A comparative study on hemolymph sampling methods: Monthly variations in hemocyte characteristics of farmed oyster...

Immune bivalve mollusk response is closely related to environmental conditions (i.e. seawater temperature and salinity, availability and food quality); including routine handling that occurs during sea farming, such as manipulation for size sorting and cleaning epibionts from shells. Storage (hypoxia, dry conditions, etc.), transportation, and acclimatization to laboratory conditions of wild or reared organisms are also known to exert significant changes in bivalve mollusk immune cell responses. Therefore, the aim of the present study is to assess the effect of hemolymph withdrawal handled in two different conditions (field vs. laboratory) in monthly variations of both morphological and functional hemocyte characteristics in oyster C. corteziensis. Hemolymph samples were extracted from oysters immediately sampled at the farming site (field), and from oysters stored in dry-cold conditions and terrestrially transported during 2 h to the laboratory facilities. Total (THC) and differential (DHC) hemocyte counts, hemocyte mortality (HM), and anion superoxide production (ASO/hemocyte) were assessed in hemolymph samples obtained from oysters in both handled conditions (field vs. laboratory). Sea surface temperature (SST), salinity, pH, seston (total, inorganic, and organic particulate matter), and chlorophyll-a (Chl-a) were monthly assessed in seawater samples. THC, DHC, HM, and ASO/hemocyte production were not different between hemolymph samples withdrawal from oysters handled in both conditions. All immune parameters assessed were significantly different between sampling dates, but most were not significantly correlated with physicochemical seawater parameters assessed, except THC and hyalinocyte count (HC), which were positively correlated with phytoplankton availability (i.e. Chl-a) that increased during winter, and granulocytes that increased with SST rise but negatively correlated with OCI, along with seston availability. HM decreased with THC and HC increase, suggesting a link with hemocyte proliferation but without compromising immune response. Hemocyte oxidative capacity (i.e. ASO/hemocyte) increased significantly with oyster condition, which coincided with the increase of phytoplankton availability (i.e. Chl-a). Although increasing energy allocation improves oyster physiological condition -including immune response- circulating hemocyte increased and their respective oxidative capacity were more likely to be due to a response to availability and consumption on certain toxin-producing phytoplankton species (dinoflagellates). Hemolymph withdrawal from oysters directly in field could be advantageous, mainly when no other tissues than immune cells and plasma are required to be analyzed, avoiding whole oyster transportation.