Evidence of immunosurveillance to the pharynx entry by external granulocytes in ascidians (Urochordata)

The body of ascidians is covered by the tunic, which is produced by epidermis and extends also into the siphons as far as the tentacular ring. In botryllids, at the surface exposed to the seawater, the tunic forms a thin dense cuticle bearing numerous small papillate protrusions. In the siphons, the cutiular surface is well developed owing to folding and indentations of the tunic and various cell types, particularly granulocytes, can be found in tunic matrix, some of them were seen to cross the epidermis coming from blood. In the oral siphon of some botryllid species, we observed, as an unusual and interesting feature, the presence of external amoeboid granulocytes completely exposed to seawater flow. These granulocytes were in the deepest region of the siphon, especially located closely to the tentacular ring. They contacted the cuticle protrusions by means long filopodia, which often penetrated into the narrow spaces of the tunic folds. At the electron microscope, granulocytes appeared various in both shape and size: mononucleate, they possessed numerous round granules, mostly homogeneous and strongly dense, but other heterogeneous in content. Granules were positive to the acid phosphatase activity. In all the cases, the ulttrastructure revealed a good preservetion of these cells suggestive of a normal physiological state. With the aim to verify the functional role of these ganulocytes, we exposed, for various times, filtering colonies of Botryllus schlosseri to carmine or Bacillus subtilis spores, suspended in seawater. These colonies were then embedded in paraffin or Epon and cut serially to examine the siphon area at liht and electron microscope, respectively. Beginning from 5 min of exposition, the external granulocytes revealed the presence of bacteria in heterophagic vacuoles. This feature was observed at every exposition time. However, the number of external granulocytes decreased significantly with time and, in some cases, they were no more recognisable after 60 min of exposure. Moreover, bacteria were also seen in some granulocytes in the tunic, while the epidermis around the siphon continued to be crossed by granulocytes coming from blood. Therefore, all the findings gave evidence that, in botryllids, external granulocytes represent "sentinel-cells" which are exposed to seawater and play a role of immunosurveillance to the pharynx entry, engulfing potential pathogens by phagocytosis.