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

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.

Keywords: allograft inflammatory factor-1; apple snail; hemocyanin; image analysis; immunity; inflammation; invertebrate; mollusc; phagocytosis; transglutaminase.

Figure 1

Clodronate liposome effects on circulating hemocytes of P. canaliculata. For each condition, 1 animal representative of a group of 10 is represented. Flow cytometry-based analysis of (A) control hemolymph, (B) 6 h after the injection of clodronate liposomes, and (C) 24 h after the injection of clodronate liposomes. The R1 gate included all the circulating hemocytes, excluding debris collected with the hemolymph. Clodronate liposome effects on circulating hemocytes could be observed only at 6 h after the injection and mainly on cells with a higher FSC value (B). (D) Number of events gated within R1 region at different timepoints. * p < 0,05 according to Dunnett’s test. The microscopical analysis of hemocytes from clodronate liposome-injected snails indicated an increase in the relative presence of small/intermediate hemocytes (Figure S2).

Figure 2

Automated granular hemocyte identification and count using the in-house routine. (A) non-regenerating and (B) clodronate liposome-injected regenerating tentacles at 12 hpa. Examples of cells recognized as large and granular hemocytes are indicated (green arrowheads). (C) Magnified image representing a detail of Figure (B). Examples of cells with a morphology recognized by the computer-assisted hemocyte count are indicated by green arrowheads. During the setup of the in-house developed protocol in MATLAB^® environment, the validation step was performed via a manual inspection, to visually check if the automated counting was appropriate [30]. (D) Computer-assisted hemocyte count in non-regenerating control, regenerating tentacles (12 hpa), and clodronate liposome-injected regenerating tentacles (12 hpa). Data from regenerating tentacles (12 hpa) have already been presented in a previous publication [11]; hence, they arereported in a different color. * p < 0.05 according to the Tukey–Kramer test.

Figure 3

Wound closure and blastema formation were delayed steps in the tentacle regeneration of clodronate liposome-injected P. canaliculata. For each experimental condition and time, 1 animal representative of a group of 3 is represented. Non-injected snails showed (A) a well-recognizable blastema (bl), (C) a newly formed epithelium (e) on the wound surface, and (E) a smaller blastema under the re-epithelialized wound (brackets) at 12, 24, and 48 hpa, respectively. Clodronate liposome-injected animals, (B) still presented an open wound (arrowhead) at 12 hpa. (D) The blastema (bl) was well recognizable at 24 hpa (brackets), under a closed but not completely re-epithelialized wound. (F) Re-epithelialization (e) was completed at 48 hpa. Abbreviations: connective tissue (c), muscle (m), nerve (n). Magnified details of the wounds represented in each panel are presented in Figure S3.

Figure 4

RT-qPCR analysis of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1 expressions in regenerating tentacles of P. canaliculata. (A) Schematic representation of RT-qPCR results: ns: no significant difference, ↑: significant increase; ↓: significant decrease. Data were analyzed with either Student’s t-test (^t) or Wilcoxon test (^W) (p < 0.05). (B) Left: Pc-Hemocyanin, Pc-TG, and Pc-AIF-1 basal (0 h) and amputation-modified expressions (12 and 24 h) in regenerating tentacles. (B) Right: Pc-Hemocyanin, Pc-TG, and Pc-AIF-1 basal (0 h) and post-amputation expressions (12 and 24 h) in regenerating tentacles from either control (non-injected) or clodronate liposome-injected snails. * p < 0.05 according to Student’s t-test.