A New Protocol of Computer-Assisted Image Analysis Highlights the Presence of Hemocytes in the Regenerating Cephalic Tentacles of Adult Pomacea canaliculata

Abstract

In humans, injuries and diseases can result in irreversible tissue or organ loss. This well-known fact has prompted several basic studies on organisms capable of adult regeneration, such as amphibians, bony fish, and invertebrates. These studies have provided important biological information and helped to develop regenerative medicine therapies, but important gaps concerning the regulation of tissue and organ regeneration remain to be elucidated. To this aim, new models for studying regenerative biology could prove helpful. Here, the description of the cephalic tentacle regeneration in the adult of the freshwater snail Pomacea canaliculata is presented. In this invasive mollusk, the whole tentacle is reconstructed within 3 months. Regenerating epithelial, connective, muscular and neural components are already recognizable 72 h post-amputation (hpa). Only in the early phases of regeneration, several hemocytes are retrieved in the forming blastema. In view of quantifying the hemocytes retrieved in regenerating organs, granular hemocytes present in the tentacle blastema at 12 hpa were counted, with a new and specific computer-assisted image analysis protocol. Since it can be applied in absence of specific cell markers and after a common hematoxylin-eosin staining, this protocol could prove helpful to evidence and count the hemocytes interspersed among regenerating tissues, helping to unveil the role of immune-related cells in sensory organ regeneration.

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

“…The involvement of circulating hemocytes in cell-mediated immune responses and the synthesis of immune-related molecules has already been confirmed in P. canaliculata [13,15,21,28,36]. As the number of physiological responses regulated by, or associated with, immune-related cells and molecules is increasing [37], the focus of subsequent experiments has been to investigate whether the chemical depletion of phagocytic immune-related cells could affect complex organ regeneration, in the absence of a specific immune challenge [11,38]. In previous experiments, it was demonstrated that adult P. canaliculata could regenerate the cephalic tentacle, and the presence of granular and large hemocytes therein was quantified via an in-house developed routine for semi-automated computer-assisted image analysis [11].…”

“…(D) Computerassisted 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.…”

“…In this context, the regeneration of the Pomacea canaliculata cephalic tentacles has been documented. After the experimental amputation, the cephalic tentacles rapidly regrew, starting from a blastema that contained numerous granular hemocytes [10,11]. P. canaliculata is a freshwater snail that, due to its invasiveness and relevance to human health, has been the subject of molecular, behavioral, physiological, and immunological studies [12][13][14][15][16][17][18].…”

Pomacea canaliculata Ampullar Proteome: A Nematode-Based Bio-Pesticide Induces Changes in Metabolic and Stress-Related Pathways

“…The interest towards this model is due to its invasiveness [28], neurotoxicity [29], human parasite hosting [30] and observations about its capability to regenerate [31][32][33][34]. The systemic immune response in this snail involves circulating and tissue-resident haemocytes, together with different organs [11,16,[18][19][20][21]31,35,36]. The ampulla has been described as a capacious expansion of the anterior aorta, acting as a potential compensation chamber for the haemolymph when the animal is forced to retract into its shell [34].…”

Invertebrate Models in Innate Immunity and Tissue Remodeling Research

“…Bergamini et al, 2021 [ 6 ] provided fascinating outcomes in their survey on a regenerative event in Pomacea canaliculata . They followed three months of the regeneration process for an adult cephalic tentacle, normally used in food searching, co-specific recognition and orientation.…”