Parameters of hemocyte populations have been considered as relevant indicators of bivalve health and are currently used in immunotoxicological studies. Hemocytes in hemolymph can be collected by puncturing either the pericardial cavity or the adductor muscle sinus with a syringe. Flow cytometry is a methodological approach that is increasingly being used in laboratories for the study of hemocyte parameters in aquatic invertebrates. However, various protocols for hemocyte processing in laboratories equipped with different types of cytometers have been published. In this context, two flow cytometers (EPICS XL4®, Beckman Coulter and FacsCalibur®, Becton Dickinson) and two sites of hemocyte collection (pericardial cavity and adductor muscle sinus) were compared for the analysis of hemocyte parameters in the Pacific oyster, Crassostrea gigas. Hemolymph cells were analyzed in terms of their number and organelle contents. Cell mortality, phagocytosis, non specific esterase, extension of the lysosomal compartment and production of reactive oxygen species were quantified. The results showed that the phagocytic index was higher for hemocytes obtained in the muscle sinus hemolymph. The results are discussed with respect to the potential use of flow cytometry as a tool for hemocyte studies in bivalves.

The main purpose of this study was to establish the pattern of variability of some hemato-immunological parameters in the mangrove oyster, Crassostrea rhizophorae, from 3 different environmental conditions in Santa Catarina Island: two natural habitats (mangrove and rocks of the coastal bay) and one oyster culture station. The water quality was adequate in all localities. The animals were collected seasonally over a period of one year (n = 30, per locality). The oyster hemolymph comprised two basic cell populations, hyaline (HH) and granular hemocytes (GH). Both cell populations contained carbohydrates and glycogen in their cytoplasm (periodic acid Schiff reaction, PAS staining) and the occurrence of lysosomes was suggested by the detection of acid phosphatase (Gomori's method). Both hemocyte populations were able of phagocytosis of zymosan particles in vitro and producing cytotoxic molecules, such as the superoxide anions (nitroblue-tetrazolium, NBT reduction). The oyster hemograms significantly differed in the different habitats and also among seasons. The total circulating hemocyte counts (THC) and the percentage of GH in the mangrove and rock oysters always differed from each other, but not from those of the cultured oysters. The highest THC and the lowest percentage of GH were always found in summer. The total plasma protein concentration (PC) exhibited a similar seasonal pattern in all oyster populations, with a marked decrease in spring. The PC of the cultured oysters was almost always significantly lower than that of the other oyster populations. The plasma agglutinating titer was higher in the cultured oysters regardless of season. The histological organization of the oyster digestive gland and gills was basically similar to that of other oyster species. Their structural aspect did not show any detectable alteration, corroborating that the oysters were in good health. The results of this study will serve as a basis for further analyses on the monitoring of C. rhizophorae health status and environmental quality in different aquatic habitats.