Clodronate Liposome-Mediated Phagocytic Hemocyte Depletion Affects the Regeneration of the Cephalic Tentacle of the Invasive Snail, Pomacea canaliculata

SIMPLE SUMMARY: In the adult freshwater snail, Pomacea canaliculata, their cephalic sensory tentacles can regenerate after experimental amputation. Immediately after the amputation, the wound closes and a hemocyte-rich blastema is formed, but whether hemocytes play a role in blastema formation and the regeneration process is not known. Here, we have analyzed the effects of the chemical depletion of phagocytic hemocytes on tentacle regeneration. The transient depletion of phagocytic hemocytes was achieved by injecting the snails with clodronate liposomes before tentacle amputation. Flow cytometry demonstrated the effects of clodronate liposomes on molluscan phagocytic hemocytes. Histological analysis, followed by an application of an in-house developed semi-automated hemocyte count protocol, documented that in phagocytic hemocyte-depleted snails, the regeneration process was significantly delayed. When the number of circulating phagocytic hemocytes was restored to the control values, the regeneration process recovered. The expression in the blastema of genes associated with hemocyte-mediated functions, like oxygen transport, clotting and inflammation, was evaluated using RT-qPCR. Consistent with flow cytometry and histochemical observations, the blastema from clodronate liposome-injected snails, presented significantly modified gene expression levels, thus reinforcing the hypothesis of an active role of hemocytes in the onset of tentacle regeneration. ABSTRACT: After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates.