et al, 2012). The upregulation of Mm-LGBP mRNA after Vibrio anguillarum challenge showed that Mm-LGBP plays a pivotal role in antibacterial immunity, further confirming the presence of the LPS recognition and binding site in Mm-LGBP.…”