Immunocompetence of bivalve hemocytes as evaluated by a miniaturized phagocytosis assay

Abstract

Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 x 10(6) hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play.

Effects of a major municipal effluent on the St. Lawrence River: A case study

“…Hemocytes of bivalves exposed for 62 days in cages at sites located downstream of the effluent plume in the SLR displayed a significant decrease in immunocompetence in relation to the upstream site (Blaise et al 2002), corroborating the 96-h laboratory exposure.…”

Aquatic ecotoxicology: what has been accomplished and what lies ahead? An Eastern Canada historical perspective

“…45 By measuring the responses of a microplatebased phagocytosis assay appraising the capacity of mussel hemocytes to ingest fluorescent bacteria, results showed that phagocytosis activity was significantly suppressed in hemocytes after the 96 h-exposure to high concentrations (≥25% v/v) of the WWTP effluent, as well as after the two month caging experiment for mussels exposed downstream of the WWTP emission. 34 Similarly, phagocytosis measurements generated with endemic Mya arenaria soft-shell clams collected from intertidal zones of the Saguenay Fjord, a major affluent of the SLR estuary (Figure 2), also displayed immunosuppression in animals located in zones impacted by urban effluents. 46 Immunosuppression in hemocytes has also been observed in other urban effluents to which caged bivalves were exposed.…”

“…For example, WOE studies can include toxicity testing on whole organisms, multiple biomarker measurements, and chemistry on water, sediment and tissue, as well as ecological parameters. 25 With continuing development in ecotoxicological research that led to increasing numbers of biomarkers, they have often been grouped into different categories based on the type of ecotoxicity information that they engender (Table 1) 21,[26][27][28][29][30][31][32][33][34][35][36][37][38][39] . Of notable interest are biomarkers that can intimate exposure to a specific class of chemicals.…”

Études de biomarqueurs chez la mye commune (Mya arenaria) du fjord du Saguenay : bilan de recherches (1997 à 2006)

“…La phagocytose, entre autres, constitue un important moyen par lequel les hémocytes -cellules de l'hémolymphe composées de granulocytes et de hyalinocytes -peuvent gérer la prise en charge et l'élimination de microorganismes indésirables (GLINSKI et JAROSZ, 1997). Lors de la campagne de juin 1997, nous avons documenté l'immunocompétence de myes récoltés à plusieurs sites du fjord à l'aide d'une technique développée en microplaque qui mesure la capacité des hémocytes à phagocyter des bactéries fluorescentes (BLAISE et al, 2002b). Ce biomarqueur d'immunocompétence nous a signalé, comparativement aux sites de références ASE et BMB, une augmentation significative de l'activité de phagocytose aux sites AE, BE et ASJ, au sujet desquels des effets indésirables chez la mye ont déjà été observés précédemment, et qui se trouvent à plus grande proximité des sources industrielles et urbaines provenant de l'amont ( Figure 1).…”