Cytological alterations in Mytilus galloprovincialis granulocytes in response to chemical stress exposure

INTRODUCTION: Bivalve mollusks and specially mussels are widely used as sentinel organisms in marine environmental quality assessment (Goldberg et al., 1978). Pollutant exposure biological responses (i.e. biomarkers) are increasingly investigated in mussel tissues and their measurements largely used in marine environmental monitoring. Mussels emocytes play very important role in mollusk physiology, mainly in the internal defense, and used for general biomarker determination such as lysosomal membrane stability. The aim of the present work was to investigate possible morphological alterations in Mytilus galloprovincialis haemocytes, in particular granulocytes, which represent the main defense system of the organisms, following pollutant exposure either in laboratory or in field conditions. MATERIAL AND METHODS: As regards laboratory exposure, an homogeneous stock of M. galloprovincialis was divided in two groups: the control one and the other one exposed to cadmium chloride (600 g l-1) for 14 days. At time 0, 3, 7 and 14 days haemolymph samples were collected. As regards field exposure an homogeneous stock of M. galloprovincialis was divided in two caged groups, translocated in a reference and in a polluted sites respectively. After 30 days exposure haemolymph samples were collected from both groups. M. galloprovincialis haemolymph samples were stained with Diff Quick (Dade Behring, Newark -USA), a rapid staining solution kit, to date utilized in the diagnostic field (Jorundsson et al, 1999). Diff Quick allowed the staining of haemolymph cells, in particular granulocytes, whose morphology including cytoplasm, nucleus and acidophilic granules appeared clearly defined. Diff Quick stained granulocytes were investigated by optical microscopy and image analysis. RESULTS AND DISCUSSION: Granulocytes from cadmium exposed mussels showed a significant increase of their surface, suggesting an activation of the granulocytes defence system in this organism after pollutant exposure. Moreover, a significant decrease of the perimeter/area ratio of granulocytes bidimentional images was observed, probably due to a cadmium induced reduction of the microfilament and microspine number. Interestingly, these findings were confirmed by results obtained with granulocytes from in field exposed mussels, which also showed a significant increase in their acidophilic granules. Results obtained suggest that the observed cytological alterations in mussel granulocytes can be proposed as potential biomarker of chemical stress exposure suitable to be used in a biomarker battery in marine environmental monitoring programmes. Moreover, the Diff Quick staining protocol proposed for mussel morphological alteration measurements, being rapid, sensitive, inexpensive and easy to be used, responds to the needs of the field biomonitoring application.